Evidence in favor of the symbiotic origin of chloroplasts: primary structure and evolution of tobacco glyceraldehyde-3-phosphate dehydrogenases

We report nucleotide sequences of cDNAs for the nuclear genes encoding chloroplast (GapA and GapB) and cytosolic (GapC) glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from N. tabacum. Comparison of nucleotide sequences indicates that the GapA and GapB genes evolved following duplication of an ancestral gene about 450 million years ago. However, the divergence of GapA/B and GapC occurred much earlier in evolution than the divergence of GapC and GAPDH genes of animals and fungi, suggesting that chloroplast and cytosolic GAPDHs evolved from different lineages. Comparison of amino acid sequences shows that the chloroplast GAPDHs are related to GAPDHs found in thermophilic bacteria, while the cytosolic GAPDH is related to the GAPDH found in mesophilic prokaryotes. These results strongly support the symbiotic origin of chloroplasts.     

The thioredoxin-independent isoform of chloroplastic glyceraldehyde-3-phosphate dehydrogenase is selectively regulated by glutathionylation

In animal cells, many proteins have been shown to undergo glutathionylation under conditions of oxidative stress. By contrast, very little is known about this post-translational modification in plants. In the present work, we showed, using mass spectrometry, that the recombinant chloroplast A(4)-glyceraldehyde-3-phosphate dehydrogenase (A(4)-GAPDH) from Arabidopsis thaliana is glutathionylated with either oxidized glutathione or reduced glutathione and H(2)O(2). The formation of a mixed disulfide between glutathione and A(4)-GAPDH resulted in the inhibition of enzyme activity. A(4)-GAPDH was also inhibited by oxidants such as H(2)O(2). However, the effect of glutathionylation was reversed by reductants, whereas oxidation resulted in irreversible enzyme inactivation. On the other hand, the major isoform of photosynthetic GAPDH of higher plants (i.e. the A(n)B(n)-GAPDH isozyme in either A(2)B(2) or A(8)B(8) conformation) was sensitive to oxidants but did not seem to undergo glutathionylation significantly. GAPDH catalysis is based on Cys149 forming a covalent intermediate with the substrate 1,3-bisphosphoglycerate. In the presence of 1,3-bisphosphoglycerate, A(4)-GAPDH was fully protected from either oxidation or glutathionylation. Site-directed mutagenesis of Cys153, the only cysteine located in close proximity to the GAPDH active-site Cys149, did not affect enzyme inhibition by glutathionylation or oxidation. Catalytic Cys149 is thus suggested to be the target of both glutathionylation and thiol oxidation. Glutathionylation could be an important mechanism of regulation and protection of chloroplast A(4)-GAPDH from irreversible oxidation under stress.     

Reversible post-translational modification of proteins by nitrated fatty acids in vivo

Nitric oxide ((*)NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon beta to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro-linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9- and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys-149). GAPDH inhibition displayed an IC(50) of approximately 3 microM for both nitroalkenes, an IC(50) equivalent to the potent thiol oxidant peroxynitrite (ONOO(-)) and an IC(50) 30-fold less than H(2)O(2), indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiol-reversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The occurrence of these electrophilic nitroalkylation reactions in vivo indicates that this reversible post-translational protein modification represents a new pathway for redox regulation of enzyme function, cell signaling, and protein trafficking.     

Functional Divergence and Convergent Evolution in the Plastid-Targeted Glyceraldehyde-3-Phosphate Dehydrogenases of Diverse Eukaryotic Algae

Background

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme of the glycolytic pathway, reversibly catalyzing the sixth step of glycolysis and concurrently reducing the coenzyme NAD⁺ to NADH. In photosynthetic organisms a GAPDH paralog (Gap2 in Cyanobacteria, GapA in most photosynthetic eukaryotes) functions in the Calvin cycle, performing the reverse of the glycolytic reaction and using the coenzyme NADPH preferentially. In a number of photosynthetic eukaryotes that acquired their plastid by the secondary endosymbiosis of a eukaryotic red alga (Alveolates, haptophytes, cryptomonads and stramenopiles) GapA has been apparently replaced with a paralog of the host’s own cytosolic GAPDH (GapC1). Plastid GapC1 and GapA therefore represent two independent cases of functional divergence and adaptations to the Calvin cycle entailing a shift in subcellular targeting and a shift in binding preference from NAD⁺ to NADPH.

Methods

We used the programs FunDi, GroupSim, and Difference Evolutionary-Trace to detect sites involved in the functional divergence of these two groups of GAPDH sequences and to identify potential cases of convergent evolution in the Calvin-cycle adapted GapA and GapC1 families. Sites identified as being functionally divergent by all or some of these programs were then investigated with respect to their possible roles in the structure and function of both glycolytic and plastid-targeted GAPDH isoforms.

Conclusions

In this work we found substantial evidence for convergent evolution in GapA/B and GapC1. In many cases sites in GAPDHs of these groups converged on identical amino acid residues in specific positions of the protein known to play a role in the function and regulation of plastid-functioning enzymes relative to their cytosolic counterparts. In addition, we demonstrate that bioinformatic software like FunDi are important tools for the generation of meaningful biological hypotheses that can then be tested with direct experimental techniques.     

The active site cysteine of the proapoptotic protein glyceraldehyde-3-phosphate dehydrogenase is essential in oxidative stress-induced aggregation and cell death

Recent studies have revealed that the redox-sensitive glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is involved in neuronal cell death that is triggered by oxidative stress. GAPDH is locally deposited in disulfide-bonded aggregates at lesion sites in certain neurodegenerative diseases. In this study, we investigated the molecular mechanism that underlies oxidative stress-induced aggregation of GAPDH and the relationship between structural abnormalities in GAPDH and cell death. Under nonreducing in vitro conditions, oxidants induced oligomerization and insoluble aggregation of GAPDH via the formation of intermolecular disulfide bonds. Because GAPDH has four cysteine residues, including the active site Cys(149), we prepared the cysteine-substituted mutants C149S, C153S, C244A, C281S, and C149S/C281S to identify which is responsible for disulfide-bonded aggregation. Whereas the aggregation levels of C281S were reduced compared with the wild-type enzyme, neither C149S nor C149S/C281S aggregated, suggesting that the active site cysteine plays an essential role. Oxidants also caused conformational changes in GAPDH concomitant with an increase in beta-sheet content; these abnormal conformations specifically led to amyloid-like fibril formation via disulfide bonds, including Cys(149). Additionally, continuous exposure of GAPDH-overexpressing HeLa cells to oxidants produced disulfide bonds in GAPDH leading to both detergent-insoluble and thioflavin-S-positive aggregates, which were associated with oxidative stress-induced cell death. Thus, oxidative stresses induce amyloid-like aggregation of GAPDH via aberrant disulfide bonds of the active site cysteine, and the formation of such abnormal aggregates promotes cell death.     

Glyceraldehyde-3-phosphate dehydrogenase gene diversity in eubacteria and eukaryotes: evidence for intra- and inter-kingdom gene transfer.

Cyanobacteria contain up to three highly divergent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes: gap1, gap2, and gap3. Genes gap1 and gap2 are closely related at the sequence level to the nuclear genes encoding cytosolic and chloroplast GAPDH of higher plants and have recently been shown to play distinct key roles in catabolic and anabolic carbon flow, respectively, of the unicellular cyanobacterium Synechocystis sp. PCC6803. In the present study, sequences of 10 GAPDH genes distributed across the cyanobacteria Prochloron didemni, Gloeobacter violaceus PCC7421, and Synechococcus PCC7942 and the alpha-proteobacterium Paracoccus denitrificans and the beta-proteobacterium Ralstonia solanacearum were determined. Prochloron didemni possesses homologs to the gap2 and gap3 genes from Anabaena, Gloeobacter harbors gap1 and gap2 homologs, and Synechococcus possesses gap1, gap2, and gap3. Paracoccus harbors two highly divergent gap genes that are related to gap3, and Ralstonia possesses a homolog of the gap1 gene. Phylogenetic analyses of these sequences in the context of other eubacterial and eukaryotic GAPDH genes reveal that divergence across eubacterial gap1, and gap2, and gap3 genes is greater than that between eubacterial gap1 and eukaroytic glycolytic GapC or between eubacterial gap2 and eukaryotic Calvin cycle GapAB. These data strongly support previous analyses which suggested that eukaryotes acquired their nuclear genes for GapC and GapAB via endosymbiotic gene transfer from the antecedents of mitochondria and chloroplasts, and extend the known range of sequence diversity of the antecedent eubacterial genes. Analyses of available GAPDH sequences from other eubacterial sources indicate that the glycosomal gap gene from trypanosomes (cytosolic in Euglena) and the gap gene from the spirochete Treponema pallidum are each other's closest relatives. This specific relationship can therefore not reflect organismal evolution but must be the result of an interkingdom gene transfer, the direction of which cannot be determined with certainty at present. Contrary to this, the origin of the cytosolic Gap gene from trypanosomes can now be clearly defined as gamma-proteobacterial, since the newly established Ralstonia sequence (beta-proteobacteria) branches basally to the gamma-proteobacterial/trypanosomal assemblage.     

