SITE-SPECIFIC GLYCOSYLATION OF BOVINE BUTYROPHILIN

Our previous studies showed that the N-linked sugar chains of most bov ine glycoproteins from milk fat globule membranes (MFGM) contain the G alNAc beta 1-->4GlcNAc group [Sato et al, (1993) J. Biochem, 114, 890- 900], Since expression of the disaccharide structure is influenced by peptide sequences near the glycosylation sites [Smith and Baenziger (1 992) Proc. Natl. Acad Sci. USA 89, 329-333], the site-specificity of t he N-acetylgalactosaminylated sugar chains was investigated using bovi ne butyrophilin, a major MFGM glycoprotein with known primary structur e. Two glycopeptide fragments which contained the N-linked sugar chain s linked to either Asn-55 or Asn-215 residue were obtained by digestio n of the protein with Achromobacter protease I. The sugar chains relea sed from each glycopeptide by hydrazinolysis were reduced with (NaBH4) -H-3. Structural analyses of the oligosaccharides by sequential exogly cosidase digestion and methylation analysis revealed that only complex -type sugar chains with the GalNAc beta 1-->4GlcNAc structure are incl uded in Asn-55-linked oligosaccharides, while only novel hybrid-type s ugar chains detected previously in bovine MFGM glycoproteins are inclu ded in Asn-215-linked oligosaccharides. The results show that the glyc osylation of butyrophilin occurs in a site-specific manner.