Probing the action of Clostridium toxins B and exoenzyme C3 for detection of Rho-like motifs of alfalfa proteins

Small GTP-binding Rho proteins are involved in signalling, cell polarity, m embrane outgrowths and actin stabilization in eukaryotes. Known plant homol ogues represent essentially the Rac subfamily and an original Rop (Rho in p ollen). Mammalian Rho proteins are preferential targets of clostridial toxi ns. In alfalfa (Medicago saliva L.) cells, Clostridium botulinum C3-exoenzy me (C3) provoked disassembly of the actin cytoskeleton, similar to its effe ct in mammalian cells. In alfalfa proteins, several epitopes appear to be r ecognized by commercial antibodies raised against peptides characteristic f or human Rho. One similar to 40-kDa band was detected immunologically by an ti-RhoB: a protein of this size was ADP-ribosylated by C3 and glucosylated in vitro by Clostridium difficile toxin B, without interference between the two nor from phosphatidyl inositide. C3 was also active upon a 34-kDa band which contained protein(s) immunoreactive with anti-Rac2 and which bound [ gamma(35)S]-GTP, but was glucosylated by neither toxin B nor Clostridium so rdellii Lethal Toxin. An 18-kDa band detected by [gamma(35)S]-GTP overlay w as immunologically recognized by anti-Rad. Anti-Cdc42Hs recognized a 54-kDa band. Substrates to toxin B and C3 were purified from alfalfa cell culture and partially sequenced: they included two proteins, P40 and P41, of simil ar to 40 kDa (by SDS-electrophoresis). P40 appears to constitute a tetramer ic aldolase (160 kDa by gel filtration; EC 4.1.2.13) whose activity is part ially inhibited by toxin B and the anti-RhoB. (C) 1999 Editions scientifiqu es et medicales Elsevier SAS.