20 S proteasome from Saccharomyces cerevisiae is responsive to redox modifications and is S-glutathionylated

The 20 S proteasome core purified from Saccharomyces cerevisiae is inhibited by reduced glutathione (GSH), cysteine (Cys), or the GSH precursor gamma-glutamylcysteine. Chymotrypsin-like activity was more affected by GSH than trypsin-like activity, whereas the peptidylglutamyl-hydrolyzing activity (caspase-like) was not inhibited by GSH. Cys-sulfenic acid formation in the 20 S core was demonstrated by spectral characterization of the Cys-S(O)-4-nitrobenzo-2-oxa-1,3-diazole adduct, indicating that 20 S proteasome Cys residues might react with reduced sulfhydryls (GSH, Cys, and gamma-glutamylcysteine) through the oxidized Cys-sulfenic acid form. S-Glutahionylation of the 20 S core was demonstrated in vitro by GSH-biotin incorporation and by decreased alkylation with monobromobimane. Compounds such as N-ethylmaleimide (-S-sulfhydril H alkylating), dimedone (-SO sulfenic acid H reactant), or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (either -SH or -SOH reactant) highly inhibited proteasomal chymotrypsin-like activity. In vivo experiments revealed that 20 S proteasome extracted from H(2)O(2)-treated cells showed decreased chymotrypsin-like activity accompanied by S-glutathionylation as demonstrated by GSH release from the 20 S core after reduction with NaBH(4). Moreover, cells pretreated with H(2)O(2) showed decreased reductive capacity assessed by determination of the GSH/oxidized glutathione ratio and increased protein carbonyl levels. The present results indicate that at the physiological level the yeast 20 S proteasome is regulated by its sulfhydryl content, thereby coupling intracellular redox signaling to proteasome-mediated proteolysis.     

金黄色葡萄球菌重组GapC蛋白的GAPDH活性及免疫原性分析

为研究金黄色葡萄球菌(Staphylococcus aureus)表面GapC蛋白的GAPDH活性、免疫原性及免疫保护作用, 应用PCR方法扩增出S. aureus的gapC基因, 插入到pQE-30载体相应位点, 构建重组质粒pQE/gapC。将其导入宿主菌E.coli M15(pREP4)后, IPTG诱导表达。重组蛋白纯化后进行GAPDH活性检测, 并与灭活全菌体分别免疫健康家兔。然后, 应用ELISA方法检测血清中IgG抗体水平及IFN-g、IL-4细胞因子浓度, 并用1.0×108CFU/mL S. aureus菌株Wood46对免疫家兔攻毒。SDS-PAGE结果显示, GapC蛋白在E. coli M15(pREP4)中获得表达; 经GAPDH活性检测及Western Blot检测, 重组蛋白具有较高的GAPDH活性和抗原特异性; 经ELISA检测, GapC蛋白及全菌体组兔血清中IgG抗体水平迅速升高, 并在加强免疫后第28天达到最高(1:64 000), 加强免疫后第14 d, 血清中细胞因子IFN-g和IL-4浓度与对照组相比, 显著升高(P<0.05), 而全菌体免疫组升高不明显(P>0.05); 攻毒结果为蛋白免疫组家兔获得一定的免疫保护(4/5)。以上结果表明, 表达的重组GapC蛋白具有GAPDH活性、较好的免疫原性及免疫保护力, 可作为深入研究S. aureus基因工程疫苗的良好靶向。     

