Cécile Herve

Pierre-Olivier De Franco

Agnès Groisillier

Thierry Tonon

Catherine Boyen
email: boyen@sb-roscoff.fr

New members of the Glutathione transferase family discovered in Red and Brown algae

Keywords: CDNB, 1-chloro-2,4-dinitrobenzene, EST, expressed sequence tag, FDNB, 1-fluoro-2,4-dinitrobenzene, GSH, glutathione, GST, glutathione transferase, 4-HNE Glutathione transferase, Chondrus crispus, Laminaria digitata, phylogenetic classification, recombinant proteins, methyl jasmonate

The GSTs (glutathione transferases) are involved in the detoxification of a wide variety of hydrophobic substrates. These enzymes have been found in virtually all types of organisms, including plants, animals, nematodes and bacteria. In the present study, we report the molecular and biochemical characterization of algal GSTs. Phylogenetic analysis showed that most of them were distinct from previously described GST classes but were most closely related to the sigma class. Profiling of GST genes from the red alga Chondrus crispus and brown alga Laminaria digitata was undertaken after different chemical treatments and showed that they displayed contrasting patterns of transcription. Recombinant algal GST from both species showed transferase activities against the common substrates aryl halides, but also on the α,β-unsaturated carbonyl 4hydroxynonenal. As well, they exhibit significant peroxidation towards organic hydroperoxides, including oxygenated derivatives of polyunsaturated fatty acids. Among a range of compounds tested, cibacron blue was the most efficient inhibitor of algal GSTs identified.

INTRODUCTION

Among the cellular defense systems, GSTs (EC 2.5.1.18) form a highly diverse family of proteins with functions ranging from detoxification to cell signaling. They are named after their ability to catalyze the nucleophilic attack of GSH (glutathione) on electrophilic substrates. In this manner, they contribute to the metabolism of drugs, chemical carcinogens, pesticides and other xenobiotics [START_REF] Dixon | Plant glutathione transferases[END_REF]. Endogenous products formed as secondary metabolites during oxidative stress are also substrates for GST enzymes. In addition, several GSTs act as GSH-dependent peroxidases by catalyzing the reduction of organic hydroperoxides to the less-toxic monohydroxy alcohols [2].

Beyond detoxification and involvement in various defense responses, there is increasing evidence indicating that GSTs may be implicated in many other physiological processes including isomerization of specific metabolites [START_REF] Thom | The structure of a zeta class glutathione S-transferase from Arabidopsis thaliana: characterisation of a GST with novel active-site architecture and a putative role in tyrosine catabolism[END_REF], transportation of endogenous substrates [START_REF] Mueller | AN9, a Petunia Glutathione S-Transferase Required for Anthocyanin Sequestration, Is a Flavonoid-Binding Protein[END_REF], plant cell growth and development [START_REF] Gong | Expression of glutathione-S-transferase and its role in plant growth and development in vivo and shoot morphogenesis in vitro[END_REF].

These proteins are found in almost all species and are divided into classes, based primarily on sequence similarity. Currently recognized classes of cytosolic GSTs in mammals include the Alpha, Mu, Omega, Pi, Sigma, Theta and Zeta classes [START_REF] Pearson | Phylogenies of Glutathione Transferase Families[END_REF]. GSTs from the last two classes have also been identified in plants and other organisms. Other distinct groups have been identified only in insects (Delta and Epsilon; [START_REF] Ranson | Identification of a novel class of insect glutathione S-transferases involved in resistance to DDT in the malaria vector Anopheles gambiae[END_REF]), plants (Lambda, Phi, Tau; [START_REF] Dixon | Plant glutathione transferases[END_REF]), or prokaryotes (Beta; [8]). A Kappa class regroups mitochondrial GSTs, and a family of microsomal GSTs comprises the membraneassociated proteins designated MAPEG.

GST enzymes have been extensively purified from mammals, plants and invertebrates [START_REF] Armstrong | Structure, catalytic mechanism, and evolution of the glutathione transferases[END_REF]. In contrast, the occurrence of GSTs has not been explored in some taxa, such as the marine algae. Algae currently encompass eight groups of photosynthetic eukaryotes which have evolved independently in the early history of life.

