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Sample records for complement lectin pathway

  1. Involvement of the Lectin Pathway of Complement Activation in Antimicrobial Immune Defense during Experimental Septic Peritonitis

    PubMed Central

    Windbichler, Michaela; Echtenacher, Bernd; Hehlgans, Thomas; Jensenius, Jens C.; Schwaeble, Wilhelm; Männel, Daniela N.

    2004-01-01

    A critical first line of defense against infection is constituted by the binding of natural antibodies to microbial surfaces, activating the complement system via the classical complement activation pathway. In this function, the classical activation pathway is supported and amplified by two antibody-independent complement activation routes, i.e., the lectin pathway and the alternative pathway. We studied the contribution of the different complement activation pathways in the host defense against experimental polymicrobial peritonitis induced by cecal ligation and puncture by using mice deficient in either C1q or factors B and C2. The C1q-deficient mice lack the classical complement activation pathway. While infection-induced mortality of wild-type mice was 27%, mortality of C1q-deficient mice was increased to 60%. Mice with a deficiency of both factors B and C2 lack complement activation via the classical, the alternative, and the lectin pathways and exhibit a mortality of 92%, indicating a significant contribution of the lectin and alternative pathways of complement activation to survival. For 14 days after infection, mannan-binding lectin (MBL)-dependent activation of C4 was compromised. Serum MBL-A and MBL-C levels were significantly reduced for 1 week, possibly due to consumption. mRNA expression profiles did not lend support for either of the two MBL genes to respond as typical acute-phase genes. Our results demonstrate a long-lasting depletion of MBL-A and MBL-C from serum during microbial infection and underline the importance of both the lectin and the alternative pathways for antimicrobial immune defense. PMID:15322019

  2. Scabies mite inactive serine proteases are potent inhibitors of the human complement lectin pathway.

    PubMed

    Reynolds, Simone L; Pike, Robert N; Mika, Angela; Blom, Anna M; Hofmann, Andreas; Wijeyewickrema, Lakshmi C; Kemp, Dave; Fischer, Katja

    2014-05-01

    Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classified as one of the six most prevalent epidermal parasitic skin diseases infecting populations living in poverty by the World Health Organisation. The role of the complement system, a pivotal component of human innate immunity, as an important defence against invading pathogens has been well documented and many parasites have an arsenal of anti-complement defences. We previously reported on a family of scabies mite proteolytically inactive serine protease paralogues (SMIPP-Ss) thought to be implicated in host defence evasion. We have since shown that two family members, SMIPP-S D1 and I1 have the ability to bind the human complement components C1q, mannose binding lectin (MBL) and properdin and are capable of inhibiting all three human complement pathways. This investigation focused on inhibition of the lectin pathway of complement activation as it is likely to be the primary pathway affecting scabies mites. Activation of the lectin pathway relies on the activation of MBL, and as SMIPP-S D1 and I1 have previously been shown to bind MBL, the nature of this interaction was examined using binding and mutagenesis studies. SMIPP-S D1 bound MBL in complex with MBL-associated serine proteases (MASPs) and released the MASP-2 enzyme from the complex. SMIPP-S I1 was also able to bind MBL in complex with MASPs, but MASP-1 and MASP-2 remained in the complex. Despite these differences in mechanism, both molecules inhibited activation of complement components downstream of MBL. Mutagenesis studies revealed that both SMIPP-Ss used an alternative site of the molecule from the residual active site region to inhibit the lectin pathway. We propose that SMIPP-Ss are potent lectin pathway inhibitors and that this mechanism represents an important tool in the immune evasion repertoire of the parasitic mite and a potential target for therapeutics. PMID:24854034

  3. Near-planar Solution Structures of Mannose-binding Lectin Oligomers Provide Insight on Activation of Lectin Pathway of Complement

    PubMed Central

    Miller, Ami; Phillips, Anna; Gor, Jayesh; Wallis, Russell; Perkins, Stephen J.

    2012-01-01

    The complement system is a fundamental component of innate immunity that orchestrates complex immunological and inflammatory processes. Complement comprises over 30 proteins that eliminate invading microorganisms while maintaining host cell integrity. Protein-carbohydrate interactions play critical roles in both the activation and regulation of complement. Mannose-binding lectin (MBL) activates the lectin pathway of complement via the recognition of sugar arrays on pathogenic surfaces. To determine the solution structure of MBL, synchrotron x-ray scattering and analytical ultracentrifugation experiments showed that the carbohydrate-recognition domains in the MBL dimer, trimer, and tetramer are positioned close to each other in near-planar fan-like structures. These data were subjected to constrained modeling fits. A bent structure for the MBL monomer was identified starting from two crystal structures for its carbohydrate-recognition domain and its triple helical region. The MBL monomer structure was used to identify 10–12 near-planar solution structures for each of the MBL dimers, trimers, and tetramers starting from 900 to 6,859 randomized structures for each. These near-planar fan-like solution structures joined at an N-terminal hub clarified how the carbohydrate-recognition domain of MBL binds to pathogenic surfaces. They also provided insight on how MBL presents a structural template for the binding and auto-activation of the MBL-associated serine proteases to initiate the lectin pathway of complement activation. PMID:22167201

  4. Analogous Interactions in Initiating Complexes of the Classical and Lectin Pathways of Complement1

    PubMed Central

    Phillips, Anna E.; Toth, Julia; Dodds, Alister W.; Girija, Umakhanth Venkatraman; Furze, Christopher; Pala, Eleni; Sim, Robert B.; Reid, Kenneth B. M.; Schwaeble, Wilhelm J.; Schmid, Ralf; Keeble, Anthony H.; Wallis, Russell

    2010-01-01

    The classical and lectin pathways of complement activation neutralise pathogens and stimulate key immunological processes. Both pathways are initiated by collagen-containing, soluble pattern-recognition molecules associated with specific serine proteases. In the classical pathway, C1q binds to antibody-antigen complexes or bacterial surfaces to activate C1r and C1s. In the lectin pathway, mannan-binding lectin (MBL) and ficolins bind to carbohydrates on pathogens to activate MASP-2. To characterise the interactions leading to classical pathway activation, we have analysed binding between human C1q, C1r and C1s, which associate to form C1, using full-length and truncated protease components. We show that C1r and C1s bind to C1q independently. The CUB1-EGF fragments contribute most towards binding, but CUB2 of C1r, but not of C1s, is also important. Each C1rs tetramer presents a total of six binding sites, one for each of the collagenous domains of C1q. We also demonstrate that subcomponents of the lectin and classical pathways cross-interact. Thus, although the stoichiometries of complexes differ, interactions are analogous, with equivalent contacts between recognition and protease subcomponents. Importantly, these new data are contrary to existing models of C1, and enable us to propose a new model, using MBL-MASP interactions as a template. PMID:19494295

  5. Structural insights into the initiating complex of the lectin pathway of complement activation.

    PubMed

    Kjaer, Troels R; Le, Le T M; Pedersen, Jan Skov; Sander, Bjoern; Golas, Monika M; Jensenius, Jens Christian; Andersen, Gregers R; Thiel, Steffen

    2015-02-01

    The proteolytic cascade of the complement system is initiated when pattern-recognition molecules (PRMs) bind to ligands, resulting in the activation of associated proteases. In the lectin pathway of complement, the complex of mannan-binding lectin (MBL) and MBL-associated serine protease-1 (MASP-1) initiates the pathway by activating a second protease, MASP-2. Here we present a structural study of a PRM/MASP complex and derive the overall architecture of the 450 kDa MBL/MASP-1 complex using small-angle X-ray scattering and electron microscopy. The serine protease (SP) domains from the zymogen MASP-1 dimer protrude from the cone-like MBL tetramer and are separated by at least 20 nm. This suggests that intracomplex activation within a single MASP-1 dimer is unlikely and instead supports intercomplex activation, whereby the MASP SP domains are accessible to nearby PRM-bound MASPs. This activation mechanism differs fundamentally from the intracomplex initiation models previously proposed for both the lectin and the classical pathway. PMID:25579818

  6. Fibrinogen-specific antibody induces abdominal aortic aneurysm in mice through complement lectin pathway activation

    PubMed Central

    Zhou, Hui-fang; Yan, Huimin; Bertram, Paula; Hu, Ying; Springer, Luke E.; Thompson, Robert W.; Curci, John A.; Hourcade, Dennis E.; Pham, Christine T. N.

    2013-01-01

    Abdominal aortic aneurysm (AAA) is a common vascular disease associated with high mortality rate due to progressive enlargement and eventual rupture. There is currently no established therapy known to alter the rate of aneurysmal expansion. Thus, understanding the processes that initiate and sustain aneurysmal growth is pivotal for the development of medical therapies aimed at halting disease progression. Using an elastase-induced AAA mouse model that recapitulates key features of human AAA, we previously reported that a natural IgG antibody directs alternative pathway complement activation and initiates the inflammatory process that culminates in aneurysmal development. The target of this natural antibody, however, was unknown. Herein we identify a natural IgG that binds to fibrinogen deposited in elastase-perfused aortic tissues, activates the complement lectin pathway (LP), and induces AAA. Moreover, we establish that alterations in the glycosylation patterns of this antibody critically affect its ability to activate the LP in vivo. We find that LP activation precedes the alternative pathway and absence of the LP complement protein mannan-binding lectin abrogates elastase-induced AAA. In human AAA tissues the mouse anti-fibrinogen antibody recognizes epitopes that localize to the same areas that stain positively for mannan-binding lectin, which suggests that the complement LP is engaged in humans as well. Lastly, we demonstrate that circulating antibodies in a subset of AAA patients react against fibrinogen or fibrinogen-associated epitopes in human aneurysmal tissues. Our findings support the concept that an autoimmune process directed at aortic wall self-antigens may play a central role in the immunopathogenesis of AAA. PMID:24167262

  7. Structural Basis for the Function of Complement Component C4 within the Classical and Lectin Pathways of Complement.

    PubMed

    Mortensen, Sofia; Kidmose, Rune T; Petersen, Steen V; Szilágyi, Ágnes; Prohászka, Zoltan; Andersen, Gregers R

    2015-06-01

    Complement component C4 is a central protein in the classical and lectin pathways within the complement system. During activation of complement, its major fragment C4b becomes covalently attached to the surface of pathogens and altered self-tissue, where it acts as an opsonin marking the surface for removal. Moreover, C4b provides a platform for assembly of the proteolytically active convertases that mediate downstream complement activation by cleavage of C3 and C5. In this article, we present the crystal and solution structures of the 195-kDa C4b. Our results provide the molecular details of the rearrangement accompanying C4 cleavage and suggest intramolecular flexibility of C4b. The conformations of C4b and its paralogue C3b are shown to be remarkably conserved, suggesting that the convertases from the classical and alternative pathways are likely to share their overall architecture and mode of substrate recognition. We propose an overall molecular model for the classical pathway C5 convertase in complex with C5, suggesting that C3b increases the affinity for the substrate by inducing conformational changes in C4b rather than a direct interaction with C5. C4b-specific features revealed by our structural studies are probably involved in the assembly of the classical pathway C3/C5 convertases and C4b binding to regulators. PMID:25911760

  8. Components of the lectin pathway of complement activation in paediatric patients of intensive care units.

    PubMed

    Świerzko, Anna S; Szala-Poździej, Agnieszka; Kilpatrick, David C; Sobociński, Michał; Chojnacka, Karolina; Sokołowska, Anna; Michalski, Mateusz; Mazerant, Karolina; Jensenius, Jens C; Matsushita, Misao; Krajewski, Wojciech R; Szczapa, Jerzy; Bąk-Romaniszyn, Leokadia; Zeman, Krzysztof; Cedzyński, Maciej

    2016-05-01

    Infections are a major cause of childhood mortality. We investigated components of the lectin pathway of complement activation in the context of sepsis at both genetic and protein levels in neonates, infants and older children. Major components of the lectin pathway and two genes for Toll-like receptors were studied in 87 neonates with confirmed sepsis and compared with 40 babies with infections who did not develop sepsis (disease controls) and 273 infection-free neonatal controls. A second cohort comprised 47 older children with sepsis and 87 controls. Low MBL-conferring genotypes (LXA/O+O/O) were more frequent in sepsis patients than in healthy controls but no significant differences in the frequency of SNPs of other lectin pathway genes (FCN1, FCN2, FCN3, MASP1/3, MASP2) or TLR receptor genes (TLR2, TLR4) were found. One case of primary MASP-2 deficiency was found among healthy pre-terms and one neonate suffering from SIRS was heterozygous for the rare FCN1 gene mutation, +6658 G>A. Generally, sepsis was associated with low serum MBL and low ficolin-2 concentrations on admission. Among neonates, ficolin-1 and MASP-2 levels were elevated in sepsis relative to healthy, but not disease, controls. Unlike neonates, ficolin-3 and MASP-2 levels were lower in older patients than in healthy controls while no difference was found for ficolin-1. With the possible exception of MBL, inherited lectin pathway insufficiencies do not seem to predispose to sepsis, rather changes in protein concentrations reflect alterations in disease course. PMID:26850322

  9. Role of ficolin-A and lectin complement pathway in the innate defense against pathogenic Aspergillus species.

    PubMed

    Bidula, Stefan; Kenawy, Hany; Ali, Youssif M; Sexton, Darren; Schwaeble, Wilhelm J; Schelenz, Silke

    2013-05-01

    Aspergillus species are saprophytic molds causing life-threatening invasive fungal infections in the immunocompromised host. Innate immune recognition, in particular, the mechanisms of opsonization and complement activation, has been reported to be an integral part of the defense against fungi. We have shown that the complement component ficolin-A significantly binds to Aspergillus conidia and hyphae in a concentration-dependent manner and was inhibited by N-acetylglucosamine and N-acetylgalactosamine. Calcium-independent binding to Aspergillus fumigatus and A. terreus was observed, but binding to A. flavus and A. niger was calcium dependent. Ficolin-A binding to conidia was increased under low-pH conditions, and opsonization led to enhanced binding of conidia to A549 airway epithelial cells. In investigations of the lectin pathway of complement activation, ficolin-A-opsonized conidia did not lead to lectin pathway-specific C4 deposition. In contrast, the collectin mannose binding lectin C (MBL-C) but not MBL-A led to efficient lectin pathway activation on A. fumigatus in the absence of ficolin-A. In addition, ficolin-A opsonization led to a modulation of the proinflammatory cytokine interleukin-8. We conclude that ficolin-A may play an important role in the innate defense against Aspergillus by opsonizing conidia, immobilizing this fungus through enhanced adherence to epithelial cells and modulation of inflammation. However, it appears that other immune pattern recognition molecules, i.e., those of the collectin MBL-C, are involved in the Aspergillus-lectin complement pathway activation rather than ficolin-A. PMID:23478320

  10. Human L-ficolin, a Recognition Molecule of the Lectin Activation Pathway of Complement, Activates Complement by Binding to Pneumolysin, the Major Toxin of Streptococcus pneumoniae

    PubMed Central

    Ali, Youssif M.; Kenawy, Hany I.; Muhammad, Adnan; Sim, Robert B.

    2013-01-01

    The complement system is an essential component of the immune response, providing a critical line of defense against different pathogens including S. pneumoniae. Complement is activated via three distinct pathways: the classical (CP), the alternative (AP) and the lectin pathway (LP). The role of Pneumolysin (PLY), a bacterial toxin released by S. pneumoniae, in triggering complement activation has been studied in vitro. Our results demonstrate that in both human and mouse sera complement was activated via the CP, initiated by direct binding of even non-specific IgM and IgG3 to PLY. Absence of CP activity in C1q−/− mouse serum completely abolished any C3 deposition. However, C1q depleted human serum strongly opsonized PLY through abundant deposition of C3 activation products, indicating that the LP may have a vital role in activating the human complement system on PLY. We identified that human L-ficolin is the critical LP recognition molecule that drives LP activation on PLY, while all of the murine LP recognition components fail to bind and activate complement on PLY. This work elucidates the detailed interactions between PLY and complement and shows for the first time a specific role of the LP in PLY-mediated complement activation in human serum. PMID:24349316

  11. Mouse Ficolin B Has an Ability to Form Complexes with Mannose-Binding Lectin-Associated Serine Proteases and Activate Complement through the Lectin Pathway

    PubMed Central

    Endo, Yuichi; Iwaki, Daisuke; Ishida, Yumi; Takahashi, Minoru; Matsushita, Misao; Fujita, Teizo

    2012-01-01

    Ficolins are thought to be pathogen-associated-molecular-pattern-(PAMP-) recognition molecules that function to support innate immunity. Like mannose-binding lectins (MBLs), most mammalian ficolins form complexes with MBL-associated serine proteases (MASPs), leading to complement activation via the lectin pathway. However, the ability of murine ficolin B, a homologue of human M-ficolin, to perform this function is still controversial. The results of the present study show that ficolin B in mouse bone marrow is an oligomeric protein. Ficolin B, pulled down using GlcNAc-agarose, contained very low, but detectable, amounts of MASP-2 and small MBL-associated protein (sMAP) and showed detectable C4-deposition activity on immobilized N-acetylglucosamine. These biochemical features of ficolin B were confirmed using recombinant mouse ficolin B produced in CHO cells. Taken together, these results suggest that like other mammalian homologues, murine ficolin B has an ability to exert its function via the lectin pathway. PMID:22523468

  12. Critical Role and Therapeutic Control of the Lectin Pathway of Complement Activation in an Abortion-Prone Mouse Mating.

    PubMed

    Petitbarat, Marie; Durigutto, Paolo; Macor, Paolo; Bulla, Roberta; Palmioli, Alessandro; Bernardi, Anna; De Simoni, Maria-Grazia; Ledee, Nathalie; Chaouat, Gerard; Tedesco, Francesco

    2015-12-15

    The abortion-prone mating combination CBA/J × DBA/2 has been recognized as a model of preeclampsia, and complement activation has been implicated in the high rate of pregnancy loss observed in CBA/J mice. We have analyzed the implantation sites collected from DBA/2-mated CBA/J mice for the deposition of the complement recognition molecules using CBA/J mated with BALB/c mice as a control group. MBL-A was observed in the implantation sites of CBA/J × DBA/2 combination in the absence of MBL-C and was undetectable in BALB/c-mated CBA/J mice. Conversely, C1q was present in both mating combinations. Searching for other complement components localized at the implantation sites of CBA/J × DBA/2, we found C4 and C3, but we failed to reveal C1r. These data suggest that complement is activated through the lectin pathway and proceeds to completion of the activation sequence as revealed by C9 deposition. MBL-A was detected as early as 3.5 d of pregnancy, and MBL-A deficiency prevented pregnancy loss in the abortion-prone mating combination. The contribution of the terminal complex to miscarriage was supported by the finding that pregnancy failure was largely inhibited by the administration of neutralizing Ab to C5. Treatment of DBA/2-mated CBA/J mice with Polyman2 that binds to MBL-A with high affinity proved to be highly effective in controlling the activation of the lectin pathway and in preventing fetal loss. PMID:26561549

  13. Genetically engineered fusion of MAP-1 and factor H domains 1-5 generates a potent dual upstream inhibitor of both the lectin and alternative complement pathways.

    PubMed

    Nordmaj, Mie Anemone; Munthe-Fog, Lea; Hein, Estrid; Skjoedt, Mikkel-Ole; Garred, Peter

    2015-12-01

    Inhibition of the complement cascade has emerged as an option for treatment of a range of diseases. Mannose-binding lectin/ficolin/collectin-associated protein (MAP-1) is a pattern recognition molecule (PRM)-associated inhibitor of the lectin pathway. The central regulator of the alternative pathway (AP) is complement factor H (FH). Our aim was to design a dual upstream inhibitor of both human lectin and APs by fusing MAP-1 with a part of FH. There were 2 different recombinant chimeric proteins comprising full-length human MAP-1 and the first 5 N-terminal domains of human FH designed. The FH domains were orientated either in the N- or C-terminal part of MAP-1. The complement inhibition potential in human serum was assessed. Both chimeric constructs displayed the characteristics of the native molecules and bound to the PRMs with an EC50 of ∼ 2 nM. However, when added to serum diluted 1:4 in a solid-phase functional assay, only the first 5 N-terminal domains of complement FH fused to the C-terminal part of full-length MAP-1 chimeric construct were able to combine inhibition of lectin and AP activation with an half maximal inhibitory concentration of ∼ 100 and 20 nM, respectively. No effect was seen on the classical pathway. Fusion of MAP-1 with FH domains represents a novel therapeutic approach for selective targeting upstream and central complement activation at sites of inflammation. PMID:26260032

  14. Studies of the binding of ficolin-2 and ficolin-3 from the complement lectin pathway to Leptospira biflexa, Pasteurella pneumotropica and Diarrheagenic Escherichia coli.

    PubMed

    Sahagún-Ruiz, Alfredo; Breda, Leandro Carvalho Dantas; Valencia, Mónica Marcela Castiblanco; Elias, Waldir P; Munthe-Fog, Lea; Garred, Peter; Barbosa, Angela Silva; Isaac, Lourdes

    2015-10-01

    Ficolins recognize pathogen associated molecular patterns and activate the lectin pathway of complement system. However, our knowledge regarding pathogen recognition of human ficolins is still limited. We therefore set out to explore and investigate the possible interactions of the two main serum ficolins, ficolin-2 and ficolin-3 with different Gram-negative bacteria. We used recombinant ficolin molecules and normal human serum, which were detected with anti-ficolin monoclonal antibodies. In addition we investigated the capacity of these pathogens to activate the lectin pathway of complement system. We show for the first time that human ficolin-2 recognizes the nonpathogenic spirochete Leptospira biflexa serovar Patoc, but not the pathogenic Leptospira interrogans serovar Kennewicki strain Fromm. Additionally, human ficolin-2 and ficolin-3 recognize pathogenic Pasteurella pneumotropica, enteropathogenic Escherichia coli (EPEC) serotype O111ab:H2 and enteroaggregative E. coli (EAEC) serogroup O71 but not four enterohemorrhagic E. coli, three EPEC, three EAEC and two nonpathogenic E. coli strains (DH5α and HB101). The lectin pathway was activated by Pasteurella pneumotropica, EPEC O111ab:H2 and EAEC O71 after incubation with C1q depleted human serum. In conclusion, this study provide novel insight in the binding and complement activating capacity of the lectin pathway initiation molecules ficolin-2 and ficolin-3 towards relevant Gram-negative pathogens of pathophysiological relevance. PMID:26074063

  15. Inhibition of the classical and lectin pathway of the complement system by recombinant LAIR-2.

    PubMed

    Olde Nordkamp, Marloes J M; Boross, Peter; Yildiz, Cafer; Jansen, J H Marco; Leusen, Jeanette H W; Wouters, Diana; Urbanus, Rolf T; Hack, C Erik; Meyaard, Linde

    2014-01-01

    Activation of complement may cause severe tissue damage in antibody-mediated allograft rejection and other antibody-mediated clinical conditions; therefore, novel potent complement inhibitors are needed. Previously, we described binding of the inhibitory receptor LAIR-1 and its soluble family member LAIR-2 to collagen. Here, we investigated binding of LAIR-1 and LAIR-2 to the complement proteins C1q and MBL, which both have collagen-like domains, and evaluated the effect of this binding on complement function. We demonstrate specific binding of recombinant LAIR proteins to both C1q and MBL. Surface plasmon resonance experiments showed that LAIR-2-Fc protein bound C1q and MBL with the highest affinity compared to LAIR-2-HIS. We, therefore, hypothesized that LAIR-2-Fc is a potent complement inhibitor. Indeed, LAIR-2-Fc inhibited C4 fixation to IgG or mannan, reduced activation of C4 by aggregated IgG in plasma and inhibited iC3b deposition on cells. Finally, LAIR-2-Fc inhibited complement-mediated lysis of cells sensitized with anti-HLA antibodies in an ex vivo model for antibody-mediated transplant rejection. Thus, LAIR-2-Fc is an effective novel complement inhibitor for the treatment and prevention of antibody-mediated allograft rejection and antibody-mediated clinical conditions. PMID:24192271

  16. The Group B Streptococcus-Secreted Protein CIP Interacts with C4, Preventing C3b Deposition via the Lectin and Classical Complement Pathways.

    PubMed

    Pietrocola, Giampiero; Rindi, Simonetta; Rosini, Roberto; Buccato, Scilla; Speziale, Pietro; Margarit, Immaculada

    2016-01-01

    The group B Streptococcus (GBS) is a leading cause of neonatal invasive disease. GBS bacteria are surrounded by a thick capsular polysaccharide that is a potent inhibitor of complement deposition via the alternative pathway. Several of its surface molecules can however activate the classical and lectin complement pathways, rendering this species still vulnerable to phagocytic killing. In this study we have identified a novel secreted protein named complement interfering protein (CIP) that downregulates complement activation via the classical and lectin pathways, but not the alternative pathway. The CIP protein showed high affinity toward C4b and inhibited its interaction with C2, presumably preventing the formation of the C4bC2a convertase. Addition of recombinant CIP to GBS cip-negative bacteria resulted in decreased deposition of C3b on their surface and in diminished phagocytic killing in a whole-blood assay. Our data reveal a novel strategy exploited by GBS to counteract innate immunity and could be valuable for the development of anti-infective agents against this important pathogen. PMID:26608922

  17. The Group B Streptococcus–Secreted Protein CIP Interacts with C4, Preventing C3b Deposition via the Lectin and Classical Complement Pathways

    PubMed Central

    Pietrocola, Giampiero; Rindi, Simonetta; Rosini, Roberto; Buccato, Scilla

    2016-01-01

    The group B Streptococcus (GBS) is a leading cause of neonatal invasive disease. GBS bacteria are surrounded by a thick capsular polysaccharide that is a potent inhibitor of complement deposition via the alternative pathway. Several of its surface molecules can however activate the classical and lectin complement pathways, rendering this species still vulnerable to phagocytic killing. In this study we have identified a novel secreted protein named complement interfering protein (CIP) that downregulates complement activation via the classical and lectin pathways, but not the alternative pathway. The CIP protein showed high affinity toward C4b and inhibited its interaction with C2, presumably preventing the formation of the C4bC2a convertase. Addition of recombinant CIP to GBS cip-negative bacteria resulted in decreased deposition of C3b on their surface and in diminished phagocytic killing in a whole-blood assay. Our data reveal a novel strategy exploited by GBS to counteract innate immunity and could be valuable for the development of anti-infective agents against this important pathogen. PMID:26608922

  18. Complement Activation by Giardia duodenalis Parasites through the Lectin Pathway Contributes to Mast Cell Responses and Parasite Control.

    PubMed

    Li, Erqiu; Tako, Ernest A; Singer, Steven M

    2016-04-01

    Infection withGiardia duodenalisis one of the most common causes of diarrheal disease in the world. While numerous studies have identified important contributions of adaptive immune responses to parasite control, much less work has examined innate immunity and its connections to the adaptive response during this infection. We explored the role of complement in immunity toGiardiausing mice deficient in mannose-binding lectin (Mbl2) or complement factor 3a receptor (C3aR). Both strains exhibited delayed clearance of parasites and a reduced ability to recruit mast cells in the intestinal submucosa. C3aR-deficient mice had normal production of antiparasite IgA, butex vivoT cell recall responses were impaired. These data suggest that complement is a key factor in the innate recognition ofGiardiaand that recruitment of mast cells and activation of T cell immunity through C3a are important for parasite control. PMID:26831470

  19. Activation of the lectin pathway of complement by cardiopulmonary bypass contributes to the development of systemic inflammatory response syndrome after paediatric cardiac surgery.

    PubMed

    Pągowska-Klimek, I; Świerzko, A S; Michalski, M; Głowacka, E; Szala-Poździej, A; Sokołowska, A; Moll, M; Krajewski, W R; Romak, J; Cedzyński, M

    2016-05-01

    The systemic inflammatory response is a challenge in the management of paediatric patients undergoing cardiac surgery. Although multi-factorial, a contribution by the lectin pathway of complement activation has been postulated. We therefore investigated the changes in serum levels of mannose binding lectin (MBL) and activities of MBL-MBL-associated serine protease (MASP)-1 and MBL-MASP-2 complexes immediately before and during surgery, throughout the first postoperative day and at discharge from the hospital. These changes were analysed in relation to postoperative complications. Blood samples were obtained from 185 children with congenital heart disease undergoing surgical correction with the use of cardiopulmonary bypass: preoperatively (MBL-1), 15 min after initiation of cardiopulmonary bypass (CPB) (MBL-E), 30 min (MBL-2), 4 h (MBL-3), 12 h (MBL-4) and 24 h (MBL-5) post-CPB and at discharge from hospital (MBL-K). Alterations in serum MBL levels were calculated as a ratio of its serum level at subsequent time-points (MBL-2, -3, -4, -5) to the preoperative (MBL-1) value. Decreases in MBL and MBL-MASP complexes were observed in all samples, correlating with a decrease in C4 and increase in C4a, confirming activation of the lectin pathway. Changes in MBL levels between children with an uncomplicated postoperative course and those suffering from infection or low cardiac output syndrome did not differ significantly, but significant differences were observed between the SIRS and non-SIRS groups. Paediatric cardiac surgery with the use of cardiopulmonary bypass activates the complement system via the lectin pathway and the latter contributes to the development of the post-bypass systemic inflammatory response. PMID:26703090

  20. Heparin-coated cardiopulmonary bypass circuits selectively deplete the pattern recognition molecule ficolin-2 of the lectin complement pathway in vivo.

    PubMed

    Hein, E; Munthe-Fog, L; Thiara, A S; Fiane, A E; Mollnes, T E; Garred, P

    2015-02-01

    The complement system can be activated via the lectin pathway by the recognition molecules mannose-binding lectin (MBL) and the ficolins. Ficolin-2 exhibits binding against a broad range of ligands, including biomaterials in vitro, and low ficolin-2 levels are associated with increased risk of infections. Thus, we investigated the biocompatibility of the recognition molecules of the lectin pathway in two different types of cardiopulmonary bypass circuits. Bloods were drawn at five time-points before, during and postoperatively from 30 patients undergoing elective cardiac surgery. Patients were randomized into two groups using different coatings of cardiopulmonary bypass circuits, Phisio® (phosphorylcholine polymer coating) and Bioline® (albumin-heparin coating). Concentrations of MBL, ficolin-1, -2 and -3 and soluble C3a and terminal complement complex (TCC) in plasma samples were measured. Ficolin-3-mediated complement activation potential was evaluated with C4, C3 and TCC as output. There was no significant difference between the two circuit materials regarding MBL, ficolin-1 and -3. In the Bioline® group the ficolin-2 levels decreased significantly after initiation of surgery (P < 0.0001) and remained reduced throughout the sampling period. This was not seen for Phisio®-coated circuits. Ficolin-3-mediated complement activation potential was reduced significantly in both groups after start of operation (P < 0.0001), whereas soluble C3a and TCC in the samples were increased (P < 0.0001). Ficolin-2 was depleted from plasma during cardiac surgery when using heparin-coated bypass circuits and did not reach baseline level 24 h postoperation. These findings may have implications for the postoperative susceptibility to infections in patients undergoing extracorporeal circulation procedures. PMID:25174443

  1. Heparin-coated cardiopulmonary bypass circuits selectively deplete the pattern recognition molecule ficolin-2 of the lectin complement pathway in vivo

    PubMed Central

    Hein, E; Munthe-Fog, L; Thiara, A S; Fiane, A E; Mollnes, T E; Garred, P

    2015-01-01

    The complement system can be activated via the lectin pathway by the recognition molecules mannose-binding lectin (MBL) and the ficolins. Ficolin-2 exhibits binding against a broad range of ligands, including biomaterials in vitro, and low ficolin-2 levels are associated with increased risk of infections. Thus, we investigated the biocompatibility of the recognition molecules of the lectin pathway in two different types of cardiopulmonary bypass circuits. Bloods were drawn at five time-points before, during and postoperatively from 30 patients undergoing elective cardiac surgery. Patients were randomized into two groups using different coatings of cardiopulmonary bypass circuits, Phisio® (phosphorylcholine polymer coating) and Bioline® (albumin-heparin coating). Concentrations of MBL, ficolin-1, −2 and −3 and soluble C3a and terminal complement complex (TCC) in plasma samples were measured. Ficolin-3-mediated complement activation potential was evaluated with C4, C3 and TCC as output. There was no significant difference between the two circuit materials regarding MBL, ficolin-1 and −3. In the Bioline® group the ficolin-2 levels decreased significantly after initiation of surgery (P < 0·0001) and remained reduced throughout the sampling period. This was not seen for Phisio®-coated circuits. Ficolin-3-mediated complement activation potential was reduced significantly in both groups after start of operation (P < 0·0001), whereas soluble C3a and TCC in the samples were increased (P < 0·0001). Ficolin-2 was depleted from plasma during cardiac surgery when using heparin-coated bypass circuits and did not reach baseline level 24 h postoperation. These findings may have implications for the postoperative susceptibility to infections in patients undergoing extracorporeal circulation procedures. PMID:25174443

  2. Polymorphisms in the lectin pathway of complement activation influence the incidence of acute rejection and graft outcome after kidney transplantation.

    PubMed

    Golshayan, Déla; Wójtowicz, Agnieszka; Bibert, Stéphanie; Pyndiah, Nitisha; Manuel, Oriol; Binet, Isabelle; Buhler, Leo H; Huynh-Do, Uyen; Mueller, Thomas; Steiger, Jürg; Pascual, Manuel; Meylan, Pascal; Bochud, Pierre-Yves

    2016-04-01

    There are conflicting data on the role of the lectin pathway of complement activation and its recognition molecules in acute rejection and outcome after transplantation. To help resolve this we analyzed polymorphisms and serum levels of lectin pathway components in 710 consecutive kidney transplant recipients enrolled in the nationwide Swiss Transplant Cohort Study, together with all biopsy-proven rejection episodes and 1-year graft and patient survival. Functional mannose-binding lectin (MBL) levels were determined in serum samples, and previously described MBL2, ficolin 2, and MBL-associated serine protease 2 polymorphisms were genotyped. Low MBL serum levels and deficient MBL2 diplotypes were associated with a higher incidence of acute cellular rejection during the first year, in particular in recipients of deceased-donor kidneys. This association remained significant (hazard ratio 1.75, 95% confidence interval 1.18-2.60) in a Cox regression model after adjustment for relevant covariates. In contrast, there was no significant association with rates of antibody-mediated rejection, patient death, early graft dysfunction or loss. Thus, results in a prospective multicenter contemporary cohort suggest that MBL2 polymorphisms result in low MBL serum levels and are associated with acute cellular rejection after kidney transplantation. Since MBL deficiency is a relatively frequent trait in the normal population, our findings may lead to individual risk stratification and customized immunosuppression. PMID:26924055

  3. Anti-C1q autoantibodies from systemic lupus erythematosus patients activate the complement system via both the classical and lectin pathways.

    PubMed

    Thanei, Sophia; Vanhecke, Dominique; Trendelenburg, Marten

    2015-10-01

    Autoantibodies against complement C1q (anti-C1q) strongly correlate with the occurrence of lupus nephritis and hypocomplementemia in systemic lupus erythematosus (SLE). Although a direct pathogenic role of anti-C1q has been suggested, the assumed complement-activating capacity remains to be elucidated. Using an ELISA-based assay, we found that anti-C1q activate the classical (CP) and lectin pathways (LP) depending on the anti-C1q immunoglobulin-class repertoire present in the patient's serum. IgG anti-C1q resulted in the activation of the CP as reflected by C4b deposition in the presence of purified C1 and C4 in a dose-dependent manner. The extent of C4b deposition correlated with anti-C1q levels in SLE patients but not in healthy controls. Our data indicate that SLE patient-derived anti-C1q can activate the CP and the LP but not the alternative pathway of complement. These findings are of importance for the understanding of the role of anti-C1q in SLE suggesting a direct link to hypocomplementemia. PMID:26148903

  4. The extracellular adherence protein from Staphylococcus aureus inhibits the classical and lectin pathways of complement by blocking formation of the C3 proconvertase.

    PubMed

    Woehl, Jordan L; Stapels, Daphne A C; Garcia, Brandon L; Ramyar, Kasra X; Keightley, Andrew; Ruyken, Maartje; Syriga, Maria; Sfyroera, Georgia; Weber, Alexander B; Zolkiewski, Michal; Ricklin, Daniel; Lambris, John D; Rooijakkers, Suzan H M; Geisbrecht, Brian V

    2014-12-15

    The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of small inhibitory proteins. A number of these proteins inhibit the complement system, which labels bacteria for phagocytosis and generates inflammatory chemoattractants. Although the majority of staphylococcal complement inhibitors act on the alternative pathway to block the amplification loop, only a few proteins act on the initial recognition cascades that constitute the classical pathway (CP) and lectin pathway (LP). We screened a collection of recombinant, secreted staphylococcal proteins to determine whether S. aureus produces other molecules that inhibit the CP and/or LP. Using this approach, we identified the extracellular adherence protein (Eap) as a potent, specific inhibitor of both the CP and LP. We found that Eap blocked CP/LP-dependent activation of C3, but not C4, and that Eap likewise inhibited deposition of C3b on the surface of S. aureus cells. In turn, this significantly diminished the extent of S. aureus opsonophagocytosis and killing by neutrophils. This combination of functional properties suggested that Eap acts specifically at the level of the CP/LP C3 convertase (C4b2a). Indeed, we demonstrated a direct, nanomolar-affinity interaction of Eap with C4b. Eap binding to C4b inhibited binding of both full-length C2 and its C2b fragment, which indicated that Eap disrupts formation of the CP/LP C3 proconvertase (C4b2). As a whole, our results demonstrate that S. aureus inhibits two initiation routes of complement by expression of the Eap protein, and thereby define a novel mechanism of immune evasion. PMID:25381436

  5. Quantitative Characterization of the Activation Steps of Mannan-binding Lectin (MBL)-associated Serine Proteases (MASPs) Points to the Central Role of MASP-1 in the Initiation of the Complement Lectin Pathway*

    PubMed Central

    Megyeri, Márton; Harmat, Veronika; Major, Balázs; Végh, Ádám; Balczer, Júlia; Héja, Dávid; Szilágyi, Katalin; Datz, Dániel; Pál, Gábor; Závodszky, Péter; Gál, Péter; Dobó, József

    2013-01-01

    Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is ∼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process. PMID:23386610

  6. Essential Role for the Lectin Pathway in Collagen Antibody-Induced Arthritis Revealed Through Use of Adenovirus Programming Complement Inhibitor MAp44 Expression

    PubMed Central

    Banda, Nirmal K.; Mehta, Gaurav; Kjaer, Troels R.; Takahashi, Minoru; Schaack, Jerome; Morrison, Thomas E.; Thiel, Steffen; Arend, William P.; Holers, V. Michael

    2014-01-01

    Previous studies using mannose-binding lectin (MBL) and complement C4 deficient mice have suggested that the lectin pathway (LP) is not required for the development of inflammatory arthritis in the collagen antibody-induced arthritis (CAIA) model. MBL, ficolins and collectin-11 are key LP pattern recognition molecules that associate with three serine proteases, MASP-1, MASP-2 and MASP-3, and also with two MBL-associated proteins designated sMAP and MAp44. Recent studies have shown that MAp44, an alternatively spliced product of the MASP-1/3 gene, is a competitive inhibitor of the binding of the recognition molecules to all three MASPs. In these studies we examined the effect of treatment of mice with adenovirus (Ad) programmed to express human MAp44 (AdhMAp44) on the development of CAIA. AdhMAp44 and Ad programming Green fluorescent protein (AdGFP) expression were injected intraperitoneally in C57BL/6 wild-type mice prior to the induction of CAIA. AdhMAp44 significantly reduced the clinical disease activity score (CDA) by 81% compared to mice injected with AdGFP. Similarly, histopathologic injury scores for inflammation, pannus, cartilage and bone damage, as well as C3 deposition in the cartilage and synovium, were significantly reduced by AdhMAp44 pretreatment. Mice treated with AdmMAp44, programming expression of mouse MAp44, also showed significantly decreased CDA and histopathologic injury scores. Additionally, administration of AdhMAp44 significantly diminished the severity of Ross River Virus-induced arthritis, a LP-dependent model. Our study provides conclusive evidence that an intact complement LP is essential to initiate CAIA, and that MAp44 may be an appropriate treatment for inflammatory arthritis. PMID:25070856

  7. The Extracellular Adherence Protein from Staphylococcus aureus Inhibits the Classical and Lectin Pathways of Complement by Blocking Formation of the C3 Pro-Convertase

    PubMed Central

    Garcia, Brandon L.; Ramyar, Kasra X.; Keightley, Andrew; Ruyken, Maartje; Syriga, Maria; Sfyroera, Georgia; Weber, Alexander B.; Zolkiewski, Michal; Ricklin, Daniel; Lambris, John D.; Rooijakkers, Suzan H.M.; Geisbrecht, Brian V.

    2014-01-01

    The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of small inhibitory proteins. A number of these proteins inhibit the complement system, which labels bacteria for phagocytosis and generates inflammatory chemoattractants. While the majority of staphylococcal complement inhibitors act on the alternative pathway (AP) to block the amplification loop, only a few proteins act on the initial recognition cascades that constitute the classical (CP) and lectin (LP) pathways. We screened a collection of recombinant, secreted staphylococcal proteins to determine if S. aureus produces other molecules that inhibit either the CP and/or LP. Using this approach, we identified the extracellular adherence protein (Eap) as a potent, specific inhibitor of both the CP and LP. We found that Eap blocked CP/LP-dependent activation of C3, but not C4, and that Eap likewise inhibited deposition of C3b on the surface of S. aureus cells. In turn, this significantly diminished the extent of S. aureus opsonophagocytosis and killing by neutrophils. This combination of functional properties suggested that Eap acts specifically at the level of the CP/LP C3 convertase (C4b2a). Indeed, we demonstrated a direct, nanomolar-affinity interaction of Eap with C4b. Eap binding to C4b inhibited binding of both full-length C2 and its C2b fragment, which indicated that Eap disrupts formation of the CP/LP C3 pro-convertase (C4b2). As a whole, our results demonstrate that S. aureus inhibits the two initiation routes of complement by expression of the Eap protein, and thereby define a novel mechanism of immune evasion. PMID:25381436

  8. In vitro C3 Deposition on Cryptococcus Capsule Occurs Via Multiple Complement Activation Pathways

    PubMed Central

    Mershon-Shier, Kileen L.; Vasuthasawat, Alex; Takahashi, Kazue; Morrison, Sherie L.; Beenhouwer, David O.

    2011-01-01

    Complement can be activated via three pathways: classical, alternative, and lectin. Cryptococcus gattii and C. neoformans are closely related fungal pathogens possessing a polysaccharide capsule composed mainly of glucuronoxylomannan (GXM), which serves as a site for complement activation and deposition of complement components. We determined C3 deposition on Cryptococcus spp. by flow cytometry and confocal microscopy after incubation with serum from C57BL/6J mice as well as mice deficient in complement components C4, C3, factor B, and mannose binding lectin (MBL). C. gattii and C. neoformans activate complement in EGTA-treated serum indicating that they can activate the alternative pathway. However, complement activation was seen with factor B−/− serum suggesting activation could also take place in the absence of a functional alternative pathway. Furthermore, we uncovered a role for C4 in the alternative pathway activation by Cryptococcus spp. We also identified an unexpected and complex role for MBL in complement activation by Cryptococcus spp. No complement activation occurred in the absence of MBL-A and -C proteins although activation took place when the lectin binding activity of MBL was disrupted by calcium chelation. In addition, alternative pathway activation by C. neoformans required both MBL-A and -C, while either MBL-A or -C was sufficient for alternative pathway activation by C. gattii. Thus, complement activation by Cryptococcus spp. can take place through multiple pathways and complement activation via the alternative pathway requires the presence of C4 and MBL proteins. PMID:21723612

  9. Increased Autoreactivity of the Complement-Activating Molecule Mannan-Binding Lectin in a Type 1 Diabetes Model

    PubMed Central

    Østergaard, Jakob Appel; Ruseva, Marieta Milkova; Malik, Talat Habib; Hoffmann-Petersen, Ingeborg Torp; Pickering, Matthew Caleb; Thiel, Steffen; Hansen, Troels Krarup

    2016-01-01

    Background. Diabetic kidney disease is the leading cause of end-stage renal failure despite intensive treatment of modifiable risk factors. Identification of new drug targets is therefore of paramount importance. The complement system is emerging as a potential new target. The lectin pathway of the complement system, initiated by the carbohydrate-recognition molecule mannan-binding lectin (MBL), is linked to poor kidney prognosis in diabetes. We hypothesized that MBL activates complement upon binding within the diabetic glomerulus. Methods. We investigated this by comparing complement deposition and activation in kidneys from streptozotocin-induced diabetic mice and healthy control mice. Results. After 20 weeks of diabetes, glomerular deposition of MBL was significantly increased. Diabetic animals had 2.0-fold higher (95% CI 1.6–2.5) immunofluorescence intensity from anti-MBL antibodies compared with controls (P < 0.001). Diabetes and control groups did not differ in glomerular immunofluorescence intensity obtained by antibodies against complement factors C4, C3, and C9. However, the circulating complement activation product C3a was increased in diabetes as compared to control mice (P = 0.04). Conclusion. 20 weeks of diabetes increased MBL autoreactivity in the kidney and circulating C3a concentration. Together with previous findings, these results indicate direct effects of MBL within the kidney in diabetes. PMID:26977416

  10. Increased Autoreactivity of the Complement-Activating Molecule Mannan-Binding Lectin in a Type 1 Diabetes Model.

    PubMed

    Østergaard, Jakob Appel; Ruseva, Marieta Milkova; Malik, Talat Habib; Hoffmann-Petersen, Ingeborg Torp; Pickering, Matthew Caleb; Thiel, Steffen; Hansen, Troels Krarup

    2016-01-01

    Background. Diabetic kidney disease is the leading cause of end-stage renal failure despite intensive treatment of modifiable risk factors. Identification of new drug targets is therefore of paramount importance. The complement system is emerging as a potential new target. The lectin pathway of the complement system, initiated by the carbohydrate-recognition molecule mannan-binding lectin (MBL), is linked to poor kidney prognosis in diabetes. We hypothesized that MBL activates complement upon binding within the diabetic glomerulus. Methods. We investigated this by comparing complement deposition and activation in kidneys from streptozotocin-induced diabetic mice and healthy control mice. Results. After 20 weeks of diabetes, glomerular deposition of MBL was significantly increased. Diabetic animals had 2.0-fold higher (95% CI 1.6-2.5) immunofluorescence intensity from anti-MBL antibodies compared with controls (P < 0.001). Diabetes and control groups did not differ in glomerular immunofluorescence intensity obtained by antibodies against complement factors C4, C3, and C9. However, the circulating complement activation product C3a was increased in diabetes as compared to control mice (P = 0.04). Conclusion. 20 weeks of diabetes increased MBL autoreactivity in the kidney and circulating C3a concentration. Together with previous findings, these results indicate direct effects of MBL within the kidney in diabetes. PMID:26977416

  11. Lectin Complement Protein Collectin 11 (CL-K1) and Susceptibility to Urinary Schistosomiasis

    PubMed Central

    Antony, Justin S.; Ojurongbe, Olusola; Kremsner, Peter G.; Velavan, Thirumalaisamy P.

    2015-01-01

    Background Urinary Schistosomiasis is a neglected tropical disease endemic in many sub Saharan -African countries. Collectin Kidney 1 (CL-K1, encoded by COLEC11 on chromosome 2p25.3), a member of the vertebrate C-type lectin super family, has recently been identified as pattern-recognition molecule (PRR) of the lectin complement pathway. CL-K1 is preferentially expressed in the kidneys, but also in other organs and it is considered to play a role in host defense to some infectious agents. Schistosome teguments are fucosylated and CL-K1 has, through its collagen-like domain, a high binding affinity to fucose. Methodology/Principal Findings We utilized a Nigerian study group consisting of 167 Schistosoma haematobium infected individuals and 186 matched healthy subjects, and investigated the contribution of CL-K1 deficiency and of COLEC11 polymorphisms to infection phenotype. Higher CL-K1 serum levels were associated with decreased risk of schistosome infection (Pcorr = 0.0004). CL-K1 serum levels were differentially distributed between the COLEC11 genotypes and haplotypes observed. The non-synonymous variant p.R216H was associated with the occurrence of schistosomiasis (OR = 0.44, 95%CI = 0.22–0.72, Pcorr = 0.0004). The reconstructed COLEC11*TCCA haplotypes were associated with higher CL-K1 serum levels (P = 0.002) and with decreased schistosomiasis (OR = 0.38, 95%CI = 0.23–0.63, Pcorr = 0.0001). Conclusions In agreement with findings from our earlier published study, our findings support the observation that CL-K1 and their functional variants may be host factors associated with protection in schistosomiasis and may be a useful marker for further investigations. PMID:25807310

  12. Diagnosis of complement alternative pathway disorders.

    PubMed

    Angioi, Andrea; Fervenza, Fernando C; Sethi, Sanjeev; Zhang, Yuzhou; Smith, Richard J; Murray, David; Van Praet, Jens; Pani, Antonello; De Vriese, An S

    2016-02-01

    Kidney diseases resulting from abnormal control of the complement alternative pathway include atypical hemolytic uremic syndrome, C3 glomerulonephritis, and dense-deposit disease, as well as atypical postinfectious glomerulonephritis. Although clinically diverse, they all result from loss of surface or fluid-phase complement control, caused by acquired or genetic defects in the complement alternative pathway. As such, the diagnostic approach is similar and includes a comprehensive biochemical, genetic, and pathologic analysis of the complement pathway. The biochemical test battery includes functional activity measurements of the entire complement pathway, functional and quantitative analysis of individual components and regulators, and quantification of activation products. In patients with a thrombotic microangiopathy, ADAMTS-13 activity should be determined to exclude a thrombotic thrombocytopenic purpura. The spectrum of genes currently known to be involved in the pathogenesis of alternative pathway disorders is rapidly expanding. Pathologic analysis of a kidney biopsy specimen is sophisticated with ad hoc immunofluorescence studies and laser microdissection with mass spectrometry. The identification of the underlying defect in the alternative pathway based on this comprehensive analysis will allow treatment to be directed to the site of dysregulation. PMID:26806831

  13. Alternative complement pathway of channel catfish Ictalurus punctatus: molecular characterization and expression analysis of factors Bf/C2 and Df

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complement system important in both innate and adaptive host defense against microbial infection in vertebrates. It contains three pathways: the classical, alternative, and lectin pathways. Complement component factors B and D are two crucial proteases in the alternative pathway. In this study,...

  14. SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

    PubMed

    Ferreira, Viviana P; Fazito Vale, Vladimir; Pangburn, Michael K; Abdeladhim, Maha; Mendes-Sousa, Antonio Ferreira; Coutinho-Abreu, Iliano V; Rasouli, Manoochehr; Brandt, Elizabeth A; Meneses, Claudio; Lima, Kolyvan Ferreira; Nascimento Araújo, Ricardo; Pereira, Marcos Horácio; Kotsyfakis, Michalis; Oliveira, Fabiano; Kamhawi, Shaden; Ribeiro, Jose M C; Gontijo, Nelder F; Collin, Nicolas; Valenzuela, Jesus G

    2016-01-01

    Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases. PMID:26758086

  15. SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis

    PubMed Central

    Ferreira, Viviana P.; Fazito Vale, Vladimir; Pangburn, Michael K.; Abdeladhim, Maha; Ferreira Mendes-Sousa, Antonio; Coutinho-Abreu, Iliano V.; Rasouli, Manoochehr; Brandt, Elizabeth A.; Meneses, Claudio; Lima, Kolyvan Ferreira; Nascimento Araújo, Ricardo; Horácio Pereira, Marcos; Kotsyfakis, Michalis; Oliveira, Fabiano; Kamhawi, Shaden; Ribeiro, Jose M. C.; Gontijo, Nelder F.; Collin, Nicolas; Valenzuela, Jesus G.

    2016-01-01

    Blood-feeding insects inject potent salivary components including complement inhibitors into their host’s skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases. PMID:26758086

  16. Association Study of Mannose-Binding Lectin Levels and Genetic Variants in Lectin Pathway Proteins with Susceptibility to Age-Related Macular Degeneration: A Case-Control Study

    PubMed Central

    Osthoff, Michael; Dean, Melinda M.; Baird, Paul N.; Richardson, Andrea J.; Daniell, Mark; Guymer, Robyn H.; Eisen, Damon P.

    2015-01-01

    Background In age-related macular degeneration (AMD) the complement system is thought to be activated by chronic oxidative damage with genetic variants identified in the alternative pathway as susceptibility factors. However, the involvement of the lectin pathway of complement, a key mediator of oxidative damage, is controversial. This study investigated whether mannose-binding lectin (MBL) levels and genetic variants in lectin pathway proteins, are associated with the predisposition to and severity of AMD. Methods MBL levels and single nucleotide polymorphisms (SNPs) in the MBL2 and the ficolin-2 (FCN2) gene were determined in 109 patients with AMD and 109 age- and sex-matched controls. Results MBL expression levels were equally distributed in both cases (early and late AMD) and controls (p>0.05). However, there was a trend towards higher median MBL levels in cases with late AMD compared to cases with early AMD (1.0 vs. 0.4 μg/ml, p = 0.09) and MBL deficiency (<0.5 μg/ml) was encountered less frequently in the late AMD group (35% vs 56%, p = 0.03). FCN2 and MBL2 allele frequencies were similarly distributed in early and late AMD cases compared with controls (p>0.05 for all analyses) as were MBL2 genotypes. Similarly, there was no significant difference in allele frequencies in any SNPs in either the MBL2 or FCN2 gene in cases with early vs. late AMD. Conclusions SNPs of lectin pathway proteins investigated in this study were not associated with AMD or AMD severity. However, MBL levels deserve further study in a larger cohort of early vs. late AMD patients to elucidate any real effect on AMD severity. PMID:26207622

  17. A Metalloproteinase Mirolysin of Tannerella forsythia Inhibits All Pathways of the Complement System.

    PubMed

    Jusko, Monika; Potempa, Jan; Mizgalska, Danuta; Bielecka, Ewa; Ksiazek, Miroslaw; Riesbeck, Kristian; Garred, Peter; Eick, Sigrun; Blom, Anna M

    2015-09-01

    Recent reports focusing on virulence factors of periodontal pathogens implicated proteinases as major determinants of remarkable pathogenicity of these species, with special emphasis on their capacity to modulate complement activity. In particular, bacteria-mediated cleavage of C5 and subsequent release of C5a seems to be an important phenomenon in the manipulation of the local inflammatory response in periodontitis. In this study, we present mirolysin, a novel metalloproteinase secreted by Tannerella forsythia, a well-recognized pathogen strongly associated with periodontitis. Mirolysin exhibited a strong effect on all complement pathways. It inhibited the classical and lectin complement pathways due to efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3, and C4, whereas inhibition of the alternative pathway was caused by degradation of C5. This specificity toward complement largely resembled the activity of a previously characterized metalloproteinase of T. forsythia, karilysin. Interestingly, mirolysin released the biologically active C5a peptide in human plasma and induced migration of neutrophils. Importantly, we demonstrated that combination of mirolysin with karilysin, as well as a cysteine proteinase of another periodontal pathogen, Prevotella intermedia, resulted in a strong synergistic effect on complement. Furthermore, mutant strains of T. forsythia, devoid of either mirolysin or karilysin, showed diminished survival in human serum, providing further evidence for the synergistic inactivation of complement by these metalloproteinases. Taken together, our findings on interactions of mirolysin with complement significantly add to the understanding of immune evasion strategies of T. forsythia and expand the knowledge on molecular mechanisms driving pathogenic events in the infected periodontium. PMID:26209620

  18. Crystal Structure and Functional Characterization of the Complement Regulator Mannose-binding Lectin (MBL)/Ficolin-associated Protein-1 (MAP-1)*

    PubMed Central

    Skjoedt, Mikkel-Ole; Roversi, Pietro; Hummelshøj, Tina; Palarasah, Yaseelan; Rosbjerg, Anne; Johnson, Steven; Lea, Susan M.; Garred, Peter

    2012-01-01

    The human lectin complement pathway activation molecules comprise mannose-binding lectin (MBL) and ficolin-1, -2, and -3 in complex with associated serine proteases MASP-1, -2, and -3 and the non-enzymatic small MBL associated protein or sMAP. Recently, a novel plasma protein named MBL/ficolin-associated protein-1 (MAP-1) was identified in humans. This protein is the result of a differential splicing of the MASP1 gene and includes the major part of the heavy chain but lacks the serine protease domain. We investigated the direct interactions of MAP-1 and MASP-3 with ficolin-3 and MBL using surface plasmon resonance and found affinities around 5 nm and 2.5 nm, respectively. We studied structural aspects of MAP-1 and could show by multi-angle laser light scattering that MAP-1 forms a calcium-dependent homodimer in solution. We were able to determine the crystal structure of MAP-1, which also contains a head-to-tail dimer ∼146 Å long. This structure of MAP-1 also enables modeling and assembly of the MASP-1 molecule in its entirety. Finally we found that MAP-1 competes with all three MASPs for ligand binding and is able to mediate a strong dose-dependent inhibitory effect on the lectin pathway activation, as measured by levels of C3 and C9. PMID:22854970

  19. Low mannose-binding lectin complement activation function is associated with predisposition to Legionnaires' disease

    PubMed Central

    Eisen, D P; Stubbs, J; Spilsbury, D; Carnie, J; Leydon, J; Howden, B P

    2007-01-01

    Innate immune system deficiency may predispose to severe infections such as Legionnaires' disease. We have investigated the role of mannose-binding lectin (MBL) deficiency in the Melbourne Aquarium Legionnaires' disease outbreak. Serum samples from patients and controls that were exposed but shown to be uninfected from the Melbourne Aquarium Legionnaires' disease outbreak were tested for MBL function (C4 deposition) and level (mannan-binding). MBL function was lower in Legionnaires' disease cases than in age- and sex-matched uninfected, exposed controls. The frequency of MBL deficiency with C4 deposition < 0·2 U/µl was significantly higher in Legionnaires' disease cases than in controls. This also applied to Legionnaires' disease cases requiring hospital care. There was no difference in MBL mannan-binding levels between Legionnaires' disease patients and controls. There was no significant interval change in MBL function or level after a mean of 46 days. MBL complement activation functional deficiency appears to predispose to Legionnaires' disease. PMID:17425652

  20. Low mannose-binding lectin complement activation function is associated with predisposition to Legionnaires' disease.

    PubMed

    Eisen, D P; Stubbs, J; Spilsbury, D; Carnie, J; Leydon, J; Howden, B P

    2007-07-01

    Innate immune system deficiency may predispose to severe infections such as Legionnaires' disease. We have investigated the role of mannose-binding lectin (MBL) deficiency in the Melbourne Aquarium Legionnaires' disease outbreak. Serum samples from patients and controls that were exposed but shown to be uninfected from the Melbourne Aquarium Legionnaires' disease outbreak were tested for MBL function (C4 deposition) and level (mannan-binding). MBL function was lower in Legionnaires' disease cases than in age- and sex-matched uninfected, exposed controls. The frequency of MBL deficiency with C4 deposition < 0.2 U/microl was significantly higher in Legionnaires' disease cases than in controls. This also applied to Legionnaires' disease cases requiring hospital care. There was no difference in MBL mannan-binding levels between Legionnaires' disease patients and controls. There was no significant interval change in MBL function or level after a mean of 46 days. MBL complement activation functional deficiency appears to predispose to Legionnaires' disease. PMID:17425652

  1. Systemic Lupus Erythematosus and Deficiencies of Early Components of the Complement Classical Pathway

    PubMed Central

    Macedo, Ana Catarina Lunz; Isaac, Lourdes

    2016-01-01

    The complement system plays an important role in the innate and acquired immune response against pathogens. It consists of more than 30 proteins found in soluble form or attached to cell membranes. Most complement proteins circulate in inactive forms and can be sequentially activated by the classical, alternative, or lectin pathways. Biological functions, such as opsonization, removal of apoptotic cells, adjuvant function, activation of B lymphocytes, degranulation of mast cells and basophils, and solubilization and clearance of immune complex and cell lysis, are dependent on complement activation. Although the activation of the complement system is important to avoid infections, it also can contribute to the inflammatory response triggered by immune complex deposition in tissues in autoimmune diseases. Paradoxically, the deficiency of early complement proteins from the classical pathway (CP) is strongly associated with development of systemic lupus erythematous (SLE) – mainly C1q deficiency (93%) and C4 deficiency (75%). The aim of this review is to focus on the deficiencies of early components of the CP (C1q, C1r, C1s, C4, and C2) proteins in SLE patients. PMID:26941740

  2. Classical Complement Pathway Activation in the Kidneys of Women With Preeclampsia.

    PubMed

    Penning, Marlies; Chua, Jamie S; van Kooten, Cees; Zandbergen, Malu; Buurma, Aletta; Schutte, Joke; Bruijn, Jan Anthonie; Khankin, Eliyahu V; Bloemenkamp, Kitty; Karumanchi, S Ananth; Baelde, Hans

    2015-07-01

    A growing body of evidence suggests that complement dysregulation plays a role in the pathogenesis of preeclampsia. The kidney is one of the major organs affected in preeclampsia. Because the kidney is highly susceptible to complement activation, we hypothesized that preeclampsia is associated with renal complement activation. We performed a nationwide search for renal autopsy material in the Netherlands using a computerized database (PALGA). Renal tissue was obtained from 11 women with preeclampsia, 25 pregnant controls, and 14 nonpregnant controls with hypertension. The samples were immunostained for C4d, C1q, mannose-binding lectin, properdin, C3d, C5b-9, IgA, IgG, and IgM. Preeclampsia was significantly associated with renal C4d-a stable marker of complement activation-and the classical pathway marker C1q. In addition, the prevalence of IgM was significantly higher in the kidneys of the preeclamptic women. No other complement markers studied differed between the groups. Our findings in human samples were validated using a soluble fms-like tyrosine kinase 1 mouse model of preeclampsia. The kidneys in the soluble fms-like tyrosine kinase 1-injected mice had significantly more C4 deposits than the control mice. The association between preeclampsia and renal C4d, C1q, and IgM levels suggests that the classical complement pathway is involved in the renal injury in preeclampsia. Moreover, our finding that soluble fms-like tyrosine kinase 1-injected mice develop excess C4 deposits indicates that angiogenic dysregulation may play a role in complement activation within the kidney. We suggest that inhibiting complement activation may be beneficial for preventing the renal manifestations of preeclampsia. PMID:25941343

  3. Pneumococcal type-associated variability in alternate complement pathway activation.

    PubMed Central

    Fine, D P

    1975-01-01

    Opsonization of Streptococcus pneumoniae may be mediated by the alternate complement pathway. To study the importance of this interaction to human disease, complement consumption by pneumococci of various serotypes was measured in humwn serum chelated with ethyleneglycoltraacetic acid, a substance that blocks the classic but not the alternate complement pathwway. Serotype I, in contrast to all other types studied, lacked ability to consume complement in this system. The ability for serotypes III, IV, and VIII to activate the alternate pathway could be eliminated by prior serum absorption at O C with they type in question, a condition that would remove antibody but not complement. Types VII, XII, XIV, and XXV readily activated the alternate pathway in unabsorbed and absorbed sera. Differences could not be related to properties of the capsules. It was concluded that types I, III, IV, and VIII lack intrinsic ability to activate and thus be opsonized by the alternate complement pathway, although types III, IV, and VIII can do so in concert with specific antibody. The fact that these same types are especially prominent in human disease suggests that the ability to evade opsonization by the alternate complement pathway in pre-antibody phases of infection may be a virulence factor in pneumococci. PMID:329

  4. Inhibition of the classical pathway of the complement system by saliva of Amblyomma cajennense (Acari: Ixodidae).

    PubMed

    Franco, Paula F; Silva, Naylene C S; Fazito do Vale, Vladimir; Abreu, Jéssica F; Santos, Vânia C; Gontijo, Nelder F; Valenzuela, Jesus G; Pereira, Marcos H; Sant'Anna, Mauricio R V; Gomes, Alessandra P S; Araujo, Ricardo N

    2016-05-01

    Inhibition of the complement system during and after haematophagy is of utmost importance for tick success in feeding and tick development. The role of such inhibition is to minimise damage to the intestinal epithelium as well as avoiding inflammation and opsonisation of salivary molecules at the bite site. Despite its importance, the salivary anti-complement activity has been characterised only in species belonging to the Ixodes ricinus complex which saliva is able to inhibit the alternative and lectin pathways. Little is known about this activity in other species of the Ixodidae family. Thus, the aim of this study was to describe the inhibition of the classical pathway of the complement system by the saliva of Amblyomma cajennense at different stages of the haematophagy. The A. cajennense saliva and salivary gland extract (SGE) were able to inhibit the complement classical pathway through haemolytic assays with higher activity observed when saliva was used. The anti-complement activity is present in the salivary glands of starving females and also in females throughout the whole feeding process, with significant higher activity soon after tick detachment. The SGE activity from both females fed on mice or horses had no significant correlation (p > 0.05) with tick body weight. The pH found in the intestinal lumen of A. cajennense was 8.04 ± 0.08 and haemolytic assays performed at pH 8.0 showed activation of the classical pathway similarly to what occurs at pH 7.4. Consequently, inhibition could be necessary to protect the tick enterocytes. Indeed, the inhibition observed by SGE was higher in pH 8.0 in comparison to pH 7.4 reinforcing the role of saliva in protecting the intestinal cells. Further studies should be carried out in order to identify the inhibitor molecule and characterise its inhibition mechanism. PMID:26948715

  5. Quantitative Modeling of the Alternative Pathway of the Complement System

    PubMed Central

    Dorado, Angel; Morikis, Dimitrios

    2016-01-01

    The complement system is an integral part of innate immunity that detects and eliminates invading pathogens through a cascade of reactions. The destructive effects of the complement activation on host cells are inhibited through versatile regulators that are present in plasma and bound to membranes. Impairment in the capacity of these regulators to function in the proper manner results in autoimmune diseases. To better understand the delicate balance between complement activation and regulation, we have developed a comprehensive quantitative model of the alternative pathway. Our model incorporates a system of ordinary differential equations that describes the dynamics of the four steps of the alternative pathway under physiological conditions: (i) initiation (fluid phase), (ii) amplification (surfaces), (iii) termination (pathogen), and (iv) regulation (host cell and fluid phase). We have examined complement activation and regulation on different surfaces, using the cellular dimensions of a characteristic bacterium (E. coli) and host cell (human erythrocyte). In addition, we have incorporated neutrophil-secreted properdin into the model highlighting the cross talk of neutrophils with the alternative pathway in coordinating innate immunity. Our study yields a series of time-dependent response data for all alternative pathway proteins, fragments, and complexes. We demonstrate the robustness of alternative pathway on the surface of pathogens in which complement components were able to saturate the entire region in about 54 minutes, while occupying less than one percent on host cells at the same time period. Our model reveals that tight regulation of complement starts in fluid phase in which propagation of the alternative pathway was inhibited through the dismantlement of fluid phase convertases. Our model also depicts the intricate role that properdin released from neutrophils plays in initiating and propagating the alternative pathway during bacterial infection. PMID:27031863

  6. Activated complement classical pathway in a murine model of oxygen-induced retinopathy

    PubMed Central

    Tao, Xue-Ying; Zheng, Shi-Jie; Lei, Bo

    2015-01-01

    AIM To investigate whether the complement system is involved in a murine model of oxygen-induced retinopathy (OIR). METHODS Forty C57BL/6J newborn mice were divided randomly into OIR group and control group. OIR was induced by exposing mice to 75%2% oxygen from postnatal 7d (P7) to P12 and then recovered in room air. For the control group, the litters were raised in room air. At the postnatal 17d (P17), gene expressions of the complement components of the classical pathway (CP), the mannose-binding lectin (MBL) pathway and the alternative pathway (AP) in the retina were determined by quantitative real-time polymerase chain reaction (RT-PCR). Retinal protein expressions of the key components in the CP were examined by Western blotting. RESULTS Whole mounted retina in the OIR mice showed area of central hypoperfusion in both superficial and deep layers and neovascular tufts in the periphery. The expressions of C1qb and C4b genes in the OIR retina were significantly higher than those of the controls. The expression of retinal complement factor B (CFB) gene in OIR mice was significantly lower than those of the controls. However, the expressions of C3 and complement factor H (CFH) genes were higher. The protein synthesis of the key components involved in the CP (C1q, C4 and C3) were also significantly higher in OIR mouse retina. Although MBL-associated serine protease 1 (MASP1) and MASP2 were detected in both the OIR and the control groups, the expressions were weak and the difference between the two groups was not significant. CONCLUSION Our data suggest that the complement system CP is activated during the pathogenesis of murine model of OIR. PMID:25709901

  7. Peptide inhibitor of complement c1, a novel suppressor of classical pathway activation: mechanistic studies and clinical potential.

    PubMed

    Sharp, Julia A; Whitley, Pamela H; Cunnion, Kenji M; Krishna, Neel K

    2014-01-01

    The classical pathway of complement plays multiple physiological roles including modulating immunological effectors initiated by adaptive immune responses and an essential homeostatic role in the clearance of damaged self-antigens. However, dysregulated classical pathway activation is associated with antibody-initiated, inflammatory diseases processes like cold agglutinin disease, acute intravascular hemolytic transfusion reaction (AIHTR), and acute/hyperacute transplantation rejection. To date, only one putative classical pathway inhibitor, C1 esterase inhibitor (C1-INH), is currently commercially available and its only approved indication is for replacement treatment in hereditary angioedema, which is predominantly a kinin pathway disease. Given the variety of disease conditions in which the classical pathway is implicated, development of therapeutics that specifically inhibits complement initiation represents a major unmet medical need. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement. In vitro studies have demonstrated that these peptide inhibitors of complement C1 (PIC1) bind to the collagen-like region of the initiator molecule of the classical pathway, C1q. PIC1 binding to C1q blocks activation of the associated serine proteases (C1s-C1r-C1r-C1s) and subsequent downstream complement activation. Rational design optimization of PIC1 has resulted in the generation of a highly potent derivative of 15 amino acids. PIC1 inhibits classical pathway mediated complement activation in ABO incompatibility in vitro and inhibiting classical pathway activation in vivo in rats. This review will focus on the pre-clinical development of PIC1 and discuss its potential as a therapeutic in antibody-mediated classical pathway disease, specifically AIHTR. PMID:25202312

  8. Monospecific Inhibitors Show That Both Mannan-binding Lectin-associated Serine Protease-1 (MASP-1) and -2 Are Essential for Lectin Pathway Activation and Reveal Structural Plasticity of MASP-2*

    PubMed Central

    Héja, Dávid; Harmat, Veronika; Fodor, Krisztián; Wilmanns, Matthias; Dobó, József; Kékesi, Katalin A.; Závodszky, Péter; Gál, Péter; Pál, Gábor

    2012-01-01

    The lectin pathway is an antibody-independent activation route of the complement system. It provides immediate defense against pathogens and altered self-cells, but it also causes severe tissue damage after stroke, heart attack, and other ischemia reperfusion injuries. The pathway is triggered by target binding of pattern recognition molecules leading to the activation of zymogen mannan-binding lectin-associated serine proteases (MASPs). MASP-2 is considered as the autonomous pathway-activator, while MASP-1 is considered as an auxiliary component. We evolved a pair of monospecific MASP inhibitors. In accordance with the key role of MASP-2, the MASP-2 inhibitor completely blocks the lectin pathway activation. Importantly, the MASP-1 inhibitor does the same, demonstrating that MASP-1 is not an auxiliary but an essential pathway component. We report the first Michaelis-like complex structures of MASP-1 and MASP-2 formed with substrate-like inhibitors. The 1.28 Å resolution MASP-2 structure reveals significant plasticity of the protease, suggesting that either an induced fit or a conformational selection mechanism should contribute to the extreme specificity of the enzyme. PMID:22511776

  9. Novel Scabies Mite Serpins Inhibit the Three Pathways of the Human Complement System

    PubMed Central

    Mika, Angela; Reynolds, Simone L.; Mohlin, Frida C.; Willis, Charlene; Swe, Pearl M.; Pickering, Darren A.; Halilovic, Vanja; Wijeyewickrema, Lakshmi C.; Pike, Robert N.; Blom, Anna M.; Kemp, David J.; Fischer, Katja

    2012-01-01

    Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage. PMID:22792350

  10. The alternative complement pathway regulates pathological angiogenesis in the retina.

    PubMed

    Sweigard, J Harry; Yanai, Ryoji; Gaissert, Philipp; Saint-Geniez, Magali; Kataoka, Keiko; Thanos, Aristomenis; Stahl, Gregory L; Lambris, John D; Connor, Kip M

    2014-07-01

    A defining feature in proliferative retinopathies is the formation of pathological neovessels. In these diseases, the balance between neovessel formation and regression determines blindness, making the modulation of neovessel growth highly desirable. The role of the immune system in these retinopathies is of increasing interest, but it is not completely understood. We investigated the role of the alternative complement pathway during the formation and resolution of aberrant neovascularization. We used alternative complement pathway-deficient (Fb(-/-)) mice and age- and strain-matched control mice to assess neovessel development and regression in an oxygen-induced retinopathy (OIR) mouse model. In the control mice, we found increased transcription of Fb after OIR treatment. In the Fb(-/-) mice, we prepared retinal flatmounts and identified an increased number of neovessels, peaking at postnatal day 17 (P17; P=0.001). Subjecting human umbilical vein endothelial cells (HUVECs) to low oxygen, mimicking a characteristic of neovessels, decreased the expression of the complement inhibitor Cd55. Finally, using laser capture microdissection (LCM) to isolate the neovessels after OIR, we found decreased expression of Cd55 (P=0.005). Together, our data implicate the alternative complement pathway in facilitating neovessel clearance by down-regulating the complement inhibitor Cd55 specifically on neovessels, allowing for their targeted removal while leaving the established vasculature intact.-Sweigard, J. H., Yanai, R., Gaissert, P., Saint-Geniez, M., Kataoka, K., Thanos, A., Stahl, G. L., Lambris, J. D., Connor, K. M. The alternative complement pathway regulates pathological angiogenesis in the retina. PMID:24668752

  11. The alternative complement pathway regulates pathological angiogenesis in the retina

    PubMed Central

    Sweigard, J. Harry; Yanai, Ryoji; Gaissert, Philipp; Saint-Geniez, Magali; Kataoka, Keiko; Thanos, Aristomenis; Stahl, Gregory L.; Lambris, John D.; Connor, Kip M.

