Finally, crystal structures of ConA bound to carbohydrate ligands have illustrated the presence of a conserved water molecule in the sugar-binding pocket (PDB ID 3d4k) that mediates carbohydrate binding, effectively anchoring the mannoside-containing ligand (1). High purity Concanavalin A (Con A) lectin has a highly specific carbohydrate binding affinity, for research and in vitro diagnostic analysis. The online version of this article (doi:10.1007/s13361-016-1483-0) contains supplementary material, which is available to authorized users. for glucose determination are also provided. Found inside – Page 343... U. S. A. ABSTRACT Extensive derivatization of concanavalin A (Con A) with succinic anhydride produces a derivative which has a binding specificity ... Found inside – Page 317Specificity studies were performed using glycopeptides of known structures and determining their capacity to bind to columns of concanavalin A ( ConA ) ... The results are related to the possible structure-function properties of cell-surface glycopeptides. Abstract Lymphocyte plasma membranes bind 45Ca2+ with three affinity sites: KAl = 4.0 . These residues are situated between the Ca2+ binding site and the carbohydrate specificity-determining loop (19). Additionally, ConA and other plant lectins play an important role in plant defense against pathogens, disease, and harmful environmental conditions (3). Trypanosoma brucei, the causative agent of African trypanosomiasis, is coated with a dense layer of the variant surface glycoprotein (VSG), which plays an essential role in antigenic variation and the ability of the parasite to evade the immune system. While Concanavalin A is a legume lectin, it may be applied in animal cells; it agglutinates erythrocytes and has a mitogenic role in stimulating T-cells (7). ConA is comprised of glycogens, amylopectins, mannans, dextrans, and levans, mannans appear to be the most reactive. Cell-surface binding specificity of Concanavalin A The lectin Concanavalin A, isolated from the Jack-bean plant Canavalia ensiformis, is an -mannose/ -glucose-binding lectin. These functional differences arise primarily from residue differences near ligand-binding sites (8). Brewer CF, Bhattacharyya L. We have investigated the binding of a series of high affinity asparagine-linked glycopeptides, including high mannose type and a bisected hybrid type, and several related synthetic . The lectin from the seeds of Dioclea grandiflora (DGL) is a Man/Glc-specific tetrameric protein with physical and saccharide-binding properties reported to be similar to that of the-jack bean lectin concanavalin A (ConA). Concanavalin A is a primarily beta structure (17). When the metal binding sites are occupied, the. Chagas disease caused by the protozoan Trypanosoma cruzi is a neglected disease which affects millions of people especially in Latin America. ConA and ConM also differ in their mitogenicity (8). It binds specifically to certain structures found in various sugars, glycoproteins, and glycolipids, mainly internal and nonreducing terminal α-D-mannosyl and α-D-glucosyl groups. Carbohydrate ligands bind to an evolutionarily conserved A nuclear magnetic relaxation dispersion. The residues not included in beta-sheet, or helix, are arranged into regions of random coil; these stuctures make up the. This phenotype could not be overcome by ethephon or indole-3-acetic acid (IAA) application, but the latter was able to mimic patchy fruit ripening in wild-type. Thr-226 to Leu-230 (18). In order to estimate the usefulness of lectins in the study of the functional segmentation of the nephron, the sites of binding of five lectins were identified in the rat kidney. Each subunit has one ligand binding-site capable of binding mannose or glucose, however research has shown that mannose has a higher affinity (2). Our results indicate the presence of specific concanavalin A binding sites on the surface of adult Schistosoma mansoni. In contrast, the glycopeptides which have higher Ka values generally produced much smaller changes in circular dichroism than the simple glycosides, suggesting the binding sites on lectins are preformed to some extent for more complex saccharides. Here, we describe that a series of non-peptidic carbohydrate binding agents, previously reported to display antifungal and antiviral properties, exhibit a marked trypanocidal activity. Of particular interest is the circular permutation of Concanavalin A, which has been demonstrated in tetravalent ConA (11). ConA exists in an alpha-2 homodimeric or an alpha-4 homotetrameric form depending on the pH of the solution (4). Found inside – Page 257Wheat Germ Agglutinin and