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Sample records for carbohydrate binding specificities

  1. The Multiple Carbohydrate Binding Specificities of Helicobacter pylori

    NASA Astrophysics Data System (ADS)

    Teneberg, Susann

    Persistent colonization of the human stomach by Helicobacter pylori is a risk factor for the development of peptic ulcer disease and gastric cancer. Adhesion of microbes to the target tissue is an important determinant for successful initiation, establishment and maintenance of infection, and a variety of different candidate carbohydrate receptors for H. pylori have been identified. Here the different the binding specifities, and their potential role in adhesion to human gastric epithelium are described. Finally, recent findings on the roles of sialic acid binding SabA adhesin in interactions with human neutrophils and erythrocytes are discussed.

  2. Engineered xyloglucan specificity in a carbohydrate-binding module.

    PubMed

    Gunnarsson, Lavinia Cicortas; Zhou, Qi; Montanier, Cedric; Karlsson, Eva Nordberg; Brumer, Harry; Ohlin, Mats

    2006-12-01

    The field of plant cell wall biology is constantly growing and consequently so is the need for more sensitive and specific probes for individual wall components. Xyloglucan is a key polysaccharide widely distributed in the plant kingdom in both structural and storage tissues that exist in both fucosylated and non-fucosylated variants. Presently, the only xyloglucan marker available is the monoclonal antibody CCRC-M1 that is specific to terminal alpha-1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides. As a viable alternative to searches for natural binding proteins or creation of new monoclonal antibodies, an approach to select xyloglucan-specific binding proteins from a combinatorial library of the carbohydrate-binding module, CBM4-2, from xylanase Xyn10A of Rhodothermus marinus is described. Using phage display technology in combination with a chemoenzymatic method to anchor xyloglucan to solid supports, the selection of xyloglucan-binding modules with no detectable residual wild-type xylan and beta-glucan-binding ability was achieved.

  3. Correlation between carbohydrate-binding specificity and amino acid sequence of carbohydrate-binding regions of Cytisus-type anti-H(O) lectins.

    PubMed

    Konami, Y; Yamamoto, K; Osawa, T; Irimura, T

    1992-06-15

    A carbohydrate-binding peptide of the di-N-acetylchitobiose-binding Cytisus sessilifolius anti-H(O) lectin I (CSA-I) was isolated from the endoproteinase Asp-N digest of CSA-I by affinity chromatography on a column of N-acetyl-D-glucosamine oligomer-Sepharose (GlcNAc oligomer-Sepharose). The amino acid sequence of the carbohydrate-binding peptide of CSA-I was determined to be DTYFGKTYNPW using a gas-phase protein sequencer. This sequence corresponds to the sequence from Asp-129 to Trp-139 based on the primary structure of CSA-I, and shows a high degree of homology to those of the putative carbohydrate-binding peptide of the Laburnum alpinum lectin I (LAA-I) (DTYFGKAYNPW) and of the Ulex europaeus lectin II (UEA-II) (DSYFGKTYNPW). The binding of these three anti-H(O) lectins is known to be inhibited by di-N-acetylchitobiose but not by L-fucose. These results strongly suggest that there is a good correlation between the carbohydrate-binding specificity and the amino acid sequence of the carbohydrate-binding regions of di-N-acetylchitobiose-binding lectins.

  4. Alteration of the carbohydrate-binding specificity of a C-type lectin CEL-I mutant with an EPN carbohydrate-binding motif.

    PubMed

    Hatakeyama, Tomomitsu; Ishimine, Tomohiro; Baba, Tomohiro; Kimura, Masanari; Unno, Hideaki; Goda, Shuichiro

    2013-07-01

    CEL-I is a Gal/GalNAc-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-recognition domains (CRDs) with the carbohydrate-recognition motif QPD (Gln-Pro- Asp), which is generally known to exist in galactose-specific C-type CRDs. In the present study, a mutant CEL-I with EPN (Glu-Pro-Asn) motif, which is thought to be responsible for the carbohydrate-recognition of mannose-specific Ctype CRDs, was produced in Escherichia coli, and its effects on the carbohydrate-binding specificity were examined using polyamidoamine dendrimer (PD) conjugated with carbohydrates. Although wild-type CEL-I effectively formed complexes with N-acetylgalactosamine (GalNAc)-PD but not with mannose-PD, the mutant CEL-I showed relatively weak but definite affinity for mannose-PD. These results indicated that the QPD and EPN motifs play a significant role in the carbohydrate-recognition mechanism of CEL-I, especially in the discrimination of galactose and mannose. Additional mutations in the recombinant CEL-I binding site may further increase its specificity for mannose, and should provide insights into designing novel carbohydrate-recognition proteins.

  5. Binding specificity of mannose-specific carbohydrate-binding protein from the cell surface of Trypanosoma cruzi.

    PubMed

    Bonay, P; Molina, R; Fresno, M

    2001-09-01

    The sugar binding specificity of the recently described mannose-specific carbohydrate-binding proteins (CBP) isolated to homogeneity from both the epimastigote and trypomastigote stages of the pathogenic protozoa Trypanosoma cruzi has been studied by quantitative hapten inhibition of the biotinylated CBPs to immobilized thyroglobulin using model oligosaccharides. The results clearly show a differential specificity toward high-mannose glycans between the CBPs from the two developmental stages. Thus, the isolated CBP from epimastigotes exhibited stronger affinity for higher mannose oligomers containing the Manalpha1-2Manalpha1-6Manalpha1-6 structure. Its affinity decreased, as did the number of mannose residues on the oligomer or removal of the terminal Manalpha1-2-linked mannose. By contrast the CBP isolated from the trypomastigote stage showed about 400-fold lower avidity than the epimastigote form, and contrary to it, it was slightly more specific toward Man5GlcNAc than Man9GlcNAc. Analysis of the interaction of epimastigote-Man-CBP with its ligands by UV difference spectroscopy indicates the existence of an extended binding site in that protein with a large enthalpic contribution to the binding. The thermodynamic parameters of binding were obtained by isothermal titration calorimetry and been found that the DeltaH values to be in good agreement with the van't Hoff values. The binding reactions are mainly enthalpically driven and exhibit enthalpy-enthropy compensation. In addition, analysis of the high-mannose glycans from different parts of the digestive tract of the reduviid insect vector of T. cruzi suggest a role of the CBP in the retention of the epimastigote stage in the anterior portion of the gut.

  6. Structural basis for the unusual carbohydrate-binding specificity of jacalin towards galactose and mannose.

    PubMed Central

    Bourne, Yves; Astoul, Corinne Houlès; Zamboni, Véronique; Peumans, Willy J; Menu-Bouaouiche, Laurence; Van Damme, Els J M; Barre, Annick; Rougé, Pierre

    2002-01-01

    Evidence is presented that the specificity of jacalin, the seed lectin from jack fruit (Artocarpus integrifolia), is not directed exclusively against the T-antigen disaccharide Galbeta1,3GalNAc, lactose and galactose, but also against mannose and oligomannosides. Biochemical analyses based on surface-plasmon-resonance measurements, combined with the X-ray-crystallographic determination of the structure of a jacalin-alpha-methyl-mannose complex at 2 A resolution, demonstrated clearly that jacalin is fully capable of binding mannose. Besides mannose, jacalin also interacts readily with glucose, N-acetylneuraminic acid and N-acetylmuramic acid. Structural analyses demonstrated that the relatively large size of the carbohydrate-binding site enables jacalin to accommodate monosaccharides with different hydroxyl conformations and provided unambiguous evidence that the beta-prism structure of jacalin is a sufficiently flexible structural scaffold to confer different carbohydrate-binding specificities to a single lectin. PMID:11988090

  7. Carbohydrate binding activities of Bradyrhizobium japonicum. II. Isolation and characterization of a galactose-specific lectin

    PubMed Central

    1990-01-01

    Extracts of Bradyrhizobium japonicum were fractionated on Sepharose columns covalently derivatized with lactose. Elution of the material that was specifically bound to the affinity column with lactose yielded a protein of Mr approximately 38,000. Isoelectric focusing of this sample yielded two spots with pI values of 6.4 and 6.8. This protein specifically bound to galactose-containing glycoconjugates, but did not bind either to glucose or mannose. Derivatives of galactose at the C-2 position showed much weaker binding; there was an 18-fold difference in the relative binding affinities of galactose versus N-acetyl-D- galactosamine. These results indicate that we have purified a newly identified carbohydrate-binding protein from Bradyrhizobium japonicum, that can exquisitely distinguish galactose from its derivatives at the C-2 position. PMID:2211830

  8. Differential recognition of plant cell walls by microbial xylan-specific carbohydrate-binding modules.

    PubMed

    McCartney, Lesley; Blake, Anthony W; Flint, James; Bolam, David N; Boraston, Alisdair B; Gilbert, Harry J; Knox, J Paul

    2006-03-21

    Glycoside hydrolases that degrade plant cell walls have complex molecular architectures in which one or more catalytic modules are appended to noncatalytic carbohydrate-binding modules (CBMs). CBMs promote binding to polysaccharides and potentiate enzymic hydrolysis. Although there are diverse sequence-based families of xylan-binding CBMs, these modules, in general, recognize both decorated and unsubstituted forms of the target polysaccharide, and thus the evolutionary rationale for this diversity is unclear. Using immunohistochemistry to interrogate the specificity of six xylan-binding CBMs for their target polysaccharides in cell walls has revealed considerable differences in the recognition of plant materials between these protein modules. Family 2b and 15 CBMs bind to xylan in secondary cell walls in a range of dicotyledon species, whereas family 4, 6, and 22 CBMs display a more limited capability to bind to secondary cell walls. A family 35 CBM, which displays more restricted ligand specificity against purified xylans than the other five protein modules, reveals a highly distinctive binding pattern to plant material including the recognition of primary cell walls of certain dicotyledons, a feature shared with CBM15. Differences in the specificity of the CBMs toward walls of wheat grain and maize coleoptiles were also evident. The variation in CBM specificity for ligands located in plant cell walls provides a biological rationale for the repertoire of structurally distinct xylan-binding CBMs present in nature, and points to the utility of these modules in probing the molecular architecture of cell walls.

  9. Modular Glucuronoxylan-Specific Xylanase with a Family CBM35 Carbohydrate-Binding Module

    PubMed Central

    Valenzuela, Susana Valeria; Diaz, Pilar

    2012-01-01

    Xyn30D from the xylanolytic strain Paenibacillus barcinonensis has been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing a Km of 14.72 mg/ml and a kcat value of 1,510 min−1. The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (β/α)8 barrel linked to a side-associated β-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side β-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized. PMID:22447606

  10. Binding specificity and thermodynamics of a family 9 carbohydrate-binding module from Thermotoga maritima xylanase 10A.

    PubMed

    Boraston, A B; Creagh, A L; Alam, M M; Kormos, J M; Tomme, P; Haynes, C A; Warren, R A; Kilburn, D G

    2001-05-29

    The C-terminal family 9 carbohydrate-binding module of xylanase 10A from Thermotoga maritima (CBM9-2) binds to amorphous cellulose, crystalline cellulose, and the insoluble fraction of oat spelt xylan. The association constants (K(a)) for adsorption to insoluble polysaccharides are 1 x 10(5) to 3 x 10(5) M(-1). Of the soluble polysaccharides tested, CBM9-2 binds to barley beta-glucan, xyloglucan, and xylan. CBM9-2 binds specifically to the reducing ends of cellulose and soluble polysaccharides, a property that is currently unique to this CBM. CBM9-2 also binds glucose, xylose, galactose, arabinose, cellooligosaccharides, xylooligosaccharides, maltose, and lactose, with affinities ranging from 10(3) M(-1) for monosaccharides to 10(6) M(-1) for disaccharides and oligosaccharides. Cellooligosaccharides longer than two glucose units do not bind with improved affinity, indicating that cellobiose is sufficient to occupy the entire binding site. In general, the binding reaction is dominated by favorable changes in enthalpy, which are partially compensated by unfavorable entropy changes.

  11. Understanding How Noncatalytic Carbohydrate Binding Modules Can Display Specificity for Xyloglucan*

    PubMed Central

    Luís, Ana S.; Venditto, Immacolata; Temple, Max J.; Rogowski, Artur; Baslé, Arnaud; Xue, Jie; Knox, J. Paul; Prates, José A.M.; Ferreira, Luís M. A.; Fontes, Carlos M. G. A.; Najmudin, Shabir; Gilbert, Harry J.

    2013-01-01

    Plant biomass is central to the carbon cycle and to environmentally sustainable industries exemplified by the biofuel sector. Plant cell wall degrading enzymes generally contain noncatalytic carbohydrate binding modules (CBMs) that fulfil a targeting function, which enhances catalysis. CBMs that bind β-glucan chains often display broad specificity recognizing β1,4-glucans (cellulose), β1,3-β1,4-mixed linked glucans and xyloglucan, a β1,4-glucan decorated with α1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the β1,4-glucan backbone and only accommodate the xylose decorations. Here we show that two closely related CBMs, CBM65A and CBM65B, derived from EcCel5A, a Eubacterium cellulosolvens endoglucanase, bind to a range of β-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a β-sandwich fold. The ligand binding site comprises the β-sheet that forms the concave surface of the proteins. Binding to the backbone chains of β-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize β-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln106 is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which β-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated β-glucans. PMID:23229556

  12. How nature can exploit nonspecific catalytic and carbohydrate binding modules to create enzymatic specificity

    PubMed Central

    Cuskin, Fiona; Flint, James E.; Gloster, Tracey M.; Morland, Carl; Baslé, Arnaud; Henrissat, Bernard; Coutinho, Pedro M.; Strazzulli, Andrea; Solovyova, Alexandra S.; Davies, Gideon J.; Gilbert, Harry J.

    2012-01-01

    Noncatalytic carbohydrate binding modules (CBMs) are components of glycoside hydrolases that attack generally inaccessible substrates. CBMs mediate a two- to fivefold elevation in the activity of endo-acting enzymes, likely through increasing the concentration of the appended enzymes in the vicinity of the substrate. The function of CBMs appended to exo-acting glycoside hydrolases is unclear because their typical endo-binding mode would not fulfill a targeting role. Here we show that the Bacillus subtilis exo-acting β-fructosidase SacC, which specifically hydrolyses levan, contains the founding member of CBM family 66 (CBM66). The SacC-derived CBM66 (BsCBM66) targets the terminal fructosides of the major fructans found in nature. The crystal structure of BsCBM66 in complex with ligands reveals extensive interactions with the terminal fructose moiety (Fru-3) of levantriose but only limited hydrophobic contacts with Fru-2, explaining why the CBM displays broad specificity. Removal of BsCBM66 from SacC results in a ∼100-fold reduction in activity against levan. The truncated enzyme functions as a nonspecific β-fructosidase displaying similar activity against β-2,1– and β-2,6–linked fructans and their respective fructooligosaccharides. Conversely, appending BsCBM66 to BT3082, a nonspecific β-fructosidase from Bacteroides thetaiotaomicron, confers exolevanase activity on the enzyme. We propose that BsCBM66 confers specificity for levan, a branched fructan, through an “avidity” mechanism in which the CBM and the catalytic module target the termini of different branches of the same polysaccharide molecule. This report identifies a unique mechanism by which CBMs modulate enzyme function, and shows how specificity can be tailored by integrating nonspecific catalytic and binding modules into a single enzyme. PMID:23213210

  13. Mutated Leguminous Lectin Containing a Heparin-Binding like Motif in a Carbohydrate-Binding Loop Specifically Binds to Heparin

    PubMed Central

    Abo, Hirohito; Soga, Keisuke; Tanaka, Atsuhiro; Tateno, Hiroaki; Hirabayashi, Jun; Yamamoto, Kazuo

    2015-01-01

    We previously introduced random mutations in the sugar-binding loops of a leguminous lectin and screened the resulting mutated lectins for novel specificities using cell surface display. Screening of a mutated peanut agglutinin (PNA), revealed a mutated PNA with a distinct preference for heparin. Glycan microarray analyses using the mutated lectin fused to the Fc region of human immunoglobulin, revealed that a particular sulfated glycosaminoglycan (GAG), heparin, had the highest binding affinity for mutated PNA among 97 glycans tested, although wild-type PNA showed affinity towards Galβ1-3GalNAc and similar galactosylated glycans. Further analyses of binding specificity using an enzyme-linked immunoadsorbent assay demonstrated that the mutated PNA specifically binds to heparin, and weakly to de-2-O-sulfated heparin, but not to other GAG chains including de-6-O-sulfated and de-N-sulfated heparins. The mutated PNA had six amino acid substitutions within the eight amino acid-long sugar-binding loop. In this loop, the heparin-binding like motif comprised three arginine residues at positions 124, 128, and 129, and a histidine at position 125 was present. Substitution of each arginine or histidine residue to alanine reduced heparin-binding ability, indicating that all of these basic amino acid residues contributed to heparin binding. Inhibition assay demonstrated that heparin and dextran sulfate strongly inhibited mutated PNA binding to heparin in dose-dependent manner. The mutated PNA could distinguish between CHO cells and proteoglycan-deficient mutant cells. This is the first report establishing a novel leguminous lectin that preferentially binds to highly sulfated heparin and may provide novel GAG-binding probes to distinguish between heterogeneous GAG repeating units. PMID:26714191

  14. Specificity of O-glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules.

    PubMed

    Chen, Liqun; Drake, Matthew R; Resch, Michael G; Greene, Eric R; Himmel, Michael E; Chaffey, Patrick K; Beckham, Gregg T; Tan, Zhongping

    2014-05-27

    The majority of biological turnover of lignocellulosic biomass in nature is conducted by fungi, which commonly use Family 1 carbohydrate-binding modules (CBMs) for targeting enzymes to cellulose. Family 1 CBMs are glycosylated, but the effects of glycosylation on CBM function remain unknown. Here, the effects of O-mannosylation are examined on the Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase at three glycosylation sites. To enable this work, a procedure to synthesize glycosylated Family 1 CBMs was developed. Subsequently, a library of 20 CBMs was synthesized with mono-, di-, or trisaccharides at each site for comparison of binding affinity, proteolytic stability, and thermostability. The results show that, although CBM mannosylation does not induce major conformational changes, it can increase the thermolysin cleavage resistance up to 50-fold depending on the number of mannose units on the CBM and the attachment site. O-Mannosylation also increases the thermostability of CBM glycoforms up to 16 °C, and a mannose disaccharide at Ser3 seems to have the largest themostabilizing effect. Interestingly, the glycoforms with small glycans at each site displayed higher binding affinities for crystalline cellulose, and the glycoform with a single mannose at each of three positions conferred the highest affinity enhancement of 7.4-fold. Overall, by combining chemical glycoprotein synthesis and functional studies, we show that specific glycosylation events confer multiple beneficial properties on Family 1 CBMs.

  15. Specificity of O-glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

    PubMed Central

    Chen, Liqun; Drake, Matthew R.; Resch, Michael G.; Greene, Eric R.; Himmel, Michael E.; Chaffey, Patrick K.; Beckham, Gregg T.; Tan, Zhongping

    2014-01-01

    The majority of biological turnover of lignocellulosic biomass in nature is conducted by fungi, which commonly use Family 1 carbohydrate-binding modules (CBMs) for targeting enzymes to cellulose. Family 1 CBMs are glycosylated, but the effects of glycosylation on CBM function remain unknown. Here, the effects of O-mannosylation are examined on the Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase at three glycosylation sites. To enable this work, a procedure to synthesize glycosylated Family 1 CBMs was developed. Subsequently, a library of 20 CBMs was synthesized with mono-, di-, or trisaccharides at each site for comparison of binding affinity, proteolytic stability, and thermostability. The results show that, although CBM mannosylation does not induce major conformational changes, it can increase the thermolysin cleavage resistance up to 50-fold depending on the number of mannose units on the CBM and the attachment site. O-Mannosylation also increases the thermostability of CBM glycoforms up to 16 °C, and a mannose disaccharide at Ser3 seems to have the largest themostabilizing effect. Interestingly, the glycoforms with small glycans at each site displayed higher binding affinities for crystalline cellulose, and the glycoform with a single mannose at each of three positions conferred the highest affinity enhancement of 7.4-fold. Overall, by combining chemical glycoprotein synthesis and functional studies, we show that specific glycosylation events confer multiple beneficial properties on Family 1 CBMs. PMID:24821760

  16. Specific tracking of xylan using fluorescent-tagged carbohydrate-binding module 15 as molecular probe.

    PubMed

    Khatri, Vinay; Hébert-Ouellet, Yannick; Meddeb-Mouelhi, Fatma; Beauregard, Marc

    2016-01-01

    Xylan has been identified as a physical barrier which limits cellulose accessibility by covering the outer surface of fibers and interfibrillar space. Therefore, tracking xylan is a prerequisite for understanding and optimizing lignocellulosic biomass processes. In this study, we developed a novel xylan tracking approach using a two-domain probe called OC15 which consists of a fusion of Cellvibrio japonicus carbohydrate-binding domain 15 with the fluorescent protein mOrange2. The new probe specifically binds to xylan with an affinity similar to that of CBM15. The sensitivity of the OC15-xylan detection approach was compared to that of standard methods such as X-ray photoelectron spectroscopy (XPS) and chemical composition analysis (NREL/TP-510-42618). All three approaches were used to analyze the variations of xylan content of kraft pulp fibers. XPS, which allows for surface analysis of fibers, did not clearly indicate changes in xylan content. Chemical composition analysis responded to the changes in xylan content, but did not give any specific information related to the fibers surface. Interestingly, only the OC15 probe enabled the highly sensitive detection of xylan variations at the surface of kraft pulp fibers. At variance with the other methods, the OC15 probe can be used in a high throughput format. We developed a rapid and high throughput approach for the detection of changes in xylan exposure at the surface of paper fibers. The introduction of this method into the lignocellulosic biomass-based industries should revolutionize the understanding and optimization of most wood biomass processes.

  17. Carbohydrate-binding specificity of the daffodil (Narcissus pseudonarcissus) and amaryllis (Hippeastrum hybr.) bulb lectins.

    PubMed

    Kaku, H; Van Damme, E J; Peumans, W J; Goldstein, I J

    1990-06-01

    The carbohydrate binding specificity of the daffodil (Narcissus pseudonarcissus; NPA) and amaryllis (Hippeastrum hybr.; HHA) lectins, isolated from extracts of their bulbs by affinity chromatography on immobilized mannose, was studied by quantitative precipitation, sugar hapten inhibition, and affinity chromatography on the immobilized lectins. These lectins gave strong precipitation reactions with several yeast mannans, but did not precipitate with alpha-D-glucans (e.g., dextrans and glycogen). Interestingly, both lectins reacted strongly with yeast galactomannans having multiple nonreducing terminal alpha-D-galactosyl groups, a synthetic linear alpha-1,6-mannan, and an alpha-1,3-mannan (DP = 30). Treatment of the linear alpha-1,3-mannan with periodate, resulting in oxidation of the terminal, nonreducing mannosyl group, did not reduce its reactivity with NPA or HHA. Taken together, these observations suggest that NPA and HHA react not only with terminal but also with internal alpha-D-mannosyl residues. Sugar hapten inhibition studies showed these lectins to possess the greatest specific activity for alpha-D-mannosyl units whereas D-Glc and D-GlcNAc did not inhibit either lectin precipitation system. Of the oligosaccharides tested, the best inhibitor of NPA interaction was alpha-1,6-linked mannotriose, which was twice as good an inhibitor as Man alpha 1,6Man alpha-O-Me and 10 times better than methyl alpha-D-mannoside. On the other hand, oligosaccharides containing either 1,3- or 1,6-linked mannosyl units were good inhibitors of the HHA-mannan precipitation system (6- to 20-fold more active than D-Man). These results indicate that both lectins appear to possess an extended binding site(s) complementary to at least three 1,6-linked alpha-mannosyl units. Various glycosylasparagine glycopeptides which contain alpha-1,6-Man units were retarded on the immobilized NPA column. On the other hand, those containing either alpha-1,3- or alpha-1,6-mannosyl residues were

  18. Carbohydrate-binding specificities of potential probiotic Lactobacillus strains in porcine jejunal (IPEC-J2) cells and porcine mucin.

    PubMed

    Valeriano, Valerie Diane; Bagon, Bernadette B; Balolong, Marilen P; Kang, Dae-Kyung

    2016-07-01

    Bacterial lectins are carbohydrate-binding adhesins that recognize glycoreceptors in the gut mucus and epithelium of hosts. In this study, the contribution of lectin-like activities to adhesion of Lactobacillus mucosae LM1 and Lactobacillus johnsonii PF01, which were isolated from swine intestine, were compared to those of the commercial probiotic Lactobacillus rhamnosus GG. Both LM1 and PF01 strains have been reported to have good adhesion ability to crude intestinal mucus of pigs. To confirm this, we quantified their adhesion to porcine gastric mucin and intestinal porcine enterocytes isolated from the jejunum of piglets (IPEC-J2). In addition, we examined their carbohydrate-binding specificities by suspending bacterial cells in carbohydrate solutions prior to adhesion assays. We found that the selected carbohydrates affected the adherences of LM1 to IPEC-J2 cells and of LGG to mucin. In addition, compared to adhesion to IPEC-J2 cells, adhesion to mucin by both LM1 and LGG was characterized by enhanced specific recognition of glycoreceptor components such as galactose, mannose, and N-acetylglucosamine. Hydrophobic interactions might make a greater contribution to adhesion of PF01. A similar adhesin profile between a probiotic and a pathogen, suggest a correlation between shared pathogen-probiotic glycoreceptor recognition and the ability to exclude enteropathogens such as Escherichia coli K88 and Salmonella Typhimurium KCCM 40253. These findings extend our understanding of the mechanisms of the intestinal adhesion and pathogen-inhibition abilities of probiotic Lactobacillus strains.

  19. Binding of Sperm to the Zona Pellucida Mediated by Sperm Carbohydrate-Binding Proteins is not Species-Specific in vitro between Pigs and Cattle

    PubMed Central

    Takahashi, Kazuya; Kikuchi, Kazuhiro; Uchida, Yasuomi; Kanai-Kitayama, Saeko; Suzuki, Reiichiro; Sato, Reiko; Toma, Kazunori; Geshi, Masaya; Akagi, Satoshi; Nakano, Minoru; Yonezawa, Naoto

    2013-01-01

    Carbohydrates are candidates for the basis of species-selective interaction of gametes during mammalian fertilization. In this study, we sought to clarify the roles of sugar residues in the species-selective, sperm–oocyte interaction in pigs and cattle. Acrosome-intact porcine and bovine sperm exhibited their strongest binding affinities for β-Gal and α-Man residues, respectively. Porcine-sperm specificity changed from β-Gal to α-Man after the acrosome reaction, while bovine-sperm specificity did not. Binding of acrosome-intact and acrosome-reacted sperm decreased after trypsinization, indicating that the carbohydrate-binding components are proteins. While immature oocytes bound homologous sperm preferentially to heterologous sperm, oocytes matured in vitro bound similar numbers of homologous and heterologous sperm. Lectin staining revealed the aggregation of α-Man residues on the outer surface of the porcine zona during maturation. In both species, zona-free, mature oocytes bound homologous sperm preferentially to heterologous sperm. The lectin-staining patterns of the zona pellucida and zona-free oocytes coincided with the carbohydrate-binding specificities of acrosome-intact and acrosome-reacted sperm, respectively, supporting the involvement of carbohydrates in gamete recognition in pigs and cattle. These results also indicate that sperm-zona pellucida and sperm–oolemma bindings are not strictly species-specific in pigs and cattle, and further suggest that sperm penetration into the zona and/or fusion with oolemma may be species-specific between pigs and cattle. PMID:24970158

  20. Affinity electrophoresis as a method for determining substrate-binding specificity of carbohydrate-active enzymes for soluble polysaccharides.

    PubMed

    Moraïs, Sarah; Lamed, Raphael; Bayer, Edward A

    2012-01-01

    Affinity electrophoresis is a simple and rapid tool for the analysis of protein-binding affinities to soluble polysaccharides. This approach is particularly suitable for the characterization of the carbohydrate-active enzymes that contain a carbohydrate-binding module and for their mutants and chimeras. Knowledge of the binding characteristics of these enzymes can be the first step to elucidate the enzymatic activity of a putative enzyme; moreover in some cases, enzymes are able to bind polysaccharides targets other than their specified substrate, and this knowledge can be essential to understand the basics of the intrinsic mechanism of these enzymes in their natural environment.

  1. A Novel, Noncatalytic Carbohydrate-binding Module Displays Specificity for Galactose-containing Polysaccharides through Calcium-mediated Oligomerization*

    PubMed Central

    Montanier, Cedric Y.; Correia, Márcia A. S.; Flint, James E.; Zhu, Yanping; Baslé, Arnaud; McKee, Lauren S.; Prates, José A. M.; Polizzi, Samuel J.; Coutinho, Pedro M.; Lewis, Richard J.; Henrissat, Bernard; Fontes, Carlos M. G. A.; Gilbert, Harry J.

    2011-01-01

    The enzymic degradation of plant cell walls plays a central role in the carbon cycle and is of increasing environmental and industrial significance. The catalytic modules of enzymes that catalyze this process are generally appended to noncatalytic carbohydrate-binding modules (CBMs). CBMs potentiate the rate of catalysis by bringing their cognate enzymes into intimate contact with the target substrate. A powerful plant cell wall-degrading system is the Clostridium thermocellum multienzyme complex, termed the “cellulosome.” Here, we identify a novel CBM (CtCBM62) within the large C. thermocellum cellulosomal protein Cthe_2193 (defined as CtXyl5A), which establishes a new CBM family. Phylogenetic analysis of CBM62 members indicates that a circular permutation occurred within the family. CtCBM62 binds to d-galactose and l-arabinopyranose in either anomeric configuration. The crystal structures of CtCBM62, in complex with oligosaccharides containing α- and β-galactose residues, show that the ligand-binding site in the β-sandwich protein is located in the loops that connect the two β-sheets. Specificity is conferred through numerous interactions with the axial O4 of the target sugars, a feature that distinguishes galactose and arabinose from the other major sugars located in plant cell walls. CtCBM62 displays tighter affinity for multivalent ligands compared with molecules containing single galactose residues, which is associated with precipitation of these complex carbohydrates. These avidity effects, which confer the targeting of polysaccharides, are mediated by calcium-dependent oligomerization of the CBM. PMID:21454512

  2. The family 42 carbohydrate-binding module of family 54 α-L-arabinofuranosidase specifically binds the arabinofuranose side chain of hemicellulose

    PubMed Central

    Miyanaga, Akimasa; Koseki, Takuya; Miwa, Yozo; Mese, Yuichiro; Nakamura, Sachiko; Kuno, Atsushi; Hirabayashi, Jun; Matsuzawa, Hiroshi; Wakagi, Takayoshi; Shoun, Hirofumi; Fushinobu, Shinya

    2006-01-01

    α-L-Arabinofuranosidase catalyses the hydrolysis of the α-1,2-, α-1,3-, and α-1,5-L-arabinofuranosidic bonds in L-arabinose-containing hemicelluloses such as arabinoxylan. AkAbf54 (the glycoside hydrolase family 54 α-L-arabinofuranosidase from Aspergillus kawachii) consists of two domains, a catalytic and an arabinose-binding domain. The latter has been named AkCBM42 [family 42 CBM (carbohydrate-binding module) of AkAbf54] because homologous domains are classified into CBM family 42. In the complex between AkAbf54 and arabinofuranosyl-α-1,2-xylobiose, the arabinose moiety occupies the binding pocket of AkCBM42, whereas the xylobiose moiety is exposed to the solvent. AkCBM42 was found to facilitate the hydrolysis of insoluble arabinoxylan, because mutants at the arabinose binding site exhibited markedly decreased activity. The results of binding assays and affinity gel electrophoresis showed that AkCBM42 interacts with arabinose-substituted, but not with unsubstituted, hemicelluloses. Isothermal titration calorimetry and frontal affinity chromatography analyses showed that the association constant of AkCBM42 with the arabinose moiety is approximately 103 M−1. These results indicate that AkCBM42 binds the non-reducing-end arabinofuranosidic moiety of hemicellulose. To our knowledge, this is the first example of a CBM that can specifically recognize the side-chain monosaccharides of branched hemicelluloses. PMID:16846393

  3. Molecular Basis for the Selectivity and Specificity of Ligand Recognition by the Family 16 Carbohydrate-binding Modules from Thermoanaerobacterium polysaccharolyticum ManA

    SciTech Connect

    Bae, Brian; Ohene-Adjei, Samuel; Kocherginskaya, Svetlana; Mackie, Roderick I.; Spies, M. Ashley; Cann, Isaac K.O.; Nair, Satish K.

    2008-06-06

    Enzymes that hydrolyze complex polysaccharides into simple sugars are modular in architecture and consist of single or multiple catalytic domains fused to targeting modules called carbohydrate-binding modules (CBMs). CBMs bind to their ligands with high affinity and increase the efficiency of the catalytic components by targeting the enzymes to its substrate. Here we utilized a multidisciplinary approach to characterize each of the two family 16 carbohydrate-binding domain components of the highly active mannanase from the thermophile Thermoanaerobacterium polysaccharolyticum. These represent the first crystal structures of family 16 CBMs. Calorimetric analysis showed that although these CBMs demonstrate high specificity toward {beta}-1,4-linked sugars, they can engage both cello- and mannopolysaccharides. To elucidate the molecular basis for this specificity and selectivity, we have determined high resolution crystal structures of each of the two CBMs, as well as of binary complexes of CBM16-1 bound to either mannopentaose or cellopentaose. These results provide detailed molecular insights into ligand recognition and yield a framework for rational engineering experiments designed to expand the natural repertoire of these targeting modules.

  4. A platform to screen for C-type lectin receptor-binding carbohydrates and their potential for cell-specific targeting and immune modulation.

    PubMed

    Maglinao, Maha; Eriksson, Magdalena; Schlegel, Mark K; Zimmermann, Stephanie; Johannssen, Timo; Götze, Sebastian; Seeberger, Peter H; Lepenies, Bernd

    2014-02-10

    Myeloid C-type lectin receptors (CLRs) in innate immunity represent a superfamily of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-antigens. The primary interaction of an antigen-presenting cell and a pathogen shapes the following immune response. Therefore, the identification of CLR ligands that can either enhance or modulate the immune response is of interest. We have developed a screening platform based on glycan arrays to identify immune modulatory carbohydrate ligands of CLRs. A comprehensive library of CLRs was expressed by fusing the extracellular part of each respective CLR, the part containing the carbohydrate-recognition domain (CRD), to the Fc fragment of human IgG1 molecules. CLR-Fc fusion proteins display the CRD in a dimeric form, are properly glycosylated, and can be detected by a secondary antibody with a conjugated fluorophore. Thus, they are valuable tools for high-throughput screening. We were able to identify novel carbohydrate binders of CLRs using the glycan array technology. These CLR-binding carbohydrates were then covalently attached to the model antigen ovalbumin. The ovalbumin neoglycoconjugates were used in a dendritic cell/T cell co-culture assay to stimulate transgenic T cells in vitro. In addition, mice were immunized with these conjugates to analyze the immune modulatory properties of the CLR ligands in vivo. The CLR ligands induced an increased Th1 cytokine production in vitro and modulated the humoral response in vivo. The platform described here allows for the identification of CLR ligands, as well as the evaluation of each ligand's cell-specific targeting and immune modulatory properties.

  5. Effects of lectins with various carbohydrate binding specificities on lipid metabolism in isolated rat and hamster adipocytes.

    PubMed

    Ng, T B; Li, W W; Yeung, H W

    1989-01-01

    1. Mannose-binding and N-acetylglucosamine binding lectins exhibited potent antilipolytic and lipogenic activities. 2. Fucose-binding lectins had minimal lipogenic activity but possessed antilipolytic activity. 3. Most galactose-binding and N-acetylgalactosamine-binding lectins were devoid of significant antilipolytic and lipogenic activities. Notable exceptions were lectins from Momordica charantia, Wisteria floribunda, Vicia villosa, Codium fragile and the Siberian pine tree. lipogenic activity but lacked antilipolytic activity. 5. The galactose-binding horse gram and osage orange lectins exerted antilipolytic activity in hamster but not in rat adipocytes.

  6. Use of substructure-specific carbohydrate binding modules to track changes in cellulose accessibility and surface morphology during the amorphogenesis step of enzymatic hydrolysis.

    PubMed

    Gourlay, Keith; Arantes, Valdeir; Saddler, Jack N

    2012-07-24

    Cellulose amorphogenesis, described as the non-hydrolytic "opening up" or disruption of a cellulosic substrate, is becoming increasingly recognized as one of the key steps in the enzymatic deconstruction of cellulosic biomass when used as a feedstock for fuels and chemicals production. Although this process is thought to play a major role in facilitating hydrolysis, the lack of quantitative techniques capable of accurately describing the molecular-level changes occurring in the substrate during amorphogenesis has hindered our understanding of this process. In this work, techniques for measuring changes in cellulose accessibility are reviewed and a new quantitative assay method is described. Carbohydrate binding modules (CBMs) with specific affinities for crystalline (CBM2a) or amorphous (CBM44) cellulose were used to track specific changes in the surface morphology of cotton fibres during amorphogenesis. The extents of phosphoric acid-induced and Swollenin-induced changes to cellulose accessibility were successfully quantified using this technique. The adsorption of substructure-specific CBMs can be used to accurately quantify the extent of changes to cellulose accessibility induced by non-hydrolytic disruptive proteins. The technique provided a quick, accurate and quantitative measure of the accessibility of cellulosic substrates. Expanding the range of CBMs used for adsorption studies to include those specific for such compounds as xylan or mannan should also allow for the accurate quantitative tracking of the accessibility of these and other polymers within the lignocellulosic biomass matrix.

  7. Use of substructure-specific carbohydrate binding modules to track changes in cellulose accessibility and surface morphology during the amorphogenesis step of enzymatic hydrolysis

    PubMed Central

    2012-01-01

    Background Cellulose amorphogenesis, described as the non-hydrolytic “opening up” or disruption of a cellulosic substrate, is becoming increasingly recognized as one of the key steps in the enzymatic deconstruction of cellulosic biomass when used as a feedstock for fuels and chemicals production. Although this process is thought to play a major role in facilitating hydrolysis, the lack of quantitative techniques capable of accurately describing the molecular-level changes occurring in the substrate during amorphogenesis has hindered our understanding of this process. Results In this work, techniques for measuring changes in cellulose accessibility are reviewed and a new quantitative assay method is described. Carbohydrate binding modules (CBMs) with specific affinities for crystalline (CBM2a) or amorphous (CBM44) cellulose were used to track specific changes in the surface morphology of cotton fibres during amorphogenesis. The extents of phosphoric acid-induced and Swollenin-induced changes to cellulose accessibility were successfully quantified using this technique. Conclusions The adsorption of substructure-specific CBMs can be used to accurately quantify the extent of changes to cellulose accessibility induced by non-hydrolytic disruptive proteins. The technique provided a quick, accurate and quantitative measure of the accessibility of cellulosic substrates. Expanding the range of CBMs used for adsorption studies to include those specific for such compounds as xylan or mannan should also allow for the accurate quantitative tracking of the accessibility of these and other polymers within the lignocellulosic biomass matrix. PMID:22828270

  8. Bivalent Carbohydrate Binding Is Required for Biological Activity of Clitocybe nebularis Lectin (CNL), the N,N′-Diacetyllactosediamine (GalNAcβ1–4GlcNAc, LacdiNAc)-specific Lectin from Basidiomycete C. nebularis*

    PubMed Central

    Pohleven, Jure; Renko, Miha; Magister, Špela; Smith, David F.; Künzler, Markus; Štrukelj, Borut; Turk, Dušan; Kos, Janko; Sabotič, Jerica

    2012-01-01

    Lectins are carbohydrate-binding proteins that exert their biological activity by binding to specific cell glycoreceptors. We have expressed CNL, a ricin B-like lectin from the basidiomycete Clitocybe nebularis in Escherichia coli. The recombinant lectin, rCNL, agglutinates human blood group A erythrocytes and is specific for the unique glycan N,N′-diacetyllactosediamine (GalNAcβ1–4GlcNAc, LacdiNAc) as demonstrated by glycan microarray analysis. We here describe the crystal structures of rCNL in complex with lactose and LacdiNAc, defining its interactions with the sugars. CNL is a homodimeric lectin, each of whose monomers consist of a single ricin B lectin domain with its β-trefoil fold and one carbohydrate-binding site. To study the mode of CNL action, a nonsugar-binding mutant and nondimerizing monovalent CNL mutants that retain carbohydrate-binding activity were prepared. rCNL and the mutants were examined for their biological activities against Jurkat human leukemic T cells and the hypersensitive nematode Caenorhabditis elegans mutant strain pmk-1. rCNL was toxic against both, although the mutants were inactive. Thus, the bivalent carbohydrate-binding property of homodimeric CNL is essential for its activity, providing one of the rare pieces of evidence that certain activities of lectins are associated with their multivalency. PMID:22298779

  9. Bridging lectin binding sites by multivalent carbohydrates.

    PubMed

    Wittmann, Valentin; Pieters, Roland J

    2013-05-21

    Carbohydrate-protein interactions are involved in a multitude of biological recognition processes. Since individual protein-carbohydrate interactions are usually weak, multivalency is often required to achieve biologically relevant binding affinities and selectivities. Among the possible mechanisms responsible for binding enhancement by multivalency, the simultaneous attachment of a multivalent ligand to several binding sites of a multivalent receptor (i.e. chelation) has been proven to have a strong impact. This article summarizes recent examples of chelating lectin ligands of different size. Covered lectins include the Shiga-like toxin, where the shortest distance between binding sites is ca. 9 Å, wheat germ agglutinin (WGA) (shortest distance between binding sites 13-14 Å), LecA from Pseudomonas aeruginosa (shortest distance 26 Å), cholera toxin and heat-labile enterotoxin (shortest distance 31 Å), anti-HIV antibody 2G12 (shortest distance 31 Å), concanavalin A (ConA) (shortest distance 72 Å), RCA120 (shortest distance 100 Å), and Erythrina cristagalli (ECL) (shortest distance 100 Å). While chelating binding of the discussed ligands is likely, experimental proof, for example by X-ray crystallography, is limited to only a few cases.

  10. Tissue-specific response of carbohydrate-responsive element binding protein (ChREBP) to mammalian hibernation in 13-lined ground squirrels.

    PubMed

    Logan, Samantha M; Storey, Kenneth B

    2016-10-01

    Mammalian hibernation is characterized by a general suppression of energy expensive processes and a switch to lipid oxidation as the primary fuel source. Glucose-responsive carbohydrate responsive element binding protein (ChREBP) has yet to be studied in hibernating organisms, which prepare for the cold winter months by feeding until they exhibit an obesity-like state that is accompanied by naturally-induced and completely reversible insulin resistance. Studying ChREBP expression and activity in the hibernating 13-lined ground squirrel is important to better understand the molecular mechanisms that regulate energy metabolism under cellular stress. Immunoblotting was used to determine the relative expression level and subcellular localization of ChREBP, as well as serine phosphorylation at 95 kDa, comparing euthermic and late torpid ground squirrel liver, kidney, heart and muscle. DNA-binding ELISAs and RT-PCR were used to explore ChREBP transcriptional activity during cold stress. ChREBP activity seemed generally suppressed in liver and kidney. During torpor, ChREBP total protein levels decreased to 44% of EC in liver, phosphoserine levels increased 2.1-fold of EC in kidney, and downstream Fasn/Pkl transcript levels decreased to <60% of EC in liver. By contrast, ChREBP activity generally increased during torpor in cardiac and skeletal muscle, where ChREBP total protein levels increased over 1.5-fold and 5-fold of EC in muscle and heart, respectively; where DNA-binding increased by ∼2-fold of EC in muscle; and where Fasn transcript levels increased over 3-fold and 7-fold in both muscle and heart, respectively. In summary, ChREBP has a tissue-specific role in regulating energy metabolism during hibernation.

  11. Overall carbohydrate-binding properties of Castanea crenata agglutinin (CCA).

    PubMed

    Nomura, Keiichi; Takahashi, Nobuyuki; Hirose, Masaaki; Nakamura, Sachiko; Yagi, Fumio

    2005-09-05

    The carbohydrate-binding properties of Castanea crenata agglutinin (CCA) were investigated by an enzyme-linked lectin absorbent assay. The binding ability of each carbohydrate was compared using IC(50) values. CCA exhibited mannose/glucose specificity, as observed with many mannose-binding jacalin-related lectins. For oligosaccharides containing glucose, it has been shown that the degree of polymerization and the linkage mode of glucose residues have no effect on CCA-carbohydrate interaction; thus, only the non-reducing end glucose unit in glucooligosaccharides may be involved in the interaction with CCA. Among mannooligosaccharides, CCA strongly recognized alpha-(1-->3)-D-Man-[alpha-D-Man-(1-->6)]-D-Man, which is a core in N-linked carbohydrate chains. By considering the results with glycoproteins, it is likely that CCA binds preferentially to mono- or non-sialylated biantennary carbohydrate chains. We also obtained K(d) values by analysis of the dependency of the IC(50) on CCA concentration, based on the hypothesis that CCA has a single binding site or two equivalent binding sites. The estimated K(d) values for mannose, glucose and alpha-(1-->3)-D-Man-[alpha-D-Man-(1-->6)]-D-Man were 2.39, 7.19 and 0.483 mM, respectively. The relative binding abilities showed good agreement with the relative inhibition intensities. Isothermal calorimetric titration was carried out to directly estimate the dissociation constants of CCA for mannose and for alpha-D-Man-(1-->3)-D-Man. The values were 2.34 mM for mannose and 0.507 mM alpha-D-Man-(1-->3)-D-Man. These results suggest that the relative inhibition intensity represents the ratio of K(d) values and that CCA has a single or two equivalent binding sites.

  12. Using structure to inform carbohydrate binding module function.

    PubMed

    Abbott, D Wade; van Bueren, Alicia Lammerts

    2014-10-01

    Generally, non-catalytic carbohydrate binding module (CBM) specificity has been shown to parallel the catalytic activity of the carbohydrate active enzyme (CAZyme) module it is appended to. With the rapid expansion in metagenomic sequence space for the potential discovery of new CBMs in addition to the recent emergence of several new CBM families that display diverse binding profiles and novel functions, elucidating the function of these protein modules has become a much more challenging task. This review summarizes several approaches that have been reported for using primary structure to inform CBM specificity and streamlining their biophysical characterization. In addition we discuss general trends in binding site architecture and several newly identified functions for CBMs. Streams of investigation that will facilitate the development and refinement of sequence-based prediction tools are suggested.

  13. Amino acid sequence and carbohydrate-binding analysis of the N-acetyl-D-galactosamine-specific C-type lectin, CEL-I, from the Holothuroidea, Cucumaria echinata.

    PubMed

    Hatakeyama, Tomomitsu; Matsuo, Noriaki; Shiba, Kouhei; Nishinohara, Shoichi; Yamasaki, Nobuyuki; Sugawara, Hajime; Aoyagi, Haruhiko

    2002-01-01

    CEL-I is one of the Ca2+-dependent lectins that has been isolated from the sea cucumber, Cucumaria echinata. This protein is composed of two identical subunits held by a single disulfide bond. The complete amino acid sequence of CEL-I was determined by sequencing the peptides produced by proteolytic fragmentation of S-pyridylethylated CEL-I. A subunit of CEL-I is composed of 140 amino acid residues. Two intrachain (Cys3-Cys14 and Cys31-Cys135) and one interchain (Cys36) disulfide bonds were also identified from an analysis of the cystine-containing peptides obtained from the intact protein. The similarity between the sequence of CEL-I and that of other C-type lectins was low, while the C-terminal region, including the putative Ca2+ and carbohydrate-binding sites, was relatively well conserved. When the carbohydrate-binding activity was examined by a solid-phase microplate assay, CEL-I showed much higher affinity for N-acetyl-D-galactosamine than for other galactose-related carbohydrates. The association constant of CEL-I for p-nitrophenyl N-acetyl-beta-D-galactosaminide (NP-GalNAc) was determined to be 2.3 x 10(4) M(-1), and the maximum number of bound NP-GalNAc was estimated to be 1.6 by an equilibrium dialysis experiment.

  14. Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module.

    PubMed

    Cicortas Gunnarsson, Lavinia; Montanier, Cedric; Tunnicliffe, Richard B; Williamson, Mike P; Gilbert, Harry J; Nordberg Karlsson, Eva; Ohlin, Mats

    2007-09-01

    Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.

  15. Carbohydrate-binding modules: fine-tuning polysaccharide recognition

    PubMed Central

    2004-01-01

    The enzymic degradation of insoluble polysaccharides is one of the most important reactions on earth. Despite this, glycoside hydrolases attack such polysaccharides relatively inefficiently as their target glycosidic bonds are often inaccessible to the active site of the appropriate enzymes. In order to overcome these problems, many of the glycoside hydrolases that utilize insoluble substrates are modular, comprising catalytic modules appended to one or more non-catalytic CBMs (carbohydrate-binding modules). CBMs promote the association of the enzyme with the substrate. In view of the central role that CBMs play in the enzymic hydrolysis of plant structural and storage polysaccharides, the ligand specificity displayed by these protein modules and the mechanism by which they recognize their target carbohydrates have received considerable attention since their discovery almost 20 years ago. In the last few years, CBM research has harnessed structural, functional and bioinformatic approaches to elucidate the molecular determinants that drive CBM–carbohydrate recognition. The present review summarizes the impact structural biology has had on our understanding of the mechanisms by which CBMs bind to their target ligands. PMID:15214846

  16. Comparison of the carbohydrate-binding specificities of cholera toxin and Escherichia coli heat-labile enterotoxins LTh-I, LT-IIa, and LT-IIb.

    PubMed Central

    Fukuta, S; Magnani, J L; Twiddy, E M; Holmes, R K; Ginsburg, V

    1988-01-01

    The heat-labile enterotoxins of Vibrio cholerae and Escherichia coli are related in structure and function. They are oligomers consisting of A and B polypeptide subunits. They bind to gangliosides, and they activate adenylate cyclase. The toxins form two antigenically distinct groups; members of each group cross-react but are not necessarily identical. Serogroup I includes cholera toxin (CT) and type I heat-labile enterotoxin (LT-I) of E. coli. LTh-I and LTp-I are antigenic variants of LT-I produced by strains of E. coli from humans and pigs, respectively. Serogroup II contains the type II heat-labile enterotoxin (LT-II) of E. coli. Two antigenic variants designated LT-IIa and LT-IIb have been described. The binding of CT, LTh-I, LT-IIa, and LT-IIb to gangliosides was analyzed by immunostaining thin-layer chromatograms and by solid-phase radioimmunoassay. The four toxins have different glycolipid-binding specificities. LTh-I and CT bind strongly to ganglioside GM1 and less strongly to ganglioside GD1b. However, LTh-I, unlike CT, also binds weakly to GM2 and asialo GM1. LTh-I, like CT, probably binds to the terminal sugar sequence Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)Gal . . ., where GalNAc is N-acetylgalactosamine and NeuAc is N-acetylneuraminic acid. LT-IIa probably binds to the same sugar sequence to which CT and LTh-I bind, with the additional contribution to binding of a second NeuAc as in GD1b and GD2. Also, LT-IIa must bind the Gal beta 1-3GalNAc . . . sequence in such a way that its binding is relatively unaffected by attachment of NeuAc to the terminal galactose residue as in GD1a, GT1b, and GQ1b. LT-IIb probably binds to the terminal sugar sequence NeuAc alpha 2-3Gal beta 1-4GalNAc . . ., as it binds to gangliosides GD1a and GT1b but not to GM1. Images PMID:3290106

  17. Xylan-specific carbohydrate-binding module belonging to family 6 enhances the catalytic performance of a GH11 endo-xylanase.

    PubMed

    Hoffmam, Zaira B; Zanphorlin, Letícia M; Cota, Junio; Diogo, José A; Almeida, Gabriela B; Damásio, André R L; Squina, Fabio; Murakami, Mario T; Ruller, Roberto

    2016-06-25

    Xylanases catalyze the hydrolysis of β-1,4-linked xylosyl moieties from xylan chains, one of the most abundant hemicellulosic polysaccharides found in plant cell walls. These enzymes can exist either as single catalytic domains or as modular proteins composed of one or more carbohydrate-binding modules (CBMs) appended to the catalytic core. However, the molecular mechanisms governing the synergistic effects between catalytic domains and their CBMs are not fully understood. Thus, the goal of this study was to evaluate the functional effects of the fusion of a CBM belonging to family 6, which exhibits high affinity to xylan, with the GH11 xylanase from Bacillus subtilis, which does not have a CBM in its wild-type form. The wild-type enzyme (BsXyl11) and the chimeric protein (BsXyl11-CBM6) were heterologously produced in Escherichia coli and purified to homogeneity for biochemical characterization. The molecular fusion did not alter the pH and temperature dependence, but kinetic data revealed an increase of 65% in the catalytic efficiency of the chimeric enzyme. Furthermore, the BsXyl11-CBM6 chimera was used to supplement the commercial cocktail Accellerase® 1500 and improved the reducing sugar release by 17% from pretreated sugarcane bagasse. These results indicate that CBM6 can be used as a molecular tool to enhance the catalytic performance of endo-xylanases (GH11) and provide a new strategy for the development of optimized biocatalysts for biotechnological applications.

  18. Oxidized cellulose binding to allergens with a carbohydrate-binding module attenuates allergic reactions.

    PubMed

    Shani, Nir; Shani, Ziv; Shoseyov, Oded; Mruwat, Rufayda; Shoseyov, David

    2011-01-15

    Grass and mite allergens are of the main causes of allergy and asthma. A carbohydrate-binding module (CBM) represents a common motif to groups I (β-expansin) and II/III (expansin-like) grass allergens and is suggested to mediate allergen-IgE binding. House dust mite group II allergen (Der p 2 and Der f 2) structures bear strong similarity to expansin's CBM, suggesting their ability to bind carbohydrates. Thus, this study proposes the design of a carbohydrate-based treatment in which allergen binding to carbohydrate particles will promote allergen airway clearance and prevent allergic reactions. The aim of the study was to identify a polysaccharide with high allergen-binding capacities and to explore its ability to prevent allergy. Oxidized cellulose (OC) demonstrated allergen-binding capacities toward grass and mite allergens that surpassed those of any other polysaccharide examined in this study. Furthermore, inhalant preparations of OC microparticles attenuated allergic lung inflammation in rye grass-sensitized Brown Norway rats and OVA-sensitized BALB/c mice. Fluorescently labeled OC efficiently cleared from the mouse airways and body organs. Moreover, long-term administration of OC inhalant to Wistar rats did not result in toxicity. In conclusion, many allergens, such as grass and dust mite, contain a common CBM motif. OC demonstrates a strong and relatively specific allergen-binding capacity to CBM-containing allergens. OC's ability to attenuate allergic inflammation, together with its documented safety record, forms a firm basis for its application as an alternative treatment for prevention and relief of allergy and asthma.

  19. Deciphering Ligand Specificity of a Clostridium thermocellum Family 35 Carbohydrate Binding Module (CtCBM35) for Gluco- and Galacto- Substituted Mannans and Its Calcium Induced Stability

    PubMed Central

    Ghosh, Arabinda; Luís, Ana Sofia; Brás, Joana L. A.; Pathaw, Neeta; Chrungoo, Nikhil K.; Fontes, Carlos M. G. A.; Goyal, Arun

    2013-01-01

    This study investigated the role of CBM35 from Clostridium thermocellum (CtCBM35) in polysaccharide recognition. CtCBM35 was cloned into pET28a (+) vector with an engineered His6 tag and expressed in Escherichia coli BL21 (DE3) cells. A homogenous 15 kDa protein was purified by immobilized metal ion chromatography (IMAC). Ligand binding analysis of CtCBM35 was carried out by affinity electrophoresis using various soluble ligands. CtCBM35 showed a manno-configured ligand specific binding displaying significant association with konjac glucomannan (Ka = 14.3×104 M−1), carob galactomannan (Ka = 12.4×104 M−1) and negligible association (Ka = 12 µM−1) with insoluble mannan. Binding of CtCBM35 with polysaccharides which was calcium dependent exhibited two fold higher association in presence of 10 mM Ca2+ ion with konjac glucomannan (Ka = 41×104 M−1) and carob galactomannan (Ka = 30×104 M−1). The polysaccharide binding was further investigated by fluorescence spectrophotometric studies. On binding with carob galactomannan and konjac glucomannan the conformation of CtCBM35 changed significantly with regular 21 nm peak shifts towards lower quantum yield. The degree of association (Ka) with konjac glucomannan and carob galactomannan, 14.3×104 M−1 and 11.4×104 M−1, respectively, corroborated the findings from affinity electrophoresis. The association of CtCBM35with konjac glucomannan led to higher free energy of binding (ΔG) −25 kJ mole−1 as compared to carob galactomannan (ΔG) −22 kJ mole−1. On binding CtCBM35 with konjac glucomannan and carob galactomannan the hydrodynamic radius (RH) as analysed by dynamic light scattering (DLS) study, increased to 8 nm and 6 nm, respectively, from 4.25 nm in absence of ligand. The presence of 10 mM Ca2+ ions imparted stiffer orientation of CtCBM35 particles with increased RH of 4.52 nm. Due to such stiffer orientation CtCBM35 became more thermostable and its melting temperature was shifted

  20. Diversified Carbohydrate-Binding Lectins from Marine Resources

    PubMed Central

    Ogawa, Tomohisa; Watanabe, Mizuki; Naganuma, Takako; Muramoto, Koji

    2011-01-01

    Marine bioresources produce a great variety of specific and potent bioactive molecules including natural organic compounds such as fatty acids, polysaccharides, polyether, peptides, proteins, and enzymes. Lectins are also one of the promising candidates for useful therapeutic agents because they can recognize the specific carbohydrate structures such as proteoglycans, glycoproteins, and glycolipids, resulting in the regulation of various cells via glycoconjugates and their physiological and pathological phenomenon through the host-pathogen interactions and cell-cell communications. Here, we review the multiple lectins from marine resources including fishes and sea invertebrate in terms of their structure-activity relationships and molecular evolution. Especially, we focus on the unique structural properties and molecular evolution of C-type lectins, galectin, F-type lectin, and rhamnose-binding lectin families. PMID:22312473

  1. Possible role for cell-surface carbohydrate-binding molecules in lymphocyte recirculation

    PubMed Central

    1983-01-01

    We are investigating the hypothesis that carbohydrate-binding molecules on the cell surface are involved in the recirculation of lymphocytes from the bloodstream into lymphoid organs. This phenomenon requires the specific attachment of circulating lymphocytes to the endothelial cells of postcapillary venules. Using an in vitro assay to measure the adhesive interaction between lymphocytes and postcapillary venules, we have found that L-fucose, D mannose, and the L-fucose-rich, sulfated polysaccharide fucoidin specifically inhibit this binding interaction. L-fucose shows stereo-selective inhibitory activity at concentrations greater than 18 mM while fucoidin produces 50% inhibition at approximately 1-5 X 10(-8) M. Fucoidin appears to interact with the lymphocyte, and not the postcapillary venule, to inhibit binding. These data suggest that cell surface carbohydrates (fucoselike) and carbohydrate-binding molecules (cell surface lectins) may contribute to the specific attachment of lymphocytes to postcapillary venules. PMID:6833380

  2. Ligand-mediated dimerization of a carbohydrate-binding molecule reveals a novel mechanism for protein-carbohydrate recognition.

    PubMed

    Flint, James; Nurizzo, Didier; Harding, Stephen E; Longman, Emma; Davies, Gideon J; Gilbert, Harry J; Bolam, David N

    2004-03-19

    The structural and thermodynamic basis for carbohydrate-protein recognition is of considerable importance. NCP-1, which is a component of the Piromyces equi cellulase/hemicellulase complex, presents a provocative model for analyzing how structural and mutational changes can influence the ligand specificity of carbohydrate-binding proteins. NCP-1 contains two "family 29" carbohydrate-binding modules designated CBM29-1 and CBM29-2, respectively, that display unusually broad specificity; the proteins interact weakly with xylan, exhibit moderate affinity for cellulose and mannan, and bind tightly to the beta-1,4-linked glucose-mannose heteropolymer glucomannan. The crystal structure of CBM29-2 in complex with cellohexaose and mannohexaose identified key residues involved in ligand recognition. By exploiting this structural information and the broad specificity of CBM29-2, we have used this protein as a template to explore the evolutionary mechanisms that can lead to significant changes in ligand specificity. Here, we report the properties of the E78R mutant of CBM29-2, which displays ligand specificity that is different from that of wild-type CBM29-2; the protein retains significant affinity for cellulose but does not bind to mannan or glucomannan. Significantly, E78R exhibits a stoichiometry of 0.5 when binding to cellohexaose, and both calorimetry and ultracentrifugation show that the mutant protein displays ligand-mediated dimerization in solution. The three-dimensional structure of E78R in complex with cellohexaose reveals the intriguing molecular basis for this "dimeric" binding mode that involves the lamination of the oligosaccharide between two CBM molecules. The 2-fold screw axis of the ligand is mirrored in the orientation of the two protein domains with adjacent sugar rings stacking against the equivalent aromatic residues in the binding site of each protein molecule of the molecular sandwich. The sandwiching of an oligosaccharide chain between two protein

  3. Carbohydrate-binding motif in Chitinase 3-like 1 (CHI3L1/YKL-40) specifically activates Akt signaling pathway in colonic epithelial cells

    PubMed Central

    Chen, Chun-Chuan; Llado, Victoria; Eurich, Katrin; Tran, Hoa T.; Mizoguchi, Emiko

    2011-01-01

    Host-microbial interactions play a key role during the development of colitis. We have previously shown that chinase 3-like 1 (CHI3L1) is an inducible molecule overexpressed in colonic epithelial cells (CECs) under inflammatory conditions. In this study, we found that chitin-binding motif (CBM) of CHI3L1 is specifically associated with the CHI3L1-mediated activation of the Akt-signaling in CEC by transfecting the CBM-mutant CHI3L1 vectors in SW480 CECs. Downstream, CHI3L1 enhanced the secretion of IL-8 and TNFα in a dose-dependent manner. We previously show that 325 through 339 amino-acids in CBM are crucial for the biological function of CHI3L1. Here we demonstrated that 325th–339th residues of CBM in CHI3L1 is a critical region for the activation of Akt, IL-8 production, and for a specific cellular localization of CHI3L1. In conclusion, CBM region of CHI3L1 is critical in activating Akt signaling in CECs, and the activation may be associated with the development of chronic colitis. PMID:21546314

  4. Lectin binding patterns and carbohydrate mediation of sperm binding to llama oviductal cells in vitro.

    PubMed

    Apichela, Silvana A; Valz-Gianinet, Jorge N; Schuster, Stefanie; Jiménez-Díaz, María A; Roldán-Olarte, Eugenia M; Miceli, Dora C

    2010-04-01

    Sperm binding to oviductal epithelium would be involved in sperm reservoir formation in the utero tubal junction (UTJ). Although in other mammals sperm-oviduct interaction has been proved to be mediated by carbohydrate-recognition mechanisms, the factors implicated in the sperm adhesion to oviductal epithelium of llama are still unknown. In order to assess the role of carbohydrates present in the mucosa surface, we examined the distribution of glycoconjugates in the llama oviduct by confocal lectin-histochemistry. Mannosyl, glucosyl, N-acetylglucosaminyl, galactosyl, N-acetylgalactosaminyl and sialic acid residues were detected in the oviductal mucose glycocalyx. By incubation of UTJ oviductal explants with LCA, DBA, UEA-1 or PNA lectin previous to co-culture with sperm, we observed a significant decrease in sperm binding only with LCA lectin. In the mucosa surface there were numerous d-glucosyl and D-manosyl residues, which were spotted by this lectin. Probably, this fact promotes the whole covering of the oviduct luminal surface by the sugar-lectin complex, preventing sperm access and adhesion of further residues. However, sperm incubation with mannose or glucose does not significantly prevent binding, which means that glucose and mannose would not be involved in a specific sperm-oviduct interaction. On the other hand, we observed a high reduction in sperm binding to UTJ explants with N-acetylgalactosamine and galactose (p<0.001). Coincidentally, binding sites for N-acetylgalactosamine-PAA-FITC conjugate were observed on the whole surface of the sperm, supporting the concept that llama sperm have lectin-like molecules in their surface, as is the case in other mammals. Probably, these lectin-like molecules, by means of N-acetylgalactosamine and galactose recognition, could link the sperm to the oviductal mucosa with the purpose of forming storing sites in the UTJ. Our results support the idea that more than one carbohydrate could participate in sperm reservoir

  5. Computational Investigation of Glycosylation Effects on a Family 1 Carbohydrate-binding Module*

    PubMed Central

    Taylor, Courtney B.; Talib, M. Faiz; McCabe, Clare; Bu, Lintao; Adney, William S.; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T.

    2012-01-01

    Carbohydrate-binding modules (CBMs) are ubiquitous components of glycoside hydrolases, which degrade polysaccharides in nature. CBMs target specific polysaccharides, and CBM binding affinity to cellulose is known to be proportional to cellulase activity, such that increasing binding affinity is an important component of performance improvement. To ascertain the impact of protein and glycan engineering on CBM binding, we use molecular simulation to quantify cellulose binding of a natively glycosylated Family 1 CBM. To validate our approach, we first examine aromatic-carbohydrate interactions on binding, and our predictions are consistent with previous experiments, showing that a tyrosine to tryptophan mutation yields a 2-fold improvement in binding affinity. We then demonstrate that enhanced binding of 3–6-fold over a nonglycosylated CBM is achieved by the addition of a single, native mannose or a mannose dimer, respectively, which has not been considered previously. Furthermore, we show that the addition of a single, artificial glycan on the anterior of the CBM, with the native, posterior glycans also present, can have a dramatic impact on binding affinity in our model, increasing it up to 140-fold relative to the nonglycosylated CBM. These results suggest new directions in protein engineering, in that modifying glycosylation patterns via heterologous expression, manipulation of culture conditions, or introduction of artificial glycosylation sites, can alter CBM binding affinity to carbohydrates and may thus be a general strategy to enhance cellulase performance. Our results also suggest that CBM binding studies should consider the effects of glycosylation on binding and function. PMID:22147693

  6. Computational Investigation of Glycosylation Effects on a Family 1 Carbohydrate-Binding Module

    SciTech Connect

    Taylor, C. B.; Talib, M. F.; McCabe, C.; Bu, L.; Adney, W. S.; Himmel, M. E.; Crowley, M. F.; Beckham, G. T.

    2012-01-27

    Carbohydrate-binding modules (CBMs) are ubiquitous components of glycoside hydrolases, which degrade polysaccharides in nature. CBMs target specific polysaccharides, and CBM binding affinity to cellulose is known to be proportional to cellulase activity, such that increasing binding affinity is an important component of performance improvement. To ascertain the impact of protein and glycan engineering on CBM binding, we use molecular simulation to quantify cellulose binding of a natively glycosylated Family 1 CBM. To validate our approach, we first examine aromatic-carbohydrate interactions on binding, and our predictions are consistent with previous experiments, showing that a tyrosine to tryptophan mutation yields a 2-fold improvement in binding affinity. We then demonstrate that enhanced binding of 3-6-fold over a nonglycosylated CBM is achieved by the addition of a single, native mannose or a mannose dimer, respectively, which has not been considered previously. Furthermore, we show that the addition of a single, artificial glycan on the anterior of the CBM, with the native, posterior glycans also present, can have a dramatic impact on binding affinity in our model, increasing it up to 140-fold relative to the nonglycosylated CBM. These results suggest new directions in protein engineering, in that modifying glycosylation patterns via heterologous expression, manipulation of culture conditions, or introduction of artificial glycosylation sites, can alter CBM binding affinity to carbohydrates and may thus be a general strategy to enhance cellulase performance. Our results also suggest that CBM binding studies should consider the effects of glycosylation on binding and function.

  7. The effect of structural differences in the reducing terminus of sugars on the binding affinity of carbohydrates and proteins analyzed using photoaffinity labeling.

    PubMed

    Ohtsuka, Isao; Sadakane, Yutaka; Higuchi, Mari; Hada, Noriyasu; Hada, Junko; Kakiuchi, Nobuko; Sakushima, Akiyo

    2011-01-15

    Because carbohydrates and proteins bind with such low affinity, the nature of their interactions is not clear. Photoaffinity labeling with diazirin groups is useful for elucidating the roles of carbohydrates in these binding processes. However, when carbohydrate probes are synthesized according to this conventional method, the reducing terminus of the sugar is opened to provide an acyclic structure. Because greater elucidation of carbohydrate-protein interactions requires a closed-ring carbohydrate in addition to the photoreactive group, we synthesized new molecular tools. The carbohydrate ligands were synthesized in three steps (glycosylation with allyl alcohol, deprotection, and ozonolysis). Specific binding proteins for carbohydrate ligands were obtained by photoaffinity labeling. Closed ring-type carbohydrate ligands, in which the reducing sugar is closed, bound to lectins more strongly than open ring-type sugars. Carbohydrate to protein binding was observed using AFM.

  8. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes.

    PubMed

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data.

  9. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    PubMed Central

    Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  10. Modeling of Carbohydrate Binding Modules Complexed to Cellulose

    SciTech Connect

    Nimlos, M. R.; Beckham, G. T.; Bu, L.; Himmel, M. E.; Crowley, M. F.; Bomble, Y. J.

    2012-01-01

    Modeling results are presented for the interaction of two carbohydrate binding modules (CBMs) with cellulose. The family 1 CBM from Trichoderma reesei's Cel7A cellulase was modeled using molecular dynamics to confirm that this protein selectively binds to the hydrophobic (100) surface of cellulose fibrils and to determine the energetics and mechanisms for locating this surface. Modeling was also conducted of binding of the family 4 CBM from the CbhA complex from Clostridium thermocellum. There is a cleft in this protein, which may accommodate a cellulose chain that is detached from crystalline cellulose. This possibility is explored using molecular dynamics.

  11. Biophysical studies on calcium and carbohydrate binding to carbohydrate recognition domain of Gal/GalNAc lectin from Entamoeba histolytica: insights into host cell adhesion.

    PubMed

    Yadav, Rupali; Verma, Kuldeep; Chandra, Mintu; Mukherjee, Madhumita; Datta, Sunando

    2016-09-01

    Entamoeba histolytica, an enteric parasite expresses a Gal/GalNAc-specific lectin that contributes to its virulence by establishing adhesion to host cell. In this study, carbohydrate recognition domain of Hgl (EhCRD) was purified and biophysical studies were conducted to understand the thermodynamic basis of its binding to carbohydrate and Ca(++) Here, we show that carbohydrate recognition domain (CRD) of the lectin binds to calcium through DPN motif. To decipher the role of calcium in carbohydrate binding and host cell adhesion, biophysical and cell-based studies were carried out. We demonstrated that the presence of the cation neither change the affinity of the lectin for carbohydrates nor alters its conformation. Mutation of the calcium-binding motif in EhCRD resulted in complete loss of ability to bind calcium but retained its affinity for carbohydrates. Purified EhCRD significantly diminished adhesion of the amebic trophozoites to Chinese Hamster Ovary (CHO) cells as well as triggered red blood cell agglutination. The calcium-binding defective mutant abrogated amebic adhesion to CHO cells similar to the wild-type protein, but it failed to agglutinate RBCs suggesting a differential role of the cation in these two processes. This study provides the first molecular description of the role of calcium in Gal/GalNAc mediated host cell adhesion.

  12. Prediction of carbohydrate binding sites on protein surfaces with 3-dimensional probability density distributions of interacting atoms.

    PubMed

    Tsai, Keng-Chang; Jian, Jhih-Wei; Yang, Ei-Wen; Hsu, Po-Chiang; Peng, Hung-Pin; Chen, Ching-Tai; Chen, Jun-Bo; Chang, Jeng-Yih; Hsu, Wen-Lian; Yang, An-Suei

    2012-01-01

    Non-covalent protein-carbohydrate interactions mediate molecular targeting in many biological processes. Prediction of non-covalent carbohydrate binding sites on protein surfaces not only provides insights into the functions of the query proteins; information on key carbohydrate-binding residues could suggest site-directed mutagenesis experiments, design therapeutics targeting carbohydrate-binding proteins, and provide guidance in engineering protein-carbohydrate interactions. In this work, we show that non-covalent carbohydrate binding sites on protein surfaces can be predicted with relatively high accuracy when the query protein structures are known. The prediction capabilities were based on a novel encoding scheme of the three-dimensional probability density maps describing the distributions of 36 non-covalent interacting atom types around protein surfaces. One machine learning model was trained for each of the 30 protein atom types. The machine learning algorithms predicted tentative carbohydrate binding sites on query proteins by recognizing the characteristic interacting atom distribution patterns specific for carbohydrate binding sites from known protein structures. The prediction results for all protein atom types were integrated into surface patches as tentative carbohydrate binding sites based on normalized prediction confidence level. The prediction capabilities of the predictors were benchmarked by a 10-fold cross validation on 497 non-redundant proteins with known carbohydrate binding sites. The predictors were further tested on an independent test set with 108 proteins. The residue-based Matthews correlation coefficient (MCC) for the independent test was 0.45, with prediction precision and sensitivity (or recall) of 0.45 and 0.49 respectively. In addition, 111 unbound carbohydrate-binding protein structures for which the structures were determined in the absence of the carbohydrate ligands were predicted with the trained predictors. The overall

  13. Prediction of Carbohydrate Binding Sites on Protein Surfaces with 3-Dimensional Probability Density Distributions of Interacting Atoms

    PubMed Central

    Tsai, Keng-Chang; Jian, Jhih-Wei; Yang, Ei-Wen; Hsu, Po-Chiang; Peng, Hung-Pin; Chen, Ching-Tai; Chen, Jun-Bo; Chang, Jeng-Yih; Hsu, Wen-Lian; Yang, An-Suei

    2012-01-01

    Non-covalent protein-carbohydrate interactions mediate molecular targeting in many biological processes. Prediction of non-covalent carbohydrate binding sites on protein surfaces not only provides insights into the functions of the query proteins; information on key carbohydrate-binding residues could suggest site-directed mutagenesis experiments, design therapeutics targeting carbohydrate-binding proteins, and provide guidance in engineering protein-carbohydrate interactions. In this work, we show that non-covalent carbohydrate binding sites on protein surfaces can be predicted with relatively high accuracy when the query protein structures are known. The prediction capabilities were based on a novel encoding scheme of the three-dimensional probability density maps describing the distributions of 36 non-covalent interacting atom types around protein surfaces. One machine learning model was trained for each of the 30 protein atom types. The machine learning algorithms predicted tentative carbohydrate binding sites on query proteins by recognizing the characteristic interacting atom distribution patterns specific for carbohydrate binding sites from known protein structures. The prediction results for all protein atom types were integrated into surface patches as tentative carbohydrate binding sites based on normalized prediction confidence level. The prediction capabilities of the predictors were benchmarked by a 10-fold cross validation on 497 non-redundant proteins with known carbohydrate binding sites. The predictors were further tested on an independent test set with 108 proteins. The residue-based Matthews correlation coefficient (MCC) for the independent test was 0.45, with prediction precision and sensitivity (or recall) of 0.45 and 0.49 respectively. In addition, 111 unbound carbohydrate-binding protein structures for which the structures were determined in the absence of the carbohydrate ligands were predicted with the trained predictors. The overall

  14. Photonic crystal borax competitive binding carbohydrate sensing motif.

    PubMed

    Cui, Qingzhou; Ward Muscatello, Michelle M; Asher, Sanford A

    2009-05-01

    We developed a photonic crystal sensing method for diol containing species such as carbohydrates based on a poly(vinyl alcohol) (PVA) hydrogel containing an embedded crystalline colloidal array (CCA). The polymerized CCA (PCCA) diffracts visible light. We show that in the presence of borax the diffraction wavelength shifts as the concentration of glucose changes. The diffraction shifts result from the competitive binding of glucose to borate, which reduces the concentration of borate bound to the PVA diols.

