Glycoprotein receptors for a heat-stable enterotoxin (STh) produced by enterotoxigenic Escherichia coli

T Hirayama, A Wada, N Iwata, S Takasaki, Y Shimonishi, Y Takeda
1992 Infection and Immunity  
Glycoprotein receptors for heat-stable enterotoxin STh of enterotoxigenic Escherichia colfi in the rat intestinal cell membrane were identified and characterized. Incubation of rat intestinal cell membranes with radioiodinated followed by photolysis resulted in specific radiolabeling of two distinct proteins with M,s of 200,000 (designated STR-200A and STR-200B). STR-200A was found to be composed of two molecules of a protein with an Mr of 70,000 (70-kDa protein), whereas STR-200B was composed
more » ... f two different protein molecules with Mrs of 53,000 (53-kDa protein) and 77,000 (77-kDa protein). These proteins showed no guanylate cyclase activity. The 70-kDa protein was labeled most with 125I-ANB-STh[5-191, suggesting that STR-200A is the main receptor protein in the rat intestinal cell membrane. The carbohydrate moieties of STR-200A and STR-200B were examined by enzymatic deglycosylation. The 70-kDa protein of STR-200A was found to contain N-linked high-mannose-type and/or hybrid-type oligosaccharides, and results suggested that it possesses at least three N glycosylation sites. The 53-kDa protein of STR-200B was found to have an N-linked complex-type oligosaccharide side chain. The deglycosylated 70-kDa protein retained activity for binding to STh, suggesting that the carbohydrate moieties of these receptor proteins are not important for binding with STh. Heat-stable enterotoxins (STs) produced by enterotoxigenic Escherichia coli are composed of 18 (STp [40]) or 19 (STh [1]) amino acid residues and cause secretory diarrhea in humans and animals (32, 37). In STh, the Cys-6 to Cys-18 region has a cyclindrical structure formed by three disulfide bonds between Cys-6 and Cys-11, Cys-7 and Cys-15, and Cys-10 and Cys-18 (35) and is its active domain (11, 35, 45) . There are reports that other enteropathogens, such as Yersinia enterocolitica (42), Vibrio cholerae non-Ol (41), Vibrio mimicus (34), and Citrobacter freundii (14) , also produce STs that are structurally similar to STh. These STs probably have biological activities similar to that of STh in the intestinal lumen. Structural details of the STs have been elucidated by protein chemical studies (1, 11, 14, 29, 34, 35, (40) (41) (42) 45) and DNA sequencing (28, 38), but the molecular mechanism by which STs induce secretory diarrhea is still not well understood. The initial step in the biological action of ST is its interaction with specific high-affinity receptors (3, 7, 9, 23, 29) . The binding of ST to the epithelial cell membranes of intestinal cells of rabbits (7, 30) and rats (6, 13, 15, 23) through these receptors stimulates membrane-bound guanylate cyclase in the cells, leading to an increase in the intracellular concentration of cyclic GMP, followed by activation of cyclic GMP-dependent protein kinase (4, 17), and culminating in the final biological reaction, inhibition of Na+ absorption and stimulation of Clsecretion (7). Scatchard analysis of stoichiometry by Franz et al. (9) and Cohen et al. (3) indicated a single receptor for STh on rat intestinal epithelial cells and membranes. On the other hand, Kuno et al. (23) demonstrated the presence of three specific,
doi:10.1128/iai.60.10.4213-4220.1992 fatcat:4prulsxxdjg3fhnoxogsd2zdr4