This research will characterize the mechanism of action of the E. coli heat-stable enterotoxin (STa) which specifically activates particulate guanylate cyclase of small intestinal tissue. It will be determined if the specific high affinity STa receptor and guanylate cyclase are separate proteins, or a single transmembrane glycoprotein. Photo-active GTP derivatives and 125I-STa will be used to label the active site of particulate guanylate cyclase and the STa receptor, respectively. Homobifunctional and heterobifunctional crosslinking reagents will be used to characterize protein-protein interactions of the STa receptor, guanylate cyclase, and other closely associated membrane proteins. Particulate rat intestinal guanylate cyclase will be extracted from intestinal membranes with detergents. The enzyme will be purified and characterized with respect to chemical properties, subunit structure, and antigenic properties. The mechanism of processing and secretion of STa and E. coli heat-labile enterotoxin (LT) will be established. Pulse labeling techniques will be used to detect precursors of STa and LT subunits and determine if secretion occurs via a co-translational or post-translational mechanism. An in vitro bacterial secretory system will be used to characterize the synthesis and secretion of STa and LT subunits. Hybridoma cell cultures and monoclonal antibody technology will be used to characterize the antigenic determinants of LTs produced by human (LThs) and porcine (LTps) strains of enterotoxigenic E. coli (ETEC). Reactivity of monoclonal antibodies with the A and B subunits of LTps and LThs and cholera toxin will be determined. Monoclonal antibodies to the B subunits will be grouped according to their neutralization capacity against homologous and heterologous LT-Bs and used to map different epitopes. Epitopes present in protease digests of LT-B will be isolated by immunoaffinity chromatography. Manual sequence analysis will be used to locate the epitope within the LT-B polypeptide. Also, amino acid residues involved in binding of LT to Gml ganglioside will be identified. The E. coli enterotoxin with biological activity in piglets, but not suckling mice, (STb) will be purified and characterized if an in vitro assay can be developed.
| Amirmozafari, N; Robertson, D C (1993) Nutritional requirements for synthesis of heat-stable enterotoxin by Yersinia enterocolitica. Appl Environ Microbiol 59:3314-20 |
| Jaso-Friedmann, L; Dreyfus, L A; Whipp, S C et al. (1992) Effect of age on activation of porcine intestinal guanylate cyclase and binding of Escherichia coli heat-stable enterotoxin (STa) to porcine intestinal cells and brush border membranes. Am J Vet Res 53:2251-8 |
