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

Oligosaccharyltransferase (OST) catalyzes the transfer of a branched oligosaccharide 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 bovine pancreas and thyroid. Steady-state kinetic parameters comparing the two Dol-PP-OS to a shorter dolichylpyrophosphate disaccharide (DolPP-DS) previously 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 solubilized OST and, for the first time, saturation kinetics were observed for all substrates. The kinetic data provide a basis for analyzing quantitatively the individual contributions of oligosaccharide donor and peptide acceptor substrates to OST-catalyzed glycosylation.