Expression, purification, enzymatic characterization and crystallization of glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi, the first one from phylum Percolozoa.

Naegleria gruberi had its genome sequenced by Fritz-Laylin and collaborators in 2010. It is not pathogenic, but has characteristics similar to those of Naegleria fowleri, opportunistic pathogen that can cause fatal encephalitis in humans. N. gruberi genome has contributed to a better understanding of the primitive eukaryotic metabolism and revealed the complexity of several metabolic pathways. In this paper we describe the expression, purification, enzyme characterization and crystallization of N. gruberi GAPDH, the first one for an organism belonging to phylum Percolozoa. The results indicated that 10 mM, 8.0 and 25 °C are the optimum arsenate concentration, pH and temperature, respectively. The enzyme presents allosteric positive cooperativity for substrates NAD(+) and G3P as indicated by the Hill coefficients. The phylogenetic proximity between N. fowleri and N. gruberi suggests that contributions from the study of the latter might provide information to assist the search for treatments of Primary Amebic Meningoencephalitis, especially, in this work, taking into account that GAPDH is identified as a therapeutic target.

Glyceraldehyde-3-phosphate dehydrogenase as a surface associated antigen on human breast cancer cell lines MACL-1 and MGSO-3.

Breast cancer is a major health burden, responsible for >10% of all cases of cancer worldwide. Advances in breast cancer diagnosis and treatment have contributed to an improved rate of survival, although mortality rates remains significantly high. The establishment of breast cancer cell lines is an important model for understanding biological processes involved in this disease and for identifying potential therapeutic targets. The novel human breast cancer cell lines, MACL-1 and MGSO-3, were used in this study to identify possible surface antigens by antibodies directed against two commercial breast cancer cell lines MCF-7 and MDA-MB-231. We purified a 37 kDa antigen by affinity chromatography from MDA-MB-231, and its N-terminal amino acid sequence was homologous to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Therefore, immunohistochemical experiments, using specific monoclonal antibodies, evidenced a co-localization of GAPDH and Na+/K+-ATPase on the surface of commercially available and recently established breast cancer cell lines. It is of note that Na+/K+-ATPase was used as a plasma membrane marker. This finding opens new perspectives for breast cancer diagnosis and treatment since GAPDH could be used as a biomarker or as a potential therapeutic target in breast cancer.

Heterologous expression of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Triticum aestivum and Arabidopsis thaliana.

Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) plays a key metabolic role in higher plants. Purification to homogeneity of enzymes found in relatively low abundance in plants represents a major technical challenge that can be solved by molecular gene cloning and heterologous expression. To apply this strategy to np-Ga3PDHase we performed the cloning of the gapN gene from Arabidopsis thaliana and Triticum aestivum, followed by the heterologous expression in Escherichia coli by two different strategies. Soluble expression of the Arabidopsis enzyme in the pET32c+ vector required a chaperone co-expression system (pGro7). The system using E. coli BL21-CodonPlus cells and the pRSETB vector was successful for expression of a soluble His(6)-taged recombinant wheat enzyme producing 2.5 mg of electrophoretically pure protein per liter of cell culture after a single chromatographic purification step. Both systems were effective for the expression of functional plant np-Ga3PDHases, however the expression of the Arabidopsis enzyme in pRSETB was affordable but not as optimal as for the wheat protein. This would be associated with a different codon usage preference between this specific plant and E. coli. Considering the relevant role played by np-Ga3PDHase in plant metabolism, it is experimentally valuable the development of a procedure to obtain adequate amounts of highly purified enzyme, which envisages the viability to perform studies of structure-to-function relationships to better understand the enzyme kinetics and regulation, as well as carbon and energy metabolism in higher plants.

Discovery of novel Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase inhibitors.

Based on its essential role in the life cycle of Trypanosoma cruzi, the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has been considered a promising target for the development of novel chemotherapeutic agents for the treatment of Chagas' disease. In the course of our research program to discover novel inhibitors of this trypanosomatid enzyme, we have explored a combination of structure and ligand-based virtual screening techniques as a complementary approach to a biochemical screening of natural products using a standard biochemical assay. Seven natural products, including anacardic acids, flavonoid derivatives, and one glucosylxanthone were identified as novel inhibitors of T. cruzi GAPDH. Promiscuous inhibition induced by nonspecific aggregation has been discarded as specific inhibition was not reversed or affected in all cases in the presence of Triton X-100, demonstrating the ability of the assay to find authentic inhibitors of the enzyme. The structural diversity of this series of promising natural products is of special interest in drug design, and should therefore be useful in future medicinal chemistry efforts aimed at the development of new GAPDH inhibitors having increased potency.

Phosphorylated non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from heterotrophic cells of wheat interacts with 14-3-3 proteins.

