OLIGOSACCHARYL TRANSFERASE IS A CONSTITUTIVE COMPONENT OF AN OLIGOMERIC PROTEIN COMPLEX FROM PIG-LIVER ENDOPLASMIC-RETICULUM

Oligosaccharyl transferase (OST), an intrinsic component of the endopl asmic reticulum membrane, catalyses the N-glycosylation of specific as paragine residues in nascent polypeptide chains. We have purified the enzyme from crude pig liver microsomes by a procedure involving salt/d etergent extraction, concanavalin-A precipitation, S-Sepharose, MonoP and concanavalin-A-Sepharose chromatographies. A highly purified OST p reparation exerting catalytic activity, contained two protein subunits of 48 kDa and 65 kDa, from which the 66-kDa species was identified by immunoblotting as ribophorin I. The function of ribophorin I in this dimeric protein complex is unknown. The high degree of similarity betw een its transmembrane region and a putative dolichol-recognition conse nsus sequence suggests that ribophorin I could be involved in glycolip id binding and delivery. Several lines of evidence indicate that the c atalytically active 48-kDa/66-kDa polypeptides are associated in the e ndoplasmic reticulum membrane with other proteins, including ribophori n II and a 40-kDa glycoprotein. The implication of ribophorins I and I I in the translocation machinery and their apparent association with t he OST activity point to a close relationship between polypeptide synt hesis, translocation and N-glycosylation, both spacially and temporall y. Kinetic studies with the MonoP-purified oligosaccharyl transferase showed that the enzyme transfers dolichyl-diphosphate-linked GlcNAc(2) to synthetic tripeptides and hexapeptides, containing the Asn-Xaa-Thr motif, at a comparable rate. The glycosylation reaction was found to have a pH optimum close to 7 and to require divalent metal ions, with Mn2+ being most effective. Substitution of threonine in the N-glycosyl ation motif by serine impairs its function as an acceptor, measured by V-max/K-m, by approximately 17-fold, consisting of a 7.3-fold increas e in K-m and a 2.3-fold decrease in V-max. This indicates that the sid e chain structure of the hydroxyamino acid influences both binding and catalysis, consistent with previous studies highlighting its particip ation in the catalytic mechanism of transglycosylation. The K-m values of peptide accepters improved significantly when dolichyl-phosphate-b ound oligosaccharides were used instead of lipid-linked GlcNAc(2) as t he glycosyl donor. We conclude from this observation that the sugar re sidues on the outer branches of the glycolipid donor induce conformati onal changes in the active site of the oligosaccharyl transferase, thu s influencing the association constant of the peptide substrate.