The Pasteurella multocida toxin (PMT) is a potent mitogen for fibroblasts [1]. PMT also affects bone cells, acting as a mitogen for osteoblasts and stimulating bone resorption in vitro [2–5]. PMT stimulates members of the G_q and G₁₂ families of heterotrimeric G-proteins [6–9], and these signalling proteins are thought likely to be the molecular targets of PMT. Their activation by PMT leads to numerous downstream sequelae including activation of phospholipase Cβ [6,10] and Rho-linked signalling [11,12].

PMT is the only toxin known to activate members of the G_q and G₁₂ G-protein families and is thus likely to become a useful tool for studying pathways linked to these important signalling molecules. We have already used PMT to investigate the function of tyrosine phosphorylation in the activation of G_q [8] and to identify the role of the Rho GTPase in osteoblast function [5].

PMT is an intracellularly-acting toxin of 1285 amino acids (146 kDa), which comprises a receptor binding domain at the N-terminus, followed by a translocation domain, and an active domain at the C-terminus [13–15]. We have previously analysed the role of cysteine residues in the toxin molecule and identified an essential role for C1165 [16]. Mutation of this residue completely abrogated activity without grossly affecting the toxin structure. Orth and co-workers subsequently suggested that 2 histidine residues within the same region (H1205 and H1223) were essential for activity [17]. Recently, Kitadokoro and co-workers have crystallised the C-terminal domain and suggest that it comprises 3 subdomains, including a domain (the C3 domain) that contains a catalytic triad that consists of C1165, H1205 and D1220 (PDB identifier 2EBF) [18]. We have analysed the role of all 4 histidine residues in the C3 subdomain and show here that H1205 is critical for activity, whereas mutation of H1223 has only a partial effect that is dependent on which amino acid replaces H1223.

The four published sequences of the PMT gene show a high degree of conservation and the four histidine residues analysed here are conserved and in the same context in each sequence. Histidine point mutants of PMT (PMT^H1202L, PMT^H1205L, PMT^H1223L, PMT^H1228L, PMT^H1202Y, PMT^H1205Y, PMT^H1223Y and PMT^H1228Y) were prepared as described in Methods. Sequencing confirmed that at least one isolate of each had the desired mutation. The 8 mutant toxins were all expressed in similar amounts to wild-type PMT (not shown). The relative activities of these mutant toxins were assessed in a number of assays. The ability of purified toxins to induce DNA synthesis in quiescent Swiss 3T3 cells was compared with normal PMT. PMT^H1205L and PMT^H1205Y were completely inactive up to a concentration of 100 ng ml^-1, whilst wild-type toxin stimulated DNA synthesis at as little as 0.3 ng ml^-1 (Fig. 1). PMT^H1202Y was as active as wild type toxin, whereas PMT^H1228Y was attenuated about 3-fold. Interestingly, PMT^H1223Y was around 30 times less active than wild-type toxin. For each of the 3 pairs of mutations, the associated leucine mutants were attenuated around a further 30-50 fold compared to the tyrosine substitutions. This meant that PMT^H1223L was at least 1000-fold less active than wild-type PMT

Fig. (1). Induction of DNA synthesis in quiescent Swiss 3T3 fibroblasts by PMT and mutants. The results are expressed as a percentage of the counts obtained following stimulation with 10 % foetal bovine serum. The results presented are the means and standard errors from six determinations at each point

The effect of the amino acid substitutions on the ability to induce cytoskeletal rearrangements was determined. PMT induced the formation of thick, parallel stress fibres, whereas untreated cells contained disorganised actin fibres (Fig. 2A). Only the three histidine mutants that demonstrated the least activity in the thymidine incorporation assay, PMT^H1205L, PMT^H1205Y and PMT^H1223L, failed to induce actin stress fibre formation under these conditions. The effect of the mutations on the activation of G_q was tested by assessing the ability of the mutant toxins to stimulate production of inositol trisphosphate (IP₃). PMT^H1205L, PMT^H1205Y and PMT^H1223L did not significantly induce inositol phosphate formation (Fig.2B), showing that they failed to activate G_q. The other mutants were all partially active. These results are summarised in Table 1.