These organisms have acquired their plastids through different endosymbiotic events. A first event involving a cyanobacterial ancestor gave rise to the red algae (Rhodophytae) and the green plants (green algae and land plants). Brown algae (Phaeophyceae) and diatoms stem from a secondary endosymbiotic event, and together with the oomycetes, belong to the Heterokonts [10]. So far, there has been virtually no report on the molecular characterization of GSTs in algae, particularly seaweed, mostly due to a lack of genomic information in these organisms. Recently, we analyzed cDNA catalogs from thallus and protoplasts of the red alga Chondrus crispus and of the brown alga Laminaria digitata, and provided the first genetic evidence of cytosolic GST sequences in both organisms [START_REF] Collén | An expressed sequence tag analysis of thallus and regenerating protoplasts of Chondrus crispus (Gigartinales, Rhodophyceae)[END_REF][START_REF] Roeder | Identification of stress genes transcripts in Laminaria digitata (Phaeophyceae) protoplast cultures by expressed sequence tag analysis[END_REF]. In the present study, phylogenetic analyses demonstrate an unexpected link between macroalgal GSTs and a large animal GST cluster including the Sigma class GSTs. We thus propose that these red and brown algal GSTs, together with oomycetes representatives, define a new group of GSTs closely-related to the Sigma class. In addition expression profiling of these genes showed that red and brown algal GSTs exhibit different patterns of transcription according to chemical treatments. Transferase and peroxidase activities of the recombinant proteins from both algae were determined in presence of different categories of substrates, and cibacronblue was determined as the most effective inhibitor among the molecules tested.

EXPERIMENTAL Plant material cultivation and chemical treatments

Gametophytic Chondrus crispus unialgal isolates, and sporophytic Laminaria digitata unialgal isolates, were cultivated at 13°C in enriched seawater medium SFC [START_REF] Correa | Endophytic algae of Chondrus crispus (Rhodophyta). II. Acrochaete heteroclada sp. nov., A. operculata sp. nov., and Phaeophyla dendroides (Chlorophyta)[END_REF], using a photoperiod of 10 hours of light and 14 hours of darkness and a photon flux density of 40 µmol m -2 s -1 . Oligoguluronates were produced by enzyme degradation of sodium alginate [START_REF] Boyen | Preparation of alginate lyases from marine molluscs for protoplast isolation in brown algae[END_REF].

To perform chemical treatments, algae were first maintained in Petri dishes with seawater for at least 18 hours. Final concentrations of chemicals were as follows: 10 mM H 2 O 2 ; 500 µM each tested pesticide; 100 or 200 µM each heavy metal tested in L.

Identification of cDNA and corresponding genes encoding GST

EST encoding GSTs were identified in C. crispus and L. digitata through analysis of corresponding cDNA libraries [START_REF] Collén | An expressed sequence tag analysis of thallus and regenerating protoplasts of Chondrus crispus (Gigartinales, Rhodophyceae)[END_REF][START_REF] Collén | Expression profiling of Chondrus crispus (Rhodophyta) after exposure to methyl jasmonate[END_REF]. To obtain the genomic sequence of CcGST1, CcGST2, CcGST3, LdGST1, LdGST2 and LdGST3 genes, PCR amplifications were performed on genomic DNA using specific primers flanking the coding region of each gene. The genomic sequence of LdGST4 could not be amplified in a single PCR, due to the presence of large introns. All the primers used in this study are reported in Supplementary Table S1. PCR conditions consisted of an initial denaturation of 5 min at 95°C, followed by 35 cycles of 45 s at 95°C, 60 s at 58°C and 100 s at 72°C, and a final extension of 10 min at 72°C. PCR products were recovered from agarose gels, purified using the Mini Elute Purification kit (Qiagen), and sequenced in both directions.

Bioinformatic analysis

Protein alignment was initially performed using the The robustness of the branches was evaluated using bootstrap replicates.

Sequences selected from public databases and used for the phylogenetic analysis are listed in Supplementary Table S3.

Real-time PCR

Total RNA were reverse transcribed using the Superscript II RT kit (Invitrogen), and real-time PCR was performed in a GeneAmp 5700 sequence detection system using the SYBR Green PCR master kit (Applied Biosystems) according to the manufacturer's instructions. The primers used in the reaction were recommended by Primers Express The relative changes in the GST mRNA expression were calculated as x-fold changes relative to the appropriate control experiment for the different chemical treatments.

Heterologous expression and protein purification

The open reading frames of the GST transcripts from C. crispus and L. digitata were amplified by PCR using the primers listed in Supplementary Table S1. These primers were designed to incorporate BamHI and EcoRI restriction sites into the 5' and 3' ends, respectively. PCR conditions consisted of an initial denaturation of 5 min at 94°C, followed by 30 cycles of 30 s at 94°C, 30 s at 50°C and 2 min at 72°C, and a final extension step of 10 min at 72°C. The resulting PCR products were purified, digested with the appropriate restriction enzymes and subcloned into the pFO4 expression vector, a vector modified from pET15 (Novagen) to be compatible with the BamHI/EcoRI and BamHI/MfeI ligation strategies.

Recombinant plasmids were used to transform Escherichia coli strain BL21(DE3). Transformed colonies were grown at 37°C overnight in a Luria-Bertani medium containing ampicillin. The culture was diluted 1:100 with auto-inducible ZYP medium [START_REF] Studier | Protein production by auto-induction in high density shaking cultures[END_REF] containing ampicillin and subjected to further incubation at 20°C until the culture density reached saturation. After centrifugation, pelleted bacteria were resuspended in Tris-HCl pH7.5 buffer containing 300 mM NaCl and 60 mM imidazole and disrupted using a French press prior to centrifugation. The resulting supernatant was applied onto a Chelating Fast Flow Sepharose column charged with 100 mM NiSO 4 (Amersham Biosciences). After washing, the bound proteins were eluted with a linear gradient of imidazole ranging from 60 mM to 300 mM. The protein eluted was collected and dialyzed against a buffer containing 20 mM Tris-HCl pH7.

Enzyme assays

GST enzyme activity assays were carried out in a microplate Safire² UV spectrophotometer reader (Tecan), except for 4-HNE and trans-2-nonenal that used a UV-2401-PC spectrophotometer (Shimadzu). In all instances, the non-enzymatic reaction was measured and subtracted from the overall reaction rate. Assays were conducted at 30°C and measures were performed during 10 min after addition of second substrate. Ethacrynic acid, trans-4-phenyl-3-buten-2-one, para-nitrophenyl acetate and cumene hydroperoxide were generously provided by Fabrice Morel (U620 Inserm, Rennes, France). Except the PUFA hydroperoxides and the 4-HNE obtained from

Cayman chemicals, all chemicals were purchased from Sigma.

Glutathione transferase and glutathione peroxidase activities were measured as reported by Thomson et al. [START_REF] Thomson | Sequence, catalytic properties and expression of chicken glutathione-dependent prostaglandin D2 synthase, a novel class Sigma glutathione S-transferase[END_REF]. Enzyme activity with aldehydes 4-HNE and trans-2nonenal was assessed as reported by Alin et al. [START_REF] Alin | 4-Hydroxyalk-2-enals are substrates for glutathione transferase[END_REF]. Substrate concentrations for each test are given in the Supplementary Table S2. A unit of enzyme activity is defined as the amount of enzyme that catalyses the turnover of 1 µmol substrate per min. Specific activity is expressed in µmol min -1 per mg of enzyme. The apparent Km value for GSH

RESULTS

Sequence analysis of cDNA-encoding algal GSTs

Seven putative cytosolic GSTs were identified through analysis of algal EST libraries:

three from the rhodophyte C. crispus (CcGST1, CcGST2, CcGST3) and four from the phaeophyte L. digitata (LdGST1, LdGST2, LdGST3, LdGST4). Their length ranged from 201 to 230 amino acids. In both protoplast libraries, these GST genes were among the most represented sequences. The cDNA sequences corresponding to the complete coding regions of these GSTs have been deposited in GenBank under the following accession numbers: EF423557 for CcGST1, EF423558 for CcGST2, EF423559 for CcGST3, EF422836 for LdGST1, EF424837 for LdGST2, EF422838 for LdGST3 and EF422839 for LdGST4.

The GenBank database was searched using the protein blast program and revealed that CcGST1, CcGST2, LdGST1, LdGST2, LdGST3 and LdGST4 were most similar to Sigma-class enzymes, with a maximum of 33 % identity. Conserved residues usually encountered in GST classes from mammals were also found in the algal sequences. Of significance was the highly conserved catalytic tyrosine found in the early N-terminal portion of most mammalian GSTs (Figure 1; [START_REF] Thomson | Sequence, catalytic properties and expression of chicken glutathione-dependent prostaglandin D2 synthase, a novel class Sigma glutathione S-transferase[END_REF]). In the mammalian Pi, Mu and Sigma classes, the glutamine and serine residues just prior to the α3-helixe are also involved in the interaction with GSH [START_REF] Armstrong | Structure, catalytic mechanism, and evolution of the glutathione transferases[END_REF]. Compared to other Sigma-class GST members, the C-terminal domain of the algal sequences were highly divergent, which may indicate different substrate selectivities and specificities of the algal GSTs. In contrast to the other GSTs identified in this study, CcGST3 shared higher identities (25-residues are found in the early N-terminal portion of the CcGST3 sequence and may be involved in the GSH binding (see Supplementary Figure S1).

The similarity of amino acids between the six most-related algal sequences was 28-63 %, indicating a high degree of heterogeneity. The highest identities were observed between CcGST1 and CcGST2 (51 %), and between LdGST1, LdGST2 and LdGST3 (50-63 %). LdGST4 was the most divergent among the L. digitata sequences, as CcGST3 was among the C. crispus sequences.

While classification in different GST families can be based on sequence similarities alone, distinct gene structure provides additional supporting evidence [START_REF] Dixon | Plant glutathione transferases[END_REF][START_REF] Frova | Glutathione transferases in the genomics era: New insights and perspectives. Biomolecular Engineering 23, 149-169 A new class of glutathione S-transferase from the hepatopancreas of the red sea bream Pagrus major[END_REF]. Therefore, the cDNA sequences of C. crispus and L. digitata GSTs were compared with their corresponding genomic DNA sequences. The genes encoding CcGST3, LdGST1, LdGST2 and LdGST3 were intronless. CcGST1 holds an intron of 93 bp, whose size and position coincides exactly with those of the unique intron of 100 bp encountered in CcGST2 (Figure 2). The LdGST4 genomic sequence contains four exons and three introns. Their length could not be determined accurately due to the low PCRproduct yield for this gene. This could be attributed to the presence of large introns in the sequence.

Phylogenetic position of algal GSTs

In order to elucidate phylogenetic relationships between the identified algal GSTs with sequences from other organisms, we searched for additional GST genes using genomic resources available for micro-and macroalgae. Among the sequences identified, 7 full-length putative cytosolic GST genes that are well supported by ESTs were considered for further analyses. These sequences were as follow: two from Gracilaria tenuistipitata (Rhodophyta), one from the green macroalga Ulva linza (Chlorophyta) and four for the brown macroalga Ectocarpus siliculosus (Phaeophyceae). They will be made available upon request to the authors. Sequences from the oomycetes Phytophthora sojae and Phytophthora ramorum were also taken into account. Two other previously identified GST sequences were also included: CspGST1 from the green unicellular alga Coocomyxa sp.

[24] and PiGST1 from the oomycete Phytophthora infestans [START_REF] Bryant | Cloning and characterization of a theta class glutathione transferase from the potato pathogen Phytophthora infestans[END_REF]. The red alga CcGST3 sequence was not included due to its highly divergent pattern. In total, 155 sequences were integrated in a multiple protein alignment. Several optimal trees were then generated from both distance and maximum likelihood methods, and their topologies were essentially the same, with slight differences observed in the bootstrap values supporting the nodes. The most reliable tree is presented in Figure 3. Two major groups of GST proteins can be established, in agreement with previous studies [START_REF] Marco | Evolutionary and Structural Analyses of GDAP1, Involved in Charcot-Marie-Tooth Disease, Characterize a Novel Class of Glutathione Transferase-Related Genes[END_REF][START_REF] Snyder | Molecular phylogeny of glutathione-S-transferases[END_REF].

The first group contains proteins specific of different eukaryotic organisms, as plants (Tau, Lambda and Phi classes), animals (Omega class) and insects (Delta and Epsilon classes). The Zeta and Theta classes have counterparts in plants and animals, and several sequences from the oomycetes P. sojae and P. ramorum can also be found close to these classes. This phylogeny confirms that the recently described Rho class, thought to be fish-specific, also comprises green algal sequences [24]. The second major group includes the Alpha, Pi, Mu and Sigma classes, primarily encountered in mammals. Most of the newly isolated sequences from this study fall into this group and are closely related to the Sigma GST members. Because of this unexpected distribution, we undertook a more focused phylogenetic analysis, restricted to GSTs of the Alpha, Mu, Pi and Sigma classes and including the "algae-oomycetes" sequences (see Supplementary Figure S2). The overall topology already observed in the previous phylogenetic tree was confirmed and a more detailed analyze is discussed on line.

Different transcription patterns for brown and red algal GSTs

In both C. crispus and L. digitata, GST sequences were identified through analysis of protoplast cDNA libraries and were among the most represented genes, suggesting a possible role of these enzymes in detoxification processes during protoplast generation.

Changes in expression of these genes were further investigated under different chemical treatments and after short exposure times (Figure 4). for the C20 derivatives.

In the brown alga L. digitata, elicitors released after wounding and degradation of the cell wall such as oligoguluronates are recognized as defense signals and induce an oxidative burst in the alga [START_REF] Kupper | Oligoguluronates elicit an oxidative burst in the brown algal kelp Laminaria digitata[END_REF]. LdGST genes were all induced by the oligoguluronates (4 to 24-fold), LdGST4 being the most up-regulated gene (Figure 4B). A similar pattern of variation was obtained in presence of copper, with LdGST2 being the most upregulated (30-fold). In presence of fatty acid derivatives, slight changes of transcript levels were observed with 15(S)-HETE, while MeJA caused a repression with a maximum of a 6-fold decrease for LdGST1.

Biochemical studies of algal GST

In order to investigate the biochemical features and to determine the catalytic properties of the GST enzymes from both algae, the GST genes presently described were assayed for heterologous expression in Escherichia coli. Only CcGST2 and LdGST3 were selected for further investigation since they were the most highly expressed in the soluble fraction. Migration on SDS-PAGE gels of these enzymes showed that the purified proteins gave single bands of 26 kDa. Gel-filtration chromatography analysis showed a single peak with molecular mass of 52 kDa for both GSTs, strongly suggesting that the active form of these enzymes exhibits homodimeric structures (see Supplementary Figure S3).

The catalytic specificities of CcGST2 and LdGST3 were explored using a range of GST substrates. In presence of the commonly used CDNB, the two algal GSTs exhibited moderate transferase activity (Table 1). Higher transferase activities were measured towards several model aryl halide substrates, as illustrated by the results obtained with CcGST2 in presence of 1-fluoro-2,4-dinitrobenzene (FDNB; 20.34 µmol min -1 mg -1 ). Slight transferase activities were also observed with one aldehyde of the α,β-unsaturated carbonyls, the 4-HNE. No activity was detected with the paranitrophenyl acetate substrate. The algal GSTs were also incubated in presence of the synthetic cumene hydroperoxide and t-butyl hydroperoxide substrates to ascertain their glutathione peroxidase activity. Results show that the recombinant enzymes exhibited similar moderate activity to reduce cumene hydroperoxide (Table 1). In addition, both proteins had activity towards hydroperoxides of C18 polyunsaturated fatty acids (13-HpODE and 13-HpOTE).

The Km values of CcGST2 and LdGST3 for GSH in presence of CDNB were calculated to be 0.9 and 1.1 mM respectively (see Supplementary Table S4).

Measurement of GST activity through a wide range of pH for the two purified recombinant enzymes indicated that optimal pH was 8 and 10 for CcGST2 and LdGST3 respectively (see Supplementary Figure S4).

To complete these data, inhibiting assays were also performed and results are presented in Table 2. Cibacron blue, a typical ligandin-type inhibitor, showed the most potent inhibition effect (IC 50 of 0.5 and 0.1 µM for CcGST2 and LdGST3 respectively), whereas chenodeoxycholic acid, lithocholic acid, indomethacin and acetyl salicylic acid did not show any marked inhibition effect on the activity of either enzyme.

