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J. Biol. Chem., Vol. 277, Issue 14, 11941-11948, April 5, 2002

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The ADP-ribosylating Mosquitocidal Toxin from Bacillus sphaericus
PROTEOLYTIC ACTIVATION, ENZYME ACTIVITY, AND CYTOTOXIC EFFECTS^*

Jörg Schirmer, Ingo Just, and Klaus Aktories^§

From the Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, D-79104 Freiburg, Germany

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. Glu¹⁹⁷ of MTX^30-264 was identified as the "catalytic" glutamate that is conserved in all ADP-ribosyltransferases. Whereas mutated MTX^30-264E197Q has neither ADP-ribosyltransferase nor NAD glycohydrolase activity, mutated MTX^30-264E195Q 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^30-264E197Q mutant, indicating that the MTX enzyme activity is essential for the cytotoxicity in mammalian cells.

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