The linkage of teleost skin keratan sulfate to protein

The linkage of teleost skin keratan sulfate to protein was investigated. Afeter its exhaustive digestion with pronase, peptidokeratan sulfate was obtained with aspartic acid as the predominant amino acid. The N-terminal of the amino acid residues of the preparation was dansylated, and the carbohydrate-peptide linkage fragment was isolated, with the aid of fluorescence, by sequential digestion with Flavobacterium endo-β-galactosidase, β-galactosidase, β-N-acetylhexosaminidase and endo-β-N-acetylglucosaminidase D, followed by Bio-Gel p-4 column chromatography. The structure of the dansylated fragment thus obtained was identified dansylated asparaginyl N-acetyl-D-glucosamine. Treatment of the dansylated keratan sulfate peptide with almond glycopeptidase, which specially cleaves thet asparaginyl N-acetyl-D-glucosamine linkage in the glycoproteins, also showed asparaginyl N-aceytyl-D-glucosamine linkage to be in the core region of this keratan sulfate. We conclude that teleost skin keratan sulfate is bound to protein via an N-glycosyl linkage between N-acetyl-D-glucosamine and asparagine. The keratan sulfate core apparently consist of trimannosyl-N,N′-diacetylchitobiose units, considering the specificity of endo-β-N-acetylglucosaminidase D.