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Sample records for 2-macroglobulin receptor recognition

  1. Binding of receptor-recognized forms of alpha2-macroglobulin to the alpha2-macroglobulin signaling receptor activates phosphatidylinositol 3-kinase.

    PubMed

    Misra, U K; Pizzo, S V

    1998-05-29

    Ligation of the alpha2-macroglobulin (alpha2M) signaling receptor by receptor-recognized forms of alpha2M (alpha2M*) initiates mitogenesis secondary to increased intracellular Ca2+. We report here that ligation of the alpha2M signaling receptor also causes a 1. 5-2.5-fold increase in wortmannin-sensitive phosphatidylinositol 3-kinase (PI3K) activity as measured by the quantitation of phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 formation was alpha2M* concentration-dependent with a maximal response at approximately 50 pM ligand concentration. The peak formation of PIP3 occurred at 10 min of incubation. The alpha2M receptor binding fragment mutant K1370R which binds to the alpha2M signaling receptor activating the signaling cascade, increased PIP3 formation by 2-fold. The mutant K1374A, which binds very poorly to the alpha2M signaling receptor, did not cause any increase in PIP3 formation. alpha2M*-induced DNA synthesis was inhibited by wortmannin. 1, 2Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethylester a chelator of intracellular Ca2+, drastically reduced alpha2M*-induced increases in PIP3 formation. We conclude that PI3K is involved in alpha2M*-induced mitogenesis in macrophages and intracellular Ca2+ plays a role in PI3K activation. PMID:9593670

  2. Gene transfer mediated by alpha2-macroglobulin.

    PubMed Central

    Schneider, H; Huse, K; Birkenmeier, G; Otto, A; Scholz, G H

    1996-01-01

    alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein. PMID:8871570

  3. The monomeric receptor binding domain of tetrameric α2-macroglobulin binds to cell surface GRP78 triggering equivalent activation of signaling cascades.

    PubMed

    Misra, Uma Kant; Payne, Sturgis; Pizzo, Salvatore Vincent

    2013-06-11

    α2-Macroglobulin (α2M) is a broad spectrum proteinase inhibitor that when activated by proteinases (α2M*) undergoes a major conformational change exposing receptor recognition sites in each of its four subunits. These complexes bind to two distinct receptors, namely, the low-density lipoprotein receptor-related protein (LRP) and cell surface glucose-regulated protein [Mr ∼ 78000 (GRP78)]. The latter is a very high affinity receptor (Kd = 50-100 pM) whose ligation triggers pro-proliferative and anti-apoptotic signaling cascades. Despite its four binding sites, Scatchard analysis of binding of α2M* to cells does not yield a cooperative plot. We, therefore, hypothesize that a monomeric cloned and expressed α2M receptor binding domain (RBD) should trigger comparable signaling events. Indeed, RBD or its K1370A mutant that binds to GRP78 but cannot bind to LRP regulates DNA and protein synthesis by human prostate cancer cells in a manner comparable to that of α2M*. Akt and mTORC1 activation and signaling are also comparably upregulated by α2M*, RBD, or mutant K1370A. Antibodies directed against the carboxyl-terminal domain of GRP78 are antagonists that block α2M*-mediated effects on pro-proliferative and anti-apoptotic signaling cascades and protein and DNA synthesis. The effects of RBD and its mutant were similarly blocked by these antibodies. Finally, proteolysis of α2M at pH values from 5.7 to 7.0 causes production of free RBD and RBD-containing fragments. Thus, while α2M* ligates only one GRP78 receptor molecule per α2M*, it may potentially serve as a reservoir for release of up to four binding fragments per molecule. PMID:23721263

  4. Regulation of macrophage alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein by lipopolysaccharide and interferon-gamma.

    PubMed Central

    LaMarre, J; Wolf, B B; Kittler, E L; Quesenberry, P J; Gonias, S L

    1993-01-01

    alpha 2-Macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2M-R/LRP) is a broad specificity receptor that may function in lipoprotein metabolism, proteinase regulation, and growth factor regulation. In this study, we demonstrated that alpha 2M-R/LRP expression in macrophages can be markedly decreased by LPS and by IFN-gamma. Regulation of alpha 2M-R/LRP in RAW 264.7 cells was demonstrated at the mRNA, antigen, and receptor-function levels. In receptor-function studies, the decrease in alpha 2M-R/LRP expression was detected as a 90% decrease in the Bmax or maximum receptor binding capacity for activated alpha 2M after treatment with LPS or IFN-gamma. Western blot analysis of whole cell lysates demonstrated significant loss of alpha 2M-R/LRP heavy-chain. Northern blot analysis of poly(A)+ RNA revealed a marked decrease in alpha 2M-R/LRP mRNA after treatment with LPS (79% decrease) or IFN-gamma (70% decrease). Other cytokines, including tumor necrosis factor-alpha, transforming growth factor-beta-1, and interleukin-6 did not regulate alpha 2M-R/LRP. The ability of LPS and IFN-gamma to regulate alpha 2M-R/LRP was confirmed in experiments with primary cultures of murine bone marrow macrophages. These studies demonstrate that macrophage alpha 2M-R/LRP is subject to significant downregulation by physiologically significant cytokines and signaling macromolecules. Images PMID:7680664

  5. The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor regulates cell surface plasminogen activator activity on human trophoblast cells.

    PubMed

    Zhang, J C; Sakthivel, R; Kniss, D; Graham, C H; Strickland, D K; McCrae, K R

    1998-11-27

    The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP/alpha2MR) mediates the internalization of numerous ligands, including prourokinase (pro-UK) and complexes between two-chain urokinase (tc-u-PA) and plasminogen activator inhibitor type-1 (PAI-1). It has been suggested that through its ability to internalize these ligands, LRP/alpha2MR may regulate the expression of plasminogen activator activity on cell surfaces; this hypothesis, however, has not been experimentally confirmed. To address this issue, we assessed the ability of LRP/alpha2MR to regulate plasminogen activator activity on human trophoblast cells, which express both LRP/alpha2MR and the urokinase receptor (uPAR). Trophoblasts internalized and degraded exogenous 125I-pro-UK (primarily following its conversion to tc-u-PA and incorporation into tc-u-PA.PAI complexes) in an LRP/alpha2MR-dependent manner, which was inhibited by the LRP/alpha2MR receptor-associated protein. Receptor-associated protein also caused a approximately 50% reduction in cell surface plasminogen activator activity and delayed the regeneration of unoccupied uPAR by cells on which uPAR were initially saturated with pro-UK. Identical effects were caused by anti-LRP/alpha2MR antibodies. These results demonstrate that LRP/alpha2MR promotes the expression of cell surface plasminogen activator activity on trophoblasts by facilitating the clearance of tc-u-PA.PAI complexes and regeneration of unoccupied cell surface uPAR. PMID:9822706

  6. NR4A receptors up-regulate the antiproteinase alpha-2 macroglobulin (A2M) and modulate MMP-2 and MMP-9 in vascular smooth muscle cells.

    PubMed

    Rodríguez-Calvo, Ricardo; Ferrán, Beatriz; Alonso, Judith; Martí-Pàmies, Ingrid; Aguiló, Silvia; Calvayrac, Olivier; Rodríguez, Cristina; Martínez-González, José

    2015-06-01

    Matrix metalloproteinases (MMPs) are associated with tissue remodelling and repair. In non-vascular tissues, NR4A receptors have been involved in the regulation of MMPs by transcriptional repression mechanisms. Here, we analyse alternative mechanisms involving NR4A receptors in the modulation of MMP activity in vascular smooth muscle cells (VSMC). Lentiviral overexpression of NR4A receptors (NOR-1, Nurr1 and Nur77) in human VSMC strongly decreased MMP-2 and MMP-9 activities (analysed by zymography and DQ-gelatin assays) and protein levels. NR4A receptors also down-regulated MMP-2 mRNA levels. Real-time PCR analysis evidenced that alpha-2-macroglobulin (A2M), but not other MMP inhibitors (TIMP-1 and TIMP-2) were up-regulated in NR4A-transduced cells. Interestingly, A2M was expressed in human vascular tissues including the smooth muscle media layer. While NR4A receptors increased A2M expression and secretion in VSMC, NR4A knockdown significantly reduced basal A2M expression in these cells. The direct transcriptional regulation of the human A2M promoter by NR4A receptors was characterised in luciferase reporter assays, electrophoretic mobility shift assays and by chromatin immunoprecipitation, identifying a NGFI-B response element (NBRE-71/-64) essential for the NR4A-mediated induction. The blockade of A2M partially prevented the reduction of MMPs activity observed in NR4A-transduced cells. Although mouse A2M promoter was unresponsive to NR4A receptors, vascular MMP expression was attenuated in transgenic mice over-expressing human NOR-1 in VSMC challenged with lipopolysaccharide. Our results show that the pan-proteinase inhibitor A2M is expressed in the vasculature and that NR4A receptors modulate VSMC MMP activity by several mechanisms including the up-regulation of A2M. PMID:25809189

  7. Peroxisome proliferator-activated receptor alpha (PPARalpha) agonists down-regulate alpha2-macroglobulin expression by a PPARalpha-dependent mechanism.

    PubMed

    González, María del Carmen; Corton, J Christopher; Cattley, Russell C; Herrera, Emilio; Bocos, Carlos

    2009-08-01

    Fibrates are peroxisome proliferator-activated receptor alpha (PPARalpha) ligands used to normalize lipid and glucose parameters and exert anti-inflammatory effects. The acute-phase response (APR) is an important inflammatory process. One of the most important acute-phase proteins in rats is alpha2-macroglobulin (A2Mg). Whereas normal adult rats present low serum levels, pregnant rats display high amounts. Therefore, we used pregnant rats to detect the effect of fenofibrate on hepatic A2Mg expression by RT-PCR and Northern blot. Virgin rats were used as controls. The expression of other APR genes, a known fibrate-responder gene, gamma-chain fibrinogen (gamma-Fib), and one gene from the same family as A2Mg, complement component 3 (C3), were also measured in liver. In order to determine whether the fibrate-effects were mediated by PPARalpha, wild-type mice and PPARalpha-null mice were also used and treated with WY-14,643 (WY) or di-2-ethylhexyl phthalate (DEHP). Fenofibrate depressed A2Mg expression in virgin rats, but expression was decreased more sharply in pregnant rats. Expression of C3 and gamma-Fib was diminished after treatment only in pregnant rats. On the other hand, WY, but not DEHP, reduced A2Mg and gamma-Fib expression in the livers of wild-type mice, without any effect in PPARalpha-null mice. WY or DEHP did not affect C3 expression. Therefore, A2Mg expression is modified by PPARalpha agonists not only in pregnant rats under augmented APR protein synthesis, but also in virgin rats and mice under basal conditions. Interestingly, our results also identify A2Mg as a novel PPARalpha agonist-regulated gene. PMID:19497347

  8. Effects of a recombinant complement component C3b functional fragment α2MR (α2-macroglobulin receptor) additive on the immune response of juvenile orange-spotted grouper (Epinephelus coioides) after the exposure to cold shock challenge.

    PubMed

    Luo, Sheng-Wei; Cai, Luo; Qi, Zeng-Hua; Wang, Cong; Liu, Yuan; Wang, Wei-Na

    2015-08-01

    The effects of Ec-α2MR (Epinephelus coiodes-α2-macroglobulin receptor) on growth performance, enzymatic activity, respiratory burst, MDA level, total antioxidant capacity, DPPH radical scavenging percentage and immune-related gene expressions of the juvenile orange-spotted grouper were evaluated. The commercial diet supplemented with α2MR additive was used to feed the orange-spotted grouper for six weeks. Although a slight increase was observed in the specific growth rate, survival rate and weight gain, no significance was observed among different group. After the feeding trial, the groupers were exposed to cold stress. Respiratory burst activity and MDA level decreased significantly in α2MR additive group by comparing with the control and additive control group, while a sharp increase of ACP activity, ALP activity, total antioxidant capacity and DPPH radial scavenging percentage was observed in α2MR additive group. qRT-PCR analyses confirmed that the up-regulated mRNA expressions of C3, TNF1, TNF2, IL-6, CTL, LysC, SOD1 and SOD2 were observed in α2MR additive group at 20 °C. These results showed that α2MR additive may moderate the immune response in grouper following cold shock challenge. PMID:25917969

  9. Peroxisome proliferator-activated receptor alpha (PPARalpha) agonists down-regulate alpha2-macroglobulin expression by a PPARalpha-dependent mechanism.

    EPA Science Inventory

    Peroxisome proliferator-activated receptor alpha (PPARα) regulates transcription of genes involved both in lipid and glucose metabolism as well as inflammation. Fibrates are PPARα ligands used to normalize lipid and glucose parameters and exert anti-inflammatory effects. Fibrates...

  10. 21 CFR 866.5620 - Alpha-2-macroglobulin immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alpha-2-macroglobulin immunological test system....5620 Alpha-2-macroglobulin immunological test system. (a) Identification. An alpha-2-macroglobulin... the alpha-2-macroglobulin (a serum protein) in plasma. Measurement of alpha-2-macroglobulin may aid...

  11. 21 CFR 866.5620 - Alpha-2-macroglobulin immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alpha-2-macroglobulin immunological test system....5620 Alpha-2-macroglobulin immunological test system. (a) Identification. An alpha-2-macroglobulin... the alpha-2-macroglobulin (a serum protein) in plasma. Measurement of alpha-2-macroglobulin may aid...

  12. Functions of alpha 2 macroglobulins in pregnancy.

    PubMed

    Tayade, Chandrakant; Esadeg, Souad; Fang, Yuan; Croy, B A

    2005-12-21

    The alpha 2 macroglobulins (A2M) are a family of abundant plasma proteins produced predominantly by the mammalian liver. Pregnancy zone proteins (PZP) of humans and rats are A2M family members that bind a wide variety of macromolecules including the important pregnancy-associated molecules such as vascular endothelial growth factor, placenta growth factor and glycodelin (also called PP14). Recently, a mouse gene analogous to PZP (A2M of pregnancy or A2Mp) was cloned. A2Mp has a unique pattern of expression in reproductive and cardiovascular tissues and, unexpectedly, is not expressed by liver. Since changes in heart function and remodeling of renal and uterine vasculature are amongst the earliest maternal responses to pregnancy, the product of the A2Mp gene has been postulated to systemically regulate these changes. A2Ms with and without non-covalently bound ligands also down regulate immune cell activation but promote immune cell migration, additional features associated with gestational success. Here, we review the A2M gene families of mice and humans, the predicted structural relationships between A2M and its pregnancy induced forms and the postulated roles for this gene family in normal pregnancy. PMID:16297527

  13. 21 CFR 866.5620 - Alpha-2-macroglobulin immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... the alpha-2-macroglobulin (a serum protein) in plasma. Measurement of alpha-2-macroglobulin may aid in... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alpha-2-macroglobulin immunological test system....5620 Alpha-2-macroglobulin immunological test system. (a) Identification. An...

  14. [Alpha-2-macroglobulin ligands and their biotransport mechanisms].

    PubMed

    Zorina, V N; Zorin, N A; Lykova, O F; Konysheva, T V; Zorina, R M

    2007-01-01

    Alpha-2-macroglobulin (MG) is a high-molecular weight glycoprotein that possesses a wide range of regulatory functions. Earlier it has been shown that covalent binding of MG with proteinases results in conformational transformation of MG, which enables MG to transport some additional types of cytokines linked by noncovalent interactions. The results of our study have demonstrated that the range of proteins, with the ability for additional binding with transformed MG is variable and comprises IgG, IgA, IgM, albumin, both types of lipoprotein chain, plasmin, some cytokines and even pregnancy associated alpha-2-glycoprotein (structured MG homolog). The major ligands are found to be albumin, IgG, plasmin and, to a lesser degree, lipoproteins. MG interactions with both acidic and low-alkaline proteinases contribute to neutralization of total charge of the formed complex at neutral pH, typical for internal fluids of the organism, and that the addition of low-density lipoprotein receptor-related protein (LRP) increases the amount of electroneutral complexes at pH 7.4. We suppose, that this mechanism enables the transformed MG (or may be its complex with other regulatory proteins) to rapidly precipitate rapidly on cellular surface and then, after binding with LRP and secondary neutralization of the total charge under physiological pH conditions, to pass through cellular membrane and to realize its own regulatory functions. PMID:17639717

  15. Characterization of alpha-2-macroglobulin from groupers.

    PubMed

    Chuang, Wen-Hsiao; Lee, Kuo-Kau; Liu, Ping-Chung

    2013-08-01

    Alpha-2-macroglobulin (α-2-M) is a protease inhibitor broadly present in the plasma of vertebrates and invertebrates, and is an important non-specific humoral factor in defence system of the animals. This study conducted the immuno-analysis and mass spectrometric analysis methods to investigate the characteristics of the protease inhibitor, α-2-M, among groupers and related species. Rabbit antiserum to the purified α-2-M of Epinephelus coioides was used in different immunological methods to determine the immune cross-reactions of the α-2-M in samples. Plasma of Epinephelus bruneus, Epinephelus fuscoguttatus, Epinephelus lanceolatus, and Epinephelus quoyanus exhibited high protease inhibitory activities by BAPNA-trypsin assay. To purify the α-2-M protein, plasma protein of grouper E. coioides was first precipitated by using PEG 6000, then Blue Sepharose 6 Fast Flow, DEAE Sephacel, Con A Separose 4B and Phenyl Sepharose High Performance columns were used on FPLC system for purification. The molecular mass of grouper plasma α-2-M was determined as a 180 kDa protein on non-reduced SDS-PAGE. In addition, it was determined as 97 and 80 kDa protein on reduced SDS-PAGE. Enzymatic and chemical deglycosylation of glycogen revealed that the contents of glycogen in 97 and 80 kDa subunits were 12.4% and 15%, respectively, and were all belonging to N-linked type. Only one precipitation arc was visualized in all plasma of Epinephelus spp. using the rabbit antiserum to the purified α-2-M of E. coioides, on crossed immunoelectrophoresis (CIE) gels. The plasma of Epinephelus spp. and seawater fish species showed stronger responses than freshwater fish species while that of other animal species showed no response by dot-blot assay. One single band was detected on Native PAGE-Western blotting assay, one single 180 kDa band was detected on non-reduced SDS-PAGE-Western blotting, and four bands (80, 97, 160, 250 kDa) were detected on reduced SDS-PAGE when various grouper plasma

  16. Bacterial α2-macroglobulins: colonization factors acquired by horizontal gene transfer from the metazoan genome?

    PubMed Central

    Budd, Aidan; Blandin, Stephanie; Levashina, Elena A; Gibson, Toby J

    2004-01-01

    Background Invasive bacteria are known to have captured and adapted eukaryotic host genes. They also readily acquire colonizing genes from other bacteria by horizontal gene transfer. Closely related species such as Helicobacter pylori and Helicobacter hepaticus, which exploit different host tissues, share almost none of their colonization genes. The protease inhibitor α2-macroglobulin provides a major metazoan defense against invasive bacteria, trapping attacking proteases required by parasites for successful invasion. Results Database searches with metazoan α2-macroglobulin sequences revealed homologous sequences in bacterial proteomes. The bacterial α2-macroglobulin phylogenetic distribution is patchy and violates the vertical descent model. Bacterial α2-macroglobulin genes are found in diverse clades, including purple bacteria (proteobacteria), fusobacteria, spirochetes, bacteroidetes, deinococcids, cyanobacteria, planctomycetes and thermotogae. Most bacterial species with bacterial α2-macroglobulin genes exploit higher eukaryotes (multicellular plants and animals) as hosts. Both pathogenically invasive and saprophytically colonizing species possess bacterial α2-macroglobulins, indicating that bacterial α2-macroglobulin is a colonization rather than a virulence factor. Conclusions Metazoan α2-macroglobulins inhibit proteases of pathogens. The bacterial homologs may function in reverse to block host antimicrobial defenses. α2-macroglobulin was probably acquired one or more times from metazoan hosts and has then spread widely through other colonizing bacterial species by more than 10 independent horizontal gene transfers. yfhM-like bacterial α2-macroglobulin genes are often found tightly linked with pbpC, encoding an atypical peptidoglycan transglycosylase, PBP1C, that does not function in vegetative peptidoglycan synthesis. We suggest that YfhM and PBP1C are coupled together as a periplasmic defense and repair system. Bacterial α2-macroglobulins might

  17. Cultured Ito cells of rat liver express the alpha 2-macroglobulin gene.

    PubMed

    Andus, T; Ramadori, G; Heinrich, P C; Knittel, T; Meyer zum Büschenfelde, K H

    1987-11-01

    Ito cells were isolated from rat liver and kept in culture for up to 13 days. The capability of the Ito cells to synthesize alpha 2-macroglobulin was analyzed at different times after isolation and by pulse-chase experiments. Newly synthesized alpha 2-macroglobulin was determined by immunoprecipitation followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. alpha 2-Macroglobulin synthesis was hardly detectable in Ito cells and their media 3 days after plating. However, 5-11 days after the isolation of the cells, increasing amounts of alpha 2-macroglobulin were synthesized. The results of pulse-chase experiments performed on day 7 showed that radioactively labeled alpha 2-macroglobulin decreased in the intracellular compartment and increased in the culture medium. alpha 2-Macroglobulin was identified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions. Furthermore, when unlabeled alpha 2-macroglobulin was added during the immunoprecipitation, a competition was observed. Incubation of pancreatic elastase with culture medium of rat Ito cells or rat hepatocytes led to the same cleavage products as found with alpha 2-macroglobulin. alpha 2-Macroglobulin-specific mRNA could be demonstrated by Northern blot analysis of total RNA extracted from rat Ito cells. Under the conditions where alpha 2-macroglobulin was synthesized in Ito cells, no synthesis of alpha 1-macroglobulin, alpha 1-inhibitor 3, alpha 1-proteinase inhibitor, alpha 1-acid glycoprotein, alpha 1-acute-phase globulin (T-kininogen) and albumin could be demonstrated. It is concluded that alpha 2-macroglobulin is a true secretory protein of rat Ito cells in culture. This could be of importance for collagen metabolism in liver diseases. PMID:2444437

  18. 21 CFR 866.5620 - Alpha-2-macroglobulin immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alpha-2-macroglobulin immunological test system. 866.5620 Section 866.5620 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5620 Alpha-2-macroglobulin...

  19. 21 CFR 866.5620 - Alpha-2-macroglobulin immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alpha-2-macroglobulin immunological test system. 866.5620 Section 866.5620 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5620 Alpha-2-macroglobulin...

  20. Shrimp Alpha-2-Macroglobulin Prevents the Bacterial Escape by Inhibiting Fibrinolysis of Blood Clots

    PubMed Central

    Chaikeeratisak, Vorrapon; Somboonwiwat, Kunlaya; Tassanakajon, Anchalee

    2012-01-01

    Proteomic analysis of the hemocytic proteins of Penaeus monodon (Pm) has previously shown that alpha-2-macroglobulin (A2M) was among the proteins that showed substantially altered expression levels upon Vibrio harveyi infection. Therefore, in this study its potentially important role in the response of shrimp to bacterial infection was further characterized. The yeast two-hybrid system revealed that the receptor binding domain of PmA2M interacted with the carboxyl-terminus of one or both of the transglutaminase type II isoforms, which are key enzymes involved in the shrimp clotting system. In accord with this, PmA2M was found to be localized on the extracellular blood clots and to colocalize with clottable proteins. RNA interference (RNAi)-mediated knockdown of A2M transcript levels reduced the PmA2M transcript levels (∼94%) and significantly reduced the bacterial seizing ability of the clotting system, resulting in an up to 3.3-fold higher number of V. harveyi that systemically disseminated into the circulatory system at 5 min post-infection before subsequent clearance by the immune system. Furthermore, an appearance of PmA2M depleted clots in the presence of V. harveyi strikingly demonstrated fibrinolysis zones surrounding the bacteria. This study provides the first evidence of the vital role of PmA2M in enhancing bacterial sequestration by protecting blood clots against fibrinolysis. PMID:23082160

  1. Hepcidin, the hormone of iron metabolism, is bound specifically to alpha-2-macroglobulin in blood.

    PubMed

    Peslova, Gabriela; Petrak, Jiri; Kuzelova, Katerina; Hrdy, Ivan; Halada, Petr; Kuchel, Philip W; Soe-Lin, Shan; Ponka, Prem; Sutak, Robert; Becker, Erika; Huang, Michael Li-Hsuan; Suryo Rahmanto, Yohan; Richardson, Des R; Vyoral, Daniel

    2009-06-11

    Hepcidin is a major regulator of iron metabolism. Hepcidin-based therapeutics/diagnostics could play roles in hematology in the future, and thus, hepcidin transport is crucial to understand. In this study, we identify alpha2-macroglobulin (alpha2-M) as the specific hepcidin-binding molecule in blood. Interaction of 125I-hepcidin with alpha2-M was identified using fractionation of plasma proteins followed by native gradient polyacrylamide gel electrophoresis and mass spectrometry. Hepcidin binding to nonactivated alpha2-M displays high affinity (Kd 177 +/- 27 nM), whereas hepcidin binding to albumin was nonspecific and displayed nonsaturable kinetics. Surprisingly, the interaction of hepcidin with activated alpha2-M exhibited a classical sigmoidal binding curve demonstrating cooperative binding of 4 high-affinity (Kd 0.3 microM) hepcidin-binding sites. This property probably enables efficient sequestration of hepcidin and its subsequent release or inactivation that may be important for its effector functions. Because alpha2-M rapidly targets ligands to cells via receptor-mediated endocytosis, the binding of hepcidin to alpha2-M may influence its functions. In fact, the alpha2-M-hepcidin complex decreased ferroportin expression in J774 cells more effectively than hepcidin alone. The demonstration that alpha2-M is the hepcidin transporter could lead to better understanding of hepcidin physiology, methods for its sensitive measurement and the development of novel drugs for the treatment of iron-related diseases. PMID:19380872

  2. Alpha-2-macroglobulin as a radioprotective agent: a review

    PubMed Central

    Chen, Xueying; Kong, Xiangbo; Zhang, Zhaoqiang; Chen, Wei; Chen, Jieyu; Li, Huanyang; Cao, Wanting; Ge, Yaping

    2014-01-01

    Radiation is an important modality in cancer treatment, and eighty percent of cancer patients need radiotherapy at some point during their clinical management. However, radiation-induced damage to normal tissues restricts the therapeutic doses of radiation that can be delivered to tumours and thereby limits the effectiveness of the treatment. The use of radioprotectors represents an obvious strategy to obtain better tumour control using a higher dose in radiotherapy. However, most of the synthetic radioprotective compounds studied have shown inadequate clinical efficacy owing to their inherent toxicity and high cost. Hence, the development of radioprotective agents with lower toxicity and an extended window of protection has attracted a great deal of attention, and the identification of alternative agents that are less toxic and highly effective is an absolute necessity. Recent studies have shown that alpha-2-macroglobulin (α2M) possesses radioprotective effects. α2M is a tetrameric, disulfide-rich plasma glycoprotein that functions as a non-selective inhibitor of different types of non-specific proteases and as a carrier of cytokines, growth factors, and hormones. α2M induces protein factors whose interplay underlies radioprotection, which supports the idea that α2M is the central effector of natural radioprotection in the rat. Pretreatment with α2M has also induced a significant reduction of irradiation-induced DNA damage and the complete restoration of liver and body weight. Mihailović et al. concluded that the radioprotection provided by α2M was in part mediated through cytoprotection of new blood cells produced in the bone marrow; these authors also indicated that an important aspect of the radioprotective effect of amifostine was the result of the induction of the endogenous cytoprotective capability of α2M. The radioprotective effects of α2M are possibly due to antioxidant, anti-fibrosis, and anti-inflammatory functions, as well as the maintenance

  3. Alpha-2-macroglobulin as a radioprotective agent: a review.

    PubMed

    Chen, Xueying; Kong, Xiangbo; Zhang, Zhaoqiang; Chen, Wei; Chen, Jieyu; Li, Huanyang; Cao, Wanting; Ge, Yaping; Fang, Silian

    2014-10-01

    Radiation is an important modality in cancer treatment, and eighty percent of cancer patients need radiotherapy at some point during their clinical management. However, radiation-induced damage to normal tissues restricts the therapeutic doses of radiation that can be delivered to tumours and thereby limits the effectiveness of the treatment. The use of radioprotectors represents an obvious strategy to obtain better tumour control using a higher dose in radiotherapy. However, most of the synthetic radioprotective compounds studied have shown inadequate clinical efficacy owing to their inherent toxicity and high cost. Hence, the development of radioprotective agents with lower toxicity and an extended window of protection has attracted a great deal of attention, and the identification of alternative agents that are less toxic and highly effective is an absolute necessity. Recent studies have shown that alpha-2-macroglobulin (α2M) possesses radioprotective effects. α2M is a tetrameric, disulfide-rich plasma glycoprotein that functions as a non-selective inhibitor of different types of non-specific proteases and as a carrier of cytokines, growth factors, and hormones. α2M induces protein factors whose interplay underlies radioprotection, which supports the idea that α2M is the central effector of natural radioprotection in the rat. Pretreatment with α2M has also induced a significant reduction of irradiation-induced DNA damage and the complete restoration of liver and body weight. Mihailović et al. concluded that the radioprotection provided by α2M was in part mediated through cytoprotection of new blood cells produced in the bone marrow; these authors also indicated that an important aspect of the radioprotective effect of amifostine was the result of the induction of the endogenous cytoprotective capability of α2M. The radioprotective effects of α2M are possibly due to antioxidant, anti-fibrosis, and anti-inflammatory functions, as well as the maintenance

  4. Surfactant Protein D Interacts with α2-Macroglobulin and Increases Its Innate Immune Potential*

    PubMed Central

    Craig-Barnes, Hayley A.; Doumouras, Barbara S.; Palaniyar, Nades

    2010-01-01

    Surfactant protein D (SP-D) is an innate immune collectin that recognizes microbes via its carbohydrate recognition domains, agglutinates bacteria, and forms immune complexes. During microbial infections, proteases, such as elastases, cleave the carbohydrate recognition domains and can inactivate the innate immune functions of SP-D. Host responses to counterbalance the reduction of SP-D-mediated innate immune response under these conditions are not clearly understood. We have unexpectedly identified that SP-D could interact with protein fractions containing ovomucin and ovomacroglobulin. Here, we show that SP-D interacts with human α2-macroglobulin (A2M), a protease inhibitor present in the lungs and serum. Using enzyme-linked immunosorbent assays, surface plasmon resonance, and carbohydrate competition assays, we show that SP-D interacts with A2M both in solid phase (KD of 7.33 nm) and in solution via lectin-carbohydrate interactions under physiological calcium conditions. Bacterial agglutination assays further show that SP-D·A2M complexes increase the ability of SP-D to agglutinate bacteria. Western blot analyses show that SP-D, but not A2M, avidly binds bacteria. Interestingly, intact and activated A2M also protect SP-D against elastase-mediated degradation, and the cleaved A2M still interacts with SP-D and is able to enhance its agglutination abilities. We also found that SP-D and A2M can interact with each other in the airway-lining fluid. Therefore, we propose that SP-D utilizes a novel mechanism in which the collectin interacts with protease inhibitor A2M to decrease its degradation and to concurrently increase its innate immune function. These interactions particularly enhance bacterial agglutination and immune complex formation. PMID:20207732

  5. Separation of Plasma Thromboplastin Antecedent from Kallikrein by the Plasma α2-Macroglobulin, Kallikrein Inhibitor

    PubMed Central

    Harpel, Peter C.

    1971-01-01

    Plasma thromboplastin antecedent (PTA, factor XI) is an important intermediate in the intrinsic coagulation system, and plasma kallikrein has been implicated as a mediator of the inflammatory process. Whereas their biologic activities are functionally distinct, their identity as separate entities in plasma has not been fully established, and the nature of their plasma inhibitors has not been completely characterized. A partially purified preparation containing the clotting, tosyl arginine methyl ester (TAMe) esterase and kinin-producing activities of these substances has been prepared by DEAE-cellulose chromatography of a Celite eluate obtained from acid-treated human plasma. These activities were not separable by acrylamide gel electrophoresis nor by isoelectric focusing, their pI being approximately 8.7. Human plasma α2-macroglobulin has been shown to inhibit the proteolytic activity of kallikrein and to inhibit partially its TAMe esterase activity. An α2-macroglobulin, PTA, kallikrein incubation mixture was separated by gel filtration chromatography. The α2-macroglobulin formed a high molecular weight complex with kallikrein and appeared in early chromatographic fractions. The PTA-clotting activity was not inhibited by the α2-macroglobulin; 64% of the initial PTA activity was isolated in later fractions free of kallikrein-induced kinin-like activity. In contrast, clotting, TAMe esterase, and kinin-forming activities were inhibited after gel filtration chromatography of an incubation mixture of these activities and partially purified C1̄ inactivator (C1 esterase inhibitor). Electrofocusing of an incubation mixture of an activated PTA, kallikrein preparation, and α2-macroglobulin resulted in the isolation of a PTA fraction free of kallikrein proteolytic activity, and with 4% of the original TAMe esterase activity. In this manner, activated PTA and plasma kallikrein have been shown to be distinct substances, and methods have been introduced for the further

  6. Evidence of an alpha 2-macroglobulin-like molecule in plasma of Salamandra salamandra. Structural and functional similarity with human alpha 2-macroglobulin.

    PubMed

    Sallenave, J M; Bellot, R

    1987-07-13

    A high-Mr (Mr 750,000) alpha 1-macroglobulin, obtained from Salamandra salamandra, is described. Salamander alpha 1-macroglobulin is composed of two monomers of equal Mr, which are composed of two polypeptide chains, each of Mr 180,000, linked by disulfide bonds. The molecular parameters of this protein, its binding to trypsin and inactivation by methylamine suggest that salamander alpha 1-macroglobulin is closely related to human alpha 2-macroglobulin and to other related proteins described in the animal kingdom. PMID:2439383

  7. Binding of tumor necrosis factor alpha to activated forms of human plasma alpha 2 macroglobulin.

    PubMed Central

    Wollenberg, G. K.; LaMarre, J.; Rosendal, S.; Gonias, S. L.; Hayes, M. A.

    1991-01-01

    We tested the hypothesis that human plasma alpha 2 macroglobulin (alpha 2M) is a latent binding glycoprotein for human tumor necrosis factor alpha (TNF-alpha). Human recombinant 125I-TNF-alpha was incubated for 2 hours (37 degrees C) with purified native alpha 2M and with alpha 2M that was modified by reaction with methylamine or various proteinases. 125I-TNF-alpha/alpha 2M complexes were detected by nondenaturing polyacrylamide gel electrophoresis after autoradiography or by liquid chromatography on Superose-6. 125I-TNF-alpha bound strongly but noncovalently to alpha 2M-plasmin and alpha 2M-methylamine. There was minimal binding of 125I-TNF-alpha to native alpha 2M, alpha 2M-trypsin, or alpha 2M-thrombin. A 10(6) molar excess of porcine heparin did not reduce the binding of 125I-TNF-alpha to alpha 2M-methylamine or alpha 2M-plasmin. alpha 2M-plasmin or alpha 2M-methylamine added to human plasma or serum preferentially bound 125I-TNF-alpha in the presence of native alpha 2M. 125I-TNF-alpha also bound to 'fast' alpha-macroglobulins in methylamine-reacted human, rat, mouse, swine, equine, and bovine plasma. However, TNF-alpha, preincubated with either alpha 2M-plasmin or alpha 2M-methylamine, remained a potent necrogen for cultured L929 cells. Purified 125I-TNF-alpha/alpha 2M-plasmin complex injected intravenously in CD-1 mice rapidly cleared from the circulation, unless the alpha 2M-receptor pathway was blocked by coinjection of excess alpha 2M-trypsin. These findings demonstrate that alpha 2M is a latent plasmin-activated binding glycoprotein for TNF-alpha and that TNF-alpha/alpha 2M-plasmin complexes can be removed from the circulation by the alpha 2M-receptor pathway. This suggests that alpha 2M may be an important regulator of the activity and distribution of TNF-alpha in vivo. Images Figure 1 Figure 3 PMID:1704186

  8. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    PubMed Central

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-01-01

    Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macro­globulin, this protease-activation mechanism is likely to operate across the diverse members of this group. PMID:26143919

  9. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    SciTech Connect

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-06-30

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

  10. α-2-Macroglobulin in Saliva Is Associated with Glycemic Control in Patients with Type 2 Diabetes Mellitus

    PubMed Central

    Aitken, Juan Pablo; Ortiz, Carolina; Morales-Bozo, Irene; Rojas-Alcayaga, Gonzalo; Baeza, Mauricio; Beltran, Caroll

    2015-01-01

    Background. Subjects with type 2 diabetes mellitus (DM2) require an adequate glycemic control to avoid diabetic complications. Currently, saliva biomarkers are used as a diagnostic tool and can be indicative of the degree of progression and control of various diseases. Several studies indicate that α-2-macroglobulin levels are elevated in diabetic patients. Methods. 120 subjects with DM2 were enrolled and classified into two groups according to their glycemic control (percentage of glycated hemoglobin-A1c (HbA1c), <7% adequate glycemic control group; >7% inadequate glycemic control group). The relationship between α-2-macroglobulin levels from saliva samples and HbA1c was subsequently evaluated. Results. We found a positive correlation between α-2-macroglobulin and HbA1c (r = 0.778 and P < 0.0001). Area under the receivers operating characteristic (ROC) curve of α-2-macroglobulin indicated a positive discrimination threshold of α-2-macroglobulin (AUC = 0.903, CI 95%: 0.847–0.959, P < 0.0001) to diagnose glycemic control. Conclusions. Our data strongly suggest that the level of saliva α-2-macroglobulin is an indicator for the degree of glycemic control in diabetic patients and represents a promising alternative method to evaluate this parameter. PMID:25821337

  11. Pattern recognition receptors in antifungal immunity.

    PubMed

    Plato, Anthony; Hardison, Sarah E; Brown, Gordon D

    2015-03-01

    Receptors of the innate immune system are the first line of defence against infection, being able to recognise and initiate an inflammatory response to invading microorganisms. The Toll-like (TLR), NOD-like (NLR), RIG-I-like (RLR) and C-type lectin-like receptors (CLR) are four receptor families that contribute to the recognition of a vast range of species, including fungi. Many of these pattern recognition receptors (PRRs) are able to initiate innate immunity and polarise adaptive responses upon the recognition of fungal cell wall components and other conserved molecular patterns, including fungal nucleic acids. These receptors induce effective mechanisms of fungal clearance in normal hosts, but medical interventions, immunosuppression or genetic predisposition can lead to susceptibility to fungal infections. In this review, we highlight the importance of PRRs in fungal infection, specifically CLRs, which are the major PRR involved. We will describe specific PRRs in detail, the importance of receptor collaboration in fungal recognition and clearance, and describe how genetic aberrations in PRRs can contribute to disease pathology. PMID:25420452

  12. [Role of alpha 1-antitrypsin and alpha-2 macroglobulin in hepatopathies].

    PubMed

    Triolo, L; Mian, G; Magris, D; Novello, E; D'Agnolo, B

    1979-02-18

    Two pictures of alpha 1-antitrypsin deficiency, one associated with alpha 2-macroglobulin deficiency and one isolated case of the latter deficiency have been observed in three patients suffering from cirrhosis of the liver and/or hepatoma. On the basis of these cases, the literature on the subject is reviewed. The unusually high incidence of such anti-enzymatic deficiencies (three cases in the first eleven patients studied) in severe liver pathology, calls for a reassessment of such research and suggests that these tests should be carried out on a routine basis in cases of cryptogenetic cirrhosis and probably for long-term prognosis in cases of viral hepatitis. PMID:86175

  13. Lipopolysaccharide recognition, CD14, and lipopolysaccharide receptors.

    PubMed

    Ingalls, R R; Heine, H; Lien, E; Yoshimura, A; Golenbock, D

    1999-06-01

    The ability of a host to sense invasion by a pathogenic organism, and to respond appropriately to control infection, is paramount to survival. To that end, an array of receptors and binding proteins has evolved as part of the innate immune system to detect Gram-negative bacteria. This article reviews the role of CD14, other LPS binding proteins, and the Toll family of receptors in the innate recognition of bacterial lipopolysaccharide. PMID:10340170

  14. Antigen Recognition By Variable Lymphocyte Receptors

    SciTech Connect

    Han, B.W.; Herrin, B.R.; Cooper, M.D.; Wilson, I.A.

    2009-05-18

    Variable lymphocyte receptors (VLRs) rather than antibodies play the primary role in recognition of antigens in the adaptive immune system of jawless vertebrates. Combinatorial assembly of leucine-rich repeat (LRR) gene segments achieves the required repertoire for antigen recognition. We have determined a crystal structure for a VLR-antigen complex, VLR RBC36 in complex with the H-antigen trisaccharide from human blood type O erythrocytes, at 1.67 angstrom resolution. RBC36 binds the H-trisaccharide on the concave surface of the LRR modules of the solenoid structure where three key hydrophilic residues, multiple van der Waals interactions, and the highly variable insert of the carboxyl-terminal LRR module determine antigen recognition and specificity. The concave surface assembled from the most highly variable regions of the LRRs, along with diversity in the sequence and length of the highly variable insert, can account for the recognition of diverse antigens by VLRs.