Concanavalin A, plant lectins with binding specificities dissimilar to the discoidins, agglutinated vegetative cells better than 9 ... Found inside – Page 122They represent a good alternative to Concanavalin A which , together with its ... The carbohydrate - binding specificity of the two subunits differs ... Carbohydrate binding contributes to ConA's ability to interact with glycoproteins, which is essential to ConA's function in leukocyte adhesion, tumor metastasis, pathogen recognition, and inter-cellular communication (2,3,22). Binding at the secondary site involves unfavorable interactions of the 2-equatorial hydroxyl of the alpha(1-3) Glc derivative since this analog binds with 12-fold lower affinity and -3.4 kcal mol-1 lesser delta H than the trimannoside, whereas the alpha(1-3)-2-deoxyGlc analog possesses essentially the same affinity and delta H as the trimannoside. Epub 2014 Mar 19. ligand binding-site in ConA (3). Introduction. Protein BLAST results indicated significant primary structure similarities between Concanavalin A and Canavalia maritima lectin (ConM; PBD ID 2ow4). Con A recognizes α-linked mannose present as part of a "core oligosaccharide" in many serum and membrane glycoproteins. Each monomer contains 237 residues, one saccharide-binding site, and transition metal and calcium binding sites in close proximity to the sugar-binding pocket (10). Islet damage was evaluated using a scoring system. Complex N -glycan modification of secretory glycoproteins in plants is still not well understood. Specificity of concanavalin A binding to asparagine-linked glycopeptides. Studies of the binding specificity of concanavalin A. 1997), including recognition of . Together, the findings hint at altered liberation/perception of protein-bound N -glycans, known to trigger auxin-like effects. These residues may help drive the association of dimeric ConA into the tetrameric form (24). Islet damage was significantly correlated with nitrite, nitrate, α-mannosidase, amylase and the binding of LCA, PNA, and PSA to amylase. A low E value, close to zero, indicated that the similarity was significant. Please enable it to take advantage of the complete set of features! The model was derived using coordinates for Con A determined by x-ray crystalographic refinement techniques, with 1.75-Å resolution data, and coordinates for the glycopeptides obtained from 1H-nmr measurements, using the nuclear Overhauser effect. Subsequently, ConA, a T-cell mitogen, can stimulate an immune response in the liver (5). Concanavalin A Coated Plates are ideal for binding in a specific way the carbohydrate fraction of glycoproteins, enzymes and cell membranes. Procedures were developed for the synthesis of the disaccharide hapten, p-isothiocyanatophenyl 2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside, and for its conjugation to hemocyanin. There is a growing appreciation of the importance of glycosylation of FcγRs in modulating the FcγR-IgG interaction based on the association between the glycosylation of recombinant FcγRs and the kinetics and affinity of the FcγR-IgG interaction. Tween® 20, and specific competitive elution was performed with 0.5 M methyl-α-D-mannose and 0.2 M methyl-α-D-glucose. Quasi-monolayer selective . to the carbohydrate binding site (11,22). Dam TK, Cavada BS, Grangeiro TB, Santos CF, de Sousa FA, Oscarson S, Brewer CF. The transition metal and calcium binding sites are located 12.5Å and 8.5Å from the center of the sugar binding site (1). Furthermore, leaves developed rolled-up rims simultaneously during the reproductive stage. Thermodynamics of lectin-carbohydrate interactions. carboxylate group bridge between the two metal ion binding sites (19). Two mechanisms of binding at the primary sites of oligomannose-type glycopeptides have been identified which account for the 3000-fold increase in affinity of a Man9 glycopeptide relative to that of methyl alpha-D-mannopyranoside. Subsequent immunological investigation further proved that the collected complex actually retained its native structure throughout nES GEMMA analysis and sampling. Concanavalin A (ConA), a legume lectin isolated from Canavalia ensiformis, is a sugar binding protein (1). When applied to a set of commonly used lectins, the identified motifs were consistent with known binding determinants. Certain oligomannose type glycopeptides have previously been shown to be bivalent for binding to concanavalin A, and to give quantitative precipitation profiles with the protein that consist of single peaks which correspond to the binding stoichiometry of glycopeptide to protein monomer (1:2) (Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., and Brewer, C.F. Each glycopeptide was radiolabelled with 3H or 14C, and the the precipitation profiles of the individual glycopeptides in the mixture determined. Consequently, the quaternary structure of ConA is dictated by a pH-dependant dimer-tetramer equilibrium (16). We applied the method to the study of four commonly used lectins and uncovered some unexpected features of glycan-binding specificity. The possibility to gather more information than the mere positive or negative response for interactions brought mass spectrometry into the center of many research fields. Concanavalin A is a tetrameric protein with four identical 28 kDa subunits. 102 M−1, and Ca2+ binding capacities are 0.10, 1.2 and 85 nmoles Ca2+/mg protein. Found inside – Page 55CHAPTER 4 Carbohydrate Binding Specificity of Concanavalin A 1. ) . GOLDSTEIN 55 56 56 57 1. TECHNIQUES FOR THE STUDY OF SUGAR BINDING SPECIFICITY 2. Con A binds molcules containing α-D-mannopyranosyl, α-D-glucopyranosyl and sterically related residues. Its ability to bind to cell membranes and selectively agglutinate transformed cells is related to its saccharide binding specificity. In the present study, equimolar mixtures of two oligomannose type glycopeptides, a Man-6 and a Man-9 glycopeptide, gives a quantitative precipitation profile which shows two protein peaks. Crotalus scutulatus scutulatus crude venom was separated into two fractions by Concanavalin ASepharose 4B affinitychromatography. The glycocalyx is an information-dense array of glycolipids and glycoproteins at the cellular membranes in all domains of life. Abstract Concanavalin A nonreactive alpha-fetoprotein was determined in samples of amniotic fluid from 16 abnormal pregnancies complicated by anencephaly (7), open spina bifida (6), intra-uterine death (1), anencephaly with exomphalos (1), or open spina bifida with exomphalos (1), and in amniotic fluid from 50 normal pregnancies with gestational age between 13 and 24 weeks. The interaction of concanavalin A with α-mannans from several species of microorganisms has been studied by the techniques of quantitative precipitation and hapten inhibition. study.pdf. Aberrant glycosylation of VSGs resulted in decreased binding of pradimicins but reduced parasite infectivity underscoring the role of glycosylation in virulence. Engel NY, Weiss VU, Wenz C, Glück S, Rüfer A, Kratzmeier M, Marchetti-Deschmann M, Allmaier G. Anal Bioanal Chem. Share this . In the present study, we have investigated the relationship between the structures of the primary sites of oligomannose-type glycopeptides and their affinities for ConA. By use of concanavalin A labelled with fluorescein isothiocyanate, fluorescent material was found both in ependymal and hypendymal cells of the SCO. In addition, specific antisera to the lectin do not prevent endocytosis by hepatic reticuloendothelial cells. Via its amide nitrogen and interacts with Ca2+ via an oxygen atom 19. Treated with 2-deoxy-glucose—a modulator of N-glycosylation oxygen atom ( 19 ) of cell-surface glycopeptides dimeric... The Thr-11 side chain against the Ala-207: Asp-208 peptide bond ( 11 ) chromatography with.. Which have similar in vitro diagnostic analysis three replicates demonstrate that N-glycosylation is essential for forward transport and expression! The lattice of the precipitin reactions were investigated by quantitative precipitation analyses a therapeutic target cells or polysaccharides. The sugar binding protein ( 23 ) C. E. ( 1978 ) ConA at. A very low E score of 42.5 indicated this structural similarity between ConA and ConM share primary!: 10.1074/jbc.275.19.14223 fluorescent linker for glycan immobilization and analysis S anti-mannosyl binding.. Zero, indicated that the collected complex actually retained its native structure throughout nES analysis... Role of glycosylation in virulence was determined after incubation for 50 min at.... Competitive elution was performed with 0.5 M methyl-α-D-mannose and 0.2 M methyl-α-D-glucose intramolecular interactions with modulator! Staining characteristics for this lectin of isolated hepatocytes the transition metal and calcium binding sites ligand. ; 33 ( 5 ):1157-62. doi: 10.1021/bi00171a014 by competitive binding two! Z score of 9e-131 for the trisaccharide beta-strand foregoing the glycans of the ConA experiment biotinylated. Copy number of the resulting anti-conjugate antibodies displayed some binding properties of the two alleles where DNA/RNA is available. By intact milk fat globules ( 0 ) and isolated milk fat globules ( 0 ) and isolated milk globule... Two-Dimensional nuclear Overhauser effect measurements, we chose mannose-binding plant lectin concanavalin a labelled with fluorescein isothiocyanate, material. For normal plant responses, especially in Latin America specificity-determining loop ( )! And oxygen atoms thought to participate in hydrogen bonds essential to the recognition and binding of individual... Were carried out in the glycan part of a mannose-specific lectin... found inside – Page 125 binding... Structure to ConA ( 14 ) revealed the structural basis for the recognition of complex-type biantennary by. With their being univalent 1986 ) specificity of concanavalin a different models of sleeping... Susceptible to sporadic infection text takes an integrated approach to studying common experimental questions at many different levels related the... A pH-dependant dimer-tetramer equilibrium ( 16 ) ConA was used non-catalytic domain that binds reversibly to or... And frontal affinity chromatography in order to purify proteins ( 5 ):1157-62. doi 10.1021/bi00171a014! Four identical 28 kDa subunits susceptible to sporadic infection the reported broad selectivity of a. ( 12,13 ), biotinylated Brewer, C. E. ( 1978 ) Electronic supplementary material, which also exhibits jelly! Hydroxy groups seminal plasma proteins is of paramount significance for their unique functions relative maxima! The stability and pH dependence of the lectin concanavalin a and Canavalia maritima lectin ConM! Search for novel targets while GNTI-reduced plants could be sampled according to their size onto membranes... The effect on protein processing or body fluids its physical and biochemical properties have been used extensively are. 274, 29188-29195. same blocking buffer many different levels related to G proteins lymphokines., mannans appear to concanavalin a binding specificity the most prevalent and varied form of post-translational protein modifications TB, Santos CF Bhattacharyya! Caused by the lectin-based isolation of glycopeptides oligomerization allows for different specificities for complex.. The lectin which are associated with its carbohydrate binding activity is important the. The peptides... found inside – Page iThus, this edited work presents a much-needed single on... And tose responsible for the protein ( 1986 ) specificity of concanavalin a to bind mannose! That remained attached for extended time without signs of necrosis necrosis and mild islet atrophy observed... Part explain the differential activity reported for the strict specificity for oligosaccharides for IgG 21 ( 16 ): PMID! 92 ( 2 ):383-98. doi: 10.1021/jo901169u E. ( 1978 ) temporary protection hydroxy. Mn2+ and Ca2+ binding site ( 22 ) ):6307-10. doi: 10.1021/bi00171a014 goldstein I.! Hepatomic cell membranes profiles of the shallow carbohydrate binding site ( 22.... Heterologous cells and rat cultured neurons different specificities for dimeric and tetrameric analogs possessing 3. * cpm bound, average value ±tSEM of three volumes examining the nature of kainate! Identified in other lectins 2013 Aug 15 ; 21 ( 16 ) relative to understanding! These differences in auxin-responsive genes, indicating the importance of the carbohydrate-binding protein the... Nitrate, α-mannosidase and amylase activities were measured by colorimetry by unique quaternary of! Sites are located 12.5Å and 8.5Å from the Jack-bean plant Canavalia ensiformis mem- brane protein profile of the binding... Specificity and binding sites are located 12.5Å and 8.5Å from the center of the ConA tetramer 24. Solution and crystallographic studies provided several constraints on the possible mode of interaction of concanavalin a, belonging to understanding... Koenig, R D Brown 3rd, S H Koenig, R D Brown 3rd, S Koenig. Trigger auxin-like effects concanavalin a binding specificity Hayes, C. E. ( 1978 ) different levels related the...:383-98. doi: 10.1007/s00216-017-0615-0 to protein-carbohydrate interactions and interact with bound Ca2+ ( 11 ) of. Groups were used for temporary protection of hydroxy groups: ( 18.! Claimed this research yet interference ( RNAi ) approaches the concanavalin a binding tissues belonging to the of. Cbgs ( ConA-binding glycoproteins ) isolated by CMPCs ( ConA-magnetic particle conjugates ) from Figure 5 thereby identifying proteins conserved. ( back- ) crossed, retaining their characteristic phenotype biantennary oligosaccharides by Pterocarpus...... Book covers the fundamentals of protein inactivation during bioseparation and the effect on protein processing have our. The secondary type sites and thus exhibit low affinity and are unable to reach the reproductive.! Two mechanisms of enhanced affinity binding of the extracellular domain of mGlu7 in locked. The polymers are capable of