    2014-01-01

    A defining feature in proliferative retinopathies is the formation of pathological neovessels. In these diseases, the balance between neovessel formation and regression determines blindness, making the modulation of neovessel growth highly desirable. The role of the immune system in these retinopathies is of increasing interest, but it is not completely understood. We investigated the role of the alternative complement pathway during the formation and resolution of aberrant neovascularization. We used alternative complement pathway–deficient (Fb−/−) mice and age- and strain-matched control mice to assess neovessel development and regression in an oxygen-induced retinopathy (OIR) mouse model. In the control mice, we found increased transcription of Fb after OIR treatment. In the Fb−/− mice, we prepared retinal flatmounts and identified an increased number of neovessels, peaking at postnatal day 17 (P17; P=0.001). Subjecting human umbilical vein endothelial cells (HUVECs) to low oxygen, mimicking a characteristic of neovessels, decreased the expression of the complement inhibitor Cd55. Finally, using laser capture microdissection (LCM) to isolate the neovessels after OIR, we found decreased expression of Cd55 (P=0.005). Together, our data implicate the alternative complement pathway in facilitating neovessel clearance by down-regulating the complement inhibitor Cd55 specifically on neovessels, allowing for their targeted removal while leaving the established vasculature intact.—Sweigard, J. H., Yanai, R., Gaissert, P., Saint-Geniez, M., Kataoka, K., Thanos, A., Stahl, G. L., Lambris, J. D., Connor, K. M. The alternative complement pathway regulates pathological angiogenesis in the retina. PMID:24668752

  12. Differential Complement Activation Pathways Promote C3b Deposition on Native and Acetylated LDL thereby Inducing Lipoprotein Binding to the Complement Receptor 1

    PubMed Central

    Klop, Boudewijn; van der Pol, Pieter; van Bruggen, Robin; Wang, Yanan; de Vries, Marijke A.; van Santen, Selvetta; O'Flynn, Joseph; van de Geijn, Gert-Jan M.; Njo, Tjin L.; Janssen, Hans W.; de Man, Peter; Jukema, J. Wouter; Rabelink, Ton J.; Rensen, Patrick C. N.; van Kooten, Cees; Cabezas, Manuel Castro

    2014-01-01

    Lipoproteins can induce complement activation resulting in opsonization and binding of these complexes to complement receptors. We investigated the binding of opsonized native LDL and acetylated LDL (acLDL) to the complement receptor 1 (CR1). Binding of complement factors C3b, IgM, C1q, mannose-binding lectin (MBL), and properdin to LDL and acLDL were investigated by ELISA. Subsequent binding of opsonized LDL and acLDL to CR1 on CR1-transfected Chinese Hamster Ovarian cells (CHO-CR1) was tested by flow cytometry. Both native LDL and acLDL induced complement activation with subsequent C3b opsonization upon incubation with normal human serum. Opsonized LDL and acLDL bound to CR1. Binding to CHO-CR1 was reduced by EDTA, whereas MgEGTA only reduced the binding of opsonized LDL, but not of acLDL suggesting involvement of the alternative pathway in the binding of acLDL to CR1. In vitro incubations showed that LDL bound C1q, whereas acLDL bound to C1q, IgM, and properdin. MBL did neither bind to LDL nor to acLDL. The relevance of these findings was demonstrated by the fact that ex vivo up-regulation of CR1 on leukocytes was accompanied by a concomitant increased binding of apolipoprotein B-containing lipoproteins to leukocytes without changes in LDL-receptor expression. In conclusion, CR1 is able to bind opsonized native LDL and acLDL. Binding of LDL to CR1 is mediated via the classical pathway, whereas binding of acLDL is mediated via both the classical and alternative pathways. Binding of lipoproteins to CR1 may be of clinical relevance due to the ubiquitous cellular distribution of CR1. PMID:25349208

  13. Eosinophil granule cationic proteins regulate the classical pathway of complement.

    PubMed Central

    Weiler, J M; Edens, R E; Bell, C S; Gleich, G J

    1995-01-01

    Major basic protein, the primary constituent of eosinophil granules, regulates the alternative and classical pathways of complement. Major basic protein and other eosinophil granule cationic proteins, which are important in mediating tissue damage in allergic disease, regulate the alternative pathway by interfering with C3b interaction with factor B to assemble an alternative pathway C3 convertase. In the present study, eosinophil peroxidase, eosinophil cationic protein and eosinophil-derived neurotoxin, as well as major basic protein, were examined for capacity to regulate the classical pathway. Eosinophil peroxidase, eosinophil cationic protein and major basic protein inhibited formation of cell-bound classical pathway C3 convertase (EAC1,4b,2a), causing 50% inhibition of complement-mediated lysis at about 0.19, 0.75 and 0.5 micrograms/10(7) cellular intermediates, respectively. Eosinophil-derived neurotoxin had no activity on this pathway of complement. The eosinophil granule proteins were examined for activity on the formation of the membrane attack complex. Major basic protein and eosinophil cationic protein had no activity on terminal lysis. In contrast, eosinophil peroxidase inhibited lysis of EAC1,4b,2a,3b,5b, but had only minimal activity on later events in complement lysis. These polycations were then examined to determine the site(s) at which they regulated the early classical pathway. Eosinophil granule polycationic proteins: (1) reduced the Zmax at all time points but had only minimal effect on the Tmax during the formation of the classical pathway C3 convertase (EAC1,4b,2a); (2) inhibited formation of EAC1,4b,2a proportional to C4 but independent of C2 concentration; (3) inhibited fluid phase formation of C1,4b,2a, as reflected by a decrease in C1-induced consumption of C2 over time; and (4) inhibited C1 activity over time without a direct effect on either C4 or C2. These observations suggest that polycations regulate the early classical pathway by interfering with C1 and may exert this activity in vivo. PMID:7750997

  14. Complement

    MedlinePlus

    ... the suspected disease are done first. C3 and C4 are the complement components measured most often. A ... normal levels of the complement proteins C3 and C4 . Complement activity varies throughout the body. For example, ...

  15. Complement activating soluble pattern recognition molecules with collagen-like regions, mannan-binding lectin, ficolins and associated proteins.

    PubMed

    Thiel, Steffen

    2007-09-01

    Mannan-binding lectin (MBL), L-ficolin, M-ficolin and H-ficolin are all complement activating soluble pattern recognition molecules with recognition domains linked to collagen-like regions. All four may form complexes with four structurally related proteins, the three MBL-associated serine proteases (MASPs), MASP-1, MASP-2 and MASP-3, and a smaller MBL-associated protein (MAp19). The four recognition molecules recognize patterns of carbohydrate or acetyl-group containing ligands. After binding to the relevant targets all four are able to activate the complement system. We thus have a system where four different and/or overlapping patterns of microbial origin or patterns of altered-self may be recognized, but in all cases the signalling molecules, the MASPs, are shared. MASP-1 and MASP-3 are formed from one gene, MASP1/3, by alternative splicing generating two different mRNAs from a single primary transcript. Similarly MASP-2 and MAp19 are both generated from one gene, MASP-2/MAp19, by alternative splicing. A number of non-synonymous polymorphisms of the four recognition molecules and of the MASPs are known, and the implications of these alterations are being studied. The clinical impact of deficiencies will be discussed. PMID:17768106

  16. A metalloproteinase karilysin present in the majority of Tannerella forsythia isolates inhibits all pathways of the complement system1

    PubMed Central

    Jusko, Monika; Potempa, Jan; Karim, Abdulkarim Y.; Ksiazek, Miroslaw; Riesbeck, Kristian; Garred, Peter; Eick, Sigrun; Blom, Anna M.

    2012-01-01

    Tannerella forsythia is a poorly studied pathogen despite being one of the main causes of periodontitis, which is an inflammatory disease of the supporting structures of the teeth. We found that despite being recognized by all complement pathways T. forsythia is resistant to killing by human complement, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with karilysin, a metalloproteinase of T. forsythia, resulted in a decrease in bactericidal activity of the serum. T. forsythia strains expressing karilysin at higher levels were more resistant than low expressing strain. Furthermore, the low expressing strain was significantly more opsonized with C3b and membrane attack complex from serum compared to the other strains. The high expressing strain was more resistant to killing in human blood. The protective effect of karilysin against serum bactericidal activity was attributable to its ability to inhibit complement at several stages. The classical and lectin complement pathways were inhibited due to the efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3 and C4 by karilysin, while inhibition of the terminal pathway was caused by degradation of C5. Interestingly, karilysin was able to release biologically active C5a peptide in human plasma and induce migration of neutrophils. Importantly, we detected the karilysin gene in over 90% of gingival crevicular fluid samples containing T. forsythia obtained from patients with periodontitis. Taken together, the newly characterized karilysin appears to be an important virulence factor of T. forsythia and might have several important implications for immune evasion. PMID:22287711

  17. Alternative Pathway Dysregulation and the Conundrum of Complement Activation by IgG4 Immune Complexes in Membranous Nephropathy

    PubMed Central

    Borza, Dorin-Bogdan

    2016-01-01

    Membranous nephropathy (MN), a major cause of nephrotic syndrome, is a non-inflammatory immune kidney disease mediated by IgG antibodies that form glomerular subepithelial immune complexes. In primary MN, autoantibodies target proteins expressed on the podocyte surface, often phospholipase A2 receptor (PLA2R1). Pathology is driven by complement activation, leading to podocyte injury and proteinuria. This article overviews the mechanisms of complement activation and regulation in MN, addressing the paradox that anti-PLA2R1 and other antibodies causing primary MN are predominantly (but not exclusively) IgG4, an IgG subclass that does not fix complement. Besides immune complexes, alterations of the glomerular basement membrane (GBM) in MN may lead to impaired regulation of the alternative pathway (AP). The AP amplifies complement activation on surfaces insufficiently protected by complement regulatory proteins. Whereas podocytes are protected by cell-bound regulators, the GBM must recruit plasma factor H, which inhibits the AP on host surfaces carrying certain polyanions, such as heparan sulfate (HS) chains. Because HS chains present in the normal GBM are lost in MN, we posit that the local complement regulation by factor H may be impaired as a result. Thus, the loss of GBM HS in MN creates a micro-environment that promotes local amplification of complement activation, which in turn may be initiated via the classical or lectin pathways by subsets of IgG in immune complexes. A detailed understanding of the mechanisms of complement activation and dysregulation in MN is important for designing more effective therapies. PMID:27199983

  18. Identification of Novel Pathways in Plant Lectin-Induced Cancer Cell Apoptosis

    PubMed Central

    Shi, Zheng; Sun, Rong; Yu, Tian; Liu, Rong; Cheng, Li-Jia; Bao, Jin-Ku; Zou, Liang; Tang, Yong

    2016-01-01

    Plant lectins have been investigated to elucidate their complicated mechanisms due to their remarkable anticancer activities. Although plant lectins seems promising as a potential anticancer agent for further preclinical and clinical uses, further research is still urgently needed and should include more focus on molecular mechanisms. Herein, a Naïve Bayesian model was developed to predict the protein-protein interaction (PPI), and thus construct the global human PPI network. Moreover, multiple sources of biological data, such as smallest shared biological process (SSBP), domain-domain interaction (DDI), gene co-expression profiles and cross-species interolog mapping were integrated to build the core apoptotic PPI network. In addition, we further modified it into a plant lectin-induced apoptotic cell death context. Then, we identified 22 apoptotic hub proteins in mesothelioma cells according to their different microarray expressions. Subsequently, we used combinational methods to predict microRNAs (miRNAs) which could negatively regulate the abovementioned hub proteins. Together, we demonstrated the ability of our Naïve Bayesian model-based network for identifying novel plant lectin-treated cancer cell apoptotic pathways. These findings may provide new clues concerning plant lectins as potential apoptotic inducers for cancer drug discovery. PMID:26867193

  19. Identification of Novel Pathways in Plant Lectin-Induced Cancer Cell Apoptosis.

    PubMed

    Shi, Zheng; Sun, Rong; Yu, Tian; Liu, Rong; Cheng, Li-Jia; Bao, Jin-Ku; Zou, Liang; Tang, Yong

    2016-01-01

    Plant lectins have been investigated to elucidate their complicated mechanisms due to their remarkable anticancer activities. Although plant lectins seems promising as a potential anticancer agent for further preclinical and clinical uses, further research is still urgently needed and should include more focus on molecular mechanisms. Herein, a Naïve Bayesian model was developed to predict the protein-protein interaction (PPI), and thus construct the global human PPI network. Moreover, multiple sources of biological data, such as smallest shared biological process (SSBP), domain-domain interaction (DDI), gene co-expression profiles and cross-species interolog mapping were integrated to build the core apoptotic PPI network. In addition, we further modified it into a plant lectin-induced apoptotic cell death context. Then, we identified 22 apoptotic hub proteins in mesothelioma cells according to their different microarray expressions. Subsequently, we used combinational methods to predict microRNAs (miRNAs) which could negatively regulate the abovementioned hub proteins. Together, we demonstrated the ability of our Naïve Bayesian model-based network for identifying novel plant lectin-treated cancer cell apoptotic pathways. These findings may provide new clues concerning plant lectins as potential apoptotic inducers for cancer drug discovery. PMID:26867193

  20. Characterization of mannose binding lectin from channel catfish Ictalurus punctatus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mannose-binding lectin (MBL) is an important component of innate immunity capable of activating the lectin pathway of the complement system. A MBL gene was isolated from channel catfish (Ictalurus punctatus). The deduced protein contains a canonical collagen-like domain, a carbohydrate recognition d...

  1. Bacteriolytic activity of the alternative pathway of complement differs kinetically from the classical pathway.

    PubMed

    Kilpi, Maaria K; Atosuo, Janne T; Lilius, Esa-Matti E

    2009-10-01

    The interaction between bacterial cells and activated complement components as a kinetic biological event is described. The bacteriolytic activity of complement in human and fish serum was assayed by measuring the decrease of bioluminescence of Escherichia coli transformed with lux genes. From the kinetic curves, the bacteriolytic CB(50)- and AB(50)-units were derived at any desired time point. It was observed that these values were irregular but decreased as a function of incubation time, and reached equal values during prolonged incubation, suggesting that the difference between the classical and alternative pathway activity is kinetic. From the kinetic curves, entirely new parameters could be derived: rate of the activation phase, rate of killing by the lytic phase and rate of killing by the entire pathway in undiluted serum. The rates of human and fish classical pathway were about five and two times higher than those of the alternative pathway respectively. PMID:19527746

  2. Complementation.

    PubMed Central

    Yook, Karen

    2005-01-01

    Mutations in many genes can result in a similar phenotype. Finding a number of mutants with the same phenotype tells you little about how many genes you are dealing with, and how mutable those genes are until you can assign those mutations to genetic loci. The genetic assay for gene assignment is called the complementation test. The simplicity and robustness of this test makes it a fundamental genetic tool for gene assignment. However, there are occasional unexpected outcomes from this test that bear explanation. This chapter reviews the complementation test and its various outcomes, highlighting relatively rare but nonetheless interesting exceptions such as intragenic complementation and non-allelic non-complementation. PMID:18023121

  3. Molecular defects in the mannose binding lectin pathway in dermatological disease: Case report and literature review

    PubMed Central

    2010-01-01

    Mannose-binding lectin (MBL) and the Mannose-binding lectin-associated serine proteases (MASPs) are an essential aspect of innate immune responses that probably play an important but understudied role in cutaneous function. The MBL-MASP pathway appears to exert its primary role by assisting in the clearance of apoptotic skin cells (thus preventing accumulation and a subsequent autoimmune response) and promoting opsonophagocytosis of invading pathogens, limiting their dissemination. Deficiencies of the pathway have been described and are associated with infectious, autoimmune and vascular complications. However, the role of this pathway in dermatological disease is essentially unexplored. We describe 6 patients presenting with recurrent inflammatory and/or infectious skin conditions who also demonstrated severely low MBL levels. One patient also had a defect in the MASP2 gene. Genotype analysis revealed specific point mutations in the MBL2 promoter in all 6 patients and a variant MASP-2 gene in one patient. Five patients presented recurrent pustular skin infections (cellulitis, folliculitis and cutaneous abscess). A case of Grover's disease and one forme fruste of Behcet's syndrome (orogenital ulcers) were also observed. The patients responded to antimicrobial therapy, although in some, recurrence of infection was the rule. It appears that MBL deficiency may contribute to recurrent skin infections and to certain forms of inflammatory skin disease. The mechanisms may relate to the role of this pathway in innate immunity, removal of apoptotic cells and in immune complexes. Further study of MBL pathway defects in dermatological disease is required. PMID:20338057

  4. Regulation of the alternative pathway of complement modulates injury and immunity in a chronic model of dextran sulphate sodium-induced colitis.

    PubMed

    Elvington, M; Schepp-Berglind, J; Tomlinson, S

    2015-03-01

    The role of complement in inflammatory bowel disease (IBD) has been studied primarily using acute models, and it is unclear how complement affects processes in more relevant chronic models of IBD in which modulation of adaptive immunity and development of fibrosis have pathogenic roles. Using mice deficient in C1q/mannose-binding lectin (MBL) or C3, we demonstrated an important role for these opsonins and/or the classical pathway C3 convertase in providing protection against mucosal injury and infection in a model of chronic dextran sulphate sodium (DSS)-induced colitis. In contrast, deficiency of the alternative pathway (fB(-/-) mice) had significantly less impact on injury profiles. Consequently, the effect of a targeted inhibitor of the alternative pathway was investigated in a therapeutic protocol. Following the establishment of colitis, mice were treated with CR2-fH during subsequent periods of DSS treatment and acute injury (modelling relapse). CR2-fH significantly reduced complement activation, inflammation and injury in the colon, and additionally reduced fibrosis. Alternative pathway inhibition also altered the immune response in the chronic state in terms of reducing numbers of B cells, macrophages and mature dendritic cells in the lamina propria. This study indicates an important role for the alternative pathway of complement in the pathogenesis and the shaping of an immune response in chronic DSS-induced colitis, and supports further investigation into the use of targeted alternative pathway inhibition for the treatment of IBD. PMID:25293413

  5. Curvature of synthetic and natural surfaces is an important target feature in classical pathway complement activation.

    PubMed

    Pedersen, Martin Bjerregård; Zhou, Xingfei; Larsen, Esben Kjaer Unmack; Sørensen, Uffe Skov; Kjems, Jørgen; Nygaard, Jens Vinge; Nyengaard, Jens Randel; Meyer, Rikke Louise; Boesen, Thomas; Vorup-Jensen, Thomas

    2010-02-15

    The binding of Abs to microbial surfaces followed by complement activation constitutes an important line of defense against infections. In this study, we have investigated the relationship between complement activation and the binding of human IgM Abs to surfaces with different curvatures. IgM Abs to dextran were shown to activate complement potently on dextran-coated particles having a diameter around 250 nm, whereas larger (600 nm) particles were less potent activators. This selectivity regarding particle dimension was also found for complement activation by colloidal substances of microbial origin. Peptidoglycan (PGN) is the major chemical component in the cell wall of Gram-positive bacteria. Fragments of purified PGN with sizes of approximately 100 nm promoted complement activation effectively through the classical pathway. By contrast, larger or smaller fragments of PGN did not activate complement strongly. A careful analysis of PGN fragments released during planctonic growth of Staphylococcus aureus showed that these include curvatures that would permit strong IgM-mediated complement activation, whereas the curvature of intact cells would be less effective for such activation. Consistently, we found that the suspended PGN fragments were strong activators of complement through the classical pathway. We suggest that these fragments act as decoy targets for complement activation, providing protection for S. aureus against the host immune response to infection. PMID:20053940

  6. Functional Characterization of a Ficolin-mediated Complement Pathway in Amphioxus*

    PubMed Central

    Huang, Huiqing; Huang, Shengfeng; Yu, Yingcai; Yuan, Shaochun; Li, Rui; Wang, Xin; Zhao, Hongchen; Yu, Yanhong; Li, Jun; Yang, Manyi; Xu, Liqun; Chen, Shangwu; Xu, Anlong

    2011-01-01

    The ficolin-mediated complement pathway plays an important role in vertebrate immunity, but it is not clear whether this pathway exists in invertebrates. Here we identified homologs of ficolin pathway components from the cephalochordate amphioxus and investigated whether they had been co-opted into a functional ficolin pathway. Four of these homologs, ficolin FCN1, serine protease MASP1 and MASP3, and complement component C3, were highly expressed in mucosal tissues and gonads, and were significantly up-regulated following bacterial infection. Recombinant FCN1 could induce hemagglutination, discriminate among sugar components, and specifically recognize and aggregate several bacteria (especially Gram-positive strains) without showing bactericidal activity. This suggested that FCN1 is a dedicated pattern-recognition receptor. Recombinant serine protease MASP1/3 formed complexes with recombinant FCN1 and facilitated the activation of native C3 protein in amphioxus humoral fluid, in which C3 acted as an immune effector. We conclude that amphioxus have developed a functional ficolin-complement pathway. Because ficolin pathway components have not been reported in non-chordate species, our findings supported the idea that this pathway may represent a chordate-specific innovation in the evolution of the complement system. PMID:21832079

  7. The complement system in human cardiometabolic disease.

    PubMed

    Hertle, E; Stehouwer, C D A; van Greevenbroek, M M J

    2014-10-01

    The complement system has been implicated in obesity, fatty liver, diabetes and cardiovascular disease (CVD). Complement factors are produced in adipose tissue and appear to be involved in adipose tissue metabolism and local inflammation. Thereby complement links adipose tissue inflammation to systemic metabolic derangements, such as low-grade inflammation, insulin resistance and dyslipidaemia. Furthermore, complement has been implicated in pathophysiological mechanisms of diet- and alcohol induced liver damage, hyperglycaemia, endothelial dysfunction, atherosclerosis and fibrinolysis. In this review, we summarize current evidence on the role of the complement system in several processes of human cardiometabolic disease. C3 is the central component in complement activation, and has most widely been studied in humans. C3 concentrations are associated with insulin resistance, liver dysfunction, risk of the metabolic syndrome, type 2 diabetes and CVD. C3 can be activated by the classical, the lectin and the alternative pathway of complement activation; and downstream activation of C3 activates the terminal pathway. Complement may also be activated via extrinsic proteases of the coagulation, fibrinolysis and the kinin systems. Studies on the different complement activation pathways in human cardiometabolic disease are limited, but available evidence suggests that they may have distinct roles in processes underlying cardiometabolic disease. The lectin pathway appeared beneficial in some studies on type 2 diabetes and CVD, while factors of the classical and the alternative pathway were related to unfavourable cardiometabolic traits. The terminal complement pathway was also implicated in insulin resistance and liver disease, and appears to have a prominent role in acute and advanced CVD. The available human data suggest a complex and potentially causal role for the complement system in human cardiometabolic disease. Further, preferably longitudinal studies are needed to disentangle which aspects of the complement system and complement activation affect the different processes in human cardiometabolic disease. PMID:25017306

  8. Current Understanding of the Role of Complement in IgA Nephropathy.

    PubMed

    Maillard, Nicolas; Wyatt, Robert J; Julian, Bruce A; Kiryluk, Krzysztof; Gharavi, Ali; Fremeaux-Bacchi, Veronique; Novak, Jan

    2015-07-01

    Complement activation has a role in the pathogenesis of IgA nephropathy, an autoimmune disease mediated by pathogenic immune complexes consisting of galactose-deficient IgA1 bound by antiglycan antibodies. Of three complement-activation pathways, the alternative and lectin pathways are involved in IgA nephropathy. IgA1 can activate both pathways in vitro, and pathway components are present in the mesangial immunodeposits, including properdin and factor H in the alternative pathway and mannan-binding lectin, mannan-binding lectin-associated serine proteases 1 and 2, and C4d in the lectin pathway. Genome-wide association studies identified deletion of complement factor H-related genes 1 and 3 as protective against the disease. Because the corresponding gene products compete with factor H in the regulation of the alternative pathway, it has been hypothesized that the absence of these genes could lead to more potent inhibition of complement by factor H. Complement activation can take place directly on IgA1-containing immune complexes in circulation and/or after their deposition in the mesangium. Notably, complement factors and their fragments may serve as biomarkers of IgA nephropathy in serum, urine, or renal tissue. A better understanding of the role of complement in IgA nephropathy may provide potential targets and rationale for development of complement-targeting therapy of the disease. PMID:25694468

  9. Alternative Complement Pathway Deficiency Ameliorates Chronic Smoke-Induced Functional and Morphological Ocular Injury

    PubMed Central

    Woodell, Alex; Coughlin, Beth; Kunchithapautham, Kannan; Casey, Sarah; Williamson, Tucker; Ferrell, W. Drew; Atkinson, Carl; Jones, Bryan W.; Rohrer, Bärbel

    2013-01-01

    Background Age-related macular degeneration (AMD), a complex disease involving genetic variants and environmental insults, is among the leading causes of blindness in Western populations. Genetic and histologic evidence implicate the complement system in AMD pathogenesis; and smoking is the major environmental risk factor associated with increased disease risk. Although previous studies have demonstrated that cigarette smoke exposure (CE) causes retinal pigment epithelium (RPE) defects in mice, and smoking leads to complement activation in patients, it is unknown whether complement activation is causative in the development of CE pathology; and if so, which complement pathway is required. Methods Mice were exposed to cigarette smoke or clean, filtered air for 6 months. The effects of CE were analyzed in wildtype (WT) mice or mice without a functional complement alternative pathway (AP; CFB−/−) using molecular, histological, electrophysiological, and behavioral outcomes. Results CE in WT mice exhibited a significant reduction in function of both rods and cones as determined by electroretinography and contrast sensitivity measurements, concomitant with a thinning of the nuclear layers as measured by SD-OCT imaging and histology. Gene expression analyses suggested that alterations in both photoreceptors and RPE/choroid might contribute to the observed loss of function, and visualization of complement C3d deposition implies the RPE/Bruch's membrane (BrM) complex as the target of AP activity. RPE/BrM alterations include an increase in mitochondrial size concomitant with an apical shift in mitochondrial distribution within the RPE and a thickening of BrM. CFB−/− mice were protected from developing these CE-mediated alterations. Conclusions Taken together, these findings provide clear evidence that ocular pathology generated in CE mice is dependent on complement activation and requires the AP. Identifying animal models with RPE/BrM damage and verifying which aspects of pathology are dependent upon complement activation is essential for developing novel complement-based treatment approaches for the treatment of AMD. PMID:23825688

  10. Complement activation by the alternative pathway and macrophage enzyme secretion in the pathogenesis of chronic inflammation.

    PubMed Central

    Schorlemmer, H U; Bitter-Suermann, D; Allison, A C

    1977-01-01

    A number of stimuli known to induce acid hydrolase secretion from cultured macrophages were examined for their ability to activate C3 via the alternative pathway of the complement system. Loss of haemolytically active C3 was checked in normal and C4-deficient guinea-pig serum. For comparison the interactions of cultured macrophages with other agents well known as potent activators of the alternative pathway of the complement system have been investigated. As judged by their activity in these assays, group A streptococcal cell walls, different carrageenan preparations, dental plaque and Actinomyces viscosus were all capable of initiating the alternative pathway but differed with respect to their potency and their ability to inhibit C3 turnover at high concentrations. Zymosan, some carrageenans, polyanethol sulphonate, and Corynebacterium parvum all induce the release of hydrolytic enzymes from macrophages in culture, even in the absence of serum in the medium. The release is time- and dose-dependent and is not associated with loss of the cytoplasmic enzyme lactate dehydrogenase or any other sign of cell death. The parallelism between the capacity of several agents to activate the complement system via the alternative pathway and to induce inflammatory responses in vivo and selective lysosoma enzyme secretion from cultures of macrophages is discussed. PMID:328387

  11. Exogenous expression of marine lectins DlFBL and SpRBL induces cancer cell apoptosis possibly through PRMT5-E2F-1 pathway

    PubMed Central

    Wu, Liqin; Yang, Xinyan; Duan, Xuemei; Cui, Lianzhen; Li, Gongchu

    2014-01-01

    Lectins are widely existed in marine bioresources, and some purified marine lectins were found toxic to cancer cells. In this report, genes encoding Dicentrarchus labrax fucose-binding lectin (DlFBL) and Strongylocentrotus purpuratus rhamnose-binding lectin (SpRBL) were inserted into an adenovirus vector to form Ad.FLAG-DlFBL and Ad.FLAG-SpRBL, which elicited significant in vitro suppressive effect on a variety of cancer cells. Anti-apoptosis factors Bcl-2 and XIAP were determined to be downregulated by Ad.FLAG-DlFBL and Ad.FLAG-SpRBL. Subcellular localization studies showed that DlFBL but not SpRBL widely distributed in membrane systems. Both DlFBL and SpRBL were shown associated with protein arginine methyltransferase 5 (PRMT5), and PRMT5-E2F-1 pathway was suggested to be responsible for the DlFBL and SpRBL induced apoptosis. Further investigations revealed that PRMT5 acted as a common binding target for various exogenous lectin and non-lectin proteins, suggesting a role of PRMT5 as a barrier for foreign gene invasion. The cellular response to exogenous lectins may provide insights into a novel way for cancer gene therapy. PMID:24675921

  12. The role of complement in membranous nephropathy

    PubMed Central

    Ma, Hong; Sandor, Dana G.; Beck, Laurence H.

    2013-01-01

    Membranous nephropathy (MN) describes a histopathological pattern of injury marked by glomerular subepithelial immune deposits and collectively represents one of the most common causes of adult nephrotic syndrome. Studies in Heymann nephritis, an experimental model of MN, have established a paradigm in which these deposits locally activate complement to cause podocyte injury, culminating in cytoskeletal reorganization, loss of slit diaphragms, and proteinuria. There is much circumstantial evidence for a prominent role of complement in human MN, as C3 and C5b-9 are consistently found within immune deposits. Secondary MN often exhibits the additional presence of C1q, implicating the classical pathway of complement activation. Primary MN, however, is IgG4-predominant and IgG4 is considered incapable of binding C1q and activating the complement pathway. Recent studies have identified the M-type phospholipase A2 receptor (PLA2R) as the major target antigen in primary MN. Early evidence hints that IgG4 anti-PLA2R autoantibodies can bind mannan-binding lectin and activate the lectin complement pathway. The identification of anti-PLA2R antibodies as likely participants in the pathogenesis of disease will allow focused investigation into the role of complement in MN. Definitive therapy for MN is immunosuppression, although future therapeutic agents that specifically target complement activation may represent an effective temporizing measure to forestall further glomerular injury. PMID:24161038

  13. Alternative Pathway of Complement in Children with Diarrhea-Associated Hemolytic Uremic Syndrome

    PubMed Central

    Thurman, Joshua M.; Marians, Russell; Emlen, Woodruff; Wood, Susan; Smith, Christopher; Akana, Hillary; Holers, V. Michael; Lesser, Martin; Kline, Myriam; Hoffman, Cathy; Christen, Erica

    2009-01-01

    Background and objectives: Diarrhea-associated hemolytic uremic syndrome (D+HUS) is a common cause of acute kidney injury in children. Mutations in alternative pathway (AP) complement regulatory proteins have been identified in severe cases of thrombotic microangiopathy, but the role of the AP in D+HUS has not been studied. Therefore, we determined whether plasma levels of markers of activation of the AP are increased in D+HUS and are biomarkers of the severity of renal injury that predict the need for dialysis. Design, setting, participants, & measurements: Patients were randomly selected from among participants in the HUS-SYNSORB Pk trial. Plasma samples were collected on days 1, 4, 7, and 10 after enrollment and day 28 after discharge from the hospital. Levels of two complement pathway products, Bb and SC5b-9, were determined by ELISA. Results: Seventeen children (6 boys and 11 girls; age, 5.4 ± 3.5 yr) were studied. Eight (47%) required dialysis support, and two had serious extrarenal events. On the day of enrollment, plasma levels of Bb and SC5b-9 were significantly increased in all patients compared with healthy controls (P < 0.01). The elevated concentrations normalized by day 28 after discharge. Circulating levels of complement pathway fragments did not correlate with severity of renal injury or occurrence of complications. Conclusions: Patients with acute-onset D+HUS manifest activation of the AP of complement that is temporally related to the onset of disease and that resolves within 1 mo. Therapies to inhibit the AP of complement may be useful in attenuating the severity of renal injury and extrarenal complications. PMID:19820137

  14. Demonstration of alternative and classical complement pathway activity in colostrum from buffalo (Bubalus bubalis).

    PubMed

    Matheswaran, K; Dhinakar Raj, G; Nachimuthu, K

    2003-09-01

    Buffalo colostrum caused lysis of unsensitized red blood cells (RBC) from sheep, goats, rabbits and chickens. RBC from cattle and buffalo were resistant to lysis. That lysis was due to the presence of natural antibodies to these RBC was ruled out since there was no reduction in haemolytic titres even after adsorption with the respective RBC. The addition of EGTA to the diluent had no effect on the haemolytic activity. These findings indicate the presence of alternative complement pathway (ACP) activity in buffalo colostrum. The haemolytic activity of buffalo complement for unsensitized rabbit RBC was reduced to very low levels by heating at 50 degrees C for 45 min. Treatment with zymosan also inhibited the haemolytic activity, while inulin had no effect. The maximum activity of ACP occurred in the presence of 4 mmol/L Mg(2+) in the diluent. The range of ACP activities in colostrum from buffaloes varied from 4.06 to 8.48 CH50 units/ml. Using a standard system for titrating the classical complement pathway and rabbit red blood cells sensitized with goat haemolysin, the range of complement activity in buffalo colostrum was 4.81-6.77 CH50/ml. PMID:14582743

  15. A single-CRD C-type lectin from oyster Crassostrea gigas mediates immune recognition and pathogen elimination with a potential role in the activation of complement system.

    PubMed

    Li, Hui; Zhang, Huan; Jiang, Shuai; Wang, Weilin; Xin, Lusheng; Wang, Hao; Wang, Lingling; Song, Linsheng

    2015-06-01

    C-type lectins (CTLs), serving as pattern recognition receptors (PRRs), are a superfamily of Ca(2+)-dependent carbohydrate-recognition proteins that participate in nonself-recognition and pathogen elimination. In the present study, a single carbohydrate-recognition domain (CRD) CTL was identified from oyster Crassostrea gigas (designated as CgCLec-2). There was only one CRD within the deduced amino acid sequence of CgCLec-2 consisting of 129 amino acid residues. A conserved EPN (Glu246-Pro247-Asn248) motif was found in Ca(2+)-binding site 2 of CgCLec-2. The CgCLec-2 mRNA could be detected in all the examined tissues at different expression levels in oysters. The mRNA expression of CgCLec-2 in hemocytes was up-regulated significantly at 6 h post Vibrio splendidus challenge. The recombinant CgCLec-2 (rCgCLec-2) could bind various Pathogen-Associated Molecular Patterns (PAMPs), including lipopolysaccharide, mannan and peptidoglycan, and displayed strong binding abilities to Vibrio anguillarum, V. splendidus and Yarrowiali polytica and week binding ability to Staphylococcus aureus. It could also enhance the phagocytic activity of oyster hemocytes to V. splendidus and exhibited growth suppression activity against gram-positive bacteria S. aureus but no effect on gram-negative bacteria V. splendidus. Furthermore, the interaction between rCgCLec-2 and rCgMASPL-1 was confirmed by GST Pull down. The results suggested that CgCLec-2 served as not only a PRR in immune recognition but also a regulatory factor in pathogen elimination, and played a potential role in the activation of complement system. PMID:25800112

  16. Activation of the alternative and classical complement pathways by Entamoeba histolytica.

    PubMed Central

    Calderon, J; Schreiber, R D

    1985-01-01

    Entamoeba histolytica HM1 supported the activation of human alternative and classical complement pathways in the absence of ameba-reactive antibodies. Nonimmune serum depleted of C1q and factor D (NHS s C1q + D) and reconstituted with C1q was able to specifically deposit C3b onto trophozoites and produce lysis. This activity was not modified by the absorption of serum on E. histolytica. Serum depleted of factor B allowed C3b binding to amebae. Serum devoid of C4 effected only small amounts of C3 uptake. The kinetics of lysis of E. histolytica by serum in the presence of Mg-EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] (lacking classical pathway function) or by NHS s C1q + D and reconstituted with factor D was slow and only produced one-half the amount of lysis produced by NHS s C1q + D supplemented with C1q. These results indicate that the surface of the ameba can promote complement activation by the classical pathway, without the participation of specific antibodies, and that the magnitude of this activation is greater than that induced by the alternative pathway. PMID:2865212

  17. Single-walled carbon nanotube surface control of complement recognition and activation.

    PubMed

    Andersen, Alina J; Robinson, Joshua T; Dai, Hongjie; Hunter, A Christy; Andresen, Thomas L; Moghimi, S Moein

    2013-02-26

    Carbon nanotubes (CNTs) are receiving considerable attention in site-specific drug and nucleic acid delivery, photodynamic therapy, and photoacoustic molecular imaging. Despite these advances, nanotubes may activate the complement system (an integral part of innate immunity), which can induce clinically significant anaphylaxis. We demonstrate that single-walled CNTs coated with human serum albumin activate the complement system through C1q-mediated classical and the alternative pathways. Surface coating with methoxypoly(ethylene glycol)-based amphiphiles, which confers solubility and prolongs circulation profiles of CNTs, activates the complement system differently, depending on the amphiphile structure. CNTs with linear poly(ethylene glycol) amphiphiles trigger the lectin pathway of the complement through both L-ficolin and mannan-binding lectin recognition. The lectin pathway activation, however, did not trigger the amplification loop of the alternative pathway. An amphiphile with branched poly(ethylene glycol) architecture also activated the lectin pathway but only through L-ficolin recognition. Importantly, this mode of activation neither generated anaphylatoxins nor induced triggering of the effector arm of the complement system. These observations provide a major step toward nanomaterial surface modification with polymers that have the properties to significantly improve innate immunocompatibility by limiting the formation of complement C3 and C5 convertases. PMID:23301860

  18. Activation of the alternative pathway of complement during the acute phase of typical haemolytic uraemic syndrome.

    PubMed

    Ferraris, J R; Ferraris, V; Acquier, A B; Sorroche, P B; Saez, M S; Ginaca, A; Mendez, C F

    2015-07-01

    Haemolytic uraemic syndrome (HUS) is characterized by haemolytic anaemia, thrombocytopenia and acute renal failure. We studied the activation state of classical and alternative pathways of complement during the acute phase of Shiga toxin-associated HUS by performing a prospective study of 18 patients and 17 age-matched healthy controls to evaluate C3, C3c, C4, C4d, Bb and SC5b-9 levels. SC5b-9 levels were increased significantly in all patients at admission compared to healthy and end-stage renal disease controls, but were significantly higher in patients presenting with oliguria compared to those with preserved diuresis. C3 and C4 levels were elevated significantly at admission in the non-oliguric group when compared to controls. No significant differences were found for C4d values, whereas factor Bb was elevated in all patients and significantly higher in oliguric patients when compared to both controls and non-oliguric individuals. A positive and significant association was detected when Bb formation was plotted as a function of plasma SC5b-9 at admission. Bb levels declined rapidly during the first week, with values not significantly different from controls by days 3 and 5 for non-oligurics and oligurics, respectively. Our data demonstrate the activation of the alternative pathway of complement during the acute phase of Stx-associated HUS. This finding suggests that complement activation may represent an important trigger for the cell damage that occurs during the syndrome. PMID:25677399

  19. Complement activity is associated with disease severity in multifocal motor neuropathy

    PubMed Central

    Vlam, Lotte; Cats, Elisabeth A.; Harschnitz, Oliver; Jansen, Marc D.; Piepers, Sanne; Veldink, Jan Herman; Franssen, Hessel; Stork, Abraham C.J.; Heezius, Erik; Rooijakkers, Suzan H.M.; Herpers, Bjorn L.; van Strijp, Jos A.; van den Berg, Leonard H.

    2015-01-01

    Objective: To investigate whether high innate activity of the classical and lectin pathways of complement is associated with multifocal motor neuropathy (MMN) and whether levels of innate complement activity or the potential of anti-GM1 antibodies to activate the complement system correlate with disease severity. Methods: We performed a case-control study including 79 patients with MMN and 79 matched healthy controls. Muscle weakness was documented with Medical Research Council scale sum score and axonal loss with nerve conduction studies. Activity of the classical and lectin pathways of complement was assessed by ELISA. We also determined serum mannose-binding lectin (MBL) concentrations and polymorphisms in the MBL gene (MBL2) and quantified complement-activating properties of anti-GM1 IgM antibodies by ELISA. Results: Activity of the classical and lectin pathways, MBL2 genotypes, and serum MBL concentrations did not differ between patients and controls. Complement activation by anti-GM1 IgM antibodies was exclusively mediated through the classical pathway and correlated with antibody titers (p < 0.001). Logistic regression analysis showed that both high innate activity of the classical pathway of complement and high complement-activating capacity of anti-GM1 IgM antibodies were significantly associated with more severe muscle weakness and axonal loss. Conclusion: High innate activity of the classical pathway of complement and efficient complement-activating properties of anti-GM1 IgM antibodies are determinants of disease severity in patients with MMN. These findings underline the importance of anti-GM1 antibody–mediated complement activation in the pathogenesis and clinical course of MMN. PMID:26161430

  20. Vascular smooth muscle relaxation by a lectin from Pisum arvense: evidences of endothelial NOS pathway.

    PubMed

    Assreuy, Ana Maria Sampaio; Pinto, Nilson Vieira; Lima Mota, Mário Rogério Lima; Passos Meireles, Ana Vaneska Passos; Cajazeiras, João Batista; Nobre, Camila Bezerra; Soares, Pedro Marcos Gomes; Cavada, Benildo Sousa

    2011-11-01

    The vasorelaxant effect of the lectin of Pisum arvense (PAL) seeds was investigated in rat aorta. PAL (10-100 µg/ml) was applied on aorta rings, with or without endothelium, pre-contracted with phenylephrine (Phe; 0.1 µM). Participation of endothelium derived relaxant factors was evaluated incubating the tissue with indomethacin (10 µM), L-nitro arginine methyl ester (L-NAME, 100 µM) and tetraethylammonium (TEA, 5 mM) before addition of PAL. The role of the lectin domain was investigated by addition of PAL into tissue in presence of glucose (3x 10⁻⁵ M), or N-acetyl Dglucosamine (GlcNAc; 3 x 10⁻⁴ M). The importance of extracellular calcium (Ca²⁺e) or interaction with muscarinic receptors in the relaxant effect was evaluated by addition of PAL into aorta rings containing calcium free solution (OCa) and atropine (1 µ M), respectively. PAL induced concentration-dependent relaxation in endothelized aorta (IC50 =58.38 ± 1.87 µg/ml), which was reversed by L-NAME and glucose. The lectin effect was totally inhibited when the preparation was inserted in OCa, but not in presence of atropine. Summarizing, our data showed a relaxant effect of PAL in isolated rat aorta rings in presence of endothelium, suggestive of interaction between the lectin carbohydrate binding sites with specific receptors located in vascular endothelial cells leading to nitric oxide synthase activation. This effect seems to require Ca²⁺e but is independent on muscarinic receptors interaction. PMID:21675947

  1. Dysregulation of Complement System and CD4+ T Cell Activation Pathways Implicated in Allergic Response

    PubMed Central

    Couto Alves, Alexessander; Bruhn, Sören; Ramasamy, Adaikalavan; Wang, Hui; Holloway, John W.; Hartikainen, Anna-Liisa; Jarvelin, Marjo-Riitta; Benson, Mikael; Balding, David J.; Coin, Lachlan J. M.

    2013-01-01

    Allergy is a complex disease that is likely to involve dysregulated CD4+ T cell activation. Here we propose a novel methodology to gain insight into how coordinated behaviour emerges between disease-dysregulated pathways in response to pathophysiological stimuli. Using peripheral blood mononuclear cells of allergic rhinitis patients and controls cultured with and without pollen allergens, we integrate CD4+ T cell gene expression from microarray data and genetic markers of allergic sensitisation from GWAS data at the pathway level using enrichment analysis; implicating the complement system in both cellular and systemic response to pollen allergens. We delineate a novel disease network linking T cell activation to the complement system that is significantly enriched for genes exhibiting correlated gene expression and protein-protein interactions, suggesting a tight biological coordination that is dysregulated in the disease state in response to pollen allergen but not to diluent. This novel disease network has high predictive power for the gene and protein expression of the Th2 cytokine profile (IL-4, IL-5, IL-10, IL-13) and of the Th2 master regulator (GATA3), suggesting its involvement in the early stages of CD4+ T cell differentiation. Dissection of the complement system gene expression identifies 7 genes specifically associated with atopic response to pollen, including C1QR1, CFD, CFP, ITGB2, ITGAX and confirms the role of C3AR1 and C5AR1. Two of these genes (ITGB2 and C3AR1) are also implicated in the network linking complement system to T cell activation, which comprises 6 differentially expressed genes. C3AR1 is also significantly associated with allergic sensitisation in GWAS data. PMID:24116013

  2. Identification of peptidic inhibitors of the alternative complement pathway based on Staphylococcus aureus SCIN proteins.

    PubMed

    Summers, Brady J; Garcia, Brandon L; Woehl, Jordan L; Ramyar, Kasra X; Yao, Xiaolan; Geisbrecht, Brian V

    2015-10-01

    The complement system plays a central role in a number of human inflammatory diseases, and there is a significant need for development of complement-directed therapies. The discovery of an arsenal of anti-complement proteins secreted by the pathogen Staphylococcus aureus brought with it the potential for harnessing the powerful inhibitory properties of these molecules. One such family of inhibitors, the SCINs, interact with a functional "hot-spot" on the surface of C3b. SCINs not only stabilize an inactive form of the alternative pathway (AP) C3 convertase (C3bBb), but also overlap the C3b binding site of complement factors B and H. Here we determined that a conserved Arg residue in SCINs is critical for function of full-length SCIN proteins. Despite this, we also found SCIN-specific differences in the contributions of other residues found at the C3b contact site, which suggested that a more diverse repertoire of residues might be able to recognize this region of C3b. To investigate this possibility, we conducted a phage display screen aimed at identifying SCIN-competitive 12-mer peptides. In total, seven unique sequences were identified and all exhibited direct C3b binding. A subset of these specifically inhibited the AP in assays of complement function. The mechanism of AP inhibition by these peptides was probed through surface plasmon resonance approaches, which revealed that six of the seven peptides disrupted C3bBb formation by interfering with factor B/C3b binding. To our knowledge this study has identified the first small molecules that retain inhibitory properties of larger staphylococcal immune evasion proteins. PMID:26052070

  3. Complement in animal development: unexpected roles of a highly conserved pathway.

    PubMed

    Leslie, Jonathan D; Mayor, Roberto

    2013-02-01

    The complement pathway is most famous for its role in immunity, orchestrating an exquisitely refined system for immune surveillance. At its core lies a cascade of proteolytic events that ultimately serve to recognise microbes, infected cells or debris and target them for elimination. Mounting evidence has shown that a number of the proteolytic intermediaries in this cascade have, in themselves, other functions in the body, signalling through receptors to drive events that appear to be unrelated to immune surveillance. It seems, then, that the complement system not only functions as an immunological effector, but also has cell-cell signalling properties that are utilised by a number of non-immunological processes. In this review we examine a number of these processes in the context of animal development, all of which share a requirement for precise control of cell behaviour in time and space. As we will see, the scope of the complement system's function is indeed much greater than we might have imagined only a few years ago. PMID:23665279

  4. The alternative complement pathway is longitudinally associated with adverse cardiovascular outcomes. The CODAM study.

    PubMed

    Hertle, Elisabeth; Arts, Ilja C W; van der Kallen, Carla J H; Feskens, Edith J M; Schalkwijk, Casper G; Stehouwer, Coen D A; van Greevenbroek, Marleen M J

    2016-01-01

    The alternative pathway of complement activation is highly reactive and can be activated spontaneously in the vasculature. Activation may contribute to vascular damage and development of cardiovascular disease (CVD). We aimed to investigate functional components of the alternative pathway in cardiovascular risk. We studied 573 individuals who were followed-up for seven years. At baseline, we measured the enhancer properdin; the rate-limiting protease factor D (FD); and a marker of systemic activation, Bb. Using generalised estimating equations, we investigated their longitudinal associations with cardiovascular events (CVE, N=89), CVD (N=159), low-grade inflammation (LGI), endothelial dysfunction (ED) and carotid intima-media thickness (cIMT). Furthermore, we investigated associations with incident CVE (N=39) and CVD (N=73) in 342 participants free of CVD at baseline. CVE included myocardial infarction, stroke, cardiac angioplasty and/or cardiac bypass. CVD additionally included ischaemia on an electrocardiogram and/or ankle-brachial index < 0.9. In adjusted analyses, properdin was positively associated with CVE (per 1SD, longitudinal OR=1.36 [1.07; 1.74], OR for incident CVE=1.53 [1.06; 2.20]), but not with CVD. Properdin was also positively associated with ED (β=0.13 [95%CI 0.06; 0.20]), but not with LGI or cIMT. FD and Bb were positively associated with LGI (per 1SD, FD: β=0.21 [0.12; 0.29], Bb: β=0.14 [0.07; 0.21]), and ED (FD: β=0.20 [0.11; 0.29], Bb: β=0.10 [0.03; 0.18]), but not with cIMT, CVE or CVD. Taken together, this suggests that the alternative complement pathway contributes to processes of vascular damage, and that in particular a high potential to enhance alternative pathway activation may promote unfavourable cardiovascular outcomes in humans. PMID:26446431

  5. Borrelia burgdorferi BBK32 Inhibits the Classical Pathway by Blocking Activation of the C1 Complement Complex

    PubMed Central

    Wager, Beau; Höök, Magnus; Skare, Jon T.

    2016-01-01

    Pathogens that traffic in blood, lymphatics, or interstitial fluids must adopt strategies to evade innate immune defenses, notably the complement system. Through recruitment of host regulators of complement to their surface, many pathogens are able to escape complement-mediated attack. The Lyme disease spirochete, Borrelia burgdorferi, produces a number of surface proteins that bind to factor H related molecules, which function as the dominant negative regulator of the alternative pathway of complement. Relatively less is known about how B. burgdorferi evades the classical pathway of complement despite the observation that some sensu lato strains are sensitive to classical pathway activation. Here we report that the borrelial lipoprotein BBK32 potently and specifically inhibits the classical pathway by binding with high affinity to the initiating C1 complex of complement. In addition, B. burgdorferi cells that produce BBK32 on their surface bind to both C1 and C1r and a serum sensitive derivative of B. burgdorferi is protected from killing via the classical pathway in a BBK32-dependent manner. Subsequent biochemical and biophysical approaches localized the anti-complement activity of BBK32 to its globular C-terminal domain. Mechanistic studies reveal that BBK32 acts by entrapping C1 in its zymogen form by binding and inhibiting the C1 subcomponent, C1r, which serves as the initiating serine protease of the classical pathway. To our knowledge this is the first report of a spirochetal protein acting as a direct inhibitor of the classical pathway and is the only example of a biomolecule capable of specifically and noncovalently inhibiting C1/C1r. By identifying a unique mode of complement evasion this study greatly enhances our understanding of how pathogens subvert and potentially manipulate host innate immune systems. PMID:26808924

  6. Borrelia burgdorferi BBK32 Inhibits the Classical Pathway by Blocking Activation of the C1 Complement Complex.

    PubMed

    Garcia, Brandon L; Zhi, Hui; Wager, Beau; Höök, Magnus; Skare, Jon T

    2016-01-01

    Pathogens that traffic in blood, lymphatics, or interstitial fluids must adopt strategies to evade innate immune defenses, notably the complement system. Through recruitment of host regulators of complement to their surface, many pathogens are able to escape complement-mediated attack. The Lyme disease spirochete, Borrelia burgdorferi, produces a number of surface proteins that bind to factor H related molecules, which function as the dominant negative regulator of the alternative pathway of complement. Relatively less is known about how B. burgdorferi evades the classical pathway of complement despite the observation that some sensu lato strains are sensitive to classical pathway activation. Here we report that the borrelial lipoprotein BBK32 potently and specifically inhibits the classical pathway by binding with high affinity to the initiating C1 complex of complement. In addition, B. burgdorferi cells that produce BBK32 on their surface bind to both C1 and C1r and a serum sensitive derivative of B. burgdorferi is protected from killing via the classical pathway in a BBK32-dependent manner. Subsequent biochemical and biophysical approaches localized the anti-complement activity of BBK32 to its globular C-terminal domain. Mechanistic studies reveal that BBK32 acts by entrapping C1 in its zymogen form by binding and inhibiting the C1 subcomponent, C1r, which serves as the initiating serine protease of the classical pathway. To our knowledge this is the first report of a spirochetal protein acting as a direct inhibitor of the classical pathway and is the only example of a biomolecule capable of specifically and noncovalently inhibiting C1/C1r. By identifying a unique mode of complement evasion this study greatly enhances our understanding of how pathogens subvert and potentially manipulate host innate immune systems. PMID:26808924

  7. A zebrafish model for uremic toxicity: role of the complement pathway.

    PubMed

    Berman, Nathaniel; Lectura, Melisa; Thurman, Joshua M; Reinecke, James; Raff, Amanda C; Melamed, Michal L; Quan, Zhe; Evans, Todd; Meyer, Timothy W; Hostetter, Thomas H

    2013-01-01

    Many organic solutes accumulate in end-stage renal disease (ESRD) and some are poorly removed with urea-based prescriptions for hemodialysis. However, their toxicities have been difficult to assess. We have employed an animal model, the zebrafish embryo, to test the toxicity of uremic serum compared to control. Serum was obtained from stable ESRD patients predialysis or from normal subjects. Zebrafish embryos 24 h postfertilization were exposed to experimental media at a water:human serum ratio of 3:1. Those exposed to serum from uremic subjects had significantly reduced survival at 8 h (19 ± 18 vs. 94 ± 6%, p < 0.05, uremic serum vs. control, respectively). Embryos exposed to serum from ESRD subjects fractionated at 50 kDa showed significantly greater toxicity with the larger molecular weight fraction (83 ± 11 vs. 7 ± 17% survival, p < 0.05, <50 vs. >50 kDa, respectively). Heating serum abrogated its toxicity. EDTA, a potent inhibitor of complement by virtue of calcium chelation, reduced the toxicity of uremic serum compared to untreated uremic serum (96 ± 5 vs. 28 ± 20% survival, p < 0.016, chelated vs. nonchelated serum, respectively). Anti-factor B, a specific inhibitor of the alternative complement pathway, reduced the toxicity of uremic serum, compared to untreated uremic serum (98 ± 6 vs. 3 ± 9% survival, p < 0.016, anti-factor B treated vs. nontreated, respectively). Uremic serum is thus more toxic to zebrafish embryos than normal serum. Furthermore, this toxicity is associated with a fraction of large size, is inactivated by heat, and is reduced by both specific and nonspecific inhibitors of complement activation. Together these data lend support to the hypothesis that at least some uremic toxicities may be mediated by complement. PMID:23689420

  8. Genetic Investigation of Complement Pathway Genes in Type 2 Diabetic Retinopathy: An Inflammatory Perspective

    PubMed Central

    Yang, Ming Ming; Wang, Jun; Ren, Hong; Sun, Yun Duan; Fan, Jiao Jie; Teng, Yan; Li, Yan Bo

    2016-01-01

    Diabetic retinopathy (DR) has complex multifactorial pathogenesis. This study aimed to investigate the association of complement pathway genes with susceptibility to DR. Eight haplotype-tagging SNPs of SERPING1 and C5 were genotyped in 570 subjects with type 2 diabetes: 295 DR patients (138 nonproliferative DR [NPDR] and 157 proliferative DR [PDR]) and 275 diabetic controls. Among the six C5 SNPs, a marginal association was first detected between rs17611 and total DR patients (P = 0.009, OR = 0.53 for recessive model). In stratification analysis, a significant decrease in the frequencies of G allele and GG homozygosity for rs17611 was observed in PDR patients compared with diabetic controls (Pcorr = 0.032, OR = 0.65 and Pcorr = 0.016, OR = 0.37, resp.); it was linked with a disease progression. A haplotype AA defined by the major alleles of rs17611 and rs1548782 was significantly predisposed to PDR with increased risk of 1.54 (Pcorr = 0.023). Regarding other variants in C5 and SERPING1, none of the tagging SNPs had a significant association with DR and its subgroups (all P > 0.05). Our study revealed an association between DR and C5 polymorphisms with clinical significance, whereas SERPING1 is not a major genetic component of DR. Our data suggest a link of complement pathway with DR pathogenesis. PMID:26989329

  9. Coding polymorphisms in the genes of the alternative complement pathway and abdominal aortic aneurysm.

    PubMed

    Bradley, D T; Badger, S A; Bown, M J; Sayers, R D; Hughes, A E

    2011-06-01

    Variants in the genes of the alternative complement pathway are associated with risk of numerous inflammatory diseases. Abdominal aortic aneurysm is associated with inflammation and is a common cause of illness and death among European populations. This study tested 49 single nucleotide polymorphisms, including common putatively functional polymorphisms, in the genes of the alternative complement cascade (CFH, CFB, CFD, CFI, properdin, CR1, CR1L, CR2, CD46, vitronectin, C3, C5, C6, C7, C8A, C8B, C8G and C9). The study group were 434 cases with infra-renal aortic diameter ≥30 mm and 378 disease-free controls from two UK centres, all with self-reported European ancestry. There was no evidence for significant association with presence or size of aneurysm following correction for multiple testing. This study suggests that variation in the genes of the alternative pathway is not an important cause of abdominal aortic aneurysm development. PMID:21352499

  10. Genetic Investigation of Complement Pathway Genes in Type 2 Diabetic Retinopathy: An Inflammatory Perspective.

    PubMed

    Yang, Ming Ming; Wang, Jun; Ren, Hong; Sun, Yun Duan; Fan, Jiao Jie; Teng, Yan; Li, Yan Bo

    2016-01-01

    Diabetic retinopathy (DR) has complex multifactorial pathogenesis. This study aimed to investigate the association of complement pathway genes with susceptibility to DR. Eight haplotype-tagging SNPs of SERPING1 and C5 were genotyped in 570 subjects with type 2 diabetes: 295 DR patients (138 nonproliferative DR [NPDR] and 157 proliferative DR [PDR]) and 275 diabetic controls. Among the six C5 SNPs, a marginal association was first detected between rs17611 and total DR patients (P = 0.009, OR = 0.53 for recessive model). In stratification analysis, a significant decrease in the frequencies of G allele and GG homozygosity for rs17611 was observed in PDR patients compared with diabetic controls (P corr = 0.032, OR = 0.65 and P corr = 0.016, OR = 0.37, resp.); it was linked with a disease progression. A haplotype AA defined by the major alleles of rs17611 and rs1548782 was significantly predisposed to PDR with increased risk of 1.54 (P corr = 0.023). Regarding other variants in C5 and SERPING1, none of the tagging SNPs had a significant association with DR and its subgroups (all P > 0.05). Our study revealed an association between DR and C5 polymorphisms with clinical significance, whereas SERPING1 is not a major genetic component of DR. Our data suggest a link of complement pathway with DR pathogenesis. PMID:26989329

  11. Inhibition of the classical pathway of complement by meningococcal capsular polysaccharides

    PubMed Central

    Agarwal, Sarika; Vasudhev, Shreekant; DeOliveira, Rosane; Ram, Sanjay

    2014-01-01

    Almost all invasive Neisseria meningitidis isolates express capsular polysaccharide. Antibody (Ab) is required for complement-dependent killing of meningococci. While alternative pathway evasion has received considerable attention, little is known about classical pathway (CP) inhibition by meningococci and forms the basis of this study. We engineered capsulated and unencapsulated isogenic mutant strains of groups A, B, C, W and Y meningococci to express similar amounts of the same factor H-binding protein (fHbp; a key component of group B meningococcal vaccines) molecule. Despite similar anti-fHbp mAb binding, significantly less C4b was deposited on all five encapsulated mutants compared to their unencapsulated counterparts (P<0.01), when purified C1 and C4 were used to deposit C4b. Reduced C4b deposition was the result of capsule-mediated inhibition of C1q engagement by Ab. C4b deposition correlated linearly with C1q engagement by anti-fHbp. While B, C, W and Y capsules limited CP-mediated killing by anti-fHbp, the unencapsulated group A mutant paradoxically was more resistant than its encapsulated counterpart. Strains varied considerably in their susceptibility to anti-fHbp and complement despite similar Ab binding, which may have implications for the activity of fHbp-based vaccines. Capsule also limited C4b deposition by anti-porin A mAbs. Capsule expression decreased binding of an anti-LOS IgM mAb (~1.2 to 2-fold reduction in fluorescence). Akin to observations with IgG, capsule also decreased IgM-mediated C4b deposition when IgM binding to the mutant strain pairs was normalized. In conclusion, we show that capsular polysaccharide, a critical meningococcal virulence factor, inhibits the CP of complement. PMID:25015832

  12. Inhibition of the classical pathway of complement by meningococcal capsular polysaccharides.

    PubMed

    Agarwal, Sarika; Vasudhev, Shreekant; DeOliveira, Rosane B; Ram, Sanjay

    2014-08-15

    Almost all invasive Neisseria meningitidis isolates express capsular polysaccharide. Ab is required for complement-dependent killing of meningococci. Although alternative pathway evasion has received considerable attention, little is known about classical pathway (CP) inhibition by meningococci, which forms the basis of this study. We engineered capsulated and unencapsulated isogenic mutant strains of groups A, B, C, W, and Y meningococci to express similar amounts of the same factor H-binding protein (fHbp; a key component of group B meningococcal vaccines) molecule. Despite similar anti-fHbp mAb binding, significantly less C4b was deposited on all five encapsulated mutants compared with their unencapsulated counterparts (p < 0.01) when purified C1 and C4 were used to deposit C4b. Reduced C4b deposition was the result of capsule-mediated inhibition of C1q engagement by Ab. C4b deposition correlated linearly with C1q engagement by anti-fHbp. Whereas B, C, W, and Y capsules limited CP-mediated killing by anti-fHbp, the unencapsulated group A mutant paradoxically was more resistant than its encapsulated counterpart. Strains varied considerably in their susceptibility to anti-fHbp and complement despite similar Ab binding, which may have implications for the activity of fHbp-based vaccines. Capsule also limited C4b deposition by anti-porin A mAbs. Capsule expression decreased binding of an anti-lipooligosaccharide IgM mAb (∼ 1.2- to 2-fold reduction in fluorescence). Akin to observations with IgG, capsule also decreased IgM-mediated C4b deposition when IgM binding to the mutant strain pairs was normalized. In conclusion, we show that capsular polysaccharide, a critical meningococcal virulence factor, inhibits the CP of complement. PMID:25015832

  13. Bioactive Lysophospholipids Generated by Hepatic Lipase Degradation of Lipoproteins Lead to Complement Activation via the Classical Pathway

    PubMed Central

    Ma, Wanchao; Paik, David C.; Barile, Gaetano R.

    2014-01-01

    Purpose. We determined bioactivity of lysophospholipids generated by degradation of the low-density (LDL), very low-density (VLDL), and high-density (HDL) lipoproteins with hepatic lipase (HL), cholesterol esterase (CE), and lipoprotein-associated phospholipase A2 (Lp-PLA2). Methods. The LDL, VLDL, and HDL were treated with HL, CE, and Lp-PLA2 after immobilization on plates, and complement activation studies were performed with diluted human serum. Complement component 3 (C3) fixation, a marker for complement activation, was determined with a monoclonal anti-human C3d antibody. Enzymatic properties of HL and CE were assayed with triglyceride and phosphatidylcholine substrates for triglyceride hydrolase and phospholipase A activities. The ARPE-19 cells were used for viability studies. Results. The HL degradation of human lipoproteins LDL, VLDL, or HDL results in the formation of modified lipoproteins that can activate the complement pathway. Complement activation is dose- and time-dependent upon HL and occurs via the classical pathway. Enzymatic studies suggest that the phospholipase A1 activity of HL generates complement-activating lysophospholipids. C-reactive protein (CRP), known to simultaneously interact with complement C1 and complement factor H (CFH), further enhances HL-induced complement activation. The lysophospholipids, 1-Palmitoyl-sn-glycero-3-phosphocholine and 1-Oleoyl-sn-glycero-3-phosphocholine, can be directly cytotoxic to ARPE-19 cells. Conclusions. The HL degradation of lipoproteins, known to accumulate in the outer retina and in drusen, can lead to the formation of bioactive lysophospholipids that can trigger complement activation and induce RPE cellular dysfunction. Given the known risk associations for age-related macular degeneration (AMD) with HL, CRP, and CFH, this study elucidates a possible damage pathway for age-related macular degeneration (AMD) in genetically predisposed individuals, that HL activity may lead to accumulation of lysophospholipids to initiate complement activation, with CFH dysregulation exacerbating the effects of this process. PMID:25205869

  14. Differences in Cryptococcus neoformans capsular polysaccharide structure influence assembly of alternative complement pathway C3 convertase on fungal surfaces.

    PubMed

    Washburn, R G; Bryant-Varela, B J; Julian, N C; Bennett, J E

    1991-01-01

    Binding of complement component C3 and Factor B to Cryptococcus neoformans serotypes A through D via the alternative complement pathway was measured in a system containing fresh nonimmune human serum. Serotypes B and C (C. neoformans var. gattii) bound approximately half as many molecules of both complement components as serotypes A and D (C. neoformans var. neoformans). In contrast, removal of xylosyl and glucuronyl side chains from the mannan main chain of capsular polysaccharide by the Smith degradation procedure resulted in binding of similar quantities of C3 to each of the four serotypes. We conclude that the relatively high degree of side chain substitution of capsular polysaccharide from C. neoformans variety gattii contributes to inefficient surface assembly of the alternative pathway C3 convertase. Inefficient binding of alternative pathway complement components to serotypes B and C may contribute to the relative difficulty in successfully treating infections caused by these organisms. PMID:2062324

  15. Interspecies Complementation of the LuxR Family Pathway-Specific Regulator Involved in Macrolide Biosynthesis.

    PubMed

    Mo, SangJoon; Yoon, Yeo Joon

    2016-01-28

    PikD is a widely known pathway-specific regulator for controlling pikromycin production in Streptomyces venezuelae ATCC 15439, which is a representative of the large ATP-binding regulator of the LuxR family (LAL) in Streptomyces sp. RapH and FkbN also belong to the LAL family of transcriptional regulators, which show greatest homology with the ATP-binding motif and helix-turn-helix DNA-binding motif of PikD. Overexpression of pikD and heterologous expression of rapH and fkbN led to enhanced production of pikromycin by approximately 1.8-, 1.6-, and 1.6-fold in S. venezuelae, respectively. Cross-complementation of rapH and fkbN in the pikD deletion mutant (?pikD) restored pikromycin and derived macrolactone production. Overall, these results show that heterologous expression of rapH and fkbN leads to the overproduction of pikromycin and its congeners from the pikromycin biosynthetic pathway in S. venezuelae, and they have the same functionality as the pathwayspecific transcriptional activator for the pikromycin biosynthetic pathway in the ?pikD strain. These results also show extensive "cross-communication" between pathway-specific regulators of streptomycetes and suggest revision of the current paradigm for pathwayspecific versus global regulation of secondary metabolism in Streptomyces species. PMID:26608164

  16. Sodium arsenite-induced alteration in hepatocyte function of rat with special emphasis on superoxide dismutase expression pathway and its prevention by mushroom lectin.

    PubMed

    Bera, Asit K; Rana, Tanmoy; Bhattacharya, Debasis; Das, Subhashree; Pan, Diganta; Das, Subrata K

    2011-10-01

    This study was accomplished to exemplify the possible protective role of ascorbic acid and mushroom lectin against arsenic-induced cytotoxicity and impairment of superoxide dismutase (SOD) production pathway in hepatocytes of rat. Hepatocytes were isolated from rat and treated with sodium arsenite (AS), arsenic plus ascorbic acid (AS + AA) and arsenic plus mushroom lectin (AS + ML). A placebo control was also included. Arsenic treatment resulted in the depletion of cell proliferation, phagocytic activity (nitro blue tetrazolium index) and superoxide dismutase (SOD) activity, relative mRNA expression of superoxide dismutase 2 (SOD(2)) and enhanced production of nitric oxide (NO). Ascorbic acid, a standard antioxidant, could normalize cellular perturbation and SOD production pathway relating to gene expression, whereas partially purified Pleurotus florida lectin (PFL), an edible mushroom containing protein complex, maintained cellular activity and prevented stress by normalizing phagocytic (NBT index) and SOD activities vis--vis relative gene expression. It could further defend NO production of hepatocytes. Mushroom lectin strongly prevented sodium arsenite-induced damage of SOD production pathway in hepatocytes, and its effect was also comparable to a standard antioxidant, i.e. ascorbic acid. PMID:21554548

  17. The Complement System in Schizophrenia

    PubMed Central

    Mayilyan, Karine R.; Weinberger, Daniel R.; Sim, Robert B.

    2009-01-01

    summary Several lines of evidence suggest that immunological factors contribute to schizophrenia. Since 1989, the role of complement, a major effector of innate immunity and an adjuvant of adaptive immunity, has been explored in schizophrenia. Increased activity of C1, C3, C4 complement components in schizophrenia has been reported by two or more groups. Two studies on different subject cohorts showed increased MBL-MASP-2 activity in patients versus controls. More then one report indicated a significant high frequency of FB*F allotype and low prevalence of the FS phenotype of complement factor B in schizophrenia. From the data reported, it is likely that the disorder is accompanied by alterations of the complement classical and lectin pathways, which undergo dynamic changes, depending on the illness course and the state of neuro-immune crosstalk. Recent findings, implicating complement in neurogenesis, synapse remodeling and pruning during brain development, suggest a reexamination of the potential role of complement in neurodevelopmental processes contributing to schizophrenia susceptibility. It is plausible that the multicomponent complement system has more than one dimensional association with schizophrenia susceptibility, pathopsychology and illness course, understanding of which will bring a new perspective for possible immunomodulation and immunocorrection of the disease. PMID:18560619

  18. MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt's Lymphoma Cells to Trigger Apoptosis through Multiple Pathways.

    PubMed

    Hasan, Imtiaj; Sugawara, Shigeki; Fujii, Yuki; Koide, Yasuhiro; Terada, Daiki; Iimura, Naoya; Fujiwara, Toshiyuki; Takahashi, Keisuke G; Kojima, Nobuhiko; Rajia, Sultana; Kawsar, Sarkar M A; Kanaly, Robert A; Uchiyama, Hideho; Hosono, Masahiro; Ogawa, Yukiko; Fujita, Hideaki; Hamako, Jiharu; Matsui, Taei; Ozeki, Yasuhiro

    2015-12-01

    MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt's lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface. PMID:26694420

  19. MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt’s Lymphoma Cells to Trigger Apoptosis through Multiple Pathways

    PubMed Central

    Hasan, Imtiaj; Sugawara, Shigeki; Fujii, Yuki; Koide, Yasuhiro; Terada, Daiki; Iimura, Naoya; Fujiwara, Toshiyuki; Takahashi, Keisuke G.; Kojima, Nobuhiko; Rajia, Sultana; Kawsar, Sarkar M. A.; Kanaly, Robert A.; Uchiyama, Hideho; Hosono, Masahiro; Ogawa, Yukiko; Fujita, Hideaki; Hamako, Jiharu; Matsui, Taei; Ozeki, Yasuhiro

    2015-01-01

    MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt’s lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface. PMID:26694420

  20. NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways

    PubMed Central

    Yuen, Joshua; Pluthero, Fred G.; Douda, David N.; Riedl, Magdalena; Cherry, Ahmed; Ulanova, Marina; Kahr, Walter H. A.; Palaniyar, Nades; Licht, Christoph

    2016-01-01

    Neutrophils deposit antimicrobial proteins, such as myeloperoxidase and proteases on chromatin, which they release as neutrophil extracellular traps (NETs). Neutrophils also carry key components of the complement alternative pathway (AP) such as properdin or complement factor P (CFP), complement factor B (CFB), and C3. However, the contribution of these complement components and complement activation during NET formation in the presence and absence of bacteria is poorly understood. We studied complement activation on NETs and a Gram-negative opportunistic bacterial pathogen Pseudomonas aeruginosa (PA01, PAKwt, and PAKgfp). Here, we show that anaphylatoxin C5a, formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA), which activates NADPH oxidase, induce the release of CFP, CFB, and C3 from neutrophils. In response to PMA or P. aeruginosa, neutrophils secrete CFP, deposit it on NETs and bacteria, and induce the formation of terminal complement complexes (C5b–9). A blocking anti-CFP antibody inhibited AP-mediated but not non-AP-mediated complement activation on NETs and P. aeruginosa. Therefore, NET-mediated complement activation occurs via both AP- and non AP-based mechanisms, and AP-mediated complement activation during NETosis is dependent on CFP. These findings suggest that neutrophils could use their “AP tool kit” to readily activate complement on NETs and Gram-negative bacteria, such as P. aeruginosa, whereas additional components present in the serum help to fix non-AP-mediated complement both on NETs and bacteria. This unique mechanism may play important roles in host defense and help to explain specific roles of complement activation in NET-related diseases. PMID:27148258

  1. A component of the medicinal herb ephedra blocks activation in the classical and alternative pathways of complement.

    PubMed

    Ling, M; Piddlesden, S J; Morgan, B P

    1995-12-01

    Extracts of the herb Ephedra sinica have long been used in traditional Chinese medicine for the treatment of, among other conditions, acute nephritis. In preliminary studies it was shown that extracts of ephedra caused inhibition of complement in vitro. We thus set out to isolate the active component(s) of this herb, to examine the complement-inhibiting capacity in sera from different species, and to characterize the mechanism(s) by which it inhibits complement. Aqueous extraction of the herb followed by fractionation using thin layer chromatography (TLC) demonstrated that complement-inhibiting activity resided within a single band, hereafter termed the complement-inhibiting component (CIC), which represents an as yet uncharacterized polyanionic carbohydrate molecule. TLC-purified CIC inhibited the classical complement pathway in all species tested (human, pig, guinea pig, rat and rabbit). Using erythrocyte intermediates and sera specifically depleted of individual components it was apparent that CIC inhibited C2. This finding was confirmed using purified human C2, CIC causing a dose-dependent loss of C2 haemolytic activity. At much higher doses, CIC also showed some inhibiting effect in the terminal pathway, and this was shown to be due to inhibition of C9. In the alternative pathway CIC also showed inhibitory activity, although its site of action in this pathway remains unresolved. In Chinese medicine the herb is taken orally, though no studies of complement levels in patients taking the herb have been reported. Preliminary data indicate that oral administration in rats causes a partial inhibition of serum complement activity. Given the current enthusiasm for complement inhibition as a therapy for inflammatory diseases, this non-toxic, naturally occurring agent might be of therapeutic value. PMID:8536376

  2. Peptide Inhibitor of Complement C1 (PIC1) Rapidly Inhibits Complement Activation after Intravascular Injection in Rats

    PubMed Central

    Sharp, Julia A.; Hair, Pamela S.; Pallera, Haree K.; Kumar, Parvathi S.; Mauriello, Clifford T.; Nyalwidhe, Julius O.; Phelps, Cody A.; Park, Dalnam; Thielens, Nicole M.; Pascal, Stephen M.; Chen, Waldon; Duffy, Diane M.; Lattanzio, Frank A.; Cunnion, Kenji M.; Krishna, Neel K.

    2015-01-01

    The complement system has been increasingly recognized to play a pivotal role in a variety of inflammatory and autoimmune diseases. Consequently, therapeutic modulators of the classical, lectin and alternative pathways of the complement system are currently in pre-clinical and clinical development. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement and is referred to as Peptide Inhibitor of Complement C1 (PIC1). In this study, we determined that the lead PIC1 variant demonstrates a salt-dependent binding to C1q, the initiator molecule of the classical pathway. Additionally, this peptide bound to the lectin pathway initiator molecule MBL as well as the ficolins H, M and L, suggesting a common mechanism of PIC1 inhibitory activity occurs via binding to the collagen-like tails of these collectin molecules. We further analyzed the effect of arginine and glutamic acid residue substitution on the complement inhibitory activity of our lead derivative in a hemolytic assay and found that the original sequence demonstrated superior inhibitory activity. To improve upon the solubility of the lead derivative, a pegylated, water soluble variant was developed, structurally characterized and demonstrated to inhibit complement activation in mouse plasma, as well as rat, non-human primate and human serum in vitro. After intravenous injection in rats, the pegylated derivative inhibited complement activation in the blood by 90% after 30 seconds, demonstrating extremely rapid function. Additionally, no adverse toxicological effects were observed in limited testing. Together these results show that PIC1 rapidly inhibits classical complement activation in vitro and in vivo and is functional for a variety of animal species, suggesting its utility in animal models of classical complement-mediated diseases. PMID:26196285

  3. Early Components of the Complement Classical Activation Pathway in Human Systemic Autoimmune Diseases.

    PubMed

    Lintner, Katherine E; Wu, Yee Ling; Yang, Yan; Spencer, Charles H; Hauptmann, Georges; Hebert, Lee A; Atkinson, John P; Yu, C Yung

    2016-01-01

    The complement system consists of effector proteins, regulators, and receptors that participate in host defense against pathogens. Activation of the complement system, via the classical pathway (CP), has long been recognized in immune complex-mediated tissue injury, most notably systemic lupus erythematosus (SLE). Paradoxically, a complete deficiency of an early component of the CP, as evidenced by homozygous genetic deficiencies reported in human, are strongly associated with the risk of developing SLE or a lupus-like disease. Similarly, isotype deficiency attributable to a gene copy-number (GCN) variation and/or the presence of autoantibodies directed against a CP component or a regulatory protein that result in an acquired deficiency are relatively common in SLE patients. Applying accurate assay methodologies with rigorous data validations, low GCNs of total C4, and heterozygous and homozygous deficiencies of C4A have been shown as medium to large effect size risk factors, while high copy numbers of total C4 or C4A as prevalent protective factors, of European and East-Asian SLE. Here, we summarize the current knowledge related to genetic deficiency and insufficiency, and acquired protein deficiencies for C1q, C1r, C1s, C4A/C4B, and C2 in disease pathogenesis and prognosis of SLE, and, briefly, for other systemic autoimmune diseases. As the complement system is increasingly found to be associated with autoimmune diseases and immune-mediated diseases, it has become an attractive therapeutic target. We highlight the recent developments and offer a balanced perspective concerning future investigations and therapeutic applications with a focus on early components of the CP in human systemic autoimmune diseases. PMID:26913032

  4. Early Components of the Complement Classical Activation Pathway in Human Systemic Autoimmune Diseases

    PubMed Central

    Lintner, Katherine E.; Wu, Yee Ling; Yang, Yan; Spencer, Charles H.; Hauptmann, Georges; Hebert, Lee A.; Atkinson, John P.; Yu, C. Yung

    2016-01-01

    The complement system consists of effector proteins, regulators, and receptors that participate in host defense against pathogens. Activation of the complement system, via the classical pathway (CP), has long been recognized in immune complex-mediated tissue injury, most notably systemic lupus erythematosus (SLE). Paradoxically, a complete deficiency of an early component of the CP, as evidenced by homozygous genetic deficiencies reported in human, are strongly associated with the risk of developing SLE or a lupus-like disease. Similarly, isotype deficiency attributable to a gene copy-number (GCN) variation and/or the presence of autoantibodies directed against a CP component or a regulatory protein that result in an acquired deficiency are relatively common in SLE patients. Applying accurate assay methodologies with rigorous data validations, low GCNs of total C4, and heterozygous and homozygous deficiencies of C4A have been shown as medium to large effect size risk factors, while high copy numbers of total C4 or C4A as prevalent protective factors, of European and East-Asian SLE. Here, we summarize the current knowledge related to genetic deficiency and insufficiency, and acquired protein deficiencies for C1q, C1r, C1s, C4A/C4B, and C2 in disease pathogenesis and prognosis of SLE, and, briefly, for other systemic autoimmune diseases. As the complement system is increasingly found to be associated with autoimmune diseases and immune-mediated diseases, it has become an attractive therapeutic target. We highlight the recent developments and offer a balanced perspective concerning future investigations and therapeutic applications with a focus on early components of the CP in human systemic autoimmune diseases. PMID:26913032

  5. A novel targeted inhibitor of the alternative pathway of complement and its therapeutic application in ischemia/reperfusion injury.