  15. Expression of human. alpha. sub 2 -macroglobulin cDNA in baby hamster kidney fibroblasts: Secretion of high levels of active. alpha. sub 2 -macroglobulin

    SciTech Connect

    Boel, E.; Mortensen, S.B. ); Kristensen, T.; Sottrup-Jensen, L. ); Petersen, C.M. )

    1990-05-01

    Human {alpha}{sub 2}-macroglobulin ({alpha}{sub 2}M) is a unique 720-kDa proteinase inhibitor with a broad specificity. Unlike most other proteinase inhibitors, it does not inhibit proteolytic activity by blocking the active site of the proteinase. During complex formation with a proteinase {alpha}{sub 2}M entraps the proteinase molecule in a reaction that involves large conformational changes in {alpha}{sub 2}M. The authors describe the molecular cloning of {alpha}{sub 2}M cDNA from the human hepatoblastoma cell line HepG2. The cDNA was subcloned under control of the adenovirus major late promoter in a mammalian expression vector and introduced into the baby hamster kidney (BHK) cell line. Transformed clones were isolated and tested for production of human {alpha}{sub 2}M with a specific enzyme-linked immunosorbent assay. Human recombinant {alpha}{sub 2}M (r{alpha}{sub 2}M), secreted and purified form isolated transfected BHK cell lines, was structurally and functionally compared to {alpha}{sub 2}M purified from human serum. The results show that r{alpha}{sub 2}M was secreted from the BHK cells as an active proteinase-binding tetramer with functional thiol esters. Cleavage reactions of r{alpha}{sub 2}M with methylamine and trypsin showed that the recombinant product, which was correctly processed at the N-terminus, exhibited molecular characteristics similar to those of the human serum derived reference.

  16. Molecular cloning of α-2-macroglobulin from hemocytes of common periwinkle Littorina littorea.

    PubMed

    Borisova, Elena A; Gorbushin, Alexander M

    2014-08-01

    We report the sequence of the proteinase inhibitor with a wide inhibition spectrum, α-2-macroglobulin (α2M), belonging to the thioester superfamily of proteins. This is the first α2M sequence from coenogastropod prosobranch snails. The full-length cDNA was cloned by RACE method, spans 7897 bp and contains an open reading frame of 5460 bp. The ORF encodes a protein of 1819 amino acids. The deduced mature protein contains 1795 amino acids with a molecular weight of 200 kDa and isoelectric point of 5.00. Littorina littorea α2M bears 4 conserved α2M domains and one internal thioester. Phylogenetic analysis showed that the sequence forms well supported cluster with Mollusca species and other representatives of Lophotrochozoa. PMID:24830774

  17. Discovery, structural characterization and functional analysis of alpha-2-macroglobulin, a novel immune-related molecule from Holothuria atra.

    PubMed

    Qian, Jing; Ren, Chunhua; Xia, Jianjun; Chen, Ting; Yu, Zonghe; Hu, Chaoqun

    2016-07-10

    The non-specific protease inhibitor alpha-2-macroglobulin (A2M) is a key macromolecular glycoprotein that involved in host immune defense against pathogens in vertebrates and invertebrates. However, no research regarding A2M has been developed in echinoderms to date. In this study, the full-length cDNA of A2M was cloned from the sea cucumber (Holothuria atra), which is a tropical species widely distributed along the coasts of the South China Sea and designated HaA2M. HaA2M possesses all three conserved functional domains of known A2M proteins, including the bait region domain, thioester domain and receptor-binding domain. Compared to fish and shrimp A2Ms, the histidine residue from the catalytical regions is well conserved in HaA2M. HaA2M mRNA was predominantly expressed in coelomocytes and, to a lesser extent, in the body wall, intestine and respiratory tree. A2M activity was detected in the coelomic fluids of H. atra. The mRNA expression and activity levels were investigated in the major immune tissues and coelomic fluids of H. atra after challenge with inactivated Vibrio alginolyticus or polyriboinosinic polyribocytidylic acid [Poly (I: C)]. RNA interference (RNAi)-mediated knockdown of HaA2M resulted in a significant reduction of HaA2M gene transcript level (86%). RNAi-mediated silencing of HaA2M gene significantly decreased the A2M activity (38%) and increased the number of viable bacteria (2.8-fold) in the coelomic fluids of H. atra infected by V. alginolyticus. Our study, as a whole, supplied the evidences for HaA2M as an immune-relevant molecule and it might have multiple functions in the innate immune system of H. atra. PMID:27033585

  18. An alpha-2 macroglobulin in the pearl oyster Pinctada fucata: Characterization and function in hemocyte phagocytosis of Vibrio alginolyticus.

    PubMed

    Wang, Zhongliang; Wang, Bei; Chen, Gang; Lu, Yishan; Jian, Jichang; Wu, Zaohe

    2016-08-01

    Alpha-2 macroglobulin (α2M) is a ubiquitous protease inhibitor and considered to be an evolutionarily conserved constituent of innate host defence system. Here, an α2M gene (designated as Pfα2M) was obtained from the pearl oyster Pinctada fucata by RT-PCR, PCR walking and rapid amplification of cDNA ends (RACE). The Pfα2M cDNA consists of 6394 bp with an open reading frame (ORF) of 5745 bp encoding a protein of 1914 amino acids with a 19 residues signal peptide. Pfα2M sequence contains three putative functional domains, including a bait region, a thiol ester domain and a receptor-binding domain. Phylogenetic analysis revealed that Pfα2M is closely related to the α2Ms from other molluscs. Pfα2M was expressed in all tested tissues including digestive gland, gill, adductor muscle, mantle and foot, while the highest expression was found in hemocytes. Following challenge with Vibrio alginolyticus, Pfα2M expression in hemocytes was significantly up-regulated at 2 h and then returned to the original level at 48 h. Knockdown of Pfα2M by RNA interference significantly reduced the phagocytosis of V. alginolyticus by hemocytes in vivo, and similar results were obtained upon chemical inactivation of the reactive thioester bond in Pfα2M by methylamine treatment. Taken together, it is suggested that Pfα2M is an immune-relevant molecule and involved in phagocytosis of V. alginolyticus by P. fucata hemocytes, and the function of Pfα2M in phagocytosis is dependent on the active thioester bond. PMID:27346151

  19. Destruction of articular cartilage by alpha2 macroglobulin elastase complexes: role in rheumatoid arthritis

    PubMed Central

    Moore, A.; Appelboam, A.; Kawabata, K.; Da Silva, J. A P; D'Cruz, D.; Gowland, G.; Willoughby, D.

    1999-01-01

    OBJECTIVE—Neutrophil elastase accounts for the ability of some fresh rheumatoid synovial fluids to degrade cartilage matrix in vitro. The aim of this study was to determine if enzyme activity could result from depletion of synovial fluid inhibitors or protection of the enzyme from inhibition.
METHODS—The ability of synovial fluids to inhibit porcine pancreatic elastase was investigated together with chemical pretreatments capable of inactivating alpha1 protease inhibitor (α1PI) or preventing formation of alpha2 macroglobulin (α2M) elastase complexes. Subsequently, complexes of human neutrophil elastase with α2M were prepared and applied to frozen sections of cartilage. Proteoglycan loss was quantified by alcian blue staining and scanning and integrating microdensitometry. Parallel studies were carried out using a low molecular weight chromogenic elastase substrate. The effects of α1PI and SF on these systems were investigated. Finally, synovial fluids were subjected to gel filtration and the fractions assayed for elastase activity. High molecular weight fractions were pooled, concentrated, and tested for their ability to degrade cartilage sections.
RESULTS—All synovial fluids reduced the activity of porcine pancreatic elastase, the inhibition mainly being attributable to α1PI, whereas remaining activity resulted from complexes of elastase with α2M. Complexes of human neutrophil elastase with α2M were shown to cause proteoglycan degradation in frozen sections of human articular cartilage. Alpha1PI prevented α2M elastase complexes from degrading cartilage but not the chromogenic substrate. The data suggested that α1PI does not inhibit elastase bound to α2M but sterically hinders the complex. However, only one of five synovial fluids was able to completely block the actions of α2M elastase complexes against cartilage. Gel filtration of rheumatoid synovial fluids showed elastase and cartilage degrading activity to be associated with fractions that

  20. Pattern-Recognition Receptors and Gastric Cancer

    PubMed Central

    Castaño-Rodríguez, Natalia; Kaakoush, Nadeem O.; Mitchell, Hazel M.

    2014-01-01

    Chronic inflammation has been associated with an increased risk of several human malignancies, a classic example being gastric adenocarcinoma (GC). Development of GC is known to result from infection of the gastric mucosa by Helicobacter pylori, which initially induces acute inflammation and, in a subset of patients, progresses over time to chronic inflammation, gastric atrophy, intestinal metaplasia, dysplasia, and finally intestinal-type GC. Germ-line encoded receptors known as pattern-recognition receptors (PRRs) are critical for generating mature pro-inflammatory cytokines that are crucial for both Th1 and Th2 responses. Given that H. pylori is initially targeted by PRRs, it is conceivable that dysfunction within genes of this arm of the immune system could modulate the host response against H. pylori infection, and subsequently influence the emergence of GC. Current evidence suggests that Toll-like receptors (TLRs) (TLR2, TLR3, TLR4, TLR5, and TLR9), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) (NOD1, NOD2, and NLRP3), a C-type lectin receptor (DC-SIGN), and retinoic acid-inducible gene (RIG)-I-like receptors (RIG-I and MDA-5), are involved in both the recognition of H. pylori and gastric carcinogenesis. In addition, polymorphisms in genes involved in the TLR (TLR1, TLR2, TLR4, TLR5, TLR9, and CD14) and NLR (NOD1, NOD2, NLRP3, NLRP12, NLRX1, CASP1, ASC, and CARD8) signaling pathways have been shown to modulate the risk of H. pylori infection, gastric precancerous lesions, and/or GC. Further, the modulation of PRRs has been suggested to suppress H. pylori-induced inflammation and enhance GC cell apoptosis, highlighting their potential relevance in GC therapeutics. In this review, we present current advances in our understanding of the role of the TLR and NLR signaling pathways in the pathogenesis of GC, address the involvement of other recently identified PRRs in GC, and discuss the potential implications of PRRs in GC immunotherapy

  1. Alpha-2-macroglobulin loaded microcapsules enhance human leukocyte functions and innate immune response

    PubMed Central

    Canova, Donata Federici; Pavlov, Anton M.; Norling, Lucy V.; Gobbetti, Thomas; Brunelleschi, Sandra; Le Fauder, Pauline; Cenac, Nicolas; Sukhorukov, Gleb B.; Perretti, Mauro

    2015-01-01

    Synthetic microstructures can be engineered to deliver bioactive compounds impacting on their pharmacokinetics and pharmacodynamics. Herein, we applied dextran-based layer-by-layer (LbL) microcapsules to deliver alpha-2-macroglobulin (α2MG), a protein with modulatory properties in inflammation. Extending recent observations made with dextran-microcapsules loaded with α2MG in experimental sepsis, we focused on the physical and chemical characteristics of these microstructures and determined their biology on rodent and human cells. We report an efficient encapsulation of α2MG into microcapsules, which enhanced i) human leukocyte recruitment to inflamed endothelium and ii) human macrophage phagocytosis: in both settings microcapsules were more effective than soluble α2MG or empty microcapsules (devoid of active protein). Translation of these findings revealed that intravenous administration of α2MG-microcapsules (but not empty microcapsules) promoted neutrophil migration into peritoneal exudates and augmented macrophage phagocytic functions, the latter response being associated with alteration of bioactive lipid mediators as assessed by mass spectrometry. The present study indicates that microencapsulation can be an effective strategy to harness the complex biology of α2MG with enhancing outcomes on fundamental processes of the innate immune response paving the way to potential future development in the control of sepsis. PMID:26385167

  2. Alpha-2-macroglobulin loaded microcapsules enhance human leukocyte functions and innate immune response.

    PubMed

    Federici Canova, Donata; Pavlov, Anton M; Norling, Lucy V; Gobbetti, Thomas; Brunelleschi, Sandra; Le Fauder, Pauline; Cenac, Nicolas; Sukhorukov, Gleb B; Perretti, Mauro

    2015-11-10

    Synthetic microstructures can be engineered to deliver bioactive compounds impacting on their pharmacokinetics and pharmacodynamics. Herein, we applied dextran-based layer-by-layer (LbL) microcapsules to deliver alpha-2-macroglobulin (α2MG), a protein with modulatory properties in inflammation. Extending recent observations made with dextran-microcapsules loaded with α2MG in experimental sepsis, we focused on the physical and chemical characteristics of these microstructures and determined their biology on rodent and human cells. We report an efficient encapsulation of α2MG into microcapsules, which enhanced i) human leukocyte recruitment to inflamed endothelium and ii) human macrophage phagocytosis: in both settings microcapsules were more effective than soluble α2MG or empty microcapsules (devoid of active protein). Translation of these findings revealed that intravenous administration of α2MG-microcapsules (but not empty microcapsules) promoted neutrophil migration into peritoneal exudates and augmented macrophage phagocytic functions, the latter response being associated with alteration of bioactive lipid mediators as assessed by mass spectrometry. The present study indicates that microencapsulation can be an effective strategy to harness the complex biology of α2MG with enhancing outcomes on fundamental processes of the innate immune response paving the way to potential future development in the control of sepsis. PMID:26385167

  3. Prostate cancer serum biomarker discovery through proteomic analysis of alpha-2 macroglobulin protein complexes

    PubMed Central

    Burgess, Earle F.; Ham, Amy-Joan L.; Tabb, David L.; Billheimer, Dean; Roth, Bruce J.; Chang, Sam S.; Cookson, Michael S.; Hinton, Timothy J.; Cheek, Kristin L.; Hill, Salisha; Pietenpol, Jennifer A.

    2010-01-01

    Alpha-2 macroglobulin (A2M) functions as a universal protease inhibitor in serum and is capable of binding various cytokines and growth factors. In this study, we investigated if immunoaffinity enrichment and proteomic analysis of A2M protein complexes from human serum could improve detection of biologically relevant and novel candidate protein biomarkers in prostate cancer. Serum samples from six patients with androgen-independent, metastatic prostate cancer and six control patients without malignancy were analyzed by immunoaffinity enrichment of A2M protein complexes and MS identification of associated proteins. Known A2M substrates were reproducibly identified from patient serum in both cohorts, as well as proteins previously undetected in human serum. One example is heat shock protein 90 alpha (HSP90α), which was identified only in the serum of cancer patients in this study. Using an ELISA, the presence of HSP90α in human serum was validated on expanded test cohorts and found to exist in higher median serum concentrations in prostate cancer (n = 18) relative to control (n = 13) patients (median concentrations 50.7 versus 27.6 ng/mL, respectively, p = 0.001). Our results demonstrate the technical feasibility of this approach and support the analysis of A2M protein complexes for proteomic-based serum biomarker discovery. PMID:20107526

  4. Copper is taken up efficiently from albumin and α2-macroglobulin by cultured human cells by more than one mechanism

    PubMed Central

    Moriya, Mizue; Ho, Yi-Hsuan; Grana, Anne; Nguyen, Linh; Alvarez, Arrissa; Jamil, Rita; Ackland, M. Leigh; Michalczyk, Agnes; Hamer, Pia; Ramos, Danny; Kim, Stephen; Mercer, Julian F. B.; Linder, Maria C.

    2008-01-01

    Ionic copper entering blood plasma binds tightly to albumin and the macroglobulin transcuprein. It then goes primarily to the liver and kidney except in lactation, where a large portion goes directly to the mammary gland. Little is known about how this copper is taken up from these plasma proteins. To examine this, the kinetics of uptake from purified human albumin and α2-macroglobulin, and the effects of inhibitors, were measured using human hepatic (HepG2) and mammary epithelial (PMC42) cell lines. At physiological concentrations (3–6 μM), both cell types took up copper from these proteins independently and at rates similar to each other and to those for Cu-dihistidine or Cu-nitrilotriacetate (NTA). Uptakes from α2-macroglobulin indicated a single saturable system in each cell type, but with different kinetics, and 65–80% inhibition by Ag(I) in HepG2 cells but not PMC42 cells. Uptake kinetics for Cu-albumin were more complex and also differed with cell type (as was the case for Cu-histidine and NTA), and there was little or no inhibition by Ag(I). High Fe(II) concentrations (100–500 μM) inhibited copper uptake from albumin by 20–30% in both cell types and that from α2-macroglobulin by 0–30%, and there was no inhibition of the latter by Mn(II) or Zn(II). We conclude that the proteins mainly responsible for the plasma-exchangeable copper pool deliver the metal to mammalian cells efficiently and by several different mechanisms. α2-Macroglobulin delivers it primarily to copper transporter 1 in hepatic cells but not mammary epithelial cells, and additional as-yet-unidentified copper transporters or systems for uptake from these proteins remain to be identified. PMID:18579803

  5. Receptor Recognition Mechanisms of Coronaviruses: a Decade of Structural Studies

    PubMed Central

    2014-01-01

    Receptor recognition by viruses is the first and essential step of viral infections of host cells. It is an important determinant of viral host range and cross-species infection and a primary target for antiviral intervention. Coronaviruses recognize a variety of host receptors, infect many hosts, and are health threats to humans and animals. The receptor-binding S1 subunit of coronavirus spike proteins contains two distinctive domains, the N-terminal domain (S1-NTD) and the C-terminal domain (S1-CTD), both of which can function as receptor-binding domains (RBDs). S1-NTDs and S1-CTDs from three major coronavirus genera recognize at least four protein receptors and three sugar receptors and demonstrate a complex receptor recognition pattern. For example, highly similar coronavirus S1-CTDs within the same genus can recognize different receptors, whereas very different coronavirus S1-CTDs from different genera can recognize the same receptor. Moreover, coronavirus S1-NTDs can recognize either protein or sugar receptors. Structural studies in the past decade have elucidated many of the puzzles associated with coronavirus-receptor interactions. This article reviews the latest knowledge on the receptor recognition mechanisms of coronaviruses and discusses how coronaviruses have evolved their complex receptor recognition pattern. It also summarizes important principles that govern receptor recognition by viruses in general. PMID:25428871

  6. Hepcidin bound to α2-macroglobulin reduces ferroportin-1 expression and enhances its activity at reducing serum iron levels.

    PubMed

    Huang, Michael Li-Hsuan; Austin, Christopher J D; Sari, Marie-Agnès; Rahmanto, Yohan Suryo; Ponka, Prem; Vyoral, Daniel; Richardson, Des R

    2013-08-30

    Hepcidin regulates iron metabolism by down-regulating ferroportin-1 (Fpn1). We demonstrated that hepcidin is complexed to the blood transport protein, α2-macroglobulin (α2M) (Peslova, G., Petrak, J., Kuzelova, K., Hrdy, I., Halada, P., Kuchel, P. W., Soe-Lin, S., Ponka, P., Sutak, R., Becker, E., Huang, M. L., Suryo Rahmanto, Y., Richardson, D. R., and Vyoral, D. (2009) Blood 113, 6225-6236). However, nothing is known about the mechanism of hepcidin binding to α2M or the effects of the α2M·hepcidin complex in vivo. We show that decreased Fpn1 expression can be mediated by hepcidin bound to native α2M and also, for the first time, hepcidin bound to methylamine-activated α2M (α2M-MA). Passage of high molecular weight α2M·hepcidin or α2M-MA·hepcidin complexes (≈725 kDa) through a Sephadex G-25 size exclusion column retained their ability to decrease Fpn1 expression. Further studies using ultrafiltration indicated that hepcidin binding to α2M and α2M-MA was labile, resulting in some release from the protein, and this may explain its urinary excretion. To determine whether α2M-MA·hepcidin is delivered to cells via the α2M receptor (Lrp1), we assessed α2M uptake and Fpn1 expression in Lrp1(-/-) and Lrp1(+/+) cells. Interestingly, α2M·hepcidin or α2M-MA·hepcidin demonstrated similar activities at decreasing Fpn1 expression in Lrp1(-/-) and Lrp1(+/+) cells, indicating that Lrp1 is not essential for Fpn1 regulation. In vivo, hepcidin bound to α2M or α2M-MA did not affect plasma clearance of α2M/α2M-MA. However, serum iron levels were reduced to a significantly greater extent in mice treated with α2M·hepcidin or α2M-MA·hepcidin relative to unbound hepcidin. This effect could be mediated by the ability of α2M or α2M-MA to retard kidney filtration of bound hepcidin, increasing its half-life. A model is proposed that suggests that unlike proteases, which are irreversibly bound to activated α2M, hepcidin remains labile and available to down

  7. Activated α2-macroglobulin binding to human prostate cancer cells triggers insulin-like responses.

    PubMed

    Misra, Uma Kant; Pizzo, Salvatore Vincent

    2015-04-10

    Ligation of cell surface GRP78 by activated α2-macroglobulin (α2M*) promotes cell proliferation and suppresses apoptosis. α2M*-treated human prostate cancer cells exhibit a 2-3-fold increase in glucose uptake and lactate secretion, an effect similar to insulin treatment. In both α2M* and insulin-treated cells, the mRNA levels of SREBP1-c, SREBP2, fatty-acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, and Glut-1 were significantly increased together with their protein levels, except for SREBP2. Pretreatment of cells with α2M* antagonist antibody directed against the carboxyl-terminal domain of GRP78 blocks these α2M*-mediated effects, and silencing GRP78 expression by RNAi inhibits up-regulation of ATP citrate lyase and fatty-acid synthase. α2M* induces a 2-3-fold increase in lipogenesis as determined by 6-[(14)C]glucose or 1-[(14)C]acetate incorporation into free cholesterol, cholesterol esters, triglycerides, free fatty acids, and phosphatidylcholine, which is blocked by inhibitors of fatty-acid synthase, PI 3-kinase, mTORC, or an antibody against the carboxyl-terminal domain of GRP78. We also assessed the incorporation of [(14)CH3]choline into phosphatidylcholine and observed similar effects. Lipogenesis is significantly affected by pretreatment of prostate cancer cells with fatostatin A, which blocks sterol regulatory element-binding protein proteolytic cleavage and activation. This study demonstrates that α2M* functions as a growth factor, leading to proliferation of prostate cancer cells by promoting insulin-like responses. An antibody against the carboxyl-terminal domain of GRP78 may have important applications in prostate cancer therapy. PMID:25720493

  8. Synthesis of acute-phase alpha 2-macroglobulin during inflammation and pregnancy.

    PubMed

    Panrucker, D E; Lorscheider, F L

    1983-01-01

    A recent investigation of acute-phase alpha 2-macroglobulin (AP alpha 2M) concentration in the rat during pregnancy demonstrated a bimodal distribution, for which we suggested a maternal source of AP alpha 2M in early gestation and a fetal source in late gestation. This interpretation was supported by the findings of the present study, which employed organ culture techniques, incorporation of [35S]methionine, immunoprecipitation of radioactivity, and fluorography to measure synthesis of AP alpha 2M in specific fetal, adult, and maternal tissues. Preliminary results indicated that in adult male rats treated with croton oil (compared with nontreated males), AP alpha 2M was synthesized in kidney, spleen, thymus, and lymphocytes by 48 hr post induction, but synthesis in the liver was not evident. In the pregnant rat from 12 to 16 days (compared with nonpregnant females), synthesis of AP alpha 2M was high in metrial gland, moderate in spleen, thymus and lymphocytes, and absent in liver; at 21 days, synthesis of AP alpha 2M in these four maternal tissues had declined. Fetal synthesis of AP alpha 2M in yolk sac (12 to 16 days) and in liver (15 to 16 days) was significantly elevated, and at 21 days fetal liver still displayed marked synthesis. These data are consistent with the interpretation that an early maternal source of AP alpha 2M synthesis is the metrial gland and that in the fetus both yolk sac and liver are major sources of AP alpha 2M, the latter tissue continuing synthesis into late gestation. Lymphopoietic and lymph-containing tissues appear to be major sites of AP alpha 2M synthesis during inflammation and pregnancy. PMID:6200027

  9. Zinc-binding proteins (metallothionein and alpha-2 macroglobulin) and immunosenescence.

    PubMed

    Mocchegiani, Eugenio; Costarelli, Laura; Giacconi, Robertina; Cipriano, Catia; Muti, Elisa; Malavolta, Marco

    2006-11-01

    Zinc is a relevant trace element for the efficiency of the entire immune system. The binding of zinc with some proteins, such as metallothioneins (MT) and alpha-2 macroglobulin (alpha-2M) is crucial for the immune efficiency during ageing and in age-related diseases, because these proteins may be involved in antagonistic pleiotropic effects. Indeed, the presence of chronic inflammation during ageing, generally, induces overexpression of these proteins that, due to their original biological function in fighting stressor agents, continuously sequester intracellular zinc. As a consequence, a low zinc ion availability may appear in aged organisms leading to impairments of the immune response at thymic and extrathymic levels with the risk of the appearance of age-related diseases. Therefore, MT and alpha-2M turn from protective in "young-adult age" to harmful agents in "ageing" following the basic assumption of an evolutionary theory of ageing, named the "antagonistic pleiotropy", which suggests that a trade off between early beneficial effects and late negative outcomes can occur at a genetic and molecular level. On the other hand, some polymorphisms of MT (MT2A) and alpha-2M have been associated with atherosclerosis or Alzheimer disease, respectively. Physiological zinc supplementation in elderly restores the thymic endocrine activity and innate immune response (NK cell cytotoxicity) and increases the survival rate in old mice. Therefore, zinc supplementation is useful to achieve health longevity because these zinc-binding proteins may regain their original protective task against oxidative damage with, thus, a beneficial impact on immune response. PMID:17030107

  10. Activated α2-Macroglobulin Binding to Human Prostate Cancer Cells Triggers Insulin-like Responses

    PubMed Central

    Misra, Uma Kant; Pizzo, Salvatore Vincent

    2015-01-01

    Ligation of cell surface GRP78 by activated α2-macroglobulin (α2M*) promotes cell proliferation and suppresses apoptosis. α2M*-treated human prostate cancer cells exhibit a 2–3-fold increase in glucose uptake and lactate secretion, an effect similar to insulin treatment. In both α2M* and insulin-treated cells, the mRNA levels of SREBP1-c, SREBP2, fatty-acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, and Glut-1 were significantly increased together with their protein levels, except for SREBP2. Pretreatment of cells with α2M* antagonist antibody directed against the carboxyl-terminal domain of GRP78 blocks these α2M*-mediated effects, and silencing GRP78 expression by RNAi inhibits up-regulation of ATP citrate lyase and fatty-acid synthase. α2M* induces a 2–3-fold increase in lipogenesis as determined by 6-[14C]glucose or 1-[14C]acetate incorporation into free cholesterol, cholesterol esters, triglycerides, free fatty acids, and phosphatidylcholine, which is blocked by inhibitors of fatty-acid synthase, PI 3-kinase, mTORC, or an antibody against the carboxyl-terminal domain of GRP78. We also assessed the incorporation of [14CH3]choline into phosphatidylcholine and observed similar effects. Lipogenesis is significantly affected by pretreatment of prostate cancer cells with fatostatin A, which blocks sterol regulatory element-binding protein proteolytic cleavage and activation. This study demonstrates that α2M* functions as a growth factor, leading to proliferation of prostate cancer cells by promoting insulin-like responses. An antibody against the carboxyl-terminal domain of GRP78 may have important applications in prostate cancer therapy. PMID:25720493

  11. Fibroblastic synoviocytes secrete plasma proteins via α2 -macroglobulins serving as intracellular and extracellular chaperones.

    PubMed

    Zhao, Ke-Wei; Murray, Elsa J Brochmann; Murray, Samuel S

    2015-11-01

    Changes in plasma protein levels in synovial fluid (SF) have been implicated in osteoarthritis and rheumatoid arthritis. It was previously thought that the presence of plasma proteins in SF reflected ultrafiltration or extravasation from the vasculature, possibly due to retraction of inflamed endothelial cells. Recent proteomic analyses have confirmed the abundant presence of plasma proteins in SF from control and arthritic patients. Systematic depletion of high-abundance plasma proteins from SF and conditioned media from synoviocytes cultured in serum, and protein analysis under denaturing/reducing conditions have limited our understanding of sources and the native structures of "plasma protein" complexes in SF. Using Western blotting, qPCR, and mass spectrometry, we found that Hig-82 lapine fibroblastic synovicytes cultured under serum-free conditions expressed and secreted plasma proteins, including the cytokine-binding protein secreted phosphoprotein 24 kDa (Spp24) and many of the proteases and protease inhibitors found in SF. Treating synoviocytes with TGF-β1 or BMP-2 for 24 h upregulated the expression of plasma proteins, including Spp24, α2 -HS-glycoprotein, α1 -antitrypsin, IGF-1, and C-reactive protein. Furthermore, many of the plasma proteins of mass <151 kDa were secreted as disulfide-bound complexes with members of the α2 -macroglobulin (A2M) family, which serve as intracellular and extracellular chaperones, not protease inhibitors. Using brefeldin A to block vesicular traffic and protease inhibitors to inhibit endogenous activation of naïve A2M, we demonstrated that the complexes were formed in the endoplasmic reticulum lumen and that Ca(2+) cysteine protease-dependent processes are involved. PMID:25900303

  12. Activated α2-macroglobulin binding to cell surface GRP78 induces T-loop phosphorylation of Akt1 by PDK1 in association with Raptor.

    PubMed

    Misra, Uma Kant; Pizzo, Salvatore Vincent

    2014-01-01

    PDK1 phosphorylates multiple substrates including Akt by PIP3-dependent mechanisms. In this report we provide evidence that in prostate cancer cells stimulated with activated α2-macroglobulin (α2M*) PDK1 phosphorylates Akt in the T-loop at Thr(308) by using Raptor in the mTORC1 complex as a scaffold protein. First we demonstrate that PDK1, Raptor, and mTOR co-immunoprecipitate. Silencing the expression, not only of PDK1, but also Raptor by RNAi nearly abolished Akt phosphorylation at Akt(Thr308) in Raptor-immunoprecipitates of α2M*-stimulated prostate cancer cells. Immunodepleting Raptor or PDK from cell lysates of cells treated with α2M* drastically reduced Akt phosphorylation at Thr(308), which was recovered by adding the supernatant of Raptor- or PDK1-depleted cell lysates, respectively. Studies of insulin binding to its receptor on prostate cancer cells yielded similar results. We thus demonstrate that phosphorylating the T-loop Akt residue Thr(308) by PDK1 requires Raptor of the mTORC1 complex as a platform or scaffold protein. PMID:24516643

  13. The essentiality of alpha-2-macroglobulin in human salivary innate immunity against new H1N1 swine origin influenza A virus

    PubMed Central

    Chen, Chao-Hsuan; Zhang, Xing-Quan; Lo, Chih-Wei; Liu, Pei-Feng; Liu, Yu-Tsueng; Gallo, Richard L.; Hsieh, Ming-Fa; Schooley, Robert T.; Huang, Chun-Ming

    2010-01-01

    A novel strain of influenza A H1N1 emerged in the spring of 2009 and has spread rapidly throughout the world. Although vaccines have recently been developed that are expected to be protective, their availability was delayed until well into the influenza season. While anti-influenza drugs such as neuraminidase inhibitors can be effective, resistance to these drugs has already been reported. Although human saliva was known to inhibit viral infection and may thus prevent viral transmission, the components responsible for this activity on influenza virus, in particular, influenza A swine origin influenza A virus (S-OIV), have not yet been defined. By using a proteomics approach in conjunction with beads that bind alpha 2,6-sialylated glycoprotein, we determined that an alpha-2-macroglobulin (A2M) and a A2M-like protein are essential components in salivary innate immunity against hemagglutination mediated by a clinical isolate of S-OIV [San Diego/01/09 (SD/H1N1-S-OIV)]. A model of an A2M-based “double-edged sword” on competition of alpha 2,6-sialylated glycoprotein receptors and inactivation of host proteases is proposed. We emphasize that endogenous A2M in human innate immunity functions as a natural inhibitor against S-OIV. PMID:20391540

  14. Purification, characterization and molecular cloning of alpha-2-macroglobulin in cobia, Rachycentron canadum.

    PubMed

    Chuang, Wen-Hsiao; Liu, Ping-Chung; Hung, Chia-Yu; Lee, Kuo-Kau

    2014-12-01

    Alpha-2-macroglobulin (α-2-M) is a broad spectrum protease inhibitor which is abundant in the plasma of vertebrates and several invertebrates. The α-2-M was purified from cobia (Rachycentron canadum) plasma by a four-step procedure: poly ethylene glycol fractionation, affinity chromatography, hydrophobic interaction chromatography and ion exchange chromatography on Fast Protein liquid chromatography system in the present study. It migrated as one protein band with a molecular mass of about 360 kDa in the native state, whereas in SDS-PAGE it was about 180 kDa under non-reducing condition. This result revealed that the native protein was a dimer. In addition, it was cleaved into two different fragments of molecular mass about 93 and 87 kDa when reduced by dithiothreitol (DTT). The anti-protease activity of the purified α-2-M was apparently decreased as temperature elevated above 50 °C. The α-2-M exhibited highest protease inhibitory activity at pH 9. The results indicate that the α-2-M is a heat-labile and alkaline protease inhibitor. The purified α-2-M exhibited more than 50% protease inhibitory activity against extracellular products (ECP) of Vibrio alginolytius isolated from diseased cobia. It seems that the protease activities in ECP may be affected by the plasma α-2-M. The protease inhibitory activities of cobia plasma or purified α-2-M were decreased when incubated with 10 mM methylamine for 30 min. The α-2-M cDNA consisted of 4611 bp with an open reading frame of 4374 bp had been cloned from cobia liver. This sequence contained thioester domain (GCGEQ) and thirteen predicted N-linked glycosylation sites. In addition, the amino acid sequence of thioester domain and genes of adjacent regions of cobia α-2-M were further compared with sequences of known fish species in GenBank. The unweighted pair group method using arithmetic average (UPGMA) was employed to construct the phylogenetic trees of α-2-M among different fish species (freshwater fish, sea

  15. Levels of acute inflammatory biomarkers in advanced prostate cancer patients with α2-macroglobulin deficiency.

    PubMed

    Kanoh, Yuhsaku; Ohtani, Hideki; Egawa, Shin; Baba, Shiro; Akahoshi, Tohru

    2011-12-01

    C-reactive protein (CRP), serum amyloid A (SAA), interleukin-6 (IL-6), α1-antitrypsin (α1AT), α1-acid glycoprotein (α1AG) and ceruloplasmin (CP) are acute inflammatory biomarkers that increase in various conditions including infection, inflammation, malignancy and tissue disturbance. In contrast, α2-macroglobulin (α2M) is involved in inflammation through its function as a carrier protein of IL-6. We had previously reported on advanced prostate cancer (PCa) patients with multiple distant bone metastases in whom serum α2M levels were markedly decreased (α2M deficiency). However, the relationship between serum levels of α2M and acute inflammatory biomarkers in PCa patients with or without α2M deficiency has not been demonstrated. In the present study, we examined serum levels of CRP, SAA, IL-6, α1AT, α1AG and CP in PCa patients with or without α2M deficiency to establish clinical significance and changes in these biomarkers during PCa disease progression. We found that upon addition of recombinant IL-6 (rIL-6) to serum from PCa patients with α2M deficiency, since a function of α2M is to bind and stabilize IL-6, the α2M-IL-6 complex and free endogenous IL-6 were not detectable. Serum levels of the α2M-independent markers, α1AT, α1AG and CP, in all PCa patients regardless of α2M deficiency were significantly higher than in healthy controls, but those of the α2M-dependent molecules, CRP, SAA and IL-6, were not increased in PCa patients with α2M deficiency. Therefore, quantitation of both α2M-dependent (CRP, SAA and IL-6) and α2M-independent (α1AT, α1AG and CP) acute inflammatory biomarkers in advanced PCa patients may be an auxiliary indicator, together with prostate-specific antigen (PSA), to monitor PCa disease progression. PMID:21894431

  16. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Perception of pathogen-associated molecular patterns (PAMPs) by surface-localised pattern-recognition receptors (PRRs) is a key component of plant innate immunity. Most known plant PRRs are receptor kinases and initiation of PAMP-triggered immunity (PTI) signalling requires phosphorylation of the PR...

  17. The electrophoretically 'slow' and 'fast' forms of the alpha 2-macroglobulin molecule.

    PubMed Central

    Barrett, A J; Brown, M A; Sayers, C A

    1979-01-01

    alpha 2-Macroglobulin (alpha 2M) was isolated from human plasma by a four-step procedure: poly(ethylene glyco) fractionation, gel chromatography, euglobulin precipitation and immunoadsorption. No contaminants were detected in the final preparations by electrophoresis or immunoprecipitation. The protein ran as a single slow band in gel electrophoresis, and was designated 'S-alpha 2M'. S-alpha 2M bound about 2 mol of trypsin/mol. Treatment of S-alpha 2M with a proteinase or ammonium salts produced a form of the molecule more mobile in electrophoresis, and lacking proteinase-binding activity (F-alpha 2M). The electrophoretic mobility of the F-alpha 2M resulting from reaction with NH4+ salts was identical with that of proteinase complexes. We attribute the change in electrophoretic mobility of the alpha 2M to a conformation change, but there was no evidence of a change in pI or Strokes radius. Electrophoresis of S-alpha 2M in the presence of sodium dodecylsulphate gave results consistent with the view that the alpha 2M molecule is a tetramer of identical subunits, assembled as a non-covalent pair of disulphide-linked dimers. Some of the subunits seemed to be 'nicked' into two-thires-length and one-third-length chains, however. This was not apparent with F-alpha 2M produced by ammonium salts. F-alpha 2M produced by trypsin showed two new bands attributable to cleavage of the subunit polypeptide chain near the middle. Immunoassays of F-alpha 2M gave 'rockets' 12-29% lower than those with S-alpha 2M. The nature of the interactions between subunits in S-alpha 2M and F-alpha 2M was investigated by treating each form with glutaraldehyde before electrophoresis in the presence of sodium dodecyl sulphate. A much greater degree of cross-linking was observed with the F-alpha 2M, indicating that the subunits interact most closely in this form of the molecule. Exposure of S-alpha 2M to 3 M-urea or pH3 resulted in dissociation to the disulphide-bonded half-molecules; these did not

  18. Anti-lymphoproliferative activity of alpha-2-macroglobulin in the plasma of hibernating 13-lined ground squirrels and woodchucks.

    PubMed

    Sieckmann, Donna G; Jaffe, Howard; Golech, Susanne; Cai, DeCheng; Hallenbeck, John M; McCarron, Richard M

    2014-09-15

    Plasma from hibernating (HIB) woodchucks (Marmota monax) or 13-lined ground squirrels (Ictidomys tridecemlineatus) suppressed (3)H-thymidine uptake in mouse spleen cell cultures stimulated with Concanavalin A (ConA); plasma from non-hibernating animals were only slightly inhibitory. Maximum inhibition occurred when HIB plasma was added to the cultures prior to ConA. After HPLC size exclusion chromatography of the HIB ground squirrel plasma, a single fraction (fraction-14) demonstrated inhibitory activity. Assay of fraction-14 from 8 HIB squirrels showed inhibition ranging from 13 to 95%; inhibition was correlated to the time the squirrels were exposed to cold prior to hibernation. Western blot analysis showed the factor to be a large molecular weight protein (>300 kDa), and mass spectrometry identified sequences that were 100% homologous with alpha-2-macroglobulin from humans and other species. These findings indicate a hibernation-related protein that may be responsible for immune system down regulation. PMID:25113962

  19. Anion recognition by oligo-(thio)urea-based receptors.

    PubMed

    Jia, Chuandong; Zuo, Wei; Zhang, Dan; Yang, Xiao-Juan; Wu, Biao

    2016-07-26

    Oligo-(thio)ureas have proven to be a promising class of receptors that are widely applied in anion recognition. This article aims to present some recent progress in the construction of oligoureas and their anion coordination (recognition) chemistry. Typical examples of metal-coordination assisted and covalently connected oligo-(thio)urea receptors are summarized, with focus on geometry characteristics required for achieving complementary binding of a target anion. Special emphasis is given to ortho-phenylene-connected oligoureas in the application of anion binding and the self-assembly of important supramolecular architectures, including helicates, tetrahedral cages, and so on. PMID:27352298

  20. The structural basis for receptor recognition of human interleukin-18

    PubMed Central

    Tsutsumi, Naotaka; Kimura, Takeshi; Arita, Kyohei; Ariyoshi, Mariko; Ohnishi, Hidenori; Yamamoto, Takahiro; Zuo, Xiaobing; Maenaka, Katsumi; Park, Enoch Y.; Kondo, Naomi; Shirakawa, Masahiro; Tochio, Hidehito; Kato, Zenichiro

    2014-01-01

    Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors’ recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is unique among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-18 activity. PMID:25500532

  1. The structural basis for receptor recognition of human interleukin-18

    DOE PAGESBeta

    Tsutsumi, Naotaka; Kimura, Takeshi; Arita, Kyohei; Ariyoshi, Mariko; Ohnishi, Hidenori; Yamamoto, Takahiro; Zuo, Xiaobing; Maenaka, Katsumi; Park, Enoch Y.; Kondo, Naomi; et al

    2014-12-15

    Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors’ recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is uniquemore » among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-8 activity.« less

  2. The structural basis for receptor recognition of human interleukin-18

    SciTech Connect

    Tsutsumi, Naotaka; Kimura, Takeshi; Arita, Kyohei; Ariyoshi, Mariko; Ohnishi, Hidenori; Yamamoto, Takahiro; Zuo, Xiaobing; Maenaka, Katsumi; Park, Enoch Y.; Kondo, Naomi; Shirakawa, Masahiro; Tochio, Hidehito; Kato, Zenichiro

    2014-12-15

    Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor α (Rα) and β (Rβ) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors’ recognition mode for IL-18 is similar to IL-1β; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is unique among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-8 activity.

  3. Sulfate recognition by a hexaaza cryptand receptor.

    PubMed

    Mateus, Pedro; Delgado, Rita; André, Vânia; Teresa Duarte, M

    2015-01-21

    A hexamine macrobicycle with pyrrolyl spacers was evaluated as an anion receptor in its protonated forms. The protonation constants of the receptor, as well as its association constants with Cl(-), NO3(-), AcO(-), ClO4(-), H2PO4(-), and SO4(2-) were determined by potentiometry at 298.2 ± 0.1 K in H2O-MeOH (50 : 50 v/v) and at an ionic strength of 0.10 ± 0.01 M in KTsO. These studies revealed that the Hnpyrr(n+) receptor has a very high effective association constant value for the SO4(2-) at pH 4.0 (log Keff = 6.42), and it is selective for the uptake of this anion in the presence of the other studied anionic substrates. In particular, the receptor showed very high SO4(2-)/NO3(-) selectivity. Using the indicator-displacement approach the receptor is able to signal the presence of sulfate by a change of color. Single crystal X-ray diffraction determination of [(H6pyrr)(SO4)(H2O)3](SO4)2·9.3H2O revealed the presence of one sulfate anion inside the receptor cavity and showed that the encapsulation of the anion is favored by an array of nine hydrogen bonding interactions, including N-HO, C-HO and water-mediated ones. PMID:25407639

  4. α2-Macroglobulin Can Crosslink Multiple Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Molecules and May Facilitate Adhesion of Parasitized Erythrocytes

    PubMed Central

    Stevenson, Liz; Laursen, Erik; Cowan, Graeme J.; Bandoh, Betty; Barfod, Lea; Cavanagh, David R.; Andersen, Gregers R.; Hviid, Lars

    2015-01-01

    Rosetting, the adhesion of Plasmodium falciparum-infected erythrocytes to uninfected erythrocytes, involves clonal variants of the parasite protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) and soluble serum factors. While rosetting is a well-known phenotypic marker of parasites associated with severe malaria, the reason for this association remains unclear, as do the molecular details of the interaction between the infected erythrocyte (IE) and the adhering erythrocytes. Here, we identify for the first time a single serum factor, the abundant serum protease inhibitor α2-macroglobulin (α2M), which is both required and sufficient for rosetting mediated by the PfEMP1 protein HB3VAR06 and some other rosette-mediating PfEMP1 proteins. We map the α2M binding site to the C terminal end of HB3VAR06, and demonstrate that α2M can bind at least four HB3VAR06 proteins, plausibly augmenting their combined avidity for host receptors. IgM has previously been identified as a rosette-facilitating soluble factor that acts in a similar way, but it cannot induce rosetting on its own. This is in contrast to α2M and probably due to the more limited cross-linking potential of IgM. Nevertheless, we show that IgM works synergistically with α2M and markedly lowers the concentration of α2M required for rosetting. Finally, HB3VAR06+ IEs share the capacity to bind α2M with subsets of genotypically distinct P. falciparum isolates forming rosettes in vitro and of patient parasite isolates ex vivo. Together, our results are evidence that P. falciparum parasites exploit α2M (and IgM) to expand the repertoire of host receptors available for PfEMP1-mediated IE adhesion, such as the erythrocyte carbohydrate moieties that lead to formation of rosettes. It is likely that this mechanism also affects IE adhesion to receptors on vascular endothelium. The study opens opportunities for broad-ranging immunological interventions targeting the α2M—(and IgM-) binding domains of PfEMP1

  5. Pattern Recognition Receptors in Innate Immunity, Host Defense, and Immunopathology

    ERIC Educational Resources Information Center

    Suresh, Rahul; Mosser, David M.

    2013-01-01

    Infection by pathogenic microbes initiates a set of complex interactions between the pathogen and the host mediated by pattern recognition receptors. Innate immune responses play direct roles in host defense during the early stages of infection, and they also exert a profound influence on the generation of the adaptive immune responses that ensue.…

  6. Pattern recognition receptors and central nervous system repair

    PubMed Central

    Kigerl, Kristina A.; de Rivero Vaccari, Juan Pablo; Dietrich, W. Dalton

    2016-01-01

    Pattern recognition receptors (PRRs) are part of the innate immune response and were originally discovered for their role in recognizing pathogens by ligating specific pathogen associated molecular patterns (PAMPs) expressed by microbes. Now the role of PRRs in sterile inflammation is also appreciated, responding to endogenous stimuli referred to as “damage associated molecular patterns” (DAMPs) instead of PAMPs. The main families of PRRs include Toll-like receptors (TLRs), Nod-like receptors (NLRs), RIG-like receptors (RLRs), AIM2-like receptors (ALRs), and C-type lectin receptors. Broad expression of these PRRs in the CNS and the release of DAMPs in and around sites of injury suggest an important role for these receptor families in mediating post-injury inflammation. Considerable data now show that PRRs are among the first responders to CNS injury and activation of these receptors on microglia, neurons, and astrocytes triggers an innate immune response in the brain and spinal cord. Here we discuss how the various PRR families are activated and can influence injury and repair processes following CNS injury. PMID:25017883

  7. Characterization of a novel positive transcription regulatory element that differentially regulates the alpha-2-macroglobulin gene in replicative senescence.

    PubMed

    Li, Renzhong; Ma, Liwei; Huang, Yu; Zhang, Zongyu; Tong, Tanjun

    2011-12-01

    Alpha-2-macroglobulin (α2M), a protease inhibitor, is implicated in Alzheimer's disease, atherosclerosis, and other age-related diseases. The elevated level of α2M mRNA has been described in replicative senescence and it could be used as a biomarker of the aging cells. However, the mechanism responsible for the up-regulation of its expression is still unclear. This report identified a novel transcriptional regulatory element, the α2M transcription enhancement element (ATEE), within the α2M promoter. This element differentially activates α2M expression in senescent versus young fibroblasts. Electrophoretic mobility shift assays revealed abundant complexes in senescent cell nuclear extracts compared with young cell nuclear extracts. The DNase I footprint revealed the protein-binding core sequence through which the protein binds the ATEE. Mutation within ATEE selectively abolished α2M promoter activity in senescent (but not young) cells. These results indicated the ATEE, as a positive transcription regulatory element, contributes to the up-regulation of α2M during replicative senescence. PMID:21541797

  8. Activated α2-Macroglobulin Regulates Transcriptional Activation of c-MYC Target Genes through Cell Surface GRP78 Protein.

    PubMed

    Gopal, Udhayakumar; Gonzalez-Gronow, Mario; Pizzo, Salvatore Vincent

    2016-05-13

    Activated α2-macroglobulin (α2M*) signals predominantly through cell surface GRP78 (CS-GRP78) to promote proliferation and survival of cancer cells; however, the molecular mechanism remains obscure. c-MYC is an essential transcriptional regulator that controls cell proliferation. We hypothesize that α2M*/CS-GRP78-evoked key signaling events are required for transcriptional activation of c-MYC target genes. Activation of CS-GRP78 by α2M* requires ligation of the GRP78 primary amino acid sequence (Leu(98)-Leu(115)). After stimulation with α2M*, CS-GRP78 signaling activates 3-phosphoinositide-dependent protein kinase-1 (PDK1) to induce phosphorylation of PLK1, which in turn induces c-MYC transcription. We demonstrate that PLK1 binds directly to c-MYC and promotes its transcriptional activity by phosphorylating Ser(62) Moreover, activated c-MYC is recruited to the E-boxes of target genes FOSL1 and ID2 by phosphorylating histone H3 at Ser(10) In addition, targeting the carboxyl-terminal domain of CS-GRP78 with a mAb suppresses transcriptional activation of c-MYC target genes and impairs cell proliferation. This work demonstrates that α2M*/CS-GRP78 acts as an upstream regulator of the PDK1/PLK1 signaling axis to modulate c-MYC transcription and its target genes, suggesting a therapeutic strategy for targeting c-MYC-associated malignant progression. PMID:27002159

  9. Alteration of prolyl oligopeptidase and activated α-2-macroglobulin in multiple sclerosis subtypes and in the clinically isolated syndrome.

    PubMed

    Tenorio-Laranga, Jofre; Peltonen, Iida; Keskitalo, Salla; Duran-Torres, Gilberto; Natarajan, Renuka; Männistö, Pekka T; Nurmi, Antti; Vartiainen, Nina; Airas, Laura; Elovaara, Irina; García-Horsman, J Arturo

    2013-06-15

    Prolyl oligopeptidase (PREP) has been considered as a drug target for the treatment of neurodegenerative diseases. In plasma, PREP has been found altered in several disorders of the central nervous system including multiple sclerosis (MS). Oxidative stress and the levels of an endogenous plasma PREP inhibitor have been proposed to decrease PREP activity in MS. In this work, we measured the circulating levels of PREP in patients suffering of relapsing remitting (RR), secondary progressive (SP), primary progressive (PP) MS, and in subjects with clinically isolated syndrome (CIS). We found a significantly lower PREP activity in plasma of RRMS as well as in PPMS patients and a trend to reduced activity in subjects diagnosed with CIS, compared to controls. No signs of oxidative inactivation of PREP, and no correlation with the endogenous PREP inhibitor, identified as activated α-2-macroglobulin (α2M*), were observed in any of the patients studied. However, a significant decrease of α2M* was recorded in MS. In cell cultures, we found that PREP specifically stimulates immune active cells possibly by modifying the levels of fibrinogen β, thymosin β4, and collagen. Our results open new lines of research on the role of PREP and α2M* in MS, aiming to relate them to the diagnosis and prognosis of this devastating disease. PMID:23643808

  10. Alpha2-macroglobulin from an Atlantic shrimp: biochemical characterization, sub-cellular localization and gene expression upon fungal challenge.

    PubMed

    Perazzolo, Luciane Maria; Bachère, Evelyne; Rosa, Rafael Diego; Goncalves, Priscila; Andreatta, Edemar Roberto; Daffre, Sirlei; Barracco, Margherita Anna

    2011-12-01

    In this study, we report on the isolation and characterization of an alpha2-macroglobulin (α2M) from the plasma of the pink shrimp Farfantepenaeus paulensis, its sub-cellular localization and transcriptional changes after infection by fungi. The molecular mass of the α2M was estimated at 389 kDa by gel filtration and 197 kDa by SDS-PAGE, under reducing conditions, suggesting that α2M from F. paulensis consists of two identical sub-units, covalently linked by disulphide bonds. The N-terminal amino acid sequence of the α2M from F. paulensis was very similar to those of other penaeid shrimps, crayfish and lobster (70-90% identity) and to a less extent with that of freshwater prawn (40% identity). A monoclonal antibody raised against the Marsupenaeus japonicus α2M made it possible to demonstrate that α2M of F. paulensis is stored in the vesicles of the shrimp granular hemocytes (through immunogold assay). Quantitative real-time PCR (qPCR) analysis showed that α2M mRNA transcripts significantly increased 24 h after an experimental infection with the shrimp pathogen Fusarium solani and it returned to the basal levels at 48 h post-injection. This is the first report on a α2M characterization in an Atlantic penaeid species and its expression profile upon a fungal infection. PMID:21888978

  11. Molecular Recognition of Natural Products by Resorc[4]arene Receptors.

    PubMed

    D'Acquarica, Ilaria; Ghirga, Francesca; Quaglio, Deborah; Cerreto, Antonella; Ingallina, Cinzia; Tafi, Andrea; Botta, Bruno

    2016-01-01

    This review is aimed at providing an overview of the up-to-now published literature on resorc[4]arene macrocycles exploited as artificial receptors for the molecular recognition of some classes of natural products. A concise illustration of the main synthetic strategies developed to afford the resorc[4]arene scaffold is followed by a report on the principles of the gas-phase investigation of recognition phenomena by mass spectrometry (MS). Emphasis is placed on gas-phase studies of diastereoisomeric complexes generated inside a Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer by resorc[4]arene receptors towards a series of natural products, namely amino acids, amphetamine, ethanolamine neurotransmitters, dipeptides, vinca alkaloids and nucleosides. The literature outcomes discussed here, taken largely from our own revisited work, have been completed by references to other studies, in order to draw a broader picture of this rapidly evolving field of research. PMID:26654589

  12. Analysis of Alpha-2 Macroglobulin from the Long-Lived and Cancer-Resistant Naked Mole-Rat and Human Plasma

    PubMed Central

    Thieme, René; Kurz, Susanne; Kolb, Marlen; Debebe, Tewodros; Holtze, Susanne; Morhart, Michaela; Huse, Klaus; Szafranski, Karol; Platzer, Matthias; Hildebrandt, Thomas B.; Birkenmeier, Gerd

    2015-01-01

    Background The naked mole-rat (NMR) is a long-lived and cancer resistant species. Identification of potential anti-cancer and age related mechanisms is of great interest and makes this species eminent to investigate anti-cancer strategies and understand aging mechanisms. Since it is known that the NMR expresses higher liver mRNA-levels of alpha 2-macroglobulin than mice, nothing is known about its structure, functionality or expression level in the NMR compared to the human A2M. Results Here we show a comprehensive analysis of NMR- and human plasma-A2M, showing a different prediction in glycosylation of NMR-A2M, which results in a higher molecular weight compared to human A2M. Additionally, we found a higher concentration of A2M (8.3±0.44 mg/mL vs. and 4.4±0.20 mg/mL) and a lower total plasma protein content (38.7±1.79 mg/mL vs. 61.7±3.20 mg/mL) in NMR compared to human. NMR-A2M can be transformed by methylamine and trypsin resulting in a conformational change similar to human A2M. NMR-A2M is detectable by a polyclonal antibody against human A2M. Determination of tryptic and anti-tryptic activity of NMR and human plasma revealed a higher anti-tryptic activity of the NMR plasma. On the other hand, less proteolytic activity was found in NMR plasma compared to human plasma. Conclusion We found transformed NMR-A2M binding to its specific receptor LRP1. We could demonstrate lower protein expression of LRP1 in the NMR liver tissue compared to human but higher expression of A2M. This was accompanied by a higher EpCAM protein expression as central adhesion molecule in cancer progression. NMR-plasma was capable to increase the adhesion in human fibroblast in vitro most probably by increasing CD29 protein expression. This is the first report, demonstrating similarities as well as distinct differences between A2M in NMR and human plasma. This might be directly linked to the intriguing phenotype of the NMR and suggests that A2M might probably play an important role in anti

  13. Alpha2-plasmin inhibitor and alpha2-macroglobulin-plasmin complexes in plasma. Quantitation by an enzyme-linked differential antibody immunosorbent assay.

    PubMed Central

    Harpel, P C

    1981-01-01

    An enzyme-linked differential antibody immunosorbent assay has been developed for the quantification of alpha2-plasmin inhibitor-plasmin and alpha2-macroglobulin-plasmin complexes. In this method the inhibitor-plasmin complex is bound to a surface by an inhibitor-specific antibody, and the plasmin bound to the inhibitor is quantified by a second antibody, rabbit antiplasminogen F(ab')2, labeled with alkaline phosphatase. The hydrolysis of p-nitrophenyl phosphate by the alkaline phosphatase is expressed in femtomoles of plasminogen per milliliter, by reference to a standard plasminogen curve. Inhibitor-enzyme complexes were generated in plasma by the addition of plasmin or of urokinase. The concentration of plasmin added was well below the plasma concentration of alpha2-plasmin inhibitor (1 microM) or of alpha2-macroglobulin (3.5 microM), so that neither inhibitor would be fully saturated with enzyme. Under these conditions increasing amounts of plasmin generated an increase in both alpha2-plasmin inhibitor-plasmin and alpha2-macroglobulin-plasmin complexes. Varying amounts of plasmin were incubated with each of the purified inhibitors in the concentration found in plasma, and the complexes. Varying amounts of plasmin were incubated with each of the purified inhibitors in the concentration found in plasma, and the complexes that formed were quantified by immunoassay. These studies made it possible to quantify the distribution of plasmin between the two inhibitors in plasmin or urokinase-treated plasma. In plasmin-treated plasma, 10% or less of the plasmin bound to both inhibitors was in complex with alpha2-macroglobulin. In contrast, between 19 and 51% of the plasmin generated in urokinase-activated plasma was bound to alpha2-macroglobulin. Thus, major changes in the distribution of plasma were observed, according to whether plasmin was added to plasma or whether plasminogen was activated endogenously. The pattern of inhibitor plasmin complexes generated in vivo by

  14. Gibberellin Perception by the Gibberellin Receptor and its Effector Recognition

    NASA Astrophysics Data System (ADS)

    Hakoshima, Toshio; Murase, Kohji; Hirano, Yoshinori; Sun, Tai-Ping

    Gibberellins control a diverse range of growth and developmental processes in higher plants and have been widely utilized in the agricultural industry. By binding to a nuclear receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1), gibberellins regulate gene expression by promoting degradation of the transcriptional regulator DELLA proteins. The precise manner in which GID1 discriminates and becomes activated by bioactive gibberellins for specific binding to DELLA proteins remains unclear. We present the crystal structure of a ternary complex of Arabidopsis thaliana GID1A, a bioactive gibberellin and the N-terminal DELLA domain of GAI. In this complex, GID1a occludes gibberellin in a deep binding pocket covered by its N-terminal helical switch region, which in turn interacts with the DELLA domain containing DELLA, VHYNP and LExLE motifs. Our results establish a structural model of a plant hormone receptor which is distinct from the hormone-perception mechanism and effector recognition of the known auxin receptors.

  15. Carbohydrate Recognition by the Mannose 6-phosphate Receptors

    PubMed Central

    Kim, Jung-Ja P.; Olson, Linda J.; Dahms, Nancy M.

    2009-01-01

    Summary The two P-type lectins, the 46 kDa cation-dependent mannose 6-phosphate (Man-6-P) receptor (CD-MPR) and the 300 kDa cation-independent Man-6-P receptor (CI-MPR), are the founding members of the growing family of mannose 6-phosphate receptor homology (MRH) proteins. A major cellular function of the MPRs is to transport Man-6-P-containing acid hydrolases from the Golgi to endosomal/lysosomal compartments. Recent advances in the structural analyses of both CD-MPR and CI-MPR have revealed the structural basis for phosphomannosyl recognition by these receptors and provided insights into how the receptors load and unload their cargo. A surprising finding is that the CD-MPR is dynamic, with at least two stable quaternary states, the open (ligand bound) and closed (ligand free) conformations, similar to those of hemoglobin. Ligand binding stabilizes the open conformation; changes in the pH of the environment at the cell surface and in endosomal compartments weaken the ligand-receptor interaction and/or weaken the electrostatic interactions at the subunit interface, resulting in the closed conformation. PMID:19801188

  16. Studies on human pregnancy-associated plasma protein A. Purification by affinity chromatography and structural comparisons with alpha 2-macroglobulin.

    PubMed Central

    Sutcliffe, R G; Kukulska-Langlands, B M; Coggins, J R; Hunter, J B; Gore, C H

    1980-01-01

    Pregnancy-associated plasma protein-A (PAPP-A) has been purified by a combination of methods including antibody-affinity chromatography. The resultant protein, obtained in 16% yield from maternal serum, appeared as a single major component on non-denaturing polyacrylamide and SDS/polyacrylamide gel electrophoresis. The protein showed a single component when analysed by isoelectric focusing under denaturing conditions in the presence and absence of reduction and had a pI of 4.34 and 4.42 respectively. These pI values were indistinguishable from those of alpha 2-macroglobulin (alpha 2M). The molecular weight of the PAPP-A polypeptide as shown by SDS/polyacrylamide-gel electrophoresis was 187000, with a minor component of mol.wt. 82500 that was attributed to proteolysis. Since native PAPP-A had a molecular weight on gel chromatography very similar to that of alpha 2M (620000--820000), it was concluded that PAPP-A was a homotetramer. In the absence of reduction, a high-molecular-weight (420000) protomer of PAPP-A was found. It was deduced that PAPP-A, like alpha 2M, is a dinner, whose protomers are composed of disulphide-linked polypeptide chains. It was found that the molecular weight of the PAPP-A polypeptide exceeded that of alpha 2M by 3.3%, but that the total carbohydrate content of PAPP-A exceeded that of alpha 2M by 10% and that its neutral carbohydrate content exceeded that of alpha 2M by between 7.4 and 9.0%. The significance of the estimated molecular weights of alpha 2M (181000) and its major tryptic fragments is discussed in the light of published values. A tryptic fragment alpha 2M (82500 mol.wt.) was apparently the same size as the major tryptic fragment of PAPP-A. Images Fig. 1. Fig. 4. Fig. 6. PMID:6169340

  17. Recognition of Bacterial Signal Peptides by Mammalian Formyl Peptide Receptors

    PubMed Central

    Bufe, Bernd; Schumann, Timo; Kappl, Reinhard; Bogeski, Ivan; Kummerow, Carsten; Podgórska, Marta; Smola, Sigrun; Hoth, Markus; Zufall, Frank

    2015-01-01

    Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system. PMID:25605714

  18. Molecular Recognition of Insulin by a Synthetic Receptor

    SciTech Connect

    Chinai, Jordan M.; Taylor, Alexander B.; Ryno, Lisa M.; Hargreaves, Nicholas D.; Morris, Christopher A.; Hart, P. John; Urbach, Adam R.

    2011-08-29

    The discovery of molecules that bind tightly and selectively to desired proteins continues to drive innovation at the interface of chemistry and biology. This paper describes the binding of human insulin by the synthetic receptor cucurbit[7]uril (Q7) in vitro. Isothermal titration calorimetry and fluorescence spectroscopy experiments show that Q7 binds to insulin with an equilibrium association constant of 1.5 x 10{sup 6} M{sup -1} and with 50-100-fold selectivity versus proteins that are much larger but lack an N-terminal aromatic residue, and with >1000-fold selectivity versus an insulin variant lacking the N-terminal phenylalanine (Phe) residue. The crystal structure of the Q7{center_dot}insulin complex shows that binding occurs at the N-terminal Phe residue and that the N-terminus unfolds to enable binding. These findings suggest that site-selective recognition is based on the properties inherent to a protein terminus, including the unique chemical epitope presented by the terminal residue and the greater freedom of the terminus to unfold, like the end of a ball of string, to accommodate binding. Insulin recognition was predicted accurately from studies on short peptides and exemplifies an approach to protein recognition by targeting the terminus.

  19. Evaluation of plasma alpha-2-macroglobulin and interactions with tumour necrosis factor-alpha in horses with endotoxemic signs.

    PubMed Central

    Coté, N; Trout, D R; Hayes, A M

    1996-01-01

    The electrophoretic position and behavior of the native and activated forms of equine plasma alpha-2-macroglobulin (alpha 2M) were characterized and compared to human alpha 2M by nondenaturing polyacrylamide-gel electrophoresis (PAGE). Plasma alpha 2M was also compared between 6 normal horses and 6 horses with clinical signs of colic and endotoxemia due to volvulus or enteritis. Native and activated forms of alpha 2M were quantified by PAGE and densitometry. Binding of radio-labeled recombinant human tumour necrosis factor-alpha (125I-rhTNF-alpha) to native and activated forms of equine alpha 2M was also evaluated by autoradiography and densitometry of PAGE. Equine plasma alpha 2M migrated as a single band at a position equivalent to native human alpha 2M. Methylamine-reacted equine plasma samples resulted in faster migration of alpha 2M in a similar position to activated human alpha 2M. However, in methylamine-reacted equine plasma, an intermediate alpha 2M band was consistently present between the bands corresponding to native and activated alpha 2M. Amounts of plasma alpha 2M were similar in normal and endotoxemic horses, and remained in the electrophoretically slow or unreacted native form. The vast majority of 125I-rHuTNF-alpha did not bind to alpha 2M or other equine plasma proteins. 125I-rHuTNF-alpha bound weakly to both native and fast methylamine-reacted equine forms of alpha 2M, although binding was better to the activated form. This study indicates that: (1) equine plasma alpha 2M behaves similarly to human alpha 2M on PAGE, (2) plasma alpha 2M of horses can be activated to electrophoretically fast forms, but it is neither activated nor depleted during endotoxemia, and (3) the binding interactions between equine alpha 2M and TNF-alpha are too low to implicate equine alpha 2M as a regulator of TNF-alpha during endotoxemia in horses. Images Figure 1. Figure 2. Figure 3. PMID:8785722

  20. Inhibition of ADAMTS-7 and ADAMTS-12 Degradation of Cartilage Oligomeric Matrix Protein by Alpha-2-Macroglobulin

    PubMed Central

    Luan, Yi; Kong, Li; Howell, Damani R.; Ilalov, Kiril; Fajardo, Marc; Bai, Xiao-hui; Di Cesare, Paul E.; Goldring, Mary B.; Abramson, Steven B.; Liu, Chuan-ju

    2008-01-01

    Objective As we previously reported, ADAMTS-7 and ADAMTS-12, two members of ADAMTS (adisintegrin and metalloprotease with thrombospondin motifs) family, degrade COMP in vitro and are significantly induced in the cartilage and synovium of arthritic patients. The purpose of this study was to determine 1) whether cleavage activity of ADAMTS-7 and -12 of COMP are associated with COMP degradation in osteoarthritis; 2) whether a2M is a novel substrate for ADAMTS-7 and -12; and 3) whether a2M inhibits ADAMTS-7 or -12 cleavage of COMP. Methods An in vitro digestion assay was used to examine the degradation of COMP by ADAMTS-7 and ADAMTS-12 in the cartilage of OA patients; in cartilage explants incubated with TNF-α or IL-1β with or without blocking antibodies; and in human chondrocytes treated with specific siRNA to knock down ADAMTS-7 or/and-12. Digestion of alpha-2-macroglobulin (a2M) by ADAMTS-7 and -12 in vitro and the inhibition of ADAMTS-7 or -12-mediated digestion of COMP by α2M were also analyzed. Results The molecular mass of the COMP fragments produced by either ADAMTS-7 or ADAMTS-12 were similar to those observed in OA patients. Specific blocking antibodies against ADAMTS-7 and ADAMTS-12 dramatically inhibited TNF-α- or IL-1β-induced COMP degradation in the cultured cartilage explants. The suppression of ADAMTS-7 or ADAMTS-12 expression by siRNA silencing in the human chondrocytes also prevented TNF-α- or IL-1β-induced COMP degradation. Both ADAMTS-7 and ADAMTS-12 were able to cleave α2M, giving rise to 180 and 105 kDa cleavage products, respectively. Furthermore, α2M inhibited both ADAMTS-7- and ADAMTS-12-mediated COMP degradation in a concentration (or dose)-dependent manner. Conclusion Our observations demonstrate the importance of COMP degradation by ADAMTS-7 and ADAMTS-12 in vivo. Furthermore, α2M is a novel substrate for ADAMTS-7 and ADAMTS-12. More significantly, α2M represents the first endogenous inhibitor of ADAMTS-7 and ADAMTS-12. PMID

  1. Interaction of transforming growth factor-beta-1 with alpha-2-macroglobulin from normal and inflamed equine joints.

    PubMed Central

    Coté, N; Trout, D R; Hayes, M A

    1998-01-01

    Binding between equine plasma alpha-2-macroglobulin (alpha 2M) and several cytokines known to participate in inflammatory reactions in other species was initially examined. Plasma was obtained from 5 horses with various abnormalities. Samples, both untreated and after reaction with methylamine, were incubated with exogenous, radiolabeled, porcine-derived transforming growth factor-beta-1 (125I-TGF-beta 1), recombinant human interleukin-1-beta (125I-IL-1 beta), and recombinant human tumor necrosis factor-alpha (125I-rhTNF-alpha). They were then subjected to nondenaturing polyacrylamide gel electrophoresis (PAGE). Binding of the native (slow) and activated (fast) forms of alpha 2M to each cytokine was subjectively evaluated with autoradiography. Equine alpha 2M bound 125I-TGF-beta 1. However, poor or no binding was observed between alpha 2M and either of 125I-rhTNF-alpha or 125I-IL-1 beta. Synovial fluid was then obtained from 6 normal horses, 6 horses with septic arthritis, and 6 horses with degenerative joint disease. Untreated and methylamine-reacted samples were quantitatively examined for binding with 125I-TGF-beta 1, using the autoradiographic techniques described above and densitometry. Native and activated alpha 2M were also quantified by densitometry of PAGE gels. Native alpha 2M was significantly elevated in septic arthritis (6.4% to 29.5% of total protein detected) and degenerative joint disease (2.8% to 12.3%), compared to normal joints (0.9% to 4.2%). Activated alpha 2M, however, was not detected in untreated synovial fluid samples. In all plasma and joint fluid samples, whether untreated or reacted with methylamine, 125I-TGF-beta 1 bound predominantly to alpha 2M, and preferentially to the activated form of alpha 2M. In synovial fluid, the amount of 125I-TGF-beta 1 binding was proportional to the quantity of alpha 2M present. These results indicate that: 1) equine alpha 2M binds TGF-beta 1; 2) the native form of alpha 2M is present in both equine plasma

  2. Kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin as measured by small-angle neutron scattering.

    PubMed

    Sjöberg, B; Pap, S; Järnberg, S E; Mortensen, K

    1991-09-01

    The kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin into two half-molecular fragments was investigated at 21.0 degrees C by using small-angle neutron scattering. The relative change in molecular mass that occurs upon dissociation was monitored by recording the forward scattering of neutrons as a function of time. All these kinetic data can be explained by a reaction that is first-order with respect to the concentration of undissociated alpha 2-macroglobulin. The velocity constant is a function of urea concentration and it varies within wide limits. For instance, the half-life of the reaction at the lowest concentration of [2H]urea studied (2.70 M) is 328 h, whereas the same value at the highest concentration of [2H]urea (6.24 M) is only 8 min. Measurements were made both with [1H]urea in 1H2O and with [2H]urea in 99% 2H2O, and it was found that there is a pronounced kinetic isotope effect, i.e. the dissociation is 4 times faster in the 1H-containing medium as compared with the 2H-containing medium at the same molar concentration of urea. From the angular dependence of the neutron scattering it can be concluded that the dissociation is associated with a drastic change in structure. This is directly shown by the radius of gyration, which increases from about 7.4 nm immediately after the addition of urea up to about 9.4 nm when the protein is fully dissociated. A structural analysis shows that the scattering curve of urea-dissociated alpha 2-macroglobulin can best be explained by that of a Gaussian coil with a radius of gyration equal to 9.44 nm. These data indicate that the so-called non-covalent interaction of alpha 2-macroglobulin probably is more complicated than just a pure hydrophobic interaction. Finally, it is also shown that the dissociation is accompanied by a loss in trypsin-binding activity, which is directly related to the fraction of dissociated protein. PMID:1716880

  3. [New immunology--immunology of pattern recognition receptors].

    PubMed

    Lebedev, K A; Poniakina, I D

    2006-01-01

    Pattern recognition receptors (PRRs) have been found on all cells of the body--cells of the innate and adaptive immune systems, epithelial and endothelial cells, keratinocytes, etc. PRRs can recognize specific molecular structures of microorganisms as well as allergens and other substances. The interaction with ligands of foreign microorganisms activates PRRs, after which host cells start to produce cytokines to both specifically activate innate immunity and to control adaptive immune reactions. On the other hand, no immune response develops against microorganisms of the normal microflora. Practically, the development of all immune responses is controlled by PRRs. These responses start in epithelial cells, skin cells, and vascular epithelial cells, which meet alien first. The immune system uses these cells to control the composition of normal microflora. Accordingly, the definition of immune system functions should be complemented by the regulation of body's microflora in addition to the protection from alien and altered self. PMID:17086961

  4. Multivalent recognition of peptides by modular self-assembled receptors.

    PubMed

    Reczek, Joseph J; Kennedy, Aimee A; Halbert, Brian T; Urbach, Adam R

    2009-02-18

    Developing nontraditional approaches to the synthesis and characterization of multivalent compounds is critical to our efforts to study and interface with biological systems and to build new noncovalent materials. This paper demonstrates a biomimetic approach to the construction of discrete, modular, multivalent receptors via molecular self-assembly in aqueous solution. Scaffolds presenting 1-3 viologen groups recruit a respective 1-3 copies of the synthetic host, cucurbit[8]uril, in a noncooperative manner and with a consistent equilibrium association constant (K(a)) value of 2 x 10(6) M(-1) per binding site. The assembled mono-, di-, and trivalent receptors bind to their cognate target peptides containing 1-3 Trp residues with K(a) values in the range 1.7 x 10(4)-4.7 x 10(6) M(-1) and in predetermined mono- or multivalent binding modes with 31-280-fold enhancements in affinity and additive enthalpies due to multivalency. The extent of valency was determined directly by measuring the visible charge-transfer absorptivity due to the viologen-indole pair. The predictable behavior of this system and its ease of synthesis and analysis make it well suited to serve as a model for multivalent binding and for the multivalent recognition of peptides by design. PMID:19199617

  5. Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products.

    PubMed

    Lien, E; Sellati, T J; Yoshimura, A; Flo, T H; Rawadi, G; Finberg, R W; Carroll, J D; Espevik, T; Ingalls, R R; Radolf, J D; Golenbock, D T

    1999-11-19

    Toll-like receptors (TLRs) 2 and 4 are signal transducers for lipopolysaccharide, the major proinflammatory constituent in the outer membrane of Gram-negative bacteria. We observed that membrane lipoproteins/lipopeptides from Borrelia burgdorferi, Treponema pallidum, and Mycoplasma fermentans activated cells heterologously expressing TLR2 but not those expressing TLR1 or TLR4. These TLR2-expressing cells were also stimulated by living motile B. burgdorferi, suggesting that TLR2 recognition of lipoproteins is relevant to natural Borrelia infection. Importantly, a TLR2 antibody inhibited bacterial lipoprotein/lipopeptide-induced tumor necrosis factor release from human peripheral blood mononuclear cells, and TLR2-null Chinese hamster macrophages were insensitive to lipoprotein/lipopeptide challenge. The data suggest a role for the native protein in cellular activation by these ligands. In addition, TLR2-dependent responses were seen using whole Mycobacterium avium and Staphylococcus aureus, demonstrating that this receptor can function as a signal transducer for a wide spectrum of bacterial products. We conclude that diverse pathogens activate cells through TLR2 and propose that this molecule is a central pattern recognition receptor in host immune responses to microbial invasion. PMID:10559223

  6. CD14: a soluble pattern recognition receptor in milk.

    PubMed

    Vidal, Karine; Donnet-Hughes, Anne

    2008-01-01

    An innate immune system capable of distinguishing among self, non-self, and danger is a prerequisite for health. Upon antigenic challenge, pattern recognition receptors (PRRs), such as the Toll-like receptor (TLR) family of proteins, enable this system to recognize and interact with a number of microbial components and endogenous host proteins. In the healthy host, such interactions culminate in tolerance to self-antigen, dietary antigen, and commensal microorganisms but in protection against pathogenic attack. This duality implies tightly regulated control mechanisms that are not expected of the inexperienced neonatal immune system. Indeed, the increased susceptibility of newborn infants to infection and to certain allergens suggests that the capacity to handle certain antigenic challenges is not inherent. The observation that breast-fed infants experience a lower incidence of infections, inflammation, and allergies than formula-fed infants suggests that exogenous factors in milk may play a regulatory role. There is increasing evidence to suggest that upon exposure to antigen, breast milk educates the neonatal immune system in the decision-making processes underlying the immune response to microbes. Breast milk contains a multitude of factors such as immunoglobulins, glycoproteins, glycolipids, and antimicrobial peptides that, qualitatively or quantitatively, may modulate how neonatal cells perceive and respond to microbial components. The specific role of several of these factors is highlighted in other chapters in this book. However, an emerging concept is that breast milk influences the neonatal immune system's perception of "danger." Here we discuss how CD14, a soluble PRR in milk, may contribute to this education. PMID:18183930

  7. Inhibition of pattern recognition receptor-mediated inflammation by bioactive phytochemicals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Emerging evidence reveals that pattern-recognition receptors (PRRs), Toll-like receptors (TLRs) and Nucleotide-binding oligomerization domain proteins (NODs) mediate both infection-induced and sterile inflammation by recognizing pathogen-associated molecular patterns (PAMPs) and endogenous molecules...

  8. Molecular recognition of organic ammonium ions in solution using synthetic receptors

    PubMed Central

    Späth, Andreas

    2010-01-01

    Summary Ammonium ions are ubiquitous in chemistry and molecular biology. Considerable efforts have been undertaken to develop synthetic receptors for their selective molecular recognition. The type of host compounds for organic ammonium ion binding span a wide range from crown ethers to calixarenes to metal complexes. Typical intermolecular interactions are hydrogen bonds, electrostatic and cation–π interactions, hydrophobic interactions or reversible covalent bond formation. In this review we discuss the different classes of synthetic receptors for organic ammonium ion recognition and illustrate the scope and limitations of each class with selected examples from the recent literature. The molecular recognition of ammonium ions in amino acids is included and the enantioselective binding of chiral ammonium ions by synthetic receptors is also covered. In our conclusion we compare the strengths and weaknesses of the different types of ammonium ion receptors which may help to select the best approach for specific applications. PMID:20502608

  9. The other side of scavenger receptors: pattern recognition for host defense.

    PubMed

    Krieger, M

    1997-10-01

    Scavenger receptors bind modified lipoproteins and may play an important role both in normal and in pathological lipid metabolism. A number of different classes of scavenger receptors have been identified and several of these are multiligand receptors. Studies, both in vitro and in vivo, have indicated that at least some of these scavenger receptors may serve as pattern recognition receptors because they are able to bind a wide variety of pathogens. As a consequence, they may play key roles in innate immunity and host defense. PMID:9335951

  10. Recognition of bacterial capsular polysaccharides and lipopolysaccharides by the macrophage mannose receptor.

    PubMed

    Zamze, Susanne; Martinez-Pomares, Luisa; Jones, Hannah; Taylor, Philip R; Stillion, Richard J; Gordon, Siamon; Wong, Simon Y C

    2002-11-01

    The in vitro binding of the macrophage mannose receptor to a range of different bacterial polysaccharides was investigated. The receptor was shown to bind to purified capsular polysaccharides from Streptococcus pneumoniae and to the lipopolysaccharides, but not capsular polysaccharides, from Klebsiella pneumoniae. Binding was Ca(2+)-dependent and inhibitable with d-mannose. A fusion protein of the mannose receptor containing carbohydrate recognition domains 4-7 and a full-length soluble form of the mannose receptor containing all domains external to the transmembrane region both displayed very similar binding specificities toward bacterial polysaccharides, suggesting that domains 4-7 are sufficient for recognition of these structures. Surprisingly, no direct correlation could be made between polysaccharide structure and binding to the mannose receptor, suggesting that polysaccharide conformation may play an important role in recognition. The full-length soluble form of the mannose receptor was able to bind simultaneously both polysaccharide via the carbohydrate recognition domains and sulfated oligosaccharide via the cysteine-rich domain. The possible involvement of the mannose receptor, either cell surface or soluble, in the innate and adaptive immune responses to bacterial polysaccharides is discussed. PMID:12196537

  11. Modification of the erythrocyte surface in rats bearing Yoshida ascites sarcoma is brought about by a tumour variant of alpha2-macroglobulin.

    PubMed Central

    Sanjay, A; Kalraiya, R D; Mehta, N G

    1997-01-01

    Erythrocytes from the circulation of rats bearing Yoshida ascites sarcoma exhibit higher concanavalin A (ConA)-mediated agglutinability than those from normal animals. A tetrameric glycoprotein of subunit molecular mass 170 kDa, purified from the cell-free ascites fluid, was found to confer higher ConA-mediated agglutinability on erythrocytes in vitro. An antiserum to this tumour-derived protein failed to detect any cross-reactive component in normal rat plasma or in any of the normal tissues examined. An immunoreactive protein was, however, detected in blood plasma when the acute-phase reaction was stimulated by injection of turpentine. The cross-reactive acute-phase protein was purified by ConA-affinity, gel-filtration and ion-exchange chromatography, and identified as alpha2-macroglobulin. The acute-phase protein and the protein obtained from the ascites fluid have identical or very similar native and subunit molecular masses, subunit arrangement and pI. They both are able to inhibit trypsin and, as a consequence, acquire greater mobility in native PAGE. In addition, the two proteins bind to rat erythrocytes non-specifically, and in similar amounts. However, despite these similarities, the acute-phase protein is unable to enhance the agglutinability of erythrocytes. The two proteins differ in their carbohydrate content, but this differential glycosylation is not the cause of the difference in their surface modification activity. The chemically deglycosylated proteins show a small but consistent difference in the size of their polypeptides. Their tryptic peptide maps, although largely similar, show some differences, as do their amino acid compositions. It is probable that the proteins are independent members of the same (alpha-macroglobulin) family. The rat embryo is also found to express a soluble protein consisting of a 170 kDa polypeptide that cross-reacts with the antibody to the tumour-derived protein. The purified embryo protein is able to alter the Con

  12. Galectins as Pattern Recognition Receptors: Structure, Function, and Evolution

    PubMed Central

    Vasta, Gerardo R.

    2012-01-01

    Galectins constitute an evolutionary conserved family of β-galactoside-binding proteins, ubiquitous in mammals and other vertebrate taxa, invertebrates, and fungi. Since their discovery in the 1970s, their biological roles, initially understood as limited to recognition of carbohydrate ligands in embryogenesis and development, have expanded in recent years by the discovery of their immunoregulatory activities. A gradual paradigm shift has taken place in the past few years through the recognition that galectins also bind glycans on the surface of potentially pathogenic microbes, and function as recognition and effector factors in innate immunity. Further, an additional level of functional complexity has emerged with the most recent findings that some parasites “subvert” the recognition roles of the vector/host galectins for successful attachment or invasion. PMID:21948360

  13. Variable Lymphocyte Receptor Recognition of the Immunodominant Glycoprotein of Bacillus anthracis Spores

    SciTech Connect

    Kirchdoerfer, Robert N.; Herrin, Brantley R.; Han, Byung Woo; Turnbough, Jr., Charles L.; Cooper, Max D.; Wilson, Ian A.

    2012-07-25

    Variable lymphocyte receptors (VLRs) are the adaptive immune receptors of jawless fish, which evolved adaptive immunity independent of other vertebrates. In lieu of the immunoglobulin fold-based T and B cell receptors, lymphocyte-like cells of jawless fish express VLRs (VLRA, VLRB, or VLRC) composed of leucine-rich repeats and are similar to toll-like receptors (TLRs) in structure, but antibodies (VLRB) and T cell receptors (VLRA and VLRC) in function. Here, we present the structural and biochemical characterization of VLR4, a VLRB, in complex with BclA, the immunodominant glycoprotein of Bacillus anthracis spores. Using a combination of crystallography, mutagenesis, and binding studies, we delineate the mode of antigen recognition and binding between VLR4 and BclA, examine commonalities in VLRB recognition of antigens, and demonstrate the potential of VLR4 as a diagnostic tool for the identification of B. anthracis spores.

  14. Structural basis for molecular recognition at serotonin receptors.

    PubMed

    Wang, Chong; Jiang, Yi; Ma, Jinming; Wu, Huixian; Wacker, Daniel; Katritch, Vsevolod; Han, Gye Won; Liu, Wei; Huang, Xi-Ping; Vardy, Eyal; McCorvy, John D; Gao, Xiang; Zhou, X Edward; Melcher, Karsten; Zhang, Chenghai; Bai, Fang; Yang, Huaiyu; Yang, Linlin; Jiang, Hualiang; Roth, Bryan L; Cherezov, Vadim; Stevens, Raymond C; Xu, H Eric

    2013-05-01

    Serotonin or 5-hydroxytryptamine (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. We report the crystal structures of the human 5-HT1B G protein-coupled receptor bound to the agonist antimigraine medications ergotamine and dihydroergotamine. The structures reveal similar binding modes for these ligands, which occupy the orthosteric pocket and an extended binding pocket close to the extracellular loops. The orthosteric pocket is formed by residues conserved in the 5-HT receptor family, clarifying the family-wide agonist activity of 5-HT. Compared with the structure of the 5-HT2B receptor, the 5-HT1B receptor displays a 3 angstrom outward shift at the extracellular end of helix V, resulting in a more open extended pocket that explains subtype selectivity. Together with docking and mutagenesis studies, these structures provide a comprehensive structural basis for understanding receptor-ligand interactions and designing subtype-selective serotonergic drugs. PMID:23519210

  15. Pattern Recognition Receptors and Cytokines in Mycobacterium tuberculosis Infection—The Double-Edged Sword?

    PubMed Central

    Hossain, Md. Murad; Norazmi, Mohd-Nor

    2013-01-01

    Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis (Mtb), remains a major cause of human death worldwide. Innate immunity provides host defense against Mtb. Phagocytosis, characterized by recognition of Mtb by macrophages and dendritic cells (DCs), is the first step of the innate immune defense mechanism. The recognition of Mtb is mediated by pattern recognition receptors (PRRs), expressed on innate immune cells, including toll-like receptors (TLRs), complement receptors, nucleotide oligomerization domain like receptors, dendritic cell-specific intercellular adhesion molecule grabbing nonintegrin (DC-SIGN), mannose receptors, CD14 receptors, scavenger receptors, and FCγ receptors. Interaction of mycobacterial ligands with PRRs leads macrophages and DCs to secrete selected cytokines, which in turn induce interferon-γ- (IFNγ-) dominated immunity. IFNγ and other cytokines like tumor necrosis factor-α (TNFα) regulate mycobacterial growth, granuloma formation, and initiation of the adaptive immune response to Mtb and finally provide protection to the host. However, Mtb can evade destruction by antimicrobial defense mechanisms of the innate immune system as some components of the system may promote survival of the bacteria in these cells and facilitate pathogenesis. Thus, although innate immunity components generally play a protective role against Mtb, they may also facilitate Mtb survival. The involvement of selected PRRs and cytokines on these seemingly contradictory roles is discussed. PMID:24350246

  16. The role of histamine receptors in the consolidation of object recognition memory.

    PubMed

    da Silveira, Clarice Krás Borges; Furini, Cristiane R G; Benetti, Fernando; Monteiro, Siomara da Cruz; Izquierdo, Ivan

    2013-07-01

    Findings have shown that histamine receptors in the hippocampus modulate the acquisition and extinction of fear motivated learning. In order to determine the role of hippocampal histaminergic receptors on recognition memory, adult male Wistar rats with indwelling infusion cannulae stereotaxically placed in the CA1 region of dorsal hippocampus were trained in an object recognition learning task involving exposure to two different stimulus objects in an enclosed environment. In the test session, one of the objects presented during training was replaced by a novel one. Recognition memory retention was assessed 24 h after training by comparing the time spent in exploration (sniffing and touching) of the known object with that of the novel one. When infused in the CA1 region immediately, 30, 120 or 360 min posttraining, the H1-receptor antagonist, pyrilamine, the H2-receptor antagonist, ranitidine, and the H3-receptor agonist, imetit, blocked long-term memory retention in a time dependent manner (30-120 min) without affecting general exploratory behavior, anxiety state or hippocampal function. Our data indicate that histaminergic system modulates consolidation of object recognition memory through H1, H2 and H3 receptors. PMID:23583502

  17. Distinctive Recognition of Flagellin by Human and Mouse Toll-Like Receptor 5

    PubMed Central

    Forstnerič, Vida; Ivičak-Kocjan, Karolina; Ljubetič, Ajasja; Jerala, Roman; Benčina, Mojca

    2016-01-01

    Toll-like receptor 5 (TLR5) is a receptor of the innate immune system that recognizes flagellin from certain bacterial species and triggers an inflammatory response. The Salmonella dublin flagellin in complex with zebrafish TLR5 has been crystallized previously. In the present study, we extrapolate the structure of this complex using structure-guided mutagenesis to determine the recognition modes of human and mouse TLR5 receptors and demonstrate species-specific differences in flagellin recognition. In general, the recognition mode of the mouse receptor can be said to be more robust in comparison to that of the human receptor. All-atom molecular dynamics simulation showed differences between the two receptors within the primary binding region. Using a functional motility assay, we show that although the highly conserved area of the flagellin analyzed in this study encompasses key structural requirements for flagella formation, a direct correlation between immune recognition and structure on the level of amino acid residues is not observed. PMID:27391968

  18. Biochemical study of multiple drug recognition sites on central benzodiazepine receptors

    SciTech Connect

    Trifiletti, R.R.

    1986-01-01

    The benzodiazepine receptor complex of mammalian brain possesses recognition sites which mediate (at least in part) the pharmacologic actions of the 1,4-benzodiazepines and barbiturates. Evidence is provided suggesting the existence of least seven distinct drug recognition sites on this complex. Interactions between the various recognition sites have been explored using radioligand binding techniques. This information is utilized to provide a comprehensive scheme for characterizing receptor-active drugs on an anxiolytic-anticonvulsant/proconvulsant continuum using radioligand binding techniques, as well as a comprehensive program for identifying potential endogenous receptor-active substances. Further evidence is provided here supporting the notion of benzodiazepine recognition site heterogeneity. Classical 1,4-benzodiazepines do not appear to differentiate two populations of benzodiazepine receptors in an equilibrium sense, but appear to do so in a kinetic sense. An apparent physical separation of the two receptor subtypes can be achieved by differential solubilization. The benzodiazepine binding subunit can be identified by photoaffinity labeling with the benzodiazepine agonist (/sup 3/H)flunitrazepan. Conditions for reproducible partial proteolytic mapping of (/sup 3/H)flunitrazepam photoaffinity labeled receptors are established. From these maps, it is concluded that there are probably no major differences in the primary sequence of the benzodiazepine binding subunit in various regions of the rat central nervous system.

  19. Alpha 2 macroglobulin is a maternally-derived immune factor in amphioxus embryos: New evidence for defense roles of maternal immune components in invertebrate chordate.

    PubMed

    Pathirana, Anjalika; Diao, Mingyue; Huang, Shibo; Zuo, Lingling; Liang, Yujun

    2016-03-01

    In fish, a series of maternal derived immune components have been identified in their eggs or embryos at very early stages, which are proposed to provide protections to themselves against pathogenic attacks from hostile environment. The phenomenon of maternal immunity has been also recorded in several invertebrate species, however, so far, very limited information about the maternal immune molecules are available. In this study, it was demonstrated maternal alpha2 macroglobulin (A2m) protein, an important innate immune factor, exists in the fertilized eggs of amphioxus Branchiostoma japonicum, an invertebrate chordate. Maternal mRNA of A2m was also detected in amphioxus embryos at very early developing stages. In addition, it was recorded that the egg lysate prepared from the newly fertilized eggs can inhibit the growth of both Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus in a concentration dependent manner. The bacteriostatic activity can be reduced notably after precipitated A2m with anti-A2m antibody. Thus maternal A2m is partly attributed to the bacteriostatic activity. It was further demonstrated that recombinant A2m can bind to E. coli cells directly. All these points come to a result that A2m is a maternal immune factor existing in eggs of invertebrate chordate, which may be involved in defense their embryos against harmful microbes' attacks. PMID:26796816

  20. Purification of α2-macroglobulin from Cohn Fraction IV by immobilized metal affinity chromatography: A promising method for the better utilization of plasma.

    PubMed

    Huangfu, Chaoji; Ma, Yuyuan; Lv, Maomin; Jia, Junting; Zhao, Xiong; Zhang, Jingang

    2016-07-01

    As an abundant plasma protein, α2-macroglobulin (α2-M) participates widely in physiological and pathological activities including coagulation regulation, antitumor activities, and regulation of cytokines. It also presents a therapeutic potential for radiation injury. A two-step isolation method for the purification of α2-M from Cohn Fraction IV is described. This process includes a salting-out method and immobilized metal affinity chromatography. The LC-ESI-MS/MS analysis and a comparison of the amino acid composition demonstrated that the final product was α2-M. The final protein, with a purity of approximately 95% and a yield of nearly 45%, was obtained from Cohn Fraction IV regardless of plasma haptoglobin type, although all but type 1-1 have previously been considered unfavorable for α2-M preparation. The effects of temperature, pH, and methylamine on α2-M activity were evaluated to avoid activity loss during preparation and preservation. The results suggested that α2-M activity could be readily inactivated at temperatures above 50°C, at pH levels above 9.0 or below 4.0, or in the presence of methylamine. Cohn Fraction IV is usually discarded as a biological waste product in the human serum albumin production process; because the simple process developed in this study is relatively inexpensive, the preparation of α2-M from Cohn Fraction IV may better utilize human plasma, a valuable resource. PMID:27214605

  1. Gels based on anion recognition between triurea receptor and phosphate anion.

    PubMed

    Yang, Cuiling; Wu, Biao; Chen, Yongming; Zhang, Ke

    2015-04-01

    Anion recognition between the triurea receptor and phosphate anion is demonstrated as the cross-linkage to build supramolecular polymer gels for the first time. A novel multi-block copolymer (3) is designed to have functional triurea groups as cross-linking units along the polymer main chain. By virtue of anion coordination between the triurea receptor and phosphate anion with a binding mode of 2:1, supramolecular polymer gels are then prepared based on anion recognition using 3 as the building block. PMID:25694389

  2. Pathogen recognition receptors crosstalk in respiratory syncytial virus sensing: host and cell type perspective

    PubMed Central

    Marr, Nico; Turvey, Stuart E.

    2015-01-01

    Human respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infection in young children, immunocompromized adults and the elderly. The innate immune response plays a pivotal role in host defense against RSV, but whether severe outcomes following RSV infection result from excessive or poor innate immune recognition remains unclear. Recent research suggests a situation in which crosstalk between families of pattern recognition receptors (PRRs) occurs in a cell type-dependent manner. The current challenge to empower novel therapeutic approaches and vaccine development is to confirm the role of the individual receptors in RSV pathogenesis in humans. PMID:24119913

  3. Paired immunoglobulin-like receptors and their MHC class I recognition.

    PubMed

    Takai, Toshiyuki

    2005-08-01

    The immunoglobulin-like receptors provide positive and negative regulation of immune cells upon recognition of various ligands, thus enabling those cells to respond properly to extrinsic stimuli. Murine paired immunoglobulin-like receptor (PIR)-A and PIR-B, a typical receptor pair of the immunoglobulin-like receptor family, are expressed on a wide range of cells in the immune system, such as B cells, mast cells, macrophages and dendritic cells, mostly in a pair-wise fashion. The PIR-A requires the homodimeric Fc receptor common gamma chain for its efficient cell-surface expression and for the delivery of an activation signal. In contrast, PIR-B inhibits receptor-mediated activation signals in vitro upon engagement with other activating-type receptors, such as the antigen receptor on B cells and the high-affinity Fc receptor for immunoglobulin E on mast cells. Recent identification of major histocompatibility complex (MHC) class I molecules as the physiological ligands for PIR has enabled us to attribute various immunological phenotypes observed in PIR-B-deficient mice to the consequences of the absence of a balanced interaction between PIR and MHC class I molecules expressed ubiquitously. Thus, PIR-A and PIR-B constitute a novel and physiologically important MHC class I recognition system. PMID:16011512

  4. [Probable mechanism of recognition of cholinergic ligands by acetylcholine receptors].

    PubMed

    Demushkin, V P; Kotelevtsev, Iu V; Pliashkevich, Iu G; Khramtsov, N V

    1982-01-01

    Dryding's models were used for the conformational analysis of compounds affecting muscarin-specific acetylcholine receptor and nicotin-specific acetylcholine receptor. Ammonium group and ether oxygen (3.6 A apart from the ammonium group) specifically oriented to each other were shown to be necessary structural elements to reveal muscarin-type cholinergic activity. Ammonium group along with carbonyl oxygen or its substituent (5 A distance) are the necessary structural units providing nicotin-type cholinergic activity. The presence of two hydrophobic substituents (one in the ammonium area and the other neighbouring the second active grouping) is the additional factor. The developed principles were justified by the use of a series of synthetic samples. The compounds were obtained likely favouring affinitive modification of acetylcholine receptor (dissociation constants of acetylcholine receptor complexes equalling to 10(-4)--10(-7) M-1). PMID:7070378

  5. A tricatecholic receptor for carbohydrate recognition: synthesis and binding studies.

    PubMed

    Cacciarini, Martina; Cordiano, Elisa; Nativi, Cristina; Roelens, Stefano

    2007-05-11

    A new tripodal receptor bearing three catechol subunits on a benzene platform has been synthesized in four steps from 1,3,5-triethylbenzene and pyrogallol. The binding ability of the tricatecholic receptor was investigated toward several monosaccharides in CDCl3, where multiple equilibria were detected, and compared to that of a previously reported trisureidic receptor of analogous structure. Association constants were measured by 1H NMR titrations, and the corresponding affinities were assessed through the BC50 parameter, a binding descriptor univocally defining the affinity of a host for a guest in multi-equilibrium systems. Results show that the tripodal catecholic receptor binds the octyl glycosides with affinities ranging from 0.87 to 5.2 mM and with a 6-fold selectivity factor for the alpha-mannoside over the beta-glucoside. Although the affinity for glycosides was not appreciably improved with respect to the ureidic receptor, a significant change in selectivity was obtained by the H-bonding group replacement. PMID:17444686

  6. Principles of agonist recognition in Cys-loop receptors

    PubMed Central

    Lynagh, Timothy; Pless, Stephan A.

    2014-01-01

    Cys-loop receptors are ligand-gated ion channels that are activated by a structurally diverse array of neurotransmitters, including acetylcholine, serotonin, glycine, and GABA. After the term “chemoreceptor” emerged over 100 years ago, there was some wait until affinity labeling, molecular cloning, functional studies, and X-ray crystallography experiments identified the extracellular interface of adjacent subunits as the principal site of agonist binding. The question of how subtle differences at and around agonist-binding sites of different Cys-loop receptors can accommodate transmitters as chemically diverse as glycine and serotonin has been subject to intense research over the last three decades. This review outlines the functional diversity and current structural understanding of agonist-binding sites, including those of invertebrate Cys-loop receptors. Together, this provides a framework to understand the atomic determinants involved in how these valuable therapeutic targets recognize and bind their ligands. PMID:24795655

  7. Oxoanion Recognition by Benzene-based Tripodal Pyrrolic Receptors

    SciTech Connect

    Bill, Nathan; Kim, Dae-Sik; Kim, Sung Kuk; Park, Jung Su; Lynch, Vincent M.; Young, Neil J; Hay, Benjamin; Yang, Youjun; Anslyn, Eric; Sessler, Jonathan L.

    2012-01-01

    Two new tripodal receptors based on pyrrole- and dipyrromethane-functionalised derivatives of a sterically geared precursor, 1,3,5-tris(aminomethyl)-2,4,6-triethylbenzene, are reported; these systems, compounds 1 and 2, display high affinity and selectivity for tetrahedral anionic guests, in particular dihydrogen phosphate, pyrophosphate and hydrogen sulphate, in acetonitrile as inferred from isothermal titration calorimetry measurements. Support for the anion-binding ability of these systems comes from theoretical calculations and a single-crystal X-ray diffraction structure of the 2:2 (host:guest) dihydrogen phosphate complex is obtained in the case of the pyrrole-based receptor system, 1. Keywords anion receptors, dihydrogen phosphate, hydrogen sulphate, X-ray structure, theoretical calculations.

  8. Molecular cloning of the mouse gene coding for {alpha}{sub 2}-macroglobulin and targeting of the gene in embryonic stem cells

    SciTech Connect

    Umans, L.; Serneels, L.; Hilliker, C.

    1994-08-01

    The authors have cloned the mouse gene coding for {alpha}{sub 2}-macroglobulin in overlapping {lambda} clones and have analyzed its structure. The gene contains 36 exons, coding for the 4.8-kb cDNA that we cloned previously. Including putative control elements in the 5{prime} flanking region, the gene covers about 45 kb. A region of 3.8 kb, stretching from 835 bases upstream of the cDNA start site to exon 4, including all intervening sequences, was sequenced completely. The analysis demonstrated that the putative promoter region of the mouse A2M gene differed considerably from the known promoter sequences of the human A2M gene and of the rat acute-phas A2M gene. Comparison of the exon-intron structure of all known genes of the A2M family confirmed that the rat acute phase A2M gene is more closely related to the human gene than to the mouse A2M gene. To generate mice with the A2M gene inactivated, an insertion type of construct containing 7.5 kb of genomic DNA of the mouse strain 129/J, encompassing exons 16 to 19, was synthesized. A hygromycin marker gene was embedded in intron 17. After electroporation, 198 hygromycin-resistant ES cell lines were isolated and analyzed by Southern blotting. Five ES cell lines were obtained with one allele of the mouse A2M gene targeted by this insertion construct, demonstrating that the position and the characteristics of the vector served the intended goal.

  9. Differential Levels of Alpha-2-Macroglobulin, Haptoglobin and Sero-Transferrin as Adjunct Markers for TB Diagnosis and Disease Progression in the Malnourished Tribal Population of Melghat, India.

    PubMed

    Bapat, Prachi R; Satav, Ashish R; Husain, Aliabbas A; Shekhawat, Seema D; Kawle, Anuja P; Chu, Justin J; Purohit, Hemant J; Daginawala, Hatim F; Taori, Girdhar M; Kashyap, Rajpal S

    2015-01-01

    Lack of diagnostic capacity has been a crucial barrier preventing an effective response to the challenges of malnutrition and tuberculosis (TB). Point-of-care diagnostic tests for TB in immuno-incompetent, malnourished population are thus needed to ensure rapid and accurate detection. The aim of the study was to identify potential biomarkers specific for TB infection and progression to overt disease in the malnourished population of Melghat. A prospective cohort study was conducted in the year 2009 through 2011 in six villages of the Melghat region. 275 participants consisting of malnourished cases with a) active TB (n = 32), b) latent TB infection (n = 90), c) with no clinical or bacteriological signs of active or latent TB (n = 130) and healthy control subjects (n = 23) were recruited for the study. The proteome changes of the host serum in response to Mycobacterium tuberculosis (M.tb) infection were investigated using one dimensional electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Three most differentially expressed proteins; alpha-2-macroglobulin (A-2-M), sero-transferrin and haptoglobin were identified by MALDI-TOF MS analysis, which were up-regulated in the malnourished patients with active TB and down-regulated in the malnourished patients compared with the healthy controls. Additionally, follow-up studies indicated that the expression of these proteins increased to nearly two folds in patients who developed active disease from latent state. Our preliminary results suggest that A-2-M, sero-transferrin and haptoglobin may be clinically relevant host biomarkers for TB diagnosis and disease progression in the malnourished population. This study provides preliminary framework for an in-depth analysis of the biomarkers in larger well-characterized cohorts. Evaluation of these biomarkers in follow-up cases may further aid in improving TB diagnosis. PMID:26241963

  10. Analysis of serum β-amyloid peptides, α2-macroglobulin, complement factor H, and clusterin levels in APP/PS1 transgenic mice during progression of Alzheimer's disease.

    PubMed

    Wang, Dejiang; Di, Xiangjun; Fu, Lu; Li, Yingnan; Han, Xiao; Wu, Hui; Cai, Linjun; Meng, Xiangyu; Jiang, Chunlai; Kong, Wei; Su, Weiheng

    2016-10-19

    As a progressive age-related neurodegenerative disorder, Alzheimer's disease (AD) is a global health concern. Despite the availability of psychological testing, neuroimaging, genetic testing, and biochemical assays of cerebrospinal fluid, convenient and accurate blood biomarkers for the prediction, diagnosis, and preclinical studies of AD are still lacking. The present study aims to longitudinally evaluate the feasibility of β-amyloid proteins, α2-macroglobulin (α-2M), complement factor H (CFH), and clusterin as blood biomarkers of AD. Using APP/PS1 transgenic and wild-type mice, cognitive impairment and amyloid plaque counts in the brain were evaluated over a range of ages using the Morris water maze test and immunohistochemistry methods, respectively. Serum Aβ40, Aβ42, α-2M, CFH, and clusterin levels were measured by enzyme-linked immunosorbent assay and correlated with progression of AD. APP/PS1 transgenic mice presented progressive AD characteristics at the ages of 3, 6, 9, and 12 months. Serum Aβ42 levels and Aβ42/Aβ40 ratios increased significantly in transgenic 3- and 6-month-old mice compared with controls. Serum CFH levels decreased significantly in 3- and 6-month-old transgenic mice compared with controls. Meanwhile, serum clusterin levels increased significantly in 12-month-old transgenic mice compared with controls. The α-2M level was not significantly different between transgenic and wild-type mice. The APP/PS1 transgenic mouse is a model of familial AD. The present study indicated that the serum Aβ42 level, Aβ42/Aβ40 ratio, and CFH level are potential biomarkers in preclinical and early stages of AD, whereas serum clusterin level is a potential biomarker in the late stage of AD. PMID:27541273

  11. Differential Levels of Alpha-2-Macroglobulin, Haptoglobin and Sero-Transferrin as Adjunct Markers for TB Diagnosis and Disease Progression in the Malnourished Tribal Population of Melghat, India

    PubMed Central

    Bapat, Prachi R.; Satav, Ashish R.; Husain, Aliabbas A.; Shekhawat, Seema D.; Kawle, Anuja P.; Chu, Justin J.; Purohit, Hemant J.; Daginawala, Hatim F.; Taori, Girdhar M.; Kashyap, Rajpal S.

    2015-01-01

    Lack of diagnostic capacity has been a crucial barrier preventing an effective response to the challenges of malnutrition and tuberculosis (TB). Point-of-care diagnostic tests for TB in immuno-incompetent, malnourished population are thus needed to ensure rapid and accurate detection. The aim of the study was to identify potential biomarkers specific for TB infection and progression to overt disease in the malnourished population of Melghat. A prospective cohort study was conducted in the year 2009 through 2011 in six villages of the Melghat region. 275 participants consisting of malnourished cases with a) active TB (n = 32), b) latent TB infection (n = 90), c) with no clinical or bacteriological signs of active or latent TB (n = 130) and healthy control subjects (n = 23) were recruited for the study. The proteome changes of the host serum in response to Mycobacterium tuberculosis (M.tb) infection were investigated using one dimensional electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Three most differentially expressed proteins; alpha-2-macroglobulin (A-2-M), sero-transferrin and haptoglobin were identified by MALDI-TOF MS analysis, which were up-regulated in the malnourished patients with active TB and down-regulated in the malnourished patients compared with the healthy controls. Additionally, follow-up studies indicated that the expression of these proteins increased to nearly two folds in patients who developed active disease from latent state. Our preliminary results suggest that A-2-M, sero-transferrin and haptoglobin may be clinically relevant host biomarkers for TB diagnosis and disease progression in the malnourished population. This study provides preliminary framework for an in-depth analysis of the biomarkers in larger well-characterized cohorts. Evaluation of these biomarkers in follow-up cases may further aid in improving TB diagnosis. PMID:26241963

  12. Structural basis for collagen recognition by the immune receptor OSCAR

    PubMed Central

    Zhou, Long; Hinerman, Jennifer M.; Blaszczyk, Michal; Miller, Jeanette L. C.; Conrady, Deborah G.; Barrow, Alexander D.; Chirgadze, Dimitri Y.; Bihan, Dominique; Farndale, Richard W.

    2016-01-01

    The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 μM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2. PMID:26552697

  13. Structural basis for collagen recognition by the immune receptor OSCAR.

    PubMed

    Zhou, Long; Hinerman, Jennifer M; Blaszczyk, Michal; Miller, Jeanette L C; Conrady, Deborah G; Barrow, Alexander D; Chirgadze, Dimitri Y; Bihan, Dominique; Farndale, Richard W; Herr, Andrew B

    2016-02-01

    The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 μM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2. PMID:26552697

  14. Polymorphisms in pattern recognition receptors and their relationship to infectious disease susceptibility in pigs

    PubMed Central

    2011-01-01

    Background Pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), are censoring receptors for molecules derived from bacteria, viruses, and fungi. The PRR system is a prerequisite for proper responses to pathogens, for example by cytokine production, resulting in pathogen eradication. Many cases of polymorphisms in PRR genes affecting the immune response and disease susceptibility are known in humans and mice. Methods We surveyed polymorphisms in pig genes encoding PRRs and investigated the relationship between some of the detected polymorphisms and molecular function or disease onset. Results Nonsynonymous polymorphisms abounded in pig TLR genes, particularly in the region corresponding to the ectodomains of TLRs expressed on the cell surface. Intracellular TLRs such as TLR3, TLR7, and TLR8, and other intracellular PRRs, such as the peptidoglycan receptor NOD2 and viral RNA receptors RIG-I and MDA5, also possessed nonsynonymous polymorphisms. Several of the polymorphisms influenced molecular functions such as ligand recognition. Polymorphisms in the PRR genes may be related to disease susceptibility in pigs: pigs with a particular allele of TLR2 showed an increased tendency to contract pneumonia. Conclusions We propose the possibility of pig breeding aimed at disease resistance by the selection of PRR gene alleles that affect pathogen recognition. PMID:21645307

  15. Recognition and Accommodation at the Androgen Receptor Coactivator Binding Interface

    PubMed Central

    Hur, Eugene; Pfaff, Samuel J; Payne, E. Sturgis; Grøn, Hanne; Buehrer, Benjamin M

    2004-01-01

    Prostate cancer is a leading killer of men in the industrialized world. Underlying this disease is the aberrant action of the androgen receptor (AR). AR is distinguished from other nuclear receptors in that after hormone binding, it preferentially responds to a specialized set of coactivators bearing aromatic-rich motifs, while responding poorly to coactivators bearing the leucine-rich “NR box” motifs favored by other nuclear receptors. Under normal conditions, interactions with these AR-specific coactivators through aromatic-rich motifs underlie targeted gene transcription. However, during prostate cancer, abnormal association with such coactivators, as well as with coactivators containing canonical leucine-rich motifs, promotes disease progression. To understand the paradox of this unusual selectivity, we have derived a complete set of peptide motifs that interact with AR using phage display. Binding affinities were measured for a selected set of these peptides and their interactions with AR determined by X-ray crystallography. Structures of AR in complex with FxxLF, LxxLL, FxxLW, WxxLF, WxxVW, FxxFF, and FxxYF motifs reveal a changing surface of the AR coactivator binding interface that permits accommodation of both AR-specific aromatic-rich motifs and canonical leucine-rich motifs. Induced fit provides perfect mating of the motifs representing the known family of AR coactivators and suggests a framework for the design of AR coactivator antagonists. PMID:15328534

  16. Role of Toll-Like Receptor 13 in Innate Immune Recognition of Group B Streptococci

    PubMed Central

    Signorino, Giacomo; Mohammadi, Nastaran; Patanè, Francesco; Buscetta, Marco; Venza, Mario; Venza, Isabella; Mancuso, Giuseppe; Midiri, Angelina; Alexopoulou, Lena; Teti, Giuseppe; Beninati, Concetta

    2014-01-01

    Murine Toll-like receptor 13 (TLR13), an endosomal receptor that is not present in humans, is activated by an unmethylated motif present in the large ribosomal subunit of bacterial RNA (23S rRNA). Little is known, however, of the impact of TLR13 on antibacterial host defenses. Here we examined the role of this receptor in the context of infection induced by the model pathogen group B streptococcus (GBS). To this end, we used bacterial strains masked from TLR13 recognition by virtue of constitutive expression of the ErmC methyltransferase, which results in dimethylation of the 23S rRNA motif at a critical adenine residue. We found that TLR13-mediated rRNA recognition was required for optimal induction of tumor necrosis factor alpha and nitrous oxide in dendritic cell and macrophage cultures stimulated with heat-killed bacteria or purified bacterial RNA. However, TLR13-dependent recognition was redundant when live bacteria were used as a stimulus. Moreover, masking bacterial rRNA from TLR13 recognition did not increase the ability of GBS to avoid host defenses and replicate in vivo. In contrast, increased susceptibility to infection was observed under conditions in which signaling by all endosomal TLRs was abolished, i.e., in mice with a loss-of-function mutation in the chaperone protein UNC93B1. Our data lend support to the conclusion that TLR13 participates in GBS recognition, although blockade of the function of this receptor can be compensated for by other endosomal TLRs. Lack of selective pressure by bacterial infections might explain the evolutionary loss of TLR13 in humans. However, further studies using different bacterial species are needed to prove this hypothesis. PMID:25225249

  17. Toxicities of chimeric antigen receptor T cells: recognition and management.

    PubMed

    Brudno, Jennifer N; Kochenderfer, James N

    2016-06-30

    Chimeric antigen receptor (CAR) T cells can produce durable remissions in hematologic malignancies that are not responsive to standard therapies. Yet the use of CAR T cells is limited by potentially severe toxicities. Early case reports of unexpected organ damage and deaths following CAR T-cell therapy first highlighted the possible dangers of this new treatment. CAR T cells can potentially damage normal tissues by specifically targeting a tumor-associated antigen that is also expressed on those tissues. Cytokine release syndrome (CRS), a systemic inflammatory response caused by cytokines released by infused CAR T cells can lead to widespread reversible organ dysfunction. CRS is the most common type of toxicity caused by CAR T cells. Neurologic toxicity due to CAR T cells might in some cases have a different pathophysiology than CRS and requires different management. Aggressive supportive care is necessary for all patients experiencing CAR T-cell toxicities, with early intervention for hypotension and treatment of concurrent infections being essential. Interleukin-6 receptor blockade with tocilizumab remains the mainstay pharmacologic therapy for CRS, though indications for administration vary among centers. Corticosteroids should be reserved for neurologic toxicities and CRS not responsive to tocilizumab. Pharmacologic management is complicated by the risk of immunosuppressive therapy abrogating the antimalignancy activity of the CAR T cells. This review describes the toxicities caused by CAR T cells and reviews the published approaches used to manage toxicities. We present guidelines for treating patients experiencing CRS and other adverse events following CAR T-cell therapy. PMID:27207799

  18. Labeled Protein Recognition at a Membrane Bilayer Interface by Embedded Synthetic Receptors

    PubMed Central

    2015-01-01

    Self-folding deep cavitands embedded in a supported lipid bilayer are capable of recognizing suitably labeled proteins at the bilayer interface. The addition of a choline derived binding “handle” to a number of different proteins allows their selective noncovalent recognition, with association constants on the order of 105 M–1. The proteins are displayed at the water:bilayer interface, and a single binding handle allows recognition of the large, charged protein by a small molecule synthetic receptor via complementary shape and charge interactions. PMID:25130415

  19. Gibberellin Receptor GID1: Gibberellin Recognition and Molecular Evolution

    NASA Astrophysics Data System (ADS)

    Kato, Hiroaki; Sato, Tomomi; Ueguchi-Tanaka, Miyako

    Gibberellins (GAs) are phytohormones essential for many developmental processes in plants. We analyzed the crystal structure of a nuclear GA receptor, GIBBERELLIN INSENSITIVE DWARF 1 (GID1) from Oryza sativa. As it was proposed from the sequence similarity, the overall structure of GID1 shows an α/β-hydrolase fold similar to that of the hormone-sensitive lipases (HSLs) except for an amino-terminal lid. The GA-binding site corresponds to the substrate-binding site of HSLs. Almost residues assigned for GA binding showed very little or no activity when they were replaced with Ala. The substitution of the residues corresponding to those of the lycophyte GID1s caused an increase in the binding affinity for GA34, a 2β-hydroxylated GA4. These findings indicate that GID1 originated from HSL and was tinkered to have the specificity for bioactive GAs in the course of plant evolution.

  20. Comparative binding of biotinylated neurotrophins to alpha(2)-macroglobulin family of proteins: relationship between cytokine-binding and neuro-modulatory activities of the macroglobulins.

    PubMed

    Skornicka, Erin L; Shi, Xiaoqing; Koo, Peter H

    2002-02-01

    Human alpha(2)-macroglobulin (alpha(2)M), pregnancy zone protein (PZP), rat alpha(1)M and acute-phase rat alpha(2)M belong to the alpha(2)M gene family of proteins, which can react covalently with nucleophilic monoamines to yield monoamine-activated (MA) macroglobulins. The MA forms of human alpha(2)M, PZP and rat alpha(2)M have been demonstrated previously to inhibit various neurotrophin-promoted neuronal activities, whereas MA-alpha(1)M is neurostimulatory and all native macroglobulins are generally inactive. The mechanism of neuromodulation is unknown, but it has been postulated that MA macroglobulins might inhibit neurons via their binding and sequestration of neurotrophins. This study employed a novel biotinylation-Western blot technique to compare the neurotrophin-binding properties of the four macroglobulins, and to correlate their binding activities with their known neuro-modulatory activities. In comparison with their respective native counterparts, human and rat MA-alpha(2)M bound slightly more NGF, but significantly less BDNF or NT-3. Native human alpha(2)M and PZP in general have no neuro-modulatory activity, but native PZP bound significantly more NGF, BDNF or NT-3 than either native alpha(2)M or MA-alpha(2)M, which is neuro-inhibitory. It is known that MA-PZP is neuro-inhibitory, but it fails to bind more NGF, BDNF, or NT-3 than native PZP. MA-alpha(1)M is the only macroglobulin known to stimulate NGF-promoted neurite outgrowth, but it bound NGF with similar affinities as native alpha(1)M and rat alpha(2)M; in addition, it bound significantly less BDNF or NT-3 than native alpha(1)M. All the bindings were non-covalent and appeared specific. In conclusion, PZP and rat macroglobulins are versatile carriers of neurotrophins with diverse binding capacities, and the neurotrophin-binding property does not appear to mediate the neuro-modulatory activity of these human and rat macroglobulins. PMID:11813239

  1. Non-classical amine recognition evolved in a large clade of olfactory receptors

    PubMed Central

    Li, Qian; Tachie-Baffour, Yaw; Liu, Zhikai; Baldwin, Maude W; Kruse, Andrew C; Liberles, Stephen D

    2015-01-01

    Biogenic amines are important signaling molecules, and the structural basis for their recognition by G Protein-Coupled Receptors (GPCRs) is well understood. Amines are also potent odors, with some activating olfactory trace amine-associated receptors (TAARs). Here, we report that teleost TAARs evolved a new way to recognize amines in a non-classical orientation. Chemical screens de-orphaned eleven zebrafish TAARs, with agonists including serotonin, histamine, tryptamine, 2-phenylethylamine, putrescine, and agmatine. Receptors from different clades contact ligands through aspartates on transmembrane α-helices III (canonical Asp3.32) or V (non-canonical Asp5.42), and diamine receptors contain both aspartates. Non-classical monoamine recognition evolved in two steps: an ancestral TAAR acquired Asp5.42, gaining diamine sensitivity, and subsequently lost Asp3.32. Through this transformation, the fish olfactory system dramatically expanded its capacity to detect amines, ecologically significant aquatic odors. The evolution of a second, alternative solution for amine detection by olfactory receptors highlights the tremendous structural versatility intrinsic to GPCRs. DOI: http://dx.doi.org/10.7554/eLife.10441.001 PMID:26519734

  2. Non-classical amine recognition evolved in a large clade of olfactory receptors.

    PubMed

    Li, Qian; Tachie-Baffour, Yaw; Liu, Zhikai; Baldwin, Maude W; Kruse, Andrew C; Liberles, Stephen D

    2015-01-01

    Biogenic amines are important signaling molecules, and the structural basis for their recognition by G Protein-Coupled Receptors (GPCRs) is well understood. Amines are also potent odors, with some activating olfactory trace amine-associated receptors (TAARs). Here, we report that teleost TAARs evolved a new way to recognize amines in a non-classical orientation. Chemical screens de-orphaned eleven zebrafish TAARs, with agonists including serotonin, histamine, tryptamine, 2-phenylethylamine, putrescine, and agmatine. Receptors from different clades contact ligands through aspartates on transmembrane α-helices III (canonical Asp(3.32)) or V (non-canonical Asp(5.42)), and diamine receptors contain both aspartates. Non-classical monoamine recognition evolved in two steps: an ancestral TAAR acquired Asp(5.42), gaining diamine sensitivity, and subsequently lost Asp(3.32). Through this transformation, the fish olfactory system dramatically expanded its capacity to detect amines, ecologically significant aquatic odors. The evolution of a second, alternative solution for amine detection by olfactory receptors highlights the tremendous structural versatility intrinsic to GPCRs. PMID:26519734

  3. International Union of Basic and Clinical Pharmacology. XCVI. Pattern Recognition Receptors in Health and Disease

    PubMed Central

    Orr, Selinda; Ferguson, Brian; Symmons, Martyn F.; Boyle, Joseph P.; Monie, Tom P.

    2015-01-01

    Since the discovery of Toll, in the fruit fly Drosophila melanogaster, as the first described pattern recognition receptor (PRR) in 1996, many families of these receptors have been discovered and characterized. PRRs play critically important roles in pathogen recognition to initiate innate immune responses that ultimately link to the generation of adaptive immunity. Activation of PRRs leads to the induction of immune and inflammatory genes, including proinflammatory cytokines and chemokines. It is increasingly clear that many PRRs are linked to a range of inflammatory, infectious, immune, and chronic degenerative diseases. Several drugs to modulate PRR activity are already in clinical trials and many more are likely to appear in the near future. Here, we review the different families of mammalian PRRs, the ligands they recognize, the mechanisms of activation, their role in disease, and the potential of targeting these proteins to develop the anti-inflammatory therapeutics of the future. PMID:25829385

  4. Bacterial and fungal pattern recognition receptors in homologous innate signaling pathways of insects and mammals.

    PubMed

    Stokes, Bethany A; Yadav, Shruti; Shokal, Upasana; Smith, L C; Eleftherianos, Ioannis

    2015-01-01

    In response to bacterial and fungal infections in insects and mammals, distinct families of innate immune pattern recognition receptors (PRRs) initiate highly complex intracellular signaling cascades. Those cascades induce a variety of immune functions that restrain the spread of microbes in the host. Insect and mammalian innate immune receptors include molecules that recognize conserved microbial molecular patterns. Innate immune recognition leads to the recruitment of adaptor molecules forming multi-protein complexes that include kinases, transcription factors, and other regulatory molecules. Innate immune signaling cascades induce the expression of genes encoding antimicrobial peptides and other key factors that mount and regulate the immune response against microbial challenge. In this review, we summarize our current understanding of the bacterial and fungal PRRs for homologous innate signaling pathways of insects and mammals in an effort to provide a framework for future studies. PMID:25674081

  5. Serotonin 2a Receptor and Serotonin 1a Receptor Interact Within the Medial Prefrontal Cortex During Recognition Memory in Mice.

    PubMed

    Morici, Juan F; Ciccia, Lucia; Malleret, Gaël; Gingrich, Jay A; Bekinschtein, Pedro; Weisstaub, Noelia V

    2015-01-01

    Episodic memory, can be defined as the memory for unique events. The serotonergic system one of the main neuromodulatory systems in the brain appears to play a role in it. The serotonin 2a receptor (5-HT2aR) one of the principal post-synaptic receptors for 5-HT in the brain, is involved in neuropsychiatric and neurological disorders associated with memory deficits. Recognition memory can be defined as the ability to recognize if a particular event or item was previously encountered and is thus considered, under certain conditions, a form of episodic memory. As human data suggest that a constitutively decrease of 5-HT2A signaling might affect episodic memory performance we decided to compare the performance of mice with disrupted 5-HT2aR signaling (htr2a (-/-)) with wild type (htr2a (+/+)) littermates in different recognition memory and working memory tasks that differed in the level of proactive interference. We found that ablation of 5-HT2aR signaling throughout development produces a deficit in tasks that cannot be solved by single item strategy suggesting that 5-HT2aR signaling is involved in interference resolution. We also found that in the absence of 5-HT2aR signaling serotonin has a deleterious effect on recognition memory retrieval through the activation of 5-HT1aR in the medial prefrontal cortex. PMID:26779016

  6. Structural basis for gibberellin recognition by its receptor GID1.

    PubMed

    Shimada, Asako; Ueguchi-Tanaka, Miyako; Nakatsu, Toru; Nakajima, Masatoshi; Naoe, Youichi; Ohmiya, Hiroko; Kato, Hiroaki; Matsuoka, Makoto

    2008-11-27

    Gibberellins (GAs) are phytohormones essential for many developmental processes in plants. A nuclear GA receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1), has a primary structure similar to that of the hormone-sensitive lipases (HSLs). Here we analyse the crystal structure of Oryza sativa GID1 (OsGID1) bound with GA(4) and GA(3) at 1.9 A resolution. The overall structure of both complexes shows an alpha/beta-hydrolase fold similar to that of HSLs except for an amino-terminal lid. The GA-binding pocket corresponds to the substrate-binding site of HSLs. On the basis of the OsGID1 structure, we mutagenized important residues for GA binding and examined their binding activities. Almost all of them showed very little or no activity, confirming that the residues revealed by structural analysis are important for GA binding. The replacement of Ile 133 with Leu or Val-residues corresponding to those of the lycophyte Selaginella moellendorffii GID1s-caused an increase in the binding affinity for GA(34), a 2beta-hydroxylated GA(4). These observations indicate that GID1 originated from HSL and was further modified to have higher affinity and more strict selectivity for bioactive GAs by adapting the amino acids involved in GA binding in the course of plant evolution. PMID:19037316

  7. Structural basis of transcobalamin recognition by human CD320 receptor

    PubMed Central

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S.; Zenobi, Renato; Locher, Kaspar P.

    2016-01-01

    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway. PMID:27411955

  8. Structural basis of transcobalamin recognition by human CD320 receptor.

    PubMed

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S; Zenobi, Renato; Locher, Kaspar P

    2016-01-01

    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway. PMID:27411955

  9. Structural basis of transcobalamin recognition by human CD320 receptor

    NASA Astrophysics Data System (ADS)

    Alam, Amer; Woo, Jae-Sung; Schmitz, Jennifer; Prinz, Bernadette; Root, Katharina; Chen, Fan; Bloch, Joël S.; Zenobi, Renato; Locher, Kaspar P.

    2016-07-01

    Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway.

  10. On the logic of restrictive recognition of peptide by the T-cell antigen receptor

    PubMed Central

    2011-01-01

    This essay provides an analysis of the inadequacy of the current view of restrictive recognition of peptide by the T-cell antigen receptor. A competing model is developed, and the experimental evidence for the prevailing model is reinterpreted in the new framework. The goal is to contrast the two models with respect to their consistency, coverage of the data, explanatory power, and predictability. PMID:20931295

  11. Common polymorphism in the oxytocin receptor gene (OXTR) is associated with human social recognition skills

    PubMed Central

    Skuse, David H.; Lori, Adriana; Cubells, Joseph F.; Lee, Irene; Conneely, Karen N.; Puura, Kaija; Lehtimäki, Terho; Binder, Elisabeth B.; Young, Larry J.

    2014-01-01

    The neuropeptides oxytocin and vasopressin are evolutionarily conserved regulators of social perception and behavior. Evidence is building that they are critically involved in the development of social recognition skills within rodent species, primates, and humans. We investigated whether common polymorphisms in the genes encoding the oxytocin and vasopressin 1a receptors influence social memory for faces. Our sample comprised 198 families, from the United Kingdom and Finland, in whom a single child had been diagnosed with high-functioning autism. Previous research has shown that impaired social perception, characteristic of autism, extends to the first-degree relatives of autistic individuals, implying heritable risk. Assessments of face recognition memory, discrimination of facial emotions, and direction of gaze detection were standardized for age (7–60 y) and sex. A common SNP in the oxytocin receptor (rs237887) was strongly associated with recognition memory in combined probands, parents, and siblings after correction for multiple comparisons. Homozygotes for the ancestral A allele had impairments in the range −0.6 to −1.15 SD scores, irrespective of their diagnostic status. Our findings imply that a critical role for the oxytocin system in social recognition has been conserved across perceptual boundaries through evolution, from olfaction in rodents to visual memory in humans. PMID:24367110

  12. NMDA Receptor Antagonist Ketamine Distorts Object Recognition by Reducing Feedback to Early Visual Cortex.

    PubMed

    van Loon, Anouk M; Fahrenfort, Johannes J; van der Velde, Bauke; Lirk, Philipp B; Vulink, Nienke C C; Hollmann, Markus W; Scholte, H Steven; Lamme, Victor A F

    2016-05-01

    It is a well-established fact that top-down processes influence neural representations in lower-level visual areas. Electrophysiological recordings in monkeys as well as theoretical models suggest that these top-down processes depend on NMDA receptor functioning. However, this underlying neural mechanism has not been tested in humans. We used fMRI multivoxel pattern analysis to compare the neural representations of ambiguous Mooney images before and after they were recognized with their unambiguous grayscale version. Additionally, we administered ketamine, an NMDA receptor antagonist, to interfere with this process. Our results demonstrate that after recognition, the pattern of brain activation elicited by a Mooney image is more similar to that of its easily recognizable grayscale version than to the pattern evoked by the identical Mooney image before recognition. Moreover, recognition of Mooney images decreased mean response; however, neural representations of separate images became more dissimilar. So from the neural perspective, unrecognizable Mooney images all "look the same", whereas recognized Mooneys look different. We observed these effects in posterior fusiform part of lateral occipital cortex and in early visual cortex. Ketamine distorted these effects of recognition, but in early visual cortex only. This suggests that top-down processes from higher- to lower-level visual areas might operate via an NMDA pathway. PMID:25662715

  13. Two Redundant Receptor-Like Cytoplasmic Kinases Function Downstream of Pattern Recognition Receptors to Regulate Activation of SA Biosynthesis.

    PubMed

    Kong, Qing; Sun, Tongjun; Qu, Na; Ma, Junling; Li, Meng; Cheng, Yu-Ti; Zhang, Qian; Wu, Di; Zhang, Zhibin; Zhang, Yuelin

    2016-06-01

    Salicylic acid (SA) serves as a critical signaling molecule in plant defense. Two transcription factors, SARD1 and CBP60g, control SA biosynthesis through regulating pathogen-induced expression of Isochorismate Synthase1, which encodes a key enzyme for SA biosynthesis. Here, we report that Pattern-Triggered Immunity Compromised Receptor-like Cytoplasmic Kinase1 (PCRK1) and PCRK2 function as key regulators of SA biosynthesis. In the pcrk1 pcrk2 double mutant, pathogen-induced expression of SARD1, CBP60g, and ICS1 is greatly reduced. The pcrk1 pcrk2 double mutant, but neither of the single mutants, exhibits reduced accumulation of SA and enhanced disease susceptibility to bacterial pathogens. Both PCRK1 and PCRK2 interact with the pattern recognition receptor FLS2, and treatment with pathogen-associated molecular patterns leads to rapid phosphorylation of PCRK2. Our data suggest that PCRK1 and PCRK2 function downstream of pattern recognition receptor in a signal relay leading to the activation of SA biosynthesis. PMID:27208222

  14. An Overview of Pathogen Recognition Receptors for Innate Immunity in Dental Pulp

    PubMed Central

    Jang, Ji-Hyun; Shin, Hee Woong; Lee, Jung Min; Lee, Hyeon-Woo; Kim, Eun-Cheol; Park, Sang Hyuk

    2015-01-01

    Pathogen recognition receptors (PRRs) are a class of germ line-encoded receptors that recognize pathogen-associated molecular patterns (PAMPs). The activation of PRRs is crucial for the initiation of innate immunity, which plays a key role in first-line defense until more specific adaptive immunity is developed. PRRs differ in the signaling cascades and host responses activated by their engagement and in their tissue distribution. Currently identified PRR families are the Toll-like receptors (TLRs), the C-type lectin receptors (CLRs), the nucleotide-binding oligomerization domain-like receptors (NLRs), the retinoic acid-inducible gene-I-like receptors (RLRs), and the AIM2-like receptor (ALR). The environment of the dental pulp is substantially different from that of other tissues of the body. Dental pulp resides in a low compliance root canal system that limits the expansion of pulpal tissues during inflammatory processes. An understanding of the PRRs in dental pulp is important for immunomodulation and hence for developing therapeutic targets in the field of endodontics. Here we comprehensively review recent finding on the PRRs and the mechanisms by which innate immunity is activated. We focus on the PRRs expressed on dental pulp and periapical tissues and their role in dental pulp inflammation. PMID:26576076

  15. Dopamine D1 receptor stimulation modulates the formation and retrieval of novel object recognition memory: Role of the prelimbic cortex

    PubMed Central

    Pezze, Marie A.; Marshall, Hayley J.; Fone, Kevin C.F.; Cassaday, Helen J.

    2015-01-01

    Previous studies have shown that dopamine D1 receptor antagonists impair novel object recognition memory but the effects of dopamine D1 receptor stimulation remain to be determined. This study investigated the effects of the selective dopamine D1 receptor agonist SKF81297 on acquisition and retrieval in the novel object recognition task in male Wistar rats. SKF81297 (0.4 and 0.8 mg/kg s.c.) given 15 min before the sampling phase impaired novel object recognition evaluated 10 min or 24 h later. The same treatments also reduced novel object recognition memory tested 24 h after the sampling phase and when given 15 min before the choice session. These data indicate that D1 receptor stimulation modulates both the encoding and retrieval of object recognition memory. Microinfusion of SKF81297 (0.025 or 0.05 μg/side) into the prelimbic sub-region of the medial prefrontal cortex (mPFC) in this case 10 min before the sampling phase also impaired novel object recognition memory, suggesting that the mPFC is one important site mediating the effects of D1 receptor stimulation on visual recognition memory. PMID:26277743

  16. Impact of Pattern Recognition Receptors on the Prognosis of Breast Cancer Patients Undergoing Adjuvant Chemotherapy.

    PubMed

    Vacchelli, Erika; Enot, David P; Pietrocola, Federico; Zitvogel, Laurence; Kroemer, Guido

    2016-06-01

    Pattern recognition receptors allow the innate immune system to perceive the presence of microbial products and to launch the first steps of the defense response. Some pattern recognition receptors also sense endogenous ligands that are released from uninfected dying cells, thereby activating immune responses against dead-cell antigens. This applies to toll-like receptors 3 and 4 (TLR3, TLR4), which sense double-stranded RNA and high-mobility group protein B1 (HMGB1), respectively, as well as to formyl peptide receptor-1 (FPR1), which interacts with Annexin A1 (ANXA1) from dead cells. Breast cancer patients who bear loss-of-function alleles in TLR3, TLR4, and FPR1 exhibit a reduced metastasis-free and overall survival after treatment with anthracycline-based adjuvant chemotherapy. These genetic defects are epistatic with respect to each other, suggesting that they act on the same pathway, linking chemotherapy to a therapeutically relevant anticancer immune response. Loss-of-function alleles in TLR4 and FPR1 also affect the prognosis of colorectal cancer patients treated with oxaliplatin-based chemotherapy. Altogether, these results support the idea that conventional anticancer treatments rely on stimulation of anticancer immune responses to become fully efficient. Cancer Res; 76(11); 3122-6. ©2016 AACR. PMID:27197163

  17. An Update on PYRIN Domain-Containing Pattern Recognition Receptors: From Immunity to Pathology

    PubMed Central

    Ratsimandresy, Rojo A.; Dorfleutner, Andrea; Stehlik, Christian

    2013-01-01

    Cytosolic pattern recognition receptors (PRRs) sense a wide range of endogenous danger-associated molecular patterns as well as exogenous pathogen-associated molecular patterns. In particular, Nod-like receptors containing a pyrin domain (PYD), called NLRPs, and AIM2-like receptors (ALRs) have been shown to play a critical role in host defense by facilitating clearance of pathogens and maintaining a healthy gut microflora. NLRPs and ALRs both encode a PYD, which is crucial for relaying signals that result in an efficient innate immune response through activation of several key innate immune signaling pathways. However, mutations in these PRRs have been linked to the development of auto-inflammatory and autoimmune diseases. In addition, they have been implicated in metabolic diseases. In this review, we summarize the function of PYD-containing NLRPs and ALRs and address their contribution to innate immunity, host defense, and immune-linked diseases. PMID:24367371

  18. Involvement of hippocampal NMDA receptors in retrieval of spontaneous object recognition memory in rats.

    PubMed

    Iwamura, Etsushi; Yamada, Kazuo; Ichitani, Yukio

    2016-07-01

    The involvement of hippocampal N-methyl-d-aspartate (NMDA) receptors in the retrieval process of spontaneous object recognition memory was investigated. The spontaneous object recognition test consisted of three phases. In the sample phase, rats were exposed to two identical objects several (2-5) times in the arena. After the sample phase, various lengths of delay intervals (24h-6 weeks) were inserted (delay phase). In the test phase in which both the familiar and the novel objects were placed in the arena, rats' novel object exploration behavior under the hippocampal treatment of NMDA receptor antagonist, AP5, or vehicle was observed. With 5 exposure sessions in the sample phase (experiment 1), AP5 treatment in the test phase significantly decreased discrimination ratio when the delay was 3 weeks but not when it was one week. On the other hand, with 2 exposure sessions in the sample phase (experiment 2) in which even vehicle-injected control animals could not discriminate the novel object from the familiar one with a 3 week delay, AP5 treatment significantly decreased discrimination ratio when the delay was one week, but not when it was 24h. Additional experiment (experiment 3) showed that the hippocampal treatment of an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, NBQX, decreased discrimination ratio with all delay intervals tested (24h-3 weeks). Results suggest that hippocampal NMDA receptors play an important role in the retrieval of spontaneous object recognition memory especially when the memory trace weakens. PMID:27036649

  19. Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor.

    PubMed

    Booe, Jason M; Walker, Christopher S; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A; Warner, Margaret L; Bill, Roslyn M; Harris, Paul W; Brimble, Margaret A; Poyner, David R; Hay, Debbie L; Pioszak, Augen A

    2015-06-18

    Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes. PMID:25982113

  20. Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor

    PubMed Central

    Booe, Jason M.; Walker, Christopher S.; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A.; Warner, Margaret L.; Bill, Roslyn M.; Harris, Paul W.; Brimble, Margaret A.; Poyner, David R.; Hay, Debbie L.; Pioszak, Augen A.

    2015-01-01

    Summary Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes. PMID:25982113

  1. Native Serotonin Membrane Receptors Recognize 5-Hydroxytryptophan-Functionalized Substrates: Enabling Small-Molecule Recognition

    PubMed Central

    2010-01-01

    Recognition of small diffusible molecules by large biomolecules is ubiquitous in biology. To investigate these interactions, it is important to be able to immobilize small ligands on substrates; however, preserving recognition by biomolecule-binding partners under these circumstances is challenging. We have developed methods to modify substrates with serotonin, a small-molecule neurotransmitter important in brain function and psychiatric disorders. To mimic soluble serotonin, we attached its amino acid precursor, 5-hydroxytryptophan, via the ancillary carboxyl group to oligo(ethylene glycol)-terminated alkanethiols self-assembled on gold. Anti-5-hydroxytryptophan antibodies recognize these substrates, demonstrating bioavailability. Interestingly, 5-hydroxytryptophan-functionalized surfaces capture membrane-associated serotonin receptors enantiospecifically. By contrast, surfaces functionalized with serotonin itself fail to bind serotonin receptors. We infer that recognition by biomolecules evolved to distinguish small-molecule ligands in solution requires tethering of the latter via ectopic moieties. Membrane proteins, which are notoriously difficult to isolate, or other binding partners can be captured for identification, mapping, expression, and other purposes using this generalizable approach. PMID:22778841

  2. A Temporally Distinct Role for Group I and Group II Metabotropic Glutamate Receptors in Object Recognition Memory

    ERIC Educational Resources Information Center

    Brown, Malcolm Watson; Warburton, Elizabeth Clea; Barker, Gareth Robert Isaac; Bashir, Zafar Iqbal

    2006-01-01

    Recognition memory, involving the ability to discriminate between a novel and familiar object, depends on the integrity of the perirhinal cortex (PRH). Glutamate, the main excitatory neurotransmitter in the cortex, is essential for many types of memory processes. Of the subtypes of glutamate receptor, metabotropic receptors (mGluRs) have received…

  3. Metal Oxide Nanosensors Using Polymeric Membranes, Enzymes and Antibody Receptors as Ion and Molecular Recognition Elements

    PubMed Central

    Willander, Magnus; Khun, Kimleang; Ibupoto, Zafar Hussain

    2014-01-01

    The concept of recognition and biofunctionality has attracted increasing interest in the fields of chemistry and material sciences. Advances in the field of nanotechnology for the synthesis of desired metal oxide nanostructures have provided a solid platform for the integration of nanoelectronic devices. These nanoelectronics-based devices have the ability to recognize molecular species of living organisms, and they have created the possibility for advanced chemical sensing functionalities with low limits of detection in the nanomolar range. In this review, various metal oxides, such as ZnO-, CuO-, and NiO-based nanosensors, are described using different methods (receptors) of functionalization for molecular and ion recognition. These functionalized metal oxide surfaces with a specific receptor involve either a complex formation between the receptor and the analyte or an electrostatic interaction during the chemical sensing of analytes. Metal oxide nanostructures are considered revolutionary nanomaterials that have a specific surface for the immobilization of biomolecules with much needed orientation, good conformation and enhanced biological activity which further improve the sensing properties of nanosensors. Metal oxide nanostructures are associated with certain unique optical, electrical and molecular characteristics in addition to unique functionalities and surface charge features which shows attractive platforms for interfacing biorecognition elements with effective transducing properties for signal amplification. There is a great opportunity in the near future for metal oxide nanostructure-based miniaturization and the development of engineering sensor devices. PMID:24841244

  4. The peroxisomal receptor Pex19p forms a helical mPTS recognition domain

    PubMed Central

    Schueller, Nicole; Holton, Simon J; Fodor, Krisztian; Milewski, Morlin; Konarev, Petr; Stanley, Will A; Wolf, Janina; Erdmann, Ralf; Schliebs, Wolfgang; Song, Young-Hwa; Wilmanns, Matthias

    2010-01-01

    The protein Pex19p functions as a receptor and chaperone of peroxisomal membrane proteins (PMPs). The crystal structure of the folded C-terminal part of the receptor reveals a globular domain that displays a bundle of three long helices in an antiparallel arrangement. Complementary functional experiments, using a range of truncated Pex19p constructs, show that the structured α-helical domain binds PMP-targeting signal (mPTS) sequences with about 10 μM affinity. Removal of a conserved N-terminal helical segment from the mPTS recognition domain impairs the ability for mPTS binding, indicating that it forms part of the mPTS-binding site. Pex19p variants with mutations in the same sequence segment abolish correct cargo import. Our data indicate a divided N-terminal and C-terminal structural arrangement in Pex19p, which is reminiscent of a similar division in the Pex5p receptor, to allow separation of cargo-targeting signal recognition and additional functions. PMID:20531392

  5. Molecular Mechanism for Fungal Cell Wall Recognition by Rice Chitin Receptor OsCEBiP.

    PubMed

    Liu, Simiao; Wang, Jizong; Han, Zhifu; Gong, Xinqi; Zhang, Heqiao; Chai, Jijie

    2016-07-01

    Chitin is the major component of fungal cell wall and serves as a molecular pattern that can be recognized by the receptor OsCEBiP in rice, a lysine motif (LysM) receptor-like protein (RLP), to trigger immune responses. The molecular mechanisms underlying chitin recognition remain elusive. Here we report the crystal structures of the ectodomain of OsCEBiP (OsCEBiP-ECD) in free and chitin-bound forms. The structures reveal that OsCEBiP-ECD contains three tandem LysMs followed by a novel structure fold of cysteine-rich domain. The structures showed that chitin binding induces no striking conformational changes in OsCEBiP. Structural comparison among N-acetylglucosamine (NAG) oligomer-bound LysMs revealed a highly conserved recognition mechanism, which is expected to facilitate study of other LysM-containing proteins for their NAG binding. Modeling study showed that chitin induces OsCEBiP homodimerization in a "sliding mode". Our data provide insights into rice chitin receptor-mediated immunity triggered by fungal cell wall. PMID:27238968

  6. Human blood dendritic cell subsets exhibit discriminative pattern recognition receptor profiles

    PubMed Central

    Lundberg, Kristina; Rydnert, Frida; Greiff, Lennart; Lindstedt, Malin

    2014-01-01

    Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human DCs have been identified; however their expression of PRRs is not fully clarified. Expressions of CLRs by DC subpopulations, in particular, remain elusive. This study aimed to identify and compare PRR expressions on human blood DC subsets, including CD1c+, CD141+ and CD16+ myeloid DCs and CD123+ plasmacytoid DCs, in order to understand their capacity to recognize different antigens as well as their responsiveness to PRR-directed targeting. Whole blood was obtained from 13 allergic and six non-allergic individuals. Mononuclear cells were purified and multi-colour flow cytometry was used to assess the expression of 10 CLRs and two TLRs on distinct DC subsets. PRR expression levels were shown to differ between DC subsets for each PRR assessed. Furthermore, principal component analysis and random forest test demonstrated that the PRR profiles were discriminative between DC subsets. Interestingly, CLEC9A was expressed at lower levels by CD141+ DCs from allergic compared with non-allergic donors. The subset-specific PRR expression profiles suggests individual responsiveness to PRR-targeting and supports functional specialization. PMID:24444310

  7. Human blood dendritic cell subsets exhibit discriminative pattern recognition receptor profiles.

    PubMed

    Lundberg, Kristina; Rydnert, Frida; Greiff, Lennart; Lindstedt, Malin

    2014-06-01

    Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human DCs have been identified; however their expression of PRRs is not fully clarified. Expressions of CLRs by DC subpopulations, in particular, remain elusive. This study aimed to identify and compare PRR expressions on human blood DC subsets, including CD1c(+) , CD141(+) and CD16(+) myeloid DCs and CD123(+) plasmacytoid DCs, in order to understand their capacity to recognize different antigens as well as their responsiveness to PRR-directed targeting. Whole blood was obtained from 13 allergic and six non-allergic individuals. Mononuclear cells were purified and multi-colour flow cytometry was used to assess the expression of 10 CLRs and two TLRs on distinct DC subsets. PRR expression levels were shown to differ between DC subsets for each PRR assessed. Furthermore, principal component analysis and random forest test demonstrated that the PRR profiles were discriminative between DC subsets. Interestingly, CLEC9A was expressed at lower levels by CD141(+) DCs from allergic compared with non-allergic donors. The subset-specific PRR expression profiles suggests individual responsiveness to PRR-targeting and supports functional specialization. PMID:24444310

  8. Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor

    PubMed Central

    Maqbool, A; Saitoh, H; Franceschetti, M; Stevenson, CEM; Uemura, A; Kanzaki, H; Kamoun, S; Terauchi, R; Banfield, MJ

    2015-01-01

    Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops. DOI: http://dx.doi.org/10.7554/eLife.08709.001 PMID:26304198

  9. Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor

    SciTech Connect

    Whitelaw, M.; Pongratz, I.; Wilhelmsson, A.; Gustafsson, J.; Poellinger, L. )

    1993-04-01

    Signal transduction by dioxins is mediated by the intracellular dioxin or aryl hydrocarbon receptor. This receptor binds dioxin and its planar aromatic congeners in a saturable manner with high affinity. The extreme toxicity of dioxin has been demonstrated in animals but not in humans. In animals, dioxin causes thymic wasting, immune suppression, severe epithelial disorders and tumor promotion. On a molecular level, dioxins are inducers of transcription of a battery of target genes encoding xenobiotic metabolizing enzymes. Dioxin also appears to transcriptionally regulate the expression of the growth modulatory genes for interleukin-1 Beta and plasminogen activator inhibitor-2. The dioxin induction response is mediated by single or multiple copies of dioxin-inducible transcriptional control elements in target promoters. The research data detailed in this paper examines the ligand-dependent recruitment of the Arnt coregulator which determines DNA recognition by the dioxin receptor. This data suggests that dioxin receptor activity is governed by a complex pattern of combinatorial regulation involving repression by hsp90 and then by ligand-dependent recruitment of the positive coregulator Arnt and that the dioxin receptor system provides the first example of signal-controlled dimerization of bHLH factors.

  10. Molecular recognition of parathyroid hormone by its G protein-coupled receptor

    SciTech Connect

    Pioszak, Augen A.; Xu, H. Eric

    2008-08-07

    Parathyroid hormone (PTH) is central to calcium homeostasis and bone maintenance in vertebrates, and as such it has been used for treating osteoporosis. It acts primarily by binding to its receptor, PTH1R, a member of the class B G protein-coupled receptor (GPCR) family that also includes receptors for glucagon, calcitonin, and other therapeutically important peptide hormones. Despite considerable interest and much research, determining the structure of the receptor-hormone complex has been hindered by difficulties in purifying the receptor and obtaining diffraction-quality crystals. Here, we present a method for expression and purification of the extracellular domain (ECD) of human PTH1R engineered as a maltose-binding protein (MBP) fusion that readily crystallizes. The 1.95-{angstrom} structure of PTH bound to the MBP-PTH1R-ECD fusion reveals that PTH docks as an amphipathic helix into a central hydrophobic groove formed by a three-layer {alpha}-{beta}-{beta}{alpha} fold of the PTH1R ECD, resembling a hot dog in a bun. Conservation in the ECD scaffold and the helical structure of peptide hormones emphasizes this hot dog model as a general mechanism of hormone recognition common to class B GPCRs. Our findings reveal critical insights into PTH actions and provide a rational template for drug design that targets this hormone signaling pathway.

  11. Platelet receptor recognition and cross-talk in collagen-induced activation of platelets.

    PubMed

    Farndale, R W; Slatter, D A; Siljander, P R-M; Jarvis, G E

    2007-07-01

    Comprehensive mapping of protein-binding sites within human collagen III has allowed the recognition motifs for integrin alpha(2)beta(1) and VWF A3 domain to be identified. Glycoprotein VI-binding sites are understood, although less well defined. This information, together with recent developments in understanding collagen fiber architecture, and crystal structures of the receptor collagen-binding domains, allows a coherent model for the interaction of collagen with the platelet surface to be developed. This complements our understanding of the orchestration of receptor presentation by membrane microdomains, such that the polyvalent collagen surface may stabilize signaling complexes within the heterogeneous receptor composition of the lipid raft. The ensuing interactions lead to the convergence of signals from each of the adhesive receptors, mediated by FcR gamma-chain and/or FcgammaRIIa, leading to concerted and co-operative platelet activation. Each receptor has a shear-dependent role, VWF/GpIb essential at high shear, and alpha(2)beta(1) at low and intermediate shear, whilst GpVI provides core signals that contribute to enhanced integrin affinity, tighter binding to collagen and consequent platelet activation. PMID:17635730

  12. Evidence for a specific role for muscarinic receptors in crossmodal object recognition in rats.

    PubMed

    Jacklin, Derek L; Kelly, Patrick; Bianchi, Cristina; MacDonald, Tyler; Traquair, Hugh; Winters, Boyer D

    2015-02-01

    Acetylcholine (ACh) has been implicated in numerous cognitive functions, including multisensory feature binding. In the present study, we systematically assessed the involvement of cholinergic muscarinic receptors in several variations of an object recognition task for rats. In the standard spontaneous object recognition (SOR) task, tactile and visual properties of objects were freely available throughout the sample and choice phases. In the tactile- and visual-only unimodal SOR tasks, exploration in both phases was restricted to tactile and visual information, respectively. For the basic crossmodal object recognition (CMOR) task, sample object exploration was limited to tactile features, whereas choice objects were available only in the visual domain. In Experiment 1, pre-sample systemic administration of scopolamine (0.2mg/kg) disrupted performance on standard SOR, both unimodal SOR tasks, and basic CMOR, consistent with a role for muscarinic receptors in memory encoding. Conversely, in Experiment 2, pre-choice systemic scopolamine selectively impaired object recognition on the CMOR task. For Experiment 3, the inclusion of multimodal, but not unimodal pre-exposure to the to-be-remembered objects prevented scopolamine from disrupting performance on the CMOR task when given prior to the choice phase. These results suggest that ACh is necessary during the choice phase of the CMOR task to facilitate the binding of object features across sensory modalities, a function that is not required for the other tasks assessed. Multimodal object pre-exposure might preclude the requisite contribution of ACh in the choice phase by allowing rats to bind important visual and tactile object information prior to testing. PMID:25490059

  13. Molecular Recognition Analyzed by Docking Simulations: The Aspartate Receptor and Isocitrate Dehydrogenase from Escherichia coli

    NASA Astrophysics Data System (ADS)

    Stoddard, Barry L.; Koshland, Daniel E., Jr.

    1993-02-01

    Protein docking protocols are used for the prediction of both small molecule binding to DNA and protein macromolecules and of complexes between macromolecules. These protocols are becoming increasingly automated and powerful tools for computer-aided drug design. We review the basic methodologies and strategies used for analyzing molecular recognition by computer docking algorithms and discuss recent experiments in which (i) substrate and substrate analogues are docked to the active site of isocitrate dehydrogenase and (ii) maltose binding protein is docked to the extracellular domain of the receptor, which signals maltose chemotaxis.

  14. Chiral recognition in adrenergic receptor binding mimics prepared by molecular imprinting.

    PubMed

    Ramström, O; Yu, C; Mosbach, K

    1996-01-01

    Molecularly imprinted polymers were prepared against the adrenomimetic agents ephedrine and pseudoephedrine. These compounds each incorporate two chiral centres. The polymers were evaluated with respect to enantiodiscrimination of various adrenergic ligands. The selectivity of the polymeric binding sites for the imprinted molecules was very high, and it was found that binding of both the enantiomeric and diastereomeric isomers of the imprint species were effectively obstructed, it was found that these polymers could selectively recognize the enantiomers of the endogenous adrenergic ligand epinephrine as well as several beta-adrenergic blockers. These observations suggest that these polymers effectively mimic the recognition patterns exhibited by natural adrenergic receptors. PMID:9174958

  15. The INs and OUTs of pattern recognition receptors at the cell surface.

    PubMed

    Beck, Martina; Heard, William; Mbengue, Malick; Robatzek, Silke

    2012-08-01

    Pattern recognition receptors (PRRs) enable plants to sense non-self molecules displayed by microbes to mount proper defense responses or establish symbiosis. In recent years the importance of PRR subcellular trafficking to plant immunity has become apparent. PRRs traffic through the endoplasmatic reticulum (ER) and the Golgi apparatus to the plasma membrane, where they recognize their cognate ligands. At the plasma membrane, PRRs can be recycled or internalized via endocytic pathways. By using genetic and biochemical tools in combination with bioimaging, the trafficking pathways and their role in PRR perception of microbial molecules are now being revealed. PMID:22664220

  16. Oxytocin and the oxytocin receptor underlie intra-strain, but not inter-strain, social recognition

    PubMed Central

    Macbeth, Abbe H.; Lee, Heon-Jin; Edds, Jennifer; Young, W. Scott

    2009-01-01

    We studied three lines of oxytocin (Oxt) and oxytocin receptor (Oxtr) knockout (KO) male mice (Oxt−/−, total Oxtr−/−, and partial-forebrain Oxtr (OxtrFB/FB)) with established deficits in social recognition to further refine our understanding of their deficits with regard to stimulus female's strain. We used a modified social discrimination paradigm in which subjects are singly housed only for the duration of the test. Additionally, stimulus females are singly-housed throughout testing and are presented within corrals for rapid comparison of investigation by subject males. Wildtype (WT) males from all three lines discriminated between familiar and novel females of three different strains (C57BL/6, Balb/c, Swiss-Webster). No KO males discriminated between familiar and novel Balb/c or C57BL/6 females. Male Oxt−/− and Oxtr−/− mice, but not OxtrFB/FB mice, discriminated between familiar and novel Swiss-Webster females. As this might indicate a global deficit in individual recognition for OxtrFB/FB males, we examined their ability to discriminate between females from different strains and compared performance with Oxtr−/− males. WT and KO males from both lines were able to distinguish between familiar and novel females from different strains, indicating the social recognition deficit is not universal. Instead, we hypothesize that the Oxtr is involved in “fine” intra-strain recognition, but is less important in “broad” inter-strain recognition. We also present the novel finding of decreased investigation across tests, which is likely an artifact of repeated testing and not due to stimulus female's strain or age of subject males. PMID:19531157

  17. Direct ubiquitination of pattern recognition receptor FLS2 attenuates plant innate immunity

    PubMed Central

    Lu, Dongping; Lin, Wenwei; Gao, Xiquan; Wu, Shujing; Cheng, Cheng; Avila, Julian; Heese, Antje; Devarenne, Timothy P.; He, Ping; Shan, Libo

    2011-01-01

    Innate immune responses are triggered by the activation of pattern-recognition receptors (PRRs). The Arabidopsis PRR FLS2 senses bacterial flagellin and initiates immune signaling by association with BAK1. The molecular mechanisms underlying the attenuation of FLS2 activation are largely unknown. We report that flagellin induces recruitment of two closely related U-box E3 ubiquitin ligases PUB12 and PUB13 to FLS2 receptor complex in Arabidopsis. BAK1 phosphorylates PUB12/13 and is required for FLS2-PUB12/13 association. PUB12/13 polyubiquitinate FLS2 and promote flagellin-induced FLS2 degradation, and the pub12 and pub13 mutants displayed elevated immune responses to flagellin treatment. Our study has revealed a unique regulatory circuit of direct ubiquitination and turnover of FLS2 by BAK1-mediated phosphorylation and recruitment of specific E3 ligases for attenuation of immune signaling. PMID:21680842

  18. Ameliorative effect of membrane-associated estrogen receptor G protein coupled receptor 30 activation on object recognition memory in mouse models of Alzheimer's disease.

    PubMed

    Kubota, Takashi; Matsumoto, Hiroshi; Kirino, Yutaka

    2016-07-01

    Membrane-associated estrogen receptor "G protein-coupled receptor 30" (GPR30) has been implicated in spatial recognition memory and protection against neuronal death. The present study investigated the role of GPR30 in object recognition memory in an Alzheimer's disease (AD) mouse model (5XFAD) by using novel object recognition (NOR) test. Impairment of long-term (24 h) recognition memory was observed in both male and female 5XFAD mice. Selective GPR30 agonist, G-1, ameliorated this impairment in female 5XFAD mice, but not in male mice. Our study demonstrated the ameliorative role of GPR30 in NOR memory impaired by AD pathology in female mice. PMID:27423484

  19. Reptile Toll-like receptor 5 unveils adaptive evolution of bacterial flagellin recognition

    PubMed Central

    Voogdt, Carlos G. P.; Bouwman, Lieneke I.; Kik, Marja J. L.; Wagenaar, Jaap A.; van Putten, Jos P. M.

    2016-01-01

    Toll-like receptors (TLR) are ancient innate immune receptors crucial for immune homeostasis and protection against infection. TLRs are present in mammals, birds, amphibians and fish but have not been functionally characterized in reptiles despite the central position of this animal class in vertebrate evolution. Here we report the cloning, characterization, and function of TLR5 of the reptile Anolis carolinensis (Green Anole lizard). The receptor (acTLR5) displays the typical TLR protein architecture with 22 extracellular leucine rich repeats flanked by a N- and C-terminal leucine rich repeat domain, a membrane-spanning region, and an intracellular TIR domain. The receptor is phylogenetically most similar to TLR5 of birds and most distant to fish TLR5. Transcript analysis revealed acTLR5 expression in multiple lizard tissues. Stimulation of acTLR5 with TLR ligands demonstrated unique responsiveness towards bacterial flagellin in both reptile and human cells. Comparison of acTLR5 and human TLR5 using purified flagellins revealed differential sensitivity to Pseudomonas but not Salmonella flagellin, indicating development of species-specific flagellin recognition during the divergent evolution of mammals and reptiles. Our discovery of reptile TLR5 fills the evolutionary gap regarding TLR conservation across vertebrates and provides novel insights in functional evolution of host-microbe interactions. PMID:26738735

  20. Reptile Toll-like receptor 5 unveils adaptive evolution of bacterial flagellin recognition.

    PubMed

    Voogdt, Carlos G P; Bouwman, Lieneke I; Kik, Marja J L; Wagenaar, Jaap A; van Putten, Jos P M

    2016-01-01

    Toll-like receptors (TLR) are ancient innate immune receptors crucial for immune homeostasis and protection against infection. TLRs are present in mammals, birds, amphibians and fish but have not been functionally characterized in reptiles despite the central position of this animal class in vertebrate evolution. Here we report the cloning, characterization, and function of TLR5 of the reptile Anolis carolinensis (Green Anole lizard). The receptor (acTLR5) displays the typical TLR protein architecture with 22 extracellular leucine rich repeats flanked by a N- and C-terminal leucine rich repeat domain, a membrane-spanning region, and an intracellular TIR domain. The receptor is phylogenetically most similar to TLR5 of birds and most distant to fish TLR5. Transcript analysis revealed acTLR5 expression in multiple lizard tissues. Stimulation of acTLR5 with TLR ligands demonstrated unique responsiveness towards bacterial flagellin in both reptile and human cells. Comparison of acTLR5 and human TLR5 using purified flagellins revealed differential sensitivity to Pseudomonas but not Salmonella flagellin, indicating development of species-specific flagellin recognition during the divergent evolution of mammals and reptiles. Our discovery of reptile TLR5 fills the evolutionary gap regarding TLR conservation across vertebrates and provides novel insights in functional evolution of host-microbe interactions. PMID:26738735

  1. Molecular Recognition of Corticotropin releasing Factor by Its G protein-coupled Receptor CRFR1

    SciTech Connect

    Pioszak, Augen A.; Parker, Naomi R.; Suino-Powell, Kelly; Xu, H. Eric

    2009-01-15

    The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observed for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.

  2. Probability model for molecular recognition in biological receptor repertoires: significance to the olfactory system.

    PubMed

    Lancet, D; Sadovsky, E; Seidemann, E

    1993-04-15

    A generalized phenomenological model is presented for stereospecific recognition between biological receptors and their ligands. We ask what is the distribution of binding constants psi(K) between an arbitrary ligand and members of a large receptor repertoire, such as immunoglobulins or olfactory receptors. For binding surfaces with B potential subsite and S different types of subsite configurations, the number of successful elementary interactions obeys a binomial distribution. The discrete probability function psi(K) is then derived with assumptions on alpha, the free energy contribution per elementary interaction. The functional form of psi(K) may be universal, although the parameter values could vary for different ligand types. An estimate of the parameter values of psi(K) for iodovanillin, an analog of odorants and immunological haptens, is obtained by equilibrium dialysis experiments with nonimmune antibodies. Based on a simple relationship, predicted by the model, between the size of a receptor repertoire and its average maximal affinity toward an arbitrary ligand, the size of the olfactory receptor repertoire (Nolf) is calculated as 300-1000, in very good agreement with recent molecular biological studies. A very similar estimate, Nolf = 500, is independently derived by relating a theoretical distribution of maxima for psi(K) with published human olfactory threshold variations. The present model also has implications to the question of olfactory coding and to the analysis of specific anosmias, genetic deficits in perceiving particular odorants. More generally, the proposed model provides a better understanding of ligand specificity in biological receptors and could help in understanding their evolution. PMID:8475121

  3. Recognition of bacterial signal peptides by mammalian formyl peptide receptors: a new mechanism for sensing pathogens.

    PubMed

    Bufe, Bernd; Schumann, Timo; Kappl, Reinhard; Bogeski, Ivan; Kummerow, Carsten; Podgórska, Marta; Smola, Sigrun; Hoth, Markus; Zufall, Frank

    2015-03-20

    Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system. PMID:25605714

  4. Probability model for molecular recognition in biological receptor repertoires: significance to the olfactory system.

    PubMed Central

    Lancet, D; Sadovsky, E; Seidemann, E

    1993-01-01

    A generalized phenomenological model is presented for stereospecific recognition between biological receptors and their ligands. We ask what is the distribution of binding constants psi(K) between an arbitrary ligand and members of a large receptor repertoire, such as immunoglobulins or olfactory receptors. For binding surfaces with B potential subsite and S different types of subsite configurations, the number of successful elementary interactions obeys a binomial distribution. The discrete probability function psi(K) is then derived with assumptions on alpha, the free energy contribution per elementary interaction. The functional form of psi(K) may be universal, although the parameter values could vary for different ligand types. An estimate of the parameter values of psi(K) for iodovanillin, an analog of odorants and immunological haptens, is obtained by equilibrium dialysis experiments with nonimmune antibodies. Based on a simple relationship, predicted by the model, between the size of a receptor repertoire and its average maximal affinity toward an arbitrary ligand, the size of the olfactory receptor repertoire (Nolf) is calculated as 300-1000, in very good agreement with recent molecular biological studies. A very similar estimate, Nolf = 500, is independently derived by relating a theoretical distribution of maxima for psi(K) with published human olfactory threshold variations. The present model also has implications to the question of olfactory coding and to the analysis of specific anosmias, genetic deficits in perceiving particular odorants. More generally, the proposed model provides a better understanding of ligand specificity in biological receptors and could help in understanding their evolution. PMID:8475121

  5. Structural basis of collagen recognition by human osteoclast-associated receptor and design of osteoclastogenesis inhibitors

    PubMed Central

    Haywood, Joel; Qi, Jianxun; Chen, Chun-Chi; Lu, Guangwen; Liu, Yingxia; Yan, Jinghua; Shi, Yi; Gao, George F.

    2016-01-01

    Human osteoclast-associated receptor (OSCAR) is an immunoglobulin (Ig)-like collagen receptor that is up-regulated on osteoclasts during osteoclastogenesis and is expressed in a range of myeloid cells. As a member of the leukocyte receptor complex family of proteins, OSCAR shares a high degree of sequence and structural homology with other collagen receptors of this family, including glycoprotein VI, leukocyte-associated Ig-like receptor-1, and leukocyte Ig-like receptor B4, but recognizes a unique collagen sequence. Here, we present the crystal structures of OSCAR in its free form and in complex with a triple-helical collagen-like peptide (CLP). These structures reveal that the CLP peptide binds only one of the two Ig-like domains, the membrane-proximal domain (domain 2) of OSCAR, with the middle and trailing chain burying a total of 661 Å2 of solvent-accessible collagen surface. This binding mode is facilitated by the unusual topography of the OSCAR protein, which displays an obtuse interdomain angle and a rotation of domain 2 relative to the membrane-distal domain 1. Moreover, the binding of the CLP to OSCAR appears to be mediated largely by tyrosine residues and conformational changes at a shallow Phe pocket. Furthermore, we investigated CLP peptides as inhibitors of osteoclastogenesis and found that a peptide length of 40 amino acids is required to ensure adequate inhibition of osteoclastogenesis in vitro. These findings provide valuable structural insights into the mode of collagen recognition by OSCAR and into the use of synthetic peptide matrikines for osteoclastogenesis inhibition. PMID:26744311

  6. Natural anti-proteases in rainbow trout, Oncorhynchus mykiss and brook charr, Salvelinus fontinalis and the in vitro neutralization of fish alpha 2-macroglobulin by the metalloprotease from the pathogenic haemoflagellate, Cryptobia salmositica.

    PubMed

    Zuo, X; Woo, P T

    1997-04-01

    Natural anti-proteases (alpha 1-protease inhibitor (alpha 1-PI; alpha 1-antitrypsin) and alpha 2-macroglobulin (alpha 2-M)) were found in the blood of rainbow trout, Oncorhynchus mykiss and brook charr, Salvelinus fontinalis. The alpha 2-M inhibited Cryptobia salmositica proteases and was significantly higher in brook charr than in rainbow trout. Under in vitro conditions it took longer for the same number of parasites to neutralize the alpha 2-M in charr than in trout blood. The haemolysis which occurred when C. salmositica was incubated in the blood of rainbow trout was due to neutralization of alpha 2-M. This in vitro study also showed that it was the metalloprotease of C. salmositica that lysed red blood cells and the plasma of the two species of fishes initially prevented haemolysis by inhibiting the proteolytic activity. We suggest that the natural plasma alpha 2-M plays an important role in defence against cryptobiosis in fishes. PMID:9107024

  7. Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

    PubMed

    Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

    2000-04-01

    Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy. PMID:10811469

  8. Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis.

    PubMed Central

    Savill, J; Hogg, N; Ren, Y; Haslett, C

    1992-01-01

    We have investigated the cell surface recognition mechanisms used by human monocyte-derived macrophages (M phi) in phagocytosis of intact aging human neutrophils (PMNs) undergoing apoptosis. This study shows that the adhesive protein thrombospondin (TSP) was present in the interaction, both associated with the M phi surface and in solution at a mean concentration of 0.59 micrograms/ml. The interaction was inhibited by treatment of M phi (but not aged PMN) with cycloheximide, but could be "rescued" by replenishment with exogenous TSP. Under control conditions, M phi recognition of aged PMNs was specifically potentiated by purified platelet TSP at 5 micrograms/ml, present either in the interaction or if preincubated with either cell type, suggesting that TSP might act as a "molecular bridge" between the two cell types. In support, both aged PMN and M phi were found to adhere to TSP, and phagocytosis of aged PMN was specifically inhibited by (a) excess soluble TSP; (b) antibodies to TSP that also inhibit TSP-mediated adhesion to aged PMN; and (c) down-regulation of M phi receptors for TSP by plating M phi on TSP-coated surfaces. Furthermore, inhibition with mAbs/Arg-Gly-Asp-Ser peptide of the candidate M phi receptors for TSP, CD36, and alpha v beta 3 exerted synergistic effects on both M phi recognition of aged PMN and M phi adhesion to TSP, indicating that "two point" adhesion of TSP to these M phi structures is involved in phagocytosis of aged PMN. Our findings indicate newly defined roles for TSP and CD36 in phagocytic clearance of senescent neutrophils, which may limit inflammatory tissue injury and promote resolution. Images PMID:1383273

  9. Synthetic autoantigens of immunoglobulins and T-cell receptors: their recognition in aging, infection, and autoimmunity.

    PubMed

    Marchalonis, J J; Schluter, S F; Wang, E; Dehghanpisheh, K; Lake, D; Yocum, D E; Edmundson, A B; Winfield, J B

    1994-11-01

    Immunoglobulins and their close relatives, the antigen-specific T-cell receptors, are recognition proteins that express structures which readily serve as self-immunogens. Healthy humans can produce antibodies against variable region-defined recognition structures termed idiotypes, as well as against constant region structures, and the levels of these can increase markedly in autoimmune disease; e.g., rheumatoid factors are autoantibodies directed against a conformational determinant of the gamma heavy chain. More recent analyses employing synthetic peptide technologies and construction of recombinant T-cell receptors document that autoantibodies directed against both variable and constant region markers of the alpha/beta T-cell receptor occur in healthy individuals. Alterations in levels of antibody, usage of IgM or IgG isotypes, and specificity for particular peptide-defined regions vary with natural physiological processes (aging, pregnancy), with artificial allografting, with retroviral infection, and with the inception and progression of autoimmune disease (e.g., rheumatoid arthritis, systemic lupus erythematosus). Two of the major autoimmunogeneic regions of the Tcr alpha/beta are "constitutive" markers inasmuch as all individuals tested produce antibodies against these regions. The most frequently observed autoantibodies are against Tcr V beta CDR1 and Fr3 markers. It is hypothesized that these are normally involved in immunoregulation. Autoantibodies usually are not detected against CDR2 region determinants, or the "private idiotypes" defined by the CDR3 region, or the highly conserved FR4 segment specified by the joining gene segment. However, autoantibodies against the CDR2 of the Tcr alpha chain occur in some SLE patients, and healthy pregnant women produce antibodies against the common peptide determinant expressed by the joining gene and the beginning of the C alpha or C beta domain. Although the precise role of the naturally occurring autoantibodies in

  10. Cflec-5, a pattern recognition receptor in scallop Chlamys farreri agglutinating yeast Pichia pastoris.

    PubMed

    Zhang, Huan; Kong, Pengfei; Wang, Lingling; Zhou, Zhi; Yang, Jialong; Zhang, Ying; Qiu, Limei; Song, Linsheng

    2010-07-01

    C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles as pattern recognition receptors (PRRs) in the innate immunity. In this study, the full-length cDNA of a C-type lectin was cloned from scallop Chlamys farreri (designated as Cflec-5) by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of Cflec-5 was of 1412 bp. The open reading frame encoded a polypeptide of 153 amino acids, including a signal sequence and a conserved carbohydrate-recognition domain with the EPN motif determining the mannose-binding specificity. The deduced amino acid sequence of Cflec-5 showed high similarity to members of C-type lectin superfamily. The quantitative real-time PCR was performed to investigate the tissue distribution of Cflec-5 mRNA and its temporal expression profiles in hemocytes post pathogen-associated molecular patterns (PAMPs) stimulation. In healthy scallops, the Cflec-5 mRNA was mainly detected in gill and mantle, and marginally in other tissues. The mRNA expression of Cflec-5 could be significantly induced by lipopolysaccharide (LPS) and glucan stimulation and reached the maximum level at 6 h and 12 h, respectively. But its expression level did not change significantly during peptidoglycan (PGN) stimulation. The function of Cflec-5 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta Gami (DE3). The recombinant Cflec-5 agglutinated Pichia pastoris in a calcium-independent way. The agglutinating activity could be inhibited by d-mannose, LPS and glucan, but not by d-galactose or PGN. These results collectively suggested that Cflec-5 was involved in the innate immune response of scallops and might contribute to nonself-recognition through its interaction with various PAMPs. PMID:20211738

  11. Closely Related Antibody Receptors Exploit Fundamentally Different Strategies for Steroid Recognition

    SciTech Connect

    Verdino, P.; Aldag, C.; Hilvert, D.; Wilson, I.A.

    2009-05-26

    Molecular recognition by the adaptive immune system relies on specific high-affinity antibody receptors that are generated from a restricted set of starting sequences through homologous recombination and somatic mutation. The steroid binding antibody DB3 and the catalytic Diels-Alderase antibody 1E9 derive from the same germ line sequences but exhibit very distinct specificities and functions. However, mutation of only two of the 36 sequence differences in the variable domains, Leu{sup H47}Trp and Arg{sup H100}Trp, converts 1E9 into a high-affinity steroid receptor with a ligand recognition profile similar to DB3. To understand how these changes switch binding specificity and function, we determined the crystal structures of the 1E9 Leu{sup H47}Trp/Arg{sup H100}Trp double mutant (1E9dm) as an unliganded Fab at 2.05 {angstrom} resolution and in complex with two configurationally distinct steroids at 2.40 and 2.85 {angstrom}. Surprisingly, despite the functional mimicry of DB3, 1E9dm employs a distinct steroid binding mechanism. Extensive structural rearrangements occur in the combining site, where residue H47 acts as a specificity switch and H100 adapts to different ligands. Unlike DB3, 1E9dm does not use alternative binding pockets or different sets of hydrogen-bonding interactions to bind configurationally distinct steroids. Rather, the different steroids are inserted more deeply into the 1E9dm combining site, creating more hydrophobic contacts that energetically compensate for the lack of hydrogen bonds. These findings demonstrate how subtle mutations within an existing molecular scaffold can dramatically modulate the function of immune receptors by inducing unanticipated, but compensating, mechanisms of ligand interaction.

  12. Macrophage recognition of toxic advanced glycosylation end products through the macrophage surface-receptor nucleolin.

    PubMed

    Miki, Yuichi; Dambara, Hikaru; Tachibana, Yoshihiro; Hirano, Kazuya; Konishi, Mio; Beppu, Masatoshi

    2014-01-01

    Advanced glycosylation end-products (AGEs) are non-enzymatically glycosylated proteins that play an important role in several diseases and aging processes, including angiopathy, renal failure, diabetic complications, and some neurodegenerative diseases. In particular, glyceraldehyde (GCA)- and glycolaldehyde (GOA)-derived AGEs are deemed toxic AGEs, due to their cytotoxicity. Recently, the shuttling-protein nucleolin has been shown to possess scavenger receptor-activity. Here, we investigated whether or not macrophages recognize toxic AGEs through nucleolin receptors expressed on their surface. Free amino acid groups and arginine residues found in bovine serum albumin (BSA) were time-dependently modified by incubation with GCA and GOA. In addition, average molecular size was increased by incubation with GCA and GOA. While GCA-treated BSA (GCA-BSA) and GOA-treated BSA (GOA-BSA) were recognized by thioglycollate-elicited mouse peritoneal macrophages in proportion to their respective aldehyde-modification ratios, aldehyde-untreated control-BSA was not. Surface plasmon-resonance analysis revealed that nucleolin strongly associated with GCA-BSA and GOA-BSA, but not with control-BSA. Further, pretreating macrophages with anti-nucleolin antibody, but not control-Immunoglobulin G, inhibited recognition of GCA-BSA and GOA-BSA by macrophages. Additionally, AGRO, a nucleolin-specific oligonucleotide aptamer, inhibited recognition of GCA-BSA and GOA-BSA. Moreover, nucleolin-transfected HEK293 cells recognized more GCA-BSA and GOA-BSA than control HEK cells did. Binding of nucleolin and GCA-BSA/GOA-BSA was also blocked by anti-nucleolin antibody at molecular level. These results indicate that nucleolin is a receptor that allows macrophages to recognize toxic AGEs. PMID:24818254

  13. Site-specific basicities regulate molecular recognition in receptor binding: in silico docking of thyroid hormones.

    PubMed

    Tóth, Gergő; Baska, Ferenc; Schretner, András; Rácz, Akos; Noszál, Béla

    2013-09-01

    Interactions between thyroid hormone α and β receptors and the eight protonation microspecies of each of the main thyroid hormones (thyroxine, liothyronine, and reverse liothyronine) were investigated and quantitated by molecular modeling. Flexible docking of the various protonation forms of thyroid hormones and high-affinity thyromimetics to the two thyroid receptors was carried out. In this method the role of the ionization state of each basic site could be studied in the composite process of molecular recognition. Our results quantitate at the molecular level how the ionization state and the charge distribution influence the protein binding. The anionic form of the carboxyl group (i.e., carboxylate site) is essential for protein binding, whereas the protonated form of amino group worsens the binding. The protonation state of the phenolate plays a less important role in the receptor affinity; its protonation, however, alters the electron density and the concomitant stacking propensity of the aromatic rings, resulting in a different binding score. The combined results of docking and microspeciation studies show that microspecies with the highest concentration at the pH of blood are not the strongest binding ones. The calculated binding free energy values can be well interpreted in terms of the interactions between the actual sites of the microspecies and the receptor amino acids. Our docking results were validated and compared with biological data from the literature. Since the thyroid hormone receptors influence several physiologic functions, such as metabolic rate, cholesterol and triglyceride levels, and heart frequency, our binding results provide a molecular basis for drug design and development in related therapeutic indications. PMID:23907234

  14. Differential effects of m1 and m2 receptor antagonists in perirhinal cortex on visual recognition memory in monkeys.

    PubMed

    Wu, Wei; Saunders, Richard C; Mishkin, Mortimer; Turchi, Janita

    2012-07-01

    Microinfusions of the nonselective muscarinic antagonist scopolamine into perirhinal cortex impairs performance on visual recognition tasks, indicating that muscarinic receptors in this region play a pivotal role in recognition memory. To assess the mnemonic effects of selective blockade in perirhinal cortex of muscarinic receptor subtypes, we locally infused either the m1-selective antagonist pirenzepine or the m2-selective antagonist methoctramine in animals performing one-trial visual recognition, and compared these scores with those following infusions of equivalent volumes of saline. Compared to these control infusions, injections of pirenzepine, but not of methoctramine, significantly impaired recognition accuracy. Further, similar doses of scopolamine and pirenzepine yielded similar deficits, suggesting that the deficits obtained earlier with scopolamine were due mainly, if not exclusively, to blockade of m1 receptors. The present findings indicate that m1 and m2 receptors have functionally dissociable roles, and that the formation of new visual memories is critically dependent on the cholinergic activation of m1 receptors located on perirhinal cells. PMID:22561485

  15. Differential effects of m1 and m2 receptor antagonists in perirhinal cortex on visual recognition memory in monkeys

    PubMed Central

    Wu, Wei; Saunders, Richard C.; Mishkin, Mortimer; Turchi, Janita

    2012-01-01

    Microinfusions of the nonselective muscarinic antagonist scopolamine into perirhinal cortex impairs performance on visual recognition tasks, indicating that muscarinic receptors in this region play a pivotal role in recognition memory. To assess the mnemonic effects of selective blockade in perirhinal cortex of muscarinic receptor subtypes, we locally infused either the m1-selective antagonist pirenzepine or the m2-selective antagonist methoctramine in animals performing one-trial visual recognition, and compared these scores with those following infusions of equivalent volumes of saline. Compared to these control infusions, injections of pirenzepine, but not of methoctramine, significantly impaired recognition accuracy. Further, similar doses of scopolamine and pirenzepine yielded similar deficits, suggesting that the deficits obtained earlier with scopolamine were due mainly, if not exclusively, to blockade of m1 receptors. The present findings indicate that m1 and m2 receptors have functionally dissociable roles, and that the formation of new visual memories is critically dependent on the cholinergic activation of m1 receptors located on perirhinal cells. PMID:22561485

  16. Selective recognition of neutral guests in an aqueous medium by a biomimetic calix[6]cryptamide receptor.

    PubMed

    Lascaux, Angélique; De Leener, Gaël; Fusaro, Luca; Topić, Filip; Rissanen, Kari; Luhmer, Michel; Jabin, Ivan

    2016-01-14

    The design of artificial receptors that can efficiently work in water is a challenging research area. A possible biomimetic approach for the elaboration of such receptors consists of associating a hydrophobic cavity with a polar polyfunctional binding site. On this basis, a hydrophilic calix[6]cryptamide decorated with oligo(ethylene glycol) units (i.e. 8) was synthesized through an efficient [1 + 1] macrocyclization reaction as the key-step. The complexation of neutral molecules was evaluated by NMR spectroscopy through competition experiments either in apolar or aqueous media. In both media, host 8 can bind neutral species that display H-bonding acceptor and donor groups such as amides or ureas. Interestingly, the most polar and acidic molecule is the best guest in chloroform and the worst one in an aqueous medium, highlighting the importance of the environment. As shown by NMR and X-ray diffraction data, the mode of recognition involves a complementary DAAAD-ADDDA quintuple H-bonding array between the binding partners as well as multiple CH-π interactions. A comparison of this calix[6]arene-based host-guest system with the binding site of biotin-binding proteins shows strong similarities. Besides, the acid-base control of the binding properties of receptor 8 in aqueous media is highly reminiscent of allosteric processes encountered in natural systems. PMID:26580493

  17. Recognition and sequestration of ω-fatty acids by a cavitand receptor

    PubMed Central

    Mosca, Simone; Ajami, Dariush; Rebek, Julius

    2015-01-01

    One of the largest driving forces for molecular association in aqueous solution is the hydrophobic effect, and many synthetic receptors with hydrophobic interiors have been devised for molecular recognition studies in water. Attempts to create the longer, narrower cavities appropriate for long-chain fatty acids have been thwarted by solvophobic collapse of the synthetic receptors, giving structures that have no internal spaces. The collapse generally involves the stacking of aromatic panels onto themselves. We describe here the synthesis and application of a deep cavitand receptor featuring “prestacked” aromatic panels at the upper rim of the binding pocket. The cavitand remains open and readily sequesters biologically relevant long-chain molecules—unsaturated ω-3, -6, and -9 fatty acids and derivatives such as anandamide—from aqueous media. The cavitand exists in isomeric forms with different stacking geometries and n-alkanes were used to characterize the binding modes and conformational properties. Long alkyl chains are accommodated in inverted J-shaped conformations. An analogous cavitand with electron-rich aromatic walls was prepared and comparative binding experiments indicated the role of intramolecular stacking in the binding properties of these deep container molecules. PMID:26305974

  18. Recognition and sequestration of ω-fatty acids by a cavitand receptor.

    PubMed

    Mosca, Simone; Ajami, Dariush; Rebek, Julius

    2015-09-01

    One of the largest driving forces for molecular association in aqueous solution is the hydrophobic effect, and many synthetic receptors with hydrophobic interiors have been devised for molecular recognition studies in water. Attempts to create the longer, narrower cavities appropriate for long-chain fatty acids have been thwarted by solvophobic collapse of the synthetic receptors, giving structures that have no internal spaces. The collapse generally involves the stacking of aromatic panels onto themselves. We describe here the synthesis and application of a deep cavitand receptor featuring "prestacked" aromatic panels at the upper rim of the binding pocket. The cavitand remains open and readily sequesters biologically relevant long-chain molecules-unsaturated ω-3, -6, and -9 fatty acids and derivatives such as anandamide-from aqueous media. The cavitand exists in isomeric forms with different stacking geometries and n-alkanes were used to characterize the binding modes and conformational properties. Long alkyl chains are accommodated in inverted J-shaped conformations. An analogous cavitand with electron-rich aromatic walls was prepared and comparative binding experiments indicated the role of intramolecular stacking in the binding properties of these deep container molecules. PMID:26305974

  19. Aldehyde Recognition and Discrimination by Mammalian Odorant Receptors via Functional Group-Specific Hydration Chemistry

    PubMed Central

    2015-01-01

    The mammalian odorant receptors (ORs) form a chemical-detecting interface between the atmosphere and the nervous system. This large gene family is composed of hundreds of membrane proteins predicted to form as many unique small molecule binding niches within their G-protein coupled receptor (GPCR) framework, but very little is known about the molecular recognition strategies they use to bind and discriminate between small molecule odorants. Using rationally designed synthetic analogs of a typical aliphatic aldehyde, we report evidence that among the ORs showing specificity for the aldehyde functional group, a significant percentage detect the aldehyde through its ability to react with water to form a 1,1-geminal (gem)-diol. Evidence is presented indicating that the rat OR-I7, an often-studied and modeled OR known to require the aldehyde function of octanal for activation, is likely one of the gem-diol activated receptors. A homology model based on an activated GPCR X-ray structure provides a structural hypothesis for activation of OR-I7 by the gem-diol of octanal. PMID:25181321

  20. CGP 36,742, an orally active GABAB receptor antagonist, facilitates memory in a social recognition test in rats.

    PubMed

    Mondadori, C; Moebius, H J; Zingg, M

    1996-05-01

    CGP 36,742, an orally active GABAB receptor antagonist, improves the retention performance of rats in a social recognition test. This effect is detectable over a very wide range of doses (0.03 to 300 mg/kg, p.o.). Considering its binding (32 mumol affinity for the GABAB site) the surprisingly potent activity of CGP 36,742 makes it appear quite possible that the effect is mediated by an as yet unknown receptor subtype. PMID:8762176

  1. Structural Basis for Hormone Recognition by the Human CRFR2[alpha] G Protein-coupled Receptor

    SciTech Connect

    Pal, Kuntal; Swaminathan, Kunchithapadam; Xu, H. Eric; Pioszak, Augen A.

    2012-05-09

    The mammalian corticotropin releasing factor (CRF)/urocortin (Ucn) peptide hormones include four structurally similar peptides, CRF, Ucn1, Ucn2, and Ucn3, that regulate stress responses, metabolism, and cardiovascular function by activating either of two related class B G protein-coupled receptors, CRFR1 and CRFR2. CRF and Ucn1 activate both receptors, whereas Ucn2 and Ucn3 are CRFR2-selective. The molecular basis for selectivity is unclear. Here, we show that the purified N-terminal extracellular domains (ECDs) of human CRFR1 and the CRFR2{alpha} isoform are sufficient to discriminate the peptides, and we present three crystal structures of the CRFR2{alpha} ECD bound to each of the Ucn peptides. The CRFR2{alpha} ECD forms the same fold observed for the CRFR1 and mouse CRFR2{beta} ECDs but contains a unique N-terminal {alpha}-helix formed by its pseudo signal peptide. The CRFR2{alpha} ECD peptide-binding site architecture is similar to that of CRFR1, and binding of the {alpha}-helical Ucn peptides closely resembles CRF binding to CRFR1. Comparing the electrostatic surface potentials of the ECDs suggests a charge compatibility mechanism for ligand discrimination involving a single amino acid difference in the receptors (CRFR1 Glu104/CRFR2{alpha} Pro-100) at a site proximate to peptide residue 35 (Arg in CRF/Ucn1, Ala in Ucn2/3). CRFR1 Glu-104 acts as a selectivity filter preventing Ucn2/3 binding because the nonpolar Ala-35 is incompatible with the negatively charged Glu-104. The structures explain the mechanisms of ligand recognition and discrimination and provide a molecular template for the rational design of therapeutic agents selectively targeting these receptors.

  2. Signal recognition particle receptor is a complex that contains two distinct polypeptide chains

    SciTech Connect

    Tajima, S.; Lauffer, L.; Rath, V.L.; Walter, P.

    1986-10-01

    Signal recognition particle (SRP) and SRP receptor are known to be essential components of the cellular machinery that targets nascent secretory proteins to the endoplasmic reticulum (ER) membrane. Here the authors report that the SRP receptor contains, in addition to the previously identified and sequenced 69-kD polypeptide (..cap alpha..-subunit, SR..cap alpha..), a 30-kD ..beta..-subunit SR..beta..). When SRP receptor was purified by SRP-Sepharose affinity chromatography, they observed the co-purification of two other ER membrane proteins. Both proteins are approx.30 kD in size and are immunologically distinct from each other, as well as from SR..cap alpha.. and SRP proteins. One of the 30-kD proteins (SR..beta..) forms a tight complex with SR..cap alpha.. in detergent solution that is stable to high salt and can be immunoprecipitated with antibodies to either SR..cap alpha.. or SR..beta... Both subunits are present in the ER membrane in equimolar amounts and co-fractionate in constant stoichiometry when rough and smooth liver microsomes are separated on sucrose gradients. They therefore conclude that SR..beta.. is an integral component of SRP receptor. The presence of SR..beta.. was previously masked by proteolytic breakdown products of SR..cap alpha.. observed by others and by the presence of another 30-kD ER membrane protein (mp30) which co-purifies with SR..cap alpha... Mp30 binds to SRP-Sepharose directly and is present in the ER membrane in several-fold molar excess of SR..cap alpha.. and SR..beta... The affinity of mp30 for SRP suggests that it may serve a yet unknown function in protein translocation.

  3. The selectivity of water-based pyrophosphate recognition is tuned by metal substitution in dimetallic receptors.

    PubMed

    Svane, Simon; Kjeldsen, Frank; McKee, Vickie; McKenzie, Christine J

    2015-07-14

    The three dimetallic compounds [Ga2(bpbp)(OH)2(H2O)2](ClO4)3, [In2(bpbp)(CH3CO2)2](ClO4)3 and [Zn2(bpbp)(HCO2)2](ClO4) (bpbp(-) = 2,6-bis((N,N'-bis(2-picolyl)amino)methyl)-4-tertbutylphenolate) were evaluated as stable solid state precursors for reactive solution state receptors to use for the recognition of the biologically important anion pyrophosphate in water at neutral pH. Indicator displacement assays using in situ generated complex-pyrocatechol violet adducts, {M2(bpbp)(HxPV)}(n+) M = Ga(3+), In(3+), Zn(2+), were tested for selectivity in their reactions with a series of common anions: pyrophosphate, phosphate, ATP, arsenate, nitrate, perchlorate, chloride, sulfate, formate, carbonate and acetate. The receptor employing Ga(3+) showed a slow but visually detectable response (blue to yellow) in the presence of one equivalent of pyrophosphate but no response to any other anion, even when they were present in much higher concentrations. The systems based on In(3+) or Zn(2+) show less selectivity in accord with visibly discernible responses to several of the anions. These results demonstrate a facile method for increasing anion selectivity without modification of an organic dinucleating ligand scaffold. The comfortable supramolecular recognition of pyrophosphate by the dimetallic complexes is demonstrated by the single crystal X-ray structure of [Ga2(bpbp)(HP2O7)](ClO4)2 in which the pyrophosphate is coordinated to the two gallium ions via four of its oxygen atoms. PMID:26057368

  4. Neural Androgen Receptors Modulate Gene Expression and Social Recognition But Not Social Investigation

    PubMed Central

    Karlsson, Sara A.; Studer, Erik; Kettunen, Petronella; Westberg, Lars

    2016-01-01

    The role of sex and androgen receptors (ARs) for social preference and social memory is rather unknown. In this study of mice we compared males, females and males lacking ARs specifically in the nervous system, ARNesDel, with respect to social preference, assessed with the three-chambered apparatus test, and social recognition, assessed with the social discrimination procedure. In the social discrimination test we also evaluated the tentative importance of the sex of the stimulus animal. Novel object recognition and olfaction were investigated to complement the results from the social tests. Gene expression analysis was performed to reveal molecules involved in the effects of sex and androgens on social behaviors. All three test groups showed social preference in the three-chambered apparatus test. In both social tests an AR-independent sexual dimorphism was seen in the persistence of social investigation of female conspecifics, whereas the social interest toward male stimuli mice was similar in all groups. Male and female controls recognized conspecifics independent of their sex, whereas ARNesDel males recognized female but not male stimuli mice. Moreover, the non-social behaviors were not affected by AR deficiency. The gene expression analyses of hypothalamus and amygdala indicated that Oxtr, Cd38, Esr1, Cyp19a1, Ucn3, Crh, and Gtf2i were differentially expressed between the three groups. In conclusion, our results suggest that ARs are required for recognition of male but not female conspecifics, while being dispensable for social investigation toward both sexes. In addition, the AR seems to regulate genes related to oxytocin, estrogen and William’s syndrome. PMID:27014003

  5. Atypical natural killer T-cell receptor recognition of CD1d-lipid antigens.

    PubMed

    Le Nours, Jérôme; Praveena, T; Pellicci, Daniel G; Gherardin, Nicholas A; Ross, Fiona J; Lim, Ricky T; Besra, Gurdyal S; Keshipeddy, Santosh; Richardson, Stewart K; Howell, Amy R; Gras, Stephanie; Godfrey, Dale I; Rossjohn, Jamie; Uldrich, Adam P

    2016-01-01

    Crucial to Natural Killer T (NKT) cell function is the interaction between their T-cell receptor (TCR) and CD1d-antigen complex. However, the diversity of the NKT cell repertoire and the ensuing interactions with CD1d-antigen remain unclear. We describe an atypical population of CD1d-α-galactosylceramide (α-GalCer)-reactive human NKT cells that differ markedly from the prototypical TRAV10-TRAJ18-TRBV25-1(+) type I NKT cell repertoire. These cells express a range of TCR α- and β-chains that show differential recognition of glycolipid antigens. Two atypical NKT TCRs (TRAV21-TRAJ8-TRBV7-8 and TRAV12-3-TRAJ27-TRBV6-5) bind orthogonally over the A'-pocket of CD1d, adopting distinct docking modes that contrast with the docking mode of all type I NKT TCR-CD1d-antigen complexes. Moreover, the interactions with α-GalCer differ between the type I and these atypical NKT TCRs. Accordingly, diverse NKT TCR repertoire usage manifests in varied docking strategies and specificities towards CD1d-α-GalCer and related antigens, thus providing far greater scope for diverse glycolipid antigen recognition. PMID:26875526

  6. Effects of the uncompetitive nmda receptor antagonist memantine on recognition memory in rats.

    PubMed

    Dashniani, M; Burjanadze, M; Beselia, G; Chkhikvishvili, N; Naneishvili, T

    2010-06-01

    Memantine is an NMDA receptor antagonist that has been recently approved in EU for the treatment of moderate to severe Alzheimer's disease. The previous studies have not allowed for the evaluation of the possible effects of this drug at therapeutic doses on different forms of memory. To address this question, we administered memantine to adult rats, using doses 2.5 or 5 mg/kg and evaluated the effects of these doses on open field activity and recognition memory. Memantine or saline was administered daily by intraperitoneal injection beginning on the day of behavioral testing and continuing 5 days. The main results of experiments are as follows: the memantine treatment produced a dose-related suppression of total ambulations. There was no significant impairment in detecting spatial and object novelty in the 2.5 mg/kg memantine treated rats. However, the 5 mg/kg intraperitoneal dose of memantine disrupted both recognition memory and locomotor behaviors. Our evaluation of memantine reveals that at doses lower than are required for neuroprotection disrupt memory. This raises the possibility that the beneficial effects seen in AD patients may be attributable to the interaction of memantine with other transmitter systems. PMID:20622272

  7. Atypical natural killer T-cell receptor recognition of CD1d–lipid antigens

    PubMed Central

    Le Nours, Jérôme; Praveena, T.; Pellicci, Daniel G.; Gherardin, Nicholas A.; Ross, Fiona J.; Lim, Ricky T.; Besra, Gurdyal S.; Keshipeddy, Santosh; Richardson, Stewart K.; Howell, Amy R.; Gras, Stephanie; Godfrey, Dale I.; Rossjohn, Jamie; Uldrich, Adam P.

    2016-01-01

    Crucial to Natural Killer T (NKT) cell function is the interaction between their T-cell receptor (TCR) and CD1d-antigen complex. However, the diversity of the NKT cell repertoire and the ensuing interactions with CD1d-antigen remain unclear. We describe an atypical population of CD1d–α-galactosylceramide (α-GalCer)-reactive human NKT cells that differ markedly from the prototypical TRAV10-TRAJ18-TRBV25-1+ type I NKT cell repertoire. These cells express a range of TCR α- and β-chains that show differential recognition of glycolipid antigens. Two atypical NKT TCRs (TRAV21-TRAJ8-TRBV7–8 and TRAV12-3-TRAJ27-TRBV6-5) bind orthogonally over the A′-pocket of CD1d, adopting distinct docking modes that contrast with the docking mode of all type I NKT TCR-CD1d-antigen complexes. Moreover, the interactions with α-GalCer differ between the type I and these atypical NKT TCRs. Accordingly, diverse NKT TCR repertoire usage manifests in varied docking strategies and specificities towards CD1d–α-GalCer and related antigens, thus providing far greater scope for diverse glycolipid antigen recognition. PMID:26875526

  8. Selection and identification of ligand peptides targeting a model of castrate-resistant osteogenic prostate cancer and their receptors.

    PubMed

    Mandelin, Jami; Cardó-Vila, Marina; Driessen, Wouter H P; Mathew, Paul; Navone, Nora M; Lin, Sue-Hwa; Logothetis, Christopher J; Rietz, Anna Cecilia; Dobroff, Andrey S; Proneth, Bettina; Sidman, Richard L; Pasqualini, Renata; Arap, Wadih

    2015-03-24

    We performed combinatorial peptide library screening in vivo on a novel human prostate cancer xenograft that is androgen-independent and induces a robust osteoblastic reaction in bonelike matrix and soft tissue. We found two peptides, PKRGFQD and SNTRVAP, which were enriched in the tumors, targeted the cell surface of androgen-independent prostate cancer cells in vitro, and homed to androgen receptor-null prostate cancer in vivo. Purification of tumor homogenates by affinity chromatography on these peptides and subsequent mass spectrometry revealed a receptor for the peptide PKRGFQD, α-2-macroglobulin, and for SNTRVAP, 78-kDa glucose-regulated protein (GRP78). These results indicate that GRP78 and α-2-macroglobulin are highly active in osteoblastic, androgen-independent prostate cancer in vivo. These previously unidentified ligand-receptor systems should be considered for targeted drug development against human metastatic androgen-independent prostate cancer. PMID:25762070

  9. Molecular recognition of human ephrinB2 cell surface receptor by an emergent African henipavirus.

    PubMed

    Lee, Benhur; Pernet, Olivier; Ahmed, Asim A; Zeltina, Antra; Beaty, Shannon M; Bowden, Thomas A

    2015-04-28

    The discovery of African henipaviruses (HNVs) related to pathogenic Hendra virus (HeV) and Nipah virus (NiV) from Southeast Asia and Australia presents an open-ended health risk. Cell receptor use by emerging African HNVs at the stage of host-cell entry is a key parameter when considering the potential for spillover and infection of human populations. The attachment glycoprotein from a Ghanaian bat isolate (GhV-G) exhibits <30% sequence identity with Asiatic NiV-G/HeV-G. Here, through functional and structural analysis of GhV-G, we show how this African HNV targets the same human cell-surface receptor (ephrinB2) as the Asiatic HNVs. We first characterized this virus-receptor interaction crystallographically. Compared with extant HNV-G-ephrinB2 structures, there was significant structural variation in the six-bladed β-propeller scaffold of the GhV-G receptor-binding domain, but not the Greek key fold of the bound ephrinB2. Analysis revealed a surprisingly conserved mode of ephrinB2 interaction that reflects an ongoing evolutionary constraint among geographically distal and phylogenetically divergent HNVs to maintain the functionality of ephrinB2 recognition during virus-host entry. Interestingly, unlike NiV-G/HeV-G, we could not detect binding of GhV-G to ephrinB3. Comparative structure-function analysis further revealed several distinguishing features of HNV-G function: a secondary ephrinB2 interaction site that contributes to more efficient ephrinB2-mediated entry in NiV-G relative to GhV-G and cognate residues at the very C terminus of GhV-G (absent in Asiatic HNV-Gs) that are vital for efficient receptor-induced fusion, but not receptor binding per se. These data provide molecular-level details for evaluating the likelihood of African HNVs to spill over into human populations. PMID:25825759

  10. Molecular recognition of human ephrinB2 cell surface receptor by an emergent African henipavirus

    PubMed Central

    Lee, Benhur; Pernet, Olivier; Ahmed, Asim A.; Zeltina, Antra; Beaty, Shannon M.; Bowden, Thomas A.

    2015-01-01

    The discovery of African henipaviruses (HNVs) related to pathogenic Hendra virus (HeV) and Nipah virus (NiV) from Southeast Asia and Australia presents an open-ended health risk. Cell receptor use by emerging African HNVs at the stage of host-cell entry is a key parameter when considering the potential for spillover and infection of human populations. The attachment glycoprotein from a Ghanaian bat isolate (GhV-G) exhibits <30% sequence identity with Asiatic NiV-G/HeV-G. Here, through functional and structural analysis of GhV-G, we show how this African HNV targets the same human cell-surface receptor (ephrinB2) as the Asiatic HNVs. We first characterized this virus−receptor interaction crystallographically. Compared with extant HNV-G–ephrinB2 structures, there was significant structural variation in the six-bladed β-propeller scaffold of the GhV-G receptor-binding domain, but not the Greek key fold of the bound ephrinB2. Analysis revealed a surprisingly conserved mode of ephrinB2 interaction that reflects an ongoing evolutionary constraint among geographically distal and phylogenetically divergent HNVs to maintain the functionality of ephrinB2 recognition during virus–host entry. Interestingly, unlike NiV-G/HeV-G, we could not detect binding of GhV-G to ephrinB3. Comparative structure–function analysis further revealed several distinguishing features of HNV-G function: a secondary ephrinB2 interaction site that contributes to more efficient ephrinB2-mediated entry in NiV-G relative to GhV-G and cognate residues at the very C terminus of GhV-G (absent in Asiatic HNV-Gs) that are vital for efficient receptor-induced fusion, but not receptor binding per se. These data provide molecular-level details for evaluating the likelihood of African HNVs to spill over into human populations. PMID:25825759

  11. Capturing intercellular sugar-mediated ligand-receptor recognitions via a simple yet highly biospecific interfacial system

    NASA Astrophysics Data System (ADS)

    Li, Zhen; Deng, Si-Si; Zang, Yi; Gu, Zhen; He, Xiao-Peng; Chen, Guo-Rong; Chen, Kaixian; James, Tony D.; Li, Jia; Long, Yi-Tao

    2013-07-01

    Intercellular ligand-receptor recognitions are crucial natural interactions that initiate a number of biological and pathological events. We present here the simple construction of a unique class of biomimetic interfaces based on a graphene-mediated self-assembly of glycosyl anthraquinones to a screen-printed electrode for the detection of transmembrane glycoprotein receptors expressed on a hepatoma cell line. We show that an electroactive interface confined with densely clustered galactosyl ligands is able to ingeniously recognize the asialoglycoprotein receptors on live Hep-G2 cells employing simple electrochemical techniques. The only facility used is a personal laptop in connection with a cheap and portable electrochemical workstation.

  12. 2'Fluoro Modification Differentially Modulates the Ability of RNAs to Activate Pattern Recognition Receptors.

