The Functional Research And Substrates Screening Of A New Subfamily Gene Of O-Glycosyltransferase

Glycosylation is a significant post-translational modification in cells and more than 50% of eukaryotic proteins are glycosylated. Glycans possess complex functions in a variety of biological conditions, such as intracellular protein folding and stability, lymphocyte trafficking, cell adhesion and migration, host-pathogen interaction, immune response, fertilization, and embryogenesis.Glycosylation can be classified into two common forms, N-glycosylation and O-glycosylation. The first step of mucin-type O-glycosylation is catalyzed by members of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T, EC 2.4.1.41) family and each member have unique expression pattern and substrate specificity. In this report, the function and the characteristic of a new subfamily gene of ppGalNAc-Ts, designated as Y subfamily, were researched. The Y subfamily consists of four members, they are ppGalNAc-T8, -T9, -T17, and -T18 which have significant mutations at the protein level compared with other ppGalNAc-Ts. Genetic analysis revealed that Y subfamily members only exist in vertebrate. Interestingly, all four Y subfamily members lack the classical in vitro GalNAc-transferase activity. Structural analysis by homology modeling of the defining member of Y subfamily, ppGalNAc-T18, suggested that mutations in the UDP-GalNAc binding pocket of Y subfamily may lead to the lost of their transferase activity. Furthermore, ppGalNAc-T18 localizes in ER rather than Golgi apparatus in lung carcinoma cells. We also study the biological function of Y subfamily, although the function has not been fully understood, their distinct expression indicates that they might have critical roles in O-glycosylation of vertebrate.Nowadays, the substrates research of ppGalNAc-T is a hot topic of Glycobiology research. It is significant to our understanding of the functions of O-glycosylation. Azido labeled substrate analog of glycosyltransferase is a new glycosylation detection method which is more specific and stable than traditional detection by antibody or lectin. The glycoproteins bearing azides can be selectively detected by alkyne labeled fluorescence through Click reaction. Here, we combine the azido labeled donor substrate analog of ppGalNAc-T, UDP-GalNAz, and the protein microarray technique to optimize the experimental condition on the glycosylable peptide chip, and finally construct a high-throughput ppGalNAc-T substrates screen strategy. From the 16,000 proteins on chip, we successfully found out 226 substrate candidates and some of them are transcription factors, kinase and endocytosis related proteins and so on. Our study provides a powerful strategy to systematically screen the substrates of 20 human ppGalNAc-Ts and simultaneously provides insight into the functional understanding of ppGalNAc-T and mucin-type O-glycosylation.