Alkali-labile oligosaccharides from bovine milk fat globule membrane clycoprotein

Phenol extraction of bovine milk fat globule membrane gave a glycoprotein fraction which, in sodium dodecyl sulphate electrophoresis, showed three major bands, all staining for both protein and carbohydrate. Alkaline borohydride treatment and desialylation of the glycoprotein fraction released the reduced disaccharide β-d-galactosyl(1 → 3)-$\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ (T-antigen), which was identified by gas chromatography using a standard. All of the disaccharide units in the native glycoprotein were shown to be substituted by sialic acid, and a tetrasaccharide containing the disaccharide plus two molecules of sialic acid was isolated following alkaline borohydride treatment of the glycoprotein and gel filtration. Periodate oxidation of native and desialylated glycoprotein, together with paper chromatography of alkali degraded oligosaccharide fragments, indicated that the major alkali-labile oligosaccharide of the glycoprotein fraction is a tetrasaccharide containing $\text{β-}\text{d}\text{-galactosyl(1 → 3)}\text{-N-}\text{acetyl-}\text{d}\text{-galactosamine}$ substituted by sialic acid at position C3 of the galactosyl and position C6 of the $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ residue. Evidence was also obtained for the presence of small amounts of unsubstituted alkali-labile $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ linked directly to protein in the native glycoprotein.Serological evidence using agglutinins from Vicia graminea, Arachis hypogoea and human anti-T serum confirmed the presence in the native glycoprotein of a sialic acid substituted T-antigen. Similar evidence using agglutinins from Helix pomatia and Cepaea hortensis also confirmed the presence of terminal alkali-labile $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ in the native glycoprotein.