The ADP-ribosylating Mosquitocidal Toxin from Bacillus sphaericus

Abstract

The mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1 is a ∼97-kDa protein sharing sequence homology within the N terminus with the catalytic domains of various bacterial ADP-ribosyltransferases. Here we studied the proteolytic activation of the ADP-ribosyltransferase activity of MTX. Chymotrypsin treatment of the 97-kDa MTX holotoxin (MTX 30–870) results in a 70-kDa putative binding component (MTX 265–870) and a 27-kDa enzyme component (MTX 30–264), possessing ADP-ribosyltransferase activity. Chymotryptic cleavage of an N-terminal 32-kDa fragment of MTX (MTX 30–308) also yields MTX 30–264, but the resulting ADP-ribosyltransferase activity is much greater than that of the processed MTX 30–870. Kinetic studies revealed a K m NAD value of 45 μm for the processed 32-kDa MTX fragment, and a K m NAD value of 1300 μm for the processed holotoxin. Moreover, the k cat value for the activated MTX 30–308 fragment was about 10-fold higher than that for the activated holotoxin (MTX 30–870). Precipitation analysis showed that the 70-kDa proteolytic fragment of MTX remains noncovalently bound to the N-terminal 27-kDa fragment, thereby inhibiting ADP-ribosyltransferase and NAD glycohydrolase activities. Glu 197 of MTX 30–264 was identified as the "catalytic" glutamate that is conserved in all ADP-ribosyltransferases. Whereas mutated MTX 30–264 E197Q has neither ADP-ribosyltransferase nor NAD glycohydrolase activity, mutated MTX 30–264 E195Q possesses glycohydrolase activity but not transferase activity. Transfection of HeLa cells with a vector encoding a fusion protein of MTX 30–264 with a green fluorescent protein led to cytotoxic effects characterized by cell rounding and formation of filopodia-like protrusions. These cytotoxic effects were not observed with the catalytically inactive MTX 30–264 E197Q mutant, indicating that the MTX enzyme activity is essential for the cytotoxicity in mammalian cells.