C3 exoenzyme

Bourmeyster N, Stasia M-J, Garin J et al. (1992) Copurification of rho protein and the rho-GDP dissociation inhibitor from bovine neutrophil cytosol. Effect of phosphoinosifides on rho ADP-ribosylafion by the C3 exoenzyme of Clostridium botulinum. In Biochemistry 31 12863-9... 

Saito Y, Nemoto Y, IshizakiTetal. (1995) Identification of Glu as the critical amino acid residue for the ADP-ribosyltransferase activity of Clostridium botulinum C3 exoenzyme. In FEBS Lett. 371 105—9... 

Preparation of Clostridium botulinum C3 Exoenzyme and Application of ADP-ribosylation of Rho Proteins in Biological Systems... 

The recombinant C3 exoenzyme that we used is a modified enzyme that does not contain the signal peptide, but has Met-Ala attached to Ser of the mature enzyme (Kumagai et a/., 1993). The modified gene was cloned into pET 3a vector, resulting in pET 3a C3, which allows the enzyme to be expressed under control of the bacteriophage T7 promoter. E. coli strain BL21 (DE3) cells are transformed with pET 3a C3. 

Isopropyl-1-thio- 3-D-galactopyranoside is added to a final concentration of 1 mM to induce C3 exoenzyme expression, followed by incubation at 37°C for another 3 h with agitation. 

Fig. 1. Time course of ADP-ribosylation of Rho. 1 ng of recombinant GST-Rho (fusion protein) was incubated with 20 (iM of [ P]-NAD and 3ng of the wild type ( ) or the E173Q (o) mutant of C3 exoenzyme in a total volume of 50 nl of ADP-ribosylation buffer, as described in the text, at 30°C for times indicated. The reaction was terminated by the addition of trichloroacetic acid and Na deoxycholate. After centrifugation, the pellets were subjected to SDS polyacrylamide gel electrophoresis, followed by staining with Coomassie Brilliant Blue and autoradiography. P-ADP-ribosylation of Rho was quantified by cutting out the radiolabeled GST-Rho bands and determining their radioactivities. The relative ADP-ribosylation was expressed as the ratio of the P incorporation into GST-Rho at each point to the maximal P incorporation by the wild type C3 exoenzyme... 

Figure 1 shows a time course for ADP-ribosylation using 1 g of recombinant Rho fused with glutathione S transferase and 3 ng of C3 exoenzyme. The reaction proceeds in a time-dependent manner and reaches a plateau at 30 min. In contrast, no ADP-ribosylation is observed in the reaction using the E173Q mutant (Fig. 1). The recombinant wild-type enzyme has a catalytic activity of 1.3 pmol of ADP-ribose transferred/ng/h and the Km value for NAD is 0.125 [iM (Fig. 2).

The reaction mixture consists of 100 mM Tris HCI, pH 8.0, 10 mM nicotinamide, 10 mM thymidine, 10 mM dithiothreitol, 5mM MgCb, 10 nM pP]-NAD (900 cpm/pmol) (ADP-ribosylation buffer), purified C3 exoenzyme, and recombinant Rho or a crude homogenate in a total volume of 100 jil. The mixture is incubated at 30 C. To measure the amount of Rho, the ADP-ribosylation reaction should reach a plateau. When 50 ng of C3 exoenzyme... 

The biological effects of C3 exoenzyme correlate well with the extent of in sifu ADP-ribosylation of Rho. However, the biological effect does not vary linearly with the extent of ADP-ribosylation, but is... 

Fig. 3. Autoradiogram of P-ADP-ribosylation of Rho in Swiss 3T3 cell homogenate. Swiss 3T3 cells were treated with buffer alone (lane 1), 30 ng/ml of the wild type (lane 2) or the E173Q mutant (lane 3) of C3 exoenzyme for 72 h in DMEM containing 10 % fetal bovine serum. The cells were washed twice in phosphate-buffered saline (PBS), and incubated with 0.05 % (w/v) trypsin in PBS. The detached cells were collected and suspended in ADP-ribosylation buffer containing [ P]-NAD, followed by sonication. The homogenates were incubated with 100 ng of the wild type C3 enzyme at 30°C for 2 h. The reaction was terminated by the addition of trichloroacetic acid and Na deoxycholate. The pellets were subjected to 12 % SDS polyacrylamide gel electrophoresis and autoradiography. The positions of molecular weight markers are indicated on the left. The position of ADP-ribosylated Rho is indicated by an arrow...

Fig. 4. Effects of the wild type and mutant C3 exoenzyme on the morphology of Swiss 3T3 cells. Swiss 3T3 cells were treated with buffer alone (a), the wild type (b) or the E173Q (c) mutant of C3 exoenzyme tor 72 h. Morphology was examined using a phase-contrast microscope and photographed... 

Morii N, Ohasi Y, Nemoto Y etal. (1990) Immunochemical identification of the ADP-ribosyltransferase in Botulinum Cl neurotoxin as C3 exoenzyme-like molecole. J. Biochem. 107 769- 775. 

Yamamoto M, Marui N, Sasaki T et al. (1993) ADP-ribosylation of the rho A gene product by C3 exoenzyme causes Swiss 3T3 cells to accumulate in the G1 phase of cell cycle. Oncogene 8 1449-1455. 

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