Structure of the O-Glycosidically Linked Carbohydrate Units of Fetuin

Abstract

Abstract Fetuin isolated from fetal calf serum was shown to contain 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 O-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)-β-d-galactopyranosyl-(1 →3)[N-acetylneuraminyl-(2 →6)]-N-acetylgalactosaminitol 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 was accomplished by digestion with α-N-acetylgalactosaminidase although no cleavage of this sugar was effected by the action of β-N-acetylhexosaminidase. This indicated that the alkali-labile units of fetuin are attached to the peptide chain by α-D-N-acetylgalactosaminyl-(1 →3)-serine (threonine) bonds. Of the total carbohydrate of fetuin 21%, including all three of the galactosamine residues, was shown to be located in the O-glycosidically linked units with the remainder occurring in the more alkali-stable N-glycosidically bound form. The alkaline sulfite treatment employed in this investigation produced in good yield the sulfonyl derivatives of the amino acids and amino sugar involved in the glycopeptide linkage. Since the cysteic acid and α-amino-β-hydroxybutyric acid obtained in this manner do not separate on the amino acid analyzer, a chromatographic procedure employing Dowex 1 was developed which permitted resolution of these two components.