Analysis of Congenital Disorder of glycosylation-Id in a Yeast Model System Shows Diverse Site-Specific Under-Glycosylation of Glycoproteins

J Proteome Res. 2012 Nov 2;11(11):5376-83. doi: 10.1021/pr300599f. Epub 2012 Oct 16.

Abstract

Asparagine-linked glycosylation is a common post-translational modification of proteins in eukaryotes. Mutations in the human ALG3 gene cause changed levels and altered glycan structures on mature glycoproteins and are the cause of a severe congenital disorder of glycosylation (CDG-Id). Diverse glycoproteins are also under-glycosylated in Saccharomyces cerevisae alg3 mutants. Here we analyzed site-specific glycosylation occupancy in this yeast model system using peptide-N-glycosidase F to label glycosylation sites with an asparagine-aspartate conversion that creates a new endoproteinase AspN cleavage site, followed by proteolytic digestion, and detection of peptides and glycopeptides by LC-ESI-MS/MS. We used this analytical method to identify and measure site-specific glycosylation occupancy in alg3 mutant and wild type yeast strains. We found decreased site-specific N-glycosylation occupancy in the alg3 knockout strain preferentially at Asn-Xaa-Ser sequences located in secondary structural elements, features previously associated with poor glycosylation efficiency. Furthermore, we identified 26 previously experimentally unverified glycosylation sites. Our results provide insights into the underlying mechanisms of disease in CDG-Id, and our methodology will be useful in site-specific glycosylation analysis in many model systems and clinical applications.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Wall / metabolism
  • Congenital Disorders of Glycosylation / metabolism*
  • Glycoproteins / chemistry
  • Glycoproteins / metabolism*
  • Glycosylation
  • Models, Biological
  • Molecular Sequence Data
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / metabolism
  • Proteolysis
  • Saccharomyces cerevisiae / metabolism*
  • Spectrometry, Mass, Electrospray Ionization
  • Trypsin / metabolism

Substances

  • Glycoproteins
  • Trypsin
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase