Substrate specificities and reaction kinetics of the yeast oligosaccharyltransferase isoforms
| Autor: | Jillianne, Eyring, Chia-Wei, Lin, Elsy Mankah, Ngwa, Jérémy, Boilevin, Giorgio, Pesciullesi, Kaspar P, Locher, Tamis, Darbre, Jean-Louis, Reymond, Markus, Aebi |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Saccharomyces cerevisiae Proteins
glycosylation FDR false discovery rate TAMRA tetramethylrhodamine DDM n-dodecyl-β-D-maltopyranoside Saccharomyces cerevisiae carbohydrate structure glycosylation inhibitor yeast Substrate Specificity glycoprotein biosynthesis XIC extracted ion chromatography ER endoplasmic reticulum enzyme kinetics Protein Isoforms OST oligosaccharyltransferase FA formic acid OST protein complex LLO lipid-linked oligosaccharide Membrane Proteins oligosaccharyltransferase ACN acetonitrile Kinetics Hexosyltransferases HCD higher-energy collisional dissociation CHS cholesteryl hemisuccinate membrane enzyme Research Article |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X |
| Popis: | Oligosaccharyltransferase (OST) catalyzes the central step in N-linked protein glycosylation, the transfer of a preassembled oligosaccharide from its lipid carrier onto asparagine residues of secretory proteins. The prototypic hetero-octameric OST complex from the yeast Saccharomyces cerevisiae exists as two isoforms that contain either Ost3p or Ost6p, both noncatalytic subunits. These two OST complexes have different protein substrate specificities in vivo. However, their detailed biochemical mechanisms and the basis for their different specificities are not clear. The two OST complexes were purified from genetically engineered strains expressing only one isoform. The kinetic properties and substrate specificities were characterized using a quantitative in vitro glycosylation assay with short peptides and different synthetic lipid-linked oligosaccharide (LLO) substrates. We found that the peptide sequence close to the glycosylation sequon affected peptide affinity and turnover rate. The length of the lipid moiety affected LLO affinity, while the lipid double-bond stereochemistry had a greater influence on LLO turnover rates. The two OST complexes had similar affinities for both the peptide and LLO substrates but showed significantly different turnover rates. These data provide the basis for a functional analysis of the Ost3p and Ost6p subunits. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|