  15. Photonic crystal borax competitive binding carbohydrate sensing motif†

    PubMed Central

    Cui, Qingzhou; Muscatello, Michelle M. Ward; Asher, Sanford A.

    2009-01-01

    We developed a photonic crystal sensing method for diol containing species such as carbohydrates based on a poly(vinyl alcohol) (PVA) hydrogel containing an embedded crystalline colloidal array (CCA). The polymerized CCA (PCCA) diffracts visible light. We show that in the presence of borax the diffraction wavelength shifts as the concentration of glucose changes. The diffraction shifts result from the competitive binding of glucose to borate, which reduces the concentration of borate bound to the PVA diols. PMID:19381378

  16. Sequence and structural features of carbohydrate binding in proteins and assessment of predictability using a neural network

    PubMed Central

    Malik, Adeel; Ahmad, Shandar

    2007-01-01

    Background Protein-Carbohydrate interactions are crucial in many biological processes with implications to drug targeting and gene expression. Nature of protein-carbohydrate interactions may be studied at individual residue level by analyzing local sequence and structure environments in binding regions in comparison to non-binding regions, which provide an inherent control for such analyses. With an ultimate aim of predicting binding sites from sequence and structure, overall statistics of binding regions needs to be compiled. Sequence-based predictions of binding sites have been successfully applied to DNA-binding proteins in our earlier works. We aim to apply similar analysis to carbohydrate binding proteins. However, due to a relatively much smaller region of proteins taking part in such interactions, the methodology and results are significantly different. A comparison of protein-carbohydrate complexes has also been made with other protein-ligand complexes. Results We have compiled statistics of amino acid compositions in binding versus non-binding regions- general as well as in each different secondary structure conformation. Binding propensities of each of the 20 residue types and their structure features such as solvent accessibility, packing density and secondary structure have been calculated to assess their predisposition to carbohydrate interactions. Finally, evolutionary profiles of amino acid sequences have been used to predict binding sites using a neural network. Another set of neural networks was trained using information from single sequences and the prediction performance from the evolutionary profiles and single sequences were compared. Best of the neural network based prediction could achieve an 87% sensitivity of prediction at 23% specificity for all carbohydrate-binding sites, using evolutionary information. Single sequences gave 68% sensitivity and 55% specificity for the same data set. Sensitivity and specificity for a limited galactose

  17. Closely related mammals have distinct asialoglycoprotein receptor carbohydrate specificities.

    PubMed

    Park, Eric I; Baenziger, Jacques U

    2004-09-24

    We recently reported that the rat asialoglycoprotein receptor binds oligosaccharides terminating with sialic acid (Sia) alpha2,6GalNAc. Despite a high percentage of identical amino acids in their sequences, orthologues of the asialoglycoprotein receptor (ASGP-R) in different mammals differ in their specificity for terminal Siaalpha2,6GalNAc. The recombinant subunit 1 of the ASGP-R from the rat (RHL-1 or rat hepatic lectin) and the mouse (MHL-1 or mouse hepatic lectin), which differ at only 12 positions in the amino acid sequence of their carbohydrate recognition domains, binds Siaalpha2,6GalNAcbeta1,4GlcNAcbeta1,2Man-bovine serum albumin and GalNAcbeta1,4GlcNAcbeta1,2Man-bovine serum albumin in ratios of 16:1.0 and 1.0:1.0, respectively. Mutagenesis was used to show that amino acids both in the immediate vicinity of the proposed binding site for terminal GalNAc and on the alpha2 helix that is distant from the binding site contribute to the specificity for terminal Siaalpha2,6GalNAc. Thus, multiple amino acid sequence alterations in two key locations contribute to the difference in specificity observed for the rat and mouse ASGP-Rs. We hypothesize that the altered specificity of ASPG-R orthologues in such evolutionarily closely related species reflects rapidly changing requirements for recognition of endogenous or exogenous oligosaccharides in vivo.

  18. Carbohydrate-binding protein identification by coupling structural similarity searching with binding affinity prediction.

    PubMed

    Zhao, Huiying; Yang, Yuedong; von Itzstein, Mark; Zhou, Yaoqi

    2014-11-15

    Carbohydrate-binding proteins (CBPs) are potential biomarkers and drug targets. However, the interactions between carbohydrates and proteins are challenging to study experimentally and computationally because of their low binding affinity, high flexibility, and the lack of a linear sequence in carbohydrates as exists in RNA, DNA, and proteins. Here, we describe a structure-based function-prediction technique called SPOT-Struc that identifies carbohydrate-recognizing proteins and their binding amino acid residues by structural alignment program SPalign and binding affinity scoring according to a knowledge-based statistical potential based on the distance-scaled finite-ideal gas reference state (DFIRE). The leave-one-out cross-validation of the method on 113 carbohydrate-binding domains and 3442 noncarbohydrate binding proteins yields a Matthews correlation coefficient of 0.56 for SPalign alone and 0.63 for SPOT-Struc (SPalign + binding affinity scoring) for CBP prediction. SPOT-Struc is a technique with high positive predictive value (79% correct predictions in all positive CBP predictions) with a reasonable sensitivity (52% positive predictions in all CBPs). The sensitivity of the method was changed slightly when applied to 31 APO (unbound) structures found in the protein databank (14/31 for APO versus 15/31 for HOLO). The result of SPOT-Struc will not change significantly if highly homologous templates were used. SPOT-Struc predicted 19 out of 2076 structural genome targets as CBPs. In particular, one uncharacterized protein in Bacillus subtilis (1oq1A) was matched to galectin-9 from Mus musculus. Thus, SPOT-Struc is useful for uncovering novel carbohydrate-binding proteins. SPOT-Struc is available at http://sparks-lab.org.

  19. Entirely Carbohydrate-Based Vaccines: An Emerging Field for Specific and Selective Immune Responses

    PubMed Central

    Nishat, Sharmeen; Andreana, Peter R.

    2016-01-01

    Carbohydrates are regarded as promising targets for vaccine development against infectious disease because cell surface glycans on many infectious agents are attributed to playing an important role in pathogenesis. In addition, oncogenic transformation of normal cells, in many cases, is associated with aberrant glycosylation of the cell surface glycan generating tumor associated carbohydrate antigens (TACAs). Technological advances in glycobiology have added a new dimension to immunotherapy when considering carbohydrates as key targets in developing safe and effective vaccines to combat cancer, bacterial infections, viral infections, etc. Many consider effective vaccines induce T-cell dependent immunity with satisfactory levels of immunological memory that preclude recurrence. Unfortunately, carbohydrates alone are poorly immunogenic as they do not bind strongly to the MHCII complex and thus fail to elicit T-cell immunity. To increase immunogenicity, carbohydrates have been conjugated to carrier proteins, which sometimes can impede carbohydrate specific immunity as peptide-based immune responses can negate antibodies directed at the targeted carbohydrate antigens. To overcome many challenges in using carbohydrate-based vaccine design and development approaches targeting cancer and other diseases, zwitterionic polysaccharides (ZPSs), isolated from the capsule of commensal anaerobic bacteria, will be discussed as promising carriers of carbohydrate antigens to achieve desired immunological responses. PMID:27213458

  20. Detection of weak-binding sugar activity using membrane-based carbohydrates.

    PubMed

    Yamamoto, Kazuo; Kawasaki, Norihito

    2010-01-01

    Protein-sugar interactions underlie many biological events. Although protein-sugar interactions are weak, they are regulated in physiological conditions including clustering, association with other proteins, pH condition, and so on. The elucidation of the precise specificities of sugar-binding proteins is essential for understanding their biological functions. To detect the weak-binding activity of carbohydrate-binding proteins to sugar ligands, we studied lectin tetramer binding to cell-surface carbohydrates by flow cytometry. Tetramerization of lectins enhanced their avidity for sugar ligands, and sugar chains displayed on the cell surfaces were easily accessible to such soluble lectins. In this chapter, we describe methods to (1) prepare biotinylated soluble lectin, (2) obtain R-phycoerythrin-labeled lectin tetramer, and (3) measure tetramer binding to various lectin-resistant cell lines or cells treated with sugar-processing inhibitors. This approach enabled us to detect the weak sugar-binding activity of lectins (K(a) approximately 10(4)M(-1)), especially those from animals, and also to elucidate their specificity for sugar ligands. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  1. Carbohydrates

    MedlinePlus

    ... glossary girlshealth.gov home http://www.girlshealth.gov/ Home Nutrition Nutrition basics Carbohydrates Carbohydrates Carbohydrates (say: kar-boh-HEYE-drayts) are the body's main source of energy. They are sometimes called "carbs" for short. If ...

  2. Carbohydrate specificity of a galectin from chicken liver (CG-16).

    PubMed Central

    Wu, A M; Wu, J H; Tsai, M S; Kaltner, H; Gabius, H J

    2001-01-01

    Owing to the expression of more than one type of galectin in animal tissues, the delineation of the functions of individual members of this lectin family requires the precise definition of their carbohydrate specificities. Thus, the binding properties of chicken liver galectin (CG-16) to glycoproteins (gps) and Streptococcus pneumoniae type 14 polysaccharide were studied by the biotin/avidin-mediated microtitre-plate lectin-binding assay and by the inhibition of lectin-glycan interactions with sugar ligands. Among 33 glycans tested for lectin binding, CG-16 reacted best with human blood group ABO (H) precursor gps and their equivalent gps, which contain a high density of D-galactopyranose(beta1-4)2-acetamido-2-deoxy-D-glucopyranose [Gal(beta1-4)GlcNAc] and Gal(beta1-3)GlcNAc residues at the non-reducing end, but this lectin reacted weakly or not at all with A-,H-type and sialylated gps. Among the oligosaccharides tested by the inhibition assay, the tri-antennary Gal(beta1-4)GlcNAc (Tri-II) was the best. It was 2.1x10(3) nM and 3.0 times more potent than Gal and Gal(beta1-4)GlcNAc (II)/Gal(beta1-3)GlcNAc(beta1-3)Gal(beta1-4)Glc (lacto-N-tetraose) respectively. CG-16 has a preference for the beta-anomer of Gal at the non-reducing end of oligosaccharides with a Gal(beta1-4) linkage >Gal(beta1-3)> or =Gal(beta1-6). From the results, it can be concluded that the combining site of this agglutinin should be a cavity type, and that a hydrophobic interaction in the vicinity of the binding site for sugar accommodation increases the affinity. The binding site of CG-16 is as large as a tetrasaccharide of the beta-anomer of Gal, and is most complementary to lacto-N-tetraose and Gal(beta1-4)GlcNAc related sequences. PMID:11535116

  3. Developmentally Regulated, Carbohydrate-Binding Protein in Dictyostelium discoideum

    PubMed Central

    Rosen, Steven D.; Kafka, John A.; Simpson, David L.; Barondes, Samuel H.

    1973-01-01

    A carbohydrate-binding protein assayed by its ability to agglutinate formalinized sheep erythrocytes is synthesized between 3 and 9 hr after Dictyostelium discoideum cells are deprived of food, as the cells become cohesive. Agglutination of erythrocytes by this protein was inhibited by N-acetyl-D-galactosamine, D-galactose, and L-fucose, but other monosaccharides had little or no effect. The protein bound completely to Sepharose 4B, and was isolated in highly purified form by elution with D-galactose. It appears to be present on the surface of cohesive but not vegetative slime-mold cells. The possibility that this protein may mediate intercellular adhesion in Dictyostelium is considered. Images PMID:4517669

  4. A novel family of carbohydrate-binding modules identified with Ruminococcus albus proteins.

    PubMed

    Xu, Qi; Morrison, Mark; Nelson, Karen E; Bayer, Edward A; Atamna, Nof; Lamed, Raphael

    2004-05-21

    We recently showed that some of the enzymes underpinning cellulose solubilization by Ruminococcus albus 8 lack the conventional type of dockerin module characteristic of cellulosomal proteins and instead, bear an "X" domain of unknown function at their C-termini. We have now subcloned and expressed six X domains and showed that five of them bind to xylan, chitin, microcrystalline and phosphoric-acid swollen cellulose, as well as more heterogenous substrates such as alfalfa cell walls, banana stem and wheat straw. The X domain that did not bind to these substrates was derived from a family-5 glycoside hydrolase (Cel5G), which possesses two X domains in tandem. Whereas the internal X domain failed to bind to the substrates, the recombinant dyad exhibited markedly enhanced binding relative to that observed for the C-terminal X domain alone. The evidence supports a distinctive carbohydrate-binding role of broad specificity for this type of domain, and we propose a novel family (designated family 37) of carbohydrate-binding modules that appear to be peculiar to R. albus.

  5. DOPI and PALM imaging of single carbohydrate binding modules bound to cellulose nanocrystals

    NASA Astrophysics Data System (ADS)

    Dagel, D. J.; Liu, Y.-S.; Zhong, L.; Luo, Y.; Zeng, Y.; Himmel, M.; Ding, S.-Y.; Smith, S.

    2011-03-01

    We use single molecule imaging methods to study the binding characteristics of carbohydrate-binding modules (CBMs) to cellulose crystals. The CBMs are carbohydrate specific binding proteins, and a functional component of most cellulase enzymes, which in turn hydrolyze cellulose, releasing simple sugars suitable for fermentation to biofuels. The CBM plays the important role of locating the crystalline face of cellulose, a critical step in cellulase action. A biophysical understanding of the CBM action aids in developing a mechanistic picture of the cellulase enzyme, important for selection and potential modification. Towards this end, we have genetically modified cellulose-binding CBM derived from bacterial source with green fluorescent protein (GFP), and photo-activated fluorescence protein PAmCherry tags, respectively. Using the single molecule method known as Defocused Orientation and Position Imaging (DOPI), we observe a preferred orientation of the CBM-GFP complex relative to the Valonia cellulose nanocrystals. Subsequent analysis showed the CBMs bind to the opposite hydrophobic <110> faces of the cellulose nanocrystals with a welldefined cross-orientation of about { 70°. Photo Activated Localization Microscopy (PALM) is used to localize CBMPAmCherry with a localization accuracy of { 10nm. Analysis of the nearest neighbor distributions along and perpendicular to the cellulose nanocrystal axes are consistent with single-file CBM binding along the fiber axis, and microfibril bundles consisting of close packed { 20nm or smaller cellulose microfibrils.

  6. Engineering Cel7A carbohydrate binding module and linker for reduced lignin inhibition.

    PubMed

    Strobel, Kathryn L; Pfeiffer, Katherine A; Blanch, Harvey W; Clark, Douglas S

    2016-06-01

    Non-productive binding of cellulases to lignin inhibits enzymatic hydrolysis of biomass, increasing enzyme requirements and the cost of biofuels. This study used site-directed mutagenesis of the Trichoderma Cel7A carbohydrate binding module (CBM) and linker to investigate the mechanisms of adsorption to lignin and engineer a cellulase with increased binding specificity for cellulose. CBM mutations that added hydrophobic or positively charged residues decreased the specificity for cellulose, while mutations that added negatively charged residues increased the specificity. Linker mutations that altered predicted glycosylation patterns selectively impacted lignin affinity. Beneficial mutations were combined to generate a mutant with 2.5-fold less lignin affinity while fully retaining cellulose affinity. This mutant was uninhibited by added lignin during hydrolysis of Avicel and generated 40% more glucose than the wild-type enzyme from dilute acid-pretreated Miscanthus. Biotechnol. Bioeng. 2016;113: 1369-1374. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  7. Carbohydrate-appended curdlans as a new family of glycoclusters with binding properties both for a polynucleotide and lectins.

    PubMed

    Hasegawa, Teruaki; Numata, Munenori; Okumura, Shiro; Kimura, Taro; Sakurai, Kazuo; Shinkai, Seiji

    2007-08-07

    Beta-1,3-glucans having carbohydrate-appendages (alpha-D-mannoside, N-acetyl-beta-D-glucosaminide and beta-lactoside) at the C6-position of every repeating unit can be readily prepared from curdlan (a linear beta-1,3-glucan) through regioselective bromination/azidation to afford 6-azido-6-deoxycurdlan followed by chemo-selective Cu(i)-catalyzed [3 + 2]-cycloaddition with various carbohydrate modules having a terminal alkyne. The resultant carbohydrate-appended curdlans can interact with polycytosine to form stable macromolecular complexes consistent with two polysaccharide strands and one polycytosine strand. Furthermore, these macromolecular complexes show strong and specific affinity toward carbohydrate-binding proteins (lectins). Therefore, one can utilize these carbohydrate-appended curdlans as a new family of glycoclusters.

  8. Crystallization, neutron data collection, initial structure refinement and analysis of a xyloglucan heptamer bound to an engineered carbohydrate-binding module from xylanase.

    PubMed

    Ohlin, Mats; von Schantz, Laura; Schrader, Tobias E; Ostermann, Andreas; Logan, Derek T; Fisher, S Zoë

    2015-08-01

    Carbohydrate-binding modules (CBMs) are discrete parts of carbohydrate-hydrolyzing enzymes that bind specific types of carbohydrates. Ultra high-resolution X-ray crystallographic studies of CBMs have helped to decipher the basis for specificity in carbohydrate-protein interactions. However, additional studies are needed to better understand which structural determinants confer which carbohydrate-binding properties. To address these issues, neutron crystallographic studies were initiated on one experimentally engineered CBM derived from a xylanase, X-2 L110F, a protein that is able to bind several different plant carbohydrates such as xylan, β-glucan and xyloglucan. This protein evolved from a CBM present in xylanase Xyn10A of Rhodothermus marinus. The protein was complexed with a branched xyloglucan heptasaccharide. Large single crystals of hydrogenous protein (∼1.6 mm(3)) were grown at room temperature and subjected to H/D exchange. Both neutron and X-ray diffraction data sets were collected to 1.6 Å resolution. Joint neutron and X-ray refinement using phenix.refine showed significant density for residues involved in carbohydrate binding and revealed the details of a hydrogen-bonded water network around the binding site. This is the first report of a neutron structure of a CBM and will add to the understanding of protein-carbohydrate binding interactions.

  9. Carbohydrate-binding modules recognize fine substructures of cellulose.

    PubMed

    McLean, Bradley W; Boraston, Alisdair B; Brouwer, Darren; Sanaie, Nooshafarin; Fyfe, Colin A; Warren, R Antony J; Kilburn, Douglas G; Haynes, Charles A

    2002-12-27

    Competition isotherms are used to identify the set of cellulose substructures to which cellulose binding modules (CBMs) from families 2a, 3, 4, 9, and 17 bind. The experiments are based on coupling a unique fluorescent tag to each CBM in a manner that does not alter the natural binding properties of the CBM and therefore allows the surface and solution concentrations of each CBM to be monitored as a function of time and composition. Adsorption and surface exchange of like or competing CBMs are monitored using a range of cellulose preparations varying in both crystallinity and provenance. CBMs from families 2a, 3, 4, 9, and 17 are shown to recognize different physical forms of prepared cellulose. The demonstration of the very fine binding specificity of cellulose-specific CBMs implies that the polysaccharide targets of CBMs extend down to the resolution of cellulose microstructures.

  10. Carbohydrate-binding module assisting glycosynthase-catalysed polymerizations.

    PubMed

    Codera, Victoria; Gilbert, Harry J; Faijes, Magda; Planas, Antoni

    2015-08-15

    Carbohydrate-binding modules (CBMs) are found within multi-modular polysaccharide degrading enzymes [glycoside hydrolases (GHs)]. CBMs play a critical role in the recognition of plant cell-wall polysaccharides and enhance the hydrolase activity of their cognate catalytic domains by increasing enzyme substrate proximity. Mimicking their role in Nature, we, in the present study, propose that CBMs may assist in vitro glycosynthase-catalysed polymerization reactions to produce artificial polysaccharides. Glycosynthases are GHs that have been engineered to catalyse glycoside bond formation for the synthesis of oligosaccharides, glycoconjugates and glycans. The degree of polymerization (DP) of the glycans generated is limited by the solubility of the polymeric product. In the present study, we have targeted the synthesis of artificial 1,3-1,4-β-glucans with a regular sequence using the glycosynthase E(134)S derived from a Bacillus licheniformis lichenase. We show that the addition of CBM11, which binds mixed-linked β-glucans, either as an isolated protein or fused to the glycosynthase E(134)S, has an effect on the DP of the polysaccharide products that is dependent on the rate of polymerization. The mechanism by which CBM influences the DP of the synthesized glycans is discussed.

  11. Binding Preferences, Surface Attachment, Diffusivity, and Orientation of a Family 1 Carbohydrate-Binding Module on Cellulose

    SciTech Connect

    Nimlos, M. R.; Beckham, G. T.; Matthews, J. F.; Bu, L.; Himmel, M. E.; Crowley, M. F.

    2012-06-08

    Cellulase enzymes often contain carbohydrate-binding modules (CBMs) for binding to cellulose. The mechanisms by which CBMs recognize specific surfaces of cellulose and aid in deconstruction are essential to understand cellulase action. The Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase, Cel7A, is known to selectively bind to hydrophobic surfaces of native cellulose. It is most commonly suggested that three aromatic residues identify the planar binding face of this CBM, but several recent studies have challenged this hypothesis. Here, we use molecular simulation to study the CBM binding orientation and affinity on hydrophilic and hydrophobic cellulose surfaces. Roughly 43 {mu}s of molecular dynamics simulations were conducted, which enables statistically significant observations. We quantify the fractions of the CBMs that detach from crystal surfaces or diffuse to other surfaces, the diffusivity along the hydrophobic surface, and the overall orientation of the CBM on both hydrophobic and hydrophilic faces. The simulations demonstrate that there is a thermodynamic driving force for the Cel7A CBM to bind preferentially to the hydrophobic surface of cellulose relative to hydrophilic surfaces. In addition, the simulations demonstrate that the CBM can diffuse from hydrophilic surfaces to the hydrophobic surface, whereas the reverse transition is not observed. Lastly, our simulations suggest that the flat faces of Family 1 CBMs are the preferred binding surfaces. These results enhance our understanding of how Family 1 CBMs interact with and recognize specific cellulose surfaces and provide insights into the initial events of cellulase adsorption and diffusion on cellulose.

  12. Norwalk Virus–specific Binding to Oyster Digestive Tissues

    PubMed Central

    Loisy, Fabienne; Atmar, Robert L.; Hutson, Anne M.; Estes, Mary K.; Ruvoën-Clouet, Nathalie; Pommepuy, Monique; Le Pendu, Jacques

    2006-01-01

    The primary pathogens related to shellfishborne gastroenteritis outbreaks are noroviruses. These viruses show persistence in oysters, which suggests an active mechanism of virus concentration. We investigated whether Norwalk virus or viruslike particles bind specifically to oyster tissues after bioaccumulation or addition to tissue sections. Since noroviruses attach to carbohydrates of the histo-blood group family, tests using immunohistochemical analysis were performed to evaluate specific binding of virus or viruslike particles to oyster tissues through these ligands. Viral particles bind specifically to digestive ducts (midgut, main and secondary ducts, and tubules) by carbohydrate structures with a terminal N-acetylgalactosamine residue in an α linkage (same binding site used for recognition of human histo-blood group antigens). These data show that the oyster can selectively concentrate a human pathogen and that conventional depuration will not eliminate noroviruses from oyster tissue. PMID:16707048

  13. Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules

    SciTech Connect

    Walker, Johnnie A.; Takasuka, Taichi E.; Deng, Kai; Bianchetti, Christopher M.; Udell, Hannah S.; Prom, Ben M.; Kim, Hyunkee; Adams, Paul D.; Northen, Trent R.; Fox, Brian G.

    2015-12-21

    Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed. CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolytic activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass. In conclusion, we have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.

  14. Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules

    DOE PAGES

    Walker, Johnnie A.; Takasuka, Taichi E.; Deng, Kai; ...

    2015-12-21

    Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed. CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolyticmore » activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass. In conclusion, we have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.« less

  15. Carbohydrates

    MedlinePlus

    Carbohydrates are one of the main types of nutrients. They are the most important source of energy for your body. Your digestive system changes carbohydrates into glucose (blood sugar). Your body uses this ...

  16. Giardia cyst wall-specific carbohydrate: evidence for the presence of galactosamine.

    PubMed

    Jarroll, E L; Manning, P; Lindmark, D G; Coggins, J R; Erlandsen, S L

    1989-01-15

    Gas chromatographic (GC), mass spectrometric (MS), lectin binding and enzymatic analyses of the carbohydrates from Giardia cyst walls, intact cysts and trophozoites were performed to investigate the carbohydrate composition of Giardia cyst walls and to test the hypothesis that the Giardia cyst wall is composed largely of chitin. Galactosamine, verified by MS, was present in Giardia cyst walls and intact cysts (ca. 47 nmol 10(-6) cysts). Since not even trace amounts of it were detected in trophozoites by either GC or lectin binding, galactosamine is hypothesized to be a cyst wall-specific amino hexose. Based on the putative binding affinity of Phaseolus limensis lectin, galactosamine may be present in cyst walls as N-acetylgalactosamine. Neither glucosamine nor sialic acid were detected in as much as 11 mg dry weight of cysts, cyst walls, or trophozoites. Glucose, the most abundant carbohydrate, and ribose were detected in Giardia cysts and trophozoites. Galactose (ca. 10 nmol 10(-6) cysts) was detected in cysts but not in trophozoites. The lack of detectable levels of (1) glucosamine in cyst wall hydrolysates, (2) cyst staining by Calcofluor M2R, (3) endogenous chitinase activity and (4) N-acetylglucosamine when cysts served as a substrate for exogenous chitinase suggests that the Giardia cyst wall is not composed largely of chitin as previously reported. beta-N-Acetylgalactosaminidase, EC 3.2.1.32, activity was detected in cysts and trophozoites and represents the first carbohydrate splitting hydrolase detected in Giardia.

  17. Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

    DOE PAGES

    Ng, Simon; Lin, Edith; Kitov, Pavel I.; ...

    2015-04-10

    Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 108 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3 outmore » of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.« less

  18. Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

    SciTech Connect

    Ng, Simon; Lin, Edith; Kitov, Pavel I.; Tjhung, Katrina F.; Gerlits, Oksana O.; Deng, Lu; Kasper, Brian; Sood, Amika; Paschal, Beth M.; Zhang, Ping; Ling, Chang-Chun; Klassen, John S.; Noren, Christopher J.; Mahal, Lara K.; Woods, Robert J.; Coates, Leighton; Derda, Ratmir

    2015-04-10

    Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 108 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3 out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.

  19. Antibodies and carbohydrate ligands binding to DC-SIGN differentially modulate receptor trafficking.

    PubMed

    Tacken, Paul J; Ter Huurne, Menno; Torensma, Ruurd; Figdor, Carl G

    2012-08-01

    DCs are regarded as key APCs that initiate humoral and cellular immune responses. Consequently, targeted delivery of Ag toward DC-specific receptors enhances vaccine efficacy. DC-SIGN is a C-type lectin receptor that facilitates DC-specific delivery of Ag. This is accomplished by conjugating Ag to receptor-specific Ab or carbohydrate ligands that bind to its carbohydrate recognition domain. Here, we investigated the fate of DC-SIGN following receptor triggering with Ab. Both whole and single-chain Ab induced rapid internalization of about half of the surface receptor molecules. Biochemical studies showed that about half of the receptor molecules were still intracellular after 3 h, while minimal or no resurfacing of internalized or newly synthesized unbound DC-SIGN molecules was observed. Prolonged exposure of DCs to DC-SIGN Ab, but not carbohydrate ligands, resulted in reduced receptor expression levels, which lasted up to 2 days following removal of the Ab. In addition, exposure to DC-SIGN Ab reduced the ability of the receptor to internalize. Consequently, DC-SIGN showed a poor ability to accumulate targeting Abs within DCs. Vaccine efficacy may therefore be enhanced by strategies increasing the amount of Ag entering via a single receptor molecule, such as the use of targeting moieties allowing DC-SIGN recycling or Ab-coated vaccine carriers.

  20. High Sensitive Detection of Carbohydrate Binding Proteins in an ELISA-Solid Phase Assay Based on Multivalent Glyconanoparticles

    PubMed Central

    Chiodo, Fabrizio; Marradi, Marco; Tefsen, Boris; Snippe, Harm; van Die, Irma; Penadés, Soledad

    2013-01-01

    Improved detection of anti-carbohydrate antibodies is a need in clinical identification of biomarkers for cancer cells or pathogens. Here, we report a new ELISA approach for the detection of specific immunoglobulins (IgGs) against carbohydrates. Two nanometer gold glyconanoparticles bearing oligosaccharide epitopes of HIV or Streptococcus pneumoniae were used as antigens to coat ELISA-plates. A ~3,000-fold improved detection of specific IgGs in mice immunized against S. pneumoniae respect to the well known BSA-glycoconjugate ELISA was achieved. Moreover, these multivalent glyconanoparticles have been employed in solid phase assays to detect the carbohydrate-dependent binding of human dendritic cells and the lectin DC-SIGN. Multivalent glyconanoparticles in ELISA provide a versatile, easy and highly sensitive method to detect and quantify the binding of glycan to proteins and to facilitate the identification of biomarkers. PMID:24014084

  1. Probing the mechanism of ligand recognition in family 29 carbohydrate-binding modules.

    PubMed

    Flint, James; Bolam, David N; Nurizzo, Didier; Taylor, Edward J; Williamson, Michael P; Walters, Christopher; Davies, Gideon J; Gilbert, Harry J

    2005-06-24

    The recycling of photosynthetically fixed carbon, by the action of microbial plant cell wall hydrolases, is integral to one of the major geochemical cycles and is of considerable industrial importance. Non-catalytic carbohydrate-binding modules (CBMs) play a key role in this degradative process by targeting hydrolytic enzymes to their cognate substrate within the complex milieu of polysaccharides that comprise the plant cell wall. Family 29 CBMs have, thus far, only been found in an extracellular multienzyme plant cell wall-degrading complex from the anaerobic fungus Piromyces equi, where they exist as a CBM29-1:CBM29-2 tandem. Here we present both the structure of the CBM29-1 partner, at 1.5 A resolution, and examine the importance of hydrophobic stacking interactions as well as direct and solvent-mediated hydrogen bonds in the binding of CBM29-2 to different polysaccharides. CBM29 domains display unusual binding properties, exhibiting specificity for both beta-manno- and beta-gluco-configured ligands such as mannan, cellulose, and glucomannan. Mutagenesis reveals that "stacking" of tryptophan residues in the n and n+2 subsites plays a critical role in ligand binding, whereas the loss of tyrosine-mediated stacking in the n+4 subsite reduces, but does not abrogate, polysaccharide recognition. Direct hydrogen bonds to ligand, such as those provided by Arg-112 and Glu-78, play a pivotal role in the interaction with both mannan and cellulose, whereas removal of water-mediated interactions has comparatively little effect on carbohydrate binding. The interactions of CBM29-2 with the O2 of glucose or mannose contribute little to binding affinity, explaining why this CBM displays dual gluco/manno specificity.

  2. THE INDIVIDUAL ANTIGENIC SPECIFICITY OF ANTIBODIES TO STREPTOCOCCAL CARBOHYDRATES

    PubMed Central

    Braun, Dietmar G.; Krause, Richard M.

    1968-01-01

    Although a single electrophoretically uniform antibody component with specificity for the group carbohydrate may comprise the bulk of the γ-globulin in rabbits immunized with streptococcal vaccines, this is not always the case. Not infrequently, electrophoresis may reveal multiple antibody components. Nevertheless, it has been feasible by various preparative procedures to isolate from a single antiserum at least two antibody components with similar reactivity for the carbohydrate both of which are electrophoretically monodisperse. Light chains from such antibodies reveal a restricted pattern when examined by disc electrophoresis. Antibodies to streptococcal carbohydrates have been examined for their individual antigenic specificity. Goats were immunized with isolated Group C and Group A-variant antibodies raised in rabbits. Individual antigenic specificity of these antibodies was brought out by absorption of the goat anti-antiserum with Fr II of pooled normal rabbit sera. Additional absorption of the goat anti-antisera with Fr II diminished but did not eliminate the reactivity for the homologous antibody. Immunoelectrophoretic studies with papain fragments of purified streptococcal antibodies localized the specificity to the Fab fragment. Specificity was not confined to the isolated light chains of the antibody. PMID:4176226

  3. Flow cytometric analysis of lectin binding to in vitro-cultured Perkinsus marinus surface carbohydrates

    USGS Publications Warehouse

    Gauthier, J.D.; Jenkins, J.A.; La Peyre, Jerome F.

    2004-01-01

    Parasite surface glycoconjugates are frequently involved in cellular recognition and colonization of the host. This study reports on the identification of Perkinsus marinus surface carbohydrates by flow cytometric analyses of fluorescein isothiocyanate-conjugated lectin binding. Lectin-binding specificity was confirmed by sugar inhibition and Kolmogorov-Smirnov statistics. Clear, measurable fluorescence peaks were discriminated, and no parasite autofluorescence was observed. Parasites (GTLA-5 and Perkinsus-1 strains) harvested during log and stationary phases of growth in a protein-free medium reacted strongly with concanavalin A and wheat germ agglutinin, which bind to glucose-mannose and N-acetyl-D-glucosamine (GlcNAc) moieties, respectively. Both P. marinus strains bound with lower intensity to Maclura pomifera agglutinin, Bauhinia purpurea agglutinin, soybean agglutinin (N-acetyl-D-galactosamine-specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffonia simplicifolia II (GlcNAc specific). Only background fluorescence levels were detected with Ulex europaeus agglutinin I (L-fucose specific) and Limulus polyphemus agglutinin (sialic acid specific). The lectin-binding profiles were similar for the 2 strains except for a greater relative binding intensity of PNA for Perkinsus-1 and an overall greater lectin-binding capacity of Perkinsus-1 compared with GTLA-5. Growth stage comparisons revealed increased lectin-binding intensities during stationary phase compared with log phase of growth. This is the first report of the identification of surface glycoconjugates on a Perkinsus spp. by flow cytometry and the first to demonstrate that differential surface sugar expression is growth phase and strain dependent. ?? American Society of Parasitologists 2004.

  4. Glycosylated aniline polymer sensor: Amine to imine conversion on protein–carbohydrate binding

    PubMed Central

    Wang, Zhe; Sun, Chunyan; Vegesna, Giri; Liu, Haiying; Liu, Yang; Li, Jinghong; Zeng, Xiangqun

    2013-01-01

    In this report, functionalized mannosylated aniline polymer (manno-PANI) was investigated as an electrochemical platform to study carbohydrate–protein interactions by exploiting the conductivity change of manno-PANI when the specific lectin binding occurs. A systematic study was performed to characterize the interconversion of polyaniline content (from amine to imine) in manno-PANI by UV–vis spectroscopy during its binding with concanavalin A (Con A). Both X-ray photoelectron spectrometry (XPS) and UV–vis results suggest that Con A binding with the manno-PANI film triggers the switching of amine functionalities in the polyaniline backbone, converting them to imine forms. Electrochemical impedance spectroscopy (EIS) was used to quantify the specific interactions between Con A and mannose by measuring the impedance change of manno-PANI film for the detection of Con A. A linear relationship between the impedance and Con A concentration was obtained, and the detection limit reaches to 0.12 nM Con A in a buffer solution (pH=7.4), whereas the addition of nonspecific control lectins to the same manno-PANI film gave very little impedance variations. Stability characterization of the manno-PANI film over 20 weeks shows a maximum drift of only 3% from the original signal. Thus, the uniquely constructed carbohydrate–PANI hybrid is a promising new carbohydrate recognition moiety for studying carbohydrate-protein interactions, presumably leading to a new electrochemical method for characterization of carbohydrate–protein interactions and carbohydrate-mediated intercellular recognitions. PMID:23563436

  5. Structural characterization of the Streptococcus pneumoniae carbohydrate substrate-binding protein SP0092

    PubMed Central

    Tang, Minzhe

    2017-01-01

    Streptococcus pneumoniae is an opportunistic respiratory pathogen that remains a major cause of morbidity and mortality globally, with infants and the elderly at the highest risk. S. pneumoniae relies entirely on carbohydrates as a source of carbon and dedicates a third of all uptake systems to carbohydrate import. The structure of the carbohydrate-free substrate-binding protein SP0092 at 1.61 Å resolution reveals it to belong to the newly proposed subclass G of substrate-binding proteins, with a ligand-binding pocket that is large enough to accommodate complex oligosaccharides. SP0092 is a dimer in solution and the crystal structure reveals a domain-swapped dimer with the monomer subunits in a closed conformation but in the absence of carbohydrate ligand. This closed conformation may be induced by dimer formation and could be used as a mechanism to regulate carbohydrate uptake. PMID:28045395

  6. Multidomain Carbohydrate-binding Proteins Involved in Bacteroides thetaiotaomicron Starch Metabolism.

    PubMed

    Cameron, Elizabeth A; Maynard, Mallory A; Smith, Christopher J; Smith, Thomas J; Koropatkin, Nicole M; Martens, Eric C

    2012-10-05

    Human colonic bacteria are necessary for the digestion of many dietary polysaccharides. The intestinal symbiont Bacteroides thetaiotaomicron uses five outer membrane proteins to bind and degrade starch. Here, we report the x-ray crystallographic structures of SusE and SusF, two outer membrane proteins composed of tandem starch specific carbohydrate-binding modules (CBMs) with no enzymatic activity. Examination of the two CBMs in SusE and three CBMs in SusF reveals subtle differences in the way each binds starch and is reflected in their K(d) values for both high molecular weight starch and small maltooligosaccharides. Thus, each site seems to have a unique starch preference that may enable these proteins to interact with different regions of starch or its breakdown products. Proteins similar to SusE and SusF are encoded in many other polysaccharide utilization loci that are possessed by human gut bacteria in the phylum Bacteroidetes. Thus, these proteins are likely to play an important role in carbohydrate metabolism in these abundant symbiotic species. Understanding structural changes that diversify and adapt related proteins in the human gut microbial community will be critical to understanding the detailed mechanistic roles that they perform in the complex digestive ecosystem.

  7. Screening method of carbohydrate-binding proteins in biological sources by capillary affinity electrophoresis and its application to determination of Tulipa gesneriana agglutinin in tulip bulbs.

    PubMed

    Nakajima, Kazuki; Kinoshita, Mitsuhiro; Oda, Yasuo; Masuko, Takashi; Kaku, Hanae; Shibuya, Naoto; Kakehi, Kazuaki

    2004-09-01

    We developed capillary affinity electrophoresis (CAE) to analyze the molecular interaction between carbohydrate chains and proteins in solution state. A mixture of oligosaccharides derived from a glycoprotein was labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS), and used as glycan library without isolation. Interaction of a carbohydrate-binding protein with each oligosaccharide in the mixture could be simultaneously observed, and relative affinities of oligosaccharides toward the protein were accurately determined. In this study, we applied CAE to detect the presence of lectins in some plants (Japanese elderberry bark and tulip bulb). In the crude extract of the elderberry bark, binding activity toward sialo-carbohydrate chains could be easily detected. We also examined the presence of lectins in the crude extract of tulip bulbs and determined the detailed carbohydrate-binding specificity of Tulipa gesneriana agglutinin (TGA), one of the lectins from tulip bulbs. Kinetic studies demonstrated that TGA showed novel carbohydrate-binding specificity and preferentially recognized triantennary oligosaccharides with Gal residues at nonreducing termini and a Fuc residue linked through alpha(1-6) linkage at chitobiose portion of the reducing termini but not tetraantennary carbohydrates. The results described here indicate that CAE will be a valuable method for both screening of lectins in natural sources and determination of their detailed carbohydrate-binding specificities.

  8. Nutritional therapy in pediatric Crohn disease: the specific carbohydrate diet.

    PubMed

    Suskind, David L; Wahbeh, Ghassan; Gregory, Nila; Vendettuoli, Heather; Christie, Dennis

    2014-01-01

    Crohn disease is characterized by chronic intestinal inflammation in the absence of a recognized etiology. Nutritional therapy in the form of exclusive enteral nutrition (EEN) has an established role within pediatric Crohn disease. Following exclusive enteral nutrition's success, many dietary therapies focusing on the elimination of specific complex carbohydrates have been anecdotally reported to be successful. Many of these therapies have not been evaluated scientifically; therefore, we reviewed the medical records of our patients with Crohn disease on the specific carbohydrate diet (SCD). Seven children with Crohn disease receiving the SCD and no immunosuppressive medications were retrospectively evaluated. Duration of the dietary therapy ranged from 5 to 30 months, with an average of 14.6±10.8 months. Although the exact time of symptom resolution could not be determined through chart review, all symptoms were notably resolved at a routine clinic visit 3 months after initiating the diet. Each patient's laboratory indices, including serum albumin, C-reactive protein, hematocrit, and stool calprotectin, either normalized or significantly, improved during follow-up clinic visits. This chart review suggests that the SCD and other low complex carbohydrate diets may be possible therapeutic options for pediatric Crohn disease. Further prospective studies are required to fully assess the safety and efficacy of the SCD, or any other low complex SCDs in pediatric patients with Crohn disease.

  9. Circular Permutation Provides an Evolutionary Link between Two Families of Calcium-dependent Carbohydrate Binding Modules*

    PubMed Central

    Montanier, Cedric; Flint, James E.; Bolam, David N.; Xie, Hefang; Liu, Ziyuan; Rogowski, Artur; Weiner, David P.; Ratnaparkhe, Supriya; Nurizzo, Didier; Roberts, Shirley M.; Turkenburg, Johan P.; Davies, Gideon J.; Gilbert, Harry J.

    2010-01-01

    The microbial deconstruction of the plant cell wall is a critical biological process, which also provides important substrates for environmentally sustainable industries. Enzymes that hydrolyze the plant cell wall generally contain non-catalytic carbohydrate binding modules (CBMs) that contribute to plant cell wall degradation. Here we report the biochemical properties and crystal structure of a family of CBMs (CBM60) that are located in xylanases. Uniquely, the proteins display broad ligand specificity, targeting xylans, galactans, and cellulose. Some of the CBM60s display enhanced affinity for their ligands through avidity effects mediated by protein dimerization. The crystal structure of vCBM60, displays a β-sandwich with the ligand binding site comprising a broad cleft formed by the loops connecting the two β-sheets. Ligand recognition at site 1 is, exclusively, through hydrophobic interactions, whereas binding at site 2 is conferred by polar interactions between a protein-bound calcium and the O2 and O3 of the sugar. The observation, that ligand recognition at site 2 requires only a β-linked sugar that contains equatorial hydroxyls at C2 and C3, explains the broad ligand specificity displayed by vCBM60. The ligand-binding apparatus of vCBM60 displays remarkable structural conservation with a family 36 CBM (CBM36); however, the residues that contribute to carbohydrate recognition are derived from different regions of the two proteins. Three-dimensional structure-based sequence alignments reveal that CBM36 and CBM60 are related by circular permutation. The biological and evolutionary significance of the mechanism of ligand recognition displayed by family 60 CBMs is discussed. PMID:20659893

  10. Metabolic inhibition of galectin-1-binding carbohydrates accentuates antitumor immunity.

    PubMed

    Cedeno-Laurent, Filiberto; Opperman, Matthew J; Barthel, Steven R; Hays, Danielle; Schatton, Tobias; Zhan, Qian; He, Xiaoying; Matta, Khushi L; Supko, Jeffrey G; Frank, Markus H; Murphy, George F; Dimitroff, Charles J

    2012-02-01

    Galectin-1 (Gal-1) has been shown to play a major role in tumor immune escape by inducing apoptosis of effector leukocytes and correlating with tumor aggressiveness and disease progression. Thus, targeting the Gal-1/Gal-1 ligand axis represents a promising cancer therapeutic approach. Here, to test the Gal-1-mediated tumor immune evasion hypothesis and demonstrate the importance of Gal-1-binding N-acetyllactosamines in controlling the fate and function of antitumor immune cells, we treated melanoma- or lymphoma-bearing mice with peracetylated 4-fluoro-glucosamine (4-F-GlcNAc), a metabolic inhibitor of N-acetyllactosamine biosynthesis, and analyzed tumor growth and immune profiles. We found that 4-F-GlcNAc spared Gal-1-mediated apoptosis of T cells and natural killer (NK) cells by decreasing their expression of Gal-1-binding determinants. 4-F-GlcNAc enhanced tumor lymphocytic infiltration and promoted elevations in tumor-specific cytotoxic T cells and IFN-γ levels, while lowering IL-10 production. Collectively, our data suggest that metabolic lowering of Gal-1-binding N-acetyllactosamines may attenuate tumor growth by boosting antitumor immune cell levels, representing a promising approach for cancer immunotherapy.

  11. O-linked carbohydrate of recombinant von Willebrand factor influences ristocetin-induced binding to platelet glycoprotein 1b.

    PubMed Central

    Carew, J A; Quinn, S M; Stoddart, J H; Lynch, D C

    1992-01-01

    By transfecting the full-length cDNA for human von Willebrand factor (vWf) into a line of Chinese hamster ovary cells with a defect in carbohydrate metabolism, we have prepared recombinant vWf specifically lacking O-linked carbohydrates. We have compared this under-glycosylated protein to fully glycosylated recombinant vWf with respect to several structural and binding properties. vWf deficient in O-linked glycans was synthesized, assembled into multimers, and secreted in an apparently normal manner and was not prone to degradation in the extracellular milieu. It did not differ from fully glycosylated vWf in ability to bind to heparin or to collagen type I but did interact less well with glycoprotein 1b on formalin-fixed platelets. This decreased interaction was evidenced in both a lessened overall binding to platelets and in diminished capacity to promote platelet agglutination, in the presence of ristocetin. In contrast, no difference was seen in platelet binding in the presence of botrocetin. These data indicate a possible role for O-linked carbohydrates in the vWf-glycoprotein 1b interaction promoted by ristocetin and suggest that abnormalities in carbohydrate modification might contribute to the altered ristocetin-dependent reactivity between vWf and platelets described for some variant forms of von Willebrand disease. Images PMID:1469086

  12. Novel Fold and Carbohydrate Specificity of the Potent Anti-HIV Cyanobacterial Lectin from Oscillatoria agardhii*

    PubMed Central

    Koharudin, Leonardus M. I.; Furey, William; Gronenborn, Angela M.

    2011-01-01

    Oscillatoria agardhii agglutinin (OAA) is a recently discovered cyanobacterial lectin that exhibits potent anti-HIV activity. Up to now, only its primary structure and carbohydrate binding data have been available. To elucidate the structural basis for the antiviral mechanism of OAA, we determined the structure of this lectin by x-ray crystallography at 1.2 Å resolution and mapped the specific carbohydrate recognition sites of OAA by NMR spectroscopy. The overall architecture of OAA comprises 10 β-strands that fold into a single, compact, β-barrel-like domain, creating a unique topology compared with all known protein structures in the Protein Data Bank. OAA sugar binding was tested against Man-9 and various disaccharide components of Man-9. Two symmetric carbohydrate-binding sites were located on the protein, and a preference for Manα(1–6)Man-linked sugars was found. Altogether, our structural results explain the antiviral activity OAA and add to the growing body of knowledge about antiviral lectins. PMID:20961847

  13. Binding energy calculations for hevein-carbohydrate interactions using expanded ensemble molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Koppisetty, Chaitanya A. K.; Frank, Martin; Lyubartsev, Alexander P.; Nyholm, Per-Georg

    2015-01-01

    Accurate estimation of protein-carbohydrate binding energies using computational methods is a challenging task. Here we report the use of expanded ensemble molecular dynamics (EEMD) simulation with double decoupling for estimation of binding energies of hevein, a plant lectin with its monosaccharide and disaccharide ligands GlcNAc and (GlcNAc)2, respectively. In addition to the binding energies, enthalpy and entropy components of the binding energy are also calculated. The estimated binding energies for the hevein-carbohydrate interactions are within the range of ±0.5 kcal of the previously reported experimental binding data. For comparison, binding energies were also estimated using thermodynamic integration, molecular dynamics end point calculations (MM/GBSA) and the expanded ensemble methodology is seen to be more accurate. To our knowledge, the method of EEMD simulations has not been previously reported for estimating biomolecular binding energies.

  14. [Lectin-binding analysis of the biofilm exopolymeric matrix carbohydrate composition of corrosion-aggressive bacteria].

    PubMed

    Purish, L M; Asaulenko, L G; Abdulina, D R; Voĭtchuk, S I; Iutinskaia, G A

    2013-01-01

    The carbohydrate components of biofilms of corrosion-aggressive bacteria were studied by transmisstion electron microscopy using lectins labeled with colloidal gold. N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and neutral carbohydrates D-glucose and D-mannose were found within the exopolymeric matrix. Lectins with equal carbohydrate specificity demonstrated different degrees of interaction with the carbohydrate components of bacterial biofilms. To identify N-acetyl-D-galactosamine in biofilms of Desulfovibrio sp. 10 and Bacillus subtilis 36, the LBA lectin appeared to be most specific; in the case of N-acetyl-D-glucosamine in biofilms of B. subtilis 36 and Pseudomonas aeruginosa 27, the WGA lectin. During visualization of neutral carbohydrates in the studied cultures, the PSA lectin was most specific. We have shown that lectins labeled with colloidal gold could be used as an express method for the identification and localization of carbohydrates in glycopolymers of the biofilm exopolymeric matrix.

  15. AMPK beta subunits display isoform specific affinities for carbohydrates.

    PubMed

    Koay, Ann; Woodcroft, Ben; Petrie, Emma J; Yue, Helen; Emanuelle, Shane; Bieri, Michael; Bailey, Michael F; Hargreaves, Mark; Park, Jong-Tae; Park, Kwan-Hwa; Ralph, Stuart; Neumann, Dietbert; Stapleton, David; Gooley, Paul R

    2010-08-04

    AMP-activated protein kinase (AMPK) is a heterotrimer of catalytic (alpha) and regulatory (beta and gamma) subunits with at least two isoforms for each subunit. AMPK beta1 is widely expressed whilst AMPK beta2 is highly expressed in muscle and both beta isoforms contain a mid-molecule carbohydrate-binding module (beta-CBM). Here we show that beta2-CBM has evolved to contain a Thr insertion and increased affinity for glycogen mimetics with a preference for oligosaccharides containing a single alpha-1,6 branched residue. Deletion of Thr-101 reduces affinity for single alpha-1,6 branched oligosaccharides by 3-fold, while insertion of this residue into the equivalent position in the beta1-CBM sequence increases affinity by 3-fold, confirming the functional importance of this residue. Copyright (c) 2010. Published by Elsevier B.V.

  16. Cooperative binding of LysM domains determines the carbohydrate affinity of a bacterial endopeptidase protein.

    PubMed

    Wong, Jaslyn E M M; Alsarraf, Husam M A B; Kaspersen, Jørn Døvling; Pedersen, Jan Skov; Stougaard, Jens; Thirup, Søren; Blaise, Mickaël

    2014-02-01

    Cellulose, chitin and peptidoglycan are major long-chain carbohydrates in living organisms, and constitute a substantial fraction of the biomass. Characterization of the biochemical basis of dynamic changes and degradation of these β,1-4-linked carbohydrates is therefore important for both functional studies of biological polymers and biotechnology. Here, we investigated the functional role of multiplicity of the carbohydrate-binding lysin motif (LysM) domain that is found in proteins involved in bacterial peptidoglycan synthesis and remodelling. The Bacillus subtilis peptidoglycan-hydrolysing NlpC/P60 D,L-endopeptidase, cell wall-lytic enzyme associated with cell separation, possesses four LysM domains. The contribution of each LysM domain was determined by direct carbohydrate-binding studies in aqueous solution with microscale thermophoresis. We found that bacterial LysM domains have affinity for N-acetylglucosamine (GlcNac) polymers in the lower-micromolar range. Moreover, we demonstrated that a single LysM domain is able to bind carbohydrate ligands, and that LysM domains act additively to increase the binding affinity. Our study reveals that affinity for GlcNAc polymers correlates with the chain length of the carbohydrate, and suggests that binding of long carbohydrates is mediated by LysM domain cooperativity. We also show that bacterial LysM domains, in contrast to plant LysM domains, do not discriminate between GlcNAc polymers, and recognize both peptidoglycan fragments and chitin polymers with similar affinity. Finally, an Ala replacement study suggested that the carbohydrate-binding site in LysM-containing proteins is conserved across phyla. © 2013 FEBS.

  17. Isolation by cell-column chromatography of immunoglobulins specific for cell surface carbohydrates

    PubMed Central

    1977-01-01

    A new method of affinity chromatography using glutaraldehyde-fixed cells immobilized on Sephadex beads has been used to isolate immunoglobulins (Ig's) specific for cell surface glycoproteins. Ig's that specifically bound and agglutinated the same cells as those originally fixed on the columns were isolated from nonimmune sera of various species. Periodate treatment of the cell-columns and the free cells destroyed their ability to bind the Ig's, and the binding of the Ig's to untreated cells was inhibited by monosaccharides such as D- galactose and sialic acid. The binding of antibodies directed against cell surfaces obtained by immunizing animals with the same mouse tumor cell lines used on the columns (P388 and EL4) was not inhibited by various saccharides. Surface glycoproteins obtained from the mouse tumor cells by immunoprecipitation with the column-isolated Ig's yielded specific electrophoretic patterns that differed from those obtained using Ig's from the sera of rabbits immunized with the tumor cells. The data suggest that the Ig's isolated by cell-column chromatography were directed against carbohydrates, probably those in terminal positions of the polysaccharide portions of the tumor cell surface glycoproteins. Column-isolated Ig's specific for carbohydrates were also useful in studies of cell interactions in nonmammalian systems including Dictyostelium discoideum and Saccharomyces cerevisiae. The cell-column method appears to be adaptable to the isolation of a variety of molecules in addition to antibodies. PMID:833547

  18. Carbohydrate malabsorption in patients with non-specific abdominal complaints

    PubMed Central

    Born, Peter

    2007-01-01

    Non-specific abdominal complaints are a considerable problem worldwide. Many patients are affected and many differential diagnoses have to be considered. Among these, carbohydrate malabsorption seems to play an important role. However, so far, only incomplete absorption of lactose is broadly accepted, while the malabsorption of fructose and sorbitol is still underestimated, although in many parts of the world it is much more frequent. Despite the success of dietary interventions in many patients, there are still a lot of unanswered questions that make further investigations necessary. PMID:17963293

  19. Carbohydrates.

    PubMed

    Cocinero, Emilio J; Çarçabal, Pierre

    2015-01-01

    Although carbohydrates represent one of the most important families of biomolecules, they remain under-studied in comparison to the other biomolecular families (peptides, nucleobases). Beyond their best-known function of energy source in living systems, they act as mediator of molecular recognition processes, carrying molecular information in the so-called "sugar code," just to name one of their countless functions. Owing to their high conformational flexibility, they encode extremely rich information conveyed via the non-covalent hydrogen bonds within the carbohydrate and with other biomolecular assemblies, such as peptide subunits of proteins. Over the last decade there has been tremendous progress in the study of the conformational preferences of neutral oligosaccharides, and of the interactions between carbohydrates and various molecular partners (water, aromatic models, and peptide models), using vibrational spectroscopy as a sensitive probe. In parallel, other spectroscopic techniques have recently become available to the study of carbohydrates in the gas phase (microwave spectroscopy, IRMPD on charged species).

  20. A role for carbohydrate recognition in mammalian sperm-egg binding

    SciTech Connect

    Clark, Gary F.

    2014-08-01

    Highlights: • Mammalian sperm-egg binding as a carbohydrate dependent species recognition event. • The role of carbohydrate recognition in human, mouse and pig sperm-egg binding. • Historical perspective and future directions for research focused on gamete binding. - Abstract: Mammalian fertilization usually requires three sequential cell–cell interactions: (i) initial binding of sperm to the specialized extracellular matrix coating the egg known as the zona pellucida (ZP); (ii) binding of sperm to the ZP via the inner acrosomal membrane that is exposed following the induction of acrosomal exocytosis; and (iii) adhesion of acrosome-reacted sperm to the plasma membrane of the egg cell, enabling subsequent fusion of these gametes. The focus of this review is on the initial binding of intact sperm to the mammalian ZP. Evidence collected over the past fifty years has confirmed that this interaction relies primarily on the recognition of carbohydrate sequences presented on the ZP by lectin-like egg binding proteins located on the plasma membrane of sperm. There is also evidence that the same carbohydrate sequences that mediate binding also function as ligands for lectins on lymphocytes that can inactivate immune responses, likely protecting the egg and the developing embryo up to the stage of blastocyst hatching. The literature related to initial sperm-ZP binding in the three major mammalian models (human, mouse and pig) is discussed. Historical perspectives and future directions for research related to this aspect of gamete adhesion are also presented.

  1. An Adenovirus Vector Incorporating Carbohydrate Binding Domains Utilizes Glycans for Gene Transfer

    PubMed Central

    Nakayama, Masaharu; Ak, Ferhat; Ugai, Hideyo; Curiel, David T.

    2013-01-01

    Background Vectors based on human adenovirus serotype 5 (HAdV-5) continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting. Methodology/Principal Findings As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4). This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells. Conclusions/Significance These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers. PMID:23383334

  2. Ion mobility studies of carbohydrates as group I adducts: isomer specific collisional cross section dependence on metal ion radius.

    PubMed

    Huang, Yuting; Dodds, Eric D

    2013-10-15

    Carbohydrates play numerous critical roles in biological systems. Characterization of oligosaccharide structures is essential to a complete understanding of their functions in biological processes; nevertheless, their structural determination remains challenging in part due to isomerism. Ion mobility spectrometry provides the means to resolve gas phase ions on the basis of their shape-to-charge ratios, thus providing significant potential for separation and differentiation of carbohydrate isomers. Here, we report on the determination of collisional cross sections for four groups of isomeric carbohydrates (including five isomeric disaccharides, four isomeric trisaccharides, two isomeric pentasaccharides, and two isomeric hexasaccharides) as their group I metal ion adducts (i.e., [M + Li](+), [M + Na](+), [M + K](+), [M + Rb](+), and [M + Cs](+)). In all, 65 collisional cross sections were measured, the great majority of which have not been previously reported. As anticipated, the collisional cross sections of the carbohydrate metal ion adducts generally increase with increasing metal ion radius; however, the collisional cross sections were found to scale with the group I cation size in isomer specific manners. Such measurements are of substantial analytical value, as they illustrate how the selection of charge carrier influences carbohydrate ion mobility determinations. For example, certain pairs of isomeric carbohydrates assume unique collisional cross sections upon binding one metal ion, but not another. On the whole, these data suggest a role for the charge carrier as a probe of carbohydrate structure and thus have significant implications for the continued development and application of ion mobility spectrometry for the distinction and resolution of isomeric carbohydrates.

  3. Carbohydrates act as receptors for the periodontitis-associated bacterium Porphyromonas gingivalis: a study of bacterial binding to glycolipids.

    PubMed

    Hellström, Ulrika; Hallberg, Eva C; Sandros, Jens; Rydberg, Lennart; Bäcker, Annika E

    2004-06-01

    In this study we show for the first time the use of carbohydrate chains on glycolipids as receptors for the periodontitis-associated bacterium Porphyromonas gingivalis. Previous studies have shown that this bacterium has the ability to adhere to and invade the epithelial lining of the dental pocket. Which receptor(s) the adhesin of P. gingivalis exploit in the adhesion to epithelial cells has not been shown. Therefore, the binding preferences of this specific bacterium to structures of carbohydrate origin from more than 120 different acid and nonacid glycolipid fractions were studied. The bacteria were labeled externally with (35)S and used in a chromatogram binding assay. To enable detection of carbohydrate receptor structures for P. gingivalis, the bacterium was exposed to a large number of purified total glycolipid fractions from a variety of organs from different species and different histo-blood groups. P. gingivalis showed a preference for fractions of human and pig origin for adhesion. Both nonacid and acid glycolipids were used by the bacterium, and a preference for shorter sugar chains was noticed. Bacterial binding to human acid glycolipid fractions was mainly obtained in the region of the chromatograms where sulfated carbohydrate chains usually are found. However, the binding pattern to nonacid glycolipid fractions suggests a core chain of lactose bound to the ceramide part as a tentative receptor structure. The carbohydrate binding of the bacterium might act as a first step in the bacterial invasion process of the dental pocket epithelium, subsequently leading to damage to periodontal tissue and tooth loss.

  4. Ligand identification of carbohydrate-binding proteins employing a biotinylated glycan binding assay and tandem mass spectrometry.

    PubMed

    Wuhrer, Manfred; van Remoortere, Alexandra; Balog, Crina I A; Deelder, André M; Hokke, Cornelis H

    2010-11-15

    Characterization of protein-carbohydrate interactions at the molecular level is important for understanding many glycan-mediated processes. Here we present a method for the identification of glycan ligands of carbohydrate-binding proteins. The glycans released from natural sources are labeled with biotinamidocaproyl hydrazide (BACH) and subsequently fractionated by high-performance liquid chromatography. Glycan fractions are screened for binding to carbohydrate-binding proteins (CBPs) using a microtitration plate binding assay; CBPs are immobilized, BACH-glycan fractions are added, and bound BACH-glycans are detected using alkaline phosphatase-conjugated streptavidin. The glycan structures in binding fractions are studied by (tandem) mass spectrometry, exoglycosidase treatment, and rechromatography, thereby revealing the glycan motifs recognized by the CBPs. Subsequent surface plasmon resonance experiments using a reverse setup with immobilization of the BACH-glycan ligands on streptavidin-coated surfaces provide more information on glycan-CBP interactions via association and dissociation curves. The presented method is easy and fast, and the required instrumentation is available in many laboratories. The assay is very sensitive given that both the mass spectrometric analysis and the microtitration plate binding assay can be performed on femtomole amounts of BACH-glycans. This approach should be generally applicable to study and structurally identify carbohydrate ligands of anti-glycan antibodies and lectins.

  5. Application of surface plasmon resonance for the detection of carbohydrates, glycoconjugates, and measurement of the carbohydrate-specific interactions: a comparison with conventional analytical techniques. A critical review.

    PubMed

    Safina, Gulnara

    2012-01-27

    Carbohydrates (glycans) and their conjugates with proteins and lipids contribute significantly to many biological processes. That makes these compounds important targets to be detected, monitored and identified. The identification of the carbohydrate content in their conjugates with proteins and lipids (glycoforms) is often a challenging task. Most of the conventional instrumental analytical techniques are time-consuming and require tedious sample pretreatment and utilising various labeling agents. Surface plasmon resonance (SPR) has been intensively developed during last two decades and has received the increasing attention for different applications, from the real-time monitoring of affinity bindings to biosensors. SPR does not require any labels and is capable of direct measurement of biospecific interaction occurring on the sensing surface. This review provides a critical comparison of modern analytical instrumental techniques with SPR in terms of their analytical capabilities to detect carbohydrates, their conjugates with proteins and lipids and to study the carbohydrate-specific bindings. A few selected examples of the SPR approaches developed during 2004-2011 for the biosensing of glycoforms and for glycan-protein affinity studies are comprehensively discussed.

  6. Generating novel recombinant prokaryotic lectins with altered carbohydrate binding properties through mutagenesis of the PA-IL protein from Pseudomonas aeruginosa.

    PubMed

    Keogh, Damien; Thompson, Roisin; Larragy, Ruth; McMahon, Kenneth; O'Connell, Michael; O'Connor, Brendan; Clarke, Paul

    2014-06-01

    Prokaryotic lectins offer significant advantages over eukaryotic lectins for the development of enhanced glycoselective tools. Amenability to recombinant expression in Escherichia coli simplifies their production and presents opportunities for further genetic manipulation to create novel recombinant prokaryotic lectins (RPLs) with altered or enhanced carbohydrate binding properties. This study explored the potential of the α-galactophilic PA-IL lectin from Pseudomonas aeruginosa for use as a scaffold structure for the generation of novel RPLs. Specific amino acid residues in the carbohydrate binding site of a recombinant PA-IL protein were randomly substituted by site-directed mutagenesis. The resulting expression clones were then functionally screened to identify clones expressing rPA-IL proteins with altered carbohydrate binding properties. This study generated RPLs exhibiting diverse carbohydrate binding activities including specificity and high affinity for β-linked galactose and N-acetyl-lactosamine (LacNAc) displayed by N-linked glycans on glycoprotein targets. Key amino acid substitutions were identified and linked with specific carbohydrate binding activities. Ultimately, the utility of these novel RPLs for glycoprotein analysis and for selective fractionation and isolation of glycoproteins and their glycoforms was demonstrated. The carbohydrate binding properties of the PA-IL protein can be significantly altered using site-directed mutagenesis strategies to generate novel RPLs with diverse carbohydrate binding properties. The novel RPLs reported would find a broad range of applications in glycobiology, diagnostics and in the analysis of biotherapeutics. The ability to readily produce these RPLs in gram quantities could enable them to find larger scale applications for glycoprotein or biotherapeutic purification. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. O-glycosylation effects on family 1 carbohydrate-binding module solution structures.

    PubMed

    Happs, Renee M; Guan, Xiaoyang; Resch, Michael G; Davis, Mark F; Beckham, Gregg T; Tan, Zhongping; Crowley, Michael F

    2015-11-01

    Family 1 carbohydrate-binding modules (CBMs) are ubiquitous components of multimodular fungal enzymes that degrade plant cell wall polysaccharides and bind specifically to cellulose. Native glycosylation of family 1 CBMs has been shown to substantially impact multiple physical properties, including thermal and proteolytic stability and cellulose binding affinity. To gain molecular insights into the changes in CBM properties upon glycosylation, solution structures of two glycoforms of a Trichoderma reesei family 1 CBM were studied by NMR spectroscopy: a glycosylated family 1 CBM with a mannose group attached to both Thr1 and Ser3 and a second family 1 CBM with single mannose groups attached to Thr1, Ser3 and Ser14. The structures clearly reveal that monosaccharides at both Ser3 and Ser14 on family 1 CBMs present additional cellulose binding platforms, similar to well-characterized aromatic residues at the binding interface, which align to the cellulose surface. These results are in agreement with previous experimental work demonstrating that glycans at Ser3 and Ser14 impart significant improvements in binding affinity. Additionally, detailed analysis of the NMR structures and molecular simulations indicates that the protein backbone of the CBM is not significantly altered by attachment of monosaccharides, and that the mannose attached to Ser14 may be more flexible than the mannose at Ser3. Overall, the present study reveals how family 1 CBM structures are affected by covalent attachment of monosaccharides, which are likely important post-translational modifications of these common subdomains of fungal plant cell wall degrading enzymes. Structural data have been deposited in the RCSB Protein Data Bank (PDB codes: 2MWJ and 2MWK) and the BioMagRes Bank (BMRB codes: 25331 and 25332) for CBM_M2 and CBM_M3, respectively. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

  8. The Structural Basis of Alpha-Glucan Recognition by a Family 41 Carbohydrate-Binding Module from Therotoga Maritima

    SciTech Connect

    van Bueren,A.; Boraston, A.

    2006-01-01

    Starch recognition by carbohydrate-binding modules (CBMs) is important for the activity of starch-degrading enzymes. The N-terminal family 41 CBM, TmCBM41 (from pullulanase PulA secreted by Thermotoga maritima) was shown to have {alpha}-glucan binding activity with specificity for {alpha}-1, 4-glucans but was able to tolerate the {alpha}-1, 6-linkages found roughly every three or four glucose units in pullulan. Using X-ray crystallography, the structures were solved for TmCBM41 in an uncomplexed form and in complex with maltotetraose and 63-{alpha}-d-glucosyl-maltotriose (GM3). Ligand binding was facilitated by stacking interactions between the {alpha}-faces of the glucose residues and two tryptophan side-chains in the two main subsites of the carbohydrate-binding site. Overall, this mode of starch binding is quite well conserved by other starch-binding modules. The structure in complex with GM3 revealed a third binding subsite with the flexibility to accommodate an {alpha}-1, 4- or an {alpha}-1, 6-linked glucose.

  9. Divergent modes of glycan recognition by a new family of carbohydrate-binding modules.

    PubMed

    Gregg, Katie J; Finn, Ron; Abbott, D Wade; Boraston, Alisdair B

    2008-05-02

    The genomes of myonecrotic Clostridium perfringens isolates contain genes encoding a large and fascinating array of highly modular glycoside hydrolase enzymes. Although the catalytic activities of many of these enzymes are somewhat predictable based on their amino acid sequences, the functions of their abundant ancillary modules are not and remain poorly studied. Here, we present the structural and functional analysis of a new family of ancillary carbohydrate-binding modules (CBMs), CBM51, which was previously annotated in data bases as the novel putative CBM domain. The high resolution crystal structures of two CBM51 members, GH95CBM51 and GH98CBM51, from a putative family 95 alpha-fucosidase and from a family 98 blood group A/B antigen-specific endo-beta-galactosidase, respectively, showed them to have highly similar beta-sandwich folds. However, GH95CBM51 was shown by glycan microarray screening, isothermal titration calorimetry, and x-ray crystallography to bind galactose residues, whereas the same analyses of GH98CBM51 revealed specificity for the blood group A/B antigens through non-conserved interactions. Overall, this work identifies a new family of CBMs with many members having apparent specificity for eukaryotic glycans, in keeping with the glycan-rich environment C. perfringens would experience in its host. However, a wider bioinformatic analysis of this CBM family also indicated a large number of members in non-pathogenic environmental bacteria, suggesting a role in the recognition of environmental glycans.

  10. CD44 receptor unfolding enhances binding by freeing basic amino acids to contact carbohydrate ligand.

    PubMed

    Favreau, Amanda J; Faller, Christina E; Guvench, Olgun

    2013-09-03

    The extracellular carbohydrate-binding domain of the Type I transmembrane receptor CD44 is known to undergo affinity switching, where change in conformation leads to enhanced binding of its carbohydrate ligand hyaluronan. Separate x-ray crystallographic and NMR experiments have led to competing explanations, with the former supporting minor conformational changes at the binding site and the latter a major order-to-disorder unfolding transition distant from the binding site. Here, all-atom explicit-solvent molecular dynamics studies employing adaptive biasing force sampling revealed a substantial favorable free-energy change associated with contact formation between the Arg(41) side chain and hyaluronan at the binding site, independent of whether the distant site was ordered or disordered. Analogous computational experiments on Arg(41)Ala mutants showed loss of this favorable free-energy change, consistent with existing experimental data. More provocatively, the simulation data revealed the molecular mechanism by which the order-to-disorder transition enhances hyaluronan binding: in the disordered state, a number of basic residues gain sufficient conformational freedom-lacking in the ordered state-to spontaneously form side-chain contacts with hyaluronan. Mutation of these residues to Ala had been known to decrease binding affinity, but there had previously been no structural explanation, given their lack of proximity to the carbohydrate-binding site in existing structures of the complex.

  11. Identification of Oligosaccharides in Human Milk Bound onto the Toxin A Carbohydrate Binding Site of Clostridium difficile.

    PubMed

    Nguyen, Thi Thanh Hanh; Kim, Jong Woon; Park, Jun-Seong; Hwang, Kyeong Hwan; Jang, Tae-Su; Kim, Chun-Hyung; Kim, Doman

    2016-04-28

    The oligosaccharides in human milk constitute a major innate immunological mechanism by which breastfed infants gain protection against infectious diarrhea. Clostridium difficile is the most important cause of nosocomial diarrhea, and the C-terminus of toxin A with its carbohydrate binding site, TcdA-f2, demonstrates specific abolishment of cytotoxicity and receptor binding activity upon diethylpyrocarbonate modification of the histidine residues in TcdA. TcdA-f2 was cloned and expressed in E. coli BL21 (DE3). A human milk oligosaccharide (HMO) mixture displayed binding with TcdA-f2 at 38.2 respond units (RU) at the concentration of 20 μg/ml, whereas the eight purified HMOs showed binding with the carbohydrate binding site of TcdA-f2 at 3.3 to 14 RU depending on their structures via a surface plasma resonance biosensor. Among them, Lacto-N-fucopentaose V (LNFPV) and Lacto-N-neohexaose (LNnH) demonstrated tight binding to TcdA-f2 with docking energy of -9.48 kcal/mol and -12.81 kcal/mol, respectively. It displayed numerous hydrogen bonding and hydrophobic interactions with amino acid residues of TcdA-f2.