DISCUSSION

The soluble glutathione transferases constitute a widespread enzyme superfamily that has been subdivided into an ever-increasing number of classes [START_REF] Sheehan | Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily[END_REF]. While there are no clearly established criteria concerning the extent of sequence similarity required for placing a GST in a particular class, it is generally accepted that GSTs of the same class share more than ca. 50% sequence identity, while GSTs of different classes share less than ca. 30% identity [START_REF] Frova | Glutathione transferases in the genomics era: New insights and perspectives. Biomolecular Engineering 23, 149-169 A new class of glutathione S-transferase from the hepatopancreas of the red sea bream Pagrus major[END_REF]30]. Some of these classes are ubiquitous all the way up to the kingdom level, whereas others are exclusively found in particular groups of organisms such as mammals, plants or insects. As large-scale EST and genomic approaches are being applied to previously uninvestigated phyla, new GST genes and even new GST classes are being discovered. Here we identified and characterized GST genes from two independent eukaryotic lineages, the Rhodophyta (red algae) and the Heterokonta (brown algae and oomycetes).

The phylogenetic analyses showed that the GST sequences from the red and brown algae, along with some oomycetes, fall into a distinctive, newly identified group.

Within this new cluster, sequences may be assigned to several sub-groups: GtGST sequences are closely related to CcGST sequences, while EsGST3, EsGST4 and LdGST4 group together; LdGST1, LdGST2, LdGST3 and EsGST1 appear to form another group. This grouping is further supported by evidence for distinct gene structure. The LdGST1, LdGST2 and LdGST3 genes are intronless, while CcGST1 and CcGST2 genes contain a single intron, and the LdGST4 gene has three introns. In addition, it is interesting to note that the brown algal sequences appear to be more closely related to the red algal enzymes than to the P. sojae sequences. Considering the common evolutionary history of oomycetes and brown algae in the heterokont lineage, their respective GST members were expected to group in the same cluster. The divergence among the sequences identified in this work is also illustrated by long branch lengths, of great magnitude compared to the Alpha-, Mu-and Pi-class clusters whose members are much more closely related to each other. This higher evolutionary rate may also reflect an adaptive response to habitat. It is tempting to suggest that this unexpected topology results from convergent evolution of algal enzymes in response to a specific adaptation to the marine environment.

This new set of GSTs are closely related to the Sigma GST enzymes which is encountered in both vertebrates and invertebrates, while the Alpha, Pi and Mu members are only found in vertebrates [31]. It was initially suggested that the Alpha-class enzymes were the oldest members of this clade and that they had been subsequently lost in invertebrates [START_REF] Armstrong | Structure, catalytic mechanism, and evolution of the glutathione transferases[END_REF][START_REF] Sheehan | Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily[END_REF]31]. In contrast, Frova [START_REF] Frova | Glutathione transferases in the genomics era: New insights and perspectives. Biomolecular Engineering 23, 149-169 A new class of glutathione S-transferase from the hepatopancreas of the red sea bream Pagrus major[END_REF] recently proposed that the Sigmaclass GSTs diverged before the Alpha, Mu and Pi group and has evolved distinct functional properties in the diverse lineages. The occurrence of sequences closely linked to the Sigma enzymes in algae and oomycetes supports this last hypothesis suggesting that the entire Sigma class emerged before the eukaryotes split and diverged extensively later in independent lineages. Biotic stress and chemicals are known to affect the expression of GSTs at the transcriptional level [START_REF] Dixon | Plant glutathione transferases[END_REF], transcript accumulations for C. crispus and L. digitata genes were determined in response to various treatments. The seven identified genes were constitutively expressed in the two algae. The basal levels of expression were lower for the LdGST genes than for the CcGST genes. CcGST2 was expressed at the same level as the housekeeping gene actin, indicating that this GST protein plays a major role in the basic metabolism of C. crispus. CcGST genes were highly induced by the herbicides atrazine and metolachlor and the insecticide dichlorvos. This increase in gene expression was correlated with a higher GST activity in total extracts of the red alga treated during 24 hours with metolachlor and dichlorvos (data not shown). The red macroalgae may therefore have the potential to remove xenobiotic pollutants from their environment.

