REGULATION OF MUCIN GLYCOPROTEIN BIOSYNTHESIS

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The long-term goal of this program is to understand how the biosynthesis of mucin-glycoproteins (mucins) is regulated. Mucins are the principal organic constituent of mucus, the slimy viscoelastic coat which protects all mucosal surfaces of the body. The addition of N-acetylgalactosamine (GalNAc) to the hydroxyamino acids within the protein backbone of mucin (apomucin) represents the first committed step in mucin biosynthesis. This reaction is catalyzed by a UDP-GalNAc polypeptide:N- acetylgalactosaminyltransferase (ppGaNTase). During the current program period the rat submandibular gland (RSMG) apomucin was cloned. It is architecturally similar to human salivary mucin MG2 and is composed of three distinct regions. Given the diversity of potential O-glycosylation sites within RSMG apomucin, we reasoned that there were multiple forms of ppGaNTase. Through molecular cloning and biochemical analysis, evidence for a family of ppGaNTases has been found. We hypothesize that the glycosylation of apomucin requires the coordinated action of discrete isoforms of ppGaNTase. To test this hypothesis, our first specific aim will be to: identify and characterize the isoforms of ppGaNTase which are expressed in the RSMG. To provide evidence for the functional importance of ppGaNTase isoforms, our second specific aim will be to: correlate sites of RSMG mucin O-glycosylation with the substrate specificities of the repertoire of ppGaNTases found in the RSMG. Despite the central role played by the ppGaNTase in regulating the initiation of mucin glycosylation, we know very little about how this enzyme functions. To address the gaps in our basic understanding of ppGaNTase, our third specific aim will be to: map the catalytic domain and Golgi localization signal of ppGaNTase T-1. Our current understanding of mucin function is based on indirect evidence. We hypothesize that salivary mucins play a significant role in protecting the hard and soft tissues of the mouth. To test this hypothesis, our fourth specific aim will be to: develop methods to modulate the expression of mucin in vivo.