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

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.

Deglycosylation systematically improves N-glycoprotein identification in liquid chromatography–tandem mass spectrometry proteomics for analysis of cell wall stress responses in Saccharomyces cerevisiae lacking Alg3p

“…This sample contains many N-and O-glycoproteins, as well as abundant non-glycosylated proteins [27][28][29][30][31]. This analysis robustly identified 26 proteins, with 19 identified after both AspN and trypsin digest, four identified only by trypsin and three only by AspN (Supplementary Table S1, Supplementary material).…”

“…Preparation of proteins covalently linked to the yeast polysaccharide cell wall was performed as previously described [27,28]. Biological triplicates of cells grown in 50 mL of YPD to mid log phase were harvested by centrifugation at 4000 rcf for 10 min, resuspended in 50 mM Tris-HCl buffer, pH 7.5, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 x Complete protease inhibitor cocktail (Roche, Basel, Switzerland) and lysed by agitation using a Mo Bio Laboratories (Carlsbad, CA, USA) Vortex-Genie 2 at maximum speed with 425-600 µm glass beads for 1 h at 4˚C.…”

“…Peptides were desalted using C18 ZipTips (Millipore, Billerica, Massachusetts, USA) and analyzed by LC-ESI-MS/MS using a Prominence nanoLC system (Shimadzu, Kyoto, Japan) direct on-line with a TripleTof 5600 mass spectrometer with a Nanospray III interface (AB SCIEX, Washington, D.C., USA) as previously described [27]. The Prominence nanoLC system consisted of a DGU- mDa; dynamic exclusion of former target ions for 30 s after 2 occurrences.…”

“…This analysis also allowed putative identification of sites of N-glycosylation, by the conversion of previously glycosylated asparagine to aspartate by PNGase F deglycosylation (Fig. 2) [27]. However, validation of these glycosylation sites requires a negative control lacking PNGase F enzyme to ensure the observed deamidation events are not spontaneous [35], which occurs at asparagines in a sequence-dependent manner [36,37].…”

“…The activity of OTase is affected by the structure of glycan present, as OTase has high affinity for the mature lipid linked glycan, which limits transfer of the biosynthetic intermediates that are also present on the ER-lumenal face of the ER membrane. This means that OTase has reduced activity in the presence of incomplete glycan, for instance in Δalg3 cells lacking the Alg3p protein [27,39]. We have previously shown that Δalg3 cells show site-specific underglycosylation, with the subset of normally glycosylated asparagine residues that are located in NxS sequons and in secondary structural elements tending to be under-glycosylated, compared with wild type yeast cells [27].…”