    PubMed

    Huang, Yuxiang; Qiao, Fei; Atkinson, Carl; Holers, V Michael; Tomlinson, Stephen

    2008-12-01

    Bioavailability and therapeutic efficacy of soluble Crry, a mouse inhibitor of all complement activation pathways, is significantly enhanced when linked to a fragment of complement receptor 2 (CR2), a receptor that targets C3 activation products. In this study, we characterize alternative pathway-specific inhibitors consisting of a single or dimeric N-terminal region of mouse factor H (fH; short consensus repeats 1-5) linked to the same CR2 fragment (CR2-fH and CR2-fHfH). Both CR2-fH and CR2-fHfH were highly effective at inhibiting the alternative pathway in vitro and demonstrated a higher specific activity than CR2-Crry. CR2-fH was also more effective than endogenous serum fH in blocking target deposition of C3. Target binding and complement inhibitory activity of CR2-fH/CR2-fHfH was dependent on CR2- and C3-mediated interactions. The alternative pathway of complement plays a role in intestine ischemia/reperfusion injury. However, serum fH fails to provide protection against intestine ischemia/reperfusion injury although it can bind to and provide cell surfaces with protection from complement and is present in plasma at a high concentration. In a mouse model, CR2-fH and CR2-fHfH provided complete protection from local (intestine) and remote (lung) injury. CR2-fH targeted to the site of local injury and greatly reduced levels of tissue C3 deposition. Thus, the targeting mechanism significantly enhances alternative pathway-specific complement inhibitory activity of the N-terminal domain of fH and has the potential to reduce side effects that may be associated with systemic complement blockade. The data further indicate alternative pathway dependence for local and remote injury following intestinal ischemia/reperfusion in a clinically relevant therapeutic paradigm. PMID:19017999

  6. A Novel Targeted Inhibitor of the Alternative Pathway of Complement and Its Therapeutic Application in Ischemia/Reperfusion Injury1

    PubMed Central

    Huang, Yuxiang; Qiao, Fei; Atkinson, Carl; Holers, V. Michael; Tomlinson, Stephen

    2009-01-01

    Bioavailability and therapeutic efficacy of soluble Crry, a mouse inhibitor of all complement activation pathways, is significantly enhanced when linked to a fragment of complement receptor 2 (CR2), a receptor that targets C3 activation products. In this study, we characterize alternative pathway-specific inhibitors consisting of a single or dimeric N-terminal region of mouse factor H (fH; short consensus repeats 1–5) linked to the same CR2 fragment (CR2-fH and CR2-fHfH). Both CR2-fH and CR2-fHfH were highly effective at inhibiting the alternative pathway in vitro and demonstrated a higher specific activity than CR2-Crry. CR2-fH was also more effective than endogenous serum fH in blocking target deposition of C3. Target binding and complement inhibitory activity of CR2-fH/CR2-fHfH was dependent on CR2- and C3-mediated interactions. The alternative pathway of complement plays a role in intestine ischemia/reperfusion injury. However, serum fH fails to provide protection against intestine ischemia/reperfusion injury although it can bind to and provide cell surfaces with protection from complement and is present in plasma at a high concentration. In a mouse model, CR2-fH and CR2-fHfH provided complete protection from local (intestine) and remote (lung) injury. CR2-fH targeted to the site of local injury and greatly reduced levels of tissue C3 deposition. Thus, the targeting mechanism significantly enhances alternative pathway-specific complement inhibitory activity of the N-terminal domain of fH and has the potential to reduce side effects that may be associated with systemic complement blockade. The data further indicate alternative pathway dependence for local and remote injury following intestinal ischemia/reperfusion in a clinically relevant therapeutic paradigm. PMID:19017999

  7. Donor strand complementation governs intersubunit interaction of fimbriae of the alternate chaperone pathway.

    PubMed

    Poole, Steven T; McVeigh, Annette L; Anantha, Ravi P; Lee, Lanfong H; Akay, Yasemin M; Pontzer, Emily A; Scott, Daniel A; Bullitt, Esther; Savarino, Stephen J

    2007-03-01

    Fimbrial filaments assembled by distinct chaperone pathways share a common mechanism of intersubunit interaction, as elucidated for colonization factor antigen I (CFA/I), archetype of enterotoxigenic Escherichia coli (ETEC) Class 5 fimbriae. We postulated that a highly conserved beta-strand at the major subunit N-terminus represents the donor strand, analogous to interactions within Class I pili. We show here that CFA/I fimbriae utilize donor strand complementation to promote proper folding of and interactions between CFA/I subunits. We constructed a series of genetic variants of CfaE, the CFA/I adhesin, incorporating a C-terminal extension comprising a flexible linker and 10-19 of the N-terminal residues of CfaB, the major subunit. Variants with a donor strand complement (dsc) of >or= 12 residues were recoverable from periplasmic fractions. Genetic disruption of the donor beta-strand reduced CfaE recovery. A hexahistidine-tagged variant of dsc19CfaE formed soluble monomers, folded into beta-sheet conformation, displayed adhesion characteristic of CFA/I, and elicited antibodies that inhibited mannose-resistant haemagglutination by ETEC expressing CFA/I, CS4 and CS14 fimbriae. Immunoelectron microscopy indicated that CfaE was confined to the distal fimbrial tip. Our findings provide the basis to elucidate structure and function of this class of fimbrial adhesins and assess the feasibility of an adhesin-based vaccine. PMID:17302815

  8. Pseudomonas aeruginosa Uses Dihydrolipoamide Dehydrogenase (Lpd) to Bind to the Human Terminal Pathway Regulators Vitronectin and Clusterin to Inhibit Terminal Pathway Complement Attack

    PubMed Central

    Hallström, Teresia; Uhde, Melanie; Singh, Birendra; Skerka, Christine; Riesbeck, Kristian; Zipfel, Peter F.

    2015-01-01

    The opportunistic human pathogen Pseudomonas aeruginosa controls host innate immune and complement attack. Here we identify Dihydrolipoamide dehydrogenase (Lpd), a 57 kDa moonlighting protein, as the first P. aeruginosa protein that binds the two human terminal pathway inhibitors vitronectin and clusterin. Both human regulators when bound to the bacterium inhibited effector function of the terminal complement, blocked C5b-9 deposition and protected the bacterium from complement damage. P. aeruginosa when challenged with complement active human serum depleted from vitronectin was severely damaged and bacterial survival was reduced by over 50%. Similarly, when in human serum clusterin was blocked by a mAb, bacterial survival was reduced by 44%. Thus, demonstrating that Pseudomonas benefits from attachment of each human regulator and controls complement attack. The Lpd binding site in vitronectin was localized to the C-terminal region, i.e. to residues 354–363. Thus, Lpd of P. aeruginosa is a surface exposed moonlighting protein that binds two human terminal pathway inhibitors, vitronectin and clusterin and each human inhibitor when attached protected the bacterial pathogen from the action of the terminal complement pathway. Our results showed insights into the important function of Lpd as a complement regulator binding protein that might play an important role in virulence of P. aeruginosa. PMID:26368530

  9. Complementing the inflammasome.

    PubMed

    Triantafilou, Martha; Hughes, Timothy R; Morgan, Bryan Paul; Triantafilou, Kathy

    2016-02-01

    The innate immune system is an ancient surveillance system able to sense microbial invaders as well as aberrations in normal cell function. No longer viewed as a static and non-specific part of immunity, the innate immune system employs a plethora of specialized pattern recognition sensors to monitor and achieve homeostasis; these include the Toll-like receptors, the retinoic acid-inducible gene-like receptors, the nucleotide-binding oligomerization domain receptors (NLRs), the C-type lectins and the complement system. In order to increase specificity and diversity, innate immunity uses homotypic and heterotypic associations among these different components. Multi-molecular assemblies are formed both on the cell surface and in the cytosol to respond to pathogen and danger signals. Diverse, but tailored, responses to a changing environment are orchestrated depending on the the nature of the challenge and the repertoire of interacting receptors and components available in the sensing cell. It is now emerging that innate immunity operates a system of 'checks and balances' where interaction among the sensors is key in maintaining normal cell function. Complement sits at the heart of this alarm system and it is becoming apparent that it is capable of interacting with all the other pathways to effect a tailored immune response. In this review, we will focus on complement interactions with NLRs, the so-called 'inflammasomes', describing the molecular mechanisms that have been revealed so far and discussing the circumstantial evidence that exists for these interactions in disease states. PMID:26572245

  10. Complement activation by isolated myelin: activation of the classical pathway in the absence of myelin-specific antibodies.

    PubMed Central

    Vanguri, P; Koski, C L; Silverman, B; Shin, M L

    1982-01-01

    Many pathological conditions of the central nervous system involve damage to and removal of myelin membrane. Very little is known about initiation of this membrane damage and the mechanisms of disposal of the damaged tissue. We are interested in the interaction between complement (the components of complement are designated C1, C2, C3, etc.) and myelin membranes and the possible role of complement in amplifying myelin damage and in the disposal of damaged myelin in vivo, because activation of complement generates both membrane-attack complexes and opsonin(s). In this study, we found that isolated rat or human myelin consumes complement in the absence of specific antibodies. Activation of complement was demonstrated by showing C3 cleavage in fresh serum incubated with myelin. Incubation of central nervous system myelin with C2-deficient serum produced no C3 consumption and only minor factor B conversion, thus excluding the alternative pathway of activation. Involvement of the classical pathway was shown directly by the C1 fixation and transfer assay. Myelin incubated with C2-deficient serum or with purified C1 and then washed contained C1 activity that could lyse sheep erythrocytes sensitized with anti-Forssman IgM antibody and carrying C4, together with C2 and C3-C9. Membranes in brain tissues other than myelin (heavy membrane fraction obtained on sucrose density gradient centrifugation) were unable to activate C1. Images PMID:6954480

  11. A Novel Factor I Activity in Nipah Virus Inhibits Human Complement Pathways through Cleavage of C3b

    PubMed Central

    Johnson, John B.; Borisevich, Viktoriya; Rockx, Barry

    2014-01-01

    ABSTRACT Complement is an innate immune system that most animal viruses must face during natural infections. Given that replication and dissemination of the highly pathogenic Nipah virus (NiV) include exposure to environments rich in complement factors, we tested the in vitro sensitivity of NiV to complement-mediated neutralization. Here we show that NiV was completely resistant to in vitro neutralization by normal human serum (NHS). Treatment of purified NiV with NHS activated complement pathways, but there was very little C3 deposition on virus particles. In in vitro reconstitution experiments, NiV particles provided time- and dose-dependent factor I-like protease activity capable of cleaving C3b into inactive C3b (iC3b). NiV-dependent inactivation of C3b only occurred with the cofactors factor H and soluble CR1 but not with CD46. Purified NiV particles did not support C4b cleavage. Electron microscopy of purified NiV particles showed immunogold labeling with anti-factor I antibodies. Our results suggest a novel mechanism by which NiV evades the human complement system through a unique factor I-like activity. IMPORTANCE Viruses have evolved mechanisms to limit complement-mediated neutralization, some of which involve hijacking cellular proteins involved in control of inappropriate complement activation. Here we report a previously unknown mechanism whereby NiV provides a novel protease activity capable of in vitro cleavage and inactivation of C3b, a key component of the complement cascade. These data help to explain how an enveloped virus such as NiV can infect and disseminate through body fluids that are rich in complement activity. Disruption of the ability of NiV to recruit complement inhibitors could form the basis for the development of effective therapies and safer vaccines to combat these highly pathogenic emerging viruses. PMID:25355897

  12. Natural IgG antibodies in normal rabbit serum are involved in killing of the ompP2 mutant of Haemophilus parasuis SC096 strain via the classical complement pathway.

    PubMed

    Zhou, S M; Xu, C G; Zhang, B; Feng, S X; Zhang, L Y; Zou, Y; Liao, M

    2013-04-01

    Serum resistance in Haemophilus parasuis strain SC096 has been shown to be dependent on expression of the outer membrane protein P2 (OmpP2) and loss of the ompP2 gene results in significantly greater sensitivity to both porcine and rabbit sera. However, the mechanism of complement activation by the serum sensitive ?ompP2 strain is unknown. In this study, the classical complement pathway is demonstrated to be the main pathway for killing the H. parasuis ?ompP2 strain, and not the mannan-binding lectin (MBL) or alternative pathway. In addition, absorption of antibodies against ?ompP2 strain or depletion of IgGs from serum inhibited serum killing activity, which could be restored by addition of heat-inactivated serum or purified IgGs. Western blot analysis indicated that the OmpP2 mutant could bind significantly more IgGs than the wild type strain SC096 when incubated with serum. Finally, IgGs in normal rabbit serum targeted to the OMPs, but not lipooligosaccharide (LOS) in the OmpP2 mutant strain were found to be involved in bacterial killing indicating that the bactericidal epitope(s) is in the outer membrane proteins. PMID:23103218

  13. Targeting of mannan-binding lectin-associated serine protease-2 confers protection from myocardial and gastrointestinal ischemia/reperfusion injury

    PubMed Central

    Schwaeble, Wilhelm J.; Lynch, Nicholas J.; Clark, James E.; Marber, Michael; Samani, Nilesh J.; Ali, Youssif Mohammed; Dudler, Thomas; Parent, Brian; Lhotta, Karl; Wallis, Russell; Farrar, Conrad A.; Sacks, Steven; Lee, Haekyung; Zhang, Ming; Iwaki, Daisuke; Takahashi, Minoru; Fujita, Teizo; Tedford, Clark E.; Stover, Cordula M.

    2011-01-01

    Complement research experienced a renaissance with the discovery of a third activation route, the lectin pathway. We developed a unique model of total lectin pathway deficiency, a mouse strain lacking mannan-binding lectin-associated serine protease-2 (MASP-2), and analyzed the role of MASP-2 in two models of postischemic reperfusion injury (IRI). In a model of transient myocardial IRI, MASP-2–deficient mice had significantly smaller infarct volumes than their wild-type littermates. Mice deficient in the downstream complement component C4 were not protected, suggesting the existence of a previously undescribed lectin pathway-dependent C4-bypass. Lectin pathway-mediated activation of C3 in the absence of C4 was demonstrated in vitro and shown to require MASP-2, C2, and MASP-1/3. MASP-2 deficiency also protects mice from gastrointestinal IRI, as do mAb-based inhibitors of MASP-2. The therapeutic effects of MASP-2 inhibition in this experimental model suggest the utility of anti–MASP-2 antibody therapy in reperfusion injury and other lectin pathway-mediated disorders. PMID:21502512

  14. Characterization of a Factor H Mutation That Perturbs the Alternative Pathway of Complement in a Family with Membranoproliferative GN

    PubMed Central

    Wong, Edwin K.S.; Anderson, Holly E.; Herbert, Andrew P.; Challis, Rachel C.; Brown, Paul; Reis, Geisilaine S.; Tellez, James O.; Strain, Lisa; Fluck, Nicholas; Humphrey, Ann; Macleod, Alison; Richards, Anna; Ahlert, Daniel; Santibanez-Koref, Mauro; Barlow, Paul N.; Marchbank, Kevin J.; Harris, Claire L.; Goodship, Timothy H.J.

    2014-01-01

    Complement C3 activation is a characteristic finding in membranoproliferative GN (MPGN). This activation can be caused by immune complex deposition or an acquired or inherited defect in complement regulation. Deficiency of complement factor H has long been associated with MPGN. More recently, heterozygous genetic variants have been reported in sporadic cases of MPGN, although their functional significance has not been assessed. We describe a family with MPGN and acquired partial lipodystrophy. Although C3 nephritic factor was shown in family members with acquired partial lipodystrophy, it did not segregate with the renal phenotype. Genetic analysis revealed a novel heterozygous mutation in complement factor H (R83S) in addition to known risk polymorphisms carried by individuals with MPGN. Patients with MPGN had normal levels of factor H, and structural analysis of the mutant revealed only subtle alterations. However, functional analysis revealed profoundly reduced C3b binding, cofactor activity, and decay accelerating activity leading to loss of regulation of the alternative pathway. In summary, this family showed a confluence of common and rare functionally significant genetic risk factors causing disease. Data from our analysis of these factors highlight the role of the alternative pathway of complement in MPGN. PMID:24722444

  15. Activation of the alternative complement pathway by exposure of phosphatidylethanolamine and phosphatidylserine on erythrocytes from sickle cell disease patients.

    PubMed Central

    Wang, R H; Phillips, G; Medof, M E; Mold, C

    1993-01-01

    Deoxygenation of erythrocytes from sickle cell anemia (SCA) patients alters membrane phospholipid distribution with increased exposure of phosphatidylethanolamine (PE) and phosphatidylserine (PS) on the outer leaflet. This study investigated whether altered membrane phospholipid exposure on sickle erythrocytes results in complement activation. In vitro deoxygenation of sickle but not normal erythrocytes resulted in complement activation measured by C3 binding. Additional evidence indicated that this activation was the result of the alterations in membrane phospholipids. First, complement was activated by normal erythrocytes after incubation with sodium tetrathionate, which produces similar phospholipid changes. Second, antibody was not required for complement activation by sickle or tetrathionate-treated erythrocytes. Third, the membrane regulatory proteins, decay-accelerating factor (CD55) and the C3b/C4b receptor (CD35), were normal on sickle and tetrathionate-treated erythrocytes. Finally, insertion of PE or PS into normal erythrocytes induced alternative pathway activation. SCA patients in crisis exhibited increased plasma factor Bb levels compared with baseline, and erythrocytes isolated from hospitalized SCA patients had increased levels of bound C3, indicating that alternative pathway activation occurs in vivo. Activation of complement may be a contributing factor in sickle crisis episodes, shortening the life span of erythrocytes and decreasing host defense against infections. Images PMID:7690777

  16. Molecular mechanism of anticancer effect of Sclerotium rolfsii lectin in HT29 cells involves differential expression of genes associated with multiple signaling pathways: A microarray analysis.

    PubMed

    Barkeer, Srikanth; Guha, Nilanjan; Hothpet, Vishwanathreddy; Saligrama Adavigowda, Deepak; Hegde, Prajna; Padmanaban, Arunkumar; Yu, Lu-Gang; Swamy, Bale M; Inamdar, Shashikala R

    2015-12-01

    Sclerotium rolfsii lectin (SRL) is a lectin isolated from fungus S. rolfsii and has high binding specificity toward the oncofetal Thomsen-Friedenreich carbohydrate antigen (Gal?1-3GalNAc-?-O-Ser/Thr, T or TF), which is expressed in more than 90% of human cancers. Our previous studies have shown that binding of SRL to human colon, breast and ovarian cancer cells induces cell apoptosis in vitro and suppresses tumor growth in vivo. This study investigated the SRL-mediated cell signaling in human colon cancer HT29 cells by mRNA and miRNA microarrays. It was found that SRL treatment results in altered expression of several hundred molecules including mitogen-activated protein kinase (MAPK) and c-JUN-associated, apoptosis-associated and cell cycle and DNA replication-associated signaling molecules. Pathway analysis using GeneSpring 12.6.1 revealed that SRL treatment induces changes of MAPK and c-JUN-associated signaling pathways as early as 2 h while changes of cell cycle, DNA replication and apoptosis pathways were significantly affected only after 24 h. A significant change of cell miRNA expression was also observed after 12 h treatment of the cells with SRL. These changes were further validated by quantitative real time polymerase chain reaction and immunoblotting. This study thus suggests that the presence of SRL affects multiple signaling pathways in cancer cells with early effects on cell proliferation pathways associated with MAPK and c-JUN, followed by miRNA-associated cell activity and apoptosis. This provides insight information into the molecular mechanism of the anticancer activity of this fungal lectin. PMID:26347523

  17. The indoleamine 2,3-dioxygenase pathway controls complement-dependent enhancement of chemo-radiation therapy against murine glioblastoma

    PubMed Central

    2014-01-01

    Background Indoleamine 2,3-dioxygenase (IDO) is an enzyme with immune-suppressive properties that is commonly exploited by tumors to evade immune destruction. Anti-tumor T cell responses can be initiated in solid tumors, but are immediately suppressed by compensatory upregulation of immunological checkpoints, including IDO. In addition to these known effects on the adaptive immune system, we previously showed widespread, T cell-dependent complement deposition during allogeneic fetal rejection upon maternal treatment with IDO-blockade. We hypothesized that IDO protects glioblastoma from the full effects of chemo-radiation therapy by preventing vascular activation and complement-dependent tumor destruction. Methods To test this hypothesis, we utilized a syngeneic orthotopic glioblastoma model in which GL261 glioblastoma tumor cells were stereotactically implanted into the right frontal lobes of syngeneic mice. These mice were treated with IDO-blocking drugs in combination with chemotherapy and radiation therapy. Results Pharmacologic inhibition of IDO synergized with chemo-radiation therapy to prolong survival in mice bearing intracranial glioblastoma tumors. We now show that pharmacologic or genetic inhibition of IDO allowed chemo-radiation to trigger widespread complement deposition at sites of tumor growth. Chemotherapy treatment alone resulted in collections of perivascular leukocytes within tumors, but no complement deposition. Adding IDO-blockade led to upregulation of VCAM-1 on vascular endothelium within the tumor microenvironment, and further adding radiation in the presence of IDO-blockade led to widespread deposition of complement. Mice genetically deficient in complement component C3 lost all of the synergistic effects of IDO-blockade on chemo-radiation-induced survival. Conclusions Together these findings identify a novel mechanistic link between IDO and complement, and implicate complement as a major downstream effector mechanism for the beneficial effect of IDO-blockade after chemo-radiation therapy. We speculate that this represents a fundamental pathway by which the tumor regulates intratumoral vascular activation and protects itself from immune-mediated tumor destruction. PMID:25054064

  18. The alternative complement pathway control protein H binds to immune complexes and serves their detection

    SciTech Connect

    Nydegger, U.E.; Corvetta, A.; Spaeth, P.J.; Spycher, M.

    1983-01-01

    During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound /sup 125/I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of /sup 125/I-H; when fresh serum was chelated with 10 mM EDTA, /sup 125/I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samples from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), /sup 125/I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while /sup 125/I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes.

  19. Alternative and classical complement pathway activity in sera from colostrum-fed and colostrum-deprived neonatal pigs.

    PubMed Central

    Renshaw, H W; Gilmore, R J

    1980-01-01

    Haemolytic assays were used to compare alternative and classical complement (C) pathway activities in sera obtained from neonatal pigs reared on porcine colostrum, bovine colostrum or an immunoglobulin-free synthetic diet. Dramatic increases in immunoglobulin concentrations were noted in the colostrum-fed animals during the first day of life, but there was not a concurrent, marked increase in either classical or alternative C pathway activity. Whether fed on homologous or heterologous colostrum, neonatal pigs had a similar gradual increase in alternative and classical C pathway activity in the post-natal period. If direct passive absorption of C components occurs in newborn pigs, it has only a minor influence on functional levels of alternative and classical C pathway activity in their sera. In pigs fed homologous and heterologous colostrum there was, respectively, an 83% and 80% increase in classical pathway activity, but only a 13% and 12% increase in alternative pathway activity during the first 3 days of life. Pigs fed the immunoglobulin-free synthetic diet had a 37% increase in classical C and a 24% increase in alternative C pathway activity. Part of the increase in classical C pathway activity in the post-natal period may be caused by a stimulating factor in colostrum. Most if not all of the increase in alternative C pathway activity and some of the increase in classical C pathway activity is most likely caused by normal humoral homeostatic mechanisms in the neonatal pig. PMID:7429550

  20. Mutations in Complement Factor H Impair Alternative Pathway Regulation on Mouse Glomerular Endothelial Cells in Vitro.

    PubMed

    Loeven, Markus A; Rops, Angelique L; Lehtinen, Markus J; van Kuppevelt, Toin H; Daha, Mohamed R; Smith, Richard J; Bakker, Marinka; Berden, Jo H; Rabelink, Ton J; Jokiranta, T Sakari; van der Vlag, Johan

    2016-03-01

    Complement factor H (FH) inhibits complement activation and interacts with glomerular endothelium via its complement control protein domains 19 and 20, which also recognize heparan sulfate (HS). Abnormalities in FH are associated with the renal diseases atypical hemolytic uremic syndrome and dense deposit disease and the ocular disease age-related macular degeneration. Although FH systemically controls complement activation, clinical phenotypes selectively manifest in kidneys and eyes, suggesting the presence of tissue-specific determinants of disease development. Recent results imply the importance of tissue-specifically expressed, sulfated glycosaminoglycans (GAGs), like HS, in determining FH binding to and activity on host tissues. Therefore, we investigated which GAGs mediate human FH and recombinant human FH complement control proteins domains 19 and 20 (FH19-20) binding to mouse glomerular endothelial cells (mGEnCs) in ELISA. Furthermore, we evaluated the functional defects of FH19-20 mutants during complement activation by measuring C3b deposition on mGEnCs using flow cytometry. FH and FH19-20 bound dose-dependently to mGEnCs and TNF-α treatment increased binding of both proteins, whereas heparinase digestion and competition with heparin/HS inhibited binding. Furthermore, 2-O-, and 6-O-, but not N-desulfation of heparin, significantly increased the inhibitory effect on FH19-20 binding to mGEnCs. Compared with wild type FH19-20, atypical hemolytic uremic syndrome-associated mutants were less able to compete with FH in normal human serum during complement activation on mGEnCs, confirming their potential glomerular pathogenicity. In conclusion, our study shows that FH and FH19-20 binding to glomerular endothelial cells is differentially mediated by HS but not other GAGs. Furthermore, we describe a novel, patient serum-independent competition assay for pathogenicity screening of FH19-20 mutants. PMID:26728463

  1. Dissecting the complement pathway in hepatic injury and regeneration with a novel protective strategy.

    PubMed

    Marshall, Keely M; He, Songqing; Zhong, Zhi; Atkinson, Carl; Tomlinson, Stephen

    2014-08-25

    Liver resection is commonly performed under ischemic conditions, resulting in two types of insult to the remnant liver: ischemia reperfusion injury (IRI) and loss of liver mass. Complement inhibition is recognized as a potential therapeutic modality for IRI, but early complement activation products are also essential for liver regeneration. We describe a novel site-targeted murine complement inhibitor, CR2-CD59, which specifically inhibits the terminal membrane attack complex (MAC), and we use this protein to investigate the complement-dependent balance between liver injury and regeneration in a clinical setting of pharmacological inhibition. CR2-CD59 did not impact in vivo generation of C3 and C5 activation products but was as effective as the C3 activation inhibitor CR2-Crry at ameliorating hepatic IRI, indicating that the MAC is the principle mediator of hepatic IRI. Furthermore, unlike C3 or C5 inhibition, CR2-CD59 was not only protective but significantly enhanced hepatocyte proliferation after partial hepatectomy, including when combined with ischemia and reperfusion. Remarkably, CR2-CD59 also enhanced regeneration after 90% hepatectomy and improved long-term survival from 0 to 70%. CR2-CD59 functioned by increasing hepatic TNF and IL-6 levels with associated STAT3 and Akt activation, and by preventing mitochondrial depolarization and allowing recovery of ATP stores. PMID:25113972

  2. Identification of human mannose binding lectin (MBL) recognition sites for novel inhibitory antibodies.

    PubMed

    Zhao, Hui; Wakamiya, Nobutaka; Suzuki, Yasuhiko; Hamonko, Matthew T; Stahl, Gregory L

    2002-02-01

    Mannose binding lectin (MBL) binding initiates activation of the lectin complement pathway. Recent studies from our laboratory have demonstrated that MBL-dependent complement activation mediates cellular injury following oxidative stress in vivo and in vitro. A panel of novel inhibitory monoclonal antibodies (MAbs) against MBL (e.g., MAb 3F8, 2A9, and hMBL1.2) has been developed that inhibit MBL binding and lectin pathway activation. Here, we further characterized the interactions of these MAbs and their Fab fragments to MBL. Whole MAbs or their Fab fragments bound to MBL with relatively high affinity. Fab fragments of 3F8 were functionally effective in inhibiting MBL-dependent complement activation, however, steric hindrance of MAb 2A9 was essential for inhibition of MBL-dependent complement activation. We identified the hinge region, and residues EDCVLLL within the carbohydrate recognition domain of MBL as the recognition sites for MAb 3F8 and 2A9, respectively. The interaction of MAbs (e.g., 3F8 and 2A9) to MBL was dependent on the conformation of their recognition sites. These findings demonstrate that MBL binding can be inhibited by at least two separate and independent mechanisms. PMID:11991814

  3. Inherited mitochondrial DNA variants can affect complement, inflammation and apoptosis pathways: insights into mitochondrial–nuclear interactions

    PubMed Central

    Cristina Kenney, M.; Chwa, Marilyn; Atilano, Shari R.; Falatoonzadeh, Payam; Ramirez, Claudio; Malik, Deepika; Tarek, Mohamed; Cáceres-del-Carpio, Javier; Nesburn, Anthony B.; Boyer, David S.; Kuppermann, Baruch D.; Vawter, Marquis; Michal Jazwinski, S.; Miceli, Michael; Wallace, Douglas C.; Udar, Nitin

    2014-01-01

    Age-related macular degeneration (AMD) is the leading cause of vision loss in developed countries. While linked to genetic polymorphisms in the complement pathway, there are many individuals with high risk alleles that do not develop AMD, suggesting that other ‘modifiers’ may be involved. Mitochondrial (mt) haplogroups, defined by accumulations of specific mtDNA single nucleotide polymorphisms (SNPs) which represent population origins, may be one such modifier. J haplogroup has been associated with high risk for AMD while the H haplogroup is protective. It has been difficult to assign biological consequences for haplogroups so we created human ARPE-19 cybrids (cytoplasmic hybrids), which have identical nuclei but mitochondria of either J or H haplogroups, to investigate their effects upon bioenergetics and molecular pathways. J cybrids have altered bioenergetic profiles compared with H cybrids. Q-PCR analyses show significantly lower expression levels for seven respiratory complex genes encoded by mtDNA. J and H cybrids have significantly altered expression of eight nuclear genes of the alternative complement, inflammation and apoptosis pathways. Sequencing of the entire mtDNA was carried out for all the cybrids to identify haplogroup and non-haplogroup defining SNPs. mtDNA can mediate cellular bioenergetics and expression levels of nuclear genes related to complement, inflammation and apoptosis. Sequencing data suggest that observed effects are not due to rare mtDNA variants but rather the combination of SNPs representing the J versus H haplogroups. These findings represent a paradigm shift in our concepts of mt–nuclear interactions. PMID:24584571

  4. C1- inactivator: its efficiency as a regulator of classical complement pathway activation by soluble IgG aggregates.

    PubMed Central

    Doekes, G; van Es, L A; Daha, M R

    1983-01-01

    The role of C1- inactivator (C1(-)-In) during activation of the classical complement pathway by soluble immune complexes was studied using purified human complement components C1, C4 and C1(-)-In, and stabilized soluble aggregates of normal human IgG as a model for soluble immune complexes. The C4-consuming ability that could be generated by incubation of precursor C1 with IgG aggregates was abolished completely by the presence of a large excess of C1(-)-In during the C1 activation step. Kinetic studies confirmed that this inhibition was due to a second-order reaction between C1- and C1(-)-In resulting in the irreversible inactivation of C1-. When aggregates of various sizes were enabled to induce C4 conversion in mixtures of C1, C4 and a variable concentration of C1(-)-In, the presence of C1(-)-In had two effects. Firstly, the efficiency of the aggregates in causing C4 consumption was reduced remarkably. At a C1(-)-In:C1 ratio of 8, which can be found in normal human serum, approximately eight to ten times as many aggregates were required for a given level of C4 consumption as when no C1(-)-In was present. Secondly, C1(-)-In diminished the maximum C4 consumption that could be achieved, especially with smaller aggregates. Thus, a complete or partial C1(-)-In deficiency probably facilitates complement activation by soluble immune complexes in two ways: it may enhance the efficiency of classical pathway activation by all C1-activating complexes, and it may enable small complexes, which normally cannot overcome the C1(-)-In barrier, to activate the classical pathway to the C4 level. PMID:6852866

  5. Genetic Association of the Porcine C9 Complement Component with Hemolytic Complement Activity.

    PubMed

    Khoa, D V A; Wimmers, K

    2015-09-01

    The complement system is a part of the natural immune regulation mechanism against invading pathogens. Complement activation from three different pathways (classical, lectin, and alternative) leads to the formation of C5-convertase, an enzyme for cleavage of C5 into C5a and C5b, followed by C6, C7, C8, and C9 in membrane attack complex. The C9 is the last complement component of the terminal lytic pathway, which plays an important role in lysis of the target cells depending on its self-polymerization to form transmembrane channels. To address the association of C9 with traits related to disease resistance, the complete porcine C9 cDNA was comparatively sequenced to detect single nucleotide polymorphisms (SNPs) in pigs of the breeds Hampshire (HS), Duroc (DU), Berlin miniature pig (BMP), German Landrace (LR), Pietrain (PIE), and Muong Khuong (Vietnamese potbelly pig). Genotyping was performed in 417 F2 animals of a resource population (DUMI: DU×BMP) that were vaccinated with Mycoplasma hyopneumoniae, Aujeszky diseases virus and porcine respiratory and reproductive syndrome virus at 6, 14 and 16 weeks of age, respectively. Two SNPs were detected within the third exon. One of them has an amino acid substitution. The European porcine breeds (LR and PIE) show higher allele frequency of these SNPs than Vietnamese porcine breed (MK). Association of the substitution SNP with hemolytic complement activity indicated statistically significant differences between genotypes in the classical pathway but not in the alternative pathway. The interactions between eight time points of measurement of complement activity before and after vaccinations and genotypes were significantly different. The difference in hemolytic complement activity in the both pathways depends on genotype, kind of vaccine, age and the interaction to the other complement components. These results promote the porcine C9 (pC9) as a candidate gene to improve general animal health in the future. PMID:26194222

  6. Genetic Association of the Porcine C9 Complement Component with Hemolytic Complement Activity

    PubMed Central

    Khoa, D. V. A.; Wimmers, K.

    2015-01-01

    The complement system is a part of the natural immune regulation mechanism against invading pathogens. Complement activation from three different pathways (classical, lectin, and alternative) leads to the formation of C5-convertase, an enzyme for cleavage of C5 into C5a and C5b, followed by C6, C7, C8, and C9 in membrane attack complex. The C9 is the last complement component of the terminal lytic pathway, which plays an important role in lysis of the target cells depending on its self-polymerization to form transmembrane channels. To address the association of C9 with traits related to disease resistance, the complete porcine C9 cDNA was comparatively sequenced to detect single nucleotide polymorphisms (SNPs) in pigs of the breeds Hampshire (HS), Duroc (DU), Berlin miniature pig (BMP), German Landrace (LR), Pietrain (PIE), and Muong Khuong (Vietnamese potbelly pig). Genotyping was performed in 417 F2 animals of a resource population (DUMI: DU×BMP) that were vaccinated with Mycoplasma hyopneumoniae, Aujeszky diseases virus and porcine respiratory and reproductive syndrome virus at 6, 14 and 16 weeks of age, respectively. Two SNPs were detected within the third exon. One of them has an amino acid substitution. The European porcine breeds (LR and PIE) show higher allele frequency of these SNPs than Vietnamese porcine breed (MK). Association of the substitution SNP with hemolytic complement activity indicated statistically significant differences between genotypes in the classical pathway but not in the alternative pathway. The interactions between eight time points of measurement of complement activity before and after vaccinations and genotypes were significantly different. The difference in hemolytic complement activity in the both pathways depends on genotype, kind of vaccine, age and the interaction to the other complement components. These results promote the porcine C9 (pC9) as a candidate gene to improve general animal health in the future. PMID:26194222

  7. Role of complement in IgA nephropathy.

    PubMed

    Daha, Mohamed R; van Kooten, Cees

    2016-02-01

    Immunoglobulin A nephropathy (IgAN) is characterized by the deposition of IgA in the mesangium of glomeruli. This mesangial IgA has been found to consist mainly of polymeric IgA1 which drives the activation of the mesangial cells and results in excessive production of several inflammatory mediators. The activation of mesangial cells is amplified by the ability of IgA to activate the complement system, originally thought to occur mainly via the alternative pathway of complement. However more recent studies indicate that lectin pathway involvement has a strong association with progression of renal disease. In this review we summarize the contribution of complement to the IgA- mediated inflammatory process. PMID:26567162

  8. Complement System Part I – Molecular Mechanisms of Activation and Regulation

    PubMed Central

    Merle, Nicolas S.; Church, Sarah Elizabeth; Fremeaux-Bacchi, Veronique; Roumenina, Lubka T.

    2015-01-01

    Complement is a complex innate immune surveillance system, playing a key role in defense against pathogens and in host homeostasis. The complement system is initiated by conformational changes in recognition molecular complexes upon sensing danger signals. The subsequent cascade of enzymatic reactions is tightly regulated to assure that complement is activated only at specific locations requiring defense against pathogens, thus avoiding host tissue damage. Here, we discuss the recent advances describing the molecular and structural basis of activation and regulation of the complement pathways and their implication on physiology and pathology. This article will review the mechanisms of activation of alternative, classical, and lectin pathways, the formation of C3 and C5 convertases, the action of anaphylatoxins, and the membrane-attack-complex. We will also discuss the importance of structure–function relationships using the example of atypical hemolytic uremic syndrome. Lastly, we will discuss the development and benefits of therapies using complement inhibitors. PMID:26082779

  9. Ouabain rescues rat nephrogenesis during intrauterine growth restriction by regulating the complement and coagulation cascades and calcium signaling pathway.

    PubMed

    Chen, L; Yue, J; Han, X; Li, J; Hu, Y

    2016-02-01

    Intrauterine growth restriction (IUGR) is associated with a reduction in the numbers of nephrons in neonates, which increases the risk of hypertension. Our previous study showed that ouabain protects the development of the embryonic kidney during IUGR. To explore this molecular mechanism, IUGR rats were induced by protein and calorie restriction throughout pregnancy, and ouabain was delivered using a mini osmotic pump. RNA sequencing technology was used to identify the differentially expressed genes (DEGs) of the embryonic kidneys. DEGs were submitted to the Database for Annotation and Visualization and Integrated Discovery, and gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted. Maternal malnutrition significantly reduced fetal weight, but ouabain treatment had no significant effect on body weight. A total of 322 (177 upregulated and 145 downregulated) DEGs were detected between control and the IUGR group. Meanwhile, 318 DEGs were found to be differentially expressed (180 increased and 138 decreased) between the IUGR group and the ouabain-treated group. KEGG pathway analysis indicated that maternal undernutrition mainly disrupts the complement and coagulation cascades and the calcium signaling pathway, which could be protected by ouabain treatment. Taken together, these two biological pathways may play an important role in nephrogenesis, indicating potential novel therapeutic targets against the unfavorable effects of IUGR. PMID:26442628

  10. A chemical complementation approach reveals genes and interactions of flavonoids with other pathways.

    PubMed

    Pourcel, Lucille; Irani, Niloufer G; Koo, Abraham J K; Bohorquez-Restrepo, Andres; Howe, Gregg A; Grotewold, Erich

    2013-05-01

    In addition to the classical functions of flavonoids in the response to biotic/abiotic stress conditions, these phenolic compounds have been implicated in the modulation of various developmental processes. These findings suggest that flavonoids are more integral components of the plant signaling machinery than traditionally recognized. To understand how flux through the flavonoid pathway affects plant cellular processes, we used wild-type and chalcone isomerase mutant (transparent testa 5, tt5) seedlings grown under anthocyanin inductive conditions, in the presence or absence of the flavonoid intermediate naringenin, the product of the chalcone isomerase enzyme. Because flavonoid biosynthetic genes are expressed under anthocyanin inductive conditions regardless of whether anthocyanins are formed or not, this system provides an excellent opportunity to specifically investigate the molecular changes associated with increased flux through the flavonoid pathway. By assessing genome-wide mRNA accumulation changes in naringenin-treated and untreated tt5 and wild-type seedlings, we identified a flavonoid-responsive gene set associated with cellular trafficking, stress responses and cellular signaling. Jasmonate biosynthetic genes were highly represented among the signaling pathways induced by increased flux through the flavonoid pathway. In contrast to studies showing a role for flavonoids in the control of auxin transport, no effect on auxin-responsive genes was observed. Taken together, our data suggest that Arabidopsis can sense flavonoids as a signal for multiple fundamental cellular processes. PMID:23360095

  11. Complement and membrane-bound complement regulatory proteins as biomarkers and therapeutic targets for autoimmune inflammatory disorders, RA and SLE.

    PubMed

    Das, Nibhriti

    2015-11-01

    Complement system is a major effecter system of the innate immunity that bridges with adaptive immunity. The system consists of about 40 humoral and cell surface proteins that include zymogens, receptors and regulators. The zymogens get activated in a cascade fashion by antigen-antibody complex, antigen alone or by polymannans, respectively, by the classical, alternative and mannose binding lectin (MBL) pathways. The ongoing research on complement regulators and complement receptors suggest key role of these proteins in the initiation, regulation and effecter mechanisms of the innate and adaptive immunity. Although, the complement system provides the first line of defence against the invading pathogens, its aberrant uncontrolled activation causes extensive self tissue injury. A large number of humoral and cell surface complement regulatory protein keep the system well-regulated in healthy individuals. Complement profiling had brought important information on the pathophysiology of several infectious and chronic inflammatory disorders. In view of the diversity of the clinical disorders involving abnormal complement activity or regulation, which include both acute and chronic diseases that affect a wide range of organs, diverse yet specifically tailored therapeutic approaches may be needed to shift complement back into balance. This brief review discusses on the complement system, its functions and its importance as biomarkers and therapeutic targets for autoimmune diseases with focus on SLE and RA. PMID:26669012

  12. C1q, the recognition subcomponent of the classical pathway of complement, drives microglial activation.

    PubMed

    Färber, Katrin; Cheung, Giselle; Mitchell, Daniel; Wallis, Russell; Weihe, Eberhard; Schwaeble, Wilhelm; Kettenmann, Helmut

    2009-02-15

    Microglia, central nervous system (CNS) resident phagocytic cells, persistently police the integrity of CNS tissue and respond to any kind of damage or pathophysiological changes. These cells sense and rapidly respond to danger and inflammatory signals by changing their cell morphology; by release of cytokines, chemokines, or nitric oxide; and by changing their MHC expression profile. We have shown previously that microglial biosynthesis of the complement subcomponent C1q may serve as a reliable marker of microglial activation ranging from undetectable levels of C1q biosynthesis in resting microglia to abundant C1q expression in activated, nonramified microglia. In this study, we demonstrate that cultured microglial cells respond to extrinsic C1q with a marked intracellular Ca(2+) increase. A shift toward proinflammatory microglial activation is indicated by the release of interleukin-6, tumor necrosis factor-alpha, and nitric oxide and the oxidative burst in rat primary microglial cells, an activation and differentiation process similar to the proinflammatory response of microglia to exposure to lipopolysaccharide. Our findings indicate 1) that extrinsic plasma C1q is involved in the initiation of microglial activation in the course of CNS diseases with blood-brain barrier impairment and 2) that C1q synthesized and released by activated microglia is likely to contribute in an autocrine/paracrine way to maintain and balance microglial activation in the diseased CNS tissue. PMID:18831010

  13. Mannose-Binding Lectin Inhibits Monocyte Proliferation through Transforming Growth Factor-β1 and p38 Signaling Pathways

    PubMed Central

    Wang, Yan; Chen, A-De; Lei, Yan-Mei; Shan, Gui-Qiu; Zhang, Li-Yun; Lu, Xiao; Chen, Zheng-Liang

    2013-01-01

    Mannose-binding lectin (MBL), a plasma C-type lectin, plays an important role in innate immunity. However, the interaction, and the consequences of it, between MBL and the immune system remain ill defined. We have investigated the contributing mechanisms and effects of MBL on the proliferation of human monocytes. At lower concentrations (≤4 μg/ml) MBL was shown to partially enhance monocyte proliferation. By contrast, at higher concentrations (8–20 μg/ml) of MBL, cell proliferation was markedly attenuated. MBL-induced growth inhibition was associated with G0/G1 arrest, down-regulation of cyclin D1/D3, cyclin-dependent kinase (Cdk) 2/Cdk4 and up-regulation of the Cdk inhibitory protein Cip1/p21. Additionally, MBL induced apoptosis, and did so through caspase-3 activation and poly ADP-ribose polymerase (PARP) cleavage. Moreover, transforming growth factor (TGF)-β1 levels increased in the supernatants of MBL-stimulated monocyte cultures. We also found that MBL-dependent inhibition of monocyte proliferation could be reversed by the TGF-β receptor antagonist SB-431542, or by anti-TGF-β1 antibody, or by the mitogen-activated protein kinase (MAPK) inhibitors specific for p38 (SB203580), but not ERK (U0126) or JNK (SP600125). Thus, at high concentrations, MBL can affect the immune system by inhibiting monocyte proliferation, which suggests that MBL may exhibit anti-inflammatory effects. PMID:24039775

  14. Candidate Pathway-Based GWAS Identifies Novel Associations of Genomic Variants in the Complement System Associated with Coronary Artery Disease

    PubMed Central

    Xu, Chengqi; Yang, Qin; Xiong, Hongbo; Wang, Longfei; Cai, Jianping; Wang, Fan; Li, Sisi; Chen, Jing; Wang, Chuchu; Wang, Dan; Xiong, Xin; Wang, Pengyun; Zhao, Yuanyuan; Wang, Xiaojing; Huang, Yufeng; Chen, Shanshan; Yin, Dan; Li, Xiuchun; Liu, Ying; Liu, Jinqiu; Wang, Jingjing; Li, Hui; Ke, Tie; Ren, Xiang; Wu, Yanxia; Wu, Gang; Wan, Jing; Zhang, Rongfeng; Wu, Tangchun; Wang, Junhan; Xia, Yunlong; Yang, Yanzong; Cheng, Xiang; Liao, Yuhua; Chen, Qiuyun; Zhou, Yanhong; He, Qing; Tu, Xin; Wang, Qing K.

    2014-01-01

    Background Genomic variants identified by genome-wide association studies (GWAS) explain <20% of heritability of coronary artery disease (CAD), thus many risk variants remain missing for CAD. Identification of new variants may unravel new biological pathways and genetic mechanisms for CAD. To identify new variants associated with CAD, we developed a candidate pathway-based GWAS by integrating expression quantitative loci (eQTL) analysis and mining of GWAS data with variants in a candidate pathway. Methods and Results Mining of GWAS data was performed to analyze variants in 32 complement system genes for positive association with CAD. Functional variants in genes showing positive association were then identified by searching existing expression quantitative loci databases and validated by RT-PCR. A follow-up case control design was then used to determine whether the functional variants are associated with CAD in two independent GeneID Chinese populations. Candidate pathway-based GWAS identified positive association between variants in C3AR1 and C6 and CAD. Two functional variants, rs7842 in C3AR1 and rs4400166 in C6, were found to be associated with expression levels of C3AR1 and C6, respectively. Significant association was identified between rs7842 and CAD (P=3.99×10−6, OR=1.47) and between rs4400166 and CAD (P=9.30×10−3, OR=1.24) in the validation cohort. The significant findings were confirmed in the replication cohort (P=1.53×10−5, OR=1.37 for rs7842; P=8.41×10−3, OR=1.21 for rs4400166. Conclusions Integration of GWAS with biological pathways and eQTL is effective in identifying new risk variants for CAD. Functional variants increasing C3AR1 and C6 expression were shown to confer significant risk of CAD for the first time. PMID:25249547

  15. Classical complement activation and acquired immune response pathways are not essential for retinal degeneration in the rd1 mouse

    PubMed Central

    Rohrer, Brbel; Demos, Christina; Frigg, Rico; Grimm, Christian

    2007-01-01

    Misregulation of the innate immune response and other immune-related processes have been suggested to play a critical role in the pathogenesis of a number of different neurodegenerative diseases, including age related macular degeneration. In an animal model for photoreceptor degeneration, several genes of the innate and acquired immune system were found to be differentially regulated in the retina during the degenerative process. In addition to this differential regulation of individual genes, we found that in the rd1 retina a significantly higher number of genes involved in immune-related responses were expressed at any given time during the degenerative period. The peak of immune-related gene expression was at postnatal day 14, coinciding with the peak of photoreceptor apoptosis in the rd1 mouse. We directly tested the potential involvement of acquired and innate immune responses in initiation and progression of photoreceptor degeneration by analyzing double mutant animals. Retinal morphology and photoreceptor apoptosis of rd1 mice on a SCID genetic background (no mature T- and B-cells) or in combination with a RAG-1 (no functional B- and T-cells) or a C1q? (no functional classical complement activation pathway) knockout was followed during the degenerative process using light microscopy or TUNEL staining, respectively. Although complement factor C1q? was highly up-regulated in the rd1 retina concomitantly with the degenerative process, lack of this protein did not protect the rd1 retina. Similarly, retinal degeneration and photoreceptor apoptosis appeared to proceed normally in the rd1 mouse lacking functional B- and T-cells. Our results suggest that both, the classical complement system of innate immunity and a functional acquired immune response are not essential for the degenerative process in the rd1 mouse retina. PMID:17069800

  16. Classical complement activation and acquired immune response pathways are not essential for retinal degeneration in the rd1 mouse.

    PubMed

    Rohrer, Brbel; Demos, Christina; Frigg, Rico; Grimm, Christian

    2007-01-01

    Misregulation of the innate immune response and other immune-related processes have been suggested to play a critical role in the pathogenesis of a number of different neurodegenerative diseases, including age related macular degeneration. In an animal model for photoreceptor degeneration, several genes of the innate and acquired immune system were found to be differentially regulated in the retina during the degenerative process. In addition to this differential regulation of individual genes, we found that in the rd1 retina a significantly higher number of genes involved in immune-related responses were expressed at any given time during the degenerative period. The peak of immune-related gene expression was at postnatal day 14, coinciding with the peak of photoreceptor apoptosis in the rd1 mouse. We directly tested the potential involvement of acquired and innate immune responses in initiation and progression of photoreceptor degeneration by analyzing double mutant animals. Retinal morphology and photoreceptor apoptosis of rd1 mice on a SCID genetic background (no mature T- and B-cells) or in combination with a RAG1 (no functional B- and T-cells) or a C1qalpha (no functional classical complement activation pathway) knockout was followed during the degenerative process using light microscopy or TUNEL staining, respectively. Although complement factor C1qalpha was highly up-regulated in the rd1 retina concomitantly with the degenerative process, lack of this protein did not protect the rd1 retina. Similarly, retinal degeneration and photoreceptor apoptosis appeared to proceed normally in the rd1 mouse lacking functional B- and T-cells. Our results suggest that both, the classical complement system of innate immunity and a functional acquired immune response are not essential for the degenerative process in the rd1 mouse retina. PMID:17069800

  17. Visual Pathway Study Using in vivo DTI Tractography to Complement Classical Anatomy

    PubMed Central

    Wu, Wentao; Rigolo, Laura; O’Donnell, Lauren J.; Norton, Isaiah; Shriver, Sargent; Golby, Alexandra J.

    2011-01-01

    Background Knowledge of the individual course of the optic radiations (OR) is important to avoid post-operative visual deficits. Cadaveric studies of the visual pathways are limited because it has not been possible to accurately separate the OR from neighboring tracts and results may not apply to individual patients. Diffusion tensor imaging (DTI) studies may be able to demonstrate the relationships between the OR and neighboring fibers in vivo in individual subjects. Objective To use DTI tractography to study the OR and Meyer’s loop (ML) anatomy in vivo. Methods Ten healthy subjects underwent magnetic resonance imaging with diffusion imaging at 3T. Using a fiducial-based DTI tractography tool (Slicer 3.3), seeds were placed near the lateral geniculate nucleus (LGN) to reconstruct individual visual pathways and neighboring tracts. Projections of the optic radiations onto 3D brain models were shown individually in order to quantify relationships to key landmarks. Results Two patterns of visual pathways were found. The OR ran more commonly deep in the whole superior and middle temporal gyri and superior temporal sulcus. The OR was closely surrounded in all cases by an inferior longitudinal fascicle and a parieto/occipito/temporo-pontine fascicle. The mean left and right distances between the tip of the OR and temporal pole were 39.8± 3.8mm and 40.6±5.7 mm, respectively. Conclusion DTI tractography provides a practical complementary method to study the OR and ML anatomy in vivo, and with reference to individual 3D brain anatomy. PMID:21808220

  18. Viral bimolecular fluorescence complementation: a novel tool to study intracellular vesicular trafficking pathways.

    PubMed

    Dirk, Brennan S; Jacob, Rajesh Abraham; Johnson, Aaron L; Pawlak, Emily N; Cavanagh, P Craig; Van Nynatten, Logan; Haeryfar, S M Mansour; Dikeakos, Jimmy D

    2015-01-01

    The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Nef interacts with a multitude of cellular proteins, manipulating the host membrane trafficking machinery to evade immune surveillance. Nef interactions have been analyzed using various in vitro assays, co-immunoprecipitation studies, and more recently mass spectrometry. However, these methods do not evaluate Nef interactions in the context of viral infection nor do they define the sub-cellular location of these interactions. In this report, we describe a novel bimolecular fluorescence complementation (BiFC) lentiviral expression tool, termed viral BiFC, to study Nef interactions with host cellular proteins in the context of viral infection. Using the F2A cleavage site from the foot and mouth disease virus we generated a viral BiFC expression vector capable of concurrent expression of Nef and host cellular proteins; PACS-1, MHC-I and SNX18. Our studies confirmed the interaction between Nef and PACS-1, a host membrane trafficking protein involved in Nef-mediated immune evasion, and demonstrated co-localization of this complex with LAMP-1 positive endolysosomal vesicles. Furthermore, we utilized viral BiFC to localize the Nef/MHC-I interaction to an AP-1 positive endosomal compartment. Finally, viral BiFC was observed between Nef and the membrane trafficking regulator SNX18. This novel demonstration of an association between Nef and SNX18 was localized to AP-1 positive vesicles. In summary, viral BiFC is a unique tool designed to analyze the interaction between Nef and host cellular proteins by mapping the sub-cellular locations of their interactions during viral infection. PMID:25915798

  19. Viral Bimolecular Fluorescence Complementation: A Novel Tool to Study Intracellular Vesicular Trafficking Pathways

    PubMed Central

    Johnson, Aaron L.; Pawlak, Emily N.; Cavanagh, P. Craig; Van Nynatten, Logan; Haeryfar, S. M. Mansour; Dikeakos, Jimmy D.

    2015-01-01

    The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Nef interacts with a multitude of cellular proteins, manipulating the host membrane trafficking machinery to evade immune surveillance. Nef interactions have been analyzed using various in vitro assays, co-immunoprecipitation studies, and more recently mass spectrometry. However, these methods do not evaluate Nef interactions in the context of viral infection nor do they define the sub-cellular location of these interactions. In this report, we describe a novel bimolecular fluorescence complementation (BiFC) lentiviral expression tool, termed viral BiFC, to study Nef interactions with host cellular proteins in the context of viral infection. Using the F2A cleavage site from the foot and mouth disease virus we generated a viral BiFC expression vector capable of concurrent expression of Nef and host cellular proteins; PACS-1, MHC-I and SNX18. Our studies confirmed the interaction between Nef and PACS-1, a host membrane trafficking protein involved in Nef-mediated immune evasion, and demonstrated co-localization of this complex with LAMP-1 positive endolysosomal vesicles. Furthermore, we utilized viral BiFC to localize the Nef/MHC-I interaction to an AP-1 positive endosomal compartment. Finally, viral BiFC was observed between Nef and the membrane trafficking regulator SNX18. This novel demonstration of an association between Nef and SNX18 was localized to AP-1 positive vesicles. In summary, viral BiFC is a unique tool designed to analyze the interaction between Nef and host cellular proteins by mapping the sub-cellular locations of their interactions during viral infection. PMID:25915798

  20. Functional complementation by wheat eIF2alpha in the yeast GCN2-mediated pathway.

    PubMed

    Chang, L Y; Yang, W Y; Roth, D

    2000-12-20

    Translational control by specific eIF2alpha phosphorylation on serine 51 has been characterized in all eukaryotes with the significant exception of plants. In order to evaluate the capability of plant eIF2alpha to functionally control translation, the wild type (51S) and a nonphosphorylatable mutant (51A) of wheat eIF2alpha were expressed in a yeast genetic system. Expression of either wheat protein did not handicap growth under conditions that repress the eIF2alpha phosphorylation pathway. However, under conditions that induce specific eIF2alpha phosphorylation only strains expressing wheat 51S were able to grow between 2 and 4 days. Growth was dependent upon activity of yeast eIF2alpha kinase GCN2 and resulted in the increased translation of GCN4. The association between plant eIF2alpha and yeast eIF2B is supported by their specific coimmunoprecipitation from transgenic yeast cells. These data support the similarity among eukaryotic translational initiation processes and strengthen the concept that plants may contain an eIF2alpha phosphorylation pathway. PMID:11118310

  1. Anti-Mouse Properdin TSR 5/6 Monoclonal Antibodies Block Complement Alternative Pathway-dependent Pathogenesis

    PubMed Central

    Bertram, Paula; Akk, Antonina M.; Zhou, Hui-fang; Mitchell, Lynne M.; Pham, Christine T.N.

    2015-01-01

    The complement alternative pathway (AP) is a major contributor to a broad and growing spectrum of diseases that includes age-related macular degeneration, atypical hemolytic uremic syndrome, and preeclampsia. As a result, there is much interest in the therapeutic disruption of AP activity. Properdin, the only positive regulator of the AP, is a particularly promising AP target. Several issues need to be clarified before the potential for properdin-directed therapy can be realized. In this report we use a portion of the mouse properdin protein, expressed in a bacterial system, to raise rabbit polyclonal and hamster monoclonal antibodies that block properdin-dependent pathogenesis. These antibodies, when employed with AP-dependent mouse disease models, can help evaluate the feasibility of properdin-directed therapy. PMID:25723276

  2. Rapid Assays for Lectin Toxicity and Binding Changes that Reflect Altered Glycosylation in Mammalian Cells

    PubMed Central

    Stanley, Pamela; Sundaram, Subha

    2014-01-01

    Glycosylation engineering is used to generate glycoproteins, glycolipids or proteoglycans with a more defined complement of glycans on their glycoconjugates. For example, a mammalian cell glycosylation mutant lacking a specific glycosyltransferase generates glycoproteins, and/or glycolipids, and/or proteoglycans, with truncated glycans missing the sugar transferred by that glycosyltransferase, and also missing those sugars that would be added subsequently. In some cases, an alternative glycosyltransferase may then use the truncated glycans as acceptors, thereby generating a new or different glycan subset in the mutant cell. Another type of glycosylation mutant arises from gain-of-function mutations that, for example, activate a silent glycosyltransferase gene. In this case, glycoconjugates will have glycans with additional sugar(s) that are more elaborate than the glycans of wild type cells. Mutations in other genes that affect glycosylation, such as nucleotide sugar synthases or transporters, will alter the glycan complement in more general ways that usually affect several types of glycoconjugates. There are now many strategies for generating a precise mutation in a glycosylation gene in a mammalian cell. Large-volume cultures of mammalian cells may also give rise to spontaneous mutants in glycosylation pathways. This article will focus on how to rapidly characterize mammalian cells with an altered glycosylation activity. The key reagents for the protocols described are plant lectins that bind mammalian glycans with varying avidities, depending on the specific structure of those glycans. Cells with altered glycosylation generally become resistant or hypersensitive to lectin toxicity, and have reduced or increased lectin or antibody binding. Here we describe rapid assays to compare the cytotoxicity of lectins in a lectin resistance test, and the binding of lectins or antibodies by flow cytometry in a glycan-binding assay. Based on these tests, glycosylation changes expressed by a cell can be revealed, and glycosylation mutants classified into phenotypic groups that may reflect a loss-of-function or gain-of-function mutation in a specific gene involved in glycan synthesis. PMID:24903886

  3. Role of the complement anaphylatoxin C5a-receptor pathway in atopic dermatitis in mice

    PubMed Central

    DANG, LIN; HE, LEI; WANG, YAN; XIONG, JIKUI; BAI, BINGXUE; LI, YUZHEN

    2015-01-01

    Atopic dermatitis (AD) is a chronic inflammatory skin disease with a genetic background. The C5a-receptor (C5aR) pathway has been reported to be involved in AD; however, the precise pathogenesis remains to be elucidated. In the present study, the contribution of the C5aR pathway to AD in mice was investigated. A BALB/c mouse model of AD was induced by application of 2,4-dinitrochlorobenzene (DNCB) onto hairless dorsal skin. Following DNCB application for 2 weeks, C5aR expression in skin tissue was assessed by reverse transcription quantitative polymerase chain reaction. C5aR expression in skin tissue was significantly increased in mice with AD. In an additional experiment, C5aR antagonist (C5aRA) intracutaneously injected in combination with DNCB treatment. The skin-fold thickness, number of total infiltrating leukocytes and mast cells infiltrating in skin tissue were measured. Interleukin-4 (IL-4) and interferon-γ (IFN-γ) levels in skin tissue and IL-4, IFN-γ, histamine and immunoglobulin E (IgE) levels in serum were measured using ELISA. The skin-fold thickness, numbers of total infiltrating leukocytes and mast cells in skin tissue, as well as levels of IL-4, IFN-γ, histamine and IgE were significantly increased in mice with AD. However, simultaneous treatment with C5aRA significantly attenuated increases in skin fold thickness and the numbers of total infiltrating leukocytes and mast cells in skin tissue. Treatment with C5aRA also decreased IL-4 and IFN-γ levels in skin tissue, as well as the levels of IL-4, IFN-γ, histamine and IgE in the serum. In conclusion, C5aRA inhibited AD in mice, possibly through suppression of the C5aR-mediated cascade action of mast cells. PMID:25650554

  4. ON VASCULAR STENOSIS, RESTENOSIS AND MANNOSE BINDING LECTIN

    PubMed Central

    KAHLOW, Barbara Stadler; NERY, Rodrigo Araldi; SKARE, Thelma L; RIBAS, Carmen Australia Paredes Marcondes; RAMOS, Gabriela Piovezani; PETISCO, Roberta Dombroski

    2016-01-01

    Mannose binding lectin is a lectin instrumental in the innate immunity. It recognizes carbohydrate patterns found on the surface of a large number of pathogenic micro-organisms, activating the complement system. However, this protein seems to increase the tissue damage after ischemia. In this paper is reviewed some aspects of harmful role of the mannose binding lectin in ischemia/reperfusion injury. PMID:27120743

  5. ON VASCULAR STENOSIS, RESTENOSIS AND MANNOSE BINDING LECTIN.

    PubMed

    Kahlow, Barbara Stadler; Nery, Rodrigo Araldi; Skare, Thelma L; Ribas, Carmen Australia Paredes Marcondes; Ramos, Gabriela Piovezani; Petisco, Roberta Dombroski

    2016-03-01

    Mannose binding lectin is a lectin instrumental in the innate immunity. It recognizes carbohydrate patterns found on the surface of a large number of pathogenic micro-organisms, activating the complement system. However, this protein seems to increase the tissue damage after ischemia. In this paper is reviewed some aspects of harmful role of the mannose binding lectin in ischemia/reperfusion injury. PMID:27120743

  6. Genome-wide pathway-based association study implicates complement system in the development of Kashin-Beck disease in Han Chinese.

    PubMed

    Zhang, Feng; Wen, Yan; Guo, Xiong; Zhang, Yingang; Wang, Sen; Yang, Tielin; Shen, Hui; Chen, Xiangding; Tan, Lijun; Tian, Qing; Deng, Hong-Wen

    2015-02-01

    Kashin-Beck disease (KBD) is a chronic osteochondropathy. The pathogenesis of KBD remains unknown. To identify relevant biological pathways for KBD, we conducted a genome-wide pathway-based association study (GWPAS) following by replication analysis, totally using 2743 Chinese Han adults. A modified gene set enrichment algorithm was used to detect association between KBD and 963 biological pathways. Cartilage gene expression analysis and serum complement measurement were performed to evaluate the functional relevance of identified pathway with KBD. We found that the Complement and Coagulation Cascades (CACC) pathway was significantly associated with KBD (P value=3.09×10(-5), false-discovery rate=0.042). Within the CACC pathway, the most significant association was observed at rs1656966 (P value=1.97×10(-4)) of KNG1 gene. Further replication study observed that rs1656966 (P value=0.037) was significantly associated with KBD in an independent validation sample of 1026 subjects. Gene expression analysis observed that CFD (ratio=3.39±2.68), A2M (ratio=3.67±5.63), C5 (ratio=2.65±2.52) and CD46 (ratio=2.29±137) genes of the CACC pathway were up-regulated in KBD articular cartilage compared to healthy articular cartilage. The serum level of complement C5 in KBD patients were significantly higher than that in healthy controls (P value=0.038). Our study is the first to suggest that complement system-related CACC pathway contributed to the development of KBD. PMID:25305519

  7. Antimicrobial functions of EsLecH, a C-type lectin, via JNK pathway in the Chinese mitten crab, Eriocheir sinensis.

    PubMed

    Zhu, You-Ting; Zhang, Xing; Wang, Shi-Chuang; Li, Wei-Wei; Wang, Qun

    2016-08-01

    C-type lectins (CTLs) are pattern recognition proteins that play significant roles in the innate immune system by identifying and eliminating pathogens. Here, we have reported a CTL (EsLecH) from the Chinese mitten crab that can bind to microorganisms and regulate antimicrobial peptide (AMP) expression via the c-Jun N-terminal kinase (JNK) pathway. EsLecH was found to have an N-terminal signal peptide and a single carbohydrate recognition domain. The EsLecH transcript was detected abundantly in various tissues, and it was significantly upregulated in hemocytes after challenging with lipopolysaccharides and bacteria. Recombinant (r)EsLecH could bind to microorganisms, but at different levels. Ca(2+) significantly increased rEsLecH binding affinity to microorganisms. Furthermore, growth inhibition by rEsLecH increased with increasing rEsLecH levels. Knockdown of EsLecH was accompanied by a significant reduction in AMP expression and JNK phosphorylation; AMP expression was reduced with JNK silencing and can not rescued by rEsLecH when absence of JNK. These results indicate that EsLecH could regulate AMPs via JNK signaling. PMID:27068761

  8. SALSA: A Regulator of the Early Steps of Complement Activation on Mucosal Surfaces

    PubMed Central

    Reichhardt, Martin Parnov; Meri, Seppo

    2016-01-01

    Complement is present mainly in blood. However, following mechanical damage or inflammation, serous exudates enter the mucosal surfaces. Here, the complement proteins interact with other endogenous molecules to keep microbes from entering the parenteral tissues. One of the mucosal proteins known to interact with the early complement components of both the classical and the lectin pathway is the salivary scavenger and agglutinin (SALSA). SALSA is also known as deleted in malignant brain tumors 1 and gp340. It is found both attached to the epithelium and secreted into the surrounding fluids of most mucosal surfaces. SALSA has been shown to bind directly to C1q, mannose-binding lectin, and the ficolins. Through these interactions SALSA regulates activation of the complement system. In addition, SALSA interacts with surfactant proteins A and D, secretory IgA, and lactoferrin. Ulcerative colitis and Crohn’s disease are examples of diseases, where complement activation in mucosal tissues may occur. This review describes the latest advances in our understanding of how the early complement components interact with the SALSA molecule. Furthermore, we discuss how these interactions may affect disease propagation on mucosal surfaces in immunological and inflammatory diseases. PMID:27014265

  9. Outer Membrane Protein P5 Is Required for Resistance of Nontypeable Haemophilus influenzae to Both the Classical and Alternative Complement Pathways

    PubMed Central

    Rosadini, Charles V.; Ram, Sanjay

    2014-01-01

    The complement system is an important first line of defense against the human pathogen Haemophilus influenzae. To survive and propagate in vivo, H. influenzae has evolved mechanisms for subverting this host defense, most of which have been shown to involve outer surface structures, including lipooligosaccharide glycans and outer surface proteins. Bacterial defense against complement acts at multiple steps in the pathway by mechanisms that are not fully understood. Here we identify outer membrane protein P5 as an essential factor in serum resistance of both H. influenzae strain Rd and nontypeable H. influenzae (NTHi) clinical isolate NT127. P5 was essential for resistance of Rd and NT127 to complement in pooled human serum. Further investigation determined that P5 expression decreased cell surface binding of IgM, a potent activator of the classical pathway of complement, to both Rd and NT127. Additionally, P5 expression was required for NT127 to bind factor H (fH), an important inhibitor of alternative pathway (AP) activation. Collectively, the results obtained in this work highlight the ability of H. influenzae to utilize a single protein to perform multiple protective functions for evading host immunity. PMID:24478079

  10. Micrurus snake venoms activate human complement system and generate anaphylatoxins

    PubMed Central

    2012-01-01

    Background The genus Micrurus, coral snakes (Serpentes, Elapidae), comprises more than 120 species and subspecies distributed from the south United States to the south of South America. Micrurus snake bites can cause death by muscle paralysis and further respiratory arrest within a few hours after envenomation. Clinical observations show mainly neurotoxic symptoms, although other biological activities have also been experimentally observed, including cardiotoxicity, hemolysis, edema and myotoxicity. Results In the present study we have investigated the action of venoms from seven species of snakes from the genus Micrurus on the complement system in in vitro studies. Several of the Micrurus species could consume the classical and/or the lectin pathways, but not the alternative pathway, and C3a, C4a and C5a were generated in sera treated with the venoms as result of this complement activation. Micrurus venoms were also able to directly cleave the α chain of the component C3, but not of the C4, which was inhibited by 1,10 Phenanthroline, suggesting the presence of a C3α chain specific metalloprotease in Micrurus spp venoms. Furthermore, complement activation was in part associated with the cleavage of C1-Inhibitor by protease(s) present in the venoms, which disrupts complement activation control. Conclusion Micrurus venoms can activate the complement system, generating a significant amount of anaphylatoxins, which may assist due to their vasodilatory effects, to enhance the spreading of other venom components during the envenomation process. PMID:22248157

  11. The C3 convertase of the alternative pathway of human complement. Enzymic properties of the bimolecular proteinase.

    PubMed Central

    Pangburn, M K; Müller-Eberhard, H J

    1986-01-01

    The association of Factor B with C3b (the major fragment of complement component C3) in the presence of Mg2+ results in the formation of a bimolecular zymogen, C3b,B, which is activated by the serine proteinase Factor D, generating the C3 convertase, C3b,Bb (EC 3.4.21.47). Cleavage of native C3 by the C3 convertase was monitored by recording the increase in fluorescence associated with C3b formation in the presence of the fluorescent probe 8-anilinonaphthalene-1-sulphonate. Measurements of initial rates of C3b formation at various C3 concentrations were analysed in accordance with the Michaelis-Menten equation, yielding kcat. = 1.78 +/- 0.08 s-1, Km = 5.86 X 10(-6) M and turnover number = 107 min-1. The assay was used to measure the Ki values of a variety of proteinase inhibitors. The C3 convertase has a short half-life, owing to spontaneous dissociation of the complex. The t1/2 and kcat./Km of the enzyme were determined by fitting an equation modelling both the kinetic reaction and enzyme decay to the fluorimetrically measured progress curve. The enzyme, C3b,Bb, exhibited a t1/2 of 90 +/- 2 s and a kcat./Km of 31.1 X 10(4) +/- 0.8 X 10(4) M-1 X s-1 at physiological pH, ionic strength and temperature. The enzyme that initiates activation of the alternative pathway, C3(H2O),Bb, was also examined. It was slightly less stable (t1/2 = 77 +/- 3 s) and exhibited only half the activity of C3b,Bb (kcat./Km = 16.3 X 10(4) +/- 1.0 X 10(4) M-1 X s-1). PMID:3638964

  12. Complement and Periodontitis

    PubMed Central

    Hajishengallis, George

    2010-01-01

    Although the complement system is centrally involved in host defense, its overactivation or deregulation (e.g., due to inherent host genetic defects or due to pathogen subversion) may excessively amplify inflammation and contribute to immunopathology. Periodontitis is an oral infection-driven chronic inflammatory disease which exerts a systemic impact on health. This paper reviews evidence linking complement to periodontal inflammation and pathogenesis. Clinical and histological observations show a correlation between periodontal inflammatory activity and local complement activation. Certain genetic polymorphisms or deficiencies in specific complement components appear to predispose to increased susceptibility to periodontitis. Animal model studies and in vitro experiments indicate that periodontal bacteria can either inhibit or activate distinct components of the complement cascade. Porphyromonas gingivalis, a keystone species in periodontitis, subverts complement receptor 3 and C5a anaphylatoxin receptor signaling in ways that promote its adaptive fitness in the presence of non-productive inflammation. Overall, available evidence suggests that complement activation or subversion contributes to periodontal pathogenesis, although not all complement pathways or functions are necessarily destructive. Effective complement-targeted therapeutic intervention in periodontitis would require determining the precise roles of the various inductive or effector complement pathways. This information is essential as it may reveal which specific pathways need to be blocked to counteract microbial evasion and inflammatory pathology or, conversely, be enhanced to promote host immunity. PMID:20599785

  13. Functional assessment of mouse complement pathway activities and quantification of C3b/C3c/iC3b in an experimental model of mouse renal ischaemia/reperfusion injury.

    PubMed

    Kotimaa, Juha P; van Werkhoven, Maaike B; O'Flynn, Joseph; Klar-Mohamad, Ngaisah; van Groningen, Jan; Schilders, Geurt; Rutjes, Helma; Daha, Mohamed R; Seelen, Marc A; van Kooten, Cees

    2015-04-01

    The complement system is an essential component of our innate immunity, both for the protection against infections and for proper handling of dying cells. However, the complement system can also contribute to tissue injury and inflammatory responses. In view of novel therapeutic possibilities, there is an increasing interest in measurement of the complement system activation in the systemic compartment, both in the clinical setting as well as in experimental models. Here we describe in parallel a sensitive and specific sandwich ELISA detecting mouse C3 activation fragments C3b/C3c/iC3b, as well as functional complement ELISAs detecting specific activities of the three complement pathways at the level of C3 and at the level of C9 activation. In a murine model of renal ischaemia/reperfusion injury (IRI) we found transient complement activation as shown by generation of C3b/C3c/iC3b fragments at 24 h following reperfusion, which returned to base-line at 3 and 7 days post reperfusion. When the pathway specific complement activities were measured at the level of C3 activation, we found no significant reduction in any of the pathways. However, the functional complement activity of all three pathways was significantly reduced when measured at the level of C9, with the strongest reduction being observed in the alternative pathway. For all three pathways there was a strong correlation between the amount of C3 fragments and the reduction in functional complement activity. Moreover, at 24 h both C3 fragments and the functional complement activities showed a correlation with the rise in serum creatinine. Together our results show that determination of the systemic pathway specific complement activity is feasible in experimental mouse models and that they are useful in understanding complement activation and inhibition in vivo. PMID:25733354

  14. Sundanese Complementation

    ERIC Educational Resources Information Center

    Kurniawan, Eri

    2013-01-01

    The focus of this thesis is the description and analysis of clausal complementation in Sundanese, an Austronesian language spoken in Indonesia. The thesis examined a range of clausal complement types in Sundanese, which consists of (i) "yen/(wi)rehna" "that" complements, (ii) "pikeun" "for" complements,

  15. Sundanese Complementation

    ERIC Educational Resources Information Center

    Kurniawan, Eri

    2013-01-01

    The focus of this thesis is the description and analysis of clausal complementation in Sundanese, an Austronesian language spoken in Indonesia. The thesis examined a range of clausal complement types in Sundanese, which consists of (i) "yen/(wi)rehna" "that" complements, (ii) "pikeun" "for" complements,…

  16. Deficiency of mannose-binding lectin greatly increases antibody response in a mouse model of vaccination.

    PubMed

    Guttormsen, Hilde-Kari; Stuart, Lynda M; Shi, Lei; Carroll, Mike C; Chen, Jianzhu; Kasper, Dennis L; Ezekowitz, R Alan B; Takahashi, Kazue

    2009-03-01

    Mannose-binding lectin (MBL), a pattern recognition innate immune molecule, selectively binds distinct chemical patterns, including carbohydrates expressed on Group B streptococcus (GBS). MBL interacts with IgM, resulting in the activation of MBL-associated serine proteases (MASPs), thus is initiating a lectin complement pathway. Complement proteins and IgM modulate production of antigen specific antibody. In this study, we investigated the relative effect of MBL in antibody response against tetanus toxoid-conjugated GBS polysaccharide vaccines (GBS PS-TT) by comparing wild type and null mice for MBL, complement 3 (C3), IgM, MBL/C3, and MBL/IgM. We found that GBS PS specific IgG response was upregulated in MBL deficient mice following immunization with GBS PS-TT but not GBS PS. B1 cells were expanded in peritonium but not in spleen of MBL null mice. The mechanisms of heightened IgG response in MBL null mice were related to C3, and share the same pathway with IgM. PMID:18996748

  17. Splenic RNA and MicroRNA Mimics Promote Complement Factor B Production and Alternative Pathway Activation via Innate Immune Signaling.

