Regulation of glutamate dehydrogenase by reversible ADP-ribosylation in mitochondria

Mitochondrial ADP-ribosylation leads to modification of two proteins of approximately 26 and 53 kDA: The nature of these proteins and, hence, the physiological consequences of their modification have remained unknown. Here, a 55 kDa protein, glutamate dehydrogenase (GDH), was established as a specific acceptor for enzymatic, cysteine-specific ADP-ribosylation in mitochondria. The modified protein was isolated from the mitochondrial preparation and identified as GDH by N-terminal sequencing and mass spectrometric analyses of tryptic digests. Incubation of human hepatoma cells with [14C]adenine demonstrated the occurrence of the modification in vivo. Purified GDH was ADP-ribosylated in a cysteine residue in the presence of the mitochondrial activity that transferred the ADP-ribose from NAD+ onto the acceptor site. ADP- ribosylation of GDH led to substantial inhibition of its catalytic activity. The stoichiometry between incorporated ADP-ribose and GDH subunits suggests that modification of one subunit per catalytically active homohexamer causes the inactivation of the enzyme. Isolated, ADP-ribosylated GDH was reactivated by an Mg2+-dependent mitochondrial ADP-ribosylcysteine hydrolase. GDH, a highly regulated enzyme, is the first mitochondrial protein identified whose activity may be modulated by ADP-ribosylation.     

Amino acid-specific ADP-ribosylation: structural characterization and chemical differentiation of ADP-ribose-cysteine adducts formed nonenzymatically and in a pertussis toxin-catalyzed reaction.

ADP-ribosylation is a posttranslational modification of proteins by amino acid-specific ADP-ribosyltransferases. Both pertussis toxin and eukaryotic enzymes ADP-ribosylate cysteine residues in proteins and also, it has been suggested, free cysteine. Analysis of the reaction mechanisms of cysteine-specific ADP-ribosyltransferases revealed that free ADP-ribose combined nonenzymatically with cysteine. L- and D-cysteine, L-cysteine methyl ester, and cysteamine reacted with ADP-ribose, but alanine, serine, lysine, arginine, N-acetyl-L-cysteine, 2-mercaptoethanol, dithiothreitol, and glutathione did not. The 1H NMR spectrum of the product, along with the requirement for both free sulfhydryl and amino groups of cysteine, suggested that the reaction produced a thiazolidine linkage. ADP-ribosylthiazolidine was labile to hydroxylamine and mercuric ion, unlike the ADP-ribosylcysteine formed by pertussis toxin and NAD in guanine nucleotide-binding (G-) proteins, which is labile to mercuric ion but stable in hydroxylamine. In the absence of G-proteins but in the presence of NAD and cysteine, pertussis toxin generated a hydroxylamine-sensitive product, suggesting that a free ADP-ribose intermediate, expected to be formed by the NADase activity of the toxin, reacted with cysteine. Chemical analysis, or the use of alternative thiol acceptors lacking a free amine, is necessary to distinguish the enzymatic formation of ADP-ribosylcysteine from nonenzymatic formation of ADP-ribosylthiazolidine, thereby differentiating putative NAD:cysteine ADP-ribosyltransferases from NAD glycohydrolases.     

Nitric oxide and NAD-dependent protein modification

Nitric oxide (NO) has been suggested to act as a regulator of endogenous intracellular ADP-ribosylation, based on radiolabelling of proteins in tissue homogenates incubated with [³²P]NAD and No. After the NO-stimulated modification was replicated in a defined system containing only the purified acceptor protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the hypothesis of NO-stimulation of an endogenous ADP-ribosyltransferase became moot. The NO-stimulated, NAD-dependent modification of GAPDH was recently characterized as covalent binding of the whole NAD molecule to the enzyme, not ADP-ribosylation. With this result, along with the knowledge that GAPDH is stoichiometrically S-nitrosylated, the role of NO in protein modification with NAD may be viewed as the conferring of an unexpected chemical reactivity upon GAPDH, possibly due to nitrosylation of a cysteine in the enzyme active site.     

Effects of nucleotides on activity of a purified ADP-ribosyltransferase from turkey erythrocytes

A transferase purified from turkey erythrocytes catalyzed the NAD-dependent ADP-ribosylation of proteins in the supernatant, particulate, and detergent-solubilized fractions of bovine thymus as well as several purified proteins. Nucleoside triphosphates increased the rate of ADP-ribosylation of multiple soluble proteins from thymus and several purified proteins by about twofold. With lysozyme as substrate and 10 mm nucleotide, the order of effectiveness was ATP > ITP = GTP > CTP = UTP. Half-maximal stimulation of ADP-ribose incorporation into lysozyme was observed with 2.5 mm ATP. App(NH)p and inorganic tri- and tetrapolyphosphate were less effective than ATP; ADP, AMP, cAMP, and inorganic pyrophosphate were ineffective. Enhancement of transferase-catalyzed ADP-ribosylation by ATP was observed only at low (20–200 μm) NAD concentrations; with lysozyme as substrate, however, the effect of ATP was not due to prevention of NAD hydrolysis during the assay, nor was it due to an effect on ionic strength. The transferase catalyzed the ADP-ribosylation of several purified proteins and, depending on the protein substrate, ATP either increased, decreased, or did not alter the rate of ADP-ribosylation. It appears that ADP-ribosylation of cellular proteins by endogenous ADP-ribosyltransferases may be subject to regulation by nucleoside triphosphates.     

CTP-dependent endogenous ADP-ribosylation of a 38 kDa protein in HL-60 cell membranes

Incubation of membranes of human promyelocytic leukemia HL-60 cells with [32P]NAD led to ADP-ribosylation of several proteins including a 38 kDa protein by endogenous ADP-ribosyltransferases. The ADP-ribosylation of the 38 kDa protein was distinctly different from others on the basis of pH dependency and heat stability at 50 degrees C, suggesting that there are at least two endogenous ADP-ribosyltransferases. It was enhanced by CTP, but not affected by ATP, GTP and UTP, whereas it was inhibited by GTP gamma S. [alpha-32P]CTP bound to the 38 kDa protein immobilized on a nitrocellulose sheet, indicating that the 38 kDa protein which bound CTP is strongly ADP-ribosylated by an endogenous ADP-ribosyltransferase.     

Demonstration and partial characterization of ADP-ribosylation in Pseudomonas maltophilia.

ADP-ribosylation of proteins occurs in many eukaryotes, and it is also the mechanism of action of a growing number of important bacterial toxins. To date, however, there is only one well-characterized ADP-ribosylation system where the ADP-ribosyltransferase and the substrate protein are both bacterial in origin, namely within the nitrogen-fixing bacterium Rhodospirillum rubrum. The present paper demonstrates the endogenous ADP-ribosylation of two proteins of Mr 32,000 and 20,000 within Pseudomonas maltophilia, a Gram-negative aerobe. The proteins have been partially purified: two apparently separate species of modified protein can be separated by ion-exchange chromatography and gel filtration (V0 and Mr 158,000 - Vi). The substrate protein(s) either has, or is co-eluted with, NAD+ glycohydrolase activity. The modification is mono-ADP-ribosyl in nature. The linkage between the acceptor amino acid and the ADP-ribose moiety is alkali-labile and stable to hydroxylamine, possibly indicating an S-glycosidic bond. The activity appears to be a true ADP-ribosylation reaction and not an NAD+ glycohydrolase activity followed by non-enzymic addition of ADP-ribose to protein. The results presented here indicate that ADP-ribosylation may have a wider significance within prokaryotic systems than previously thought.     

Endogenous ADP-ribosylation of the G protein beta subunit prevents the inhibition of type 1 adenylyl cyclase

Mono-ADP-ribosylation is a post-translational modification of cellular proteins that has been implicated in the regulation of signal transduction, muscle cell differentiation, protein trafficking, and secretion. In several cell systems we have observed that the major substrate of endogenous mono-ADP-ribosylation is a 36-kDa protein. This ADP-ribosylated protein was both recognized in Western blotting experiments and selectively immunoprecipitated by a G protein beta subunit-specific polyclonal antibody, indicating that this protein is the G protein beta subunit. The ADP-ribosylation of the beta subunit was due to a plasma membrane-associated enzyme, was sensitive to treatment with hydroxylamine, and was inhibited by meta-iodobenzylguanidine, indicating that the involved enzyme is an arginine-specific mono-ADP-ribosyltransferase. By mutational analysis, the target arginine was located in position 129. The ADP-ribosylated beta subunit was also deribosylated by a cytosolic hydrolase. This ADP-ribosylation/deribosylation cycle might be an in vivo modulator of the interaction of betagamma with specific effectors. Indeed, we found that the ADP-ribosylated betagamma subunit is unable to inhibit calmodulin-stimulated type 1 adenylyl cyclase in cell membranes and that the endogenous ADP-ribosylation of the beta subunit occurs in intact Chinese hamster ovary cells, where the NAD(+) pool was labeled with [(3)H]adenine. These results show that the ADP-ribosylation of the betagamma subunit could represent a novel cellular mechanism in the regulation of G protein-mediated signal transduction.     

