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Stephen G. Withers - One of the best experts on this subject based on the ideXlab platform.
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characterization of a β n acetylhexosaminidase and a β n acetylglucosaminidase β glucosidase from cellulomonas fimi
FEBS Journal, 2006Co-Authors: Dominik Stoll, Karen Rupitz, R A J Warren, Christoph Mayer, David J Vocadlo, Melanie Mah, Stephen G. WithersAbstract:The Gram-positive soil bacterium Cellulomonas fimi is shown to produce at least two intracellular β-N-acetylglucosaminidases, a family 20 β-N-acetylhexosaminidase (Hex20), and a novel family 3-β-N-acetylglucosaminidase/β-glucosidase (Nag3), through screening of a genomic expression library, cloning of genes and analysis of their sequences. Nag3 exhibits broad substrate specificity for substituents at the C2 position of the glycone: kcat/Km values at 25 °C were 0.066 s−1·mm−1 and 0.076 s−1·mm−1 for 4′-nitrophenyl β-N-acetyl-d-glucosaminide and 4′-nitrophenyl β-d-glucoside, respectively. The first glycosidase with this broad specificity to be described, Nag3, suggests an interesting evolutionary link between β-N-acetylglucosaminidases and β-glucosidases of family 3. Reaction by a double-displacement mechanism was confirmed for Nag3 through the identification of a glycosyl–enzyme species trapped with the slow substrate 2′,4′-dinitrophenyl 2-deoxy-2-fluoro-β-d-glucopyranoside. Hex20 requires the Acetamido Group at C2 of the substrate, being unable to cleave β-glucosides, since its mechanism involves an oxazolinium ion intermediate. However, it is broad in its specificity for the d-glucosyl/d-galactosyl configuration of the glycone: Km and kcat values were 53 µm and 482.3 s−1 for 4′-nitrophenyl β-N-acetyl-d-glucosaminide and 66 µm and 129.1 s−1 for 4′-nitrophenyl β-N-acetyl-d-galactosaminide.
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subsite structure of the endo type chitin deacetylase from a deuteromycete colletotrichum lindemuthianum an investigation using steady state kinetic analysis and ms
Biochemical Journal, 2003Co-Authors: Omid Hekmat, Ken Tokuyasu, Stephen G. WithersAbstract:The endo-type chitin deacetylase (EC 3.5.1.41) from a Deuteromycete, Colletotrichum lindemuthianum (ATCC 56676), catalyses the hydrolysis of the Acetamido Group of GlcNAc (2-Acetamido-2-deoxy-d-glucose) residues in chitin or chito-oligosaccharides with a degree of polymerization ( n ) equal to or greater than 2. The steady-state kinetic parameters for the initial deacetylation reactions of (GlcNAc) 2–6 were determined using a direct, continuous spectrophotometric assay in combination with ESI-MS (electrospray ionization MS) analysis of the products. The dependence of the observed K m and k cat / K m on n suggests the presence of four enzyme subsites (−2, −1, 0 and +1) that interact with GlcNAc residues from the non-reducing end to the reducing end of the substrate. The turnover number ( k cat , 7 s −1 ) is independent of n and represents the intrinsic rate constant ( k int ) for the hydrolysis of the Acetamido Group in subsite 0. The subsite affinities for the GlcNAc residues were calculated from the observed k cat / K m values ( A −2 , −11.0; A −1 , −1.5; A 0 , −7.7; A +1 , −12.5 kJ·mol −1 ). The increments in the subsite affinities due to the recognition of the Acetamido Groups were calculated [ΔΔ G (N-acetyl) =3.3, 0, 4.0 and 0 kJ·mol −1 for subsites −2, −1, 0 and +1 respectively]. The steady-state kinetic parameters for the second deacetylation reaction of (GlcNAc) 4 were also determined using (GlcNAcGlcNAcGlcNGlcNAc) as the substrate. The comparison of the experimental and theoretical values (calculated using the subsite affinities) suggests that the mono-deacetylated substrate binds strongly in a non-productive mode occupying all four subsites, thereby inhibiting the second deacetylation reaction.