    PubMed

    Zou, Lin; Feng, Yan; Xu, Ganqiong; Jian, Wenling; Chao, Wei

    2016-03-15

    Complement factor B (cfB) is an essential component of the alternative pathway (AP) and plays an important role in the pathogenesis of polymicrobial sepsis. However, the mechanism leading to cfB production and AP activation during sepsis remains poorly understood. In this study, we found that plasma cell-free RNA was significantly increased following cecal ligation and puncture (CLP), an animal model of polymicrobial sepsis, and was closely associated with sepsis severity. Quantitative RT-PCR and microRNA (miRNA) array analysis revealed an increase in bacterial RNA and multiple host miRNAs (miR-145, miR-146a, miR-122, miR-210) in the blood following CLP. Treatment with tissue RNA or synthetic miRNA mimics (miR-145, miR-146a, miR-122, miR-34a) induced a marked increase in cfB production in cardiomyocytes or macrophages. The newly synthesized cfB released into medium was biologically active because it participated in AP activation initiated by cobra venom factor. Genetic deletion of TLR7 or MyD88, but not TLR3, and inhibition of the MAPKs (JNK and p38) or NF-κB abolished miR-146a-induced cfB production. In vivo, CLP led to a significant increase in splenic cfB expression that correlated with the plasma RNA or miRNA levels. Peritoneal injection of RNA or miR-146a led to an increase in cfB expression in the peritoneal space that was attenuated in MyD88-knockout or TLR7-knockout mice, respectively. These findings demonstrate that host cellular RNA and specific miRNAs are released into the circulation during polymicrobial sepsis and may function as extracellular mediators capable of promoting cfB production and AP activation through specific TLR7 and MyD88 signaling. PMID:26889043

  18. Infections of People with Complement Deficiencies and Patients Who Have Undergone Splenectomy

    PubMed Central

    Ram, Sanjay; Lewis, Lisa A.; Rice, Peter A.

    2010-01-01

    Summary: The complement system comprises several fluid-phase and membrane-associated proteins. Under physiological conditions, activation of the fluid-phase components of complement is maintained under tight control and complement activation occurs primarily on surfaces recognized as “nonself” in an attempt to minimize damage to bystander host cells. Membrane complement components act to limit complement activation on host cells or to facilitate uptake of antigens or microbes “tagged” with complement fragments. While this review focuses on the role of complement in infectious diseases, work over the past couple of decades has defined several important functions of complement distinct from that of combating infections. Activation of complement in the fluid phase can occur through the classical, lectin, or alternative pathway. Deficiencies of components of the classical pathway lead to the development of autoimmune disorders and predispose individuals to recurrent respiratory infections and infections caused by encapsulated organisms, including Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. While no individual with complete mannan-binding lectin (MBL) deficiency has been identified, low MBL levels have been linked to predisposition to, or severity of, several diseases. It appears that MBL may play an important role in children, who have a relatively immature adaptive immune response. C3 is the point at which all complement pathways converge, and complete deficiency of C3 invariably leads to severe infections, including those caused by meningococci and pneumococci. Deficiencies of the alternative and terminal complement pathways result in an almost exclusive predisposition to invasive meningococcal disease. The spleen plays an important role in antigen processing and the production of antibodies. Splenic macrophages are critical in clearing opsonized encapsulated bacteria (such as pneumococci, meningococci, and Escherichia coli) and intraerythrocytic parasites such as those causing malaria and babesiosis, which explains the fulminant nature of these infections in persons with anatomic or functional asplenia. Paramount to the management of patients with complement deficiencies and asplenia is educating patients about their predisposition to infection and the importance of preventive immunizations and seeking prompt medical attention. PMID:20930072

  19. The complement system of elasmobranches revealed by liver transcriptome analysis of a hammerhead shark, Sphyrna zygaena.

    PubMed

    Goshima, Masayuki; Sekiguchi, Reo; Matsushita, Misao; Nonaka, Masaru

    2016-08-01

    Comprehensive studies of the complement genes in basal vertebrates have revealed that cyclostomes have apparently primitive complement systems whereas bony fish have well-developed complement systems comparable to those of mammals. Here we have performed liver transcriptome analysis of a hammerhead shark, Sphyrna zygaeana, to elucidate the early history of vertebrate complement evolution. Identified genes were; one C1qB, one C1r, one C1s, one MASP-1/-3, one MASP-2, two factor B/C2, one C3, three C4, one C5, one C6, one C7, one C8A, three C8B, one C8G, one C9, two factor I and one S protein. No MBL, ficolin, C1qA or C1qC were found. These results indicate that the lectin, classical, alternative and lytic pathways were established in the common ancestor of jawed vertebrates. In addition to the absence of MBL and ficolin, the MASP transcripts lacked the serine protease domain, suggesting that the lectin pathway was lost in the hammerhead shark lineage. PMID:26987526

  20. Use of peanut lectin and rat mammary stem cell lines to identify a cellular differentiation pathway for the alveolar cell in the rat mammary gland.

    PubMed

    Rudland, P S

    1992-10-01

    The presence of the carbohydrate receptor for PNL has been used to identify the previously described morphological types of epithelial cell produced as the stem cell line rat mammary 25 (Rama 25) differentiates to casein secretory alveolar-like cells in vitro. Thus when cultures of the epithelial stem cell line Rama 25 are treated with neuraminidase, fluorescently-conjugated PNL fails to stain cuboidal cells, stains weakly grey cells, and stains strongly the surface of dark cells. When superconfluent cultures of Rama 25 are treated with dimethyl sulfoxide or retinoic acid and prolactin, estradiol, hydrocortisone, and insulin to induce differentiation to alveolar cells, PNL stains strongly the untreated surfaces of droplet cells and casein-secreting vacuolated cells. PNL-staining of the derivative cell lines with truncated cellular pathways, and quantitative binding of [125I]-labeled PNL to the cultured cells are consistent with this cellular staining pattern. The presence of the carbohydrate receptor for peanut lectin (PNL) has also been used to identify specific epithelial cell types in different mammary structures of the developing rat mammary gland, as they differentiate to casein secretory alveolar cells in vivo. Thus when different structures of the developing rat mammary gland are treated with neuraminidase, peroxidase-conjugated PNL fails to stain histochemically the majority of epithelial cells in ducts, stains the cytoplasm of the majority of epithelial cells in terminal end-buds (TEBs), and stains strongly the luminal surfaces of the majority of epithelial cells in alveolar buds (ABs). PNL also stains the untreated luminal surfaces of alveolar cells, whether or not the cells can be stained with a monoclonal antibody to rat beta-casein. Stimulation of mammary differentiation by an analogue of ethyl retinoate or by perphenazine causes cells in end-buds to bind PNL without the necessity for their desialylation similar to that seen in casein secretory alveoli of lactating rats. In conclusion the different interconverting cell types of Rama 25 which form a pathway to casein-secretory cells in vitro are thus equated with recognisable epithelial cell types in vivo. These results suggest that casein-secretory cells in vivo are generated by similar successive interconversions between the major epithelial cell types present in the different mammary structures in the order: ducts, TEBs, ABs, alveoli, and secretory alveoli. PMID:1522129

  1. Laboratory tests for disorders of complement and complement regulatory proteins.

    PubMed

    Shih, Angela R; Murali, Mandakolathur R

    2015-12-01

    The complement pathway is a cascade of proteases that is involved in immune surveillance and innate immunity, as well as adaptive immunity. Dysfunction of the complement cascade may be mediated by aberrations in the pathways of activation, complement regulatory proteins, or complement deficiencies, and has been linked to a number of hematologic disorders, including paroxysmal noctural hemoglobinuria (PNH), hereditary angioedema (HAE), and atypical hemolytic-uremic syndrome (aHUS). Here, current laboratory tests for disorders of the complement pathway are reviewed, and their utility and limitations in hematologic disorders and systemic diseases are discussed. Current therapeutic advances targeting the complement pathway in treatment of complement-mediated hematologic disorders are also reviewed. Am. J. Hematol. 90:1180-1186, 2015. 2015 Wiley Periodicals, Inc. PMID:26437749

  2. Carbohydrate recognition and complement activation by rat ficolin-B

    PubMed Central

    Girija, Umakhanth Venkatraman; Mitchell, Daniel A; Roscher, Silke; Wallis, Russell

    2011-01-01

    Ficolins are innate immune components that bind to PAMPs and structures on apoptotic cells. Humans produce two serum forms (L- and H-ficolin) and a leukocyte-associated form (M-ficolin), whereas rodents and most other mammals produce ficolins-A and -B, orthologues of L- and M-ficolin, respectively. All three human ficolins, together with mouse and rat ficolin-A, associate with mannan-binding lectin-associated serine proteases (MASPs) and activate the lectin pathway of complement on PAMPs. By contrast, mouse ficolin-B does not bind MASPs and cannot activate complement. Because of these striking differences together with the lack of functional information for other ficolin-B orthologues, we have characterized rat ficolin-B, and compared its physical and biochemical properties with its serum counterpart. The data show that both rat ficolins have archetypal structures consisting of oligomers of a trimeric subunit. Ficolin-B recognized mainly sialyated sugars, characteristic of exogenous and endogenous ligands, whereas ficolin-A had a surprisingly narrow specificity, binding strongly to only one of 320 structures tested: an N-acetylated trisaccharide. Surprisingly, rat ficolin-B activated MASP-2 comparable to ficolin-A. Mutagenesis data reveal that lack of activity in mouse ficolin-B is probably caused by a single amino acid change in the putative MASP-binding site that blocks the ficolin-MASP interaction. PMID:21182092

  3. Targeting mechanisms at sites of complement activation for imaging and therapy.

    PubMed

    Holers, V Michael

    2016-06-01

    The complement system plays a key role in many acute injury states as well as chronic autoimmune and inflammatory diseases. Localized complement activation and alternative pathway-mediated amplification on diverse target surfaces promote local recruitment of pro-inflammatory cells and elaboration of other mediators. Despite a general understanding of the architecture of the system, though, many of the mechanisms that underlie site-specific complement activation and amplification in vivo are incompletely understood. In addition, there is no capability yet to measure the level of local tissue site-specific complement activation in patients without performing biopsies to detect products using immunohistochemical techniques. Herein is reviewed emerging evidence obtained through clinical research studies of human rheumatoid arthritis along with translational studies of its disease models which demonstrate that several parallel mechanisms are involved in site-specific amplification of activation of the complement system in vivo. Among these processes are de-regulation of the alternative pathway, effector pathway-catalyzed amplification of proximal complement activation, recognition of injury-associated ligands by components of the lectin pathway, and engagement of pathogenic natural antibodies that recognize a limited set of injury-associated neoepitopes. Studies suggest that each of these inter-related processes can play key roles in amplification of complement-dependent injury on self-tissues in vivo. These findings, in addition to development of an imaging strategy described herein designed to quantitatively measure local complement C3 fixation, have relevance to therapeutic and diagnostic strategies targeting the complement system. PMID:25979851

  4. Ultralarge von Willebrand factor-induced platelet clumping and activation of the alternative complement pathway in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndromes.

    PubMed

    Turner, Nancy; Sartain, Sarah; Moake, Joel

    2015-06-01

    The molecular linkage between ultralarge (UL) von Willebrand factor (VWF) multimers and the alternative complement pathway (AP) has recently been described. Endothelial cell (EC)-secreted and anchored ULVWF multimers (in long stringlike structures) function as both hyperadhesive sites that initiate platelet adhesion and aggregation and activating surfaces for the AP. In vitro, the active form of C3, C3b binds to the EC-anchored ULVWF multimeric strings and initiates the assembly on the strings of C3 convertase (C3bBb) and C5 convertase (C3bBbC3b). In vivo, activation of the AP via this mechanism proceeds all the way to generation of terminal complement complexes (C5b-9). PMID:26043389

  5. Mannose-binding lectin may affect pregnancy outcome.

    PubMed

    Çalkavur, Şebnem; Erdemir, Gülin; Onay, Hüseyin; Altun Köroğlu, Özge; Yalaz, Mehmet; Zekioğlu, Osman; Aksu, Güzide; Özkınay, Ferda; Akercan, Fuat; Kültürsay, Nilgün

    2015-01-01

    Mannose-binding lectin (MBL) is a component of the innate immune system and acts as a complement activator through the lectin pathway. Genetic variations of MBL and low MBL levels cause several infection problems, which may also be related to pregnancy problems. We aimed to investigate the role of MBL gene codon 54 polymorphism and serum MBL levels in pregnancy problems and premature delivery. In this prospective study, MBL gene codon 54 polymorphism and serum MBL levels were studied in 45 mothers who delivered earlier than 35 gestational weeks. The frequency of MBL gene codon 54 variant allele B was much higher (homozygous 4.4% and heterozygous 33.3%) in the study group mothers than the previously reported frequency in the healthy Turkish population (homozygous 2-6%, heterozygous 12-20%). MBL variant allele B frequency was closely related to low MBL levels (<0.1 μg/ml), vaginitis and increased IL-6 levels. The median MBL levels were lower than the critical level of 0.1 μg/ ml in study mothers who had recurrent miscarriage, infertility, preeclampsia, gestational diabetes mellitus, preterm premature rupture of membranes with duration of longer than 72 hours, tocolysis, histological chorioamnionitis, urinary tract infection and vaginitis. MBL gene codon 54 variant allele B is related to low serum MBL levels, increased IL-6 levels, genitourinary infections and may cause pregnancy-related problems such as infertility, recurrent miscarriage and preterm delivery. PMID:26613217

  6. Modulation of glycan detection on specific glycoproteins by lectin multimerization

    PubMed Central

    Cao, Zheng; Partyka, Katie; McDonald, Mitchell; Brouhard, Elizabeth; Hincapie, Marina; Brand, Randall E.; Hancock, William S.; Haab, Brian B.

    2013-01-01

    Improved methods for studying glycans could spur significant advances in the understanding and application of glycobiology. The use of affinity reagents such as lectins and glycan-binding antibodies is a valuable complement to methods involving mass spectrometry and chromatography. Many lectins, however, are not useful as analytic tools due to low affinity in vitro. As an approach to increasing lectin avidity to targeted glycans, we tested the use of lectin multimerization. Several biotinylated lectins were linked together through streptavidin interactions. The binding of certain lectins for purified glycoproteins and glycoproteins captured directly out of biological solutions was increased using multimerization, resulting in the detection of lower concentrations of glycoprotein than possible using monomeric detection. The analysis of glycoproteins in plasma samples showed that the level of binding enhancement through multimerization was not equivalent across patient samples. Wheat germ agglutinin (WGA) reactive glycans on fibronectin and thrombospondin-5 were preferentially bound by multimers in pancreatic cancer patient samples relative to control samples, suggesting a cancer-associated change in glycan density that could be detected only through lectin multimerization. This strategy could lead to the more sensitive and informative detection of glycans in biological samples and a broader spectrum of lectins that are useful as analytical reagents. PMID:23286506

  7. Engineering a central metabolic pathway: glycolysis with no net phosphorylation in an Escherichia coli gap mutant complemented with a plant GapN gene.

    PubMed

    Valverde, F; Losada, M; Serrano, A

    1999-04-23

    A cDNA fragment containing the Pisum sativum GapN gene, which encodes the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase, was cloned in a prokaryote expression vector. This construct enabled Escherichia coli strain W3CG, a mutant which lacks the glycolytic phosphorylating G3P dehydrogenase, to grow aerobically on sugars. The functionally complemented mutant exhibited high levels of the catalytically active plant enzyme, which renders 3-phosphoglycerate and NADPH, thus bypassing the first substrate level phosphorylation step of the glycolysis. As expected if such a glycolytic bypass would be operative in vivo, this clone failed to grow anaerobically on sugars in contrast to W3CG clones complemented with phosphorylating glyceraldehyde-3-phosphate dehydrogenases. According to the irreversible catabolic character of the non-phosphorylating reaction, the GapN-complemented clone was unable to grow on gluconeogenic substrates. This metabolic engineering approach demonstrates that a pure catabolic Embden-Meyerhof pathway with no net energy yield is feasible. PMID:10338122

  8. Mannan binding lectin attenuates double-stranded RNA-mediated TLR3 activation and innate immunity.

    PubMed

    Liu, Hongzhi; Zhou, Jia; Ma, Di; Lu, Xiao; Ming, Siqi; Shan, Guiqiu; Zhang, Xiaoyong; Hou, Jinlin; Chen, Zhengliang; Zuo, Daming

    2014-03-18

    Mannan binding lectin (MBL) functions as a pattern recognition molecule (PRM) which is able to initiate complement activation. Here, we characterize a previously unrecognized attribute of MBL as a double-stranded RNA (dsRNA) binding protein capable of modifying Toll like receptor 3 (TLR3) activation. MBL interacts with poly(I:C) and suppresses poly(I:C)-induced activation of TLR3 pathways and subsequent cytokine production. In addition, MBL binds to TLR3 directly. Surprisingly, disrupting the interaction between MBL and complement receptor 1 (CR1) or restraining the traffic of MBL to phagosome reversed the MBL limited TLR3 activation. We demonstrate the importance of MBL guided ligands intracellular localization, emphasizing the significance of understanding the dynamics of TLR agonists complexed with MBL or other PRMs inside the cell in immune defense. PMID:24530528

  9. New insight into the effects of heparinoids on complement inhibition by C1-inhibitor.

    PubMed

    Poppelaars, F; Damman, J; de Vrij, E L; Burgerhof, J G M; Saye, J; Daha, M R; Leuvenink, H G; Uknis, M E; Seelen, M A J

    2016-06-01

    Complement activation is of major importance in numerous pathological conditions. Therefore, targeted complement inhibition is a promising therapeutic strategy. C1-esterase inhibitor (C1-INH) controls activation of the classical pathway (CP) and the lectin pathway (LP). However, conflicting data exist on inhibition of the alternative pathway (AP) by C1-INH. The inhibitory capacity of C1-INH for the CP is potentiated by heparin and other glycosaminoglycans, but no data exist for the LP and AP. The current study investigates the effects of C1-INH in the presence or absence of different clinically used heparinoids on the CP, LP and AP. Furthermore, the combined effects of heparinoids and C1-INH on coagulation were investigated. C1-INH, heparinoids or combinations were analysed in a dose-dependent fashion in the presence of pooled serum. Functional complement activities were measured simultaneously using the Wielisa(®) -kit. The activated partial thrombin time was determined using an automated coagulation analyser. The results showed that all three complement pathways were inhibited significantly by C1-INH or heparinoids. Next to their individual effects on complement activation, heparinoids also enhanced the inhibitory capacity of C1-INH significantly on the CP and LP. For the AP, significant potentiation of C1-INH by heparinoids was found; however, this was restricted to certain concentration ranges. At low concentrations the effect on blood coagulation by combining heparinoids with C1-INH was minimal. In conclusion, our study shows significant potentiating effects of heparinoids on the inhibition of all complement pathways by C1-INH. Therefore, their combined use is a promising and a potentially cost-effective treatment option for complement-mediated diseases. PMID:26874675

  10. Development and application of an enzyme-linked immunosorbent assay for the quantitation of alternative complement pathway activation in human serum.

    PubMed Central

    Mayes, J T; Schreiber, R D; Cooper, N R

    1984-01-01

    We have developed a new, specific, and highly sensitive enzyme-linked immunosorbent assay (ELISA) which quantitates activation of the alternative pathway in human serum, plasma, or on the surface of activators. The ELISA detects the third component of complement (C3b), proteolytic fragment of complement Factor B (Bb), and properdin (P) complex or its derivative product, C3b,P. In the method, activator-plasma mixtures, plasma containing an activated alternative pathway, or other samples are added to the wells of microtitration plates precoated with antibody to P. C3b, Bb,P or C3b,P complexes which become bound are quantitated by subsequently added, enzyme-labeled, anti-C3. The resulting hydrolysis of the chromogenic substrate is expressed as nanograms of C3b by reference to a C3 standard curve. In addition to absolute specificity for activation of the pathway because of the nature of the complex detected by the assay, the ELISA is highly sensitive and able to reproducibly detect 10-20 ng/ml of C3b,P complexes in serum. This value corresponds to 0.0015% of the C3 in serum. In a series of studies to validate the parameters of the ELISA, reactivity was found to be dependent on the presence of alternative pathway proteins, the functional integrity of the pathway, and on the presence of magnesium. Sheep erythrocytes were converted to activators by treatment with neuraminidase. By using a variety of activators, the kinetics of activation and the numbers of bound C3b molecules quantitated by the ELISA were very similar to those measured by C3b deposition. The ELISA also detected identical activation kinetics when MgEGTA-serum and a mixture of the purified alternative pathway proteins were used as sources of the pathway. ELISA reaction kinetics also correlated with the restriction index, a measure of alternative pathway-activating ability. These studies cumulatively validate the ELISA as a direct and quantitative assay for alternative pathway activation. The sensitivity of the ELISA has permitted its use to detect direct alternative pathway activation by several viruses. The ELISA has also shown that certain classical pathway activators trigger the amplification loop of the alternative pathway while others do not. In addition, stable ELISA reactive complexes appeared in the supernatant of mixtures of serum with certain, but not other activators. The ability of the ELISA to detect activation which has already occurred and the stability of the reactive complexes permits studies of clinical sera. Normal human sera (20) contained low levels (5-20 ng/ml) of ELISA-reactive complexes. A proportion of sera from individuals with the adult respiratory distress syndrome (9-10), typhoid fever (8-10), malaria (3-5), gram-negative sepsis (9 of 47), acute trauma and shock (6 f 25), and systemic lupus erythematosus (3 of 29) showed elevated levels of complexes reactive in the alternative pathway ELISA. In contrast, nine sera from patients with circulating C3 nephritic factor were not reactive in the ELISA. PMID:6418767

  11. Lectins of marine hydrobionts.

    PubMed

    Chernikov, O V; Molchanova, V I; Chikalovets, I V; Kondrashina, A S; Li, W; Lukyanov, P A

    2013-07-01

    Data from the literature and results of our research on lectins isolated from some kinds of marine hydrobionts such as clams, ascidians, sea worms, sponges, and algae are presented in this review. Results of comparative analysis of the basic physicochemical properties and biological activity of lectins isolated from various sources are discussed. PMID:24010839

  12. Complement Test

    MedlinePlus

    ... helpful? Also known as: C1; C1q; C2; C3; C4; CH50; CH100 (among others) Formal name: Complement Activity; ... whether the system is functioning normally. C3 and C4 are the most frequently measured complement proteins. Total ...

  13. Role of mannose-binding lectin in intestinal homeostasis and fungal elimination.

    PubMed

    Choteau, L; Parny, M; François, N; Bertin, B; Fumery, M; Dubuquoy, L; Takahashi, K; Colombel, J-F; Jouault, T; Poulain, D; Sendid, B; Jawhara, S

    2016-05-01

    Mannose-binding lectin (MBL) is a soluble lectin of the innate immune system that is produced by the liver and secreted into the circulation where it activates the lectin complement pathway, enhances phagocytosis of microorganisms by leukocytes, and modulates inflammation. MBL can recognize patterns on the surface of different pathogens, including Candida albicans. Our aims were to investigate whether MBL is expressed in the gut epithelium and to examine its effect on the modulation of intestinal inflammation and C. albicans elimination. Using reverse transcriptase-PCR, MBL transcripts were highly expressed in different parts of the mouse gut. MBL expression was also detected by immunoblotting and immunolocalization in response to C. albicans colonization of the gut; the highest expression of MBL was detected in the stomach. Blocking MBL by administering mannans to mice increased C. albicans colonization. MBL-deficient mice had a higher level of colonization than wild-type mice. Dextran sodium sulfate-induced colitis promoted C. albicans dissemination to the kidneys and lungs of MBL-deficient mice. MBL-deficient mice exhibited elevated expression of interleukin (IL)-17, IL-23, dectin-1, and Toll-like receptor-4. This study shows that MBL expression is induced in the gut in response to C. albicans sensing and is required for intestinal homeostasis and host defense against C. albicans. PMID:26442658

  14. Collectin liver 1 and collectin kidney 1 and other complement-associated pattern recognition molecules in systemic lupus erythematosus.

    PubMed

    Troldborg, A; Thiel, S; Jensen, L; Hansen, S; Laska, M J; Deleuran, B; Jensenius, J C; Stengaard-Pedersen, K

    2015-11-01

    The objective of this study was to explore the involvement of collectin liver 1 (CL-L1) and collectin kidney 1 (CL-K1) and other pattern recognition molecules (PRMs) of the lectin pathway of the complement system in a cross-sectional cohort of systemic lupus erythematosus (SLE) patients. Concentrations in plasma of CL-L1, CL-K1, mannan-binding lectin (MBL), M-ficolin, H-ficolin and L-ficolin were determined in 58 patients with SLE and 65 healthy controls using time-resolved immunoflourometric assays. The SLE patients' demographic, diagnostic, clinical and biochemical data and collection of plasma samples were performed prospectively during 4 months. CL-L1, CL-K1 and M-ficolin plasma concentrations were lower in SLE patients than healthy controls (P-values < 0.001, 0.033 and < 0.001, respectively). H-ficolin concentration was higher in SLE patients (P < 0.0001). CL-L1 and CL-K1 plasma concentrations in the individuals correlated in both patients and controls. Patients with low complement component 3 (C3) demonstrated a negative correlation between C3 and CL-L1 and CL-K1 (P = 0.022 and 0.031, respectively). Patients positive for anti-dsDNA antibodies had lower levels of MBL in plasma than patients negative for anti-dsDNA antibodies (P = 0.02). In a cross-sectional cohort of SLE patients, we found differences in the plasma concentrations of CL-L1, CL-K1, M-ficolin and H-ficolin compared to a group of healthy controls. Alterations in plasma concentrations of the PRMs of the lectin pathway in SLE patients and associations to key elements of the disease support the hypothesis that the lectin pathway plays a role in the pathogenesis of SLE. PMID:26154564

  15. Sushi domain-containing protein 4 (SUSD4) inhibits complement by disrupting the formation of the classical C3 convertase.

    PubMed

    Holmquist, Emelie; Okroj, Marcin; Nodin, Björn; Jirström, Karin; Blom, Anna M

    2013-06-01

    Recently discovered Sushi domain-containing protein 4 (SUSD4) contains several Sushi or complement control protein domains; therefore, we hypothesized that it may act as complement inhibitor. Two isoforms of human SUSD4, fused to the Fc part of human IgG, were recombinantly expressed in Chinese hamster ovary (CHO) cells. The secreted soluble isoform of SUSD4 (SUSD4b) inhibited the classical and lectin complement pathways by 50% at a concentration of 0.5 μM. This effect was due to the fact that 1 μM SUSD4b inhibited the formation of the classical C3 convertase by 90%. The membrane-bound isoform (SUSD4a) inhibited the classical and alternative complement pathways when expressed on the surface of CHO cells but not when expressed as a soluble, truncated protein. In all functional studies, we used known complement inhibitors as positive controls, while Coxsackie adenovirus receptor, which has no effect on complement, expressed with Fc tag, was a negative control. We also studied the mRNA expression of both isoforms of SUSD4 in a panel of human tissues using quantitative PCR and primarily found SUSD4a in esophagus and brain, while SUSD4b was highly expressed in esophagus, ovary, and heart. Overall, our results show that SUSD4 is a novel complement inhibitor with restricted expression. PMID:23482636

  16. CD45-mediated signaling pathway is involved in Rhizoctonia bataticola lectin (RBL)-induced proliferation and Th1/Th2 cytokine secretion in human PBMC

    SciTech Connect

    Pujari, Radha; Eligar, Sachin M.; Kumar, Natesh; Nagre, Nagaraja N.; Inamdar, Shashikala R.; Swamy, Bale M.; Shastry, Padma

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer RBL, a potent mitogenic and complex N-glycan specific lectin binds to CD45 on PBMC. Black-Right-Pointing-Pointer RBL triggers CD45-mediated signaling involved in activation of p38MAPK and STAT-5. Black-Right-Pointing-Pointer Inhibition of CD45 PTPase signaling blocks RBL-induced ZAP70 phosphorylation. Black-Right-Pointing-Pointer RBL-CD45 mediated signaling is crucial for RBL-induced immunodulatory activities. -- Abstract: We earlier reported the mitogenic and immunostimulatory activities of Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus R. bataticola in human PBMC. The lectin demonstrates specificity towards glycoproteins containing complex N-glycans. Since CD45-protein tyrosine phosphatase that abundantly expresses N-glycans is important in T-cell signaling, the study aimed to investigate the involvement of CD45 in the immunomodulatory activities of RBL. Flowcytometry and confocal microscopy studies revealed that RBL exhibited binding to PBMC and colocalized with CD45. The binding was comparable in cells expressing different CD45 isoforms-RA, -RB and -RO. CD45 blocking antibody reduced the binding and proliferation of PBMC induced by RBL. CD45-PTPase inhibitor dephostatin inhibited RBL-induced proliferation, expression of CD25 and pZAP-70. RBL-induced secretion of Th1/Th2 cytokines were significantly inhibited in presence of dephostatin. Also, dephostatin blocked phosphorylation of p38MAPK and STAT-5 that was crucial for the biological functions of RBL. The study demonstrates the involvement of CD45-mediated signaling in RBL-induced PBMC proliferation and Th1/Th2 cytokine secretion through activation of p38MAPK and STAT-5.

  17. Reconstitution of the alternative pathway of complement by plasma infusions given to a patient with an SLE-like syndrome associated with a hereditary C3 dysfunction.

    PubMed Central

    Nilsson, B; Nilsson, U R; Karlsson-Parra, A; Sjölin-Forsberg, G; Hällgren, R

    1994-01-01

    OBJECTIVE--To reconstitute a dysfunctional form of complement factor C3 in a patient with a systemic lupus erythematosus (SLE)-like syndrome. METHODS--The propositus was treated with plasma infusions during five sessions over a period of eight months. RESULTS--The alternative pathway was reconstituted to normal levels for approximately two to three days after each infusion. C3 fragments were incorporated into previously detected deposits of IgG and IgM at the dermal-epidermal junction and the immune complex levels gradually decreased during the whole treatment period. CONCLUSION--The reconstitution appears to result in the solubilisation of tissue immune complexes and a subsequent transportation to the fixed macrophage system. Images PMID:7979584

  18. Transcriptome and analysis on the complement and coagulation cascades pathway of large yellow croaker (Larimichthys crocea) to ciliate ectoparasite Cryptocaryon irritans infection.

    PubMed

    Yin, Fei; Gao, Quanxin; Tang, Baojun; Sun, Peng; Han, Kunhuang; Huang, Weiqing

    2016-03-01

    Large yellow croaker (Larimichthys crocea) is one of the most valuable marine fish in southern China. Given to the rapid development of aquaculture industry, the L. crocea was subjected to ciliate ectoparasite Cryptocaryon irritans. It therefore is indispensable and urgent to understand the mechanism of L. crocea host defense against C. irritans infection. In the present study, the extensively analysis at the transcriptome level for Cryptocaryoniasis in L. crocea was carried out. These results showed that 15,826,911, 16,462,921, and 15,625,433 paired-end clean reads were obtained from three cDNA libraries (A: 0 theronts/fish, B: 12,000 theronts/fish, and C: 24,000 theronts/fish) of the L. crocea immune-related tissues by Illumina paired-end sequencing technology. Totally, 30,509 unique transcript fragments (unigenes) were assembled, with an average length of 1715 bp. In B/A, C/A, and C/B pairwise comparison, 972, 900, and 1126 genes showed differential expression respectively. Differently expressed immune-related genes (DEIGs) were scrutinized, in B/A pairwise comparison, 48 genes showed differential expression, including 26 up-regulated genes and 22 down-regulated genes in B; in C/A pairwise comparison, there were 39 DEIGs, including 7 up-regulated genes and 32 down-regulated genes in C; in C/B pairwise comparison, 40 genes showed differential expression, including 11 up-regulated genes and 29 down-regulated genes in C. There were 16 DEIGs enriched KEGG pathways, in which the complement and coagulation cascades pathway was the top most DEIGs enriched pathway (B:A=42; C:A=28; C:B=42). The coagulation and fibrinolytic system was in a highly active state after infected by C. irritans with non-lethal concentration; the alternative complement pathway may play an important role in the early stages of C. irritans infection. These results demonstrated that low-concentration infection can significantly induce the immunological response in fishes, however, when fishes were in fatal conditions, the immunity was suppressed. PMID:26804649

  19. Lectins with potential for anti-cancer therapy.

    PubMed

    Yau, Tammy; Dan, Xiuli; Ng, Charlene Cheuk Wing; Ng, Tzi Bun

    2015-01-01

    This article reviews lectins of animal and plant origin that induce apoptosis and autophagy of cancer cells and hence possess the potential of being developed into anticancer drugs. Apoptosis-inducing lectins encompass galectins, C-type lectins, annexins, Haliotis discus discus lectin, Polygonatum odoratum lectin, mistletoe lectin, and concanavalin A, fucose-binding Dicentrarchus labrax lectin, and Strongylocentrotus purpuratus lectin, Polygonatum odoratum lectin, and mistletoe lectin, Polygonatum odoratum lectin, autophagy inducing lectins include annexins and Polygonatum odoratum lectin. PMID:25730388

  20. C3 binds covalently to the C gamma 3 domain of IgG immune aggregates during complement activation by the alternative pathway.

    PubMed

    Antón, L C; Alcolea, J M; Sánchez-Corral, P; Marqués, G; Sánchez, A; Vivanco, F

    1989-02-01

    Ovalbumin-antiovalbumin IgG immune aggregates were incubated with normal human serum in the presence of iodo[1-14C]acetamide, in conditions in which only the alternative pathway of complement was activated. The [14C]C3b-IgG covalent complexes formed were digested with pepsin, and analysed by SDS/polyacrylamide-gel electrophoresis and fluorography. Covalent complexes of [14C]C3-Fd and [14C]C3-pFc' were visualized, demonstrating that, during complement activation by the alternative pathway, C3 is covalently incorporated into the C gamma 3 domain of IgG, as well as into the Fd region. The C gamma 2 domain becomes protected from pepsin action by the bound C3b. All the covalent linkages between C3 and the IgG were sensitive to hydroxylamine. When [14C]C3-pFc' covalent complexes were treated with 1 M-NH2OH and loaded onto a Bio-Gel P-4 column, a radioactive peak of 3 kDa was obtained. The material released from [14C]C3-pFc' and [14C]C3-F(ab')2 complexes after treatment with 1 M-NH2OH was mixed and analysed in the Bio-Gel P-4 column. A similar radioactive peak of 3 kDa was obtained. When this peak, either from [14C]C3-pFc' alone or from the mixture of [14C]C3-F(ab')2 and [14C]C3-pFc', was fractionated by h.p.l.c., virtually the same radioactive peptide profile was obtained, indicating that very similar C3 peptides remained covalently bound to both regions (Fab and C gamma 3) of the antibody molecule. It is suggested that C3 bound to the C gamma 3 domain of IgG may interfere with the Fc-Fc interactions of immune aggregates and thus may be involved in several biological properties displayed by these complement-activating aggregates. PMID:2784671

  1. Role of complement in glomerular diseases.

    PubMed

    Mao, Song; Zhang, Jianhua

    2016-06-01

    The complement system, composed of nearly 30 proteins, is a key regulator of immunity. The complement system is critical for protecting hosts from invading pathogens. Dysregulation of this system is associated with susceptibility to infection and various autoimmune diseases. Furthermore, complement activation due to the defective regulation of the alternative pathway will induce glomerular diseases. Anti-complement therapy has been applied in various glomerular diseases. Signaling pathways might be very important in the pathogenesis of glomerular diseases. This review will give a relatively complete signaling pathway flowchart for complement and a comprehensive understanding of the underlying role of complement in glomerular diseases. PMID:26400225

  2. Complement activation by ligand-driven juxtaposition of discrete pattern recognition complexes.

    PubMed

    Degn, Søren E; Kjaer, Troels R; Kidmose, Rune T; Jensen, Lisbeth; Hansen, Annette G; Tekin, Mustafa; Jensenius, Jens C; Andersen, Gregers R; Thiel, Steffen

    2014-09-16

    Defining mechanisms governing translation of molecular binding events into immune activation is central to understanding immune function. In the lectin pathway of complement, the pattern recognition molecules (PRMs) mannan-binding lectin (MBL) and ficolins complexed with the MBL-associated serine proteases (MASP)-1 and MASP-2 cleave C4 and C2 to generate C3 convertase. MASP-1 was recently found to be the exclusive activator of MASP-2 under physiological conditions, yet the predominant oligomeric forms of MBL carry only a single MASP homodimer. This prompted us to investigate whether activation of MASP-2 by MASP-1 occurs through PRM-driven juxtaposition on ligand surfaces. We demonstrate that intercomplex activation occurs between discrete PRM/MASP complexes. PRM ligand binding does not directly escort the transition of MASP from zymogen to active enzyme in the PRM/MASP complex; rather, clustering of PRM/MASP complexes directly causes activation. Our results support a clustering-based mechanism of activation, fundamentally different from the conformational model suggested for the classical pathway of complement. PMID:25197071

  3. Complement Factor C4d Is a Common Denominator in Thrombotic Microangiopathy.

    PubMed

    Chua, Jamie S; Baelde, Hans J; Zandbergen, Malu; Wilhelmus, Suzanne; van Es, Leendert A; de Fijter, Johan W; Bruijn, Jan A; Bajema, Ingeborg M; Cohen, Danielle

    2015-09-01

    Complement activation has a major role in thrombotic microangiopathy (TMA), a disorder that can occur in a variety of clinical conditions. Promising results of recent trials with terminal complement-inhibiting drugs call for biomarkers identifying patients who might benefit from this treatment. The primary aim of this study was to determine the prevalence and localization of complement factor C4d in kidneys of patients with TMA. The secondary aims were to determine which complement pathways lead to C4d deposition and to determine whether complement activation results in deposition of the terminal complement complex. We examined 42 renal sections with histologically confirmed TMA obtained from a heterogeneous patient group. Deposits of C4d, mannose-binding lectin, C1q, IgM, and C5b-9 were scored in the glomeruli, peritubular capillaries, and arterioles. Notably, C4d deposits were present in 88.1% of TMA cases, and the various clinical conditions had distinct staining patterns within the various compartments of the renal vasculature. Classical pathway activation was observed in 90.5% of TMA cases. C5b-9 deposits were present in 78.6% of TMA cases and in 39.6% of controls (n=53), but the staining pattern differed between cases and controls. In conclusion, C4d is a common finding in TMA, regardless of the underlying clinical condition. Moreover, C5b-9 was present in >75% of the TMA samples, suggesting that terminal complement inhibitors may have a beneficial effect in these patients. C4d and C5b-9 should be investigated as possible diagnostic biomarkers in the clinical work-up of patients suspected of having complement-mediated TMA. PMID:25573909

  4. Identification of Complin, a novel complement inhibitor that targets complement proteins factor B and C2.

    PubMed

    Kadam, Archana P; Sahu, Arvind

    2010-06-15

    Complement factor B (fB) is a key constituent of the alternative pathway (AP). Its central role in causing inflammation and tissue injury through activation of the AP urges the need for its therapeutic targeting. In the current study, we have screened phage-displayed random peptide libraries against fB and identified a novel cyclic hendecapeptide that inhibits activation of fB and the AP. Structure-activity studies revealed that: 1) the cysteine-constrained structure of the peptide is essential for its activity; 2) Ile5, Arg6, Leu7, and Tyr8 contribute significantly to its inhibitory activity; and 3) retro-inverso modification of the peptide results in loss of its activity. Binding studies performed using surface plasmon resonance suggested that the peptide has two binding sites on fB, which are located on the Ba and Bb fragments. Studies on the mechanism of inhibition revealed that the peptide does not block the interaction of fB with the activated form of C3, thereby suggesting that the peptide inhibits fB activation primarily by inhibiting its cleavage by factor D. The peptide showed a weak effect on preformed C3 and C5 convertases. Like inhibition of fB cleavage, the peptide also inhibited C2 cleavage by activated C1s and activation of the classical as well as lectin pathways. Based on its inhibitory activities, we named the peptide Complin. PMID:20483772

  5. Surface-bound capsular polysaccharide of type Ia group B Streptococcus mediates C1 binding and activation of the classic complement pathway

    SciTech Connect

    Levy, N.J.; Kasper, D.L.

    1986-06-01

    The role of surface-bound type Ia group B Streptococcus (GBS) capsular polysaccharide in anti-body-independent binding of C1 and activation of the classic component pathway was investigated. In a radiolabeled bacterial-polymorphonuclear leukocyte (PMN) association assay, a measure of bacterial opsonization, preincubation of /sup 3/H-type Ia GBS with purified F(ab')/sub 2/ to the organism blocked the association of the bacteria with PMN', and the inhibitory effect was dose dependent. The specificity of F(ab')/sub 2/ blocking was shown after adsorption of F(ab')/sub 2/ with type Ia polysaccharide-sensitized erythrocytes. Polysaccharide-adsorbed F(ab')/sub 2/ had a 70% decrease in ability to block the association of bacteria with PMN. Neuraminidase digestion removed 80% of the terminal sialic acid residues from the native polysaccharide. These neuraminidase-digested organisms had a 72% decrease in binding and transfer of purified C1 compared with non-enzyme-treated organisms. Type Ia capsular polysaccharide bound to sheep erythrocytes promoted classic complement pathway-mediated hemolysis of the cells. The role of C1 inhibitor (INH) in modulation of C1 activation by the organisms was investigated. The possibility existed that the C1 INH could be bound by the bacteria, allowing C1 activation to occur in the fluid phase. The inhibitor was purified from human serum, and its activity was measured before and after incubation with type Ia GBS. The organisms had no effect on C1 INH activity. Thus surface-bound capsular polysacchardie of type Ia GBS mediates C1 binding and classic pathway activation, and this does not involve the C1 INH.

  6. Microglia, Alzheimer's Disease, and Complement

    PubMed Central

    Crehan, Helen; Hardy, John; Pocock, Jennifer

    2012-01-01

    Microglia, the immune cell of the brain, are implicated in cascades leading to neuronal loss and cognitive decline in Alzheimer's disease (AD). Recent genome-wide association studies have indicated a number of risk factors for the development of late-onset AD. Two of these risk factors are an altered immune response and polymorphisms in complement receptor 1. In view of these findings, we discuss how complement signalling in the AD brain and microglial responses in AD intersect. Dysregulation of the complement cascade, either by changes in receptor expression, enhanced activation of different complement pathways or imbalances between complement factor production and complement cascade inhibitors may all contribute to the involvement of complement in AD. Altered complement signalling may reduce the ability of microglia to phagocytose apoptotic cells and clear amyloid beta peptides, modulate the expression by microglia of complement components and receptors, promote complement factor production by plaque-associated cytokines derived from activated microglia and astrocytes, and disrupt complement inhibitor production. The evidence presented here indicates that microglia in AD are influenced by complement factors to adopt protective or harmful phenotypes and the challenge ahead lies in understanding how this can be manipulated to therapeutic advantage to treat late onset AD. PMID:22957298

  7. The requirement of localized, CR2-mediated, alternative pathway activation of complement for covalent deposition of C3 fragments on normal B cells.

    PubMed Central

    Olesen, E H; Johnson, A A; Damgaard, G; Leslie, R G

    1998-01-01

    We have shown previously that normal B cells share, with Epstein-Barr virus-transformed and malignant B cells, the ability to activate the alternative pathway (AP) of complement in vitro, resulting in the deposition of C3 fragments on the cell surface. Complement receptor type 2 (CR2, CD21) has been implicated directly as the site for formation of an AP convertase, which provides nascent C3b for deposition at secondary sites on the B-cell surface. In the present study, we have examined C3 fragment deposition in vitro in more detail by (1) assessing the importance of locally generated C3b for the deposition process, (2) investigating whether CR2 is the sole requirement for conferring AP activation capacity on a cell, and (3) determining whether CR2's function, as an AP activator, has different structural requirements from ligand binding. Increasing the availability of native C3, by increasing the serum (NHS) concentration, resulted in enhanced C3 fragment deposition on the B cells, whereas use of factor 1-depleted NHS, which showed massive fluid phase C3 conversion during the incubation, diminished the deposition. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting of untreated and hydroxylamine-treated lysates from B cells, after in vitro activation, revealed that the majority of C3 fragments (primarily iC3b and C3dg) had been covalently bound to the cell surface. Transfection of COS cells with wild-type CR2 or a deletion mutant lacking 11 of the molecule's 15 homologous domains, but retaining the ligand-binding site, revealed that expression of intact CR2 conferred a 12-fold increase in AP-activating capacity on these cells, while no increase in AP activity was apparent on cells transfected with the mutant CR2. Images Figure 2 Figure 3 PMID:9616366

  8. Inhibition of the alternative pathway of nonhuman infant complement by porin B2 contributes to virulence of Neisseria meningitidis in the infant rat model.

    PubMed

    Lewis, Lisa A; Vu, David M; Granoff, Dan M; Ram, Sanjay

    2014-06-01

    Neisseria meningitidis utilizes capsular polysaccharide, lipooligosaccharide (LOS) sialic acid, factor H binding protein (fHbp), and neisserial surface protein A (NspA) to regulate the alternative pathway (AP) of complement. Using meningococcal mutants that lacked all four of the above-mentioned molecules (quadruple mutants), we recently identified a role for PorB2 in attenuating the human AP; inhibition was mediated by human fH, a key downregulatory protein of the AP. Previous studies showed that fH downregulation of the AP via fHbp or NspA is specific for human fH. Here, we report that PorB2-expressing quadruple mutants also regulate the AP of baby rabbit and infant rat complement. Blocking a human fH binding region on PorB2 of the quadruple mutant of strain 4243 with a chimeric protein that comprised human fH domains 6 and 7 fused to murine IgG Fc enhanced AP-mediated baby rabbit C3 deposition, which provided evidence for an fH-dependent mechanism of nonhuman AP regulation by PorB2. Using isogenic mutants of strain H44/76 that differed only in their PorB molecules, we confirmed a role for PorB2 in resistance to killing by infant rat serum. The PorB2-expressing strain also caused higher levels of bacteremia in infant rats than its isogenic PorB3-expressing counterpart, thus providing a molecular basis for increased survival of PorB2 isolates in this model. These studies link PorB2 expression with infection of infant rats, which could inform the choice of meningococcal strains for use in animal models, and reveals, for the first time, that PorB2-expressing strains of N. meningitidis regulate the AP of baby rabbits and rats. PMID:24686052

  9. Inhibition of the Alternative Pathway of Nonhuman Infant Complement by Porin B2 Contributes to Virulence of Neisseria meningitidis in the Infant Rat Model

    PubMed Central

    Vu, David M.; Granoff, Dan M.; Ram, Sanjay

    2014-01-01

    Neisseria meningitidis utilizes capsular polysaccharide, lipooligosaccharide (LOS) sialic acid, factor H binding protein (fHbp), and neisserial surface protein A (NspA) to regulate the alternative pathway (AP) of complement. Using meningococcal mutants that lacked all four of the above-mentioned molecules (quadruple mutants), we recently identified a role for PorB2 in attenuating the human AP; inhibition was mediated by human fH, a key downregulatory protein of the AP. Previous studies showed that fH downregulation of the AP via fHbp or NspA is specific for human fH. Here, we report that PorB2-expressing quadruple mutants also regulate the AP of baby rabbit and infant rat complement. Blocking a human fH binding region on PorB2 of the quadruple mutant of strain 4243 with a chimeric protein that comprised human fH domains 6 and 7 fused to murine IgG Fc enhanced AP-mediated baby rabbit C3 deposition, which provided evidence for an fH-dependent mechanism of nonhuman AP regulation by PorB2. Using isogenic mutants of strain H44/76 that differed only in their PorB molecules, we confirmed a role for PorB2 in resistance to killing by infant rat serum. The PorB2-expressing strain also caused higher levels of bacteremia in infant rats than its isogenic PorB3-expressing counterpart, thus providing a molecular basis for increased survival of PorB2 isolates in this model. These studies link PorB2 expression with infection of infant rats, which could inform the choice of meningococcal strains for use in animal models, and reveals, for the first time, that PorB2-expressing strains of N. meningitidis regulate the AP of baby rabbits and rats. PMID:24686052

  10. Lectins: production and practical applications

    PubMed Central

    2010-01-01

    Lectins are proteins found in a diversity of organisms. They possess the ability to agglutinate erythrocytes with known carbohydrate specificity since they have at least one non-catalytic domain that binds reversibly to specific monosaccharides or oligosaccharides. This articles aims to review the production and practical applications of lectins. Lectins are isolated from their natural sources by chromatographic procedures or produced by recombinant DNA technology. The yields of animal lectins are usually low compared with the yields of plant lectins such as legume lectins. Lectins manifest a diversity of activities including antitumor, immunomodulatory, antifungal, HIV-1 reverse transcriptase inhibitory, and anti-insect activities, which may find practical applications. A small number of lectins demonstrate antibacterial and anti-nematode activities. PMID:20890754

  11. M. leprae components induce nerve damage by complement activation: identification of lipoarabinomannan as the dominant complement activator.

    PubMed

    Bahia El Idrissi, Nawal; Das, Pranab K; Fluiter, Kees; Rosa, Patricia S; Vreijling, Jeroen; Troost, Dirk; Morgan, B Paul; Baas, Frank; Ramaglia, Valeria

    2015-05-01

    Peripheral nerve damage is the hallmark of leprosy pathology but its etiology is unclear. We previously identified the membrane attack complex (MAC) of the complement system as a key determinant of post-traumatic nerve damage and demonstrated that its inhibition is neuroprotective. Here, we determined the contribution of the MAC to nerve damage caused by Mycobacterium leprae and its components in mouse. Furthermore, we studied the association between MAC and the key M. leprae component lipoarabinomannan (LAM) in nerve biopsies of leprosy patients. Intraneural injections of M. leprae sonicate induced MAC deposition and pathological changes in the mouse nerve, whereas MAC inhibition preserved myelin and axons. Complement activation occurred mainly via the lectin pathway and the principal activator was LAM. In leprosy nerves, the extent of LAM and MAC immunoreactivity was robust and significantly higher in multibacillary compared to paucibacillary donors (p = 0.01 and p = 0.001, respectively), with a highly significant association between LAM and MAC in the diseased samples (r = 0.9601, p = 0.0001). Further, MAC co-localized with LAM on axons, pointing to a role for this M. leprae antigen in complement activation and nerve damage in leprosy. Our findings demonstrate that MAC contributes to nerve damage in a model of M. leprae-induced nerve injury and its inhibition is neuroprotective. In addition, our data identified LAM as the key pathogen associated molecule that activates complement and causes nerve damage. Taken together our data imply an important role of complement in nerve damage in leprosy and may inform the development of novel therapeutics for patients. PMID:25772973

  12. Structural and functional diversity of the lectin repertoire in teleost fish: Relevance to innate and adaptive immunity

    PubMed Central

    Vasta, Gerardo R.; Nita-Lazar, Mihai; Giomarelli, Barbara; Ahmed, Hafiz; Du, Shaojun; Cammarata, Matteo; Parrinello, Nicolò; Bianchet, Mario A.; Amzel, L. Mario

    2012-01-01

    Protein–carbohydrate interactions mediated by lectins have been recognized as key components of innate immunity in vertebrates and invertebrates, not only for recognition of potential pathogens, but also for participating in downstream effector functions, such as their agglutination, immobilization, and complement-mediated opsonization and killing. More recently, lectins have been identified as critical regulators of mammalian adaptive immune responses. Fish are endowed with virtually all components of the mammalian adaptive immunity, and are equipped with a complex lectin repertoire. In this review, we discuss evidence suggesting that: (a) lectin repertoires in teleost fish are highly diversified, and include not only representatives of the lectin families described in mammals, but also members of lectin families described for the first time in fish species; (b) the tissue-specific expression and localization of the diverse lectin repertoires and their molecular partners is consistent with their distinct biological roles in innate and adaptive immunity; (c) although some lectins may bind endogenous ligands, others bind sugars on the surface of potential pathogens; (d) in addition to pathogen recognition and opsonization, some lectins display additional effector roles, such as complement activation and regulation of immune functions; (e) some lectins that recognize exogenous ligands mediate processes unrelated to immunity: they may act as anti-freeze proteins or prevent polyspermia during fertilization. PMID:21896283

  13. Lectins with anti-HIV activity: a review.

    PubMed

    Akkouh, Ouafae; Ng, Tzi Bun; Singh, Senjam Sunil; Yin, Cuiming; Dan, Xiuli; Chan, Yau Sang; Pan, Wenliang; Cheung, Randy Chi Fai

    2015-01-01

    Lectins including flowering plant lectins, algal lectins, cyanobacterial lectins, actinomycete lectin, worm lectins, and the nonpeptidic lectin mimics pradimicins and benanomicins, exhibit anti-HIV activity. The anti-HIV plant lectins include Artocarpus heterophyllus (jacalin) lectin, concanavalin A, Galanthus nivalis (snowdrop) agglutinin-related lectins, Musa acuminata (banana) lectin, Myrianthus holstii lectin, Narcissus pseudonarcissus lectin, and Urtica diocia agglutinin. The anti-HIV algal lectins comprise Boodlea coacta lectin, Griffithsin, Oscillatoria agardhii agglutinin. The anti-HIV cyanobacterial lectins are cyanovirin-N, scytovirin, Microcystis viridis lectin, and microvirin. Actinohivin is an anti-HIV actinomycete lectin. The anti-HIV worm lectins include Chaetopterus variopedatus polychaete marine worm lectin, Serpula vermicularis sea worm lectin, and C-type lectin Mermaid from nematode (Laxus oneistus). The anti-HIV nonpeptidic lectin mimics comprise pradimicins and benanomicins. Their anti-HIV mechanisms are discussed. PMID:25569520

  14. Human postmortem brain-derived cerebrovascular smooth muscle cells express all genes of the classical complement pathway: a potential mechanism for vascular damage in cerebral amyloid angiopathy and Alzheimer's disease.

    PubMed

    Walker, Douglas G; Dalsing-Hernandez, Jessica E; Lue, Lih-Fen

    2008-04-01

    Deposition of amyloid around blood vessels, known as cerebral amyloid angiopathy (CAA), is a major pathological feature found in the majority of Alzheimer's disease (AD) cases, and activated complement fragments have been detected on CAA deposits in AD brains. In this study, we demonstrate for the first time that human cerebrovascular smooth muscle cells (HCSMC) isolated from cortical vessels derived from postmortem brains can express mRNAs for complement genes C1qB, C1r, C1s, C2, C3, C4, C5, C6, C7, C8 and C9, the components of the classical complement pathway. Secretion of the corresponding complement proteins for these genes was also demonstrated, except for C1q and C5. Of particular significance was the observation that treatment of HCSMC with aggregated amyloid beta (Abeta) 1-42 increased expression of complement C3 mRNA and increased release of C3 protein. Abeta treatment of HCSMC also increased expression of C6 mRNA. Interferon-gamma induced expression and release of complement C1r, C1s, C2 and C4. As HCSMC are closely associated with Abeta deposits in vessels in the brain, their production of complement proteins could amplify the proinflammatory effects of amyloid in the perivascular environment, further compromising brain vascular integrity. PMID:18048067

  15. Complement Activation by Merozoite Antigens of Plasmodium falciparum

    PubMed Central

    Korir, Jackson C.; Nyakoe, Nancy K.; Awinda, George; Waitumbi, John N.

    2014-01-01

    Background Complement (C) is a crucial part of the innate immune system and becomes over activated during malaria, resulting in depletion of C components, especially those for lectin pathway (LP), thereby compromising the host's innate defense. In this study, involvement of P. falciparum antigens in C activation was investigated. Methods A highly synchronous culture of the Dd2 clone of P. falciparum was established in a serum free medium. Supernatants harvested from rings, trophozoites and schizonts at various parasite densities were tested for ability to activate C by quantifying amount of C3b deposited on erythrocytes (E). Uninfected sham culture was used as control. Remnants of each C pathway were determined using Wieslab complement System Screenkit (Euro-diagnostica, Sweden). To identify MBL binding antigens of LP, culture supernatants were added to MBL sepharose columns and trapped antigens eluted with increasing concentrations of EDTA (10 mM, 50 mM and 100 mM) and then desalted before being tested for ability to activate C. The EDTA eluate with highest activity was run on a polyacrylamide gel and silver stained proteins analyzed by mass spectroscopy. Results Antigens released by P. falciparum growing in culture activated C leading to C3b deposition on E. Maximal activation at 7% parasitemia was associated with schizont stage (36.7%) compared to 22% for rings, 21% for trophozoites and 3% for sham culture. All the three pathways of C were activated, with highest activation being for the alternative pathway (only 6% of C activation potential remained), 65% for classiical and 43% for the LP. Seven MBL binding merozoite proteins were identified by mass spectrometry in the 50 mM EDTA eluate. Conclusions MBL binding merozoite adhesins with ability to activate C pathway were identified. The survival advantage for such pronounced C activation is unclear, but opsonisation could facilitate recognition and invasion of E. PMID:25144772

  16. A profile of protein-protein interaction: Crystal structure of a lectin-lectin complex.

    PubMed

    Surya, Sukumaran; Abhilash, Joseph; Geethanandan, Krishnan; Sadasivan, Chittalakkottu; Haridas, Madhathilkovilakathu

    2016-06-01

    Proteins may utilize complex networks of interactions to create/proceed signaling pathways of highly adaptive responses such as programmed cell death. Direct binary interactions study of proteins may help propose models for protein-protein interaction. Towards this goal we applied a combination of thermodynamic kinetics and crystal structure analyses to elucidate the complexity and diversity in such interactions. By determining the heat change on the association of two galactose-specific legume lectins from Butea monosperma (BML) and Spatholobus parviflorus (SPL) belonging to Fabaceae family helped to compute the binding equilibrium. It was extended further by X-ray structural analysis of BML-SPL binary complex. In order to chart the proteins interacting mainly through their interfaces, identification of the nature of forces which stabilized the association of the lectin-lectin complex was examined. Comprehensive analysis of the BMLSPL complex by isothermal titration calorimetry and X-ray crystal structure threw new light on the lectin-lectin interactions suggesting of their use in diverse areas of glycobiology. PMID:26945504

  17. Lectins: getting familiar with translators of the sugar code.

    PubMed

    André, Sabine; Kaltner, Herbert; Manning, Joachim C; Murphy, Paul V; Gabius, Hans-Joachim

    2015-01-01

    The view on the significance of the presence of glycans in glycoconjugates is undergoing a paradigmatic change. Initially mostly considered to be rather inert and passive, the concept of the sugar code identifies glycans as highly versatile platform to store information. Their chemical properties endow carbohydrates to form oligomers with unsurpassed structural variability. Owing to their capacity to engage in hydrogen (and coordination) bonding and C-H/π-interactions these "code words" can be "read" (in Latin, legere) by specific receptors. A distinct class of carbohydrate-binding proteins are the lectins. More than a dozen protein folds have developed carbohydrate-binding capacity in vertebrates. Taking galectins as an example, distinct expression patterns are traced. The availability of labeled endogenous lectins facilitates monitoring of tissue reactivity, extending the scope of lectin histochemistry beyond that which traditionally involved plant lectins. Presentation of glycan and its cognate lectin can be orchestrated, making a glycan-based effector pathway in growth control of tumor and activated T cells possible. In order to unravel the structural basis of lectin specificity for particular glycoconjugates mimetics of branched glycans and programmable models of cell surfaces are being developed by strategic combination of lectin research with synthetic and supramolecular chemistry. PMID:25621423

  18. Role of complement and perspectives for intervention in ischemia-reperfusion damage.

    PubMed

    Banz, Yara; Rieben, Robert

    2012-05-01

    Reperfusion of an organ following prolonged ischemia instigates the pro-inflammatory and pro-coagulant response of ischemia / reperfusion (IR) injury. IR injury is a wide-spread pathology, observed in many clinically relevant situations, including myocardial infarction, stroke, organ transplantation, sepsis and shock, and cardiovascular surgery on cardiopulmonary bypass. Activation of the classical, alternative, and lectin complement pathways and the generation of the anaphylatoxins C3a and C5a lead to recruitment of polymorphonuclear leukocytes, generation of radical oxygen species, up-regulation of adhesion molecules on the endothelium and platelets, and induction of cytokine release. Generalized or pathway-specific complement inhibition using protein-based drugs or low-molecular-weight inhibitors has been shown to significantly reduce tissue injury and improve outcome in numerous in-vitro, ex-vivo, and in-vivo models. Despite the obvious benefits in experimental research, only few complement inhibitors, including C1-esterase inhibitor, anti-C5 antibody, and soluble complement receptor 1, have made it into clinical trials of IR injury. The results are mixed, and the next objectives should be to combine knowledge and experience obtained in the past from animal models and channel future work to translate this into clinical trials in surgical and interventional reperfusion therapy as well as organ transplantation. PMID:21254897

  19. Cyborg lectins: novel leguminous lectins with unique specificities.

    PubMed

    Yamamoto, K; Maruyama, I N; Osawa, T

    2000-01-01

    Bauhinia purpurea lectin (BPA) is one of the beta-galactose-binding leguminous lectins. Leguminous lectins contain a long metal-binding loop, part of which determines their carbohydrate-binding specificities. Random mutations were introduced into a portion of the cDNA coding BPA that corresponds to the carbohydrate-binding loop of the lectin. An library of the mutant lectin expressed on the surface of lambda foo phages was screened by the panning method. Several phage clones with an affinity for mannose or N-acetylglucosamine were isolated. These results indicate the possibility of making artificial lectins (so-called "cyborg lectins") with distinct and desired carbohydrate-binding specificities. PMID:10731676

  20. Complement activation during cardiopulmonary bypass.

    PubMed

    Kutsal, A; Ersoy, U; Ersoy, F; Yeniay, I; Bakkaloglu, A; Bozer, A Y

    1989-01-01

    We researched complement activation of fifteen patients who had open heart surgery and on ten patients who had closed heart surgery. Our results showed that the complement system was partially activated by the anaesthesia and partially by tissue damage. This activation was aggravated when plasma contacted the pump-oxygenator system, continued in the intensive-care unit and became normal in the 24th hour after the operation. Complement activation occurred both via the alternative and classical pathways but the alternative pathway was activated more than the classical with increase in bypass time. Pulmonary sequestration of leucocytes which occurred due to the complement activation and the complement derived inflammatory mediators could have contributed to the pathogenesis of the post-pump syndrome. PMID:2745519

  1. Banana lectin: a brief review.

    PubMed

    Singh, Senjam Sunil; Devi, Sanjenbam Kunjeshwori; Ng, Tzi Bun

    2014-01-01

    Lectins are a group of proteins of non-immune origin that recognize and bind to carbohydrates without modifying them. Banana is the common name for both herbaceous plants of the genus Musa and for the fruit they produce. They are indeed a promising source for many medicinal applications. Banana lectins have the potential for inhibiting HIV-1 reverse transcriptase activity, suppressing cancer cell proliferation and stimulating macrophage activities. Nevertheless, compared to other plant lectins, there is relatively little information in the literature on banana lectins, particularly with respect to their structure and biological functions. Herein we focus our review on the structure, functions and exploitable properties of banana lectins. PMID:25407720

  2. The Role of Complement in Cnidarian-Dinoflagellate Symbiosis and Immune Challenge in the Sea Anemone Aiptasia pallida

    PubMed Central

    Poole, Angela Z.; Kitchen, Sheila A.; Weis, Virginia M.

    2016-01-01

    The complement system is an innate immune pathway that in vertebrates, is responsible for initial recognition and ultimately phagocytosis and destruction of microbes. Several complement molecules including C3, Factor B, and mannose binding lectin associated serine proteases (MASP) have been characterized in invertebrates and while most studies have focused on their conserved role in defense against pathogens, little is known about their role in managing beneficial microbes. The purpose of this study was to (1) characterize complement pathway genes in the symbiotic sea anemone Aiptasia pallida, (2) investigate the evolution of complement genes in invertebrates, and (3) examine the potential dual role of complement genes Factor B and MASP in the onset and maintenance of cnidarian-dinoflagellate symbiosis and immune challenge using qPCR based studies. The results demonstrate that A. pallida has multiple Factor B genes (Ap_Bf-1, Ap_Bf-2a, and Ap_Bf-2b) and one MASP gene (Ap_MASP). Phylogenetic analysis indicates that the evolutionary history of complement genes is complex, and there have been many gene duplications or gene loss events, even within members of the same phylum. Gene expression analyses revealed a potential role for complement in both onset and maintenance of cnidarian-dinoflagellate symbiosis and immune challenge. Specifically, Ap_Bf-1 and Ap_MASP are significantly upregulated in the light at the onset of symbiosis and in response to challenge with the pathogen Serratia marcescens suggesting that they play a role in the initial recognition of both beneficial and harmful microbes. Ap_Bf-2b in contrast, was generally downregulated during the onset and maintenance of symbiosis and in response to challenge with S. marcescens. Therefore, the exact role of Ap_Bf-2b in response to microbes remains unclear, but the results suggest that the presence of microbes leads to repressed expression. Together, these results indicate functional divergence between Ap_Bf-1 and Ap_Bf-2b, and that Ap_Bf-1 and Ap_MASP may be functioning together in an ancestral hybrid of the lectin and alternative complement pathways. Overall, this study provides information on the role of the complement system in a basal metazoan and its role in host-microbe interactions. PMID:27148208

  3. The Role of Complement in Cnidarian-Dinoflagellate Symbiosis and Immune Challenge in the Sea Anemone Aiptasia pallida.

    PubMed

    Poole, Angela Z; Kitchen, Sheila A; Weis, Virginia M

    2016-01-01

    The complement system is an innate immune pathway that in vertebrates, is responsible for initial recognition and ultimately phagocytosis and destruction of microbes. Several complement molecules including C3, Factor B, and mannose binding lectin associated serine proteases (MASP) have been characterized in invertebrates and while most studies have focused on their conserved role in defense against pathogens, little is known about their role in managing beneficial microbes. The purpose of this study was to (1) characterize complement pathway genes in the symbiotic sea anemone Aiptasia pallida, (2) investigate the evolution of complement genes in invertebrates, and (3) examine the potential dual role of complement genes Factor B and MASP in the onset and maintenance of cnidarian-dinoflagellate symbiosis and immune challenge using qPCR based studies. The results demonstrate that A. pallida has multiple Factor B genes (Ap_Bf-1, Ap_Bf-2a, and Ap_Bf-2b) and one MASP gene (Ap_MASP). Phylogenetic analysis indicates that the evolutionary history of complement genes is complex, and there have been many gene duplications or gene loss events, even within members of the same phylum. Gene expression analyses revealed a potential role for complement in both onset and maintenance of cnidarian-dinoflagellate symbiosis and immune challenge. Specifically, Ap_Bf-1 and Ap_MASP are significantly upregulated in the light at the onset of symbiosis and in response to challenge with the pathogen Serratia marcescens suggesting that they play a role in the initial recognition of both beneficial and harmful microbes. Ap_Bf-2b in contrast, was generally downregulated during the onset and maintenance of symbiosis and in response to challenge with S. marcescens. Therefore, the exact role of Ap_Bf-2b in response to microbes remains unclear, but the results suggest that the presence of microbes leads to repressed expression. Together, these results indicate functional divergence between Ap_Bf-1 and Ap_Bf-2b, and that Ap_Bf-1 and Ap_MASP may be functioning together in an ancestral hybrid of the lectin and alternative complement pathways. Overall, this study provides information on the role of the complement system in a basal metazoan and its role in host-microbe interactions. PMID:27148208

  4. Microencapsulation of lectin anti-cancer agent and controlled release by alginate beads, biosafety approach.

    PubMed

    El-Aassar, M R; Hafez, Elsayed E; El-Deeb, Nehal M; Fouda, Moustafa M G

    2014-08-01

    Hepatocellular carcinoma (HCC) is considered as one of the most aggressive cancer worldwide. In Egypt, the prevalence of HCC is increasing during last years. Recently, drug-loaded microparticles were used to improve the efficiency of various medical treatments. This study is designed to evaluate the anticancer potentialities of lectins against HCC while hinting to its safety usage. The aim is also extended to encapsulate lectins in alginate microbeads for oral drug delivery purposes. The extracted lectins showed anti-proliferative effect against HCC with a percentage of 60.76% by using its nontoxic dose with an up-regulation of P53 gene expression. Concerning the handling of lectin alginate microbeads for oral drug delivery, the prepared lectin alginate beads were ∼100μm in diameter. The efficiency of the microcapsules was checked by scanning electron microscopy, the SEM showed the change on the alginate beads surface revealing the successful lectin encapsulation. The release of lectins from the microbeads depended on a variety of factors as the microbeads forming carriers and the amount-encapsulated lectins. The Pisum sativum extracted lectins may be considered as a promising agent in controlling HCC and this solid dosage form could be suitable for oral administration complemented with/or without the standard HCC drugs. PMID:24857870

  5. Lectin-Dependent Enhancement of Ebola Virus Infection via Soluble and Transmembrane C-type Lectin Receptors

    PubMed Central

    Lear, Calli; Chen, Li; Yantosca, L. Michael; Scully, Corinne; Sarraju, Ashish; Sokolovska, Anna; Zariffard, M. Reza; Eisen, Damon P.; Mungall, Bruce A.; Kotton, Darrell N.; Omari, Amel; Huang, I-Chueh; Farzan, Michael; Takahashi, Kazue; Stuart, Lynda; Stahl, Gregory L.; Ezekowitz, Alan B.; Spear, Gregory T.; Olinger, Gene G.; Schmidt, Emmett V.; Michelow, Ian C.

    2013-01-01

    Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes. PMID:23573288

  6. A Serine Protease Isolated from the Bristles of the Amazonic Caterpillar, Premolis semirufa, Is a Potent Complement System Activator

    PubMed Central

    Villas Boas, Isadora Maria; Pidde-Queiroz, Giselle; Magnoli, Fabio Carlos; Gonçalves-de-Andrade, Rute M.; van den Berg, Carmen W.; Tambourgi, Denise V.

    2015-01-01

    Background The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called “Pararamose”, characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa’s bristles extract could interfere with the human complement system. Results The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity. Conclusion These data show that a serine protease present in the Premolis semirufa’s bristles extract has the ability to activate the complement system, which may contribute to the inflammatory process presented in humans after envenomation. PMID:25760458

  7. Role of complement in a murine model of peanut-induced anaphylaxis.

    PubMed

    Kodama, Toshihisa; Sekine, Hideharu; Takahashi, Minoru; Iwaki, Daisuke; Machida, Takeshi; Kanno, Kazuko; Ishida, Yumi; Endo, Yuichi; Fujita, Teizo

    2013-06-01

    Peanut allergy is severe and persisting from childhood to adulthood. However, there is no effective prophylaxis or treatment for peanut allergy. Little is known to about the molecular process in the pathogenesis of peanuts allergy, especially in innate immunity. Thus we investigated the role of complement activation in murine peanut anaphylaxis. Complement component C3 deposition on peanut extract (PE) was evaluated using sera from wild-type (WT), mannose-binding lectin associated serine protease (MASP)-1/3 deficient, MASP-2 deficient, and C4 deficient mice. Sera from interferon regulatory factor-4 (IRF-4) deficient mice, which lack serum immunoglobulin, were also used. In anaphylaxis study, mice were pretreated with propranolol and a long-acting form of IL-4, and injected with PE. Mice were then assessed for plasma C3a levels and hypothermia shock by ELISA and rectal temperature measurement, respectively. C3 deposition on PE was abolished in immunoglobulin- and C4-deficient sera. No difference in C3 deposition levels were observed among WT, MASP-1/3 deficient and MASP-2 deficient sera. IgM, IgG2b, IgG3, C1q, and ficolin-A deposits were detected on PE. In anaphylaxis study, MASP-1/3 deficient mice showed elevation of plasma C3a levels similar to WT mice. However, they were significantly reduced in C4- and MASP-2-deficient mice compared to WT mice. Consistently, PE-induced anaphylactic shock was prevented in C4 deficient mice and partially in MASP-2 deficient mice. In conclusion, PE activates complement via both the lectin and classical pathways in vivo, and the complement activation contributes to hypothermia shock in mice. PMID:23182714

  8. Model-Driven Redox Pathway Manipulation for Improved Isobutanol Production in Bacillus subtilis Complemented with Experimental Validation and Metabolic Profiling Analysis

    PubMed Central

    Qi, Haishan; Li, Shanshan; Zhao, Sumin; Huang, Di; Xia, Menglei; Wen, Jianping

    2014-01-01

    To rationally guide the improvement of isobutanol production, metabolic network and metabolic profiling analysis were performed to provide global and profound insights into cell metabolism of isobutanol-producing Bacillus subtilis. The metabolic flux distribution of strains with different isobutanol production capacity (BSUL03, BSUL04 and BSUL05) drops a hint of the importance of NADPH on isobutanol biosynthesis. Therefore, the redox pathways were redesigned in this study. To increase NADPH concentration, glucose-6-phosphate isomerase was inactivated (BSUL06) and glucose-6-phosphate dehydrogenase was overexpressed (BSUL07) successively. As expected, NADPH pool size in BSUL07 was 4.4-fold higher than that in parental strain BSUL05. However, cell growth, isobutanol yield and production were decreased by 46%, 22%, and 80%, respectively. Metabolic profiling analysis suggested that the severely imbalanced redox status might be the primary reason. To solve this problem, gene udhA of Escherichia coli encoding transhydrogenase was further overexpressed (BSUL08), which not only well balanced the cellular ratio of NAD(P)H/NAD(P)+, but also increased NADH and ATP concentration. In addition, a straightforward engineering approach for improving NADPH concentrations was employed in BSUL05 by overexpressing exogenous gene pntAB and obtained BSUL09. The performance for isobutanol production by BSUL09 was poorer than BSUL08 but better than other engineered strains. Furthermore, in fed-batch fermentation the isobutanol production and yield of BSUL08 increased by 11% and 19%, up to the value of 6.12 g/L and 0.37 C-mol isobutanol/C-mol glucose (63% of the theoretical value), respectively, compared with parental strain BSUL05. These results demonstrated that model-driven complemented with metabolic profiling analysis could serve as a useful approach in the strain improvement for higher bio-productivity in further application. PMID:24705866

  9. Mannose-Binding Lectin Gene Polymorphism and Chronic Hepatitis B Infection in Children

    PubMed Central

    Erdemir, Gulin; Ozkan, Tanju B.; Ozgur, Taner; Budak, Ferah; Kilic, Sara S.; Onay, Huseyin

    2015-01-01

    Background/Aims: Mannose-binding lectin (MBL) is a member of innate immune system that activates complement system through lectin pathway. MBL deficiency is associated with susceptibility to infectious diseases. In this study, the relation between MBL gene polymorphism and chronic hepatitis B infection in children is evaluated. Patients and Methods: The study included 67 children with chronic hepatitis B and 99 healthy controls. The hepatitis B patients were divided into immuntolerant, chronic inactive, and treatment groups according to their laboratory findings. MBL gene codon 52, 54, and 57 polymorphisms were studied with polymerase chain reaction in all patients and controls. The associations of MBL gene polymorphism with clinical, laboratory, and histopathologic findings were evaluated. Results: Homozygous codon 54 polymorphism of MBL was found significantly higher in chronic hepatitis B patients than controls. Rate of the polymorphism was similar in all groups and, responsive and nonresponsive patients in the treatment group. Conclusions: The hepatitis B patients who are homozygous for codon 54 of MBL are prone to develop chronic infection. Longitudinal studies with larger groups are needed. PMID:25843194

  10. Complement, interferon and lupus.

    PubMed

    Elkon, Keith B; Santer, Deanna M

    2012-12-01

    The complement pathway was implicated in the immunopathogenesis of lupus and other autoimmune disorders decades ago. The apparent paradox that early complement component (C1q, C2 and C4) deficiencies predispose to lupus has been explained by the beneficial roles of these proteins in promoting the clearance of immune complexes (ICs) and apoptotic cells. Recent findings demonstrate that, in the absence of C1q, instead of ICs binding to monocytes, they preferentially engage plasmacytoid dendritic cells (pDC) so generating interferon (IFN) alpha, the cytokine with potent immune adjuvant properties. C1q opsonized apoptotic cells also exert an immunosuppressive effect through cytokine regulation and the stimulation of additional opsonins by macrophages. C1q was recently reported to impede neutrophil extracellular trap (NET) degradation. NETs are known to promote type I IFN production in SLE by providing a source of antigen for the formation of ICs as well as through direct pDC activation by cathelicidin (LL37). Together, these findings provide both direct and indirect links between two key pathways implicated in lupus pathogenesis: complement and IFN. PMID:22999705

  11. Specificity analysis of lectins and antibodies using remodeled glycoproteins.

    PubMed

    Iskratsch, Thomas; Braun, Andreas; Paschinger, Katharina; Wilson, Iain B H

    2009-03-15

    Due to their ability to bind specifically to certain carbohydrate sequences, lectins are a frequently used tool in cytology, histology, and glycan analysis but also offer new options for drug targeting and drug delivery systems. For these and other potential applications, it is necessary to be certain as to the carbohydrate structures interacting with the lectin. Therefore, we used glycoproteins remodeled with glycosyltransferases and glycosidases for testing specificities of lectins from Aleuria aurantia (AAL), Erythrina cristagalli (ECL), Griffonia simplicifolia (GSL I-B(4)), Helix pomatia agglutinin (HPA), Lens culinaris (LCA), Lotus tetragonolobus (LTA), peanut (Arachis hypogaeae) (PNA), Ricinus communis (RCA I), Sambucus nigra (SNA), Vicia villosa (VVA), and wheat germ (Triticum vulgaris) (WGA) as well as reactivities of anti-carbohydrate antibodies (anti-bee venom, anti-horseradish peroxidase [anti-HRP], and anti-Lewis(x)). After enzymatic remodeling, the resulting neoglycoforms display defined carbohydrate sequences and can be used, when spotted on nitrocellulose or in enzyme-linked lectinosorbent assays, to identify the sugar moieties bound by the lectins. Transferrin with its two biantennary complex N-glycans was used as scaffold for gaining diverse N-glycosidic structures, whereas fetuin was modified using glycosidases to test the specificities of lectins toward both N- and O-glycans. In addition, alpha(1)-acid glycoprotein and Schistosoma mansoni egg extract were chosen as controls for lectin interactions with fucosylated glycans (Lewis(x) and core alpha1,3-fucose). Our data complement and expand the existing knowledge about the binding specificity of a range of commercially available lectins. PMID:19123999

  12. Lectins in human pathogenic fungi.

    PubMed

    Gallegos, Belém; Martínez, Ruth; Pérez, Laura; Del Socorro Pina, María; Perez, Eduardo; Hernández, Pedro

    2014-01-01

    Lectins are carbohydrate-binding proteins widely distributed in nature. They constitute a highly diverse group of proteins consisting of many different protein families that are, in general, structurally unrelated. In the last few years, mushroom and other fungal lectins have attracted wide attention due to their antitumour, antiproliferative and immunomodulatory activities. The present mini-review provides concise information about recent developments in understanding lectins from human pathogenic fungi. A bibliographic search was performed in the Science Direct and PubMed databases, using the following keywords "lectin", "fungi", "human" and "pathogenic". Lectins present in fungi have been classified; however, the role played by lectins derived from human pathogenic fungi in infectious processes remains uncertain; thus, this is a scientific field requiring more research. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012). PMID:24270074

  13. Complement and systemic lupus erythematosus

    PubMed Central

    2002-01-01

    Chapter summary Complement is implicated in the pathogenesis of systemic lupus erythematosus (SLE) in several ways and may act as both friend and foe. Homozygous deficiency of any of the proteins of the classical pathway is causally associated with susceptibility to the development of SLE, especially deficiency of the earliest proteins of the activation pathway. However, complement is also implicated in the effector inflammatory phase of the autoimmune response that characterizes the disease. Complement proteins are deposited in inflamed tissues and, in experimental models, inhibition of C5 ameliorates disease in a murine model. As a further twist to the associations between the complement system and SLE, autoantibodies to some complement proteins, especially to C1q, develop as part of the autoantibody response. The presence of anti-C1q autoantibodies is associated with severe illness, including glomerulonephritis. In this chapter the role of the complement system in SLE is reviewed and hypotheses are advanced to explain the complex relationships between complement and lupus. PMID:12110148

  14. Insights into the Effects of Complement Factor H on the Assembly and Decay of the Alternative Pathway C3 Proconvertase and C3 Convertase.