In contrast to these results and considering what is known in higher plants [32] heavy metals such as cadmium, copper, zinc and aluminium caused a decrease in expression of the three CcGST genes. Opposite results were observed for the LdGST Interestingly, contrasting results were also obtained for GST from both algae in presence of oxylipins derived from C18 and C20 polyunsaturated fatty acids. Several oxylipin structures have been described so far in marine algae, some of them being closely related, and even identical, to those identified in terrestrial plants (C18derivatives; [33]) and mammals (C20-derivatives; [START_REF] Sajiki | Identification of eicosanoids in the red algae, Gracilaria asiatica, using high-performance liquid chromatography and electrospray ionization mass spectrometry[END_REF]). Their biological functions remain elusive, despite the fact that some of them have been demonstrated to act as potential defense and signaling mediators in red marine alga [START_REF] Bouarab | The innate immunity of a marine red alga involves oxylipins from both the eicosanoid and octadecanoid pathways[END_REF]. The present study demonstrated that C18 and C20 oxylipins greatly increased levels of CcGST transcripts, confirming previous results obtained after treatment of C. crispus with MeJA . The precise roles of CcGST during these treatments are unknown. They may be expressed as active participants in a more global defense response to detoxify by-products released during biotic stresses. However, they could also have a more dedicated function, such as a potential involvement in fatty acid and oxylipin metabolism. Since the tested oxylipins had the opposite effect on LdGST genes, these molecules may have different roles in the two algae, and/or GSTs may not drive similar processes in the two lineages.

It is also worth noting that, despite structural differences in gene organization among GST genes within a species, all GSTs from the same alga exhibited a similar transcription profile. This probably suggests functional convergence of these enzymes within a species, and their importance in specific metabolic pathways. Further experiments are required to identify the biological significance of this result.

Expression and purification of recombinant CcGST and LdGST enzymes in E. coli made it possible to characterize the properties of these proteins. The catalytic profile of CcGST2 was similar to that of LdGST3, but the red alga GST showed greater activity towards FDNB substrates. In contrast, LdGST3 exhibited a higher transferase activity in presence of 4-HNE, an electrophilic end-product of lipid peroxidation. Both enzymes have no activity on the related compound trans-2-nonenal. In addition to their glutathione transferase activity, both enzymes are able to reduce organic hydroperoxides of fatty acids. This peroxidase activity might play a role in the algae during oxidative stress, in preventing the production of cytotoxic aldehydes from the organic hydroperoxides. Algal GST Km values illustrate a moderate affinity substrate to GSH. In addition to detoxifying electrophilic compounds, there is growing evidence that GST members carry out additional functions. The GST activities are quite diverse, not only between, but also within classes, as demonstrated for members of the Sigma class. For example, in cephalopods, S-crystallin proteins are highly abundant in eye lenses, and have lost their GST activity due to insertion of 11 residues between the conserved α4 and α5 helixes [START_REF] Chang | Molecular basis for the polymerization of octopus lens S-crystallin[END_REF]. In Drosophila melanogaster, Sigma-type GSTs have considerable activities towards α,β-unsaturated carbonyl compounds derived from a secondary lipid-peroxidation [START_REF] Agianian | Structure of a Drosophila sigma class glutathione S-transferase reveals a novel active site topography suited for lipid peroxidation products[END_REF]. In vertebrates, they are known as glutathione- prostaglandin H2 to prostaglandin D2 [START_REF] Thomson | Sequence, catalytic properties and expression of chicken glutathione-dependent prostaglandin D2 synthase, a novel class Sigma glutathione S-transferase[END_REF]. This activity is also conserved in the Sigma GST from the parasitic flatworms Schistosoma [START_REF] Johnson | Crystal structure of the 28 kDa glutathione S-transferase from Schistosoma haematobium[END_REF], despite their distinct phylogenetic position.

dependent
We previously described the production of cyclopentenonic prostaglandins of the A2, B1, B2 and E2 series after various treatments in C. crispus [START_REF] Bouarab | The innate immunity of a marine red alga involves oxylipins from both the eicosanoid and octadecanoid pathways[END_REF]. In mammals, these molecules are produced by cyclooxygenases [START_REF] Funk | Prostaglandins and leukotrienes: advances in eicosanoid biology[END_REF], and pharmacological studies have shown that acetyl salicylic acid and indomethacin inhibits specifically these enzymes [40]. In the red alga, the enzymatic machinery involved in the production of prostaglandins remains elusive. However, synthesis of these cyclopentenones in C.

crispus is strongly affected in presence of cibacronblue, while no alteration was monitored in presence of either indomethacin or acetyl salicylic acid (data not shown).