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subsite structure of the endo type chitin deacetylase from a deuteromycete colletotrichum lindemuthianum an investigation using steady state kinetic analysis and ms
Biochemical Journal, 2003Co-Authors: Omid Hekmat, Ken Tokuyasu, Stephen G. WithersAbstract:The endo-type chitin deacetylase (EC 3.5.1.41) from a deuteromycete, Colletotrichum lindemuthianum (ATCC 56676), catalyses the hydrolysis of the Acetamido Group of GlcNAc (2-Acetamido-2-deoxy-D-glucose) residues in chitin or chito-oligosaccharides with a degree of polymerization (n) equal to or greater than 2. The steady-state kinetic parameters for the initial deacetylation reactions of (GlcNAc)(2-6) were determined using a direct, continuous spectrophotometric assay in combination with ESI-MS (electrospray ionization MS) analysis of the products. The dependence of the observed K(m) and k(cat)/K(m) on n suggests the presence of four enzyme subsites (-2, -1, 0 and +1) that interact with GlcNAc residues from the non-reducing end to the reducing end of the substrate. The turnover number (k (cat), 7 s(-1)) is independent of n and represents the intrinsic rate constant (k(int)) for the hydrolysis of the Acetamido Group in subsite 0. The subsite affinities for the GlcNAc residues were calculated from the observed k(cat)/K(m) values (A (-2), -11.0; A (-1), -1.5; A (0), -7.7; A (+1), -12.5 kJ x mol(-1)). The increments in the subsite affinities due to the recognition of the Acetamido Groups were calculated [DeltaDelta G ((N-acetyl))=3.3, 0, 4.0 and 0 kJ x mol(-1) for subsites -2, -1, 0 and +1 respectively]. The steady-state kinetic parameters for the second deacetylation reaction of (GlcNAc)(4) were also determined using (GlcNAcGlcNAcGlcNGlcNAc) as the substrate. The comparison of the experimental and theoretical values (calculated using the subsite affinities) suggests that the mono-deacetylated substrate binds strongly in a non-productive mode occupying all four subsites, thereby inhibiting the second deacetylation reaction.
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aspartate 313 in the streptomyces plicatus hexosaminidase plays a critical role in substrate assisted catalysis by orienting the 2 Acetamido Group and stabilizing the transition state
Journal of Biological Chemistry, 2002Co-Authors: Spencer J Williams, David J Vocadlo, Brian L Mark, Michael N G James, Stephen G. WithersAbstract:SpHex, a retaining family 20 glycosidase from Streptomyces plicatus, catalyzes the hydrolysis of N-acetyl-β-hexosaminides. Accumulating evidence suggests that the hydrolytic mechanism involves substrate-assisted catalysis wherein the 2-Acetamido substituent acts as a nucleophile to form an oxazolinium ion intermediate. The role of a conserved aspartate residue (D313) in the active site ofSpHex was investigated through kinetic and structural analyses of two variant enzymes, D313A and D313N. Three-dimensional structures of the wild-type and variant enzymes in product complexes with N-acetyl-d-glucosamine revealed substantial differences. In the D313A variant the 2-Acetamido Group was found in two conformations of which only one is able to aid in catalysis through anchimeric assistance. The mutation D313N results in a steric clash in the active site between Asn-313 and the 2-Acetamido Group preventing the 2-Acetamido Group from providing anchimeric assistance, consistent with the large reduction in catalytic efficiency and the insensitivity of this variant to chemical rescue. By comparison, the D313A mutation results in a shift in a shift in the pH optimum and a modest decrease in activity that can be rescued by using azide as an exogenous nucleophile. These structural and kinetic data provide evidence that Asp-313 stabilizes the transition states flanking the oxazoline intermediate and also assists to correctly orient the 2-Acetamido Group for catalysis. Based on analogous conserved residues in the family 18 chitinases and family 56 hyaluronidases, the roles played by the Asp-313 residue is likely general for all hexosaminidases using a mechanism involving substrate-assisted catalysis.
