Exploring the acceptor substrate recognition of the human beta-galactosidealpha 2,6-sialyltransferase

Human beta1,4-galactoside alpha2,6-sialyltransferase I (ST6GalI) recognitio n of glycoprotein accepters has been investigated using various soluble for ms of the enzyme deleted to a variable extent in the N-terminal half of the polypeptide. Full-length and truncated forms of the enzyme have been inves tigated with respect to their specificity for a variety of desialylated gly coproteins of known complex glycans as well as related proteins with differ ent carbohydrate chains. Differences in transfer efficiency have been obser ved between membrane and soluble enzymatic forms, indicating that deletion of the transmembrane fragment induces loss of acceptor preference. No diffe rence in substrate recognition could be observed when soluble enzymes of si milar peptide sequence were produced in yeast or mammalian cells, confirmin g that removal of the membrane anchor and heterologous expression do not al ter enzyme folding and activity. When tested on free oligosaccharides, solu ble ST6GalI displayed full ability to sialylate free N-glycans as well as v arious N-acetyllactosaminyl substrates. Progressive truncation of the N ter minus demonstrated that the catalytic domain can proceed with sialic acid t ransfer with increased efficiency until 80 amino acids are deleted. Fusion of the ST6GalI catalytic domain to the N-terminal half of an unrelated tran sferase (core 2 beta1,6-N-acetylglucosaminyltransferase) further showed tha t a chimeric form of broad acceptor specificity and high activity could als o be engineered in vivo, These findings therefore delineate a peptide regio n of similar to 50 amino acids within the ST6GalI stem region that governs both the preference for glycoprotein accepters and catalytic activity, ther eby suggesting that it may exert a steric control on the catalytic domain.