Study On A Putative Udp-Galnac: Polypeptide N-Acetylgalactosaminyltransferase Gene (ppGalNAc-T20): Enzyme Activity And Antibody

The polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts, EC 2.4.1.41) play a key role in mucin-type O-linked glycan biosynthesis by installing the intial GalNAc residue on the rotein scaffold. The first ppGalNAc-T activity was reported in 1967 by McQuire and Roseman. Up to now, over 16 distinct isoforms of mammalian enzymes have been biochemically characterized in this family. Each isoform exhibits a different tissue distribution and substrate specificity. Immunohistochemical studies have shown an altered expression of some ppGalNAc-Ts in the tumor development. ppGalNAc-T6 is an immunohistochemical marker for breast carcinoma cells. ppGalNAc-T13 is an informative marker for the molecular diagnosis of bone marrow involvement in neuroblastoma. ppGalNAc-T14 modulates Apo2L/TRAIL signaling in tumors through death-receptor O-glycosylation.Using the BLAST search of GenBank database, we found an expressed sequence tag which identified as homologues to ppGalNAc-T10. We obtained the complete ORF, named it ppGalNAc-T20. The deduced amino acid sequence of ppGalNAc-T20 contains all conserved motifs in ppGalNAc-T family proteins. Analysised by the phylogenetic tree of ppGalNAc-Ts, ppGalNAc-T20 is 70.7% in the homology with ppGalNAc-T10, based on the amino acid sequences. They are the closest members of the ppGalNAc-T members in the phylogenetic tree. Compared to the significant expressions of ppGalNAc-T10 in rat sublingual gland, testis, small intestine, colon and ovary, with lesser amounts in heart and brain, ppGalNAc-T20 is only detected in brain and testis in a comparatively high level. The activity of ppGalNAc-T20 is distinguished from the previously identified form to support the existence of a hierarchical network of differential enzymatic acitivity within the diversely regulated ppGalNAc-Ts family.To study the enzyme activity of ppGalNAc-T20, we prepared mono-EA2 as glycopeptides substitute for the activity detecting. Further more, two truncated ppGalNAc-T20 were constructed to detect the different enzyme activity in vitro based on various protein structure. We have also predicted the crystal protein structure by using SWISS-PORT and VMD software in a bioinformatics way. Based on the various result of enzyme activity on different types truncated ppGalNAc-T20s, we find a significant difference in protein structure, which may lead to the loss of enzyme activity. To further understand the function of ppGalNAc-T20, we produced a polyclonal antibody against ppGalNAc-T20. The anti-T20 antibody has very good specificity and sensitivity; it does not react with other homologous ppGalNAc-T. The antibody provides a good tool for studying the biofunctions of ppGalNAc-T20.In this thesis, we provided original experiments on ppGalNAc-T20, this will provide a substantial base for further study of its biological function.