A comparison of proteins and peptides as substrates for microsomal and solubilized oligosaccharyltransferase.
A chemoenzymatic synthesis of homogeneous neoglycoproteins and glycopeptides was explored using oligosaccharyltransferase isolated from yeast. Neither the microsomal form nor the solubilized form of the enzyme catalyzed the transfer of the core Glc3Man9(GlcNAc)2 oligosaccharide to chemically modified ribonuclease A or alpha-lactalbumin. Similarly, no transfer was observed to the 32-amino acid peptide hormone, calcitonin, by either the membrane-bound or soluble form of oligosaccharyltransferase. However, a 17-amino acid fragment of yeast invertase with the unusual sequence containing two overlapping glycosylation sequons proved to be a good substrate, slightly less effective than the well studied tripeptide, Bz-Asn-Leu-Thr-NH2. Product analysis using gel permeation chromatography showed that the expected glycopeptides were formed and endo H-catalyzed cleavage of the oligosaccharide portion from the glycopeptides demonstrated that the glycopeptides contained the same carbohydrate moiety.
Liu
Y L
YL
Department of Chemistry, College of Pharmacy, University of Michigan, Ann Arbor 48109-1055, USA.
Hoops
G C
GC
Coward
J K
JK
D000595:Amino Acid Sequence; D002116:Calcitonin / Q000378:metabolism; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006602:Hexosyltransferases; D008565:Membrane Proteins; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012995:Solubility; D013379:Substrate Specificity; D014166:Transferases / Q000378:metabolism*; D043324:beta-Fructofuranosidase
D008565:Membrane Proteins; D010455:Peptides; D011506:Proteins; D002116:Calcitonin; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D012260:Ribonucleases; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase