LT is an AB5-type enterotoxin (consisting of one A-subunit and five B-subunits) produced by some strains of Escherichia coli and is responsible for the characteristic secretory diarrhea associated with this organism. LT and related toxins are also known to have powerful mucosal adjuvant activity for co-administrated protein antigens. The 28 kDa A-subunit has an ADP-ribosyltransferase that is responsible for ADP-ribosylating the GTP-binding protein Gsalpha found on the cell membrane of eukaryotic cells, leading to the subsequent activation of adenylate cyclase and increasing intracellular levels of cAMP. The 55 kDa B-subunit facilitates the entry of the LT through its binding to the host cell membrane receptor, GM1. In addition to ADP-ribosylation of Gsalpha, LT also undergoes auto-ADP-ribosylation, transferring ADP-ribose from NAD to one or more of its own arginine residues. The role of auto-ADP-ribosylation of LT in the toxicity and adjuvanticity of this molecule is currently unknown. In this study we used Quadrupole tandem mass spectrometry to identify the arginine residues in the A-subunit that had been auto-ADP-ribosylated. Using site directed mutagenesis, LT mutants were constructed that corresponded to the nine arginine residues where the modification occurred (R18G, R25G, R33G, R129G, R138K, R141K, R148K, R151G & R192G). Each mutant was assessed for its structural integrity, sensitivity to proteolytic digestion, GM1 binding, enzymatic activity and auto-ADP ribosylation ability when compared with native toxin. LT mutants show different level of modification, activity, toxicity and adjuvanticity as compared with the native toxin