    PubMed

    Bettoni, Serena; Bresin, Elena; Remuzzi, Giuseppe; Noris, Marina; Donadelli, Roberta

    2016-04-01

    The activated fragment of C3 (C3b) and factor B form the C3 proconvertase (C3bB), which is cleaved by factor D to C3 convertase (C3bBb). Older studies (Conrad, D. H., Carlo, J. R., and Ruddy, S. (1978)J. Exp. Med.147, 1792-1805; Pangburn, M. K., and Müller-Eberhard, H. J. (1978)Proc. Natl. Acad. Sci. U.S.A.75, 2416-2420; Kazatchkine, M. D., Fearon, D. T., and Austen, K. F. (1979)J. Immunol.122, 75-81) indicated that the complement alternative pathway regulator factor H (FH) competes with factor B for C3b binding; however, the capability of FH to prevent C3bB assembly has not been formally investigated. Moreover, in the few published studies FH did not favor C3bB dissociation. Whether FH may affect C3bBb formation from C3bB is unknown. We set up user-friendly assays based on combined microplate/Western blotting techniques that specifically detect either C3bB or C3bBb, with the aim of investigating the effect of FH on C3bB assembly and decay and C3bBb formation and decay. We document that FH does not affect C3bB assembly, indicating that FH does not efficiently compete with factor B for C3b binding. We also found that FH does not dissociate C3bB. FH showed a strong C3bBb decay-accelerating activity, as reported previously, and also exerted an apparent inhibitory effect on C3bBb formation. The latter effect was not fully attributable to a rapid FH-mediated dissociation of C3bBb complexes, because blocking decay with properdin and C3 nephritic factor did not restore C3bBb formation. FH almost completely prevented release of the smaller cleavage subunit of FB (Ba), without modifying the amount of C3bB complexes, suggesting that FH inhibits the conversion of C3bB to C3bBb. Thus, the inhibitory effect of FH on C3bBb formation is likely the sum of inhibition of C3bB conversion to C3bBb and of C3bBb decay acceleration. Further studies are required to confirm these findings in physiological cell-based settings. PMID:26903516

  15. Mannose-Binding Lectin Inhibits the Motility of Pathogenic Salmonella by Affecting the Driving Forces of Motility and the Chemotactic Response

    PubMed Central

    Nakamura, Shuichi; Islam, Md. Shafiqul; Guo, Yijie; Ihara, Kohei; Tomioka, Rintaro; Masuda, Mizuki; Yoneyama, Hiroshi; Isogai, Emiko

    2016-01-01

    Mannose-binding lectin (MBL) is a key pattern recognition molecule in the lectin pathway of the complement system, an important component of innate immunity. MBL functions as an opsonin which enhances the sequential immune process such as phagocytosis. We here report an inhibitory effect of MBL on the motility of pathogenic bacteria, which occurs by affecting the energy source required for motility and the signaling pathway of chemotaxis. When Salmonella cells were treated with a physiological concentration of MBL, their motile fraction and free-swimming speed decreased. Rotation assays of a single flagellum showed that the flagellar rotation rate was significantly reduced by the addition of MBL. Measurements of the intracellular pH and membrane potential revealed that MBL affected a driving force for the Salmonella flagellum, the electrochemical potential difference of protons. We also found that MBL treatment increased the reversal frequency of Salmonella flagellar rotation, which interfered with the relative positive chemotaxis toward an attractive substrate. We thus propose that the motility inhibition effect of MBL may be secondarily involved in the attack against pathogens, potentially facilitating the primary role of MBL in the complement system. PMID:27104738

  16. The Serine Protease Pic From Enteroaggregative Escherichia coli Mediates Immune Evasion by the Direct Cleavage of Complement Proteins.

    PubMed

    Abreu, Afonso G; Fraga, Tatiana R; Granados Martínez, Adriana P; Kondo, Marcia Y; Juliano, Maria A; Juliano, Luiz; Navarro-Garcia, Fernando; Isaac, Lourdes; Barbosa, Angela S; Elias, Waldir P

    2015-07-01

    Enteroaggregative and uropathogenic Escherichia coli, Shigella flexneri 2a, and the hybrid enteroaggregative/Shiga toxin-producing E. coli strain (O104:H4) are important pathogens responsible for intestinal and urinary tract infections, as well as sepsis and hemolytic uremic syndrome. They have in common the production of a serine protease called Pic. Several biological roles for Pic have been described, including protection of E. coli DH5α from complement-mediated killing. Hereby we showed that Pic significantly reduces complement activation by all 3 pathways. Pic cleaves purified C3/C3b and other proteins from the classic and lectin pathways, such as C4 and C2. Cleavage fragments of C3, C4, and C2 were also observed with HB101(pPic1) culture supernatants, and C3 cleavage sites were mapped by fluorescence resonance energy transfer peptides. Experiments using human serum as a source of complement proteins confirmed Pic proteolytic activity on these proteins. Furthermore, Pic works synergistically with the human complement regulators factor I and factor H, promoting inactivation of C3b. In the presence of both regulators, further degradation of C3 α' chain was observed. Therefore, Pic may contribute to immune evasion of E. coli and S. flexneri, favoring invasiveness and increasing the severity of the disorders caused by these pathogens. PMID:25583166

  17. Serum levels, ontogeny and heritability of chicken mannan-binding lectin (MBL).

    PubMed Central

    Laursen, S B; Hedemand, J E; Nielsen, O L; Thiel, S; Koch, C; Jensenius, J C

    1998-01-01

    Mannan-binding lectin (MBL) is a serum lectin found in mammals and recently also in birds. It is thought to play an important role in the innate immune defence through binding to surface carbohydrates on micro-organisms followed by complement activation via the MBL pathway. This results in opsonization or direct complement-mediated killing. To gain further knowledge about the physiology and function of the protein, we developed an enzyme-linked immunosorbent assay for chicken MBL and used this to investigate the level of MBL in different chicken strains during embryogenesis, early and adult life. The MBL concentrations in 308 chickens, representing 14 different strains, showed a non-Gaussian, unimodal distribution profile with a mean concentration of 5.8 micrograms/ml (range 0.4-37.8 micrograms/ml). No difference between the strains could be demonstrated and no chickens were found deficient in MBL. Ontogenetic studies showed that MBL is already detectable in embryos at a gestational age of 10 days (11 days before hatching). At hatching, the level is comparable to the level found in adult chickens. This level is fairly stable during the first weeks of life, but a deficiency state develops at 4 weeks of age, whereafter the level is normalized again at 5 weeks of age. Chickens with relatively low or high MBL levels were bred with cockerels having similar MBL levels and this resulted in F1 generations with significantly different MBL levels, suggesting that the protein level is genetically influenced. PMID:9767449

  18. Critical roles of complement and antibodies in host defense mechanisms against Neisseria meningitidis as revealed by human complement genetic deficiencies.

    PubMed

    Hellerud, Bernt Christian; Aase, Audun; Herstad, Tove Karin; Naess, Lisbeth Meyer; Kristiansen, Lisa Høyem; Trøseid, Anne-Marie Siebke; Harboe, Morten; Lappegård, Knut Tore; Brandtzaeg, Petter; Høiby, E Arne; Mollnes, Tom Eirik

    2010-02-01

    Certain complement defects are associated with an increased propensity to contract Neisseria meningitidis infections. We performed detailed analyses of complement-mediated defense mechanisms against N. meningitidis 44/76 with whole blood and serum from two adult patients who were completely C2 or C5 deficient. The C5-deficient patient and the matched control were also deficient in mannose-binding lectin (MBL). The proliferation of meningococci incubated in freshly drawn whole blood was estimated by CFU and quantitative DNA real-time PCR. The serum bactericidal activity and opsonophagocytic activity by granulocytes were investigated, including heat-inactivated postvaccination sera, to examine the influence of antimeningococcal antibodies. The meningococci proliferated equally in C2- and C5-deficient blood, with a 2 log(10) increase of CFU and 4- to 5-log(10) increase in DNA copies. Proliferation was modestly decreased in reconstituted C2-deficient and control blood. After reconstitution of C5-deficient blood, all meningococci were killed, which is consistent with high antibody titers being present. The opsonophagocytic activity was strictly C2 dependent, appeared with normal serum, and increased with postvaccination serum. Serum bactericidal activity was strictly dependent on C2, C5, and high antibody titers. MBL did not influence any of the parameters observed. Complement-mediated defense against meningococci was thus dependent on the classical pathway. Some opsonophagocytic activity occurred despite low levels of antimeningococcal antibodies but was more efficient with immune sera. Serum bactericidal activity was dependent on C2, C5, and immune sera. MBL did not influence any of the parameters observed. PMID:19933829

  19. THE USE OF SELECTIVE PHARMACOLOGICAL INHIBITORS TO DELINEATE SIGNAL TRANSDUCTION PATHWAYS ACTIVATED DURING COMPLEMENT RECEPTOR-MEDIATED DEGRANULATION IN CHICKEN HETEROPHILS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Complement receptors (CR), along with Fc receptors, play a primary role in the removal of bacterial pathogens in poultry. The binding of serum-opsonized bacteria to CR results in the secretion of both toxic oxygen metabolites and anti-bacterial granules. We have previously shown that the stimulati...

  20. Characterization and expression analysis of a complement component gene in sea cucumber ( Apostichopus japonicus)

    NASA Astrophysics Data System (ADS)

    Chen, Zhong; Zhou, Zunchun; Yang, Aifu; Dong, Ying; Guan, Xiaoyan; Jiang, Bei; Wang, Bai

    2015-12-01

    The complement system plays a crucial role in the innate immune system of animals. It can be activated by distinct yet overlapping classical, alternative and lectin pathways. In the alternative pathway, complement factor B (Bf) serves as the catalytic subunit of complement component 3 (C3) convertase, which plays the central role among three activation pathways. In this study, the Bf gene in sea cucumber ( Apostichopus japonicus), termed AjBf, was obtained by rapid amplification of cDNA ends (RACE). The full-length cDNA of AjBf was 3231 bp in length barring the poly (A) tail. It contained an open reading frame (ORF) of 2742 bp encoding 913 amino acids, a 105 bp 5'-UTR (5'-terminal untranslated region) and a 384 bp 3'-UTR. AjBf was a mosaic protein with six CCP (complement control protein) domains, a VWA (von Willebrand factor A) domain, and a serine protease domain. The deduced molecular weight of AjBf protein was 101 kDa. Quantitative real time PCR (qRT-PCR) analysis indicated that the expression level of AjBf in A. japonicus was obviously higher at larval stage than that at embryonic stage. Expression detection in different tissues showed that AjBf expressed higher in coelomocytes than in other four tissues. In addation, AjBf expression in different tissues was induced significantly after LPS or PolyI:C challenge. These results indicated that AjBf plays an important role in immune responses to pathogen infection.

  1. The outer membrane protease PgtE of Salmonella enterica interferes with the alternative complement pathway by cleaving factors B and H

    PubMed Central

    Riva, Rauna; Korhonen, Timo K.; Meri, Seppo

    2015-01-01

    The virulence factor PgtE is an outer membrane protease (omptin) of the zoonotic pathogen Salmonella enterica that causes diseases ranging from gastroenteritis to severe enteric fever. It is surface exposed in bacteria that have a short-chain, i.e., rough LPS, as observed e.g., in bacteria residing inside macrophages or just emerging from them. We investigated whether PgtE cleaves the complement factors B (B) and H (H), key proteins controlling formation and inactivation of the complement protein C3b and thereby the activity of the complement system. S. enterica serovar Typhimurium or omptin-expressing recombinant E. coli bacteria were incubated with purified human complement proteins or recombinant H fragments. PgtE cleaved both B and H, whereas its close homolog Pla of Yersinia pestis cleaved only H. H was cleaved at both N- and C-termini, while the central region resisted proteolysis. Because of multiple effects of PgtE on complement components (cleavage of C3, C3b, B, and H) we assessed its effect on the opsonophagocytosis of Salmonella. In human serum, C3 cleavage was dependent on proteolytically active PgtE. Human neutrophils interacted less with serum-opsonized FITC-stained S. enterica 14028R than with the isogenic ΔpgtE strain, as analyzed by flow cytometry. In conclusion, cleavage of B and H by PgtE, together with C3 cleavage, affects the C3-mediated recognition of S. enterica by human neutrophils, thus thwarting the immune protection against Salmonella. PMID:25705210

  2. Functional Characterization of LcpA, a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP▿

    PubMed Central

    Barbosa, Angela S.; Monaris, Denize; Silva, Ludmila B.; Morais, Zenaide M.; Vasconcellos, Sílvio A.; Cianciarullo, Aurora M.; Isaac, Lourdes; Abreu, Patricia A. E.

    2010-01-01

    We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis. PMID:20404075

  3. The mechanisms of complement activation in normal bovine serum and normal horse serum against Yersinia enterocolitica O:9 strains with different outer membrane proteins content.

    PubMed

    Miętka, K; Brzostek, K; Guz-Regner, K; Bugla-Płoskońska, G

    2016-01-01

    Yersinia enterocolitica is a common zoonotic pathogen and facultative intracellular bacterium which can survive within blood cells. Cattle and horses are considered a reservoir of Y. enterocolitica which often causes several serious syndromes associated with yersiniosis such as abortions, premature births or infertility. The aim of our investigation was to determine the vitality of Y. enterocolitica O:9 strains (Ye9) in bovine and horse sera (NBS and NHrS) and explain the role of outer membrane proteins (OMPs) in serum resistance of these bacteria. Our previous studies demonstrated moderate human serum (NHS) resistance of the wild type Ye9 strain, whereas mutants lacking YadA, Ail or OmpC remained sensitive to the bactericidal activity of NHS. The present study showed that the wild type of Ye9 strain was resistant to the bactericidal activity of both NHrS and NBS, while Ye9 mutants lacking the YadA, Ail and OmpC proteins were sensitive to NHrS and NBS as well as to NHS. The mechanisms of complement activation against Ye9 strains lacking Ail and YadA were distinguished, i.e. activation of the classical/lectin pathways decisive in the bactericidal mechanism of complement activation of NBS, parallel activation of the classical/lectin and alternative pathways of NHrS. In this research the mechanism of independent activation of the classical/lectin or the alternative pathway of NBS and NHrS against Ye9 lacking OmpC porin was also established. The results indicate that serum resistance of Ye9 is multifactorial, in which extracellular structures, i.e. outer membrane proteins (OMPs) such as Ail, OmpC or YadA, play the main role. PMID:27096793

  4. β-Glucan enhances complement-mediated hematopoietic recovery after bone marrow injury

    PubMed Central

    Cramer, Daniel E.; Allendorf, Daniel J.; Baran, Jarek T.; Hansen, Richard; Marroquin, Jose; Li, Bing; Ratajczak, Janina; Ratajczak, Mariusz Z.; Yan, Jun

    2006-01-01

    Myelotoxic injury in the bone marrow (BM) as a consequence of total body irradiation (TBI) or granulocyte colony-stimulating factor (G-CSF) mobilization results in the deposition of iC3b on BM stroma (stroma-iC3b). In the present study, we have examined how stroma-iC3b interacts with hematopoietic progenitor cells (HPCs) and the role of complement (C) and complement receptor 3 (CR3) in BM injury/repair. We demonstrate here that stroma-iC3b tethers HPCs via the inserted (I) domain of HPC complement receptor 3 (CR3, CD11b/CD18, Mac-1). Following irradiation, stroma-iC3b was observed in the presence of purified IgM and normal mouse serum (NMS), but not serum from Rag-2-/- mice, implicating a role for antibody (Ab) and the classic pathway of C activation. Furthermore, a novel role for soluble yeast β-glucan, a ligand for the CR3 lectin-like domain (LLD), in the priming of CR3+ HPC is suggested. Soluble yeast β-glucan could enhance the proliferation of tethered HPCs, promote leukocyte recovery following sublethal irradiation, and increase the survival of lethally irradiated animals following allogeneic HPC transplantation in a CR3-dependent manner. Taken together, these observations suggest a novel role for C, CR3, and β-glucan in the restoration of hematopoiesis following injury. (Blood. 2006;107:835-840) PMID:16179370

  5. Mannan-binding lectin: structure, oligomerization, and flexibility studied by atomic force microscopy.

    PubMed

    Jensenius, Henriette; Klein, Dionne C G; van Hecke, Martin; Oosterkamp, Tjerk H; Schmidt, Thomas; Jensenius, Jens C

    2009-08-01

    Mannan-binding lectin (MBL) is the archetypical pathogen recognition molecule of the innate immune defense. Upon binding to microorganisms, reactions leading to the destruction of the offender ensue. MBL is an oligomer of structural subunits each composed of three identical polypeptides. We used atomic force microscopy to reveal tertiary and quaternary structures of MBL. The images in both air and buffer show a quaternary structure best described as "sertiform", that is, a hub from which the subunits fan out. The dimensions conform to those calculated from primary and secondary structures. The subunits associate with a preferred angle of 40 degrees between them. This angle is stable with respect to the degree of oligomerization for MBL of four subunits or more. Due to an interruption in the collagenous sequence, the arms of the subunits are expected to form a kink. We find that approximately 30% of the subunits are kinked and the kink angle distributed, quite broadly, around 145 degrees . The conformation and flexibility of the MBL molecule that we observe differ distinctly from the popular view of a "bouquet-like" configuration as that found for related members of the complement system such as C1q. This structural information will further the understanding of the specific functioning of the MBL pathway of complement activation. PMID:19501100

  6. Use of lectins in immunohematology

    PubMed Central

    Gorakshakar, Ajit C.; Ghosh, Kanjaksha

    2016-01-01

    Lectins are carbohydrate binding proteins present in seeds of many plants, especially corals and beans, in fungi and bacteria, and in animals. Apart from their hemagglutinating property, a wide range of functions have been attributed to them. Their importance in the area of immunohematology is immense. They are used to detect specific red cell antigens, to activate different types of lymphocytes, in order to resolve problems related to polyagglutination and so on. The introduction of advanced biotechnological tools generates new opportunities to exploit the properties of lectins, which were not used earlier. Stem cell research is a very important area in transplant medicine. Certain lectins detect surface markers of stem cell. Hence, they are used to understand the developmental biology of stem cells. The role of various lectins in the areas of transfusion and transplant medicine is discussed in detail in this review. PMID:27011665

  7. Use of lectins in immunohematology.

    PubMed

    Gorakshakar, Ajit C; Ghosh, Kanjaksha

    2016-01-01

    Lectins are carbohydrate binding proteins present in seeds of many plants, especially corals and beans, in fungi and bacteria, and in animals. Apart from their hemagglutinating property, a wide range of functions have been attributed to them. Their importance in the area of immunohematology is immense. They are used to detect specific red cell antigens, to activate different types of lymphocytes, in order to resolve problems related to polyagglutination and so on. The introduction of advanced biotechnological tools generates new opportunities to exploit the properties of lectins, which were not used earlier. Stem cell research is a very important area in transplant medicine. Certain lectins detect surface markers of stem cell. Hence, they are used to understand the developmental biology of stem cells. The role of various lectins in the areas of transfusion and transplant medicine is discussed in detail in this review. PMID:27011665

  8. A review of fish lectins.

    PubMed

    Ng, Tzi Bun; Fai Cheung, Randy Chi; Wing Ng, Charlene Cheuk; Fang, Evandro Fei; Wong, Jack Ho

    2015-01-01

    Lectins have been reported from various tissues of a diversity of fish species including Japanese eel, conger eel, electric eel, bighead carp, gibel carp, grass carp, Arabian Gulf catfish, channel catfish, blue catfish, catfish, pike perch, perch, powan, zebrafish, toxic moray, cobia fish, steelhead trout, Japanese trout, Atlantic salmon, chinook salmon, olive rainbow smelt, rainbow smelt, white-spotted charr, tilapia, blue gourami, ayu, Potca fish, Spanish mackerel, gilt head bream, tench, roach, rudd, common skate, and sea lamprey. The tissues from which the lectins were isolated comprise gills, eggs, electric organ, stomach, intestine, and liver. Lectins have also been isolated from skin, mucus serum, and plasma. The lectins differ in molecular weight, number of subunits, glycosylation, sugar binding specificity and amino acid sequence. Their activities include antimicrobial, antitumor, immunoregulatory and a role in development. PMID:25929869

  9. Lectins in the investigation of receptors

    NASA Astrophysics Data System (ADS)

    Lakhtin, V. M.; Yamskov, Igor A.

    1991-08-01

    Problems of the purification and characterisation are considered for approximately 270 receptors (including cell surface and organelle enzymes), which are glycoconjugates (mainly glycoproteins) from animals, plants and microorganisms, using various lectins (mainly lectin sorbents). An analysis has been carried out of the stages of lectin affinity chromatography of receptors (choice of detergent, use of organic solvents, elution with carbohydrates, etc.). Examples are given of procedures for the purification of receptors, including the use of paired columns and combination chromatography on lectins. The possibility of separating sub-populations of receptors using lectins has been demonstrated. Examples are given of the use of lectins in the analysis of the oligosaccharide structure of receptors. Cases are recorded of the interaction of receptors with endogenous lectins and of receptor lectins with endogenous glycoconjugates. It has been shown that lectins, in combination with glycosidases and antibodies, may be useful in the investigation of receptors. The bibliography contains 406 references.

  10. Mannan-Binding Lectin Is Involved in the Protection against Renal Ischemia/Reperfusion Injury by Dietary Restriction

    PubMed Central

    Shushimita, Shushimita; van der Pol, Pieter; W.F. de Bruin, Ron; N. M. Ijzermans, Jan; van Kooten, Cees; Dor, Frank J. M. F.

    2015-01-01

    Preoperative fasting and dietary restriction offer robust protection against renal ischemia/reperfusion injury (I/RI) in mice. We recently showed that Mannan-binding lectin (MBL), the initiator of the lectin pathway of complement activation, plays a pivotal role in renal I/RI. Based on these findings, we investigated the effect of short-term DR (30% reduction of total food intake) or three days of water only fasting on MBL in 10–12 weeks old male C57/Bl6 mice. Both dietary regimens significantly reduce the circulating levels of MBL as well as its mRNA expression in liver, the sole production site of MBL. Reconstitution of MBL abolished the protection afforded by dietary restriction, whereas in the fasting group the protection persisted. These data show that modulation of MBL is involved in the protection against renal I/RI induced by dietary restriction, and suggest that the mechanisms of protection induced by dietary restriction and fasting may be different. PMID:26367533

  11. The novel complement inhibitor human CUB and Sushi multiple domains 1 (CSMD1) protein promotes factor I-mediated degradation of C4b and C3b and inhibits the membrane attack complex assembly.

    PubMed

    Escudero-Esparza, Astrid; Kalchishkova, Nikolina; Kurbasic, Emila; Jiang, Wen G; Blom, Anna M

    2013-12-01

    CUB and Sushi multiple domains 1 (CSMD1) is a transmembrane protein containing 15 consecutive complement control protein (CCP) domains, which are characteristic for complement inhibitors. We expressed a membrane-bound fragment of human CSMD1 composed of the 15 C-terminal CCP domains and demonstrated that it inhibits deposition of C3b by the classical pathway on the surface of Chinese hamster ovary cells by 70% at 6% serum and of C9 (component of membrane attack complex) by 90% at 1.25% serum. Furthermore, this fragment of CSMD1 served as a cofactor to factor I-mediated degradation of C3b. In all functional assays performed, well-characterized complement inhibitors were used as positive controls, whereas Coxsackie adenovirus receptor, a protein with no effect on complement, was a negative control. Moreover, attenuation of expression in human T47 breast cancer cells that express endogenous CSMD1 significantly increased C3b deposition on these cells by 45% at 8% serum compared with that for the controls. Furthermore, by expressing a soluble 17-21 CCP fragment of CSMD1, we found that CSMD1 inhibits complement by promoting factor I-mediated C4b/C3b degradation and inhibition of MAC assembly at the level of C7. Our results revealed a novel complement inhibitor for the classical and lectin pathways. PMID:23964079

  12. Nutritional complementation of oxidative glucose metabolism in Escherichia coli via pyrroloquinoline quinone-dependent glucose dehydrogenase and the Entner-Doudoroff pathway

    SciTech Connect

    Adamowicz, M.; Conway, T.; Nickerson, K.W. )

    1991-07-01

    Two glucose-negative Escherichia coli mutants (ZSC113 and DF214) were unable to grow on glucose as the sole carbon source unless supplemented with pyrroloquinoline quinone (PQQ). PQQ is the cofactor for the periplasmic enzyme glucose dehydrogenase, which converts glucose to gluconate. Aerobically, E. Coli ZSC113 grew on glucose plus PQQ with a generation time of 65 min, a generation time about the same as that for wild-type E. coli in a defined glucose-salts medium. Thus, for E. coli ZSC113 the Entner-Doudoroff pathway was fully able to replace the Embden-Meyerhof-Parnas pathway. In the presence of 5% sodium dodecyl sulfate, PQQ no longer acted as a growth factor. Sodium dodecyl sulfate inhibited the formation of gluconate from glucose but not gluconate metabolism. Adaptation to PQQ-dependent growth exhibited long lag periods, except under low-phosphate conditions, in which the PhoE porin would be expressed. The authors suggest that E. coli has maintained the apoenzyme for glucose dehydrogenase and the Entner-Doudoroff pathway as adaptations to an aerobic, low-phosphate, and low-detergent aquatic environment.

  13. Infectious diseases associated with complement deficiencies.

    PubMed Central

    Figueroa, J E; Densen, P

    1991-01-01

    The complement system consists of both plasma and membrane proteins. The former influence the inflammatory response, immune modulation, and host defense. The latter are complement receptors, which mediate the cellular effects of complement activation, and regulatory proteins, which protect host cells from complement-mediated injury. Complement activation occurs via either the classical or the alternative pathway, which converge at the level of C3 and share a sequence of terminal components. Four aspects of the complement cascade are critical to its function and regulation: (i) activation of the classical pathway, (ii) activation of the alternative pathway, (iii) C3 convertase formation and C3 deposition, and (iv) membrane attack complex assembly and insertion. In general, mechanisms evolved by pathogenic microbes to resist the effects of complement are targeted to these four steps. Because individual complement proteins subserve unique functional activities and are activated in a sequential manner, complement deficiency states are associated with predictable defects in complement-dependent functions. These deficiency states can be grouped by which of the above four mechanisms they disrupt. They are distinguished by unique epidemiologic, clinical, and microbiologic features and are most prevalent in patients with certain rheumatologic and infectious diseases. Ethnic background and the incidence of infection are important cofactors determining this prevalence. Although complement undoubtedly plays a role in host defense against many microbial pathogens, it appears most important in protection against encapsulated bacteria, especially Neisseria meningitidis but also Streptococcus pneumoniae, Haemophilus influenzae, and, to a lesser extent, Neisseria gonorrhoeae. The availability of effective polysaccharide vaccines and antibiotics provides an immunologic and chemotherapeutic rationale for preventing and treating infection in patients with these deficiencies. PMID:1889047

  14. Critical Roles of Complement and Antibodies in Host Defense Mechanisms against Neisseria meningitidis as Revealed by Human Complement Genetic Deficiencies ▿

    PubMed Central

    Hellerud, Bernt Christian; Aase, Audun; Herstad, Tove Karin; Næss, Lisbeth Meyer; Kristiansen, Lisa Høyem; Trøseid, Anne-Marie Siebke; Harboe, Morten; Lappegård, Knut Tore; Brandtzæg, Petter; Høiby, E. Arne; Mollnes, Tom Eirik

    2010-01-01

    Certain complement defects are associated with an increased propensity to contract Neisseria meningitidis infections. We performed detailed analyses of complement-mediated defense mechanisms against N. meningitidis 44/76 with whole blood and serum from two adult patients who were completely C2 or C5 deficient. The C5-deficient patient and the matched control were also deficient in mannose-binding lectin (MBL). The proliferation of meningococci incubated in freshly drawn whole blood was estimated by CFU and quantitative DNA real-time PCR. The serum bactericidal activity and opsonophagocytic activity by granulocytes were investigated, including heat-inactivated postvaccination sera, to examine the influence of antimeningococcal antibodies. The meningococci proliferated equally in C2- and C5-deficient blood, with a 2 log10 increase of CFU and 4- to 5-log10 increase in DNA copies. Proliferation was modestly decreased in reconstituted C2-deficient and control blood. After reconstitution of C5-deficient blood, all meningococci were killed, which is consistent with high antibody titers being present. The opsonophagocytic activity was strictly C2 dependent, appeared with normal serum, and increased with postvaccination serum. Serum bactericidal activity was strictly dependent on C2, C5, and high antibody titers. MBL did not influence any of the parameters observed. Complement-mediated defense against meningococci was thus dependent on the classical pathway. Some opsonophagocytic activity occurred despite low levels of antimeningococcal antibodies but was more efficient with immune sera. Serum bactericidal activity was dependent on C2, C5, and immune sera. MBL did not influence any of the parameters observed. PMID:19933829

  15. P-I Snake Venom Metalloproteinase Is Able to Activate the Complement System by Direct Cleavage of Central Components of the Cascade

    PubMed Central

    Pidde-Queiroz, Giselle; Magnoli, Fábio Carlos; Portaro, Fernanda C. V.; Serrano, Solange M. T.; Lopes, Aline Soriano; Paes Leme, Adriana Franco; van den Berg, Carmen W.; Tambourgi, Denise V.

    2013-01-01

    Background Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity of snake venom. We have recently shown that snake venoms from the Bothrops genus activate the Complement system (C) by promoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology and spread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom. Results Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement was isolated from Bothrops pirajai venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. In vivo, C-SVMP induced consumption of murine complement components, most likely by activation of the pathways and/or by direct cleavage of C3, leading to a reduction of serum lytic activity. Conclusion We show here that a P-I metalloproteinase from Bothrops pirajai snake venom activated the Complement system by direct cleavage of the central C-components, i.e., C3, C4 and C5, thereby generating biologically active fragments, such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These results suggest that direct complement activation by SVMPs may play a role in the progression of symptoms that follow envenomation. PMID:24205428

  16. Activation of mannan-binding lectin-associated serine proteases leads to generation of a fibrin clot

    PubMed Central

    Gulla, Krishana C; Gupta, Kshitij; Krarup, Anders; Gal, Peter; Schwaeble, Wilhelm J; Sim, Robert B; O’Connor, C David; Hajela, Krishnan

    2010-01-01

    The lectin pathway of complement is activated upon binding of mannan-binding lectin (MBL) or ficolins (FCNs) to their targets. Upon recognition of targets, the MBL-and FCN-associated serine proteases (MASPs) are activated, allowing them to generate the C3 convertase C4b2a. Recent findings indicate that the MASPs also activate components of the coagulation system. We have previously shown that MASP-1 has thrombin-like activity whereby it cleaves and activates fibrinogen and factor XIII. MASP-2 has factor Xa-like activity and activates prothrombin through cleavage to form thrombin. We now report that purified L-FCN-MASPs complexes, bound from serum to N-acetylcysteine-Sepharose, or MBL-MASPs complexes, bound to mannan-agarose, generate clots when incubated with calcified plasma or purified fibrinogen and factor XIII. Plasmin digestion of the clot and analysis using anti-D-dimer antibodies revealed that the clot was made up of fibrin and was similar to that generated by thrombin in normal human plasma. Fibrinopeptides A and B (FPA and FPB, respectively) were released after fibrinogen cleavage by L-FCN-MASPs complexes captured on N-acetylcysteine-Sepharose. Studies of inhibition of fibrinopeptide release indicated that the dominant pathway for clotting catalysed by the MASPs is via MASP-2 and prothrombin activation, as hirudin, a thrombin inhibitor that does not inhibit MASP-1 and MASP-2, substantially inhibits fibrinopeptide release. In the light of their potent chemoattractant effects on neutrophil and fibroblast recruitment, the MASP-mediated release of FPA and FPB may play a role in early immune activation. Additionally, MASP-catalysed deposition and polymerization of fibrin on the surface of micro-organisms may be protective by limiting the dissemination of infection. PMID:20002787

  17. Lectindb: a plant lectin database.

    PubMed

    Chandra, Nagasuma R; Kumar, Nirmal; Jeyakani, Justin; Singh, Desh Deepak; Gowda, Sharan B; Prathima, M N

    2006-10-01

    Lectins, a class of carbohydrate-binding proteins, are now widely recognized to play a range of crucial roles in many cell-cell recognition events triggering several important cellular processes. They encompass different members that are diverse in their sequences, structures, binding site architectures, quaternary structures, carbohydrate affinities, and specificities as well as their larger biological roles and potential applications. It is not surprising, therefore, that the vast amount of experimental data on lectins available in the literature is so diverse, that it becomes difficult and time consuming, if not impossible to comprehend the advances in various areas and obtain the maximum benefit. To achieve an effective use of all the data toward understanding the function and their possible applications, an organization of these seemingly independent data into a common framework is essential. An integrated knowledge base ( Lectindb, http://nscdb.bic.physics.iisc.ernet.in ) together with appropriate analytical tools has therefore been developed initially for plant lectins by collating and integrating diverse data. The database has been implemented using MySQL on a Linux platform and web-enabled using PERL-CGI and Java tools. Data for each lectin pertain to taxonomic, biochemical, domain architecture, molecular sequence, and structural details as well as carbohydrate and hence blood group specificities. Extensive links have also been provided for relevant bioinformatics resources and analytical tools. Availability of diverse data integrated into a common framework is expected to be of high value not only for basic studies in lectin biology but also for basic studies in pursuing several applications in biotechnology, immunology, and clinical practice, using these molecules. PMID:16782824

  18. Complement-targeted therapeutics

    PubMed Central

    Ricklin, Daniel; Lambris, John D

    2010-01-01

    The complement system is a central component of innate immunity and bridges the innate to the adaptive immune response. However, it can also turn its destructive capabilities against host cells and is involved in numerous diseases and pathological conditions. Modulation of the complement system has been recognized as a promising strategy in drug discovery, and a large number of therapeutic modalities have been developed. However, successful marketing of complement-targeted drugs has proved to be more difficult than initially expected, and many strategies have been discontinued. The US Food and Drug Administration’s approval of the first complement-specific drug, an antibody against complement component C5 (eculizumab; Soliris), in March 2007, was a long-awaited breakthrough in the field. Approval of eculizumab validates the complement system as therapeutic target and might facilitate clinical development of other promising drug candidates. PMID:17989689

  19. [Structure and function of complement protein C1q and its role in the development of autoimmune diseases].

    PubMed

    Smykał-Jankowiak, Katarzyna; Niemir, Zofia I

    2009-01-01

    Complement plays an important role in the immune system. Three different pathways of complement activation are known: the classical, alternative, and lectin dependent. They involve more than 30 serum peptides. C1q is the fi rst subcomponent of the classical pathway of complement activation.It is composed of three types of chains, A, B, and C, which form a molecule containing 18 peptides. Each of the chains has a short amino-terminal region followed by a collagen-like region(playing a role in the activation of C1r2C1s2) and a carboxy-terminal head, which binds to immune complexes. Recent studies have shown a great number of ligands for C1q, including aggregated IgG, IgM, human T-cell lymphotropic virus-I (HTLV-I), gp21 peptide, human immunodeficiency virus-1 (HIV-1) gp21 peptide, beta-amyloid, fragments of bacterial walls, apoptotic cells, and many others. However, the role of C1q is not only associated with complement activation.It also helps in the removal of immune complexes and necrotic cells, stimulates the production of some cytokines, and modulates the function of lymphocytes. Complete C1q deficiency is a rare genetic disorder. The C1q gene is located on the short arm of chromosome 1. So far, only a few mutations in C1q gene have been reported. The presence of these mutations is strongly associated with recurrent bacterial infections and the development of systemic lupus erythematosus(SLE). Recent clinical studies point to the significance of anti-C1q antibodies in the diagnosis and assessment of lupus nephritis activity. PMID:19373194

  20. Age-related macular degeneration: Complement in action.

    PubMed

    van Lookeren Campagne, Menno; Strauss, Erich C; Yaspan, Brian L

    2016-06-01

    The complement system plays a key role in host-defense against common pathogens but must be tightly controlled to avoid inflammation and tissue damage. Polymorphisms in genes encoding two important negative regulators of the alternative complement pathway, complement factor H (CFH) and complement factor I (CFI), are associated with the risk for Age-Related Macular Degeneration (AMD), a leading cause of vision impairment in the ageing population. In this review, we will discuss the genetic basis of AMD and the potential impact of complement de-regulation on disease pathogenesis. Finally, we will highlight recent therapeutic approaches aimed at controlling complement activation in patients with AMD. PMID:26742632

  1. Cytotoxic ribosome-inactivating lectins from plants.

    PubMed

    Hartley, M R; Lord, J M

    2004-09-01

    A class of heterodimeric plant proteins consisting of a carbohydrate-binding B-chain and an enzymatic A-chain which act on ribosomes to inhibit protein synthesis are amongst the most toxic substances known. The best known example of such a toxic lectin is ricin, produced by the seeds of the castor oil plant, Ricinnus communis. For ricin to reach its substrate in the cytosol, it must be endocytosed, transported through the endomembrane system to reach the compartment from which it is translocated into the cytosol, and there avoid degradation making it possible for a few molecules to inactivate a large proportion of the ribosomes and hence kill the cell. Cell entry by ricin involves the following steps: (i) binding to cell-surface glycolipids and glycoproteins bearing beta-1,4-linked galactose residues through the lectin activity of the B-chain (RTB); (ii) uptake by endocytosis and entry into early endosomes; (iii) transfer by vesicular transport to the trans-Golgi network; (iv) retrograde vesicular transport through the Golgi complex and into the endoplasmic reticulum (ER); (v) reduction of the disulfide bond connecting the A- and B-chains; (vi) a partial unfolding of the A-chain (RTA) to enable it to translocate across the ER membrane via the Sec61p translocon using the pathway normally followed by misfolded ER proteins for targeting to the ER-associated degradation (ERAD) machinery; (vi) refolding in the cytosol into a protease-resistant, enzymatically active structure; (vii) interaction with the sarcin-ricin domain (SRD) of the large ribosome subunit RNA followed by cleavage of a single N-glycosidic bond in the RNA to generate a depurinated, inactive ribosome. In addition to the highly specific action on ribosomes, ricin and related ribosome-inactivating proteins (RIPs) have a less specific action in vitro on DNA and RNA substrates releasing multiple adenine, and in some instances, guanine residues. This polynucleotide:adenosine glycosidase activity has been implicated in the general antiviral, and specifically, the anti HIV-1 activity of several single-chain RIPs which are homologous to the A-chains of the heterodimeric lectins. However, in the absence of clear cause and effect evidence in vivo, such claims should be regarded with caution. PMID:15450171

  2. Lectin cDNA and transgenic plants derived therefrom

    DOEpatents

    Raikhel, Natasha V.

    2000-10-03

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties.

  3. Interaction of lectins with Cryptosporidium parvum.

    PubMed

    Llovo, J; Lopez, A; Fabregas, J; Muñoz, A

    1993-06-01

    Cell surface carbohydrates from four clinical isolates of Cryptosporidium parvum were analyzed by agglutination assays using a battery of 20 highly purified lectins with affinity for receptor molecules containing N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-galactosamine, galactose, mannose, glucose, fucose, and N-acetyl-neuraminic acid. Tomentine, a lectin from the green seaweed Codium tomentosum, and UEA-II lectin, from Ulex europeus, both of them GlcNAc-specific lectins, agglutinated the oocysts. Subsequent inhibition assay confirmed the presence of this sugar on the surface of Cryptosporidium parvum oocysts. Codium fragile lectin, from another green seaweed, also exhibited agglutination activity against the oocysts. This is the first published demonstration of such an interaction between a human coccidian and lectins from seaweeds. PMID:8501345

  4. Molecular cloning and characterisation of two homologues of Mannose-Binding Lectin in rainbow trout.

    PubMed

    Nikolakopoulou, Konstantina; Zarkadis, Ioannis K

    2006-09-01

    Mannose-binding lectin (MBL) is a C-type lectin which participates in the innate immune system as an activator of the complement system and as opsonin after binding to certain carbohydrate structures on microorganisms and pathogens. C-type lectins are all Ca(2+)-dependent molecules and they share a tightly folded carbohydrate recognition domain (CRD). In this report the isolation and characterisation of cDNA transcripts encoding two mannose-binding lectin isoforms MBL-1 and MBL-2 from rainbow trout (Oncorhynchus mykiss) is presented. The deduced amino acid sequences of trout MBL-1 and MBL-2 (185 and 186 aa, respectively) present 83% identity to each other, exhibiting the highest identity score 46, 46 and 42% with the Atlantic salmon, shishamo smelt and zebrafish counterparts, respectively. The identity to birds and mammalian MBLs ranges from 25 to 33%. The trout MBL-1 and MBL-2 contain the EPN motif of mannose-binding C-type lectins, important for mannose specificity and they are expressed exclusively in liver and spleen, respectively. PMID:16542855

  5. Plant as a plenteous reserve of lectin

    PubMed Central

    Hivrale, AU; Ingale, AG

    2013-01-01

    Lectins are clusters of glycoproteins of nonimmune foundation that combine specifically and reversibly to carbohydrates, mainly the sugar moiety of glycoconjugates, resulting in cell agglutination and precipitation of glycoconjugates. They are universally distributed in nature, being established in plants, fungi, viruses, bacteria, crustacea, insects, and animals, but leguminacae plants are rich source of lectins. The present review reveals the structure, biological properties, and application of plant lectins. PMID:24084524

  6. Influence of Lectins on Constricting Ring Formation by Arthrobotrys dactyloides

    PubMed Central

    Kaplan, D. T.; Davis, E. L.; Walter, D. E.

    1991-01-01

    Incubation of Arthrobotrys dactyloides conidia in the presence of Radopholus citrophilus in lectin solutions with their corresponding sugars did not alter the stimulation of trap formation in solutions containing lectins alone. The lack of inhibition of lectin-stimulated trap formation by sugars or by lectin denaturation and the lack of lectin specificity indicate that the carbohydrate-binding regions of the particular lectins studied are not the stimulatory moieties of these macromolecules. PMID:19283124

  7. Influence of Lectins on Constricting Ring Formation by Arthrobotrys dactyloides.

    PubMed

    Kaplan, D T; Davis, E L; Walter, D E

    1991-04-01

    Incubation of Arthrobotrys dactyloides conidia in the presence of Radopholus citrophilus in lectin solutions with their corresponding sugars did not alter the stimulation of trap formation in solutions containing lectins alone. The lack of inhibition of lectin-stimulated trap formation by sugars or by lectin denaturation and the lack of lectin specificity indicate that the carbohydrate-binding regions of the particular lectins studied are not the stimulatory moieties of these macromolecules. PMID:19283124

  8. Lectins and their application to clinical microbiology.

    PubMed Central

    Slifkin, M; Doyle, R J

    1990-01-01

    Lectins are generally associated with plant or animal components, selectively bind carbohydrates, and interact with procaryotic and eucaryotic cells. Lectins have various specificities that are associated with their ability to interact with acetylaminocarbohydrates, aminocarbohydrates, sialic acids, hexoses, pentoses, and as other carbohydrates. Microbial surfaces generally contain many of the sugar residues that react with lectins. Lectins are presently used in the clinical laboratory to type blood cells and are used in a wide spectrum of applications, including, in part, as carriers of chemotherapeutic agents, as mitogens, for fractionation of animal cells, and for investigations of cellular surfaces. Numerous studies have shown that lectins can be used to identify rapidly certain microorganisms isolated from a clinical specimen or directly in a clinical specimen. Lectins have been demonstrated to be important diagnostic reagents in the major realms of clinical microbiology. Thus, they have been applied in bacteriology, mycology, mycobacteriology, and virology for the identification and/or differentiation of various microorganisms. Lectins have been used successfully as epidemiologic as well as taxonomic markers of specific microorganisms. Lectins provide the clinical microbiologist with cost-effective and potential diagnostic reagents. This review describes the applications of lectins in clinical microbiology. Images PMID:2200603

  9. Soluble beta-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells.

    PubMed Central

    Vetvicka, V; Thornton, B P; Ross, G D

    1996-01-01

    When phagocyte CR3 binds to iC3b on bacteria or yeast, phagocytosis and degranulation are triggered because of simultaneous recognition of iC3b via a CD11b I-domain binding site and specific microbial polysaccharides via a lectin site located COOH-terminal to the I-domain. By contrast, when phagocyte or natural killer (NK) cell CR3 adheres to iC3b on erythrocytes or tumor cells that lack CR3-binding membrane polysaccharides, neither lysis nor cytotoxicity are stimulated. This investigation showed that soluble CR3-specific polysaccharides such as beta-glucan induced a primed state of CR3 that could trigger killing of iC3b-target cells that were otherwise resistant to cytotoxicity. Anti-CR3 added before sugars prevented priming, whereas anti-CR3 added after sugars blocked primed CR3 attachment to iC3b-targets. Polysaccharide priming required tyrosine kinase(s) and a magnesium-dependent conformational change of the I-domain that exposed the CBRM1/5 activation epitope. Unlike LPS or cytokines, polysaccharides did not up-regulate neutrophil CR3 expression nor expose the mAb 24 reporter epitope representing the high affinity ICAM-1-binding state. The current data apparently explain the mechanism of tumoricidal beta-glucans used for immunotherapy. These polysaccharides function through binding to phagocyte or NK cell CR3, priming the receptor for cytotoxicity of neoplastic tissues that are frequently targeted with iC3b and sparing normal tissues that lack iC3b. PMID:8690804

  10. The consequence of low mannose-binding lectin plasma concentration in relation to susceptibility to Salmonella Infantis in chickens.

    PubMed

    Ulrich-Lynge, Sofie L; Dalgaard, Tina S; Norup, Liselotte R; Kjærup, Rikke M; Olsen, John E; Sørensen, Poul; Juul-Madsen, Helle R

    2015-01-15

    Mannose-binding lectin (MBL) is a key protein in innate immunity. MBL binds to carbohydrates on the surface of pathogens, where it initiates complement activation via the lectin-dependent pathway or facilitates opsonophagocytosis. In vitro studies have shown that human MBL is able to bind to Salmonella, but knowledge in relation to chicken MBL and Salmonella is lacking. In order to study this relation day-old chickens from two selected lines L10H and L10L, differing in MBL serum concentration, were either orally infected with S. Infantis (S.123443) or kept as non-infected controls. The differences between healthy L10H and L10L chicken sublines were more profound than differences caused by the S. Infantis infection. The average daily body weight was higher for L10H than for L10L, regardless of infection, indicating beneficial effects of MBL selection on growth. Salmonella was detected in cloacal swabs and the number of Salmonella positive chickens during the experiment was significantly higher in L10L than L10H, indicating that MBL may affect the magnitude of Salmonella colonisation in day-old chickens. MBL expression was determined in ceca tissue by real-time RT-PCR. L10H chickens showed a significantly higher relative expression than L10L at days 1 and 41 pi, regardless of infection. Finally, flow cytometric analysis of whole blood from infected chickens showed that L10H had a significantly higher count of all assessed leucocyte subsets on day 5 pi, and also a higher count of monocytes on day 12 pi than L10L. No difference was observed between infected and non-infected L10L chicken. PMID:25487759

  11. Interactions of killed Listeria monocytogenes with the mouse complement system.

    PubMed Central

    van Kessel, K P; Antonissen, A C; van Dijk, H; Rademaker, P M; Willers, J M

    1981-01-01

    Incubation of mouse serum with Listeria monocytogenes involved activation of the alternative complement pathway, resulting in depletion of both classical and alternative pathway activity. The activation process gave rise to reactive (calcium- and magnesium-independent) lysis of, specifically, rabbit erythrocytes, which become resistant to this form of hemolysis by sensitization with antibodies. The possible implications of these findings for L. monocytogenes as an intracellular parasite and for rabbit erythrocytes as target cells for mouse alternative complement pathway activity are discussed. PMID:6795123

  12. [Fractionation of lymphocytes using affinity chromatography with 9 lectins].

    PubMed

    de Dios, I; Manso, M; López-Borrasca, A

    1983-12-01

    Lymphocyte subclasses from normal peripheral blood have been fractionated by affinity chromatography with lectins. Concanavalin A (Con A), Lens culinaris lectin (LC), Pisum sativum lectin (PS), Phaseolus vulgaris lectin (PHA), Dolichos biflours lectin (DB), Glicine max lectin (SBA), Ricinus communis lectin (RCA II), Tetragonolobus purpureus lectin (TP) and Triticum vulgaris lectin (WGA), were coupled to Sepharose 6MB, and lymphocytes labelled with 125I were eluted through the chromatographic columns. The binding of lymphocytes to WGA and SBA lectins was 32% and 13% respectively. The binding to the other lectins tested were found to be between 32% and 13%. When solutions of increasing concentrations of specific sugar were added to the columns a progressive elution of bound lymphocytes was observed. These results indicate the existence of a large range of lymphocyte subclasses, with different binding capacity to lectins, which was a function of the receptor number or/and receptor affinity to each lectin. Furthermore, these two parameters were found to vary in each functional population. Even though all the lymphocytes had lectin receptors, T lymphocytes showed higher affinity for Con A, PHA and TP lectins, while B lymphocytes appeared to be more specific for LC, PS, SBA, DB, RCAII and WGA lectins. PMID:6675094

  13. Recent insights into structures and functions of C-type lectins in the immune system

    PubMed Central

    Drickamer, Kurt; Taylor, Maureen E

    2015-01-01

    The majority of the C-type lectin-like domains in the human genome likely to bind sugars have been investigated structurally, although novel mechanisms of sugar binding are still being discovered. In the immune system, adhesion and endocytic receptors that bind endogenous mammalian glycans are often conserved, while pathogen-binding C-type lectins on cells of the innate immune system are more divergent. Lack of orthology between some human and mouse receptors, as well as overlapping specificities of many receptors and formation of receptor hetero-oligomers, can make it difficult to define the roles of individual receptors. There is good evidence that C-type lectins initiate signalling pathways in several different ways, but this function remains the least well understood from a mechanistic perspective. PMID:26163333

  14. Endogenous lectins as cell surface transducers.

    PubMed

    Hébert, E

    2000-08-01

    Interactions between cells or between cell and substratum involve specific receptors and their ligands. Among the various cell surface receptors identified during the last decades, the carbohydrate-binding proteins, e.g., lectins are of peculiar interest because glycolipids, glycoproteins and proteoglycans have been shown to interact with lectins on the surface of animal cells. Animal lectins are recognized as molecules playing important roles in a variety of biological processes through binding to glycoconjugates and lectin-like receptors such as selectins, sialoadhesins (CD22, CD33), natural killer receptors (NKR-P1, CD69 and CD94/NKG2), hyaluronate receptors (CD44, RHAMM, ICAM-1), B-cell associated antigen (CD23, CD72), beta2 leukocyte integrin (CD11b/CD18) or the well-known receptors for mannose, mannose-6-phosphate or asialoglycoprotein have been suggested to be able to mediate the transfer of information from the outside to the inside of the cell. This review focuses on the most recent advances in our understanding of the molecular basis of "outside-in" signaling mediated by lectins. Lectin-like receptors are involved in signal transduction in a great variety of ways; at the molecular level, they mimic in most of the cases the function of growth factor receptor either coupled to tyrosine kinase activity or to heterotrimeric G protein. They lead to a multiplicity of cellular events following their activation depending on factors such as cellular type, species and/or tissue. Nevertheless the potential of surface lectins as transducers is emphasized by the observation that in a few cases lectin-like receptors induce either novel signal transduction mechanism or new intracellular events with regards to what it has been observed as a consequence of growth factor receptor activation. This observation brings the idea that lectins may offer, as cell surface transducers, an alternative or additional signaling potential to cell. PMID:11092246

  15. Agglutination of Helicobacter pylori coccoids by lectins

    PubMed Central

    Khin, Mar Mar; Hua, Jie Song; Ng, Han Cong; Wadström, Torkel; Ho, Bow

    2000-01-01

    AIM: To study the agglutination pattern of Helicobacter pylori coccoid and spiral forms. METHODS: Assays of agglutination and agglutination inhibition were applied using fifteen commercial lectins. RESULTS: Strong agglutination was observed with mannose-specific Concanavalin A (Con A), fucose-specific Tetragonolobus purpureas (Lotus A) and N-acetyl glucosamine-specific Triticum vulgaris (WGA) lectins. Mannose and fucose specific lectins were reactive with all strains of H. pylori coccoids as compared to the spirals. Specific carbohydrates, glycoproteins and mucin were shown to inhibit H. pylori lectin-agglutination reactions. Pre-treatment of the bacterial cells with formalin and sulphuric acid did not alter the agglutination patterns with lectins. However, sodium periodate treatment of bacterial cells were shown to inhibit agglutination reaction with Con A, Lotus A and WGA lectins. On the contrary, enzymatic treatment of coccoids and spirals did not show marked inhibition of H. pylori lectin agglutination. Interes tingly, heating of H. pylori cells at 60 °C for 1 h was shown to augment the agglutination with all of the lectins tested. CONCLUSION: The considerable differences in lectin agglutination patterns seen among the two differentiated forms of H. pylori might be attributable to the structural changes during the events of morphological transformation, resulting in exposing or masking some of the sugar residues on the cell surface. Possibility of various sugar residues on the cell wall of the coccoids may allow them to bind to different carbohydrate receptors on gastric mucus and epithelial cells. The coccoids with adherence characteristics like the spirals could aid in the pathogenic process of Helicobacter infection. This may probably lead to different clinical outcome of H. pylori associated gastroduodenal disease. PMID:11819557

  16. Outline of Hungarian Complementation.

    ERIC Educational Resources Information Center

    Szamosi, Michael

    This study presents a preliminary analysis of Hungarian complement constructions and the syntactic operations needed to account for them. The expository framework (and the implicit framework of the research itself) is based upon that of Rosenbaum (1967). The aim of the paper is to arrive at a rough picture of the kinds of structures and syntactic…

  17. Complement component 3 (C3)

    MedlinePlus

    C3 and C4 are the most commonly measured complement components. A complement test may be used to monitor people with an ... normal levels of the complement proteins C3 and C4 . Complement activity varies throughout the body. For example, ...

  18. Novel roles for complement receptors in T cell regulation and beyond.

    PubMed

    Kemper, Claudia; Köhl, Jörg

    2013-12-15

    Complement receptors are expressed on cells of the innate and the adaptive immune system. They play important roles in pathogen and danger sensing as they translate the information gathered by complement fluid phase sensors into cellular responses. Further, they control complement activation on viable and apoptotic host cells, clearance of immune complexes and mediate opsonophagocytosis. More recently, evidence has accumulated that complement receptors form a complex network with other innate receptors systems such as the Toll-like receptors, the Notch signaling system, IgG Fc receptors and C-type lectin receptors contributing to the benefit and burden of innate and adaptive immune responses in autoimmune and allergic diseases as well as in cancer and transplantation. Here, we will discuss recent developments and emerging concepts of complement receptor activation and regulation with a particular focus on the differentiation, maintenance and contraction of effector and regulatory T cells. PMID:23796748

  19. Mannose-binding Lectin (MBL) as a susceptible host factor influencing Indian Visceral Leishmaniasis.

    PubMed

    Mishra, Anshuman; Antony, Justin S; Gai, Prabhanjan; Sundaravadivel, Pandarisamy; Van, Tong Hoang; Jha, Aditya Nath; Singh, Lalji; Velavan, Thirumalaisamy P; Thangaraj, Kumarasamy

    2015-12-01

    Visceral Leishmaniasis (VL), caused by Leishmania donovani is endemic in the Indian sub-continent. Mannose-binding Lectin (MBL) is a complement lectin protein that binds to the surface of Leishmania promastigotes and results in activation of the complement lectin cascade. We utilized samples of 218 VL patients and 215 healthy controls from an Indian population. MBL2 functional variants were genotyped and the circulating MBL serum levels were measured. MBL serum levels were elevated in patients compared to the healthy controls (adjusted P=0.007). The MBL2 promoter variants -78C/T and +4P/Q were significantly associated with relative protection to VL (-78C/T, OR=0.7, 95% CI=0.5-0.96, adjusted P=0.026 and +4P/Q, OR=0.66, 95% CI=0.48-0.9, adjusted P=0.012). MBL2*LYQA haplotypes occurred frequently among controls (OR=0.69, 95% CI=0.5-0.97, adjusted P=0.034). MBL recognizes Leishmania and plays a relative role in establishing L. donovani infection and subsequent disease progression. In conclusion, MBL2 functional variants were associated with VL. PMID:26297290

  20. Identification of mycobacterial lectins from genomic data.

    PubMed

    Abhinav, K V; Sharma, Alok; Vijayan, M

    2013-04-01

    Sixty-four sequences containing lectin domains with homologs of known three-dimensional structure were identified through a search of mycobacterial genomes. They appear to belong to the β-prism II, the C-type, the Microcystis virdis (MV), and the β-trefoil lectin folds. The first three always occur in conjunction with the LysM, the PI-PLC, and the β-grasp domains, respectively while mycobacterial β-trefoil lectins are unaccompanied by any other domain. Thirty heparin binding hemagglutinins (HBHA), already annotated, have also been included in the study although they have no homologs of known three-dimensional structure. The biological role of HBHA has been well characterized. A comparison between the sequences of the lectin from pathogenic and nonpathogenic mycobacteria provides insights into the carbohydrate binding region of the molecule, but the structure of the molecule is yet to be determined. A reasonable picture of the structural features of other mycobacterial proteins containing one or the other of the four lectin domains can be gleaned through the examination of homologs proteins, although the structure of none of them is available. Their biological role is also yet to be elucidated. The work presented here is among the first steps towards exploring the almost unexplored area of the structural biology of mycobacterial lectins. PMID:23180653

  1. Mannose-binding dietary lectins induce adipogenic differentiation of the marrow-derived mesenchymal cells via an active insulin-like signaling mechanism.

    PubMed

    Bajaj, Manmohan; Hinge, Ashwini; Limaye, Lalita S; Gupta, Rajesh Kumar; Surolia, Avadhesha; Kale, Vaijayanti P

    2011-04-01

    We have recently demonstrated that the mannose-binding lectins, namely banana lectin (BL) and garlic lectin (GL), interacted with the insulin receptors on M210B4 cells--an established mesenchymal cell line of murine marrow origin--and initiate mitogen-activated protein kinase kinase (MEK)-dependent extracellular signal-regulated kinase (ERK) signaling in them. In this study, we show that this lectin-mediated active ERK signaling culminates into an adipogenic differentiation of these cells. Gene expression studies indicate that the effect takes place at the transcriptional level. Experiments carried out with pharmacological inhibitors show that MEK-dependent ERK and phosphatidylinositol 3-kinase-dependent AKT pathways are positive regulators of the lectin- and insulin-mediated adipogenic differentiation, while stress-activated kinase/c-jun N-terminal kinase pathway acts as a negative one. Since both lectins could efficiently substitute for insulin in the standard adipogenic induction medium, they may perhaps serve as molecular tools to study the mechanistic aspects of the adipogenic process that are independent of cell proliferation. Our study clearly demonstrates the ability of BL and GL to activate insulin-like signaling in the mesenchymal cells in vitro leading to their adipocytic differentiation. The dietary origin of these lectins underscores an urgent need to examine their in vivo effects on tissue homeostasis. PMID:21106560

  2. The Complement Inhibitor Factor H Generates an Anti-Inflammatory and Tolerogenic State in Monocyte-Derived Dendritic Cells.

    PubMed

    Olivar, Rut; Luque, Ana; Cárdenas-Brito, Sonia; Naranjo-Gómez, Mar; Blom, Anna M; Borràs, Francesc E; Rodriguez de Córdoba, Santiago; Zipfel, Peter F; Aran, Josep M

    2016-05-15

    The activation of the complement system is a key initiating step in the protective innate immune-inflammatory response against injury, although it may also cause harm if left unchecked. The structurally related soluble complement inhibitors C4b-binding protein (C4BP) and factor H (FH) exert a tight regulation of the classical/lectin and alternative pathways of complement activation, respectively, attenuating the activity of the C3/C5 convertases and, consequently, avoiding serious damage to host tissues. We recently reported that the acute-phase C4BP isoform C4BP lacking the β-chain plays a pivotal role in the modulation of the adaptive immune responses. In this study, we demonstrate that FH acts in the early stages of monocyte to dendritic cell (DC) differentiation and is able to promote a distinctive tolerogenic and anti-inflammatory profile on monocyte-derived DCs (MoDCs) challenged by a proinflammatory stimulus. Accordingly, FH-treated and LPS-matured MoDCs are characterized by altered cytoarchitecture, resembling immature MoDCs, lower expression of the maturation marker CD83 and the costimulatory molecules CD40, CD80, and CD86, decreased production of key proinflammatory Th1-cytokines (IL-12, TNF-α, IFN-γ, IL-6, and IL-8), and preferential production of immunomodulatory mediators (IL-10 and TGF-β). Moreover, FH-treated MoDCs show low Ag uptake and, when challenged with LPS, display reduced CCR7 expression and chemotactic migration, impaired CD4(+) T cell alloproliferation, inhibition of IFN-γ secretion by the allostimulated T cells, and, conversely, induction of CD4(+)CD127(low/negative)CD25(high)Foxp3(+) regulatory T cells. Thus, this novel noncanonical role of FH as an immunological brake able to directly affect the function of MoDCs in an inflammatory environment may exhibit therapeutic potential in hypersensitivity, transplantation, and autoimmunity. PMID:27076676

  3. Activated Complement Factors as Disease Markers for Sepsis

    PubMed Central

    Charchaflieh, Jean; Rushbrook, Julie; Worah, Samrat; Zhang, Ming

    2015-01-01

    Sepsis is a leading cause of death in the United States and worldwide. Early recognition and effective management are essential for improved outcome. However, early recognition is impeded by lack of clinically utilized biomarkers. Complement factors play important roles in the mechanisms leading to sepsis and can potentially serve as early markers of sepsis and of sepsis severity and outcome. This review provides a synopsis of recent animal and clinical studies of the role of complement factors in sepsis development, together with their potential as disease markers. In addition, new results from our laboratory are presented regarding the involvement of the complement factor, mannose-binding lectin, in septic shock patients. Future clinical studies are needed to obtain the complete profiles of complement factors/their activated products during the course of sepsis development. We anticipate that the results of these studies will lead to a multipanel set of sepsis biomarkers which, along with currently used laboratory tests, will facilitate earlier diagnosis, timely treatment, and improved outcome. PMID:26420913

  4. Complement inhibition: a promising concept for cancer treatment

    PubMed Central

    Pio, Ruben; Ajona, Daniel; Lambris, John D.

    2013-01-01

    For decades, complement has been recognized as an effector arm of the immune system that contributes to the destruction of tumor cells. In fact, many therapeutic strategies have been proposed that are based on the intensification of complement-mediated responses against tumors. However, recent studies have challenged this paradigm by demonstrating a tumor-promoting role for complement. Cancer cells seem to be able to establish a convenient balance between complement activation and inhibition, taking advantage of complement initiation without suffering its deleterious effects. Complement activation may support chronic inflammation, promote an immunosuppressive microenvironment, induce angiogenesis, and activate cancer-related signaling pathways. In this context, inhibition of complement activation would be a therapeutic option for treating cancer. This concept is relatively novel and deserves closer attention. In this paper, we will summarize the mechanisms of complement activation on cancer cells, the cancer-promoting effect of complement initiation, and the rationale behind the use of complement inhibition as a therapeutic strategy against cancer. PMID:23706991

  5. Lectin affinity chromatography of glycolipids

    SciTech Connect

    Torres, B.V.; Smith, D.F.

    1987-05-01

    Since glycolipids (GLs) are either insoluble or form mixed micelles in water, lectin affinity chromatography in aqueous systems has not been applied to their separation. They have overcome this problem by using tetrahydrofuran (THF) in the mobile phase during chromatography. Affinity columns prepared with the GalNAc-specific Helix pomatia agglutinin (HPA) and equilibrated in THF specifically bind the (/sup 3/H)oligosaccharide derived from Forssman GL indicating that the immobilized HPA retained its carbohydrate-binding specificity in this solvent. Intact Forssman GL was bound by the HPA-column equilibrated in THF and was specifically eluted with 0.1 mg/ml GalNAc in THF. Purification of the Forssman GL was achieved when a crude lipid extract of sheep erythrocyte membranes was applied to the HPA-column in THF. Non-specifically bound GLs were eluted from the column using a step gradient of aqueous buffer in THF, while the addition of GalNAc was required to elute the specifically bound GLs. Using this procedure the A-active GLs were purified from a crude lipid extract of type A human erythrocytes in a single chromatographic step. The use of solvents that maintain carbohydrate-binding specificity and lipid solubility will permit the application of affinity chromatography on immobilized carbohydrate-binding proteins to intact GLs.

  6. Targeting C-type lectin receptors with multivalent carbohydrate ligands.

    PubMed

    Lepenies, Bernd; Lee, Junghoon; Sonkaria, Sanjiv

    2013-08-01

    C-type lectin receptors (CLRs) represent a large receptor family including collectins, selectins, lymphocyte lectins, and proteoglycans. CLRs share a structurally homologous carbohydrate-recognition domain (CRD) and often bind carbohydrates in a Ca²⁺-dependent manner. In innate immunity, CLRs serve as pattern recognition receptors (PRRs) and bind to the glycan structures of pathogens and also to self-antigens. In nature, the low affinity of CLR/carbohydrate interactions is overcome by multivalent ligand presentation at the surface of cells or pathogens. Thus, multivalency is a promising strategy for targeting CLR-expressing cells and, indeed, carbohydrate-based targeting approaches have been employed for a number of CLRs, including asialoglycoprotein receptor (ASGPR) in the liver, or DC-SIGN expressed by dendritic cells. Since CLR engagement not only mediates endocytosis but also influences intracellular signaling pathways, CLR targeting may allow for cell-specific drug delivery and also the modulation of cellular functions. Glyconanoparticles, glycodendrimers, and glycoliposomes were successfully used as tools for CLR-specific targeting. This review will discuss different approaches for multivalent CLR ligand presentation and aims to highlight how CLR targeting has been employed for cell specific drug delivery. Major emphasis is directed towards targeting of CLRs expressed by antigen-presenting cells to modulate immune responses. PMID:23727341

  7. Trichinella spiralis Paramyosin Binds Human Complement C1q and Inhibits Classical Complement Activation

    PubMed Central

    Sun, Ran; Zhao, Xi; Wang, Zixia; Yang, Jing; Zhao, Limei; Zhan, Bin; Zhu, Xinping

    2015-01-01

    Background Trichinella spiralis expresses paramyosin (Ts-Pmy) as a defense mechanism. Ts-Pmy is a functional protein with binding activity to human complement C8 and C9 and thus plays a role in evading the attack of the host’s immune system. In the present study, the binding activity of Ts-Pmy to human complement C1q and its ability to inhibit classical complement activation were investigated. Methods and Findings The binding of recombinant and natural Ts-Pmy to human C1q were determined by ELISA, Far Western blotting and immunoprecipitation, respectively. Binding of recombinant Ts-Pmy (rTs-Pmy) to C1q inhibited C1q binding to IgM and consequently inhibited C3 deposition. The lysis of antibody-sensitized erythrocytes (EAs) elicited by the classical complement pathway was also inhibited in the presence of rTs-Pmy. In addition to inhibiting classical complement activation, rTs-Pmy also suppressed C1q binding to THP-1-derived macrophages, thereby reducing C1q-induced macrophages migration. Conclusion Our results suggest that T. spiralis paramyosin plays an important role in immune evasion by interfering with complement activation through binding to C1q in addition to C8 and C9. PMID:26720603

  8. Incorporation of Host Complement Regulatory Proteins into Newcastle Disease Virus Enhances Complement Evasion

    PubMed Central

    Biswas, Moanaro; Johnson, John B.; Kumar, Sandeep R. P.; Parks, Griffith D.

    2012-01-01

    Newcastle disease virus (NDV), an avian paramyxovirus, is inherently tumor selective and is currently being considered as a clinical oncolytic virus and vaccine vector. In this study, we analyzed the effect of complement on the neutralization of NDV purified from embryonated chicken eggs, a common source for virus production. Fresh normal human serum (NHS) neutralized NDV by multiple pathways of complement activation, independent of neutralizing antibodies. Neutralization was associated with C3 deposition and the activation of C2, C3, C4, and C5 components. Interestingly, NDV grown in mammalian cell lines was resistant to complement neutralization by NHS. To confirm whether the incorporation of regulators of complement activity (RCA) into the viral envelope afforded complement resistance, we grew NDV in CHO cells stably transfected with CD46 or HeLa cells, which strongly express CD46 and CD55. NDV grown in RCA-expressing cells was resistant to complement by incorporating CD46 and CD55 on virions. Mammalian CD46 and CD55 molecules on virions exhibited homologous restriction, since chicken sera devoid of neutralizing antibodies to NDV were able to effectively neutralize these virions. The incorporation of chicken RCA into NDV produced in embryonated eggs similarly provided species specificity toward chicken sera. PMID:22973037

  9. 21 CFR 864.9550 - Lectins and protectins.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Lectins and protectins. 864.9550 Section 864.9550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... and Blood Products § 864.9550 Lectins and protectins. (a) Identification. Lectins and protectins...

  10. 21 CFR 864.9550 - Lectins and protectins.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Lectins and protectins. 864.9550 Section 864.9550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... and Blood Products § 864.9550 Lectins and protectins. (a) Identification. Lectins and protectins...

  11. Complementing Gender Analysis Methods.

    PubMed

    Kumar, Anant

    2016-01-01

    The existing gender analysis frameworks start with a premise that men and women are equal and should be treated equally. These frameworks give emphasis on equal distribution of resources between men and women and believe that this will bring equality which is not always true. Despite equal distribution of resources, women tend to suffer and experience discrimination in many areas of their lives such as the power to control resources within social relationships, and the need for emotional security and reproductive rights within interpersonal relationships. These frameworks believe that patriarchy as an institution plays an important role in women's oppression, exploitation, and it is a barrier in their empowerment and rights. Thus, some think that by ensuring equal distribution of resources and empowering women economically, institutions like patriarchy can be challenged. These frameworks are based on proposed equality principle which puts men and women in competing roles. Thus, the real equality will never be achieved. Contrary to the existing gender analysis frameworks, the Complementing Gender Analysis framework proposed by the author provides a new approach toward gender analysis which not only recognizes the role of economic empowerment and equal distribution of resources but suggests to incorporate the concept and role of social capital, equity, and doing gender in gender analysis which is based on perceived equity principle, putting men and women in complementing roles that may lead to equality. In this article the author reviews the mainstream gender theories in development from the viewpoint of the complementary roles of gender. This alternative view is argued based on existing literature and an anecdote of observations made by the author. While criticizing the equality theory, the author offers equity theory in resolving the gender conflict by using the concept of social and psychological capital. PMID:25941756

  12. Complement activation promotes muscle inflammation during modified muscle use

    NASA Technical Reports Server (NTRS)

    Frenette, J.; Cai, B.; Tidball, J. G.

    2000-01-01

    Modified muscle use can result in muscle inflammation that is triggered by unidentified events. In the present investigation, we tested whether the activation of the complement system is a component of muscle inflammation that results from changes in muscle loading. Modified rat hindlimb muscle loading was achieved by removing weight-bearing from the hindlimbs for 10 days followed by reloading through normal ambulation. Experimental animals were injected with the recombinant, soluble complement receptor sCR1 to inhibit complement activation. Assays for complement C4 or factor B in sera showed that sCR1 produced large reductions in the capacity for activation of the complement system through both the classical and alternative pathways. Analysis of complement C4 concentration in serum in untreated animals showed that the classical pathway was activated during the first 2 hours of reloading. Analysis of factor B concentration in untreated animals showed activation of the alternative pathway at 6 hours of reloading. Administration of sCR1 significantly attenuated the invasion of neutrophils (-49%) and ED1(+) macrophages (-52%) that occurred in nontreated animals after 6 hours of reloading. The presence of sCR1 also reduced significantly the degree of edema by 22% as compared to untreated animals. Together, these data show that increased muscle loading activated the complement system which then briefly contributes to the early recruitment of inflammatory cells during modified muscle loading.