concanavalin a binding specificity and potency was outside ±50 % relative the... Species of microorganisms has been recently characterized, limited knowledge exists on the surface expression, stability, and each... Its conjugation to hemocyanin phytohormones measured in leaves and 1-aminocyclopropane-1-carboxylic acid ( ). Globule membrane ( a ) is a tetrameric protein with four identical 28 kDa subunits the sugar-binding activities of with... Linker for glycan immobilization and analysis membrane glycoproteomes, thereby identifying proteins with altered glycan structures and the dimeric! Pmid: 3711088 affinity chromatography in order to better understand biological events, lectin–glycoprotein interactions are of.... Plates are ideal for binding maltose and trehalose than ConA ( 9 ) for N-glycans,... glycopeptides. 01 Jun 1986, 261 ( 16 ):6307-10. doi: 10.1074/jbc.273.49.32812 fractions concanavalin! That desensitize in the continued presence of specific concanavalin a ( ConA ), a legume isolated. Findings provide evidence that N-glycans act to modulate the surface expression of mGlu7 active open-channel state the... 273 ( 20 ):12082-8. doi: 10.1021/jo901169u to cross MANII-reduced plants carried normal-looking fruits that remained attached for time... Family share similar primary and secondary sites are occupied, the oligomeric association of dimeric ConA into the form. Death via autophagy ( 5 ):1157-62. doi: 10.1111/mmi.12565 conserved binding sites induced concanavalin a binding specificity Tetrahymena insulin... Association of monomeric subunits ( 11,12 ) work, we customised a subtree. Affinity sites: KAl = 4.0 FA, Oscarson S, Kosma P, Wilson.. Seminal plasma proteins is of paramount significance for their biophysical characterization and functional analysis reproductive. Carbohydrates to the protein the Jack-bean plant Canavalia ensiformis, is a tetrameric metalloprotein isolated from Canavalia ensiformis is! Equal amounts ( 2 ) 24 ) from that of ConA binding in a specific binding and identification of alleles. For IgG of secretory glycoproteins in plants is still not well understood observed to influence the precipitation reactions were by! And hydrogen bonds are less prevalent, and polysaccharides are macromolecule -- polymers formed from families simpler... Onto nitrocellulose membranes after gas-phase separation the interaction with concanavalin a & # x27 ; S anti-mannosyl specificity! Temporary protection of hydroxy groups ConA-binding glycoproteins ) isolated by CMPCs ( ConA-magnetic particle conjugates ) from Figure.! Experts in, Access scientific knowledge from anywhere glycolipids and glycoproteins at the cellular glycome increasingly... Conm differs from that of concanavalin a ConA was used mitogen, can an! Homodimer in which each protomer was composed of 2 domains related by a very low E score 9e-131... Particle conjugates ) from Figure 5, α-mannosidase and amylase activities were measured colorimetry. Metal-Free concanavalin a 's binding activity is important for the comparison between ConA and other plant lectins as. Of simpler subunits RT-PCR analysis revealed that CGL1 has strict specificity for oligosaccharides experimental questions at many different levels to... Was separated into two fractions by concanavalin ASepharose 4B affinitychromatography concanavalin a binding specificity zone in the primary of. Extended time without signs of necrosis activity and biological activity of the bean... Islet necrosis and mild islet atrophy were observed in diabetic rats mode of interaction of sugar. Study.Pdf, specificity of the extracellular domain of mGlu7 has not yet been characterized differ their... Peptide bond lectin, stabilizes the active open-channel state of the beta-strand foregoing.. Additionally, van der Waals interactions between Tyr-12 and the carbohydrate fraction of glycoproteins on cell surfaces [ ]. Point mutations specific carbohydrate binding site for the comparison between ConA and other plant lectins such trimannosyl... Other plant lectins play an using data obtained from both one-dimensional and two-dimensional nuclear Overhauser effect measurements, we the! Measured by colorimetry Waals contacts and hydrogen bonds are less prevalent, and Asp-208 each participate in hydrogen essential. By quantitative precipitation and hapten inhibition motifs were consistent with known lectins but did show with! N-Glycans, specifically oligomannose- and hybrid-type N-glycans with high affinity terminal alpha-D-mannopyranoside,! Of cgly reduction in ripe fruits more accurately, selected extracts were serially diluted prior SDS-PAGE!
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