PubMed · 11348163
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S Knapp, D S Myers. 2001-05-18. Alpha-GlcNAc thioconjugates.. https://doi.org/10.1021/jo010088e
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The aim of the present study was the characterization of the dominant epitope present on Schistosoma mansoni glycolipids, which causes cross-reactivity of S. mansoni and S. haematobium infection sera with keyhole-limpet haemocyanin (KLH). To this end, the monoclonal antibody M2D3H was chosen for its similar behaviour in high-performance TLC immunostaining and inhibition-ELISA to infection sera. Individual, structurally defined oligosaccharides derived from S. mansoni egg glycolipids were tested for their binding to this monoclonal antibody by immunoaffinity chromatography. A terminal fucose residue linked in the (alpha1-->3) position to N-acetylgalactosamine was found to be the common structural determinant of the four oligosaccharides binding to M2D3H. The Fuc(alpha1-->3)GalNAc-motif also appeared to be the basis for the cross-reactivity with KLH, a phenomenon used in the serodiagnosis of S. mansoni, S. haematobium and S. japonicum infections.
beta-Glucosidase from Flavobacterium meningosepticum (Fbgl) catalyses the hydrolysis of beta-1,4-glucosidic bonds via a two-step double-displacement mechanism in which two amino acid residues act as nucleophile and acid/base catalyst. Definitive identification of these two residues is provided by the two active-site-directed inactivators, 2',4'-dinitrophenyl-2-deoxy-2-fluoro-beta-d-glucoside (2FDNPG) and N-bromoacetyl-beta-d-glucosylamine (NBGN), which stoichiometrically label the nucleophile and the acid/base catalyst of Fbgl, respectively. Pseudo-first-order inactivation rate constants (k(i)) of 0.25+/-0.01 and 0.05+/-0.01 min(-1) and dissociation constants (K(i)) of 90+/-15 and 4.4+/-0.2 mM are determined for 2FDNPG and NBGN, respectively. Proteolytic digestion of the labelled proteins, followed by peptide mapping and tandem MS analysis identify Asp-247 and Glu-473 as the catalytic nucleophile and acid/base residues, respectively, of Fbgl. This study confirms that the catalytic nucleophile of family 3 glycohydrolase is conserved across sub-families. However, different sub-families may have unique general acid/base catalysts.
Immunoglobulins are glycoproteins, containing N- linked carbohydrates in the heavy chain constant regions of all isotypes and O-linked carbohydrates in the hinge regions of human IgA1 and IgD. A previous study showed that IgD synthesized in the presence of tunicamycin and lacking the three N-linked glycans on the heavy chain was not secreted (Shin, S. U., Wei, D. F., Amin, A. R., Thorbecke, G. J., and Morrison, S. L. (1992) Hum. Antibodies 3, 65-74). The contribution of each of the carbohydrates in the Fc of IgD to assembly and secretion was now analyzed by eliminating the carbohydrate addition sequence, Asn-X-Ser/Thr, through site-directed mutagenesis. Only the carbohydrate nearest the sole disulfide bond between heavy chains, which remained high mannose and appeared to be buried within the folded molecule, was found to be essential for secretion. When IgD lacked that glycan, assembly reached only the heavy/light chain half-molecule stage, and heavy chains were held inside the endoplasmic reticulum. Using benzyl 2-acetamido-2-deoxy-alpha-d-galactopyranoside (BADG) to inhibit complete O-linked glycosylation, we found that IgA1 and IgD with incomplete hinge carbohydrates were assembled and secreted from cells. Thus, one N-linked glycan plays a structural role in IgD and is required for proper assembly and secretion, but the O-linked carbohydrates in the hinge of IgD and IgA1 are not required for folding and export.