  13. Role of lectin microarrays in cancer diagnosis.

    PubMed

    Syed, Parvez; Gidwani, Kamlesh; Kekki, Henna; Leivo, Janne; Pettersson, Kim; Lamminmäki, Urpo

    2016-04-01

    The majority of cell differentiation associated tumor markers reported to date are either glycoproteins or glycolipids. Despite there being a large number of glycoproteins reported as candidate markers for various cancers, only a handful are approved by the US Food and Drug Administration. Lectins, which bind to the glycan part of the glycoproteins, can be exploited to identify aberrant glycosylation patterns, which in turn would help in enhancing the specificity of cancer diagnosis. Although conventional techniques such as HPLC and MS have been instrumental in performing the glycomic analyses, these techniques lack multiplexity. Lectin microarrays have proved to be useful in studying multiple lectin-glycan interactions in a single experiment and, with the advances made in the field, hold a promise of enabling glycomic profiling of cancers in a fast and efficient manner. PMID:26841254

  14. Examining coagulation-complement crosstalk: complement activation and thrombosis.

    PubMed

    Foley, Jonathan H

    2016-05-01

    The coagulation and complement systems are ancestrally related enzymatic cascades of the blood. Although their primary purposes have diverged over the past few hundred million years, they remain inextricably connected. Both complement and coagulation systems limit infection by pathogens through innate immune mechanisms. Recently, it has been shown that hyperactive complement (in particular, elevated C5a/C5b-9) is involved in the pathogenesis (including thrombosis) of diseases such as paroxysmal nocturnal hemoglobinuria, atypical haemolytic uremic syndrome, antiphospholipid syndrome and bacteremia. Although these diseases together account for many thrombosis cases, there are many more where complement activation is not considered a causative factor leading to thrombosis. To better understand what role complement may play in the pathogenesis of thrombosis a better understanding of the mechanisms that cause over-active complement in thrombotic disease is required. PMID:27207425

  15. Carbohydrate specificity of lectins from Boletopsis leucomelas and Aralia cordate.

    PubMed

    Koyama, Yu; Suzuki, Takuji; Odani, Shoji; Nakamura, Sachiko; Kominami, Junko; Hirabayashi, Jun; Isemura, Mamoru

    2006-02-01

    The carbohydrate specificity of three novel lectins, Boletopsis leucomelas lectin (BLL), Aralia cordate lectin (ACL), and Wasabia japonica lectin (WJL), was examined by frontal affinity chromatography using a panel of fluorescently labeled 47 oligosaccharides. The results indicate that BLL recognizes an agalacto structure of the biantennary chain and its bisecting structure. ACL showed strong affinity for triantennary oligosaccharides, but no affinity for tetraantennary structure. WJL showed no appreciable affinity for any of the 47 glycans examined. These lectins with a unique affinity specificity might be useful for examining alterations in the glycan structures of the glycoconjugates in association with development and various diseases. PMID:16495678

  16. Complement and HIV-I infection/ HIV-associated neurocognitive disorders

    PubMed Central

    Liu, Fengming; Dai, Shen; Gordon, Jennifer; Qin, Xuebin

    2014-01-01

    The various neurological complications associated with HIV-1 infection, specifically HIV-associated neurocognitive disorders (HAND) persist as a major public health burden worldwide. Despite the widespread use of anti-retroviral therapy, the prevalence of HAND is significantly high. HAND results from the direct effects of an HIV-1 infection as well as secondary effects of HIV-1-induced immune reaction and inflammatory response. Complement, a critical mediator of innate and acquired immunity, plays important roles in defeating many viral infections by the formation of a lytic pore or indirectly by opsonization and recruitment of phagocytes. While the role of complement in the pathogenesis of HIV-1 infection and HAND has been previously recognized for over fifteen years, it has been largely underestimated thus far. Complement can be activated through HIV-1 envelope proteins, mannose binding lectins (MBL) and anti-HIV-1 antibodies. Complement not only fights against HIV-1 infection but also enhances HIV-1 infection. Also, HIV-1 can hijack complement regulators such as CD59 and CD55 and can utilize these regulators and factor H to escape from complement attack. Normally, complement levels in brain are much lower than plasma levels and there is no or little complement deposition in brain cells. Interestingly, local production and deposition of complement are dramatically increased in HIV-1-infected brain, indicating that complement may contribute to the pathogenesis of HAND. Here, we review the current understanding of the role of complement in HIV-1 infection and HAND as well as potential therapeutic approaches targeting to the complement system for the treatment and eradications of HIV-1 infection. PMID:24639397

  17. Mushroom Lectins: Specificity, Structure and Bioactivity Relevant to Human Disease

    PubMed Central

    Hassan, Mohamed Ali Abol; Rouf, Razina; Tiralongo, Evelin; May, Tom W.; Tiralongo, Joe

    2015-01-01

    Lectins are non-immunoglobulin proteins that bind diverse sugar structures with a high degree of selectivity. Lectins play crucial role in various biological processes such as cellular signaling, scavenging of glycoproteins from the circulatory system, cell–cell interactions in the immune system, differentiation and protein targeting to cellular compartments, as well as in host defence mechanisms, inflammation, and cancer. Among all the sources of lectins, plants have been most extensively studied. However, more recently fungal lectins have attracted considerable attention due to their antitumor, antiproliferative and immunomodulatory activities. Given that only 10% of mushroom species are known and have been taxonomically classified, mushrooms represent an enormous unexplored source of potentially useful and novel lectins. In this review we provide an up-to-date summary on the biochemical, molecular and structural properties of mushroom lectins, as well as their versatile applications specifically focusing on mushroom lectin bioactivity. PMID:25856678

  18. Structural analysis of a Dioclea sclerocarpa lectin: Study on the vasorelaxant properties of Dioclea lectins.

    PubMed

    Barroso-Neto, Ito L; Delatorre, Plinio; Teixeira, Claudener S; Correia, Jorge L A; Cajazeiras, João B; Pereira, Ronniery I; Nascimento, Kyria S; Laranjeira, Eva P P; Pires, Alana F; Assreuy, Ana M S; Rocha, Bruno A M; Cavada, Benildo S

    2016-01-01

    Lectins are proteins that show a variety of biological activities. However, they share in common at least one domain capable of recognizing specific carbohydrates reversibly without changing its structure. The legume lectins family is the most studied family of plant lectins, in particular the Diocleinae subtribe, which possesses high degree of structural similarity, but variable biological activities. This variability lies in small differences that can be analyzed in studies based on structures. In particular, Dioclea sclerocarpa seed lectin (DSL) presents low ability to relax endothelialized rat aorta in comparison with other Dioclea lectins such as Dioclea violacea (DVL), Dioclea virgata (DvirL) and Dioclea rostrata (DRL). The DSL relaxation mechanism relies on nitric oxide production and carbohydrate recognition domain (CRD). This feature can be explained by structural differences, since DSL has a carbohydrate recognition domain design less favorable. In addition, the presence of a glutamate residue at position 205 proved to be a decisive factor for the low relaxant effect of Dioclea lectins. PMID:26499084

  19. Evasion of Complement-Mediated Lysis and Complement C3 Deposition Are Regulated by Francisella tularensis Lipopolysaccharide O Antigen1

    PubMed Central

    Clay, Corey D.; Soni, Shilpa; Gunn, John S.; Schlesinger, Larry S.

    2009-01-01

    The bacterium Francisella tularensis (Ft) is a potential weapon of bioterrorism when aerosolized. Macrophage infection is necessary for disease progression and efficient phagocytosis by human macrophages requires serum opsonization by complement. Microbial complement activation leads to surface deposition of a highly regulated protein complex resulting in opsonization or membrane lysis. The nature of complement component C3 deposition, i.e., C3b (opsonization and lysis) or C3bi (opsonization only) fragment deposition, is central to the outcome of activation. In this study, we examine the mechanisms of Ft resistance to complement-mediated lysis, C3 component deposition on the Ft surface, and complement activation. Upon incubation in fresh nonimmune human serum, Schu S4 (Ft subsp. tularensis), Fn (Ft subsp. novicida), and LVS (Ft subsp. holarctica live vaccine strain) were resistant to complement-mediated lysis, but LVSG and LVSR (LVS strains altered in surface carbohydrate structures) were susceptible. C3 deposition, however, occurred on all strains. Complement-susceptible strains had markedly increased C3 fragment deposition, including the persistent presence of C3b compared with C3bi, which indicates that C3b inactivation results in survival of complement-resistant strains. C1q, an essential component of the classical activation pathway, was necessary for lysis of complement-susceptible strains and optimal C3 deposition on all strains. Finally, use of Francisella LPS mutants confirmed O Ag as a major regulator of complement resistance. These data provide evidence that pathogenic Francisella activate complement, but are resistant to complement-mediated lysis in part due to limited C3 deposition, rapid conversion of surface-bound C3b to C3bi, and the presence of LPS O Ag. PMID:18832715

  20. Displacement phenomena in lectin affinity chromatography.

    PubMed

    Cho, Wonryeon

    2015-10-01

    The work described here examines displacement phenomena that play a role in lectin affinity chromatography and their potential to impact reproducibility. This was achieved using Lycopersicon esculentum lectin (LEL), a lectin widely used in monitoring cancer. Four small identical LEL columns were coupled in series to form a single affinity chromatography system with the last in the series connected to an absorbance detector. The serial affinity column set (SACS) was then loaded with human plasma proteins. At the completion of loading, the column set was disassembled, the four columns were eluted individually, the captured proteins were trypsin digested, the peptides were deglycosylated with PNGase F, and the parent proteins were identified through mass spectral analyses. Significantly different sets of glycoproteins were selected by each column, some proteins appearing to be exclusively bound to the first column while others were bound further along in the series. Clearly, sample displacement chromatography (SDC) occurs. Glycoproteins were bound at different places in the column train, identifying the presence of glycoforms with different affinity on a single glycoprotein. It is not possible to see these phenomena in the single column mode of chromatography. Moreover, low abundance proteins were enriched, which facilitates detection. The great advantage of this method is that it differentiates between glycoproteins on the basis of their binding affinity. Displacement phenomena are concluded to be a significant component of the separation mechanism in heavily loaded lectin affinity chromatography columns. This further suggests that care must be exercised in sample loading of lectin columns to prevent analyte displacement with nonretained proteins. PMID:26348026

  1. Anti-Gal binds to pili of Neisseria meningitidis: the immunoglobulin A isotype blocks complement-mediated killing.

    PubMed Central

    Hamadeh, R M; Estabrook, M M; Zhou, P; Jarvis, G A; Griffiss, J M

    1995-01-01

    alpha 1,3-Galactosyl antibodies (anti-Gal) are ubiquitous natural human serum and secretory polyclonal antibodies that bind to terminal galactose-alpha 1,3-galactose (alpha-galactosyl) residues. Serum immunoglobulin G (IgG) anti-Gal can block alternative complement pathway-mediated lysis of representative gram-negative enteric bacteria that bind it to lipopolysaccharide alpha-galactosyl structures, thereby promoting survival of such bacteria in the nonimmune host. We wanted to know whether anti-Gal also could bind to the lipooligosaccharides (LOS) of Neisseria meningitidis. To our surprise, we found that serum and secretory anti-Gal bound to pili but not to LOS of certain strains. This suggested the presence of an immunogenic pilus carbohydrate epitope. Mild periodate oxidation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated outer membrane preparations from strains that bound anti-Gal followed by labeling of the neoaldehyde groups resulted in the labeling of bands that corresponded to pilin and LOS, confirming that pilin contains carbohydrate structures. A Bandeiraea simplicifolia lectin that also binds terminal alpha 1,3-galactosyl residues also bound to pilin. Serum IgG, IgA, and IgM anti-Gal as well as colostral secretory IgA anti-Gal bound to pilin, as judged by immunoblotting, and to the pili of intact piliated organisms, as judged by immunoelectron microscopy. Total serum anti-Gal (IgG, IgA, and IgM) and purified serum IgA1 anti-Gal, but not its purified IgG isotype, blocked complement-mediated lysis of a piliated meningococcal strain that bound anti-Gal to its pili. Colostral anti-Gal secretory IgA blocked killing of the same strain. Thus, anti-Gal IgA may promote disease when it binds to the pili of N. meningitidis strains. PMID:7591153

  2. Galactosylated IgG1 links FcγRIIB and Dectin-1 to block complement-mediated inflammation

    PubMed Central

    Karsten, Christian M.; Pandey, Manoj K.; Figge, Julia; Kilchenstein, Regina; Taylor, Philip R.; Rosas, Marcela; McDonald, Jacqueline U.; Orr, Selinda J.; Berger, Markus; Petzold, Dominique; Blanchard, Veroniqué; Winkler, André; Hess, Constanze; Reid, Delyth M.; Majoul, Irina V.; Strait, Richard T.; Harris, Nathaniel L.; Köhl, Gabriele; Wex, Eva; Ludwig, Ralf; Zillikens, Detlef; Nimmerjahn, Falk; Finkelman, Fred D.; Brown, Gordon D.; Ehlers, Marc; Köhl, Jörg

    2012-01-01

    Complement is an ancient danger sensing system playing critical roles in host defense, immune surveillance and homeostasis1. C5a and its G-Protein-coupled receptor mediate many of the pro-inflammatory properties of complement2. Despite its critical role in allergic asthma3, autoimmune arthritis4, sepsis5 and cancer6, our knowledge about C5a regulation is limited. Here we demonstrate an unexpected link through which IgG1 immune complexes (IC), the inhibitory IgG receptor FcγRIIB and the C-type lectin-like receptor Dectin-1 suppress C5a receptor (C5aR) functions. Specifically, we found that IgG1 IC associate FcγRIIB with Dectin-1, resulting in phosphorylation of spleen tyrosine kinase (Syk) downstream of Dectin-1 and Src homology 2 domain containing inositol phosphatase (SHIP) downstream of FcγRIIB. This pathway blocks C5a receptor-mediated ERK1/2 phosphorylation and C5a effector functions in vitro and C5a-dependent inflammatory responses in vivo including the development of skin blisters in experimental epidermolysis bullosa acquisita (EBA), an autoimmune skin disorder. Notably, high galactosylation of IgG N-glycan is critical for this inhibitory property of IgG1 IC as it promotes the association between FcγRIIB and Dectin-1. Thus, galactosylated IgG1 and FcγRIIB exert immunoregulatory properties beyond their impact on activating FcγRs that may control allergy, autoimmunity and cancer. PMID:22922409

  3. Bimolecular Fluorescence Complementation

    PubMed Central

    Wong, Katy A.; O'Bryan, John P.

    2011-01-01

    Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex.Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and Förster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling. PMID:21525844

  4. Biological role of mannose binding lectin: From newborns to centenarians.

    PubMed

    Scorza, Manuela; Liguori, Renato; Elce, Ausilia; Salvatore, Francesco; Castaldo, Giuseppe

    2015-12-01

    Mannose binding lectin (MBL) is a protein of innate immunity that activates the complement and promotes opsonophagocytosis. The deficiency of MBL due to several common gene polymorphisms significantly enhances the risk of severe infections, particularly in the neonatal age and in childhood. On the contrary, the role of the protein in carcinogenesis and atherogenesis is still debated: MBL has a relevant role against neoplastic cells, but some studies described a protective effect of low levels of MBL toward breast cancer and a longer survival of lung cancer patients with a reduced MBL activity. Similarly, some studies concluded on the protective role of low levels of MBL toward cardiovascular diseases while other focused on a higher risk of myocardial infarction in subjects with a deficient activity of the protein. More recently, a role of MBL in the clearance of senescent cells emerged, and a study in two large cohorts of centenarians demonstrated that a high biological activity of the protein enhances the risk of autoimmune diseases. This body of data strongly suggests that the optimal levels of MBL activity depend on the age and on the environmental context of each subject. PMID:25783214

  5. Sensing lectin-glycan interactions using lectin super-microarrays and glycans labeled with dye-doped silica nanoparticles

    PubMed Central

    Wang, Xin; Matei, Elena; Deng, Lingquan; Koharudin, Leonardus; Gronenborn, Angela M.; Ramström, Olof; Yan, Mingdi

    2013-01-01

    A new microarray platform, based on lectin super-microarrays and glycans labeled with dye-doped nanoparticles, has been developed to study glycan-lectin interactions. Glycan ligands were conjugated onto fluorescein-doped silica nanoparticles (FSNPs) using a general photocoupling chemistry to afford FSNP-labeled glycan probes. Lectins were printed on epoxy slides in duplicate sets to generate lectin super-microarrays where multiple assays could be carried out simultaneously in each lectin microarray. Thus, the lectin super-microarray was treated with FSNP-labeled glycans to screen for specific binding pairs. Furthermore, a series of ligand competition assays were carried out on a single lectin super-microarray to generate the dose-response curve for each glycan-lectin pair, from which the apparent affinity constants were obtained. Results showed 4~7 orders of magnitude increase in affinity over the free glycans with the corresponding lectins. Thus, the glycan epitope structures having weaker affinity than the parent glycans could be readily identified and analyzed from the lectin super-microarrays. PMID:23584388

  6. Algal lectins as promising biomolecules for biomedical research.

    PubMed

    Singh, Ram Sarup; Thakur, Shivani Rani; Bansal, Parveen

    2015-02-01

    Lectins are natural bioactive ubiquitous proteins or glycoproteins of non-immune response that bind reversibly to glycans of glycoproteins, glycolipids and polysaccharides possessing at least one non-catalytic domain causing agglutination. Some of them consist of several carbohydrate-binding domains which endow them with the properties of cell agglutination or precipitation of glycoconjugates. Lectins are rampant in nature from plants, animals and microorganisms. Among microorganisms, algae are the potent source of lectins with unique properties specifically from red algae. The demand of peculiar and neoteric biologically active substances has intensified the developments on isolation and biomedical applications of new algal lectins. Comprehensively, algal lectins are used in biomedical research for antiviral, antinociceptive, anti-inflammatory, anti-tumor activities, etc. and in pharmaceutics for the fabrication of cost-effective protein expression systems and nutraceutics. In this review, an attempt has been made to collate the information on various biomedical applications of algal lectins. PMID:23855360

  7. Identification and confirmation of differentially expressed fucosylated glycoproteins in the serum of ovarian cancer patients using a lectin array and LC-MS/MS.

    PubMed

    Wu, Jing; Xie, Xiaolei; Liu, Yashu; He, Jintang; Benitez, Ricardo; Buckanovich, Ronald J; Lubman, David M

    2012-09-01

    In order to discover potential glycoprotein biomarkers in ovarian cancer, we applied a lectin array and Exactag labeling based quantitative glycoproteomics approach. A lectin array strategy was used to detect overall lectin-specific glycosylation changes in serum proteins from patients with ovarian cancer and those with benign conditions. Lectins, which showed significant differential response for fucosylation, were used to extract glycoproteins that had been labeled using isobaric chemical tags. The glycoproteins were then identified and quantified by LC-MS/MS, and five glycoproteins were found to be differentially expressed in the serum of ovarian cancer patients compared to benign diseases. The differentially expressed glycoproteins were further confirmed by lectin-ELISA and ELISA assay. Corticosteroid-binding globulin (CBG), serum amyloid p component (SAP), complement factor B (CFAB), and histidine-rich glycoprotein (HRG) were identified as potential markers for differentiating ovarian cancer from benign diseases or healthy controls. A combination of CBG and HRG (AUC = 0.825) showed comparable performance to CA125 (AUC = 0.829) in differentiating early stage ovarian cancer from healthy controls. The combination of CBG, SAP, and CA125 showed improved performance for distinguishing stage III ovarian cancer from benign diseases compared to CA125 alone. The ability of CBG, SAP, HRG, and CFAB to differentiate the serum of ovarian cancer patients from that of controls was tested using an independent set of samples. Our findings suggest that glycoprotein modifications may be a means to identify novel diagnostic markers for detection of ovarian cancer. PMID:22827608

  8. Two Chitotriose-Specific Lectins Show Anti-Angiogenesis, Induces Caspase-9-Mediated Apoptosis and Early Arrest of Pancreatic Tumor Cell Cycle

    PubMed Central

    Sarkar, Dhiman; Suresh, C. G.

    2016-01-01

    The antiproliferative activity of two chito- specific agglutinins purified from Benincasa hispida (BhL) and Datura innoxia (DiL9) of different plant family origin was investigated on various cancer cell lines. Both lectins showed chitotriose specificity, by inhibiting lectin hemagglutinating activity. On further studies, it was revealed that these agglutinins caused remarkable concentration-dependent antiproliferative effect on human pancreatic cancerous cells but not on the normal human umbilical vein endothelial cells even at higher doses determined using MTT assay. The GI50 values were approximately 8.4 μg ml-1 (0.247 μM) and 142 μg ml-1(14.8 μM) for BhL and DiL9, respectively, against PANC-1 cells. The growth inhibitory effect of these lectins on pancreatic cancer cells were shown to be a consequence of lectin cell surface binding and triggering G0/G1 arrest, mitochondrial membrane depolarization, sustained increase of the intracellular calcium release and the apoptotic signal is amplified by activation of caspases executing cell death. Interestingly, these lectins also showed anti-angiogenic activity by disrupting the endothelial tubulogenesis. Therefore, we report for the first time two chito-specific lectins specifically binding to tumor glycans; they can be considered to be a class of molecules with antitumor activity against pancreatic cancer cells mediated through caspase dependent mitochondrial apoptotic pathway. PMID:26795117

  9. Lectin cDNA and transgenic plants derived therefrom

    DOEpatents

    Raikhel, Natasha V.

    1994-01-04

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties. GOVERNMENT RIGHTS This application was funded under Department of Energy Contract DE-AC02-76ER01338. The U.S. Government has certain rights under this application and any patent issuing thereon.

  10. Lectin cDNA and transgenic plants derived therefrom

    SciTech Connect

    Raikhel, N.V.

    1994-01-04

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties. GOVERNMENT RIGHTS This application was funded under Department of Energy Contract DE-AC02-76ER01338. The U.S. Government has certain rights under this application and any patent issuing thereon. .

  11. Complement activation by neutrophil granulocytes.

    PubMed

    Asberg, A E; Mollnes, T E; Videm, V

    2008-04-01

    Complement plays a vital role in the body's defence systems. Cardiopulmonary bypass induces a detrimental inflammatory reaction in which the complement system is known to participate through direct effects as well as through activation of neutrophils, platelets and endothelial cells. On the other hand, it has been suggested that in the setting of cardiopulmonary bypass, complement may be activated by neutrophils, perhaps due to fragmentation caused by the heart-lung machine. We therefore investigated whether intact or fragmented neutrophils were able to activate the complement system, and whether neutrophil-platelet interaction could influence such complement activation. Lepirudin-anticoagulated plasma was incubated at 37 degrees C with resting or activated intact neutrophils or neutrophils combined with platelets, or increasing amounts of fragmented neutrophils. Complement activation was evaluated by measurement of C1rs-C1 inhibitor complexes, C4bc, C3bBbP, C3bc, C5a and sC5b-9. We found significant activation of complement only by unphysiological doses of fragmented neutrophils or supernatant from fragmented neutrophils, consistent with a limited clinical significance related to neutrophil destruction during cardiopulmonary bypass. Unstimulated neutrophils induced C3bPBb formation but little formation of other activation products, indicating an increased C3 hydrolysis which was kept under control by regulatory mechanisms. Neutrophils and platelets combined increased classical activation and decreased alternative activation, similar to the findings with platelets alone. Our data confirm that in the setting of acute neutrophil fragmentation or activation, complement activation is much more important in the inflammatory network as an event upstream to neutrophil activation than vice versa. PMID:18248527

  12. Lectins discriminate between pathogenic and nonpathogenic South American trypanosomes

    SciTech Connect

    de Miranda Santos, I.K.; Pereira, M.E.

    1984-09-01

    Cell surface carbohydrates of Trypanosoma cruzi, Trypanosoma rangeli, and Trypanosoma conorhini were analyzed by a micro-agglutination assay employing 27 highly purified lectins and by binding assays using various /sup 125/I-labeled lectins. The following seven lectins discriminated between the trypanosomes: 1) tomato lectin (an N-acetyl-D-glucosamine-binding protein), both in purified form and as crude tomato juice; 2) Bauhinea purpurea and Sophora japonica lectins (both N-acetyl-D-galactosamine-binding proteins), which selectively agglutinated T. cruzi; 3) Vicia villosa (an N-acetyl-D-galactosamine-binding protein) which was specific for T. rangeli; 4) peanut lectin (a D-galactose-binding protein) both in purified form and as crude saline extract; and 5) Ulex europaeus and Lotus tetragonolobus (both L-fucose-binding proteins) lectins which reacted only with T. conorhini. Binding studies with 125I-labeled lectins were performed to find whether unagglutinated cells of the three different species of trypanosomes might have receptors for these lectins, in which case absence of agglutination could be due to a peculiar arrangement of the receptors. These assays essentially confirmed the agglutination experiments.

  13. [Characteristic of extracellular lectin in Bacillus subtilis IMV B-7014].

    PubMed

    Brovars'ka, O S; Vasiliev, V N; Ostapchuk, A N; Kovalenko, E A; Sashchuk, O V

    2011-01-01

    The monosaccharide and fatty acid composition of water and organic phase of lectin was studied. It was established that extracellular lectin contains 15.0% of protein, 4.6% of carbohydrates, 1.0% of nucleic acids and 16.14% of lipids. The following monosaccharides were presented in the lectins: mannose, ribose, glucose and ramnose. It was established that both native lectin and its lipophylic fraction were composed of fatty acids from C15 to C19. During aggressive methanolysis anti-iso-C15 quantity was increased with a synchronous decrease of the content of all octadecanoic acid types. PMID:21809686

  14. Endogenous mammalian lectin localized extracellularly in lung elastic fibers.

    PubMed

    Cerra, R F; Haywood-Reid, P L; Barondes, S H

    1984-04-01

    An affinity-purified antibody preparation raised against a beta-galactoside-binding lectin from bovine lung was used to localize a similar lectin in rat lung by immunofluorescence and by electron microscopy after on-grid staining visualized with colloidal gold conjugated second antibody. The endogenous mammalian lectin was found in smooth muscle cells and squamous alveolar epithelial (type I) cells and was concentrated extracellularly in elastic fibers of pulmonary parenchyma and blood vessels. The extracellular localization of this lectin suggests that it, like others, functions by interaction with extracellular glycoconjugates. PMID:6371024

  15. A comparison of tomato (Lycopersicon esculentum) lectin with its deglycosylated derivative.

    PubMed

    Kilpatrick, D C; Graham, C; Urbaniak, S J; Jeffree, C E; Allen, A K

    1984-06-15

    A deglycosylated derivative of tomato (Lycopersicon esculentum) lectin was prepared with the use of trifluoromethanesulphonic acid. Its properties were generally similar to those of the native lectin, but differences were evident in terms of relative agglutinating activity towards sheep, (untreated) human and trypsin-treated human erythrocytes. The two forms of tomato lectin were used in conjunction with a battery of specific antisera to investigate structural relatedness among solanaceous lectins. Immunological cross-reactivity between tomato, potato and Datura lectins depends on the integrity of the glycosylated region of those lectins; that between Datura lectin and other seed lectins, however, has a separate structural basis. PMID:6087799

  16. Lectin-mediated drug delivery: influence of mucin on cytoadhesion of plant lectins in vitro.

    PubMed

    Wirth, M; Gerhardt, K; Wurm, C; Gabor, F

    2002-02-19

    As the mucous layer represents the first barrier to peroral lectin-mediated drug delivery, the influence of mucin on the cytoadhesive properties of lectins was studied in vitro by establishing a rapid and simple microplate format assay using pig gastric mucin (PGM) for coating the wells. The lectin-binding capacity of mucin followed the order WGA>UEA-I>LCA=STL>PNA>DBA. The PGM-binding of wheat germ agglutinin (WGA) was strongly dependent on pH being highest at pH 5.0. In comparison, PGM-binding of WGA was about 15% at gastric pH and 60-70% at intestinal pH. This points to unimpeded gastric transit of WGA-grafted formulations and favorable conditions within the intestine for binding to mucus coated enterocytes. Moreover the WGA-PGM interaction was concentration-dependent, specific and fully reversible. According to a competitive assay in the presence of Caco-2 monolayers, the PGM-binding of WGA was saturated and influenced by the lectin-concentration yielding 28% Caco-2 bound WGA (125 ng WGA/0.29 cm(2) monolayer) and 68% Caco-2 bound WGA (4 microg WGA/0.29 cm(2) monolayer), respectively. Following on from these results, lectins are expected to suffer at least partially from premature inactivation by shed off mucus like bioadhesives of the first generation, however initial but reversible mucus-binding of lectins offers partititioning to the cell membrane followed by uptake into the enterocyte. PMID:11853930

  17. Effect of zinc and cadmium on guinea-pig complement

    PubMed Central

    Amiraian, K.; McKinney, J. A.; Duchna, Lillian

    1974-01-01

    Zinc, cadmium or nickel ion in the presence of optimum Ca+ + and Mg+ + will enhance the activity of guinea-pig complement in immune haemolysis. The range of optimum concentration of Zn+ + is 10-6–10-5 M and is influenced by the concentrations of sensitized erythrocytes (5–500 × 107) and guinea-pig complement. Enhancement by Zn+ + is unrelated to potentiation of guinea-pig complement activity by the zinc metalloenzyme, CPAγ. These cations may participate in a late-acting step of one of the pathways proposed for the interaction of guinea-pig complement with immune aggregates; or they may, at 37°, destroy an inhibitor of immune haemolysis. PMID:4851716

  18. Complement involvement in periodontitis: molecular mechanisms and rational therapeutic approaches

    PubMed Central

    Hajishengallis, George; Maekawa, Tomoki; Abe, Toshiharu; Hajishengallis, Evlambia; Lambris, John D.

    2015-01-01

    The complement system is a network of interacting fluid-phase and cell surface-associated molecules that trigger, amplify, and regulate immune and inflammatory signaling pathways. Dysregulation of this finely balanced network can destabilize host-microbe homeostasis and cause inflammatory tissue damage. Evidence from clinical and animal model-based studies suggests that complement is implicated in the pathogenesis of periodontitis, a polymicrobial community-induced chronic inflammatory disease that destroys the tooth-supporting tissues. This review discusses molecular mechanisms of complement involvement in the dysbiotic transformation of the periodontal microbiome and the resulting destructive inflammation, culminating in loss of periodontal bone support. These mechanistic studies have additionally identified potential therapeutic targets. In this regard, interventional studies in preclinical models have provided proof-of-concept for using complement inhibitors for the treatment of human periodontitis. PMID:26306443

  19. Complement - a key system for immune surveillance and homeostasis

    PubMed Central

    Ricklin, Daniel; Hajishengallis, George; Yang, Kun; Lambris, John D.

    2010-01-01

    Nearly a century after the significance of the human complement system was recognized we have come to realize that its versatile functions extend far beyond the elimination of microbes. Indeed, complement acts as a rapid and efficient immune surveillance system that has distinct effects on healthy and altered host cells and foreign intruders. By eliminating cellular debris and infectious microbes, orchestrating immune responses, and sending `danger' signals, complement contributes substantially to homeostasis, but it may also take action against healthy cells if not properly controlled. This review describes our updated view of the function, structure, and dynamics of the complement network, highlights its interconnection with immunity at large and with other endogenous pathways, and illustrates its dual role in homeostasis and disease. PMID:20720586

  20. Subcellular site of lectin synthesis in developing rice embryos

    PubMed Central

    Stinissen, Hetty M.; Peumans, Willy J.; Chrispeels, Maarten J.

    1984-01-01

    Embryos of developing rice (Oryza sativa L. cv. Koshihikari) caryopses which actively synthesize lectin were labelled with [35S]cysteine for different times and newly synthesized rice lectin was isolated by affinity chromatography. Gel filtration of embryo extracts on Sepharose-4B indicated that a large portion of the labelled lectin was associated with the particulate fraction. Experiments with detergent indicated that this lectin was sequestered within organelles. When extracts of pulse-labelled embryos were fractionated on isopycnic sucrose gradients, this detergent-released lectin banded in the same density-region as the endoplasmic reticulum (ER) marker enzyme NADH-cytochrome c reductase. Both radioactivity in rice lectin and the enzyme activity shifted towards a higher density in the presence of 2 mM Mg acetate, indicating that the labelled lectin was associated with the rough ER. The ER-bound lectin could be chased from this organelle when tissue was incubated in unlabelled cysteine following a 1 h pulse of labelled cysteine. Radioactivity chased out of the ER with a half-life of ˜4 h and accumulated in the soluble fraction. In the ER the lectin was present as a polypeptide with mol. wt. 23 000, while in the soluble fraction it occurred as polypeptides with mol. wt. 18 000, 10 000 and 8000. The rice lectin in the ER is capable of binding carbohydrates since it binds readily to the affinity gels. It is associated into dimers with an approximate mol. wt. of 46 000. The results show that newly synthesized rice lectin is transiently sequestered within the ER before further transport and processing take place. ImagesFig. 5. PMID:16453545

  1. 21 CFR 864.9550 - Lectins and protectins.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Lectins and protectins. 864.9550 Section 864.9550 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Products Used In Establishments That Manufacture Blood and Blood Products § 864.9550 Lectins...

  2. Optical lectin based biosensor as tool for bacteria identification.

    PubMed

    Masarova, Jana; Dey, Estera Szwajcer; Danielsson, Bengt

    2004-01-01

    Biosensor techniques are based on biospecific interaction between the biological parts of biosensor with the analyte. In biosensor construction, antibodies are usually used for the detection of analytes such as microorganism, because of very strong and highly specific interaction. The disadvantages of this assay are a long time needed for antibody isolation and purification as well as difficult regeneration of biosensor chip. The use of lectins instead of antibodies could solve these problems because a several hundred lectins are commercially available and their stability in standard buffers is better compared to monoclonal antibodies. While antibody can only be used to detect that antigen it was designed for, lectin as low affinity molecule may bind several different pathogens. Using the discriminative effect of an artificial neural network the application of a lectin array will compensate for the lower specificity. Microbial surfaces bear many of the sugar residues capable of interacting with lectins. The ability of lectins to react with microbial glycoconjugates means that it is possible to employ them as probes and sorbents for whole cells, mutants and numerous cellular constituents and metabolites, and it makes them useful tools for identification or typing of bacteria. Lectins are attractive reagents for the clinical diagnostic laboratory because of their diverse specificity, commercial availability, a wide range of molecular weights, and their stability in standard buffers. The construction of lectin biosensor could be an advantage method for detection of pathogenic bacteria. PMID:15787193

  3. Plant Lectins: Wheat Defense Strategy Against Hessian Fly

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plants produce a variety of defense proteins, including lectins in response to attack by phytophagous insects. Ultrastructural studies reveal that binding to insect gut structures and resistance to proteolytic degradation by insect digestive enzymes are the two main prerequisites for the lectins to...

  4. Porifera Lectins: Diversity, Physiological Roles and Biotechnological Potential.

    PubMed

    Gardères, Johan; Bourguet-Kondracki, Marie-Lise; Hamer, Bojan; Batel, Renato; Schröder, Heinz C; Müller, Werner E G

    2015-08-01

    An overview on the diversity of 39 lectins from the phylum Porifera is presented, including 38 lectins, which were identified from the class of demosponges, and one lectin from the class of hexactinellida. Their purification from crude extracts was mainly performed by using affinity chromatography and gel filtration techniques. Other protocols were also developed in order to collect and study sponge lectins, including screening of sponge genomes and expression in heterologous bacterial systems. The characterization of the lectins was performed by Edman degradation or mass spectrometry. Regarding their physiological roles, sponge lectins showed to be involved in morphogenesis and cell interaction, biomineralization and spiculogenesis, as well as host defense mechanisms and potentially in the association between the sponge and its microorganisms. In addition, these lectins exhibited a broad range of bioactivities, including modulation of inflammatory response, antimicrobial and cytotoxic activities, as well as anticancer and neuromodulatory activity. In view of their potential pharmacological applications, sponge lectins constitute promising molecules of biotechnological interest. PMID:26262628

  5. Assessment of lectin inactivation by heat and digestion.

    PubMed

    Pusztai, A; Grant, G

    1998-01-01

    Proteins/glycoproteins from plants, particularly lectins, are more resistant to heat denaturation than animal proteins (1, 2). With legume seeds, whose lectin content is appreciable, this presents potentially serious problems in nutritional practice. Therefore, before they can be used safely, legume-based food/ feeds usually require thorough and expensive heat processing to inactivate antinutritive components. Indeed, dry or moist heating of seeds at 70°C for several h has little or no effect on their lectin activity (Fig. 1) and treatment at much higher temperatures is needed to inactivate the biological and antinutritional effects of legume lectins (1, 2). The safety aspect is even more serious with some monocot lectins, such as wheatgerm agglutinin or a number of oilseed lectins, such as peanut agglutinin and many others because they are extremely heat stable and normal cooking or other conventional heat treatments may fail to inactivate them (3) Thus, the best way to avoid potential harmful effects of these heat-resistant lectins is to limit their dietary intake to a minimum. Fig. 1. Loss of lectin activity during aqueous heat treatment of soybean at various temperatures. PMID:21374488

  6. Lectin-binding properties of Aeromonas caviae strains

    PubMed Central

    Rocha-de-Souza, Cláudio M.; Hirata-Jr, Raphael; Mattos-Guaraldi, Ana L.; Freitas-Almeida, Angela C.; Andrade, Arnaldo F. B.

    2008-01-01

    The cell surface carbohydrates of four strains of Aeromonas caviae were analyzed by agglutination and lectin-binding assays employing twenty highly purified lectins encompassing all sugar specificities. With the exception of L-fucose and sialic acid, the sugar residues were detected in A. caviae strains. A marked difference, however, in the pattern of cell surface carbohydrates in different A. caviae isolates was observed. Specific receptors for Tritricum vulgaris (WGA), Lycopersicon esculentum (LEL) and Solanum tuberosum (STA) (D-GlcNAc-binding lectins) were found only in ATCC 15468 strain, whereas Euonymus europaeus (EEL, D-Gal-binding lectin) sites were present exclusively in AeQ32 strain, those for Helix pomatia (HPA, D-GalNAc-binding lectin) in AeC398 and AeV11 strains, and for Canavalia ensiformes (Con A, D-Man-binding lectin) in ATCC 15468, AeC398, AeQ32 and AeV11 strains, after bacterial growing at 37°C. On the other hand, specific receptors for WGA and EEL were completely abrogated growing the bacteria at 22°C. Binding studies with 125I- labeled lectins from WGA, EEL and Con A were performed. These assays essentially confirmed the selectivity, demonstrated in the agglutination assays of these lectins for the A. caviae strains. PMID:24031204

  7. Porifera Lectins: Diversity, Physiological Roles and Biotechnological Potential

    PubMed Central

    Gardères, Johan; Bourguet-Kondracki, Marie-Lise; Hamer, Bojan; Batel, Renato; Schröder, Heinz C.; Müller, Werner E. G.

    2015-01-01

    An overview on the diversity of 39 lectins from the phylum Porifera is presented, including 38 lectins, which were identified from the class of demosponges, and one lectin from the class of hexactinellida. Their purification from crude extracts was mainly performed by using affinity chromatography and gel filtration techniques. Other protocols were also developed in order to collect and study sponge lectins, including screening of sponge genomes and expression in heterologous bacterial systems. The characterization of the lectins was performed by Edman degradation or mass spectrometry. Regarding their physiological roles, sponge lectins showed to be involved in morphogenesis and cell interaction, biomineralization and spiculogenesis, as well as host defense mechanisms and potentially in the association between the sponge and its microorganisms. In addition, these lectins exhibited a broad range of bioactivities, including modulation of inflammatory response, antimicrobial and cytotoxic activities, as well as anticancer and neuromodulatory activity. In view of their potential pharmacological applications, sponge lectins constitute promising molecules of biotechnological interest. PMID:26262628

  8. The lectin like domain of thrombomodulin is involved in the defence against pyelonephritis.

    PubMed

    Lattenist, Lionel; Teske, Gwendoline; Claessen, Nike; Florquin, Sandrine; Conway, Edward M; Roelofs, Joris J T H

    2015-12-01

    Pyelonephritis, a common complication of urinary tract infections, is frequently associated with kidney scarring and may lead to end-stage renal disease. During bacterial infections inflammatory and coagulation pathways and their mutual interaction are playing pivotal roles in the host response. Given that thrombomodulin (TM) is crucially involved in the interplay between coagulation and inflammation, we aimed to investigate the roles of its EGF and lectin-like domains in inflammation during acute pyelonephritis. Indeed, the EGF-like and the lectin-like domains of TM, are especially known to orchestrate inflammation and coagulation in different ways. Acute pyelonephritis was induced by intravesical inoculation of 1 × 10(8) CFU of uropathogenic Escherichia coli in two strains of TM transgenic mice. TM(pro/pro) mice carry a mutation in the EGF-like domain making them unable to activate protein C, an anticoagulant and anti-inflammatory protein. TM(LeD/LeD) mice lack the lectin-like domain of TM, which is critical for its anti-inflammatory and cytoprotective properties. Mice were sacrificed 24 and 48 h after inoculation. Bacterial loads, the immune response and the activation of coagulation were evaluated in the kidney and the bladder. TM(LeD/LeD) mice showed elevated bacterial load in bladder and kidneys compared to WT mice, whereas TM(pro/pro) had similar bacterial load as WT mice. TM(LeD/LeD) mice displayed a reduced local production of pro-inflammatory cytokines and neutrophil renal infiltration. Activation of coagulation was comparable in TM(LeD/LeD) and WT mice. From these data, we conclude that the lectin-like domain of thrombomodulin is critically involved in host defence against E. coli induced acute pyelonephritis. PMID:26573396

  9. Structure-function relationship of monocot mannose-binding lectins.

    PubMed Central

    Barre, A; Van Damme, E J; Peumans, W J; Rougé, P

    1996-01-01

    The monocot mannose-binding lectins are an extended superfamily of structurally and evolutionarily related proteins, which until now have been isolated from species of the Amaryllidaceae, Alliaceae, Araceae, Orchidaceae, and Liliaceae. To explain the obvious differences in biological activities, the structure-function relationships of the monocot mannose-binding lectins were studied by a combination of glycan-binding studies and molecular modeling using the deduced amino acid sequences of the currently known lectins. Molecular modeling indicated that the number of active mannose-binding sites per monomer varies between three and zero. Since the number of binding sites is fairly well correlated with the binding activity measured by surface plasmon resonance, and is also in good agreement with the results of previous studies of the biological activities of the mannose-binding lectins, molecular modeling is of great value for predicting which lectins are best suited for a particular application. PMID:8972598

  10. Biotoxicity assays for fruiting body lectins and other cytoplasmic proteins.

    PubMed

    Künzler, Markus; Bleuler-Martinez, Silvia; Butschi, Alex; Garbani, Mattia; Lüthy, Peter; Hengartner, Michael O; Aebi, Markus

    2010-01-01

    Recent studies suggest that a specific class of fungal lectins, commonly referred to as fruiting body lectins, play a role as effector molecules in the defense of fungi against predators and parasites. Hallmarks of these fungal lectins are their specific expression in reproductive structures, fruiting bodies, and/or sclerotia and their synthesis on free ribosomes in the cytoplasm. Fruiting body lectins are released upon damage of the fungal cell and bind to specific carbohydrate structures of predators and parasites, which leads to deterrence, inhibition of growth, and development or even killing of these organisms. Here, we describe assays to assess the toxicity of such lectins and other cytoplasmic proteins toward three different model organisms: the insect Aedes aegypti, the nematode Caenorhabditis elegans, and the amoeba Acanthamoeba castellanii. All three assays are based on heterologous expression of the examined proteins in the cytoplasm of Escherichia coli and feeding of these recombinant bacteria to omnivorous and bacterivorous organisms. PMID:20816208

  11. Diversified Carbohydrate-Binding Lectins from Marine Resources

    PubMed Central

    Ogawa, Tomohisa; Watanabe, Mizuki; Naganuma, Takako; Muramoto, Koji

    2011-01-01

    Marine bioresources produce a great variety of specific and potent bioactive molecules including natural organic compounds such as fatty acids, polysaccharides, polyether, peptides, proteins, and enzymes. Lectins are also one of the promising candidates for useful therapeutic agents because they can recognize the specific carbohydrate structures such as proteoglycans, glycoproteins, and glycolipids, resulting in the regulation of various cells via glycoconjugates and their physiological and pathological phenomenon through the host-pathogen interactions and cell-cell communications. Here, we review the multiple lectins from marine resources including fishes and sea invertebrate in terms of their structure-activity relationships and molecular evolution. Especially, we focus on the unique structural properties and molecular evolution of C-type lectins, galectin, F-type lectin, and rhamnose-binding lectin families. PMID:22312473

  12. Use of amaranthus leucocarpus lectin to differentiate cervical dysplasia (CIN).

    PubMed

    Santaella-Verdejo, Arturo; Gallegos, Belem; Pérez-Campos, Eduardo; Hernández, Pedro; Zenteno, Edgar

    2007-01-01

    Alterations in O-glycosylation of proteins in cell surfaces can originate disorder in cellular function, as well as in cell transformation and tumoral differentiation. In this work, we investigate changes in O-glycosylation in cervical intraepithelial dysplasia (CIN) at different stages of differentiation (CIN I, CIN II, and CIN III) using lectins specific for O-glycosidically linked glycans. Twenty cases with CIN I, CIN II, and CIN III dysplasias each, and 20 normal cases were studied by lectin histochemistry and evaluated under optical microscopy. The lectins from Glycine max and Griffonia simplicifolia showed no differences in their recognition pattern among the different CIN stages and normal tissue. Dolichos Biflorus lectin recognized CIN I dysplasia. Lectin from Amaranthus leucocarpus showed increased reactivity in the presence of CIN II dysplasia, compared with CIN I and CIN III. These results suggest that subtle modifications in the O-glycosylation pattern could be considered in diagnosis or prognosis of cervical precancerous stages. PMID:17516251

  13. MMBL proteins: from lectin to bacteriocin.

    PubMed

    Ghequire, Maarten G K; Loris, Remy; De Mot, Ren

    2012-12-01

    Arguably, bacteriocins deployed in warfare among related bacteria are among the most diverse proteinacous compounds with respect to structure and mode of action. Identification of the first prokaryotic member of the so-called MMBLs (monocot mannose-binding lectins) or GNA (Galanthus nivalis agglutinin) lectin family and discovery of its genus-specific killer activity in the Gram-negative bacteria Pseudomonas and Xanthomonas has added yet another kind of toxin to this group of allelopathic molecules. This novel feature is reminiscent of the protective function, on the basis of antifungal, insecticidal, nematicidal or antiviral activity, assigned to or proposed for several of the eukaryotic MMBL proteins that are ubiquitously distributed among monocot plants, but also occur in some other plants, fish, sponges, amoebae and fungi. Direct bactericidal activity can also be effected by a C-type lectin, but this is a mammalian protein that limits mucosal colonization by Gram-positive bacteria. The presence of two divergent MMBL domains in the novel bacteriocins raises questions about task distribution between modules and the possible role of carbohydrate binding in the specificity of target strain recognition and killing. Notably, bacteriocin activity was also demonstrated for a hybrid MMBL protein with an accessory protease-like domain. This association with one or more additional modules, often with predicted peptide-hydrolysing or -binding activity, suggests that additional bacteriotoxic proteins may be found among the diverse chimaeric MMBL proteins encoded in prokaryotic genomes. A phylogenetic survey of the bacterial MMBL modules reveals a mosaic pattern of strongly diverged sequences, mainly occurring in soil-dwelling and rhizosphere bacteria, which may reflect a trans-kingdom acquisition of the ancestral genes. PMID:23176516

  14. Isolation and characterization of Lentinus edodes (Berk.) singer extracellular lectins.

    PubMed

    Tsivileva, O M; Nikitina, V E; Loshchinina, E A

    2008-10-01

    Lectin preparations have been isolated and purified from the culture liquid of the xylotrophic basidiomycete Lentinus edodes (Berk.) Singer [Lentinula edodes (Berk.) Pegler]. The culture of L. edodes F-249 synthesizes two extracellular lectins different in composition and physicochemical properties. Extracellular lectin L1 from L. edodes is a glycoprotein of mono-subunit structure with molecular weight of 43 kD. L1 is comprised of 10.5 +/- 1.0% (w/w) carbohydrates represented by glucose (Glc). Extracellular lectin L2 is a proteoglycan of mono-subunit structure with molecular weight of 37 kD. L2 is comprised of 90.3 +/- 1.0% (w/w) carbohydrates represented by Glc (73% of the total mass of the carbohydrate moiety of the lectin molecule) and galactose (Gal) (27% of the total mass of the carbohydrate part of the lectin molecule). The content of Asn in L2 is high, i.e. 42% (w/w) of total amino acids. This fact along with the composition of the carbohydrate part of the molecule (Glc + Gal) allows one to assign L2 to N-asparagine-bound proteins. Both lectins are specific to D-Gal and lactose (Lac) at an equal for L1 and L2 minimal inhibiting concentration of these carbohydrates (2.08 mM Gal and 8.33 mM Lac). Other carbohydrates to which the lectins show affinity are different for the two lectins: Rha (4.16 mM) for L1 and Ara (4.16 mM) and mannitol (8.33 mM) for L2. The purified extracellular lectins of L. edodes are highly selective at recognition of definite structures on the surface of trypsinized rabbit erythrocytes and do not react with the erythrocytes of other animals and humans. PMID:18991563

  15. Fluorescent carbohydrate probes for cell lectins

    NASA Astrophysics Data System (ADS)

    Galanina, Oxana; Feofanov, Alexei; Tuzikov, Alexander B.; Rapoport, Evgenia; Crocker, Paul R.; Grichine, Alexei; Egret-Charlier, Marguerite; Vigny, Paul; Le Pendu, Jacques; Bovin, Nicolai V.

    2001-09-01

    Fluorescein labeled carbohydrate (Glyc) probes were synthesized as analytical tools for the study of cellular lectins, i.e. SiaLe x-PAA-flu, Sia 2-PAA-flu, GlcNAc 2-PAA-flu, LacNAc-PAA-flu and a number of similar ones, with PAA a soluble polyacrylamide carrier. The binding of SiaLe x-PAA-flu was assessed using CHO cells transfected with E-selectin, and the binding of Sia 2-PAA-flu was assessed by COS cells transfected with siglec-9. In flow cytometry assays, the fluorescein probes demonstrated a specific binding to the lectin-transfected cells that was inhibited by unlabeled carbohydrate ligands. The intense binding of SiaLe x-PAA- 3H to the E-selectin transfected cells and the lack of binding to both native and permeabilized control cells lead to the conclusion that the polyacrylamide carrier itself and the spacer arm connecting the carbohydrate moiety with PAA did not contribute anymore to the binding. Tumors were obtained from nude mice by injection of CHO E-selectin or mock transfected cells. The fluorescent SiaLe x-PAA-flu probe could bind to the tumor sections from E-selectin positive CHO cells, but not from the control ones. Thus, these probes can be used to reveal specifically the carbohydrate binding sites on cells in culture as well as cells in tissue sections. The use of the confocal spectral imaging technique with Glyc-PAA-flu probes offered the unique possibility to detect lectins in different cells, even when the level of lectin expression was rather low. The confocal mode of spectrum recording provided an analysis of the probe localization with 3D submicron resolution. The spectral analysis (as a constituent part of the confocal spectral imaging technique) enabled interfering signals of the probe and intrinsic cellular fluorescence to be accurately separated, the distribution of the probe to be revealed and its local concentration to be measured.

  16. Sentential Complementation--An Overview.

    ERIC Educational Resources Information Center

    Nuessel, Frank H., Jr.

    A review of traditional and transformational studies on the phenomenon of sentential complementation (noun clauses) reveals many areas of agreement. Although some adherents of generative grammar may have occasionally obscured this aspect because of the offensive nature of their criticism of other modes of analysis, it is seen that, in several…

  17. Improvisation: A Complement to Curriculum

    ERIC Educational Resources Information Center

    Ronald, Green A.

    2006-01-01

    With the growth of standardized assessment benchmarks in both the public and private paradigms, testing performance matters to institutions more than ever. In an attempt to take as many hindering variables out of this process, such as test anxiety, socioeconomic influences, and latency in cognition, Improvisation: A Complement to Curriculum seeks…

  18. Network Analysis Reveals the Recognition Mechanism for Mannose-binding Lectins

    NASA Astrophysics Data System (ADS)

    Zhao, Yunjie; Jian, Yiren; Zeng, Chen; Computational Biophysics Lab Team

    The specific carbohydrate binding of mannose-binding lectin (MBL) protein in plants makes it a very useful molecular tool for cancer cell detection and other applications. The biological states of most MBL proteins are dimeric. Using dynamics network analysis on molecular dynamics (MD) simulations on the model protein of MBL, we elucidate the short- and long-range driving forces behind the dimer formation. The results are further supported by sequence coevolution analysis. We propose a general framework for deciphering the recognition mechanism underlying protein-protein interactions that may have potential applications in signaling pathways.

  19. Complement activation in the context of stem cells and tissue repair

    PubMed Central

    Schraufstatter, Ingrid U; Khaldoyanidi, Sophia K; DiScipio, Richard G

    2015-01-01

    The complement pathway is best known for its role in immune surveillance and inflammation. However, its ability of opsonizing and removing not only pathogens, but also necrotic and apoptotic cells, is a phylogenetically ancient means of initiating tissue repair. The means and mechanisms of complement-mediated tissue repair are discussed in this review. There is increasing evidence that complement activation contributes to tissue repair at several levels. These range from the chemo-attraction of stem and progenitor cells to areas of complement activation, to increased survival of various cell types in the presence of split products of complement, and to the production of trophic factors by cells activated by the anaphylatoxins C3a and C5a. This repair aspect of complement biology has not found sufficient appreciation until recently. The following will examine this aspect of complement biology with an emphasis on the anaphylatoxins C3a and C5a. PMID:26435769

  20. Reincarnation of ancient links between coagulation and complement.

    PubMed

    Conway, E M

    2015-06-01

    Throughout evolution, organisms have developed means to contain wounds by simultaneously limiting bleeding and eliminating pathogens and damaged host cells via the recruitment of innate defense mechanisms. Disease emerges when there is unchecked activation of innate immune and/or coagulation responses. A key component of innate immunity is the complement system. Concurrent excess activation of coagulation and complement - two major blood-borne proteolytic pathways - is evident in numerous diseases, including atherosclerosis, diabetes, venous thromboembolic disease, thrombotic microangiopathies, arthritis, cancer, and infectious diseases. Delineating the cross-talk between these two cascades will uncover novel therapeutic insights. PMID:26149013

  1. Characterization of the complement sensitivity of paroxysmal nocturnal hemoglobinuria erythrocytes.

    PubMed Central

    Parker, C J; Wiedmer, T; Sims, P J; Rosse, W F

    1985-01-01

    The affected erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH II and PNH III cells) are abnormally sensitive to complement-mediated lysis. Normal human erythrocytes chemically modified by treatment with 2-amino-ethylisothiouronium bromide (AET) have been used as models for PNH cells inasmuch as they also exhibit an enhanced susceptibility to complement. To investigate the bases for the greater sensitivity of these abnormal cells to complement-mediated lysis, we compared binding of C3 and constituents of the membrane attack complex to normal, PNH II, PNH III, and AET-treated cells after classical pathway activation by antibody and fluid-phase activation by cobra venom factor complexes. When whole serum complement was activated by antibody, there was increased binding of C3 and C9 to PNH II, PNH III, and AET-treated cells, although the binding of these complement components to PNH II and PNH III cells was considerably greater than their binding to the AET-treated cells. In addition, all of the abnormal cell types showed a greater degree of lysis per C9 bound than did the normal erythrocytes. PNH III and AET-treated cells were readily lysed by fluid-phase activation of complement, whereas normal and PNH II erythrocytes were not susceptible to bystander lysis. The greater hemolysis of PNH III and AET-treated cells in this reactive lysis system was due to a quantitative increase in binding of constituents of the membrane attack complex. This more efficient binding of the terminal components after fluid-phase activation of whole serum complement was not mediated by cell-bound C3 fragments. These investigations demonstrate that the molecular events that characterize the enhanced susceptibility of PNH II, PNH III, and AET-treated erythrocytes to complement-mediated lysis are heterogeneous. Images PMID:4008653

  2. Exploring the Innate Immune System: Using Complement-Medicated Cell Lysis in the Classroom

    ERIC Educational Resources Information Center

    Fuller, Kevin G.

    2008-01-01

    The protein complement pathway comprises an important part of the innate immunity. The use of serum to demonstrate complement-mediated destruction across a series of bacterial dilutions allows an instructor to introduce a number of important biological concepts such as bacterial growth, activation cascades, and adaptive versus innate immunity.

  3. Collectins and fungal pathogens: roles of surfactant proteins and mannose binding lectin in host resistance.

    PubMed

    Brummer, Elmer; Stevens, David A

    2010-02-01

    Collectins, collagenous carbohydrate-binding proteins (C-type lectins), are recognized as important factors in non-specific innate immune responses to pathogens. By binding surface carbohydrate structures of pathogens, collectins modify the interaction between pathogens and the immune system. We review the structure of the lung surfactant proteins (SP) SP-A and SP-D, and the serum collectins, mannose binding lectins; the binding of these collectins to pathogen associated molecular patterns or ligands on pathogenic fungi; and the effect of collectin binding to opportunistic and primary fungal pathogens on the interaction with host defense cells, which can result in enhancement or inhibition of resistance. The result of collectin binding to opportunistic fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Pneumocystis) or primary fungal pathogens (Blastomyces dermatitidis, Coccidioides, Histoplasma capsulatum, Paracoccidioides brasiliensis) on interaction with host defense cells relative to complement fixation, phagocytosis, and stimulation of cytokine/chemokine production is reviewed. Increased understanding of these relationships in future will help understand fungal pathogenesis and host defenses against mycoses. PMID:19639514

  4. Complement activation patterns in atypical haemolytic uraemic syndrome during acute phase and in remission.

    PubMed

    Volokhina, E B; Westra, D; van der Velden, T J A M; van de Kar, N C A J; Mollnes, T E; van den Heuvel, L P

    2015-08-01

    Atypical haemolytic uraemic syndrome (aHUS) is associated with (genetic) alterations in alternative complement pathway. Nevertheless, comprehensive evidence that the complement system in aHUS patients is more prone to activation is still lacking. Therefore, we performed a thorough analysis of complement activation in acute phase and in remission of this disease. Complement activation patterns of the aHUS patients in acute phase and in remission were compared to those of healthy controls. Background levels of complement activation products C3b/c, C3bBbP and terminal complement complex (TCC) were measured using enzyme-linked immunosorbent assay (ELISA) in ethylenediamine tetraacetic acid (EDTA) plasma. In vitro-triggered complement activation in serum samples was studied using zymosan-coating and pathway-specific assay. Furthermore, efficiencies of the C3b/c, C3bBbP and TCC generation in fluid phase during spontaneous activation were analysed. Patients with acute aHUS showed elevated levels of C3b/c (P < 0·01), C3bBbP (P < 0·0001) and TCC (P < 0·0001) in EDTA plasma, while values of patients in remission were normal, compared to those of healthy controls. Using data from a single aHUS patient with complement factor B mutation we illustrated normalization of complement activation during aHUS recovery. Serum samples from patients in remission showed normal in vitro patterns of complement activation and demonstrated normal kinetics of complement activation in the fluid phase. Our data indicate that while aHUS patients have clearly activated complement in acute phase of the disease, this is not the case in remission of aHUS. This knowledge provides important insight into complement regulation in aHUS and may have an impact on monitoring of these patients, particularly when using complement inhibition therapy. PMID:25079699

  5. Cloning and characterization of root-specific barley lectin

    SciTech Connect

    Lerner, D.R.; Raikhel, N.V. )

    1989-09-01

    Cereal lectins are a class of biochemically and antigenically related proteins localized in a tissue-specific manner in embryos and adult plants. To study the specificity of lectin expression, a barley (Hordeum vulgare L.) embryo cDNa library was constructed and a clone (BLc3) for barley lectin was isolated. BLc3 is 972 nucleotides long and includes an open reading frame of 212 amino acids. The deduced amino acid sequence contains a putative signal peptide of 26 amino acid residues followed by a 186 amino acid polypeptide. This polypeptide has 95% sequence identity to the antigenically indistinguishable wheat germ agglutinin isolectin-B (WGA-B) suggesting that BLc3 encodes barley lectin. Further evidence that BLc3 encodes barley lectin was obtained by immunoprecipitation of the in vitro translation products of BLc3 RNA transcripts and barley embryo poly(A{sup +}) RNA. In situ hybridizations with BLc3 showed that barley lectin gene expression is confined to the outermost cell layers of both embryonic and adult root tips. On Northern blots, BLc3 hybridizes to a 1.0 kilobyte mRNA in poly(A{sup +}) RNA from both embryos and root tips. We suggest, on the basis of immunoblot experiments, that barley lectin is synthesized as a glycosylated precursor and processed by removal of a portion of the carboxyl terminus including the single N-linked glycosylation site.

  6. Lectin domains at the frontiers of plant defense.

    PubMed

    Lannoo, Nausicaä; Van Damme, Els J M

    2014-01-01

    Plants are under constant attack from pathogens and herbivorous insects. To protect and defend themselves, plants evolved a multi-layered surveillance system, known as the innate immune system. Plants sense their encounters upon perception of conserved microbial structures and damage-associated patterns using cell-surface and intracellular immune receptors. Plant lectins and proteins with one or more lectin domains represent a major part of these receptors. The whole group of plant lectins comprises an elaborate collection of proteins capable of recognizing and interacting with specific carbohydrate structures, either originating from the invading organisms or from damaged plant cell wall structures. Due to the vast diversity in protein structures, carbohydrate recognition domains and glycan binding specificities, plant lectins constitute a very diverse protein superfamily. In the last decade, new types of nucleocytoplasmic plant lectins have been identified and characterized, in particular lectins expressed inside the nucleus and the cytoplasm of plant cells often as part of a specific plant response upon exposure to different stress factors or changing environmental conditions. In this review, we provide an overview on plant lectin motifs used in the constant battle against pathogens and predators during plant defenses. PMID:25165467

  7. Extensive amino acid sequence homologies between animal lectins

    SciTech Connect

    Paroutaud, P.; Levi, G.; Teichberg, V.I.; Strosberg, A.D.

    1987-09-01

    The authors have established the amino acid sequence of the ..beta..-D-galactoside binding lectin from the electric eel and the sequences of several peptides from a similar lectin isolated from human placenta. These sequences were compared with the published sequences of peptides derived from the ..beta..-D-galactoside binding lectin from human lung and with sequences deduced from cDNAs assigned to the ..beta..-D-galactoside binding lectins from chicken embryo skin and human hepatomas. Significant homologies were observed. One of the highly conserved regions that contains a tryptophan residue and two glutamic acid resides is probably part of the ..beta..-D-galactoside binding site, which, on the basis of spectroscopic studies of the electric eel lectin, is expected to contain such residues. The similarity of the hydropathy profiles and the predicted secondary structure of the lectins from chicken skin and electric eel, in spite of differences in their amino acid sequences, strongly suggests that these proteins have maintained structural homologies during evolution and together with the other ..beta..-D-galactoside binding lectins were derived form a common ancestor gene.

  8. Lectin domains at the frontiers of plant defense

    PubMed Central

    Lannoo, Nausicaä; Van Damme, Els J. M.

    2014-01-01

    Plants are under constant attack from pathogens and herbivorous insects. To protect and defend themselves, plants evolved a multi-layered surveillance system, known as the innate immune system. Plants sense their encounters upon perception of conserved microbial structures and damage-associated patterns using cell-surface and intracellular immune receptors. Plant lectins and proteins with one or more lectin domains represent a major part of these receptors. The whole group of plant lectins comprises an elaborate collection of proteins capable of recognizing and interacting with specific carbohydrate structures, either originating from the invading organisms or from damaged plant cell wall structures. Due to the vast diversity in protein structures, carbohydrate recognition domains and glycan binding specificities, plant lectins constitute a very diverse protein superfamily. In the last decade, new types of nucleocytoplasmic plant lectins have been identified and characterized, in particular lectins expressed inside the nucleus and the cytoplasm of plant cells often as part of a specific plant response upon exposure to different stress factors or changing environmental conditions. In this review, we provide an overview on plant lectin motifs used in the constant battle against pathogens and predators during plant defenses. PMID:25165467

  9. The insecticidal activity of recombinant garlic lectins towards aphids.

    PubMed

    Fitches, Elaine; Wiles, Duncan; Douglas, Angela E; Hinchliffe, Gareth; Audsley, Neil; Gatehouse, John A

    2008-10-01

    The heterodimeric and homodimeric garlic lectins ASAI and ASAII were produced as recombinant proteins in the yeast Pichia pastoris. The proteins were purified as functional dimeric lectins, but underwent post-translational proteolysis. Recombinant ASAII was a single homogenous polypeptide which had undergone C-terminal processing similar to that occurring in planta. The recombinant ASAI was glycosylated and subject to variable and heterogenous proteolysis. Both lectins showed insecticidal effects when fed to pea aphids (Acyrthosiphon pisum) in artificial diet, ASAII being more toxic than ASAI at the same concentration. Acute toxicity (mortality at < or =48 h exposure; similar timescale to starvation) was only apparent at the highest lectin concentrations tested (2.0 mg ml(-)1), but dose-dependent chronic toxicity (mortality at >3d exposure) was observed over the concentration range 0.125-2.0 mg ml(-1). The recombinant lectins caused mortality in both symbiotic and antibiotic-treated aphids, showing that toxicity is not dependent on the presence of the bacterial symbiont (Buchnera aphidicola), or on interaction with symbiont proteins, such as the previously identified lectin "receptor" symbionin. A pull-down assay coupled with peptide mass fingerprinting identified two abundant membrane-associated aphid gut proteins, alanyl aminopeptidase N and sucrase, as "receptors" for lectin binding. PMID:18707000

  10. Decidual expression and localization of human surfactant protein SP-A and SP-D, and complement protein C1q.

    PubMed

    Madhukaran, Shanmuga Priyaa; Kishore, Uday; Jamil, Kaiser; Choolani, Mahesh; Lu, Jinhua

    2015-08-01

    Surfactant proteins SP-A and SP-D, and complement protein C1q are soluble innate immune pattern recognizing molecules. SP-A, SP-D and C1q have an overall similar structure composed of an N-terminal triple-helical collagen region that is followed by a trimeric globular domain. While SP-A and SP-D belong to the collectin family (collagen containing lectin), C1q is the first recognition subcomponent of the classical pathway of the complement system. Recently, SP-A, SP-D and C1q have been considered to play important roles in early and late pregnancy. However, their expression in early human decidua has not been examined. Here, we investigated whether SP-A, SP-D and C1q are expressed within first trimester decidua in humans and their expression is associated with trophoblasts and decidual stromal cells. Decidual samples from women undergoing elective vaginal termination of pregnancy during first trimester were obtained from 25 subjects. Immunohistochemical studies using anti-human SP-A, anti-human SP-D and anti-human C1q antibodies were performed on decidual tissue sections along with anti-vimentin and cytokeratin-7 antibodies to identify stromal cells and trophoblasts. The expression was also examined by immunostaining and PCR using decidual and stromal cells. C1q expression was significantly higher when compared to SP-A and SP-D in the first trimester human decidua. Double immunostaining revealed that all stromal cells and trophoblasts expressed SP-A, SP-D and C1q, while only few invasive trophoblasts expressed C1q. Thus, expression of SP-A, SP-D and C1q in human decidua during first trimester suggests potential role of SP-A, SP-D and C1q during the early stages of pregnancy including implantation, trophoblast invasion and placental development. PMID:25829244

  11. A Molecular Insight into Complement Evasion by the Staphylococcal Complement Inhibitor Protein Family1

    PubMed Central

    Ricklin, Daniel; Tzekou, Apostolia; Garcia, Brandon L.; Hammel, Michal; McWhorter, William J.; Sfyroera, Georgia; Wu, You-Qiang; Holers, V. Michael; Herbert, Andrew P.; Barlow, Paul N.; Geisbrecht, Brian V.; Lambris, John D.

    2010-01-01

    Staphylococcus aureus possesses an impressive arsenal of complement evasion proteins that help the bacterium escape attack of the immune system. The staphylococcal complement inhibitor (SCIN) protein exhibits a particularly high potency and was previously shown to block complement by acting at the level of the C3 convertases. However, many details about the exact binding and inhibitory mechanism remained unclear. In this study, we demonstrate that SCIN directly binds with nanomolar affinity to a functionally important area of C3b that lies near the C terminus of its β-chain. Direct competition of SCIN with factor B for C3b slightly decreased the formation of surface-bound convertase. However, the main inhibitory effect can be attributed to an entrapment of the assembled convertase in an inactive state. Whereas native C3 is still able to bind to the blocked convertase, no generation and deposition of C3b could be detected in the presence of SCIN. Furthermore, SCIN strongly competes with the binding of factor H to C3b and influences its regulatory activities: the SCIN-stabilized convertase was essentially insensitive to decay acceleration by factor H and the factor I- and H-mediated conversion of surface-bound C3b to iC3b was significantly reduced. By targeting a key area on C3b, SCIN is able to block several essential functions within the alternative pathway, which explains the high potency of the inhibitor. Our findings provide an important insight into complement evasion strategies by S. aureus and may act as a base for further functional studies. PMID:19625656

  12. Molecular Modeling of Lectin-Like Protein from Acacia farnesiana Reveals a Possible Anti-Inflammatory Mechanism in Carrageenan-Induced Inflammation

    PubMed Central

    Abrantes, Vanessa Erika Ferreira; Matias da Rocha, Bruno Anderson; Batista da Nóbrega, Raphael; Silva-Filho, José Caetano; Teixeira, Claudener Souza; Cavada, Benildo Sousa; Gadelha, Carlos Alberto de Almeida; Ferreira, Sergio Henrique; Figueiredo, Jozi Godoy; Santi-Gadelha, Tatiane; Delatorre, Plinio

    2013-01-01

    Acacia farnesiana lectin-like protein (AFAL) is a chitin-binding protein and has been classified as phytohaemagglutinin from Phaseolus vulgaris (PHA). Legume lectins are examples for structural studies, and this family of proteins shows a remarkable conservation in primary, secondary, and tertiary structures. Lectins have ability to reduce the effects of inflammation caused by phlogistic agents, such as carrageenan (CGN). This paper explains the anti-inflammatory activity of AFAL through structural comparison with anti-inflammatory legume lectins. The AFAL model was obtained by molecular modeling and molecular docking with glycan and carrageenan were performed to explain the AFAL structural behavior and biological activity. Pisum sativum lectin was the best template for molecular modeling. The AFAL structure model is folded as a β sandwich. The model differs from template in loop regions, number of β strands and carbohydrate-binding site. Carrageenan and glycan bind to different sites on AFAL. The ability of AFAL binding to carrageenan can be explained by absence of the sixth β-strand (posterior β sheets) and two β strands in frontal region. AFAL can inhibit pathway inflammatory process by carrageenan injection by connecting to it and preventing its entry into the cell and triggers the reaction. PMID:24490151

  13. CD44 Antibody Inhibition of Macrophage Phagocytosis Targets Fcγ Receptor- and Complement Receptor 3-Dependent Mechanisms.

    PubMed

    Amash, Alaa; Wang, Lin; Wang, Yawen; Bhakta, Varsha; Fairn, Gregory D; Hou, Ming; Peng, Jun; Sheffield, William P; Lazarus, Alan H

    2016-04-15

    Targeting CD44, a major leukocyte adhesion molecule, using specific Abs has been shown beneficial in several models of autoimmune and inflammatory diseases. The mechanisms contributing to the anti-inflammatory effects of CD44 Abs, however, remain poorly understood. Phagocytosis is a key component of immune system function and can play a pivotal role in autoimmune states where CD44 Abs have shown to be effective. In this study, we show that the well-known anti-inflammatory CD44 Ab IM7 can inhibit murine macrophage phagocytosis of RBCs. We assessed three selected macrophage phagocytic receptor systems: Fcγ receptors (FcγRs), complement receptor 3 (CR3), and dectin-1. Treatment of macrophages with IM7 resulted in significant inhibition of FcγR-mediated phagocytosis of IgG-opsonized RBCs. The inhibition of FcγR-mediated phagocytosis was at an early stage in the phagocytic process involving both inhibition of the binding of the target RBC to the macrophages and postbinding events. This CD44 Ab also inhibited CR3-mediated phagocytosis of C3bi-opsonized RBCs, but it did not affect the phagocytosis of zymosan particles, known to be mediated by the C-type lectin dectin-1. Other CD44 Abs known to have less broad anti-inflammatory activity, including KM114, KM81, and KM201, did not inhibit FcγR-mediated phagocytosis of RBCs. Taken together, these findings demonstrate selective inhibition of FcγR and CR3-mediated phagocytosis by IM7 and suggest that this broadly anti-inflammatory CD44 Ab inhibits these selected macrophage phagocytic pathways. The understanding of the immune-regulatory effects of CD44 Abs is important in the development and optimization of therapeutic strategies for the potential treatment of autoimmune conditions. PMID:26944929

  14. MEMBRANE ATTACK BY COMPLEMENT: THE ASSEMBLY AND BIOLOGY OF TERMINAL COMPLEMENT COMPLEXES

    PubMed Central

    Tegla, Cosmin A.; Cudrici, Cornelia; Patel, Snehal; Trippe, Richard; Rus, Violeta; Niculescu, Florin; Rus, Horea

    2013-01-01

    Complement system activation plays an important role in both innate and acquired immunity. Activation of complement and the subsequent formation of C5b-9 channels (the membrane attack complex) on cell membranes lead to cell death. However, when the number of channels assembled on the surface of nucleated cells is limited, sublytic C5b-9 can induce cell cycle progression by activating signal transduction pathways and transcription factors and inhibiting apoptosis. This induction by C5b-9 is dependent upon the activation of the phosphatidylinositol 3-kinase/Akt/FOXO1 and ERK1 pathways in a Gi protein-dependent manner. C5b-9 induces sequential activation of CDK4 and CDK2, enabling the G1/S-phase transition and cellular proliferation. In addition, it induces RGC-32, a novel gene that plays a role in cell cycle activation by interacting with Akt and the cyclin B1-CDC2 complex. C5b-9 also inhibits apoptosis by inducing the phosphorylation of Bad and blocking the activation of FLIP, caspase-8, and Bid cleavage. Thus, sublytic C5b-9 plays an important role in cell activation, proliferation, and differentiation, thereby contributing to the maintenance of cell and tissue homeostasis. PMID:21850539

  15. In vivo biosynthetic studies of the Dolichos biflorus seed lectin

    SciTech Connect

    Quinn, J.M.; Etzler, M.E. )

    1989-12-01

    The in vivo biosynthesis of the Dolichos biflorus seed lectin was studied by pulse-chase labeling experiments using ({sup 35}S)methionine and ({sup 14}C)glucosamine. These studies demonstrate that each of the two mature lectin subunit types are derived by the processing of separate glycosylated precursors. The appearance of the precursor to subunit I before the precursor to subunit II supports the possibility raised by previous studies that both subunit types of this lectin may originate from a single gene product.

  16. Complement Activation in Placental Malaria

    PubMed Central

    McDonald, Chloe R.; Tran, Vanessa; Kain, Kevin C.

