Studies on glycoproteins XI. The O-glycosidic linkage of N-acetylgalactosamine to seryl and threonyl residues in ovine submaxillary gland glycoprotein

Abstract

Ovine submaxillary glycoprotein (OSM) was digested exhaustively with pronase and the sialoglycopeptides separated by gel filtration. By subsequent neuraminidase (EC 3.2.1.18) treatment and further purification, glycopeptides of average molecular weight 660 and containing 50% of the total galactosamine of OSM were obtained. Serine and threonine were the only amino acids with a functional group in the side chain present in a concentration high enough to accommodate all hexosamine residues. Treatment of the glycopeptides with 0.5 N NaOH at 0° or 22° resulted in the release of N-acetylgalactosamine coincident with the loss of an equimolecular amount of hydroxyamino acids. Most likely the carbohydrate was released by the mechanism of β-carbonyl elimination. Some evidence for the formation of an unsaturated compound, presumably α-aminoacrylic acid, was afforded spectrophotometrically. It is concluded that in OSM at least 50% of the prosthetic groups are joined glycosidically to the hydroxyl groups of serine and threonine. When native OSM was treated at pH 8.0 and 42° for 120 h, about one-third of the prosthetic groups was released. After this treatment the isolated, non-dialyzable residual glycoprotein reacted strongly positive in the Warren test. The mechanism of the reaction is discussed.