Dataset Information

Map of the Neuronal Tissue O-glycoproteome Reveal Specific Traits in the Secretogranin Family

ABSTRACT: Protein O-glycosylation is an abundant PTM of the central nervous system and impairments in O-glycosylation cause severe developmental disorders most with prominent neurological involvement. Despite recent advancements, lack of insight into targets of protein O-glycosylation in neuronal tissues severely hampers progression in unravelling disease etiology. Here we report a comprehensive map of GalNAc-type O-glycoproteins (>800) and O-glycosites (>4,000) from neuronal tissues and cell lines compiled in a web-based resource. We identified abundant O-glycosites within major classes of proteins involved in neuroplasticity such as perineural nets, synapse formation, axon guidance, membrane remodeling and dense core granulogenesis. We demonstrate that Chromogranin A, a key player in neurotransmitter signaling, has abundant O-GalNAc as well as glycosaminoglycan glycosylation, and that these modifications are important for proper multimerization. In agreement with this, neuronal cell lines deficient in O-glycosylation exhibit higher capacity for storing the neurotransmitter noradrenaline and enlarged neurotransmitter-containing dense core granules.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Dendritic Cell, Cell Culture

SUBMITTER: Jonas Nilsson

LAB HEAD: Katrine Schjoldager

PROVIDER: PXD058040 | Pride | 2025-07-28

REPOSITORIES: Pride

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Publications

Map of the neuronal O-glycoproteome reveals driver functions in the regulated secretory pathway.

Madsen Thomas D TD Topaktas Asli B AB Dworkin Leo A LA Hintze John J Hansen Lasse H LH Nikpour Mahnaz M Lackman Jarkko J JJ Goth Christoffer K CK Larson Göran G Shiplett Emily J EJ Edmondson Andrew C AC Zhou Zhaolan Z Miller Rebecca L RL Joshi Hiren J HJ Vakhrushev Sergey Y SY Schjoldager Katrine T KT

The Journal of biological chemistry 20250529 7

Impairments in protein glycosylation, including O-GalNAc-type glycosylation, are linked to severe developmental disorders with prominent neurological involvement. However, the role of this glycosylation pathway at a cellular level is not yet fully understood. Here, we report a comprehensive map of GalNAc-type O-glycoproteins (>800) and O-glycosites (>4000) from neuronal tissues and cell lines and identify abundant O-glycosites within major classes of proteins involved in neuroplasticity, includi ...[more]

PMID: 40449597

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Project description: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.

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