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Eur J Biochem. 1995 Mar 15;228(3):689-96.

Oligosaccharyl transferase is a constitutive component of an oligomeric protein complex from pig liver endoplasmic reticulum.

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Institut für Physiologische Chemie, Bonn, Germany.


Oligosaccharyl transferase (OST), an intrinsic component of the endoplasmic reticulum membrane, catalyses the N-glycosylation of specific asparagine residues in nascent polypeptide chains. We have purified the enzyme from crude pig liver microsomes by a procedure involving salt/detergent extraction, concanavalin-A precipitation, S-Sepharose, MonoP and concanavalin-A-Sepharose chromatographies. A highly purified OST preparation exerting catalytic activity, contained two protein subunits of 48 kDa and 66 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 between its transmembrane region and a putative dolichol-recognition consensus sequence suggests that ribophorin I could be involved in glycolipid binding and delivery. Several lines of evidence indicate that the catalytically active 48-kDa/66-kDa polypeptides are associated in the endoplasmic reticulum membrane with other proteins, including ribophorin II and a 40-kDa glycoprotein. The implication of ribophorins I and II in the translocation machinery and their apparent association with the OST activity point to a close relationship between polypeptide synthesis, translocation and N-glycosylation, both spacially and temporally. Kinetic studies with the MonoP-purified oligosaccharyl transferase showed that the enzyme transfers dolichyl-diphosphate-linked GlcNAc2 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-glycosylation motif by serine impairs its function as an acceptor, measured by Vmax/Km, by approximately 17-fold, consisting of a 7.3-fold increase in Km and a 2.3-fold decrease in Vmax. This indicates that the side chain structure of the hydroxyamino acid influences both binding and catalysis, consistent with previous studies highlighting its participation in the catalytic mechanism of transglycosylation. The Km values of peptide acceptors improved significantly when dolichyl-phosphate-bound oligosaccharides were used instead of lipid-linked GlcNAc2 as the glycosyl donor. We conclude from this observation that the sugar residues on the outer branches of the glycolipid donor induce conformational changes in the active site of the oligosaccharyl transferase, thus influencing the association constant of the peptide substrate.

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