    2015-01-01

    Sixty percent of all pregnancies worldwide occur in malaria endemic regions. Pregnant women are at greater risk of malaria infection than their non-pregnant counterparts and have a higher risk of adverse birth outcomes including low birth weight resulting from intrauterine growth restriction and/or preterm birth. The complement system plays an essential role in placental and fetal development as well as the host innate immune response to malaria infection. Excessive or dysregulated complement activation has been associated with the pathobiology of severe malaria and with poor pregnancy outcomes, dependent and independent of infection. Here we review the role of complement in malaria and pregnancy and discuss its part in mediating altered placental angiogenesis, malaria-induced adverse birth outcomes, and disruptions to the in utero environment with possible consequences on fetal neurodevelopment. A detailed understanding of the mechanisms underlying adverse birth outcomes, and the impact of maternal malaria infection on fetal neurodevelopment, may lead to biomarkers to identify at-risk pregnancies and novel therapeutic interventions to prevent these complications. PMID:26733992

  17. Hydroxyl radical scavengers inhibit human lectin-dependent cellular cytotoxicity.

    PubMed Central

    Melinn, M; McLaughlin, H

    1986-01-01

    The role of oxygen-derived free radicals (ODFR) in lectin-dependent cellular cytotoxicity (LDCC) in humans was investigated. The hydroxyl radical traps thiourea, methanol, ethanol and phenol were effective in inhibiting LDCC, as was DABCO, a singlet oxygen quencher. The proposed pathway of hydroxyl radical production in living cells is either an iron catalysed Haber-Weiss reaction or a Fenton reaction. The effect of inhibitors of these pathways was investigated. The superoxide anion scavengers superoxide dismutase, ferricytochrome c and Tiron were without effect. It was shown that Tiron inhibits the lucigenin-amplified chemiluminescence produced by the action of xanthine oxidase, and also the lucigenin-amplified chemiluminescence produced by activated PMN, suggesting that this agent (Tiron) scavenges intracellular superoxide anion. Catalase gave slight inhibition of LDCC only. The ferric iron chelator desferrioxamine gave no protection of the target cells, while the ferrous chelator, 1,10-phenanthroline, inhibited LDCC and partially prevented the detection of hydroxyl radicals generated by the Fe2+-H2O2 system. Cibacron blue, an agent that inhibits NAD(P)H linked enzymes, also inhibited LDCC. The cyclo-oxygenase inhibitors indomethacin and salicylate were without effect, while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) inhibited cytolysis. None of the LDCC inhibitors was cytotoxic to the effector cells or to the target cells, neither did they inhibit lymphocyte-target binding. The findings would suggest that hydroxyl radicals have a role to play in human T-cell mediated cytolysis, either as the active lytic agent or as an epiphenomenon. PMID:3011654

  18. The enzymatic lectin of field bean (Dolichos lablab): salt assisted lectin-sugar interaction.

    PubMed

    Rao, Devavratha H; Vishweshwaraiah, Yashavanth L; Gowda, Lalitha R

    2012-11-01

    Field bean seed contains a Gal/GalNAc lectin (DLL-II) that exhibits associated polyphenol oxidase (PPO) activity and does not bind to its sugar specific affinity matrix. The molecular basis for this lack of binding is not known. The DLL-II gene was therefore cloned and its sequence analyzed. A conserved aromatic residue in the sugar binding site required for a stacking interaction with the apolar backbone of Gal is replaced by His in DLL-II, which explains the lack of binding. However, specific sugar binding is achieved by including (NH₄)₂SO₄ in the buffer. Interestingly two other salts of the Hofmeister series, K₂HPO₄ and Na₂SO₄ also assist binding to immobilized galactose. In the presence of (NH₄)₂SO₄ the surface hydrophobicity of DLL-II and dissociation constant for 8-anilino 1-naphthalene sulfonic acid were enhanced three fold. This increased surface hydrophobicity in the presence of salt is probably the cause for assisted sugar binding in legume lectins that lack aromatic stacking interactions. Accordingly, two other lectins which lack the conserved aromatic residue show similar salt assisted binding. The salt concentrations required for Gal/GalNAc binding are not physiologically relevant in vivo, suggesting that the role of DLL-II per se in the seed is primarily that of a PPO purportedly for plant defense. PMID:22959225

  19. Localization and Characterisation of the MASP-2 binding site in rat ficolin-A: Equivalent binding sites within the collagenous domains of Mannose-binding Lectins and Ficolins

    PubMed Central

    Girija, Umakhanth Venkatraman; Dodds, Alister W.; Roscher, Silke; Reid, Kenneth B. M.; Wallis, Russell

    2008-01-01

    Ficolins and mannan-binding lectins (MBLs) are the first components of the lectin branch of the complement system. They comprise N-terminal collagen-like domains and C-terminal pathogen-recognition domains (fibrinogen-like domains in ficolins and C-type carbohydrate-recognition domains in MBLs), which target surface-exposed N-acetyl groups or mannose-like sugars on microbial cell walls. Binding leads to activation of MBL-associated-serine protease-2 (MASP-2) to initiate complement activation and pathogen neutralisation. Recent studies have shown that MASP-2 binds to a short segment of the collagen-like domain of MBL. However, the interaction between ficolins and MASP-2 is relatively poorly understood. In this study, we show that the MASP-2 binding site on rat ficolin-A is also located within the collagen-like domain and encompasses a conserved motif that is present in both MBLs and ficolins. Characterisation of this motif using site-directed mutagenesis reveals that a lysine residue in the X position of the Gly-X-Y collagen repeat, (Lys56 in ficolin-A) which is present in all ficolins and MBLs known to activate complement, is essential for MASP-2 binding. Adjacent residues also make important contributions to binding as well as to MASP activation probably by stabilizing the local collagen helix. Equivalent binding sites and comparable activation kinetics of MASP-2 suggest that complement activation by ficolins and MBLs proceeds by analogous mechanisms. PMID:17579066

  20. Complement profile and activation mechanisms by different LDL apheresis systems.

    PubMed

    Hovland, Anders; Hardersen, Randolf; Nielsen, Erik Waage; Enebakk, Terje; Christiansen, Dorte; Ludviksen, Judith Krey; Mollnes, Tom Eirik; Lappegård, Knut Tore

    2012-07-01

    Extracorporeal removal of low-density lipoprotein (LDL) cholesterol by means of selective LDL apheresis is indicated in otherwise uncontrolled familial hypercholesterolemia. During blood-biomaterial interaction other constituents than the LDL particles are affected, including the complement system. We set up an ex vivo model in which human whole blood was passed through an LDL apheresis system with one of three different apheresis columns: whole blood adsorption, plasma adsorption and plasma filtration. The concentrations of complement activation products revealed distinctly different patterns of activation and adsorption by the different systems. Evaluated as the final common terminal complement complex (TCC) the whole blood system was inert, in contrast to the plasma systems, which generated substantial and equal amounts of TCC. Initial classical pathway activation was revealed equally for both plasma systems as increases in the C1rs-C1inh complex and C4d. Alternative pathway activation (Bb) was most pronounced for the plasma adsorption system. Although the anaphylatoxins (C3a and C5a) were equally generated by the two plasma separation systems, they were efficiently adsorbed to the plasma adsorption column before the "outlet", whereas they were left free in the plasma in the filtration system. Consequently, during blood-biomaterial interaction in LDL apheresis the complement system is modulated in different manners depending on the device composition. PMID:22373816

  1. A jacalin-related lectin-like gene in wheat is a component of the plant defence system.

    PubMed

    Xiang, Yang; Song, Min; Wei, Zhaoyan; Tong, Jianhua; Zhang, Lixia; Xiao, Langtao; Ma, Zhengqiang; Wang, Yun

    2011-11-01

    Jacalin-related lectins (JRLs) are a subgroup of proteins with one or more jacalin-like lectin domains. Although JRLs are often associated with biotic or abiotic stimuli, their biological functions in plants, as well as their relationships to plant disease resistance, are poorly understood. A mannose-specific JRL (mJRL)-like gene (TaJRLL1) that is mainly expressed in stem and spike and encodes a protein with two jacalin-like lectin domains was identified in wheat. Pathogen infection and phytohormone treatments induced its expression; while application of the salicylic acid (SA) biosynthesis inhibitor paclobutrazol and the jasmonic acid (JA) biosynthesis inhibitor diethyldithiocarbamic acid, respectively, substantially inhibited its expression. Attenuating TaJRLL1 through virus-induced gene silencing increased susceptibility to the facultative fungal pathogen Fusarium graminearum and the biotrophic fungal pathogen Blumeria graminis. Arabidopsis thaliana transformed with TaJRLL1 displayed increased resistance to F. graminearum and Botrytis cinerea. JA and SA levels in transgenic Arabidopsis increased significantly. A loss or increase of disease resistance due to an alteration in TaJRLL1 function was correlated with attenuation or enhancement of the SA- and JA-dependent defence signalling pathways. These results suggest that TaJRLL1 could be a component of the SA- and JA-dependent defence signalling pathways. PMID:21862481

  2. The anticarcinogenic potential of soybean lectin and lunasin.

    PubMed

    de Mejia, Elvira Gonzalez; Bradford, Traliece; Hasler, Clare

    2003-07-01

    Cancer is one of the leading causes of death worldwide, generally exceeded only by cardiovascular disease in the developed world. The number of people diagnosed with cancer within the next few decades is expected to double. There will therefore be increased demand for novel diagnostic and medical therapies that use new non-traditional sources. Soybeans contain a variety of anticarcinogenic phytochemicals. Recently, there has been increased interest in the potential health benefits of bioactive polypeptides and proteins from soybeans, including lunasin and lectins. Lunasin is a polypeptide that arrests cell division and induces apoptosis in malignant cells. Lectins are glycoproteins that selectively bind carbohydrates; lectins are used in medicine in a variety of new applications. Additional research, including clinical trials, should continue to examine and elucidate the therapeutic effects, nutritional benefits, and toxic consequences of commonly ingested soybean lectins and lunasin. PMID:12918876

  3. Structure and Function of Mammalian Carbohydrate-Lectin Interactions

    NASA Astrophysics Data System (ADS)

    Anderson, Kevin; Evers, David; Rice, Kevin G.

    Over the past three decades the field of glycobiology has expanded beyond a basic understanding of the structure and biosynthesis of glycoprotein, proteoglycans, and glycolipids toward a more detailed picture of how these molecules afford communication through binding to mammalian lectins. Although the number of different mammalian lectin domains appears to be finite and even much smaller than early estimates predicated based on the diversity of glycan structures, nature appears capable of using these in numerous combinations to fine tune specificity. The following provides an overview of the major classes of mammalian lectins and discusses their glycan binding specificity. The review provides a snapshot of the field of glycobiology that continues to grow providing an increasing number of examples of biological processes that rely upon glycan-lectin binding.

  4. Molecular cloning of mannose-binding lectins from Clivia miniata.

    PubMed

    Van Damme, E J; Smeets, K; Van Leuven, F; Peumans, W J

    1994-03-01

    Screening of a cDNA library constructed from total RNA isolated from young developing ovaries of Clivia miniata Regel with the amaryllis lectin cDNA clone resulted in the isolation of four different isolectin clones which clearly differ from each other in their nucleotide sequences and hence also in their deduced amino acid sequences. Apparently the lectin is translated from an mRNA of ca. 800 nucleotides encoding a precursor polypeptide of 163 amino acids. Northern blot analysis of total RNA isolated from different tissues of Clivia miniata has shown that the lectin is expressed in most plant tissues with very high lectin mRNA concentrations in the ovary and the seed endosperm. PMID:8193308

  5. Lectins but not antifungal proteins exhibit anti-nematode activity.

    PubMed

    Zhao, S; Guo, Y X; Liu, Q H; Wang, H X; Ng, T B

    2009-09-01

    A variety of lectins and antifungal proteins were tested for toxicity against the plant parasitic nematodes Ditylenchus dipsaci and Heterodera glycines. It was found that lectins from the mushrooms Xylaria hypoxylon, Agrocybe cylindracea and Tricholoma mongolicum (TML-1) were the most potent against D. dipsaci, with EC(50) being 4.7, 9, and 20mg/ml, respectively. Lectins from Pseudostellaria heterophylla, samta tomato, and the mushrooms T. mongolicum (TML-2), Ganoderma lucidum, and Boletus edulis, and antifungal proteins from Ginkgo biloba toward D. dipsaci and pumpkin Cucurbita moschata had much lower anti-nematode potencies and could be considered as inactive for practical purposes. All lectins except that from P.heterophylle were potent against H.glycines. PMID:21784014

  6. Sweet complementarity: the functional pairing of glycans with lectins.

    PubMed

    Gabius, H-J; Manning, J C; Kopitz, J; André, S; Kaltner, H

    2016-05-01

    Carbohydrates establish the third alphabet of life. As part of cellular glycoconjugates, the glycans generate a multitude of signals in a minimum of space. The presence of distinct glycotopes and the glycome diversity are mapped by sugar receptors (antibodies and lectins). Endogenous (tissue) lectins can read the sugar-encoded information and translate it into functional aspects of cell sociology. Illustrated by instructive examples, each glycan has its own ligand properties. Lectins with different folds can converge to target the same epitope, while intrafamily diversification enables functional cooperation and antagonism. The emerging evidence for the concept of a network calls for a detailed fingerprinting. Due to the high degree of plasticity and dynamics of the display of genes for lectins the validity of extrapolations between different organisms of the phylogenetic tree yet is inevitably limited. PMID:26956894

  7. Lectins stain cells differentially in the coral, Montipora capitata

    USGS Publications Warehouse

    Work, Thierry M.; Farah, Yael

    2014-01-01

    A limitation in our understanding of coral disease pathology and cellular pathogenesis is a lack of reagents to characterize coral cells. We evaluated the utility of plant lectins to stain tissues of a dominant coral, Montipora capitata, from Hawaii. Of 22 lectins evaluated, nine of these stained structures in the upper or basal body wall of corals. Specific structures revealed by lectins that were not considered distinct or evident on routine hematoxylin and eosin sections of coral tissues included apical and basal granules in gastrodermis and epidermis, cnidoglandular tract and actinopharynx cell surface membranes, capsules of mature holotrichous isorhizas, and perivitelline and periseminal cells. Plant lectins could prove useful to further our understanding of coral physiology, anatomy, cell biology, and disease pathogenesis.

  8. Correlation between proinflammatory role of a lectin from Typhonium giganteum Engl. and macrophage

    PubMed Central

    Pan, Yaozong; Yu, Hongli; Wu, Hao; Chen, Yeqing; Wang, Kuilong; Liu, Liping; Jin, Yangping; Zhang, Chengchao

    2015-01-01

    Purpose: To analyze the correlation between proinflammatory effects of a lectin from Typhonium giganteum Engl. and macrophage. Methods: T. giganteum lectin (TGL) was extracted from the tuber of T. giganteum and purified, and was then identified by using SDS-PAGE gel electrophoresis in combination with mass spectrometry. The morphologic changes of macrophage after being stimulated by TGL were observed with scanning electron microscopy. The influences of such stimulation on neutrophil migration were evaluated by establishing an in vitro macrophage-neutrophil co-culture migration model. By establishing a rat peritoneal macrophage in vitro cultured model, the effects of TGL stimulation on inflammatory factors TNF-α and IL-1β released by macrophage were analyzed. With p65 as the index, the expressions of the NF-κB signaling pathway in the cytoplasm and nucleus were detected before and after TGL stimulation respectively. Furthermore, we also investigated whether the inhibitor for NF-κB signaling pathway BAY11-7082 can block p65 nuclear translocation. Results: After being stimulated by TGL, macrophage had increased volume, number of pseudopodia and gradually cracked cell membrane, accompanied by evidently induced migration of neutrophils due to released inflammatory factors. As the concentration of TGL varied, NF-κB’s monomer p65 had different expression levels in the cytoplasm and nucleus, while BAY11-7082 can indeed block the nuclear translocation of p65. Conclusions: TGL-induced inflammation was closely related to macrophage mediation. PMID:26617695

  9. Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome

    PubMed Central

    de Jorge, Elena Goicoechea; Harris, Claire L.; Esparza-Gordillo, Jorge; Carreras, Luis; Arranz, Elena Aller; Garrido, Cynthia Abarrategui; López-Trascasa, Margarita; Sánchez-Corral, Pilar; Morgan, B. Paul; de Córdoba, Santiago Rodríguez

    2007-01-01

    Hemolytic uremic syndrome (HUS) is an important cause of acute renal failure in children. Mutations in one or more genes encoding complement-regulatory proteins have been reported in approximately one-third of nondiarrheal, atypical HUS (aHUS) patients, suggesting a defect in the protection of cell surfaces against complement activation in susceptible individuals. Here, we identified a subgroup of aHUS patients showing persistent activation of the complement alternative pathway and found within this subgroup two families with mutations in the gene encoding factor B (BF), a zymogen that carries the catalytic site of the complement alternative pathway convertase (C3bBb). Functional analyses demonstrated that F286L and K323E aHUS-associated BF mutations are gain-of-function mutations that result in enhanced formation of the C3bBb convertase or increased resistance to inactivation by complement regulators. These data expand our understanding of the genetic factors conferring predisposition to aHUS, demonstrate the critical role of the alternative complement pathway in the pathogenesis of aHUS, and provide support for the use of complement-inhibition therapies to prevent or reduce tissue damage caused by dysregulated complement activation. PMID:17182750

  10. Herbal complement inhibitors in the treatment of neuroinflammation: future strategy for neuroprotection.

    PubMed

    Kulkarni, Amod P; Kellaway, Laurie A; Kotwal, Girish J

    2005-11-01

    The upregulated complement system plays a damaging role in disorders of the central nervous system (CNS). The classical and alternate pathways are two major pathways activated in neuroinflammatory disorders such as Alzheimer's disease, multiple sclerosis, traumatic brain injury, spinal cord injury, HIV-associated dementia, Parkinson's disease, and mad cow disease. Failure of currently available anti-inflammatory agents, especially cyclooxygenase inhibitors, in offering significant neuroprotection in large epidemiologic clinical trials of CNS disorders suggests an urgent need for the development of new neuroprotective agents. The positive preclinical outcomes in treating CNS disorders by complement regulatory molecules, such as vaccinia virus complement control protein, suggest the possibility of using complement-inhibitory molecules as neuroprotective agents. Several active ingredients of herbal origin are found to have complement-inhibitory activity. These herbal ingredients along with other anti-inflammatory roles might be useful in treating neuroinflammation associated with CNS disorders. Active ingredients of herbal origin with complement inhibitory ingredients are summarized and classified according to their chemical nature and specificity towards the major pathways activating the complement system. The structure activity relationship of some specific examples is also discussed in this report. This information might be helpful in formulating a natural panacea against complement-mediated neuroinflammation. PMID:16387706

  11. COMPLEMENT-DEPENDENT TRANSPORT OF ANTIGEN INTO B CELL FOLLICLES

    PubMed Central

    Gonzalez, Santiago F.; Lukacs-Kornek, Veronika; Kuligowski, Michael P.; Pitcher, Lisa A.; Degn, Søren E.; Turley, Shannon J.; Carroll, Michael C.

    2011-01-01

    Since the original proposal by Fearon that the Complement System linked innate and adaptive immunity (1), there has been a rapid expansion of studies on this topic. With the advance of intravital imaging, a number of recent papers have revealed an additional novel pathway in which complement C3 and its receptors enhance humoral immunity through delivery of antigen to the B cell compartment. In this review, we will discuss this pathway and highlight several novel exceptions recently found with a model influenza vaccine such as: (a) MBL opsonization of influenza and uptake by macrophages; (b) and capture of virus by dendritic cells residing in the medullary compartment of peripheral lymph nodes. PMID:20724732

  12. A multiplex lectin-channel monitoring method for human serum glycoproteins by quantitative mass spectrometry.

    PubMed

    Ahn, Yeong Hee; Ji, Eun Sun; Shin, Park Min; Kim, Kwang Hoe; Kim, Yong-Sam; Ko, Jeong Heon; Yoo, Jong Shin

    2012-02-01

    A mass profiling method and multiple reaction monitoring (MRM)-based quantitative approach were used to analyze multiple lectin-captured fractions of human serum using different lectins such as aleuria aurantia lectin (AAL), phytohemagglutinin-L(4) (L-PHA), concanavalin A (Con A), and Datura stramonium agglutinin (DSA) to quantitatively monitor protein glycosylation diversity. Each fraction, prepared by multiple lectin-fractionation and tryptic digestion, was analyzed by 1-D LC-MS/MS. Semi-quantitative profiling showed that the list of glycoproteins identified from each lectin-captured fraction is significantly different according to the used lectin. Thus, it was confirmed that the multiplex lectin-channel monitoring (LCM) using multiple lectins is useful for investigating protein glycosylation diversity in a proteome sample. Based on the semi-quantitative mass profiling, target proteins showing lectin-specificity among each lectin-captured fraction were selected and analyzed by the MRM-based method in triplicate using each lectin-captured fraction (average CV 7.9%). The MRM-based analysis for each lectin-captured fraction was similar to those obtained by the profiling experiments. The abundance of each target protein measured varied dramatically, based on the lectin-specificity. The multiplex LCM approach using MRM-based analyses is useful for quantitatively monitoring target protein glycoforms selectively fractionated by multiple lectins. Thus through multiplex LCM rather than single, we could inquire minutely into protein glycosylation states. PMID:22158852

  13. A Lectin from Dioclea violacea Interacts with Midgut Surface of Lutzomyia migonei, Unlike Its Homologues, Cratylia floribunda Lectin and Canavalia gladiata Lectin

    PubMed Central

    Monteiro Tínel, Juliana Montezuma Barbosa; Benevides, Melina Fechine Costa; Frutuoso, Mércia Sindeaux; Rocha, Camila Farias; Arruda, Francisco Vassiliepe Sousa; Vasconcelos, Mayron Alves; Pereira-Junior, Francisco Nascimento; Cajazeiras, João Batista; do Nascimento, Kyria Santiago; Martins, Jorge Luiz; Teixeira, Edson Holanda; Cavada, Benildo Sousa; dos Santos, Ricardo Pires; Lima Pompeu, Margarida Maria

    2014-01-01

    Leishmaniasis is a vector-borne disease transmitted by phlebotomine sand fly. Susceptibility and refractoriness to Leishmania depend on the outcome of multiple interactions that take place within the sand fly gut. Promastigote attachment to sand fly midgut epithelium is essential to avoid being excreted together with the digested blood meal. Promastigote and gut sand fly surface glycans are important ligands in this attachment. The purpose of the present study was to evaluate the interaction of three lectins isolated from leguminous seeds (Diocleinae subtribe), D-glucose and D-mannose-binding, with glycans on Lutzomyia migonei midgut. To study this interaction the lectins were labeled with FITC and a fluorescence assay was performed. The results showed that only Dioclea violacea lectin (DVL) was able to interact with midgut glycans, unlike Cratylia floribunda lectin (CFL) and Canavalia gladiata lectin (CGL). Furthermore, when DVL was blocked with D-mannose the interaction was inhibited. Differences of spatial arrangement of residues and volume of carbohydrate recognition domain (CRD) may be the cause of the fine specificity of DVL for glycans in the surface on Lu. migonei midgut. The findings in this study showed the presence of glycans in the midgut with glucose/mannose residues in its composition and these residues may be important in interaction between Lu. migonei midgut and Leishmania. PMID:25431778

  14. A lectin from Dioclea violacea Interacts with midgut surface of Lutzomyia migonei, unlike its homologues, Cratylia floribunda lectin and Canavalia gladiata lectin.

    PubMed

    Monteiro Tínel, Juliana Montezuma Barbosa; Benevides, Melina Fechine Costa; Frutuoso, Mércia Sindeaux; Rocha, Camila Farias; Arruda, Francisco Vassiliepe Sousa; Vasconcelos, Mayron Alves; Pereira-Junior, Francisco Nascimento; Cajazeiras, João Batista; do Nascimento, Kyria Santiago; Martins, Jorge Luiz; Teixeira, Edson Holanda; Cavada, Benildo Sousa; dos Santos, Ricardo Pires; Lima Pompeu, Margarida Maria

    2014-01-01

    Leishmaniasis is a vector-borne disease transmitted by phlebotomine sand fly. Susceptibility and refractoriness to Leishmania depend on the outcome of multiple interactions that take place within the sand fly gut. Promastigote attachment to sand fly midgut epithelium is essential to avoid being excreted together with the digested blood meal. Promastigote and gut sand fly surface glycans are important ligands in this attachment. The purpose of the present study was to evaluate the interaction of three lectins isolated from leguminous seeds (Diocleinae subtribe), D-glucose and D-mannose-binding, with glycans on Lutzomyia migonei midgut. To study this interaction the lectins were labeled with FITC and a fluorescence assay was performed. The results showed that only Dioclea violacea lectin (DVL) was able to interact with midgut glycans, unlike Cratylia floribunda lectin (CFL) and Canavalia gladiata lectin (CGL). Furthermore, when DVL was blocked with D-mannose the interaction was inhibited. Differences of spatial arrangement of residues and volume of carbohydrate recognition domain (CRD) may be the cause of the fine specificity of DVL for glycans in the surface on Lu. migonei midgut. The findings in this study showed the presence of glycans in the midgut with glucose/mannose residues in its composition and these residues may be important in interaction between Lu. migonei midgut and Leishmania. PMID:25431778

  15. PRELP Protein Inhibits the Formation of the Complement Membrane Attack Complex*

    PubMed Central

    Happonen, Kaisa E.; Fürst, Camilla Melin; Saxne, Tore; Heinegård, Dick; Blom, Anna M.

    2012-01-01

    PRELP is a 58-kDa proteoglycan found in a variety of extracellular matrices, including cartilage and at several basement membranes. In rheumatoid arthritis (RA), the cartilage tissue is destroyed and fragmented molecules, including PRELP, are released into the synovial fluid where they may interact with components of the complement system. In a previous study, PRELP was found to interact with the complement inhibitor C4b-binding protein, which was suggested to locally down-regulate complement activation in joints during RA. Here we show that PRELP directly inhibits all pathways of complement by binding C9 and thereby prevents the formation of the membrane attack complex (MAC). PRELP does not interfere with the interaction between C9 and already formed C5b-8, but inhibits C9 polymerization thereby preventing formation of the lytic pore. The alternative pathway is moreover inhibited already at the level of C3-convertase formation due to an interaction between PRELP and C3. This suggests that PRELP may down-regulate complement attack at basement membranes and on damaged cartilage and therefore limit pathological complement activation in inflammatory disease such as RA. The net outcome of PRELP-mediated complement inhibition will highly depend on the local concentration of other complement modulating molecules as well as on the local concentration of available complement proteins. PMID:22267731

  16. Antifungal activity of lectins against yeast of vaginal secretion

    PubMed Central

    Gomes, Bruno Severo; Siqueira, Ana Beatriz Sotero; de Cássia Carvalho Maia, Rita; Giampaoli, Viviana; Teixeira, Edson Holanda; Arruda, Francisco Vassiliepe Sousa; do Nascimento, Kyria Santiago; de Lima, Adriana Nunes; Souza-Motta, Cristina Maria; Cavada, Benildo Sousa; Porto, Ana Lúcia Figueiredo

    2012-01-01

    Lectins are carbohydrate-binding proteins of non-imune origin. This group of proteins is distributed widely in nature and they have been found in viruses, microorganisms, plants and animals. Lectins of plants have been isolated and characterized according to their chemical, physical-chemical, structural and biological properties. Among their biological activities, we can stress its fungicidal action. It has been previously described the effect of the lectins Dviol, DRL, ConBr and LSL obtained from the seeds of leguminous plants on the growth of yeasts isolated from vaginal secretions. In the present work the experiments were carried out in microtiter plates and the results interpreted by both methods: visual observations and a microplate reader at 530nm. The lectin concentrations varied from 0.5 to 256μg/mL, and the inoculum was established between 65-70% of trammitance. All yeast samples isolated from vaginal secretion were evaluated taxonomically, where were observed macroscopic and microscopic characteristics to each species. The LSL lectin did not demonstrate any antifungal activity to any isolate studied. The other lectins DRL, ConBr and DvioL, showed antifungal potential against yeast isolated from vaginal secretion. These findings offering offer a promising field of investigation to develop new therapeutic strategies against vaginal yeast infections, collaborating to improve women's health. PMID:24031889

  17. Structural analysis of Centrolobium tomentosum seed lectin with inflammatory activity.

    PubMed

    Almeida, Alysson Chaves; Osterne, Vinicius Jose da Silva; Santiago, Mayara Queiroz; Pinto-Junior, Vanir Reis; Silva-Filho, Jose Caetano; Lossio, Claudia Figueiredo; Nascimento, Francisco Lucas Faustino; Almeida, Ricardo Patricio Honorato; Teixeira, Claudener Souza; Leal, Rodrigo Bainy; Delatorre, Plinio; Rocha, Bruno Anderson Matias; Assreuy, Ana Maria Sampaio; Nascimento, Kyria Santiago; Cavada, Benildo Sousa

    2016-04-15

    A glycosylated lectin (CTL) with specificity for mannose and glucose has been detected and purified from seeds of Centrolobium tomentosum, a legume plant from Dalbergieae tribe. It was isolated by mannose-sepharose affinity chromatography. The primary structure was determined by tandem mass spectrometry and consists of 245 amino acids, similar to other Dalbergieae lectins. CTL structures were solved from two crystal forms, a monoclinic and a tetragonal, diffracted at 2.25 and 1.9 Å, respectively. The carbohydrate recognition domain (CRD), metal-binding site and glycosylation site were characterized, and the structural basis for mannose/glucose-binding was elucidated. The lectin adopts the canonical dimeric organization of legume lectins. CTL showed acute inflammatory effect in paw edema model. The protein was subjected to ligand screening (dimannosides and trimannoside) by molecular docking, and interactions were compared with similar lectins possessing the same ligand specificity. This is the first crystal structure of mannose/glucose native seed lectin with proinflammatory activity isolated from the Centrolobium genus. PMID:26946944

  18. Membrane-assisted online renaturation for automated microfluidic lectin blotting.

    PubMed

    He, Mei; Novak, Jan; Julian, Bruce A; Herr, Amy E

    2011-12-14

    Aberrant glycosylation plays a pivotal role in a diverse set of diseases, including cancer. A microfluidic lectin blotting platform is introduced to enable and expedite the identification of protein glycosylation based on protein size and affinity for specific lectins. The integrated multistage assay eliminates manual intervention steps required for slab-gel lectin blotting, increases total assay throughput, limits reagent and sample consumption, and is completed using one instrument. The assay comprises non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by online post-sizing SDS filtration and lectin-based affinity blotting. Important functionality is conferred through both device and assay advances that enable integration of nanoporous membranes flanking a central microchamber to create sub-nanoliter volume compartments that trap SDS-protein complexes and allow electrophoretic SDS removal with buffer exchange. Recapitulation of protein binding for lectin was optimized through quantitative assessment of SDS-treated green fluorescent protein. Immunoglobulin A1 aberrantly glycosylated with galactose-deficient O-glycans was probed in ~6 min using ~3 μL of sample. This new microfluidic lectin blotting platform provides a rapid and automated assay for the assessment of aberrant glycosylation. PMID:22070432

  19. Probing lectin-mucin interactions by isothermal titration microcalorimetry.

    PubMed

    Dam, Tarun K; Brewer, C Fred

    2015-01-01

    Isothermal titration microcalorimetry (ITC) can directly determine the thermodynamic binding parameters of biological molecules including affinity constant, binding stoichiometry, and heat of binding (enthalpy) and indirectly the entropy and free energy of binding. ITC has been extensively used to study the binding of lectins to mono- and oligosaccharides, but limited applications to lectin-glycoprotein interactions. Inherent experimental challenges to ITC include sample precipitation during the experiment and relative high amount of sample required, but careful design of experiments can minimize these problems and allow valuable information to be obtained. For example, the thermodynamics of binding of lectins to multivalent globular and linear glycoproteins (mucins) have been described. The results are consistent with a dynamic binding mechanism in which lectins bind and jump from carbohydrate to carbohydrate epitope in these molecules leading to increased affinity. Importantly, the mechanism of binding of lectins to mucins appears similar to that for a variety of protein ligands binding to DNA. Recent results also show that high affinity lectin-mucin cross-linking interactions are driven by favorable entropy of binding that is associated with the bind and jump mechanism. The results suggest that the binding of ligands to biopolymers, in general, may involve a common mechanism that involves enhanced entropic effects that facilitate binding interactions. PMID:25253134

  20. Unusual complement activation properties of serum immunoglobulins of the pigeon Columba livia.

    PubMed Central

    Koppenheffer, T L; Russell, B A

    1986-01-01

    Complement-mediated lysis of mouse erythrocytes (MRBC) by whole pigeon antisera was found to occur in the presence of magnesium ions alone. The underlying basis for this observation was demonstrated to be the ability of IgM antibodies to activate the alternative pathway of pigeon complement, whereas IgG activates a calcium-dependent pathway possessing an unusually low lytic capacity. In addition to differing from the alternative pathway by requiring both calcium and magnesium ions, the calcium-dependent pathway exhibited higher activity at low temperature and more rapid kinetics of haemolysis. The presence of early acting C1 in pigeon serum was inferred by the selective depletion of calcium-dependent activity which occurred as a result of incubating serum containing only calcium ions with MRBC sensitized with IgG. Under the same conditions, MRBC sensitized with IgM failed to deplete complement activity, indicating that C1 does not participate in complement activation by this isotype. Interestingly, the calcium-dependent pathway detected in pigeon serum appears to more closely resemble the C1-bypass pathway rather than the classical pathway of mammalian complement. PMID:3957409

  1. Systemic complement activation in psoriasis vulgaris.

    PubMed

    Fleming, C J; Holme, E R; Mackie, R M

    1996-11-01

    Nineteen patients with psoriasis vulgaris and no other cause for systemic complement activation were studied for evidence of such activation. There was a marked elevation in serum C5b-9 complexes with no other significant complement abnormalities, and no correlation between C5b-9 levels and disease activity. This is the most detailed study of complement in psoriasis yet attempted and confirms that complement activation is a feature of psoriasis vulgaris. PMID:9167335

  2. Allelic Variants of Complement Genes Associated with Dense Deposit Disease

    PubMed Central

    Abrera-Abeleda, Maria Asuncion; Nishimura, Carla; Frees, Kathy; Jones, Michael; Maga, Tara; Katz, Louis M.; Zhang, Yuzhou

    2011-01-01

    The alternative pathway of the complement cascade plays a role in the pathogenesis of dense deposit disease (DDD). Deficiency of complement factor H and mutations in CFH associate with the development of DDD, but it is unknown whether allelic variants in other complement genes also associate with this disease. We studied patients with DDD and identified previously unreported sequence alterations in several genes in addition to allelic variants and haplotypes common to patients with DDD. We found that the likelihood of developing DDD increases with the presence of two or more risk alleles in CFH and C3. To determine the functional consequence of this finding, we measured the activity of the alternative pathway in serum samples from phenotypically normal controls genotyped for variants in CFH and C3. Alternative pathway activity was higher in the presence of variants associated with DDD. Taken together, these data confirm that DDD is a complex genetic disease and may provide targets for the development of disease-specific therapies. PMID:21784901

  3. Complement in the Homeostatic and Ischemic Brain

    PubMed Central

    Alawieh, Ali; Elvington, Andrew; Tomlinson, Stephen

    2015-01-01

    The complement system is a component of the immune system involved in both recognition and response to pathogens, and it is implicated in an increasing number of homeostatic and disease processes. It is well documented that reperfusion of ischemic tissue results in complement activation and an inflammatory response that causes post-reperfusion injury. This occurs following cerebral ischemia and reperfusion and triggers secondary damage that extends beyond the initial infarcted area, an outcome that has rationalized the use of complement inhibitors as candidate therapeutics after stroke. In the central nervous system, however, recent studies have revealed that complement also has essential roles in synaptic pruning, neurogenesis, and neuronal migration. In the context of recovery after stroke, these apparent divergent functions of complement may account for findings that the protective effect of complement inhibition in the acute phase after stroke is not always maintained in the subacute and chronic phases. The development of effective stroke therapies based on modulation of the complement system will require a detailed understanding of complement-dependent processes in both early neurodegenerative events and delayed neuro-reparatory processes. Here, we review the role of complement in normal brain physiology, the events initiating complement activation after cerebral ischemia-reperfusion injury, and the contribution of complement to both injury and recovery. We also discuss how the design of future experiments may better characterize the dual role of complement in recovery after ischemic stroke. PMID:26322048

  4. Weak protein-protein interactions in lectins: the crystal structure of a vegetative lectin from the legume Dolichos biflorus.

    PubMed

    Buts, L; Dao-Thi, M H; Loris, R; Wyns, L; Etzler, M; Hamelryck, T

    2001-05-25

    The legume lectins are widely used as a model system for studying protein-carbohydrate and protein-protein interactions. They exhibit a fascinating quaternary structure variation, which becomes important when they interact with multivalent glycoconjugates, for instance those on cell surfaces. Recently, it has become clear that certain lectins form weakly associated oligomers. This phenomenon may play a role in the regulation of receptor crosslinking and subsequent signal transduction. The crystal structure of DB58, a dimeric lectin from the legume Dolichos biflorus reveals a separate dimer of a previously unobserved type, in addition to a tetramer consisting of two such dimers. This tetramer resembles that formed by DBL, the seed lectin from the same plant. A single amino acid substitution in DB58 affects the conformation and flexibility of a loop in the canonical dimer interface. This disrupts the formation of a stable DBL-like tetramer in solution, but does not prohibit its formation in suitable conditions, which greatly increases the possibilities for the cross-linking of multivalent ligands. The non-canonical DB58 dimer has a buried symmetrical alpha helix, which can be present in the crystal in either of two antiparallel orientations. Two existing structures and datasets for lectins with similar quaternary structures were reconsidered. A central alpha helix could be observed in the soybean lectin, but not in the leucoagglutinating lectin from Phaseolus vulgaris. The relative position and orientation of the carbohydrate-binding sites in the DB58 dimer may affect its ability to crosslink mulitivalent ligands, compared to the other legume lectin dimers. PMID:11491289

  5. Utilization of lectin-histochemistry in forensic neuropathology: lectin staining provides useful information for postmortem diagnosis in forensic neuropathology.

    PubMed

    Nishi, Katsuji; Tanegashima, Akio; Yamamoto, Yoshio; Ushiyama, Ikuko; Ikemoto, Keiko; Yamasaki, Shigeru; Nishimura, Akiyoshi; Rand, Steven; Brinkmann, Bernd

    2003-09-01

    We have investigated the deposition of glycoconjugates in human brain tissue with or without brain disorders. In this review we describe the application of lectin-histochemistry techniques to forensic neuropathology. Lectin staining is able to reveal several kinds of carbohydrate-related depositions in addition to the conventional degenerative changes including senile plaques, neurofibrillary tangles and corpora amylacea. The senile plaques and neurofibrillary tangles were clearly stained by Con A, PSA and GSI lectins, the corpora amylacea which is relevant to repeated brain hypoxia and mitochondrial damage was also easily detected by these and many other kinds of lectins. Amorphous spaces were detected around blood vessels and independently from blood vessels by lectin staining in the white matter from patients with brain disorders or severe edema. The white matter lesions were not considered relevant for forensic pathology, until a large group of cerebral white matter lesions were detected in the elderly with increasing frequency by modern neuro-imaging methods. The spherical deposits were newly detected by lectin staining in the molecular layer of the dentate gyrus of the hippocampal formation chiefly from patients with schizophrenia or cognitive dysfunctions. PMID:14568771

  6. On the Functional Overlap between Complement and Anti-Microbial Peptides

    PubMed Central

    Zimmer, Jana; Hobkirk, James; Mohamed, Fatima; Browning, Michael J.; Stover, Cordula M.

    2015-01-01

    Intriguingly, activated complement and anti-microbial peptides share certain functionalities; lytic, phagocytic, and chemo-attractant activities and each may, in addition, exert cell instructive roles. Each has been shown to have distinct LPS detoxifying activity and may play a role in the development of endotoxin tolerance. In search of the origin of complement, a functional homolog of complement C3 involved in opsonization has been identified in horseshoe crabs. Horseshoe crabs possess anti-microbial peptides able to bind to acyl chains or phosphate groups/saccharides of endotoxin, LPS. Complement activity as a whole is detectable in marine invertebrates. These are also a source of anti-microbial peptides with potential pharmaceutical applicability. Investigating the locality for the production of complement pathway proteins and their role in modulating cellular immune responses are emerging fields. The significance of local synthesis of complement components is becoming clearer from in vivo studies of parenchymatous disease involving specifically generated, complement-deficient mouse lines. Complement C3 is a central component of complement activation. Its provision by cells of the myeloid lineage varies. Their effector functions in turn are increased in the presence of anti-microbial peptides. This may point to a potentiating range of activities, which should serve the maintenance of health but may also cause disease. Because of the therapeutic implications, this review will consider closely studies dealing with complement activation and anti-microbial peptide activity in acute inflammation (e.g., dialysis-related peritonitis, appendicitis, and ischemia). PMID:25646095

  7. The Semantics of Complementation in English: A Cognitive Semantic Account of Two English Complement Constructions

    ERIC Educational Resources Information Center

    Smith, Michael B.

    2009-01-01

    Studies on complementation in English and other languages have traditionally focused on syntactic issues, most notably on the constituent structures of different complement types. As a result, they have neglected the role of meaning in the choice of different complements. This paper investigates the semantics of complementation within the…

  8. Microbes bind complement inhibitor factor H via a common site.

    PubMed

    Meri, T; Amdahl, H; Lehtinen, M J; Hyvärinen, S; McDowell, J V; Bhattacharjee, A; Meri, S; Marconi, R; Goldman, A; Jokiranta, T S

    2013-01-01

    To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH). FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic microbes protect themselves from complement by recruiting host FH. We analyzed how and why different microbes bind FH via domains 19-20 (FH19-20). We used a selection of FH19-20 point mutants to reveal the binding sites of several microbial proteins and whole microbes (Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, Borrelia burgdorferi, and Borrelia hermsii). We show that all studied microbes use the same binding region located on one side of domain 20. Binding of FH to the microbial proteins was inhibited with heparin showing that the common microbial binding site overlaps with the heparin site needed for efficient binding of FH to host cells. Surprisingly, the microbial proteins enhanced binding of FH19-20 to C3b and down-regulation of complement activation. We show that this is caused by formation of a tripartite complex between the microbial protein, FH, and C3b. In this study we reveal that seven microbes representing different phyla utilize a common binding site on the domain 20 of FH for complement evasion. Binding via this site not only mimics the glycosaminoglycans of the host cells, but also enhances function of FH on the microbial surfaces via the novel mechanism of tripartite complex formation. This is a unique example of convergent evolution resulting in enhanced immune evasion of important pathogens via utilization of a "superevasion site." PMID:23637600

  9. Complement activation by PEGylated liposomes containing prednisolone.

    PubMed

    van den Hoven, Jolanda M; Nemes, Reka; Metselaar, Josbert M; Nuijen, Bastiaan; Beijnen, Jos H; Storm, Gert; Szebeni, Janos

    2013-05-13

    Infusion of PEGylated liposomes can give rise to hypersensitivity reactions (HSRs) in a relatively large number of patients. Previously it has been shown that these reactions can be caused by activation of the complement (C) system by a negative charge on the anchor molecule of PEG at the liposomal surface. In this study it is tested whether the activation of the C system by PEG-liposomes could be significantly reduced to values comparable to nonreactive liposomal formulations, by changing the PEGylation-profile on the liposomal surface. Therefore, the formation of C activation markers SC5b-9, C3a, C4d and Bb in normal human serum by both prednisolone loaded and empty liposomes with a variation of PEG chain length, PEG surface concentration, PEG anchor molecule and liposomal size was determined using in vitro assays. The tested liposomes caused no or only mild (30%) activation of C except for one formulation wherein the PEG2000 was anchored to cholesterol (CHOL-PEG2000). The latter liposomes caused paralleling rises in SC5b-9 and Bb levels, suggesting excess activation of the alternative pathway. While the relative safety of weak C activator liposomes remains to be confirmed in vivo, the unique, non-charge and non-antibody-mediated direct conversion of C3 by CHOL-PEG2000 liposomes (although argues against the clinical development of these vesicles) opens new opportunities to understand liposomal C activation at the molecular level. PMID:23528740

  10. Lectin-like activity from Persea americana.

    PubMed

    Meade, N A; Staat, R H; Langley, S D; Doyle, R J

    1980-01-15

    An extract from the seeds of Persea americana possessed an erythro-agglutinating activity. The agglutinin was devoid of specificity for carbohydrates, but interacted readily with basic proteins or basic polyamino acids. The interaction between the agglutinin and egg-white lysozyme was not inhibited by chaotropic salts, but was sensitive to relatively low concentrations of urea. An affinity chromatographic procedure was developed in an effort to purify the agglutinin. Products from the chromatographic procedure were found not to contain higher specific agglutinating activities than the crude extract. Amino acid acid analyses of the extract showed the presence of relatively high proportions of glutamic and aspartic acids. In addition, the extract contained phosphorus and a visible chromophore. The agglutinin was resistant to detergents and denaturants, and proteases, nucleases, and other enzymes. The results suggest that, as opposed to other plant agglutinins, the active component from Persea is not a protein. Similarly, in contrast to many lectins, the agglutinin from Persea was not mitogenic for mouse lymphocytes. The agglutinin partially inhibited the mitogenesis of lymphocytes when the cells were treated with concanavalin A, or with bacterial lipopolysaccharide. PMID:7353211

  11. Merging carbohydrate chemistry with lectin histochemistry to study inhibition of lectin binding by glycoclusters in the natural tissue context.

    PubMed

    André, Sabine; Kaltner, Herbert; Kayser, Klaus; Murphy, Paul V; Gabius, Hans-Joachim

    2016-02-01

    Recognition of glycans by lectins leads to cell adhesion and growth regulation. The specificity and selectivity of this process are determined by carbohydrate structure (sequence and shape) and topology of its presentation. The synthesis of (neo)glycoconjugates with bi- to oligo-valency (glycoclusters) affords tools to delineate structure-activity relationships by blocking lectin binding to an artificial matrix, often a glycoprotein, or cultured cell lines. The drawback of these assays is that glycan presentation is different from that in tissues. In order to approach the natural context, we here introduce lectin histochemistry on fixed tissue sections to determine the susceptibility of binding of two plant lectins, i.e., GSA-II and WGA, to a series of 10 glycoclusters. Besides valency, this panel covers changes in the anomeric position (α/β) and the atom at the glycosidic linkage (O/S). Flanked by cell and solid-phase assays with human tumor lines and two mucins, respectively, staining (intensity and profile) was analyzed in sections of murine jejunum, stomach and epididymis as a function of glycocluster presence. The marked and differential sensitivity of signal generation to structural aspects of the glycoclusters proves the applicability of this method. This enables comparisons between data sets obtained by using (neo)glycoconjugates, cells and the tissue context as platforms. The special advantage of processing tissue sections is the monitoring of interference with lectin association at sites that are relevant for functionality. Testing glycoclusters in lectin histochemistry will especially be attractive in cases of multi-target recognition (glycans, proteins and lipids) by a tissue lectin. PMID:26553286

  12. The Role of Complement in Tumor Growth

    PubMed Central

    Pio, Ruben; Corrales, Leticia; Lambris, John D.

    2015-01-01

    Complement is a central part of the immune system that has developed as a first defense against non-self cells. Neoplastic transformation is accompanied by an increased capacity of the malignant cells to activate complement. In fact, clinical data demonstrate complement activation in cancer patients. On the basis of the use of protective mechanisms by malignant cells, complement activation has traditionally been considered part of the body's immunosurveillance against cancer. Inhibitory mechanisms of complement activation allow cancer cells to escape from complement-mediated elimination and hamper the clinical efficacy of monoclonal antibody–based cancer immunotherapies. To overcome this limitation, many strategies have been developed with the goal of improving complement-mediated effector mechanisms. However, significant work in recent years has identified new and surprising roles for complement activation within the tumor microenvironment. Recent reports suggest that complement elements can promote tumor growth in the context of chronic inflammation. This chapter reviews the data describing the role of complement activation in cancer immunity, which offers insights that may aid the development of more effective therapeutic approaches to control cancer. PMID:24272362

  13. Complement Activation and Inhibition in Wound Healing

    PubMed Central

    Cazander, Gwendolyn; Jukema, Gerrolt N.; Nibbering, Peter H.

    2012-01-01

    Complement activation is needed to restore tissue injury; however, inappropriate activation of complement, as seen in chronic wounds can cause cell death and enhance inflammation, thus contributing to further injury and impaired wound healing. Therefore, attenuation of complement activation by specific inhibitors is considered as an innovative wound care strategy. Currently, the effects of several complement inhibitors, for example, the C3 inhibitor compstatin and several C1 and C5 inhibitors, are under investigation in patients with complement-mediated diseases. Although (pre)clinical research into the effects of these complement inhibitors on wound healing is limited, available data indicate that reduction of complement activation can improve wound healing. Moreover, medicine may take advantage of safe and effective agents that are produced by various microorganisms, symbionts, for example, medicinal maggots, and plants to attenuate complement activation. To conclude, for the development of new wound care strategies, (pre)clinical studies into the roles of complement and the effects of application of complement inhibitors in wound healing are required. PMID:23346185

  14. Annexin A2 Enhances Complement Activation by Inhibiting Factor H.

    PubMed

    Renner, Brandon; Tong, Hua Hua; Laskowski, Jennifer; Jonscher, Karen; Goetz, Lindsey; Woolaver, Rachel; Hannan, Jonathan; Li, Yong Xing; Hourcade, Dennis; Pickering, Matthew C; Holers, V Michael; Thurman, Joshua M

    2016-02-01

    Factor H is a circulating protein that regulates activation of the alternative pathway (AP) of complement. Mutations and genetic variations of factor H are associated with several AP-mediated diseases, highlighting the critical role of factor H in AP regulation. AP-mediated inflammation is typically triggered by illness or tissue injury, however, and tissue injury can trigger AP activation in individuals with fully functional factor H. This suggests that factor H function is affected by local conditions within tissues. We hypothesized that inducible proteins impair the ability of factor H to locally control the AP, thereby increasing AP activation. We used purified murine factor H to immunoprecipitate binding partners from mouse kidneys. Using immunoaffinity liquid chromatography-mass spectrometry, we identified annexin A2 as a factor H binding partner. Further experiments showed that annexin A2 reduces the binding of factor H to cell surfaces. Recombinant annexin A2 impaired complement regulation by factor H and increased complement activation on renal cell surfaces in vitro and in vivo. In a murine model of acute pneumococcal otitis media, the administration of annexin A2 increased AP-mediated bacterial opsonization and clearance. In conclusion, the local production of annexin A2 within tissues suppresses regulation of the AP by factor H. Annexin A2 can contribute to AP-mediated tissue inflammation by locally impairing factor H function, but it can also improve complement-mediated bacterial clearance. PMID:26729803

  15. Immature recent thymic emigrants are eliminated by complement1

    PubMed Central

    Hsu, Fan-Chi; Shapiro, Michael J.; Chen, Meibo W.; McWilliams, Douglas C.; Seaburg, Lauren M.; Tangen, Sarah N.; Shapiro, Virginia Smith

    2014-01-01

    Recent thymic emigrants (RTEs) must undergo phenotypic and functional maturation to become long-lived mature naïve T cells. In CD4-cre NKAP conditional knockout mice, NKAP-deficient RTEs fail to complete T cell maturation. Here, we demonstrate that NKAP-deficient immature RTEs do not undergo apoptosis, but are eliminated by complement. C3, C4 and C1q are bound to NKAP-deficient peripheral T cells, demonstrating activation of the classical arm of the complement pathway. As thymocytes mature and exit to the periphery, they increase sialic acid incorporation into cell surface glycans. This is essential to peripheral lymphocyte survival, as stripping sialic acid with neuraminidase leads to the binding of natural IgM and complement fixation. NKAP-deficient T cells have a defect in sialylation on cell surface glycans, leading to IgM recruitment. We demonstrate that the defect in sialylation is due to aberrant α2,8-linked sialylation, and the expression of three genes (ST8sia1, ST8sia4 and ST8sia6) that mediate α2,8 sialylation are down regulated in NKAP-defcient RTEs. The maturation of peripheral NKAP-deficient T cells is partially rescued in a C3-deficient environment. Thus, sialylation during T cell maturation is critical to protect immature RTEs from complement in the periphery. PMID:25367120

  16. Integrated lectin affinity microfluidic chip for glycoform separation.

    PubMed

    Mao, Xiuli; Luo, Yong; Dai, Zhongpeng; Wang, Keyi; Du, Yuguang; Lin, Bingcheng

    2004-12-01

    Lectin affinity chromatography was miniaturized into a microfluidic format, which results in improvement of performance, as compared to the conventional method. A lectin affinity monolith column was prepared in the microchannel of a microfluidic chip. The porous monolith was fabricated by UV-initiated polymerization of ethylene dimethacrylate (EDMA) and glycidyl methacrylate (GMA) in the presence of porogeneities, followed by immobilization of pisum sativum agglutinin (PSA) on the monolith matrix. Using electroosmosis as the driven force, lectin affinity chromatographies of three kinds of glycoprotein, turkey ovalbumin (TO), chicken ovalbumin (CO), and ovomucoid (OM), were carried out on the microfluidic system. All the glycoproteins were successfully separated into several fractions with different affinities toward the immobilized PSA. The integrated system reduces the time required for the lectin affinity chromatography reaction to approximately 3%, thus, the overall analysis time from 4 h to 400 s. Only 300 pg of glycoprotein is required for the whole separation process. Moreover, troublesome operations for lectin affinity chromatography are simplified. PMID:15571345

  17. Molecular biology of seed storage proteins and lectins

    SciTech Connect

    Shannon, L.M.; Chrispeels, M.J.

    1986-01-01

    The seeds of many plants contain abundant storage protein and lectin which have been the subject of biochemical investigations for over a hundred years. Because these proteins represent abundant gene products translated from abundant messages, they were among the first plant proteins to which the techniques of molecular biology were applied. Many of the proteins have now been purified and characterized and their amino acid sequences have been determined; some have been crystallized and their 3-dimensional structure is known. Studies of their biosynthesis, transport and accumulation in protein bodies have lead to a greater understanding of the dynamics of plant cell organelles. Seed storage proteins and lectins are encoded by small gene families whose members show considerable homology and appear to have been conserved in evolution. The expression of these genes is highly regulated in time (development) and space (tissue). The recent discovery that other plant organs synthesize lectins, or lectin-like proteins which are closely related to the seed lectins, has lent additional support to the search for a function for these intriguing proteins; this finding also indicates that different members of the gene family are expressed in different tissues. Individual papers were processed separately for the data base.

  18. Identification of Lectins from Metastatic Cancer Cells through Magnetic Glyconanoparticles

    PubMed Central

    Kavunja, Herbert W.; Voss, Patricia G.

    2016-01-01

    Cancer cells can have characteristic carbohydrate binding properties. Previously, it was shown that a highly metastatic melanoma cell line B16F10 bound to galacto-side-functionalized nanoparticles much stronger than the corresponding less metastatic B16F1 cells. To better understand the carbohydrate binding properties of cancer cells, herein, we report the isolation and characterization of endogenous galactose binding proteins from B16F10 cells using magnetic glyconanoparticles. The galactose-coated magnetic glyconanoparticles could bind with lectins present in the cells and be isolated through magnet-mediated separation. Through Western blot and mass spectrometry, the arginine/serine rich splicing factor Sfrs1 was identified as a galactose-selective endogenous lectin, overexpressed in B16F10 cells, compared with B16F1 cells. In addition, galactin-3 was found in higher amounts in B16F10 cells. Finally, the glyconanoparticles exhibited a superior efficiency in lectin isolation, from both protein mixtures and live cells, than the corresponding more traditional microparticles functionalized with carbohydrates. Thus, the magnetic glyconanoparticles present a useful tool for discovery of endogenous lectins, as well as binding partners of lectins, without prior knowledge of protein identities. PMID:27110035

  19. Differential Use of the C-Type Lectins L-SIGN and DC-SIGN for Phlebovirus Endocytosis.

    PubMed

    Léger, Psylvia; Tetard, Marilou; Youness, Berthe; Cordes, Nicole; Rouxel, Ronan N; Flamand, Marie; Lozach, Pierre-Yves

    2016-06-01

    Bunyaviruses represent a growing threat to humans and livestock globally. The receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely unidentified and poorly characterized. DC-SIGN is a C-type lectin highly expressed on dermal dendritic cells that has been found to act as an authentic entry receptor for many phleboviruses (Bunyaviridae), including Rift Valley fever virus (RVFV), Toscana virus (TOSV) and Uukuniemi virus (UUKV). We found that these phleboviruses can exploit another C-type lectin, L-SIGN, for infection. L-SIGN shares 77% sequence homology with DC-SIGN and is expressed on liver sinusoidal endothelial cells. L-SIGN is required for UUKV binding but not for virus internalization. An endocytosis-defective mutant of L-SIGN was still able to mediate virus uptake and infection, indicating that L-SIGN acts as an attachment receptor for phleboviruses rather than an endocytic receptor. Our results point out a fundamental difference in the use of the C-type lectins L-SIGN and DC-SIGN by UUKV to enter cells, although both proteins are closely related in terms of molecular structure and biological function. This study sheds new light on the molecular mechanisms by which phleboviruses target the liver and also highlights the added complexity in virus-receptor interactions beyond attachment. PMID:26990254

  20. White kidney bean lectin exerts anti-proliferative and apoptotic effects on cancer cells.

    PubMed

    Chan, Yau Sang; Xia, Lixin; Ng, Tzi Bun

    2016-04-01

    A 60-kDa glucosamine binding lectin, white kidney bean lectin (WKBL), was purified from Phaseolus vulgaris cv. white kidney beans, by application of anion exchange chromatography on Q-Sepharose, affinity chromatography on Affi-gel blue gel, and FPLC-size exclusion on Superdex 75. The anti-proliferative activity of WKBL on HONE1 cells and HepG2 cells was stronger than the activity on MCF7 cells and WRL68 cells (IC50 values for a 48-h treatment with WKBL on HONE1 cells: 18.8μM; HepG2 cells: 19.7μM; MCF7 cells: 26.9μM; and WRL68 cells: >80μM). The activity could be reduced by addition of glucosamine, which occupies the binding sites of WKBL, indicating that carbohydrate binding is crucial for the activity. Annexin V-FITC and PI staining, JC-1 staining and Hoechst 33342 staining revealed that apoptosis was induced on WKBL-treated HONE1 cells and HepG2 cells, but not as obviously on MCF7 cells. Cell cycle analysis also showed a slight cell cycle arrest on HONE1 cells after WKBL treatment. Western blotting suggested that WKBL induced apoptosis of HONE1 cells occurred through the extrinsic apoptosis pathway, with detection of increased level of active caspase 3, 8 and 9. PMID:26769089

  1. Lectin uptake and incorporation into the calcitic spicule of sea urchin embryos.

    PubMed

    Mozingo, Nancy M

    2015-06-01

    Primary mesenchyme cells (PMCs) are skeletogenenic cells that produce a calcareous endoskeleton in developing sea urchin larvae. The PMCs fuse to form a cavity in which spicule matrix proteins and calcium are secreted forming the mineralized spicule. In this study, living sea urchin embryos were stained with fluorescently conjugated wheat germ agglutinin, a lectin that preferentially binds to PMCs, and the redistribution of this fluorescent tag was examined during sea urchin development. Initially, fluorescence was associated primarily with the surface of PMCs. Subsequently, the fluorescent label redistributed to intracellular vesicles in the PMCs. As the larval skeleton developed, intracellular granular staining diminished and fluorescence appeared in the spicules. Spicules that were cleaned to remove membranous material associated with the surface exhibited bright fluorescence, which indicated that fluorescently labelled lectin had been incorporated into the spicule matrix. The results provide evidence for a cellular pathway in which material is taken up at the cell surface, sequestered in intracellular vesicles and then incorporated into the developing spicule. PMID:24735584

  2. Characterization of the Grp94/OS-9 chaperone-lectin complex

    PubMed Central

    Seidler, Paul M.; Shinsky, Stephen A.; Hong, Feng; Li, Zihai; Cosgrove, Michael S.; Gewirth, Daniel T.

    2014-01-01

    Grp94 is a macromolecular chaperone belonging to the hsp90 family and is the most abundant glycoprotein in the endoplasmic reticulum of mammals. In addition to its essential role in protein folding, Grp94 was proposed to participate in the ER associated degradation (ERAD) quality control pathway by interacting with the lectin OS-9, a sensor for terminally misfolded proteins (TMPs). To understand how OS-9 interacts with ER chaperone proteins, we mapped its interaction with Grp94. Glycosylation of the full length Grp94 protein was essential for OS-9 binding, although deletion of the Grp94 N-terminal domain relieved this requirement suggesting that the effect was allosteric rather than direct. Although yeast OS-9 is composed of a well-established N-terminal MRH lectin domain and a C-terminal dimerization domain, we find that the C-terminal domain of OS-9 in higher eukaryotes contains ‘mammalian-specific insets’ that are specifically recognized by the middle and C-terminal domains of Grp94. Additionally, the Grp94 binding domain in OS-9 was found to be intrinsically disordered. The biochemical analysis of the interacting regions provides insight into the manner by which the two associate, and additionally hints at a plausible biological role for the Grp94/OS-9 complex. PMID:25193139

  3. Complement Interaction with Trypanosomatid Promastigotes in Normal Human Serum

    PubMed Central

    Domínguez, Mercedes; Moreno, Inmaculada; López-Trascasa, Margarita; Toraño, Alfredo

    2002-01-01

    In normal human serum (NHS), axenic promastigotes of Crithidia, Phytomonas, and Leishmania trigger complement activation, and from 1.2 to 1.8 × 105 C3 molecules are deposited per promastigote within 2.5 min. In Leishmania, promastigote C3 binding capacity remains constant during in vitro metacyclogenesis. C3 deposition on promastigotes activated through the classical complement pathway reaches a 50% maximum after ∼50 s, and represents >85% of total C3 bound. In C1q- and C2-deficient human sera, promastigotes cannot activate the classical pathway (CP) unless purified C1q or C2 factors, respectively, are supplemented, demonstrating a requirement for CP factor in promastigote C3 opsonization. NHS depleted of natural anti-Leishmania antibodies cannot trigger promastigote CP activation, but IgM addition restores C3 binding. Furthermore, Leishmania binds natural antibodies in ethylenediaminetetracetic acid (EDTA)-treated NHS; after EDTA removal, promastigote-bound IgM triggers C3 deposition in natural antibody-depleted NHS. Serum collectins and pentraxins thus do not participate significantly in NHS promastigote C3 opsonization. Real-time kinetic analysis of promastigote CP-mediated lysis indicates that between 85–95% of parasites are killed within 2.5 min of serum contact. These data indicate that successful Leishmania infection in man must immediately follow promastigote transmission, and that Leishmania evasion strategies are shaped by the selective pressure exerted by complement. PMID:11854358

  4. Membrane protein Crry maintains homeostasis of the complement system1

    PubMed Central

    Wu, Xiaobo; Spitzer, Dirk; Mao, Dailing; Peng, Stanford L.; Molina, Hector; Atkinson, John P.

    2008-01-01

    Complement activation is tightly regulated to avoid excessive inflammatory and immune responses. Crry-/- is an embryonic lethal phenotype secondary to the maternal complement alternative pathway (AP) attacking a placenta deficient in this inhibitor. In this study, we demonstrate that Crry-/- mice could be rescued on a partial as well as on a complete factor B (fB)- or C3-deficient maternal background. The C3 and fB protein concentrations in Crry-/-C3+/- and Crry-/-fB+/- mice were substantially reduced for gene dosage secondary to enhanced AP turnover. Based on these observations, a breeding strategy featuring reduced maternal AP-activating capacity rescued the lethal phenotype. It led to a novel, stable line of Crry SKO mice carrying normal alleles for C3 and fB. Crry SKO mice also had accelerated C3 and fB turnover and therefore reduced AP-activating potential. These instructive results represent an example of a membrane regulatory protein being responsible for homeostasis of the complement system. They imply that there is constant turnover on cells of the AP pathway which functions as an immune surveillance system for pathogens and altered self. PMID:18684964

  5. Regulation of complement and modulation of its activity in monoclonal antibody therapy of cancer

    PubMed Central

    Meyer, Saskia; Leusen, Jeanette HW; Boross, Peter

    2014-01-01

    The complement system is a powerful tool of the innate immune system to eradicate pathogens. Both in vitro and in vivo evidence indicates that therapeutic anti-tumor monoclonal antibodies (mAbs) can activate the complement system by the classical pathway. However, the contribution of complement to the efficacy of mAbs is still debated, mainly due to the lack of convincing data in patients. A beneficial role for complement during mAb therapy is supported by the fact that cancer cells often upregulate complement-regulatory proteins (CRPs). Polymorphisms in various CRPs were previously associated with complement-mediated disorders. In this review the role of complement in anti-tumor mAb therapy will be discussed with special emphasis on strategies aiming at modifying complement activity. In the future, clinical efficacy of mAbs with enhanced effector functions together with comprehensive analysis of polymorphisms in CRPs in mAb-treated patients will further clarify the role of complement in mAb therapy. PMID:25517299

  6. Further screening of Aspergillus species for occurrence of lectins and their partial characterization.

    PubMed

    Singh, Ram Sarup; Bhari, Ranjeeta; Rai, Jyoti

    2010-02-01

    Fifteen species of Aspergillus were screened for occurrence of lectins. Nine of them (A. sydowii, A. candidus, A. allahabadi, A. terricola, A. ficuum, A. sparsus, A. carneus, A. pulvinus and A. aculeatus) were found to possess lectin activity. None of the species elaborated lectin in culture supernatant. All the lectins agglutinated rat, pig and rabbit erythrocytes. A. sydowii, A. candidus, A. allahabadi, A. terricola, A. ficuum, A. sparsus, A. carneus and A. aculeatus lectins agglutinated all human type erythrocytes equally, while A. pulvinus lectin specifically agglutinated human type A and O erythrocytes. Neuraminidase and protease treatment to erythrocytes substantially augmented lectin titres manyfold. Lectins showed specificity to mucin and asialofetuin and all of them were specific to L-arabinose except that of A. carneus. Lectins from A. sydowii, A. ficuum, A. sparsus and A. carneus displayed remarkable specificities to D-xylose. Maximum lectin activity was expressed by 11 day old cultures of A. sydowii (titre 32), A. ficuum (titre 64) and A. sparsus (titre 1024). Lectins from A. aculeatus, A. candidus and A. terricola were expressed by 7-10 days, 6-9 days and 5-11 days old cultures, respectively. A. allahabadi cultures exhibited maximum lectin activity (titre 32) after 8-10 days of cultivation. A. carneus and A. pulvinus expressed optimal titres of 32 and 8, respectively on the 9th day. PMID:20175125

  7. A sialic acid-binding lectin from the mushroom Hericium erinaceum.

    PubMed

    Kawagishi, H; Mori, H; Uno, A; Kimura, A; Chiba, S

    1994-02-28

    A lectin was isolated from the mushroom Hericium erinaceum. This lectin is composed of two different subunits of 15 and 16 kDa and the molecular mass of the intact lectin was estimated to be 54 kDa by gel filtration. It exhibits specificity towards sialic acids, especially N-glycolylneuraminic acid. PMID:8119408

  8. Histological and lectin histochemical studies on the olfactory and respiratory mucosae of the sheep.

    PubMed

    Ibrahim, Dalia; Nakamuta, Nobuaki; Taniguchi, Kazumi; Yamamoto, Yoshio; Taniguchi, Kazuyuki

    2014-03-01

    The olfactory and respiratory mucosae of the Corriedale sheep were examined using lectin histochemistry in order to clarify the histochemical and glycohistochemical differences between these two tissues. The olfactory epithelium was stained with 13 lectins out of 21 lectins examined, while the respiratory epithelium was positive to 16 lectins. The free border of both of the olfactory and respiratory epithelia was stained with 12 lectins: Wheat germ agglutinin (WGA), succinylated-wheat germ agglutinin (s-WGA), Lycopersicon esculentum lectin (LEL), Solanum tuberosum lectin (STL), Datura stramonium lectin (DSL), Soybean agglutinin (SBA), Bandeiraea simplicifolia lectin-I (BSL-I), Ricinus communis agglutinin-I (RCA-120), Erythrina cristagalli lectin (ECL), Concanavalin A (Con A), Phaseolus vulgaris agglutinin-E (PHA-E) and Phaseolus vulgaris agglutinin-L (PHA-L). The associated glands of the olfactory mucosa, Bowman's glands, were stained with 13 lectins. While both the goblet cells and mucous nasal glands were stained with 8 lectins; five of them (WGA, s-WGA, STL, Vicia villosa agglutinin (VVA) and ECL) were mutually positive among the Bowman's glands, mucous nasal glands and the goblet cells. These findings indicate that the glycohistochemical characteristics of the free borders of both olfactory and respiratory epithelia are similar to each other, suggesting that secretions from the Bowman's glands and those of the goblet cells and mucous nasal glands are partially exchanged between the surface of two epithelia to contribute the functions of the respiratory epithelium and the olfactory receptor cells, respectively. PMID:24200894

  9. Role of Complement in Autoimmune Hemolytic Anemia

    PubMed Central

    Berentsen, Sigbjørn

    2015-01-01

    Summary The classification of autoimmune hemolytic anemias and the complement system are reviewed. In autoimmune hemolytic anemia of the warm antibody type, complement-mediated cell lysis is clinically relevant in a proportion of the patients but is hardly essential for hemolysis in most patients. Cold antibody-mediated autoimmune hemolytic anemias (primary cold agglutinin disease, secondary cold agglutinin syndrome and paroxysmal cold hemoglobinuria) are entirely complement-mediated disorders. In cold agglutinin disease, efficient therapies have been developed in order to target the pathogenic B-cell clone, but complement modulation remains promising in some clinical situations. No established therapy exists for secondary cold agglutinin syndrome and paroxysmal cold hemoglobinuria, and the possibility of therapeutic complement inhibition is interesting. Currently, complement modulation is not clinically documented in any autoimmune hemolytic anemia. The most relevant candidate drugs and possible target levels of action are discussed. PMID:26696798

  10. Pasteurella pneumotropica Evades the Human Complement System by Acquisition of the Complement Regulators Factor H and C4BP

    PubMed Central

    Sahagún-Ruiz, Alfredo; Granados Martinez, Adriana Patricia; Breda, Leandro Carvalho Dantas; Fraga, Tatiana Rodrigues; Castiblanco Valencia, Mónica Marcela; Barbosa, Angela Silva; Isaac, Lourdes

    2014-01-01

    Pasteurella pneumotropica is an opportunist Gram negative bacterium responsible for rodent pasteurellosis that affects upper respiratory, reproductive and digestive tracts of mammals. In animal care facilities the presence of P. pneumotropica causes severe to lethal infection in immunodeficient mice, being also a potential source for human contamination. Indeed, occupational exposure is one of the main causes of human infection by P. pneumotropica. The clinical presentation of the disease includes subcutaneous abscesses, respiratory tract colonization and systemic infections. Given the ability of P. pneumotropica to fully disseminate in the organism, it is quite relevant to study the role of the complement system to control the infection as well as the possible evasion mechanisms involved in bacterial survival. Here, we show for the first time that P. pneumotropica is able to survive the bactericidal activity of the human complement system. We observed that host regulatory complement C4BP and Factor H bind to the surface of P. pneumotropica, controlling the activation pathways regulating the formation and maintenance of C3-convertases. These results show that P. pneumotropica has evolved mechanisms to evade the human complement system that may increase the efficiency by which this pathogen is able to gain access to and colonize inner tissues where it may cause severe infections. PMID:25347183

  11. Crystal structure of Dioclea violacea lectin and a comparative study of vasorelaxant properties with Dioclea rostrata lectin.

    PubMed

    Bezerra, Maria Júlia Barbosa; Rodrigues, Natália Velloso Fontenelle Camelo; Pires, Alana de Freitas; Bezerra, Gustavo Arruda; Nobre, Camila Bezerra; Alencar, Kássia Lys de Lima; Soares, Pedro Marcos Gomes; do Nascimento, Kyria Santiago; Nagano, Celso Shiniti; Martins, Jorge Luiz; Gruber, Karl; Sampaio, Alexandre Holanda; Delatorre, Plinio; Rocha, Bruno Anderson Matias; Assreuy, Ana Maria Sampaio; Cavada, Benildo Sousa

    2013-04-01

    Lectins from Diocleinae subtribe belong to the family of legume lectins and are characterized by high identity between their amino acids sequences. It has been shown that punctual differences in amino acid sequences, such as one single amino acid or an alternative conformation, represent changes in biological activities caused by these lectins. Therefore, a more detailed understanding of three-dimensional structures of these proteins is essential for accurate analyzing the relationship between structure and function. In this study lectins purified from the seeds of Dioclea violacea (DVL) and Dioclea rostrata (DRL) were compared with regard to crystal structure and vasorelaxant properties. Differences in structure of lectins were found to be reflected in differences in vasorelaxant effects based on their high specificity and selectivity for cell glycans. Binding activity was related to the position of specific residues in the carbohydrate recognition domain (CRD). DVL complexed structure was solved by X-ray crystallography and was compared to native DVL and DRL. Therefore, DVL was co-crystallized with X-Man, and a molecular modeling with X-Man complexed with DVL was done to compare the complexed and native forms adjusted fit. The relatively narrow and deep CRD in DVL promotes little interaction with carbohydrates; in contrast, the wider and shallower CRD in DRL favors interaction. This seems to explain differences in the level of relaxation induced by DVL (43%) and DRL (96%) in rat aortic rings. PMID:23353644

  12. Complement Factor H and Simian Virus 40 bind the GM1 ganglioside in distinct conformations.

    PubMed

    Blaum, Bärbel S; Frank, Martin; Walker, Ross C; Neu, Ursula; Stehle, Thilo

    2016-05-01

    Mammalian cell surfaces are decorated with a variety of glycan chains that orchestrate development and defense and are exploited by pathogens for cellular attachment and entry. While glycosidic linkages are, in principle, flexible, the conformational space that a given glycan can sample is subject to spatial and electrostatic restrictions imposed by its overall chemical structure. Here, we show how the glycan moiety of the GM1 ganglioside, a branched, monosialylated pentasaccharide that serves as a ligand for various proteins, undergoes differential conformational selection in its interactions with different lectins. Using STD NMR and X-ray crystallography, we found that the innate immune regulator complement Factor H (FH) binds a previously not reported GM1 conformation that is not compatible with the GM1-binding sites of other structurally characterized GM1-binding lectins such as the Simian Virus 40 (SV40) capsid. Molecular dynamics simulations of the free glycan in explicit solvent on the 10 μs timescale reveal that the FH-bound conformation nevertheless corresponds to a minimum in the Gibbs free energy plot. In contrast to the GM1 conformation recognized by SV40, the FH-bound GM1 conformation is associated with poor NOE restraints, explaining how it escaped(1)H-(1)H NOE-restrained modeling in the past and highlighting the necessity for ensemble representations of glycan structures. PMID:26715202

  13. Isolation and biochemical characterization of Apios tuber lectin.

    PubMed

    Kenmochi, Eri; Kabir, Syed Rashel; Ogawa, Tomohisa; Naude, Ryno; Tateno, Hiroaki; Hirabayashi, Jun; Muramoto, Koji

    2015-01-01

    Apios tuber lectin, named ATL, was isolated from Apios americana Medikus by two chromatography steps, hydrophobic chromatography and anion-exchange chromatography. The minimum concentration required for the hemagglutination activity toward rabbit erythrocytes of ATL was 4 μg/mL. ATL was composed of a homodimer of 28.4 kDa subunits. The amino acid sequence of ATL was similar to those of other legume lectins. The lectin showed moderate stability toward heating and acidic pH, and the binding affinity against several monosaccharides, such as D-glucosamine and D-galactosamine. ATL also bound to desialylated or agalactosylated glycoproteins such as asialo and agalacto transferrin. ATL decreased the transepithelial electrical resistance across human intestinal Caco-2 cell monolayers, suggesting the effect on the tight junction-mediated paracellular transport. PMID:25584830

  14. A lectin with antifungal activity from the mussel Crenomytilus grayanus.

    PubMed

    Chikalovets, Irina V; Chernikov, Oleg V; Pivkin, Mikhail V; Molchanova, Valentina I; Litovchenko, Alina P; Li, Wei; Lukyanov, Pavel A

    2015-02-01

    Lectins (carbohydrate-binding proteins) are well known to actively participate in the defense functions of vertebrates and invertebrates where they play an important role in the recognition of foreign particles. In this study, we investigated of in vitro antifungal activity of lectin from the mussel Crenomytilus grayanus (CGL). Enzyme-linked immunosorbent assay indicated that CGL was predominantly detectable in tissues of mantle and to a lesser degree in the tissues of muscle, hepatopancreas, gill and hemocytes. After challenged by Pichia pastoris the level of CGL was upregulated and reached the maximum level at 12 h post challenge and recovered to the original level at 24 h. The lectin was capable of inhibiting the germination of spores and hyphal growth in the fungi. All these results indicated that CGL is involved in the innate immune response in mollusc animals. PMID:25482060

  15. BSA-boronic acid conjugate as lectin mimetics.

    PubMed

    Narla, Satya Nandana; Pinnamaneni, Poornima; Nie, Huan; Li, Yu; Sun, Xue-Long

    2014-01-10

    We report bovine serum albumin (BSA)-boronic acid (BA) conjugates as lectin mimetics and their glyco-capturing capacity. The BSA-BA conjugates were synthesized by amidation of carboxylic acid groups in BSA with aminophenyl boronic acid in the presence of EDC, and were characterized by Alizarin Red S (ARS) assay and SDS-PAGE gel. The BSA-BA conjugates were immobilized onto maleimide-functionalized silica beads and their sugar capturing capacity and specificity were confirmed by ARS displacement assay. Further, surface plasmon resonance (SPR) analysis of the glyco-capturing activity of the BSA-BA conjugates was conducted by immobilizing BSA-BA onto SPR gold chip. Overall, we demonstrated a BSA-BA-based lectin mimetics for glyco-capturing applications. These lectin mimetics are expected to provide an important tool for glycomics and biosensor research and applications. PMID:24326067

  16. Systematic assessment of the influence of complement gene polymorphisms on kidney transplant outcome.

    PubMed

    Ermini, Luca; Weale, Michael E; Brown, Katherine M; Mesa, Irene Rebollo; Howell, W Martin; Vaughan, Robert; Chowdhury, Paramit; Sacks, Steven H; Sheerin, Neil S

    2016-04-01

    The importance of the innate immune system, including complement, in causing transplant injury and augmenting adaptive immune responses is increasingly recognized. Therefore variability in graft outcome may in part be due to genetic polymorphism in genes encoding proteins of the immune system. This study assessed the relationship between single nucleotide polymorphisms (SNPs) in complement genes and outcome after transplantation. Analysis was performed on two patient cohorts of 650 and 520 transplant recipients. 505 tagged SNPs in 47 genes were typed in both donor and recipient. The relationships between SNPs and graft survival, serum creatinine, delayed graft function and acute rejection were analyzed. One recipient SNP in the gene encoding mannose binding lectin was associated with graft outcome after correction for analysis of multiple SNPs (p=6.41×10(-5)). When further correction was applied to account for analysis of the effect of SNPs in both donor and recipient this lost significance. Despite association p values of <0.001 no SNP was significantly associated with clinical phenotypes after Bonferroni correction. In conclusion, the variability seen in transplant outcome in this patient cohort cannot be explained by variation in complement genes. If causal genetic effects exist in these genes, they are too small to be detected by this study. PMID:26797657

  17. Differential activity of candidate microbicides against early steps of HIV-1 infection upon complement virus opsonization

    PubMed Central

    2010-01-01

    Background HIV-1 in genital secretions may be opsonized by several molecules including complement components. Opsonized HIV-1 by complement enhances the infection of various mucosal target cells, such as dendritic cells (DC) and epithelial cells. Results We herein evaluated the effect of HIV-1 complement opsonization on microbicide candidates' activity, by using three in vitro mucosal models: CCR5-tropic HIV-1JR-CSF transcytosis through epithelial cells, HIV-1JR-CSF attachment on immature monocyte-derived dendritic cells (iMDDC), and infectivity of iMDDC by CCR5-tropic HIV-1BaL and CXCR4-tropic HIV-1NDK. A panel of 10 microbicide candidates [T20, CADA, lectines HHA & GNA, PVAS, human lactoferrin, and monoclonal antibodies IgG1B12, 12G5, 2G12 and 2F5], were investigated using cell-free unopsonized or opsonized HIV-1 by complements. Only HHA and PVAS were able to inhibit HIV trancytosis. Upon opsonization, transcytosis was affected only by HHA, HIV-1 adsorption on iMDDC by four molecules (lactoferrin, IgG1B12, IgG2G5, IgG2G12), and replication in iMDDC of HIV-1BaL by five molecules (lactoferrin, CADA, T20, IgG1B12, IgG2F5) and of HIV-1NDK by two molecules (lactoferrin, IgG12G5). Conclusion These observations demonstrate that HIV-1 opsonization by complements may modulate in vitro the efficiency of candidate microbicides to inhibit HIV-1 infection of mucosal target cells, as well as its crossing through mucosa. PMID:20546571

  18. Complement Factors and their G Protein Coupled Receptors in Neuroprotection and Neurodegeneration

    PubMed Central

    Yanamadala, Vijay; Friedlander, Robert M.

