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.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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Evidence ID | Analyze ID | File | Description |
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