Analyses of the mutant analogs of Escherichia coli heat-labile enterotoxin, a close relative of cholera toxin, have revealed that alterations which abrogate ADP-ribosyltransferase activity also impart conformational changes that are detectable by tryptic fragmentation patterns. However, conservative substitution at tryptophan 170 by tyrosine yields a molecule with reduced activity and apparent wild-type conformation as judged by trypsin sensitivity. This mutation, in conjunction with other previously described substitutions may be a suitable target for the generation of, mutiply substituted mutant proteins that are devoid of toxic/enzymatic activity and that retain native conformation. We are investigating various amino acid substitutions at various targets in the hope of generating mutants with the appropriate phenotypic characteristics (i.e. no enzymatic activity and no gross conformational alterations). Additional studies have been conducted to evaluate the effect of post- translational modifications on the enzymatic activity of the A subunit of LT. These studies have revealed that limited proteolytic cleavage is not absolutely necessary for the expression of enzymatic activity and that multiple auto-or self ADP-ribosylation does not detectably change the specific enzymatic activity of the toxin. These findings contrast the results of studies conducted by other groups using cholera toxin. Further studies have investigated the manner in which LT is trafficked in eukaryotic cells using mutant analogs and mutant cell lines. These studies have revealed that the toxin likely does not enter cells using a Golgi- dependent retrograde pathway and probably gains access to the target substrates (G proteins) via an early endosomal compartment.
