Structure and Function of UDP-GalNAc:polypeptide alpha-GalNAc transferases

National Institute Of Dental & Craniofacial Research

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Abstract

The family of GalNAc-Ts catalyzes the first committed step in the synthesis of O-glycans, which is an abundant and biologically important protein modification. Abnormalities in the activity of individual GalNAc-Ts can result in congenital disorders of O-glycosylation (CDG) and influence a broad array of biological functions. How site-specific O-glycans regulate biology is unclear. We have integrated chemical and enzymatic conditions that cleave O-glycosites, a higher-energy dissociation product ions-triggered electron-transfer/higher-energy collision dissociation (HCD-pd-EThcD) MS workflow, and software to study nine mouse tissues and whole blood to localize specific sites of O-glycosylation. In this manner we have identified several thousand O-glycosites from mouse glycoproteins. The O-glycosites and glycoproteins displayed consensus motifs and shared functions as classified by Gene Ontology (GO) terms. Limited overlap of O-glycosites was observed with protein O-GlcNAcylation and phosphorylation sites. 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 examined the Galnt2-null mouse model, which phenocopies CDG-GALNT2 and revealed a network of glycoproteins that lack GalNAc-T2-specific O-glycans. The known direct and indirect functions of these glycoproteins appear consistent with the complex metabolic phenotypes observed in the Galnt2-null animals. Most recently we have extended this approach to study the Galnt3-null mouse, which phenocopies CDG-GALNT3. This work revealed considerable sexual dimorphism in both submandibular and sublingual glands. It also identified glycoproteins with sites that are uniquely modified by GalNAc-T3. A number of these glycoproteins function in promoting mineralization, which is consistent with the overall phenotype of CDG-GALNT3.