Poly ADP-ribose polymerase: self-ADP-ribosylation, the stimulation by DNA, and the effects on nucleosome formation and stability.

A partially purified preparation of the enzyme poly ADP-ribose polymerase which controls the transfer of ADP-ribose from NAD has been investigated. Data presented here indicate that the enzyme ADP-ribosylates itself. The enzyme preparation can be stimulated by DNA and this stimulation is exclusively associated with an auxiliary protein which copurifies with the enzyme and which we refer to as endogenous acceptor protein. Exogenously added proteins such as histones H1, H2A, and H3, cholera toxin, and Escherichia coli DNA-dependent RNA polymerase can also act as acceptor proteins in addition to the DNA-associated labeling of the endogenous acceptor. We speculate that the self-ADP-ribosylation of enzyme and that of the endogenous acceptor may play a role in control of the extremely rapid turnover of cellular NAD. Additionally, we have used this enzyme to ADP-ribosylate histones and to determine the effect of such modification on in vitro nucleosome formation and stability. The enzyme mediated ADP-ribosylation of free histones prior to incorporation into nucleosomes affects both nucleosome formation and stability while such ADP-ribosylation of histones already incorporated into nucleosomes does not affect their stability. These observations suggest that the ADP-ribosylation of histones prior to their involvement in nucleosomes might be the site of the physiologically important ADP-ribose transfer.     

Activation of immobilized, biotinylated choleragen AI protein by a 19-kilodalton guanine nucleotide-binding protein

Cholera toxin catalyzes the ADP-ribosylation that results in activation of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system, known as Gs. The toxin also ADP-ribosylates other proteins and simple guanidino compounds and auto-ADP-ribosylates its AI protein (CTA1). All of the ADP-ribosyltransferase activities of CTAI are enhanced by 19-21-kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors, or ARFs. CTAI contains a single cysteine located near the carboxy terminus. CTAI was immobilized through this cysteine by reaction with iodoacetyl-N-biotinyl-hexylenediamine and binding of the resulting biotinylated protein to avidin-agarose. Immobilized CTAI catalyzed the ARF-stimulated ADP-ribosylation of agmatine. The reaction was enhanced by detergents and phospholipid, but the fold stimulation by purified sARF-II from bovine brain was considerably less than that observed with free CTA. ADP-ribosylation of Gsa by immobilized CTAI, which was somewhat enhanced by sARF-II, was much less than predicted on the basis of the NAD:agmatine ADP-ribosyltransferase activity. Immobilized CTAI catalyzed its own auto-ADP-ribosylation as well as the ADP-ribosylation of the immobilized avidin and CTA2, with relatively little stimulation by sARF-II. ADP-ribosylation of CTA2 by free CTAI is minimal. These observations are consistent with the conclusion that the cysteine near the carboxy terminus of the toxin is not critical for ADP-ribosyltransferase activity or for its regulation by sARF-II. Biotinylation and immobilization of the toxin through this cysteine may, however, limit accessibility to Gsa or SARF-II, or perhaps otherwise reduce interaction with these proteins whether as substrates or activator.     

Stimulation of choleragen enzymatic activities by GTP and two soluble proteins purified from bovine brain

Choleragen (cholera toxin) activates adenylate cyclase by catalyzing ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide-binding protein. It was recently found (Tsai, S.-C., Noda, M., Adamik, R., Moss, J., and Vaughan, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5139-5142) that a bovine brain membrane protein known as ADP-ribosylation factor or ARF, which enhances ADP-ribosylation of Gs alpha, also increases the GTP-dependent NAD:arginine and NAD:protein ADP-ribosyltransferase, NAD glycohydrolase, and auto-ADP-ribosylation activities of choleragen. We report here the purification and characterization of two soluble proteins from bovine brain that similarly enhance the Gs alpha-dependent and independent ADP-ribose transfer reactions catalyzed by toxin. Like membrane ARF, both soluble factors are 19-kDA proteins dependent on GTP or GTP analogues for activity. Maximal ARF effects were observed at a molar ratio of less than 2:1, ARF/toxin A subunit. Dimyristoyl phosphatidylcholine was necessary for optimal ADP-ribosylation of Gs alpha but inhibited auto-ADP-ribosylation of the choleragen A1 subunit and NAD:agmatine ADP-ribosyltransferase activity. It appears that the soluble factors directly activate choleragen in a GTP-dependent fashion. The relationships of the ARF proteins to the ras oncogene products and to the family of guanine nucleotide-binding regulatory proteins that includes Gs alpha remains to be determined.     