Glyceraldehyde-3-phosphate dehydrogenases catalyze key steps in energy and reducing power partitioning in cells of higher plants. Phosphorylated non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) present in heterotrophic cells of wheat (Triticum aestivum) was activated up to 3-fold by MgCl2. The effect was not observed with the non-phosphorylated enzyme found in leaves. The divalent cation also affected the response of the enzyme from endosperm and shoots to adenine nucleotides and inorganic pyrophosphate. Gel filtration chromatography, co-immunoprecipitation followed by immunostaining, and the use of a phosphopeptide containing a canonical binding motif showed that MgCl2 actually disrupted the interaction between GAPN and a 14-3-3 regulatory protein. After interaction with 14-3-3, phosphorylated GAPN exhibits a 3-fold lower Vmax and higher sensitivity to inhibition by ATP and pyrophosphate. Results suggest that GAPN is a target for regulation by phosphorylation, levels of divalent cations, and 14-3-3 proteins. The regulatory mechanism could be critical to maintain levels of energy and reductants in the cytoplasm of heterotrophic plant cells.

Purification and partial characterization of glyceraldehyde-phosphate dehydrogenase from electric organ of Electrophorus electricus (L.).

The glyceraldehyde-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was purified to homogeneity from electric organ of Electrophorus electricus (L.) by a hydrophobic chromatography method on deacetylcolchicine-Sepharose. The purification resulted in a 162 fold increase in specific activity of the GAPDH and final yield was approximately 37%. The purified enzyme showed a single band in SDS-PAGE, with an apparent molecular mass of 36 kDa. The purity of the colchicine-Sepharose isolated material was analysed by isoelectrophocusing and immunoblotting using a heterologous rabbit serum anti-GAPDH. Sequence analysis of the 40-N-terminal amino acids, determined by Edman degradation, revealed its identity to other GAPDHs proteins being the largest number of identical amino acids to lobster (92.5%), rabbit muscle (85%) and human liver (80%) GAPDH.

T and B cell reactivity to a 42-kDa protein is associated with human resistance to both schistosomiasis mansoni and haematobium.

Egyptian subjects living in areas endemic for Schistosoma mansoni or Schistosoma haematobium were selected on the basis of their apparent extremes of resistance or susceptibility to schistosomiasis and examined for T and B cell responses against the major electrophoretically resolved protein species from soluble adult worm extracts. A 42-kDa band was specifically recognized by a significant majority of subjects resistant to schistosomiasis. The 42-kDa species (p-42) from S. mansoni and S. haematobium were immunologically cross-reactive and induced significant protection in mice and hamsters against infection with cercariae. Amino acid sequence analysis of S. mansoni p-42 showed that it consists predominantly of glyceraldehyde 3-P dehydrogenase (G3PDH), which has been shown to be preferentially recognized by the sera of Brazilian subjects resistant to schistosomiasis mansoni. The present data extend the previous findings and imply that S. mansoni-derived G3PDH represents a target of protective T and B cell-mediated antischistosomiasis immunity in humans.

Purification and characterization of the native and the recombinant Leishmania mexicana glycosomal glyceraldehyde-3-phosphate dehydrogenase.

The gene coding for the glycosomal glyceraldehyde-3-phosphate dehydrogenase from Leishmania mexicana has been cloned into vector pET3A and expressed as a soluble and active protein in Escherichia coli BL21(DE3) in which the endogenous gene has been inactivated by mutation. The recombinant enzyme was purified to near homogeneity by ammonium sulphate precipitation, followed by hydrophobic and cation-exchange chromatography. From a 1-L culture of E. coli cells, 25 mg purified protein was obtained with a specific activity of 125 units/mg. The recombinant protein restores the natural E. coli phenotype when expressed at low level. The enzyme has also been partially purified from glycosomes of cultured L. mexicana promastigotes. The recombinant and the native proteins show identical mobilities on SDS/PAGE, and have the same isoelectric point and similar pH-activity profiles. The kinetics of both enzymes are very similar, the most important aspect being their lower apparent affinity for the cofactor NAD when compared to all other homologous enzymes studied, with the exception of glycosomal glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma brucei.

D-glyceraldehyde-3-phosphate dehydrogenase from HeLa cells--1. Purification and properties of the enzyme.

1. D-GPDH from HeLa cells was isolated and purified. 2. Some basic kinetic constants are reported. 3. Sodium dodecyl polyacrylamide gel electrophoresis gave a single band with a molecular weight of approximately 36 K. 4. ATP and NADH inhibit competitively enzyme activity. 5. Comparative catalytic properties of GPDH from normal and tumor cells were effectuated.

Muscle D-glyceraldehyde-3-phosphate dehydrogenase from Anas sp.--1. Purification and properties of the enzyme.

Muscle D-GPDH from Anas sp. ws isolated and purified. Basic kinetic constants and physico-chemical studies are reported. The enzyme is a tetramer with a mol. wt of 140,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis gave s single band with a mol. wt of approx. 36,000. The other catalytic properties of the enzyme are discussed relative to those of enzyme obtained from other sources.