PMID:7927684
| Citation |
Grant, CC, Messer, RJ and Cieplak, W Jr (1994) Role of trypsin-like cleavage at arginine 192 in the enzymatic and cytotonic activities of Escherichia coli heat-labile enterotoxin. Infect. Immun. 62:4270-8 |
|---|---|
| Abstract |
Previous studies of cholera toxin and Escherichia coli heat-labile enterotoxin have suggested that proteolytic cleavage plays an important role in the expression of ADP-ribosyltransferase activity and toxicity. Specifically, several studies have implicated a trypsin-like cleavage at arginine 192, which lies within an exposed region subtended by a disulfide bond in the intact A subunit, in toxicity. To investigate the role of this modification in the enzymatic and cytotonic properties of heat-labile enterotoxin, the response of purified, recombinant A subunit to tryptic activation and the effect of substituting arginine 192 with glycine on the activities of the holotoxin were examined. The recombinant A subunit of heat-labile enterotoxin exhibited significant levels of ADP-ribosyltransferase activity that were only nominally increased (approximately twofold) by prior limited trypsinolysis. The enzymatic activity also did not appear to be affected by auto-ADP-ribosylation that occurs during the high-level synthesis of the recombinant A subunit in E. coli. A mutant form of the holotoxin containing the arginine 192-to-glycine substitution exhibited levels of cytotonic activity for CHO cells that were similar to that of the untreated, wild-type holotoxin but exhibited a marked delay in the ability to increase intracellular levels of cyclic AMP in Caco-2 cells. The results indicate that trypsin-like cleavage of the A subunit of E. coli heat-labile enterotoxin at arginine 192 is not requisite to the expression of enzymatic activity by the A subunit and further reveal that this modification, although it enhances the biological and enzymatic activities of the toxin, is not absolutely required for the enterotoxin to elicit cytotonic effects. |
| Links | |
| Keywords |
ADP Ribose Transferases/metabolism; Adenosine Diphosphate Ribose/metabolism; Animals; Bacterial Toxins/chemistry; Bacterial Toxins/isolation & purification; Bacterial Toxins/toxicity; Base Sequence; CHO Cells; Cricetinae; Cyclic AMP/biosynthesis; Enterotoxins/chemistry; Enterotoxins/isolation & purification; Enterotoxins/toxicity; Escherichia coli/enzymology; Escherichia coli Proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Recombinant Proteins/isolation & purification; Structure-Activity Relationship; Trypsin/pharmacology |
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