Identification and sequence determination of recombinant Clostridium perfringens alpha-toxin by use of electrospray ionization mass spectrometry.

Only a few methods exist for simple, sensitive and rapid detection of alpha-toxin in clinical and biological samples. The aim of our study was to establish a procedure for the production of an antibody against a recombinant antigen with confirmed sequence identity. We applied a noble approach based on proteomics using a mass spectrometer for the conclusive identification of the recombinant alpha-toxin that was subsequently used as an antigen. The recombinant alpha-toxin was produced in Escherichia coli. A clinical isolate of Clostridium perfringens GAI 94074 was amplified by polymerase chain reaction (PCR) and subsequently, cloning was performed. Three different fragments were cloned using a pET100/D-TOPO vector. These fragments coded for a ribosome binding site, a signal peptide and the alpha-toxin gene, respectively. Recombinant pET100 plasmids were cloned into TOP 10 cells and the isolated plasmids were transferred into BL21 Star (DE3) cells. Their expression was then induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). Recombinant E. coli transformed with a plasmid encoding the alpha-toxin gene alone produced a biologically inactive protein. On the other hand, E. coli carrying the plasmid encoding the toxin sequence and its native signal peptide sequence, or the toxin sequence along with the ribosome binding sequence and the signal peptide sequence secreted an active alpha-toxin with phospholipase activity. Accordingly, the C. perfringens gene encoding the alpha-toxin protein along with its signal peptide was successfully cloned, expressed, and secreted by E. coli. Furthermore, without consideration of its activity, we used mass spectrometry to confirm that the expressed protein was indeed the alpha-toxin. Thus, the identification of alpha-toxin protein using both the biological activity testing and the mass spectrometry analysis is expected to verify the significant production of C. perfringens antibody. The study for the analysis of recombinant alpha-toxin using ESI/MS has not been reported. In this study, we report the successful cloning, expression, secretion, identification and sequence determination of the C. perfringens alpha-toxin.

Turning point in apoptosis/necrosis induced by hydrogen peroxide.

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H2O2) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 microM H2O2 exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 microM H2O2 did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 microM H2O2, but not with 50 microM H2O2, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H2O2-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 microM H2O2 condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H2O2-induced cell death is due to apoptosis or necrosis.

Proteomic characterization of oxidative dysfunction in human umbilical vein endothelial cells (HUVEC) induced by exposure to oxidized LDL.

The oxidative modification of low-density lipoprotein (LDL) and subsequent alteration of endothelial cell function are generally accepted as an important early event in the pathogenesis of atherosclerosis. To understand the mechanism by which oxidized LDL (oxLDL) causes dysfunction in endothelial cells, human umbilical vein endothelial cells (HUVEC) were exposed to oxLDL at a concentration that induces cellular dysfunction, and proteomic analysis was carried out, together with the analysis of cellular lipid peroxidation products. Time-dependent accumulation of 7-ketocholesterol and the progression of oxidative modification of peroxiredoxin 2 were observed, together with the suppression of cell proliferation. Proteomic analysis using two-dimensional gel electrophoresis (2-D gel) revealed that nucleophosmin, stathmin, and nucleolin were differentially expressed after exposure to oxLDL. Both 2-D gel and western blot analyses revealed that (1) nucleophosmin was dephosphorylated in a time-dependent manner; (2) stathmin was transiently phosphorylated at 6 h, and the unphosphorylated form was continuously down-regulated; and (3) nucleolin was identified as a 20-kDa fragment and a 76-kDa form, which were down-regulated. These observations suggest that the exposure of HUVEC to oxLDL results in the suppression of cell proliferation, which is ascribed to protein modification and/or altered expression of nucleophosmin, stathmin, and nucleolin under these oxidative stress conditions.

Cysteine-106 of DJ-1 is the most sensitive cysteine residue to hydrogen peroxide-mediated oxidation in vivo in human umbilical vein endothelial cells.

Mutation in DJ-1 gene is the cause of autosomal recessive Parkinson's disease, however, its physiological function remains unclear. The isoelectric point of DJ-1 shows an acidic shift after cells are treated with hydrogen peroxide. This suggests that DJ-1 is modified in response to oxidative stress. Here we report the structural characterization of an acidic isoform of DJ-1 using a proteomic approach with nanospray interface liquid chromatography-electrospray ionization/linear ion trap mass spectrometer. When human umbilical vein endothelial cells were exposed to hydrogen peroxide, all three cysteines in DJ-1 were oxidized to cysteine sulphonic acid. Although a small part of the Cys-46 and Cys-53 were oxidized, Cys-106 was oxidized completely at any hydrogen peroxide concentration used here. These results suggest that Cys-106 is the most sensitive among three cysteine residues to oxidative stress, and that DJ-1 function is regulated, in terms of the intracellular redox state, by oxidation of Cys-106.