Partial characterization and purification of the glycosylation site recognition component of oligosaccharyltransferase.
Oligosaccharyltransferase, the enzyme catalyzing the co-translational transfer of oligosaccharide from dolichyl-PP-GlcNAc2Man9Glc3 to -Asn-X-Ser/Thr- sequences in nascent polypeptide chains, was studied in hen oviduct microsomes using the active site-directed photoaffinity probe 125I-labeled N alpha-3-(4-hydroxyphenylpropionyl)-Asn-Lys(N epsilon-p-azidobenzoyl)-Thr-NH2. Several lines of evidence established that the tripeptide probe interacted with a 57-kDa protein of the endoplasmic reticulum that was subsequently glycosylated and converted to a 60-kDa form. The 57-kDa protein, isolated by two-dimensional gel electrophoresis, was used as immunogen to prepare polyclonal antisera. The specificity of the antibody was established on the basis of its ability to 1) recognize the 57-kDa protein by immunoblotting and 2) immunoprecipitate the photolabeled protein. The antibody also recognized photolabeled protein from different tissues and organisms. The 57-kDa protein isolated by immunoprecipitation retained its ability to interact with the photoaffinity probe but was inactive in catalyzing glycosylation of peptides. This result suggests that the 57-kDa protein is the component of oligosaccharyltransferase that recognizes the glycosylation site in polypeptides. These results are discussed in terms of possible models for the structure of oligosaccharyltransferase in the endoplasmic reticulum.
Kaplan
H A
HA
Department of Biochemistry and Molecular Biology, University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston 77030.
Naider
F
F
Lennarz
W J
WJ
D000345:Affinity Labels; D000818:Animals; D000918:Antibody Specificity; D001665:Binding Sites; D006031:Glycosylation; D006602:Hexosyltransferases; D008565:Membrane Proteins; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology; D010777:Photochemistry; D011817:Rabbits; D014166:Transferases / Q000032:analysis*
D000345:Affinity Labels; D008565:Membrane Proteins; D010446:Peptide Fragments; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase