Purification and characterization of avian oligosaccharyltransferase. Complete amino acid sequence of the 50-kDa subunit.
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.
Kumar
V
V
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030.
Heinemann
F S
FS
Ozols
J
J
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002645:Chickens; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006602:Hexosyltransferases; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D014166:Transferases / Q000737:chemistry / Q000302:isolation & purification*
D008565:Membrane Proteins; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase