PANETH CELL AND GASTROINTESTINAL HOST DEFENSE

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The objective of these studies is to define the molecular basis of mucosal host defense by investigating the synthesis of endogenous antimicrobial peptides by Paneth cells. Paneth cell defensins, or cryptdins, have been implicated as components of the mucosal barrier, because they are abundant granule components with diverse primary structures, release into the lumen, and potent microbicidal proximal region of chromosome 8 that have highly-conserved, two-exons structures. Despite diverse primary structures of mature cryptdin peptides, their prepro-coding regions and propieces are almost identical, suggesting that certain residues within Paneth cell defensin precursors have roles in the packaging of cryptdin genes along the proximal-distal axis in adult and developing small intestine, to investigate precursors of potential importance in posttranslational processing. The first aim is to determine whether individual crypts differ in the expression of specific cryptdin genes; amplified cDNAs from isolated crypts along the promixmal- distal axis will be assayed for cryptdin isoform sequences by hybridization to peptide-specific oligonucleotides. Similarly, the kinetics of cryptdin isoform mRNA appearance will be determined during intestinal development in neonatal mice to test whether cryptdin genes are induced selectively or in concert. The second aim is to test whether amino acid changes in cryptdins influence or modulate biological activity; the biochemical and antimicrobial properties of recombinant peptides corresponding to naturally-occurring and mutagenized cryptdins will be characterized. The third aim will test whether cryptdin precursors contain determinants in targeting enteric defensins to Paneth cell granules; the effect of natural and mutated cryptdin-1 propieces on the distribution of chimeric reporter gene targeting will be measured in Paneth cells from isolated crypts and compared with packaging in mouse macrophage cell lines to establish whether defensin trafficking to granules occurs by conserved or cell-specific mechanisms. These studies should characterize the molecular distribution of enteric defensins, define functional determinants for biological activity, and provide knowledge of events leading to their release into the lumen.