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

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.

Substrate specificities and reaction kinetics of the yeast oligosaccharyltransferase isoforms

“…The glycosylation rates by the OST6 complex were about five times slower than those of the OST3 complex when measured for both the peptide and LLO substrates, despite having similar substrate affinities for both the peptide and LLO. This finding corroborates with a previous report showing that an Ost3p-containing complex has a higher relative glycosylation activity than an Ost6p-containing complex in vitro , using an assay with LLO extract ( 39 ). Based on the available structures of eukaryotic OSTs ( 13 , 14 , 31 ), it is evident that the substrates used in our in vitro assay interact only with the common catalytic subunit, Stt3p, explaining the similar K M values for the peptide and the LLO substrates for the two OST enzymes.…”

“…In yeast, Ost3p- and Ost6p-containing complexes have different peptide substrate preferences in vivo , and the Ost3p-containing complex (OST3 complex) is required for efficient glycosylation of a larger subset of proteins than the Ost6p-containing complex (OST6 complex) ( 34 ). Furthermore, the OST3 complex is more abundant in yeast ( 33 , 36 , 37 ) and has a higher relative enzymatic activity than the OST6 complex in vitro ( 38 , 39 ).…”

Comparative population genomic analyses of transporters within the Asgard archaeal superphylum

“…In agreement with previous research [ 4 ], we identified homologs of the eukaryotic-specific Magnesium Transporter1 (MagT1) family (TC# 1.A.76) in all Asgard proteomes. All MagT1 homologs in these proteomes belong to subfamily 1.A.76.2, whose members are part of the oligosacharyltransferase complex responsible for the transfer of an oligosaccharide chain onto asparagine residues [ 63 , 64 ]. It is possible that these proteins also transport Mg 2+ .…”

Active site variants in STT3A cause a dominant type I congenital disorder of glycosylation with neuromusculoskeletal findings

“…This is supported by cryo-electron microscopy of the human OST-A complex, 7 as well as recent work in yeast and bacteria showing the deleterious effect of active site STT3 substitutions. 24,25 All variants identified in our cohort are in conserved regions of at least six amino acids in all eukaryotes (Figure 5B). Indeed, several of these residues (Arg 329 , Arg 405 , and Tyr 530 ) have previously been experimentally determined as essential for OST function in a yeast model.…”