The long term goals of this research are to elucidate the structure and function of porcine gastric mucin and to investigate how this glycoprotein contributes to gastric physiology, in particular to protection against acid secretion in the stomach. Two specific hypotheses will be tested in our experiments: 1) that gastric mucin peptide (apomucin), although accounting for only 20% of the molecule as against the 80% sugars, is a major determinant of the unique viscous properties of gastric mucins: 2) the ability of mucin to polymerize and form a gel is directly correlated with the protective properties of gastric mucin against secreted hydrochloric acid and other noxious agent such as proteases, oxygen radicals and alcohol.
Three specific aims are designed to test our hypotheses using a combination of molecular and biophysical techniques.
Aim 1 is to clone and sequence the major porcin gastric mucin genes. Purified porcine gastric mucin will be chemically deglycosylated, antibodies raised against this sugar-free apo-mucin will be used to screen a gastric cDNA library and positive clones will be purified and sequenced. Northern blot analysis and in situ hybridizatio will be used to localize the cells and organs of origin of these apomuci cDNA clones.
Aim 2 is to use these clones to obtain cross-hybridizing probes to rat gastric mucin, which then can be used to study the regulation of mucin gene expression in rats after physiologic and pharmacologic stimuli.
Aim 3 is to express truncated mucin genes in eukaryotic expression vectors to obtain large quantities of truncated peptides and characterize them as regards to their aggregation and gelation properties, their ability to bind lipids and retard h+ diffusion. These findings will enable us to determine the contribution of the various structural regions of apomucin to aggregation and gelation. These studies will provide unique structural, functional and physiologic data on gastric mucins. Since these molecules provide a protective barrier against H+ diffusion in the mammalian stomach, our results are of direct relevance to human peptic ulcer disease, H. pylori gastritis and gastric cytoprotection.
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