Abstract

Glycan macro- and microheterogeneity have profound impacts on protein folding and function. This heterogeneity can be regulated by physiological or environmental factors. However, unregulated heterogeneity can lead to disease, and mutations in the glycosylation process cause a growing number of Congenital Disorders of Glycosylation. We systematically studied how mutations in the N-glycosylation pathway lead to defects in mature proteins using all viable Saccharomyces cerevisiae strains with deletions in genes encoding Endoplasmic Reticulum lumenal mannosyltransferases (Alg3, Alg9, and Alg12), glucosyltransferases (Alg6, Alg8, and Die2/Alg10), or oligosaccharyltransferase subunits (Ost3, Ost5, and Ost6). To measure the changes in glycan macro- and microheterogeneity in mature proteins caused by these mutations we developed a SWATH-mass spectrometry glycoproteomics workflow. We measured glycan structures and occupancy on mature cell wall glycoproteins, and relative protein abundance, in the different mutants. All mutants showed decreased glycan occupancy and altered cell wall proteomes compared with wild-type cells. Mutations in earlier mannosyltransferase or glucosyltransferase steps of glycan biosynthesis had stronger hypoglycosylation phenotypes, but glucosyltransferase defects were more severe. ER mannosyltransferase mutants displayed substantial global changes in glycan microheterogeneity consistent with truncations in the glycan transferred to protein in these strains. Although ER glucosyltransferase and oligosaccharyltransferase subunit mutants broadly showed no change in glycan structures, ost3Δ cells had shorter glycan structures at some sites, consistent with increased protein quality control mannosidase processing in this severely hypoglycosylating mutant. This method allows facile relative quantitative glycoproteomics, and our results provide insights into global regulation of site-specific glycosylation.

Highlights

  • From the ‡School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland, 4072, Australia; §Fundacion Instituto Leloir, Avenida Patricias Argentinas 435, Ciudad Autonoma de Buenos Aires, 1405, Argentina

  • Nascent polypeptides in the Endoplasmic Reticulum (ER) are the protein acceptor substrates for N-glycosylation, as folded proteins cannot be efficiently N-glycosylated and N-glycosylation is critical for efficient protein folding

  • We focused our analysis on proteins covalently linked to the yeast cell wall, a well-defined subcellular fraction enriched in glycoproteins that has been previously extensively characterized by MSproteomics [15, 24, 27, 31]

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Summary

Introduction

From the ‡School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland, 4072, Australia; §Fundacion Instituto Leloir, Avenida Patricias Argentinas 435, Ciudad Autonoma de Buenos Aires, 1405, Argentina. LLO biosynthesis begins on the cytosolic face of the ER membrane, where Alg and the Alg13-Alg complex transfer the first and second N-acetylglucosamine (GlcNAc) residues to DolP This is followed by the sequential addition of Mannose (Man) residues by Alg, Alg, and Alg to synthesize the Man5GlcNAc2 structure. The LLO structure is completed by addition of three Glucose (Glc) residues to branch A through the sequential action of Alg, Alg, and Die2/Alg (Fig. 1A). This final Glc3Man9GlcNAc2 structure is the preferred LLO donor transferred to proteins by OTase, compared with biosynthetic intermediates. Lack of any of the nonessential subunits or mutations in essential subunits leads to inefficient N-glycosylation of diverse glycoproteins [15, 19, 20]

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