    PubMed

    Lee, Youngju; Urban, Johannes H; Xu, Li; Sullenger, Bruce A; Lee, Jaewoo

    2016-06-01

    Although the use of RNAs has enormous therapeutic potential, these RNA-based therapies can trigger unwanted inflammatory responses by the activation of pattern recognition receptors (PRRs) and cause harmful side effects. In contrast, the immune activation by therapeutic RNAs can be advantageous for treating cancers. Thus, the immunogenicity of therapeutic RNAs should be deliberately controlled depending on the therapeutic applications of RNAs. In this study, we demonstrated that RNAs containing 2'fluoro (2'F) pyrimidines differentially controlled the activation of PRRs. The activity of RNAs that stimulate toll-like receptors 3 and 7 was abrogated by the incorporation of 2'F pyrimidine. By contrast, incorporation of 2'F pyrimidines enhanced the activity of retinoic acid-inducible gene 1-stimulating RNAs. Furthermore, we found that transfection with RNAs containing 2'F pyrimidine and 5' triphosphate (5'ppp) increased cell death and interferon-β expression in human cancer cells compared with transfection with 2'hydroxyl 5'ppp RNAs, whereas RNAs containing 2'O-methyl pyrimidine and 5'ppp completely abolished the induction of cell death and cytokine expression in the cells. Our findings suggest that incorporation of 2'F and 2'O-methyl nucleosides is a facile approach to differentially control the ability of therapeutic RNAs to activate or limit immune and inflammatory responses depending on therapeutic applications. PMID:26789413

  13. Lipid activation of the signal recognition particle receptor provides spatial coordination of protein targeting

    PubMed Central

    Lam, Vinh Q.; Akopian, David; Rome, Michael; Henningsen, Doug

    2010-01-01

    The signal recognition particle (SRP) and SRP receptor comprise the major cellular machinery that mediates the cotranslational targeting of proteins to cellular membranes. It remains unclear how the delivery of cargos to the target membrane is spatially coordinated. We show here that phospholipid binding drives important conformational rearrangements that activate the bacterial SRP receptor FtsY and the SRP–FtsY complex. This leads to accelerated SRP–FtsY complex assembly, and allows the SRP–FtsY complex to more efficiently unload cargo proteins. Likewise, formation of an active SRP–FtsY GTPase complex exposes FtsY’s lipid-binding helix and enables stable membrane association of the targeting complex. Thus, membrane binding, complex assembly with SRP, and cargo unloading are inextricably linked to each other via conformational changes in FtsY. These allosteric communications allow the membrane delivery of cargo proteins to be efficiently coupled to their subsequent unloading and translocation, thus providing spatial coordination during protein targeting. PMID:20733058

  14. The phylogenetic origins of natural killer receptors and recognition:relationships, possibilities and realities

    PubMed Central

    Yoder, Jeffrey A.; Litman, Gary W.

    2013-01-01

    Natural killer (NK) cells effect a form of innate immunity that recognizes and eliminates cells that are infected with certain viruses or have undergone malignant transformation. In mammals, this recognition can be mediated through immunoglobulin- (Ig) and/or lectin-type NK receptors (NKRs). NKR genes in mammals range from minimally polymorphic single copy genes to complex multigene families that exhibit high levels of haplotypic complexity and exhibit significant interspecific variation. Certain single copy NKR genes that are present in one mammal are present as expanded multi-gene families in other mammals. These observations highlight NKRs as one of the most rapidly evolving eukaryotic gene families and likely reflect the influence of pathogens, especially viruses, on their evolution. Although well characterized in human and mice, cytotoxic cells that are functionally similar to NK cells have been identified in species ranging from birds to reptiles, amphibians and fish. Although numerous receptors have been identified in non-mammalian vertebrates that share structural relationships with mammalian NKRs, functionally defining these lower vertebrate molecules as NKRs is confounded by methodological and interpretive complexities. Nevertheless, several lines of evidence suggest that NK-type function or its equivalent has sustained a long evolutionary history throughout vertebrate species. PMID:21191578

  15. Anion recognition by simple chromogenic and chromo-fluorogenic salicylidene Schiff base or reduced-Schiff base receptors

    NASA Astrophysics Data System (ADS)

    Dalapati, Sasanka; Jana, Sankar; Guchhait, Nikhil

    2014-08-01

    This review contains extensive application of anion sensing ability of salicylidene type Schiff bases and their reduced forms having various substituents with respect to phenolic sbnd OH group. Some of these molecular systems behave as receptor for recognition or sensing of various anions in organic or aqueous-organic binary solvent mixture as well as in the solid supported test kits. Development of Schiff base or reduced Schiff base receptors for anion recognition event is commonly based on the theory of hydrogen bonding interaction or deprotonation of phenolic -OH group. The process of charge transfer (CT) or inhibition of excited proton transfer (ESIPT) or followed by photo-induced electron transfer (PET) lead to naked-eye color change, UV-vis spectral change, chemical shift in the NMR spectra and fluorescence spectral modifications. In this review we have tried to discuss about the anion sensing properties of Schiff base or reduced Schiff base receptors.

  16. [Effect of baicalin on pattern recognition receptor TLR2/4-NOD2 and its significance of druggability].

    PubMed

    Chai, Yu-Shuang; Lei, Fan; Xing, Dong-Ming; Ding, Yi; Du, Li-Jun

    2013-08-01

    Activation pattern recognition receptors can cause the startup of downstream signaling pathways, the expression of inflammatory factors, and finally immunological inflammatory reaction. Either exogenous pathogenic microorganisms or endogenous tissue components can activate these pattern recognition receptors as ligands at varying degrees, and then cause the immunological inflammatory reaction. Therefore, it is of great significance to inhibit relevant receptors, as well as the immunological inflammatory reaction, in order to avoid tissue injury during the course of disease. Baicalin is able to specifically inhibit the expression of TLR2/4-NOD2, inhibit the expression of inflammatory factors IL-1beta, IL-6 and TNF-alpha, and thereby reducing the injury of the tissue cells during the course of disease. This effect is non-specific with tissues, which is of great theoretical and practical significance in druggability. In addition, the drug metabolism and toxicity of baicalin are also discussed for its druggability in this article. PMID:24228579

  17. Roles for glycosylation of cell surface receptors involved in cellular immune recognition.

    PubMed

    Rudd, P M; Wormald, M R; Stanfield, R L; Huang, M; Mattsson, N; Speir, J A; DiGennaro, J A; Fetrow, J S; Dwek, R A; Wilson, I A

    1999-10-22

    The majority of cell surface receptors involved in antigen recognition by T cells and in the orchestration of the subsequent cell signalling events are glycoproteins. The length of a typical N-linked sugar is comparable with that of an immunoglobulin domain (30 A). Thus, by virtue of their size alone, oligosaccharides may be expected to play a significant role in the functions and properties of the cell surface proteins to which they are attached. A databank of oligosaccharide structures has been constructed from NMR and crystallographic data to aid in the interpretation of crystal structures of glycoproteins. As unambiguous electron density can usually only be assigned to the glycan cores, the remainder of the sugar is then modelled into the crystal lattice by superimposing the appropriate oligosaccharide from the database. This approach provides insights into the roles that glycosylation might play in cell surface receptors, by providing models that delineate potential close packing interactions on the cell surface. It has been proposed that the specific recognition of antigen by T cells results in the formation of an immunological synapse between the T cell and the antigen-presenting cell. The cell adhesion glycoproteins, such as CD2 and CD48, help to form a cell junction, providing a molecular spacer between opposing cells. The oligosaccharides located on the membrane proximal domains of CD2 and CD48 provide a scaffold to orient the binding faces, which leads to increased affinity. In the next step, recruitment of the peptide major histocompatibility complex (pMHC) by the T-cell receptors (TCRs) requires mobility on the membrane surface. The TCR sugars are located such that they could prevent non-specific aggregation. Importantly, the sugars limit the possible geometry and spacing of TCR/MHC clusters which precede cell signalling. We postulate that, in the final stage, the sugars could play a general role in controlling the assembly and stabilisation of the

  18. Recognition and Activation Domains Contribute to Allele-Specific Responses of an Arabidopsis NLR Receptor to an Oomycete Effector Protein

    PubMed Central

    Steinbrenner, Adam D.; Goritschnig, Sandra; Staskawicz, Brian J.

    2015-01-01

    In plants, specific recognition of pathogen effector proteins by nucleotide-binding leucine-rich repeat (NLR) receptors leads to activation of immune responses. RPP1, an NLR from Arabidopsis thaliana, recognizes the effector ATR1, from the oomycete pathogen Hyaloperonospora arabidopsidis, by direct association via C-terminal leucine-rich repeats (LRRs). Two RPP1 alleles, RPP1-NdA and RPP1-WsB, have narrow and broad recognition spectra, respectively, with RPP1-NdA recognizing a subset of the ATR1 variants recognized by RPP1-WsB. In this work, we further characterized direct effector recognition through random mutagenesis of an unrecognized ATR1 allele, ATR1-Cala2, screening for gain-of-recognition phenotypes in a tobacco hypersensitive response assay. We identified ATR1 mutants that a) confirm surface-exposed residues contribute to recognition by RPP1, and b) are recognized by and activate the narrow-spectrum allele RPP1-NdA, but not RPP1-WsB, in co-immunoprecipitation and bacterial growth inhibition assays. Thus, RPP1 alleles have distinct recognition specificities, rather than simply different sensitivity to activation. Using chimeric RPP1 constructs, we showed that RPP1-NdA LRRs were sufficient for allele-specific recognition (association with ATR1), but insufficient for receptor activation in the form of HR. Additional inclusion of the RPP1-NdA ARC2 subdomain, from the central NB-ARC domain, was required for a full range of activation specificity. Thus, cooperation between recognition and activation domains seems to be essential for NLR function. PMID:25671309

  19. Crystal structure of PXY-TDIF complex reveals a conserved recognition mechanism among CLE peptide-receptor pairs

    PubMed Central

    Zhang, Heqiao; Lin, Xiaoya; Han, Zhifu; Qu, Li-Jia; Chai, Jijie

    2016-01-01

    Plants can achieve amazing lifespans because of their continuous and repetitive formation of new organs by stem cells present within meristems. The balance between proliferation and differentiation of meristem cells is largely regulated by the CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) peptide hormones. One of the well-characterized CLE peptides, CLE41/TDIF (tracheary elements differentiation inhibitory factor), functions to suppress tracheary element differentiation and promote procambial cell proliferation, playing important roles in vascular development and wood formation. The recognition mechanisms of TDIF or other CLE peptides by their respective receptors, however, remain largely elusive. Here we report the crystal structure of TDIF in complex with its receptor PXY, a leucine-rich repeat receptor kinase (LRR-RK). Our structure reveals that TDIF mainly adopts an “Ω”-like conformation binding to the inner surface of the LRR domain of PXY. Interaction between TDIF and PXY is predominately mediated by the relatively conserved amino acids of TDIF. Structure-based sequence alignment showed that the TDIF-interacting motifs are also conserved among other known CLE receptors. Our data provide a structural template for understanding the recognition mechanism of CLE peptides by their receptors, offering an opportunity for the identification of receptors of other uncharacterized CLE peptides. PMID:27055373

  20. Crystal structure of PXY-TDIF complex reveals a conserved recognition mechanism among CLE peptide-receptor pairs.

    PubMed

    Zhang, Heqiao; Lin, Xiaoya; Han, Zhifu; Qu, Li-Jia; Chai, Jijie

    2016-05-01

    Plants can achieve amazing lifespans because of their continuous and repetitive formation of new organs by stem cells present within meristems. The balance between proliferation and differentiation of meristem cells is largely regulated by the CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) peptide hormones. One of the well-characterized CLE peptides, CLE41/TDIF (tracheary elements differentiation inhibitory factor), functions to suppress tracheary element differentiation and promote procambial cell proliferation, playing important roles in vascular development and wood formation. The recognition mechanisms of TDIF or other CLE peptides by their respective receptors, however, remain largely elusive. Here we report the crystal structure of TDIF in complex with its receptor PXY, a leucine-rich repeat receptor kinase (LRR-RK). Our structure reveals that TDIF mainly adopts an "Ω"-like conformation binding to the inner surface of the LRR domain of PXY. Interaction between TDIF and PXY is predominately mediated by the relatively conserved amino acids of TDIF. Structure-based sequence alignment showed that the TDIF-interacting motifs are also conserved among other known CLE receptors. Our data provide a structural template for understanding the recognition mechanism of CLE peptides by their receptors, offering an opportunity for the identification of receptors of other uncharacterized CLE peptides. PMID:27055373

  1. Leucine-rich Repeats of Bacterial Surface Proteins Serve as Common Pattern Recognition Motifs of Human Scavenger Receptor gp340*

    PubMed Central

    Loimaranta, Vuokko; Hytönen, Jukka; Pulliainen, Arto T.; Sharma, Ashu; Tenovuo, Jorma; Strömberg, Nicklas; Finne, Jukka

    2009-01-01

    Scavenger receptors are innate immune molecules recognizing and inducing the clearance of non-host as well as modified host molecules. To recognize a wide pattern of invading microbes, many scavenger receptors bind to common pathogen-associated molecular patterns, such as lipopolysaccharides and lipoteichoic acids. Similarly, the gp340/DMBT1 protein, a member of the human scavenger receptor cysteine-rich protein family, displays a wide ligand repertoire. The peptide motif VEVLXXXXW derived from its scavenger receptor cysteine-rich domains is involved in some of these interactions, but most of the recognition mechanisms are unknown. In this study, we used mass spectrometry sequencing, gene inactivation, and recombinant proteins to identify Streptococcus pyogenes protein Spy0843 as a recognition receptor of gp340. Antibodies against Spy0843 are shown to protect against S. pyogenes infection, but no function or host receptor have been identified for the protein. Spy0843 belongs to the leucine-rich repeat (Lrr) family of eukaryotic and prokaryotic proteins. Experiments with truncated forms of the recombinant proteins confirmed that the Lrr region is needed in the binding of Spy0843 to gp340. The same motif of two other Lrr proteins, LrrG from the Gram-positive S. agalactiae and BspA from the Gram-negative Tannerella forsythia, also mediated binding to gp340. Moreover, inhibition of Spy0843 binding occurred with peptides containing the VEVLXXXXW motif, but also peptides devoid of the XXXXW motif inhibited binding of Lrr proteins. These results thus suggest that the conserved Lrr motif in bacterial proteins serves as a novel pattern recognition motif for unique core peptides of human scavenger receptor gp340. PMID:19465482

  2. A galectin from Eriocheir sinensis functions as pattern recognition receptor enhancing microbe agglutination and haemocytes encapsulation.

    PubMed

    Wang, Mengqiang; Wang, Lingling; Huang, Mengmeng; Yi, Qilin; Guo, Ying; Gai, Yunchao; Wang, Hao; Zhang, Huan; Song, Linsheng

    2016-08-01

    Galectins are a family of β-galactoside binding lectins that function as pattern recognition receptors (PRRs) in innate immune system of both vertebrates and invertebrates. The cDNA of Chinese mitten crab Eriocheir sinensis galectin (designated as EsGal) was cloned via rapid amplification of cDNA ends (RACE) technique based on expressed sequence tags (ESTs) analysis. The full-length cDNA of EsGal was 999 bp. Its open reading frame encoded a polypeptide of 218 amino acids containing a GLECT/Gal-bind_lectin domain and a proline/glycine rich low complexity region. The deduced amino acid sequence and domain organization of EsGal were highly similar to those of crustacean galectins. The mRNA transcripts of EsGal were found to be constitutively expressed in a wide range of tissues and mainly in hepatopancreas, gill and haemocytes. The mRNA expression level of EsGal increased rapidly and significantly after crabs were stimulated by different microbes. The recombinant EsGal (rEsGal) could bind various pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan (PGN) and glucan (GLU), and exhibited strong activity to agglutinate Escherichia coli, Vibrio anguillarum, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus and Pichia pastoris, and such agglutinating activity could be inhibited by both d-galactose and α-lactose. The in vitro encapsulation assay revealed that rEsGal could enhance the encapsulation of haemocytes towards agarose beads. These results collectively suggested that EsGal played crucial roles in the immune recognition and elimination of pathogens and contributed to the innate immune response against various microbes in crabs. PMID:27095174

  3. Galectins as self/non-self recognition receptors in innate and adaptive immunity: an unresolved paradox

    PubMed Central

    Vasta, Gerardo R.; Ahmed, Hafiz; Nita-Lazar, Mihai; Banerjee, Aditi; Pasek, Marta; Shridhar, Surekha; Guha, Prasun; Fernández-Robledo, José A.

    2012-01-01

    Galectins are characterized by their binding affinity for β-galactosides, a unique binding site sequence motif, and wide taxonomic distribution and structural conservation in vertebrates, invertebrates, protista, and fungi. Since their initial description, galectins were considered to bind endogenous (“self”) glycans and mediate developmental processes and cancer. In the past few years, however, numerous studies have described the diverse effects of galectins on cells involved in both innate and adaptive immune responses, and the mechanistic aspects of their regulatory roles in immune homeostasis. More recently, however, evidence has accumulated to suggest that galectins also bind exogenous (“non-self”) glycans on the surface of potentially pathogenic microbes, parasites, and fungi, suggesting that galectins can function as pattern recognition receptors (PRRs) in innate immunity. Thus, a perplexing paradox arises by the fact that galectins also recognize lactosamine-containing glycans on the host cell surface during developmental processes and regulation of immune responses. According to the currently accepted model for non-self recognition, PRRs recognize pathogens via highly conserved microbial surface molecules of wide distribution such as LPS or peptidoglycan (pathogen-associated molecular patterns; PAMPs), which are absent in the host. Hence, this would not apply to galectins, which apparently bind similar self/non-self molecular patterns on host and microbial cells. This paradox underscores first, an oversimplification in the use of the PRR/PAMP terminology. Second, and most importantly, it reveals significant gaps in our knowledge about the diversity of the host galectin repertoire, and the subcellular targeting, localization, and secretion. Furthermore, our knowledge about the structural and biophysical aspects of their interactions with the host and microbial carbohydrate moieties is fragmentary, and warrants further investigation. PMID:22811679

  4. Molecular Recognition of Agonist and Antagonist for Peroxisome Proliferator-Activated Receptor-α Studied by Molecular Dynamics Simulations

    PubMed Central

    Liu, Mengyuan; Wang, Lushan; Zhao, Xian; Sun, Xun

    2014-01-01

    Peroxisome proliferator activated receptor-α (PPAR-α) is a ligand-activated transcription factor which plays important roles in lipid and glucose metabolism. The aim of this work is to find residues which selectively recognize PPAR-α agonists and antagonists. To achieve this aim, PPAR-α/13M and PPAR-α/471 complexes were subjected to perform molecular dynamics simulations. This research suggests that several key residues only participate in agonist recognition, while some other key residues only contribute to antagonist recognition. It is hoped that such work is useful for medicinal chemists to design novel PPAR-α agonists and antagonists. PMID:24837836

  5. Sphingosine 1-phosphate analogue recognition and selectivity at S1P4 within the endothelial differentiation gene family of receptors

    PubMed Central

    Inagaki, Yuichi; Pham, TrucChi T.; Fujiwara, Yuko; Kohno, Takayuki; Osborne, Daniel A.; Igarashi, Yasuyuki; Tigyi, Gabor; Parrill, Abby L.

    2005-01-01

    Synergistic computational and experimental studies provided previously unforeseen details concerning the structural basis of S1P (sphingosine 1-phosphate) recognition by the S1P4 G-protein-coupled receptor. Similarly to reports on the S1P1 receptor, cationic and anionic residues in the third transmembrane domain (R3.28 and E3.29 at positions 124 and 125) form ion pairs with the phosphate and ammonium of S1P, and alanine mutations at these positions abolished specific S1P binding, S1P-induced receptor activation and cell migration. Unlike findings on the S1P1 receptor, no cationic residue in the seventh transmembrane domain interacts with the phosphate. Additionally, two previously undiscovered interactions with the S1P polar headgroup have been identified. Trp186 at position 4.64 in the fourth transmembrane domain interacts by a cation-π interaction with the ammonium group of S1P. Lys204 at position 5.38 forms an ion pair with the S1P. The S1P4 and S1P1 receptors show differences in binding-pocket shape and electrostatic distributions that correlate with the published structure–activity relationships. In particular, the binding pocket of mS1P4 (mouse S1P4) has recognition sites for the anionic phosphate and cationic ammonium groups that are equidistant from the end of the non-polar tail. In contrast, the binding pocket of hS1P1 (human S1P4) places the ammonium recognition site 2 Å (1 Å=0.1 nm) closer to the end of the non-polar tail than the phosphate recognition site. PMID:15733055

  6. Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T-cells

    PubMed Central

    Hudecek, Michael; Lupo-Stanghellini, Maria-Teresa; Kosasih, Paula L.; Sommermeyer, Daniel; Jensen, Michael C.; Rader, Christoph; Riddell, Stanley R.

    2013-01-01

    Purpose The adoptive transfer of T-cells modified to express a chimeric antigen receptor (CAR) comprised of an extracellular single chain antibody (scFV) fragment specific for a tumor cell surface molecule, and linked to an intracellular signaling module has activity in advanced malignancies. ROR1 is a tumor-associated molecule expressed on prevalent B-lymphoid and epithelial cancers, and is absent on normal mature B-cells and vital tissues, making it a candidate for CAR T-cell therapy. Experimental Design We constructed ROR1-CARs from scFVs with different affinities and containing extracellular IgG4-Fc spacer domains of different lengths, and evaluated the ability of T-cells expressing each CAR to recognize ROR1+ hematopoietic and epithelial tumors in vitro, and to eliminate human mantle cell lymphoma engrafted into immunodeficient mice. Results ROR1-CARs containing a short ‘Hinge-only’ extracellular spacer conferred superior lysis of ROR1+ tumor cells and induction of T-cell effector functions compared to CARs with long ‘Hinge-CH2-CH3’ spacers. CARs derived from a higher affinity scFV conferred maximum T-cell effector function against primary CLL and ROR1+ epithelial cancer lines in vitro without inducing activation induced T-cell death. T-cells modified with an optimal ROR1-CAR were equivalently effective as CD19-CAR modified T-cells in mediating regression of JeKo-1 mantle cell lymphoma in immunodeficient mice. Conclusions Our results demonstrate that customizing spacer design and increasing affinity of ROR1-CARs enhances T-cell effector function and recognition of ROR1+ tumors. T-cells modified with an optimized ROR1-CAR have significant anti-tumor efficacy in a preclinical model in vivo, suggesting they may be useful to treat ROR1+ tumors in clinical applications. PMID:23620405

  7. Two Independent Histidines One in Human Prolactin and One in Its Receptor Are Critical for pH-dependent Receptor Recognition and Activation

    SciTech Connect

    M Kulkarni; M Tettamanzi; J Murphy; C Keeler; D Myszka; N Chayen; E Lolis; M Hodsdon

    2011-12-31

    Human prolactin (hPRL), a member of the family of hematopoietic cytokines, functions as both an endocrine hormone and autocrine/paracrine growth factor. We have previously demonstrated that recognition of the hPRL-receptor depends strongly on solution acidity over the physiologic range from pH 6 to pH 8. The hPRL-receptor binding interface contains four histidines whose protonation is hypothesized to regulate pH-dependent receptor recognition. Here, we systematically dissect its molecular origin by characterizing the consequences of His to Ala mutations on pH-dependent receptor binding kinetics, site-specific histidine protonation, and high resolution structures of the intermolecular interface. Thermodynamic modeling of the pH dependence to receptor binding affinity reveals large changes in site-specific protonation constants for a majority of interface histidines upon complexation. Removal of individual His imidazoles reduces these perturbations in protonation constants, which is most likely explained by the introduction of solvent-filled, buried cavities in the crystallographic structures without inducing significant conformational rearrangements.

  8. Functional analysis of pattern recognition receptors in miniature dachshunds with inflammatory colorectal polyps

    PubMed Central

    IGARASHI, Hirotaka; OHNO, Koichi; FUJIWARA-IGARASHI, Aki; KANEMOTO, Hideyuki; FUKUSHIMA, Kenjiro; GOTO-KOSHINO, Yuko; UCHIDA, Kazuyuki; TSUJIMOTO, Hajime

    2014-01-01

    Inflammatory colorectal polyps (ICRPs) frequently occur in miniature dachshunds (MDs) in Japan. MDs with ICRPs develop multiple polyps with severe neutrophil infiltration that respond to immunosuppressive therapy. Therefore, ICRPs are thought to constitute a novel, breed-specific form of canine inflammatory bowel disease (IBD). Pattern recognition receptors (PRRs) play a key role in the distinction of pathogens from commensal bacteria and food antigens. Dysfunction resulting from genetic disorders of PRRs have been linked to human and canine IBD. Therefore, we analyzed the reactivity of PRRs in MDs with ICRPs. Twenty-six MDs with ICRPs and 16 control MDs were recruited. Peripheral blood-derived monocytes were obtained from each dog and then stimulated with PRR ligands for 6 and 24 hr; subsequently, messenger RNA (mRNA) expression levels and protein secretion of IL-1β were quantified using quantitative real-time PCR and ELISA, respectively. The levels of IL-1β mRNA and protein secretion after stimulation with a nucleotide-binding oligomerization domain 2 (NOD2) ligand were significantly greater in monocytes from ICRP-affected MDs than in those from control MDs. In addition, IL-1β protein secretion induced by toll-like receptor (TLR) 1/2, TLR2 and TLR2/6 stimulation was also significantly greater in ICRP-affected MDs. These results suggest that reactivity against NOD2, TLR1/2, TLR2 and TLR2/6 signals is enhanced in ICRP-affected MDs and may play a role in the pathogenesis of ICRPs in MDs. Additional studies of the genetic background of these PRRs should be performed. PMID:25650150

  9. Increased Expression of Pattern Recognition Receptors and Nitric Oxide Synthase in Patients with Endometriosis

    PubMed Central

    Yeo, Seung Geun; Won, Yong Sung; Lee, Ho Yun; Kim, Young Il; Lee, Jin-Woo; Park, Dong Choon

    2013-01-01

    Objective: Endometriosis is characterized by repeated inflammatory changes and serious adhesions, inducing innate and adaptive immune responses within the abdominal cavity. To assess these immune responses, we evaluated the levels of expression of Toll-like receptors (TLR)-1, -2, -4, -5, and -9; nucleotide-binding oligomerization domains (NOD)-1 and -2; interleukins-1β, -6, -8, -10, and -12; interferon-γ; tumor necrosis factor-α; inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS); and immunoglobulins (Igs) in patients with endometriosis. Methods: The levels of TLRs, NODs, cytokines, and NOS mRNAs in peritoneal effusions were assessed by real time reverse transcription-polymerase chain reaction; and IgG, IgA and IgM concentrations were measured by enzyme-linked immunosorbent assays (ELISA) in 40 patients with and 40 without endometriosis. Findings from the two groups were compared. Results: We observed expression of all pattern recognition receptors (PRRs), cytokines, and NOS mRNAs and Igs in the effusion fluid of patients with and without endometriosis. The levels of TLR-2 and -9; NOD-1 and -2; iNOS and eNOS mRNAs and CA 125 were significantly higher in the endometriosis than in the non-endometriosis group (p<0.05 each). Moreover, PRR, cytokine, and NOS expression showed significant correlations (p<0.05). Conclusions: PRRs, cytokines, and NOS, which act cooperatively in the innate immune response, are closely associated with endometriosis. Increased expression of TLR-2, TLR -9, NOD-1, NOD-2, and NOS mRNA in peritoneal fluid may be associated with endometriosis. PMID:23935397

  10. Structural Basis of Natural Promoter Recognition by a Unique Nuclear Receptor, HNF4[alpha

    SciTech Connect

    Lu, Peng; Rha, Geun Bae; Melikishvili, Manana; Wu, Guangteng; Adkins, Brandon C.; Fried, Michael G.; Chi, Young-In

    2010-11-09

    HNF4{alpha} (hepatocyte nuclear factor 4{alpha}) plays an essential role in the development and function of vertebrate organs, including hepatocytes and pancreatic {beta}-cells by regulating expression of multiple genes involved in organ development, nutrient transport, and diverse metabolic pathways. As such, HNF4{alpha} is a culprit gene product for a monogenic and dominantly inherited form of diabetes, known as maturity onset diabetes of the young (MODY). As a unique member of the nuclear receptor superfamily, HNF4{alpha} recognizes target genes containing two hexanucleotide direct repeat DNA-response elements separated by one base pair (DR1) by exclusively forming a cooperative homodimer. We describe here the 2.0 {angstrom} crystal structure of human HNF4{alpha} DNA binding domain in complex with a high affinity promoter element of another MODY gene, HNF1{alpha}, which reveals the molecular basis of unique target gene selection/recognition, DNA binding cooperativity, and dysfunction caused by diabetes-causing mutations. The predicted effects of MODY mutations have been tested by a set of biochemical and functional studies, which show that, in contrast to other MODY gene products, the subtle disruption of HNF4{alpha} molecular function can cause significant effects in afflicted MODY patients.

  11. Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens.

    PubMed

    Cecil, Jessica D; O'Brien-Simpson, Neil M; Lenzo, Jason C; Holden, James A; Chen, Yu-Yen; Singleton, William; Gause, Katelyn T; Yan, Yan; Caruso, Frank; Reynolds, Eric C

    2016-01-01

    Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis. PMID:27035339

  12. Differential Responses of Pattern Recognition Receptors to Outer Membrane Vesicles of Three Periodontal Pathogens

    PubMed Central

    Lenzo, Jason C.; Holden, James A.; Chen, Yu-Yen; Singleton, William; Gause, Katelyn T.; Yan, Yan; Caruso, Frank; Reynolds, Eric C.

    2016-01-01

    Highly purified outer membrane vesicles (OMVs) of the periodontal pathogens, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia were produced using tangential flow ultrafiltration, ultracentrifugation and Optiprep density gradient separation. Cryo-TEM and light scattering showed OMVs to be single lipid-bilayers with modal diameters of 75 to 158 nm. Enumeration of OMVs by nanoparticle flow-cytometry at the same stage of late exponential culture indicated that P. gingivalis was the most prolific OMV producer. P. gingivalis OMVs induced strong TLR2 and TLR4-specific responses and moderate responses in TLR7, TLR8, TLR9, NOD1 and NOD2 expressing-HEK-Blue cells. Responses to T. forsythia OMVs were less than those of P. gingivalis and T. denticola OMVs induced only weak responses. Compositional analyses of OMVs from the three pathogens demonstrated differences in protein, fatty acids, lipopolysaccharide, peptidoglycan fragments and nucleic acids. Periodontal pathogen OMVs induced differential pattern recognition receptor responses that have implications for their role in chronic periodontitis. PMID:27035339

  13. Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon

    PubMed Central

    Kuhn, Patrick; Draycheva, Albena; Vogt, Andreas; Petriman, Narcis-Adrian; Sturm, Lukas; Drepper, Friedel; Warscheid, Bettina; Wintermeyer, Wolfgang

    2015-01-01

    Cotranslational protein targeting delivers proteins to the bacterial cytoplasmic membrane or to the eukaryotic endoplasmic reticulum membrane. The signal recognition particle (SRP) binds to signal sequences emerging from the ribosomal tunnel and targets the ribosome-nascent-chain complex (RNC) to the SRP receptor, termed FtsY in bacteria. FtsY interacts with the fifth cytosolic loop of SecY in the SecYEG translocon, but the functional role of the interaction is unclear. By using photo-cross-linking and fluorescence resonance energy transfer measurements, we show that FtsY–SecY complex formation is guanosine triphosphate independent but requires a phospholipid environment. Binding of an SRP–RNC complex exposing a hydrophobic transmembrane segment induces a rearrangement of the SecY–FtsY complex, which allows the subsequent contact between SecY and ribosomal protein uL23. These results suggest that direct RNC transfer to the translocon is guided by the interaction between SRP and translocon-bound FtsY in a quaternary targeting complex. PMID:26459600

  14. Structural basis for receptor recognition and pore formation of a zebrafish aerolysin-like protein.

    PubMed

    Jia, Ning; Liu, Nan; Cheng, Wang; Jiang, Yong-Liang; Sun, Hui; Chen, Lan-Lan; Peng, Junhui; Zhang, Yonghui; Ding, Yue-He; Zhang, Zhi-Hui; Wang, Xuejuan; Cai, Gang; Wang, Junfeng; Dong, Meng-Qiu; Zhang, Zhiyong; Wu, Hui; Wang, Hong-Wei; Chen, Yuxing; Zhou, Cong-Zhao

    2016-02-01

    Various aerolysin-like pore-forming proteins have been identified from bacteria to vertebrates. However, the mechanism of receptor recognition and/or pore formation of the eukaryotic members remains unknown. Here, we present the first crystal and electron microscopy structures of a vertebrate aerolysin-like protein from Danio rerio, termed Dln1, before and after pore formation. Each subunit of Dln1 dimer comprises a β-prism lectin module followed by an aerolysin module. Specific binding of the lectin module toward high-mannose glycans triggers drastic conformational changes of the aerolysin module in a pH-dependent manner, ultimately resulting in the formation of a membrane-bound octameric pore. Structural analyses combined with computational simulations and biochemical assays suggest a pore-forming process with an activation mechanism distinct from the previously characterized bacterial members. Moreover, Dln1 and its homologs are ubiquitously distributed in bony fishes and lamprey, suggesting a novel fish-specific defense molecule. PMID:26711430

  15. Structural Basis of Natural Promoter Recognition by a Unique Nuclear Receptor, HNF4α

    PubMed Central

    Lu, Peng; Rha, Geun Bae; Melikishvili, Manana; Wu, Guangteng; Adkins, Brandon C.; Fried, Michael G.; Chi, Young-In

    2008-01-01

    HNF4α (hepatocyte nuclear factor 4α) plays an essential role in the development and function of vertebrate organs, including hepatocytes and pancreatic β-cells by regulating expression of multiple genes involved in organ development, nutrient transport, and diverse metabolic pathways. As such, HNF4α is a culprit gene product for a monogenic and dominantly inherited form of diabetes, known as maturity onset diabetes of the young (MODY). As a unique member of the nuclear receptor superfamily, HNF4α recognizes target genes containing two hexanucleotide direct repeat DNA-response elements separated by one base pair (DR1) by exclusively forming a cooperative homodimer. We describe here the 2.0 Å crystal structure of human HNF4α DNA binding domain in complex with a high affinity promoter element of another MODY gene, HNF1α, which reveals the molecular basis of unique target gene selection/recognition, DNA binding cooperativity, and dysfunction caused by diabetes-causing mutations. The predicted effects of MODY mutations have been tested by a set of biochemical and functional studies, which show that, in contrast to other MODY gene products, the subtle disruption of HNF4α molecular function can cause significant effects in afflicted MODY patients. PMID:18829458

  16. T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex.

    PubMed

    Beringer, Dennis X; Kleijwegt, Fleur S; Wiede, Florian; van der Slik, Arno R; Loh, Khai Lee; Petersen, Jan; Dudek, Nadine L; Duinkerken, Gaby; Laban, Sandra; Joosten, Antoinette; Vivian, Julian P; Chen, Zhenjun; Uldrich, Adam P; Godfrey, Dale I; McCluskey, James; Price, David A; Radford, Kristen J; Purcell, Anthony W; Nikolic, Tatjana; Reid, Hugh H; Tiganis, Tony; Roep, Bart O; Rossjohn, Jamie

    2015-11-01

    Central to adaptive immunity is the interaction between the αβ T cell receptor (TCR) and peptide presented by the major histocompatibility complex (MHC) molecule. Presumably reflecting TCR-MHC bias and T cell signaling constraints, the TCR universally adopts a canonical polarity atop the MHC. We report the structures of two TCRs, derived from human induced T regulatory (iT(reg)) cells, complexed to an MHC class II molecule presenting a proinsulin-derived peptide. The ternary complexes revealed a 180° polarity reversal compared to all other TCR-peptide-MHC complex structures. Namely, the iT(reg) TCR α-chain and β-chain are overlaid with the α-chain and β-chain of MHC class II, respectively. Nevertheless, this TCR interaction elicited a peptide-reactive, MHC-restricted T cell signal. Thus TCRs are not 'hardwired' to interact with MHC molecules in a stereotypic manner to elicit a T cell signal, a finding that fundamentally challenges our understanding of TCR recognition. PMID:26437244

  17. A Novel Loop Domain in Superantigens Extends Their T Cell Receptor Recognition Site

    SciTech Connect

    Gunther,S.; Varma, A.; Moza, B.; Kasper, K.; Wyatt, A.; Zhu, P.; Nur-ur Rahman, A.; Li, Y.; Mariuzza, R.; et al.

    2007-01-01

    Superantigens (SAGs) interact with host immune receptors to induce a massive release of inflammatory cytokines that can lead to toxic shock syndrome and death. Bacterial SAGs can be classified into five distinct evolutionary groups. Group V SAGs are characterized by the {alpha}3-{beta}8 loop, a unique {approx}15 amino acid residue extension that is required for optimal T cell activation. Here, we report the X-ray crystal structures of the group V SAG staphylococcal enterotoxin K (SEK) alone and in complex with the TCR hV{beta}5.1 domain. SEK adopts a unique TCR binding orientation relative to other SAG-TCR complexes, which results in the {alpha}3-{beta}8 loop contacting the apical loop of framework region 4, thereby extending the known TCR recognition site of SAGs. These interactions are absolutely required for TCR binding and T cell activation by SEK, and dictate the TCR V{beta} domain specificity of SEK and other group V SAGs.

  18. Biomimetic Taste Receptors with Chiral Recognition by Photoluminescent Metal-Organic Frameworks Chelated with Polyaniline Helices.

    PubMed

    Lee, Tu; Lin, Tsung Yan; Lee, Hung Lin; Chang, Yun Hsuan; Tsai, Yee Chen

    2016-01-22

    The adsorption of phenylaniline (Phe) enantiomers on (+)-polyaniline (PAN)-chelated [In(OH)(bdc)]n microcrystals was carefully designed and studied by using the Job titration, circular dichroism, X-ray photoelectron spectroscopy, and photoluminescence to mimic heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors in selective, but not specific, ligand binding with chiral recognition and signal transduction. Six essential working principles across different length scales are unraveled: 1) a chiral (+)-PAN (host), 2) specific sites for Phe-(+)/PAN (guest-host) binding, 3) a conformational change of (+)-PAN after binding with Phe enantiomers, 4) different degrees of packing for (+)-PAN, 5) interactions between (+)-PAN and the underlying signal-generating framework (i.e., [In(OH)(bdc)]n microcrystals), and 6) a systematic photoluminescent signal combination by using principal-component analysis from the other three polymer-chelated metal-organic frameworkds (MOFs), such as poly(acrylic acid) (PAA), sodium alginate (SA), and polyvinylpyrrolidone (PVP) to enhance the selectivity and discrimination capabilities. PMID:26670931

  19. Structural insight into MR1-mediated recognition of the mucosal associated invariant T cell receptor

    PubMed Central

    Reantragoon, Rangsima; Kjer-Nielsen, Lars; Patel, Onisha; Chen, Zhenjun; Illing, Patricia T.; Bhati, Mugdha; Kostenko, Lyudmila; Bharadwaj, Mandvi; Meehan, Bronwyn; Hansen, Ted H.; Godfrey, Dale I.

    2012-01-01

    Mucosal-associated invariant T (MAIT) cells express a semiinvariant αβ T cell receptor (TCR) that binds MHC class I–like molecule (MR1). However, the molecular basis for MAIT TCR recognition by MR1 is unknown. In this study, we present the crystal structure of a human Vα7.2Jα33-Vβ2 MAIT TCR. Mutagenesis revealed highly conserved requirements for the MAIT TCR–MR1 interaction across different human MAIT TCRs stimulated by distinct microbial sources. Individual residues within the MAIT TCR β chain were dispensable for the interaction with MR1, whereas the invariant MAIT TCR α chain controlled specificity through a small number of residues, which are conserved across species and located within the Vα-Jα regions. Mutagenesis of MR1 showed that only two residues, which were centrally positioned and on opposing sides of the antigen-binding cleft of MR1, were essential for MAIT cell activation. The mutagenesis data are consistent with a centrally located MAIT TCR–MR1 docking that was dominated by the α chain of the MAIT TCR. This candidate docking mode contrasts with that of the NKT TCR–CD1d-antigen interaction, in which both the α and β chain of the NKT TCR is required for ligation above the F′-pocket of CD1d. PMID:22412157

  20. Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase.

    PubMed

    Mithoe, Sharon C; Ludwig, Christina; Pel, Michiel J C; Cucinotta, Mara; Casartelli, Alberto; Mbengue, Malick; Sklenar, Jan; Derbyshire, Paul; Robatzek, Silke; Pieterse, Corné M J; Aebersold, Ruedi; Menke, Frank L H

    2016-03-01

    Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN-SENSITIVE 2 (FLS2) induces the activation of mitogen-activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin-triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7-mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex. PMID:26769563

  1. Structural characteristics of the recognition site for cholinergic ligands in the nicotinic acetylcholine receptor from squid optical ganglia

    SciTech Connect

    Plyashkevich, Yu.G.; Demushkin, V.P.

    1986-01-20

    The influence of chemical modification on the parameters of the binding of cholinergic ligands by the nicotinic acetylcholine receptor of squid optical ganglia was investigated. The presence of two subpopulations of recognition sites, differing in the composition of the groups contained in them, was detected. It was established with high probability that subpopulation I contains arginine and tyrosine residues and a carboxyl group while subpopulation II contains an amino group, a thyrosine residue, and a carboxyl group. Moreover, in both subpopulations there is an amino group important only for the binding of tubocurarin. On the basis of the results obtained, a model of the recognition sites for cholinergic ligands of the nicotinic acetylcholine receptor of squid optical ganglia is proposed.

  2. The role of protein "Stability patches" in molecular recognition: A case study of the human growth hormone-receptor complex.