  12. Molecular diversity of LysM carbohydrate-binding motifs in fungi.

    PubMed

    Akcapinar, Gunseli Bayram; Kappel, Lisa; Sezerman, Osman Ugur; Seidl-Seiboth, Verena

    2015-05-01

    LysM motifs are carbohydrate-binding modules found in prokaryotes and eukaryotes. They bind to N-acetylglucosamine-containing carbohydrates, such as chitin, chitio-oligosaccharides and peptidoglycan. In this review, we summarize the features of the protein architecture of LysM-containing proteins in fungi and discuss their so far known biochemical properties, transcriptional profiles and biological functions. Further, based on data from evolutionary analyses and consensus pattern profiling of fungal LysM motifs, we show that they can be classified into a fungal-specific group and a fungal/bacterial group. This facilitates the classification and selection of further LysM proteins for detailed analyses and will contribute to widening our understanding of the functional spectrum of this protein family in fungi. Fungal LysM motifs are predominantly found in subgroup C chitinases and in LysM effector proteins, which are secreted proteins with LysM motifs but no catalytic domains. In enzymes, LysM motifs mediate the attachment to insoluble carbon sources. In plants, receptors containing LysM motifs are responsible for the perception of chitin-oligosaccharides and are involved in beneficial symbiotic interactions between plants and bacteria or fungi, as well as plant defence responses. In plant pathogenic fungi, LysM effector proteins have already been shown to have important functions in the dampening of host defence responses as well as protective functions of fungal hyphae against chitinases. However, the large number and diversity of proteins with LysM motifs that are being unravelled in fungal genome sequencing projects suggest that the functional repertoire of LysM effector proteins in fungi is only partially discovered so far.

  13. Carbohydrate-binding agents act as potent trypanocidals that elicit modifications in VSG glycosylation and reduced virulence in Trypanosoma brucei.

    PubMed

    Castillo-Acosta, Víctor M; Vidal, Antonio E; Ruiz-Pérez, Luis M; Van Damme, Els J M; Igarashi, Yasuhiro; Balzarini, Jan; González-Pacanowska, Dolores

    2013-11-01

    The surface of Trypanosoma brucei is covered by a dense coat of glycosylphosphatidylinositol-anchored glycoproteins. The major component is the variant surface glycoprotein (VSG) which is glycosylated by both paucimannose and oligomannose N-glycans. Surface glycans are poorly accessible and killing mediated by peptide lectin-VSG complexes is hindered by active endocytosis. However, contrary to previous observations, here we show that high-affinity carbohydrate binding agents bind to surface glycoproteins and abrogate growth of T. brucei bloodstream forms. Specifically, binding of the mannose-specific Hippeastrum hybrid agglutinin (HHA) resulted in profound perturbations in endocytosis and parasite lysis. Prolonged exposure to HHA led to the loss of triantennary oligomannose structures in surface glycoproteins as a result of genetic rearrangements that abolished expression of the oligosaccharyltransferase TbSTT3B gene and yielded novel chimeric enzymes. Mutant parasites exhibited markedly reduced infectivity thus demonstrating the importance of specific glycosylation patterns in parasite virulence.

  14. Evidence for a modular structure of the homologous repetitive C-terminal carbohydrate-binding sites of Clostridium difficile toxins and Streptococcus mutans glucosyltransferases.

    PubMed Central

    von Eichel-Streiber, C; Sauerborn, M; Kuramitsu, H K

    1992-01-01

    The homologous C-terminal repeats of Clostridium difficile toxins (ToxA and ToxB) and streptococcal glucosyltransferases appear to mediate protein-carbohydrate interactions at cellular binding sites with sugar moieties as substrates. A consensus sequence of 134 repeating units from gram-positive bacteria indicates that these repeats have a modular design with (i) a stretch of aromatic amino acids proposed to be involved in the primary carbohydrate-protein interaction, (ii) an amplification of this interaction by repetition of the respective sequences, and (iii) a second domain, not characterized, that is responsible for carbohydrate specificity. PMID:1307487

  15. Energetics of carbohydrate binding to Momordica charantia (bitter gourd) lectin: an isothermal titration calorimetric study.

    PubMed

    Sultan, Nabil Ali Mohammed; Swamy, Musti J

    2005-05-01

    Physico-chemical and carbohydrate binding studies have been carried out on the Momordica charantia (bitter gourd) seed lectin (MCL). The lectin activity is maximal in the pH range 7.4-11.0, but decreases steeply below pH 7.0. The lectin activity is mostly unaffected in the temperature range 4-50 degrees C, but a sharp decrease is seen between 50 and 60 degrees C, which could be correlated to changes in the structure of the protein as seen by circular dichroism and fluorescence spectroscopy. Isothermal titration calorimetric studies show that the tetrameric MCL binds two sugar molecules and the binding constants (Kb), determined at 288.15 K, for various saccharides were found to vary between 7.3 x 10(3) and 1.52 x 10(4)M(-1). The binding reactions for all the saccharides investigated were essentially enthalpy driven, with the binding enthalpies (DeltaHb) at 288.15 K being in the range of -50.99 and -43.39 kJ mol(-1), whereas the contribution to the binding reaction from the entropy of binding was negative, with values of binding entropy (DeltaSb) ranging between -99.2 and -72.0 J mol(-1)K(-1) at 288.15 K. Changes in heat capacity (DeltaCp) for the binding of disaccharides, lactose and lactulose, were significantly larger in magnitude than those obtained for the monosaccharides, methyl-beta-D-galactopyranoside, and methyl-alpha-D-galactopyranoside, and could be correlated reasonably well with the surface areas of these ligands. Enthalpy-entropy compensation was observed for all the sugars studied, suggesting that water structure plays an important role in the overall binding reaction. CD spectroscopy indicates that carbohydrate binding does not lead to significant changes in the secondary and tertiary structures of MCL, suggesting that the carbohydrate binding sites on this lectin are mostly preformed.

  16. A Structural Model for Binding of the Serine-Rich Repeat Adhesin GspB to Host Carbohydrate Receptors

    SciTech Connect

    Pyburn, Tasia M.; Bensing, Barbara A.; Xiong, Yan Q.; Melancon, Bruce J.; Tomasiak, Thomas M.; Ward, Nicholas J.; Yankovskaya, Victoria; Oliver, Kevin M.; Cecchini, Gary; Sulikowski, Gary A.; Tyska, Matthew J.; Sullam, Paul M.; Iverson, T.M.

    2014-10-02

    GspB is a serine-rich repeat (SRR) adhesin of Streptococcus gordonii that mediates binding of this organism to human platelets via its interaction with sialyl-T antigen on the receptor GPIb{alpha}. This interaction appears to be a major virulence determinant in the pathogenesis of infective endocarditis. To address the mechanism by which GspB recognizes its carbohydrate ligand, we determined the high-resolution x-ray crystal structure of the GspB binding region (GspB{sub BR}), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspB{sub BR} structure revealed that it is comprised of three independently folded subdomains or modules: (1) an Ig-fold resembling a CnaA domain from prokaryotic pathogens; (2) a second Ig-fold resembling the binding region of mammalian Siglecs; (3) a subdomain of unique fold. The disaccharide was found to bind in a pocket within the Siglec subdomain, but at a site distinct from that observed in mammalian Siglecs. Confirming the biological relevance of this binding pocket, we produced three isogenic variants of S. gordonii, each containing a single point mutation of a residue lining this binding pocket. These variants have reduced binding to carbohydrates of GPIb{alpha}. Further examination of purified GspB{sub BR}-R484E showed reduced binding to sialyl-T antigen while S. gordonii harboring this mutation did not efficiently bind platelets and showed a significant reduction in virulence, as measured by an animal model of endocarditis. Analysis of other SRR proteins revealed that the predicted binding regions of these adhesins also had a modular organization, with those known to bind carbohydrate receptors having modules homologous to the Siglec and Unique subdomains of GspBBR. This suggests that the binding specificity of the SRR family of adhesins is determined by the type and organization of discrete modules within the binding domains, which may affect the tropism of organisms for different tissues.

  17. VARIATION IN THE GROUP-SPECIFIC CARBOHYDRATE OF GROUP A STREPTOCOCCI

    PubMed Central

    McCarty, Maclyn; Lancefield, Rebecca C.

    1955-01-01

    The phenomenon of apparent loss of group-specific carbohydrate in the course of mouse passage of group A streptococci has been subjected to further study, and several additional variants showing this property have been described. The loss of group reactivity is shown to be due to an alteration in the chemical structure and serological specificity of the cell wall carbohydrate. This alteration appears to be essentially the same in each of the variants available for study. The carbohydrate of the variant strains (V) contains the same two monosaccharide components as the group A carbohydrate (A), but they are present in different proportions. Precipitating sera reactive with V carbohydrate have been prepared, and the A and V carbohydrates have been compared by qualitative and quantitative precipitin analysis. A second type of variation has been encountered during mouse passage. This variation is characterized by the occurrence of a cell wall carbohydrate (I) intermediate in chemical and serological properties between the A and V carbohydrates. The I carbohydrate reacts with both A and V antisera and does not appear to be a simple mixture of A and V carbohydrate. Similarly, antisera against the intermediate strain contain antibodies reactive with both A and V carbohydrates, and evidence is presented indicating that in part this represents antibody with double specificity. PMID:14392237

  18. Binding of polysaccharides to human galectin-3 at a noncanonical site in its carbohydrate recognition domain

    PubMed Central

    Miller, Michelle C; Ippel, Hans; Suylen, Dennis; Klyosov, Anatole A; Traber, Peter G; Hackeng, Tilman; Mayo, Kevin H

    2016-01-01

    Galectin-3 (Gal-3) is a multifunctional lectin, unique to galectins by the presence of a long N-terminal tail (NT) off of its carbohydrate recognition domain (CRD). Many previous studies have investigated binding of small carbohydrates to its CRD. Here, we used nuclear magnetic resonance spectroscopy (15N–1H heteronuclear single quantum coherence data) to assess binding of 15N-Gal-3 (and truncated 15N-Gal-3 CRD) to several, relatively large polysaccharides, including eight varieties of galactomannans (GMs), as well as a β(1 → 4)-polymannan and an α-branched mannan. Overall, we found that these polysaccharides with a larger carbohydrate footprint interact primarily with a noncanonical carbohydrate-binding site on the F-face of the Gal-3 CRD β-sandwich, and to a less extent, if at all, with the canonical carbohydrate-binding site on the S-face. While there is no evidence for interaction with the NT itself, it does appear that the NT somehow mediates stronger interactions between the Gal-3 CRD and the GMs. Significant Gal-3 resonance broadening observed during polysaccharide titrations indicates that interactions occur in the intermediate exchange regime, and analysis of these data allows estimation of affinities and stoichiometries that range from 4 × 104 to 12 × 104 M−1 per site and multiple sites per polysaccharide, respectively. We also found that lactose can still bind to the CRD S-face of GM-bound Gal-3, with the binding of one ligand attenuating affinity of the other. These data are compared with previous results on Gal-1, revealing differences and similarities. They also provide research direction to the development of these polysaccharides as galectin-targeting therapeutics in the clinic. PMID:26646771

  19. Structural energetics of protein–carbohydrate interactions: Insights derived from the study of lysozyme binding to its natural saccharide inhibitors

    PubMed Central

    García-Hernández, Enrique; Zubillaga, Rafael A.; Chavelas-Adame, Eneas A.; Vázquez-Contreras, Edgar; Rojo-Domínguez, Arturo; Costas, Miguel

    2003-01-01

    High-sensitivity isothermal titration calorimetry was used to characterize the binding of the glycohydrolitic enzyme hen egg-white lysozyme to its natural saccharide inhibitors, chitobiose and chitrotriose. Measurements were done at a pH of 4.7, in the 15°C –45°C temperature range. Using a structural-energetic parameterization derived previously for lectin-carbohydrate associations, both binding enthalpies and entropies for the present systems and for the complex of chitobiose with turkey egg-white lysozyme from the literature were correctly accounted for. These observations suggest that both lysozymes and lectins follow the same structural-energetic behavior in the binding to their ligands. From the analysis of lysozyme data in conjunction with other binding data reported in the literature, an ad hoc parameterization of ΔCp for protein–carbohydrate complexes was derived for the first time. The novel parameters for both polar and apolar surface areas differed significantly from correlations obtained previously from model compounds and protein-folding data. As ΔCp is extremely sensitive to changes in solvent structure, this finding indicates that protein–carbohydrate complexes have distinctive hydration properties. According to our analysis, the dehydration of polar groups is the major cause for the observed decrease in ΔCp, which implies that these groups behave hydrophobically. The contribution of apolar surface areas was found of the expected sign, but their specific weight is much smaller than those obtained in other correlations. This small contribution to ΔCp is consistent with Lemieux’s hypothesis of a low degree of hydration of apolar surfaces on carbohydrates. PMID:12493836

  20. Engineered carbohydrate-binding module (CBM) protein-suspended single-walled carbon nanotubes in water.

    PubMed

    Xu, Qi; Song, Qing; Ai, Xin; McDonald, Timothy J; Long, Hai; Ding, Shi-You; Himmel, Michael E; Rumbles, Garry

    2009-01-21

    Engineered protein, CtCBM4, the first carbohydrate-binding module (CBM) protein is successfully used to debundle and suspend single-walled carbon nanotubes (SWNTs) effectively in aqueous solution, which opens up a new avenue in further functionalizing and potential selectively fractionating SWNTs for diverse biology- and/or energy-related applications.

  1. Identification and Characterization of Sulfated Carbohydrate-Binding Protein from Lactobacillus reuteri

    PubMed Central

    Nishiyama, Keita; Ochiai, Ayaka; Tsubokawa, Daigo; Ishihara, Kazuhiko; Yamamoto, Yuji; Mukai, Takao

    2013-01-01

    We previously purified a putative sulfated-galactosylceramide (sulfatide)-binding protein with a molecular weight of 47 kDa from the cell surface of Lactobacillus reuteri JCM1081. The aim of this study was to identify the 47-kDa protein, examine its binding to sulfated glycolipids and mucins, and evaluate its role in bacterial adhesion to mucosal surfaces. By cloning and sequencing analysis, the 47-kDa protein was identified as elongation factor-Tu (EF-Tu). Adhesion properties were examined using 6×Histidine-fused EF-Tu (His6-EF-Tu). Surface plasmon resonance analysis demonstrated pH-dependent binding of His6-EF-Tu to sulfated glycolipids, but not to neutral or sialylated glycolipids, suggesting that a sulfated galactose residue was responsible for EF-Tu binding. Furthermore, His6-EF-Tu was found to bind to porcine gastric mucin (PGM) by enzyme-linked immunosorbent assay. Binding was markedly reduced by sulfatase treatment of PGM and in the presence of acidic and desialylated oligosaccharide fractions containing sulfated carbohydrate residues prepared from PGM, demonstrating that sulfated carbohydrate moieties mediated binding. Histochemical staining revealed similar localization of His6-EF-Tu and high iron diamine staining in porcine mucosa. These results indicated that EF-Tu bound PGM via sulfated carbohydrate moieties. To characterize the contribution of EF-Tu to the interaction between bacterial cells and PGM, we tested whether anti-EF-Tu antibodies could inhibit the interaction. Binding of L. reuteri JCM1081 to PGM was significantly blocked in a concentration-dependent matter, demonstrating the involvement of EF-Tu in bacterial adhesion. In conclusion, the present results demonstrated, for the first time, that EF-Tu bound sulfated carbohydrate moieties of sulfated glycolipids and sulfomucin, thereby promoting adhesion of L. reuteri to mucosal surfaces. PMID:24391811

  2. Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Zhang, Jingjing; Kitova, Elena N.; Li, Jun; Eugenio, Luiz; Ng, Kenneth; Klassen, John S.

    2016-01-01

    The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to localize ligand binding sites in carbohydrate-binding proteins is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin and Shiga toxin type 1 and a fragment of Clostridium difficile toxin A, and their interactions with native carbohydrate receptors, GM1 pentasaccharides (β-Gal-(1→3)-β-GalNAc-(1→4)[α-Neu5Ac-(2→3)]-β-Gal-(1→4)-Glc), Pk trisaccharide (α-Gal-(1→4)-β-Gal-(1→4)-Glc) and CD-grease (α-Gal-(1→3)-β-Gal-(1→4)-β-GlcNAcO(CH2)8CO2CH3), respectively, served as model systems for this study. Comparison of the differences in deuterium uptake for peptic peptides produced in the absence and presence of ligand revealed regions of the proteins that are protected against deuterium exchange upon ligand binding. Notably, protected regions generally coincide with the carbohydrate binding sites identified by X-ray crystallography. However, ligand binding can also result in increased deuterium exchange in other parts of the protein, presumably through allosteric effects. Overall, the results of this study suggest that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. However, a detailed interpretation of the changes in deuterium exchange upon ligand binding can be challenging because of the presence of ligand-induced changes in protein structure and dynamics.

  3. Stability, subunit interactions and carbohydrate-binding of the seed lectin from Pterocarpus angolensis.

    PubMed

    Echemendia-Blanco, Dannele; Van Driessche, Edilbert; Ncube, Ignatious; Read, John S; Beeckmans, Sonia

    2009-01-01

    From 1 kg of defatted Pterocarpus angolensis (mukwa tree) seed meal, 21.6 grams of an alpha,D-mannose/glucose-specific lectin can be purified on mannose-Sepharose. Relative affinities for several (oligo)saccharides and glycoproteins were studied by haemagglutination-inhibition. Gel filtration shows that the lectin exists as a dimer above pH 5 and as a monomer below pH 3.5. This is confirmed by studies on the release of lectin subunits that were adsorbed from solution to lectin monomers immobilized onto Eupergit-c. From the gel filtration patterns it is calculated that a residue with pK(a) of about 4.4 is involved in dimer dissociation. Titration of glutamic acids (E60, E209) is postulated to be involved. CD spectroscopy shows that the secondary structure of the lectin is unchanged between pH 1 and 12.5, and that the tertiary structure remains unchanged between pH 5 and 12. In the acid pH region, reversible spectral changes occur that may be due to the titration of one or more amino acids with a pK(a) value of 3.9-4.2, probably aspartic acid. These residues are implicated in sugar-binding but not in dimerization of the lectin. Only at pH 12.5, irreversible denaturation occurs. Mukwa lectin displays full carbohydrate-binding capacity between pH 4 and 12, as is concluded from ELLA (Enzyme Linked Lectin Assay) using ovalbumin and fetuin, and from binding of the same glycoproteins to immobilized lectin monomers. The lectin is rapidly and fully reversibly demetallized at pH 2.5 with 5 mM EDTA. The demetallized lectin is completely devoid of sugar-binding activity. Mukwa lectin is a very thermostable molecule (at least till 85 degrees C). However, addition of non-ionic detergents substantially lowers its thermostability.

  4. Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose.

    PubMed

    Ferrara, Claudia; Grau, Sandra; Jäger, Christiane; Sondermann, Peter; Brünker, Peter; Waldhauer, Inja; Hennig, Michael; Ruf, Armin; Rufer, Arne Christian; Stihle, Martine; Umaña, Pablo; Benz, Jörg

    2011-08-02

    Antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism, relies on the binding of antigen-antibody complexes to Fcγ receptors expressed on immune cells. Antibodies lacking core fucosylation show a large increase in affinity for FcγRIIIa leading to an improved receptor-mediated effector function. Although afucosylated IgGs exist naturally, a next generation of recombinant therapeutic, glycoenginereed antibodies is currently being developed to exploit this finding. In this study, the crystal structures of a glycosylated Fcγ receptor complexed with either afucosylated or fucosylated Fc were determined allowing a detailed, molecular understanding of the regulatory role of Fc-oligosaccharide core fucosylation in improving ADCC. The structures reveal a unique type of interface consisting of carbohydrate-carbohydrate interactions between glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts are weakened or nonexistent, explaining the decreased affinity for the receptor. These findings allow us to understand the higher efficacy of therapeutic antibodies lacking the core fucose and also suggest a unique mechanism by which the immune system can regulate antibody-mediated effector functions.

  5. A Novel Carbohydrate-binding Module from Sugar Cane Soil Metagenome Featuring Unique Structural and Carbohydrate Affinity Properties.

    PubMed

    Campos, Bruna Medeia; Liberato, Marcelo Vizona; Alvarez, Thabata Maria; Zanphorlin, Letícia Maria; Ematsu, Gabriela Cristina; Barud, Hernane; Polikarpov, Igor; Ruller, Roberto; Gilbert, Harry J; Zeri, Ana Carolina de Mattos; Squina, Fabio Marcio

    2016-11-04

    Carbohydrate-binding modules (CBMs) are appended to glycoside hydrolases and can contribute to the degradation of complex recalcitrant substrates such as the plant cell wall. For application in bioethanol production, novel enzymes with high catalytic activity against recalcitrant lignocellulosic material are being explored and developed. In this work, we report the functional and structural study of CBM_E1, which was discovered through a metagenomics approach and is the founding member of a novel CBM family, CBM81. CBM_E1, which is linked to an endoglucanase, displayed affinity for mixed linked β1,3-β1,4-glucans, xyloglucan, Avicel, and cellooligosaccharides. The crystal structure of CBM_E1 in complex with cellopentaose displayed a canonical β-sandwich fold comprising two β-sheets. The planar ligand binding site, observed in a parallel orientation with the β-strands, is a typical feature of type A CBMs, although the expected affinity for bacterial crystalline cellulose was not detected. Conversely, the binding to soluble glucans was enthalpically driven, which is typical of type B modules. These unique properties of CBM_E1 are at the interface between type A and type B CBMs.

  6. Carbohydrate recognition in the peripheral nervous system: a calcium- dependent membrane binding site for HNK-1 reactive glycolipids potentially involved in Schwann cell adhesion

    PubMed Central

    1993-01-01

    The carbohydrate determinants recognized by the HNK-1 antibody are potential cell-cell recognition ligands in the peripheral nervous system (PNS). The HNK-1 reactive sulfoglucuronylneolacto (SGNL) glycolipids specifically support Schwann cell adhesion, suggesting the presence of a cell surface receptor specific for SGNL-oligosaccharides. We directly probed PNS membranes for receptors complementary to SGNL determinants using a synthetic radioligand consisting of radioiodinated serum albumin derivatized with multiple SGNL-oligosaccharides. A high- affinity, saturable, calcium-dependent binding site for this ligand was found in PNS myelin membranes. Binding activity was carbohydrate- specific (most potently inhibited by SGNL-lipids compared to other glycolipids) and PNS-specific (absent from comparable central nervous system membranes). The SGNL-specific binding activity on PNS membranes reported here may be involved in peripheral myelination or myelin stabilization. PMID:8468354

  7. Secretin: specific binding to rat brain membranes

    SciTech Connect

    Fremeau, R.T. Jr.; Jensen, R.T.; Charlton, C.G.; Miller, R.L.; O'Donohue, T.L.; Moody, T.W.

    1983-08-01

    The binding of (/sup 125/I)secretin to rat brain membranes was investigated. Radiolabeled secretin bound with high affinity (KD . 0.2 nM) to a single class of noninteracting sites. Binding was specific, saturable, and reversible. Regional distribution studies indicated that the specific binding was greatest in the cerebellum, intermediate in the cortex, thalamus, striatum, hippocampus, and hypothalamus, and lowest in the midbrain and medulla/pons. Pharmacological studies indicated that only secretin, but not other peptides, inhibits binding of (/sup 125/I)secretin with high affinity. Also, certain guanine nucleotides inhibited high affinity binding. These data indicate that rat brain membranes possess high affinity binding sites specific for secretin and that with the use of (/sup 125/I) secretin the kinetics, stoichiometry, specificity, and distribution of secretin receptors can be directly investigated.

  8. Fungal Hybrid B heme peroxidases - unique fusions of a heme peroxidase domain with a carbohydrate-binding domain.

    PubMed

    Zámocký, Marcel; Janeček, Štefan; Obinger, Christian

    2017-08-24

    Heme peroxidases, essential peroxide converting oxidoreductases are divided into four independently evolved superfamilies. Within the largest one - the peroxidase-catalase superfamily - two hybrid lineages were described recently. Whereas Hybrid A heme peroxidases represent intermediate enzymes between ascorbate peroxidases and cytochrome c peroxidases, Hybrid B heme peroxidases are unique fusion proteins comprised of a conserved N-terminal heme peroxidase domain and a C-terminal domain of various sugar binding motifs. So far these peculiar peroxidases are only found in the kingdom of Fungi. Here we present a phylogenetic reconstruction of the whole superfamily with focus on Hybrid B peroxidases. We analyse the domain assembly and putative structure and function of the newly discovered oligosaccharide binding domains. Two distinct carbohydrate binding modules (CBM21 and CBM34) are shown to occur in phytopathogenic ascomycetous orthologs of Hybrid B heme peroxidases only. Based on multiple sequence alignment and homology modeling the structure-function relationships are discussed with respect to physiological function. A concerted action of peroxide cleavage with specific cell-wall carbohydrate binding can support phytopathogens survival within the plant host.

  9. Specific intermolecular interactions of conserved water molecules with amino acids in the Galectin-1 carbohydrate recognition domain

    NASA Astrophysics Data System (ADS)

    Di Lella, Santiago; Petruk, Ariel A.; Armiño, Diego J. Alonso de; Álvarez, Rosa M. S.

    2010-08-01

    Water molecules, rigidly associated to protein surfaces, play a key role in stabilizing biomolecules and participating in their biological functions. Recent studies on the solvation properties of the carbohydrate recognition domain of Galectin-1 by means of molecular dynamic simulations have revealed the existence of several water sites which were well correlated to both the bound water molecules observed in the crystal structure of the protein in the free state and to some of the hydroxyl groups of the carbohydrate ligand observed in the crystal structure of the complexed protein. In this work, we present a study using quantum mechanical methods (B3LYP/6-311++G(3df,3dp)//B3LYP/6-31+G(d)) to determine the energy involved in the binding of these water molecules to specific amino acids in the carbohydrate recognition domain of the protein. By modeling the hydroxyl groups of the carbohydrate by methanol, the energies associated to the local interactions between the ligand and the protein have been evaluated by replacing specific water molecules with methanol. The values of the binding energies have been compared to those previously obtained by the molecular dynamic method.

  10. Molecular Simulations of Carbohydrates with a Fucose-Binding Burkholderia ambifaria Lectin Suggest Modulation by Surface Residues Outside the Fucose-Binding Pocket

    PubMed Central

    Dingjan, Tamir; Imberty, Anne; Pérez, Serge; Yuriev, Elizabeth; Ramsland, Paul A.

    2017-01-01

    Burkholderia ambifaria is an opportunistic respiratory pathogen belonging to the Burkholderia cepacia complex, a collection of species responsible for the rapidly fatal cepacia syndrome in cystic fibrosis patients. A fucose-binding lectin identified in the B. ambifaria genome, BambL, is able to adhere to lung tissue, and may play a role in respiratory infection. X-ray crystallography has revealed the bound complex structures for four fucosylated human blood group epitopes (blood group B, H type 1, H type 2, and Lex determinants). The present study employed computational approaches, including docking and molecular dynamics (MD), to extend the structural analysis of BambL-oligosaccharide complexes to include four additional blood group saccharides (A, Lea, Leb, and Ley) and a library of blood-group-related carbohydrates. Carbohydrate recognition is dominated by interactions with fucose via a hydrogen-bonding network involving Arg15, Glu26, Ala38, and Trp79 and a stacking interaction with Trp74. Additional hydrogen bonds to non-fucose residues are formed with Asp30, Tyr35, Thr36, and Trp74. BambL recognition is dominated by interactions with fucose, but also features interactions with other parts of the ligands that may modulate specificity or affinity. The detailed computational characterization of the BambL carbohydrate-binding site provides guidelines for the future design of lectin inhibitors. PMID:28680402

  11. The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases*

    PubMed Central

    Crouch, Lucy I.; Labourel, Aurore; Walton, Paul H.; Davies, Gideon J.; Gilbert, Harry J.

    2016-01-01

    Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases, CfLPMO10 and TbLPMO10 from Cellulomonas fimi and Thermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducing CtCBM3a, from the Clostridium thermocellum cellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact of CtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM from CfLPMO10 or the introduction of a family 10 CBM from Cellvibrio japonicus LPMO10B into TbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations. PMID:26801613

  12. The Active Site of a Carbohydrate Esterase Displays Divergent Catalytic and Noncatalytic Binding Functions

    PubMed Central

    Pires, Virginia M. R; Flint, James E; Pinheiro, Benedita A; Goyal, Arun; Prates, José A. M; Izumi, Atsushi; Stålbrand, Henrik; Morland, Carl; Cartmell, Alan; Kolenova, Katarina; Topakas, Evangelos; Dodson, Eleanor J; Bolam, David N; Davies, Gideon J; Fontes, Carlos M. G. A; Gilbert, Harry J

    2009-01-01

    Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of “gene sharing

  13. Binding Sites for Acylated Trehalose Analogs of Glycolipid Ligands on an Extended Carbohydrate Recognition Domain of the Macrophage Receptor Mincle*

    PubMed Central

    Feinberg, Hadar; Rambaruth, Neela D. S.; Jégouzo, Sabine A. F.; Jacobsen, Kristian M.; Djurhuus, Rasmus; Poulsen, Thomas B.; Weis, William I.; Taylor, Maureen E.; Drickamer, Kurt

    2016-01-01

    The macrophage receptor mincle binds to trehalose dimycolate on the surface of Mycobacterium tuberculosis. Signaling initiated by this interaction leads to cytokine production, which underlies the ability of mycobacteria to evade the immune system and also to function as adjuvants. In previous work the mechanism for binding of the sugar headgroup of trehalose dimycolate to mincle has been elucidated, but the basis for enhanced binding to glycolipid ligands, in which hydrophobic substituents are attached to the 6-hydroxyl groups, has been the subject of speculation. In the work reported here, the interaction of trehalose derivatives with bovine mincle has been probed with a series of synthetic mimics of trehalose dimycolate in binding assays, in structural studies by x-ray crystallography, and by site-directed mutagenesis. Binding studies reveal that, rather than reflecting specific structural preference, the apparent affinity of mincle for ligands with hydrophobic substituents correlates with their overall size. Structural and mutagenesis analysis provides evidence for interaction of the hydrophobic substituents with multiple different portions of the surface of mincle and confirms the presence of three Ca2+-binding sites. The structure of an extended portion of the extracellular domain of mincle, beyond the minimal C-type carbohydrate recognition domain, also constrains the way the binding domains may interact on the surface of macrophages. PMID:27542410

  14. The primary structure of the Cytisus scoparius seed lectin and a carbohydrate-binding peptide.

    PubMed

    Konami, Y; Yamamoto, K; Osawa, T; Irimura, T

    1992-09-01

    The complete amino acid sequence of 2-acetamido-2-deoxy-D-galactose-binding Cytisus scoparius seed lectin II (CSII) was determined using a protein sequencer. After digestion of CSII with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSII with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid residues of concanavalin A (Con A) involved in the metal binding site are highly conserved among those of CSII. A carbohydrate-binding peptide of CSII was obtained from the endoproteinase Asp-N digest of CSII by affinity chromatography on a column of GalNAc-Gel. This peptide was retained on the GalNAc-Gel column and was presumed to have affinity for the column. The amino acid sequence of the retarded peptide was determined using a protein sequencer. The retarded peptide was found to correspond to the putative metal-binding region of Con A. These results strongly suggest that this peptide represents the carbohydrate-binding and metal ion-binding sites of CSII.

  15. Prediction of carbohydrate-binding proteins from sequences using support vector machines.

    PubMed

    Someya, Seizi; Kakuta, Masanori; Morita, Mizuki; Sumikoshi, Kazuya; Cao, Wei; Ge, Zhenyi; Hirose, Osamu; Nakamura, Shugo; Terada, Tohru; Shimizu, Kentaro

    2010-01-01

    Carbohydrate-binding proteins are proteins that can interact with sugar chains but do not modify them. They are involved in many physiological functions, and we have developed a method for predicting them from their amino acid sequences. Our method is based on support vector machines (SVMs). We first clarified the definition of carbohydrate-binding proteins and then constructed positive and negative datasets with which the SVMs were trained. By applying the leave-one-out test to these datasets, our method delivered 0.92 of the area under the receiver operating characteristic (ROC) curve. We also examined two amino acid grouping methods that enable effective learning of sequence patterns and evaluated the performance of these methods. When we applied our method in combination with the homology-based prediction method to the annotated human genome database, H-invDB, we found that the true positive rate of prediction was improved.

  16. Hydration Repulsion between Carbohydrate Surfaces Mediated by Temperature and Specific Ions

    NASA Astrophysics Data System (ADS)

    Chen, Hsieh; Cox, Jason R.; Ow, Hooisweng; Shi, Rena; Panagiotopoulos, Athanassios Z.

    2016-06-01

    Stabilizing colloids or nanoparticles in solution involves a fine balance between surface charges, steric repulsion of coating molecules, and hydration forces against van der Waals attractions. At high temperature and electrolyte concentrations, the colloidal stability of suspensions usually decreases rapidly. Here, we report a new experimental and simulation discovery that the polysaccharide (dextran) coated nanoparticles show ion-specific colloidal stability at high temperature, where we observed enhanced colloidal stability of nanoparticles in CaCl2 solution but rapid nanoparticle-nanoparticle aggregation in MgCl2 solution. The microscopic mechanism was unveiled in atomistic simulations. The presence of surface bound Ca2+ ions increases the carbohydrate hydration and induces strongly polarized repulsive water structures beyond at least three hydration shells which is farther-reaching than previously assumed. We believe leveraging the binding of strongly hydrated ions to macromolecular surfaces represents a new paradigm in achieving absolute hydration and colloidal stability for a variety of materials, particularly under extreme conditions.

  17. Characterization of the carbohydrate binding and ADP-ribosyltransferase activities of chemically detoxified pertussis toxins.

    PubMed

    Oh, Hokyung; Kim, Byoung-Guk; Nam, Kyung-Tak; Hong, Seung-Hwa; Ahn, Dong-Ho; Choi, Gi-Sub; Kim, Hyungjin; Hong, Jin-Tae; Ahn, Byung-Yoon

    2013-06-24

    Pertussis toxin (PTx) is an essential component of the acellular pertussis (aP) vaccine. However, because PTx in its native form is considered too toxic for human vaccine use, it must be inactivated into a stable, nontoxic form by treatment with chemical detoxifying agents or by genetic modification. Therefore, testing for the residual PTx in the aP vaccine is a major quality control step for vaccine manufacturers and regulatory authorities. The histamine sensitization test is currently the standard safety test method for all aP vaccines, regardless of the vaccine formula or the detoxification process, except for those with genetically modified PTx. However, test result variability and ethical concerns regarding animal use necessitate an alternative method. In vitro assays based on the biochemical properties of PTx have been considered as potential alternatives to the histamine sensitization test. In this study, the suitability of assays based on the ADP-ribosyltransferase and carbohydrate binding activities of PTx was assessed for PTx after treatment with formaldehyde, glutaraldehyde or both denaturants in sequence. The results indicated a distinctive pattern of the biochemical activities depending on the detoxification methods and storage conditions. These results suggest that although a more careful study is needed, these in vitro biochemical assays can be considered potential alternatives to the histamine sensitization test, as they might provide more specific safety information of aP vaccines. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Specific gonadotropin binding to Pseudomonas maltophilia.

    PubMed

    Richert, N D; Ryan, R J

    1977-03-01

    Binding of 125I-labeled human chorionic gonadotropin to Pseudomonas maltophilia is dependent on time, temperature, and pH and the binding to this procaryotic species is hormone-specific and saturable. The equilibrium dissociation constant is 2.3 X 10(-9) M. There are no cooperative interactions between binding sites (Hill coefficient, 1.05). The number of sites is estimaated as 240 fmol/100 mug of protein. NaCl and KCl, at concentrations from 1 to 10 mM, have no effect on binding. Divalent cations (Mg2+ and Ca2+) and 1 mM EDTA inhibit hormone binding. Binding is destroyed by heat or by treatment with Pronase of alpha-chymotrypsin and is increased by phospholipase C. Binding of the labeled gonadotropin is not observed with other gram-negative organisms--e.g., Escherichia coli, Pseudomonas testosteroni, Pseudomonas aeruginosa, Enterobacter aerogenes, or Enterobacter cloacae.

  19. Borrelia burgdorferi shows specificity of binding to glycosphingolipids.

    PubMed Central

    Backenson, P B; Coleman, J L; Benach, J L

    1995-01-01

    Live but not fixed or heat-killed Borrelia burgdorferi bound to galactocerebroside, lactosylceramide, and ceramide trihexoside. In addition, this organism bound to the disialoganglioside GD1a and the trisialoganglioside GT1b but not to gangliosides GM1, GD1b, GM2, and GM3 and not to asialo GM1. This adhesion pattern confirmed earlier findings of binding to galactocerebroside and places this organism within a prokaryotic group which binds to lactosylceramide. The binding to GD1a and GT1b, both of which carry terminal as well as multiple sialic acids, indicates that B. burgdorferi can show specificity of binding within a group of acidic gangliosides. Adhesion could not be inhibited by several concentrations of sugars and sialic acid, indicating more complex binding requirements than for terminal carbohydrates alone. Low-passage strains adhered to the four substrates in greater numbers than strains in culture for long periods of time. OspB mutants in general bound better or at least equally well to several of the glycosphingolipids, and preincubation of substrates with soluble recombinant and affinity-purified Osp did not inhibitor or weakly inhibited the binding of the organisms. These findings suggest that outer surface lipoproteins A and B are not directly involved in adhesion to glycosphingolipids. PMID:7622201

  20. Relationship between dietary carbohydrates intake and circulating sex hormone-binding globulin levels in postmenopausal women.

    PubMed

    Huang, Mengna; Liu, Jinjie; Lin, Xiaochen; Goto, Atsushi; Song, Yiqing; Tinker, Lesley F; Chan, Kei-Hang Katie; Liu, Simin

    2017-03-17

    Low circulating levels of sex hormone-binding globulin (SHBG) have been shown to be a direct and strong risk factor for type 2 diabetes, cardiovascular diseases, and hormone-dependent cancers, although the relationship between various aspects of dietary carbohydrates and SHBG levels remains unexplored in population studies. Among postmenopausal women with available SHBG measurements at baseline (n = 11 159) in the Women's Health Initiative, a comprehensive assessment was conducted of total dietary carbohydrates, glycemic load (GL), glycemic index (GI), fiber, sugar, and various carbohydrate-abundant foods in relation to circulating SHBG levels using multiple linear regressions adjusting for potential covariates. Linear trend was tested across quartiles of dietary variables. Benjamini and Hochberg's procedure was used to calculate the false discovery rate for multiple comparisons. Higher dietary GL and GI (both based on total and available carbohydrates) and a higher intake of sugar and sugar-sweetened beverages were associated with lower circulating SHBG concentrations (all P trend  < 0.05; Q -values = 0.04,0.01, 0.07, 0.10, 0.01, and <0.0001, respectively). In contrast, women with a greater intake of dietary fiber tended to have elevated SHBG levels (P trend  = 0.01, Q -value = 0.04). There was no significant association between total carbohydrates or other carbohydrate-abundant foods and SHBG concentrations. The findings suggest that low GL or GI diets with low sugar and high fiber content may be associated with higher serum SHBG concentrations among postmenopausal women. Future studies investigating whether lower GL or GI diets increase SHBG concentrations are warranted. © 2017 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

  1. Cell surface enzyme attachment is mediated by family 37 carbohydrate-binding modules, unique to Ruminococcus albus.

    PubMed

    Ezer, Anat; Matalon, Erez; Jindou, Sadanari; Borovok, Ilya; Atamna, Nof; Yu, Zhongtang; Morrison, Mark; Bayer, Edward A; Lamed, Raphael

    2008-12-01

    The rumen bacterium Ruminococcus albus binds to and degrades crystalline cellulosic substrates via a unique cellulose degradation system. A unique family of carbohydrate-binding modules (CBM37), located at the C terminus of different glycoside hydrolases, appears to be responsible both for anchoring these enzymes to the bacterial cell surface and for substrate binding.

  2. Cell Surface Enzyme Attachment Is Mediated by Family 37 Carbohydrate-Binding Modules, Unique to Ruminococcus albus▿ ‡

    PubMed Central

    Ezer, Anat; Matalon, Erez; Jindou, Sadanari; Borovok, Ilya; Atamna, Nof; Yu, Zhongtang; Morrison, Mark; Bayer, Edward A.; Lamed, Raphael

    2008-01-01

    The rumen bacterium Ruminococcus albus binds to and degrades crystalline cellulosic substrates via a unique cellulose degradation system. A unique family of carbohydrate-binding modules (CBM37), located at the C terminus of different glycoside hydrolases, appears to be responsible both for anchoring these enzymes to the bacterial cell surface and for substrate binding. PMID:18931104

  3. Influence of glycosidic linkage on the nature of carbohydrate binding in beta-prism I fold lectins: an X-ray and molecular dynamics investigation on banana lectin-carbohydrate complexes.

    PubMed

    Sharma, Alok; Vijayan, Mamannamana

    2011-01-01

    The three crystal structures reported here provide details of the interactions of mannose and the mannosyl-α-1,3-mannose component of a pentamannose with banana lectin and evidence for the binding of glucosyl-α-1,2-glucose to the lectin. The known structures involving the lectin include a complex with glucosyl-β-1,3-glucose. Modeling studies on the three disaccharide complexes with the reducing end and the nonreducing end at the primary binding site are also provided here. The results of the X-ray and modeling studies show that the disaccharides with an α-1,3 linkage prefer to have the nonreducing end at the primary binding site, whereas the reducing end is preferred at the site when the linkage is β-1,3 in mannose/glucose-specific β-prism I fold lectins. In the corresponding galactose-specific lectins, however, α-1,3-linked disaccharides cannot bind the lectin with the nonreducing end at the primary binding site on account of steric clashes with an aromatic residue that occurs only when the lectin is galactose-specific. Molecular dynamics simulations based on the known structures involving banana lectin enrich the information on lectin-carbohydrate interactions obtained from crystal structures. They demonstrate that conformational selection as well as induced fit operate when carbohydrates bind to banana lectin.

  4. Stability and Sugar Recognition Ability of Ricin-Like Carbohydrate Binding Domains

    SciTech Connect

    Yao, Jianzhuang; Nellas, Ricky B; Glover, Mary M; Shen, Tongye

    2011-01-01

    Lectins are a class of proteins known for their novel binding to saccharides. Understanding this sugar recognition process can be crucial in creating structure-based designs of proteins with various biological roles. We focus on the sugar binding of a particular lectin, ricin, which has two -trefoil carbohydrate-binding domains (CRDs) found in several plant protein toxins. The binding ability of possible sites of ricin-like CRD has been puzzling. The apo and various (multiple) ligand-bound forms of the sugar-binding domains of ricin were studied by molecular dynamics simulations. By evaluating structural stability, hydrogen bond dynamics, flexibility, and binding energy, we obtained a detailed picture of the sugar recognition of the ricin-like CRD. Unlike what was previously believed, we found that the binding abilities of the two known sites are not independent of each other. The binding ability of one site is positively affected by the other site. While the mean positions of different binding scenarios are not altered significantly, the flexibility of the binding pockets visibly decreases upon multiple ligand binding. This change in flexibility seems to be the origin of the binding cooperativity. All the hydrogen bonds that are strong in the monoligand state are also strong in the double-ligand complex, although the stability is much higher in the latter form due to cooperativity. These strong hydrogen bonds in a monoligand state are deemed to be the essential hydrogen bonds. Furthermore, by examining the structural correlation matrix, the two domains are structurally one entity. Galactose hydroxyl groups, OH4 and OH3, are the most critical parts in both site 1 and site 2 recognition.

  5. Mutational analysis of the pumpkin (Cucurbita maxima) phloem exudate lectin, PP2 reveals Ser-104 is crucial for carbohydrate binding.

    PubMed

    Bobbili, Kishore Babu; Bandari, Shyam; Grobe, Kay; Swamy, Musti J

    2014-07-18

    The pumpkin phloem lectin (PP2) is an RNA-binding, defense-related, chitooligosaccharide-specific, homodimeric lectin of Mr 48 kDa expressed at high concentrations in the sieve elements and companion cells of pumpkin (Cucurbita maxima). In the present study, PP2 was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway as a prerequisite for unimpaired folding and posttranslational glycosylation of recombinant PP2. Previous computational modeling and ligand docking studies predicted a putative chitooligosaccharide-binding site on the PP2 surface, which was divided into three subsites, with two amino acid residues in each subsite identified as possible candidates for interaction with chitooligosaccharides (CHOs). In this work, mutational analysis and hemagglutination assays were employed to verify the role of the predicted residues in the carbohydrate binding activity of the protein. The results obtained revealed that mutation of Ser-104 to Ala (S104A) at subsite-2 resulted in about 90% loss of agglutination activity of the protein, indicating that Ser-104 is crucial for the binding of CHOs to PP2. Also, L100A (at subsite-1) and K200A (at subsite-3) independently decreased the lectin activity by about 40%, indicating that these two residues also contribute significantly to sugar binding by PP2. Together, these findings confirm that all the three subsites contribute to varying degrees toward PP2-carbohydrate interaction, and confirm the validity of the computational model, as proposed earlier. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Analysis of the carbohydrate-binding-module from Fragaria x ananassa α-L-arabinofuranosidase 1.

    PubMed

    Sin, I N; Perini, M A; Martínez, G A; Civello, P M

    2016-10-01

    α-L-arabinofuranosidases (EC 3.2.1.55) are enzymes involved in the catabolism of several cell-wall polysaccharides such as pectins and hemicelluloses, catalyzing the hydrolysis of terminal non-reducing α-L-arabinofuranosil residues. Bioinformatic analysis of the aminoacidic sequences of Fragaria x ananassa α-L-arabinofuranosidases predict a putative carbohydrate-binding-module of the family CBM_4_9, associated to a wide range of carbohydrate affinities. In this study, we report the characterization of the binding affinity profile to different cell wall polysaccharides of the putative CBM of α-L-arabinofuranosidase 1 from Fragaria x ananassa (CBM-FaARA1). The sequence encoding for the putative CBM was cloned and expressed in Escherichia coli, and the resultant recombinant protein was purified from inclusion bodies by a Nickel affinity chromatography under denaturing conditions. The refolded recombinant protein was then subjected to binding assays and affinity gel electrophoresis, which indicated its ability to bind cellulose and also high affinity for homogalacturonans.

  7. Specific binding of GM1-binding peptides to high-density GM1 in lipid membranes.

    PubMed

    Matsubara, Teruhiko; Iijima, Kazutoshi; Nakamura, Miwa; Taki, Takao; Okahata, Yoshio; Sato, Toshinori

    2007-01-16

    The ganglioside Galbeta1-3GalNAcbeta1-4(Neu5Acalpha2-3)Galbeta1-4Glcbeta1-1'Cer (GM1) is an important receptor. We have previously identified GM1-binding peptides based on affinity selection from a random peptide library. In the present study, we determined the amino acids essential for binding GM1 and investigated the specific interaction with GM1 in the lipid membrane. Arginines and aromatic amino acids in the consensus sequence (W/F)RxL(xP/Px)xFxx(Rx/xR)xP contributed to the ability of the peptides to bind GM1. The peptide p3, VWRLLAPPFSNRLLP, having the consensus sequence, showed high affinity for GM1 with a dissociation constant of 1.2 microM. Furthermore, the density-dependent binding of p3 was investigated using mixed monolayers of GM1 and Glcbeta1-1'Cer (GlcCer). p3 binds preferentially to high-density GM1, and its interaction with GM1 was found to be cooperative based on a Hill plot. These results indicated that a lateral assembly of GM1 molecules was required for the recognition of carbohydrates by p3. The GM1-binding peptide played a role as a unique anti-GM1 probe differing from the cholera toxin B subunit or antibodies.

  8. Dual Specificity of Langerin to Sulfated and Mannosylated Glycans via a Single C-type Carbohydrate Recognition Domain*

    PubMed Central

    Tateno, Hiroaki; Ohnishi, Koji; Yabe, Rikio; Hayatsu, Norihito; Sato, Takashi; Takeya, Motohiro; Narimatsu, Hisashi; Hirabayashi, Jun

    2010-01-01

    Langerin is categorized as a C-type lectin selectively expressed in Langerhans cells, playing roles in the first line of defense against pathogens and in Birbeck granule formation. Although these functions are thought to be exerted through glycan-binding activity of the C-type carbohydrate recognition domain, sugar-binding properties of Langerin have not been fully elucidated in relation to its biological functions. Here, we investigated the glycan-binding specificity of Langerin using comprehensive glycoconjugate microarray, quantitative frontal affinity chromatography, and conventional cell biological analyses. Langerin showed outstanding affinity to galactose-6-sulfated oligosaccharides, including keratan sulfate, while it preserved binding activity to mannose, as a common feature of the C-type lectins with an EPN motif. By a mutagenesis study, Lys-299 and Lys-313 were found to form extended binding sites for sulfated glycans. Consistent with the former observation, the sulfated Langerin ligands were found to be expressed in brain and spleen, where the transcript of keratan sulfate 6-O-sulfotransferase is expressed. Moreover, such sulfated ligands were up-regulated in glioblastoma relative to normal brain tissues, and Langerin-expressing cells were localized in malignant brain tissues. Langerin also recognized pathogenic fungi, such as Candida and Malassezia, expressing heavily mannosylated glycans. These observations provide strong evidence that Langerin mediates diverse functions on Langerhans cells through dual recognition of sulfated as well as mannosylated glycans by its uniquely evolved C-type carbohydrate-recognition domain. PMID:20026605

  9. Structural Insight of a Trimodular Halophilic Cellulase with a Family 46 Carbohydrate-Binding Module

    PubMed Central

    Yao, Chaoxiang; Junaid, Muhammad; Lu, Zhenghui; Zhang, Houjin; Ma, Yanhe

    2015-01-01

    Cellulases are the key enzymes used in the biofuel industry. A typical cellulase contains a catalytic domain connected to a carbohydrate-binding module (CBM) through a flexible linker. Here we report the structure of an atypical trimodular cellulase which harbors a catalytic domain, a CBM46 domain and a rigid CBM_X domain between them. The catalytic domain shows the features of GH5 family, while the CBM46 domain has a sandwich-like structure. The catalytic domain and the CBM46 domain form an extended substrate binding cleft, within which several tryptophan residues are well exposed. Mutagenesis assays indicate that these residues are essential for the enzymatic activities. Gel affinity electrophoresis shows that these tryptophan residues are involved in the polysaccharide substrate binding. Also, electrostatic potential analysis indicates that almost the entire solvent accessible surface of CelB is negatively charged, which is consistent with the halophilic nature of this enzyme. PMID:26562160

  10. Structures of parasite calreticulins provide insights into their flexibility and dual carbohydrate/peptide-binding properties

    PubMed Central

    Moreau, Christophe; Cioci, Gianluca; Iannello, Marina; Laffly, Emmanuelle; Chouquet, Anne; Ferreira, Arturo; Thielens, Nicole M.; Gaboriaud, Christine

    2016-01-01

    Calreticulin (CRT) is a multifaceted protein, initially discovered as an endoplasmic reticulum (ER) chaperone protein, that is essential in calcium metabolism. Various implications in cancer, early development and immunology have been discovered more recently for CRT, as well as its role as a dominant ‘eat-me’ prophagocytic signal. Intriguingly, cell-surface exposure/secretion of CRT is among the infective strategies used by parasites such as Trypanosoma cruzi, Entamoeba histolytica, Taenia solium, Leishmania donovani and Schistosoma mansoni. Because of the inherent flexibility of CRTs, their analysis by X-ray crystallography requires the design of recombinant constructs suitable for crystallization, and thus only the structures of two very similar mammalian CRT lectin domains are known. With the X-ray structures of two distant parasite CRTs, insights into species structural determinants that might be harnessed to fight against the parasites without affecting the functions of the host CRT are now provided. Moreover, although the hypothesis that CRT can exhibit both open and closed conformations has been proposed in relation to its chaperone function, only the open conformation has so far been observed in crystal structures. The first evidence is now provided of a complex conformational transition with the junction reoriented towards P-domain closure. SAXS experiments also provided additional information about the flexibility of T. cruzi CRT in solution, thus complementing crystallographic data on the open conformation. Finally, regarding the conserved lectin-domain structure and chaperone function, evidence is provided of its dual carbohydrate/protein specificity and a new scheme is proposed to interpret such unusual substrate-binding properties. These fascinating features are fully consistent with previous experimental observations, as discussed considering the broad spectrum of CRT sequence conservations and differences. PMID:27840680

  11. Predicting tissue specific transcription factor binding sites

    PubMed Central

    2013-01-01

    Background Studies of gene regulation often utilize genome-wide predictions of transcription factor (TF) binding sites. Most existing prediction methods are based on sequence information alone, ignoring biological contexts such as developmental stages and tissue types. Experimental methods to study in vivo binding, including ChIP-chip and ChIP-seq, can only study one transcription factor in a single cell type and under a specific condition in each experiment, and therefore cannot scale to determine the full set of regulatory interactions in mammalian transcriptional regulatory networks. Results We developed a new computational approach, PIPES, for predicting tissue-specific TF binding. PIPES integrates in vitro protein binding microarrays (PBMs), sequence conservation and tissue-specific epigenetic (DNase I hypersensitivity) information. We demonstrate that PIPES improves over existing methods on distinguishing between in vivo bound and unbound sequences using ChIP-seq data for 11 mouse TFs. In addition, our predictions are in good agreement with current knowledge of tissue-specific TF regulation. Conclusions We provide a systematic map of computationally predicted tissue-specific binding targets for 284 mouse TFs across 55 tissue/cell types. Such comprehensive resource is useful for researchers studying gene regulation. PMID:24238150

  12. Multiplicity of carbohydrate-binding sites in beta-prism fold lectins: occurrence and possible evolutionary implications.

    PubMed

    Sharma, Alok; Chandran, Divya; Singh, Desh D; Vijayan, M

    2007-09-01

    The beta-prism II fold lectins of known structure, all from monocots, invariably have three carbohydrate-binding sites in each subunit/domain. Until recently, beta-prism I fold lectins of known structure were all from dicots and they exhibited one carbohydrate-binding site per subunit/domain. However, the recently determined structure of the beta-prism fold I lectin from banana, a monocot, has two very similar carbohydrate-binding sites. This prompted a detailed analysis of all the sequences appropriate for two-lectin folds and which carry one or more relevant carbohydrate-binding motifs. The very recent observation of a beta-prism I fold lectin, griffthsin, with three binding sites in each domain further confirmed the need for such an analysis. The analysis demonstrates substantial diversity in the number of binding sites unrelated to the taxonomical position of the plant source. However, the number of binding sites and the symmetry within the sequence exhibit reasonable correlation. The distribution of the two families of beta-prism fold lectins among plants and the number of binding sites in them, appear to suggest that both of them arose through successive gene duplication, fusion and divergent evolution of the same primitive carbohydrate-binding motif involving a Greek key. Analysis with sequences in individual Greek keys as independent units lends further support to this conclusion.It would seem that the preponderance of three carbohydrate-binding sites per domain in monocot lectins, particularly those with the beta-prism II fold, is related to the role of plant lectins in defence.

  13. Effects of a carbohydrate-electrolyte drink on specific soccer tests and performance.

    PubMed

    Ostojic, Sergej M; Mazic, Sanja

    2002-06-01

    The aim of this study was to examine the effects of a carbohydrate-electrolyte drink on specific soccer tests and performance. Twenty-two professional male soccer players volunteered to participate in the study. The players were allocated to two assigned trials ingesting carbohydrate-electrolyte drink (7% carbohydrates, sodium 24 mmol.l-1, chloride 12 mmol.l-1, potassium 3 mmol.l-1) or placebo during a 90 min on-field soccer match. The trials were matched for subjects' age, weight, height and maximal oxygen uptake. Immediately after the match, players completed four soccer-specific skill tests. Blood glucose concentration [mean (SD)] was higher at the end of the match-play in the carbohydrate-electrolyte trial than in the placebo trial (4.4 (0.3) vs. 4.0 (0.3) mmol.l-1, P < 0.05). Subjects in the carbohydrate-electrolyte trial finished the specific dribble test faster in comparison with subjects in the placebo trial (12.9 (0.4) vs. 13.6 (0.5) s, P < 0.05). Ratings of the precision test were higher in the carbohydrate-electrolyte trial as compared to the placebo trial (17.2 (4.8) vs. 15.1 (5.2), P < 0.05) but there were no differences in coordination test and power test results between trials. The main finding of the present study indicates that supplementation with carbohydrate-electrolyte solution improved soccer-specific skill performance and recovery after an on-field soccer match compared with ingestion of placebo. This suggests that soccer players should consume carbohydrate-electrolyte fluid throughout a game to help prevent deterioration in specific skill performance.

  14. Binding of human fibroblast interferon to concanavalin A-agarose. Involvement of carbohydrate recognition and hydrophobic interaction.

    PubMed

    Davey, M W; Sulkowski, E; Carter, W A

    1976-02-10

    Human fibroblast interferon binds to a concanavalin A-agarose (Con A-Sepharose) equilibrated with methyl alpha-D-mannopyranoside, or levan; in contrast, it is only partially retarded on a similar column equilibrated with ethylene glycol. Interferon does not bind, however, to a lectin column equilibrated with both methyl alpha-D-mannopyranoside and ethylene glycol. Thus, a hydrophobic interaction between fibroblast interferon and the immobilized lectin seems to account for a large portion of the binding forces involved. Other hydrophobic solutes, such as dioxane, 1, 2-propanediol, and tetraethylammonium chloride, were found equally or more efficient than ethylene glycol in displacing interferon from the lectin column. The elution pattern of interferon from a concanavalin A-agarose (Con A-Sepharose) column, at a constant ehtylene glycol concentration and with an increasing mannoside concentration, reveals the existence of four distinct interferon components. The selective adsorption to and elution from a concanavalin A-agarose (Con A-Sepharose) column resulted in about a 3000-fold purification of human fibroblast interferon and complete recovery of activity. The specific activity of the partially purified interferon preparation is about 5 X 10(7) units per mg of protein. The chromatographic behavior of human leukocyte interferon is remarkable in that it does not bind to concanavalin A-agarose at all indicating the absence of carbohydrate moieties recognizable by the lectin, or if present, their masked status. When concanavalin A was coupled to an agarose matrix (cyanogen bromide activated) at pH 8.0 and 6.0 human fibroblast interferon bound to both lectin-agarose adsorbents and could be recovered with methyl alpha-D-mannopyranoside. Concanavalin A, immobilized directly on agarose matrix at pH 8.0 and 6.0, thus displays only carbohydrate recognition toward interferon. By contrast, unless a hydrophobic solute was included in the solvent containing methyl mannoside

  15. Monoclonal antibodies, carbohydrate-binding modules, and the detection of polysaccharides in plant cell walls.

    PubMed

    Hervé, Cécile; Marcus, Susan E; Knox, J Paul

    2011-01-01

    Plant cell walls are diverse composites of complex polysaccharides. Molecular probes such as monoclonal antibodies (MABs) and carbohydrate-binding modules (CBMs) are important tools to detect and dissect cell wall structures in plant materials. We provide an account of methods that can be used to detect cell wall polysaccharide structures (epitopes) in plant materials and also describe treatments that can provide information on the masking of sets of polysaccharides that may prevent detection. These masking -phenomena may indicate potential interactions between sets of cell wall polysaccharides, and methods to uncover them are an important aspect of cell wall immunocytochemistry.

  16. Differential carbohydrate binding and cell surface glycosylation of human cancer cell lines.

    PubMed

    Arndt, Nadia X; Tiralongo, Joe; Madge, Paul D; von Itzstein, Mark; Day, Christopher J

    2011-09-01

    Currently there is only a modest level knowledge of the glycosylation status of immortalised cell lines that are commonly used in cancer biology as well as their binding affinities to different glycan structures. Through use of glycan and lectin microarray technology, this study has endeavoured to define the different bindings of cell surface carbohydrate structures to glycan-binding lectins. The screening of breast cancer MDA-MB435 cells, cervical cancer HeLa cells and colon cancer Caco-2, HCT116 and HCT116-FM6 cells was conducted to determine their differential bindings to a variety of glycan and lectin structures printed on the array slides. An inverse relationship between the number of glycan structures recognised and the variety of cell surface glycosylation was observed. Of the cell lines tested, it was found that four bound to sialylated structures in initial screening. Secondary screening in the presence of a neuraminidase inhibitor (4-deoxy-4-guanidino-Neu5Ac2en) significantly reduced sialic acid binding. The array technology has proven to be useful in determining the glycosylation signatures of various cell-lines as well as their glycan binding preferences. The findings of this study provide the groundwork for further investigation into the numerous glycan-lectin interactions that are exhibited by immortalised cell lines.

  17. GROUP-SPECIFIC CARBOHYDRATE OF GROUP C-VARIANT HEMOLYTIC STREPTOCOCCI

    PubMed Central

    Araujo, Paulo; Krause, Richard M.

    1963-01-01

    The trypsinized cell walls of Group C hemolytic streptococci are composed of the specific carbohydrate antigen and a mucopeptide matrix. Certain phage resistant strains of Group C streptococci, isolated from organisms which survive exposure to Group C bacteriophage also possess carbohydrate and mucopeptide fractions, but the carbohydrate gives a precipitin reaction with Group A-variant antiserum and not with Group C antiserum. These strains have been termed Group C-variant. The C-variant carbohydrate contains 86 per cent rhamnose and only 2 per cent galactosamine, and is thus chemically and immunologically similar to Group A-variant carbohydrate. The evidence suggests that the antigenic determinants of Groups A-variant and C-variant carbohydrates are rhamnose-rhamnose linkages. These results strongly support the hypothesis that the carbohydrates of Groups A and C streptococci are composed of a similar rhamnose moiety but that the determinant amino sugar terminal to the rhamnose-rhamnose linkages in the case of Group A is N-acetylglucosamine whereas that of Group C is N-acetylgalactosamine. PMID:14112265

  18. Molecular cloning and expression of chicken carbohydrate response element binding protein and Max-like protein X gene homologues

    USDA-ARS?s Scientific Manuscript database

    Carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c) are transcription factors that are known to be key regulators of glucose metabolism and lipid synthesis in mammals. Since ChREBP and its co-activator Max-like protein X (Mlx) have not ...

  19. Structural and Functional Insight into the Carbohydrate Receptor Binding of F4 Fimbriae-producing Enterotoxigenic Escherichia coli *

    PubMed Central

    Moonens, Kristof; Van den Broeck, Imke; De Kerpel, Maia; Deboeck, Francine; Raymaekers, Hanne; Remaut, Han; De Greve, Henri

    2015-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are important causes of intestinal disease in humans and lead to severe production losses in animal farming. A range of fimbrial adhesins in ETEC strains determines host and tissue tropism. ETEC strains expressing F4 fimbriae are associated with neonatal and post-weaning diarrhea in piglets. Three naturally occurring variants of F4 fimbriae (F4ab, F4ac, and F4ad) exist that differ in the primary sequence of their major adhesive subunit FaeG, and each features a related yet distinct receptor binding profile. Here the x-ray structure of FaeGad bound to lactose provides the first structural insight into the receptor specificity and mode of binding by the poly-adhesive F4 fimbriae. A small D′-D″-α1-α2 subdomain grafted on the immunoglobulin-like core of FaeG hosts the carbohydrate binding site. Two short amino acid stretches Phe150–Glu152 and Val166–Glu170 of FaeGad bind the terminal galactose in the lactosyl unit and provide affinity and specificity to the interaction. A hemagglutination-based assay with E. coli expressing mutant F4ad fimbriae confirmed the elucidated co-complex structure. Interestingly, the crucial D′-α1 loop that borders the FaeGad binding site adopts a different conformation in the two other FaeG variants and hints at a heterogeneous binding pocket among the FaeG serotypes. PMID:25631050

  20. Carbohydrate Recognition Specificity of Trans-sialidase Lectin Domain from Trypanosoma congolense

    PubMed Central

    Waespy, Mario; Gbem, Thaddeus T.; Elenschneider, Leroy; Jeck, André-Philippe; Day, Christopher J.; Hartley-Tassell, Lauren; Bovin, Nicolai; Tiralongo, Joe; Haselhorst, Thomas; Kelm, Sørge

    2015-01-01

    Fourteen different active Trypanosoma congolense trans-sialidases (TconTS), 11 variants of TconTS1 besides TconTS2, TconTS3 and TconTS4, have been described. Notably, the specific transfer and sialidase activities of these TconTS differ by orders of magnitude. Surprisingly, phylogenetic analysis of the catalytic domains (CD) grouped each of the highly active TconTS together with the less active enzymes. In contrast, when aligning lectin-like domains (LD), the highly active TconTS grouped together, leading to the hypothesis that the LD of TconTS modulates its enzymatic activity. So far, little is known about the function and ligand specificity of these LDs. To explore their carbohydrate-binding potential, glycan array analysis was performed on the LD of TconTS1, TconTS2, TconTS3 and TconTS4. In addition, Saturation Transfer Difference (STD) NMR experiments were done on TconTS2-LD for a more detailed analysis of its lectin activity. Several mannose-containing oligosaccharides, such as mannobiose, mannotriose and higher mannosylated glycans, as well as Gal, GalNAc and LacNAc containing oligosaccharides were confirmed as binding partners of TconTS1-LD and TconTS2-LD. Interestingly, terminal mannose residues are not acceptor substrates for TconTS activity. This indicates a different, yet unknown biological function for TconTS-LD, including specific interactions with oligomannose-containing glycans on glycoproteins and GPI anchors found on the surface of the parasite, including the TconTS itself. Experimental evidence for such a scenario is presented. PMID:26474304

  1. Evidence for Conformational Mechanism on the Binding of TgMIC4 with β-Galactose-Containing Carbohydrate Ligand.

    PubMed

    Santos, Adriano; Carvalho, Fernanda C; Roque-Barreira, Maria-Cristina; Zorzetto-Fernandes, André Luiz; Gimenez-Romero, David; Monzó, Isidro; Bueno, Paulo R

    2015-11-10

    A deeper understanding of the role of sialic/desialylated groups during TgMIC4-glycoproteins interactions has importance to better clarify the odd process of host cell invasion by members of the apicomplexan phylum. Within this context, we evaluated the interaction established by recombinant TgMIC4 (the whole molecule) with sialylated (bovine fetuin) and desialylated (asialofetuin) glycoproteins by using functionalized quartz crystal microbalance with dissipation monitoring (QCM-D). A suitable receptive surface containing recombinant TgMIC4 for monitoring β-galactose-containing carbohydrate ligand (limit of quantification ∼ 40 μM) was designed and used as biomolecular recognition platform to study the binding and conformational mechanisms of TgMIC4 during the interaction with glycoprotein containing (fetuin), or not, terminal sialic group (asialofetuin). It was inferred that the binding/interaction monitoring depends on the presence/absence of sialic groups in target protein and is possible to be differentiated through a slower binding kinetic step using QCM-D approach (which we are inferring to be thus associated with β-galactose ligand). This slower binding/interaction step is likely supposed (from mechanical energetic analysis obtained in QCM-D measurements) to be involved with Toxoplasma gondii (the causative agent of toxoplasmosis) parasitic invasion accompanied by ligand (galactose) induced binding conformational change (i.e., cell internalization process can be additionally dependent on structural conformational changes, controlled by the absence of sialic groups and to the specific binding with galactose), in addition to TgMIC4-glycoprotein solely recognition binding process.

  2. Extra tyrosine in the carbohydrate-binding module of Irpex lacteus Xyn10B enhances its cellulose-binding ability.

    PubMed

    Nishijima, Hiroto; Nozaki, Kouichi; Mizuno, Masahiro; Arai, Tsutomu; Amano, Yoshihiko

    2015-01-01

    The xylanase (Xyn10B) that strongly adsorbs on microcrystalline cellulose was isolated from Driselase. The Xyn10B contains a Carbohydrate-binding module family 1 (CBM1) (IrpCBMXyn10B) at N-terminus. The canonical essential aromatic residues required for cellulose binding were conserved in IrpCBMXyn10B; however, its adsorption ability was markedly higher than that typically observed for the CBM1 of an endoglucanase from Trametes hirsuta (ThCBMEG1). An analysis of the CBM-GFP fusion proteins revealed that the binding capacity to cellulose (7.8 μmol/g) and distribution coefficient (2.0 L/μmol) of IrpCBMXyn10B-GFP were twofold higher than those of ThCBMEG1-GFP (3.4 μmol/g and 1.2 L/μmol, respectively), used as a reference structure. Besides the canonical aromatic residues (W24-Y50-Y51) of typical CBM1-containing proteins, IrpCBMXyn10B had an additional aromatic residue (Y52). The mutation of Y52 to Ser (IrpCBMY52S-GFP) reduced these adsorption parameters to 4.4 μmol/g and 1.5 L/μmol, which were similar to those of ThCBMEG1-GFP. These results indicate that Y52 plays a crucial role in strong cellulose binding.

  3. Structural insights into the affinity of Cel7A carbohydrate-binding module for lignin.

    PubMed

    Strobel, Kathryn L; Pfeiffer, Katherine A; Blanch, Harvey W; Clark, Douglas S

    2015-09-11

    The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs.

  4. Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin*

    PubMed Central

    Strobel, Kathryn L.; Pfeiffer, Katherine A.; Blanch, Harvey W.; Clark, Douglas S.

    2015-01-01

    The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs. PMID:26209638

  5. Stability and Ligand Promiscuity of Type A Carbohydrate-binding Modules Are Illustrated by the Structure of Spirochaeta thermophila StCBM64C.

    PubMed

    Pires, Virgínia M R; Pereira, Pedro M M; Brás, Joana L A; Correia, Márcia; Cardoso, Vânia; Bule, Pedro; Alves, Victor D; Najmudin, Shabir; Venditto, Immacolata; Ferreira, Luís M A; Romão, Maria João; Carvalho, Ana Luísa; Fontes, Carlos M G A; Prazeres, Duarte Miguel

    2017-03-24

    Deconstruction of cellulose, the most abundant plant cell wall polysaccharide, requires the cooperative activity of a large repertoire of microbial enzymes. Modular cellulases contain non-catalytic type A carbohydrate-binding modules (CBMs) that specifically bind to the crystalline regions of cellulose, thus promoting enzyme efficacy through proximity and targeting effects. Although type A CBMs play a critical role in cellulose recycling, their mechanism of action remains poorly understood. Here we produced a library of recombinant CBMs representative of the known diversity of type A modules. The binding properties of 40 CBMs, in fusion with an N-terminal GFP domain, revealed that type A CBMs possess the ability to recognize different crystalline forms of cellulose and chitin over a wide range of temperatures, pH levels, and ionic strengths. A Spirochaeta thermophila CBM64, in particular, displayed plasticity in its capacity to bind both crystalline and soluble carbohydrates under a wide range of extreme conditions. The structure of S. thermophila StCBM64C revealed an untwisted, flat, carbohydrate-binding interface comprising the side chains of four tryptophan residues in a co-planar linear arrangement. Significantly, two highly conserved asparagine side chains, each one located between two tryptophan residues, are critical to insoluble and soluble glucan recognition but not to bind xyloglucan. Thus, CBM64 compact structure and its extended and versatile ligand interacting platform illustrate how type A CBMs target their appended plant cell wall-degrading enzymes to a diversity of recalcitrant carbohydrates under a wide range of environmental conditions.

  6. Fiber Type-Specific Satellite Cell Content in Cyclists Following Heavy Training with Carbohydrate and Carbohydrate-Protein Supplementation

    PubMed Central

    McKenzie, Alec I.; D'Lugos, Andrew C.; Saunders, Michael J.; Gworek, Keith D.; Luden, Nicholas D.

    2016-01-01

    The central purpose of this study was to evaluate the fiber type-specific satellite cell and myonuclear responses of endurance-trained cyclists to a block of intensified training, when supplementing with carbohydrate (CHO) vs. carbohydrate-protein (PRO). In a crossover design, endurance-trained cyclists (n = 8) performed two consecutive training periods, once supplementing with CHO (de facto “control” condition) and the other with PRO. Each training period consisted of 10 days of intensified cycle training (ICT–120% increase in average training duration) followed by 10 days of recovery (RVT–reduced volume training; 33% volume reduction vs. normal training). Skeletal muscle biopsies were obtained from the vastus lateralis before and after ICT and again following RVT. Immunofluorescent microscopy was used to quantify SCs (Pax7+), myonuclei (DAPI+), and myosin heavy chain I (MyHC I). Data are expressed as percent change ± 90% confidence limits. The 10-day block of ICTCHO increased MyHC I SC content (35 ± 28%) and myonuclear density (16 ± 6%), which remained elevated following RVTCHO (SC = 69 ± 50% vs. PRE; Nuclei = 17 ± 15% vs. PRE). MyHC II SC and myonuclei were not different following ICTCHO, but were higher following RVTCHO (SC = +33 ± 31% vs. PRE; Nuclei = 15 ± 14% vs. PRE), indicating a delayed response compared to MyHC I fibers. The MyHC I SC pool increased following ICTPRO (37 ± 37%), but without a concomitant increase in myonuclei. There were no changes in MyHC II SC or myonuclei following ICTPRO. Collectively, these trained endurance cyclists possessed a relatively large pool of SCs that facilitated rapid (MyHC I) and delayed (MyHC II) satellite cell proliferation and myonuclear accretion under carbohydrate conditions. The current findings strengthen the growing body of evidence demonstrating alterations in satellite cell number in the absence of hypertrophy. Satellite cell pool expansion is typically viewed as an advantageous response to

  7. Fiber Type-Specific Satellite Cell Content in Cyclists Following Heavy Training with Carbohydrate and Carbohydrate-Protein Supplementation.

