In vivo mapping of the mouse Galnt3-specific O-glycoproteome

• Published in: The Journal of biological chemistry 2024-09, Vol.300 (9), p.107628, Article 107628
• Main Authors: Dalal, Kruti, Yang, Weiming, Tian, E., Chernish, Aliona, McCluggage, Peggy, Lara, Alexander J., Ten Hagen, Kelly G., Tabak, Lawrence A.
• Format: Article
• Language: English
• Subjects: Animals; Fibroblast Growth Factor-23 - metabolism; GalNAc-T; Galnt; glycopeptides; Glycoproteins - genetics; Glycoproteins - metabolism; Glycosylation; glycosyltransferase; Humans; hyperphosphatemic familial tumoral calcinosis (HFTC); mass spectrometry; Mice; Mice, Knockout; N-Acetylgalactosaminyltransferases - genetics; N-Acetylgalactosaminyltransferases - metabolism; O-GalNAc; O-glycosylation; Polypeptide N-acetylgalactosaminyltransferase; Proteome - metabolism
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1016/j.jbc.2024.107628

The UDP-N-acetylgalactosamine polypeptide:N-acetylgalactosaminyltransferase (GalNAc-T) family of enzymes initiates O-linked glycosylation by catalyzing the addition of the first GalNAc sugar to serine or threonine on proteins destined to be membrane-bound or secreted. Defects in individual isoforms of the GalNAc-T family can lead to certain congenital disorders of glycosylation (CDG). The polypeptide N-acetylgalactosaminyltransferase 3 (GALNT)3-CDG, is caused by mutations in GALNT3, resulting in hyperphosphatemic familial tumoral calcinosis due to impaired glycosylation of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23) within osteocytes of the bone. Patients with hyperphosphatemia present altered bone density, abnormal tooth structure, and calcified masses throughout the body. It is therefore important to identify all potential substrates of GalNAc-T3 throughout the body to understand the complex disease phenotypes. Here, we compared the Galnt3−/− mouse model, which partially phenocopies GALNT3-CDG, with WT mice and used a multicomponent approach using chemoenzymatic conditions, a product-dependent method constructed using EThcD triggered scans in a mass spectrometry workflow, quantitative O-glycoproteomics, and global proteomics to identify 663 Galnt3-specific O-glycosites from 269 glycoproteins across multiple tissues. Consistent with the mouse and human phenotypes, functional networks of glycoproteins that contain GalNAc-T3-specific O-glycosites involved in skeletal morphology, mineral level maintenance, and hemostasis were identified. This library of in vivo GalNAc-T3-specific substrate proteins and O-glycosites will serve as a valuable resource to understand the functional implications of O-glycosylation and to unravel the underlying causes of complex human GALNT3-CDG phenotypes.