Organophosphates modulate tissue transglutaminase activity in differentiated C6 neural cells.

OBJECTIVE: The organophosphate compounds chlorpyrifos (O, O-diethyl O-[3,5,6-trichloro-2-pyridinyl] phosphorothioate, CPF) and phenyl saligenin phosphate (PSP) have been widely implicated in developmental neurotoxicity and neurodegeneration. However, the underlying mechanism remains unclear. Transglutaminase (TG)2 is a calcium ion (Ca2+)-dependent enzyme with an important role in neuronal cell outgrowth and differentiation and in neurotoxin activity and is modulated by organophosphates. MATERIALS AND METHODS: We studied TG2 activity modulation by CPO and PSP during differentiation in C6 glioma cells. We studied the effects of CPO or PSP treatment with or without the TG2 inhibitor Z-DON and identified potential TG2 protein substrates via mass spectrometry. RESULTS: PSP and CPO did not affect cell viability but affected TG2 activity in differentiating cells. Our results indicate that the organophosphate-induced amine incorporation activity of TG2 may have a direct effect on neuronal outgrowth, differentiation, and cell survival by modifying several essential microtubule proteins, including tubulin. Inhibiting TG2 reduced neurite length but not cell survival. CONCLUSIONS: TG2 inhibitors can protect against organophosphate-induced neuropathy and could be used for developing novel therapeutic strategies for treating brain cancer and neurodegenerative disorders.

Determination of Transglutaminase Activity in Plants.

Transglutaminase (TGase:E.C. 2.3.2.13) catalyzes the acyl-transfer reaction between one or two primary amino groups of polyamines and protein-bound Gln residues giving rise to post-translational modifications. One increasing the positive charge on a proteins surface and the other results in the covalent crosslinking of proteins. Pioneering studies on TGase in plants started in the middle of the 1980's but the methodology designed for use with animal extracts was not directly applicable to plant extracts. Here we describe radioactive and colorimetric methods adapted to study plant TGase, as well as protocols to analyze the involvement of TGase and polyamines in the functionality of cytoskeletal proteins.

Effects of chlorpyrifos on transglutaminase activity in differentiating rat C6 glioma cells.

The organophosphorothioate compound chlorpyrifos (CPF) is a widely used pesticide, which is known to inhibit the differentiation of mouse N2a neuroblastoma and rat C6 glioma cells. This study in focused on the possible effects of CPF in the activity and expression of tissue transglutaminase (TGase 2) in differentiating C6 cells. Cells exposed for 24 h to 10 µM CPF, which had no effect on cell viability, exhibited a significant increase in cytosolic TGase 2 activity. Western blotting analysis indicated that there was no change in the cytosolic TGase 2 protein levels, suggesting that the enzyme was activated under these conditions. When commercially available TGase 2 was incubated with CPF in vitro, an increase in activity was also observed, suggesting that CPF might interact directly with TGase 2.

Effects of phenyl saligenin phosphate on cell viability and transglutaminase activity in N2a neuroblastoma and HepG2 hepatoma cell lines.

The main aim of this study was to determine whether sub-lethal concentrations of the organophosphate compound phenyl saligenin phosphate (PSP) could disrupt the activity of the Ca(2+)-activated enzyme tissue transglutaminase (TGase 2) from cultured cell lines of neuronal (N2a) and hepatic (HepG2) origin. The results indicated that PSP added directly to cytosol extracts from healthy cells was able to inhibit TGase 2 activity by 40-60% of control levels at sub-lethal concentrations (0.1 microM) that were approximately 100-fold lower than their IC(50) values in cytotoxicity assays. Following 24h exposure of N2a cells to 0.3 and 3 microM PSP in situ, a similar reduction in activity was observed in subsequent assays of TGase 2 activity. However, significantly increased activity was observed following in situ exposure of HepG2 cells to PSP (ca. 4-fold at 3 microM). Western blotting analysis indicated slightly reduced levels of TGase 2 in N2a cells compared to the control, whereas an increase was observed in the level of TGase 2 in HepG2 cells. We suggest that TGase 2 represents a potential target of organophosphate toxicity and that its response may vary in different cellular environments, possibly affected by its expression pattern.

Cryopreservation of Helianthus tuberosus cell suspension cultures: the effect of preculture treatments on cytoskeletal proteins and transglutaminase activity.

