Dataset Information

Site-specific N-linked glycosylation of recombinant SARS-CoV-1 and -2 Receptor Binding Domain from GnTI-/- cells

ABSTRACT: Fusion proteins of the SARS-CoV-1 and SARS-CoV-2 spike receptor binding domain with a fluorescent protein were created in monomeric and trimeric form as tools for receptor binding studies in cultured cells and animal tissues. Here, site specific N-linked glycosylation in the proteins expressed from GnTI-/- cells is profiled with LC-MS/MS, using electron transfer high-energy collision dissociation

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Severe Acute Respiratory Syndrome Coronavirus 2

SUBMITTER: Joost Snijder

LAB HEAD: Joost Snijder

PROVIDER: PXD021825 | Pride | 2021-02-02

REPOSITORIES: Pride

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			Action	DRS
	SARS-CoV1_2_Skyline_glycoforms.rar	Other
	SARS-CoV1_RBD_GnTI_mon_ETHCD.raw	Raw
	SARS-CoV1_RBD_GnTI_mon_ETHCD.raw_20200926_Byonic.zip	Raw
	SARS-CoV1_RBD_GnTI_mon_EndoH_ETHCD.raw	Raw
	SARS-CoV1_RBD_GnTI_mon_EndoH_ETHCD.raw_20200925_Byonic.zip	Raw

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Project description:Human noroviruses, globally the main cause of viral gastroenteritis, show strain specific affinity for histo-blood group antigens (HBGA) and can successfully be propagated ex vivo in human intestinal enteroids (HIEs). HIEs established from jejunal stem cells of individuals with different ABO, Lewis and secretor geno- and phenotypes, show varying susceptibility to such infections. Using bottom-up glycoproteomic approaches we have defined and compared the N-linked glycans of glycoproteins of seven jejunal HIEs. Membrane proteins were extracted, trypsin digested, and glycopeptides enriched by hydrophilic interaction liquid chromatography and analyzed by nanoLC-MS/MS. The Byonic software was used for glycopeptide identification followed by hands-on verifications and interpretations. Glycan structures and attachment sites were identified from MS 2 spectra obtained by higher-energy collision dissociation through analysis of diagnostic saccharide oxonium ions (B-ions), stepwise glycosidic fragmentation of the glycans (Y-ions), and peptide sequence ions (b- and y-ions). Altogether 694 unique glycopeptides from 93 glycoproteins were identified. The N-glycans encompassed pauci- and oligomannose, hybrid- and complex-type structures. Notably, polyfucosylated HBGA-containing glycopeptides of the four glycoproteins tetraspanin-8, carcinoembryonic antigen-related cell adhesion molecule 5, sucrose-isomaltase and aminopeptidase N were especially prominent and were characterized in detail and related to donor ABO, Lewis and secretor types of each HIE. Virtually no sialylated N-glycans were identified for these glycoproteins suggesting that terminal sialylation was infrequent compared to fucosylation and HBGA biosynthesis. This approach gives unique site-specific information on the structural complexity of N-linked glycans of glycoproteins of human HIEs and provides a platform for future studies on the role of host glycoproteins in gastrointestinal infectious diseases.

Dataset Information

Site-specific N-linked glycosylation of recombinant SARS-CoV-1 and -2 Receptor Binding Domain from GnTI-/- cells

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