Structure of the O-Glycosidically Linked Carbohydrate Units of Fetuin

Abstract

Fetuin isolated from fetal calf serum was shown to contam 3 carbohydrate units which are attached to serine and threonine residues on the peptide chain and are distinct from the 3 asparagine-linked heteropolysaccharides previously reported to occur in this protein.The 0-glycosidically linked saccharide units were obtained as reduced oligosaccharides after alkaline borohydride treatment of fetuin and as glycopeptides subsequent to pronase digestion of this protein.Gel filtration and Dowex 1 chromatography of the oligosaccharides resulted in the isolation of a tri-and a tetrasaccharide which were made up of sialic acid, galactose, and N-acetylgalactosaminitol residues and could be converted after selective removal of the sialic acid to the same disaccharide (galactosyl-N-acetylgalactosaminitol).Studies employing periodate oxidation and glycosidase digestion indicated that the structure of the tetrasaccharide was N-acetylneuraminyl-(2 -+3) -P-D -galactopyranosyl-(l -+3)[N-acetylneuraminyl-(2 -+6)]-N-acetylgalactosaminiitol and that the trisaccharide differed only by the absence of the sialyl residue linked to the N-acetylgalactosaminitol.Glycopeptides containing the alkali-labile carbohydrate units were purified on Sephadex and diethylaminoethylcellulose columns.In this manner peptides distinguished by a high proline content and containing 1 tetrasaccharide, 1 trisaccharide, or 2 trisaccharides were obtained.Alkaline sulfite treatment of these glycopeptides indicated that 2 serine and 1 threonine residue in each fetuin molecule is involved in the attachment of carbohydrate units.The tetrasaccharide was found to be linked only to serine while attachment of trisaccharide units involved a serine and a threonine residue which were located in close proximity in the peptide chain.Smith periodate degradation of sialic acid-free glycopeptides resulted in removal of the galactose and yielded a product from which complete release of N-acetylgalactosamine