title: The ADP-ribosylating mosquitocidal toxin from Bacillus sphaericus: proteolytic activation, enzyme activity, and cytotoxic effects.
abstract: The mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1 is a approximately 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 microm for the processed 32-kDa MTX fragment, and a K(m) NAD value of 1300 microm 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.
authors.affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, D-79104 Freiburg, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Schirmer
authors.forename: Ingo
authors.initials: I
authors.lastname: Just
authors.forename: Klaus
authors.initials: K
authors.lastname: Aktories
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D002918:Chymotrypsin / Q000494:pharmacology; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
chemical_list: D001427:Bacterial Toxins; D011993:Recombinant Fusion Proteins; C068603:mosquitocidal toxin SSII-1; D000246:Adenosine Diphosphate Ribose; D005982:Glutathione Transferase; D002918:Chymotrypsin