Substrate specificities and reaction kinetics of the yeast oligosaccharyltransferase isoforms

Eyring, Jillianne; Lin, Chia-Wei; Ngwa, Elsy Mankah; Boilevin, Jérémy; Pesciullesi, Giorgio; Locher, Kaspar P.; Darbre, Tamis; Reymond, Jean-Louis; Aebi, Markus (2021). Substrate specificities and reaction kinetics of the yeast oligosaccharyltransferase isoforms. Journal of biological chemistry, 296, p. 100809. American Society for Biochemistry and Molecular Biology 10.1016/j.jbc.2021.100809

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

08 Faculty of Science > Department of Chemistry, Biochemistry and Pharmaceutical Sciences (DCBP)

UniBE Contributor:

Boilevin, Jérémy Mathias, Pesciullesi, Giorgio, Darbre, Tamis, Reymond, Jean-Louis

Subjects:

500 Science > 570 Life sciences; biology
500 Science > 540 Chemistry

ISSN:

0021-9258

Publisher:

American Society for Biochemistry and Molecular Biology

Language:

English

Submitter:

Sandra Tanja Zbinden Di Biase

Date Deposited:

27 Jan 2022 10:19

Last Modified:

05 Dec 2022 15:59

Publisher DOI:

10.1016/j.jbc.2021.100809

PubMed ID:

34023382

BORIS DOI:

10.48350/163006

URI:

https://boris.unibe.ch/id/eprint/163006

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