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aspartate 313 in the streptomyces plicatus hexosaminidase plays a critical role in substrate assisted catalysis by orienting the 2 Acetamido Group and stabilizing the transition state
Journal of Biological Chemistry, 2002Co-Authors: Spencer J Williams, David J Vocadlo, Brian L Mark, Michael N G James, Stephen G. WithersAbstract:Next Section Abstract SpHex, a retaining family 20 glycosidase from Streptomyces plicatus, catalyzes the hydrolysis of N-acetyl-β-hexosaminides. Accumulating evidence suggests that the hydrolytic mechanism involves substrate-assisted catalysis wherein the 2-Acetamido substituent acts as a nucleophile to form an oxazolinium ion intermediate. The role of a conserved aspartate residue (D313) in the active site ofSpHex was investigated through kinetic and structural analyses of two variant enzymes, D313A and D313N. Three-dimensional structures of the wild-type and variant enzymes in product complexes with N-acetyl-d-glucosamine revealed substantial differences. In the D313A variant the 2-Acetamido Group was found in two conformations of which only one is able to aid in catalysis through anchimeric assistance. The mutation D313N results in a steric clash in the active site between Asn-313 and the 2-Acetamido Group preventing the 2-Acetamido Group from providing anchimeric assistance, consistent with the large reduction in catalytic efficiency and the insensitivity of this variant to chemical rescue. By comparison, the D313A mutation results in a shift in a shift in the pH optimum and a modest decrease in activity that can be rescued by using azide as an exogenous nucleophile. These structural and kinetic data provide evidence that Asp-313 stabilizes the transition states flanking the oxazoline intermediate and also assists to correctly orient the 2-Acetamido Group for catalysis. Based on analogous conserved residues in the family 18 chitinases and family 56 hyaluronidases, the roles played by the Asp-313 residue is likely general for all hexosaminidases using a mechanism involving substrate-assisted catalysis.
Ronald L. Schnaar - One of the best experts on this subject based on the ideXlab platform.
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binding specificities of the sialoadhesin family of i type lectins sialic acid linkage and substructure requirements for binding of myelin associated glycoprotein schwann cell myelin protein and sialoadhesin
Journal of Biological Chemistry, 1997Co-Authors: Brian E Collins, Makoto Kiso, Akira Hasegawa, Michael B Tropak, John C Roder, Paul R Crocker, Ronald L. SchnaarAbstract:Abstract The carbohydrate binding specificities of three sialoadhesins, a subGroup of I-type lectins (immunoglobulin superfamily lectins), were compared by measuring lectin-transfected COS cell adhesion to natural and synthetic gangliosides. The neural sialoadhesins, myelin-associated glycoprotein (MAG) and Schwann cell myelin protein (SMP), had similar and stringent binding specificities. Each required an α2,3-linked sialic acid on the terminal galactose of a neutral saccharide core, and they shared the following rank-order potency of binding: GQ1bα ≫ GD1a = GT1b ≫ GM3 = GM4 ≫ GM1, GD1b, GD3, GQ1b(nonbinders). In contrast, sialoadhesin had less exacting specificity, binding to gangliosides that bear either terminal α2,3- or α2,8-linked sialic acids with the following rank-order potency of binding: GQ1bα > GD1a = GD1b = GT1b = GM3 = GM4 > GD3= GQ1b ≫ GM1 (nonbinder). CD22 did not bind to any ganglioside tested. Binding of MAG, SMP, and sialoadhesin was abrogated by chemical modification of either the sialic acid carboxylic acid Group or glycerol side chain on a target ganglioside. Synthetic ganglioside GM3 derivatives further distinguished lectin binding specificities. Deoxy and/or methoxy derivatives of the 4-, 7-, 8-, or 9-position of sialic acid attenuated or eliminated binding of MAG, as did replacement of the sialic acid Acetamido Group with a hydroxyl. In contrast, the 4- and 7-deoxysialic acid derivatives supported sialoadhesin binding at near control levels (the other derivatives did not support binding). These data are consistent with sialoadhesin binding to one face of the sialic acid moiety, whereas MAG (and SMP) may have more complex binding sites or may bind sialic acids only in the context of more restricted oligosaccharide conformations.
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binding specificities of the sialoadhesin family of i type lectins sialic acid linkage and substructure requirements for binding of myelin associated glycoprotein schwann cell myelin protein and sialoadhesin
Journal of Biological Chemistry, 1997Co-Authors: Brian E Collins, Makoto Kiso, Akira Hasegawa, Michael B Tropak, John C Roder, Paul R Crocker, Ronald L. SchnaarAbstract:The carbohydrate binding specificities of three sialoadhesins, a subGroup of I-type lectins (immunoglobulin superfamily lectins), were compared by measuring lectin-transfected COS cell adhesion to natural and synthetic gangliosides. The neural sialoadhesins, myelin-associated glycoprotein (MAG) and Schwann cell myelin protein (SMP), had similar and stringent binding specificities. Each required an α2,3-linked sialic acid on the terminal galactose of a neutral saccharide core, and they shared the following rank-order potency of binding: GQ1bα ≫ GD1a = GT1b ≫ GM3 = GM4 ≫ GM1, GD1b, GD3, GQ1b(nonbinders). In contrast, sialoadhesin had less exacting specificity, binding to gangliosides that bear either terminal α2,3- or α2,8-linked sialic acids with the following rank-order potency of binding: GQ1bα > GD1a = GD1b = GT1b = GM3 = GM4 > GD3= GQ1b ≫ GM1 (nonbinder). CD22 did not bind to any ganglioside tested. Binding of MAG, SMP, and sialoadhesin was abrogated by chemical modification of either the sialic acid carboxylic acid Group or glycerol side chain on a target ganglioside. Synthetic ganglioside GM3 derivatives further distinguished lectin binding specificities. Deoxy and/or methoxy derivatives of the 4-, 7-, 8-, or 9-position of sialic acid attenuated or eliminated binding of MAG, as did replacement of the sialic acid Acetamido Group with a hydroxyl. In contrast, the 4- and 7-deoxysialic acid derivatives supported sialoadhesin binding at near control levels (the other derivatives did not support binding). These data are consistent with sialoadhesin binding to one face of the sialic acid moiety, whereas MAG (and SMP) may have more complex binding sites or may bind sialic acids only in the context of more restricted oligosaccharide conformations.
Tomomitsu Hatakeyama - One of the best experts on this subject based on the ideXlab platform.
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novel carbohydrate recognition mode of the invertebrate c type lectin spl 1 from saxidomus purpuratus revealed by the glcnac complex crystal in the presence of ca2
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2020Co-Authors: Hideaki Unno, Shuhei Higuchi, Shuichiro Goda, Tomomitsu HatakeyamaAbstract:The C-type lectins SPL-1 and SPL-2 from the bivalve Saxidomus purpuratus are composed of A and B chains and of two B chains, respectively. They bind specific carbohydrates containing Acetamido Groups, such as N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc), in a Ca2+-independent manner. Unlike ordinary C-type lectins, which require Ca2+ ions for carbohydrate recognition, these lectins recognize specific carbohydrates mainly through interactions with the Acetamido Group without Ca2+ ions, even though Ca2+ enhances the binding affinity of these lectins, especially SPL-1. In the present study, the crystal structure of the SPL-1-GlcNAc complex in the presence of Ca2+ revealed that the binding of SPL-1 to GlcNAc is stabilized by hydrogen bonds to the water molecule(s) coordinating Ca2+, whereas in ordinary C-type lectins Ca2+ directly forms coordinate bonds to the hydroxy Groups of carbohydrates. These differences may also allow SPL-1 and SPL-2 to recognize both GlcNAc and GalNAc, which have different orientations of the 4-hydroxy Group.
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novel ca2 independent carbohydrate recognition of the c type lectins spl 1 and spl 2 from the bivalve saxidomus purpuratus
Protein Science, 2019Co-Authors: Hideaki Unno, Shuhei Higuchi, Shuichiro Goda, Shuhei Itakura, Kenichi Yamaguchi, Tomomitsu HatakeyamaAbstract:: Novel Ca2+ -independent C-type lectins, SPL-1 and SPL-2, were purified from the bivalve Saxidomus purpuratus. They are composed of dimers with either identical (SPL-2 composed of two B-chains) or distinct (SPL-1 composed of A- and B-chains) polypeptide chains, and show affinity for N-acetylglucosamine (GlcNAc)- and N-acetylgalactosamine (GalNAc)-containing carbohydrates, but not for glucose or galactose. A database search for sequence similarity suggested that they belong to the C-type lectin family. X-ray crystallographic analysis revealed definite structural similarities between their subunits and the carbohydrate-recognition domain (CRD) of the C-type lectin family. Nevertheless, these lectins (especially SPL-2) showed Ca2+ -independent binding affinity for GlcNAc and GalNAc. The crystal structure of SPL-2/GalNAc complex revealed that bound GalNAc was mainly recognized via its Acetamido Group through stacking interactions with Tyr and His residues and hydrogen bonds with Asp and Asn residues, while widely known carbohydrate-recognition motifs among the C-type CRD (the QPD [Gln-Pro-Asp] and EPN [Glu-Pro-Asn] sequences) are not involved in the binding of the carbohydrate. Carbohydrate-binding specificities of individual A- and B-chains were examined by glycan array analysis using recombinant lectins produced from Escherichia coli cells, where both subunits preferably bound oligosaccharides having terminal GlcNAc or GalNAc with α-glycosidic linkages with slightly different specificities.
