Uncoupling the hydrolysis of lipid-linked oligosaccharide from the oligosaccharyl transfer reaction by point mutations in yeast oligosaccharyltransferase

Takahiro Yamasaki, Daisuke Kohda

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

Oligosaccharyltransferase (OST) is responsible for the first step in the N-linked glycosylation, transferring an oligosaccharide chain onto asparagine residues to create glycoproteins. In the absence of an acceptor asparagine, OST hydrolyzes the oligosaccharide donor, releasing free N-glycans (FNGs) into the lumen of the endoplasmic reticulum (ER). Here, we established a purification method for mutated OSTs using a high-affinity epitope tag attached to the catalytic subunit Stt3, from yeast cells co-expressing the wild-type OST to support growth. The purified OST protein with mutations is useful for wide-ranging biochemical experiments. We assessed the effects of mutations in the Stt3 subunit on the two enzymatic activities in vitro, as well as their effects on the N-glycan attachment and FNG content levels in yeast cells. We found that mutations in the first DXD motif increased the FNG generation activity relative to the oligosaccharyl transfer activity, both in vitro and in vivo, while mutations in the DK motif had the opposite effect; the decoupling of the two activities may facilitate future deconvolution of the reaction mechanism. The isolation of the mutated OSTs also enabled us to identify different enzymatic properties in OST complexes containing either the Ost3 or Ost6 subunit and to find a 15-residue peptide as a better quality substrate than shorter peptides. This toolbox of mutants, substrates, and methods will be useful for investigations of the molecular basis and physiological roles of the OST enzymes in yeast and other organisms.

Original languageEnglish
JournalThe Journal of biological chemistry
DOIs
Publication statusE-pub ahead of print - Sep 16 2020

Fingerprint

Dive into the research topics of 'Uncoupling the hydrolysis of lipid-linked oligosaccharide from the oligosaccharyl transfer reaction by point mutations in yeast oligosaccharyltransferase'. Together they form a unique fingerprint.

Cite this