Sulphate groups are involved in the antigenicity of keratan sulphate and mask i antigen expression on their poly‐N‐acetyllactosamine backbones

Abstract

Conditions were established for desulphation of hexa‐, octa‐, deca‐ and larger oligosaccharides derived from corneal keratan sulphate after treatment with endo‐β‐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 β‐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 β‐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.