金黄色葡萄球菌重组GapC蛋白的GAPDH活性及免疫原性分析

为研究金黄色葡萄球菌(Staphylococcus aureus)表面GapC蛋白的GAPDH活性、免疫原性及免疫保护作用, 应用PCR方法扩增出S. aureus的gapC基因, 插入到pQE-30载体相应位点, 构建重组质粒pQE/gapC。将其导入宿主菌E.coli M15(pREP4)后, IPTG诱导表达。重组蛋白纯化后进行GAPDH活性检测, 并与灭活全菌体分别免疫健康家兔。然后, 应用ELISA方法检测血清中IgG抗体水平及IFN-g、IL-4细胞因子浓度, 并用1.0×108CFU/mL S. aureus菌株Wood46对免疫家兔攻毒。SDS-PAGE结果显示, GapC蛋白在E. coli M15(pREP4)中获得表达; 经GAPDH活性检测及Western Blot检测, 重组蛋白具有较高的GAPDH活性和抗原特异性; 经ELISA检测, GapC蛋白及全菌体组兔血清中IgG抗体水平迅速升高, 并在加强免疫后第28天达到最高(1:64 000), 加强免疫后第14 d, 血清中细胞因子IFN-g和IL-4浓度与对照组相比, 显著升高(P<0.05), 而全菌体免疫组升高不明显(P>0.05); 攻毒结果为蛋白免疫组家兔获得一定的免疫保护(4/5)。以上结果表明, 表达的重组GapC蛋白具有GAPDH活性、较好的免疫原性及免疫保护力, 可作为深入研究S. aureus基因工程疫苗的良好靶向。     

Protein S-glutathiolation triggered by decomposed S-nitrosoglutathione

Recombinant human brain calbindin D(28K) (rHCaBP), human Cu,Zn-superoxide dismutase (HCuZnSOD), rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and bovine serum albumin (BSA) were found to be S-glutathiolated in decomposed S-nitrosoglutathione (GSNO) solutions. Tryptic or Glu-C digestion and MALDI-TOF MS analyses of the digests are consistent with S-thiolation of Cys111 and Cys187 of HCuZnSOD and rHCaBP, respectively, upon exposure to decomposed GSNO. GAPDH activity analysis reveals that S-glutathiolation most likely occurs on the active site Cys149, and the single free Cys34 is assumed to be the site of S-glutathiolation in BSA. The yields of S-glutathiolation of rHCaBP, GAPDH, and BSA were much higher than those of HCuZnSOD. The latter is limited by the accessibility of Cys111 to the glutathiolating reagent in the HCuZnSOD dimer. Unlike decomposed GSNO, fresh GSNO, reduced glutathione (GSH), and oxidized glutathione (GSSG) are not efficient S-glutathiolating agents for the proteins examined here. On the basis of analysis by mass spectrometry and UV-visible absorption, GSNO decomposition in the dark at room temperature yields glutathione disulfide S-oxide [GS(O)SG], glutathione disulfide S-dioxide (GSO(2)SG), and GSSG as products. GS(O)SG is the efficient protein S-glutathiolating agent in GSNO solutions, not GSNO, which does not carry out efficient S-glutathiolation of rHCaBP, HCuZnSOD, or GAPDH in vitro. A hydrolysis pathway yielding GSOH and nitroxyl (HNO/NO(-)) as intermediates is proposed for GSNO decomposition in the dark. This is based on inhibition of GSNO breakdown by dimedone, a reagent specific for sulfenic acids, and on nitroxyl scavenging by metmyoglobin. The results presented here are contrary to numerous reports of protein S-thiolation by low-molecular weight S-nitrosothiols.     

Origins of plastids and glyceraldehyde-3-phosphate dehydrogenase genes in the green-colored dinoflagellate Lepidodinium chlorophorum

The dinoflagellate Lepidodinium chlorophorum possesses "green" plastids containing chlorophylls a and b (Chl a+b), unlike most dinoflagellate plastids with Chl a+c plus a carotenoid peridinin (peridinin-containing plastids). In the present study we determined 8 plastid-encoded genes from Lepidodinium to investigate the origin of the Chl a+b-containing dinoflagellate plastids. The plastid-encoded gene phylogeny clearly showed that Lepidodinium plastids were derived from a member of Chlorophyta, consistent with pigment composition. We also isolated three different glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from Lepidodinium-one encoding the putative cytosolic "GapC" enzyme and the remaining two showing affinities to the "plastid-targeted GapC" genes. In a GAPDH phylogeny, one of the plastid-targeted GapC-like sequences robustly grouped with those of dinoflagellates bearing peridinin-containing plastids, while the other was nested in a clade of the homologues of haptophytes and dinoflagellate genera Karenia and Karlodinium bearing "haptophyte-derived" plastids. Since neither host nor plastid phylogeny suggested an evolutionary connection between Lepidodinium and Karenia/Karlodinium, a lateral transfer of a plastid-targeted GapC gene most likely took place from a haptophyte or a dinoflagellate with haptophyte-derived plastids to Lepidodinium. The plastid-targeted GapC data can be considered as an evidence for the single origin of plastids in haptophytes, cryptophytes, stramenopiles, and alveolates. However, in the light of Lepidodinium GAPDH data, we need to closely examine whether the monophyly of the plastids in the above lineages inferred from plastid-targeted GapC genes truly reflects that of the host lineages.     

Novel inhibitors of glyceraldehyde-3-phosphate dehydrogenase: Covalent modification of NAD-binding site by aromatic thiols

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) is a glycolytic enzyme catalyzing the formation of 1,3-diphosphoglycerate from glyceraldehyde-3-phosphate and inorganic phosphate. In cooperation with E3 ubiquitin-kinase Siah1, GAPDH directly participates in the apoptotic death of neurons in Parkinson’s disease. Potential GAPDH inhibitors were screened in silico, and three compounds with high affinity to the NAD-binding site and theoretically capable of forming a disulfide bond with amino acid residue Cys149 were found among cysteine and glutathione derivatives. The inhibitory effect of these compounds was tested on GAPDH from rabbit muscles using isothermal calorimetry and kinetic methods. As a result of experimental screening, we selected two compounds that inhibit GAPDH by forming disulfide bonds with the Cys149 residue in the enzyme active site. Since Cys149 is the key residue not only for the catalyzed reaction, but also for interaction with Siah1, the compounds can be assumed to inhibit the formation of the proapoptotic complex GAPDH-Siah1 and therefore have potential effect against Parkinson’s disease.     

Use of site-directed mutagenesis to probe the role of Cys149 in the formation of charge-transfer transition in glyceraldehyde-3-phosphate dehydrogenase

Oligonucleotide-directed mutagenesis was employed to produce mutants of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Escherichia coli and Bacillus stearothermophilus. Three different mutants proteins--His176----Asn, Cys149----Ser, Cys149----Gly--were isolated from one or both of the enzymes. The study of the properties of these mutants has shown that Cys149 is clearly responsible for the information of a charge-transfer transition, named the Racker band, observed during the NAD+ binding to apoGAPDH. This result excludes a similarity between the Racker band and the charge-transfer transition observed following the alkylation of GAPDH by 3-chloroacetyl pyridine-adenine dinucleotide.     

ATP-dependent modulation and autophosphorylation of rapeseed 2-Cys peroxiredoxin

2-Cys peroxiredoxins (2-Cys Prx) are ubiquitous thiol-containing peroxidases that have been implicated in antioxidant defense and signal transduction. Although their biochemical features have been extensively studied, little is known about the mechanisms that link the redox activity and non-redox processes. Here we report that the concerted action of a nucleoside triphosphate and Mg(2+) on rapeseed 2-Cys Prx reversibly impairs the peroxidase activity and promotes the formation of high molecular mass species. Using protein intrinsic fluorescence in the analysis of site-directed mutants, we demonstrate that ATP quenches the emission intensity of Trp179, a residue close to the conserved Cys175. More importantly, we found that ATP facilitates the autophosphorylation of 2-Cys Prx when the protein is successively reduced with thiol-bearing compounds and oxidized with hydroperoxides or quinones. MS analyses reveal that 2-Cys Prx incorporates the phosphoryl group into the Cys175 residue yielding the sulfinic-phosphoryl [Prx-(Cys175)-SO(2)PO(3)(2-)] and the sulfonic-phosphoryl [Prx-(Cys175)-SO(3)PO(3)(2-)] anhydrides. Hence, the functional coupling between ATP and 2-Cys Prx gives novel insights into not only the removal of reactive oxygen species, but also mechanisms that link the energy status of the cell and the oxidation of cysteine residues.     

S-nitrosylation and S-glutathionylation of GAPDH: Similarities,differences, and relationships

The aim of this work was to compare the effect of reversible post-translational modifications, S-nitrosylation and S-glutathionylation, on the properties of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to reveal the mechanism of the relationship between these modifications. Comparison of S-nitrosylated and S-glutathionylated GAPDH showed that both modifications inactivate the enzyme and change its spatial structure, decreasing the thermal stability of the protein and increasing its sensitivity to trypsin cleavage. Both modifications are reversible in the presence of dithiothreitol, however, in the presence of reduced glutathione and glutaredoxin 1, the reactivation of S-glutathionylated GAPDH is much slower (10% in 2 h) compared to S-nitrosylated GAPDH (60% in 10 min). This suggests that S-glutathionylation is a much less reversible modification compared to S-nitrosylation.Incubation of HEK 293 T cells in the presence of H₂O₂ or with the NO donor diethylamine NONOate results in accumulation of sulfenated GAPDH (by data of Western blotting) and S-glutathionylated GAPDH (by data of immunoprecipitation with anti-GSH antibodies). Besides GAPDH, a protein of 45 kDa was found to be sulfenated and S-glutathionylated in the cells treated with H₂O₂ or NO. This protein was identified as beta-actin. The results of this study confirm the previously proposed hypothesis based on in vitro investigations, according to which S-nitrosylation of the catalytic cysteine residue (Cys152) of GAPDH with subsequent formation of cysteine sulfenic acid at Cys152 may promote its S-glutathionylation in the presence of cellular GSH. Presumably, the mechanism may be valid in the case of beta-actin.     

An enigmatic GAPDH gene in the symbiotic dinoflagellate genus Symbiodinium and its related species (the order Suessiales): possible lateral gene transfer between two eukaryotic algae, dinoflagellate and euglenophyte

A group of unicellular eukaryotic algae, the dinoflagellates, are known to possess two types of gene for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). An enzyme encoded by one type of gene possibly plays a key role in the glycolytic pathway of the cytosol and the other in the Calvin cycle of plastids. In the present study, an additional type of GAPDH gene (GapC3) was found in the symbiotic dinoflagellates, Symbiodinium spp. and their related species, Gymnodinium simplex and Polarella glacialis, all of which belong to the order Suessiales. Since no intracellular translocation signal is found at both amino- and carboxy-termini of its deduced amino acid sequence, the protein is predicted to function in the cytosol. However, it may not be involved in glycolysis due to the presence of an amino acid signature that allows binding for NADP⁺. It is likely that dinoflagellate species, other than Suessiales investigated in this study, lack this type of GAPDH. Phylogenetic analysis placed GapC3 from the Suessialean species firmly in the clade composed of GAPDH from spirochetes, euglenophytes (cytosolic type) and kinetoplastids (glycosomal type). Specifically, this enigmatic GAPDH gene in dinoflagellates was closely related to its cytosolic counterpart in euglenophytes. It has been previously reported that plastid-targeted (Calvin cycle) GAPDH genes of the dinoflagellates Pyrocystis spp. and that of the euglenophyte Euglena gracilis also seem to share a common ancestor. It appears highly likely that at least two genes (cytosolic and plastid-targeted GAPDH genes) have been laterally transferred between these two eukaryotic algal groups.     

Mass spectrometric analysis of nitroxyl-mediated protein modification: comparison of products formed with free and protein-based cysteines

Although biologically active, nitroxyl (HNO) remains one of the most poorly studied NO(x). Protein-based thiols are suspected targets of HNO, forming either a disulfide or sulfinamide (RSONH2) through an N-hydroxysulfenamide (RSNHOH) addition product. Electrospray ionization mass spectrometry (ESI-MS) is used here to examine the products formed during incubation of thiol proteins with the HNO donor, Angeli's salt (AS; Na2N2O3). Only the disulfide, cystine, was formed in incubates of 15 mM free Cys with equimolar AS at pH 7.0-7.4. In contrast, the thiol proteins (120-180 microM), human calbindin D(28k) (HCalB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and bovine serum albumin (BSA) gave four distinct types of derivatives in incubates containing 0.9-2.5 mM AS. Ions at M + n x 31 units were detected in the ESI mass spectra of intact HCalB (n = 1-5) and GAPDH (n = 2), indicating conversion of thiol groups on these proteins to RSONH2 (+31 units). An ion at M + 14 dominated the mass spectrum of BSA, and intramolecular sulfinamide cross-linking of Cys34 to one of its neighboring Lys or Arg residues would account for this mass increase. Low abundant M + 14 adducts were observed for HCalB, which additionally formed mixed disulfides when free Cys was present in the AS incubates. Cys149 and Cys153 formed an intramolecular disulfide in the AS/GAPDH incubates. Since AS also produces nitrite above pH 5 (HN2O3(-) --> HNO + NO2(-)), incubation with NaNO2 served to confirm that protein modification was HNO-mediated, and prior blocking with the thiol-specific reagent, N-ethylmaleimide, demonstrated that thiols are the targets of HNO. The results provide the first systematic characterization of HNO-mediated derivatization of protein thiols.