CDNA CLONING AND EXPRESSION OF A FAMILY OF UDP-N-ACETYL-D-GALACTOSAMINE-POLYPEPTIDE N-ACETYLGALACTOSAMINYLTRANSFERASE SEQUENCE HOMOLOGS FROM CAENORHABDITIS-ELEGANS

The initiation of mucin-type O-glycosylation is catalyzed by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTas e) (EC 2.4.1.41), By screening two mixed-stage Caenorhabditis elegans cDNA libraries, a total of 11 distinct sequence homologs of the ppGaNT ase gene family were cloned, sequenced, and expressed as truncated rec ombinant proteins (gly-3, gly-4, gly-5a, gly3-5b, gly-5c, gly-6a, gly- 6b, gly-6c, gly-7, gly-8, and gly-9), All clones encoded type II membr ane proteins that shaped 60-80% amino acid sequence similarity with th e catalytic domain of mammalian ppGaNTase enzymes, Two sets of cDNA cl ones (gly-5 and gly-6) contained variants that appeared to be produced by alternative message processing. gly-6c contained a reading framesh ift and premature termination codon in the C-terminal lectin-libe doma in found in most other ppGaNTase proteins, and a second clone (gly-8) racked the typical C-terminal region completely, Homogenates of nemato des and immunopurified. preparations of the recombinant GLY proteins d emonstrated that worms express functional ppGaNTase enzymes (GLY-3, GL Y-4, GLY-SA, GLY-5B, and GLY-5C), which can O-glycosylate mammalian ap omucin peptide sequences in vitro. In addition to demonstrating the ex istence of ppGaNTase enzymes in a nematode organism, the substantial d iversity of these isoforms in C. elegans suggests that mucin O-glycosy lation is catalyzed by a complex gene family, which is conserved among evolutionary-distinct organisms.