Quantitative Mapping of the in vivo O-GalNAc Glycoproteome in Mouse Tissues

ABSTRACT: A large family of GalNAc transferases (GalNAc-Ts) catalyzes the covalent attachment of N-Acetylgalactosamine (GalNAc) to serine and threonine residues on proteins that pass through the secretory pathway in the first committed step of mucin-type O-glycosylation. Abnormalities in the activity of individual GalNAc-Ts can result in congenital disorders of O-glycosylation (CDG) and influence other biological functions. Although ~85% of proteins that pass through the secretory pathway are modified with O-glycans, only 47 O-glycosylation sites (O-glycosites) from 22 mouse glycoproteins and 17 publications are present on UniProt and an O-glycoprotein database (http://www.oglyp.org/). A compilation of all in vivo O-glycosites (the O-glycoproteome) would be an invaluable step in determining the function of proteins decorated with N-Acetylgalactosamine and what role(s), if any, the O-glycans play. While approaches to delineate O-glycosites have been developed for simple, genetically engineered cell lines, there is no universal approach to mapping the O-glycosites of glycoproteins in complex tissue extracts or biological fluids. We have developed chemical and enzymatic conditions that cleave solitary O-glycans while leaving the modified core protein/peptides assayable by mass spectrometry (MS). The methodology permits the mapping of thousands of O-glycosites decorated with Tn or T antigen from tissues or biological fluids. We then integrated an HCD-pd-EThcD MS workflow and software, including MSFragger-Glyco, pGlyco3, and O-Pair, to study the mouse brain, heart, lung, liver, spleen, kidney, colon, muscle, submandibular gland, and whole blood. The integrated approach identified 2154 solitary O-glycosites from 595 glycoproteins. The O-glycosites and glycoproteins displayed consensus motifs and Gene Ontology (GO) terms for O-glycoproteins. Limited overlap of O-glycosites was observed with protein O-GlcNAcylation and phosphorylation sites. Integrating quantitative glycoproteomics and proteomics revealed a tissue-specific regulation of O-glycosites that the differential expression of Galnt isoenzymes in tissues partly contributes to. We next used established quantitative glycoproteomics and proteomics methods to identify site-specific substrates that may contribute to biologically relevant phenotypes when Galnt2 was genetically ablated in a Galnt2-null mouse model, which presents with lipid and metabolic dysregulation. Our findings suggest networks of Galnt2 direct and indirect interactions that may explain the complex metabolic phenotypes. The mouse O-glycoproteome-map and quantitative glycoproteomics/proteomics approach will provide a valuable foundation for revealing the significance of O-glycosylation biology in CDGs and other diseases and conditions.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Whole Body

DISEASE(S): Congenital Disorder Of Glycosylation

SUBMITTER: Weiming Yang  

LAB HEAD: Lawrence A. Tabak

PROVIDER: PXD040196 | Pride | 2024-05-24

REPOSITORIES: Pride