The long term goal of this program is to understand what impact mucin- type O-linked sugars have on oropharyngeal development, differentiation and function. O-linked glycoproteins are dynamic constituents of the cell surface and extracellular environment; however, the roles of O- linked sugars in defining or influencing cellular properties or interactions are not fully understood either during embryogenesis or in the formation of adult tissues. O-glycosylation begins through the action of a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyl- transferases [E.C.2.4.1.41] (ppGaNTases), which transfer the first sugar residue N-acetylgalactosamine (GalNAc) to specific threonine or serine residues of secreted or cell surface proteins. In mammals, six different ppGaNTase enzymes have been isolated, cloned and expressed; however, the expressed sequence tag database suggests that at least 14 ppGaNTase homologs may exist. While many of the cloned members of this gene family glycosylate a similar repertoire of peptide sequences, some ppGaNTase isoforms have discrete specificities for peptide sequences and have expression patterns that appear to be regulated in a tissue specific pattern during development. This observation is consistent with a model that O-glycosylation of target proteins is regulated by the coordinate expression of cognate ppGaNTase isoforms. Because mammals express a large family of ppGaNTase isoforms, we have selected a simple model organism to study the role of O-glycosylation in development. Caenorhabditis elegans was selected because a mutation in a single apomucin gene (let-653) produced a defect in the pharyngeal/head region of the nematode. This mutation is lethal, severely distorts the secretory gland cell morphology, and is the only known example of a genetic defect in an apomucin gene of either vertebrates or invertebrates (Jones and Baillie, 1995). Exon 7 of the let-653 gene encodes a threonine/serine-rich domain, in which 69 percent of the amino acid residues are potentially O-glycosylated. We hypothesize that the O-linked sugars that modify this mucin are essential of the LET653 mucin function and that a defect in the O- glycosylation apparatus will phenocopy the let-653 mutation. To test this hypothesis, our first specific aim will be to: determine the expression pattern of the let-653 apomucin gene and ppGaNTase isoforms in the C. elegans pharyngeal and head region. To determine if the O- linked sugars are essential for mucin function, our second specific aim will be to: inhibit either apomucin protein translation or O-glycan acquisition by using let-653 or ppGaNTase antisense RNA.
