Sulphate groups are involved in the antigenicity of keratan sulphate and mask i antigen expression on their poly-N-acetyllactosamine backbones. An immunochemical and chromatographic study of keratan sulphate oligosaccharides after desulphation or nitrosation.
Conditions were established for desulphation of hexa-, octa-, deca- and larger oligosaccharides derived from corneal keratan sulphate after treatment with endo-beta-galactosidase. The antigenicities of the desulphated oligosaccharides were compared with those of the native oligosaccharides in chromatogram binding, plastic-plate binding or inhibition of binding assays using a novel microimmunochemical approach with oligosaccharide-lipid conjugates (neoglycolipids). The results clearly show that sulphate residues are essential components of the antigenic determinant(s) recognised by three monoclonal antibodies to keratan sulphate, 5-D-4, 1-B-4 and MZ15, but they mask the i antigen activity of the linear poly-(N-acetyllactosamine) backbones of this glycosaminoglycan. Immunochemical assays, before and after beta-N-acetylglucosaminidase treatment of desulphated linear hexa-, octa- and decasaccharides derived from keratan sulphate, indicate that for reaction with one anti-i antibody, Den, there is an absolute requirement for the non-reducing beta-galactosyl residue of the i antigen structure to be in the terminal position, but with a second anti-i antibody, Tho, there is in addition some reactivity with the i antigen structure having an N-acetylglucosamine residue at the non-reducing end. The chromatographic properties after desulphation or nitrosation of a minor keratan sulphate oligosaccharide (a dodecasaccharide), which reacts especially well with antibody 5-D-4, have provided the first evidence for the presence of glucosamine residues that may be N-sulphated in corneal keratan sulphate.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D000939:Epitopes / Q000032:analysis*; D006025:Glycosaminoglycans / Q000276:immunology*; D006026:Glycoside Hydrolases; D006801:Humans; D007118:Immunoassay; D007202:Indicators and Reagents; D007632:Keratan Sulfate / Q000276:immunology*; D051379:Mice; D009844:Oligosaccharides / Q000032:analysis* / Q000276:immunology; D013329:Structure-Activity Relationship; D014316:Tritium
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D006025:Glycosaminoglycans; D007202:Indicators and Reagents; D009844:Oligosaccharides; D014316:Tritium; D007632:Keratan Sulfate; D006026:Glycoside Hydrolases