Dolichyldiphosphoryloligosaccharide—protein oligosaccharyltransferase: Solubilization, purification, and properties

Abstract

Dolichyldiphosphoryloligosaccharide-protein oligosaccharyltransferase was solubilized from hen oviduct rough endoplasmic reticulum by extraction with 0.2% Nonidet P40. Oligosaccharyltransferase activity was assayed in an incubation mixture containing Glcn-Manx-GlcNAc2-diphosphoryldolichol as an oligosaccharyl donor and the 125I-labeled tryptic peptide consisting of residues 29-58 from bovine .alpha.-lactalbumin as acceptor. The transferase was purified .apprx. 2000-fold by fractionation on bovine .alpha.-lactalbumin-Sepharose column; the active material bound quantitatively to the gel and was eluted by removal of divalent cation from the wash buffer. The product of the transferase activity, 125I-glycopeptide, was determined as concanavalin A(Con A)-agarose-adsorbed radioactivity by a filter disc assay method. 125I-Labeled con A-agarose-bound product was characterized as a glycopeptide as follows: gel filtration behavior on Sephadex G-50; elution from Con A agarose with 1% .alpha.-methyl mannoside; absence of affinity for ricin-Sepharose and loss of affinity for Con A-agarose after treatment with endo-.beta.-N-acetylglucosaminidase H; enzymatic synthesis of identical product upon using [3H]oligosaccharyldiphosphoryldolichol and unlabeled peptide acceptor; and digestion of 3H-labeled peptide with Pronase, resulting in the formation of lower molecular weight glycopeptide. Oligosaccharyltransferase activity exhibited an absolute requirement for divalent cations (3 mM Mn2+; Mg2+ was 30% effective), complete dependence on exogenously supplied peptide acceptor (1.33 .mu.g/ml) and oligosaccharyldiphosphoryldolichol (.apprx. 10 nmol/ml), and an optimum pH between 7 and 7.5.