STUDIES ON ATTACHMENT OF N-ACETYLNEURAMINIC ACID TO GLYCOPEPTIDES FROM ALPHA-1-ANTITRYPSIN

A detergent-solubilized sialyltransferase extracted from human liver catalyzed the addition of CMP N-acetylneuraminic acid (CMP-NeuAc) to desialylated .alpha.-1-antitrypsin, fetuin, thyroglobulin, ceruloplasmin and antifreeze glycoproteins 7 and 8. In addition, 4 unique glycopeptide fragments (A, B, C and D) isolated and purified from .alpha.-1-antitrypsin were desialylated and degraded with trypsin and pronase. These asialoglycopeptides also served as sialic acid acceptors in the reaction catalyzed by human liver sialyltransferase. As a measure of acceptor capability, both the reaction rate (Vobs) and Km value for each of the individual asialoglycopeptides was determined as a function of the extent of proteolysis. The Km values calculated for asialoglycopeptide fragments A, B, C and D were 0.53, 0.40, 0.15 and 0.67 mM, respectively. The Vobs values for asialoglycopeptide fragments A, B, C and D were 215, 80, 20 and 60 nmol/mg per h, respectively. Upon treatment with trypsin, the Km for asialotryptic B fragment increased from 0.40 to .gtoreq. 5.0 mM, while the Km for the other fragments remained relatively unchanged. Upon subsequent treatment with pronase, the Km for the asialopronase D fragment increased from 0.67-5.0 mM while the Km for the other fragments remained relatively unchanged. After pronase digestion, the Vobs of asialoglycopeptides A and C decreased from 215-80 nmol/mg per h and from 20-10 nmol/mg per h, respectively. Asialoglycopeptides A and C formed 1 class of acceptors in which only the Vobs decreased upon proteolytic digestion. Asialoglycopeptides B and D formed a 2nd class of acceptors in which the Km increased and the Vobs decreased upon proteolytic digestion. The activity of the sialyltransferase required for the addition of CMP-NeuAc to a glycopeptide acceptor apparently depends in part on its polypeptide backbone.