Intestinal alkaline phosphatase (IAP) in the rat provides a model for studying a unique mechanism of protein secretion from the enterocyte, in that IAP isomers I and II are secreted from the cell bound to a phospholipid-rich surfactant-like particle (SLP), which is distinct from other membranes. SLP also appears to play a role in the transcellular movement of lipid droplets during fat absorption, and in the binding of the papG adhesin of uropathogenic E. coli pili via a unique gal-gal containing receptor. This proposal will address unresolved issues regarding both IAP and SLP metabolism and function. IAP: 1) identify the structural and functional differences of the two IAP carboxyl-termini, including especially the role of the oligothreonine region of IAP-II in SLP production. 2) explore the intracellular trafficking and secretory pathways of the carboxyl-termini of the IAP isomers. 3) identify the two putative rat IAP genes and their predicted upstream RAR and RXR sequences. SLP: I) explore factors involved in the regulation of SLP production and metabolism and to study further the role of SLP in triacylglycerol secretion. 2) characterize colonic human and rat SLP and investigate its production by colonic cells in culture. 3) explore the binding of bacterial adhesins to SLP. 4) identify cDNA clones encoding rat SLP proteins. Reagents available for IAP studies include the unique 3'- terminal coding regions of the IAP isomers (cDNA constructs) for in situ hybridization, antisera raised against the unique carboxyl-terminal protein sequences of each IAP isomer, mutants with altered putative glycosyl-phosphatidylinositol linkage sites, a cell line that can alter glycosylation of expressed proteins, fusion proteins linking the carboxyl- terminal ends of each IAP to the PvuII site of rat or human intrinsic factor, cDNA probes for each IAP isomer, and three rat genomic libraries. Reagents available for SLP studies include antisera against rat and human small intestinal and colonic SLP, antisera against rat colonic (tissue unspecific) AP and against human and rat surfactant protein-A (enriched in colonic SLP), papG and FimH adhesins, and type P and type I pili from uropathogenic E. coli, and antiserum and a best-guess oligonucleotide identifying the amino-terminal sequence of the 40 kDa protein of small intestinal SLP. Reagents in preparation include Caco-2 cells stably transfected with cDNAs encoding the IAP isomers, antisera against Caco-2 SLPs, immuno-electron microscopy using IAP and SLP antisera, and a rat duodenal cDNA library. These studies will impact digestive health by developing a) unique secreted small intestinal and colonic protein markers that may reflect in serum the health of the mucosa, b) a new site for bacterial colonization, one that could be exploited to study agents that affect bacterial binding to the intestine, and c) a role for SLP in fat absorption, possibly leading to a serum marker for the adequacy of fat absorption and to better understanding of the process of fat absorption.
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