    PubMed

    McKenzie, Alec I; D'Lugos, Andrew C; Saunders, Michael J; Gworek, Keith D; Luden, Nicholas D

    2016-01-01

    The central purpose of this study was to evaluate the fiber type-specific satellite cell and myonuclear responses of endurance-trained cyclists to a block of intensified training, when supplementing with carbohydrate (CHO) vs. carbohydrate-protein (PRO). In a crossover design, endurance-trained cyclists (n = 8) performed two consecutive training periods, once supplementing with CHO (de facto "control" condition) and the other with PRO. Each training period consisted of 10 days of intensified cycle training (ICT-120% increase in average training duration) followed by 10 days of recovery (RVT-reduced volume training; 33% volume reduction vs. normal training). Skeletal muscle biopsies were obtained from the vastus lateralis before and after ICT and again following RVT. Immunofluorescent microscopy was used to quantify SCs (Pax7+), myonuclei (DAPI+), and myosin heavy chain I (MyHC I). Data are expressed as percent change ± 90% confidence limits. The 10-day block of ICTCHO increased MyHC I SC content (35 ± 28%) and myonuclear density (16 ± 6%), which remained elevated following RVTCHO (SC = 69 ± 50% vs. PRE; Nuclei = 17 ± 15% vs. PRE). MyHC II SC and myonuclei were not different following ICTCHO, but were higher following RVTCHO (SC = +33 ± 31% vs. PRE; Nuclei = 15 ± 14% vs. PRE), indicating a delayed response compared to MyHC I fibers. The MyHC I SC pool increased following ICTPRO (37 ± 37%), but without a concomitant increase in myonuclei. There were no changes in MyHC II SC or myonuclei following ICTPRO. Collectively, these trained endurance cyclists possessed a relatively large pool of SCs that facilitated rapid (MyHC I) and delayed (MyHC II) satellite cell proliferation and myonuclear accretion under carbohydrate conditions. The current findings strengthen the growing body of evidence demonstrating alterations in satellite cell number in the absence of hypertrophy. Satellite cell pool expansion is typically viewed as an advantageous response to exercise

  8. Carbohydrate-based particles: a new adjuvant for allergen-specific immunotherapy

    PubMed Central

    Grönlund, Hans; Vrtala, Susanne; Wiedermann, Ursula; Dekan, Gerhard; Kraft, Dietrich; Valenta, Rudolf; Van Hage-Hamsten, Marianne

    2002-01-01

    The occurrence of systemic anaphylactic side-effects in the course of allergen-specific immunotherapy has been strongly reduced by the adsorption of allergens to aluminium hydroxide, the most frequently used adjuvant in humans. Using the major timothy grass pollen allergen, Phl p 5b, in its recombinant form for immunization of mice, we demonstrate that carbohydrate-based particles (CBP) exhibit several potential advantages over aluminium-hydroxide as adjuvant for immunotherapy. Similar to alum-bound rPhl p 5b, CBP-bound rPhl p 5b induced a stronger antibody and cytokine response than unbound rPhl p 5b after subcutaneous injection in mice. The antibodies induced by CBP-bound rPhl p 5b, exhibited potentially beneficial activities as they cross-reacted with group 5 allergens from five other grass species and inhibited the binding of grass pollen allergic patients IgE to Phl p 5b. Alum-bound rPhl p 5b induced a preferential allergen-specific Th2-response characterized by high immunoglobulin G1 (IgG1) antibody levels and elevated interleukin (IL)-4 and IL-5 production in cultured splenocytes. By contrast, CBP-bound rPhl p 5b, but not rPhl p 5b alone or coadministered with CBP, induced a mixed allergen-specific T helper 1 (Th1)/Th2 immune response characterized by the additional production of allergen-specific IgG2a/b antibody responses and elevated interferon-γ production. Conjugation of rPhl p 5b to CBP yielded a stable vaccine formulation with preserved immunogenic features of the allergen and, in contrast to alum, induced no granulomatous tissue reactions. Based on these results, CBP is suggested as a potentially useful adjuvant for specific immunotherapy of IgE-mediated allergies. PMID:12460198

  9. Evaluation of Selected Classical Force Fields for Alchemical Binding Free Energy Calculations of Protein-Carbohydrate Complexes.

    PubMed

    Mishra, Sushil K; Calabró, Gaetano; Loeffler, Hannes H; Michel, Julien; Koča, Jaroslav

    2015-07-14

    Protein-carbohydrate recognition is crucial in many vital biological processes including host-pathogen recognition, cell-signaling, and catalysis. Accordingly, computational prediction of protein-carbohydrate binding free energies is of enormous interest for drug design. However, the accuracy of current force fields (FFs) for predicting binding free energies of protein-carbohydrate complexes is not well understood owing to technical challenges such as the highly polar nature of the complexes, anomerization, and conformational flexibility of carbohydrates. The present study evaluated the performance of alchemical predictions of binding free energies with the GAFF1.7/AM1-BCC and GLYCAM06j force fields for modeling protein-carbohydrate complexes. Mean unsigned errors of 1.1 ± 0.06 (GLYCAM06j) and 2.6 ± 0.08 (GAFF1.7/AM1-BCC) kcal·mol(-1) are achieved for a large data set of monosaccharide ligands for Ralstonia solanacearum lectin (RSL). The level of accuracy provided by GLYCAM06j is sufficient to discriminate potent, moderate, and weak binders, a goal that has been difficult to achieve through other scoring approaches. Accordingly, the protocols presented here could find useful applications in carbohydrate-based drug and vaccine developments.

  10. Synthesis, Binding Properties, and Differences in Cell Uptake of G-Quadruplex Ligands Based on Carbohydrate Naphthalene Diimide Conjugates.

    PubMed

    Arévalo-Ruiz, Matilde; Doria, Filippo; Belmonte-Reche, Efres; De Rache, Aurore; Campos-Salinas, Jenny; Lucas, Ricardo; Falomir, Eva; Carda, Miguel; Pérez-Victoria, José María; Mergny, Jean-Louis; Freccero, Mauro; Morales, Juan Carlos

    2017-02-10

    The G-quadruplexes (G4s) are currently being explored as therapeutic targets in cancer and other pathologies. Six carbohydrate naphthalene diimide conjugates (carb-NDIs) have been synthesized as G4 ligands to investigate their potential selectivity in G4 binding and cell penetration. Carb-NDIs have shown certain selectivity for G4 structures against DNA duplexes, but different sugar moieties do not induce a preference for a specific G4 topology. Interestingly, when monosaccharides were attached through a short ethylene linker to the NDI scaffold, their cellular uptake was two- to threefold more efficient than that when the sugar was directly attached through its anomeric position. Moreover, a correlation between more efficient cell uptake of these carb-NDIs and their higher toxicity in cancerous cell lines has been observed. Carb-NDIs seem to be mainly translocated into cancer cells through glucose transporters (GLUT), of which GLUT4 plays a major role.

  11. The carbohydrate domain of calicheamicin gamma I1 determines its sequence specificity for DNA cleavage.

    PubMed Central

    Drak, J; Iwasawa, N; Danishefsky, S; Crothers, D M

    1991-01-01

    We have investigated the DNA cleaving properties of calicheamicinone, the synthetic core aglycone of calicheamicin gamma I1, a natural product with extremely potent antitumor activity. Our experiments have shown that the synthetic analog binds and cleaves DNA, albeit without any sequence selectivity and with less efficiency than the natural compound. We propose that a key element in the sequence recognition process is the thiobenzoate ring present in the natural compound. We have demonstrated by one-dimensional NMR that there is direct hydrogen abstraction from DNA by calicheamicinone, with enhanced binding affinity contributed by the carbohydrate domain. The reduced efficiency of hydrogen abstraction from DNA by bound calicheamicinone, compared with the natural compound, implicates the carbohydrate moiety in positioning the drug for hydrogen abstraction. Images PMID:1881884

  12. A photo-cleavable biotin affinity tag for the facile release of a photo-crosslinked carbohydrate-binding protein.

    PubMed

    Chang, Tsung-Che; Adak, Avijit K; Lin, Ting-Wei; Li, Pei-Jhen; Chen, Yi-Ju; Lai, Chain-Hui; Liang, Chien-Fu; Chen, Yu-Ju; Lin, Chun-Cheng

    2016-03-15

    The use of photo-crosslinking glycoprobes represents a powerful strategy for the covalent capture of labile protein complexes and allows detailed characterization of carbohydrate-mediated interactions. The selective release of target proteins from solid support is a key step in functional proteomics. We envisaged that light activation can be exploited for releasing labeled protein in a dual photo-affinity probe-based strategy. To investigate this possibility, we designed a trifunctional, galactose-based, multivalent glycoprobe for affinity labeling of carbohydrate-binding proteins. The resulting covalent protein-probe adduct is attached to a photo-cleavable biotin affinity tag; the biotin moiety enables specific presentation of the conjugate on streptavidin-coated beads, and the photolabile linker allows the release of the labeled proteins. This dual probe promotes both the labeling and the facile cleavage of the target protein complexes from the solid surfaces and the remainder of the cell lysate in a completely unaltered form, thus eliminating many of the common pitfalls associated with traditional affinity-based purification methods.

  13. Molecular recognition of carbohydrates with artificial receptors: mimicking the binding motifs found in the crystal structures of protein-carbohydrate complexes.

    PubMed

    Mazik, Monika; Cavga, Hüseyin; Jones, Peter G

    2005-06-29

    The binding motifs found in the crystal structures of protein-carbohydrate complexes have been successfully mimicked with simple acyclic pyridine- and pyrimidine-based receptors. A full discussion of the recognition motifs observed in the crystal structures of complexes of receptors 1 and 3 with glucopyranosides 4a and 4b is provided. A remarkable similarity of these motifs to those observed in the crystal structures of sugar-binding proteins and those found by molecular modeling is shown. In addition, the recognition properties of the new pyrimidine receptor 3 toward monosaccharides 4-6 are described. This molecule has been established as a highly effective receptor for beta-glucopyranosides.

  14. Hydration Repulsion between Carbohydrate Surfaces Mediated by Temperature and Specific Ions

    PubMed Central

    Chen, Hsieh; Cox, Jason R.; Ow, Hooisweng; Shi, Rena; Panagiotopoulos, Athanassios Z.

    2016-01-01

    Stabilizing colloids or nanoparticles in solution involves a fine balance between surface charges, steric repulsion of coating molecules, and hydration forces against van der Waals attractions. At high temperature and electrolyte concentrations, the colloidal stability of suspensions usually decreases rapidly. Here, we report a new experimental and simulation discovery that the polysaccharide (dextran) coated nanoparticles show ion-specific colloidal stability at high temperature, where we observed enhanced colloidal stability of nanoparticles in CaCl2 solution but rapid nanoparticle-nanoparticle aggregation in MgCl2 solution. The microscopic mechanism was unveiled in atomistic simulations. The presence of surface bound Ca2+ ions increases the carbohydrate hydration and induces strongly polarized repulsive water structures beyond at least three hydration shells which is farther-reaching than previously assumed. We believe leveraging the binding of strongly hydrated ions to macromolecular surfaces represents a new paradigm in achieving absolute hydration and colloidal stability for a variety of materials, particularly under extreme conditions. PMID:27334145

  15. Carbohydrate composition of amphiphilic macromolecules influences physicochemical properties and binding to atherogenic scavenger receptor A.

    PubMed

    Hehir, Sarah; Plourde, Nicole M; Gu, Li; Poree, Dawanne E; Welsh, William J; Moghe, Prabhas V; Uhrich, Kathryn E

    2012-11-01

    Amphiphilic macromolecules (AMs) based on carbohydrate domains functionalized with poly(ethylene glycol) can inhibit the uptake of oxidized low density lipoprotein (oxLDL) mediated by scavenger receptor A (SR-A) and counteract foam cell formation, the characteristic "atherosclerotic" phenotype. A series of AMs was prepared by altering the carbohydrate chemistry to evaluate the influence of backbone architecture on the physicochemical and biological properties. Upon evaluating the degree of polymer-based inhibition of oxLDL uptake in human embryonic kidney cells expressing SR-A, two AMs (2a and 2c) were found to have the most efficacy. Molecular modeling and docking studies show that these same AMs have the most favorable binding energies and most close interactions with the molecular model of the SR-A collagen-like domain. Thus, minor changes in the AMs' architecture can significantly affect the physicochemical properties and inhibition of oxLDL uptake. These insights can be critical for designing optimal AM-based therapeutics for the management of cardiovascular disease. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  16. Carbohydrate Composition of Amphiphilic Macromolecules Influences Physicochemical Properties and Binding to Atherogenic Scavenger Receptor A

    PubMed Central

    Hehir, Sarah; Plourde, Nicole M.; Gu, Li; Poree, Dawanne E.; Welsh, William J.; Moghe, Prabhas V.; Uhrich, Kathryn E.

    2012-01-01

    Amphiphilic macromolecules (AMs) based on carbohydrate domains functionalized with poly(ethylene glycol) can inhibit the uptake of oxidized low density lipoprotein (oxLDL) mediated by scavenger receptor A (SR-A) and counteract foam cell formation, the characteristic “atherosclerotic” phenotype. A series of AMs were prepared by altering the carbohydrate chemistry to evaluate the influence of backbone architecture on the physicochemical and biological properties. Upon evaluating the degree of polymer-based inhibition of oxLDL uptake in human embryonic kidney cells expressing SR-A, two AMs (2a and 2c) were found to have the most efficacy. Molecular modeling and docking studies show that these same AMs have the most favorable binding energies and most close interactions with the molecular model of SR-A collagen-like domain. Thus, minor changes in the AMs architecture can significantly affect the physicochemical properties and inhibition of oxLDL uptake. These insights can be critical for designing optimal AM-based therapeutics for management of cardiovascular disease. PMID:22835678

  17. Comprehensive list of lectins: origins, natures, and carbohydrate specificities.

    PubMed

    Kobayashi, Yuka; Tateno, Hiroaki; Ogawa, Haruko; Yamamoto, Kazuo; Hirabayashi, Jun

    2014-01-01

    More than 100 years have passed since the first lectin ricin was discovered. Since then, a wide variety of lectins (lect means "select" in Latin) have been isolated from plants, animals, fungi, bacteria, as well as viruses, and their structures and properties have been characterized. At present, as many as 48 protein scaffolds have been identified as functional lectins from the viewpoint of three-dimensional structures as described in this chapter. In this chapter, representative 53 lectins are selected, and their major properties that include hemagglutinating activity, mitogen activity, blood group specificity, molecular weight, metal requirement, and sugar specificities are summarized as a comprehensive table. The list will provide a practically useful, comprehensive list for not only experienced lectin users but also many other non-expert researchers, who are not familiar to lectins and, therefore, have no access to advanced lectin biotechnologies described in other chapters.

  18. TEMPERATURE-DEPENDENT VARIATION IN THE SYNTHESIS OF GROUP-SPECIFIC CARBOHYDRATE BY STREPTOCOCCAL VARIANT STRAINS

    PubMed Central

    Ayoub, Elia M.; Dudding, Burton A.

    1973-01-01

    A temperature-dependent alteration in the synthesis of the group-specific polysaccharide was found to occur in two "variant" streptococcal strains, A-486-Var and C 121/46/4. These strains synthesize a polysaccharide with variant immunochemical characteristics when grown at 37°C. However, when these organisms are grown at lower temperatures, 22°C, an enhanced synthesis of Group A carbohydrate occurs. Other variant strains show no appreciable alteration of the cell wall carbohydrate composition when grown at lower temperatures. Studies on an intermediate strain show that this organism has a propensity for the synthesis of a polysaccharide with higher glucosamine content and enhanced Group A serological reactivity when grown at 22°C. Immunochemical studies performed on the carbohydrates produced by the A-486-Var at various temperatures revealed that the appearance of Group A serological reactivity at lower temperatures is due to the additional synthesis of a polysaccharide with Group A specificity along with the continued synthesis of a variant carbohydrate. This finding contrasts with data obtained on the carbohydrate produced by the intermediate organisms that appears to consist predominently of one molecule bearing dual A and variant antigenic determinants. PMID:4123827

  19. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    PubMed Central

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the underlying molecular mechanisms. Agonist-activated FXR inhibits glucose-induced transcription of several glycolytic genes, including the liver-type pyruvate kinase gene (L-PK), in the immortalized human hepatocyte (IHH) and HepaRG cell lines. This inhibition requires the L4L3 region of the L-PK promoter, known to bind the transcription factors ChREBP and hepatocyte nuclear factor 4α (HNF4α). FXR interacts directly with ChREBP and HNF4α proteins. Analysis of the protein complex bound to the L4L3 region reveals the presence of ChREBP, HNF4α, FXR, and the transcriptional coactivators p300 and CBP at high glucose concentrations. FXR activation does not affect either FXR or HNF4α binding to the L4L3 region but does result in the concomitant release of ChREBP, p300, and CBP and in the recruitment of the transcriptional corepressor SMRT. Thus, FXR transrepresses the expression of genes involved in glycolysis in human hepatocytes. PMID:23530060

  20. Binding of Human GII.4 Norovirus Virus-Like Particles to Carbohydrates of Romaine Lettuce Leaf Cell Wall Materials

    PubMed Central

    Esseili, Malak A.

    2012-01-01

    Norovirus (NoV) genogroup II genotype 4 (GII.4) strains are the dominant cause of the majority of food-borne outbreaks, including those that involve leafy greens, such as lettuce. Since human NoVs use carbohydrates of histo-blood group antigens as receptors/coreceptors, we examined the role of carbohydrates in the attachment of NoV to lettuce leaves by using virus-like particles (VLPs) of a human NoV/GII.4 strain. Immunofluorescence analysis showed that the VLPs attached to the leaf surface, especially to cut edges, stomata, and along minor veins. Binding was quantified using enzyme-linked immunosorbent assay (ELISA) performed on cell wall materials (CWM) from innermost younger leaves and outermost lamina of older leaves. The binding to CWM of older leaves was significantly (P < 0.05) higher (1.5- to 2-fold) than that to CWM of younger leaves. Disrupting the carbohydrates of CWM or porcine gastric mucin (PGM) (a carbohydrate control) using 100 mM sodium periodate (NaIO4) significantly decreased the binding an average of 17% in younger leaves, 43% in older leaves, and 92% for PGM. In addition, lectins recognizing GalNAc, GlcNAc, and sialic acid at 100 μg/ml significantly decreased the binding an average of 41%, 33%, and 20% on CWM of older leaves but had no effect on younger leaves. Lectins recognizing α-d-Gal, α-d-Man/α-d-Glc, and α-l-Fuc showed significant inhibition on CWM of older leaves as well as that of younger leaves. All lectins, except for the lectin recognizing α-d-Gal, significantly inhibited NoV VLP binding to PGM. Collectively, our results indicate that NoV VLPs bind to lettuce CWM by utilizing multiple carbohydrate moieties. This binding may enhance virus persistence on the leaf surface and prevent effective decontamination. PMID:22138991

  1. The carbohydrate-binding module family 20--diversity, structure, and function.

    PubMed

    Christiansen, Camilla; Abou Hachem, Maher; Janecek, Stefan; Viksø-Nielsen, Anders; Blennow, Andreas; Svensson, Birte

    2009-09-01

    Starch-active enzymes often possess starch-binding domains (SBDs) mediating attachment to starch granules and other high molecular weight substrates. SBDs are divided into nine carbohydrate-binding module (CBM) families, and CBM20 is the earliest-assigned and best characterized family. High diversity characterizes CBM20s, which occur in starch-active glycoside hydrolase families 13, 14, 15, and 77, and enzymes involved in starch or glycogen metabolism, exemplified by the starch-phosphorylating enzyme glucan, water dikinase 3 from Arabidopsis thaliana and the mammalian glycogen phosphatases, laforins. The clear evolutionary relatedness of CBM20s to CBM21s, CBM48s and CBM53s suggests a common clan hosting most of the known SBDs. This review surveys the diversity within the CBM20 family, and makes an evolutionary comparison with CBM21s, CBM48s and CBM53s, discussing intrafamily and interfamily relationships. Data on binding to and enzymatic activity towards soluble ligands and starch granules are summarized for wild-type, mutant and chimeric fusion proteins involving CBM20s. Noticeably, whereas CBM20s in amylolytic enzymes confer moderate binding affinities, with dissociation constants in the low micromolar range for the starch mimic beta-cyclodextrin, recent findings indicate that CBM20s in regulatory enzymes have weaker, low millimolar affinities, presumably facilitating dynamic regulation. Structures of CBM20s, including the first example of a full-length glucoamylase featuring both the catalytic domain and the SBD, are summarized, and distinct architectural and functional features of the two SBDs and roles of pivotal amino acids in binding are described. Finally, some applications of SBDs as affinity or immobilization tags and, recently, in biofuel and in planta bioengineering are presented.

  2. The Effects of Noncellulosic Compounds on the Nanoscale Interaction Forces Measured between Carbohydrate-Binding Module and Lignocellulosic Biomass.

    PubMed

    Arslan, Baran; Colpan, Mert; Ju, Xiaohui; Zhang, Xiao; Kostyukova, Alla; Abu-Lail, Nehal I

    2016-05-09

    The lack of fundamental understanding of the types of forces that govern how cellulose-degrading enzymes interact with cellulosic and noncellulosic components of lignocellulosic surfaces limits the design of new strategies for efficient conversion of biomass to bioethanol. In a step to improve our fundamental understanding of such interactions, nanoscale forces acting between a model cellulase-a carbohydrate-binding module (CBM) of cellobiohydrolase I (CBH I)-and a set of lignocellulosic substrates with controlled composition were measured using atomic force microscopy (AFM). The three model substrates investigated were kraft (KP), sulfite (SP), and organosolv (OPP) pulped substrates. These substrates varied in their surface lignin coverage, lignin type, and xylan and acetone extractives' content. Our results indicated that the overall adhesion forces of biomass to CBM increased linearly with surface lignin coverage with kraft lignin showing the highest forces among lignin types investigated. When the overall adhesion forces were decoupled into specific and nonspecific component forces via the Poisson statistical model, hydrophobic and Lifshitz-van der Waals (LW) forces dominated the binding forces of CBM to kraft lignin, whereas permanent dipole-dipole interactions and electrostatic forces facilitated the interactions of lignosulfonates to CBM. Xylan and acetone extractives' content increased the attractive forces between CBM and lignin-free substrates, most likely through hydrogen bonding forces. When the substrates treated differently were compared, it was found that both the differences in specific and nonspecific forces between lignin-containing and lignin-free substrates were the least for OPP. Therefore, cellulase enzymes represented by CBM would weakly bind to organosolv lignin. This will facilitate an easy enzyme recovery compared to other substrates treated with kraft or sulfite pulping. Our results also suggest that altering the surface hydrophobicity

  3. Carbohydrate-Aromatic Interactions in Proteins.

    PubMed

    Hudson, Kieran L; Bartlett, Gail J; Diehl, Roger C; Agirre, Jon; Gallagher, Timothy; Kiessling, Laura L; Woolfson, Derek N

    2015-12-09

    Protein-carbohydrate interactions play pivotal roles in health and disease. However, defining and manipulating these interactions has been hindered by an incomplete understanding of the underlying fundamental forces. To elucidate common and discriminating features in carbohydrate recognition, we have analyzed quantitatively X-ray crystal structures of proteins with noncovalently bound carbohydrates. Within the carbohydrate-binding pockets, aliphatic hydrophobic residues are disfavored, whereas aromatic side chains are enriched. The greatest preference is for tryptophan with an increased prevalence of 9-fold. Variations in the spatial orientation of amino acids around different monosaccharides indicate specific carbohydrate C-H bonds interact preferentially with aromatic residues. These preferences are consistent with the electronic properties of both the carbohydrate C-H bonds and the aromatic residues. Those carbohydrates that present patches of electropositive saccharide C-H bonds engage more often in CH-π interactions involving electron-rich aromatic partners. These electronic effects are also manifested when carbohydrate-aromatic interactions are monitored in solution: NMR analysis indicates that indole favorably binds to electron-poor C-H bonds of model carbohydrates, and a clear linear free energy relationships with substituted indoles supports the importance of complementary electronic effects in driving protein-carbohydrate interactions. Together, our data indicate that electrostatic and electronic complementarity between carbohydrates and aromatic residues play key roles in driving protein-carbohydrate complexation. Moreover, these weak noncovalent interactions influence which saccharide residues bind to proteins, and how they are positioned within carbohydrate-binding sites.

  4. Recognition of the Helical Structure of β-1,4-Galactan by a New Family of Carbohydrate-binding Modules*

    PubMed Central

    Cid, Melissa; Pedersen, Henriette Lodberg; Kaneko, Satoshi; Coutinho, Pedro M.; Henrissat, Bernard; Willats, William G. T.; Boraston, Alisdair B.

    2010-01-01

    The microbial enzymes that depolymerize plant cell wall polysaccharides, ultimately promoting energy liberation and carbon recycling, are typically complex in their modularity and often contain carbohydrate-binding modules (CBMs). Here, through analysis of an unknown module from a Thermotoga maritima endo-β-1,4-galactanase, we identify a new family of CBMs that are most frequently found appended to proteins with β-1,4-galactanase activity. Polysaccharide microarray screening, immunofluorescence microscopy, and biochemical analysis of the isolated module demonstrate the specificity of the module, here called TmCBM61, for β-1,4-linked galactose-containing ligands, making it the founding member of family CBM61. The ultra-high resolution x-ray crystal structures of TmCBM61 (0.95 and 1.4 Å resolution) in complex with β-1,4-galactotriose reveal the molecular basis of the specificity of the CBM for β-1,4-galactan. Analysis of these structures provides insight into the recognition of an unexpected helical galactan conformation through a mode of binding that resembles the recognition of starch. PMID:20826814

  5. Characterization of the Carbohydrate Binding Module 18 gene family in the amphibian pathogen Batrachochytrium dendrobatidis.

    PubMed

    Liu, Peng; Stajich, Jason E

    2015-04-01

    Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis responsible for worldwide decline in amphibian populations. Previous analysis of the Bd genome revealed a unique expansion of the carbohydrate-binding module family 18 (CBM18) predicted to be a sub-class of chitin recognition domains. CBM expansions have been linked to the evolution of pathogenicity in a variety of fungal species by protecting the fungus from the host. Based on phylogenetic analysis and presence of additional protein domains, the gene family can be classified into 3 classes: Tyrosinase-, Deacetylase-, and Lectin-like. Examination of the mRNA expression levels from sporangia and zoospores of nine of the cbm18 genes found that the Lectin-like genes had the highest expression while the Tyrosinase-like genes showed little expression, especially in zoospores. Heterologous expression of GFP-tagged copies of four CBM18 genes in Saccharomyces cerevisiae demonstrated that two copies containing secretion signal peptides are trafficked to the cell boundary. The Lectin-like genes cbm18-ll1 and cbm18-ll2 co-localized with the chitinous cell boundaries visualized by staining with calcofluor white. In vitro assays of the full length and single domain copies from CBM18-LL1 demonstrated chitin binding and no binding to cellulose or xylan. Expressed CBM18 domain proteins were demonstrated to protect the fungus, Trichoderma reeseii, in vitro against hydrolysis from exogenously added chitinase, likely by binding and limiting exposure of fungal chitin. These results demonstrate that cbm18 genes can play a role in fungal defense and expansion of their copy number may be an important pathogenicity factor of this emerging infectious disease of amphibians.

  6. Isopropylamino and isobutylamino groups as recognition sites for carbohydrates: acyclic receptors with enhanced binding affinity toward β-galactosides.

    PubMed

    Mazik, Monika; Sonnenberg, Claudia

    2010-10-01

    Binding motifs observed in the crystal structures of protein-carbohydrate complexes, in particular the participation of the isopropyl/isobutyl side chain of valine/leucine in the formation of van der Waals contacts, have inspired the design of new artificial carbohydrate receptors. The new compounds, containing a trisubstituted triethylbenzene core, were expected to recognize sugar molecules through a combination of NH···O and OH···N hydrogen bonds, CH···π interactions, and numerous van der Waals contacts. (1)H NMR spectroscopic titrations in competitive and noncompetitive media, as well as binding studies in two-phase systems, such as dissolution of solid carbohydrates in apolar media and phase transfer of sugars from aqueous into organic solvents, revealed effective recognition of neutral carbohydrates and β- vs α-anomer binding preferences in the recognition of glycosides as well as significantly increased binding affinity of the receptors toward β-galactoside in comparison with the previously described receptors.

  7. Specific binding of phorbol ester tumor promoters

    PubMed Central

    Driedger, Paul E.; Blumberg, Peter M.

    1980-01-01

    [20-3H]Phorbol 12,13-dibutyrate bound to particulate preparations from chicken embryo fibroblasts in a specific, saturable, reversible fashion. Equilibrium binding occurred with a Kd of 25 nM; this value is very close to the 50% effective dose (ED50), 50 nM, previously determined for the biological response (induction of fibronectin loss) in growing chicken embryo fibroblasts. At saturation, 1.4 pmol of [20-3H]phorbol 12,13-dibutyrate was bound per mg of protein (approximately 7 × 104 molecules per cell). Binding was inhibited by phorbol 12-myristate 13-acetate (Ki = 2 nM), mezerein (Ki = 180 nM), phorbol 12,13-dibenzoate (Ki = 180 nM), phorbol 12,13-diacetate (Ki = 1.7 μM), phorbol 12,13,20-triacetate (Ki = 39 μM), and phorbol 13-acetate (Ki = 120 μM). The measured Ki values are all within a factor of 3.5 of the ED50 values of these derivatives for inducing loss of fibronectin in intact cells. Binding was not inhibited by the inactive compounds phorbol (10 μg/ml) and 4α-phorbol 12,13-didecanoate (10 μg/ml) or by the inflammatory but nonpromoting phorbol-related diterpene esters resiniferatoxin (100 ng/ml) and 12-deoxyphorbol 13-isobutyrate 20-acetate (100 ng/ml). These data suggest that biological responses to the phorbol esters in chicken embryo fibroblasts are mediated by this binding activity and that the binding activity corresponds to the phorbol ester target in mouse skin involved in tumor promotion. Binding was not inhibited by the nonphorbol promoters anthralin (1 μM), phenol (1 mM), iodoacetic acid (1.7 μM), and cantharidin (75 μM), or by epidermal growth factor (100 ng/ml), dexamethasone acetate (2 μM), retinoic acid (10 μM), or prostaglandin E2 (1 μM). These agents thus appear to act at a target distinct from that of the phorbol esters. PMID:6965793

  8. An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

    SciTech Connect

    Wong, Jaslyn E. M. M.; Midtgaard, Søren Roi; Gysel, Kira; Thygesen, Mikkel B.; Sørensen, Kasper K.; Jensen, Knud J.; Stougaard, Jens; Thirup, Søren; Blaise, Mickaël

    2015-03-01

    The crystal and solution structures of the T. thermophilus NlpC/P60 d, l-endopeptidase as well as the co-crystal structure of its N-terminal LysM domains bound to chitohexaose allow a proposal to be made regarding how the enzyme recognizes peptidoglycan. LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.

  9. Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

    SciTech Connect

    Li, Ming V.; Chen, Weiqin; Harmancey, Romain N.; Nuotio-Antar, Alli M.; Imamura, Minako; Saha, Pradip; Taegtmeyer, Heinrich; Chan, Lawrence

    2010-05-07

    Carbohydrate response element binding protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here, we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose-phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP.

  10. An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase.

    PubMed

    Wong, Jaslyn E M M; Midtgaard, Søren Roi; Gysel, Kira; Thygesen, Mikkel B; Sørensen, Kasper K; Jensen, Knud J; Stougaard, Jens; Thirup, Søren; Blaise, Mickaël

    2015-03-01

    LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.

  11. An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

    PubMed Central

    Wong, Jaslyn E. M. M.; Midtgaard, Søren Roi; Gysel, Kira; Thygesen, Mikkel B.; Sørensen, Kasper K.; Jensen, Knud J.; Stougaard, Jens; Thirup, Søren; Blaise, Mickaël

    2015-01-01

    LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed. PMID:25760608

  12. Modeling protein recognition of carbohydrates.

    PubMed

    Laederach, Alain; Reilly, Peter J

    2005-09-01

    We have a limited understanding of the details of molecular recognition of carbohydrates by proteins, which is critical to a multitude of biological processes. Furthermore, carbohydrate-modifying proteins such as glycosyl hydrolases and phosphorylases are of growing importance as potential drug targets. Interactions between proteins and carbohydrates have complex thermodynamics, and in general the specific positioning of only a few hydroxyl groups determines their binding affinities. A thorough understanding of both carbohydrate and protein structures is thus essential to predict these interactions. An atomic-level view of carbohydrate recognition through structures of carbohydrate-active enzymes complexed with transition-state inhibitors reveals some of the distinctive molecular features unique to protein-carbohydrate complexes. However, the inherent flexibility of carbohydrates and their often water-mediated hydrogen bonding to proteins makes simulation of their complexes difficult. Nonetheless, recent developments such as the parameterization of specific force fields and docking scoring functions have greatly improved our ability to predict protein-carbohydrate interactions. We review protein-carbohydrate complexes having defined molecular requirements for specific carbohydrate recognition by proteins, providing an overview of the different computational techniques available to model them. Copyright 2005 Wiley-Liss, Inc.

  13. The carbohydrate-binding module of xylanase from Nonomuraea flexuosa decreases its non-productive adsorption on lignin

    PubMed Central

    2013-01-01

    Background The enzymatic hydrolysis step converting lignocellulosic materials into fermentable sugars is recognized as one of the major limiting steps in biomass-to-ethanol process due to the low efficiency of enzymes and their cost. Xylanases have been found to be important in the improvement of the hydrolysis of cellulose due to the close interaction of cellulose and xylan. In this work, the effects of carbohydrate-binding module (CBM family II) of the xylanase 11 from Nonomuraea flexuosa (Nf Xyn11) on the adsorption and hydrolytic efficiency toward isolated xylan and lignocellulosic materials were investigated. Results The intact family 11 xylanase of N. flexuosa clearly adsorbed on wheat straw and lignin, following the Langmuir-type isotherm. The presence of the CBM in the xylanase increased the adsorption and hydrolytic efficiency on insoluble oat spelt xylan. But the presence of the CBM did not increase adsorption on pretreated wheat straw or isolated lignin. On the contrary, the CBM decreased the adsorption of the core protein to lignin containing substrates, indicating that the CBM of N. flexuosa xylanase did not contribute to the non-productive adsorption. Conclusion The CBM of the N. flexuosa xylanase was shown to be a xylan-binding module, which had low affinity on cellulose. The CBM of the N. flexuosa xylanase reduced the non-specific adsorption of the core protein to lignin and showed potential for improving the hydrolysis of lignocellulosic materials to platform sugars. PMID:23363927

  14. The carbohydrate-binding module of xylanase from Nonomuraea flexuosa decreases its non-productive adsorption on lignin.

    PubMed

    Zhang, Junhua; Moilanen, Ulla; Tang, Ming; Viikari, Liisa

    2013-01-30

    The enzymatic hydrolysis step converting lignocellulosic materials into fermentable sugars is recognized as one of the major limiting steps in biomass-to-ethanol process due to the low efficiency of enzymes and their cost. Xylanases have been found to be important in the improvement of the hydrolysis of cellulose due to the close interaction of cellulose and xylan. In this work, the effects of carbohydrate-binding module (CBM family II) of the xylanase 11 from Nonomuraea flexuosa (Nf Xyn11) on the adsorption and hydrolytic efficiency toward isolated xylan and lignocellulosic materials were investigated. The intact family 11 xylanase of N. flexuosa clearly adsorbed on wheat straw and lignin, following the Langmuir-type isotherm. The presence of the CBM in the xylanase increased the adsorption and hydrolytic efficiency on insoluble oat spelt xylan. But the presence of the CBM did not increase adsorption on pretreated wheat straw or isolated lignin. On the contrary, the CBM decreased the adsorption of the core protein to lignin containing substrates, indicating that the CBM of N. flexuosa xylanase did not contribute to the non-productive adsorption. The CBM of the N. flexuosa xylanase was shown to be a xylan-binding module, which had low affinity on cellulose. The CBM of the N. flexuosa xylanase reduced the non-specific adsorption of the core protein to lignin and showed potential for improving the hydrolysis of lignocellulosic materials to platform sugars.

  15. Carbohydrate-binding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects.

    PubMed

    Hervé, Cécile; Rogowski, Artur; Blake, Anthony W; Marcus, Susan E; Gilbert, Harry J; Knox, J Paul

    2010-08-24

    Cell wall degrading enzymes have a complex molecular architecture consisting of catalytic modules and noncatalytic carbohydrate-binding modules (CBMs). The function of CBMs in cell wall degrading processes is poorly understood. Here, we have evaluated the potential enzyme-targeting function of CBMs in the context of intact primary and secondary cell wall deconstruction. The capacity of a pectate lyase to degrade pectic homogalacturonan in primary cell walls was potentiated by cellulose-directed CBMs but not by xylan-directed CBMs. Conversely, the arabinofuranosidase-mediated removal of side chains from arabinoxylan in xylan-rich and cellulose-poor wheat grain endosperm cell walls was enhanced by a xylan-binding CBM but less so by a crystalline cellulose-specific module. The capacity of xylanases to degrade xylan in secondary cell walls was potentiated by both xylan- and cellulose-directed CBMs. These studies demonstrate that CBMs can potentiate the action of a cognate catalytic module toward polysaccharides in intact cell walls through the recognition of nonsubstrate polysaccharides. The targeting actions of CBMs therefore have strong proximity effects within cell wall structures, explaining why cellulose-directed CBMs are appended to many noncellulase cell wall hydrolases.

  16. Targeting the Cryptococcus neoformans var. grubii cell wall using lectins: study of the carbohydrate-binding domain.

    PubMed

    de Brito Ximenes, Pamella; Beltrão, Eduardo Isidoro Carneiro; Macêdo, Danielle Patrícia Cerqueira; Buonafina, Maria Daniela Silva; de Lima-Neto, Reginaldo Gonçalves; Neves, Rejane Pereira

    2015-02-25

    Cryptococcus neoformans var. grubii is considered to be the major cause of cryptococcosis in immunosuppressed patients. Understanding cell wall glycoproteins using lectins is of medical interest and can contribute to specific therapy. The aim of this study was to evaluate the carbohydrates on the cell wall of Cryptococcus neoformans var. grubii clinical isolates, using a fluorescein isothiocyanate-lectin binding protocol. Thirty yeast strains stocked in the culture collection were cultivated for 2 days at 30 °C with shaking. Cells were obtained by centrifugation, washed in phosphate-buffered saline, and a suspension of 107 cells/mL was obtained. To determine the binding profile of lectins, concanavalin A (Con A), wheat germ agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), and peanut agglutinin (PNA) conjugated to fluorescein were used. All the tested clinical isolates of Cryptococcus neoformans var. grubii were intensely stained by WGA, moderately stained by Con A, and weakly stained by PNA and UEA-I. Thus, Cryptococcus can be detected in clinical specimens such as blood and cerebrospinal fluid using the fluorescent lectin WGA, which may be considered as an option for detection in cases of suspected cryptococcosis with low laboratory sensitivity. Future applications may be developed using this basic tool.

  17. Carbohydrate-binding module 74 is a novel starch-binding domain associated with large and multidomain α-amylase enzymes.

    PubMed

    Valk, Vincent; Lammerts van Bueren, Alicia; van der Kaaij, Rachel M; Dijkhuizen, Lubbert

    2016-06-01

    Microbacterium aurum B8.A is a bacterium that originates from a potato starch-processing plant and employs a GH13 α-amylase (MaAmyA) enzyme that forms pores in potato starch granules. MaAmyA is a large and multi-modular protein that contains a novel domain at its C terminus (Domain 2). Deletion of Domain 2 from MaAmyA did not affect its ability to degrade starch granules but resulted in a strong reduction in granular pore size. Here, we separately expressed and purified this Domain 2 in Escherichia coli and determined its likely function in starch pore formation. Domain 2 independently binds amylose, amylopectin, and granular starch but does not have any detectable catalytic (hydrolytic or oxidizing) activity on α-glucan substrates. Therefore, we propose that this novel starch-binding domain is a new carbohydrate-binding module (CBM), the first representative of family CBM74 that assists MaAmyA in efficient pore formation in starch granules. Protein sequence-based BLAST searches revealed that CBM74 occurs widespread, but in bacteria only, and is often associated with large and multi-domain α-amylases containing family CBM25 or CBM26 domains. CBM74 may specifically function in binding to granular starches to enhance the capability of α-amylase enzymes to degrade resistant starches (RSs). Interestingly, the majority of family CBM74 representatives are found in α-amylases originating from human gut-associated Bifidobacteria, where they may assist in resistant starch degradation. The CBM74 domain thus may have a strong impact on the efficiency of RS digestion in the mammalian gastrointestinal tract. © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  18. Monitoring lectin interactions with carbohydrates.

    PubMed

    de Bentzmann, Sophie; Varrot, Annabelle; Imberty, Anne

    2014-01-01

    Protein-carbohydrate interactions are often involved in the first step of infection and Pseudomonas aeruginosa produces several proteins that are able to bind specifically to glycan epitopes present on host epithelia. The experimental approaches for studying protein-carbohydrate interaction have been inspired, with some adaptations, from those commonly used for protein-protein or protein-ligand interactions. A range of methods are described herein for detecting lectin activity, screening for monosaccharide or oligosaccharide specificity, determining the affinity of binding together with thermodynamics and kinetics parameters, and producing crystal of lectin-carbohydrate complexes for further structural studies.

  19. Cellulases without carbohydrate-binding modules in high consistency ethanol production process.

    PubMed

    Pakarinen, Annukka; Haven, Mai Ostergaard; Djajadi, Demi Tristan; Várnai, Anikó; Puranen, Terhi; Viikari, Liisa

    2014-02-21

    Enzymes still comprise a major part of ethanol production costs from lignocellulose raw materials. Irreversible binding of enzymes to the residual substrate prevents their reuse and no efficient methods for recycling of enzymes have so far been presented. Cellulases without a carbohydrate-binding module (CBM) have been found to act efficiently at high substrate consistencies and to remain non-bound after the hydrolysis. High hydrolysis yields could be obtained with thermostable enzymes of Thermoascus aurantiacus containing only two main cellulases: cellobiohydrolase I (CBH I), Cel7A and endoglucanase II (EG II), Cel5A. The yields were decreased by only about 10% when using these cellulases without CBM. A major part of enzymes lacking CBM was non-bound during the most active stage of hydrolysis and in spite of this, produced high sugar yields. Complementation of the two cellulases lacking CBM with CBH II (CtCel6A) improved the hydrolysis. Cellulases without CBM were more sensitive during exposure to high ethanol concentration than the enzymes containing CBM. Enzymes lacking CBM could be efficiently reused leading to a sugar yield of 90% of that with fresh enzymes. The applicability of cellulases without CBM was confirmed under industrial ethanol production conditions at high (25% dry matter (DM)) consistency. The results clearly show that cellulases without CBM can be successfully used in the hydrolysis of lignocellulose at high consistency, and that this approach could provide new means for better recyclability of enzymes. This paper provides new insight into the efficient action of CBM-lacking cellulases. The relationship of binding and action of cellulases without CBM at high DM consistency should, however, be studied in more detail.

  20. Cellulases without carbohydrate-binding modules in high consistency ethanol production process

    PubMed Central

    2014-01-01

    Background Enzymes still comprise a major part of ethanol production costs from lignocellulose raw materials. Irreversible binding of enzymes to the residual substrate prevents their reuse and no efficient methods for recycling of enzymes have so far been presented. Cellulases without a carbohydrate-binding module (CBM) have been found to act efficiently at high substrate consistencies and to remain non-bound after the hydrolysis. Results High hydrolysis yields could be obtained with thermostable enzymes of Thermoascus aurantiacus containing only two main cellulases: cellobiohydrolase I (CBH I), Cel7A and endoglucanase II (EG II), Cel5A. The yields were decreased by only about 10% when using these cellulases without CBM. A major part of enzymes lacking CBM was non-bound during the most active stage of hydrolysis and in spite of this, produced high sugar yields. Complementation of the two cellulases lacking CBM with CBH II (CtCel6A) improved the hydrolysis. Cellulases without CBM were more sensitive during exposure to high ethanol concentration than the enzymes containing CBM. Enzymes lacking CBM could be efficiently reused leading to a sugar yield of 90% of that with fresh enzymes. The applicability of cellulases without CBM was confirmed under industrial ethanol production conditions at high (25% dry matter (DM)) consistency. Conclusions The results clearly show that cellulases without CBM can be successfully used in the hydrolysis of lignocellulose at high consistency, and that this approach could provide new means for better recyclability of enzymes. This paper provides new insight into the efficient action of CBM-lacking cellulases. The relationship of binding and action of cellulases without CBM at high DM consistency should, however, be studied in more detail. PMID:24559384

  1. Probing the Functions of Carbohydrate Binding Modules in the CBEL Protein from the Oomycete Phytophthora parasitica

    PubMed Central

    Martinez, Thomas; Texier, Hélène; Nahoum, Virginie; Lafitte, Claude; Cioci, Gianluca; Heux, Laurent; Dumas, Bernard; O’Donohue, Michael

    2015-01-01

    Oomycetes are microorganisms that are distantly related to true fungi and many members of this phylum are major plant pathogens. Oomycetes express proteins that are able to interact with plant cell wall polysaccharides, such as cellulose. This interaction is thought to be mediated by carbohydrate-binding modules that are classified into CBM family 1 in the CAZy database. In this study, the two CBMs (1–1 and 1–2) that form part of the cell wall glycoprotein, CBEL, from Phytophthora parasitica have been submitted to detailed characterization, first to better quantify their interaction with cellulose and second to determine whether these CBMs can be useful for biotechnological applications, such as biomass hydrolysis. A variety of biophysical techniques were used to study the interaction of the CBMs with various substrates and the data obtained indicate that CBEL’s CBM1-1 exhibits much greater cellulose binding ability than CBM1-2. Engineering of the family 11 xylanase from Talaromyces versatilis (TvXynB), an enzyme that naturally bears a fungal family 1 CBM, has produced two variants. The first one lacks its native CBM, whereas the second contains the CBEL CBM1-1. The study of these enzymes has revealed that wild type TvXynB binds to cellulose, via its CBM1, and that the substitution of its CBM by oomycetal CBM1-1 does not affect its activity on wheat straw. However, intriguingly the addition of CBEL during the hydrolysis of wheat straw actually potentiates the action of TvXynB variant lacking a CBM1. This suggests that the potentiating effect of CBM1-1 might not require the formation of a covalent linkage to TvXynB. PMID:26390127

  2. Exploring the free-energy landscape of carbohydrate-protein complexes: development and validation of scoring functions considering the binding-site topology

    NASA Astrophysics Data System (ADS)

    Eid, Sameh; Saleh, Noureldin; Zalewski, Adam; Vedani, Angelo

    2014-12-01

    Carbohydrates play a key role in a variety of physiological and pathological processes and, hence, represent a rich source for the development of novel therapeutic agents. Being able to predict binding mode and binding affinity is an essential, yet lacking, aspect of the structure-based design of carbohydrate-based ligands. We assembled a diverse data set comprising 273 carbohydrate-protein crystal structures with known binding affinity and evaluated the prediction accuracy of a large collection of well-established scoring and free-energy functions, as well as combinations thereof. Unfortunately, the tested functions were not capable of reproducing binding affinities in the studied complexes. To simplify the complex free-energy surface of carbohydrate-protein systems, we classified the studied proteins according to the topology and solvent exposure of the carbohydrate-binding site into five distinct categories. A free-energy model based on the proposed classification scheme reproduced binding affinities in the carbohydrate data set with an r 2 of 0.71 and root-mean-squared-error of 1.25 kcal/mol ( N = 236). The improvement in model performance underlines the significance of the differences in the local micro-environments of carbohydrate-binding sites and demonstrates the usefulness of calibrating free-energy functions individually according to binding-site topology and solvent exposure.

  3. The Interaction of a Carbohydrate-Binding Module from a Clostridium perfringens N-Acetyl-beta-hexosaminidase with its Carbohydrate Receptor

    SciTech Connect

    Ficko-Blean,E.; Boraston, A.

    2006-01-01

    Clostridium perfringens is a notable colonizer of the human gastrointestinal tract. This bacterium is quite remarkable for a human pathogen by the number of glycoside hydrolases found in its genome. The modularity of these enzymes is striking as is the frequent occurrence of modules having amino acid sequence identity with family 32 carbohydrate-binding modules (CBMs), often referred to as F5/8 domains. Here we report the properties of family 32 CBMs from a C. perfringens N-acetyl-{beta}-hexosaminidase. Macroarray, UV difference, and isothermal titration calorimetry binding studies indicate a preference for the disaccharide LacNAc ({beta}-d-galactosyl-1,4-{beta}-d-N-acetylglucosamine). The molecular details of the interaction of this CBM with galactose, LacNAc, and the type II blood group H-trisaccharide are revealed by x-ray crystallographic studies at resolutions of 1.49, 2.4, and 2.3 Angstroms, respectively.

  4. Novel Carbohydrate-Binding Module of β-1,3-Xylanase from a Marine Bacterium, Alcaligenes sp. Strain XY-234

    PubMed Central

    Okazaki, Fumiyoshi; Tamaru, Yutaka; Hashikawa, Shinnosuke; Li, Yu-Teh; Araki, Toshiyoshi

    2002-01-01

    A β-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp. strain XY-234, has been cloned and sequenced. txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the β-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed β-1,3-xylan but not other polysaccharides such as β-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or β-1,4-mannan. TxyA was capable of binding specifically to β-1,3-xylan. The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the Kd and the maximum amount of protein bound to β-1,3-xylan were 4.2 μM and 18.2 μmol/g of β-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of β-1,3-xylan. PMID:11948152

  5. Two carbohydrate binding sites in the H(CC)-domain of tetanus neurotoxin are required for toxicity.

    PubMed

    Rummel, Andreas; Bade, Steffen; Alves, Jürgen; Bigalke, Hans; Binz, Thomas

    2003-02-21

    Tetanus neurotoxin binds via its carboxyl-terminal H(C)-fragment selectively to neurons mediated by complex gangliosides. We investigated the lactose and sialic acid binding pockets of four recently discovered potential binding sites employing site-directed mutagenesis. Substitution of residues in the lactose binding pocket drastically decreased the binding of the H(C)-fragment to immobilized gangliosides and to rat brain synaptosomes as well as the inhibitory action of recombinant full length tetanus neurotoxin on exocytosis at peripheral nerves. The conserved motif of S(1287)XWY(1290) em leader G(1300) assisted by N1219, D1222, and H1271 within the lactose binding site comprises a typical sugar binding pocket, as also present, for example, in cholera toxin. Replacement of the main residue of the sialic acid binding site, R1226, again caused a dramatic decline in binding affinity and neurotoxicity. Since the structural integrity of the H(C)-fragment mutants was verified by circular dichroism and fluorescence spectroscopy, these data provide the first biochemical evidence that two carbohydrate interaction sites participate in the binding and uptake process of tetanus neurotoxin. The simultaneous binding of one ganglioside molecule to each of the two binding sites was demonstrated by mass spectroscopy studies, whereas ganglioside-mediated linkage of native tetanus neurotoxin molecules was ruled out by size exclusion chromatography. Hence, a subsequent displacement of one ganglioside by a glycoprotein receptor is discussed.

  6. Advanced glycation end products increase carbohydrate responsive element binding protein expression and promote cancer cell proliferation.

    PubMed

    Chen, Hanbei; Wu, Lifang; Li, Yakui; Meng, Jian; Lin, Ning; Yang, Dianqiang; Zhu, Yemin; Li, Xiaoyong; Li, Minle; Xu, Ye; Wu, Yuchen; Tong, Xuemei; Su, Qing

    2014-09-01

    Diabetic patients have increased levels of advanced glycation end products (AGEs) and the role of AGEs in regulating cancer cell proliferation is unclear. Here, we found that treating colorectal and liver cancer cells with AGEs promoted cell proliferation. AGEs stimulated both the expression and activation of a key transcription factor called carbohydrate responsive element binding protein (ChREBP) which had been shown to promote glycolytic and anabolic activity as well as proliferation of colorectal and liver cancer cells. Using siRNAs or the antagonistic antibody for the receptor for advanced glycation end-products (RAGE) blocked AGEs-induced ChREBP expression or cell proliferation in cancer cells. Suppressing ChREBP expression severely impaired AGEs-induced cancer cell proliferation. Taken together, these results demonstrate that AGEs-RAGE signaling enhances cancer cell proliferation in which AGEs-mediated ChREBP induction plays an important role. These findings may provide new explanation for increased cancer progression in diabetic patients. Copyright © 2014. Published by Elsevier Ireland Ltd.

  7. Contribution of the carbohydrate-binding ability of Vatairea guianensis lectin to induce edematogenic activity.

    PubMed

    Marques, Gabriela F O; Osterne, Vinicius J S; Almeida, Livia M; Oliveira, Messias V; Brizeno, Luiz A C; Pinto-Junior, Vanir R; Santiago, Mayara Q; Neco, Antonio H B; Mota, Mario R L; Souza, Luiz A G; Nascimento, Kyria S; Pires, Alana F; Cavada, Benildo S; Assreuy, Ana M S

    2017-09-01

    Vatairea guianensis lectin (VGL), Dalbergiae tribe, is a N-acetyl-galactosamine (GalNAc)/Galactose (Gal) lectin previously purified and characterized. In this work, we report its structural features, obtained from bioinformatics tools, and its inflammatory effect, obtained from a rat paw edema model. The VGL model was obtained by homology with the lectin of Vatairea macrocarpa (VML) as template, and we used it to demonstrate the common characteristics of legume lectins, such as the jellyroll motif and presence of a metal-binding site in the vicinity of the carbohydrate-recognition domain (CRD). Protein-ligand docking revealed favorable interactions with N-acetyl-d-galactosamine, d-galactose and related sugars as well as several biologically relevant N- and O-glycans. In vivo testing of paw edema revealed that VGL induces edematogenic effect involving prostaglandins, interleukins and VGL CRD. Taken together, these data corroborate with previous reports showing that VGL interacts with N- and/or O-glycans of molecular targets, particularly in those presenting galactosides in their structure, contributing to the lectin inflammatory effect. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  8. The Role of Carbohydrate Response Element Binding Protein in Intestinal and Hepatic Fructose Metabolism.

    PubMed

    Iizuka, Katsumi

    2017-02-22

    Many articles have discussed the relationship between fructose consumption and the incidence of obesity and related diseases. Fructose is absorbed in the intestine and metabolized in the liver to glucose, lactate, glycogen, and, to a lesser extent, lipids. Unabsorbed fructose causes bacterial fermentation, resulting in irritable bowl syndrome. Therefore, understanding the mechanisms underlying intestinal and hepatic fructose metabolism is important for the treatment of metabolic syndrome and fructose malabsorption. Carbohydrate response element binding protein (ChREBP) is a glucose-activated transcription factor that controls approximately 50% of de novo lipogenesis in the liver. ChREBP target genes are involved in glycolysis (Glut2, liver pyruvate kinase), fructolysis (Glut5, ketohexokinase), and lipogenesis (acetyl CoA carboxylase, fatty acid synthase). ChREBP gene deletion protects against high sucrose diet-induced and leptin-deficient obesity, because Chrebp(-/-) mice cannot consume fructose or sucrose. Moreover, ChREBP contributes to some of the physiological effects of fructose on sweet taste preference and glucose production through regulation of ChREBP target genes, such as fibroblast growth factor-21 and glucose-6-phosphatase catalytic subunits. Thus, ChREBP might play roles in fructose metabolism. Restriction of excess fructose intake will be beneficial for preventing not only metabolic syndrome but also irritable bowl syndrome.

  9. The Role of Carbohydrate Response Element Binding Protein in Intestinal and Hepatic Fructose Metabolism

    PubMed Central

    Iizuka, Katsumi

    2017-01-01

    Many articles have discussed the relationship between fructose consumption and the incidence of obesity and related diseases. Fructose is absorbed in the intestine and metabolized in the liver to glucose, lactate, glycogen, and, to a lesser extent, lipids. Unabsorbed fructose causes bacterial fermentation, resulting in irritable bowl syndrome. Therefore, understanding the mechanisms underlying intestinal and hepatic fructose metabolism is important for the treatment of metabolic syndrome and fructose malabsorption. Carbohydrate response element binding protein (ChREBP) is a glucose-activated transcription factor that controls approximately 50% of de novo lipogenesis in the liver. ChREBP target genes are involved in glycolysis (Glut2, liver pyruvate kinase), fructolysis (Glut5, ketohexokinase), and lipogenesis (acetyl CoA carboxylase, fatty acid synthase). ChREBP gene deletion protects against high sucrose diet-induced and leptin-deficient obesity, because Chrebp−/− mice cannot consume fructose or sucrose. Moreover, ChREBP contributes to some of the physiological effects of fructose on sweet taste preference and glucose production through regulation of ChREBP target genes, such as fibroblast growth factor-21 and glucose-6-phosphatase catalytic subunits. Thus, ChREBP might play roles in fructose metabolism. Restriction of excess fructose intake will be beneficial for preventing not only metabolic syndrome but also irritable bowl syndrome. PMID:28241431

  10. Analysis of exposed cellulose surfaces in pretreated wood biomass using carbohydrate-binding module (CBM)-cyan fluorescent protein (CFP).

    PubMed

    Kawakubo, Takeshi; Karita, Shuichi; Araki, Yuko; Watanabe, Shota; Oyadomari, Masafumi; Takada, Rie; Tanaka, Fumio; Abe, Kentaro; Watanabe, Takahito; Honda, Yoichi; Watanabe, Takashi

    2010-02-15

    In enzymatic saccharification of lignocellulosics, the access of the enzymes to exposed cellulose surfaces is a key initial step in triggering hydrolysis. However, knowledge of the structure-hydrolyzability relationship of the pretreated biomass is still limited. Here we used fluorescent-labeled recombinant carbohydrate-binding modules (CBMs) from Clostridium josui as specific markers for crystalline cellulose (CjCBM3) and non-crystalline cellulose (CjCBM28) to analyze the complex surfaces of wood tissues pretreated with NaOH, NaOH-Na(2)S (kraft pulping), hydrothermolysis, ball-milling, and organosolvolysis. Japanese cedar wood, one of the most recalcitrant softwood species was selected for the analysis. The binding analysis clarified the linear dependency of the exposure of crystalline and non-crystalline cellulose surfaces for enzymatic saccharification yield by the organosolv and kraft delignification processes. Ball-milling for 5-30 min increased saccharification yield up to 77%, but adsorption by the CjCBM-cyan fluorescent proteins (CFPs) was below 5%. Adsorption of CjCBM-CFPs on the hydrothermolysis pulp were less than half of those for organosolvolysis pulp, in coincidence with low saccharification yields. For all the pretreated wood, crystallinity index was not directly correlated with the overall saccharification yield. Fluorescent microscopy revealed that CjCBM3-CFP and CjCBM28-CFP were site-specifically adsorbed on external fibrous structures and ruptured or distorted fiber surfaces. The assay system with CBM-CFPs is a powerful measure to estimate the initiation sites of hydrolysis and saccharification yields from chemically delignified wood pulps.

  11. Highly effective recognition of carbohydrates by phenanthroline-based receptors: alpha- versus beta-anomer binding preference.

    PubMed

    Mazik, Monika; Hartmann, Andrè; Jones, Peter G

    2009-09-14

    (1)H NMR spectroscopic titrations in competitive and non-competitive media, as well as binding studies in two-phase systems, such as phase transfer of sugars from aqueous into organic solvents and dissolution of solid carbohydrates in apolar media revealed both highly effective recognition of neutral carbohydrates and interesting binding preferences of an acyclic phenanthroline-based receptor 1. Compared to the previously described acyclic receptors, compound 1 displays significantly higher binding affinities, the rare capability to extract sugars from water into non-polar organic solutions and alpha- versus beta-anomer binding preference in the recognition of glycosides, which differs from those observed for other receptor systems. X-ray crystallographic investigations revealed the presence of water molecules in the binding pocket of 1 that are engaged in the formation of hydrogen-bonding motifs similar to those suggested by molecular modelling for the sugar OH groups in the receptor-sugar complexes. The molecular modelling calculations, synthesis, crystal structure and binding properties of 1 are described and compared with those of the previously described receptors.

  12. Carbohydrate-Binding Non-Peptidic Pradimicins for the Treatment of Acute Sleeping Sickness in Murine Models

    PubMed Central

    Castillo-Acosta, Víctor M.; Ruiz-Pérez, Luis M.; Reichardt, Niels C.; Igarashi, Yasuhiro; Liekens, Sandra; Balzarini, Jan

    2016-01-01

    Current treatments available for African sleeping sickness or human African trypanosomiasis (HAT) are limited, with poor efficacy and unacceptable safety profiles. Here, we report a new approach to address treatment of this disease based on the use of compounds that bind to parasite surface glycans leading to rapid killing of trypanosomes. Pradimicin and its derivatives are non-peptidic carbohydrate-binding agents that adhere to the carbohydrate moiety of the parasite surface glycoproteins inducing parasite lysis in vitro. Notably, pradimicin S has good pharmaceutical properties and enables cure of an acute form of the disease in mice. By inducing resistance in vitro we have established that the composition of the sugars attached to the variant surface glycoproteins are critical to the mode of action of pradimicins and play an important role in infectivity. The compounds identified represent a novel approach to develop drugs to treat HAT. PMID:27662652

  13. Fluorosugar Chain Termination Agents as Probes of the Sequence Specificity of a Carbohydrate Polymerase

    PubMed Central

    Brown, Christopher D.; Rusek, Max S.; Kiessling, Laura L.

    2012-01-01

    Naturally occurring carbohydrate polymers are ubiquitous. They are assembled by polymerizing glycosyltransferases, which can generate polysaccharide products with repeating sequence patterns. The fidelity of enzymes of this class is unknown. We report a method for testing the fidelity of carbohydrate polymerase pattern deposition: we synthesized fluorosugar donors and used them as chain termination agents. The requisite nucleotide fluorosugars could be produced from a single intermediate using the Jacobsen catalyst in a kinetically controlled separation of diastereomers. The resulting fluorosugar donors were used by galactofuranosyltransferase GlfT2 from Myco-bacterium tuberculosis (M. tb), and the data indicate that this enzyme mediates the cell wall galactan production through a sequence-specific polymerization. PMID:22458542

  14. The ion dependence of carbohydrate binding of CBM36: an MD and 3D-RISM study

    NASA Astrophysics Data System (ADS)

    Tanimoto, Shoichi; Higashi, Masahiro; Yoshida, Norio; Nakano, Haruyuki

    2016-09-01

    The molecular recognition process of the carbohydrate-binding module family 36 (CBM36) was examined theoretically. The mechanism of xylan binding by CBM36 and the role of Ca2+ were investigated by the combined use of molecular dynamics simulations and the 3D reference interaction site model method. The CBM36 showed affinity for xylan after Ca2+ binding, but not after Mg2+ binding. Free-energy component analysis of the xylan-binding process revealed that the major factor for xylan-binding affinity is the electrostatic interaction between the Ca2+ and the hydroxyl oxygens of xylan. The van der Waals interaction between the hydrophobic side chain of CBM36 and the glucopyranose ring of xylan also contributes to the stabilization of the xylan-binding state. Dehydration on the formation of the complex has the opposite effect on these interactions. The affinity of CBM36 for xylan results from a balance of the interactions between the binding ion and solvents, hydrophilic residues around xylan, and the hydroxyl oxygens of xylan. When CBM binds Ca2+, these interactions are well balanced; in contrast, when CBM binds Mg2+, the dehydration penalty is excessively large.

  15. Processing Binding Relations in Specific Language Impairment

    PubMed Central

    Hestvik, Arild; Seiger-Gardner, Liat; Almodovar, Diana

    2016-01-01

    Purpose This sentence processing experiment examined the abilities of children with specific language impairment (SLI) and children with typical language development (TD) to establish relations between pronouns or reflexives and their antecedents in real time. Method Twenty-two children with SLI and 24 age-matched children with TD (7;3–10;11 [years;months]) participated in a cross-modal picture priming experiment to determine whether they selectively activated the correct referent at the pronoun or reflexive in sentences. Triplets of auditory sentences, identical except for the presence of a pronoun, a reflexive, or a noun phrase along with a picture probe were used. Results The children with TD were slightly more accurate in their animacy judgments of pictures, but the groups exhibited the same reaction time (RT) pattern. Both groups were slower for sentences with pronouns than with reflexives or noun phrases. The children with SLI had longer RTs than their peers with TD. Conclusions Children with SLI activated only the appropriate antecedent at the pronoun or reflexive, reflecting intact core knowledge of binding as was true for their TD peers. The overall slower RT for children with SLI suggests that any deficit may be the result of processing deficits, perhaps attributable to interference effects. PMID:27788275

  16. Processing Binding Relations in Specific Language Impairment.

    PubMed

    Schwartz, Richard G; Hestvik, Arild; Seiger-Gardner, Liat; Almodovar, Diana

    2016-12-01

    This sentence processing experiment examined the abilities of children with specific language impairment (SLI) and children with typical language development (TD) to establish relations between pronouns or reflexives and their antecedents in real time. Twenty-two children with SLI and 24 age-matched children with TD (7;3-10;11 [years;months]) participated in a cross-modal picture priming experiment to determine whether they selectively activated the correct referent at the pronoun or reflexive in sentences. Triplets of auditory sentences, identical except for the presence of a pronoun, a reflexive, or a noun phrase along with a picture probe were used. The children with TD were slightly more accurate in their animacy judgments of pictures, but the groups exhibited the same reaction time (RT) pattern. Both groups were slower for sentences with pronouns than with reflexives or noun phrases. The children with SLI had longer RTs than their peers with TD. Children with SLI activated only the appropriate antecedent at the pronoun or reflexive, reflecting intact core knowledge of binding as was true for their TD peers. The overall slower RT for children with SLI suggests that any deficit may be the result of processing deficits, perhaps attributable to interference effects.

  17. A monoclonal antibody recognizes undifferentiation-specific carbohydrate moieties expressed on cell surface of the human dental pulp cells.

    PubMed

    Kang, Kyung-Jung; Ko, Seon-Yle; Ryu, Chun-Jeih; Jang, Young-Joo

    2017-05-01

    Human dental pulp cells are obtained from dental pulp tissue, and have the ability to form dentin and a pulp-like complex. Although adult stem cells have been identified from the primary culture by using specific cell surface markers, the identity of surface markers for the purification of stem cells within the dental pulp population are still unclear. Previously, we had constructed monoclonal antibodies against the undifferentiated cell-specific surface markers of human dental pulp cells (hDPCs) by performing decoy immunization. Among them, a monoclonal antibody against the cell surface antigen of the undifferentiated hDPCs (named UPSA-1) was purified and its heavy and light chain consensus regions were analyzed. The cell surface binding affinity of UPSA-1 mAb on the undifferentiated hDPCs was stronger than that on the differentiated cells. When tunicamycin was applied to hDPSCs during culture, the cell surface binding affinity of the antibody was dramatically decreased, and dentinogenic differentiation was reduced. The purified UPSA-1 antigen band resulting from immunoprecipitation disappeared or shifted down on the SDS-PAGE by deglycosylation. These data suggested that glycosylation on the cell surface might be a marker of an undifferentiated state, and that UPSA-1 mAb might be useful for identifying the carbohydrate moiety on the cell surface of undifferentiated pulp cells. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Involvement of cross-reactive carbohydrate determinants-specific IgE in pollen allergy testing

    PubMed Central

    Yoshitake, Hiroshi; Matsumoto, Yuma; Kawada, Michitsugu; Takato, Yoshiki; Shinagawa, Kiyomi; Sakurai, Hiroyuki; Saito, Koichiro

    2017-01-01

    Background Specific IgE antibodies against the low-molecular-weight carbohydrate antigen that does not bridge IgE molecules on mast cells are not associated with clinical symptoms. Cross reactivity can be determined in allergen-specific IgE detection assays when the carbohydrate structures between pollen allergens and plant derived food allergens are similar; in such cases, false positive results for grain or legume allergens can be reported for pollen allergic patients who are not sensitized to those allergens. This phenomenon arises owing to the presence of cross-reactive carbohydrate determinants (CCDs). Objective This study aimed to assess the impact of CCD interference on the results for pollen allergen-specific IgE antibodies in the general adult population and to perform CCD inhibition tests evaluating the involvement of CCD on samples positive to pollen allergens. Methods Serum samples from 322 subjects were tested for IgE antibodies to pollens and CCD. The research subjects were given questionnaires about pollen allergic symptoms to help assess the presence of allergies. Allergen IgE antibodies for Japanese cedar, Japanese cypress, orchard grass, ragweed, MUXF, bromelain, horseradish peroxidase (HRP), and ascorbate oxidase (ASOD) were analyzed. Results It was observed that among individuals who tested positive to any of the pollen allergens, the positive ratio of CCD-specific IgE antibody was the highest for HRP (13.5%–50.0%). The results from the inhibition tests revealed that CCD was marginally present. Although IgE antibodies for cedar pollen did not react with CCD, IgE antibodies for Japanese cypress, orchard grass, and ragweed might be detected by the presence of CCD. Conclusion The results of the inhibition tests revealed the obvious presence of CCD suggesting its involvement. Considering these findings, careful evaluation of patient IgE results should be performed for Japanese cypress, orchard grass, and ragweed. PMID:28154803

  19. Different binding affinities of Pb2+ and Cu2+ to glycosylation variants of human serum transferrin interfere with the detection of carbohydrate-deficient transferrin.

    PubMed

    Luo, Lian-Zhong; Jin, Hong-Wei; Huang, Lin; Huang, He-Qing

    2011-12-01

    Carbohydrate-deficient transferrin (CDT) is a specific biomarker of alcohol abuse, and for diagnosis of chronic alcohol, abuse is often determined using isoelectric focusing (IEF) and chromatographic techniques. To allow this method to be used for the diagnosis of alcohol abuse, inferences of various physical and chemical factors with the detection of CDT have been investigated. However, few reports have focused thus far on whether different metal ions have different binding affinities to CDT and HTf variants or further interfere in the detection of CDT. Here, in order to figure out whether and how metal ions such as Pb(2+) and Cu(2+) bind to holo-human serum transferrin (holo-HTf) and further interfere in CDT detection, the binding characteristics and the binding parameters of holo-HTf with metal ions such as Pb(2+) and Cu(2+) were investigated using UV-visible spectroscopy, Fluorescence spectroscopy, and ICP-MS. Moreover, whether the metal ions such as Pb(2+) and Cu(2+) will reduce the diagnostic accuracy of CDT in clinic was investigated using IEF. The present study demonstrates that Pb(2+) and Cu(2+) have different binding affinities to holo-HTf variants and produce different changes in the relative amounts of each glycosylation isoforms of HTf. Accordingly, the glycosylation chains of HTf will affect the binding affinities of glycosylation isoforms with Pb(2+) and Cu(2+), causing further interferences in CDT detection.

  20. Biochemical and Domain Analyses of FSUAxe6B, a Modular Acetyl Xylan Esterase, Identify a Unique Carbohydrate Binding Module in Fibrobacter succinogenes S85▿ †

    PubMed Central

    Yoshida, Shosuke; Mackie, Roderick I.; Cann, Isaac K. O.

    2010-01-01

    Acetyl xylan esterase (EC 3.1.1.72) is a member of a set of enzymes required to depolymerize hemicellulose, especially xylan that is composed of a main chain of β-1,4-linked xylopyranoside residues decorated with acetyl side groups. Fibrobacter succinogenes S85 Axe6B (FSUAxe6B) is an acetyl xylan esterase encoded in the genome of this rumen bacterium. The enzyme is a modular protein comprised of an esterase domain, a carbohydrate-binding module, and a region of unknown function. Sequences that are homologous to the region of unknown function are paralogously distributed, thus far, only in F. succinogenes. Therefore, the sequences were designated Fibrobacter succinogenes-specific paralogous module 1 (FPm-1). The FPm-1s are associated with at least 24 polypeptides in the genome of F. succinogenes S85. A bioinformatics search showed that most of the FPm-1-appended polypeptides are putative carbohydrate-active enzymes, suggesting a potential role in carbohydrate metabolism. Truncational analysis of FSUAxe6B, together with catalytic and substrate binding studies, has allowed us to delineate the functional modules in the polypeptide. The N-terminal half of FSUAxe6B harbors the activity that cleaves side chain acetyl groups from xylan-like substrates, and the binding of insoluble xylan was determined to originate from FPm-1. Site-directed mutagenesis studies of highly conserved active-site residues in the esterase domain suggested that the esterase activity is derived from a tetrad composed of Ser44, His273, Glu194, and Asp270, with both Glu194 and Asp270 functioning as helper acids, instead of a single carboxylate residue proposed to initiate catalysis. PMID:19897648

  1. Trimethoxybenzene- and trimethylbenzene-based compounds bearing imidazole, indole and pyrrole groups as recognition units: synthesis and evaluation of the binding properties towards carbohydrates.

    PubMed

    Rosien, Jan-Ruven; Seichter, Wilhelm; Mazik, Monika

    2013-10-14

    The aim of the study was to evaluate the potential of trimethoxybenzene- and trimethylbenzene-based compounds bearing imidazole or indole groups as recognition sites in the complexation of carbohydrates. Representatives of these compounds were prepared and their binding properties toward selected carbohydrates evaluated. The results of the binding studies were compared with those obtained for the prepared pyrrole bearing analogues and for the previously described triethylbenzene-based receptors.

  2. Energy Landscape for the Interaction of the Family 1 Carbohydrate-Binding Module and the Cellulose Surface is Altered by Hydrolyzed Glycosidic Bonds

    SciTech Connect

    Bu, L.; Beckham, G. T.; Crowley, M. F.; Chang, C. H.; Matthews, J. F.; Bomble, Y. J.; Adney, W. S.; Himmel, M. E.; Nimlos, M. R.

    2009-01-01

    A multiscale simulation model is used to construct potential and free energy surfaces for the carbohydrate-binding module [CBM] from an industrially important cellulase, Trichoderma reesei cellobiohydrolase I, on the hydrophobic face of a coarse-grained cellulose I{beta} polymorph. We predict from computation that the CBM alone exhibits regions of stability on the hydrophobic face of cellulose every 5 and 10 {angstrom}, corresponding to a glucose unit and a cellobiose unit, respectively. In addition, we predict a new role for the CBM: specifically, that in the presence of hydrolyzed cellulose chain ends, the CBM exerts a thermodynamic driving force to translate away from the free cellulose chain ends. This suggests that the CBM is not only required for binding to cellulose, as has been known for two decades, but also that it has evolved to both assist the enzyme in recognizing a cellulose chain end and exert a driving force on the enzyme during processive hydrolysis of cellulose.

  3. Anti-tumor agent calixarene 0118 targets human galectin-1 as an allosteric inhibitor of carbohydrate binding

    PubMed Central

    Dings, Ruud P.M.; Miller, Michelle C.; Nesmelova, Irina; Astorgues-Xerri, Lucile; Kumar, Nigam; Serova, Maria; Chen, Xuimei; Raymond, Eric; Hoye, Thomas R.; Mayo, Kevin H.

