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.