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

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.