    2012-01-01

    Calix[4]arene compound 0118 is an angiostatic agent that inhibits tumor growth in mice. Although 0118 is a topomimetic of galectin-1-targeting angiostatic amphipathic peptide anginex, we had yet to prove that 0118 targets galectin-1. Galectin-1 is involved in pathological disorders like tumor endothelial cell adhesion and migration and therefore presents a relevant target for therapeutic intervention against cancer. Here, 15N-1H HSQC NMR spectroscopy demonstrates that 0118 indeed targets galectin-1 at a site away from the lectin’s carbohydrate binding site, and thereby attenuates lactose binding to the lectin. Flow cytometry and agglutination assays show that 0118 attenuates binding of galectin-1 to cell surface glycans, and the inhibition of cell proliferation by 0118 is found to be correlated with the cellular expression of the lectin. In general, our data indicate that 0118 targets galectin-1 as an allosteric inhibitor of glycan/carbohydrate binding. This work contributes to the clinical development of anti-tumor calixarene compound 0118. PMID:22575017

  4. Carbohydrate Malabsorption and Putative Carbohydrate-Specific Small Intestinal Bacterial Overgrowth: Prevalence and Diagnostic Overlap Observed in an Austrian Outpatient Center.

    PubMed

    Enko, Dietmar; Kriegshäuser, Gernot; Kimbacher, Christine; Stolba, Robert; Mangge, Harald; Halwachs-Baumann, Gabriele

    2015-01-01

    While lactose malabsorption is a well-investigated condition, few epidemiologic data are available for fructose and sorbitol malabsorption. The aim of this study was to assess the prevalence rates for primary lactose malabsorption, fructose and sorbitol malabsorption, and carbohydrate-specific small intestinal bacterial overgrowth (cs-SIBO) in an Austrian outpatient center. In total, 306 adult patients, who were primarily referred with suspected carbohydrate malabsorption by general practitioners to our outpatient clinic, underwent genetic testing (C/T-13910 polymorphism) for primary lactose malabsorption, and a combined hydrogen (H2)/methane (CH4) breath test for fructose (25 g) and sorbitol (12.5 g) malabsorption. Cohen's kappa (κ) was calculated for agreement between positive breath test results and self-reported symptoms during the test. Seventy-eight (25.49%) patients were C/C-13910 homozygotes, indicating primary lactose malabsorption. Thirty-four (11.11%) and 57 (18.63%) patients were classified as fructose and sorbitol malabsorbers. Cohen's κ measuring agreements between positive fructose and sorbitol breath test results and self-reported symptoms during the test were 0.33 and 0.49, respectively. Twenty-nine (9.50%) patients with an early H2/CH4 peak (i.e. within 60 minutes after fructose and/or sorbitol ingestion) were diagnosed with cs-SIBO. In Austria, carbohydrate malabsorption is a frequent condition in patients referred by general practitioners to carbohydrate malabsorption testing. © 2015 S. Karger AG, Basel.

  5. The type of carbohydrates specifically selects microbial community structures and fermentation patterns.

    PubMed

    Chatellard, Lucile; Trably, Eric; Carrère, Hélène

    2016-12-01

    The impact on dark fermentation of seven carbohydrates as model substrates of lignocellulosic fractions (glucose, cellobiose, microcrystalline cellulose, arabinose, xylose, xylan and wheat straw) was investigated. Metabolic patterns and bacterial communities were characterized at the end of batch tests inoculated with manure digestate. It was found that hydrogen production was linked to the sugar type (pentose or hexose) and the degree of polymerisation. Hexoses produced less hydrogen, with a specific selection of lactate-producing bacterial community structures. Maximal hydrogen production was five times higher on pentose-based substrates, with specific bacterial community structures producing acetate and butyrate as main metabolites. Low hydrogen amounts accumulated from complex sugars (cellulose, xylan and wheat straw). A relatively high proportion of the reads was affiliated to Ruminococcaceae suggesting an efficient hydrolytic activity. Knowing that the bacterial community structure is very specific to a particular substrate offers new possibilities to design more efficient H2-producing biological systems.

  6. Species-specificity of amphibia carbohydrate chains: the Bufo viridis case study.

    PubMed

    Coppin, Alexandra; Maes, Emmanuel; Strecker, Gérard

    2002-02-05

    The jelly coat surrounding the eggs of amphibia is composed of oviducal mucins and plays an important role in the fertilization process. From a structural and chemical point of view, these jellies are very different from one species to another. Bufo viridis is the 13th amphibia species studied in term of carbohydrate structural analysis. The oligosaccharides have been released from the oviducal mucins by reductive beta elimination, purified by various chromatography procedures and analyzed by (1)H and (13)C 1D-2D NMR spectroscopy. Among the 15 compounds, ten have novel structures, although they possess some well-known structural patterns as blood group epitopes (Le(x), Le(y)) or other sequences already observed in other amphibia species. These results reinforce our hypothesis about the strict species-specificity of these carbohydrate chains. It must be noted that such species-specificity does not depend on one particular monosaccharide but it is rather due to a set of particular tri- or tetrasaccharide sequences. Hence, B. viridis species could be characterized by the simultaneous presence of a 2,3,6-trisubstituted galactosyl residue, the GlcNAc(beta 1-3)[Fuc(alpha 1-4)]GlcNAc beta sequence and the Le(x), Le(y) or Cad determinants. The anionic charge of the oligosaccharides is carried only by sialic acid alpha-(2-->6)-linked to GalNAc-ol residue as in Bufo bufo or in Bufo arenarum.

  7. Domain Analysis of a Modular α-l-Arabinofuranosidase with a Unique Carbohydrate Binding Strategy from the Fiber-Degrading Bacterium Fibrobacter succinogenes S85 ▿ †

    PubMed Central

    Yoshida, Shosuke; Hespen, Charles W.; Beverly, Robert L.; Mackie, Roderick I.; Cann, Isaac K. O.

    2010-01-01

    Family 43 glycoside hydrolases (GH43s) are known to exhibit various activities involved in hemicellulose hydrolysis. Thus, these enzymes contribute to efficient plant cell wall degradation, a topic of much interest for biofuel production. In this study, we characterized a unique GH43 protein from Fibrobacter succinogenes S85. The recombinant protein showed α-l-arabinofuranosidase activity, specifically with arabinoxylan. The enzyme is, therefore, an arabinoxylan arabinofuranohydrolase (AXH). The F. succinogenes AXH (FSUAXH1) is a modular protein that is composed of a signal peptide, a GH43 catalytic module, a unique β-sandwich module (XX domain), a family 6 carbohydrate-binding module (CBM6), and F. succinogenes-specific paralogous module 1 (FPm-1). Truncational analysis and site-directed mutagenesis of the protein revealed that the GH43 domain/XX domain constitute a new form of carbohydrate-binding module and that residue Y484 in the XX domain is essential for binding to arabinoxylan, although protein structural analyses may be required to confirm some of the observations. Kinetic studies demonstrated that the Y484A mutation leads to a higher kcat for a truncated derivative of FSUAXH1 composed of only the GH43 catalytic module and the XX domain. However, an increase in the Km for arabinoxylan led to a 3-fold decrease in catalytic efficiency. Based on the knowledge that most XX domains are found only in GH43 proteins, the evolutionary relationships within the GH43 family were investigated. These analyses showed that in GH43 members with a XX domain, the two modules have coevolved and that the length of a loop within the XX domain may serve as an important determinant of substrate specificity. PMID:20709893

  8. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* carbohydrate-binding protein of the human rotavirus strain Wa

    SciTech Connect

    Kraschnefski, Mark J.; Scott, Stacy A.; Holloway, Gavan; Coulson, Barbara S.; Itzstein, Mark von; Blanchard, Helen

    2005-11-01

    The carbohydrate-binding component (VP8*{sub 64–223}) of the human Wa rotavirus spike protein has been overexpressed in E. coli, purified and crystallized in two different crystal forms. X-ray diffraction data have been collected that have enabled determination of the Wa VP8*{sub 64–223} structure by molecular replacement. Rotaviruses exhibit host-specificity and the first crystallographic information on a rotavirus strain that infects humans is reported here. Recognition and attachment to host cells, leading to invasion and infection, is critically linked to the function of the outer capsid spike protein of the rotavirus particle. In some strains the VP8* component of the spike protein is implicated in recognition and binding of sialic-acid-containing cell-surface carbohydrates, thereby enabling infection by the virus. The cloning, expression, purification, crystallization and initial X-ray diffraction analysis of the VP8* core from human Wa rotavirus is reported. Two crystal forms (trigonal P3{sub 2}21 and monoclinic P2{sub 1}) have been obtained and X-ray diffraction data have been collected, enabling determination of the VP8*{sub 64–223} structure by molecular replacement.

  9. Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

    SciTech Connect

    Su, Xiaoyun; Agarwal, Vinayak; Dodd, Dylan; Bae, Brian; Mackie, Roderick I.; Nair, Satish K.; Cann, Isaac K.O.

    2010-11-22

    Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.

  10. Fiber type-specific muscle glycogen sparing due to carbohydrate intake before and during exercise.

    PubMed

    De Bock, K; Derave, W; Ramaekers, M; Richter, E A; Hespel, P

    2007-01-01

    The effect of carbohydrate intake before and during exercise on muscle glycogen content was investigated. According to a randomized crossover study design, eight young healthy volunteers (n = 8) participated in two experimental sessions with an interval of 3 wk. In each session subjects performed 2 h of constant-load bicycle exercise ( approximately 75% maximal oxygen uptake). On one occasion (CHO), they received carbohydrates before ( approximately 150 g) and during (1 g.kg body weight(-1).h(-1)) exercise. On the other occasion they exercised after an overnight fast (F). Fiber type-specific relative glycogen content was determined by periodic acid Schiff staining combined with immunofluorescence in needle biopsies from the vastus lateralis muscle before and immediately after exercise. Preexercise glycogen content was higher in type IIa fibers [9.1 +/- 1 x 10(-2) optical density (OD)/microm(2)] than in type I fibers (8.0 +/- 1 x 10(-2) OD/microm(2); P < 0.0001). Type IIa fiber glycogen content decreased during F from 9.6 +/- 1 x 10(-2) OD/microm(2) to 4.5 +/- 1 x 10(-2) OD/microm(2) (P = 0.001), but it did not significantly change during CHO (P = 0.29). Conversely, in type I fibers during CHO and F the exercise bout decreased glycogen content to the same degree. We conclude that the combination of carbohydrate intake both before and during moderate- to high-intensity endurance exercise results in glycogen sparing in type IIa muscle fibers.

  11. Verticillium dahliae manipulates plant immunity by glycoside hydrolase 12 proteins in conjunction with carbohydrate-binding module 1.

    PubMed

    Gui, Yue-Jing; Chen, Jie-Yin; Zhang, Dan-Dan; Li, Nan-Yang; Li, Ting-Gang; Zhang, Wen-Qi; Wang, Xin-Yan; Short, Dylan P G; Li, Lei; Guo, Wei; Kong, Zhi-Qiang; Bao, Yu-Ming; Subbarao, Krishna V; Dai, Xiao-Feng

    2017-05-01

    Glycoside hydrolase 12 (GH12) proteins act as virulence factors and pathogen-associated molecular patterns (PAMPs) in oomycetes. However, the pathogenic mechanisms of fungal GH12 proteins have not been characterized. In this study, we demonstrated that two of the six GH12 proteins produced by the fungus Verticillium dahliae Vd991, VdEG1 and VdEG3 acted as PAMPs to trigger cell death and PAMP-triggered immunity (PTI) independent of their enzymatic activity in Nicotiana benthamiana. A 63-amino-acid peptide of VdEG3 was sufficient for cell death-inducing activity, but this was not the case for the corresponding peptide of VdEG1. Further study indicated that VdEG1 and VdEG3 trigger PTI in different ways: BAK1 is required for VdEG1- and VdEG3-triggered immunity, while SOBIR1 is specifically required for VdEG1-triggered immunity in N. benthamiana. Unlike oomycetes, which employ RXLR effectors to suppress host immunity, a carbohydrate-binding module family 1 (CBM1) protein domain suppressed GH12 protein-induced cell death. Furthermore, during infection of N. benthamiana and cotton, VdEG1 and VdEG3 acted as PAMPs and virulence factors, respectively indicative of host-dependent molecular functions. These results suggest that VdEG1 and VdEG3 associate differently with BAK1 and SOBIR1 receptor-like kinases to trigger immunity in N. benthamiana, and together with CBM1-containing proteins manipulate plant immunity. © 2017 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

  12. Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d

    PubMed Central

    2010-01-01

    Background Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB) for coplanar aromatic motifs similar to those found in known glycan-binding proteins. Results The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO) enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192) in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry. Conclusions Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure. PMID:20678238

  13. Monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-HIV activity

    SciTech Connect

    Moulaei, Tinoush; Shenoy, Shilpa R.; Giomarelli, Barbara; Thomas, Cheryl; McMahon, James B.; Dauter, Zbigniew; O'Keefe, Barry R.; Wlodawer, Alexander

    2010-10-28

    Mutations were introduced to the domain-swapped homodimer of the antiviral lectin griffithsin (GRFT). Whereas several single and double mutants remained dimeric, insertion of either two or four amino acids at the dimerization interface resulted in a monomeric form of the protein (mGRFT). Monomeric character of the modified proteins was confirmed by sedimentation equilibrium ultracentrifugation and by their high resolution X-ray crystal structures, whereas their binding to carbohydrates was assessed by isothermal titration calorimetry. Cell-based antiviral activity assays utilizing different variants of mGRFT indicated that the monomeric form of the lectin had greatly reduced activity against HIV-1, suggesting that the antiviral activity of GRFT stems from crosslinking and aggregation of viral particles via multivalent interactions between GRFT and oligosaccharides present on HIV envelope glycoproteins. Atomic resolution crystal structure of a complex between mGRFT and nonamannoside revealed that a single mGRFT molecule binds to two different nonamannoside molecules through all three carbohydrate-binding sites present on the monomer.

  14. Monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-HIV activity.

    PubMed

    Moulaei, Tinoush; Shenoy, Shilpa R; Giomarelli, Barbara; Thomas, Cheryl; McMahon, James B; Dauter, Zbigniew; O'Keefe, Barry R; Wlodawer, Alexander

    2010-09-08

    Mutations were introduced to the domain-swapped homodimer of the antiviral lectin griffithsin (GRFT). Whereas several single and double mutants remained dimeric, insertion of either two or four amino acids at the dimerization interface resulted in a monomeric form of the protein (mGRFT). Monomeric character of the modified proteins was confirmed by sedimentation equilibrium ultracentrifugation and by their high resolution X-ray crystal structures, whereas their binding to carbohydrates was assessed by isothermal titration calorimetry. Cell-based antiviral activity assays utilizing different variants of mGRFT indicated that the monomeric form of the lectin had greatly reduced activity against HIV-1, suggesting that the antiviral activity of GRFT stems from crosslinking and aggregation of viral particles via multivalent interactions between GRFT and oligosaccharides present on HIV envelope glycoproteins. Atomic resolution crystal structure of a complex between mGRFT and nonamannoside revealed that a single mGRFT molecule binds to two different nonamannoside molecules through all three carbohydrate-binding sites present on the monomer.

  15. Polycomb silencing mediated by specific DNA-binding recruiters.

    PubMed

    March, Eduardo; Farrona, Sara

    2017-09-27

    Regulation of epigenetic factors through their recruitment to specific genomic regions is still poorly understood. A recent study demonstrates a global mechanism of tethering Polycomb group (PcG) proteins through sequence-specific DNA-binding factors.

  16. Effect of carbohydrate structure on the binding of ligands to the hepatic galactose/N-acetylgalactosamine lectin of mammalian hepatocytes

    SciTech Connect

    Hardy, M.R.

    1987-01-01

    A hierarchy of affinities, monoantennary << biantennary << triantennary approx. tetraantennary, was observed when a series of branched carbohydrates were tested for their ability to inhibit binding of /sup 125/I-asialoorosomucoid (/sup 125/I-ASOR) or /sup 125/I-Tyr-asialo-triantennary glycopeptide from ..cap alpha..-1-protease inhibitor (/sup 125/I-..cap alpha..-1-PI TRI) to rabbit hepatocytes. The difference in affinity between a monoantennary ligand and a triantennary ligand was one million fold, with affinity of ..cap alpha..-1-PI being ca. 1 nM. It was proposed that the cell surface high affinity oligosaccharide binding unit consists of three GAl-binding sites at the vertices of a triangle of sides 15, 22, and 25 A. Secondly, the binding properties of ASOR, ..cap alpha..-1-PI TRI, and ..cap alpha.., ..beta..-diaspartamide of tris ((..beta..-lactosyloxy)methyl)(6-aminohexanamido)-methane (di-tris-lac) to isolated rabbit hepatocytes were compared. A model was developed that explained the different binding properties of the ligands on the basis of a two-dimensional lattice of trivalent clusters of Gal-binding sites. Thirdly, TRI glycopeptides from bovine fetuin were purified and characterized.

  17. Carbohydrate specificity of the recognition of diverse glycolipids by natural killer T cells.

    PubMed

    Zajonc, Dirk M; Kronenberg, Mitchell

    2009-07-01

    Most T lymphocytes recognize peptide antigens bound to or presented by molecules encoded in the major histocompatibility complex (MHC). The CD1 family of antigen-presenting molecules is related to the MHC-encoded molecules, but CD1 proteins present lipid antigens, mostly glycolipids. Here we review T-lymphocyte recognition of glycolipids, with particular emphasis on the subpopulation known as natural killer T (NKT) cells. NKT cells influence many immune responses, they have a T-cell antigen receptor (TCR) that is restricted in diversity, and they share properties with cells of the innate immune system. NKT cells recognize antigens presented by CD1d with hexose sugars in alpha-linkage to lipids, although other, related antigens are known. The hydrophobic alkyl chains are buried in the CD1d groove, with the carbohydrate exposed for TCR recognition, together with the surface of the CD1d molecule. Therefore, understanding the biochemical basis for antigen recognition by NKT cells requires an understanding of how the trimolecular complex of CD1d, glycolipid, and the TCR is formed, which is in part a problem of carbohydrate recognition by the TCR. Recent investigations from our laboratories as well as studies from other groups have provided important information on the structural basis for NKT-cell specificity.

  18. Specific albumin binding to microvascular endothelium in culture

    SciTech Connect

    Schnitzer, J.E.; Carley, W.W.; Palade, G.E. )

    1988-03-01

    The specific binding of rat serum albumin (RSA) to confluent microvascular endothelial cells in culture derived from the vasculature of the rat epididymal fat pad was studied at 4{degree}C by radioassay and immunocytochemistry. Radioiodinated RSA ({sup 125}I-RSA) binding to the cells reached equilibrium at {approximately} 20 min incubation. Albumin binding was a slowly saturating function over concentrations ranging from 0.01 to 50 mg/ml. Specific RSA binding with a moderate apparent affinity constant of 1.0 mg/ml and with a maximum binding concentration of 90 ng/cm{sup 2} was immunolocalized with anti-RSA antibody to the outer (free) side of the enothelium. Scatchard analysis of the binding yielded a nonlinear binding curve with a concave-upward shape. Dissociation rate analysis supports negative cooperativity of albumin binding, but multiple binding sites may also be present. Albumin binding fulfilled many requirements for ligand specificity including saturability, reversibility, competibility, and dependence on both cell type and cell number. The results are discussed in terms of past in situ investigations on the localization of albumin binding to vascular endothelium and its effect on transendothelial molecular transport.

  19. Nucleic acid-binding specificity of human FUS protein

    PubMed Central

    Wang, Xueyin; Schwartz, Jacob C.; Cech, Thomas R.

    2015-01-01

    FUS, a nuclear RNA-binding protein, plays multiple roles in RNA processing. Five specific FUS-binding RNA sequence/structure motifs have been proposed, but their affinities for FUS have not been directly compared. Here we find that human FUS binds all these sequences with Kdapp values spanning a 10-fold range. Furthermore, some RNAs that do not contain any of these motifs bind FUS with similar affinity. FUS binds RNA in a length-dependent manner, consistent with a substantial non-specific component to binding. Finally, investigation of FUS binding to different nucleic acids shows that it binds single-stranded DNA with three-fold lower affinity than ssRNA of the same length and sequence, while binding to double-stranded nucleic acids is weaker. We conclude that FUS has quite general nucleic acid-binding activity, with the various proposed RNA motifs being neither necessary for FUS binding nor sufficient to explain its diverse binding partners. PMID:26150427

  20. Significant conformational changes in an antigenic carbohydrate epitope upon binding to a monoclonal antibody

    SciTech Connect

    Glaudemans, C.P.J.; Lerner, L.; Daves, G.D. Jr.; Kovac, P.; Bax, A. ); Venable, R. )

    1990-12-01

    Transferred nulcear Overhauser enhancement spectroscopy (TRNOE) was used to observe changes in a ligand's conformation upon binding to its specific antibody. The ligands studied were methyl O-{beta}-D-galactopyranosyl(1{yields}6)-4-deoxy-4-fluoro-{beta}-D galactopyranoside (me4FGal{sub 2}) and its selectively deuteriated analogue, methyl O-{beta}-D-galactopyranosyl(1{yields}6)-4-deoxy-2-deuterio-4-fluoro-{beta}-D-galactopyranoside (me4F2dGal{sub 2}). The monoclonal antibody was mouse IgA X24. The solution conformation of the free ligand me4F2dGal{sub 2} was inferred from measurements of vicinal {sup 1}H-{sup 1}H coupling constants, long-range {sup 1}H-{sup 13}C coupling constants, and NOE cross-peak intensities. For free ligand, both galactosyl residues adopt a regular chair conformation, but the NMR spectra are incompatible with a single unique conformation of the glycosidic linkage. Analysis of {sup 1}H-{sup 1}H and {sup 1}H-{sup 13}C constants indicates that the major conformer has an extended conformation. TRNOE measurements on me4FGal{sub 2} and me4F2dGal{sub 2} in the presence of the specific antibody indicate that the pyranose ring pucker of each galactose ring remains unchanged, but rotations about the glycosidic linkage occur upon binding to X24. Computer calculations indicate that there are two sets of torsion angles that satisfy the observed NMR constraints. A new method, based on changes in the fluorine longitudinal relaxation rate, is used to measure the ligand-antibody dissociation rate constant.

  1. The impact of Trichoderma reesei Cel7A carbohydrate binding domain mutations on its binding to a cellulose surface: a molecular dynamics free energy study.

    PubMed

    Li, Tong; Yan, Shihai; Yao, Lishan

    2012-04-01

    A critical role of the Family 7 cellobiohydrolase (Cel7A) carbohydrate binding domain (CBD) is to bind to a cellulose surface and increase the enzyme concentration on the surface. Several residues of Trichoderma reesei Cel7A CBD, including Y5, N29, Y31, Y32 and Q34, contribute to cellulose binding, as revealed by early experimental studies. To investigate the interactions between these important residues and cellulose, we applied a thermodynamic integration method to calculate the cellulose-Cel7A CBD binding free energy changes caused by Y5A, N29A, Y31A, Y32A and Q34A mutations. The experimental binding trend was successfully predicted, proving the effectiveness of the complex model. For the two polar residue mutants N29A and Q34A, the changes in the electrostatics are comparable to those of van der Waals, while for three Y to A mutants, the free energy differences mainly come from van der Waals interactions. However, in both cases, the electrostatics dominates the interactions between individual residues and cellulose. The side chains of these residues are rigidified after the complex is formed. The binding free energy changes for the two mutants Y5W and Y31W were also determined, and for these the van der Waals interaction was strengthened but the electrostatics was weakened.

  2. Two additional carbohydrate-binding sites of beta-amylase from Bacillus cereus var. mycoides are involved in hydrolysis and raw starch-binding.

    PubMed

    Ye, Zhengmao; Miyake, Hideo; Tatsumi, Maki; Nishimura, Shigenori; Nitta, Yasunori

    2004-03-01

    In the previous X-ray crystallographic study, it was found that beta-amylase from Bacillus cereus var. mycoides has three carbohydrate-binding sites aside from the active site: two (Site2 and Site3) in domain B and one (Site1) in domain C. To investigate the roles of these sites in the catalytic reaction and raw starch-binding, Site1 and Site2 were mutated. From analyses of the raw starch-binding of wild-type and mutant enzymes, it was found that Site1 contributes to the binding affinity to raw-starch more than Site2, and that the binding capacity is maintained when either Site1 or Site2 exists. The raw starch-digesting ability of this enzyme was poor. From inhibition studies by maltitol, GGX and alpha-CD for hydrolyses of maltopentaose (G5) and amylose ( (n) = 16) catalyzed by wild-type and mutant enzymes, it was found that alpha-CD is a competitive inhibitor, while, maltitol behaves as a mixed-type or competitive inhibitor depending on the chain length of the substrate and the mutant enzyme. From the analysis of the inhibition mechanism, we conclude that the bindings of maltitol and GGX to Site2 in domain B form an abortive ESI complex when amylose ( (n) = 16) is used as a substrate.

  3. Tryptophan introduction can change β-glucan binding ability of the carbohydrate-binding module of endo-1,3-β-glucanase.

    PubMed

    Miki, Ayako; Inaba, Satomi; Maruno, Takahiro; Kobayashi, Yuji; Oda, Masayuki

    2017-05-01

    Endo-1,3-β-glucanase from Cellulosimicrobium cellulans DK-1 has a carbohydrate-binding module (CBM-DK) at the C-terminal side of a catalytic domain. Out of the imperfect tandem α-, β-, and γ-repeats in CBM-DK, the α-repeat primarily contributes to β-glucan binding. This unique feature is derived from Trp273 in α-repeat, whose corresponding residues in β- and γ-repeats are Asp314 and Gly358, respectively. In this study, we generated Trp-switched mutants, W273A/D314W, D270A/W273A/D314W, W273A/G358W, and D270A/W273A/G358W, and analyzed their binding abilities toward laminarioligosaccharides and laminarin. While the binding affinities of D270A/W273A and W273A mutants were either lost or much lower than that of the wild-type, those of Trp-switched mutants recovered, indicating that a Trp introduction in β- or γ-repeat can substitute the α-repeat by primarily contributing to β-glucan binding. Thus, we have successfully engineered a CBM-DK that binds to laminarin by a mechanism different from that of the wild-type, but with similar affinity.

  4. Cation specific binding with protein surface charges

    PubMed Central

    Hess, Berk; van der Vegt, Nico F. A.

    2009-01-01

    Biological organization depends on a sensitive balance of noncovalent interactions, in particular also those involving interactions between ions. Ion-pairing is qualitatively described by the law of “matching water affinities.” This law predicts that cations and anions (with equal valence) form stable contact ion pairs if their sizes match. We show that this simple physical model fails to describe the interaction of cations with (molecular) anions of weak carboxylic acids, which are present on the surfaces of many intra- and extracellular proteins. We performed molecular simulations with quantitatively accurate models and observed that the order K+ < Na+ < Li+ of increasing binding affinity with carboxylate ions is caused by a stronger preference for forming weak solvent-shared ion pairs. The relative insignificance of contact pair interactions with protein surfaces indicates that thermodynamic stability and interactions between proteins in alkali salt solutions is governed by interactions mediated through hydration water molecules. PMID:19666545

  5. Analysis of selected blood and immune cell responses to carbohydrate-dependent surface binding of proto- and chimera-type galectins.

    PubMed

    Timoshenko, Alexander V; Gorudko, Irina V; Maslakova, Olga V; André, Sabine; Kuwabara, Ichiro; Liu, Fu-Tong; Kaltner, Herbert; Gabius, Hans-Joachim

    2003-08-01

    Cell surface glycans present docking sites to endogenous lectins. With growing insight into the diversity of lectin families it becomes important to answer the question on the activity profiles of individual family members. Focusing on galectins (beta-galactoside-binding proteins without Ca(2+)-requirement sharing the jelly-roll-like folding pattern), this study was performed to assess the potency of proto-type galectins (galectins-1 and -7 and CG-16) and the chimera-type galectin-3 to elicit selected cell responses by carbohydrate-dependent surface binding and compare the results. The galectins, except for galectin-1, were found to enhance detergent (SDS)-induced hemolysis of human erythrocytes to different degrees. Their ability to confer increased membrane osmofragility thus differs. Aggregation of neutrophils, thymocytes and platelets was induced by the proto-type galectin-1 but not -7, by CG-16 and also galectin-3. Cell-type-specific quantitative differences and the importance of the fine-specificity of the galectin were clearly apparent. In order to detect cellular responses based on galectin binding and bridging of cells the formation of haptenic-sugar-resistant (HSR) intercellular contacts (an indicator of post-binding signaling) was monitored. It was elicited by CG-16 and galectin-1 but not galectin-3, revealing another level at which activities of individual galectins can differ. Acting as potent elicitor of neutrophil aggregation, CG-16-dependent post-binding effects were further analyzed. Carbohydrate-dependent binding to the neutrophils' surface led to a sustained increase of cytoplasmic Ca2+ concentration in a dose-dependent manner. The ability of CG-16 to activate H2O2 generation by human peripheral blood neutrophils was primed by the Ca(2+)-ionophor ionomycin and by cytochalasin B. In a general context, these results emphasize that--besides plant lectins as laboratory tools--animal lectins can trigger cell reaction cascades, implying potential in

  6. Structural Insights into the Carbohydrate Binding Ability of an α-(1→2) Branching Sucrase from Glycoside Hydrolase Family 70*

    PubMed Central

    Brison, Yoann; Malbert, Yannick; Czaplicki, Georges; Mourey, Lionel; Remaud-Simeon, Magali; Tranier, Samuel

    2016-01-01

    The α-(1→2) branching sucrase ΔN123-GBD-CD2 is a transglucosylase belonging to glycoside hydrolase family 70 (GH70) that catalyzes the transfer of d-glucosyl units from sucroseto dextrans or gluco-oligosaccharides via the formation of α-(1→2) glucosidic linkages. The first structures of ΔN123-GBD-CD2 in complex with d-glucose, isomaltosyl, or isomaltotriosyl residues were solved. The glucose complex revealed three glucose-binding sites in the catalytic gorge and six additional binding sites at the surface of domains B, IV, and V. Soaking with isomaltotriose or gluco-oligosaccharides led to structures in which isomaltosyl or isomaltotriosyl residues were found in glucan binding pockets located in domain V. One aromatic residue is systematically identified at the bottom of these pockets in stacking interaction with one glucosyl moiety. The carbohydrate is also maintained by a network of hydrogen bonds and van der Waals interactions. The sequence of these binding pockets is conserved and repeatedly present in domain V of several GH70 glucansucrases known to bind α-glucans. These findings provide the first structural evidence of the molecular interaction occurring between isomalto-oligosaccharides and domain V of the GH70 enzymes. PMID:26865636

  7. Structural Insights into the Carbohydrate Binding Ability of an α-(1→2) Branching Sucrase from Glycoside Hydrolase Family 70.

    PubMed

    Brison, Yoann; Malbert, Yannick; Czaplicki, Georges; Mourey, Lionel; Remaud-Simeon, Magali; Tranier, Samuel

    2016-04-01

    The α-(1→2) branching sucrase ΔN123-GBD-CD2 is a transglucosylase belonging to glycoside hydrolase family 70 (GH70) that catalyzes the transfer ofd-glucosyl units from sucroseto dextrans or gluco-oligosaccharides via the formation of α-(1→2) glucosidic linkages. The first structures of ΔN123-GBD-CD2 in complex withd-glucose, isomaltosyl, or isomaltotriosyl residues were solved. The glucose complex revealed three glucose-binding sites in the catalytic gorge and six additional binding sites at the surface of domains B, IV, and V. Soaking with isomaltotriose or gluco-oligosaccharides led to structures in which isomaltosyl or isomaltotriosyl residues were found in glucan binding pockets located in domain V. One aromatic residue is systematically identified at the bottom of these pockets in stacking interaction with one glucosyl moiety. The carbohydrate is also maintained by a network of hydrogen bonds and van der Waals interactions. The sequence of these binding pockets is conserved and repeatedly present in domain V of several GH70 glucansucrases known to bind α-glucans. These findings provide the first structural evidence of the molecular interaction occurring between isomalto-oligosaccharides and domain V of the GH70 enzymes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Structure binding relationship of galactosylated Glycoclusters toward Pseudomonas aeruginosa lectin LecA using a DNA-based carbohydrate microarray.

    PubMed

    Gerland, Béatrice; Goudot, Alice; Ligeour, Caroline; Pourceau, Gwladys; Meyer, Albert; Vidal, Sébastien; Gehin, Thomas; Vidal, Olivier; Souteyrand, Eliane; Vasseur, Jean-Jacques; Chevolot, Yann; Morvan, François

    2014-02-19

    Pseudomonas aeruginosa (PA) is a major public health issue due to its impact on nosocomial infections as well as its impact on cystic fibrosis patient mortality. One of the main concerns is its ability to develop antibiotic resistance. Therefore, inhibition of PA virulence has been proposed as an alternative strategy to tackle PA based infections. LecA (or PA-IL), a galactose binding lectin from PA, is involved in its virulence. Herein, we aimed at designing high affinity synthetic ligands toward LecA for its inhibition and at understanding the key parameters governing the binding of multivalent galactosylated clusters. Twenty-five glycoclusters were synthesized and their bindings were studied on a carbohydrate microarray. Monosaccharide centered clusters and linear comb-like clusters were synthesized with different linkers separating the core and the galactosyl residues. Their length, flexibility, and aromaticity were varied. Our results showed that the binding profile of LecA to galactosylated clusters was dependent on both the core and the linker and also that the optimal linker was different for each core. Nevertheless, an aryl group in the linker structure drastically improved the binding to LecA. Our results also suggest that optimal distances are preferred between the core and the aromatic group and the core and the galactose.

  9. Simulation analysis of the cellulase Cel7A carbohydrate binding module on the surface of the cellulose Iβ

    SciTech Connect

    Alekozai, Emal M.; GhattyVenkataKrishna, Pavan K.; Uberbacher, Edward C.; Crowley, Michael F.; Smith, Jeremy C.; Cheng, Xiaolin

    2013-08-22

    The Family 7 cellobiohydrolase (Cel7A) from Trichoderma reesei consists of a carbohydrate-binding module (CBM) joined by a linker to a catalytic domain. Cellulose hydrolysis is limited by the accessibility of Cel7A to crystalline substrates, which is perceived to be primarily mediated by the CBM. The binding of CBM to the cellulose I fiber is characterized by combined Brownian dynamics (BD) and molecular dynamics (MD) simulations. Our results confirm that CBM prefers to dock to the hydrophobic than to the hydrophilic fiber faces. Both electrostatic (ES) and van der Waals (VDW) interactions are required for achieving the observed binding preference. The VDW interactions play a more important role in stabilizing the CBM-fiber binding, whereas the ES interactions contribute through the formation of a number of hydrogen bonds between the CBM and the fiber. At long distances, an ES steering effect is also observed that tends to align the CBM in an antiparallel manner relative to the fiber axis. Moreover, the MD results reveal hindered diffusion of the CBM on all fiber surfaces. The binding of the CBM to the hydrophobic surfaces is found to involve partial dewetting at the CBM-fiber interface coupled with local structural arrangements of the protein. The present simulation results complement and rationalize a large body of previous work and provide detailed insights into the mechanism of the CBM-cellulose fiber interactions.

  10. Carbohydrate bioavailability.

    PubMed

    Englyst, Klaus N; Englyst, Hans N

    2005-07-01

    There is consensus that carbohydrate foods, in the form of fruit, vegetables and whole-grain products, are beneficial to health. However, there are strong indications that highly processed, fibre-depleted, and consequently rapidly digestible, energy-dense carbohydrate food products can lead to over-consumption and obesity-related diseases. Greater attention needs to be given to carbohydrate bioavailability, which is determined by the chemical identity and physical form of food. The objective of the present concept article is to provide a rational basis for the nutritional characterisation of dietary carbohydrates. Based on the properties of carbohydrate foods identified to be of specific relevance to health, we propose a classification and measurement scheme that divides dietary carbohydrates into glycaemic carbohydrates (digested and absorbed in the small intestine) and non-glycaemic carbohydrates (enter the large intestine). The glycaemic carbohydrates are characterised by sugar type, and by the likely rate of digestion described by in vitro measurements for rapidly available glucose and slowly available glucose. The main type of non-glycaemic carbohydrates is the plant cell-wall NSP, which is a marker of the natural fibre-rich diet recognised as beneficial to health. Other non-glycaemic carbohydrates include resistant starch and the resistant short-chain carbohydrates (non-digestible oligosaccharides), which should be measured and researched in their own right. The proposed classification and measurement scheme is complementary to the dietary fibre and glycaemic index concepts in the promotion of healthy diets with low energy density required for combating obesity-related diseases.

  11. Chemical and photophysical mechanism of fluorescence enhancement of 3-quinolineboronic acid upon change of pH and binding with carbohydrates.

    PubMed

    Shen, Qian Jin; Jin, Wei Jun

    2011-01-01

    The free 3-quinolineboronic acid (3-QBA) with the lowest (n-π*) excited singlet is non- or weakly fluorescent while protonated 3-QBA has the lowest (π-π*) excited singlet state and is highly fluorescent. The hybridization of boronic atom or charge transfer from aromatic ring to boronic acid group plays a secondary role in affecting fluorescence intensity. Binding with carbohydrate at a proper acidity, the hybridization of boron atom changes from sp(2) to sp(3) and the nitrogen atom in the quinoline ring is partially protonated, resulting in large enhancement of fluorescence. Meanwhile, the fluorescent lifetime of 3-QBA produces obvious change by binding with carbohydrates. Quinoline boronic acid is an important water-soluble fluorescence sensor for carbohydrate recognition. Both the remarkable changes in intensity and lifetime of 3-QBA can act as working parameters in recognition of carbohydrates at physiological pH.

  12. Specific binding of beta-endorphin to normal human erythrocytes

    SciTech Connect

    Chenet, B.; Hollis, V. Jr.; Kang, Y.; Simpkins, C.

    1986-03-05

    Beta-endorphin (BE) exhibits peripheral functions which may not be mediated by interactions with receptors in the brain. Recent studies have demonstrated binding of BE to both opioid and non-opioid receptors on lymphocytes and monocytes. Abood has reported specific binding of /sup 3/H-dihydromorphine in erythrocytes. Using 5 x 10/sup -11/M /sup 125/I-beta-endorphin and 10/sup -5/M unlabeled BE, they have detected 50% specific binding to human erythrocytes. This finding is supported by results from immunoelectron microscopy using rabbit anti-BE antibody and biotinylated secondary antibody with avidin-biotin complexes horseradish peroxidase. Binding is clearly observed and is confined to only one side of the cells. Conclusions: (1) BE binding to human erythrocytes was demonstrated by radioreceptor assay and immunoelectron microscopy, and (2) BE binding sites exist on only one side of the cells.

  13. THE INHERITANCE OF INDIVIDUAL ANTIGENIC SPECIFICITIES OF RABBIT ANTIBODIES TO STREPTOCOCCAL CARBOHYDRATES

    PubMed Central

    Eichmann, Klaus; Kindt, Thomas J.

    1971-01-01

    The inheritance of individual antigenic specificities (IAS) of rabbit antibodies to the Group C streptococcal carbohydrate was demonstrated in a selectively bred rabbit family. The IAS of the antibodies from 3 proband rabbits were also observed in the Group C antibodies in as many as 7 out of 42 related rabbits, but in none of the Group C antibodies from 48 unrelated rabbits, Immunodiffusion analyses and quantitative radioprecipitin experiments revealed that this cross-specificity may be either partial or complete. Quantitative inhibition of the precipitin reaction between the proband antibody and its antiserum by preimmune IgG revealed 30-fold differences in the proportion of molecules with cross-specificity for the proband antibody. This proportion is higher in the preimmune IgG of the proband rabbit and of those relatives which produced cross-precipitating antibodies than it is in the IgG of rabbits which had the same group a allotype, but did not produce cross-precipitating antibodies. The proportion is much lower in the IgG of rabbits with a group a allotype different from that of the proband antibody. These data suggest that serologically detected individual antigenic specificities are inherited markers of immunoglobulins. PMID:4104426

  14. Processing Binding Relations in Specific Language Impairment

    ERIC Educational Resources Information Center

    Schwartz, Richard G.; Hestvik, Arild; Seiger-Gardner, Liat; Almodovar, Diana

    2016-01-01

    Purpose: This sentence processing experiment examined the abilities of children with specific language impairment (SLI) and children with typical language development (TD) to establish relations between pronouns or reflexives and their antecedents in real time. Method: Twenty-two children with SLI and 24 age-matched children with TD (7;3-10;11…

  15. Processing Binding Relations in Specific Language Impairment

    ERIC Educational Resources Information Center

    Schwartz, Richard G.; Hestvik, Arild; Seiger-Gardner, Liat; Almodovar, Diana

    2016-01-01

    Purpose: This sentence processing experiment examined the abilities of children with specific language impairment (SLI) and children with typical language development (TD) to establish relations between pronouns or reflexives and their antecedents in real time. Method: Twenty-two children with SLI and 24 age-matched children with TD (7;3-10;11…

  16. Examining the response of larch needle carbohydrates to climate using compound-specific δ13C and concentration analyses

    NASA Astrophysics Data System (ADS)

    Rinne, Katja T.; Saurer, Matthias; Kirdyanov, Alexander V.; Bryukhanova, Marina V.; Prokushkin, Anatoly S.; Churakova Sidorova, Olga V.; Siegwolf, Rolf T. W.

    2016-04-01

    Little is known about the dynamics of concentrations and carbon isotope ratios of individual carbohydrates in leaves in response to climatic and physiological factors. Improved knowledge of the isotopic ratio in sugars will enhance our understanding of the tree ring isotope ratio and will help to decipher environmental conditions in retrospect more reliably. Carbohydrate samples from larch (Larix gmelinii) needles of two sites in the continuous permafrost zone of Siberia with differing growth conditions were analysed with the Compound-Specific Isotope Analysis (CSIA). We compared concentrations and carbon isotope values (δ13C) of sucrose, fructose, glucose and pinitol combined with phenological data. The results for the variability of the needle carbohydrates show high dynamics with distinct seasonal characteristics between and within the studied years with a clear link to the climatic conditions, particularly vapour pressure deficit. Compound-specific differences in δ13C values as a response to climate were detected. The δ13C of pinitol, which contributes up to 50% of total soluble carbohydrates, was almost invariant during the whole growing season. Our study provides the first in-depth characterization of compound-specific needle carbohydrate isotope variability, identifies involved mechanisms and shows the potential of such results for linking tree physiological responses to different climatic conditions.

  17. Differences in DNA Binding Specificity of Floral Homeotic Protein Complexes Predict Organ-Specific Target Genes.

    PubMed

    Smaczniak, Cezary; Muiño, Jose M; Chen, Dijun; Angenent, Gerco C; Kaufmann, Kerstin

    2017-08-01

    Floral organ identities in plants are specified by the combinatorial action of homeotic master regulatory transcription factors. However, how these factors achieve their regulatory specificities is still largely unclear. Genome-wide in vivo DNA binding data show that homeotic MADS domain proteins recognize partly distinct genomic regions, suggesting that DNA binding specificity contributes to functional differences of homeotic protein complexes. We used in vitro systematic evolution of ligands by exponential enrichment followed by high-throughput DNA sequencing (SELEX-seq) on several floral MADS domain protein homo- and heterodimers to measure their DNA binding specificities. We show that specification of reproductive organs is associated with distinct binding preferences of a complex formed by SEPALLATA3 and AGAMOUS. Binding specificity is further modulated by different binding site spacing preferences. Combination of SELEX-seq and genome-wide DNA binding data allows differentiation between targets in specification of reproductive versus perianth organs in the flower. We validate the importance of DNA binding specificity for organ-specific gene regulation by modulating promoter activity through targeted mutagenesis. Our study shows that intrafamily protein interactions affect DNA binding specificity of floral MADS domain proteins. Differential DNA binding of MADS domain protein complexes plays a role in the specificity of target gene regulation. © 2017 American Society of Plant Biologists. All rights reserved.

  18. Specific binding of lactoferrin to brush-border membrane: Ontogeny and effect of glycan chain

    SciTech Connect

    Davidson, L.A.; Loennerdal, B. )

    1988-04-01

    Bioavailability of iron from human milk is exceptionally high. It has been suggested that lactoferrin, the major iron-binding protein in human milk, may participate in this high iron bioavailability from milk. The authors examined the interaction of lactoferrin with the intestinal brush-border membrane using the rhesus monkeys as a model. Brush-border membrane vesicles were prepared from monkeys of various ages. Binding studies with {sup 59}Fe-labeled human and monkey lactoferrin were performed to examine interaction of lactoferrin with the brush-border membrane. Specific saturable binding of lactoferrin was found at all ages studied. The dissociation constant for lactoferrin-receptor binding was 9 {times} 10{sup {minus}6} M. In contrast, no binding of serum transferrin or bovine lactoferrin occurred. Removal of fucose from the lactoferrin glycans resulted in a significant decrease in binding. It was concluded that lactoferrin in milk may function in the process of iron absorption through interaction with a small intestinal receptor and that fucosylated glycans on the carbohydrate chain of lactoferrin are necessary for receptor recognition.

  19. Diverse modes of galacto-specific carbohydrate recognition by a family 31 glycoside hydrolase from Clostridium perfringens

    PubMed Central

    Grondin, Julie M.; Duan, Da; Kirlin, Alyssa C.; Abe, Kento T.; Chitayat, Seth; Spencer, Holly L.; Spencer, Craig; Campigotto, Alisha; Houliston, Scott; Arrowsmith, Cheryl H.; Allingham, John S.; Boraston, Alisdair B.; Smith, Steven P.

    2017-01-01

    Clostridium perfringens is a commensal member of the human gut microbiome and an opportunistic pathogen whose genome encodes a suite of putative large, multi-modular carbohydrate-active enzymes that appears to play a role in the interaction of the bacterium with mucin-based carbohydrates. Among the most complex of these is an enzyme that contains a presumed catalytic module belonging to glycoside hydrolase family 31 (GH31). This large enzyme, which based on its possession of a GH31 module is a predicted α-glucosidase, contains a variety of non-catalytic ancillary modules, including three CBM32 modules that to date have not been characterized. NMR-based experiments demonstrated a preference of each module for galacto-configured sugars, including the ability of all three CBM32s to recognize the common mucin monosaccharide GalNAc. X-ray crystal structures of the CpGH31 CBM32s, both in apo form and bound to GalNAc, revealed the finely-tuned molecular strategies employed by these sequentially variable CBM32s in coordinating a common ligand. The data highlight that sequence similarities to previously characterized CBMs alone are insufficient for identifying the molecular mechanism of ligand binding by individual CBMs. Furthermore, the overlapping ligand binding profiles of the three CBMs provide a fail-safe mechanism for the recognition of GalNAc among the dense eukaryotic carbohydrate networks of the colonic mucosa. These findings expand our understanding of ligand targeting by large, multi-modular carbohydrate-active enzymes, and offer unique insights into of the expanding ligand-binding preferences and binding site topologies observed in CBM32s. PMID:28158290

  20. The FGGY Carbohydrate Kinase Family: Insights into the Evolution of Functional Specificities

    PubMed Central

    Zhang, Ying; Zagnitko, Olga; Rodionova, Irina; Osterman, Andrei; Godzik, Adam

    2011-01-01

    Function diversification in large protein families is a major mechanism driving expansion of cellular networks, providing organisms with new metabolic capabilities and thus adding to their evolutionary success. However, our understanding of the evolutionary mechanisms of functional diversity in such families is very limited, which, among many other reasons, is due to the lack of functionally well-characterized sets of proteins. Here, using the FGGY carbohydrate kinase family as an example, we built a confidently annotated reference set (CARS) of proteins by propagating experimentally verified functional assignments to a limited number of homologous proteins that are supported by their genomic and functional contexts. Then, we analyzed, on both the phylogenetic and the molecular levels, the evolution of different functional specificities in this family. The results show that the different functions (substrate specificities) encoded by FGGY kinases have emerged only once in the evolutionary history following an apparently simple divergent evolutionary model. At the same time, on the molecular level, one isofunctional group (L-ribulokinase, AraB) evolved at least two independent solutions that employed distinct specificity-determining residues for the recognition of a same substrate (L-ribulose). Our analysis provides a detailed model of the evolution of the FGGY kinase family. It also shows that only combined molecular and phylogenetic approaches can help reconstruct a full picture of functional diversifications in such diverse families. PMID:22215998

  1. The immunodominant outer membrane antigen of Actinobacillus actinomycetemcomitans is located in the serotype-specific high-molecular-mass carbohydrate moiety of lipopolysaccharide.

    PubMed Central

    Page, R C; Sims, T J; Engel, L D; Moncla, B J; Bainbridge, B; Stray, J; Darveau, R P

    1991-01-01

    Most patients with juvenile periodontitis manifest serum antibodies, sometimes at very high titers, to antigens of Actinobacillus actinomycetemcomitans, but the antigens inducing the immune response have been only partly characterized. We separated A. actinomycetemcomitans serotype b cells into protein, lipopolysaccharide (LPS), and soluble polysaccharide fractions and characterized them. Coomassie blue- and silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels were used to detect protein and LPS components, and gas-liquid chromatography was used to determine their carbohydrate and fatty acid composition. Western blots, dot blots, and enzyme-linked immunosorbent assay inhibition with high-titer sera from juvenile periodontitis patients revealed which components were highest in antibody binding activity. These results showed that the major portion of the immunoglobulin G binding activity resides in the purified mannan-free LPS, with lesser amounts in the total protein fraction. Using Sephacryl S-300 chromatography, we separated LPS into high-molecular-mass components with high carbohydrate contents by gas-liquid chromatography and a low-molecular-mass component consisting mainly of lipid A and the inner core sugar heptulose. The results of quantitative dot blot assays and enzyme-linked immunosorbent assay inhibition show that the serotype-specific antibody binding activity is highly concentrated in the high-molecular-mass carbohydrate-rich LPS fraction and is almost completely absent in the low-molecular-weight lipid-rich fraction. Our observations contrast with previous reports that the predominant serotype antigen of A. actinomycetemcomitans resides in a mannan-rich polysaccharide isolated from spent culture medium. These observations support the conclusion that the immunodominant antigen of the outer membrane is the O antigen of the LPS. Images PMID:1716610

  2. Carbohydrate-dependent binding of langerin to SodC, a cell wall glycoprotein of Mycobacterium leprae.

    PubMed

    Kim, Hee Jin; Brennan, Patrick J; Heaslip, Darragh; Udey, Mark C; Modlin, Robert L; Belisle, John T

    2015-02-01

    Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells.

  3. Overexpression of the carbohydrate binding module from Solanum lycopersicum expansin 1 (Sl-EXP1) modifies tomato fruit firmness and Botrytis cinerea susceptibility.

    PubMed

    Perini, M A; Sin, I N; Villarreal, N M; Marina, M; Powell, A L T; Martínez, G A; Civello, P M

    2017-04-01

    Firmness, one of the major determinants of postharvest quality and shelf life of fruits is determined by the mechanical resistance imposed by the plant cell wall. Expansins (EXP) are involved in the non-hydrolytic metabolic disassembly of plant cell walls, particularly in processes where relaxation of the wall is necessary, such as fruit development and ripening. As many carbohydrate-associated proteins, expansins have a putative catalytic domain and a carbohydrate-binding module (CBM). Several strategies have been pursued to control the loss of fruit firmness during storage. Most of the approaches have been to suppress the expression of key enzymes involved in the cell wall metabolism, but this is the first time that a CBM was overexpressed in a fruit aimed to control cell wall degradation and fruit softening. We report the constitutive overexpression of the CBM of Solanum lycopersicum expansin 1 (CBM-SlExp1) in the cell wall of tomato plants, and its effects on plant and fruit phenotype. Overexpression of CBM-SlExp1 increased the mechanical resistance of leaves, whereas it did not modify plant growth and general phenotype. However, transgenic plants showed delayed softening and firmer fruits. In addition, fruits were less susceptible to Botrytis cinerea infection, and the "in vitro" growth of the fungus on media containing AIR from the pericarp of transgenic fruits was lower than controls. The possibility of overexpressing a CBM of a fruit-specific expansin to control cell wall degradation and fruit softening is discussed.

  4. Carbohydrate recognition by the rhamnose-binding lectin SUL-I with a novel three-domain structure isolated from the venom of globiferous pedicellariae of the flower sea urchin Toxopneustes pileolus.

    PubMed

    Hatakeyama, Tomomitsu; Ichise, Ayaka; Unno, Hideaki; Goda, Shuichiro; Oda, Tatsuya; Tateno, Hiroaki; Hirabayashi, Jun; Sakai, Hitomi; Nakagawa, Hideyuki

    2017-08-01

    The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contains several biologically active proteins. We have cloned the cDNA of one of the toxin components, SUL-I, which is a rhamnose-binding lectin (RBL) that acts as a mitogen through binding to carbohydrate chains on target cells. Recombinant SUL-I (rSUL-I) was produced in Escherichia coli cells, and its carbohydrate-binding specificity was examined with the glycoconjugate microarray analysis, which suggested that potential target carbohydrate structures are galactose-terminated N-glycans. rSUL-I exhibited mitogenic activity for murine splenocyte cells and toxicity against Vero cells. The three-dimensional structure of the rSUL-I/l-rhamnose complex was determined by X-ray crystallographic analysis at a 1.8 Å resolution. The overall structure of rSUL-I is composed of three distinctive domains with a folding structure similar to those of CSL3, a RBL from chum salmon (Oncorhynchus keta) eggs. The bound l-rhamnose molecules are mainly recognized by rSUL-I through hydrogen bonds between its 2-, 3-, and 4-hydroxy groups and Asp, Asn, and Glu residues in the binding sites, while Tyr and Ser residues participate in the recognition mechanism. It was also inferred that SUL-I may form a dimer in solution based on the molecular size estimated via dynamic light scattering as well as possible contact regions in its crystal structure. © 2017 The Protein Society.

  5. Specific binding of antigen onto human T lymphocytes

    SciTech Connect

    Durandy, A.; Fischer, A.; Charron, D.; Griscelli, C.

    1986-05-01

    Human T lymphocytes sensitized to Candida albicans (CA) were shown to proliferate in cultures induced with mannan, a ramified polysaccharide extracted from the cell well of CA. We presently describe that, when we used strongly labeled (/sup 3/H)mannan, antigen-specific T blast cells were able to bind the labeled mannan on their membrane. The observations that irrelevant blast cells did not bind (/sup 3/H)mannan, and that mannan-specific blast cells did not bind tritiated pneumococcal polysaccharide SIII, indicate the specificity of mannan binding. Mannan binding was reversible and saturable. Mannan binding on T blast cells was inhibited by preincubation with monoclonal antibodies to T3 but not to other T cell-related molecules. The characteristics of this receptor suggest its identity with the T cell receptor for antigen. The direct binding of mannan could be either due to a cross-linking of the receptor by multivalent mannan or to a recognition of mannan in association with HLA-DQ molecules, as suggested by partial blocking of mannan binding using anti-HLA-DQ monoclonal antibodies.

  6. Impact of Module-X2 and Carbohydrate Binding Module-3 on the catalytic activity of associated glycoside hydrolases towards plant biomass.

    PubMed

    Pasari, Nandita; Adlakha, Nidhi; Gupta, Mayank; Bashir, Zeenat; Rajacharya, Girish H; Verma, Garima; Munde, Manoj; Bhatnagar, Rakesh; Yazdani, Syed Shams

    2017-06-16

    Cellulolytic enzymes capable of hydrolyzing plant biomass are secreted by microbial cells specifically in response to the carbon substrate present in the environment. These enzymes consist of a catalytic domain, generally appended to one or more non-catalytic Carbohydrate Binding Module (CBM), which enhances their activity towards recalcitrant biomass. In the present study, the genome of a cellulolytic microbe Paenibacillus polymyxa A18 was annotated for the presence of CBMs and analyzed their expression in response to the plant biomass and model polysaccharides Avicel, CMC and xylan using quantitative PCR. A gene that encodes X2-CBM3 was found to be maximally induced in response to the biomass and crystalline substrate Avicel. Association of X2-CBM3 with xyloglucanase and endoglucanase led to up to 4.6-fold increase in activity towards insoluble substrates. In the substrate binding study, module X2 showed a higher affinity towards biomass and phosphoric acid swollen cellulose, whereas CBM3 showed a higher affinity towards Avicel. Further structural modeling of X2 also indicated its potential role in substrate binding. Our findings highlighted the role of module X2 along with CBM3 in assisting the enzyme catalysis of agricultural residue and paved the way to engineer glycoside hydrolases for superior activity.

  7. Specific binding of angiogenin to calf pulmonary artery endothelial cells.

    PubMed

    Badet, J; Soncin, F; Guitton, J D; Lamare, O; Cartwright, T; Barritault, D

    1989-11-01

    Specific binding of angiogenin (ANG) to calf pulmonary artery endothelial cells was demonstrated. Cellular binding at 4 degrees C of 125I-labeled human recombinant ANG was time and concentration dependent, reversible, and saturable in the presence of increasing amounts of the unlabeled molecules. The interaction was shown to be specific since a large excess of unlabeled ANG reduced labeled ANG binding by 80%, whereas similar doses of RNase A, a structurally related protein, had no effect. Scatchard analyses of binding data revealed two apparent components. High-affinity sites with an apparent dissociation constant of 5 x 10(-9) M were shown to represent cell-specific interactions. The second component, comprising low-affinity/high-capacity sites with an apparent dissociation constant of 0.2 x 10(-6) M, was essentially associated with pericellular components. High-affinity ANG binding sites varied with cell density and were found on other endothelial cells from bovine aorta, cornea, and adrenal cortex capillary but not on Chinese hamster lung fibroblasts. Divalent copper, a modulator of angiogenesis, was found to induce a severalfold increase in specific cell-bound radioactivity. Placental ribonuclease inhibitor, a tight-binding inhibitor of both ribonucleolytic and angiogenic activities of ANG, abolished 125I-labeled human recombinant ANG binding only in the absence of copper.

  8. Carbohydrate specificities of the murine DC-SIGN homologue mSIGNR1.

    PubMed

    Koppel, Estella A; Ludwig, Irene S; Appelmelk, Ben J; van Kooyk, Yvette; Geijtenbeek, Teunis B H

    2005-01-01

    C-type lectins are important receptors expressed by antigen presenting cells that are involved in cellular communications as well as in pathogen uptake. An important C-type lectin family is represented by DC-SIGN and its homologues in human and mouse. Here we have investigated the carbohydrate specificity of cellular mSIGNR1 and compared it with DC-SIGN and L-SIGN. mSIGNR1 has a similar specificity as human DC-SIGN for high mannose-containing ligands present on both cellular and pathogen ligands. However, the DC-SIGN molecules differ in their recognition of Lewis antigens; mSIGNR1 interacts not only with Le(x/y) and Le(a/b) antigens similar to DC-SIGN, but also with sialylated Lex, a ligand for selectins. The differential recognition of Lewis antigens suggests differences between mSIGNR1 and DC-SIGN in the recognition of cellular ligands and pathogens that express Lewis epitopes.

  9. Carbohydrate modified catanionic vesicles: probing multivalent binding at the bilayer interface.

    PubMed

    Thomas, Glen B; Rader, Lenea H; Park, Juhee; Abezgauz, Ludmila; Danino, Dganit; DeShong, Philip; English, Douglas S

    2009-04-22

    This article reports on the synthesis, characterization, and binding studies of surface-functionalized, negatively charged catanionic vesicles. These studies demonstrate that the distribution of glycoconjugates in the membrane leaflet can be controlled by small alterations of the chemical structure of the conjugate. The ability to control the glycoconjugate concentration in the membrane provides a method to explore the relationship between ligand separation distance and multivalent lectin binding at the bilayer interface. The binding results using the O-linked glucosyl conjugate were consistent with a simple model in which binding kinetics are governed by the density of noninteracting glucose ligands, whereas the N-linked glycoconjugate exhibited binding kinetics consistent with interacting or clustering conjugates. From the noninteracting ligand model, an effective binding site separation of the sugar sites for concanavalin A of 3.6-4.3 nm was determined and a critical ligand density above which binding kinetics are zeroth order with respect to the amount of glycoconjugate present at the bilayer was observed. We also report cryo-transmission electron microscopy (cryo-TEM) images of conjugated vesicles showing morphological changes (multilayering) upon aggregation of unilamellar vesicles with concanavalin A.

  10. Specific insulin binding in bovine chromaffin cells; demonstration of preferential binding to adrenalin-storing cells

    SciTech Connect

    Serck-Hanssen, G.; Soevik, O.

    1987-12-28

    Insulin binding was studied in subpopulations of bovine chromaffin cells enriched in adrenalin-producing cells (A-cells) or noradrenalin-producing cells (NA-cells). Binding of /sup 125/I-insulin was carried out at 15/sup 0/C for 3 hrs in the absence or presence of excess unlabeled hormone. Four fractions of cells were obtained by centrifugation on a stepwise bovine serum albumin gradient. The four fractions were all shown to bind insulin in a specific manner and the highest binding was measured in the cell layers of higher densities, containing mainly A-cells. The difference in binding of insulin to the four subpopulations of chromaffin cells seemed to be related to differences in numbers of receptors as opposed to receptor affinities. The authors conclude that bovine chromaffin cells possess high affinity binding sites for insulin and that these binding sites are mainly confined to A-cells. 24 references, 2 figures, 1 table.

  11. Pulmonary surfactant protein A (SP-A) specifically binds dipalmitoylphosphatidylcholine

    SciTech Connect

    Kuroki, Y.; Akino, T. )

    1991-02-15

    Phospholipids are the major components of pulmonary surfactant. Dipalmitoylphosphatidylcholine is believed to be especially essential for the surfactant function of reducing the surface tension at the air-liquid interface. Surfactant protein A (SP-A) with a reduced denatured molecular mass of 26-38 kDa, characterized by a collagen-like structure and N-linked glycosylation, interacts strongly with a mixture of surfactant-like phospholipids. In the present study the direct binding of SP-A to phospholipids on a thin layer chromatogram was visualized using 125I-SP-A as a probe, so that the phospholipid specificities of SP-A binding and the structural requirements of SP-A and phospholipids for the binding could be examined. Although 125I-SP-A bound phosphatidylcholine and sphingomyeline, it was especially strong in binding dipalmitoylphosphatidylcholine, but failed to bind phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. Labeled SP-A also exhibited strong binding to distearoylphosphatidylcholine, but weak binding to dimyristoyl-, 1-palmitoyl-2-linoleoyl-, and dilinoleoylphosphatidylcholine. Unlabeled SP-A readily competed with labeled SP-A for phospholipid binding. SP-A strongly bound dipalmitoylglycerol produced by phospholipase C treatment of dipalmitoylphosphatidylcholine, but not palmitic acid. This protein also failed to bind lysophosphatidylcholine produced by phospholipase A2 treatment of dipalmitoylphosphatidylcholine. 125I-SP-A shows almost no binding to dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylethanolamine. The addition of 10 mM EGTA into the binding buffer reduced much of the 125I-SP-A binding to phospholipids. Excess deglycosylated SP-A competed with labeled SP-A for binding to dipalmitoylphosphatidylcholine, but the excess collagenase-resistant fragment of SP-A failed.

  12. Crystallization and preliminary crystallographic studies of a novel noncatalytic carbohydrate-binding module from the Ruminococcus flavefaciens cellulosome.

    PubMed

    Venditto, Immacolata; Goyal, Arun; Thompson, Andrew; Ferreira, Luis M A; Fontes, Carlos M G A; Najmudin, Shabir

    2015-01-01

    Microbial degradation of the plant cell wall is a fundamental biological process with considerable industrial importance. Hydrolysis of recalcitrant polysaccharides is orchestrated by a large repertoire of carbohydrate-active enzymes that display a modular architecture in which a catalytic domain is connected via linker sequences to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus potentiating catalysis. The genome of the most abundant ruminal cellulolytic bacterium, Ruminococcus flavefaciens strain FD-1, provides an opportunity to discover novel cellulosomal proteins involved in plant cell-wall deconstruction. It encodes a modular protein comprising a glycoside hydrolase family 9 catalytic module (GH9) linked to two unclassified tandemly repeated CBMs (termed CBM-Rf6A and CBM-Rf6B) and a C-terminal dockerin. The novel CBM-Rf6A from this protein has been crystallized and data were processed for the native and a selenomethionine derivative to 1.75 and 1.5 Å resolution, respectively. The crystals belonged to orthorhombic and cubic space groups, respectively. The structure was solved by a single-wavelength anomalous dispersion experiment using the CCP4 program suite and SHELXC/D/E.

  13. Crystallization and preliminary crystallographic studies of a novel noncatalytic carbohydrate-binding module from the Ruminococcus flavefaciens cellulosome

    PubMed Central

    Venditto, Immacolata; Goyal, Arun; Thompson, Andrew; Ferreira, Luis M. A.; Fontes, Carlos M. G. A.; Najmudin, Shabir

    2015-01-01

    Microbial degradation of the plant cell wall is a fundamental biological process with considerable industrial importance. Hydrolysis of recalcitrant polysaccharides is orchestrated by a large repertoire of carbohydrate-active enzymes that display a modular architecture in which a catalytic domain is connected via linker sequences to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus potentiating catalysis. The genome of the most abundant ruminal cellulolytic bacterium, Ruminococcus flavefaciens strain FD-1, provides an opportunity to discover novel cellulosomal proteins involved in plant cell-wall deconstruction. It encodes a modular protein comprising a glycoside hydrolase family 9 catalytic module (GH9) linked to two unclassified tandemly repeated CBMs (termed CBM-Rf6A and CBM-Rf6B) and a C-terminal dockerin. The novel CBM-Rf6A from this protein has been crystallized and data were processed for the native and a selenomethionine derivative to 1.75 and 1.5 Å resolution, respectively. The crystals belonged to orthorhombic and cubic space groups, respectively. The structure was solved by a single-wavelength anomalous dispersion experiment using the CCP4 program suite and SHELXC/D/E. PMID:25615967

  14. Preparative chromatography for specific δ13C isotopic analysis of individual carbohydrates in environmental samples

    NASA Astrophysics Data System (ADS)

    Nouara, Amel; Panagiotopoulos, Christos; Balesdent, Jérôme; Sempéré, Richard

    2017-04-01

    Carbohydrates are among the most abundant organic molecules on the Earth and are present in all geochemical systems. Despite their high abundance in the environment, very few studies assessed their origin using molecular carbohydrate isotopic analyses. In contrast with bulk stable isotope analysis (BSIA), which gives the isotopic signature of the entire sample without any specification about its chemical composition, compound specific 13C isotopic analysis of individual sugars (CSIA) offers valuable information about the origin of single molecules. Previous investigations used gas or liquid chromatography coupled with isotope ratio mass spectroscopy (GC-IRMS; HPLC-IRMS) for CSIA of sugars however the former requires δ13C corrections due to the carbon added to the sugar (derivatization) while the later does not provide always adequate separations among monosaccharides. Here we used cation preparative chromatography (Ca2+, Pb2+ and Na+) with refractive index detection in order to produce pure monosaccharide targets for subsequent EA-IRMS analyses. Milli-Q water was used as eluant at a flow rate 0.6 ml min-1. In general, three successive purifications (Ca2+, Pb2+, Ca2+) were sufficient to produce pure compounds. Pure monosaccharides were compared with authentic monosaccharide standards using 1H NMR and/or mass spectroscopy. The detection limit of our technique was about 1µM/sugar with a precision of 10% (n=6). Blanks run with Milli-Q water after three successive purifications resulted in carbon content of 0.13 to 2.77 µgC per collected sugar. These values are much lower than the minimum required amount (5 µgC) of the EA-IRSMS system with a precision of ± 0.35 ‰. Application of our method to environmental samples resulted in δ13C values of glucose, fructose, and levoglucosan in the range of -24 to -26 ‰ (PM10 atmospheric particles), and -15‰ to -22 ‰ for arabinose, glucose, and xylose (marine high molecular dissolved organic matter). These results fall in

  15. TAL Effector DNA-Binding Principles and Specificity.

    PubMed

    Richter, Annekatrin; Streubel, Jana; Boch, Jens

    2016-01-01

    Transcription activator-like effectors (TALEs) are proteins with a unique DNA-binding domain that confers both a predictable and programmable specificity. The DNA-binding domain consists typically of 34-amino acid near-identical repeats. The repeats form a right-handed superhelical structure that wraps around the DNA double helix and exposes the variable amino acids at position 13 of each repeat to the sense strand DNA bases. Each repeat binds one base in a highly specific, non-overlapping, and comma-free fashion. Although TALE specificities are encoded in a simple way, sophisticated rules can be taken into account to build highly efficient DNA-binding modules for biotechnological use.

  16. Carbohydrate-protein recognition: molecular dynamics simulations and free energy analysis of oligosaccharide binding to concanavalin A.

    PubMed Central

    Bryce, R A; Hillier, I H; Naismith, J H

    2001-01-01

    Carbohydrate ligands are important mediators of biomolecular recognition. Microcalorimetry has found the complex-type N-linked glycan core pentasaccharide beta-GlcNAc-(1-->2)-alpha-Man-(1-->3)-[beta-GlcNAc-(1-->2)-alpha-Man-(1-->6)]-Man to bind to the lectin, Concanavalin A, with almost the same affinity as the trimannoside, Man-alpha-(1-->6)-[Man-alpha-(1-->3)]-Man. Recent determination of the structure of the pentasaccharide complex found a glycosidic linkage psi torsion angle to be distorted by 50 degrees from the NMR solution value and perturbation of some key mannose-protein interactions observed in the structures of the mono- and trimannoside complexes. To unravel the free energy contributions to binding and to determine the structural basis for this degeneracy, we present the results of a series of nanosecond molecular dynamics simulations, coupled to analysis via the recently developed MM-GB/SA approach (Srinivasan et al., J. Am. Chem. Soc. 1998, 120:9401-9409). These calculations indicate that the strength of key mannose-protein interactions at the monosaccharide site is preserved in both the oligosaccharides. Although distortion of the pentasaccharide is significant, the principal factor in reduced binding is incomplete offset of ligand and protein desolvation due to poorly matched polar interactions. This analysis implies that, although Concanavalin A tolerates the additional 6 arm GlcNAc present in the pentasaccharide, it does not serve as a key recognition determinant. PMID:11509352

  17. Low or No Inhibitory Potency of the Canonical Galectin Carbohydrate-binding Site by Pectins and Galactomannans*

    PubMed Central

    Lepur, Adriana; Kahl-Knutson, Barbro; Aguilar-Moncayo, Matilde; Klyosov, Anatole A.; Field, Robert A.; Oredsson, Stina; Nilsson, Ulf J.

    2016-01-01

    Some complex plant-derived polysaccharides, such as modified citrus pectins and galactomannans, have been shown to have promising anti-inflammatory and anti-cancer effects. Most reports propose or claim that these effects are due to interaction of the polysaccharides with galectins because the polysaccharides contain galactose-containing side chains that might bind this class of lectin. However, their direct binding to and/or inhibition of the evolutionarily conserved galactoside-binding site of galectins has not been demonstrated. Using a well established fluorescence anisotropy assay, we tested the direct interaction of several such polysaccharides with physiological concentrations of a panel of galectins. The bioactive pectic samples tested were very poor inhibitors of the canonical galactoside-binding site for the tested galectins, with IC50 values >10 mg/ml for a few or in most cases no inhibitory activity at all. The galactomannan Davanat® was more active, albeit not a strong inhibitor (IC50 values ranging from 3 to 20 mg/ml depending on the galectin). Pure synthetic oligosaccharide fragments found in the side chains and backbone of pectins and galactomannans were additionally tested. The most commonly found galactan configuration in pectins had no inhibition of the galectins tested. Galactosylated tri- and pentamannosides, representing the structure of Davanat®, had an inhibitory effect of galectins comparable with that of free galactose. Further evaluation using cell-based assays, indirectly linked to galectin-3 inhibition, showed no inhibition of galectin-3 by the polysaccharides. These data suggest that the physiological effects of these plant polysaccharides are not due to inhibition of the canonical galectin carbohydrate-binding site. PMID:27129206

  18. Low or No Inhibitory Potency of the Canonical Galectin Carbohydrate-binding Site by Pectins and Galactomannans.

    PubMed

    Stegmayr, John; Lepur, Adriana; Kahl-Knutson, Barbro; Aguilar-Moncayo, Matilde; Klyosov, Anatole A; Field, Robert A; Oredsson, Stina; Nilsson, Ulf J; Leffler, Hakon

    2016-06-17

    Some complex plant-derived polysaccharides, such as modified citrus pectins and galactomannans, have been shown to have promising anti-inflammatory and anti-cancer effects. Most reports propose or claim that these effects are due to interaction of the polysaccharides with galectins because the polysaccharides contain galactose-containing side chains that might bind this class of lectin. However, their direct binding to and/or inhibition of the evolutionarily conserved galactoside-binding site of galectins has not been demonstrated. Using a well established fluorescence anisotropy assay, we tested the direct interaction of several such polysaccharides with physiological concentrations of a panel of galectins. The bioactive pectic samples tested were very poor inhibitors of the canonical galactoside-binding site for the tested galectins, with IC50 values >10 mg/ml for a few or in most cases no inhibitory activity at all. The galactomannan Davanat® was more active, albeit not a strong inhibitor (IC50 values ranging from 3 to 20 mg/ml depending on the galectin). Pure synthetic oligosaccharide fragments found in the side chains and backbone of pectins and galactomannans were additionally tested. The most commonly found galactan configuration in pectins had no inhibition of the galectins tested. Galactosylated tri- and pentamannosides, representing the structure of Davanat®, had an inhibitory effect of galectins comparable with that of free galactose. Further evaluation using cell-based assays, indirectly linked to galectin-3 inhibition, showed no inhibition of galectin-3 by the polysaccharides. These data suggest that the physiological effects of these plant polysaccharides are not due to inhibition of the canonical galectin carbohydrate-binding site. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Thermodynamic and kinetic analysis of carbohydrate binding to the basic lectin from winged bean (Psophocarpus tetragonolobus).

    PubMed

    Khan, M I; Sastry, M V; Surolia, A

    1986-03-05

    A basic lectin (pI approximately 10.0) was purified to homogeneity from the seeds of winged bean (Psophocarpus tetragonolobus) by affinity chromatography on Sepharose 6-aminocaproyl-D-galactosamine. The lectin agglutinated trypsinized rabbit erythrocytes and had a relative molecular mass of 58,000 consisting of two subunits of Mr 29,000. The lectin binds to N-dansylgalactosamine, leading to a 15-fold increase in dansyl fluorescence with a concomitant 25-nm blue shift in the emission maximum. The lectin has two binding sites/dimer for this sugar and an association constant of 4.17 X 10(5) M-1 at 25 degrees C. The strong binding to N-dansylgalactosamine is due to a relatively positive entropic contribution as revealed by the thermodynamic parameters: delta H = -33.62 kJ mol-1 and delta S0 = -5.24 J mol-1 K-1. Binding of this sugar to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent in alpha-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are critical for sugar binding to this lectin. Lectin difference absorption spectra in the presence of N-acetylgalactosamine indicate perturbation of tryptophan residues on sugar binding. The results of stopped flow kinetics with N-dansylgalactosamine and the lectin are consistent with a simple one-step mechanism for which k+1 = 1.33 X 10(4) M-1 s-1 and k-1 = 3.2 X 10(-2) s-1 at 25 degrees C. This k-1 is slower than any reported for a lectin-monosaccharide complex so far. The activation parameters indicate an enthalpically controlled association process.

  20. DNA-binding specificities of human transcription factors.

    PubMed

    Jolma, Arttu; Yan, Jian; Whitington, Thomas; Toivonen, Jarkko; Nitta, Kazuhiro R; Rastas, Pasi; Morgunova, Ekaterina; Enge, Martin; Taipale, Mikko; Wei, Gonghong; Palin, Kimmo; Vaquerizas, Juan M; Vincentelli, Renaud; Luscombe, Nicholas M; Hughes, Timothy R; Lemaire, Patrick; Ukkonen, Esko; Kivioja, Teemu; Taipale, Jussi

    2013-01-17

    Although the proteins that read the gene regulatory code, transcription factors (TFs), have been largely identified, it is not well known which sequences TFs can recognize. We have analyzed the sequence-specific binding of human TFs using high-throughput SELEX and ChIP sequencing. A total of 830 binding profiles were obtained, describing 239 distinctly different binding specificities. The models represent the majority of human TFs, approximately doubling the coverage compared to existing systematic studies. Our results reveal additional specificity determinants for a large number of factors for which a partial specificity was known, including a commonly observed A- or T-rich stretch that flanks the core motifs. Global analysis of the data revealed that homodimer orientation and spacing preferences, and base-stacking interactions, have a larger role in TF-DNA binding than previously appreciated. We further describe a binding model incorporating these features that is required to understand binding of TFs to DNA. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Solution structure and binding specificity of the p63 DNA binding domain

    PubMed Central

    Enthart, Andreas; Klein, Christian; Dehner, Alexander; Coles, Murray; Gemmecker, Gerd; Kessler, Horst; Hagn, Franz

    2016-01-01

    p63 is a close homologue of p53 and, together with p73, is grouped into the p53 family of transcription factors. p63 is known to be involved in the induction of controlled apoptosis important for differentiation processes, germ line integrity and development. Despite its high homology to p53, especially within the DNA binding domain (DBD), p63-DBD does not show cooperative DNA binding properties and is significantly more stable against thermal and chemical denaturation. Here, we determined the solution structure of p63-DBD and show that it is markedly less dynamic than p53-DBD. In addition, we also investigate the effect of a double salt bridge present in p53-DBD, but not in p63-DBD on the cooperative binding behavior and specificity to various DNA sites. Restoration of the salt bridges in p63-DBD by mutagenesis leads to enhanced binding affinity to p53-specific, but not p63-specific response elements. Furthermore, we show that p63-DBD is capable of binding to anti-apoptotic BclxL via its DNA binding interface, a feature that has only been shown for p53 so far. These data suggest that all p53 family members - despite alterations in the specificity and binding affinity - are capable of activating pro-apoptotic pathways in a tissue specific manner. PMID:27225672

  2. Solution structure and binding specificity of the p63 DNA binding domain.

    PubMed

    Enthart, Andreas; Klein, Christian; Dehner, Alexander; Coles, Murray; Gemmecker, Gerd; Kessler, Horst; Hagn, Franz

    2016-05-26

    p63 is a close homologue of p53 and, together with p73, is grouped into the p53 family of transcription factors. p63 is known to be involved in the induction of controlled apoptosis important for differentiation processes, germ line integrity and development. Despite its high homology to p53, especially within the DNA binding domain (DBD), p63-DBD does not show cooperative DNA binding properties and is significantly more stable against thermal and chemical denaturation. Here, we determined the solution structure of p63-DBD and show that it is markedly less dynamic than p53-DBD. In addition, we also investigate the effect of a double salt bridge present in p53-DBD, but not in p63-DBD on the cooperative binding behavior and specificity to various DNA sites. Restoration of the salt bridges in p63-DBD by mutagenesis leads to enhanced binding affinity to p53-specific, but not p63-specific response elements. Furthermore, we show that p63-DBD is capable of binding to anti-apoptotic BclxL via its DNA binding interface, a feature that has only been shown for p53 so far. These data suggest that all p53 family members - despite alterations in the specificity and binding affinity - are capable of activating pro-apoptotic pathways in a tissue specific manner.

  3. Potential anti-obesogenic properties of non-digestible carbohydrates: specific focus on resistant dextrin.

    PubMed

    Hobden, Mark R; Guérin-Deremaux, Laetitia; Rowland, Ian; Gibson, Glenn R; Kennedy, Orla B

    2015-08-01

    Alterations in the composition and metabolic activity of the gut microbiota appear to contribute to the development of obesity and associated metabolic diseases. However, the extent of this relationship remains unknown. Modulating the gut microbiota with non-digestible carbohydrates (NDC) may exert anti-obesogenic effects through various metabolic pathways including changes to appetite regulation, glucose and lipid metabolism and inflammation. The NDC vary in physicochemical structure and this may govern their physical properties and fermentation by specific gut bacterial populations. Much research in this area has focused on established prebiotics, especially fructans (i.e. inulin and fructo-oligosaccharides); however, there is increasing interest in the metabolic effects of other NDC, such as resistant dextrin. Data presented in this review provide evidence from mechanistic and intervention studies that certain fermentable NDC, including resistant dextrin, are able to modulate the gut microbiota and may alter metabolic process associated with obesity, including appetite regulation, energy and lipid metabolism and inflammation. To confirm these effects and elucidate the responsible mechanisms, further well-controlled human intervention studies are required to investigate the impact of NDC on the composition and function of the gut microbiota and at the same time determine concomitant effects on host metabolism and physiology.

  4. The ingestion of combined carbohydrates does not alter metabolic responses or performance capacity during soccer-specific exercise in the heat compared to ingestion of a single carbohydrate.

    PubMed

    Clarke, N D; Campbell, I T; Drust, B; Evans, L; Reilly, T; Maclaren, D P M

    2012-01-01

    This study was designed to investigate the effect of ingesting a glucose plus fructose solution on the metabolic responses to soccer-specific exercise in the heat and the impact on subsequent exercise capacity. Eleven male soccer players performed a 90 min soccer-specific protocol on three occasions. Either 3 ml · kg(-1) body mass of a solution containing glucose (1 g · min(-1) glucose) (GLU), or glucose (0.66 g · min(-1)) plus fructose (0.33 g · min(-1)) (MIX) or placebo (PLA) was consumed every 15 minutes. Respiratory measures were undertaken at 15-min intervals, blood samples were drawn at rest, half-time and on completion of the protocol, and muscle glycogen concentration was assessed pre- and post-exercise. Following the soccer-specific protocol the Cunningham and Faulkner test was performed. No significant differences in post-exercise muscle glycogen concentration (PLA, 62.99 ± 8.39 mmol · kg wet weight(-1); GLU 68.62 ± 2.70; mmol · kg wet weight(-1) and MIX 76.63 ± 6.92 mmol · kg wet weight(-1)) or exercise capacity (PLA, 73.62 ± 8.61 s; GLU, 77.11 ± 7.17 s; MIX, 83.04 ± 9.65 s) were observed between treatments (P > 0.05). However, total carbohydrate oxidation was significantly increased during MIX compared with PLA (P < 0.05). These results suggest that when ingested in moderate amounts, the type of carbohydrate does not influence metabolism during soccer-specific intermittent exercise or affect performance capacity after exercise in the heat.

  5. Applications of synthetic carbohydrates to chemical biology.

    PubMed

    Lepenies, Bernd; Yin, Jian; Seeberger, Peter H

    2010-06-01

    Access to synthetic carbohydrates is an urgent need for the development of carbohydrate-based drugs, vaccines, adjuvants as well as novel drug delivery systems. Besides traditional synthesis in solution, synthetic carbohydrates have been generated by chemoenzymatic methods as well as automated solid-phase synthesis. Synthetic oligosaccharides have proven to be useful for identifying ligands of carbohydrate-binding proteins such as C-type lectins and siglecs using glycan arrays. Furthermore, glyconanoparticles and glycodendrimers have been used for specific targeting of lectins of the immune system such as selectins, DC-SIGN, and CD22. This review focuses on how diverse carbohydrate structures can be synthetically derived and highlights the benefit of synthetic carbohydrates for glycobiology.

  6. Human Immunodeficiency Virus Envelope (gp120) Binding to DC-SIGN and Primary Dendritic Cells Is Carbohydrate Dependent but Does Not Involve 2G12 or Cyanovirin Binding Sites: Implications for Structural Analyses of gp120-DC-SIGN Binding

    PubMed Central

    Hong, Patrick W.-P.; Flummerfelt, Karen B.; de Parseval, Aymeric; Gurney, Kevin; Elder, John H.; Lee, Benhur

    2002-01-01

    The calcium-dependent lectin, DC-SIGN, binds to human immunodeficiency virus (HIV) (and simian immunodeficiency virus) gp120 and mediates the binding and transfer of HIV from monocyte-derived dendritic cells (MDDCs) to permissive T cells. However, it has been recently reported that DC-SIGN binding to HIV gp120 may be carbohydrate independent. Here, we formally demonstrate that gp120 binding to DC-SIGN and MDDCs is largely if not wholly carbohydrate dependent. Endo-β-N-glucosaminidase H (EndoH) treatment of gp120-Fc under conditions that maintained wild-type CD4 binding—and the full complement of complex glycans—significantly decreased (>90%) binding to DC-SIGN expressing cell lines, as well as to MDDCs. Any residual binding of EndoH-treated gp120-Fc to DC-SIGN was completely competed off with mannan. Mutational analysis indicated that no single glycosylation site affected the ability of gp120-Fc to bind DC-SIGN. To further guide our efforts in mapping the DC-SIGN binding sites on gp120, we used two well-characterized HIV inhibitory agents (2G12 monoclonal antibody and cyanovirin) that bind to high-mannose sugars on gp120. We showed that 2G12 and DC-SIGN bound to nonoverlapping sites in gp120 because (i) 2G12 did not block soluble gp120 or virion binding to DC-SIGN, (ii) 2G12 bound to gp120-Fc that was prebound to cell surface DC-SIGN, and (iii) gp120-Fc mutants that lack glycosylation sites involved in 2G12's epitope were also fully capable of binding DC-SIGN. These data were substantiated by the inability of cyanovirin to block gp120-Fc binding to DC-SIGN. Cyanovirin has been shown to effectively compete for 2G12 binding to gp120. Indeed, high concentrations of cyanovirin dramatically enhanced gp120-Fc binding to cell surfaces in the presence or absence of DC-SIGN. We provide evidence that this enhancement may be due to cyanovirin's ability to bridge gp120 to mannosylated cell surface proteins. These results have implications for antiviral therapeutics and

  7. Pathogen recognition of a novel C-type lectin from Marsupenaeus japonicus reveals the divergent sugar-binding specificity of QAP motif

    PubMed Central

    Alenton, Rod Russel R.; Koiwai, Keiichiro; Miyaguchi, Kohei; Kondo, Hidehiro; Hirono, Ikuo

    2017-01-01

    C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins known to assist the innate immune system as pattern recognition receptors (PRRs). The binding specificity of CTLs lies in the motif of their carbohydrate recognition domain (CRD), the tripeptide motifs EPN and QPD bind to mannose and galactose, respectively. However, variants of these motifs were discovered including a QAP sequence reported in shrimp believed to have the same carbohydrate specificity as QPD. Here, we characterized a novel C-type lectin (MjGCTL) possessing a CRD with a QAP motif. The recombinant MjGCTL has a calcium-dependent agglutinating capability against both Gram-negative and Gram-positive bacteria, and its sugar specificity did not involve either mannose or galactose. In an encapsulation assay, agarose beads coated with rMjGCTL were immediately encapsulated from 0 h followed by melanization at 4 h post-incubation with hemocytes. These results confirm that MjGCTL functions as a classical CTL. The structure of QAP motif and carbohydrate-specificity of rMjGCTL was found to be different to both EPN and QPD, suggesting that QAP is a new motif. Furthermore, MjGCTL acts as a PRR binding to hemocytes to activate their adherent state and initiate encapsulation. PMID:28374848

  8. Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models.

    PubMed

    Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

    2016-05-06

    Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers.

  9. Predicting DNA-Binding Specificities of Eukaryotic Transcription Factors

    PubMed Central

    Schröder, Adrian; Eichner, Johannes; Supper, Jochen; Eichner, Jonas; Wanke, Dierk; Henneges, Carsten; Zell, Andreas

    2010-01-01

    Today, annotated amino acid sequences of more and more transcription factors (TFs) are readily available. Quantitative information about their DNA-binding specificities, however, are hard to obtain. Position frequency matrices (PFMs), the most widely used models to represent binding specificities, are experimentally characterized only for a small fraction of all TFs. Even for some of the most intensively studied eukaryotic organisms (i.e., human, rat and mouse), roughly one-sixth of all proteins with annotated DNA-binding domain have been characterized experimentally. Here, we present a new method based on support vector regression for predicting quantitative DNA-binding specificities of TFs in different eukaryotic species. This approach estimates a quantitative measure for the PFM similarity of two proteins, based on various features derived from their protein sequences. The method is trained and tested on a dataset containing 1 239 TFs with known DNA-binding specificity, and used to predict specific DNA target motifs for 645 TFs with high accuracy. PMID:21152420

  10. Non-DNA-binding cofactors enhance DNA-binding specificity of a transcriptional regulatory complex

    PubMed Central

    Siggers, Trevor; Duyzend, Michael H; Reddy, Jessica; Khan, Sidra; Bulyk, Martha L

    2011-01-01

    Recruitment of cofactors to specific DNA sites is integral for specificity in gene regulation. As a model system, we examined how targeting and transcriptional control of the sulfur metabolism genes in Saccharomyces cerevisiae is governed by recruitment of the transcriptional co-activator Met4. We developed genome-scale approaches to measure transcription factor (TF) DNA-binding affinities and cofactor recruitment to >1300 genomic binding site sequences. We report that genes responding to the TF Cbf1 and cofactor Met28 contain a novel ‘recruitment motif' (RYAAT), adjacent to Cbf1 binding sites, which enhances the binding of a Met4–Met28–Cbf1 regulatory complex, and that abrogation of this motif significantly reduces gene induction under low-sulfur conditions. Furthermore, we show that correct recognition of this composite motif requires both non-DNA-binding cofactors Met4 and Met28. Finally, we demonstrate that the presence of an RYAAT motif next to a Cbf1 site, rather than Cbf1 binding affinity, specifies Cbf1-dependent sulfur metabolism genes. Our results highlight the need to examine TF/cofactor complexes, as novel specificity can result from cofactors that lack intrinsic DNA-binding specificity. PMID:22146299

  11. Specific binding of atrial natriuretic factor in brain microvessels

    SciTech Connect

    Chabrier, P.E.; Roubert, P.; Braquet, P.

    1987-04-01

    Cerebral capillaries constitute the blood-brain barrier. Studies of specific receptors (neurotransmitters or hormones) located on this structure can be performed by means of radioligand-binding techniques on isolated brain microvessels. The authors examined on pure bovine cerebral microvessel preparations the binding of atrial natriuretic factor (ANF), using /sup 125/I-labeled ANF. Saturation and competition experiments demonstrated the presence of a single class of ANF-binding sites with high affinity and with a binding capacity of 58 fmol/mg of protein. The binding of /sup 125/I-labeled ANF to brain microvessels is specific, reversible, and time dependent, as is shown by association-dissociation experiments. The demonstration of specific ANF-binding sites on brain microvessels supposes a physiological role of ANF on brain microvasculature. The coexistence of ANF and angiotensin II receptors on this cerebrovascular tissue suggests that the two circulating peptides may act as mutual antagonists in the regulation of brain microcirculation and/or blood-brain barrier function.

  12. Surfactant-free Colloidal Particles with Specific Binding Affinity.

    PubMed

    van der Wel, Casper; Bossert, Nelli; Mank, Quinten J; Winter, Marcel G T; Heinrich, Doris; Kraft, Daniela J

    2017-09-26

    Colloidal particles with specific binding affinity are essential for in vivo and in vitro biosensing, targeted drug delivery, and micrometer-scale self-assembly. Key to these techniques are surface functionalizations that provide high affinities to specific target molecules. For stabilization in physiological environments, current particle coating methods rely on adsorbed surfactants. However, spontaneous desorption of these surfactants typically has an undesirable influence on lipid membranes. To address this issue and create particles for targeting molecules in lipid membranes, we present here a surfactant-free coating method that combines high binding affinity with stability at physiological conditions. After activating charge-stabilized polystyrene microparticles with EDC/Sulfo-NHS, we first coat the particles with a specific protein and subsequently covalently attach a dense layer of poly(ethyelene) glycol. This polymer layer provides colloidal stability at physiological conditions as well as antiadhesive properties, while the protein coating provides the specific affinity to the targeted molecule. We show that NeutrAvidin-functionalized particles bind specifically to biotinylated membranes and that Concanavalin A-functionalized particles bind specifically to the glycocortex of Dictyostelium discoideum cells. The affinity of the particles changes with protein density, which can be tuned during the coating procedure. The generic and surfactant-free coating method reported here transfers the high affinity and specificity of a protein onto colloidal polystyrene microparticles.

  13. Probing Carbohydrate Product Expulsion from a Processive Cellulase with Multiple Absolute Binding Free Energy Methods*

    PubMed Central

    Bu, Lintao; Beckham, Gregg T.; Shirts, Michael R.; Nimlos, Mark R.; Adney, William S.; Himmel, Michael E.; Crowley, Michael F.

    2011-01-01

    Understanding the enzymatic mechanism that cellulases employ to degrade cellulose is critical to efforts to efficiently utilize plant biomass as a sustainable energy resource. A key component of cellulase action on cellulose is product inhibition from monosaccharide and disaccharides in the product site of cellulase tunnel. The absolute binding free energy of cellobiose and glucose to the product site of the catalytic tunnel of the Family 7 cellobiohydrolase (Cel7A) of Trichoderma reesei (Hypocrea jecorina) was calculated using two different approaches: steered molecular dynamics (SMD) simulations and alchemical free energy perturbation molecular dynamics (FEP/MD) simulations. For the SMD approach, three methods based on Jarzynski's equality were used to construct the potential of mean force from multiple pulling trajectories. The calculated binding free energies, −14.4 kcal/mol using SMD and −11.2 kcal/mol using FEP/MD, are in good qualitative agreement. Analysis of the SMD pulling trajectories suggests that several protein residues (Arg-251, Asp-259, Asp-262, Trp-376, and Tyr-381) play key roles in cellobiose and glucose binding to the catalytic tunnel. Five mutations (R251A, D259A, D262A, W376A, and Y381A) were made computationally to measure the changes in free energy during the product expulsion process. The absolute binding free energies of cellobiose to the catalytic tunnel of these five mutants are −13.1, −6.0, −11.5, −7.5, and −8.8 kcal/mol, respectively. The results demonstrated that all of the mutants tested can lower the binding free energy of cellobiose, which provides potential applications in engineering the enzyme to accelerate the product expulsion process and improve the efficiency of biomass conversion. PMID:21454590

  14. Characterization of Paenibacillus curdlanolyticus B-6 Xyn10D, a Xylanase That Contains a Family 3 Carbohydrate-Binding Module ▿ †

    PubMed Central

    Sakka, Makiko; Higashi, Yurika; Kimura, Tetsuya; Ratanakhanokchai, Khanok; Sakka, Kazuo

    2011-01-01

    Paenibacillus curdlanolyticus B-6 Xyn10D is a xylanase containing a family 3 carbohydrate-binding module (CBM3). Biochemical analyses using recombinant proteins derived from Xyn10D suggested that the CBM3 polypeptide has an affinity for cellulose and xylan and that CBM3 in Xyn10D is important for hydrolysis of insoluble arabinoxylan and natural biomass. PMID:21498754

  15. MULTIMERIN2 binds VEGF-A primarily via the carbohydrate chains exerting an angiostatic function and impairing tumor growth

    PubMed Central

    Andreuzzi, Eva; Paulitti, Alice; Tarticchio, Giulia; Todaro, Federico; Colombatti, Alfonso; Mongiat, Maurizio

    2016-01-01

    Angiogenesis is a key process occurring under both physiological and pathological conditions and is a hallmark of cancer. We have recently demonstrated that the extracellular matrix (ECM) molecule MULTIMERIN2 exerts an angiostatic function through the binding to VEGF-A. In this study we identify the region of the molecule responsible for the binding and demonstrate that the interaction involves the carbohydrate chains. MULTIMERIN2 interacts with other VEGF-A isoforms and VEGF family members such as VEGF-B, -C, -D and PlGF-1 suggesting that the molecule may function as a reservoir for different cytokines. In response to VEGF-A165, we show that MULTIMERIN2 impairs the phosphorylation of VEGFR2 at both Y1175 and Y1214 residues, halts SAPK2/p38 activation and negatively affects endothelial cell motility. In addition, MULTIMERIN2 and its active deletion mutant decrease the availability of the VEGFR2 receptor at the EC plasma membrane. The ectopic expression of MULTIMERIN2 or its active deletion mutant led to a striking reduction of tumor-associated angiogenesis and tumor growth. In conclusion, these data pinpoint MULTIMERIN2 as a key angiostatic molecule and disclose the possibility to develop new prognostic tools and improve the management of cancer patients. PMID:26655500

  16. Exploring the interactions between bacteriophage-encoded glycan binding proteins and carbohydrates.

    PubMed

    Simpson, David J; Sacher, Jessica C; Szymanski, Christine M

    2015-10-01

    There is an unprecedented interest in glycobiology due to the increasing appreciation of its impact on all aspects of life. Likewise, bacteriophage biology is enjoying a new renaissance as the post-antibiotic era fuels the search for novel ways to control harmful bacteria. Phages have spent the last 3 billion years developing ways of recognizing and manipulating bacterial surface glycans. Therefore, phages comprise a massive reservoir of glycan-binding and -hydrolyzing proteins with the potential to be exploited for glycan analysis, bacterial diagnostics and therapeutics. We discuss phage tail proteins that recognize bacterial surface polysaccharides, endolysins that bind and cleave peptidoglycan, Ig-like proteins that attach to mucin glycans, and phage effector proteins that recognize both bacterial and eukaryotic oligosaccharides.

  17. Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module from family 64 (StX)

    PubMed Central

    Campos, Bruna Medeia; Liberato, Marcelo Vizona; Polikarpov, Igor; Zeri, Ana Carolina de Mattos; Squina, Fabio Marcio

    2015-01-01

    In recent years, biofuels have attracted great interest as a source of renewable energy owing to the growing global demand for energy, the dependence on fossil fuels, limited natural resources and environmental pollution. However, the cost-effective production of biofuels from plant biomass is still a challenge. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases to polysaccharides, is crucial for enzyme development. Aiming at the structural and functional characterization of novel CBMs involved in plant polysaccharide deconstruction, an analysis of the CAZy database was performed and CBM family 64 was chosen owing to its capacity to bind with high specificity to microcrystalline cellulose and to the fact that is found in thermophilic microorganisms. In this communication, the CBM-encoding module named StX was expressed, purified and crystallized, and X-ray diffraction data were collected from native and derivatized crystals to 1.8 and 2.0 Å resolution, respectively. The crystals, which were obtained by the hanging-drop vapour-diffusion method, belonged to space group P3121, with unit-cell parameters a = b = 43.42, c = 100.96 Å for the native form. The phases were found using the single-wavelength anomalous diffraction method. PMID:25760706

  18. Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module from family 64 (StX).

    PubMed

    Campos, Bruna Medeia; Liberato, Marcelo Vizona; Polikarpov, Igor; Zeri, Ana Carolina de Mattos; Squina, Fabio Marcio

    2015-03-01

    In recent years, biofuels have attracted great interest as a source of renewable energy owing to the growing global demand for energy, the dependence on fossil fuels, limited natural resources and environmental pollution. However, the cost-effective production of biofuels from plant biomass is still a challenge. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases to polysaccharides, is crucial for enzyme development. Aiming at the structural and functional characterization of novel CBMs involved in plant polysaccharide deconstruction, an analysis of the CAZy database was performed and CBM family 64 was chosen owing to its capacity to bind with high specificity to microcrystalline cellulose and to the fact that is found in thermophilic microorganisms. In this communication, the CBM-encoding module named StX was expressed, purified and crystallized, and X-ray diffraction data were collected from native and derivatized crystals to 1.8 and 2.0 Å resolution, respectively. The crystals, which were obtained by the hanging-drop vapour-diffusion method, belonged to space group P3121, with unit-cell parameters a = b = 43.42, c = 100.96 Å for the native form. The phases were found using the single-wavelength anomalous diffraction method.

  19. Multivalent interaction based carbohydrate biosensors for signal amplification

    PubMed Central

    Wang, Yanyan; Chalagalla, Srinivas; Li, Tiehai; Sun, Xue-long; Zhao, Wei; Wang, Peng; Zeng, Xiangqun

    2010-01-01

    Multivalent interaction between boronic acids immobilized on Quartz Crystal Microbalance (QCM) sensor surface and the carbohydrates modified Au - nanoparticle (AuNP) has been demonstrated for the development of a sensitive carbohydrate biosensor. Briefly, a boronic acid - containing polymer (boropolymer) as multivalent carbohydrate receptor was oriented immobilized on the cysteamine coated electrode through isourea bond formation. Carbohydrates were conjugated to AuNPs to generate a multivalent carbohydrates moiety to amplify the response signal. Thus, the binding of the carbohydrate conjugated AuNPs to the boropolymer surface are multivalent which could simultaneously increase the binding affinity and specificity. We systematically studied the binding between five carbohydrate conjugated AuNPs and the boropolymer. Our studies show that the associate constant (Ka) was in the order of fucose < glucose < mannose < galactose < maltose. A linear response in the range from 23 µM to 3.83 mM was observed for mannose conjugated AuNPs and the boropolymer recognition elements, with the lower detection limit of 1.5 µM for the carbohydrate analytes. Furthermore, the multivalent binding between carbohydrates and boronic acids are reversible and allow the regeneration of boropolymer surface by using 1M acetic acid so as to sequentially capture and release the carbohydrate analytes. PMID:20863680

  20. Binding of an oligopeptide to a specific plane of ice.

    PubMed

    Houston, M E; Chao, H; Hodges, R S; Sykes, B D; Kay, C M; Sönnichsen, F D; Loewen, M C; Davies, P L

    1998-05-08

    The alpha-helical antifreeze protein (AFP) from winter flounder inhibits ice growth by binding to a specific set of pyramidal surface planes that are not otherwise macroscopically expressed. The 37-residue AFP contains three 11-amino acid repeats that make a stereo-specific fit to the ice lattice along the <01-12> direction of the (20-21) and equivalent binding planes. When the AFP was shortened to delete two of the three 11-amino acid ice-binding repeats, the resulting 15-residue peptide and its variants were less helical and showed no antifreeze activity. However, when the helicity of the peptide was reinforced by an internal lactam bridge between Glu-7 and Lys-11, the minimized AFP was able to stably express the pyramidal plane (20-21) on the surface of growing ice crystals. This dynamic shaping of the ice surface by a single ice-binding repeat provides evidence that AFP adsorption to the ice lattice is not an "all-or-nothing" interaction. Instead, a partial interaction can help develop the binding site on ice to which the remainder of the AFP (or other AFP molecules) can orient and bind.

  1. Crystal structure of an enzymatically inactive trans-sialidase-like lectin from Trypanosoma cruzi: the carbohydrate binding mechanism involves residual sialidase activity.

    PubMed

    Oppezzo, Pablo; Obal, Gonzalo; Baraibar, Martín A; Pritsch, Otto; Alzari, Pedro M; Buschiazzo, Alejandro

    2011-09-01

    Trans-sialidases are surface-located proteins in Trypanosoma cruzi that participate in key parasite-host interactions and parasite virulence. These proteins are encoded by a large multigenic family, with tandem-repeated and individual genes dispersed throughout the genome. While a large number of genes encode for catalytically active enzyme isoforms, many others display mutations that involve catalytic residues. The latter ultimately code for catalytically inactive proteins with very high similarity to their active paralogs. These inactive members have been shown to be lectins, able to bind sialic acid and galactose in vitro, although their cellular functions are yet to be fully established. We now report structural and biochemical evidence extending the current molecular understanding of these lectins. We have solved the crystal structure of one such catalytically inactive trans-sialidase-like protein, after soaking with a specific carbohydrate ligand, sialyl-α2,3-lactose. Instead of the expected trisaccharide, the binding pocket was observed occupied by α-lactose, strongly suggesting that the protein retains residual hydrolytic activity. This hypothesis was validated by enzyme kinetics assays, in comparison to fully active wild-type trans-sialidase. Surface plasmon resonance also confirmed that these trans-sialidase-like lectins are not only able to bind small oligosaccharides, but also sialylated glycoproteins, which is relevant in the physiologic scenario of parasite infection. Inactive trans-sialidase proteins appear thus to be β-methyl-galactosyl-specific lectins, evolved within an exo-sialidase scaffold, thus explaining why their lectin activity is triggered by the presence of terminal sialic acid.

  2. Binding studies of a sialic acid-specific lectin from the horseshoe crab Carcinoscorpius rotunda cauda with various sialoglycoproteins.

    PubMed Central

    Mohan, S; Thambi Dorai, D; Srimal, S; Bachhawat, B K

    1982-01-01

    Interaction of the sialic acid-specific lectin carcinoscorpin with various sialoglycoproteins was studied by using radioiodinated lectin. The binding of carcinoscorpin was dependent not only on sialic acid content but also on the type of glycosidic linkage and form (branched or linear) of the carbohydrate chains. Carcinoscorpin has different classes of binding sites, and binding follows a phenomenon of positive co-operativity. The effect of Ca2+ concentration on the binding was studied, and the optimal concentration was found to be 0.02 M. Effect of pH, temperature and other bivalent metal ions are also reported. From haemagglutination- and precipitation-inhibition studies, it was concluded that carcinoscorpin has multispecificity towards acidic sugars, and its relation to the biological role of the lectin in the horseshoe crab is discussed. PMID:7103938

  3. Properties of a family 56 carbohydrate-binding module and its role in the recognition and hydrolysis of β-1,3-glucan.

    PubMed

    Hettle, Andrew; Fillo, Alexander; Abe, Kento; Massel, Patricia; Pluvinage, Benjamin; Langelaan, David N; Smith, Steven P; Boraston, Alisdair B

    2017-08-21

    BH0236 from Bacillus halodurans is a multimodular β-1,3-glucanase comprising an N-terminal family 81 glycoside hydrolase catalytic module, an internal family 6 carbohydrate-binding module (CBM) that binds the nonreducing end of β-1,3-glucans chains, and an uncharacterized C-terminal module classified into CBM family 56. Here, we determined that this latter CBM, BhCBM56, bound the soluble β-1,3-glucan laminarin with a dissociation constant (Kd) of ~26 μM, displayed higher affinity for insoluble β-1,3-glucans with Kds of ~2-10 μM, but lacked affinity for β-1,3-glucooligosaccharides. The X-ray crystal structure of BhCBM56 and NMR-derived chemical shift mapping of the binding site revealed a β-sandwich fold, with the face of one β-sheet possessing the β-1,3-glucan-binding surface. On the basis of the functional and structural properties of BhCBM56, we propose that it binds a quaternary polysaccharide structure, most likely the triple helix adopted by polymerized β-1,3-glucans. Consistent with the BhCBM56 and BhCBM6/56 binding profiles, deletion of the CBM56 from BH0236 decreased activity of the enzyme on the insoluble β-1,3-glucan curdlan but not on soluble laminarin; additional deletion of the CBM6 also did not affect laminarin degradation but further decreased curdlan hydrolysis. The pseudo-atomic solution structure of BH0236 determined by small angle X-ray scattering (SAXS) revealed structural insights into the nature of avid binding by the BhCBM6/56 pair and how the orientation of the active site in the catalytic module factors into recognition and degradation of β-1,3-glucans. Our findings reinforce the notion that catalytic modules and their cognate CBMs have complementary specificities, including targeting of polysaccharide quaternary structure. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  4. Meta-analysis of studies of a specific delivery mode for a modified-carbohydrate diet.

    PubMed

    Shikany, J M; Desmond, R; McCubrey, R; Allison, D B

    2011-12-01

    Obesity is highly prevalent throughout the world. Although modified-carbohydrate diets (MCDs) comprise one popular approach, questions remain about their utility for weight loss. The objective of the present study was to conduct a meta-analysis of randomised controlled trials (RCTs) of a specific MCD compared with various control diets on weight loss. Data from four RCTs (three obtained from the sponsor and one indentified through literature searches) were included. Intent-to-treat analyses were conducted using multiple imputation to handle missing data, where possible. Because inter-study heterogeneity was demonstrated with fixed-effects meta-analysis, a random-effects meta-analysis also was conducted. When considered separately, all four studies showed greater reduction in body weight with the MCD compared to control diets at 12-week follow-up; the results at 24 weeks (available for three of the studies) were not as consistent. Results for body mass index (BMI) were similar. Greater reductions in waist circumference with the MCD were seen at either time point in only one study. When fixed-effects meta-analysis was applied, significantly greater reductions in weight, BMI and waist circumference with the MCD at both 12 weeks (1.66 kg, 0.53 kg m(-2) and 1.02 cm, respectively) and 24 weeks (1.20 kg, 0.43 kg m(-2) and 0.69 cm, respectively) were evident. Random-effects meta-analysis revealed similar results; however, the 24-week difference for a reduction in waist circumference was no longer statistically significant. Meta-analysis of individual RCT results demonstrated consistent benefits of this MCD compared to control diets on weight loss up to 24 weeks and waist circumference up to 12 weeks. © 2011 The Authors. Journal of Human Nutrition and Dietetics © 2011 The British Dietetic Association Ltd.

  5. Ipomoelin, a Jacalin-Related Lectin with a Compact Tetrameric Association and Versatile Carbohydrate Binding Properties Regulated by Its N Terminus

    PubMed Central

    Chang, Wei-Chieh; Liu, Kai-Lun; Hsu, Fang-Ciao; Jeng, Shih-Tong; Cheng, Yi-Sheng

    2012-01-01

    Many proteins are induced in the plant defense response to biotic stress or mechanical wounding. One group is lectins. Ipomoelin (IPO) is one of the wound-inducible proteins of sweet potato (Ipomoea batatas cv. Tainung 57) and is a Jacalin-related lectin (JRL). In this study, we resolved the crystal structures of IPO in its apo form and in complex with carbohydrates such as methyl α-D-mannopyranoside (Me-Man), methyl α-D-glucopyranoside (Me-Glc), and methyl α-D-galactopyranoside (Me-Gal) in different space groups. The packing diagrams indicated that IPO might represent a compact tetrameric association in the JRL family. The protomer of IPO showed a canonical β-prism fold with 12 strands of β-sheets but with 2 additional short β-strands at the N terminus. A truncated IPO (ΔN10IPO) by removing the 2 short β-strands of the N terminus was used to reveal its role in a tetrameric association. Gel filtration chromatography confirmed IPO as a tetrameric form in solution. Isothermal titration calorimetry determined the binding constants (KA) of IPO and ΔN10IPO against various carbohydrates. IPO could bind to Me-Man, Me-Glc, and Me-Gal with similar binding constants. In contrast, ΔN10IPO showed high binding ability to Me-Man and Me-Glc but could not bind to Me-Gal. Our structural and functional analysis of IPO revealed that its compact tetrameric association and carbohydrate binding polyspecificity could be regulated by the 2 additional N-terminal β-strands. The versatile carbohydrate binding properties of IPO might play a role in plant defense. PMID:22808208

  6. Cyclin D1 inhibits hepatic lipogenesis via repression of carbohydrate response element binding protein and hepatocyte nuclear factor 4α.

    PubMed

    Hanse, Eric A; Mashek, Douglas G; Becker, Jennifer R; Solmonson, Ashley D; Mullany, Lisa K; Mashek, Mara T; Towle, Howard C; Chau, Anhtung T; Albrecht, Jeffrey H

    2012-07-15

    Following acute hepatic injury, the metabolic capacity of the liver is altered during the process of compensatory hepatocyte proliferation by undefined mechanisms. In this study, we examined the regulation of de novo lipogenesis by cyclin D1, a key mediator of hepatocyte cell cycle progression. In primary hepatocytes, cyclin D1 significantly impaired lipogenesis in response to glucose stimulation. Cyclin D1 inhibited the glucose-mediated induction of key lipogenic genes, and similar effects were seen using a mutant (D1-KE) that does not activate cdk4 or induce cell cycle progression. Cyclin D1 (but not D1-KE) inhibited the activity of the carbohydrate response element-binding protein (ChREBP) by regulating the glucose-sensing motif of this transcription factor. Because changes in ChREBP activity could not fully explain the effect of cyclin D1, we examined hepatocyte nuclear factor 4α (HNF4α), which regulates numerous differentiated functions in the liver including lipid metabolism. We found that both cyclins D1 and D1-KE bound to HNF4α and significantly inhibited its recruitment to the promoter region of lipogenic genes in hepatocytes. Conversely, knockdown of cyclin D1 in the AML12 hepatocyte cell line promoted HNF4α activity and lipogenesis. In mouse liver, HNF4α bound to a central domain of cyclin D1 involved in transcriptional repression. Cyclin D1 inhibited lipogenic gene expression in the liver following carbohydrate feeding. Similar findings were observed in the setting of physiologic cyclin D1 expression in the regenerating liver. In conclusion, these studies demonstrate that cyclin D1 represses ChREBP and HNF4α function in hepatocytes via Cdk4-dependent and -independent mechanisms. These findings provide a direct link between the cell cycle machinery and the transcriptional control of metabolic function of the liver.

  7. An oligosaccharide-based HIV-1 2G12 mimotope vaccine induces carbohydrate-specific antibodies that fail to neutralize HIV-1 virions.

    PubMed

    Joyce, Joseph G; Krauss, Isaac J; Song, Hong C; Opalka, David W; Grimm, Karen M; Nahas, Deborah D; Esser, Mark T; Hrin, Renee; Feng, Meizhen; Dudkin, Vadim Y; Chastain, Michael; Shiver, John W; Danishefsky, Samuel J

    2008-10-14

    The conserved oligomannose epitope, Man(9)GlcNAc(2), recognized by the broadly neutralizing human mAb 2G12 is an attractive prophylactic vaccine candidate for the prevention of HIV-1 infection. We recently reported total chemical synthesis of a series of glycopeptides incorporating one to three copies of Man(9)GlcNAc(2) coupled to a cyclic peptide scaffold. Surface plasmon resonance studies showed that divalent and trivalent, but not monovalent, compounds were capable of binding 2G12. To test the efficacy of the divalent glycopeptide as an immunogen capable of inducing a 2G12-like neutralizing antibody response, we covalently coupled the molecule to a powerful immune-stimulating protein carrier and evaluated immunogenicity of the conjugate in two animal species. We used a differential immunoassay to demonstrate induction of high levels of carbohydrate-specific antibodies; however, these antibodies showed poor recognition of recombinant gp160 and failed to neutralize a panel of viral isolates in entry-based neutralization assays. To ascertain whether antibodies produced during natural infection could recognize the mimetics, we screened a panel of HIV-1-positive and -negative sera for binding to gp120 and the synthetic antigens. We present evidence from both direct and competitive binding assays that no significant recognition of the glycopeptides was observed, although certain sera did contain antibodies that could compete with 2G12 for binding to recombinant gp120.

  8. Enhanced Polysaccharide Binding and Activity on Linear β-Glucans through Addition of Carbohydrate-Binding Modules to Either Terminus of a Glucooligosaccharide Oxidase.

    PubMed

    Foumani, Maryam; Vuong, Thu V; MacCormick, Benjamin; Master, Emma R

    2015-01-01

    The gluco-oligosaccharide oxidase from Sarocladium strictum CBS 346.70 (GOOX) is a single domain flavoenzyme that favourably oxidizes gluco- and xylo- oligosaccharides. In the present study, GOOX was shown to also oxidize plant polysaccharides, including cellulose, glucomannan, β-(1→3,1→4)-glucan, and xyloglucan, albeit to a lesser extent than oligomeric substrates. To improve GOOX activity on polymeric substrates, three carbohydrate binding modules (CBMs) from Clostridium thermocellum, namely CtCBM3 (type A), CtCBM11 (type B), and CtCBM44 (type B), were separately appended to the amino and carboxy termini of the enzyme, generating six fusion proteins. With the exception of GOOX-CtCBM3 and GOOX-CtCBM44, fusion of the selected CBMs increased the catalytic activity of the enzyme (kcat) on cellotetraose by up to 50%. All CBM fusions selectively enhanced GOOX binding to soluble and insoluble polysaccharides, and the immobilized enzyme on a solid cellulose surface remained stable and active. In addition, the CBM fusions increased the activity of GOOX on soluble glucomannan by up to 30% and on insoluble crystalline as well as amorphous cellulose by over 50%.

  9. Enhanced Polysaccharide Binding and Activity on Linear β-Glucans through Addition of Carbohydrate-Binding Modules to Either Terminus of a Glucooligosaccharide Oxidase

    PubMed Central

    Foumani, Maryam; Vuong, Thu V.; MacCormick, Benjamin; Master, Emma R.

    2015-01-01

    The gluco-oligosaccharide oxidase from Sarocladium strictum CBS 346.70 (GOOX) is a single domain flavoenzyme that favourably oxidizes gluco- and xylo- oligosaccharides. In the present study, GOOX was shown to also oxidize plant polysaccharides, including cellulose, glucomannan, β-(1→3,1→4)-glucan, and xyloglucan, albeit to a lesser extent than oligomeric substrates. To improve GOOX activity on polymeric substrates, three carbohydrate binding modules (CBMs) from Clostridium thermocellum, namely CtCBM3 (type A), CtCBM11 (type B), and CtCBM44 (type B), were separately appended to the amino and carboxy termini of the enzyme, generating six fusion proteins. With the exception of GOOX-CtCBM3 and GOOX-CtCBM44, fusion of the selected CBMs increased the catalytic activity of the enzyme (kcat) on cellotetraose by up to 50%. All CBM fusions selectively enhanced GOOX binding to soluble and insoluble polysaccharides, and the immobilized enzyme on a solid cellulose surface remained stable and active. In addition, the CBM fusions increased the activity of GOOX on soluble glucomannan by up to 30 % and on insoluble crystalline as well as amorphous cellulose by over 50 %. PMID:25932926

  10. Probing the fibrate binding specificity of rat liver fatty acid binding protein.

    PubMed

    Chuang, Sara; Velkov, Tony; Horne, James; Wielens, Jerome; Chalmers, David K; Porter, Christopher J H; Scanlon, Martin J

    2009-09-10

    Liver-fatty acid binding protein (L-FABP) is found in high levels in enterocytes and is involved in cytosolic solubilization of fatty acids. In addition, L-FABP has been shown to bind endogenous and exogenous lipophilic compounds, suggesting that it may also play a role in modulating their absorption and disposition within enterocytes. Previously, we have described binding of L-FABP to a range of drugs, including a series of fibrates. In the present study, we have generated structural models of L-FABP-fibrate complexes and undertaken thermodynamic analysis of the binding of fibrates containing either a carboxylic acid or ester functionality. Analysis of the current data reveals that both the location and the energetics of binding are different for fibrates that contain a carboxylate compared to those that do not. As such, the data presented in this study suggest potential mechanisms that underpin molecular recognition and dictate specificity in the interaction between fibrates and L-FABP.

  11. pH-dependent specific binding and combing of DNA.

    PubMed Central

    Allemand, J F; Bensimon, D; Jullien, L; Bensimon, A; Croquette, V

    1997-01-01

    Recent developments in the rapid sequencing, mapping, and analysis of DNA rely on the specific binding of DNA to specially treated surfaces. We show here that specific binding of DNA via its unmodified extremities can be achieved on a great variety of surfaces by a judicious choice of the pH. On hydrophobic surfaces the best binding efficiency is reached at a pH of approximately 5.5. At that pH a approximately 40-kbp DNA is 10 times more likely to bind by an extremity than by a midsegment. A model is proposed to account for the differential adsorption of the molecule extremities and midsection as a function of pH. The pH-dependent specific binding can be used to align anchored DNA molecules by a receding meniscus, a process called molecular combing. The resulting properties of the combed molecules will be discussed. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 6 FIGURE 7 PMID:9336201

  12. Damage-specific DNA-binding proteins from human cells

    SciTech Connect

    Kanjilal, S.

    1992-01-01

    The primary objective of the study was to detect and characterize factors from human cells that bind DNA damaged by ultraviolet radiation. An application of the gel-shift assay was devised in which a DNA probe was UV-irradiated and compared with non-irradiated probe DNA for the ability to bind to such factors in cell extracts. UV-dose dependent binding proteins were identified. Formation of the DNA-protein complexes was independent of the specific sequence, form or source of the DNA. There was a marked preference for lesions on double stranded DNA over those on single stranded DNA. DNA irradiated with gamma rays did not compete with UV-irradiated DNA for the binding activities. Cell lines from patients with genetic diseases associated with disorders of the DNA repair system were screened for the presence of damaged-DNA-binding activities. Simultaneous occurrence of the clinical symptoms of some of these diseases had been previously documented and possible links between the syndromes proposed. However, supporting biochemical or molecular evidence for such associations were lacking. The data from the present investigations indicate that some cases of Xeroderma Pigmentosum group A, Cockayne's Syndrome, Bloom's Syndrome and Ataxia Telangiectasia, all of which exhibit sensitivity to UV or gamma radiation, share an aberrant damaged-DNA-binding factor. These findings support the hypothesis that some of the repair disorder diseases are closely related and may have arisen from a common defect. Partial purification of the binding activities from HeLa cells was achieved. Size-exclusion chromatography resolved the activities into various peaks, one of which was less damage-specific than the others as determined by competition studies using native or UV-irradiated DNA. Some of the activities were further separated by ion-exchange chromatography. On using affinity chromatography methods, the major damage-binding factor could be eluted in the presence of 2 M KCl and 1% NP-40.

  13. Human DC-SIGN binds specific human milk glycans.

    PubMed

    Noll, Alexander J; Yu, Ying; Lasanajak, Yi; Duska-McEwen, Geralyn; Buck, Rachael H; Smith, David F; Cummings, Richard D

    2016-05-15

    Human milk glycans (HMGs) are prebiotics, pathogen receptor decoys and regulators of host physiology and immune responses. Mechanistically, human lectins (glycan-binding proteins, hGBP) expressed by dendritic cells (DCs) are of major interest, as these cells directly contact HMGs. To explore such interactions, we screened many C-type lectins and sialic acid-binding immunoglobulin-like lectins (Siglecs) expressed by DCs for glycan binding on microarrays presenting over 200 HMGs. Unexpectedly, DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) showed robust binding to many HMGs, whereas other C-type lectins failed to bind, and Siglec-5 and Siglec-9 showed weak binding to a few glycans. By contrast, most hGBP bound to multiple glycans on other microarrays lacking HMGs. An α-linked fucose residue was characteristic of HMGs bound by DC-SIGN. Binding of DC-SIGN to the simple HMGs 2'-fucosyl-lactose (2'-FL) and 3-fucosyl-lactose (3-FL) was confirmed by flow cytometry to beads conjugated with 2'-FL or 3-FL, as well as the ability of the free glycans to inhibit DC-SIGN binding. 2'-FL had an IC50 of ∼1 mM for DC-SIGN, which is within the physiological concentration of 2'-FL in human milk. These results demonstrate that DC-SIGN among the many hGBP expressed by DCs binds to α-fucosylated HMGs, and suggest that such interactions may be important in influencing immune responses in the developing infant. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  14. Mapping the membrane proteome of anaerobic gut fungi identifies a wealth of carbohydrate binding proteins and transporters

    SciTech Connect

    Seppala, Susanna; Solomon, Kevin V.; Gilmore, Sean P.; Henske, John K.; O’Malley, Michelle A.

    2016-12-20

    Here, engineered cell factories that convert biomass into value-added compounds are emerging as a timely alternative to petroleum-based industries. Although often overlooked, integral membrane proteins such as solute transporters are pivotal for engineering efficient microbial chassis. Anaerobic gut fungi, adapted to degrade raw plant biomass in the intestines of herbivores, are a potential source of valuable transporters for biotechnology, yet very little is known about the membrane constituents of these non-conventional organisms. Here, we mined the transcriptome of three recently isolated strains of anaerobic fungi to identify membrane proteins responsible for sensing and transporting biomass hydrolysates within a competitive and rather extreme environment. Using sequence analyses and homology, we identified membrane protein-coding sequences from assembled transcriptomes from three strains of anaerobic gut fungi: Neocallimastix californiae, Anaeromyces robustus, and Piromyces finnis. We identified nearly 2000 transporter components: about half of these are involved in the general secretory pathway and intracellular sorting of proteins; the rest are predicted to be small-solute transporters. Unexpectedly, we found a number of putative sugar binding proteins that are associated with prokaryotic uptake systems; and approximately 100 class C G-protein coupled receptors (GPCRs) with non-canonical putative sugar binding domains. In conclusion, we report the first comprehensive characterization of the membrane protein machinery of biotechnologically relevant anaerobic gut fungi. Apart from identifying conserved machinery for protein sorting and secretion, we identify a large number of putative solute transporters that are of interest for biotechnological applications. Notably, our data suggests that the fungi display a plethora of carbohydrate binding domains at their surface, perhaps as a means to sense and sequester some of the sugars that their

  15. Enhanced catalytic efficiency of endo-β-agarase I by fusion of carbohydrate-binding modules for agar prehydrolysis.

    PubMed

    Alkotaini, Bassam; Han, Nam Soo; Kim, Beom Soo

    2016-11-01

    Recently, Microbulbifer thermotolerans JAMB-A94 endo-β-agarase I was expressed as catalytic domain (GH16) without a carbohydrate-binding module (CBM). In this study, we successfully constructed different fusions of GH16 with its original CBM6 and CBM13 derived from Catenovulum agarivorans. The optimum temperature and pH for fusions GH16-CBM6, GH16-CBM13, GH16-CBM6-CBM13 and GH16-CBM13-CBM6 were similar to GH16, at 55°C and pH 7. All the constructed fusions significantly enhanced the GH16 affinity (Km) and the catalytic efficiency (Kcat/Km) toward agar. Among them, GH16-CBM6-CBM13 exhibited the highest agarolytic activity, for which Km decreased from 3.67 to 2.11mg/mL and Kcat/Km increased from 98.6 (mg/mL)(-1)sec(-1) to 400.6 (mg/mL)(-1)sec(-1). Moreover, all fusions selectively increased GH16 binding ability to agar, in which the highest binding ability of 95% was obtained with fusion GH16-CBM6-CBM13. Melted agar was prehydrolyzed with GH16-CBM6-CBM13, resulting in a degree of liquefaction of 45.3% and reducing sugar yield of 14.2%. Further addition of Saccharophagus degradans agarolytic enzymes resulted in mono-sugar yields of 35.4% for galactose and 31.5% for 3,6-anhydro-l-galactose. There was no pH neutralization step required and no 5-hydroxymethylfurfural detected, suggesting the potential of a new enzymatic prehydrolysis process for efficient production of bio-products such as biofuels.

  16. Mapping the membrane proteome of anaerobic gut fungi identifies a wealth of carbohydrate binding proteins and transporters

    DOE PAGES

    Seppala, Susanna; Solomon, Kevin V.; Gilmore, Sean P.; ...

    2016-12-20

    Here, engineered cell factories that convert biomass into value-added compounds are emerging as a timely alternative to petroleum-based industries. Although often overlooked, integral membrane proteins such as solute transporters are pivotal for engineering efficient microbial chassis. Anaerobic gut fungi, adapted to degrade raw plant biomass in the intestines of herbivores, are a potential source of valuable transporters for biotechnology, yet very little is known about the membrane constituents of these non-conventional organisms. Here, we mined the transcriptome of three recently isolated strains of anaerobic fungi to identify membrane proteins responsible for sensing and transporting biomass hydrolysates within a competitive andmore » rather extreme environment. Using sequence analyses and homology, we identified membrane protein-coding sequences from assembled transcriptomes from three strains of anaerobic gut fungi: Neocallimastix californiae, Anaeromyces robustus, and Piromyces finnis. We identified nearly 2000 transporter components: about half of these are involved in the general secretory pathway and intracellular sorting of proteins; the rest are predicted to be small-solute transporters. Unexpectedly, we found a number of putative sugar binding proteins that are associated with prokaryotic uptake systems; and approximately 100 class C G-protein coupled receptors (GPCRs) with non-canonical putative sugar binding domains. In conclusion, we report the first comprehensive characterization of the membrane protein machinery of biotechnologically relevant anaerobic gut fungi. Apart from identifying conserved machinery for protein sorting and secretion, we identify a large number of putative solute transporters that are of interest for biotechnological applications. Notably, our data suggests that the fungi display a plethora of carbohydrate binding domains at their surface, perhaps as a means to sense and sequester some of the sugars that their biomass

  17. Mapping the membrane proteome of anaerobic gut fungi identifies a wealth of carbohydrate binding proteins and transporters.

    PubMed

    Seppälä, Susanna; Solomon, Kevin V; Gilmore, Sean P; Henske, John K; O'Malley, Michelle A

    2016-12-20

    Engineered cell factories that convert biomass into value-added compounds are emerging as a timely alternative to petroleum-based industries. Although often overlooked, integral membrane proteins such as solute transporters are pivotal for engineering efficient microbial chassis. Anaerobic gut fungi, adapted to degrade raw plant biomass in the intestines of herbivores, are a potential source of valuable transporters for biotechnology, yet very little is known about the membrane constituents of these non-conventional organisms. Here, we mined the transcriptome of three recently isolated strains of anaerobic fungi to identify membrane proteins responsible for sensing and transporting biomass hydrolysates within a competitive and rather extreme environment. Using sequence analyses and homology, we identified membrane protein-coding sequences from assembled transcriptomes from three strains of anaerobic gut fungi: Neocallimastix californiae, Anaeromyces robustus, and Piromyces finnis. We identified nearly 2000 transporter components: about half of these are involved in the general secretory pathway and intracellular sorting of proteins; the rest are predicted to be small-solute transporters. Unexpectedly, we found a number of putative sugar binding proteins that are associated with prokaryotic uptake systems; and approximately 100 class C G-protein coupled receptors (GPCRs) with non-canonical putative sugar binding domains. We report the first comprehensive characterization of the membrane protein machinery of biotechnologically relevant anaerobic gut fungi. Apart from identifying conserved machinery for protein sorting and secretion, we identify a large number of putative solute transporters that are of interest for biotechnological applications. Notably, our data suggests that the fungi display a plethora of carbohydrate binding domains at their surface, perhaps as a means to sense and sequester some of the sugars that their biomass degrading, extracellular enzymes

  18. Low-carbohydrate diets and all-cause and cause-specific mortality: Two cohort Studies

    PubMed Central

    Fung, Teresa T.; van Dam, Rob M.; Hankinson, Susan E.; Stampfer, Meir; Willett, Walter C.; Hu, Frank B.

    2010-01-01

    Background Data on the long-term association between low-carbohydrate diets and mortality are sparse. Objective To examine the association of low-carbohydrate diets with mortality during 26 years of follow-up in women and 20 years in men. Design A prospective cohort study of women and men, followed from 1980 (women) or 1986 (men) until 2006. Low-carbohydrate diets, either animal-based (emphasizing animal sources of fat and protein), or vegetable-based (emphasizing vegetable sources of fat and protein) were computed from multiple validated food frequency questionnaire assessed during follow-up. Setting Nurses' Health Study and Health Professionals' Follow-up Study Participants 85,168 women (aged 34-59 years at baseline) and 44,548 men (aged 40-75 years at baseline) without heart disease, cancer, or diabetes. Measurement Investigator documented 12,555 deaths (2,458 cardiovascular, 5,780 cancer) in women and 8,678 deaths (2,746 cardiovascular, 2,960 cancer) in men. Results The overall low-carbohydrate score was associated with a modest increase in overall mortality in pooled analysis (Hazard Ratio, HR, comparing extreme deciles=1.12 (95% CI=1.01-1.24, p-trend=0.14). The animal low-carbohydrate score was associated with a higher all-cause mortality (pooled HR comparing extreme deciles=1.23, 95% CI=1.11-1.37, p-trend=0.05), cardiovascular mortality (corresponding HR=1.14, 95% CI=1.01-1.29, p-trend=0.029), and cancer mortality (corresponding HR=1.28, 95% CI 1.02-1.60, p for trend = 0.09). In contrast, a higher vegetable low-carbohydrate score was associated with lower all-cause (HR=0.80, 95% CI=0.75-0.85, p-trend<0.001) and cardiovascular mortality (HR=0.77, 95% CI=0.68-0.87, p-trend<0.001). Limitations Diet and lifestyle characteristics were assessed with some degree of error, however, sensitivity analyses indicated that results were not unlikely to be substantially affected by residual or confounding or an unmeasured confounder. In addition, participants were not a

  19. Specificity in Transition State Binding: The Pauling Model Revisited

    PubMed Central

    Amyes, Tina L.; Richard, John P.

    2013-01-01

    Linus Pauling proposed that the large rate accelerations for enzymes are due to the high specificity of the protein catalyst for binding the reaction transition state. The observation that stable analogs of the transition states for enzymatic reactions often act as tight-binding binding inhibitors provided early support for this simple and elegant proposal. We review experimental results which support the proposal that Pauling’s model provides a satisfactory explanation for the rate accelerations for many heterolytic enzymatic reactions through high energy reaction intermediates, such as proton transfer and decarboxylation. Specificity in transition state binding is obtained when the total intrinsic binding energy of the substrate is significantly larger than the binding energy observed at the Michaelis complex. The results of recent studies to characterize the specificity in binding of the enolate oxygen at the transition state for the 1,3-isomerization reaction catalyzed by ketosteroid isomerase are reviewed. Interactions between pig heart succinyl-CoA:3-oxoacid coenzyme A transferase (SCOT) and the nonreacting portions of CoA are responsible for a rate increase of 3 × 1012-fold, which is close to the estimated total 5 × 1013-fold enzymatic rate acceleration. Studies that partition the interactions between SCOT and CoA into their contributing parts are reviewed. Interactions of the protein with the substrate phosphodianion group provide a ca. 12 kcal/mol stabilization of the transition state for the reactions catalyzed by triosephosphate isomerase, orotidine 5′-monophosphate decarboxylase and α-glycerol phosphate dehydrogenase. The interactions of these enzymes with the substrate piece phosphite dianion provide a 6 – 8 kcal/mol stabilization of the transition state for reaction of the appropriate truncated substrate. Enzyme activation by phosphite dianion reflects the higher dianion affinity for binding to the enzyme-transition state complex compared

  20. Sequence specific binding of chlamydial histone H1-like protein.

    PubMed Central

    Kaul, R; Allen, M; Bradbury, E M; Wenman, W M

    1996-01-01

    Chlamydia trachomatis is one of the few prokaryotic organisms known to contain proteins that bear homology to eukaryotic histone H1. Changes in macromolecular conformation of DNA mediated by the histone H1-like protein (Hc1) appear to regulate stage specific differentiation. We have developed a cross-linking immunoprecipitation protocol to examine in vivo protein-DNA interaction by immune precipitating chlamydial Hc1 cross linked to DNA. Our results strongly support the presence of sequence specific binding sites on the chlamydial plasmid and hc1 gene upstream of its open reading frame. The preferential binding sites were mapped to 520 bp BamHI-XhoI and 547 bp BamHI-DraI DNA fragments on the plasmid and hc1 respectively. Comparison of these two DNA sequences using Bestfit program has identified a 24 bp region with >75% identity that is unique to the chlamydial genome. Double-stranded DNA prepared by annealing complementary oligonucleotides corresponding to the conserved 24 bp region bind Hc1, in contrast to control sequences with similar A+T ratios. Further, Hc1 binds to DNA in a strand specific fashion, with preferential binding for only one strand. The site specific affinity to plasmid DNA was also demonstrated by atomic force microscopy data images. Binding was always followed by coiling, shrinking and aggregation of the affected DNA. Very low protein-DNA ratio was required if incubations were carried out in solution. However, if DNA was partially immobilized on mica substrate individual strands with dark foci were still visible even after the addition of excess Hc1. PMID:8760883

  1. A putative carbohydrate-binding domain of the lactose-binding Cytisus sessilifolius anti-H(O) lectin has a similar amino acid sequence to that of the L-fucose-binding Ulex europaeus anti-H(O) lectin.

    PubMed

    Konami, Y; Yamamoto, K; Osawa, T; Irimura, T

    1995-04-01

    The complete amino acid sequence of a lactose-binding Cytisus sessilifolius anti-H(O) lectin II (CSA-II) was determined using a protein sequencer. After digestion of CSA-II with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSA-II with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid sequence of a putative carbohydrate-binding domain of CSA-II was found to be similar to those of several anti-H(O) leguminous lectins, especially to that of the L-fucose-binding Ulex europaeus lectin I (UEA-I).

  2. Computational redesign of endonuclease DNA binding and cleavage specificity

    NASA Astrophysics Data System (ADS)

    Ashworth, Justin; Havranek, James J.; Duarte, Carlos M.; Sussman, Django; Monnat, Raymond J.; Stoddard, Barry L.; Baker, David

    2006-06-01

    The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein-DNA recognition, and has considerable practical relevance for biotechnology and medicine. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI using a physically realistic atomic-level forcefield. Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling. A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site ~10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.

  3. Binding specificity of a monoclonal anti-DNA antibody.

    PubMed

    Pisetsky, D S; Caster, S A

    1982-05-01

    To investigate the interaction of DNA and anti-DNA antibodies in the immune complex disease of systemic lupus erythematosus, the fine specificity of binding of a monoclonal anti-DNA antibody was determined. This antibody, termed Cll, was derived from the fusion of spleen cells from an autoimmune MRL-lpr/lpr mouse with the myeloma cell line M45. In a solid-phase ELISA assay to measure anti-DNA activity, Cll showed preference for single stranded compared to double stranded DNA of animal origin. The Cll antibody also bound some deoxyribohomopolymers as well as ribohomopolymers, but failed to bind synthetic DNA duplexes. Defined size oligonucleotides with a size range of 2-(12-18) failed to inhibit the binding of Cll to single stranded DNA. This pattern of binding is consistent with the recognition of a unique structural determinant that can be represented by a variety of nucleic acids. The absence of antigenic activity among the oligonucleotides suggests that an extended polynucleotide structure is required for antibody binding, possibly because of a bivalent or 'monogamous' mode of interaction. The binding properties of Cll further suggest that its ability to participate in immune complex formation may be limited by the nature of the available DNA antigen.

  4. Study Of The Specificity Of Xanthene Dye Binding To Mitochondria

    NASA Astrophysics Data System (ADS)

    Bunting, James R.; Kamali, Eleanor; Phan, Trung V.; Dowben, Robert M.; Matthews, J. Lester

    1989-03-01

    The binding of Rhodamine 123 (Rh123), Rhodamine 6G (R6G), and Rhodamine B (RhB) (from the cationic xanthene series) to isolated rat liver mitochondria maintained in State IV respiration in the presence of rotenone (NADH oxidase inhibitor) was monitored by following changes in the fluorescence signal of the dyes. Rh123 and Rh6G bind strongly with quenching, to 0.25 and 0.20, respectively, and red shift of emission maxima by 10 nm. RhB binds much less potently with slight emission enhancement of 1.2. For Rh123 added to 0.5 mg/ml mitochondria' protein, a sigmoidal relationship is obtained between percentage fluorescence quenching and log of Rh123 concentration with a 50% inflection point of 3.5x10-6M, estimating an apparent association constant of 2.9x 105M-1 for Rh123 binding. Addition of 7 uM RhB during Rh123 titration moves the sigmoidal inflection point to higher Rh123 concentrations, suggesting either RhB enhancement of binding of Rh123 fluorescence quenching by energy transfer to RhB bound. These results suggest that, to a great degree, the binding of the xanthene dyes to mitochondrial sites is specific, competitive, and probably cooperative.

  5. Serum carbohydrate-binding IgM are present in Vietnamese striped catfish (Pangasianodon hypophthalmus) but not in North African catfish (Clarias gariepinus).

    PubMed

    Giang, Duong Thi Huong; Van Driessche, Edilbert; Beeckmans, Sonia

    2012-02-01

    Pangasianodon hypophthalmus serum was fractionated by affinity chromatography on 12 different Sepharose-carbohydrate columns and proteins eluted by the corresponding sugar. Binding to the affinity matrices is dependent on Ca(2+) ions. Upon gel filtration using Superose-12, essentially one fraction was obtained, eluting as a protein with a molecular mass of about 900 kDa. SDS-PAGE in reducing conditions revealed the presence of large (72 kDa) subunits (H-chains) and one up to three small (24, 26 and/or 28-29 kDa) subunits (L-chains). The isolated proteins were shown to be IgM since they bind monoclonal anti-P. hypophthalmus IgM antibodies. Rabbit polyclonal anti-galactose-binding IgM only cross-react with some sugar-binding IgM. The H-chains of the anti-carbohydrate IgM are glycosylated. Circular dichroism studies revealed that the IgMs have an "all-β" type of structure, and that Ca(2+) ions, though essential for carbohydrate-binding activity, are not required for the structural integrity of the molecules. In non-reducing SDS-PAGE, only monomers and halfmers were obtained, showing that there are no disulfide bonds linking the monomers, and that a disulfide bond connecting both H-chains within one monomer is only present in 45% of the molecules. Both the monomers and the halfmers display molecular mass heterogeneity which is indicative for redox forms at the level of the intradomain disulfide bonds. The native carbohydrate-binding IgMs agglutinate erythrocytes from different animals, as well as fish pathogenic bacteria. Similar proteins could not be isolated from another catfish, Clarias gariepinus.

  6. Structural Analysis of Semi-specific Oligosaccharide Recognition by a Cellulose-binding Protein of Thermotoga maritima Reveals Adaptations for Functional Diversification of the Oligopeptide Periplasmic Binding Protein Fold

    SciTech Connect

    Cuneo, Matthew J.; Beese, Lorena S.; Hellinga, Homme W.

    2010-05-25

    Periplasmic binding proteins (PBPs) constitute a protein superfamily that binds a wide variety of ligands. In prokaryotes, PBPs function as receptors for ATP-binding cassette or tripartite ATP-independent transporters and chemotaxis systems. In many instances, PBPs bind their cognate ligands with exquisite specificity, distinguishing, for example, between sugar epimers or structurally similar anions. By contrast, oligopeptide-binding proteins bind their ligands through interactions with the peptide backbone but do not distinguish between different side chains. The extremophile Thermotoga maritima possesses a remarkable array of carbohydrate-processing metabolic systems, including the hydrolysis of cellulosic polymers. Here, we present the crystal structure of a T. maritima cellobiose-binding protein (tm0031) that is homologous to oligopeptide-binding proteins. T. maritima cellobiose-binding protein binds a variety of lengths of {beta}(1 {yields} 4)-linked glucose oligomers, ranging from two rings (cellobiose) to five (cellopentaose). The structure reveals that binding is semi-specific. The disaccharide at the nonreducing end binds specifically; the other rings are located in a large solvent-filled groove, where the reducing end makes several contacts with the protein, thereby imposing an upper limit of the oligosaccharides that are recognized. Semi-specific recognition, in which a molecular class rather than individual species is selected, provides an efficient solution for the uptake of complex mixtures.

  7. Specific binding sites for muramyl peptides on murine macrophages

    SciTech Connect

    Silverman, D.H.S.; Krueger, J.M.; Karnovsky, M.L.

    1986-03-15

    Two radiolabeled (/sup 125/I) muramyl peptide derivatives of high specific activity were prepared: a tripeptide with an iodinated C-terminal tyrosine methyl ester (Ligand I), and a muramyl tripeptide with a C-terminal lysine derivatized with Bolton-Hunter reagent (Ligand II). These were used to characterize binding of muramyl peptides to monolayers of murine macrophages. Saturable high-affinity binding to resident, caseinate-elicited, and Listeria-activated peritoneal cells was observed with both radioligands. Binding affinities varied with the state of activation of the macrophages, and K/sub D/ values ranged from 48 +/- 33 pM (for resident macrophages, Ligand I) to 1020 +/- 90 pM (for activated macrophages, Ligand II). Specific binding sites were also found on a macrophage-derived cell line. The ability of several unlabeled muramyl peptides to compete with Ligands I and II for their binding sites was tested. Competition was stereospecific and correlated with known biological activities of these compounds (i.e., immunoadjuvanticity, pyrogenicity, and somnogenicity). The sites identified here for Ligands I and II may mediate some of the effects that muramyl peptides have previously been demonstrated to have on macrophages.

  8. Importance of DNA stiffness in protein-DNA binding specificity

    NASA Astrophysics Data System (ADS)

    Hogan, M. E.; Austin, R. H.

    1987-09-01

    From the first high-resolution structure of a repressor bound specifically to its DNA recognition sequence1 it has been shown that the phage 434 repressor protein binds as a dimer to the helix. Tight, local interactions are made at the ends of the binding site, causing the central four base pairs (bp) to become bent and overtwisted. The centre of the operator is not in contact with protein but repressor binding affinity can be reduced at least 50-fold in response to a sequence change there2. This observation might be explained should the structure of the intervening DNA segment vary with its sequence, or if DNA at the centre of the operator resists the torsional and bending deformation necessary for complex formation in a sequence dependent fashion. We have considered the second hypothesis by demonstrating that DNA stiffness is sequence dependent. A method is formulated for calculating the stiffness of any particular DNA sequence, and we show that this predicted relationship between sequence and stiffness can explain the repressor binding data in a quantitative manner. We propose that the elastic properties of DNA may be of general importance to an understanding of protein-DNA binding specificity.

  9. 5-HT manipulation and dietary choice: variable carbohydrate (Polycose) suppression demonstrated only under specific experimental conditions.

    PubMed

    Lawton, C L; Blundell, J E

    1993-01-01

    The effects of six 5-HT anorectic agents, d-fenfluramine (5-HT releaser and reuptake inhibitor), fluoxetine (5-HT reuptake inhibitor), mCPP (5-HT1B/5-HT1C receptor agonist), RU24969 (5-HT1A/5-HT1B receptor agonist), MK212 (5-HT1C receptor agonist) and DOI (5-HT2/5-HT1C receptor agonist), and two non-5-HT anorectic agents, salbutamol (beta 2-adrenergic agonist) and d-amphetamine (catecholaminergic agonist), were examined in an experimental procedure designed to disclose selective effects on carbohydrate consumption. In this procedure, a revised version of what we have termed "The Classic Sclafani Paradigm", animals are presented with powdered Polycose as an optional carbohydrate supplement to hydrated chow (nutritionally complete diet). All drugs produced significant reductions in total (hydrated chow plus powdered Polycose) intake. However, only the 5-HT drugs DOI and fluoxetine exerted significantly stronger anorectic effects on intake of powdered Polycose than on intake of hydrated chow. d-Fenfluramine also showed a tendency to selectively suppress Polycose intake but this effect marginally failed to reach significance. These results suggest that when experimental conditions are favourable, what appears to be selective carbohydrate (Polycose) suppression can be demonstrated with certain 5-HT drugs. They also suggest that a selective effect on carbohydrate intake is not the most prominent feeding response to 5-HT drugs.

  10. Glycosphingolipid Binding Specificities of Rotavirus: Identification of a Sialic Acid-Binding Epitope

    PubMed Central

    Delorme, Cécile; Brüssow, Harald; Sidoti, Josette; Roche, Niamh; Karlsson, Karl-Anders; Neeser, Jean-Richard; Teneberg, Susann

    2001-01-01

    The glycosphingolipid binding specificities of neuraminidase-sensitive (simian SA11 and bovine NCDV) and neuraminidase-insensitive (bovine UK) rotavirus strains were investigated using the thin-layer chromatogram binding assay. Both triple-layered and double-layered viral particles of SA11, NCDV, and UK bound to nonacid glycosphingolipids, including gangliotetraosylceramide (GA1; also called asialo-GM1) and gangliotriaosylceramide (GA2; also called asialo-GM2). Binding to gangliosides was observed with triple-layered particles but not with double-layered particles. The neuraminidase-sensitive and neuraminidase-insensitive rotavirus strains showed distinct ganglioside binding specificities. All three strains bound to sialylneolactotetraosylceramide and GM2 and GD1a gangliosides. However, NeuAc-GM3 and the GM1 ganglioside were recognized by rotavirus strain UK but not by strains SA11 and NCDV. Conversely, NeuGc-GM3 was bound by rotaviruses SA11 and NCDV but not by rotavirus UK. Thus, neuraminidase-sensitive strains bind to external sialic acid residues in gangliosides, while neuraminidase-insensitive strains recognize gangliosides with internal sialic acids, which are resistant to neuraminidase treatment. By testing a panel of gangliosides with triple-layered particles of SA11 and NCDV, the terminal sequence sialyl-galactose (NeuGc/NeuAcα3-Galβ) was identified as the minimal structural element required for the binding of these strains. The binding of triple-layered particles of SA11 and NCDV to NeuGc-GM3, but not to NeuAc-GM3, suggested that the sequence NeuGcα3Galβ is preferred to NeuAcα3Galβ. Further dissection of this binding epitope showed that the carboxyl group and glycerol side chain of sialic acid played an important role in the binding of such triple-layered particles. PMID:11160731

  11. LHRH-pituitary plasma membrane binding: the presence of specific binding sites in other tissues.

    PubMed

    Marshall, J C; Shakespear, R A; Odell, W D

    1976-11-01

    Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.