    PubMed

    Osman, Roman; Mezei, Mihaly; Engel, Stanislav

    2016-04-15

    Dynamic characteristics of protein surfaces are among the factors determining their functional properties, including their potential participation in protein-protein interactions. The presence of clusters of static residues-"stability patches" (SPs)-is a characteristic of protein surfaces involved in intermolecular recognition. The mechanism, by with SPs facilitate molecular recognition, however, remains unclear. Analyzing the surface dynamic properties of the growth hormone and of its high-affinity variant we demonstrated that reshaping of the SPs landscape may be among the factors accountable for the improved affinity of this variant to the receptor. We hypothesized that SPs facilitate molecular recognition by moderating the conformational entropy of the unbound state, diminishing enthalpy-entropy compensation upon binding, and by augmenting the favorable entropy of desolvation. SPs mapping emerges as a valuable tool for investigating the structural basis of the stability of protein complexes and for rationalizing experimental approaches, such as affinity maturation, aimed at improving it. PMID:26691434

  3. Human-specific evolution of killer cell immunoglobulin-like receptor recognition of major histocompatibility complex class I molecules.

    PubMed

    Parham, Peter; Norman, Paul J; Abi-Rached, Laurent; Guethlein, Lisbeth A

    2012-03-19

    In placental mammals, natural killer (NK) cells are a population of lymphocytes that make unique contributions to immune defence and reproduction, functions essential for survival of individuals, populations and species. Modulating these functions are conserved and variable NK-cell receptors that recognize epitopes of major histocompatibility complex (MHC) class I molecules. In humans, for example, recognition of human leucocyte antigen (HLA)-E by the CD94:NKG2A receptor is conserved, whereas recognition of HLA-A, B and C by the killer cell immunoglobulin-like receptors (KIRs) is diversified. Competing demands of the immune and reproductive systems, and of T-cell and NK-cell immunity-combined with the segregation on different chromosomes of variable NK-cell receptors and their MHC class I ligands-drive an unusually rapid evolution that has resulted in unprecedented levels of species specificity, as first appreciated from comparison of mice and humans. Counterparts to human KIR are present only in simian primates. Observed in these species is the coevolution of KIR and the four MHC class I epitopes to which human KIR recognition is restricted. Unique to hominids is the emergence of the MHC-C locus as a supplier of specialized and superior ligands for KIR. This evolutionary trend is most highly elaborated in the chimpanzee. Unique to the human KIR locus are two groups of KIR haplotypes that are present in all human populations and subject to balancing selection. Group A KIR haplotypes resemble chimpanzee KIR haplotypes and are enriched for genes encoding KIR that bind HLA class I, whereas group B KIR haplotypes are enriched for genes encoding receptors with diminished capacity to bind HLA class I. Correlating with their balance in human populations, B haplotypes favour reproductive success, whereas A haplotypes favour successful immune defence. Evolution of the B KIR haplotypes is thus unique to the human species. PMID:22312047

  4. Receptor architecture of visual areas in the face and word-form recognition region of the posterior fusiform gyrus.

    PubMed

    Caspers, Julian; Palomero-Gallagher, Nicola; Caspers, Svenja; Schleicher, Axel; Amunts, Katrin; Zilles, Karl

    2015-01-01

    Recently, two extrastriate visual areas on the posterior fusiform gyrus, areas FG1 and FG2, were identified based on cytoarchitectonical criteria (Caspers et al. in Brain Struct Funct 218:511-526, 2013a). They are located within the object-related ventral visual stream at the transition between early and higher-order (category-specific) visual areas. FG2 has a topographical position which is best comparable to the face or visual word-form recognition area. However, the precise function of FG2 is presently unknown. Since transmitter receptors are key molecules of neurotransmission, we analysed the regional and laminar distribution of 15 different receptor binding sites by means of quantitative in vitro receptor autoradiography. Significant differences between receptor densities of both areas were found for NMDA, GABAB, M3, nicotinic α4/β2 and 5-HT1A receptors as well as for GABAA associated benzodiazepine binding sites. These results support the cytoarchitectonic segregation of FG1 and FG2 into two distinct cortical areas. In addition, principal component and hierarchical cluster analyses of the multireceptor data of both fusiform areas and 24 visual, auditory, somatosensory and multimodal association areas not only revealed the typical receptor architectonic characteristics of visual areas for FG1 and FG2, but also suggest their putative function as object recognition regions due to the similarity of their receptor fingerprints with those of areas of the ventral visual stream. Furthermore, FG1 and FG2 build a cluster with the multimodal association areas of the inferior parietal lobule. This underlines their hierarchically high position in the visual system of the human cerebral cortex. PMID:24126835

  5. Structural basis of RNA recognition and activation by innate immune receptor RIG-I

    SciTech Connect

    Jiang, Fuguo; Ramanathan, Anand; Miller, Matthew T.; Tang, Guo-Qing; Gale, Jr., Michael; Patel, Smita S.; Marcotrigiano, Joseph

    2012-05-29

    Retinoic-acid-inducible gene-I (RIG-I; also known as DDX58) is a cytoplasmic pathogen recognition receptor that recognizes pathogen-associated molecular pattern (PAMP) motifs to differentiate between viral and cellular RNAs. RIG-I is activated by blunt-ended double-stranded (ds)RNA with or without a 5'-triphosphate (ppp), by single-stranded RNA marked by a 5'-ppp and by polyuridine sequences. Upon binding to such PAMP motifs, RIG-I initiates a signalling cascade that induces innate immune defences and inflammatory cytokines to establish an antiviral state. The RIG-I pathway is highly regulated and aberrant signalling leads to apoptosis, altered cell differentiation, inflammation, autoimmune diseases and cancer. The helicase and repressor domains (RD) of RIG-I recognize dsRNA and 5'-ppp RNA to activate the two amino-terminal caspase recruitment domains (CARDs) for signalling. Here, to understand the synergy between the helicase and the RD for RNA binding, and the contribution of ATP hydrolysis to RIG-I activation, we determined the structure of human RIG-I helicase-RD in complex with dsRNA and an ATP analogue. The helicase-RD organizes into a ring around dsRNA, capping one end, while contacting both strands using previously uncharacterized motifs to recognize dsRNA. Small-angle X-ray scattering, limited proteolysis and differential scanning fluorimetry indicate that RIG-I is in an extended and flexible conformation that compacts upon binding RNA. These results provide a detailed view of the role of helicase in dsRNA recognition, the synergy between the RD and the helicase for RNA binding and the organization of full-length RIG-I bound to dsRNA, and provide evidence of a conformational change upon RNA binding. The RIG-I helicase-RD structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA. The structure yields unprecedented insight into innate immunity and has a broader impact on other areas of biology, including RNA

  6. Self-regulation and cross-regulation of pattern-recognition receptor signalling in health and disease.

    PubMed

    Cao, Xuetao

    2016-01-01

    In the initiation of innate immune responses against pathogens, pattern-recognition receptors (PRRs) have an essential role in recognizing specific components of microorganisms and triggering responses that eliminate the invading microorganisms. However, inappropriate activation of PRRs can lead to prolonged inflammation and even to autoimmune and inflammatory diseases. Thus, PRR-triggered responses are regulated through the degradation or translocation of the innate receptors themselves and through the involvement of intracellular regulators or amplifiers. In addition, a complex interplay between PRRs and/or other immune pathways finely tunes the outcome of host immune defence responses. In this Review, I describe many of the numerous distinct mechanisms for the self-regulation and cross-regulation of innate immune receptor signalling. PMID:26711677

  7. Enantioselective Recognition for Many Different Kinds of Chiral Guests by One Chiral Receptor Based on Tetraphenylethylene Cyclohexylbisurea.

    PubMed

    Xiong, Jia-Bin; Xie, Wen-Zhao; Sun, Jian-Ping; Wang, Jin-Hua; Zhu, Zhi-Hua; Feng, Hai-Tao; Guo, Dong; Zhang, Hui; Zheng, Yan-Song

    2016-05-01

    A neutral chiral receptor based on TPE cyclohexylbisurea was synthesized and could discriminate the enantiomers of many different kinds of chiral reagents, including chiral acidic compounds, basic compounds, amino acids, and even neutral alcohols. The (1)H NMR spectra disclosed that the ability of chiral recognition could be ascribed to the multiple hydrogen bonds and CH-π interactions between the TPE urea receptor and the enantiomer of the chiral guest, which led to the selective aggregation of the receptor with one of the two enantiomers. This result exhibited a great potential in enantiomer discernment and high-throughput analysis of enantiomer composition of these chiral analytes by one chiral AIE molecule. PMID:27032054

  8. Sequence variability of the pattern recognition receptor Mermaid mediates specificity of marine nematode symbioses

    PubMed Central

    Bulgheresi, Silvia; Gruber-Vodicka, Harald R; Heindl, Niels R; Dirks, Ulrich; Kostadinova, Maria; Breiteneder, Heimo; Ott, Joerg A

    2011-01-01

    Selection of a specific microbial partner by the host is an all-important process. It guarantees the persistence of highly specific symbioses throughout host generations. The cuticle of the marine nematode Laxus oneistus is covered by a single phylotype of sulfur-oxidizing bacteria. They are embedded in a layer of host-secreted mucus containing the mannose-binding protein Mermaid. This Ca2+-dependent lectin mediates symbiont aggregation and attachment to the nematode. Here, we show that Stilbonema majum—a symbiotic nematode co-occurring with L. oneistus in shallow water sediment—is covered by bacteria phylogenetically distinct to those covering L. oneistus. Mermaid cDNA analysis revealed extensive protein sequence variability in both the nematode species. We expressed three recombinant Mermaid isoforms, which based on the structural predictions display the most different carbohydrate recognition domains (CRDs). We show that the three CRDs (DNT, DDA and GDA types) possess different affinities for L. oneistus and S. majum symbionts. In particular, the GDA type, exclusively expressed by S. majum, displays highest agglutination activity towards its symbionts and lowest towards its L. oneistus symbionts. Moreover, incubation of L. oneistus in the GDA type does not result in complete symbiont detachment, whereas incubation in the other types does. This indicates that the presence of particular Mermaid isoforms on the nematode surface has a role in the attachment of specific symbionts. This is the first report of the functional role of sequence variability in a microbe-associated molecular patterns receptor in a beneficial association. PMID:21228893

  9. A fibrinogen-related protein identified from hepatopancreas of crayfish is a potential pattern recognition receptor.

    PubMed

    Chen, Qiming; Bai, Suhua; Dong, Chaohua

    2016-09-01

    Fibrinogen-related protein (FREP) family is a large group of proteins containing fibrinogen-like (FBG) domain and plays multiple physiological roles in animals. However, their immune functions in crayfish are not fully explored. In the present study, a novel fibrinogen-like protein (designated as PcFBN1) was identified and characterized from hepatopancreas of red swamp crayfish Procambarus clarkii. The cDNA sequence of PcFBN1 contains an open reading frame (ORF) of 1353 bp encoding a protein of 450 amino acids. Sequence and structural analysis indicated that PcFBN1 contains an FBG domain in C-terminal and a putative signal peptide of 19 amino acids in N-terminal. Semi-quantitative PCR revealed that the main expression of PcFBN1 was observed in hepatopancreas and hemocyte. Temporal expression analysis exhibited that PcFBN1 expression could be significantly induced by heat-killed Aeromonas hydrophila. Tissue distribution and temporal change of PcFBN1 suggested that PcFBN1 may be involved in immune responses of red swamp crayfish. Recombinant PcFBN1 protein binds and agglutinates both gram-negative bacteria Escherichia coli and gram-positive bacteria Micrococcus lysodeikticus. Moreover, binding and agglutination is Ca(2+) dependent. Further analysis indicated that PcFBN1 recognizes some acetyl group-containing substance LPS and PGN. RNAi experiment revealed that PcFBN1 is required for bacterial clearance and survival from A. hydrophila infection. Reduction of PcFBN1 expression significantly decreased the survival and enhanced the number of A. hydrophila in the hemolymph. These results indicated that PcFBN1 plays an important role in the innate immunity of red swamp crayfish as a potential pattern recognition receptor. PMID:27417229

  10. Biopolymeric receptor for peptide recognition by molecular imprinting approach--synthesis, characterization and application.

    PubMed

    Singh, Lav Kumar; Singh, Monika; Singh, Meenakshi

    2014-12-01

    The present work is focused on the development of a biocompatible zwitterionic hydrogel for various applications in analytical chemistry. Biopolymer chitosan was derivatized to obtain a series of zwitterionic hydrogel samples. Free amino groups hanging on the biopolymeric chain were reacted with γ-butyrolactone to quaternize the N-centers of polymeric chain. N,N-methylene-bis-acrylamide acts as a crosslinker via Michael-type addition in the subsequent step and facilitated gelation of betainized chitosan. These biopolymeric hydrogel samples were fully characterized by FTIR, (1)H NMR, (13)C NMR spectra, SEM and XRD. Hydrogels were further characterized for their swelling behavior at varying parameters. The extent of swelling was perceived to be dictated by solvent composition such as pH, ionic strength and temperature. This valuable polymeric format is herein chosen to design an artificial receptor for dipeptide 'carnosine', which has adequate societal significance to be analytically determined, by molecular imprinting. Electrostatic interactions along with complementary H-bonding and other hydrophobic interactions inducing additional synergetic effect between the template (carnosine) and the imprinted polymer led to the formation of imprinted sites. The MIP was able to selectively and specifically take up carnosine from aqueous solution quantitatively. Thus prepared MIPs were characterized by FTIR spectroscopy, SEM providing evidence for the quality and quantity of imprinted gels. The binding studies showed that the MIP illustrated good recognition for carnosine as compared to non-imprinted polymers (NIPs). Detection limit was estimated as 3.3 μg mL(-1). Meanwhile, selectivity experiments demonstrated that imprinted gel had a high affinity to carnosine in the presence of close structural analogues (interferrants). PMID:25491843

  11. Expression of pathogen recognition receptors and pro-inflammatory cytokine transcripts in clinical and sub-clinical endometritis cows.

    PubMed

    Loyi, Tumnyak; Kumar, Harendra; Nandi, Sukdeb; Patra, Manas Kumar

    2015-01-01

    The present study was carried out to examine the expression profile of pathogen recognition receptors (CD14 and toll-like receptor 4) and pro-inflammatory cytokines (IL-1β, IL-6, IL-8, and TNFα) in endometrial tissue of cows with endometritis at different stages of estrous cycle. Genital tracts were collected from 60 cows at slaughter from the killing village. The genitalia were examined for clinical endometritis (CE) and subclinical endometritis (SCE) through physical examination, white side test of cervico-vaginal mucus, endometrial cytology and histopathology. The stage of estrous cycle for each genitalia was determined by visual examination of both the ovaries and classified as either follicular (F) or luteal (L). Depending on the degree of inflammation and stage of estrous cycle, the genitalia were categorized in four groups i.e., FCE, FSCE, LCE, and LSCE with six genitalia in each group. Furthermore, 12 healthy genitalia comprise of six each of follicular (FN) and luteal (LN) were included as control. Endometrial tissue scrapings were collected ex vivo from all the genitalia. Total RNA was extracted and cDNA was transcribed for each sample and relative quantification of mRNA of target genes was done by real-time PCR. The results revealed a significant up-regulation of CD14 (11 fold) and IL-8 (13 fold) in follicular stage and IL-6 (8 fold) and TNFα (29 fold) in luteal stages in SCE cows. However, the majority of pro-inflammatory cytokine and pathogen recognition receptors expressed at significant higher level in both follicular and luteal stages in cows with CE. Thus, it is concluded that the endometrial transcripts of pathogen recognition receptors and pro-inflammatory cytokines expressed differentially in cows with endometritis, whereas the fold change is dependent on the severity of inflammation and the stage of cyclicity. Therefore, endometrial transcript profile with a defined threshold level could be used as a possible diagnostic marker in cows with

  12. Structural Analysis of an Avr4 Effector Ortholog Offers Insight into Chitin Binding and Recognition by the Cf-4 Receptor.

    PubMed

    Kohler, Amanda C; Chen, Li-Hung; Hurlburt, Nicholas; Salvucci, Anthony; Schwessinger, Benjamin; Fisher, Andrew J; Stergiopoulos, Ioannis

    2016-08-01

    Chitin is a key component of fungal cell walls and a potent inducer of innate immune responses. Consequently, fungi may secrete chitin-binding lectins, such as the Cf-Avr4 effector protein from the tomato pathogen Cladosporium fulvum, to shield chitin from host-derived chitinases during infection. Homologs of Cf-Avr4 are found throughout Dothideomycetes, and despite their modest primary sequence identity, many are perceived by the cognate tomato immune receptor Cf-4. Here, we determined the x-ray crystal structure of Pf-Avr4 from the tomato pathogen Pseudocercospora fuligena, thus providing a three-dimensional model of an Avr4 effector protein. In addition, we explored structural, biochemical, and functional aspects of Pf-Avr4 and Cf-Avr4 to further define the biology of core effector proteins and outline a conceptual framework for their pleiotropic recognition by single immune receptors. We show that Cf-Avr4 and Pf-Avr4 share functional specificity in binding (GlcNAc)6 and in providing protection against plant- and microbial-derived chitinases, suggesting a broader role beyond deregulation of host immunity. Furthermore, structure-guided site-directed mutagenesis indicated that residues in Pf-Avr4 important for binding chitin do not directly influence recognition by Cf-4 and further suggested that the property of recognition is structurally separated or does not fully overlap with the virulence function of the effector. PMID:27401545

  13. Two Redundant Receptor-Like Cytoplasmic Kinases Function Downstream of Pattern Recognition Receptors to Regulate Activation of SA Biosynthesis1[OPEN

    PubMed Central

    Kong, Qing; Qu, Na; Ma, Junling; Li, Meng; Cheng, Yu-ti; Zhang, Qian; Wu, Di; Zhang, Zhibin; Zhang, Yuelin

    2016-01-01

    Salicylic acid (SA) serves as a critical signaling molecule in plant defense. Two transcription factors, SARD1 and CBP60g, control SA biosynthesis through regulating pathogen-induced expression of Isochorismate Synthase1, which encodes a key enzyme for SA biosynthesis. Here, we report that Pattern-Triggered Immunity Compromised Receptor-like Cytoplasmic Kinase1 (PCRK1) and PCRK2 function as key regulators of SA biosynthesis. In the pcrk1 pcrk2 double mutant, pathogen-induced expression of SARD1, CBP60g, and ICS1 is greatly reduced. The pcrk1 pcrk2 double mutant, but neither of the single mutants, exhibits reduced accumulation of SA and enhanced disease susceptibility to bacterial pathogens. Both PCRK1 and PCRK2 interact with the pattern recognition receptor FLS2, and treatment with pathogen-associated molecular patterns leads to rapid phosphorylation of PCRK2. Our data suggest that PCRK1 and PCRK2 function downstream of pattern recognition receptor in a signal relay leading to the activation of SA biosynthesis. PMID:27208222

  14. Differential Involvement of Dopamine D1 Receptor and MEK Signaling Pathway in the Ventromedial Prefrontal Cortex in Consolidation and Reconsolidation of Recognition Memory

    ERIC Educational Resources Information Center

    Maroun, Mouna; Akirav, Irit

    2009-01-01

    We investigated MEK and D1 receptors in the ventromedial prefrontal cortex (vmPFC) in consolidation and reconsolidation of recognition memory in rats nonhabituated to the experimental context (NH) or with reduced arousal due to extensive prior habituation (H). The D1 receptor antagonist enhanced consolidation and impaired reconsolidation in NH but…

  15. Recognition of Human Erythrocyte Receptors by the Tryptophan-Rich Antigens of Monkey Malaria Parasite Plasmodium knowlesi

    PubMed Central

    Tyagi, Kriti; Gupta, Deepali; Saini, Ekta; Choudhary, Shilpa; Jamwal, Abhishek; Alam, Mohd. Shoeb; Zeeshan, Mohammad; Tyagi, Rupesh K.; Sharma, Yagya D.

    2015-01-01

    Background The monkey malaria parasite Plasmodium knowlesi also infect humans. There is a lack of information on the molecular mechanisms that take place between this simian parasite and its heterologous human host erythrocytes leading to this zoonotic disease. Therefore, we investigated here the binding ability of P. knowlesi tryptophan-rich antigens (PkTRAgs) to the human erythrocytes and sharing of the erythrocyte receptors between them as well as with other commonly occurring human malaria parasites. Methods Six PkTRAgs were cloned and expressed in E.coli as well as in mammalian CHO-K1 cell to determine their human erythrocyte binding activity by cell-ELISA, and in-vitro rosetting assay, respectively. Results Three of six PkTRAgs (PkTRAg38.3, PkTRAg40.1, and PkTRAg67.1) showed binding to human erythrocytes. Two of them (PkTRAg40.1 and PkTRAg38.3) showed cross-competition with each other as well as with the previously described P.vivax tryptophan-rich antigens (PvTRAgs) for human erythrocyte receptors. However, the third protein (PkTRAg67.1) utilized the additional but different human erythrocyte receptor(s) as it did not cross-compete for erythrocyte binding with either of these two PkTRAgs as well as with any of the PvTRAgs. These three PkTRAgs also inhibited the P.falciparum parasite growth in in-vitro culture, further indicating the sharing of human erythrocyte receptors by these parasite species and the biological significance of this receptor-ligand interaction between heterologous host and simian parasite. Conclusions Recognition and sharing of human erythrocyte receptor(s) by PkTRAgs with human parasite ligands could be part of the strategy adopted by the monkey malaria parasite to establish inside the heterologous human host. PMID:26393350

  16. Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14

    SciTech Connect

    Albright, Seth; Chen Bin; Holbrook, Kristen; Jain, Nitin U.

    2008-04-04

    CD14 functions as a key pattern recognition receptor for a diverse array of Gram-negative and Gram-positive cell-wall components in the host innate immune response by binding to pathogen-associated molecular patterns (PAMPs) at partially overlapping binding site(s). To determine the potential contribution of CD14 residues in this pattern recognition, we have examined using solution NMR spectroscopy, the binding of three different endotoxin ligands, lipopolysaccharide, lipoteichoic acid, and a PGN-derived compound, muramyl dipeptide to a {sup 15}N isotopically labeled 152-residue N-terminal fragment of sCD14 expressed in Pichia pastoris. Mapping of NMR spectral changes upon addition of ligands revealed that the pattern of residues affected by binding of each ligand is partially similar and partially different. This first direct structural observation of the ability of specific residue combinations of CD14 to differentially affect endotoxin binding may help explain the broad specificity of CD14 in ligand recognition and provide a structural basis for pattern recognition. Another interesting finding from the observed spectral changes is that the mode of binding may be dynamically modulated and could provide a mechanism for binding endotoxins with structural diversity through a common binding site.

  17. Rational design, synthesis, and application of a new receptor for the molecular recognition of tricarboxylate salts in aqueous media.

    PubMed

    Frontera, Antonio; Morey, Jeroni; Oliver, Antonia; Piña, M Neus; Quiñonero, David; Costa, Antoni; Ballester, Pablo; Deyà, Pere M; Anslyn, Eric V

    2006-09-15

    A rational design of a tripodal receptor for the molecular recognition of tricarboxylate salts in aqueous media, based on squaramide, has been performed using high-level DFT calculations (RI-BP86/SVP level of theory) in solution using the COSMO treatment, including some preliminary ab initio calculations at the higher RI-MP2/TZVP level of theory, comparing the ability of squaramide to bind carboxylate salts with two widely used guanidinium salts. The tripodal receptor has been synthesized using a new methodology that has been recently reported by some of us, and its capability of recognizing several mono-, di-, and tricarboxylate salts has been studied experimentally by means of microcalorimetry experiments in a very high competitive media, H(2)O:EtOH 1:3. These experiments give enthalpic and entropic data, which are unfortunately scarce in the literature of molecular recognition of anions. Finally, a fluorimetric ensemble of the receptor with fluorescein has been found to be useful for the fluorimetric determination of zinc citrate in a commercial toothpaste using competition assays. PMID:16958511

  18. The macromolecular assembly of pathogen-recognition receptors is impelled by serine proteases, via their complement control protein modules.

    PubMed

    Le Saux, Agnès; Ng, Patricia Miang Lon; Koh, Joanne Jing Yun; Low, Diana Hooi Ping; Leong, Geraldine E-Ling; Ho, Bow; Ding, Jeak Ling

    2008-03-28

    Although the innate immune response is triggered by the formation of a stable assembly of pathogen-recognition receptors (PRRs) onto the pathogens, the driving force that enables this PRR-PRR interaction is unknown. Here, we show that serine proteases, which are activated during infection, participate in associating with the PRRs. Inhibition of serine proteases gravely impairs the PRR assembly. Using yeast two-hybrid and pull-down methods, we found that two serine proteases in the horseshoe crab Carcinoscorpius rotundicauda are able to bind to the following three core members of PRRs: galactose-binding protein, Carcinolectin-5 and C-reactive protein. These two serine proteases are (1) Factor C, which activates the coagulation pathway, and (2) C2/Bf, a protein from the complement pathway. By systematic molecular dissection, we show that these serine proteases interact with the core "pathogen-recognition complex" via their complement control protein modules. PMID:18279891

  19. Transcription of pattern recognition receptors and abortive agents induced chemokines in the bovine pregnant uterus.

    PubMed

    Silva, Ana Patrícia Carvalho; Costa, Erica Azevedo; Macêdo, Auricélio Alves; Martins, Telma da Mata; Borges, Alan Maia; Paixão, Tatiane Alves; Santos, Renato Lima

    2012-01-15

    Pattern recognition receptors (PRRs) are important components of the innate immune system whose ligands are specific pathogen associated molecular patterns (PAMPs). Considering the scarcity of studies on transcription of PRRs in the pregnant uterus of cows, and its response to PAMPs and microorganisms that cause abortion in cattle, this study aimed to characterize the transcription of TLR1-10, NOD1, NOD2 and MD2 in bovine uterus throughout gestation and to investigate the sensitivity of different uterine tissues at third trimester of pregnancy to purified TLR ligands or heat-killed Brucella abortus, Salmonella enterica serotype Dublin (S. Dublin), Listeria monocytogenes, and Aspergillus fumigatus, by assessing chemokine transcription. RNA extracted from endometrium, placentome and intercotiledonary region of cows at the first (n=6), second (n=6), and third (n=6) trimesters of pregnancy were subjected to real time RT-PCR. After stimulation of endometrium and intercotiledonary regions with purified TLR ligands or heat-killed microorganisms, gene transcription was assessed by real time RT-PCR. In the placentome, there was no significant variation in TLRs transcription throughout the three trimesters of pregnancy. In the endometrium, there was significant variation in TLR4 and TLR5 transcription during the three stages of gestation; i.e. TLR4 transcription was higher during the third trimester, whereas TLR5 transcription was higher during the last two trimesters. In the intercotiledonary region, there was significant variation in transcription of TLR1/6, TLR7, and TLR8, which were more strongly expressed during the first trimester of pregnancy. At the third trimester of gestation, significant transcription of CXCL6 and CXCL8 was detected mostly in endometrial tissues in response to purified TLR4 and TLR2 ligands. Transcription of these chemokines was induced in the endometrium and intercotiledonary region at the third trimester of pregnancy when stimulated with heat

  20. Receptor-Mediated Recognition and Uptake of Iron from Human Transferrin by Staphylococcus aureus and Staphylococcus epidermidis

    PubMed Central

    Modun, Belinda; Evans, Robert W.; Joannou, Christopher L.; Williams, Paul

    1998-01-01

    provide evidence to suggest that there is a primary receptor recognition site on the N-lobe of human transferrin. PMID:9673237

  1. Differences in human skin between the epidermal growth factor receptor distribution detected by EGF binding and monoclonal antibody recognition.

    PubMed

    Green, M R; Couchman, J R

    1985-09-01

    Two methods have been used to examine epidermal growth factor (EGF) receptor distribution in human scalp and foreskin. The first employed [125I]EGF viable explants and autoradiography to determine the EGF binding pattern while the second used a monoclonal antibody to the human EGF receptor to map the distribution on frozen skin sections of an extracellular epitope on the EGF receptor. The [125I]EGF binding experiments showed accessible, unoccupied EGF receptors to be present on the epidermal basal cells (with reduced binding to spinous cells), the basal cells of the hair shaft and sebaceous gland, the eccrine sweat glands, capillary system, and the hair follicle outer root sheath, generally similar in pattern to that previously reported for full-thickness rat skin and human epidermis. The same areas also bound EGF-R1 but in addition the monoclonal antibody recognized a cone of melanin containing presumptive cortex cells, excluding the medulla, lying around and above the upper dermal papilla of anagen hair follicles, epithelial cells around the lower dermal papilla region, and in some tissue samples the cell margins of the viable differentiating layers of the epidermis. In a control study, to clarify whether EGF-R1 could recognize molecules unrelated to the EGF receptor, the EGF binding and EGF-R1 recognition profiles were compared on cultures of SVK14 cells, a SV40 transformed human keratinocyte cell line. EGF binding and EGF-R1 monoclonal antibody distribution on these cells was found to be similar, indicating that, at least for SVK14 cells, EGF-R1 binding provides a reliable marker for EGF binding. Explanations for the discrepancies between these two methods for determining EGF receptor distribution in human skin are discussed, including the possibility that latent EGF receptors, unable to bind [125I]EGF, may be present in some differentiating epithelial compartments. PMID:2411822

  2. The Structural Basis for Class II Cytokine Receptor Recognition by JAK1.

    PubMed

    Ferrao, Ryan; Wallweber, Heidi J A; Ho, Hoangdung; Tam, Christine; Franke, Yvonne; Quinn, John; Lupardus, Patrick J

    2016-06-01

    JAK1 is a member of the Janus kinase (JAK) family of non-receptor tyrosine kinases that are activated in response to cytokines and interferons. Here, we present two crystal structures of the human JAK1 FERM and SH2 domains bound to peptides derived from the class II cytokine receptors IFN-λ receptor 1 and IL-10 receptor 1 (IFNLR1 and IL10RA). These structures reveal an interaction site in the JAK1 FERM that accommodates the so-called "box1" membrane-proximal receptor peptide motif. Biophysical analysis of the JAK1-IFNLR1 interaction indicates that the receptor box1 is the primary driver of the JAK1 interaction, and identifies residues conserved among class II receptors as important for binding. In addition, we demonstrate that a second "box2" receptor motif further stabilizes the JAK1-IFNLR1 complex. Together, these data identify a conserved JAK binding site for receptor peptides and elucidate the mechanism by which class II cytokine receptors interact with JAK1. PMID:27133025

  3. Analysis of Influenza Virus Hemagglutinin Receptor Binding Mutants with Limited Receptor Recognition Properties and Conditional Replication Characteristics▿

    PubMed Central

    Bradley, Konrad C.; Galloway, Summer E.; Lasanajak, Yi; Song, Xuezheng; Heimburg-Molinaro, Jamie; Yu, Hai; Chen, Xi; Talekar, Ganesh R.; Smith, David F.; Cummings, Richard D.; Steinhauer, David A.

    2011-01-01

    To examine the range of selective processes that potentially operate when poorly binding influenza viruses adapt to replicate more efficiently in alternative environments, we passaged a virus containing an attenuating mutation in the hemagglutinin (HA) receptor binding site in mice and characterized the resulting mutants with respect to the structural locations of mutations selected, the replication phenotypes of the viruses, and their binding properties on glycan microarrays. The initial attenuated virus had a tyrosine-to-phenylalanine mutation at HA1 position 98 (Y98F), located in the receptor binding pocket, but viruses that were selected contained second-site pseudoreversion mutations in various structural locations that revealed a range of molecular mechanisms for modulating receptor binding that go beyond the scope that is generally mapped using receptor specificity mutants. A comparison of virus titers in the mouse respiratory tract versus MDCK cells in culture showed that the mutants displayed distinctive replication properties depending on the system, but all were less attenuated in mice than the Y98F virus. An analysis of receptor binding properties confirmed that the initial Y98F virus bound poorly to several different species of erythrocytes, while all mutants reacquired various degrees of hemagglutination activity. Interestingly, both the Y98F virus and pseudoreversion mutants were shown to bind very inefficiently to standard glycan microarrays containing an abundance of binding substrates for most influenza viruses that have been characterized to date, provided by the Consortium for Functional Glycomics. The viruses were also examined on a recently developed microarray containing glycans terminating in sialic acid derivatives, and limited binding to a potentially interesting subset of glycans was revealed. The results are discussed with respect to mechanisms for HA-mediated receptor binding, as well as regarding the species of molecules that may act

  4. Linking pattern recognition and salicylic acid responses in Arabidopsis through ACCELERATED CELL DEATH6 and receptors

    PubMed Central

    Tateda, Chika; Zhang, Zhongqin; Greenberg, Jean T

    2015-01-01

    The Arabidopsis membrane protein ACCELERATED CELL DEATH 6 (ACD6) and the defense signal salicylic acid (SA) are part of a positive feedback loop that regulates the levels of at least 2 pathogen-associated molecular patterns (PAMP) receptors, including FLAGELLIN SENSING 2 (FLS2) and CHITIN ELICITOR RECEPTOR (LYSM domain receptor-like kinase 1, CERK1). ACD6- and SA-mediated regulation of these receptors results in potentiation of responses to FLS2 and CERK1 ligands (e.g. flg22 and chitin, respectively). ACD6, FLS2 and CERK1 are also important for callose induction in response to an SA agonist even in the absence of PAMPs. Here, we report that another receptor, EF-Tu RECEPTOR (EFR) is also part of the ACD6/SA signaling network, similar to FLS2 and CERK1. PMID:26442718

  5. Structure of the Neisserial outer membrane protein Opa₆₀: loop flexibility essential to receptor recognition and bacterial engulfment.

    PubMed

    Fox, Daniel A; Larsson, Per; Lo, Ryan H; Kroncke, Brett M; Kasson, Peter M; Columbus, Linda

    2014-07-16

    The structure and dynamics of Opa proteins, which we report herein, are responsible for the receptor-mediated engulfment of Neisseria gonorrheae or Neisseria meningitidis by human cells and can offer deep understanding into the molecular recognition of pathogen-host receptor interactions. Such interactions are vital to understanding bacterial pathogenesis as well as the mechanism of foreign body entry to a human cell, which may provide insights for the development of targeted pharmaceutical delivery systems. The size and dynamics of the extracellular loops of Opa60 required a hybrid refinement approach wherein membrane and distance restraints were used to generate an initial NMR structural ensemble, which was then further refined using molecular dynamics in a DMPC bilayer. The resulting ensemble revealed that the extracellular loops, which bind host receptors, occupy compact conformations, interact with each other weakly, and are dynamic on the nanosecond time scale. We predict that this conformational sampling is critical for enabling diverse Opa loop sequences to engage a common set of receptors. PMID:24813921

  6. Structure of the Neisserial Outer Membrane Protein Opa60: Loop Flexibility Essential to Receptor Recognition and Bacterial Engulfment

    PubMed Central

    2015-01-01

    The structure and dynamics of Opa proteins, which we report herein, are responsible for the receptor-mediated engulfment of Neisseria gonorrheae or Neisseria meningitidis by human cells and can offer deep understanding into the molecular recognition of pathogen–host receptor interactions. Such interactions are vital to understanding bacterial pathogenesis as well as the mechanism of foreign body entry to a human cell, which may provide insights for the development of targeted pharmaceutical delivery systems. The size and dynamics of the extracellular loops of Opa60 required a hybrid refinement approach wherein membrane and distance restraints were used to generate an initial NMR structural ensemble, which was then further refined using molecular dynamics in a DMPC bilayer. The resulting ensemble revealed that the extracellular loops, which bind host receptors, occupy compact conformations, interact with each other weakly, and are dynamic on the nanosecond time scale. We predict that this conformational sampling is critical for enabling diverse Opa loop sequences to engage a common set of receptors. PMID:24813921

  7. The CRF1 and the CRF2 receptor mediate recognition memory deficits and vulnerability induced by opiate withdrawal.

    PubMed

    Morisot, Nadège; Contarino, Angelo

    2016-06-01

    Opiate use disorders are associated with impaired cognitive function and altered stress-responsive systems. The corticotropin-releasing factor (CRF) system mediates stress responses via CRF1 and CRF2 receptors and may be implicated in substance use disorders. However, the specific role for each of the two known CRF receptor subtypes in cognitive impairment induced by opiate administration and withdrawal remains to be elucidated. In the present study, CRF1-/-, CRF2-/- and their respective wild-type mice are injected with escalating doses of morphine and cognitive function assessed by the novel object recognition (NOR) memory task throughout relatively long periods of opiate withdrawal. Early (2 days) phases of opiate withdrawal impair NOR memory in wild-type, CRF1-/- and CRF2-/- mice. However, the duration of opiate withdrawal-induced NOR memory deficits is prolonged in CRF1-/- but shortened in CRF2-/- mice, as compared to their respective wild-type mice, indicating opposite roles for the two CRF receptor subtypes. Nevertheless, following apparent recovery, exposure to an environmental stressor induces the reemergence of NOR memory deficits in long-term opiate-withdrawn wild-type but not CRF1-/- or CRF2-/- mice, indicating an essential role for both CRF receptor subtypes in stress vulnerability. These findings bring initial evidence of a complex physiopathological role for the CRF system in cognitive deficits and the long-lasting vulnerability induced by opiate drugs. PMID:26907806

  8. Exchanging ligand-binding specificity between a pair of mouse olfactory receptor paralogs reveals odorant recognition principles.

    PubMed

    Baud, Olivia; Yuan, Shuguang; Veya, Luc; Filipek, Slawomir; Vogel, Horst; Pick, Horst

    2015-01-01

    A multi-gene family of ~1000 G protein-coupled olfactory receptors (ORs) constitutes the molecular basis of mammalian olfaction. Due to the lack of structural data its remarkable capacity to detect and discriminate thousands of odorants remains poorly understood on the structural level of the receptor. Using site-directed mutagenesis we transferred ligand specificity between two functionally related ORs and thereby revealed amino acid residues of central importance for odorant recognition and discrimination of the two receptors. By exchanging two of three residues, differing at equivalent positions of the putative odorant binding site between the mouse OR paralogs Olfr73 (mOR-EG) and Olfr74 (mOR-EV), we selectively changed ligand preference but remarkably also signaling activation strength in both ORs. Computer modeling proposed structural details at atomic resolution how the very same odorant molecule might interact with different contact residues to induce different functional responses in two related receptors. Our findings provide a mechanistic explanation of how the olfactory system distinguishes different molecular aspects of a given odorant molecule, and unravel important molecular details of the combinatorial encoding of odorant identity at the OR level. PMID:26449412

  9. Exchanging ligand-binding specificity between a pair of mouse olfactory receptor paralogs reveals odorant recognition principles

    PubMed Central

    Baud, Olivia; Yuan, Shuguang; Veya, Luc; Filipek, Slawomir; Vogel, Horst; Pick, Horst

    2015-01-01

    A multi-gene family of ~1000 G protein-coupled olfactory receptors (ORs) constitutes the molecular basis of mammalian olfaction. Due to the lack of structural data its remarkable capacity to detect and discriminate thousands of odorants remains poorly understood on the structural level of the receptor. Using site-directed mutagenesis we transferred ligand specificity between two functionally related ORs and thereby revealed amino acid residues of central importance for odorant recognition and discrimination of the two receptors. By exchanging two of three residues, differing at equivalent positions of the putative odorant binding site between the mouse OR paralogs Olfr73 (mOR-EG) and Olfr74 (mOR-EV), we selectively changed ligand preference but remarkably also signaling activation strength in both ORs. Computer modeling proposed structural details at atomic resolution how the very same odorant molecule might interact with different contact residues to induce different functional responses in two related receptors. Our findings provide a mechanistic explanation of how the olfactory system distinguishes different molecular aspects of a given odorant molecule, and unravel important molecular details of the combinatorial encoding of odorant identity at the OR level. PMID:26449412

  10. Linking ligand perception by PEPR pattern recognition receptors to cytosolic Ca2+ elevation and downstream immune signaling in plants

    PubMed Central

    Walker, Robin K.; Zhao, Yichen; Berkowitz, Gerald A.

    2012-01-01

    Little is known about molecular steps linking perception of pathogen invasion by cell surface sentry proteins acting as pattern recognition receptors (PRRs) to downstream cytosolic Ca2+ elevation, a critical step in plant immune signaling cascades. Some PRRs recognize molecules (such as flagellin) associated with microbial pathogens (pathogen-associated molecular patterns, PAMPs), whereas others bind endogenous plant compounds (damage-associated molecular patterns, DAMPs) such as peptides released from cells upon attack. This work focuses on the Arabidopsis DAMPs plant elicitor peptides (Peps) and their receptors, PEPR1 and PEPR2. Pep application causes in vivo cGMP generation and downstream signaling that is lost when the predicted PEPR receptor guanylyl cyclase (GC) active site is mutated. Pep-induced Ca2+ elevation is attributable to cGMP activation of a Ca2+ channel. Some differences were identified between Pep/PEPR signaling and the Ca2+-dependent immune signaling initiated by the flagellin peptide flg22 and its cognate receptor Flagellin-sensing 2 (FLS2). FLS2 signaling may have a greater requirement for intracellular Ca2+ stores and inositol phosphate signaling, whereas Pep/PEPR signaling requires extracellular Ca2+. Maximal FLS2 signaling requires a functional Pep/PEPR system. This dependence was evidenced as a requirement for functional PEPR receptors for maximal flg22-dependent Ca2+ elevation, H2O2 generation, defense gene [WRKY33 and Plant Defensin 1.2 (PDF1.2)] expression, and flg22/FLS2-dependent impairment of pathogen growth. In a corresponding fashion, FLS2 loss of function impaired Pep signaling. In addition, a role for PAMP and DAMP perception in bolstering effector-triggered immunity (ETI) is reported; loss of function of either FLS2 or PEPR receptors impaired the hypersensitive response (HR) to an avirulent pathogen. PMID:23150556