Dolichylpyrophosphate oligosaccharides: Large-scale isolation and evaluation as oligosaccharyltransferase substrates

-Oligosaccharyltransferase (OST) catalyzes the transfer of a branched oligo saccharide from a dolichylpyrophosphate oligosaccharide (Dol-PP-OS) to the asparagine of a nascent polypeptide chain in vivo and peptide substrates in vitro. Here we report the isolation and purification of Dol-PP-Os from bov ine pancreas and thyroid. Steady-state kinetic parameters comparing the two Dol-PP-OS to a shorter dolichylpyrophosphate disaccharide (DolPP-DS) previ ously synthesized in our laboratory are reported. These were determined for Dol-PP-OS, Dol-PP-DS, and the tripeptide Bz-Asn-Leu-Thr-NH2 with solubiliz ed OST and, for the first time, saturation kinetics were observed for all s ubstrates. The kinetic data provide a basis for analyzing quantitatively th e individual contributions of oligosaccharide donor and peptide acceptor su bstrates to OST-catalyzed glycosylation. (C) 1999 Elsevier Science Ltd. All rights reserved.