  12. Carbohydrate-Based Nanocarriers Exhibiting Specific Cell Targeting with Minimum Influence from the Protein Corona.

    PubMed

    Kang, Biao; Okwieka, Patricia; Schöttler, Susanne; Winzen, Svenja; Langhanki, Jens; Mohr, Kristin; Opatz, Till; Mailänder, Volker; Landfester, Katharina; Wurm, Frederik R

    2015-06-15

    Whenever nanoparticles encounter biological fluids like blood, proteins adsorb on their surface and form a so-called protein corona. Although its importance is widely accepted, information on the influence of surface functionalization of nanocarriers on the protein corona is still sparse, especially concerning how the functionalization of PEGylated nanocarriers with targeting agents will affect protein corona formation and how the protein corona may in turn influence the targeting effect. Herein, hydroxyethyl starch nanocarriers (HES-NCs) were prepared, PEGylated, and modified on the outer PEG layer with mannose to target dendritic cells (DCs). Their interaction with human plasma was then studied. Low overall protein adsorption with a distinct protein pattern and high specific affinity for DC binding were observed, thus indicating an efficient combination of "stealth" and targeting behavior.

  13. Quantification of specific bindings of biomolecules by magnetorelaxometry

    PubMed Central

    Eberbeck, Dietmar; Bergemann, Christian; Wiekhorst, Frank; Steinhoff, Uwe; Trahms, Lutz

    2008-01-01

    The binding reaction of the biomolecules streptavidin and anti-biotin antibody, both labelled by magnetic nanoparticles (MNP), to biotin coated on agarose beads, was quantified by magnetorelaxometry (MRX). Highly sensitive SQUID-based MRX revealed the immobilization of the MNP caused by the biotin-streptavidin coupling. We found that about 85% of streptavidin-functionalised MNP bound specifically to biotin-agarose beads. On the other hand only 20% of antibiotin-antibody functionalised MNP were specifically bound. Variation of the suspension medium revealed in comparison to phosphate buffer with 0.1% bovine serum albumin a slight change of the binding behaviour in human serum, probably due to the presence of functioning (non heated) serum proteins. Furthermore, in human serum an additional non-specific binding occurs, being independent from the serum protein functionality. The presented homogeneous bead based assay is applicable in simple, uncoated vials and it enables the assessment of the binding kinetics in a volume without liquid flow. The estimated association rate constant for the MNP-labelled streptavidin is by about two orders of magnitude smaller than the value reported for free streptavidin. This is probably due to the relatively large size of the magnetic markers which reduces the diffusion of streptavidin. Furthermore, long time non-exponential kinetics were observed and interpreted as agglutination of the agarose beads. PMID:18334023

  14. Specific binding of a basic peptide from HIV-1 Rev.

    PubMed Central

    Kjems, J; Calnan, B J; Frankel, A D; Sharp, P A

    1992-01-01

    Human immunodeficiency virus type I (HIV-1) encodes a regulatory protein, Rev, which is required for cytoplasmic expression of incompletely spliced viral mRNA. Rev activity is mediated through specific binding to a cis-acting Rev responsive element (RRE) located within the env region of HIV-1. A monomer Rev binding site corresponding to 37 nucleotides of the RRE (IIB RNA) was studied by RNA footprinting, modification interference experiments and mutational analysis. Surprisingly, a 17 amino acid peptide, corresponding to the basic domain of Rev, binds specifically to this site at essentially identical nucleotides and probably induces additional base pairing. The Rev protein and related peptide interact primarily with two sets of nucleotides located at the junction of single and double stranded regions, and at an additional site located within a helix. This suggests that the domains of proteins responsible for specific RNA binding can be remarkably small and that the interaction between RNA and protein can probably induce structure in both constituents. Images PMID:1547776

  15. Universal protein binding microarrays for the comprehensive characterization of the DNA binding specificities of transcription factors

    PubMed Central

    Berger, Michael F.; Bulyk, Martha L.

    2010-01-01

    Protein binding microarray (PBM) technology provides a rapid, high-throughput means of characterizing the in vitro DNA binding specificities of transcription factors (TFs). Using high-density, custom-designed microarrays containing all 10-mer sequence variants, one can obtain comprehensive binding site measurements for any TF, regardless of its structural class or species of origin. Here, we present a protocol for the examination and analysis of TF binding specificities at high resolution using such ‘all 10-mer’ universal PBMs. This procedure involves double-stranding a commercially synthesized DNA oligonucleotide array, binding a TF directly to the double-stranded DNA microarray, and labeling the protein-bound microarray with a fluorophore-conjugated antibody. We describe how to computationally extract the relative binding preferences of the examined TF for all possible contiguous and gapped 8-mers over the full range of affinities, from highest affinity sites to nonspecific sites. Multiple proteins can be tested in parallel in separate chambers on a single microarray, enabling the processing of a dozen or more TFs in a single day. PMID:19265799

  16. Determination of Carbohydrate Structure Recognized by Prostate-specific F77 Monoclonal Antibody through Expression Analysis of Glycosyltransferase Genes*

    PubMed Central

    Nonaka, Motohiro; Fukuda, Michiko N.; Gao, Chao; Li, Zhen; Zhang, Hongtao; Greene, Mark I.; Peehl, Donna M.; Feizi, Ten; Fukuda, Minoru

    2014-01-01

    This study reports the determination of the carbohydrate epitope of monoclonal antibody F77 previously raised against human prostate cancer PC-3 cells (Zhang, G., Zhang, H., Wang, Q., Lal, P., Carroll, A. M., de la Llera-Moya, M., Xu, X., and Greene, M. I. (2010) Proc. Natl. Acad. Sci. U. S. A. 107, 732–737). We performed a series of co-transfections using mammalian expression vectors encoding specific glycosyltransferases. We thereby identified branching enzymes and FUT1 (required for Fucα1→2Gal linkage) as being essential for F77 antigen formation. When immortalized normal prostate 267B1 cells were transfected with FUT1 alone, cells showed weak expression of F77 antigen. By contrast, cells co-transfected with FUT1 plus either GCNT1, GCNT2, or GCNT3 (an enzyme required to form GlcNAcβ1→6Gal/GalNAc) showed robust F77 antigen expression, suggesting that F77 specifically binds to Fucα1→2Galβ1→4GlcNAcβ1→6Gal/GalNAc. RT-PCR for FUT1, GCNT1, GCNT2, and GCNT3 showed that F77-positive cell lines indeed express transcripts encoding FUT1 plus one GCNT. F77-positive prostate cancer cells transfected with siRNAs targeting FUT1, GCNT2, and GCNT3 showed significantly reduced F77 antigen, confirming the requirement of these enzymes for epitope synthesis. We also found that hypoxia induces F77 epitope expression in immortalized prostate RWPE1 cells, which express F77 antigen moderately under normoxia but at an elevated level under hypoxia. Quantitative RT-PCR demonstrated up-regulation of FUT1, GCNT2, and GCNT3 transcripts in RWPE1 cells under hypoxia, suggesting that hypoxia up-regulates glycosyltransferase expression required for F77 antigen synthesis. These results define the F77 epitope and provide a potential mechanism for F77 antigen synthesis in malignant prostate cancer. PMID:24753248

  17. Stage specific kinetoplast DNA-binding proteins in Trypanosoma cruzi.

    PubMed

    Zavala-Castro, J E; Acosta-Viana, K; Guzmán-Marín, E; Rosado-Barrera, M E; Rosales-Encina, J L

    2000-09-18

    Knowledge regarding kinetoplast DNA organization in all members of the Trypanosomatid family is incomplete. Recently, the presence of kinetoplast-associated proteins in condensing kDNA networks in Crithidia fasciculata has been described and a role for these proteins in the maintenance of these complex structures was suggested. To investigate the presence of protein components in Trypanosoma cruzi kinetoplast, we previously described seven epimastigote kinetoplast-associated proteins. We report here the existence of kinetoplast binding proteins in amastigote and trypomastigote stages of T. cruzi, which could bind both mini and maxicircles components with a stage specific elements for every infective form of the parasite. We propose three major classes of kinetoplast-associated proteins related to the basic processes of this intricate disc structure and suggest a possible function of these binding proteins in the T. cruzi mitochondrial DNA organization.

  18. Cellotriose-hydrolyzing activity conferred by truncating the carbohydrate-binding modules of Cel5 from Hahella chejuensis.

    PubMed

    Lee, Hee Jin; Kim, In Jung; Youn, Hak Jin; Yun, Eun Ju; Choi, In-Geol; Kim, Kyoung Heon

    2017-02-01

    Processivity is a typical characteristic of cellobiohydrolases (CBHs); it enables the enzyme to successively hydrolyze the ends of cellulose chains and to produce cellobiose as the major product. Some microbes, which do not have CBHs, utilize endoglucanases (EGs) that exhibit processivity, commonly referred to as processive EGs. A processive EG identified from Hahella chejuensis, HcCel5, has a catalytic domain (CD) belonging to the glycoside hydrolase family 5 (GH5) and two carbohydrate-binding modules (CBM6s). In this study, we compared HcCel5-CD with the CD of Saccharophagus degradans Cel5H (SdCel5H-CD), which is a processive EG reported previously. Our results showed that in comparison to SdCel5H-CD, HcCel5-CD has more suitable characteristics for cellulose hydrolysis, such as higher hydrolytic activity, thermostability (40-80 °C), and processivity. Noticeably, HcCel5-CD is capable of hydrolyzing cellotriose, unlike HcCel5. These features of HcCel5-CD for cellulose hydrolysis could be employed for efficient saccharification of lignocellulose to produce cellobiose and glucose, which may be used to produce renewable fuels and chemicals.

  19. Sensing carbohydrate-protein interactions at picomolar concentrations using cantilever arrays.

    PubMed

    Gruber, Kathrin; Hermann, Bianca A; Seeberger, Peter H

    2011-09-05

    Carbohydrates are important mediators of many biological processes that underlie cellular communication and disease mechanisms. Therapeutic agents include carbohydrate-based vaccines and the potent anti-viral protein Cyanovirin-N (CV-N). CV-N acts by specifically binding the carbohydrate structures decorating the cell surface of deadly viruses including human immunodeficiency virus (HI-V) or Ebola. In search for new carbohydrate-binding proteins and the development of sensors that exploit carbohydrate-protein interactions the label-free cantilever array technique can provides a fast, parallel and low-cost approach.

  20. NAD+ Modulates p53 DNA Binding Specificity and Function

    PubMed Central

    McLure, Kevin G.; Takagi, Masatoshi; Kastan, Michael B.

    2004-01-01

    DNA damage induces p53 DNA binding activity, which affects tumorigenesis, tumor responses to therapies, and the toxicities of cancer therapies (B. Vogelstein, D. Lane, and A. J. Levine, Nature 408:307-310, 2000; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Both transcriptional and transcription-independent activities of p53 contribute to DNA damage-induced cell cycle arrest, apoptosis, and aneuploidy prevention (M. B. Kastan et al., Cell 71:587-597, 1992; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Small-molecule manipulation of p53 DNA binding activity has been an elusive goal, but here we show that NAD+ binds to p53 tetramers, induces a conformational change, and modulates p53 DNA binding specificity in vitro. Niacinamide (vitamin B3) increases the rate of intracellular NAD+ synthesis, alters radiation-induced p53 DNA binding specificity, and modulates activation of a subset of p53 transcriptional targets. These effects are likely due to a direct effect of NAD+ on p53, as a molecule structurally related to part of NAD+, TDP, also inhibits p53 DNA binding, and the TDP precursor, thiamine (vitamin B1), inhibits intracellular p53 activity. Niacinamide and thiamine affect two p53-regulated cellular responses to ionizing radiation: rereplication and apoptosis. Thus, niacinamide and thiamine form a novel basis for the development of small molecules that affect p53 function in vivo, and these results suggest that changes in cellular energy metabolism may regulate p53. PMID:15509798

  1. Aminoglycoside antibiotics: A-site specific binding to 16S

    NASA Astrophysics Data System (ADS)

    Baker, Erin Shammel; Dupuis, Nicholas F.; Bowers, Michael T.

    2009-06-01

    The A-site of 16S rRNA, which is a part of the 30S ribosomal subunit involved in prokaryotic translation, is a well known aminoglycoside binding site. Full characterization of the conformational changes undergone at the A-site upon aminoglycoside binding is essential for development of future RNA/drug complexes; however, the massiveness of 16S makes this very difficult. Recently, studies have found that a 27 base RNA construct (16S27) that comprises the A-site subdomain of 16S behaves similarly to the whole A-site domain. ESI-MS, ion mobility and molecular dynamics methods were utilized in this study to analyze the A-site of 16S27 before and after the addition of ribostamycin (R), paromomycin (P) and lividomycin (L). The ESI mass spectrum for 16S27 alone illustrated both single-stranded 16S27 and double-stranded (16S27)2 complexes. Upon aminoglycoside addition, the mass spectra showed that only one aminoglycoside binds to 16S27, while either one or two bind to (16S27)2. Ion mobility measurements and molecular dynamics calculations were utilized in determining the solvent-free structures of the 16S27 and (16S27)2 complexes. These studies found 16S27 in a hairpin conformation while (16S27)2 existed as a cruciform. Only one aminoglycoside binds to the single A-site of the 16S27 hairpin and this attachment compresses the hairpin. Since two A-sites exist for the (16S27)2 cruciform, either one or two aminoglycosides may bind. The aminoglycosides compress the A-sites causing the cruciform with just one aminoglycoside bound to be larger than the cruciform with two bound. Non-specific binding was not observed in any of the aminoglycoside/16S27 complexes.

  2. Immobilized sialyloligo-macroligand and its protein binding specificity.

    PubMed

    Narla, Satya Nandana; Sun, Xue-Long

    2012-05-14

    We report a chemoenzymatic synthesis of chain-end functionalized sialyllactose-containing glycopolymers with different linkages and their oriented immobilization for glycoarray and SPR-based glyco-biosensor applications. Specifically, O-cyanate chain-end functionalized sialyllactose-containing glycopolymers were synthesized by enzymatic α2,3- and α2,6-sialylation of a lactose-containing glycopolymer that was synthesized by cyanoxyl-mediated free radical polymerization. (1)H NMR showed almost quantitative α2,3- and α2,6-sialylation. The O-cyanate chain-end functionalized sialyllactose-containing glycopolymers were printed onto amine-functionalized glass slides via isourea bond formation for glycoarray formation. Specific protein binding activity of the arrays was confirmed with α2,3- and α2,6-sialyl specific binding lectins together with inhibition assays. Further, immobilizing O-cyanate chain-end functionalized sialyllactose-containing glycopolymers onto amine-modified SPR chip via isourea bond formation afforded SPR-based glyco-biosensor, which showed specific binding activity for lectins and influenza viral hemagglutinins (HA). These sialyloligo-macroligand derived glycoarray and SPR-based glyco-biosensor are closely to mimic 3D nature presentation of sialyloligosaccharides and will provide important high-throughput tools for virus diagnosis and potential antiviral drug candidates screening applications.

  3. Evaluation of a liquid chromatography method for compound-specific δ13C analysis of plant carbohydrates in alkaline media.

    PubMed

    Rinne, Katja T; Saurer, Matthias; Streit, Kathrin; Siegwolf, Rolf T W

    2012-09-30

    Isotope analysis of carbohydrates is important for improved understanding of plant carbon metabolism and plant physiological response to the environment. High-performance liquid chromatography/isotope ratio mass spectrometry (HPLC/IRMS) for direct compound-specific δ(13)C measurements of soluble carbohydrates has recently been developed, but the still challenging sample preparation and the fact that no single method is capable of separating all compounds of interest hinder its wide-spread application. Here we tested in detail a chromatography method in alkaline media. We examined the most suitable chromatographic conditions for HPLC/IRMS analysis of carbohydrates in aqueous conifer needle extracts using a CarboPac PA20 anion-exchange column with NaOH eluent, paying specific attention to compound yields, carbon isotope fractionation processes and the reproducibility of the method. Furthermore, we adapted and calibrated sample preparation methods for HPLC/IRMS analysis. OnGuard II cartridges were used for sample purification. Good peak separation and highly linear and reproducible concentration and δ(13)C measurements were obtained. The alkaline eluent was observed to induce isomerization of hexoses, detected as reduced yields and (13)C fractionation of the affected compounds. A reproducible pre-purification method providing ~100% yield for the carbohydrate compounds of interest was calibrated. The good level of peak separation obtained in this study is reflected in the good precision and linearity of concentration and δ(13)C results. The data provided crucial information on the behaviour of sugars in LC analysis with alkaline media. The observations highlight the importance for the application of compound-matched standard solution for the detection and correction of instrumental biases in concentration and δ(13)C analysis performed under identical chromatographic conditions. The calibrated pre-purification method is well suited for studies with complex matrices

  4. Sex-specific interactions between the IRS1 polymorphism and intakes of carbohydrates and fat on incident type 2 diabetes.

    PubMed

    Ericson, Ulrika; Rukh, Gull; Stojkovic, Ivana; Sonestedt, Emily; Gullberg, Bo; Wirfält, Elisabet; Wallström, Peter; Orho-Melander, Marju

    2013-01-01

    The minor T allele of rs2943641 near the gene encoding for insulin receptor substrate 1 (IRS1) has been associated with decreased risk of type 2 diabetes (T2D) and adiposity in genome-wide association studies. Dietary intake can influence the regulation of IRS1, and studies have indicated sex-specific associations between IRS1 and adiposity. The objective was to examine the interaction between IRS1 rs2943641 and macronutrient intakes on incident T2D and percentage body fat in the Malmö Diet and Cancer cohort. The study included 15,227 women and 9614 men aged 45-74 y without prevalent diabetes. Dietary data were collected with a modified diet history method. During 12 y of follow-up, 1567 incident T2D cases were identified. The T allele was associated with lower incidence of T2D (P-trend = 0.003) and, in men, with higher percentage body fat (P-trend = 0.00002). We observed 3-way interactions between sex, rs2943641, and carbohydrate intake (P = 0.01) as well as between sex, rs2943641, and fat intake (P = 0.01) on incident T2D. Among women, the T allele was associated with decreased risk only in the lower tertiles of carbohydrate intake (P-trend = 0.01, P-interaction = 0.01). In contrast, among men, the T allele was associated with decreased risk in the lowest tertile of fat intake (P-trend = 0.01, P-interaction = 0.02). No interaction was observed between macronutrient intakes and rs2943641 on percentage body fat. Our results indicate that IRS1 rs2943641 interacts with carbohydrate and fat intakes on incident T2D in a sex-specific fashion. A protective association between the rs2943641 T allele and T2D was restricted to women with low carbohydrate intake and to men with low fat intake.

  5. Visualization of specific binding sites of benzodiazepine in human brain

    SciTech Connect

    Shinotoh, H.; Yamasaki, T.; Inoue, O.; Itoh, T.; Suzuki, K.; Hashimoto, K.; Tateno, Y.; Ikehira, H.

    1986-10-01

    Using 11C-labeled Ro15-1788 and positron emission tomography, studies of benzodiazepine binding sites in the human brain were performed on four normal volunteers. Rapid and high accumulation of 11C activity was observed in the brain after i.v. injection of (11C)Ro15-1788, the maximum of which was within 12 min. Initial distribution of 11C activity in the brain was similar to the distribution of the normal cerebral blood flow. Ten minutes after injection, however, a high uptake of 11C activity was observed in the cerebral cortex and moderate uptake was seen in the cerebellar cortex, the basal ganglia, and the thalamus. The accumulation of 11C activity was low in the brain stem. This distribution of 11C activity was approximately parallel to the known distribution of benzodiazepine receptors. Saturation experiments were performed on four volunteers with oral administration of 0.3-1.8 mg/kg of cold Ro15-1788 prior to injection. Initial distribution of 11C activity following injection peaked within 2 min and then the accumulation of 11C activity decreased rapidly and remarkably throughout the brain. The results indicated that (11C) Ro15-1788 associates and dissociates to specific and nonspecific binding sites rapidly and has a high ratio of specific receptor binding to nonspecific binding in vivo. Carbon-11 Ro15-1788 is a suitable radioligand for the study of benzodiazepine receptors in vivo in humans.

  6. Looking into DNA recognition: zinc finger binding specificity

    PubMed Central

    Paillard, Guillaume; Deremble, Cyril; Lavery, Richard

    2004-01-01

    We present a quantitative, theoretical analysis of the recognition mechanisms used by two zinc finger proteins: Zif268, which selectively binds to GC-rich sequences, and a Zif268 mutant, which binds to a TATA box site. This analysis is based on a recently developed method (ADAPT), which allows binding specificity to be analyzed via the calculation of complexation energies for all possible DNA target sequences. The results obtained with the zinc finger proteins show that, although both mainly select their targets using direct, pairwise protein–DNA interactions, they also use sequence-dependent DNA deformation to enhance their selectivity. A new extension of our methodology enables us to determine the quantitative contribution of these two components and also to measure the contributions of individual residues to overall specificity. The results show that indirect recognition is particularly important in the case of the TATA box binding mutant, accounting for 30% of the total selectivity. The residue-by-residue analysis of the protein–DNA interaction energy indicates that the existence of amino acid–base contacts does not necessarily imply sequence selectivity, and that side chains without contacts can nevertheless contribute to defining the protein's target sequence. PMID:15613596

  7. Factor G utilizes a carbohydrate-binding cleft that is conserved between horseshoe crab and bacteria for the recognition of beta-1,3-D-glucans.

    PubMed

    Ueda, Yuki; Ohwada, Shuhei; Abe, Yoshito; Shibata, Toshio; Iijima, Manabu; Yoshimitsu, Yukiko; Koshiba, Takumi; Nakata, Munehiro; Ueda, Tadashi; Kawabata, Shun-ichiro

    2009-09-15

    In the horseshoe crab, the recognition of beta-1,3-D-glucans by factor G triggers hemolymph coagulation. Factor G contains a domain of two tandem xylanase Z-like modules (Z1-Z2), each of which recognizes beta-1,3-D-glucans. To gain an insight into the recognition of beta-1,3-D-glucans from a structural view point, recombinants of Z1-Z2, the C-terminal module Z2, Z2 with a Cys to Ala substitution (Z2A), and its tandem repeat Z2A-Z2A were characterized. Z2 and Z1-Z2, but not Z2A and Z2A-Z2A, formed insoluble aggregates at higher concentrations more than approximately 30 and 3 microM, respectively. Z1-Z2 and Z2A-Z2A bound more strongly to an insoluble beta-1,3-D-glucan (curdlan) than Z2A. The affinity of Z2A for a soluble beta-1,3-D-glucan (laminarin) was equivalent to those of Z1-Z2, Z2A-Z2A, and native factor G, suggesting that the binding of a single xylanase Z-like module prevents the subsequent binding of another module to laminarin. Interestingly, Z2A as well as intact factor G exhibited fungal agglutinating activity, and fungi were specifically detected with fluorescently tagged Z2A by microscopy. The chemical shift perturbation of Z2A induced by the interaction with laminaripentaose was analyzed by nuclear magnetic resonance spectroscopy. The ligand-binding site of Z2A was located in a cleft on a beta-sheet in a predicted beta-sandwich structure, which was superimposed onto cleft B in a cellulose-binding module of endoglucanase 5A from the soil bacterium Cellvibrio mixtus. We conclude that the pattern recognition for beta-1,3-D-glucans by factor G is accomplished via a carbohydrate-binding cleft that is evolutionally conserved between horseshoe crab and bacteria.

  8. Carbohydrate functionalized carbon nanotubes and their applications.

    PubMed

    Gorityala, Bala Kishan; Ma, Jimei; Wang, Xin; Chen, Peng; Liu, Xue-Wei

    2010-08-01

    Carbon nanotubes (CNTs) have attracted tremendous attention in biomedical applications due to their molecular size and unique properties. This tutorial review summarizes the strategies to functionalize CNTs with bioactive carbohydrates, which improve their solubility, biocompatibility and biofunctionalities while preserving their desired properties. In addition, studies on the usage of carbohydrate functionalized CNTs to detect bacteria, to bind to specific lectins, to deliver glycomimetic drug molecules into cells and to probe cellular activities as biosensors are reviewed. Improvement in biocompatibility and introduction of bio-functionalities by integration of carbohydrate with CNTs are paving the way to glyconanotechnology and may provide new tools for glycobiological studies.

  9. Specific glucocorticoid receptor binding to DNA reconstituted in a nucleosome.

    PubMed Central

    Perlmann, T; Wrange, O

    1988-01-01

    We have reconstituted a nucleosome with core histones from rat liver using a restriction fragment containing a sequence from the mouse mammary tumour virus (MTV) long terminal repeat (LTR). This sequence harbours glucocorticoid responsive elements (GREs) which mediate glucocorticoid hormone induction of transcription from the MTV promoter via glucocorticoid receptor (GR) binding. Exonuclease III and DNase I footprinting demonstrated that the reconstituted nucleosome was specifically located between positions -219 and -76. A nucleosome was previously shown to be located at a similar or identical position in the MTV promoter in situ and to be structurally altered upon glucocorticoid hormone induction. We demonstrated, by DNase I footprinting, that GR is able to bind sequence specifically to the DNA in the in vitro assembled nucleosome. No evidence for unfolding of the nucleosome was obtained, but the DNase I footprinting pattern demonstrated GR induced local alterations in the DNA. Images PMID:2846275

  10. Influence of a family 29 carbohydrate binding module on the activity of galactose oxidase from Fusarium graminearum.

    PubMed

    Mollerup, Filip; Parikka, Kirsti; Vuong, Thu V; Tenkanen, Maija; Master, Emma

    2016-02-01

    Galactose oxidase (GaO) selectively oxidizes the primary hydroxyl of galactose to a carbonyl, facilitating targeted chemical derivatization of galactose-containing polysaccharides, leading to renewable polymers with tailored physical and chemical properties. Here we investigate the impact of a family 29 glucomannan binding module on the activity and binding of GaO towards various polysaccharides. Specifically, CBM29-1-2 from Piromyces equi was separately linked to the N- and C-termini of GaO. Both GaO-CBM29 and CBM29-GaO were successfully expressed in Pichia pastoris, and demonstrated enhanced binding to galactomannan, galactoglucomannan and galactoxyloglucan. The position of the CBM29 fusion affected the enzyme function. Particularly, C-terminal fusion led to greatest increases in galactomannan binding and catalytic efficiency, where relative to wild-type GaO, kcat/Km values increased by 7.5 and 19.8 times on guar galactomannan and locust bean galactomannan, respectively. The fusion of CBM29 also induced oligomerization of GaO-CBM29. Similar to impacts of cellulose-binding modules associated with cellulolytic enzymes, increased substrate binding impeded the action of GaO fusions on more concentrated preparations of galactomannan, galactoglucomannan and galactoxyloglucan; this was especially true for GaO-CBM29. Given the N-terminal positioning of the native galactose-binding CBM32 in GaO, the varying impacts of N-terminal versus C-terminal fusion of CBM29-1-2 may reflect competing action of neighboring CBMs. This study thoroughly examines and discusses the effects of CBM fusion to non-lignocellulytic enzymes on soluble polysaccharides. Herein kinetics of GaO on galactose containing polysaccharides is presented for the first time. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Species-Specific Chitin-Binding Module 18 Expansion in the Amphibian Pathogen Batrachochytrium dendrobatidis

    PubMed Central

    Abramyan, John; Stajich, Jason E.

    2012-01-01

    ABSTRACT Batrachochytrium dendrobatidis is the causative agent of chytridiomycosis, which is considered one of the driving forces behind the worldwide decline in populations of amphibians. As a member of the phylum Chytridiomycota, B. dendrobatidis has diverged significantly to emerge as the only pathogen of adult vertebrates. Such shifts in lifestyle are generally accompanied by various degrees of genomic modifications, yet neither its mode of pathogenicity nor any factors associated with it have ever been identified. Presented here is the identification and characterization of a unique expansion of the carbohydrate-binding module family 18 (CBM18), specific to B. dendrobatidis. CBM (chitin-binding module) expansions have been likened to the evolution of pathogenicity in a variety of fungus species, making this expanded group a prime candidate for the identification of potential pathogenicity factors. Furthermore, the CBM18 expansions are confined to three categories of genes, each having been previously implicated in host-pathogen interactions. These correlations highlight this specific domain expansion as a potential key player in the mode of pathogenicity in this unique fungus. The expansion of CBM18 in B. dendrobatidis is exceptional in its size and diversity compared to other pathogenic species of fungi, making this genomic feature unique in an evolutionary context as well as in pathogenicity. PMID:22718849

  12. The C-terminal domains of two homologous Oleaceae β-1,3-glucanases recognise carbohydrates differently: Laminarin binding by NMR.

    PubMed

    Zamora-Carreras, Héctor; Torres, María; Bustamante, Noemí; Macedo, Anjos L; Rodríguez, Rosalía; Villalba, Mayte; Bruix, Marta

    2015-08-15

    Ole e 9 and Fra e 9 are two allergenic β-1,3-glucanases from olive and ash tree pollens, respectively. Both proteins present a modular structure with a catalytic N-terminal domain and a carbohydrate-binding module (CBM) at the C-terminus. Despite their significant sequence resemblance, they differ in some functional properties, such as their catalytic activity and the carbohydrate-binding ability. Here, we have studied the different capability of the recombinant C-terminal domain of both allergens to bind laminarin by NMR titrations, binding assays and ultracentrifugation. We show that rCtD-Ole e 9 has a higher affinity for laminarin than rCtD-Fra e 9. The complexes have different exchange regimes on the NMR time scale in agreement with the different affinity for laminarin observed in the biochemical experiments. Utilising NMR chemical shift perturbation data, we show that only one side of the protein surface is affected by the interaction and that the binding site is located in the inter-helical region between α1 and α2, which is buttressed by aromatic side chains. The binding surface is larger in rCtD-Ole e 9 which may account for its higher affinity for laminarin relative to rCtD-Fra e 9.

  13. Carbohydrate ingestion and pre-cooling improves exercise capacity following soccer-specific intermittent exercise performed in the heat.

    PubMed

    Clarke, N D; Maclaren, D P M; Reilly, T; Drust, B

    2011-07-01

    Ingestion of carbohydrate and reducing core body temperature pre-exercise, either separately or combined, may have ergogenic effects during prolonged intermittent exercise in hot conditions. The aim of this investigation was to examine the effect of carbohydrate ingestion and pre-cooling on the physiological responses to soccer-specific intermittent exercise and the impact on subsequent high-intensity exercise performance in the heat. Twelve male soccer players performed a soccer-specific intermittent protocol for 90 min in the heat (30.5°C and 42.2% r.h.) on four occasions. On two occasions, the participants underwent a pre-cooling manoeuvre. During these sessions either a carbohydrate-electrolyte solution (CHOc) or a placebo was consumed at (PLAc). During the remaining sessions either the carbohydrate-electrolyte solution (CHO) or placebo (PLA) was consumed. At 15-min intervals throughout the protocol participants performed a mental concentration test. Following the soccer-specific protocol participants performed a self-chosen pace test and a test of high-intensity exercise capacity. The period of pre-cooling significantly reduced core temperature, muscle temperature and thermal sensation (P < 0.05). Self-chosen pace was greater with CHOc (12.5 ± 0.5 km h(-1)) compared with CHO (11.3 ± 0.4 km h(-1)), PLA (11.3 ± 0.4 km h(-1)) and PLAc (11.6 ± 0.5 km h(-1)) (P < 0.05). High-intensity exercise capacity was improved with CHOc and CHO when compared with PLA (CHOc; 79.8 ± 7 s, CHO; 72.1 ± 5 s, PLAc; 70.1 ± 8 s, PLA; 57.1 ± 5 s; P < 0.05). Mental concentration during the protocol was also enhanced during CHOc compared with PLA (P < 0.05). These results suggest pre-cooling in conjunction with the ingestion of carbohydrate during exercise enhances exercise capacity and helps maintain mental performance during intermittent exercise in hot conditions.

  14. A supramolecular bioactive surface for specific binding of protein.

    PubMed

    Hu, Changming; Qu, Yangcui; Zhan, Wenjun; Wei, Ting; Cao, Limin; Yu, Qian; Chen, Hong

    2017-04-01

    Bioactive surfaces with immobilized bioactive molecules aimed specifically at promoting or supporting particular interactions are of great interest for application of biosensors and biological detection. In this work, we fabricated a supramolecular bioactive surface with specific protein binding capability using two noncovalent interactions as the driving forces. The substrates were first layer-by-layer (LbL) deposited with a multilayered polyelectrolyte film containing "guest" adamantane groups via electrostatic interactions, followed by incorporation of "host" β-cyclodextrin derivatives bearing seven biotin units (CD-B) into the films via host-guest interactions. The results of fluorescence microscopy and quartz crystal microbalance measurement demonstrated that these surfaces exhibited high binding capacity and high selectivity for avidin due to the high density of biotin residues. Moreover, since host-guest interactions are inherently reversible, the avidin-CD-B complex is easily released by treatment with the sodium dodecyl sulfate, and the "regenerated" surfaces, after re-introducing fresh CD-B, can be used repeatedly for avidin binding. Given the generality and versatility of this approach, it may pave a way for development of re-usable biosensors for the detection and measurement of specific proteins.

  15. Isolation of an Aptamer that Binds Specifically to E. coli

    PubMed Central

    Cleto, Fernanda; Krieger, Marco Aurélio; Cardoso, Josiane

    2016-01-01

    Escherichia coli is a bacterial species found ubiquitously in the intestinal flora of animals, although pathogenic variants cause major public health problems. Aptamers are short oligonucleotides that bind to targets with high affinity and specificity, and have great potential for use in diagnostics and therapy. We used cell-based Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) to isolate four single stranded DNA (ssDNA) aptamers that bind strongly to E. coli cells (ATCC generic strain 25922), with Kd values in the nanomolar range. Fluorescently labeled aptamers label the surface of E. coli cells, as viewed by fluorescent microscopy. Specificity tests with twelve different bacterial species showed that one of the aptamers–called P12-31—is highly specific for E. coli. Importantly, this aptamer binds to Meningitis/sepsis associated E. coli (MNEC) clinical isolates, and is the first aptamer described with potential for use in the diagnosis of MNEC-borne pathologies. PMID:27104834

  16. Family 13 carbohydrate-binding module of alginate lyase from Agarivorans sp. L11 enhances its catalytic efficiency and thermostability, and alters its substrate preference and product distribution.

    PubMed

    Li, Shangyong; Yang, Xuemei; Bao, Mengmeng; Wu, Ying; Yu, Wengong; Han, Feng

    2015-05-01

    The carbohydrate-binding module (CBM) in polysaccharide hydrolases plays a key role in the hydrolysis of cellulose, xylan and chitin. However, the function of CBM in alginate lyases has not been elucidated. A new alginate lyase gene, alyL2, was cloned from the marine bacterium Agarivorans sp. L11 by using degenerate and site-finding PCR. The alginate lyase, AlyL2, contained an N-terminal CBM13 and a C-terminal catalytic family 7 polysaccharide lyase (PL7) module. To better understand the function of CBM13 in alginate lyase AlyL2, the full-length enzyme (AlyL2-FL) and its catalytic module (AlyL2-CM) were expressed in Escherichia coli and characterized. The specific activity and catalytic efficiency of AlyL2-FL were approximately twice those of AlyL2-CM. The half-lives of AlyL2-FL were 4.7-6.6 times those of AlyL2-CM at 30-50°C. In addition, the presence of CBM13 in AlyL2 changed its substrate preference and increased the percentage of disaccharides from 50.5% to 64.6% in the total products. This first report of the function of CBM13 in alginate lyase provides new insights into the degradation of alginate by marine microorganisms.

  17. X-ray structure of a protease-resistant mutant form of human galectin-9 having two carbohydrate recognition domains with a metal-binding site.

    PubMed

    Yoshida, Hiromi; Nishi, Nozomu; Wada, Kenji; Nakamura, Takanori; Hirashima, Mitsuomi; Kuwabara, Naoyuki; Kato, Ryuichi; Kamitori, Shigehiro

    2017-09-02

    Galectin-9 (G9) is a tandem-repeat type β-galactoside-specific animal lectin having N-terminal and C-terminal carbohydrate recognition domains (N-CRD and C-CRD, respectively) joined by a linker peptide that is involved in the immune system. G9 is divalent in glycan binding, and structural information about the spatial arrangement of the two CRDs is very important for elucidating its biological functions. As G9 is protease sensitive due to the long linker, the protease-resistant mutant form of G9 (G9Null) was developed by modification of the linker peptide, while retaining its biological functions. The X-ray structure of a mutant form of G9Null with the replacement of Arg221 by Ser (G9Null_R221S) having two CRDs was determined. The structure of G9Null_R221S was compact to associate the two CRDs in the back-to-back orientation with a large interface area, including hydrogen bonds and hydrophobic interactions. A metal ion was newly found in the galectin structure, possibly contributing to the stable structure of protein. The presented X-ray structure was thought to be one of the stable structures of G9, which likely occurs in solution. This was supported by structural comparisons with other tandem-repeated galectins and the analyses of protein thermostability by CD spectra measurements. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Probing of exopolysaccharides with green fluorescence protein-labeled carbohydrate-binding module in Escherichia coli biofilms and flocs induced by bcsB overexpression.

    PubMed

    Nguyen, Minh Hong; Ojima, Yoshihiro; Sakka, Makiko; Sakka, Kazuo; Taya, Masahito

    2014-10-01

    Polysaccharides are major structural constituents to develop the three-dimensional architecture of Escherichia coli biofilms. In this study, confocal laser scanning microscopy was applied in combination with a fluorescent probe to analyze the location and arrangement of exopolysaccharide (EPSh) in microcolonies of E. coli K-12 derived strains, formed as biofilms on solid surfaces and flocs in the liquid phase. For this purpose, a novel fluorescent probe was constructed by conjugating a carbohydrate-binding module 3, from Paenibacillus curdlanolyticus, with the green fluorescence protein (GFP-CBM3). The GFP-CBM3 fused protein exhibited strong affinity to microcrystalline cellulose. Moreover, GFP-CBM3 specifically bound to cell-dense microcolonies in the E. coli biofilms, and to their flocs induced by bcsB overexpression. Therefore, the fused protein presents as a novel marker for EPSh produced by E. coli cells. Overexpression of bcsB was associated with abundant EPSh production and enhanced E. coli biofilm formation, which was similarly detectable by GFP-CBM3 probing.

  19. Specific binding of gibberellic acid by cytokinin-specific binding proteins: a new aspect of plant hormone-binding proteins with the PR-10 fold.

    PubMed

    Ruszkowski, Milosz; Sliwiak, Joanna; Ciesielska, Agnieszka; Barciszewski, Jakub; Sikorski, Michal; Jaskolski, Mariusz

    2014-07-01

    Pathogenesis-related proteins of class 10 (PR-10) are a family of plant proteins with the same fold characterized by a large hydrophobic cavity that allows them to bind various ligands, such as phytohormones. A subfamily with only ~20% sequence identity but with a conserved canonical PR-10 fold have previously been recognized as Cytokinin-Specific Binding Proteins (CSBPs), although structurally the binding mode of trans-zeatin (a cytokinin phytohormone) was found to be quite diversified. Here, it is shown that two CSBP orthologues from Medicago truncatula and Vigna radiata bind gibberellic acid (GA3), which is an entirely different phytohormone, in a conserved and highly specific manner. In both cases a single GA3 molecule is found in the internal cavity of the protein. The structural data derived from high-resolution crystal structures are corroborated by isothermal titration calorimetry (ITC), which reveals a much stronger interaction with GA3 than with trans-zeatin and pH dependence of the binding profile. As a conclusion, it is postulated that the CSBP subfamily of plant PR-10 proteins should be more properly linked with general phytohormone-binding properties and termed phytohormone-binding proteins (PhBP).

  20. The influence of a 12% carbohydrate-electrolyte beverage on self-paced soccer-specific exercise performance.

    PubMed

    Harper, Liam D; Stevenson, Emma J; Rollo, Ian; Russell, Mark

    2017-04-21

    To assess the physiological and performance effects of a 12% carbohydrate-electrolyte beverage consumed at practically applicable time-points (i.e., before each half) throughout simulated soccer match-play. Randomised, counterbalanced, crossover. Fed players (n=15) performed 90-min of soccer-specific exercise (including self-paced exercise at the end of each half). Players consumed carbohydrate-electrolyte (CHO; 60g×500ml(-1), Na(+) 205mg×500ml(-1)), placebo-electrolyte (PL) or water (Wat) beverages at the end of the warm-up (250ml) and half-time (250ml plus ad-libitum water). Blood was drawn before each half and every 15-min during exercise. Physical (15-m sprinting, countermovement jumps, self-paced distance, acceleration/deceleration count), technical (dribbling) and cognitive (memory, attention, decision-making) performance was assessed. Ratings of perceived exertion (RPE) and abdominal discomfort were measured. Against Wat and PL, CHO increased (all p<0.05) mean accelerations >1.5m·s(-2) during self-paced exercise (>+25%) and dribbling speed from 60-min onwards (>+3%). Mean sprinting speed improved (+2.7%) in CHO versus Wat. Blood glucose increased before and during each half in CHO versus PL and Wat (all p<0.05). A 27% decline in glycaemia occurred at 60-min in CHO. RPE was comparable between trials. Cognition reduced post-exercise (p<0.05); this decline was not attenuated by CHO. Abdominal discomfort increased during exercise but was similar between trials. Using more realistic fluid ingestion timings than have been examined previously, consuming a 12% carbohydrate-electrolyte beverage increased blood glucose, self-paced exercise performance, and improved dribbling speed in the final 30-min of exercise compared to water and placebo. Carbohydrates did not attenuate post-exercise reductions in cognition. Copyright © 2017 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

  1. Structural and Functional Studies of Peptide-Carbohydrate Mimicry

    NASA Astrophysics Data System (ADS)

    Johnson, Margaret A.; Pinto, B. Mario

    Certain peptides act as molecular mimics of carbohydrates in that they are specifically recognized by carbohydrate-binding proteins. Peptides that bind to anti-carbohydrate antibodies, carbohydrate-processing enzymes, and lectins have been identified. These peptides are potentially useful as vaccines and therapeutics; for example, immunologically functional peptide molecular mimics (mimotopes) can strengthen or modify immune responses induced by carbohydrate antigens. However, peptides that bind specifically to carbohydrate-binding proteins may not necessarily show the corresponding biological activity, and further selection based on biochemical studies is always required. The degree of structural mimicry required to generate the desired biological activity is therefore an interesting question. This review will discuss recent structural studies of peptide-carbohydrate mimicry employing NMR spectroscopy, X-ray crystallography, and molecular modeling, as well as relevant biochemical data. These studies provide insights into the basis of mimicry at the molecular level. Comparisons with other carbohydrate-mimetic compounds, namely proteins and glycopeptides, will be drawn. Finally, implications for the design of new therapeutic compounds will also be presented.

  2. Improving antibody binding affinity and specificity for therapeutic development.

    PubMed

    Bostrom, Jenny; Lee, Chingwei V; Haber, Lauric; Fuh, Germaine

    2009-01-01

    Affinity maturation is an important part of the therapeutic antibody development process as in vivo activity often requires high binding affinity. Here, we describe a targeted approach for affinity improvement of therapeutic antibodies. Sets of CDR residues that are solvent accessible and relatively diverse in natural antibodies are targeted for diversification. Degenerate oligonucleotides are used to generate combinatorial phage-displayed antibody libraries with varying degree of diversity at randomized positions from which high-affinity antibodies can be selected. An advantage of using antibodies for therapy is their exquisite target specificity, which enables selective antigen binding and reduces off-target effects. However, it can be useful, and often it is necessary, to generate cross-reactive antibodies binding to not only the human antigen but also the corresponding non-human primate or rodent orthologs. Such cross-reactive antibodies can be used to validate the therapeutic targeting and examine the safety profile in preclinical animal models before committing to a costly development track. We show how affinity improvement and cross-species binding can be achieved in a one-step process.

  3. The Carbohydrate-Binding Site in Galectin-3 Is Preorganized To Recognize a Sugarlike Framework of Oxygens: Ultra-High-Resolution Structures and Water Dynamics

    PubMed Central

    2011-01-01

    The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate–protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultra-high-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 Å at 100 K, 1.25 Å at 298 K) and in complex with lactose (0.86 Å) or glycerol (0.9 Å). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of β-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design. PMID:22111949

  4. Tissue Specific Effects of Dietary Carbohydrates and Obesity on ChREBPα and ChREBPβ Expression.

    PubMed

    Stamatikos, Alexis D; da Silva, Robin P; Lewis, Jamie T; Douglas, Donna N; Kneteman, Norman M; Jacobs, René L; Paton, Chad M

    2016-01-01

    Carbohydrate response element binding protein (ChREBP) regulates insulin-independent de novo lipogenesis. Recently, a novel ChREBPβ isoform was identified. The purpose of the current study was to define the effect of dietary carbohydrates (CHO) and obesity on the transcriptional activity of ChREBP isoforms and their respective target genes. Mice were subjected to fasting-refeeding of high-CHO diets. In all three CHO-refeeding groups, mice failed to induce ChREBPα, yet ChREBPβ increased 10- to 20-fold. High-fat fed mice increased hepatic ChREBPβ mRNA expression compared to chow-fed along with increased protein expression. To better assess the independent effect of fructose on ChREBPα/β activity, HepG2 cells were treated with fructose ± a fructose-1,6-bisphosphatase inhibitor to suppress gluconeogenesis. Fructose treatment in the absence of gluconeogenesis resulted in increased ChREBP activity. To confirm the existence of ChREBPβ in human tissue, primary hepatocytes were incubated with high-glucose and the expression of ChREBPα and -β was determined. As with the animal models, glucose induced ChREBPβ expression while ChREBPα was decreased. Taken together, ChREBPβ is more responsive to changes in dietary CHO availability than the -α isoform. Diet-induced obesity increases basal expression of ChREBPβ, which may increase the risk of developing hepatic steatosis, and fructose-induced activation is independent of gluconeogenesis.

  5. Specific volume-hole volume correlations in amorphous carbohydrates: effect of temperature, molecular weight, and water content.

    PubMed

    Townrow, Sam; Roussenova, Mina; Giardiello, Maria-Isabelle; Alam, Ashraf; Ubbink, Job

    2010-02-04

    The specific volume and the nanostructure of the free volume of amorphous blends of maltose with a narrow molecular weight distribution maltopolymer were systematically studied as a function of temperature, water content, pressure, and blend composition. Correlations between the hole free volume and the specific volume were investigated in the glassy and rubbery phases and in solution using positron annihilation lifetime spectroscopy (PALS) and pressure-volume-temperature (PVT) measurements, with the aim to provide a consolidated mechanistic understanding of the relation between changes in molecular packing and at the molecular level and the behavior of the specific volume at the macrolevel. Both specific volume and hole volume show a linear dependence on the temperature, but with a slope which is higher in the rubbery state than in the glassy state. As a function of temperature, the hole volume and the specific volume are linearly related, with no discontinuity at the glass transition temperature (T(g)). In the glassy state, both the specific volume and the hole volume decrease nonlinearly with the addition of maltose to the maltopolymer matrix, due to a more efficient molecular packing. For variations in carbohydrate composition, a linear dependence between the hole volume and the specific volume was again observed. The role of water was found to be significantly more complex, with increasing water content causing an increase in density in both the glassy and rubbery phases indicating that water exists in a highly dispersed state with a significantly lower specific molar volume than in bulk water. At very low water contents, the hole volume and the specific volume both decrease with increasing water content, which suggests that water acts as both a hole filler and a plasticizer. In the glassy state at slightly higher water contents, the specific volume continues to slowly decrease, but the hole size passes through a minimum before it starts to increase. This

  6. The SEB-1 Transcription Factor Binds to the STRE Motif in Neurospora crassa and Regulates a Variety of Cellular Processes Including the Stress Response and Reserve Carbohydrate Metabolism

    PubMed Central

    Freitas, Fernanda Zanolli; Virgilio, Stela; Cupertino, Fernanda Barbosa; Kowbel, David John; Fioramonte, Mariana; Gozzo, Fabio Cesar; Glass, N. Louise; Bertolini, Maria Célia

    2016-01-01

    When exposed to stress conditions, all cells induce mechanisms resulting in an attempt to adapt to stress that involve proteins which, once activated, trigger cell responses by modulating specific signaling pathways. In this work, using a combination of pulldown assays and mass spectrometry analyses, we identified the Neurospora crassa SEB-1 transcription factor that binds to the Stress Response Element (STRE) under heat stress. Orthologs of SEB-1 have been functionally characterized in a few filamentous fungi as being involved in stress responses; however, the molecular mechanisms mediated by this transcription factor may not be conserved. Here, we provide evidences for the involvement of N. crassa SEB-1 in multiple cellular processes, including response to heat, as well as osmotic and oxidative stress. The Δseb-1 strain displayed reduced growth under these conditions, and genes encoding stress-responsive proteins were differentially regulated in the Δseb-1 strain grown under the same conditions. In addition, the SEB-1-GFP protein translocated from the cytosol to the nucleus under heat, osmotic, and oxidative stress conditions. SEB-1 also regulates the metabolism of the reserve carbohydrates glycogen and trehalose under heat stress, suggesting an interconnection between metabolism control and this environmental condition. We demonstrated that SEB-1 binds in vivo to the promoters of genes encoding glycogen metabolism enzymes and regulates their expression. A genome-wide transcriptional profile of the Δseb-1 strain under heat stress was determined by RNA-seq, and a broad range of cellular processes was identified that suggests a role for SEB-1 as a protein interconnecting these mechanisms. PMID:26994287

  7. Studies on a novel macrophage-specific calmodulin binding glycoprotein

    SciTech Connect

    Orlow, S.J.

    1986-01-01

    The murine macrophage-like cell line J774 and peritoneal exudate cells elicited with thioglycollate or starch contain a major calmodulin-binding protein which is absent in trifluoperazine-resistant variants of J774, resident peritoneal macrophages and these elicited with concanavalin A, lipopolysaccharide, proteose peptone or Bacillus Clamette Guerin. Resident murine peritoneal cells maintained in tissue culture for 3 days begin to accumulate this protein as do human peripheral blood monocytes after 7 days of culture. A specific competitive displacement radioimmunoassay was developed using a rabbit antiserum raised to the partially purified calmodulin binding protein and (/sup 125/I) calmodulin covalently crosslinked to the principal calmodulin binding protein in the preparation. The radioimmunoassay confirmed the unique cellular distribution of this protein suggesting that it may be a marker for certain stages of macrophage differentiation. Monoclonal antibodies were prepared and one of these was used to further purify the protein by immunoaffinity chromatography. A protein of molecular weight 50,000 to 60,000 was isolated. It could be selectively adsorbed to wheat germ agglutinin agarose and subsequently eluted with N-acetyl glucosamine. This property plus its sensitivity to endoglycosidase F led to the conclusion that it is a glycoprotein. The cellular distribution, subcellular localization and evidence of glycosylation suggest that this protein may be a macrophage-specific receptor with a high affinity for calcium-calmodulin.

  8. The Atomic Structure of the Phage Tuc2009 Baseplate Tripod Suggests that Host Recognition Involves Two Different Carbohydrate Binding Modules

    PubMed Central

    Legrand, Pierre; Collins, Barry; Blangy, Stéphanie; Murphy, James; Spinelli, Silvia; Gutierrez, Carlos; Richet, Nicolas; Kellenberger, Christine; Desmyter, Aline; Mahony, Jennifer

    2016-01-01

    ABSTRACT The Gram-positive bacterium Lactococcus lactis, used for the production of cheeses and other fermented dairy products, falls victim frequently to fortuitous infection by tailed phages. The accompanying risk of dairy fermentation failures in industrial facilities has prompted in-depth investigations of these phages. Lactococcal phage Tuc2009 possesses extensive genomic homology to phage TP901-1. However, striking differences in the baseplate-encoding genes stimulated our interest in solving the structure of this host’s adhesion device. We report here the X-ray structures of phage Tuc2009 receptor binding protein (RBP) and of a “tripod” assembly of three baseplate components, BppU, BppA, and BppL (the RBP). These structures made it possible to generate a realistic atomic model of the complete Tuc2009 baseplate that consists of an 84-protein complex: 18 BppU, 12 BppA, and 54 BppL proteins. The RBP head domain possesses a different fold than those of phages p2, TP901-1, and 1358, while the so-called “stem” and “neck” domains share structural features with their equivalents in phage TP901-1. The BppA module interacts strongly with the BppU N-terminal domain. Unlike other characterized lactococcal phages, Tuc2009 baseplate harbors two different carbohydrate recognition sites: one in the bona fide RBP head domain and the other in BppA. These findings represent a major step forward in deciphering the molecular mechanism by which Tuc2009 recognizes its saccharidic receptor(s) on its host. PMID:26814179

  9. Molecular modeling suggests induced fit of Family I carbohydrate-binding modules with a broken-chain cellulose surface.

    PubMed

    Nimlos, Mark R; Matthews, James F; Crowley, Michael F; Walker, Ross C; Chukkapalli, Giridhar; Brady, John W; Adney, William S; Cleary, Joseph M; Zhong, Linghao; Himmel, Michael E

    2007-04-01

    Cellobiohydrolases are the most effective single component of fungal cellulase systems; however, their molecular mode of action on cellulose is not well understood. These enzymes act to detach and hydrolyze cellodextrin chains from crystalline cellulose in a processive manner, and the carbohydrate-binding module (CBM) is thought to play an important role in this process. Understanding the interactions between the CBM and cellulose at the molecular level can assist greatly in formulating selective mutagenesis experiments to confirm the function of the CBM. Computational molecular dynamics was used to investigate the interaction of the CBM from Trichoderma reesei cellobiohydrolase I with a model of the (1,0,0) cellulose surface modified to display a broken chain. Initially, the CBM was located in different positions relative to the reducing end of this break, and during the simulations it appeared to translate freely and randomly across the cellulose surface, which is consistent with its role in processivity. Another important finding is that the reducing end of a cellulose chain appears to induce a conformational change in the CBM. Simulations show that the tyrosine residues on the hydrophobic surface of the CBM, Y5, Y31 and Y32 align with the cellulose chain adjacent to the reducing end and, importantly, that the fourth tyrosine residue in the CBM (Y13) moves from its internal position to form van der Waals interactions with the cellulose surface. As a consequence of this induced change near the surface, the CBM straddles the reducing end of the broken chain. Interestingly, all four aromatic residues are highly conserved in Family I CBM, and thus this recognition mechanism may be universal to this family.

  10. Specificity in transition state binding: the Pauling model revisited.

    PubMed

    Amyes, Tina L; Richard, John P

    2013-03-26

    Linus Pauling proposed that the large rate accelerations for enzymes are caused by the high specificity of the protein catalyst for binding the reaction transition state. The observation that stable analogues of the transition states for enzymatic reactions often act as tight-binding inhibitors provided early support for this simple and elegant proposal. We review experimental results that support the proposal that Pauling's model provides a satisfactory explanation for the rate accelerations for many heterolytic enzymatic reactions through high-energy reaction intermediates, such as proton transfer and decarboxylation. Specificity in transition state binding is obtained when the total intrinsic binding energy of the substrate is significantly larger than the binding energy observed at the Michaelis complex. The results of recent studies that aimed to characterize the specificity in binding of the enolate oxygen at the transition state for the 1,3-isomerization reaction catalyzed by ketosteroid isomerase are reviewed. Interactions between pig heart succinyl-coenzyme A:3-oxoacid coenzyme A transferase (SCOT) and the nonreacting portions of coenzyme A (CoA) are responsible for a rate increase of 3 × 10(12)-fold, which is close to the estimated total 5 × 10(13)-fold enzymatic rate acceleration. Studies that partition the interactions between SCOT and CoA into their contributing parts are reviewed. Interactions of the protein with the substrate phosphodianion group provide an ~12 kcal/mol stabilization of the transition state for the reactions catalyzed by triosephosphate isomerase, orotidine 5'-monophosphate decarboxylase, and α-glycerol phosphate dehydrogenase. The interactions of these enzymes with the substrate piece phosphite dianion provide a 6-8 kcal/mol stabilization of the transition state for reaction of the appropriate truncated substrate. Enzyme activation by phosphite dianion reflects the higher dianion affinity for binding to the enzyme

  11. The family 11 carbohydrate-binding module of Clostridium thermocellum Lic26A-Cel5E accommodates beta-1,4- and beta-1,3-1,4-mixed linked glucans at a single binding site.

    PubMed

    Carvalho, Ana L; Goyal, Arun; Prates, José A M; Bolam, David N; Gilbert, Harry J; Pires, Virgínia M R; Ferreira, Luís M A; Planas, Antoni; Romão, Maria J; Fontes, Carlos M G A

    2004-08-13

    Modular glycoside hydrolases that attack recalcitrant polymers generally contain noncatalytic carbohydrate-binding modules (CBMs), which play a critical role in the action of these enzymes by localizing the appended catalytic domains onto the surface of insoluble polysaccharide substrates. Type B CBMs, which recognize single polysaccharide chains, display ligand specificities that are consistent with the substrates hydrolyzed by the associated catalytic domains. In enzymes that contain multiple catalytic domains with distinct substrate specificities, it is unclear how these different activities influence the evolution of the ligand recognition profile of the appended CBM. To address this issue, we have characterized the properties of a family 11 CBM (CtCBM11) in Clostridium thermocellum Lic26A-Cel5E, an enzyme that contains GH5 and GH26 catalytic domains that display beta-1,4- and beta-1,3-1,4-mixed linked endoglucanase activity, respectively. Here we show that CtCBM11 binds to both beta-1,4- and beta-1,3-1,4-mixed linked glucans, displaying K(a) values of 1.9 x 10(5), 4.4 x 10(4), and 2 x 10(3) m(-1) for Glc-beta1,4-Glc-beta1,4-Glc-beta1,3-Glc, Glc-beta1,4-Glc-beta1,4-Glc-beta1,4-Glc, and Glc-beta1,3-Glc-beta1,4-Glc-beta1,3-Glc, respectively, demonstrating that CBMs can display a preference for mixed linked glucans. To determine whether these ligands are accommodated in the same or diverse sites in CtCBM11, the crystal structure of the protein was solved to a resolution of 1.98 A. The protein displays a beta-sandwich with a concave side that forms a potential binding cleft. Site-directed mutagenesis revealed that Tyr(22), Tyr(53), and Tyr(129), located in the putative binding cleft, play a central role in the recognition of all the ligands recognized by the protein. We propose, therefore, that CtCBM11 contains a single ligand-binding site that displays affinity for both beta-1,4- and beta-1,3-1,4-mixed linked glucans.

  12. T antigen origin-binding domain of simian virus 40: determinants of specific DNA binding.

    PubMed

    Bradshaw, Elizabeth M; Sanford, David G; Luo, Xuelian; Sudmeier, James L; Gurard-Levin, Zachary A; Bullock, Peter A; Bachovchin, William W

    2004-06-08

    To better understand origin recognition and initiation of DNA replication, we have examined by NMR complexes formed between the origin-binding domain of SV40 T antigen (T-ag-obd), the initiator protein of the SV40 virus, and cognate and noncognate DNA oligomers. The results reveal two structural effects associated with "origin-specific" binding that are absent in nonspecific DNA binding. The first is the formation of a hydrogen bond (H-bond) involving His 203, a residue that genetic studies have previously identified as crucial to both specific and nonspecific DNA binding in full-length T antigen. In free T-ag-obd, the side chain of His 203 has a pK(a) value of approximately 5, titrating to the N(epsilon)(1)H tautomer at neutral pH (Sudmeier, J. L., et al. (1996) J. Magn. Reson., Ser. B 113, 236-247). In complexes with origin DNA, His 203 N(delta)(1) becomes protonated and remains nontitrating as the imidazolium cation at all pH values from 4 to 8. The H-bonded N(delta1)H resonates at 15.9 ppm, an unusually large N-H proton chemical shift, of a magnitude previously observed only in the catalytic triad of serine proteases at low pH. The formation of this H-bond requires the middle G/C base pair of the recognition pentanucleotide, GAGGC. The second structural effect is a selective distortion of the A/T base pair characterized by a large (0.6 ppm) upfield chemical-shift change of its Watson-Crick proton, while nearby H-bonded protons remain relatively unaffected. The results indicate that T antigen, like many other DNA-binding proteins, may employ "catalytic" or "transition-state-like" interactions in binding its cognate DNA (Jen-Jacobson, L. (1997) Biopolymers 44, 153-180), which may be the solution to the well-known paradox between the relatively modest DNA-binding specificity exhibited by initiator proteins and the high specificity of initiation.

  13. Demonstration of specific binding of cocaine to human spermatozoa

    SciTech Connect

    Yazigi, R.A.; Odem, R.R.; Polakoski, K.L. )

    1991-10-09

    Exposure of males to cocaine has been linked to abnormal development of their offspring. To investigate the possible role of sperm, this study examined the interaction of cocaine with human spermatozoa. Washed sperm were incubated with tritiated cocaine and the samples were filtered and the remaining radioactivity quantitated. The specific binding was optimal at 20 minutes and 23C. Competition studies with tritiated cocaine indicated the presence of approximately 3.6 {times} 10{sup 3} binding sites per cell, with a high affinity receptor dissociation constant. Cocaine concentrations as high as 670 {mu}mol/L had no detectable effect on either the motility or viability of the cells. These results support the hypothesis that the sperm may act as a vector to transport cocaine into an ovum. This novel mechanism could be involved in the abnormal development of offspring of cocaine-exposed males.

  14. Specific mutagenesis of a chlorophyll-binding protein. Progress report.

    SciTech Connect

    Eaton-Rye, Dr., Julian; Shen, Gaozhong

    1990-01-01

    During the first phase of the project regarding specific mutagenesis of the chlorophyll-binding protein CP47 in photosystem II (PS II) most of the time has been devoted to (1) establishment of an optimal procedure for the reintroduction of psbB (the gene encoding CP47) carrying a site-directed mutation into the experimental organism, the cyanobacterium Synechocystis sp. PCC 6803, (2) preparations for site-directed mutagenesis, and (3) creation and analysis of chimaeric spinach/cyanobacterial CP47 mutants of Synechocystis. In the coming year, psbB constructs with site-directed mutations in potential chlorophyll-binding regions of CP47 will be introduced into the Synechocystis genome, and site-directed mutants will be characterized according to procedures described in the original project description. In addition, analysis of chimaeric CP47 mutants will be continued.

  15. Specific and non-specific folate binding protein in normal and malignant human tissues

    PubMed Central

    Corrocher, R.; De Sandre, G.; Ambrosetti, A.; Pachor, M. L.; Bambara, L. M.; Hoffbrand, A. V.

    1978-01-01

    Binding of tritiated folic acid by supernatants prepared from extracts of normal and leukaemic leucocytes, normal mucosa, and malignant tumours from different parts of the gastrointestinal tract has been measured using Sephadex-gel filtration and albumin-coated charcoal techniques. Non-specific binding (measured by Sephadex G-75 gel filtration) was almost invariably greater than specific binding measured by albumin-coated charcoal separation of bound and unbound folate. In nine normal leucocyte extracts, binding measured by Sephadex G-75 filtration ranged from 1·3 to 18·2 (mean 8·2) pg/mg protein and by albumin-coated charcoal from 1·0 to 14·8 (mean 6·7) pg/mg protein. Raised specific binding was found in the extracts from leucocytes of eight of 14 patients with chronic granulocytic leukaemia, in four substantially so (389, 121, 108, 59·7 pg/mg protein), but was only marginally increased in one of eight cases of acute myeloid leukaemia and in two of five cases of chronic lymphocytic leukaemia. Binding was normal in the extracts of all three cases of acute lymphoblastic leukaemia tested. Among the tissues of the gastrointestinal tract binding was greatest by the duodenal mucosa and liver. Extracts of carcinoma of the stomach and colon bound greater amounts of 3H-folic acid than the corresponding normal mucosal extracts but the differences were not large. Sephadex G-200 gel chromatography showed more than one binding peak in the extracts of liver and duodenum but only one peak in the other tissues of the gastrointestinal tract, and only one peak, of molecular weight either about 50 000 or over 200 000, in the leucocyte extracts. PMID:670421

  16. Cell specificity in DNA binding and repair of chemical carcinogens.

    PubMed Central

    Swenberg, J A; Rickert, D E; Baranyi, B L; Goodman, J I

    1983-01-01

    Many animal models for organ specific neoplasia have been developed and used to study the pathogenesis of cancer. Morphologic studies have usually concentrated on the response of target cells, whereas biochemical investigations have usually employed whole organ homogenates. Since hepatocytes comprise nearly 90% of the liver's mass and 70-80% of its DNA, alterations in DNA replication, covalent binding and DNA repair of nonparenchymal cells are usually obscured when whole organ homogenates are used. By utilizing cell separation methods, we have been able to demonstrate differences between hepatocyte and nonparenchymal cell replication. DNA damage and repair following exposure to a variety of hepatocarcinogen. Differences in removal of simple O6-alkylguanine and DNA replication correlate with cell specific carcinogenesis of simply alkylating agents. For several other procarcinogens, including 2-acetylaminofluorene and dinitroluene, cell specificity appears to reside primarily in the differential metabolic competence of hepatocytes and nonparenchymal cells. This results in greater covalent binding of the carcinogen to hepatocyte DNA, although the DNA adducts are removed at a similar rate in both cell types. Images FIGURE 1. PMID:6832089

  17. Thermodynamics of multivalent carbohydrate-lectin cross-linking interactions: importance of entropy in the bind and jump mechanism.

    PubMed

    Dam, Tarun K; Gerken, Thomas A; Brewer, C Fred

    2009-05-12

    The high affinity (K(d) = 0.2 nM) of the soybean agglutinin (SBA), a tetrameric GalNAc specific lectin, for a modified form of porcine submaxillary mucin, a linear glycoprotein, with a molecular mass of approximately 10(6) Da and approximately 2300 GalNAcalpha1-O-Ser/Thr residues (Tn-PSM) has been ascribed to an internal diffusion mechanism that involves binding and jumping of the lectin from GalNAc to GalNAc residue of the mucin [Dam, T. K., et al. (2007) J. Biol. Chem. 282, 28256-28263]. Hill plot analysis of the raw ITC data shows increasing negative cooperativity, which correlates with an increasing number of lectin-mucin cross-linking interactions and decreasing favorable binding entropies. However, the affinity of bound SBA for other Tn-PSM molecules during cross-linking is much higher than that of free SBA for GalNAcalpha1-O-Ser, a monovalent analogue. The high affinity of bound SBA for GalNAc residues on other Tn-PSM molecules appears to be due to the favorable entropy of binding associated with the internal diffusion mechanism. Furthermore, the increasing negative cooperativity of SBA binding to Tn-PSM correlates with a decreasing level of internal diffusion of the lectin on the mucin as cross-linking occurs. These findings indicate the importance of the internal diffusion mechanism in generating large, favorable entropies of binding that drive lectin-mucin cross-linking interactions. The results are important for understanding the energetics of lectin-mucin cross-linking interactions that are associated with biological signaling on the surface of cells and the role of the internal diffusion mechanism in ligand-biopolymer interactions in general.

  18. The size, shape and specificity of the sugar-binding site of the jacalin-related lectins is profoundly affected by the proteolytic cleavage of the subunits.

    PubMed Central

    Houlès Astoul, Corinne; Peumans, Willy J; van Damme, Els J M; Barre, Annick; Bourne, Yves; Rougé, Pierre

    2002-01-01

    Mannose-specific lectins with high sequence similarity to jacalin and the Maclura pomifera agglutinin have been isolated from species belonging to the families Moraceae, Convolvulaceae, Brassicaceae, Asteraceae, Poaceae and Musaceae. Although these novel mannose-specific lectins are undoubtedly related to the galactose-specific Moraceae lectins there are several important differences. Apart from the obvious differences in specificity, the mannose- and galactose-specific jacalin-related lectins differ in what concerns their biosynthesis and processing, intracellular location and degree of oligomerization of the protomers. Taking into consideration that the mannose-specific lectins are widely distributed in higher plants, whereas their galactose-specific counterparts are confined to a subgroup of the Moraceae sp. one can reasonably assume that the galactose-specific Moraceae lectins are a small-side group of the main family. The major change that took place in the structure of the binding site of the diverging Moraceae lectins concerns a proteolytic cleavage close to the N-terminus of the protomer. To corroborate the impact of this change, the specificity of jacalin was re-investigated using surface plasmon resonance analysis. This approach revealed that in addition to galactose and N -acetylgalactosamine, the carbohydrate-binding specificity of jacalin extends to mannose, glucose, N -acetylmuramic acid and N -acetylneuraminic acid. Owing to this broad carbohydrate-binding specificity, jacalin is capable of recognizing complex glycans from plant pathogens or predators. PMID:12169094

  19. E2F in vivo binding specificity: Comparison of consensus versus nonconsensus binding sites

    PubMed Central

    Rabinovich, Alina; Jin, Victor X.; Rabinovich, Roman; Xu, Xiaoqin; Farnham, Peggy J.

    2008-01-01

    We have previously shown that most sites bound by E2F family members in vivo do not contain E2F consensus motifs. However, differences between in vivo target sites that contain or lack a consensus E2F motif have not been explored. To understand how E2F binding specificity is achieved in vivo, we have addressed how E2F family members are recruited to core promoter regions that lack a consensus motif and are excluded from other regions that contain a consensus motif. Using chromatin immunoprecipitation coupled with DNA microarray analysis (ChIP-chip) assays, we have shown that the predominant factors specifying whether E2F is recruited to an in vivo binding site are (1) the site must be in a core promoter and (2) the region must be utilized as a promoter in that cell type. We have tested three models for recruitment of E2F to core promoters lacking a consensus site, including (1) indirect recruitment, (2) looping to the core promoter mediated by an E2F bound to a distal motif, and (3) assisted binding of E2F to a site that weakly resembles an E2F motif. To test these models, we developed a new in vivo assay, termed eChIP, which allows analysis of transcription factor binding to isolated fragments. Our findings suggest that in vivo (1) a consensus motif is not sufficient to recruit E2Fs, (2) E2Fs can bind to isolated regions that lack a consensus motif, and (3) binding can require regions other than the best match to the E2F motif. PMID:18836037

  20. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone.

    PubMed

    Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G; Ribeiro, José M C; Andersen, John F

    2017-09-15

    Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary "long" D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

  1. Albumin binds self-assembling dyes as specific polymolecular ligands.

    PubMed

    Stopa, Barbara; Rybarska, Janina; Drozd, Anna; Konieczny, Leszek; Król, Marcin; Lisowski, Marek; Piekarska, Barbara; Roterman, Irena; Spólnik, Paweł; Zemanek, Grzegorz

    2006-12-15

    Self-assembling dyes with a structure related to Congo red (e.g. Evans blue) form polymolecular complexes with albumin. The dyes, which are lacking a self-assembling property (Trypan blue, ANS) bind as single molecules. The supramolecular character of dye ligands bound to albumin was demonstrated by indicating the complexation of dye molecules outnumbering the binding sites in albumin and by measuring the hydrodynamic radius of albumin which is growing upon complexation of self-assembling dye in contrast to dyes lacking this property. The self-assembled character of Congo red was also proved using it as a carrier introducing to albumin the intercalated nonbonding foreign compounds. Supramolecular, ordered character of the dye in the complex with albumin was also revealed by finding that self-assembling dyes become chiral upon complexation. Congo red complexation makes albumin less resistant to low pH as concluded from the facilitated N-F transition, observed in studies based on the measurement of hydrodynamic radius. This particular interference with protein stability and the specific changes in digestion resulted from binding of Congo red suggest that the self-assembled dye penetrates the central crevice of albumin.

  2. Optimizing molecular electrostatic interactions: Binding affinity and specificity

    NASA Astrophysics Data System (ADS)

    Kangas, Erik

    The design of molecules that bind tightly and specifically to designated target molecules is an important goal in many fields of molecular science. While the shape of the molecule to be designed is a relatively well defined problem with an intuitive answer, determination of the distribution of electrostatic charge that it should have in order to possess high affinity and/or specificity for a target is a subtle problem involving a tradeoff between an unfavorable electrostatic desolvation penalty incurred due to the removal of solvent from the interacting surfaces of the reactants, and the generally favorable intermolecular interactions made in the bound state. In this thesis, a theoretical formalism based on a continuum electrostatic approximation is developed in which charge distributions leading to optimal affinity and/or high specificity may be obtained. Methods for obtaining these charge distributions are developed in detail and analytical solutions are obtained in several special cases (where the molecules are shaped as infinite membranes, spheres, and spheroids). Their existence and non-uniqueness are also shown, and it is proven that the resulting optimized electrostatic binding free energies are favorable (negative) in many cases of physical interest. Affinity and specificity optimization is then applied to the chorismate mutase family of enzymes, including the catalytic antibody 1F7. It is shown that affinity optimization can be used to suggest better molecular inhibitors and that specificity optimization can be used to help elucidate molecular function and possibly aid in the creation of improved haptens. (Copies available exclusively from MIT Libraries, Rm. 14-0551, Cambridge, MA 02139-4307. Ph. 617-253-5668; Fax 617-253-1690.)

  3. Identification and quantitative analysis of stage-specific carbohydrates in loblolly pine (Pinus taeda) zygotic embryo and female gametophyte tissues.

    PubMed

    Pullman, Gerald S; Buchanan, Mike

    2008-07-01

    Stage-specific analyses of starch and 18 sugars, including pentoses, hexoses, disaccharides, trisaccharides, oligosaccharides and sugar alcohols, were made throughout seed development for zygotic embryo and female gametophyte (FG) tissues of loblolly pine (Pinus taeda L.). Tissue was most often analyzed in triplicate from two open-pollinated families grown in different locations and sampled in different years. Carbohydrates were analyzed by enzymatic assay, high performance liquid chromatography or gas chromatography/mass spectrometry. For all carbohydrates quantified, peak concentrations were higher in embryo tissue than in FG tissue. Significant changes in starch and sugar concentrations occurred over time, with both seed collections showing similar trends in temporal changes. Although concentrations were not always similar, embryo and FG tissues generally showed similar patterns of change in starch and sugar concentrations over time. Total starch concentration was highest during early seed development and decreased as development progressed. The major sugars contributing to osmotic potential during early seed development were D-pinitol, sucrose, fructose and glucose. During mid-seed development, D-pinitol, sucrose, fructose, glucose, melibiose and raffinose provided major contributions to the osmotic environment. During late seed development, sucrose, raffinose, melibiose, stachyose and fructose were the major contributors to osmotic potential. These data suggest stage-specific media composition for each step in the somatic embryogenesis protocol.