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characteristic recognition of n acetylgalactosamine by an invertebrate c type lectin cel i revealed by x ray crystallographic analysis
Journal of Biological Chemistry, 2004Co-Authors: Hajime Sugawara, Haruhiko Aoyagi, Masami Kusunoki, Genji Kurisu, Tokiko Fujimoto, Tomomitsu HatakeyamaAbstract:Abstract CEL-I is a C-type lectin, purified from the sea cucumber Cucumaria echinata, that shows a high specificity for N-acetylgalactosamine (GalNAc). We determined the crystal structures of CEL-I and its complex with GalNAc at 2.0 and 1.7 A resolution, respectively. CEL-I forms a disulfide-linked homodimer and contains two intramolecular disulfide bonds, although it lacks one intramolecular disulfide bond that is widely conserved among various C-type carbohydrate recognition domains (CRDs). Although the sequence similarity of CEL-I with other C-type CRDs is low, the overall folding of CEL-I was quite similar to those of other C-type CRDs. The structure of the complex with GalNAc revealed that the basic recognition mode of GalNAc was very similar to that for the GalNAc-binding mutant of the mannose-binding protein. However, the Acetamido Group of GalNAc appeared to be recognized more strongly by the combination of hydrogen bonds to Arg115 and van der Waals interaction with Gln70. Mutational analyses, in which Gln70 and/or Arg115 were replaced by alanine, confirmed that these residues contributed to GalNAc recognition in a cooperative manner.
Makoto Kiso - One of the best experts on this subject based on the ideXlab platform.
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total synthesis of a cholinergic neuron specific ganglioside gt1a alpha a high affinity ligand for myelin associated glycoprotein mag
Glycoconjugate Journal, 1999Co-Authors: Hiromi Ito, Hideharu Ishida, Hatsue Waki, Susum Ando, Makoto KisoAbstract:An efficient total synthesis of a cholinergic neuron-specific ganglioside GT1a alpha (IV3NeuAcIII6NeuAcII3NeuAc-GgOse4Cer) is described. The suitably protected sialyl-alpha(2-->6)-gangliotriose (III6NeuAc-GgOse3) derivative was glycosylated with the phenyl 2-thioglycoside of sialic acid in the presence of N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethane-sulfonate (TMSOTf) in acetonitrile medium, giving the disialogangliotriose (III6NeuAcII3NeuAc-GgOse3) derivative which contains both sialyl-alpha(2-->6)-GalNAc and sialyl-alpha(2-->3)-Gal structures (Route I). This pentasaccharide was efficiently synthesized also by the coupling of (methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galacto -2-nonulopyranosylonate)-(2-->6)-2-deoxy-3,4-O-isopropylidene-2-ph thalimido-D-galactopyranosyl trichloroacetimidate with 2-(trimethylsilyl)ethyl (methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galacto -2-nonulopyranosylonate)-(2-->3)-(2,6-di-O-benzyl-beta-D-galactopy ranosyl)-(1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside, followed by conversion of the phthalimido Group to the Acetamido Group (Route II). O-Deisopropylidenation and further glycosylation with methyl (methyl 5-Acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-gala ct-2-nonulopyranosylonate)-(2-->3)-2,4,6-tri-O-benzoyl-1-thio-b eta-D-galactopyranoside, promoted by dimethyl(methylthio)sulfonium triflate (DMTST), gave the desired trisialogangliotetraose (IV3NeuAcIII6NeuAcII3NeuAc-GgOse4) derivative, which was converted stepwise into the title ganglioside GT1a alpha by the introduction of the ceramide part and then complete deprotection. The ganglioside obtained was shown to be identical with the native GT1a alpha on TLC-immunostaining.