Amino acid-specific ADP-ribosylation. Sensitivity to hydroxylamine of [cysteine(ADP-ribose)]protein and [arginine(ADP-ribose)]protein linkages

Hydroxylamine stability has been used to classify (ADP-ribose)protein bonds into sensitive and resistant linkages, with the former representing (ADP-ribose)glutamate, and the latter, (ADP-ribose)arginine. Recently, it was shown that cysteine also serves as an ADP-ribose acceptor. The hydroxylamine stability of [cysteine([32P]ADP-ribose)]protein and [arginine([32P] ADP-ribose)]protein bonds was compared. In transducin, pertussis toxin catalyzes the ADP-ribosylation of a cysteine residue, whereas choleragen (cholera toxin) modifies an arginine moiety. The (ADP-ribose)cysteine bond formed by pertussis toxin was more stable to hydroxylamine than was the (ADP-ribose)arginine bond formed by choleragen. The (ADP-ribose)cysteine bond apparently represents a third class of ADP-ribose bonds. Pertussis toxin ADP-ribosylates the inhibitory guanyl nucleotide-binding regulatory protein (Gi) of adenylate cyclase, whereas choleragen modifies the stimulatory guanyl nucleotide-binding regulatory protein (Gs). These (ADP-ribose)protein linkages are identical in stability to those formed in transducin by the two toxins, consistent with the probability that cysteine and arginine are modified in Gi and Gs, respectively. Bonds exhibiting differences in hydroxylamine-stability were found in membranes from various non-intoxicated mammalian cells following incubation with [32P]NAD, which may reflect the presence of endogenous NAD:protein-ADP-ribosyl-transferases.     

Eukaryotic mono(ADP-ribosyl)transferase that ADP-ribosylates GTP-binding regulatory Gi protein

An NAD:cysteine ADP-ribosyltransferase designated ADP-ribosyltransferase C was purified approximately 35,000-fold from human erythrocytes with an 11% yield. The purified ADP-ribosyltransferase C exhibited one predominant protein band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight (Mr) of 28,500. The Km values for NAD and cysteine methyl ester were determined to be 65 and 4,400 microM, respectively. By using human erythrocyte inside-out membrane vesicles, the transferase C was found to ADP-ribosylate the alpha subunit (Mr = 41,000) of Gi, which is a substrate for pertussis toxin. The ADP-ribosylation of Gi alpha catalyzed by ADP-ribosyltransferase C was inhibited by pre-ADP-ribosylation with pertussis toxin. The linkage of ADP-ribose-Gi alpha in the membranes formed by ADP-ribosyltransferase C was as stable to hydroxylamine as that formed by pertussis toxin. These data represent the first demonstration that eukaryotic cells contain an ADP-ribosyltransferase which can catalyze the ADP-ribosylation of a cysteine residue in Gi alpha.     

Inhibition of the transport of citrate during ADP-ribosylation of inner membrane proteins of the mitochondria

The ability of rat liver submitochondrial particles to catalyze NAD+ hydrolysis with a transfer of ADP-ribose residues to protein membranes has been demonstrated ADP-ribosylation is directly dependent on NAD+ concentration upon saturation with 1 mM NAD+ and is inhibited by physiological compounds (e.g., ATP, 10 mM; nicotinamide, 10 mM); besides, it is an artificial acceptor of ADP-ribose, arginine methyl ester. It was found that ADP-ribose is accepted by inner mitochondrial membrane protein, whose molecular masses amount to 25-30 kDa. The fact that 5'-AMP is a product of ADP-ribose degradation by snake venom phosphodiesterase suggests that the inner membrane vesiculate proteins are modified by mono(ADP-ribose). Covalent modification of membrane proteins by ADP-ribose leads to citrate transport inhibition in inner membrane vesicles the [14C]citrate uptake is significantly decreased thereby. The ability of ADP-ribosylation inhibitors to restore the citrate transport rate is suggestive of a direct regulatory effect of NAD+-dependent ADP-ribosylation on the activity of citrate-translocating system of inner mitochondrial membranes.     

Metabolism of NAD+ in the nuclei of human placenta

It has long been known that the major function of NAD+ is as an electron carrier in various biological oxidation-reduction systems. From many papers it is evident that NAD+ is involved as substrate in ADP-ribosylation reactions. We have focused our attention on two chromatin enzymes: NMN-adenylyltransferase that catalyzes reversible synthesis of NAD+ utilizing ATP and NMN, and poly(ADP-ribose)polymerase that covalently modifies nucleosomal proteins through poly ADP-ribosylation reactions. Here we provided evidence of these activities in a system of isolated nuclei from human placenta. The data presented in this report show that purified nuclei might be useful to study the nuclear location of these enzymes and their reciprocal interactions.     

A novel approach to detect toxin-catalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin

Certain microbial toxins are ADP-ribosyltransferases, acting on specific substrate proteins. Although these toxins have been of great utility in studies of cellular regulatory processes, a simple procedure to directly study toxin-catalyzed ADP-ribosylation in intact cells has not been described. Our approach was to use [2-3H]adenine to metabolically label the cellular NAD+ pool. Labeled proteins were then denatured with SDS, resolved by PAGE, and detected by flurography. In this manner, we show that pertussis toxin, after a dose-dependent lag period, [3H]-labeled a 40-kD protein intact cells. Furthermore, incubation of the gel with trichloroacetic acid at 95 degrees C before fluorography caused the release of label from bands other than the pertussis toxin substrate, thus, allowing its selective visualization. The modification of the 40-kD protein was ascribed to ADP-ribosylation of a cysteine residue on the basis of inhibition of labeling by nicotinamide and the release of [3H]ADP-ribose from the labeled protein by mercuric acetate. Cholera toxin catalyzed the [3H]-labeling of a 46-kD protein in the [2-3H]adenine-labeled cells. Pretreatment of the cells with pertussis toxin before the labeling of NAD+ with [2-3H]adenine blocked [2-3H]ADP-ribosylation catalyzed by pertussis toxin, but not that by cholera toxin. Thus, labeling with [2-3H]adenine permits the study of toxin-catalyzed ADP-ribosylation in intact cells. Pasteurella multocida toxin has recently been described as a novel and potent mitogen for Swiss 3T3 cell and acts to stimulate the phospholipase C-mediated hydrolysis of polyphosphoinositides. The basis of the action of the toxin is not known. Using the methodology described here, P. multocida toxin was not found to act by ADP-ribosylation.     

A new detection method for arginine-specific ADP-ribosylation of protein -- a combinational use of anti-ADP-ribosylarginine antibody and ADP-ribosylarginine hydrolase

Arginine-specific ADP-ribosylation is one of the posttranslational modifications of proteins by transferring one ADP-ribose moiety of NAD to arginine residues of target proteins. This modification, catalyzed by ADP-ribosyltransferase (Art), is reversed by ADP-ribosylarginine hydrolase (AAH).

In this study, we describe a new method combining an anti-ADP-ribosylarginine antibody (ADP-R-Arg Ab) and AAH for detection of the target protein of ADP-ribosylation. We have raised ADP-R-Arg Ab with ADP-ribosylated histone and examined the reactivity of the antibody with proteins treated by Art and/or AAH, as well as in situ ADP-ribosylation system with mouse T cells. Our results indicate that the detection of ADP-ribosylated protein with ADP-R-Arg Ab and AAH is a useful tool to explore the target proteins of ADP-ribosylation. We applied the method to search endogenously ADP-ribosylated protein in the rat, and detected possible target proteins in the skeletal muscle, which has high Art activity.     

Acetylation-dependent ADP-ribosylation by Trypanosoma brucei Sir2

Sirtuins are a highly conserved family of proteins implicated in diverse cellular processes such as gene silencing, aging, and metabolic regulation. Although many sirtuins catalyze a well characterized protein/histone deacetylation reaction, there are a number of reports that suggest protein ADP-ribosyltransferase activity. Here we explored the mechanisms of ADP-ribosylation using the Trypanosoma brucei Sir2 homologue TbSIR2rp1 as a model for sirtuins that reportedly display both activities. Steady-state kinetic analysis revealed a highly active histone deacetylase (k cat = 0.1 s(-1), with Km values of 42 microm and for NAD+ and 65 microm for acetylated substrate). A series of biochemical assays revealed that TbSIR2rp1 ADP-ribosylation of protein/histone requires an acetylated substrate. The data are consistent with two distinct ADP-ribosylation pathways that involve an acetylated substrate, NAD+ and TbSIR2rp1 as follows: 1) a noncatalytic reaction between the deacetylation product O-acetyl-ADP-ribose (or its hydrolysis product ADP-ribose) and histones, and 2) a more efficient mechanism involving interception of an ADP-ribose-acetylpeptide-enzyme intermediate by a side-chain nucleophile from bound histone. However, the sum of both ADP-ribosylation reactions was approximately 5 orders of magnitude slower than histone deacetylation under identical conditions. The biological implications of these results are discussed.     

Identification of a botulinum C3-like enzyme in bovine brain that catalyzes ADP-ribosylation of GTP-binding proteins.

A novel enzyme activity was found in bovine brain cytosol that transfers the ADP-ribosyl moiety of NAD to proteins with Mr values of 22,000 and 25,000. The substrates were the same GTP-binding proteins serving as the substrate of an ADP-ribosyltransferase C3 which was produced by a type C strain of Clostridium botulinum. The brain enzyme was partially purified from the cytosol and had a molecular mass of approximately 20,000 on a gel filtration column. The brain endogenous enzyme displayed unique properties similar to those observed with botulinum C3 enzyme. The enzyme activity was markedly stimulated by a protein factor that had been initially found in the cytosol as an activator for botulinum C3-catalyzed ADP-ribosylation (Ohtsuka, T., Nagata, K., Iiri, T., Nozawa, Y., Ueno, K., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 15000-15005). The activity of the brain enzyme was also affected by certain types of detergents or phospholipids. The substrate of the brain enzyme was specific for GTP-binding proteins serving as the substrate of botulinum C3 enzyme; the alpha-subunits of trimeric GTP-binding proteins which served as the substrate of cholera or pertussis toxin were not ADP-ribosylated by the endogenous enzyme. Thus, this is the first report showing an endogenous enzyme in mammalian cells that catalyzes ADP-ribosylation of small molecular weight GTP-binding proteins.     

Modification of plasma membrane protein cysteine residues by ADP-ribose in vivo

Proteins can be post-translationally modified by ADP-ribose. Previously, two classes of ADP-ribosyl protein linkages have been detected in vivo which have chemical properties indistinguishable from ADP-ribosyl arginine and ADP-ribosyl glutamate or aspartate. Reported here is the detection of a third class of endogenous ADP-ribosyl protein linkage. This class is chemically indistinguishable from ADP-ribose linked to cysteine residues by a thioglycosidic bond. The distribution of ADP-ribosyl cysteine residues was studied in subcellular fractions of rat liver. Proteins modified on cysteine were detected only in the plasma membrane fraction. Pertussis toxin is known to disrupt signal transduction of ADP-ribosylation of cysteine residues of plasma membrane GTP binding proteins. The results described here raise the interesting possibility that the endogenous modification of plasma membrane protein cysteine residues may be involved in signal transduction.     

Entamoeba histolytica: ADP-ribosylation of secreted glyceraldehyde-3-phosphate dehydrogenase

In addition to its classic glycolytic role, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been implicated in many activities unrelated to glycolysis, such as membrane fusion, binding to host proteins and signal transduction. GAPDH can be the target of several modifications that allow incorporation to membranes and possible regulation of its activity; among these modifications is mono-ADP-ribosylation. This post-translational modification is important for the regulation of many cellular processes and is the mechanism of action of several bacterial toxins. In a previous study, we observed the extracellular ADP-ribosylation of a 37-kDa ameba protein. We report here that GAPDH and cysteine synthase A are the main ADP-ribosylated proteins in Entamoeba histolytica extracellular medium, GAPDH is secreted from ameba at 37 degrees C in a time-dependent manner, and its enzymatic activity is not inhibited by ADP-ribosylation. Extracellular GAPDH from ameba may play an important role in the survival of this human pathogen or in interaction with host molecules, as occurs in other organisms.