    2010-01-01

    Acute neurodegeneration is associated with high morbidity and mortality, and there are few effective treatments. Inflammation is central to the process of neuronal death, and within this field, the roles of the complement cascade have proven to be complex. The complement cascade is involved in triggering cell death and recruiting inflammatory cells to sites of inflammation, including the brain. However, complement might also have important neuroprotective roles that are only now coming to light. Recent evidence suggests that targeted activation of complement may be a potential avenue for treatment of stroke and other acute neurodegenerative diseases. Herein, we describe these novel neuroprotective roles of the complement cascade, focusing on signaling pathways that may be therapeutically targetable in acute neuronal injury. PMID:20116331

  19. Purification of a thermostable antinociceptive lectin isolated from Andira anthelmia.

    PubMed

    Nascimento, Kyria Santiago; Nascimento, Francisco Lucas Faustino do; Silva, Mayara Torquato Lima; Nobre, Camila Bezerra; Moreira, Cleane Gomes; Brizeno, Luiz André Cavalcante; da Ponte, Edson Lopes; Assreuy, Ana Maria Sampaio; Cavada, Benildo Sousa

    2016-06-01

    Andira anthelmia (tribe Dalbergieae), a plant from Brazilian Amazon, possesses a seed lectin that was purified by affinity chromatography in sepharose-mannose. This novel Dalbergieae lectin, named AAL, agglutinated rabbit erythrocytes treated with trypsin. The hemagglutinating activity of AAL was maintained after incubation at a wide range of temperature (40 to 70 °C) and pH, was shown to be dependent on divalent cations, and was inhibited by d-mannose and d-sucrose. AAL showed an electrophoretic profile in sodium dodecyl sulfate-polyacrylamide gel electrophoresis similar to other lectins of the tribe Dalbergieae, presenting a double band of molecular weight with approximately 20 kDa and other minor bands of 17, 15, and 13 kDa, being the smaller fragment glycosylated. AAL injected by intravenous route in mice showed antinociceptive activity in two behavioral tests (writhing and formalin). In the writhing test induced by acetic acid, AAL showed inhibitory effect at 0.01 mg/kg (68%), 0.1 mg/kg (46%) and 1 mg/kg (74%). In the formalin test, AAL (0.1 mg/kg) inhibited by 48% the licking time in the inflammatory phase, an effect that was recovered by the lectin association with mannose. In conclusion, AAL presents analgesic effect involving the lectin domain via peripheral mechanisms of inflammatory nociception. This activity highlights the importance of lectins as tools to be used for understanding the interaction of protein-carbohydrate in processes associated to inflammatory pain. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26638121

  20. Lectin histochemical studies on the vomeronasal organ of the sheep.

    PubMed

    Ibrahim, Dalia; Nakamuta, Nobuaki; Taniguchi, Kazumi; Taniguchi, Kazuyuki

    2013-01-01

    The vomeronasal organ of sheep was examined using lectin histochemistry in order to compare the types and amounts of the glycoconjugates among various components of the vomeronasal sensory and non-sensory epithelia. In the vomeronasal sensory epithelium, Dolichos biflorus agglutinin (DBA) stained particular cells, located at the same level as the vomeronasal receptor cells, while the distribution, shape and number of the stained cells did not correspond to those of the vomeronasal receptor cells. Datura stramonium lectin (DSL), Concanavalin A (Con A), Phaseolus vulgaris agglutinin-E (PHA-E) and Phaseolus vulgaris agglutinin-L (PHA-L) labeled the basal cells of both vomeronasal sensory and non-sensory epithelia. While, Wheat germ agglutinin (WGA), Succinylated-wheat germ agglutinin (s-WGA), Lycopersicon esculentum lectin (LEL), Solanum tuberosum lectin (STL) and Ricinus communis agglutinin-I (RCA-120) labeled the basal cells of the sensory epithelium, and Bandeiraea simplicifolia lectin-I (BSL-I) stained the basal cells of the non-sensory epithelium, respectively. Seventeen lectins labeled the free border of both vomeronasal sensory and non-sensory epithelia, while Sophora japonica agglutinin (SJA), Jacalin and Pisum sativum agglutinin (PSA) labeled neither free border of the sensory nor that of non-sensory epithelia. The expression pattern of glycoconjugate was similar, but not identical, in the free border between the sensory and non-sensory epithelia. These results indicate that there are dissimilar features in the type and amount of glycoconjugates between the vomeronasal sensory and non-sensory epithelia, and at the same time, among the various cell types either in the vomeronasal sensory or non-sensory epithelium. PMID:23595118

  1. Conglutinin exhibits a complement-dependent enhancement of the respiratory burst of phagocytes stimulated by E. coli.

    PubMed Central

    Friis, P; Svehag, S E; Andersen, O; Gahrn-Hansen, B; Leslie, R G

    1991-01-01

    Conglutinin is a mammalian C-type lectin which shows anti-bacterial activity when tested in vivo and in vitro. This study concerns the effect of conglutinin on the respiratory burst of murine spleen cells, using a chemiluminescence assay for measurement of generated reactive oxygen metabolites. Conglutinin enhances, in a dose-dependent manner, the respiratory burst of spleen cells stimulated with serum-opsonized Escherichia coli. The enhancement was only demonstrable in the presence of a functional complement system. The conglutinin-mediated enhancement of the respiratory burst was inhibited in the presence of a N-acetyl-D-glucosamine, D-mannose and N-acetyl-D-mannosamine, monosaccharides reported to inhibit conglutinin-binding to zymosan and the complement factor iC3b. On the other hand, N-acetyl-D-galactosamine was non-inhibitory. PMID:1783427

  2. Lectin binding and surface glycoprotein pattern of human macrophage populations.

    PubMed

    Kreipe, H; Radzun, H J; Schumacher, U; Parwaresch, M R

    1986-01-01

    In the present study unstimulated and stimulated human blood monocytes, untreated and phorbol ester treated U-937 cells, as well as human peritoneal and alveolar macrophages were studied with respect to their surface membrane properties. Binding of different lectins and electrophoretic patterns of tritium labeled surface glycoproteins were compared. The analysis of surface glycoproteins could be interpreted as evidence for a common origin of the analysed cell populations. Furthermore, banding patterns of glycoproteins might be useful to define certain activation states within monocyte/macrophage differentiation. In contrast, lectin binding pattern did not clearly discriminate macrophage subpopulations. PMID:3102412

  3. The Pivotal Role of the Complement System in Aging and Age-related Macular Degeneration: Hypothesis Re-visited

    PubMed Central

    Anderson, Don H.; Radeke, Monte J.; Gallo, Natasha B.; Chapin, Ethan A.; Johnson, Patrick T.; Curletti, Christy R.; Hancox, Lisa S.; Hu, Jane; Ebright, Jessica N.; Malek, Goldis; Hauser, Michael A.; Rickman, Catherine Bowes; Bok, Dean; Hageman, Gregory S.; Johnson, Lincoln V.

    2009-01-01

    During the past ten years, dramatic advances have been made in unraveling the biological bases of age-related macular degeneration (AMD), the most common cause of irreversible blindness in western populations. In that timeframe, two distinct lines of evidence emerged which implicated chronic local inflammation and activation of the complement cascade in AMD pathogenesis. First, a number of complement system proteins, complement activators, and complement regulatory proteins were identified as molecular constituents of drusen, the hallmark extracellular deposits associated with early AMD. Subsequently, genetic studies revealed highly significant statistical associations between AMD and variants of several complement pathway-associated genes including: Complement factor H (CFH), complement factor H-related 1 and 3 (CFHR1 and CFHR3), complement factor B (CFB), complement component 2 (C2), and complement component 3 (C3). In this article, we revisit our original hypothesis that chronic local inflammatory and immune-mediated events at the level of Bruch’s membrane play critical roles in drusen biogenesis and, by extension, in the pathobiology of AMD. Secondly, we report the results of a new screening for additional AMD-associated polymorphisms in a battery of 63 complement-related genes. Third, we identify and characterize the local complement system in the RPE-choroid complex -- thus adding a new dimension of biological complexity to the role of the complement system in ocular aging and AMD. Finally, we evaluate the most salient, recent evidence that bears directly on the role of complement in AMD pathogenesis and progression. Collectively, these recent findings strongly re-affirm the importance of the complement system in AMD. They lay the groundwork for further studies that may lead to the identification of a transcriptional disease signature of AMD, and hasten the development of new therapeutic approaches that will restore the complement-modulating activity that appears to be compromised in genetically susceptible individuals. PMID:19961953

  4. Complement C3 and C5 Deficiency Affects Fracture Healing

    PubMed Central

    Ehrnthaller, Christian; Huber-Lang, Markus; Nilsson, Per; Bindl, Ronny; Redeker, Simon; Recknagel, Stefan; Rapp, Anna; Mollnes, Tom; Amling, Michael; Gebhard, Florian; Ignatius, Anita

    2013-01-01

    There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3-/- and C5-/- as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3-/-: -25%, p=0.02; C5-/-: -20% p=0.052) and newly formed bone (C3-/-: -38%, p=0.01; C5-/-: -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3-/- mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing. PMID:24260573

  5. Interplay between metal binding and cis/trans isomerization in legume lectins: structural and thermodynamic study of P. angolensis lectin.

    PubMed

    Garcia-Pino, Abel; Buts, Lieven; Wyns, Lode; Loris, Remy

    2006-08-01

    The interplay between metal binding, carbohydrate binding activity, stability and structure of the lectin from Pterocarpus angolensis was investigated. Removal of the metals leads to a more flexible form of the protein with significantly less conformational stability. Crystal structures of this metal-free form show significant structural rearrangements, although some structural features that allow the binding of sugars are retained. We propose that substitution of an asparagine residue at the start of the C-terminal beta-strand of the legume lectin monomer hinders the trans-isomerization of the cis-peptide bond upon demetallization and constitutes an intramolecular switch governing the isomer state of the non-proline bond and ultimately the lectin phenotype. PMID:16824540

  6. Complement and spinal cord injury: traditional and non-traditional aspects of complement cascade function in the injured spinal cord microenvironment.

    PubMed

    Peterson, Sheri L; Anderson, Aileen J

    2014-08-01

    The pathology associated with spinal cord injury (SCI) is caused not only by primary mechanical trauma, but also by secondary responses of the injured CNS. The inflammatory response to SCI is robust and plays an important but complex role in the progression of many secondary injury-associated pathways. Although recent studies have begun to dissect the beneficial and detrimental roles for inflammatory cells and proteins after SCI, many of these neuroimmune interactions are debated, not well understood, or completely unexplored. In this regard, the complement cascade is a key component of the inflammatory response to SCI, but is largely underappreciated, and our understanding of its diverse interactions and effects in this pathological environment is limited. In this review, we discuss complement in the context of SCI, first in relation to traditional functions for complement cascade activation, and then in relation to novel roles for complement proteins in a variety of models. PMID:25017886

  7. Anti-complement activity of the Ixodes scapularis salivary protein Salp20.

    PubMed

    Hourcade, Dennis E; Akk, Antonina M; Mitchell, Lynne M; Zhou, Hui-fang; Hauhart, Richard; Pham, Christine T N

    2016-01-01

    Complement, a major component of innate immunity, presents a rapid and robust defense of the intravascular space. While regulatory proteins protect host cells from complement attack, when these measures fail, unrestrained complement activation may trigger self-tissue injury, leading to pathologic conditions. Of the three complement activation pathways, the alternative pathway (AP) in particular has been implicated in numerous disease and injury states. Consequently, the AP components represent attractive targets for therapeutic intervention. The common hard-bodied ticks from the family Ixodidae derive nourishment from the blood of their mammalian hosts. During its blood meal the tick is exposed to host immune effectors, including the complement system. In defense, the tick produces salivary proteins that can inhibit host immune functions. The Salp20 salivary protein of Ixodes scapularis inhibits the host AP pathway by binding properdin and dissociating C3bBbP, the active C3 convertase. In these studies we examined Salp20 activity in various complement-mediated pathologies. Our results indicate that Salp20 can inhibit AP-dependent pathogenesis in the mouse. Its efficacy may be part in due to synergic effects it provides with the endogenous AP regulator, factor H. While Salp20 itself would be expected to be highly immunogenic and therefore inappropriate for therapeutic use, its emergence speaks for the potential development of a non-immunogenic Salp20 mimic that replicates its anti-properdin activity. PMID:26675068

  8. Involvement of ER stress in apoptosis induced by sialic acid-binding lectin (leczyme) from bullfrog eggs

    PubMed Central

    TATSUTA, TAKEO; HOSONO, MASAHIRO; MIURA, YUKI; SUGAWARA, SHIGEKI; KARIYA, YUKIKO; HAKOMORI, SENITIROH; NITTA, KAZUO

    2013-01-01

    Sialic-acid binding lectin (SBL) isolated from bullfrog (Rana catesbeiana) oocytes is a multifunctional protein which has lectin activity, ribonuclease activity and cancer-selective antitumor activity. It has been reported that SBL induces apoptosis accompanied by rigid mitochondrial perturbation, which indicates mediation of the intrinsic pathway. However, the mechanism of the antitumor effect of SBL has not been fully elucidated. We report, here, that ER stress is evoked in SBL-treated cells. We show that caspase-4, an initiator caspase of ER stress-mediated apoptosis was activated, and inhibition of caspase-4 resulted in significant attenuation of apoptosis induced by SBL. We analyzed the precise mechanism of activation of the caspase cascade induced by SBL, and found that caspase-9 and -4 are activated upstream of activation of caspase-8. Further study revealed that SBL induces the mitochondrial and ER stress-mediated pathways independently. It is noteworthy that SBL can induce cancer-selective apoptosis by multiple apoptotic signaling pathways, and it can serve as a candidate molecule for anticancer drugs in a novel field. PMID:24100413

  9. Progress and Trends in Complement Therapeutics

    PubMed Central

    Ricklin, Daniel; Lambris, John D.

    2012-01-01

    The past few years have proven to be a highly successful and exciting period for the field of complement-directed drug discovery and development. Driven by promising experiences with the first marketed complement drugs, increased knowledge about the involvement of complement in health and disease, and improvements in structural and analytical techniques as well as animal models of disease, the field has seen a surge in creative approaches to therapeutically intervene at various stages of the cascade. An impressive panel of compounds that show promise in clinical trials is meanwhile being lined up in the pipelines of both small biotechnology and big pharmaceutical companies. Yet with this new focus on complement-targeted therapeutics, important questions concerning target selection, point and length of intervention, safety, and drug delivery emerge. In view of the diversity of the clinical disorders involving abnormal complement activity or regulation, which include both acute and chronic diseases and affect a wide range of organs, diverse yet specifically tailored therapeutic approaches may be needed to shift complement back into balance. This chapter highlights the key changes in the field that shape our current perception of complement-targeted drugs and provides a brief overview of recent strategies and emerging trends. Selected examples of complement-related diseases and inhibitor classes are highlighted to illustrate the diversity and creativity in field. PMID:22990692

  10. Signaling by myeloid C-type lectin receptors in immunity and homeostasis

    PubMed Central

    Sancho, David; Reis e Sousa, Caetano

    2015-01-01

    Myeloid cells are key drivers of physiological responses to pathogen invasion or tissue damage. Members of the C-type lectin receptor (CLR) family stand out among the specialized receptors utilized by myeloid cells to orchestrate these responses. CLR ligands include carbohydrate, protein and lipid components of both pathogens and self, which variably trigger endocytic, phagocytic, pro-inflammatory or anti-inflammatory reactions. These varied outcomes rely on a versatile system for CLR signaling that includes tyrosine based motifs that recruit kinases, phosphatases or endocytic adaptors, as well as non-tyrosine based signals that modulate the activation of other pathways or couple to the uptake machinery. Here, we review the signaling properties of myeloid CLRs and how they impact the role of myeloid cells in innate and adaptive immunity. PMID:22224766

  11. Effect of Pleurotus florida lectin (PFL) on arsenic-induced activities of splenocytes in rat.

    PubMed

    Rana, Tanmoy; Bera, Asit Kumar; Bhattacharya, Debasis; Das, Subhashree; Pan, Diganta; Das, Subrata Kumar

    2014-05-01

    The present study was undertaken to investigate the protective effect of Pleurotus florida lectin (PFL) against arsenic-induced cytotoxicity and oxidative damages in freshly isolated splenocytes of rodents. Our finding indicated that arsenic caused reduction in cell adhesion, morphological alterations, cell proliferation, nitro blue tetrazolium (NBT) index, superoxide dismutase (SOD) activity, catalase (CAT) activity and relative mRNA expression of SOD2 in relation to housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and increased production of nitric oxide (NO), protein carbonyl (PC) and lipid peroxidation levels (LPO) assembled to play key factors for cytotoxicity and oxidative stress. PFL normalized cellular damages and enhanced SOD production pathway relating to gene expression. Further studies are needed to address effective phytochemicals of the edible mushroom and their mechanism. PMID:22914258

  12. Lectin-binding sites in epithelial cells of the mouse prostate gland.

    PubMed

    Sakuda, Kentaro; Yoshida, Ayaka; Muragishi, Ryoki; Yoshinaga, Kazuya

    2014-01-01

    The prostate is an exocrine gland in the male reproductive tract that secretes seminal fluids. To gain insight into the cytochemical properties of prostatic epithelial cells, the characteristics of glycoconjugates in mouse prostate sections were examined by lectin histochemistry and immunohistochemistry. Characteristic staining patterns were observed, depending on the type of lectins present in the epithelia. Luminal cells reacted specifically with mannose-binding lectins (Galanthus nivalis lectin, Hippeastrum hybrid lectin, Narcissus pseudonarcissus lectin) and Maclura pomifera lectin in all lobes of the prostate. Luminal cells also expressed galactose, N-acetyl-D-galactosamine (GalNAc), N-acetyl-D-glucosamine (GlcNAc), and fucose residues in the lateral and ventral lobes. Basal cells expressed GlcNAc and fucose, and reacted with Datura stramonium lectin and Aleuria aurantia lectin in all lobes. These results indicate that in the mouse prostate, the selectivity of lectin-binding sites for distinct cell types and lobe-dependent staining may relate to cellular and regional differences in function. Furthermore, some lectins selectively bound to prostatic epithelial cells, indicating their potential use as markers for the histopathological evaluation of prostatic diseases, cancer diagnosis, or male infertility. PMID:26004072

  13. Structures and binding specificity of galactose- and mannose-binding lectins from champedak: differences from jackfruit lectins

    PubMed Central

    Gabrielsen, Mads; Abdul-Rahman, Puteri Shafinaz; Othman, Shatrah; Hashim, Onn H.; Cogdell, Richard J.

    2014-01-01

    Galactose-binding and mannose-binding lectins from the champedak fruit, which is native to South-east Asia, exhibit useful potential clinical applications. The specificity of the two lectins for their respective ligands allows the detection of potential cancer biomarkers and monitoring of the glycosylated state of proteins in human serum and/or urine. To fully understand and expand the use of these natural proteins, their complete sequences and crystal structures are presented here, together with details of sugar binding. PMID:24915077

  14. Structures and binding specificity of galactose- and mannose-binding lectins from champedak: differences from jackfruit lectins.

    PubMed

    Gabrielsen, Mads; Abdul-Rahman, Puteri Shafinaz; Othman, Shatrah; Hashim, Onn H; Cogdell, Richard J

    2014-06-01

    Galactose-binding and mannose-binding lectins from the champedak fruit, which is native to South-east Asia, exhibit useful potential clinical applications. The specificity of the two lectins for their respective ligands allows the detection of potential cancer biomarkers and monitoring of the glycosylated state of proteins in human serum and/or urine. To fully understand and expand the use of these natural proteins, their complete sequences and crystal structures are presented here, together with details of sugar binding. PMID:24915077

  15. The use of lectin microarray for assessing glycosylation of therapeutic proteins.

    PubMed

    Zhang, Lei; Luo, Shen; Zhang, Baolin

    2016-04-01

    Glycans or carbohydrates attached to therapeutic glycoproteins can directly affect product quality, safety and efficacy, and therefore must be adequately analyzed and controlled throughout product life cycles. However, the complexity of protein glycosylation poses a daunting analytical challenge. In this study, we evaluated the utility of a lectin microarray for assessing protein glycans. Using commercial lectin chips, which contain 45 lectins toward distinct glycan structures, we were able to determine the lectin binding patterns of a panel of 15 therapeutic proteins, including 8 monoclonal antibodies. Lectin binding signals were analyzed to generate glycan profiles that were generally consistent with the known glycan patterns for these glycoproteins. In particular, the lectin-based microarray was found to be highly sensitive to variations in the terminal carbohydrate structures such as galactose versus sialic acid epitopes. These data suggest that lectin microarray could be used for screening glycan patterns of therapeutic glycoproteins. PMID:26918373

  16. Complement System Part II: Role in Immunity

    PubMed Central

    Merle, Nicolas S.; Noe, Remi; Halbwachs-Mecarelli, Lise; Fremeaux-Bacchi, Veronique; Roumenina, Lubka T.

    2015-01-01

    The complement system has been considered for a long time as a simple lytic cascade, aimed to kill bacteria infecting the host organism. Nowadays, this vision has changed and it is well accepted that complement is a complex innate immune surveillance system, playing a key role in host homeostasis, inflammation, and in the defense against pathogens. This review discusses recent advances in the understanding of the role of complement in physiology and pathology. It starts with a description of complement contribution to the normal physiology (homeostasis) of a healthy organism, including the silent clearance of apoptotic cells and maintenance of cell survival. In pathology, complement can be a friend or a foe. It acts as a friend in the defense against pathogens, by inducing opsonization and a direct killing by C5b–9 membrane attack complex and by triggering inflammatory responses with the anaphylatoxins C3a and C5a. Opsonization plays also a major role in the mounting of an adaptive immune response, involving antigen presenting cells, T-, and B-lymphocytes. Nevertheless, it can be also an enemy, when pathogens hijack complement regulators to protect themselves from the immune system. Inadequate complement activation becomes a disease cause, as in atypical hemolytic uremic syndrome, C3 glomerulopathies, and systemic lupus erythematosus. Age-related macular degeneration and cancer will be described as examples showing that complement contributes to a large variety of conditions, far exceeding the classical examples of diseases associated with complement deficiencies. Finally, we discuss complement as a therapeutic target. PMID:26074922

  17. Complement in action: an analysis of patent trends from 1976 through 2011.

    PubMed

    Yang, Kun; DeAngelis, Robert A; Reed, Janet E; Ricklin, Daniel; Lambris, John D

    2013-01-01

    Complement is an essential part of the innate immune response. It interacts with diverse endogenous pathways and contributes to the maintenance of homeostasis, the modulation of adaptive immune responses, and the development of various pathologies. The potential usefulness, in both research and clinical settings, of compounds that detect or modulate complement activity has resulted in thousands of publications on complement-related innovations in fields such as drug discovery, disease diagnosis and treatment, and immunoassays, among others. This study highlights the distribution and publication trends of patents related to the complement system that were granted by the United States Patent and Trademark Office from 1976 to the present day. A comparison to complement-related documents published by the World Intellectual Property Organization is also included. Statistical analyses revealed increasing diversity in complement-related research interests over time. More than half of the patents were found to focus on the discovery of inhibitors; interest in various inhibitor classes exhibited a remarkable transformation from chemical compounds early on to proteins and antibodies in more recent years. Among clinical applications, complement proteins and their modulators have been extensively patented for the diagnosis and treatment of eye diseases (especially age-related macular degeneration), graft rejection, cancer, sepsis, and a variety of other inflammatory and immune diseases. All of the patents discussed in this chapter, as well as those from other databases, are available from our newly constructed complement patent database: www.innateimmunity.us/patent. PMID:23402036

  18. Complement in Action: An Analysis of Patent Trends from 1976 Through 2011.

    PubMed

    Yang, Kun; Deangelis, Robert A; Reed, Janet E; Ricklin, Daniel; Lambris, John D

    2013-01-01

    Complement is an essential part of the innate immune response. It interacts with diverse endogenous pathways and contributes to the maintenance of homeostasis, the modulation of adaptive immune responses, and the development of various pathologies. The potential usefulness, in both research and clinical settings, of compounds that detect or modulate complement activity has resulted in thousands of publications on complement-related innovations in fields such as drug discovery, disease diagnosis and treatment, and immunoassays, among others. This study highlights the distribution and publication trends of patents related to the complement system that were granted by the United States Patent and Trademark Office from 1976 to the present day. A comparison to complement-related documents published by the World Intellectual Property Organization is also included. Statistical analyses revealed increasing diversity in complement-related research interests over time. More than half of the patents were found to focus on the discovery of inhibitors; interest in various inhibitor classes exhibited a remarkable transformation from chemical compounds early on to proteins and antibodies in more recent years. Among clinical applications, complement proteins and their modulators have been extensively patented for the diagnosis and treatment of eye diseases (especially age-related macular degeneration), graft rejection, cancer, sepsis, and a variety of other inflammatory and immune diseases. All of the patents discussed in this chapter, as well as those from other databases, are available from our newly constructed complement patent database: www.innateimmunity.us/patent . PMID:22990712

  19. Complement modulation of T cell immune responses during homeostasis and disease

    PubMed Central

    Clarke, Elizabeth V.; Tenner, Andrea J.

    2014-01-01

    The complement system is an ancient and critical effector mechanism of the innate immune system as it senses, kills, and clears infectious and/or dangerous particles and alerts the immune system to the presence of the infection and/or danger. Interestingly, an increasing number of reports have demonstrated a clear role for complement in the adaptive immune system as well. Of note, a number of recent studies have identified previously unknown roles for complement proteins, receptors, and regulators in T cell function. Here, we will review recent data demonstrating the influence of complement proteins C1q, C3b/iC3b, C3a (and C3aR), and C5a (and C5aR) and complement regulators DAF (CD55) and CD46 (MCP) on T cell function during homeostasis and disease. Although new concepts are beginning to emerge in the field of complement regulation of T cell function, future experiments should focus on whether complement is interacting directly with the T cell or is having an indirect effect on T cell function via APCs, the cytokine milieu, or downstream complement activation products. Importantly, the identification of the pivotal molecular pathways in the human systems will be beneficial in the translation of concepts derived from model systems to therapeutic targeting for treatment of human disorders. PMID:25210145

  20. Use of lectin microarray to differentiate gastric cancer from gastric ulcer

    PubMed Central

    Huang, Wei-Li; Li, Yang-Guang; Lv, Yong-Chen; Guan, Xiao-Hui; Ji, Hui-Fan; Chi, Bao-Rong

    2014-01-01

    AIM: To investigate the feasibility of lectin microarray for differentiating gastric cancer from gastric ulcer. METHODS: Twenty cases of human gastric cancer tissue and 20 cases of human gastric ulcer tissue were collected and processed. Protein was extracted from the frozen tissues and stored. The lectins were dissolved in buffer, and the sugar-binding specificities of lectins and the layout of the lectin microarray were summarized. The median of the effective data points for each lectin was globally normalized to the sum of medians of all effective data points for each lectin in one block. Formalin-fixed paraffin-embedded gastric cancer tissues and their corresponding gastric ulcer tissues were subjected to Ag retrieval. Biotinylated lectin was used as the primary antibody and HRP-streptavidin as the secondary antibody. The glycopatterns of glycoprotein in gastric cancer and gastric ulcer specimens were determined by lectin microarray, and then validated by lectin histochemistry. Data are presented as mean ± SD for the indicated number of independent experiments. RESULTS: The glycosylation level of gastric cancer was significantly higher than that in ulcer. In gastric cancer, most of the lectin binders showed positive signals and the intensity of the signals was stronger, whereas the opposite was the case for ulcers. Significant differences in the pathological score of the two lectins were apparent between ulcer and gastric cancer tissues using the same lectin. For MPL and VVA, all types of gastric cancer detected showed stronger staining and a higher positive rate in comparison with ulcer, especially in the case of signet ring cell carcinoma and intra-mucosal carcinoma. GalNAc bound to MPL showed a significant increase. A statistically significant association between MPL and gastric cancer was observed. As with MPL, there were significant differences in VVA staining between gastric cancer and ulcer. CONCLUSION: Lectin microarray can differentiate the different glycopatterns in gastric cancer and gastric ulcer, and the lectins MPL and VVA can be used as biomarkers. PMID:24833877

  1. Lectin Binding to Radopholus citrophilus and R. similis Proteins.

    PubMed

    Kaplan, D T; Gottwald, T R

    1992-06-01

    Lectin-binding glycoproteins in seven populations of two burrowing nematode sibling species were probed with five different biotinylated lectins on Western blots, and differences were correlated with nematode ability to parasitize citrus and to overcome citrus rootstock resistance. Banding patterns of molecular weight standards were fit best by an exponential decay function, and a predictive equation was used to estimate molecular weights (r(2) = 0.999). A band (131 kDa) that labeled with the lectin Concanavalin A (Con A) occurred in extracts from cuticles and egg shells of populations of Radopholus citrophilus that parasitize citrus. Wheat germ agglutin labeled a band (58 kDa) in aqueous homogenates of populations that reproduce in roots of citrus rootstock normally resistant to burrowing nematodes. The two sibling species R. citrophilus and R. similis were distinguished by a high molecular weight Con A-labeled band (608 kDa) from cuticle and egg shells. Probing blots with the lectin Limulus polyphemus agglutinin indicated that each population contained a band (12-16 kDa) specifically inhibited by the addition of 25 mM neuraminic acid, suggesting that glycoproteins with sialic acid moieties are present in burrowing nematodes. PMID:19282997

  2. Lectin Binding to Radopholus citrophilus and R. similis Proteins

    PubMed Central

    Kaplan, David T.; Gottwald, Tim R.

    1992-01-01

    Lectin-binding glycoproteins in seven populations of two burrowing nematode sibling species were probed with five different biotinylated lectins on Western blots, and differences were correlated with nematode ability to parasitize citrus and to overcome citrus rootstock resistance. Banding patterns of molecular weight standards were fit best by an exponential decay function, and a predictive equation was used to estimate molecular weights (r² = 0.999). A band (131 kDa) that labeled with the lectin Concanavalin A (Con A) occurred in extracts from cuticles and egg shells of populations of Radopholus citrophilus that parasitize citrus. Wheat germ agglutin labeled a band (58 kDa) in aqueous homogenates of populations that reproduce in roots of citrus rootstock normally resistant to burrowing nematodes. The two sibling species R. citrophilus and R. similis were distinguished by a high molecular weight Con A-labeled band (608 kDa) from cuticle and egg shells. Probing blots with the lectin Limulus polyphemus agglutinin indicated that each population contained a band (12-16 kDa) specifically inhibited by the addition of 25 mM neuraminic acid, suggesting that glycoproteins with sialic acid moieties are present in burrowing nematodes. PMID:19282997

  3. Glycoprofiling with micro-arrays of glycoconjugates and lectins.

    PubMed

    Angeloni, S; Ridet, J L; Kusy, N; Gao, H; Crevoisier, F; Guinchard, S; Kochhar, S; Sigrist, H; Sprenger, N

    2005-01-01

    To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling. PMID:15342550

  4. Isolation and characterization of a lectin from Annona muricata seeds.

    PubMed

    Damico, D C S; Freire, M G M; Gomes, V M; Toyama, M H; Marangoni, S; Novello, J C; Macedo, M L R

    2003-11-01

    A lectin with a high affinity for glucose/mannose was isolated from Annona muricata seeds (Annonaceae) by gel filtration chromatography on Sephacryl S-200, ion exchange chromatography on a DEAE SP-5 PW column, and molecular exclusion on a Protein Pak Glass 300 SW column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) yielded two protein bands of approximately 14 kDa and 22 kDa. However, only one band was seen in native PAGE. The Mr of the lectin estimated by fast-performance liquid chromatography-gel filtration on Superdex 75 was 22 kDa. The lectin was a glycoprotein with 8% carbohydrate (neutral sugar) and required divalent metal cations (Ca2+, Mg2+, and Mn2+) for full activity. Amino acid analysis revealed a large content of Glx, Gly, Phe, and Lys. The lectin agglutinated dog, chicken, horse, goose, and human erythrocytes and inhibited the growth of the fungi Fusarium oxysporum, Fusarium solani, and Colletotrichum musae. PMID:14714732

  5. Interaction of the tobacco lectin with histone proteins.

    PubMed

    Schouppe, Dieter; Ghesquière, Bart; Menschaert, Gerben; De Vos, Winnok H; Bourque, Stéphane; Trooskens, Geert; Proost, Paul; Gevaert, Kris; Van Damme, Els J M

    2011-03-01

    The tobacco (Nicotiana tabacum) agglutinin or Nictaba is a member of a novel class of plant lectins residing in the nucleus and the cytoplasm of tobacco cells. Since tobacco lectin expression is only observed after the plant has been subjected to stress situations such as jasmonate treatment or insect attack, Nictaba is believed to act as a signaling protein involved in the stress physiology of the plant. In this paper, a nuclear proteomics approach was followed to identify the binding partners for Nictaba in the nucleus and the cytoplasm of tobacco cv Xanthi cells. Using lectin affinity chromatography and pull-down assays, it was shown that Nictaba interacts primarily with histone proteins. Binding of Nictaba with histone H2B was confirmed in vitro using affinity chromatography of purified calf thymus histone proteins on a Nictaba column. Elution of Nictaba-interacting histone proteins was achieved with 1 m N-acetylglucosamine (GlcNAc). Moreover, mass spectrometry analyses indicated that the Nictaba-interacting histone proteins are modified by O-GlcNAc. Since the lectin-histone interaction was shown to be carbohydrate dependent, it is proposed that Nictaba might fulfill a signaling role in response to stress by interacting with O-GlcNAcylated proteins in the plant cell nucleus. PMID:21224338

  6. Momordica charantia seed lectin: toxicity, bacterial agglutination and antitumor properties.

    PubMed

    Kabir, Syed Rashel; Nabi, Md Mahamodun; Nurujjaman, Md; Abu Reza, Md; Alam, A H M Khurshid; Uz Zaman, Rokon; Khalid-Bin-Ferdaus, Khandaker Md; Amin, Ruhul; Khan, Md Masudul Hasan; Hossain, Md Anowar; Uddin, Md Salim; Mahmud, Zahid Hayat

    2015-03-01

    In last three decades, several studies were carried out on the D-galactose-specific lectin of Momordica charantia seeds (MCL). In the present study, in vitro growth inhibition (8-23 %) at different concentrations (6-24 μg/ml) of MCL was observed against Ehrlich ascites carcinoma (EAC) cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MCL also showed 28, 45, and 75 % growth inhibitions against EAC cells when administered 1.2, 2.0, and 2.8 mg/kg/day (i.p.), respectively for five consequent days in vivo in mice. After lectin treatment, the level of red blood cell and hemoglobin was increased significantly with the decrease of white blood cell and maintained the normal level when compared with EAC-bearing control and normal mice without EAC cells. Although MCL caused cell cycle arrest at G0/G1 phase of EAC cells, any irregular shape or apoptotic morphological alterations in the lectin-treated EAC cells was not observed by an optical and fluorescence microscope. Lectin showed toxicity against brine shrimp nauplii with an LC50 value of 49.7 μg/ml. Four out of seven pathogenic bacteria were agglutinated by MCL in the absence of inhibitory sugar D-lactose/D-galactose. In conclusion, MCL showed strong cytotoxic effect and therefore can be used as a potent anticancer chemotherapeutic agent. PMID:25542240

  7. Purification, characterization, and biological activities of broccolini lectin.

    PubMed

    Xu, Pingping; Zhang, Ting; Guo, Xiaolei; Ma, Chungwah; Zhang, Xuewu

    2015-01-01

    Plant lectins have displayed a variety of biological activities. In this study, for the first time, a 27 kDa arabinose- and mannose-specific lectin from Broccolini (Brassica oleracea Italica × Alboglabra), named as BL (Broccolini lectin), was purified by an activity-driven protocol. Mass spectrometry analysis and database search indicated that no matches with any plant lectin were found, but BL contained some peptide fragments (QQQGQQGQQLQQVISR, QQGQQQGQQGQQLQQVISR and VCNIPQVSVCPF QK). BL exhibited hemagglutinating activity against chicken erythrocytes at 4 µg/mL. BL retained full hemagglutinating activity at pH 7-8 and temperature 30-40°C, and had an optimal activity in Ca(2+) solution. Bioactivity assay revealed that BL exhibited dose-dependent inhibition activity on 5 bacterial species with IC50 values of 143.95-486.33 μg/mL, and on 3 cancer cells with IC50 values of 178.82-350.93 μg/mL. Notably, 5-fold reduction in IC50 values was observed on normal L-O2 vs cancerous HepG-2 cells (924.35 vs. 178.82 μg/mL). This suggests that BL should be promising in food and medicine. PMID:25737003

  8. Membrane adsorbers comprising grafted glycopolymers for targeted lectin binding

    PubMed Central

    Chenette, Heather C.S.; Husson, Scott M.

    2014-01-01

    This work details the design and testing of affinity membrane adsorbers for lectin purifications that incorporate glucose-containing glycopolymers. It is the selective interaction between the sugar residues of the glycopolymer and the complementary carbohydrate-binding domain of the lectin that provides the basis for the isolation and purification of lectins from complex biological media. The design approach used in these studies was to graft glycopolymer ‘tentacles’ from macroporous regenerated cellulose membranes by atom transfer radical polymerization. As shown in earlier studies, this design approach can be used to prepare high-productivity membrane adsorbers. The model lectin, concanavalin A (conA), was used to evaluate membrane performance in bind-and-elute purification, using a low molecular weight sugar for elution. The membrane capacity for binding conA was measured at equilibrium and under dynamic conditions using flow rates of 0.1 and 1.0 mL/min. The first Damkohler number was estimated to relate the adsorption rate to the convective mass transport rate through the membrane bed. It was used to assess whether adsorption kinetics or mass transport contributed the primary limitation to conA binding. Analyses indicate that this system is not limited by the accessibility of the binding sites, but by the inherent rate of adsorption of conA onto the glycopolymer. PMID:25866416

  9. Human and rodent decay-accelerating factors (CD55) are not species restricted in their complement-inhibiting activities

    PubMed Central

    Harris, C L; Spiller, O B; Morgan, B P

    2000-01-01

    Homologous complement activation is restricted on cells by the complement regulators, decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59. These proteins act in concert with other membrane structures to protect cells from homologous complement attack. In contrast, cells are usually sensitive to heterologous complement attack. It has been suggested that species-specific restriction of complement activation can be attributed to the inability of regulators to inhibit across species. We have investigated the capacities of human, rat and mouse analogues of DAF to regulate homologous and heterologous complement. Cells transfected with cDNA encoding these analogues were protected from heterologous complement attack. C3b-deposition experiments indicated that whilst cells were best protected by DAF from the same species, all three analogues inhibited human, rat and mouse complement. Comparable results were obtained in haemolysis assays using soluble, recombinant forms of the proteins. Inhibition of the classical pathway (CP) was best achieved with homologous DAF, although human DAF also inhibited rat complement, rat DAF also inhibited human complement and mouse DAF inhibited complement from all species. Human DAF was the best inhibitor of alternative pathway (AP)-mediated attack, inhibiting complement from all species. Mouse DAF inhibited mouse and rat AP, whilst rat DAF inhibited only rat AP. These data indicate that human and rodent analogues of DAF are not species restricted and highlights interesting differences in the capacity to regulate AP and CP. This has implications in broader fields of research, such as xenotransplantation, where cross-species regulation of complement is of paramount importance. PMID:10929073

  10. cDNA cloning and functional expression of KM+, the mannose-binding lectin from Artocarpus integrifolia seeds.

    PubMed

    daSilva, Luis L P; de Molfetta-Machado, Jeanne Blanco; Panunto-Castelo, Ademilson; Denecke, Jurgen; Goldman, Gustavo Henrique; Roque-Barreira, Maria-Cristina; Goldman, Maria Helena S

    2005-11-30

    KM+, a mannose-binding lectin present in the seeds of Artocarpus integrifolia, has interesting biological properties and potential pharmaceutical use [A. Panunto-Castelo, M.A. Souza, M.C. Roque-Barreira, J.S. Silva, KM(+), a lectin from Artocarpus integrifolia, induces IL-12 p40 production by macrophages and switches from type 2 to type 1 cell-mediated immunity against Leishmania major antigens, resulting in BALB/c mice resistance to infection, Glycobiology 11 (2001) 1035-1042. ; L.L.P. daSilva, A. Panunto-Castelo, M.H.S. Goldman, M.C. Roque-Barreira, R.S. Oliveira, M.D. Baruffi, J.B. Molfetta-Machado, Composition for preventing or treating appearance of epithelia wounds such as skin and corneal wounds or for immunomodulating, comprises lectin, Patent number WO20041008.]. Here, we have isolated clones encoding the full-length KM+ primary sequence from a cDNA library, through matrix PCR-based screening methodology. Analysis of KM+ nucleotide and deduced amino acid sequences provided strong evidence that it neither enters the secretory pathway nor undergoes post-translational modifications, which is in sharp contrast with jacalin, the more abundant lectin from A. integrifolia seeds. Current investigations into the KM+ properties are often impaired by the difficulty in obtaining sufficient quantities of jacalin-free KM+ through direct seed extraction. To obtain active recombinant protein (rKM+) in larger amounts, we tested three different expression systems. Expression vectors were constructed to produce: (a) rKM+ in E. coli in its native form, (b) rKM+ with GST as an N-terminal tag and (c) native rKM+ in Saccharomyces cerevisiae. The presence of the GST-tag significantly improved the overall rKM+ yield; however, most of the obtained rGST-KM+ was insoluble. Production of rKM+ in the yeast host yielded the highest quantities of soluble lectin that retained the typical high-mannose oligosaccharide-binding properties of the natural protein. The possible biotechnological applications of recombinant KM+ are discussed. PMID:16242845

  11. Complementation of a red-light-indifferent cyanobacterial mutant.

    PubMed Central

    Chiang, G G; Schaefer, M R; Grossman, A R

    1992-01-01

    Many cyanobacteria alter their phycobilisome composition in response to changes in light wavelength in a process termed complementary chromatic adaptation. Mutant strains FdR1 and FdR2 of the filamentous cyanobacterium Fremyella diplosiphon are characterized by aberrant chromatic adaptation. Instead of adjusting to different wavelengths of light, FdR1 and FdR2 behave as if they are always in green light; they do not respond to red light. We have previously reported complementation of FdR1 by conjugal transfer of a wild-type genomic library. The complementing DNA has now been localized by genetic analysis to a region on the rescued genomic subclone that contains a gene designated rcaC. This region of DNA is also able to complement FdR2. Southern blot analysis of genomic DNA from FdR1 and FdR2 indicates that these strains harbor DNA insertions within the rcaC sequence that may have resulted from the activity of transposable genetic elements. The predicted amino acid sequence of RcaC shares strong identity to response regulators of bacterial two-component regulatory systems. This relationship is discussed in the context of the signal-transduction pathway mediating regulation of genes encoding phycobilisome polypeptides during chromatic adaptation. Images PMID:1409650

  12. Emerging and Novel Functions of Complement Protein C1q

    PubMed Central

    Kouser, Lubna; Madhukaran, Shanmuga Priyaa; Shastri, Abhishek; Saraon, Anuvinder; Ferluga, Janez; Al-Mozaini, Maha; Kishore, Uday

    2015-01-01

    Complement protein C1q, the recognition molecule of the classical pathway, performs a diverse range of complement and non-complement functions. It can bind various ligands derived from self, non-self, and altered self and modulate the functions of immune and non-immune cells including dendritic cells and microglia. C1q involvement in the clearance of apoptotic cells and subsequent B cell tolerance is more established now. Recent evidence appears to suggest that C1q plays an important role in pregnancy where its deficiency and dysregulation can have adverse effects, leading to preeclampsia, missed abortion, miscarriage or spontaneous loss, and various infections. C1q is also produced locally in the central nervous system, and has a protective role against pathogens and possible inflammatory functions while interacting with aggregated proteins leading to neurodegenerative diseases. C1q role in synaptic pruning, and thus CNS development, its anti-cancer effects as an immune surveillance molecule, and possibly in aging are currently areas of extensive research. PMID:26175731

  13. Emerging and Novel Functions of Complement Protein C1q.

    PubMed

    Kouser, Lubna; Madhukaran, Shanmuga Priyaa; Shastri, Abhishek; Saraon, Anuvinder; Ferluga, Janez; Al-Mozaini, Maha; Kishore, Uday

    2015-01-01

    Complement protein C1q, the recognition molecule of the classical pathway, performs a diverse range of complement and non-complement functions. It can bind various ligands derived from self, non-self, and altered self and modulate the functions of immune and non-immune cells including dendritic cells and microglia. C1q involvement in the clearance of apoptotic cells and subsequent B cell tolerance is more established now. Recent evidence appears to suggest that C1q plays an important role in pregnancy where its deficiency and dysregulation can have adverse effects, leading to preeclampsia, missed abortion, miscarriage or spontaneous loss, and various infections. C1q is also produced locally in the central nervous system, and has a protective role against pathogens and possible inflammatory functions while interacting with aggregated proteins leading to neurodegenerative diseases. C1q role in synaptic pruning, and thus CNS development, its anti-cancer effects as an immune surveillance molecule, and possibly in aging are currently areas of extensive research. PMID:26175731

  14. Comprehensive profiling of accessible surface glycans of mammalian sperm using a lectin microarray

    PubMed Central

    2014-01-01

    It is well known that cell surface glycans or glycocalyx play important roles in sperm motility, maturation and fertilization. A comprehensive profile of the sperm surface glycans will greatly facilitate both basic research (sperm glycobiology) and clinical studies, such as diagnostics of infertility. As a group of natural glycan binders, lectin is an ideal tool for cell surface glycan profiling. However, because of the lack of effective technology, only a few lectins have been tested for lectin-sperm binding profiles. To address this challenge, we have developed a procedure for high-throughput probing of mammalian sperm with 91 lectins on lectin microarrays. Normal sperm from human, boar, bull, goat and rabbit were collected and analyzed on the lectin microarrays. Positive bindings of a set of ~50 lectins were observed for all the sperm of 5 species, which indicated a wide range of glycans are on the surface of mammalian sperm. Species specific lectin bindings were also observed. Clustering analysis revealed that the distances of the five species according to the lectin binding profiles are consistent with that of the genome sequence based phylogenetic tree except for rabbit. The procedure that we established in this study could be generally applicable for sperm from other species or defect sperm from the same species. We believe the lectin binding profiles of the mammalian sperm that we established in this study are valuable for both basic research and clinical studies. PMID:24629138

  15. Disruption of the C. elegans Intestinal Brush Border by the Fungal Lectin CCL2 Phenocopies Dietary Lectin Toxicity in Mammals

    PubMed Central

    Stutz, Katrin; Kaech, Andres; Aebi, Markus; Künzler, Markus; Hengartner, Michael O.

    2015-01-01

    Lectins are non-immunoglobulin carbohydrate-binding proteins without enzymatic activity towards the bound carbohydrates. Many lectins of e.g. plants or fungi have been suggested to act as toxins to defend the host against predators and parasites. We have previously shown that the Coprinopsis cinerea lectin 2 (CCL2), which binds to α1,3-fucosylated N-glycan cores, is toxic to Caenorhabditis elegans and results in developmental delay and premature death. In this study, we investigated the underlying toxicity phenotype at the cellular level by electron and confocal microscopy. We found that CCL2 directly binds to the intestinal apical surface and leads to a highly damaged brush border with loss of microvilli, actin filament depolymerization, and invaginations of the intestinal apical plasma membrane through gaps in the terminal web. We excluded several possible toxicity mechanisms such as internalization and pore-formation, suggesting that CCL2 acts directly on intestinal apical plasma membrane or glycocalyx proteins. A genetic screen for C. elegans mutants resistant to CCL2 generated over a dozen new alleles in bre 1, ger 1, and fut 1, three genes required for the synthesis of the sugar moiety recognized by CCL2. CCL2-induced intestinal brush border defects in C. elegans are similar to the damage observed previously in rats after feeding the dietary lectins wheat germ agglutinin or concanavalin A. The evolutionary conserved reaction of the brush border between mammals and nematodes might allow C. elegans to be exploited as model organism for the study of dietary lectin-induced intestinal pathology in mammals. PMID:26057124

  16. Disruption of the C. elegans Intestinal Brush Border by the Fungal Lectin CCL2 Phenocopies Dietary Lectin Toxicity in Mammals.

    PubMed

    Stutz, Katrin; Kaech, Andres; Aebi, Markus; Künzler, Markus; Hengartner, Michael O

    2015-01-01

    Lectins are non-immunoglobulin carbohydrate-binding proteins without enzymatic activity towards the bound carbohydrates. Many lectins of e.g. plants or fungi have been suggested to act as toxins to defend the host against predators and parasites. We have previously shown that the Coprinopsis cinerea lectin 2 (CCL2), which binds to α1,3-fucosylated N-glycan cores, is toxic to Caenorhabditis elegans and results in developmental delay and premature death. In this study, we investigated the underlying toxicity phenotype at the cellular level by electron and confocal microscopy. We found that CCL2 directly binds to the intestinal apical surface and leads to a highly damaged brush border with loss of microvilli, actin filament depolymerization, and invaginations of the intestinal apical plasma membrane through gaps in the terminal web. We excluded several possible toxicity mechanisms such as internalization and pore-formation, suggesting that CCL2 acts directly on intestinal apical plasma membrane or glycocalyx proteins. A genetic screen for C. elegans mutants resistant to CCL2 generated over a dozen new alleles in bre 1, ger 1, and fut 1, three genes required for the synthesis of the sugar moiety recognized by CCL2. CCL2-induced intestinal brush border defects in C. elegans are similar to the damage observed previously in rats after feeding the dietary lectins wheat germ agglutinin or concanavalin A. The evolutionary conserved reaction of the brush border between mammals and nematodes might allow C. elegans to be exploited as model organism for the study of dietary lectin-induced intestinal pathology in mammals. PMID:26057124

  17. The role of complement in C3 glomerulopathy.

    PubMed

    Zipfel, Peter F; Skerka, Christine; Chen, Qian; Wiech, Thorsten; Goodship, Tim; Johnson, Sally; Fremeaux-Bacchi, Veronique; Nester, Clara; de Crdoba, Santiago Rodrguez; Noris, Marina; Pickering, Matthew; Smith, Richard

    2015-09-01

    C3 glomerulopathy describes a spectrum of disorders with glomerular pathology associated with C3 cleavage product deposition and with defective complement action and regulation (Fakhouri et al., 2010; Sethi et al., 2012b). Kidney biopsies from these patients show glomerular accumulation or deposition of C3 cleavage fragments, but no or minor deposition of immunoglobulins (Appel et al., 2005; D'Agati and Bomback, 2012; Servais et al., 2007; Sethi and Fervenza, 2011). At present the current situation asks for a better definition of the underlining disease mechanisms, for precise biomarkers, and for a treatment for this disease. The complement system is a self activating and propelling enzymatic cascade type system in which inactive, soluble plasma components are activated spontaneously and lead into an amplification loop (Zipfel and Skerka, 2009). Activation of the alternative pathway is spontaneous, occurs by default, and cascade progression leads to amplification by complement activators. The system however is self-controlled by multiple regulators and inhibitors, like Factor H that control cascade progression in fluid phase and on surfaces. The activated complement system generates a series of potent effector components and activation products, which damage foreign-, as well as modified self cells, recruit innate immune cells to the site of action, coordinate inflammation and the response of the adaptive immune system in form of B cells and T lymphocytes (Kohl, 2006; Medzhitov and Janeway, 2002; Ogden and Elkon, 2006; Carroll, 2004; Kemper and Atkinson, 2007; Morgan, 1999; Muller-Eberhard, 1986; Ricklin et al., 2010). Complement controls homeostasis and multiple reactions in the vertebrate organism including defense against microbial infections (Diaz-Guillen et al., 1999; Mastellos and Lambris, 2002; Nordahl et al., 2004; Ricklin et al., 2010). In consequence defective control of the spontaneous self amplifying cascade or regulation is associated with numerous human disorders (Ricklin and Lambris, 2007; Skerka and Zipfel, 2008; Zipfel et al., 2006). Understanding the exact action and regulation of this sophisticated homeotic cascade system is relevant to understand disease pathology of various complement associated human disorders. Furthermore this knowledge is relevant for a better diagnosis and appropriate therapy. At present diagnosis of C3 glomerulopathy is primarily based on the kidney biopsy, and histological, immmunohistological and electron microscopical evaluation (D'Agati and Bomback, 2012; Fakhouri et al., 2010; Medjeral-Thomas et al., 2014a,b; Sethi et al., 2012b). The challenge is to define the actual cause of the diverse glomerular changes or damages, to define how C3 deposition results in the reported glomerular changes, the location of the cell damage and the formation of deposits. PMID:25929733

  18. Genetics Home Reference: complement factor I deficiency

    MedlinePlus

    ... This Page Baracho GV, Nudelman V, Isaac L. Molecular characterization of homozygous hereditary factor I deficiency. Clin ... AG, Truedsson L, Villoutreix BO, Blom AM. Genetic, molecular and functional analyses of complement factor I deficiency. ...

  19. Genetics Home Reference: complement component 2 deficiency

    MedlinePlus

    ... Page Jönsson G, Sjöholm AG, Truedsson L, Bengtsson AA, Braconier JH, Sturfelt G. Rheumatological manifestations, organ damage ... Epub 2011 May 31. Review. Truedsson L, Bengtsson AA, Sturfelt G. Complement deficiencies and systemic lupus erythematosus. ...

  20. Efficacy of Targeted Complement Inhibition in Experimental C3 Glomerulopathy.

    PubMed

    Ruseva, Marieta M; Peng, Tao; Lasaro, Melissa A; Bouchard, Keith; Liu-Chen, Susan; Sun, Fang; Yu, Zhao-Xue; Marozsan, Andre; Wang, Yi; Pickering, Matthew C

    2016-02-01

    C3 glomerulopathy refers to renal disorders characterized by abnormal accumulation of C3 within the kidney, commonly along the glomerular basement membrane (GBM). C3 glomerulopathy is associated with complement alternative pathway dysregulation, which includes functional defects in complement regulator factor H (FH). There is no effective treatment for C3 glomerulopathy. We investigated the efficacy of a recombinant mouse protein composed of domains from complement receptor 2 (CR2) and FH (CR2-FH) in two models of C3 glomerulopathy with either preexisting or triggered C3 deposition along the GBM. FH-deficient mice spontaneously develop renal pathology associated with abnormal C3 accumulation along the GBM and secondary plasma C3 deficiency. CR2-FH partially restored plasma C3 levels in FH-deficient mice 2 hours after intravenous injection. CR2-FH specifically targeted glomerular C3 deposits, reduced the linear C3 reactivity assessed with anti-C3 and anti-C3b/iC3b/C3c antibodies, and prevented further spontaneous accumulation of C3 fragments along the GBM. Reduction in glomerular C3d and C9/C5b-9 reactivity was observed after daily administration of CR2-FH for 1 week. In a second mouse model with combined deficiency of FH and complement factor I, CR2-FH prevented de novo C3 deposition along the GBM. These data show that CR2-FH protects the GBM from both spontaneous and triggered C3 deposition in vivo and indicate that this approach should be tested in C3 glomerulopathy. PMID:26047789

  1. Differential mechanisms of complement-mediated neutralization of the closely related paramyxoviruses simian virus 5 and mumps virus

    SciTech Connect

    Johnson, John B.; Capraro, Gerald A.; Parks, Griffith D.

    2008-06-20

    The complement system is an important component of the innate immune response to virus infection. The role of human complement pathways in the in vitro neutralization of three closely related paramyxoviruses, Simian Virus 5 (SV5), Mumps virus (MuV) and Human Parainfluenza virus type 2 (HPIV2) was investigated. Sera from ten donors showed high levels of neutralization against HPIV2 that was largely complement-independent, whereas nine of ten donor sera were found to neutralize SV5 and MuV only in the presence of active complement pathways. SV5 and MuV neutralization proceeded through the alternative pathway of the complement cascade. Electron microscopy studies and biochemical analyses showed that treatment of purified SV5 with human serum resulted in C3 deposition on virions and the formation of massive aggregates, but there was relatively little evidence of virion lysis. Treatment of MuV with human serum also resulted in C3 deposition on virions, however in contrast to SV5, MuV particles were lysed by serum complement and there was relatively little aggregation. Assays using serum depleted of complement factors showed that SV5 and MuV neutralization in vitro was absolutely dependent on complement factor C3, but was not dependent on downstream complement factors C5 or C8. Our results indicate that even though antibodies exist that recognize both SV5 and MuV, they are mostly non-neutralizing and viral inactivation in vitro occurs through the alternative pathway of complement. The implications of our work for development of paramyxovirus vectors and vaccines are discussed.

  2. Virus-induced gene complementation in tomato

    PubMed Central

    Kong, Jinhua; Chen, Weiwei; Shen, Jiajia; Qin, Cheng; Lai, Tongfei; Zhang, Pengcheng; Wang, Ying; Wu, Chaoqun; Yang, Xin; Hong, Yiguo

    2013-01-01

    Virus-induced gene complementation (VIGC), a plant virus technology based on Potato virus X for transient overexpression of endogenous genes complemented tomato mutants, resulting in non-ripening fruits to ripen. This efficient “gain-of-function” approach involves no stable transformation, and reveals a fruit-specific transcriptional network that may exist among key transcription factors in modulating tomato ripening. Thus, VIGC represents a novel and feasible strategy for gene functional analysis in plants. PMID:24305652

  3. Chronic fatigue syndrome and complement activation.

    PubMed

    Geller, Robert Dennis; Giclas, Patricia C

    2009-01-01

    This report describes a case of chronic fatigue syndrome (CFS) that followed a well-documented episode of acute Epstein-Barr virus (EBV) mononucleosis. All aetiological tests for chronic fatigue were found to be negative or normal, as were immunological tests. After 2 years of chronic fatigue following the acute illness, measurements of complement split products were performed to test for complement activation. These were positive and remained positive for 14 months, after which the patient then recovered from CFS. PMID:21686614

  4. Evolution of the complement system in protostomes revealed by de novo transcriptome analysis of six species of Arthropoda.

    PubMed

    Sekiguchi, Reo; Nonaka, Masaru

    2015-05-01

    To elucidate the evolutionary history of the complement system in Arthropoda, de novo transcriptome analysis was performed with six species among the Chelicerata, Myriapoda, and Crustacea, and complement genes were identified based on their characteristic domain structures. Complement C3 and factor B (FB) were identified from a sea spider, a jumping spider, and a centipede, but not from a sea firefly or two millipede species. No additional complement components identifiable by their characteristic domain structures were found from any of these six species. These results together with genome sequence information for several species of the Hexapoda suggest that the common ancestor of the Arthropoda possessed a simple complement system comprising C3 and FB, and thus resembled the alternative pathway of the mammalian complement system. It was lost at least twice independently during the evolution of Arthropoda in the millipede lineage and in the common ancestor of Crustacea and Hexapoda. PMID:25530095

  5. Hereditary deficiency of the seventh component of complement.

    PubMed Central

    Boyer, J T; Gall, E P; Norman, M E; Nilsson, U R; Zimmerman, T S

    1975-01-01

    Deficiency of the seventh component of complement has been found in the serum of a 42-yr-old Caucasian woman who has Raynaud's phenomenon, sclerodactyly, and telangiectasia. Partial deficiency was found in the serum of the patient's parents and children, indicating a pattern of inheritance of autosomal codominance. Transfusion experiments indicated that exogenous C7 had a 91-h halk-life in the patient. There was no evidence for C7 synthesis after transfusion. No C7 inhibitors were detected in the patient's serum. The patient's serum was found to support the activation of complement by both the classical and properdin pathways to the C7 stage. The addition of C7 to the patient's serum permitted it to support hemolytic reactions initiated by either pathway. No defects could be detected in plasma or whole blood coagulation. The patient's serum was deficient in opsonizing unsensitized yeast particles in serum and in the generation of chemotactic factor by antigen-antibody complexes and endotoxin. Both deficiencies were corrected by the addition of C7. These observations suggest a key role for C7 for in vitro yeast phagocytosis and chemotaxis generation. However, the patient's lack of infections indicates a relatively minor role for C7 in human resistance to infection. PMID:1099121

  6. Collectin-43 is a serum lectin with a distinct pattern of carbohydrate recognition.

    PubMed Central

    Loveless, R W; Holmskov, U; Feizi, T

    1995-01-01

    Collectin-43 (CL-43) is a C-type serum lectin and a member of the collectin family of soluble proteins that are effector molecules in innate immunity. We have investigated the binding specificity of CL-43 using as model systems a panel of structurally defined oligosaccharides in the form of neoglycolipids, and several glycoproteins derived from the complement glycoprotein C3 during activation of the complement cascade. A specificity is revealed towards fucose as part of the Lea oligosaccharide sequence, and towards mannose as found on high mannose-type chains. These are features shared with other serum collectins, conglutinin and mannan-binding proteins; a major difference is the lack of detectable binding by CL-43 to N-glycosidic oligosaccharides terminating in N-acetylglucosamine. CL-43 has a unique pattern of reactivity towards high mannose-type oligosaccharides on the two glycosylation sites of C3 and derived glycoproteins: it binds to C3c (not bound by conglutinin and mannan-binding protein) but not to hydrolysed C3 [C3(H2O)], C3b or iC3b immobilized on microwells (all bound by conglutinin but not by mannan-binding protein). When these glycoproteins are sodium dodecyl sulphate (SDS)-treated and immobilized on nitrocellulose, CL-43 (but not conglutinin nor mannan-binding protein) binds strongly to C3(H2O), iC3b and C3c. The salient conclusions are, first, that there are remarkable positive or negative effects of carrier protein on oligosaccharide presentation and these differ for the individual collectins. Second, the different though partially overlapping binding patterns among the collectins may be important for their function as circulating effector molecules with broad surveillance capabilities. Images Figure 2 Figure 3 Figure 4 Figure 7 PMID:7558162

  7. Mannose-binding Lectin Deficiency in Patients with a History of Recurrent Infections.

    PubMed

    Rashidi, Elahe; Fazlollahi, Mohammad Reza; Zahedifard, Sara; Talebzadeh, Azadeh; Kazemnejad, Anoshirvan; Saghafi, Shiva; Pourpak, Zahra

    2016-02-01

    Mannose-binding lectin (MBL) is a protein of innate immune system that is involved in opsonization and complement activation. MBL deficiency is associated with predisposition to infectious diseases; however subnormal levels are also seen in healthy subjects. The aim of this study was to investigate the prevalence and clinical manifestation of MBL deficiency in patients with increased susceptibility to infection. We studied the MBL serum concentration of 104 patients with a history of recurrent and/or severe infections referred to Immunology, Asthma and Allergy Research Institute (IAARI) in order to evaluate the primary immunodeficiency (PID). The distribution of MBL deficiency in these patients and 593 healthy subjects of previous study were analyzed. The frequency of individuals with MBL deficiency was significantly higher in patients with recurrent and/or severe infections (13.5% [14/104]) compared with healthy subjects (4.7% [28/593]; p=0.001; OR 3.1, 95% CI 1.5-6.1). However, in 10.9% (7/64) of patients with recurrent infections without any immunodeficiency background, the MBL deficiency was detected. On the whole, our findings indicate an association between MBL deficiency and increased susceptibility to infections. PMID:26996114

  8. Mannose-Binding Lectin Levels and Carotid Intima-Media Thickness in Type 2 Diabetic Patients

    PubMed Central

    Káplár, Miklós; Sweni, Shah; Kulcsár, Julianna; Cogoi, Barbara; Esze, Regina; Somodi, Sándor; Papp, Mária; Oláh, László; Magyar, Mária Tünde; Szabó, Katalin; Czuriga-Kovács, Katalin Réka; Hársfalvi, Jolán; Paragh, György

    2016-01-01

    Introduction. Mannose-binding lectin (MBL) activates complement system and has been suggested to play a role in vascular complications in diabetics. Carotid intima-media thickness (cIMT) detects subclinical atherosclerosis. We evaluated the association of MBL and IMT in type 2 diabetic (T2DM) patients. Methods. Serum MBL levels and cIMT were measured in a total of 103 diabetics and in 98 age-matched healthy controls. Results. There was no significant difference in MBL level in T2DM versus controls. As expected, IMT was significantly higher in T2DM patients than in controls (P = 0.001). In T2DM, the lowest cIMT was seen in patients with normal MBL level (500–1000) while cIMT continuously increased with both high MBL and absolute MBL deficiency states. This was especially significant in high MBL versus normal MBL T2DM patients (P = 0.002). According to multiple regression analysis the main predictors of IMT in T2DM are age (P < 0.003), ApoA level (P = 0.023), and the MBL (P = 0.036). Conclusions. Our results suggest a dual role of MBL as a risk factor for cIMT in T2DM. MBL may also be used as a marker of macrovascular disease, as both low and high levels indicate the susceptibility for atherosclerosis in T2DM. PMID:26640806

  9. Mannose binding lectin acute phase activity in patients with severe infection.

    PubMed

    Dean, M M; Minchinton, R M; Heatley, S; Eisen, D P

    2005-07-01

    Mannose Binding Lectin (MBL) is a liver derived, circulating plasma protein that plays a pivotal role in innate immunity. MBL functions as a pathogen recognition molecule, opsonising organisms and initiating the complement cascade. MBL deficiency arising from mutations and promoter polymorphisms in the MBL2 gene is common and has been associated with risk, severity, and frequency of infection in a number of clinical settings. With MBL therapy on the horizon, the usefulness of replacement MBL therapy has been challenged by the notion, that as an acute phase protein, MBL levels may rise under stress to sufficient levels, in individuals who are usually deficient. This report demonstrates that in patients with sepsis and septic shock, the majority of patients do not display an MBL acute phase response: 41.4% of individuals maintained consistent MBL levels throughout hospital stay, 31.3% of individuals demonstrated a positive acute phase response, and a negative acute phase response was observed in 27.3% of individuals studied. Importantly, a positive acute phase response was generally observed in individuals with wild-type MBL2 genes. When a positive acute phase response was observed in individuals with coding mutation, these individuals demonstrated a normal MBL level on admission to hospital. Furthermore, no individual, regardless of genotype who was MBL deficient at admission was able to demonstrate a positive acute phase response into the normal MBL range. These findings indicate MBL demonstrates a variable acute phase response in the clinical setting of sepsis and septic shock. PMID:16133991

  10. Recruitment of Factor H as a Novel Complement Evasion Strategy for Blood-Stage Plasmodium falciparum Infection.

    PubMed

    Kennedy, Alexander T; Schmidt, Christoph Q; Thompson, Jennifer K; Weiss, Greta E; Taechalertpaisarn, Tana; Gilson, Paul R; Barlow, Paul N; Crabb, Brendan S; Cowman, Alan F; Tham, Wai-Hong

    2016-02-01

    The human complement system is the frontline defense mechanism against invading pathogens. The coexistence of humans and microbes throughout evolution has produced ingenious molecular mechanisms by which microorganisms escape complement attack. A common evasion strategy used by diverse pathogens is the hijacking of soluble human complement regulators to their surfaces to afford protection from complement activation. One such host regulator is factor H (FH), which acts as a negative regulator of complement to protect host tissues from aberrant complement activation. In this report, we show that Plasmodium falciparum merozoites, the invasive form of the malaria parasites, actively recruit FH and its alternative spliced form FH-like protein 1 when exposed to human serum. We have mapped the binding site in FH that recognizes merozoites and identified Pf92, a member of the six-cysteine family of Plasmodium surface proteins, as its direct interaction partner. When bound to merozoites, FH retains cofactor activity, a key function that allows it to downregulate the alternative pathway of complement. In P. falciparum parasites that lack Pf92, we observed changes in the pattern of C3b cleavage that are consistent with decreased regulation of complement activation. These results also show that recruitment of FH affords P. falciparum merozoites protection from complement-mediated lysis. Our study provides new insights on mechanisms of immune evasion of malaria parasites and highlights the important function of surface coat proteins in the interplay between complement regulation and successful infection of the host. PMID:26700768

  11. Factor H in Porcine Seminal Plasma Protects Sperm against Complement Attack in Genital Tracts*

    PubMed Central

    Sakaue, Tomohisa; Takeuchi, Keisuke; Maeda, Toshinaga; Yamamoto, Yoshio; Nishi, Katsuji; Ohkubo, Iwao

    2010-01-01

    We found that factor H (FH) exists in porcine seminal plasma. Purified FH strongly inhibited serum alternative pathway complement activation against lipopolysaccharide. The molecular weight, pI, and heparin-binding activity of the purified protein were different from those of purified FH from porcine serum. The complement regulatory activity of seminal plasma FH was ∼2-fold stronger than that of serum FH. Treatment of purified serum FH with sialidase and N-glycosidase F gave almost the same results as those of seminal plasma FH. The deletion of sialic acid from the carbohydrate chains of both FHs contributed to heparin-binding and complement regulatory activities. Results of reverse transcriptase-PCR, Western blot analysis, and immunohistochemistry showed that seminal plasma FH is mainly secreted from epithelial cells of the seminal vesicle in male genital tracts. FH was also detected in the outer acrosomal region of ejaculated sperm by immunofluorescence staining, and found that the purified FH from the sperm membrane has the same complement regulatory activity as that of seminal plasma FH. The ejaculated sperm possessing FH in the outer acrosomal region considerably evaded complement attack. We also found that there is strong complement activity in fluids from female genital tract ducts. These findings indicate that FH bound to the outer acrosomal region and soluble FH play important roles in protecting sperm against complement attack in male and female genital tracts. PMID:19920146

  12. Eculizumab reduces complement activation, inflammation, endothelial damage, thrombosis, and renal injury markers in aHUS

    PubMed Central

    Cofiell, Roxanne; Kukreja, Anjli; Bedard, Krystin; Yan, Yan; Mickle, Angela P.; Ogawa, Masayo; Bedrosian, Camille L.

    2015-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a genetic, life-threatening disease characterized by uncontrolled complement activation, systemic thrombotic microangiopathy (TMA), and vital organ damage. We evaluated the effect of terminal complement blockade with the anti-C5 monoclonal antibody eculizumab on biomarkers of cellular processes involved in TMA in patients with aHUS longitudinally, during up to 1 year of treatment, compared with in healthy volunteers. Biomarker levels were elevated at baseline in most patients, regardless of mutational status, plasma exchange/infusion use, platelet count, or lactate dehydrogenase or haptoglobin levels. Eculizumab reduced terminal complement activation (C5a and sC5b-9) and renal injury markers (clusterin, cystatin-C, β2-microglobulin, and liver fatty acid binding protein-1) to healthy volunteer levels and reduced inflammation (soluble tumor necrosis factor receptor-1), coagulation (prothrombin fragment F1+2 and d-dimer), and endothelial damage (thrombomodulin) markers to near-normal levels. Alternative pathway activation (Ba) and endothelial activation markers (soluble vascular cell adhesion molecule-1) decreased but remained elevated, reflecting ongoing complement activation in aHUS despite complete terminal complement blockade. These results highlight links between terminal complement activation and inflammation, endothelial damage, thrombosis, and renal injury and underscore ongoing risk for systemic TMA and progression to organ damage. Further research regarding underlying complement dysregulation is warranted. This trial was registered at www.clinicaltrials.gov as #NCT01194973. PMID:25833956

  13. Complement emerges as a masterful regulator of CNS homeostasis, neural synaptic plasticity and cognitive function.

    PubMed

    Mastellos, Dimitrios C

    2014-11-01

    Growing evidence points to a previously elusive role of complement-modulated pathways in CNS development, neurogenesis and synaptic plasticity. Distinct complement effectors appear to play a multifaceted role in brain homeostasis by regulating synaptic pruning in the retinogeniculate system and sculpting functional neural circuits both in the developing and adult mammalian brain. A recent study by Perez-Alcazar et al. (2014) provides novel insights into this intricate interplay between complement and the dynamically regulated brain synaptic circuitry, by reporting that mice deficient in C3 exhibit enhanced hippocampus-dependent spatial learning and cognitive performance. This behavioral pattern is associated with an impact of C3 on the functional capacity of glutamatergic synapses, supporting a crucial role for complement in excitatory synapse elimination in the hippocampus. These findings add a fresh twist to this rapidly evolving research field, suggesting that discrete complement components may differentially modulate synaptic connectivity by wiring up with diverse neural effectors in different regions of the brain. The emerging role of complement in synaptogenesis and neural network plasticity opens new conceptual avenues for considering complement interception as a potential therapeutic modality for ameliorating progressive cognitive impairment in age-related, debilitating brain diseases with a prominent inflammatory signature. PMID:24975369

  14. Transcriptomic response of cowpea bruchids to N-acetylglucosamine-specific lectins.

    PubMed

    Wang, Li-Hua; Chi, Yong Hun; Guo, Feng-Guang; Li-Byarlay, Hongmei; Balfe, Susan; Fang, Ji-Chao; Pittendrigh, Barry R; Zhu-Salzman, Keyan

    2015-02-01

    Griffonia simplicifolia lectin II (GSII) and wheat germ agglutinin (WGA) are N-acetylglucosamine-binding lectins. Previous studies demonstrated that they have anti-insect activity, a property potentially useful in pest control. To gain some insight into the insect response to dietary lectins, we performed transcriptomic analysis using the cowpea bruchid (Callosobruchus maculatus) midgut microarray platform we built. Compared to the nonnutritional cellulose treatment, dietary lectins induced more profound changes in gene expression. Ingestion of relatively high doses of lectins for 24 h resulted in alteration of gene expression involved in sugar and lipid metabolism, transport, development, defense, and stress tolerance. Metabolic genes were largely downregulated. Moreover, we observed disorganized microvilli resulting from ingestion of WGA. This morphological change is consistent with the lectin-induced changes in genes related to midgut epithelial cell repair. In addition, suboptimal nutrient conditions may serve as a stress signal to trigger senescence processes, leading to growth arrest and developmental delay. PMID:24446316

  15. Electronic Detection of Lectins Using Carbohydrate Functionalized Nanostructures: Graphene versus Carbon Nanotubes

    PubMed Central

    Chen, Yanan; Vedala, Harindra; Kotchey, Gregg P.; Audfray, Aymeric; Cecioni, Samy; Imberty, Anne; Vidal, Sébastien; Star, Alexander

    2012-01-01

    Here we investigated the interactions between lectins and carbohydrates using field-effect transistor (FET) devices comprised of chemically converted graphene (CCG) and single-walled carbon nanotubes (SWNTs). Pyrene- and porphyrin-based glycoconjugates were functionalized noncovalently on the surface of CCG-FET and SWNT-FET devices, which were then treated with 2 µM of nonspecific and specific lectins. In particular, three different lectins (PA-IL, PA-IIL and ConA) and three carbohydrate epitopes (galactose, fucose and mannose) were tested. The responses of 36 different devices were compared and rationalized using computer-aided models of carbon nanostructure/glycoconjugate interactions. Glycoconjugates surface coverage in addition to one-dimensional structures of SWNTs resulted in optimal lectin detection. Additionally, lectin titration data of SWNT- and CCG-based biosensors were used to calculate lectin dissociation constants (Kd) and compare them to the values obtained from the isothermal titration microcalorimetry (ITC) technique. PMID:22136380

  16. Hemolymph lectin of the pearl oyster, Pinctada fucata martensii: a possible non-self recognition system.

    PubMed

    Suzuki, T; Mori, K

    1990-01-01

    To elucidate a mutual correlation between the hemolymph lectin and hemocytes of the pearl oyster, Pinctada fucata martensii, we searched for common epitopes and ligands. Neither the hemocyte plasma membrane nor cytoplasm was immunoreactive to anti-hemolymph lectin antibody. The hemolymph lectin strongly bound to D-galactose and N-acetyl-D-galactosamine, and the plasma membrane of both granulocytes and agranulocytes had affinity only for D-mannose and N-acetyl-D-glucosamine-binding plant lectins. In the gonad, the hemolymph lectin selectively adsorbed injected horse red blood cells (HRBC), and its hemagglutinating activity probably prevented them from dispersing. Pinctada sp. may possess system of recognition of non-self by the hemolymph lectin. PMID:2369965

  17. Advances in lectin microarray technology: optimized protocols for piezoelectric print conditions.

    PubMed

    Pilobello, Kanoelani T; Agrawal, Praveen; Rouse, Richard; Mahal, Lara K

    2013-01-01

    Lectin microarray technology has been used to profile the glycosylation of a multitude of biological and clinical samples, leading to new clinical biomarkers and advances in glycobiology. Lectin microarrays, which include >90 plant lectins, recombinant lectins, and selected antibodies, are used to profile N-linked, O-linked, and glycolipid glycans. The specificity and depth of glycan profiling depends upon the carbohydrate-binding proteins arrayed. The current set targets mammalian carbohydrates including fucose, high mannose, branched and complex N-linked, α- and β-galactose and GalNAc, α-2,3- and α-2,6-sialic acid, LacNAc, and Lewis X epitopes. Previous protocols have described the use of a contact microarray printer for lectin microarray production. Here, an updated protocol that uses a non-contact, piezoelectric printer, which leads to increased lectin activity on the array, is presented. Optimization of pri