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binding specificities of the sialoadhesin family of i type lectins sialic acid linkage and substructure requirements for binding of myelin associated glycoprotein schwann cell myelin protein and sialoadhesin
Journal of Biological Chemistry, 1997Co-Authors: Brian E Collins, Makoto Kiso, Akira Hasegawa, Michael B Tropak, John C Roder, Paul R Crocker, Ronald L. SchnaarAbstract:Abstract The carbohydrate binding specificities of three sialoadhesins, a subGroup of I-type lectins (immunoglobulin superfamily lectins), were compared by measuring lectin-transfected COS cell adhesion to natural and synthetic gangliosides. The neural sialoadhesins, myelin-associated glycoprotein (MAG) and Schwann cell myelin protein (SMP), had similar and stringent binding specificities. Each required an α2,3-linked sialic acid on the terminal galactose of a neutral saccharide core, and they shared the following rank-order potency of binding: GQ1bα ≫ GD1a = GT1b ≫ GM3 = GM4 ≫ GM1, GD1b, GD3, GQ1b(nonbinders). In contrast, sialoadhesin had less exacting specificity, binding to gangliosides that bear either terminal α2,3- or α2,8-linked sialic acids with the following rank-order potency of binding: GQ1bα > GD1a = GD1b = GT1b = GM3 = GM4 > GD3= GQ1b ≫ GM1 (nonbinder). CD22 did not bind to any ganglioside tested. Binding of MAG, SMP, and sialoadhesin was abrogated by chemical modification of either the sialic acid carboxylic acid Group or glycerol side chain on a target ganglioside. Synthetic ganglioside GM3 derivatives further distinguished lectin binding specificities. Deoxy and/or methoxy derivatives of the 4-, 7-, 8-, or 9-position of sialic acid attenuated or eliminated binding of MAG, as did replacement of the sialic acid Acetamido Group with a hydroxyl. In contrast, the 4- and 7-deoxysialic acid derivatives supported sialoadhesin binding at near control levels (the other derivatives did not support binding). These data are consistent with sialoadhesin binding to one face of the sialic acid moiety, whereas MAG (and SMP) may have more complex binding sites or may bind sialic acids only in the context of more restricted oligosaccharide conformations.
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binding specificities of the sialoadhesin family of i type lectins sialic acid linkage and substructure requirements for binding of myelin associated glycoprotein schwann cell myelin protein and sialoadhesin
Journal of Biological Chemistry, 1997Co-Authors: Brian E Collins, Makoto Kiso, Akira Hasegawa, Michael B Tropak, John C Roder, Paul R Crocker, Ronald L. SchnaarAbstract:The carbohydrate binding specificities of three sialoadhesins, a subGroup of I-type lectins (immunoglobulin superfamily lectins), were compared by measuring lectin-transfected COS cell adhesion to natural and synthetic gangliosides. The neural sialoadhesins, myelin-associated glycoprotein (MAG) and Schwann cell myelin protein (SMP), had similar and stringent binding specificities. Each required an α2,3-linked sialic acid on the terminal galactose of a neutral saccharide core, and they shared the following rank-order potency of binding: GQ1bα ≫ GD1a = GT1b ≫ GM3 = GM4 ≫ GM1, GD1b, GD3, GQ1b(nonbinders). In contrast, sialoadhesin had less exacting specificity, binding to gangliosides that bear either terminal α2,3- or α2,8-linked sialic acids with the following rank-order potency of binding: GQ1bα > GD1a = GD1b = GT1b = GM3 = GM4 > GD3= GQ1b ≫ GM1 (nonbinder). CD22 did not bind to any ganglioside tested. Binding of MAG, SMP, and sialoadhesin was abrogated by chemical modification of either the sialic acid carboxylic acid Group or glycerol side chain on a target ganglioside. Synthetic ganglioside GM3 derivatives further distinguished lectin binding specificities. Deoxy and/or methoxy derivatives of the 4-, 7-, 8-, or 9-position of sialic acid attenuated or eliminated binding of MAG, as did replacement of the sialic acid Acetamido Group with a hydroxyl. In contrast, the 4- and 7-deoxysialic acid derivatives supported sialoadhesin binding at near control levels (the other derivatives did not support binding). These data are consistent with sialoadhesin binding to one face of the sialic acid moiety, whereas MAG (and SMP) may have more complex binding sites or may bind sialic acids only in the context of more restricted oligosaccharide conformations.
Brian E Collins - One of the best experts on this subject based on the ideXlab platform.
