Cloning and characterization of a ninth member of the UDP-GalNAc : polypeptide N-acetylgalactosaminyltransferase family, ppGaNTase-T9

We have cloned, expressed and characterized the gene encoding a ninth membe r of the mammalian UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGaNTase) family, termed ppGaNTase-T9. This type II membrane protein con sists of a 9-amino acid N-terminal cytoplasmic region, a 20-amino acid hydr ophobic/transmembrane region, a 94-amino acid stem region, and a 480-amino acid conserved region. Northern blot analysis revealed that the gene encodi ng this enzyme is expressed in a broadly distributed manner across many adu lt tissues. Significant levels of 5- and 4.2-kilobase transcripts were foun d in rat sublingual gland, testis, small intestine, colon, and ovary, with lesser amounts in heart, brain, spleen, lung, stomach, cervix, and uterus. In situ hybridization to mouse embryos (embryonic day 14.5) revealed signif icant hybridization in the developing mandible, maxilla, intestine, and mes encephalic ventricle. Constructs expressing this gene transiently in COS7 c ells resulted in no detectable transferase activity in vitro against a pane l of unmodified peptides, including MUC5AC (GTTPSPVPTTSTTSAP) and E6A2 (PTT DSTTPAPTTK). However, when incubated with MUC5AC and EA2 glycopeptides (obt ained by the prior action of ppGaNTase-T1), additional incorporation of Gal NAc was achieved, resulting in new hydroxyamino acid modification. The acti vity of this glycopeptide transferase is distinguished from that of ppGaNTa se-T7 in that it forms a tetra-glycopeptide species from the MUC5AC tri-gly copeptide substrate, whereas ppGaNTase-T7 forms a hexa-glycopeptide species . This isoform thus represents the second example of a glycopeptide transfe rase and is distinct from the previously identified form in enzymatic activ ity as well as expression in embryonic and adult tissues. These findings le nd further support to the existence of a hierarchical network of differenti al enzymatic activity within the diversely regulated ppGaNTase family, whic h may play a role in the various processes governing development.