  4. Generic tools to assess genuine carbohydrate specific effects on in vitro immune modulation exemplified by β-glucans.

    PubMed

    Rieder, Anne; Grimmer, Stine; Aachmann, Finn L; Westereng, Bjørge; Kolset, Svein Olav; Knutsen, Svein Halvor

    2013-02-15

    Even if carbohydrate preparations from plant/fungal sources have a high degree of purity, observed immune-stimulation may be caused by minute sample contaminations. Using the example of different β-glucans we present a range of analytical tools crucial for validation of possible immune-stimulatory effects. Two yeast (MacroGard and Zymosan) and one cereal β-glucan (CBG40) increased IL-8 secretion by HT-29 cells considerably. Degradation of the β-glucan samples with β-glucan specific enzymes did hardly influence the effect of Zymosan and CBG40 but significantly decreased the effect of MacroGard. Stimulation of IL-8 secretion by CBG40 and Zymosan was hence not due to their β-glucan content. Instead, the effect of the CBG40 sample was due to low levels of LPS despite the inability of the known LPS inhibitor Polymyxin B to supress its stimulatory effect. We conclude that targeted enzymatic degradation of samples is a powerful validation tool to investigate carbohydrate specific immune-modulation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Penicillium purpurogenum produces a family 1 acetyl xylan esterase containing a carbohydrate-binding module: characterization of the protein and its gene.

    PubMed

    Gordillo, Felipe; Caputo, Valentina; Peirano, Alessandra; Chavez, Renato; Van Beeumen, Jozef; Vandenberghe, Isabel; Claeyssens, Marc; Bull, Paulina; Ravanal, María Cristina; Eyzaguirre, Jaime

    2006-10-01

    At least three acetyl xylan esterases (AXE I, II and III) are secreted by Penicillium purpurogenum. This publication describes more detailed work on AXE I and its gene. AXE I binds cellulose but not xylan; it is glycosylated and inactivated by phenylmethylsulphonyl fluoride, showing that it is a serine esterase. The axe1 gene presents an open reading frame of 1278 bp, including two introns of 68 and 61 bp; it codes for a signal peptide of 31 residues and a mature protein of 351 amino acids (molecular weight 36,693). AXE I has a modular structure: a catalytic module at the amino terminus belonging to family 1 of the carbohydrate esterases, a linker rich in serines and threonines, and a family 1 carboxy terminal carbohydrate binding module (CBM). The CBM is similar to that of AXE from Trichoderma reesei, (with a family 5 catalytic module) indicating that the genes for catalytic modules and CBMs have evolved separately, and that they have been linked by gene fusion. The promoter sequence of axe1 contains several putative sequences for binding of gene expression regulators also found in other family 1 esterase gene promoters. It is proposed that AXE I and II act in succession in xylan degradation; first, xylan is attacked by AXE I and other xylanases possessing CBMs (which facilitate binding to lignocellulose), followed by other enzymes acting mainly on soluble substrates.

  6. Specific binding of the methyl binding domain protein 2 at the BRCA1-NBR2 locus

    PubMed Central

    Auriol, Emilie; Billard, Lise-Marie; Magdinier, Frédérique; Dante, Robert

    2005-01-01

    The methyl-CpG binding domain (MBD) proteins are key molecules in the interpretation of DNA methylation signals leading to gene silencing. We investigated their binding specificity at the constitutively methylated region of a CpG island containing the bidirectional promoter of the Breast cancer predisposition gene 1, BRCA1, and the Near BRCA1 2 (NBR2) gene. In HeLa cells, quantitative chromatin immunoprecipitation assays indicated that MBD2 is associated with the methylated region, while MeCP2 and MBD1 were not detected at this locus. MBD2 depletion (∼90%), mediated by a transgene expressing a small interfering RNA (siRNA), did not induce MeCP2 or MBD1 binding at the methylated area. Furthermore, the lack of MBD2 at the BRCA1-NBR2 CpG island is associated with an elevated level of NBR2 transcripts and with a significant reduction of induced-DNA-hypomethylation response. In MBD2 knockdown cells, transient expression of a Mbd2 cDNA, refractory to siRNA-mediated decay, shifted down the NBR2 mRNA level to that observed in unmodified HeLa cells. Variations in MBD2 levels did not affect BRCA1 expression despite its stimulation by DNA hypomethylation. Collectively, our data indicate that MBD2 has specific targets and its presence at these targets is indispensable for gene repression. PMID:16052033

  7. A carbohydrate-binding affinity ligand for the specific enrichment of glycoproteins.

    PubMed

    Chen, Chen; El Khoury, Graziella; Zhang, Peiqing; Rudd, Pauline M; Lowe, Christopher R

    2016-04-29

    One challenge facing the production of glycoprotein therapeutics is the lack of stable and selective affinity ligands for their enrichment. Synthetic affinity ligands based on the solid phase multi-component Ugi reaction represent a desirable option, particularly those incorporating benzoboroxole and its derivatives, which have been shown to enrich glycoproteins under physiological conditions. Thus, an Ugi ligand, A21C11I8, comprising 5-amino-2-hydroxymethylphenylboronic acid was synthesised on aldehyde-functionalised Sepharose™. Immobilised A21C11I8 displayed affinity for the glycosylated protein, glucose oxidase (GOx), which bound primarily through its glycan moiety. The ligand had a preference for sugar alcohols and the furanose form of the monosaccharides tested. Compared with immobilised 3-aminophenylboronic acid and Concanavalin A, the Ugi ligand was able to purify GOx from spiked Escherichia coli supernatants with retention of its maximum enzymatic activity and protein recovery. Glycan profiles of human immunoglobulin G tested on A21C11I8 columns suggested that the adsorbent possesses the potential to resolve sialylated and neutral glycoforms. The benzoboroxole-functionalised Ugi ligand may find application in selective glycoform separation.

  8. Family 46 Carbohydrate-binding Modules Contribute to the Enzymatic Hydrolysis of Xyloglucan and β-1,3–1,4-Glucans through Distinct Mechanisms*♦

    PubMed Central

    Venditto, Immacolata; Najmudin, Shabir; Luís, Ana S.; Ferreira, Luís M. A.; Sakka, Kazuo; Knox, J. Paul; Gilbert, Harry J.; Fontes, Carlos M. G. A.

    2015-01-01

    Structural carbohydrates comprise an extraordinary source of energy that remains poorly utilized by the biofuel sector as enzymes have restricted access to their substrates within the intricacy of plant cell walls. Carbohydrate active enzymes (CAZYmes) that target recalcitrant polysaccharides are modular enzymes containing noncatalytic carbohydrate-binding modules (CBMs) that direct enzymes to their cognate substrate, thus potentiating catalysis. In general, CBMs are functionally and structurally autonomous from their associated catalytic domains from which they are separated through flexible linker sequences. Here, we show that a C-terminal CBM46 derived from BhCel5B, a Bacillus halodurans endoglucanase, does not interact with β-glucans independently but, uniquely, acts cooperatively with the catalytic domain of the enzyme in substrate recognition. The structure of BhCBM46 revealed a β-sandwich fold that abuts onto the region of the substrate binding cleft upstream of the active site. BhCBM46 as a discrete entity is unable to bind to β-glucans. Removal of BhCBM46 from BhCel5B, however, abrogates binding to β-1,3–1,4-glucans while substantially decreasing the affinity for decorated β-1,4-glucan homopolymers such as xyloglucan. The CBM46 was shown to contribute to xyloglucan hydrolysis only in the context of intact plant cell walls, but it potentiates enzymatic activity against purified β-1,3–1,4-glucans in solution or within the cell wall. This report reveals the mechanism by which a CBM can promote enzyme activity through direct interaction with the substrate or by targeting regions of the plant cell wall where the target glucan is abundant. PMID:25713075

  9. An olive pollen protein with allergenic activity, Ole e 10, defines a novel family of carbohydrate-binding modules and is potentially implicated in pollen germination

    PubMed Central

    2005-01-01

    CBMs (carbohydrate-binding modules) are the most common non-catalytic modules associated with enzymes active in plant cell-wall hydrolysis. They have been frequently identified by amino acid sequence alignments, but only a few have been experimentally established to have a carbohydrate-binding activity. A small olive pollen protein, Ole e 10 (10 kDa), has been described as a major inducer of type I allergy in humans. In the present study, the ability of Ole e 10 to bind several polysaccharides has been analysed by affinity gel electrophoresis, which demonstrated that the protein bound 1,3-β-glucans preferentially. Analytical ultracentrifugation studies confirmed binding to laminarin, at a protein/ligand ratio of 1:1. The interaction of Ole e 10 with laminarin induced a conformational change in the protein, as detected by CD and fluorescence analyses, and an increase of 3.6 °C in the thermal denaturation temperature of Ole e 10 in the presence of the glycan. These results, and the absence of alignment of the sequence of Ole e 10 with that of any classified CBM, indicate that this pollen protein defines a novel family of CBMs, which we propose to name CBM43. Immunolocalization of Ole e 10 in mature and germinating pollen by transmission electron microscopy and confocal laser scanning microscopy demonstrated the co-localization of Ole e 10 and callose (1,3-β-glucan) in the growing pollen tube, suggesting a role for this protein in the metabolism of carbohydrates and in pollen tube wall re-formation during germination. PMID:15882149

  10. Carbohydrate ingestion induces sex-specific cardiac vagal inhibition, but not vascular sympathetic modulation, in healthy older women.

    PubMed

    Cao, Lei; Graham, Stuart L; Pilowsky, Paul M

    2016-07-01

    The role of vagal function in cardiovascular risk in older women remains unclear. Autonomic modulation following carbohydrate ingestion (CI) and postural stress (PS) were investigated in 14 healthy men and 21 age-matched postmenopausal women (age: 65.0 ± 2.1 vs. 64.1 ± 1.6 years), with normal and comparable insulin sensitivity. Continuous noninvasive finger arterial pressure and ECG were recorded in the lying and the standing positions before and after ingestion of a carbohydrate-rich meal (600 kcal, carbohydrate 78%, protein 13%, and fat 8%). Low-frequency (LF, 0.04-0.15 Hz) and high-frequency (HF, 0.15-0.4 Hz) components (ms(2)) of heart rate variability (HRV), low-frequency power (mmHg(2)) of systolic blood pressure variability (SBP LF power), and the sequence method for spontaneous baroreflex sensitivity (BRS, ms/mmHg) were used to quantify autonomic modulation. In response to CI and PS, mean arterial pressure maintained stable, and heart rate increased in women and men in the lying and standing positions. Following CI (60, 90, and 120 min postprandially) in the standing position, SBP LF power increased by 40% in men (P = 0.02), with unchanged HRV parameters; in contrast, in women, HRV HF power halved (P = 0.02), with unaltered SBP LF power. During PS before and after CI, similar magnitude of SBP LF power, HRV, and BRS changes was observed in men and women. In conclusion, CI induces sex-specific vascular sympathetic activation in healthy older men, and cardiac vagal inhibition in healthy older women; this CI-mediated efferent vagal inhibition may suggest differential cardiovascular risk factors in women, irrespective of insulin resistance, and impairment of autonomic control. Copyright © 2016 the American Physiological Society.

  11. Novel role for carbohydrate responsive element binding protein in the control of ethanol metabolism and susceptibility to binge drinking.

    PubMed

    Marmier, Solenne; Dentin, Renaud; Daujat-Chavanieu, Martine; Guillou, Hervé; Bertrand-Michel, Justine; Gerbal-Chaloin, Sabine; Girard, Jean; Lotersztajn, Sophie; Postic, Catherine

    2015-10-01

    Carbohydrate responsive element binding protein (ChREBP) is central for de novo fatty acid synthesis under physiological conditions and in the context of nonalcoholic fatty liver disease. We explored its contribution to alcohol-induced steatosis in a mouse model of binge drinking as acute ethanol (EtOH) intoxication has become an alarming health problem. Within 6 hours, ChREBP acetylation and its recruitment onto target gene promoters were increased in liver of EtOH-fed mice. Acetylation of ChREBP was dependent on alcohol metabolism because inhibition of alcohol dehydrogenase (ADH) activity blunted ChREBP EtOH-induced acetylation in mouse hepatocytes. Transfection of an acetylation-defective mutant of ChREBP (ChREBP(K672A) ) in HepG2 cells impaired the stimulatory effect of EtOH on ChREBP activity. Importantly, ChREBP silencing in the liver of EtOH-fed mice prevented alcohol-induced triglyceride accumulation through an inhibition of the lipogenic pathway but also led, unexpectedly, to hypothermia, increased blood acetaldehyde concentrations, and enhanced lethality. This phenotype was associated with impaired hepatic EtOH metabolism as a consequence of reduced ADH activity. While the expression and activity of the NAD(+) dependent deacetylase sirtuin 1, a ChREBP-negative target, were down-regulated in the liver of alcohol-fed mice, they were restored to control levels upon ChREBP silencing. In turn, ADH acetylation was reduced, suggesting that ChREBP regulates EtOH metabolism and ADH activity through its direct control of sirtuin 1 expression. Indeed, when sirtuin 1 activity was rescued by resveratrol pretreatment in EtOH-treated hepatocytes, a significant decrease in ADH protein content and/or acetylation was observed. our study describes a novel role for ChREBP in EtOH metabolism and unravels its protective effect against severe intoxication in response to binge drinking. © 2015 by the American Association for the Study of Liver Diseases.

  12. Glucose-specific enzyme IIA has unique binding partners in the vibrio cholerae biofilm.

    PubMed

    Pickering, Bradley S; Smith, Daniel R; Watnick, Paula I

    2012-11-06

    Glucose-specific enzyme IIA (EIIA(Glc)) is a central regulator of bacterial metabolism and an intermediate in the phosphoenolpyruvate phosphotransferase system (PTS), a conserved phosphotransfer cascade that controls carbohydrate transport. We previously reported that EIIA(Glc) activates transcription of the genes required for Vibrio cholerae biofilm formation. While EIIA(Glc) modulates the function of many proteins through a direct interaction, none of the known regulatory binding partners of EIIA(Glc) activates biofilm formation. Therefore, we used tandem affinity purification (TAP) to compare binding partners of EIIA(Glc) in both planktonic and biofilm cells. A surprising number of novel EIIA(Glc) binding partners were identified predominantly under one condition or the other. Studies of planktonic cells revealed established partners of EIIA(Glc), such as adenylate cyclase and glycerol kinase. In biofilms, MshH, a homolog of Escherichia coli CsrD, was found to be a dominant binding partner of EIIA(Glc). Further studies revealed that MshH inhibits biofilm formation. This function was independent of the Carbon storage regulator (Csr) pathway and dependent on EIIA(Glc). To explore the existence of multiprotein complexes centered on EIIA(Glc), we also affinity purified the binding partners of adenylate cyclase from biofilm cells. In addition to EIIA(Glc), this analysis yielded many of the same proteins that copurified with EIIA(Glc). We hypothesize that EIIA(Glc) serves as a hub for multiprotein complexes and furthermore that these complexes may provide a mechanism for competitive and cooperative interactions between binding partners. EIIA(Glc) is a global regulator of microbial physiology that acts through direct interactions with other proteins. This work represents the first demonstration that the protein partners of EIIA(Glc) are distinct in the microbial biofilm. Furthermore, it provides the first evidence that EIIA(Glc) may exist in multiprotein complexes with

  13. Understanding Carbohydrates

    MedlinePlus

    ... Size: A A A Listen En Español Understanding Carbohydrates How much and what type of carbohydrate foods ... glucose levels in your target range. Explore: Understanding Carbohydrates Glycemic Index and Diabetes Learn about the glycemic ...

  14. Determinants of Bacteriophage 933W Repressor DNA Binding Specificity

    PubMed Central

    Bullwinkle, Tammy J.; Samorodnitsky, Daniel; Rosati, Rayna C.; Koudelka, Gerald B.

    2012-01-01

    We reported previously that 933W repressor apparently does not cooperatively bind to adjacent sites on DNA and that the relative affinities of 933W repressor for its operators differ significantly from that of any other lambdoid bacteriophage. These findings indicate that the operational details of the lysis-lysogeny switch of bacteriophage 933W are unique among lambdoid bacteriophages. Since the functioning of the lysis-lysogeny switch in 933W bacteriophage uniquely and solely depends on the order of preference of 933W repressor for its operators, we examined the details of how 933W repressor recognizes its DNA sites. To identify the specificity determinants, we first created a molecular model of the 933W repressor-DNA complex and tested the predicted protein-DNA interactions. These results of these studies provide a picture of how 933W repressor recognizes its DNA sites. We also show that, opposite of what is normally observed for lambdoid phages, 933W operator sequences have evolved in such a way that the presence of the most commonly found base sequences at particular operator positions serves to decrease, rather than increase, the affinity of the protein for the site. This finding cautions against assuming that a consensus sequence derived from sequence analysis defines the optimal, highest affinity DNA binding site for a protein. PMID:22509323

  15. Structure and function of carbohydrate-binding module families 13 and 42 of glycoside hydrolases, comprising a β-trefoil fold.

    PubMed

    Fujimoto, Zui

    2013-01-01

    Some carbohydrate-active enzymes display a modular structure in which catalytic modules that target an insoluble substrate are often attached to one or more noncatalytic carbohydrate-binding modules (CBMs) that assist enzymatic activity. CBMs have been classified into more than 60 families based on amino acid sequence similarities. CBM family 13 (CBM13) and family 42 (CBM42) possess a β-trefoil fold and are grouped into CBM fold family 2. The β-trefoil fold contains a sequence of approximately 45 amino acid residues that is repeated 3 times, resulting in three subdomains (α, β and γ) that fold into an overall globular structure. Each subdomain is composed of four β-strands that fold into a Y-shaped β-hairpin structure. CBM13 and CBM42 have multivalent sugar-binding ability. In this review, I describe the sugar-binding mechanisms of the CBM13 and CBM42 domains of a β-xylanase, a β-L-arabinopyranosidase, and an α-L-arabinofuranosidase.

  16. Carbohydrate-binding activity of the type-2 ribosome-inactivating protein SNA-I from elderberry (Sambucus nigra) is a determining factor for its insecticidal activity.

    PubMed

    Shahidi-Noghabi, Shahnaz; Van Damme, Els J M; Smagghe, Guy

    2008-12-01

    In recent years, different classes of proteins have been reported to promote toxic effects when ingested. Type-2 ribosome-inactivating proteins (RIPs) are a group of chimeric proteins built up of an A-chain with RNA N-glycosidase activity and a B-chain with lectin activity. These proteins are thought to play a role in plant protection. Sambucus nigra agglutinin I (SNA-I) is a type-2 RIP, isolated from the bark of elderberry (S. nigra L.). This study demonstrated the insecticidal potency of SNA-I on two Hemipteran insect species using two different methods. An artificial diet supplemented with different concentrations of the purified RIP reduced survival and fecundity of pea aphids Acyrthosiphon pisum. In addition, feeding of tobacco aphids, Myzus nicotianae, on leaves from transfected plants constitutively expressing SNA-I, resulted in a delayed development and reduced adult survival and also the fertility parameters of the surviving aphids were reduced, suggesting that a population of aphids would build up significantly slower on plants expressing SNA-I. Finally, a series of experiments with transgenic lines in which a mutant RIP was expressed, revealed that the carbohydrate-binding activity of SNA-I is necessary for its insecticidal activity. In a first set of mutants, the B-chain was mutated at one position (Asp231DeltaGlu), and in the second set both carbohydrate-binding sites were mutated (Asn48DeltaSer and Asp231DeltaGlu). Mutation of one carbohydrate-binding site strongly reduced the insecticidal activity of SNA-I, whereas mutation of both lectin sites (almost) completely abolished the SNA-I effect on tobacco aphids.

  17. NMR investigations of protein-carbohydrate interactions binding studies and refined three-dimensional solution structure of the complex between the B domain of wheat germ agglutinin and N,N', N"-triacetylchitotriose.

    PubMed

    Espinosa, J F; Asensio, J L; García, J L; Laynez, J; Bruix, M; Wright, C; Siebert, H C; Gabius, H J; Cañada, F J; Jiménez-Barbero, J

    2000-07-01

    The specific interaction of the isolated B domain of wheat germ agglutinin (WGA-B) with N,N',N"-triacetylchitotriose has been analyzed by 1H-NMR spectroscopy. The association constants for the binding of WGA-B to this trisaccharide have been determined from both 1H-NMR titration experiments and microcalorimetry methods. Entropy and enthalpy of binding have been obtained. The driving force for the binding process is provided by a negative DeltaH which is partially compensated by negative DeltaS. These negative signs indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. NOESY NMR experiments in water solution provided 327 protein proton-proton distance constraints. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the refined solution conformation of this protein/carbohydrate complex. With regard to the NMR structure of the free protein, no important changes in the protein NOEs were observed, indicating that carbohydrate-induced conformational changes are small. The average backbone rmsd of the 35 refined structures was 1.05 A, while the heavy atom rmsd was 2.10 A. Focusing on the bound ligand, two different orientations of the trisaccharide within WGA-B binding site are possible. It can be deduced that both hydrogen bonds and van der Waals contacts confer stability to both complexes. A comparison of the three-dimensional structure of WGA-B in solution to that reported in the solid state and to those deduced for hevein and pseudohevein in solution has also been performed.

  18. B lymphocyte binding to E- and P-selectins is mediated through the de novo expression of carbohydrates on in vitro and in vivo activated human B cells.

    PubMed Central

    Postigo, A A; Marazuela, M; Sánchez-Madrid, F; de Landázuri, M O

    1994-01-01

    Cell adhesion to endothelium regulates the trafficking and recruitment of leukocytes towards lymphoid organs and sites of inflammation. This phenomenon is mediated by the expression of a number of adhesion molecules on both the endothelium and circulating cells. Activation of endothelial cells (EC) with different stimuli induces the expression of several adhesion molecules (E- and P-selectins, ICAM-1, VCAM-1), involved in their interaction with circulating cells. In this report, we have studied the binding of nonactivated and activated B cells to purified E- and P-selectins. Activated but not resting B cells were able to interact with both selectins. This binding capacity of activated B cells paralleled the induction of different carbohydrate epitopes (Lewisx, sialyl-Lewisx, CD57 and CDw65) as well as other molecules bearing these or related epitopes in myeloid cells (L-selectin, alpha L beta 2 and alpha X beta 2 integrins, and CD35) involved in the interaction of different cell types with selectins. B cells infiltrating inflamed tissues like in Hashimoto's thyroiditis, also expressed these selectin-binding carbohydrates in parallel with the expression of E-selectin by surrounding follicular dendritic cells. Moreover, the crosslinking of these selectin-binding epitopes resulted in an increased binding of B cells to different integrin ligands. Thus, in addition to the involvement of integrins, E- and P-selectins could play an important role in the interaction of B lymphocytes with the endothelium during B cell extravasation into lymphoid tissues and inflammatory foci as well as in their organization into lymphoid organs. Images PMID:7523454

  19. Structural characterization of a unique interface between carbohydrate response element-binding protein (ChREBP) and 14-3-3β protein.

    PubMed

    Ge, Qiang; Huang, Nian; Wynn, R Max; Li, Yang; Du, Xinlin; Miller, Bonnie; Zhang, Hong; Uyeda, Kosaku

    2012-12-07

    Carbohydrate response element-binding protein (ChREBP) is an insulin-independent, glucose-responsive transcription factor that is expressed at high levels in liver hepatocytes where it plays a critical role in converting excess carbohydrates to fat for storage. In response to fluctuating glucose levels, hepatic ChREBP activity is regulated in large part by nucleocytoplasmic shuttling of ChREBP protein via interactions with 14-3-3 proteins. The N-terminal ChREBP regulatory region is necessary and sufficient for glucose-responsive ChREBP nuclear import and export. Here, we report the crystal structure of a complex of 14-3-3β bound to the N-terminal regulatory region of ChREBP at 2.4 Å resolution. The crystal structure revealed that the α2 helix of ChREBP (residues 117-137) adopts a well defined α-helical conformation and binds 14-3-3 in a phosphorylation-independent manner that is different from all previously characterized 14-3-3 and target protein-binding modes. ChREBP α2 interacts with 14-3-3 through both electrostatic and van der Waals interactions, and the binding is partially mediated by a free sulfate or phosphate. Structure-based mutagenesis and binding assays indicated that disrupting the observed 14-3-3 and ChREBP α2 interface resulted in a loss of complex formation, thus validating the novel protein interaction mode in the 14-3-3β·ChREBP α2 complex.

  20. New structures of the O-specific polysaccharides of Proteus. 3. Polysaccharides containing non-carbohydrate organic acids.

    PubMed

    Kondakova, A N; Toukach, F V; Senchenkova, S N; Arbatsky, N P; Shashkov, A S; Knirel, Y A; Bartodziejska, B; Zych, K; Rozalski, A; Sidorczyk, Z

    2003-04-01

    Four new Proteus O-specific polysaccharides were isolated by mild acid degradation from the lipopolysaccharides of P. penneri 28 (1), P. vulgaris O44 (2), P. mirabilis G1 (O3) (3), and P. myxofaciens (4), and their structures were elucidated using NMR spectroscopy and chemical methods. They were found to contain non-carbohydrate organic acids, including ether-linked lactic acid and amide-linked amino acids, and the following structures of the repeating units were established: [Figure: see text], where (S)-Lac and (R)-aLys stand for (S)-1-carboxyethyl (residue of lactic acid) and N(epsilon)-[(R)-1-carboxyethyl]-L-lysine ("alaninolysine"), respectively. The data obtained in this work and earlier serve as the chemical basis for classification of the bacteria Proteus.

  1. Using protein-binding microarrays to study transcription factor specificity: homologs, isoforms and complexes

    PubMed Central

    Andrilenas, Kellen K.; Penvose, Ashley

    2015-01-01

    Protein–DNA binding is central to specificity in gene regulation, and methods for characterizing transcription factor (TF)–DNA binding remain crucial to studies of regulatory specificity. High-throughput (HT) technologies have revolutionized our ability to characterize protein–DNA binding by significantly increasing the number of binding measurements that can be performed. Protein-binding microarrays (PBMs) are a robust and powerful HT platform for studying DNA-binding specificity of TFs. Analysis of PBM-determined DNA-binding profiles has provided new insight into the scope and mechanisms of TF binding diversity. In this review, we focus specifically on the PBM technique and discuss its application to the study of TF specificity, in particular, the binding diversity of TF homologs and multi-protein complexes. PMID:25431149

  2. Induction, binding specificity and function of human ICOS.

    PubMed

    Beier, K C; Hutloff, A; Dittrich, A M; Heuck, C; Rauch, A; Büchner, K; Ludewig, B; Ochs, H D; Mages, H W; Kroczek, R A

    2000-12-01

    Recently, we have identified the inducible co-stimulator (ICOS), an activation-dependent, T cell-specific cell surface molecule related to CD28 and CTLA-4. Detailed analysis of human ICOS presented here shows that it is a 55-60-kDa homodimer with differently N-glycosylated subunits of 27 and 29 kDa. ICOS requires both phorbol 12-myristate 13-acetate and ionomycin for full induction, and is sensitive to Cyclosporin A. ICOS is up-regulated early on all T cells, including the CD28- subset, and continues to be expressed into later phases of T cell activation. On stimulation of T cells by antigen-presenting cells, the CD28/B7, but not the CD40 ligand/CD40 pathway is critically involved in the induction of ICOS. ICOS does not bind to B7-1 or B7-2, and CD28 does not bind to ICOS ligand; thus the CD28 and ICOS pathways do not cross-interact on the cell surface. In vivo, ICOS is expressed in the medulla of the fetal and newborn thymus, in the T cell zones of tonsils and lymph nodes, and in the apical light zones of germinal centers (predominant expression). Functionally, ICOS co-induces a variety of cytokines including IL-4, IL-5, IL-6, IFN-gamma, TNF-alpha, GM-CSF, but not IL-2, and superinduces IL-10. Furthermore, ICOS co-stimulation prevents the apoptosis of pre-activated T cells. The human ICOS gene maps to chromosome 2q33 - 34.

  3. Electrical detection of specific versus non-specific binding events in breast cancer cells

    NASA Astrophysics Data System (ADS)

    King, Benjamin C.; Clark, Michael; Burkhead, Thomas; Sethu, Palaniappan; Rai, Shesh; Kloecker, Goetz; Panchapakesan, Balaji

    2012-10-01

    Detection of circulating tumor cells (CTCs) from patient blood samples offers a desirable alternative to invasive tissue biopsies for screening of malignant carcinomas. A rigorous CTC detection method must identify CTCs from millions of other formed elements in blood and distinguish them from healthy tissue cells also present in the blood. CTCs are known to overexpress surface receptors, many of which aid them in invading other tissue, and these provide an avenue for their detection. We have developed carbon nanotube (CNT) thin film devices to specifically detect these receptors in intact cells. The CNT sidewalls are functionalized with antibodies specific to Epithelial Cell Adhesion Molecule (EpCAM), a marker overexpressed by breast and other carcinomas. Specific binding of EpCAM to anti-EpCAM antibodies causes a change in the local charge environment of the CNT surface which produces a characteristic electrical signal. Two cell lines were tested in the device: MCF7, a mammary adenocarcinoma line which overexpresses EpCAM, and MCF10A, a non-tumorigenic mammary epithelial line which does not. Introduction of MCF7s caused significant changes in the electrical conductance of the devices due to specific binding and associated charge environment change near the CNT sidewalls. Introduction of MCF10A displays a different profile due to purely nonspecific interactions. The profile of specific vs. nonspecific interaction signatures using carbon based devices will guide development of this diagnostic tool towards clinical sample volumes with wide variety of markers.

  4. Examining the response of needle carbohydrates from Siberian larch trees to climate using compound-specific δ(13) C and concentration analyses.

    PubMed

    Rinne, K T; Saurer, M; Kirdyanov, A V; Bryukhanova, M V; Prokushkin, A S; Churakova Sidorova, O V; Siegwolf, R T W

    2015-11-01

    Little is known about the dynamics of concentrations and carbon isotope ratios of individual carbohydrates in leaves in response to climatic and physiological factors. Improved knowledge of the isotopic ratio in sugars will enhance our understanding of the tree ring isotope ratio and will help to decipher environmental conditions in retrospect more reliably. Carbohydrate samples from larch (Larix gmelinii) needles of two sites in the continuous permafrost zone of Siberia with differing growth conditions were analysed with the Compound-Specific Isotope Analysis (CSIA). We compared concentrations and carbon isotope values (δ(13) C) of sucrose, fructose, glucose and pinitol combined with phenological data. The results for the variability of the needle carbohydrates show high dynamics with distinct seasonal characteristics between and within the studied years with a clear link to the climatic conditions, particularly vapour pressure deficit. Compound-specific differences in δ(13) C values as a response to climate were detected. The δ(13) C of pinitol, which contributes up to 50% of total soluble carbohydrates, was almost invariant during the whole growing season. Our study provides the first in-depth characterization of compound-specific needle carbohydrate isotope variability, identifies involved mechanisms and shows the potential of such results for linking tree physiological responses to different climatic conditions. © 2015 John Wiley & Sons Ltd.

  5. Are matrix-immobilized neoglycoproteins, plant and human lectins and carbohydrate--binding antibodies from human serum mediators of adhesion in vitro for carcinoma and lymphosarcoma cells?

    PubMed

    Wawotzny, R; André, S; Dong, X; Joshi, S S; Gabius, H J

    1995-01-01

    Mediation of cell adhesion by defined molecules can be studied by their immobilization onto a nitrocellulose matrix and incubation with cells. In order to infer the capacity of deliberately selected protein-carbohydrate interactions to establish sugar-inhibitable cell adhesion, a panel of immobilized neoglycoproteins was employed for the murine lymphosarcoma lines RAW-117 with low (P) and high (H10) metastatic capacity, a human mammary carcinoma line (DU4475) and three human colon carcinoma lines (C205, SW480, SW620). Exhibiting an otherwise rather similar behavior relative to the line with low metastatic potential, the murine line RAW117-H10 bound strongly to the matrix with carboxyl group-bearing N-acetylneuraminic acid and glucuronic acid as well as rhamnose. Whereas the analysis of carbohydrate-mediated adhesion yielded comparable results for the three colon carcinoma lines, a markedly reduced number of adherent cells was counted for matrix-attached alpha- and beta-galactosyl, alpha-mannosyl and alpha-glucosyl moieties in the case of the mammary carcinoma line, raising evidence for cell lineage-dependent alterations of this property. From the carbohydrate-binding proteins, the plant lectin, concanavalin A and Viscum album agglutinin almost invariably served well as cell adhesion molecules. Appropriate cell surface sugar receptors, probed with neoglycoproteins, and glycoconjugates, probed with lectins, thus can contribute to adhesion in this model system. The immobilized human beta-galactoside-binding lectin (Mr 14kDa) caused adhesion of the murine lines and one colon carcinoma line (SW480). Neither C-reactive protein under conditions that induce its activity as lectin nor serum amyloid P component nor a lactose-binding immunoglobulin G fraction from human serum were reactive. However, cell adhesion to the alpha-galactoside-binding immunoglobulin G fraction of human serum was seen with the murine line of low metastatic capacity and the mammary carcinoma line

  6. Binding of Haemophilus ducreyi to carbohydrate receptors is mediated by the 58.5-kDa GroEL heat shock protein.

    PubMed

    Pantzar, Martina; Teneberg, Susann; Lagergård, Teresa

    2006-08-01

    The bacterium Haemophilus ducreyi causes the sexually transmitted disease chancroid, which is characterized by the appearance of mucocutaneous, persistent ulcers on the external genitals. To identify carbohydrate receptors that mediate the attachment of this pathogen to host cells, we investigated the binding of 35S-methionine-labeled H. ducreyi strains to a panel of defined glycosphingolipids that were separated on thin layer chromatography plates. H. ducreyi bound to lactosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, neolactotetraosylceramide, the GM3 ganglioside, and sulfatide. To elucidate the role of the surface-located 58.5-kDa GroEL heat shock protein (HSP) of H. ducreyi in attachment, we investigated the binding of purified HSP to the same panel of glycosphingolipids. Our results suggest that the 58.5-kDa GroEL HSP of H. ducreyi is responsible for the attachment of this bacterium to the majority of the tested glycosphingolipids, and thus represents a potential bacterial adhesin.

  7. Photoswitchable cluster glycosides as tools to probe carbohydrate-protein interactions: synthesis and lectin-binding studies of azobenzene containing multivalent sugar ligands.

    PubMed

    Srinivas, Oruganti; Mitra, Nivedita; Surolia, Avadhesha; Jayaraman, Narayanaswamy

    2005-09-01

    Synthetic cluster glycosides have often been used to unravel mechanisms of carbohydrate-protein interactions. Although synthetic cluster glycosides are constituted on scaffolds to achieve high avidities in lectin binding, there have been no known attempts to modulate the orientations of the sugar clusters with the aid of a functional scaffold onto which the sugar units are linked. Herein, we describe synthesis, physical, and lectin-binding studies of a series of alpha-D-mannopyranoside and beta-D-galactopyranosyl-(1-->4)-beta-D-glucopyranoside glycoclusters that are attached to a photoswitchable azobenzenoid core. These glycoclusters were synthesized by the amidation of amine-tethered glycopyranosides with azobenzene carbonyl chlorides. From kinetic studies, the cis forms of the azobenzene-glycopyranoside derivative were found to be more stable in aqueous solutions than in organic solvents. Molecular modeling studies were performed to estimate the relative geometries of the photoswitchable glycoclusters in the trans- and cis-isomeric forms. Isothermal titration calorimetry (ITC) was employed to assess the binding of these glycoclusters to lectins peanut agglutinin (PNA) and concanavalin A (Con A). Although binding affinities were enhanced several orders higher as the valency of the sugar was increased, a biphasic-binding profile in ITC plots was observed during few glycoclusters lectin-binding processes. The biphasic-binding profile indicates a "cooperativity" in the binding process. An important outcome of this study is that in addition to inherent clustering of the sugar units as a molecular feature, an induced clustering emanates because of the isomerization of the trans form of the azobenzene scaffold to the cis-isomeric form.

  8. Real-time and label-free analysis of binding thermodynamics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a QCM biosensor

    PubMed Central

    Li, Xueming; Song, Siyu; Shuai, Qi; Pei, Yihan; Aastrup, Teodor; Pei, Yuxin; Pei, Zhichao

    2015-01-01

    A novel approach to the study of binding thermodynamics and kinetics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a quartz crystal microbalance (QCM) biosensor was developed, in which binding events take place at the cell surface, more closely mimicking a biologically relevant environment. In this study, colon adenocarcinoma cells (KM-12) and ovary adenocarcinoma cells (SKOV-3) grew on the optimized polystyrene-coated biosensor chip without fixation. The association and dissociation between the cell surface carbohydrates and a range of lectins, including WGA, Con A, UEA-I, GS-II, PNA and SBA, were monitored in real time and without label for evaluation of cell surface glycosylation. Furthermore, the thermodynamic and kinetic parameters of the interaction between lectins and cell surface glycan were studied, providing detailed information about the interactions, such as the association rate constant, dissociation rate constant, affinity constant, as well as the changes of entropy, enthalpy and Gibbs free energy. This application provides an insight into the cell surface glycosylation and the complex molecular recognition on the intact cell surface, which may have impacts on disease diagnosis and drug discovery. PMID:26369583

  9. Real-time and label-free analysis of binding thermodynamics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a QCM biosensor.

    PubMed

    Li, Xueming; Song, Siyu; Shuai, Qi; Pei, Yihan; Aastrup, Teodor; Pei, Yuxin; Pei, Zhichao

    2015-09-15

    A novel approach to the study of binding thermodynamics and kinetics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a quartz crystal microbalance (QCM) biosensor was developed, in which binding events take place at the cell surface, more closely mimicking a biologically relevant environment. In this study, colon adenocarcinoma cells (KM-12) and ovary adenocarcinoma cells (SKOV-3) grew on the optimized polystyrene-coated biosensor chip without fixation. The association and dissociation between the cell surface carbohydrates and a range of lectins, including WGA, Con A, UEA-I, GS-II, PNA and SBA, were monitored in real time and without label for evaluation of cell surface glycosylation. Furthermore, the thermodynamic and kinetic parameters of the interaction between lectins and cell surface glycan were studied, providing detailed information about the interactions, such as the association rate constant, dissociation rate constant, affinity constant, as well as the changes of entropy, enthalpy and Gibbs free energy. This application provides an insight into the cell surface glycosylation and the complex molecular recognition on the intact cell surface, which may have impacts on disease diagnosis and drug discovery.

  10. Ecologically Valid Carbohydrate Intake during Soccer-Specific Exercise Does Not Affect Running Performance in a Fed State.

    PubMed

    Funnell, Mark P; Dykes, Nick R; Owen, Elliot J; Mears, Stephen A; Rollo, Ian; James, Lewis J

    2017-01-05

    This study assessed the effect of carbohydrate intake on self-selected soccer-specific running performance. Sixteen male soccer players (age 23 ± 4 years; body mass 76.9 ± 7.2 kg; predicted VO2max = 54.2 ± 2.9 mL∙kg(-1)∙min(-1); soccer experience 13 ± 4 years) completed a progressive multistage fitness test, familiarisation trial and two experimental trials, involving a modified version of the Loughborough Intermittent Shuttle Test (LIST) to simulate a soccer match in a fed state. Subjects completed six 15 min blocks (two halves of 45 min) of intermittent shuttle running, with a 15-min half-time. Blocks 3 and 6, allowed self-selection of running speeds and sprint times, were assessed throughout. Subjects consumed 250 mL of either a 12% carbohydrate solution (CHO) or a non-caloric taste matched placebo (PLA) before and at half-time of the LIST. Sprint times were not different between trials (CHO 2.71 ± 0.15 s, PLA 2.70 ± 0.14 s; p = 0.202). Total distance covered in self-selected blocks (block 3: CHO 2.07 ± 0.06 km; PLA 2.09 ± 0.08 km; block 6: CHO 2.04 ± 0.09 km; PLA 2.06 ± 0.08 km; p = 0.122) was not different between trials. There was no difference between trials for distance covered (p ≥ 0.297) or mean speed (p ≥ 0.172) for jogging or cruising. Blood glucose concentration was greater (p < 0.001) at the end of half-time during the CHO trial. In conclusion, consumption of 250 mL of 12% CHO solution before and at half-time of a simulated soccer match does not affect self-selected running or sprint performance in a fed state.

  11. Compound-specific stable carbon isotopic signature of carbohydrate pyrolysis products from C3 and C4 plants.

    PubMed

    González-Pérez, José A; Jiménez-Morillo, Nicasio T; de la Rosa, José M; Almendros, Gonzalo; González-Vila, Francisco J

    2016-02-01

    Pyrolysis-compound specific isotopic analysis (Py-CSIA: Py-GC-(FID)-C-IRMS) is a relatively novel technique that allows on-line quantification of stable isotope proportions in chromatographically separated products released by pyrolysis. Validation of the Py-CSIA technique is compulsory for molecular traceability in basic and applied research. In this work, commercial sucrose from C4 (sugarcane) and C3 (sugarbeet) photosystem plants and admixtures were studied using analytical pyrolysis (Py-GC/MS), bulk δ(13)C IRMS and δ(13)C Py-CSIA. Major pyrolysis compounds were furfural (F), furfural-5-hydroxymethyl (HMF) and levoglucosan (LV). Bulk and main pyrolysis compound δ(13)C (‰) values were dependent on plant origin: C3 (F, -24.65 ± 0.89; HMF, -22.07 ± 0.41‰; LV, -21.74 ± 0.17‰) and C4 (F, -14.35 ± 0.89‰; HMF, -11.22 ± 0.54‰; LV, -11.44 ± 1.26‰). Significant regressions were obtained for δ(13)C of bulk and pyrolysis compounds in C3 and C4 admixtures. Furfural (F) was found (13)C depleted with respect to bulk and HMF and LV, indicating the incorporation of the light carbon atom in position 6 of carbohydrates in the furan ring after pyrolysis. This is the first detailed report on the δ(13)C signature of major pyrolytically generated carbohydrate-derived molecules. The information provided by Py-CSIA is valuable for identifying source marker compounds of use in food science/fraud detection or in environmental research. © 2015 Society of Chemical Industry.

  12. Functional specificity of the homeodomain protein fushi tarazu: the role of DNA-binding specificity in vivo.

    PubMed Central

    Schier, A F; Gehring, W J

    1993-01-01

    The mechanisms determining the functional specificity of Drosophila homeodomain proteins are largely unknown. Here, the role of DNA-binding specificity for the in vivo function of the homeodomain protein fushi tarazu (ftz) is analyzed. We find that specific DNA binding is an important but not sufficient determinant of the functional specificity of ftz in vivo: The ftz DNA-binding specificity mutant ftzQ50K retains partial ftz wild-type activity in gene activation and phenotypic rescue assays. Furthermore, specificity mutations in a ftz-in vivo binding site only partially reduce enhancer activity as compared to null mutations of this site. Despite bicoid-like DNA-binding specificity ftzQ50K does not activate natural or artificial bcd target genes in the realms of ftz. These results are discussed in the light of recent observations on the mechanism of action of the yeast homeodomain protein alpha 2. Images PMID:8434005

  13. A starch-binding domain identified in α-amylase (AmyP) represents a new family of carbohydrate-binding modules that contribute to enzymatic hydrolysis of soluble starch.

    PubMed

    Peng, Hui; Zheng, Yunyun; Chen, Maojiao; Wang, Ying; Xiao, Yazhong; Gao, Yi

    2014-04-02

    A novel starch-binding domain (SBD) that represents a new carbohydrate-binding module family (CBM69) was identified in the α-amylase (AmyP) of the recently established alpha-amylase subfamily GH13_37. The SBD and its homologues come mostly from marine bacteria, and phylogenetic analysis indicates that they are closely related to the CBM20 and CBM48 families. The SBD exhibited a binding preference toward raw rice starch, but the truncated mutant (AmyPΔSBD) still retained similar substrate preference. Kinetic analyses revealed that the SBD plays an important role in soluble starch hydrolysis because different catalytic efficiencies have been observed in AmyP and the AmyPΔSBD.

  14. Structures and binding specificity of galactose- and mannose-binding lectins from champedak: differences from jackfruit lectins.

    PubMed

    Gabrielsen, Mads; Abdul-Rahman, Puteri Shafinaz; Othman, Shatrah; Hashim, Onn H; Cogdell, Richard J

    2014-06-01

    Galactose-binding and mannose-binding lectins from the champedak fruit, which is native to South-east Asia, exhibit useful potential clinical applications. The specificity of the two lectins for their respective ligands allows the detection of potential cancer biomarkers and monitoring of the glycosylated state of proteins in human serum and/or urine. To fully understand and expand the use of these natural proteins, their complete sequences and crystal structures are presented here, together with details of sugar binding.

  15. Species-specific chitin-binding module 18 expansion in the amphibian pathogen Batrachochytrium dendrobatidis.

    PubMed

    Abramyan, John; Stajich, Jason E

    2012-01-01

    Batrachochytrium dendrobatidis is the causative agent of chytridiomycosis, which is considered one of the driving forces behind the worldwide decline in populations of amphibians. As a member of the phylum Chytridiomycota, B. dendrobatidis has diverged significantly to emerge as the only pathogen of adult vertebrates. Such shifts in lifestyle are generally accompanied by various degrees of genomic modifications, yet neither its mode of pathogenicity nor any factors associated with it have ever been identified. Presented here is the identification and characterization of a unique expansion of the carbohydrate-binding module family 18 (CBM18), specific to B. dendrobatidis. CBM (chitin-binding module) expansions have been likened to the evolution of pathogenicity in a variety of fungus species, making this expanded group a prime candidate for the identification of potential pathogenicity factors. Furthermore, the CBM18 expansions are confined to three categories of genes, each having been previously implicated in host-pathogen interactions. These correlations highlight this specific domain expansion as a potential key player in the mode of pathogenicity in this unique fungus. The expansion of CBM18 in B. dendrobatidis is exceptional in its size and diversity compared to other pathogenic species of fungi, making this genomic feature unique in an evolutionary context as well as in pathogenicity. Amphibian populations are declining worldwide at an unprecedented rate. Although various factors are thought to contribute to this phenomenon, chytridiomycosis has been identified as one of the leading causes. This deadly fungal disease is cause by Batrachochytrium dendrobatidis, a chytrid fungus species unique in its pathogenicity and, furthermore, its specificity to amphibians. Despite more than two decades of research, the biology of this fungus species and its deadly interaction with amphibians had been notoriously difficult to unravel. Due to the alarming rate of worldwide

  16. Human DC-SIGN Binds Specific Human Milk Glycans

    PubMed Central

    Noll, Alexander J.; Yu, Ying; Lasanajak, Yi; Duska-McEwen, Geralyn; Buck, Rachael H.; Smith, David F.; Cummings, Richard D.

    2016-01-01

    Human milk glycans (HMGs) are prebiotics, pathogen receptor decoys, and regulators of host physiology and immune responses. Mechanistically, human lectins (glycan-binding proteins, hGBPs) expressed by dendritic cells (DC) are of major interest, as these cells directly contact HMGs. To explore such interactions, we screened many C-type lectins and Siglecs expressed by DC for glycan binding on microarrays presenting over 200 HMGs. Unexpectedly, DC-SIGN showed robust binding to many HMGs, whereas other C-type lectins failed to bind, and Siglecs-5 and -9 showed weak binding to a few glycans. By contrast, most hGBPs bound to multiple glycans on other microarrays lacking HMGs. An α-linked fucose residue was characteristic of HMGs bound by DC-SIGN. Binding of DC-SIGN to the simple HMGs 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL) was confirmed by flow cytometry to beads conjugated with 2′-FL or 3-FL, as well as the ability of the free glycans to inhibit DC-SIGN binding. 2′-FL had an IC50 of ~1 mM for DC-SIGN, which is within the physiological concentration of 2′-FL in human milk. These results demonstrate that DC-SIGN among the many hGBPs expressed by DC binds to α-fucosylated HMGs, and suggest that such interactions may be important in influencing immune responses in the developing infant. PMID:26976925

  17. Modulation of DNA binding by gene-specific transcription factors.

    PubMed

    Schleif, Robert F

    2013-10-01

    The transcription of many genes, particularly in prokaryotes, is controlled by transcription factors whose activity can be modulated by controlling their DNA binding affinity. Understanding the molecular mechanisms by which DNA binding affinity is regulated is important, but because forming definitive conclusions usually requires detailed structural information in combination with data from extensive biophysical, biochemical, and sometimes genetic experiments, little is truly understood about this topic. This review describes the biological requirements placed upon DNA binding transcription factors and their consequent properties, particularly the ways that DNA binding affinity can be modulated and methods for its study. What is known and not known about the mechanisms modulating the DNA binding affinity of a number of prokaryotic transcription factors, including CAP and lac repressor, is provided.

  18. Inhibition of human GLUT1 and GLUT5 by plant carbohydrate products; insights into transport specificity

    PubMed Central

    George Thompson, Alayna M.; Iancu, Cristina V.; Nguyen, Thi Thanh Hanh; Kim, Doman; Choe, Jun-yong

    2015-01-01

    Glucose transporters GLUT1 (transports glucose) and GLUT5 (transports fructose), in addition to their functions in normal metabolism, have been implicated in several diseases including cancer and diabetes. While GLUT1 has several inhibitors, none have been described for GLUT5. By transport activity assays we found two plant products, rubusoside (from Rubus suavissimus) and astragalin-6-glucoside (a glycosylated derivative of astragalin, from Phytolacca americana) that inhibited human GLUT5. These plants are utilized in traditional medicine: R. suavissimus for weight loss and P. americana for cancer treatment, but the molecular interactions of these products are unknown. Rubusoside also inhibited human GLUT1, but astragalin-6-glucoside did not. In silico analysis of rubusoside:protein interactions pinpointed a major difference in substrate cavity between these transporters, a residue that is a tryptophan in GLUT1 but an alanine in GLUT5. Investigation of mutant proteins supported the importance of this position in ligand specificity. GLUT1W388A became susceptible to inhibition by astragalin-6-glucoside and resistant to rubusoside. GLUT5A396W transported fructose and also glucose, and maintained inhibition by rubusoside and astragalin-6-glucoside. Astragalin-6-glucoside can serve as a starting point in the design of specific inhibitors for GLUT5. The application of these studies to understanding glucose transporters and their interaction with substrates and ligands is discussed. PMID:26306809

  19. KINETICS OF MODULATORY ROLE OF Cyperus esculentus L. ON THE SPECIFIC ACTIVITY OF KEY CARBOHYDRATE METABOLIZING ENZYMES

    PubMed Central

    Sabiu, Saheed; Ajani, Emmanuel Oladipo; Sunmonu, Taofik Olatunde; Ashafa, Anofi Omotayo Tom

    2017-01-01

    Background: The continuous search for new lead compounds as viable inhibitors of specific enzymes linked to carbohydrate metabolism has intensified. Cyperus esculentus L. is one of the therapeutically implicated botanicals against several degenerative diseases including diabetes mellitus. Materials and Methods: This study evaluated the antioxidant and mechanism(s) of inhibitory potential of aqueous extract of C. esculentus on α-amylase and α-glucosidase in vitro. The extract was investigated for its radical scavenging and hypoglycaemic potentials using standard experimental procedures. Lineweaver-Burke plot was used to predict the manner in which the enzymes were inhibited. Results: The data obtained revealed that the extract moderately and potently inhibited the specific activities of α-amylase and α-glucosidase, respectively. The inhibition was concentration-related with respective IC50 values of 5.19 and 0.78 mg/mL relative to that of the control (3.72 and 3.55 mg/mL). The extract also significantly scavenged free radicals and the effects elicited could be ascribed to its phytoconstituents. Conclusion: The respective competitive and non-competitive mode of action of the extract is due to its inhibitory potentials on the activities of α-amylase and α-glucosidase. Going forward, in addition to completely characterize the exact compound(s) responsible for the elicited activity in this study, pertinent attention will be given to the in vivo evaluation of the identified constituents. PMID:28638866

  20. KINETICS OF MODULATORY ROLE OF Cyperus esculentus L. ON THE SPECIFIC ACTIVITY OF KEY CARBOHYDRATE METABOLIZING ENZYMES.

    PubMed

    Sabiu, Saheed; Ajani, Emmanuel Oladipo; Sunmonu, Taofik Olatunde; Ashafa, Anofi Omotayo Tom

    2017-01-01

    The continuous search for new lead compounds as viable inhibitors of specific enzymes linked to carbohydrate metabolism has intensified. Cyperus esculentus L. is one of the therapeutically implicated botanicals against several degenerative diseases including diabetes mellitus. This study evaluated the antioxidant and mechanism(s) of inhibitory potential of aqueous extract of C. esculentus on α-amylase and α-glucosidase in vitro. The extract was investigated for its radical scavenging and hypoglycaemic potentials using standard experimental procedures. Lineweaver-Burke plot was used to predict the manner in which the enzymes were inhibited. The data obtained revealed that the extract moderately and potently inhibited the specific activities of α-amylase and α-glucosidase, respectively. The inhibition was concentration-related with respective IC50 values of 5.19 and 0.78 mg/mL relative to that of the control (3.72 and 3.55 mg/mL). The extract also significantly scavenged free radicals and the effects elicited could be ascribed to its phytoconstituents. The respective competitive and non-competitive mode of action of the extract is due to its inhibitory potentials on the activities of α-amylase and α-glucosidase. Going forward, in addition to completely characterize the exact compound(s) responsible for the elicited activity in this study, pertinent attention will be given to the in vivo evaluation of the identified constituents.

  1. Fusion of a family 20 carbohydrate-binding module (CBM20) with cyclodextrin glycosyltransferase of Geobacillus sp. CHB1 improves catalytic efficiency.

    PubMed

    Jia, Xianbo; Guo, Yonghua; Lin, Xinjian; You, Minsheng; Lin, Chenqiang; Chen, Longjun; Chen, Jichen

    2017-06-01

    Cyclodextrin glycosyltransferase (CGTase) is an important industrial enzyme for production of cyclodextrins (CDs) from starch by intramolecular transglycosylation. CGTase consists of five domains labeled A to E. For optimizing catalytic activity of CGTase, CGTase of Geobacillus sp. was fused with the family 20 carbohydrate-binding module (CBM) of the Bacillus circulans strain 251 CGTase. The CBMbc251 that has a low binding free energy with maltohexaose, was selected by in silico design. Then the fusion enzyme, CGTΔE-CBMbc251, was constructed by fusing the CBMbc251 to the C-terminal region of CGTΔE. The fusion enzyme displayed an even greater enhancement of total α-cyclization activity (40.2%) and γ-cyclization activity (181.58%). Optimal reaction pH range was wilder and the thermal stability was better under 50 and 60 °C. Compared to the wild-type CGTase, the fusion enzyme showed a remarkable decrease in Km and a slight alteration in Vmax. The enhancement of soluble starch catalytic efficiency might be due to the changes of substrate binding ability in the critical substrate binding sites between the CBM and starch granule. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Protein−DNA binding in the absence of specific base-pair recognition

    PubMed Central

    Afek, Ariel; Schipper, Joshua L.; Horton, John; Gordân, Raluca; Lukatsky, David B.

    2014-01-01

    Until now, it has been reasonably assumed that specific base-pair recognition is the only mechanism controlling the specificity of transcription factor (TF)−DNA binding. Contrary to this assumption, here we show that nonspecific DNA sequences possessing certain repeat symmetries, when present outside of specific TF binding sites (TFBSs), statistically control TF−DNA binding preferences. We used high-throughput protein−DNA binding assays to measure the binding levels and free energies of binding for several human TFs to tens of thousands of short DNA sequences with varying repeat symmetries. Based on statistical mechanics modeling, we identify a new protein−DNA binding mechanism induced by DNA sequence symmetry in the absence of specific base-pair recognition, and experimentally demonstrate that this mechanism indeed governs protein−DNA binding preferences. PMID:25313048

  3. Carbohydrates on Proteins: Site-Specific Glycosylation Analysis by Mass Spectrometry.

    PubMed

    Zhu, Zhikai; Desaire, Heather

    2015-01-01

    Glycosylation on proteins adds complexity and versatility to these biologically vital macromolecules. To unveil the structure-function relationship of glycoproteins, glycopeptide-centric analysis using mass spectrometry (MS) has become a method of choice because the glycan is preserved on the glycosylation site and site-specific glycosylation profiles of proteins can be readily determined. However, glycopeptide analysis is still challenging given that glycopeptides are usually low in abundance and relatively difficult to detect and the resulting data require expertise to analyze. Viewing the urgent need to address these challenges, emerging methods and techniques are being developed with the goal of analyzing glycopeptides in a sensitive, comprehensive, and high-throughput manner. In this review, we discuss recent advances in glycoprotein and glycopeptide analysis, with topics covering sample preparation, analytical separation, MS and tandem MS techniques, as well as data interpretation and automation.

  4. Carbohydrates on Proteins: Site-Specific Glycosylation Analysis by Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Zhu, Zhikai; Desaire, Heather

    2015-07-01

    Glycosylation on proteins adds complexity and versatility to these biologically vital macromolecules. To unveil the structure-function relationship of glycoproteins, glycopeptide-centric analysis using mass spectrometry (MS) has become a method of choice because the glycan is preserved on the glycosylation site and site-specific glycosylation profiles of proteins can be readily determined. However, glycopeptide analysis is still challenging given that glycopeptides are usually low in abundance and relatively difficult to detect and the resulting data require expertise to analyze. Viewing the urgent need to address these challenges, emerging methods and techniques are being developed with the goal of analyzing glycopeptides in a sensitive, comprehensive, and high-throughput manner. In this review, we discuss recent advances in glycoprotein and glycopeptide analysis, with topics covering sample preparation, analytical separation, MS and tandem MS techniques, as well as data interpretation and automation.

  5. Refining small intestinal bacterial overgrowth diagnosis by means of carbohydrate specificity: a proof-of-concept study

    PubMed Central

    Enko, Dietmar; Halwachs-Baumann, Gabriele; Stolba, Robert; Mangge, Harald; Kriegshäuser, Gernot

    2015-01-01

    Background: Diagnosis of small intestinal bacterial overgrowth (SIBO) remains challenging. This study aimed at proving the diagnostic concept of carbohydrate-specific SIBO (cs-SIBO) using glucose, fructose and sorbitol hydrogen (H2)/methane (CH4) breath testing (HMBT). Methods: In this study 125 patients referred to our outpatient clinic for SIBO testing were included. All individuals underwent glucose (50 g), fructose (25 g) and sorbitol (12.5 g) HMBT at 3 consecutive days. Patients with cs-SIBO (i.e. early H2/CH4 peak) were given rifaximin (600 mg/day) in a 10-day treatment. HMBT results were reassessed in a subset of patients 3–6 months after antibiotic therapy. In view of cs-SIBO diagnosis, agreements between HMBT results obtained for different sugars were calculated using Cohen’s kappa (κ) with 95% confidence intervals (CIs). Results: A total of 59 (47.2%) patients presented an early H2/CH4 peak with one or more sugars. Among these, 21 (16.8%), 10 (8.0%) and 7 (5.6%) individuals had a positive HMBT result with either glucose, fructose or sorbitol, respectively. Another 21 (16.8%) patients with a positive glucose HMBT result were also found positive with an early H2/CH4 peak obtained after ingestion of fructose and/or sorbitol. Fair agreement was observed between glucose and fructose (κ = 0.26, p = 0.0018) and between glucose and sorbitol (κ = 0.18, p = 0.0178) HMBT results. Slight agreement was observed between fructose and sorbitol (κ = 0.03, p = 0.6955) HMBT results only. Successful antibiotic therapy with rifaximin could be demonstrated in 26/30 (86.7%) of patients as indicated by normal HMBT results and symptom remission. Conclusions: Combined glucose, fructose and sorbitol HMBT has the potential to optimize cs-SIBO diagnosis. Furthermore, the majority of patients with cs-SIBO seem to benefit from rifaximin therapy regardless of its carbohydrate specificity. PMID:27134657

  6. Allostery between two binding sites in the ion channel subunit TRIP8b confers binding specificity to HCN channels.

    PubMed

    Lyman, Kyle A; Han, Ye; Heuermann, Robert J; Cheng, Xiangying; Kurz, Jonathan E; Lyman, Reagan E; Van Veldhoven, Paul P; Chetkovich, Dane M

    2017-09-08

    Tetratricopeptide repeat (TPR) domains are ubiquitous structural motifs that mediate protein-protein interactions. For example, the TPR domains in the peroxisomal import receptor PEX5 enable binding to a range of type 1 peroxisomal targeting signal (PTS1) motifs. A homolog of PEX5, tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), binds to and functions as an auxiliary subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Given the similarity between TRIP8b and PEX5, this difference in function raises the question of what mechanism accounts for their binding specificity. In this report, we found that the cyclic nucleotide-binding domain (CNBD) and the C-terminus of the HCN channel are critical for conferring specificity to TRIP8b binding. We show that TRIP8b binds the HCN CNBD through a 37-residue domain and the HCN C-terminus through the TPR domains. Using a combination of fluorescence polarization and co-immunoprecipitation based assays, we establish that binding at either site increases affinity at the other. Thus, allosteric coupling of the TRIP8b TPR domains both promotes binding to HCN channels and limits binding to PTS1 substrates. These results raise the possibility that other TPR domains may similarly be influenced by allosteric mechanisms as a general feature of protein-protein interactions. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  7. Specific sulfation and glycosylation—a structural combination for the anticoagulation of marine carbohydrates

    PubMed Central

    Pomin, Vitor H.; Mourão, Paulo A. S.

    2014-01-01

    Based on considered achievements of the last 25 years, specific combinations of sulfation patterns and glycosylation types have been proved to be key structural players for the anticoagulant activity of certain marine glycans. These conclusions were obtained from comparative and systematic analyses on the structure-anticoagulation relationships of chemically well-defined sulfated polysaccharides of marine invertebrates and red algae. These sulfated polysaccharides are known as sulfated fucans (SFs), sulfated galactans (SGs) and glycosaminoglycans (GAGs). The structural combinations necessary for the anticoagulant activities are the 2-sulfation in α-L-SGs, the 2,4-di-sulfation in α-L-fucopyranosyl units found as composing units of certain sea-urchin and sea-cucumber linear SFs, or as branching units of the fucosylated chondroitin sulfate, a unique GAG from sea-cucumbers. Another unique GAG type from marine organisms is the dermatan sulfate isolated from ascidians. The high levels of 4-sulfation at the galactosamine units combined with certain levels of 2-sulfation at the iduronic acid units is the anticoagulant structural requirements of these GAGs. When the backbones of red algal SGs are homogeneous, the anticoagulation is proportionally dependent of their sulfation content. Finally, 4-sulfation was observed to be the structural motif required to enhance the inhibition of thrombin via heparin cofactor-II by invertebrate SFs. PMID:24639954

  8. The Baculovirus-Expressed Binding Region of Plasmodium falciparum EBA-140 Ligand and Its Glycophorin C Binding Specificity

    PubMed Central

    Rydzak, Joanna; Kaczmarek, Radoslaw; Czerwinski, Marcin; Lukasiewicz, Jolanta; Tyborowska, Jolanta; Szewczyk, Boguslaw; Jaskiewicz, Ewa

    2015-01-01

    The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum DBL family of erythrocyte binding proteins, which are considered as prospective candidates for malaria vaccine development. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They share homology of domain structure, including Region II, which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during invasion. In this report we describe, for the first time, the glycophorin C specificity of the recombinant, baculovirus-expressed binding region (Region II) of P. falciparum EBA-140 ligand. It was found that the recombinant EBA-140 Region II binds to the endogenous and recombinant glycophorin C, but does not bind to Gerbich-type glycophorin C, neither normal nor recombinant, which lacks amino acid residues 36–63 of its polypeptide chain. Our results emphasize the crucial role of this glycophorin C region in EBA-140 ligand binding. Moreover, the EBA-140 Region II did not bind either to glycophorin D, the truncated form of glycophorin C lacking the N-glycan or to desialylated GPC. These results draw attention to the role of glycophorin C glycans in EBA-140 binding. The full identification of the EBA-140 binding site on glycophorin C molecule, consisting most likely of its glycans and peptide backbone, may help to design therapeutics or vaccines that target the erythrocyte binding merozoite ligands. PMID:25588042

  9. Ganglioside-binding specificities of E. coli enterotoxin LT-IIc: Importance of long-chain fatty acyl ceramide

    PubMed Central

    Berenson, Charles S; Nawar, Hesham F; Kruzel, Ragina L; Mandell, Lorrie M; Connell, Terry D

    2013-01-01

    Bacterial heat-labile (LT) enterotoxins signal through tightly regulated interactions with host cell gangliosides. LT-IIa and LT-IIb of Escherichia coli bind preferentially to gangliosides with a NeuAcα2-3Galβ1-3GalNAc terminus, with key distinctions in specificity. LT-IIc, a newly discovered E. coli LT, is comprised of an A polypeptide with high homology, and a B polypeptide with moderate homology, to LT-IIa and LT-IIb. LT-IIc is less cytotoxic than LT-IIa and LT-IIb. We theorized that LT-IIc–host cell interaction is regulated by specific structural attributes of immune cell ganglioside receptors and designed experiments to test this hypothesis. Overlay immunoblotting to a diverse array of neural and macrophage gangliosides indicated that LT-IIc bound to a restrictive range of gangliosides, each possessing a NeuAcα2-3Galβ1-3GalNAc with a requisite terminal sialic acid. LT-IIc did not bind to GM1a with short-chain fatty acyl ceramides. Affinity overlay immunoblots, constructed to a diverse array of known ganglioside structures of murine peritoneal macrophages, established that LT-IIc bound to GM1a comprised of long-chain fatty acyl ceramides. Findings were confirmed with LT-IIc also binding to GM1a of RAW264.7 cells, comprised of a long-chain fatty acyl ceramide. Thus, LT-IIc-ganglioside binding differs distinctly from that of LT-IIa and LT-IIb. LT-IIc binding is not just dependent on carbohydrate composition, but also upon the orientation of the oligosaccharide portion of GM1a by the ceramide moiety. These studies are the first demonstration of LT-ganglioside dependence upon ceramide composition and underscore the contribution of long-chain fatty acyl ceramides to host cell interactions. PMID:22917572

  10. Ganglioside-binding specificities of E. coli enterotoxin LT-IIc: Importance of long-chain fatty acyl ceramide.

    PubMed

    Berenson, Charles S; Nawar, Hesham F; Kruzel, Ragina L; Mandell, Lorrie M; Connell, Terry D

    2013-01-01

    Bacterial heat-labile (LT) enterotoxins signal through tightly regulated interactions with host cell gangliosides. LT-IIa and LT-IIb of Escherichia coli bind preferentially to gangliosides with a NeuAcα2-3Galβ1-3GalNAc terminus, with key distinctions in specificity. LT-IIc, a newly discovered E. coli LT, is comprised of an A polypeptide with high homology, and a B polypeptide with moderate homology, to LT-IIa and LT-IIb. LT-IIc is less cytotoxic than LT-IIa and LT-IIb. We theorized that LT-IIc-host cell interaction is regulated by specific structural attributes of immune cell ganglioside receptors and designed experiments to test this hypothesis. Overlay immunoblotting to a diverse array of neural and macrophage gangliosides indicated that LT-IIc bound to a restrictive range of gangliosides, each possessing a NeuAcα2-3Galβ1-3GalNAc with a requisite terminal sialic acid. LT-IIc did not bind to GM1a with short-chain fatty acyl ceramides. Affinity overlay immunoblots, constructed to a diverse array of known ganglioside structures of murine peritoneal macrophages, established that LT-IIc bound to GM1a comprised of long-chain fatty acyl ceramides. Findings were confirmed with LT-IIc also binding to GM1a of RAW264.7 cells, comprised of a long-chain fatty acyl ceramide. Thus, LT-IIc-ganglioside binding differs distinctly from that of LT-IIa and LT-IIb. LT-IIc binding is not just dependent on carbohydrate composition, but also upon the orientation of the oligosaccharide portion of GM1a by the ceramide moiety. These studies are the first demonstration of LT-ganglioside dependence upon ceramide composition and underscore the contribution of long-chain fatty acyl ceramides to host cell interactions.

  11. Carbohydrates as allergens.

    PubMed

    Commins, Scott P

    2015-01-01

    Complex carbohydrates are effective inducers of Th2 responses, and carbohydrate antigens can stimulate the production of glycan-specific antibodies. In instances where the antigen exposure occurs through the skin, the resulting antibody production can contain IgE class antibody. The glycan-stimulated IgE may be non-specific but may also be antigen specific. This review focuses on the production of cross-reactive carbohydrate determinants, the recently identified IgE antibody response to a mammalian oligosaccharide epitope, galactose-alpha-1,3-galactose (alpha-gal), as well as discusses practical implications of carbohydrates in allergy. In addition, the biological effects of carbohydrate antigens are reviewed in setting of receptors and host recognition.

  12. Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria.

    PubMed Central

    Postma, P W; Lengeler, J W; Jacobson, G R

    1993-01-01

    Numerous gram-negative and gram-positive bacteria take up carbohydrates through the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). This system transports and phosphorylates carbohydrates at the expense of PEP and is the subject of this review. The PTS consists of two general proteins, enzyme I and HPr, and a number of carbohydrate-specific enzymes, the enzymes II. PTS proteins are phosphoproteins in which the phospho group is attached to either a histidine residue or, in a number of cases, a cysteine residue. After phosphorylation of enzyme I by PEP, the phospho group is transferred to HPr. The enzymes II are required for the transport of the carbohydrates across the membrane and the transfer of the phospho group from phospho-HPr to the carbohydrates. Biochemical, structural, and molecular genetic studies have shown that the various enzymes II have the same basic structure. Each enzyme II consists of domains for specific functions, e.g., binding of the carbohydrate or phosphorylation. Each enzyme II complex can consist of one to four different polypeptides. The enzymes II can be placed into at least four classes on the basis of sequence similarity. The genetics of the PTS is complex, and the expression of PTS proteins is intricately regulated because of the central roles of these proteins in nutrient acquisition. In addition to classical induction-repression mechanisms involving repressor and activator proteins, other types of regulation, such as antitermination, have been observed in some PTSs. Apart from their role in carbohydrate transport, PTS proteins are involved in chemotaxis toward PTS carbohydrates. Furthermore, the IIAGlc protein, part of the glucose-specific PTS, is a central regulatory protein which in its nonphosphorylated form can bind to and inhibit several non-PTS uptake systems and thus prevent entry of inducers. In its phosphorylated form, P-IIAGlc is involved in the activation of adenylate cyclase and thus in the

  13. Low-carbohydrate, high-fat diets have sex-specific effects on bone health in rats.

    PubMed

    Zengin, Ayse; Kropp, Benedikt; Chevalier, Yan; Junnila, Riia; Sustarsic, Elahu; Herbach, Nadja; Fanelli, Flaminia; Mezzullo, Marco; Milz, Stefan; Bidlingmaier, Martin; Bielohuby, Maximilian

    2016-10-01

    Studies in humans suggest that consumption of low-carbohydrate, high-fat diets (LC-HF) could be detrimental for growth and bone health. In young male rats, LC-HF diets negatively affect bone health by impairing the growth hormone/insulin-like growth factor axis (GH/IGF axis), while the effects in female rats remain unknown. Therefore, we investigated whether sex-specific effects of LC-HF diets on bone health exist. Twelve-week-old male and female Wistar rats were isoenergetically pair-fed either a control diet (CD), "Atkins-style" protein-matched diet (LC-HF-1), or ketogenic low-protein diet (LC-HF-2) for 4 weeks. In females, microcomputed tomography and histomorphometry analyses were performed on the distal femur. Sex hormones were analysed with liquid chromatography-tandem mass spectrometry, and endocrine parameters including GH and IGF-I were measured by immunoassay. Trabecular bone volume, serum IGF-I and the bone formation marker P1NP were lower in male rats fed both LC-HF diets versus CD. LC-HF diets did not impair bone health in female rats, with no change in trabecular or cortical bone volume nor in serum markers of bone turnover between CD versus both LC-HF diet groups. Pituitary GH secretion was lower in female rats fed LC-HF diet, with no difference in circulating IGF-I. Circulating sex hormone concentrations remained unchanged in male and female rats fed LC-HF diets. A 4-week consumption of LC-HF diets has sex-specific effects on bone health-with no effects in adult female rats yet negative effects in adult male rats. This response seems to be driven by a sex-specific effect of LC-HF diets on the GH/IGF system.

  14. Critical ligand binding reagent preparation/selection: when specificity depends on reagents.

    PubMed

    Rup, Bonita; O'Hara, Denise

    2007-05-11

    Throughout the life cycle of biopharmaceutical products, bioanalytical support is provided using ligand binding assays to measure the drug product for pharmacokinetic, pharmacodynamic, and immunogenicity studies. The specificity and selectivity of these ligand binding assays are highly dependent on the ligand binding reagents. Thus the selection, characterization, and management processes for ligand binding reagents are crucial to successful assay development and application. This report describes process considerations for selection and characterization of ligand binding reagents that are integral parts of the different phases of assay development. Changes in expression, purification, modification, and storage of the ligand binding reagents may have a profound effect on the ligand binding assay performance. Thus long-term management of the critical ligand binding assay reagents is addressed including suggested characterization criteria that allow ligand binding reagents to be used in as consistent a manner as possible. Examples of challenges related to the selection, modification, and characterization of ligand binding reagents are included.

  15. Acquisition of species-specific O-linked carbohydrate chains from oviducal mucins in Rana arvalis. A case study.

    PubMed

    Coppin, A; Maes, E; Flahaut, C; Coddeville, B; Strecker, G

    1999-12-01

    The extracellular matrix surrounding amphibian eggs is composed of mucin-type glycoproteins, highly O-glycosylated and plays an important role in the fertilization process. Oligosaccharide-alditols were released from the oviducal mucins of the anuran Rana arvalis by alkali-borohydride treatment in reduced conditions. Neutral and acidic oligosaccharides were fractionated by ion-exchange chromatographies and purified by HPLC. Each compound was identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) spectrometry, NMR spectroscopy, electrospray ionization-tandem mass spectroscopy (ESI-MS/MS) and permethylation analyses. This paper reports on the structures of 19 oligosaccharide-alditols, 12 of which have novel structures. These structures range in size from disaccharide to octasaccharide. Some of them are acidic, containing either a glucuronic acid or, more frequently, a sulfate group, located either at the 6 position of GlcNAc or the 3 or 4 positions of Gal. This latter sulfation is novel and has only been characterized in the species R. arvalis. This structural analysis led to the establishment of several novel carbohydrate structures, demonstrating the structural diversity and species-specificity of amphibian glycoconjugates.

  16. Ecologically Valid Carbohydrate Intake d