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binding specificities of the sialoadhesin family of i type lectins sialic acid linkage and substructure requirements for binding of myelin associated glycoprotein schwann cell myelin protein and sialoadhesin
Journal of Biological Chemistry, 1997Co-Authors: Brian E Collins, Makoto Kiso, Akira Hasegawa, Michael B Tropak, John C Roder, Paul R Crocker, Ronald L. SchnaarAbstract:Abstract The carbohydrate binding specificities of three sialoadhesins, a subGroup of I-type lectins (immunoglobulin superfamily lectins), were compared by measuring lectin-transfected COS cell adhesion to natural and synthetic gangliosides. The neural sialoadhesins, myelin-associated glycoprotein (MAG) and Schwann cell myelin protein (SMP), had similar and stringent binding specificities. Each required an α2,3-linked sialic acid on the terminal galactose of a neutral saccharide core, and they shared the following rank-order potency of binding: GQ1bα ≫ GD1a = GT1b ≫ GM3 = GM4 ≫ GM1, GD1b, GD3, GQ1b(nonbinders). In contrast, sialoadhesin had less exacting specificity, binding to gangliosides that bear either terminal α2,3- or α2,8-linked sialic acids with the following rank-order potency of binding: GQ1bα > GD1a = GD1b = GT1b = GM3 = GM4 > GD3= GQ1b ≫ GM1 (nonbinder). CD22 did not bind to any ganglioside tested. Binding of MAG, SMP, and sialoadhesin was abrogated by chemical modification of either the sialic acid carboxylic acid Group or glycerol side chain on a target ganglioside. Synthetic ganglioside GM3 derivatives further distinguished lectin binding specificities. Deoxy and/or methoxy derivatives of the 4-, 7-, 8-, or 9-position of sialic acid attenuated or eliminated binding of MAG, as did replacement of the sialic acid Acetamido Group with a hydroxyl. In contrast, the 4- and 7-deoxysialic acid derivatives supported sialoadhesin binding at near control levels (the other derivatives did not support binding). These data are consistent with sialoadhesin binding to one face of the sialic acid moiety, whereas MAG (and SMP) may have more complex binding sites or may bind sialic acids only in the context of more restricted oligosaccharide conformations.
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binding specificities of the sialoadhesin family of i type lectins sialic acid linkage and substructure requirements for binding of myelin associated glycoprotein schwann cell myelin protein and sialoadhesin
Journal of Biological Chemistry, 1997Co-Authors: Brian E Collins, Makoto Kiso, Akira Hasegawa, Michael B Tropak, John C Roder, Paul R Crocker, Ronald L. SchnaarAbstract:The carbohydrate binding specificities of three sialoadhesins, a subGroup of I-type lectins (immunoglobulin superfamily lectins), were compared by measuring lectin-transfected COS cell adhesion to natural and synthetic gangliosides. The neural sialoadhesins, myelin-associated glycoprotein (MAG) and Schwann cell myelin protein (SMP), had similar and stringent binding specificities. Each required an α2,3-linked sialic acid on the terminal galactose of a neutral saccharide core, and they shared the following rank-order potency of binding: GQ1bα ≫ GD1a = GT1b ≫ GM3 = GM4 ≫ GM1, GD1b, GD3, GQ1b(nonbinders). In contrast, sialoadhesin had less exacting specificity, binding to gangliosides that bear either terminal α2,3- or α2,8-linked sialic acids with the following rank-order potency of binding: GQ1bα > GD1a = GD1b = GT1b = GM3 = GM4 > GD3= GQ1b ≫ GM1 (nonbinder). CD22 did not bind to any ganglioside tested. Binding of MAG, SMP, and sialoadhesin was abrogated by chemical modification of either the sialic acid carboxylic acid Group or glycerol side chain on a target ganglioside. Synthetic ganglioside GM3 derivatives further distinguished lectin binding specificities. Deoxy and/or methoxy derivatives of the 4-, 7-, 8-, or 9-position of sialic acid attenuated or eliminated binding of MAG, as did replacement of the sialic acid Acetamido Group with a hydroxyl. In contrast, the 4- and 7-deoxysialic acid derivatives supported sialoadhesin binding at near control levels (the other derivatives did not support binding). These data are consistent with sialoadhesin binding to one face of the sialic acid moiety, whereas MAG (and SMP) may have more complex binding sites or may bind sialic acids only in the context of more restricted oligosaccharide conformations.
