Purification and characterization of avian oligosaccharyltransferase. Complete amino acid sequence of the 50-kDa subunit

• Published in: The Journal of biological chemistry Vol. 269; no. 18; pp. 13451 - 13457
• Main Authors: VIJAY KUMAR, HEINEMANN, F. S, OZOLS, J
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 06.05.1994
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Blotting, Western; Chickens; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Dogs; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hexosyltransferases; Membrane Proteins; Molecular Sequence Data; Sequence Homology, Amino Acid; Transferases; Transferases - chemistry; Transferases - isolation & purification; Hen; Vertebrata; Purification; Enzymatic activity; Enzyme; Transferases; Carbohydrate; Aves; Subunit; Comparative study; Aminoacid sequence
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)36853-9

We have purified oligosaccharyltransferase from hen oviduct microsomes some 850-fold. Oligosaccharyltransferase activity copurified with a 200-kDa complex consisting of two 65-kDa polypeptides and a 50-kDa polypeptide. N-terminal sequence analysis indicated that the 50-kDa subunit was the avian form of OST48, a canine pancreatic microsomal protein associated with oligosaccharyltransferase. As the first step toward reconstitution of the oligosaccharyltransferase complex, the 50-kDa subunit was purified to homogeneity under nondenaturing conditions. The complete amino acid sequence of the 50-kDa subunit was determined by sequence analysis of peptides isolated by a combination of gel filtration and high performance liquid chromatography from chemical and enzymatic digests. The protein consists of 412 residues in a single polypeptide chain. The amino acid sequence of the 50-kDa subunit of avian oligosaccharyltransferase is 92% identical to the sequence of canine OST48 protein and about 25% identical to WBP1 protein from the yeast Saccharomyces cerevisiae. The yeast WBP1 protein has been shown in vitro, as well as in vivo, to be essential for the oligosaccharyltransferase activity.