Fig. (2). Effects of histidine mutants of PMT. A On the formation of actin stress fibres in quiescent Swiss 3T3 cells. Toxins were added at 5 ng ml-1 for 16 h. Additions were as follows: (a), untreated; (b) PMTH1202L; (c) PMTH1205L; (d) PMTH1223L; (e) PMTH1228L; (f) PMT(g) PMTH1202Y; (h) PMTH1205Y; (i) PMTH1223Y; (j) PMTH1228Y. B On the induction of inositol trisphosphates. Inositol phosphates were assayed in quiescent Swiss 3T3 cells that had been labelled with 3H-inositol for 20 h, then treated for 4h with 20 ng ml-1 mutant toxins or left untreated. The results were expressed as a percentage of the counts obtained for wild-type PMT. The bars represent the mean and standard errors for six determinations at each point

The three mutants with a marked reduction in activity were tested for proteolytic resistance to the protease Glu-C to determine whether these mutations had introduced gross structural changes that affected protein stability. We have previously shown that this technique can identify subtle differences in structure that are undetectable by circular dichroism [16]. The susceptibility of these mutant toxins was tested at a range of SDS concentrations as previously described [23]. PMT^H1205Y became susceptible to proteolytic cleavage at the same SDS concentration as PMT (Fig.3), suggesting that it was structurally unaltered. PMT^H1205L and PMT^H1223L were a little more sensitive to SDS denaturation, suggesting that they possessed slightly altered structures.

Fig. (3). Resistance of PMT and selected histidine mutants to digestion with the protease, Glu-C. One microgram of PMT or mutant toxins was incubated with 25 ng Glu-C (Sigma) in the presence of different concentrations of SDS for 1 h at 37 °C. The SDS concentrations used were: lane 1, no SDS; 2, 0.005 %; 3, 0.075 %; 4, 0.01 %; 5, 0.0125 %; 6, 0.015 %; 7, 0.02 %; 8, 0.025 %. Lane 9 contains toxin with no Glu-C. The products were electrophoresed through 10 % SDS-PAGE gels, and detected by silver staining. The toxin bands of 146 kDa are shown for PMT, PMTH1205L, PMTH1205Y and PMTH1223L

The inactive mutants PMT^H1205L and PMT^H1205Y were tested for their ability to bind to cells, using the competition assay to assess whether they could block the mitogenic activity of 2 ng PMT ml^-1. Each mutant significantly inhibited the mitogenic action of PMT when added to a concentration of 6 μg ml^-1 or higher (Fig. 4).

Fig. (4). Blocking of PMT-induced DNA synthesis by PMTH1205L and PMTH1205Y. DNA synthesis was assayed in quiescent Swiss 3T3 cells with PMT at 2ng ml-1 in the presence of an excess of the mutant toxins. Results are expressed as the percentage of counts obtained with 10 % foetal bovine serum. BSA was used as a control. The blocking proteins were: (▪) PMTH1205L; (□) PMTH1205Y; (▾) BSA. The results presented are the means and standard errors from six determinations at each point.

A critical role for histidine residues in PMT activity was first suggested by Orth and co-workers [17], who proposed that H1205 and H1223 were essential for PMT function. Kitadokoro and colleagues have recently suggested that C1165, H1205 and D1220 form a catalytic triad that might facilitate either proteolytic or acyltransferase activity [18], while H1223 faces away from the putative active site.

We have analysed the role of all the histidine residues within the catalytic C3 domain and have shown here that H1223 does not have as critical a role in activity as H1205. Substitution of H1205 with either tyrosine or leucine leads to complete loss of activity, without affecting cell binding activity. In addition, the PMT^H1205Y substitution mutant shows little evidence of gross structural changes. We confirm that PMT^H1223L shows almost no activity in the most sensitive test of activity, the ability to induce thymidine incorporation, while the PMT^H1223Y mutant retained substantial but reduced activity (around 3% of the activity of wild-type toxin). Mutation of the two other histidine residues in this region had little or no effect if tyrosine was substituted, although a substantially greater effect when replaced by leucine. This is probably due to the relatively conservative nature of the histidine to tyrosine mutation, as both residues are polar and, aromatic. In contrast leucine is non-polar and smaller so is more likely to affect the structure of the toxin. We have shown here that leucine substitution leads to greater structural instability, as assessed by protease sensitivity. Similarly, PMT^C1165S is more protease sensitive [16]. Structural analysis of the catalytic domain of PMT^C1165S has confirmed that it has a slightly altered structure, which correlates with the observed increase in protease sensitivity [18]

Taken together, H1202 and H1228 appear to play no direct role in the PMT catalytic centre. Mutation of H1223 has a greater effect on activity, presumably because of its structural proximity to the catalytic centre, but our work supports the suggestion by Kitadokoro that H1223 is unlikely to be directly involved in catalysis [18]. The demonstration that mutation of H1205 has a major effect on PMT activity supports the concept that C1165, H1205 and D1220 are essential for catalysis. The identification of critical amino acid residues in the catalytic domain and the further definition of that domain should aid future studies on its function