Figure 4

New mutants of TPD modulate TNFα secretion in LPS induced septic shock in mice.

(a) Schematic representation of TPD, the red bar is the His tag and the blue bar is the TACE prodomain Asp²³-Arg²¹⁴. Marked in red is the furin cleavage site (RKR⁵⁸) and in blue the cysteine residue in the cysteine switch (C¹⁸⁴). The following mutants were created: R⁵⁸A substituting residue 58 from arginine to alanine, C¹⁸⁴A substituting cysteine 184 to alanine and a double mutant (DM) including both mutations R⁵⁸A and C¹⁸⁴A. (b) SDS PAGE analysis of the WT TPD (upper panel) and the C¹⁸⁴A TPD (lower panel) with increase concentration of reducing agent. The WT TPD exhibits a molecular mass of 40 kDa corresponding to the covalent dimer which is switch to a 20 kDa band with the increase of reducing agent. In contrast, the C¹⁸⁴A mutant display only a 20 kDa monomer independent to reducing agent concentration. (c) purified TPD WT and mutants incubated with furin protease and analyzed with SDS PAGE: TPD WT without furin (1) or with furin (2), TPD R⁵⁸A without furin (3) or with furin (4), TPD C¹⁸⁴A without furin (5) or with furin (6), TPD DM without furin (7) or with furin (8). (d) Inhibition curves of WT and mutants TPD on catalytic domain of TACE: WT (), R⁵⁸A (), C¹⁸⁴A () and DM (). (e) Serum level of TNFα in naive C57/BL mice and C57/BL mice injected with LPS and treated with control PBS or different concentrations of TPD WT and DM (1, 4 mg/Kg). *P < 0.05, n = 6 (Mean ± SEM Student’s t test).