Helianthus tuberosus cell suspension cultures were subjected to cryopreservation 24h preculture treatments with 0.5M sucrose or mannitol. Extracts were assayed for transglutaminase activity and the level of alpha-tubulin tyrosination. There was a significant reduction (compared with the non-precultured controls) in transglutaminase activity and alpha-tubulin tyrosination state after mannitol preculture treatment, whereas sucrose preculture treatment produced no significant effect. The results suggest that reduced levels of transglutaminase activity and alpha-tubulin tyrosination are associated with a lack of post-thaw recovery observed following mannitol preculture treatment of cell culture suspensions. These activities may represent useful molecular markers of the success of preculture treatments in cryopreservation protocols.

Electrospray mass spectrometry for the identification of MHC class I-associated peptides expressed on cancer cells.

Electrospray ionisation (ESI) mass spectrometry (MS) has been used extensively for the detection of peptides presented by major histocompatibility complex (MHC) molecules. This review focuses on the optimisation of electrospray mass spectrometry and the use of tandem mass spectrometry to sequence MHC class I peptides. We review the isolation of MHC class I peptides from the surface of cells with particular reference to tumour cells. In addition, we also discuss the advantages and disadvantages of the methods available to concentrate and fractionate the peptides prior to analysis by electrospray mass spectrometry.

Direct evidence that leukemic cells present HLA-associated immunogenic peptides derived from the BCR-ABL b3a2 fusion protein.

The BCR-ABL oncogene is central in the pathogenesis of chronic myeloid leukemia (CML). Here, tandem nanospray mass spectrometry was used to demonstrate cell surface HLA-associated expression of the BCR-ABL peptide KQSSKALQR on class I-negative CML cells transfected with HLA-A*0301, and on primary CML cells from HLA-A3-positive patients. These patients mounted a cytotoxic T-lymphocyte response to KQSSKALQR that also killed autologous CML cells, and tetramer staining demonstrated the presence of circulating KQSSKALQR-specific T cells. The findings are the first demonstration that CML cells express HLA-associated leukemia-specific immunogenic peptides and provide a sound basis for immunization studies against BCR-ABL.

Nano-electrospray and microbore liquid chromatography-ion trap mass spectrometry studies of copper complexation with MHC restricted peptides.

The formation of copper/peptide complex ions by nano-electrospray and microbore HPLC-electrospray mass spectrometry has been investigated for major histocompatibility complex (MHC) class I and class II restricted peptides. Post-column addition of copper(II) acetate following microbore HPLC-MS separation was carried out using a mixing T-piece or via the sheath flow inlet of the electrospray source. Optimal analytical conditions for copper complex ion formation were determined by variation of copper concentration, pH, nebulization gas supply and spray voltage. Tandem mass spectrometry of copper/peptide complex ions provides peptide sequence information and insight into the peptide chelation sites. Copper associated y fragment ions dominate the product ion spectrum for non-histidine containing peptides, but both b and y copper complex ions were observed for the histidine containing MHC class I associated peptide gp70.

Assays for the measurement of tissue transglutaminase (type II) mediated protein crosslinking via epsilon-(gamma-glutamyl) lysine and N',N'-bis (gamma-glutamyl) polyamine linkages using biotin labelled casein.

Two colorimetric assays for tissue transglutaminase (type II) activity involving the crosslinking of proteins have been developed. In one assay, biotin labelled casein is crosslinked into chemically modified casein bound to a microtiter plate by tissue transglutaminase and the biotin labelled reaction product is detected by conjugation to Extravidin peroxidase. The assay can detect activity in 10 ng of commercially available purified guinea pig liver transglutaminase and in the crude homogenate derived from 400 human endothelial cells (cell line ECV 304). A correlation (r2 = 0.977) was shown between this assay and the radiolabeled putrescine incorporation assay for the detection of transglutaminase activity. This assay measures the protein crosslinking activity of tissue transglutaminase as opposed to polyamine incorporation and offers a rapid, non-radiometric method for screening large sample numbers. Typical inter-assay variability is 13.9 +/- 1.5% (n = 8). In a second assay, the ability of tissue transglutaminase to catalyze the formation of N',N'-bis (gamma-glutamyl) polyamine bridges is measured. N',N'-dimethylcasein is bound to a microtiter plate and modified enzymatically using commercially available purified guinea pig liver transglutaminase to incorporate polyamines into glutamine residues. Biotin labelled casein is then crosslinked into the immobilized polyamines by tissue transglutaminase resulting in the formation of N',N'-bis (gamma-glutamyl) polyamine linkages.