Interestingly, similar inhibitor action has been observed on CcGST2 activity. Therefore, we are currently undertaking further experiments to determine if this GST might play a role in prostaglandin synthesis in C. crispus.

To conclude, the increasing number of genomic projects dealing with previously uninvestigated lineages will probably greatly modify our current knowledge on the evolution diversity and function of GSTs in the near future. As illustrated in the present study with GSTs characterized from C. crispus and L. digitata, the challenge lies in identifying their functions, not necessarily associated with detoxification. Further investigations on these proteins, including structural biology approaches, may provide some clues on the catalytic specificities and physiological functions of these enzymes in macroalgae.

C. Hervé was supported by a BDI fellowship from the Centre National de la Recherche Scientifique. PO de Franco was supported by the French Ministère de l'Enseignement Supérieur et de la Recherche. We thank G. Michel for technical support and helpful advice on structural alignment analyses. We are grateful to F. Morel for supplying GST substrates as well as for valuable discussion and to M. Cock for providing access to the E. siliculosus EST database. Part of this work was performed in the framework of the "Marine Genomics Europe" NoE (EC contract N° GOCE-CT-2004-505403).

SUPPLEMENTARY DATA

The following supplementary data is available for this article: Omega [START_REF] Ranson | Identification of a novel class of insect glutathione S-transferases involved in resistance to DDT in the malaria vector Anopheles gambiae[END_REF] Lambda [START_REF] Pearson | Phylogenies of Glutathione Transferase Families[END_REF] Zeta (plants) [START_REF] Pearson | Phylogenies of Glutathione Transferase Families[END_REF] PsGST1 PrGST1 PrGST2 Zeta (animals) [START_REF] Pearson | Phylogenies of Glutathione Transferase Families[END_REF] Rho Epsilon [START_REF] Collén | An expressed sequence tag analysis of thallus and regenerating protoplasts of Chondrus crispus (Gigartinales, Rhodophyceae)[END_REF] Phi [START_REF] Roeder | Identification of stress genes transcripts in Laminaria digitata (Phaeophyceae) protoplast cultures by expressed sequence tag analysis[END_REF] Mu [START_REF] Roeder | Identification of stress genes transcripts in Laminaria digitata (Phaeophyceae) protoplast cultures by expressed sequence tag analysis[END_REF] Pi [START_REF] Roeder | Identification of stress genes transcripts in Laminaria digitata (Phaeophyceae) protoplast cultures by expressed sequence tag analysis[END_REF] PGDS (Sigma) [

CuSO 4 and

 4 ZnCl 2 were purchased from Prolabo, and other chemicals from Sigma. The derivatives of polyunsaturated fatty acids 13(S)-HpODE (13S-hydroperoxyhydroperoxy-5Z,8Z,10E,14Zeicosatetraenoic acid), 15(S)-HETE (15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid), and 4-HNE (4-hydroxynonenal) were obtained from Cayman Chemicals.

  digitata and C. crispus respectively; 100 µM MeJA (methyl jasmonate); 25 or 50 µM each PUFA (polyunsaturated fatty acids) derivative in L. digitata and C. crispus respectively; and 15 µg mL -1 oligoguluronates. An equal volume of solvent was used in each corresponding control treatment. After 3 and 6 hours of incubation for C. crispus and L. digitata respectively, tissues were harvested, frozen in liquid nitrogen and stored at -80°C until use. DNA and RNA isolation Extractions of genomic DNA and total RNA from gametophyte tissues of C. crispus and sporophyte tissues of L. digitata were performed using the two-phase partitioning protocol described in Hervé et al. [15]. To improve final yields with L. digitata tissues, an additional step of polysaccharide precipitation, consisting of slow addition of 0.3 V of absolute ethanol to the extraction mixture, was included in the protocol before RNA precipitation by LiCl.

11 Biochemical

 11 GSH concentration ranging from 250 μM to 2 mM, and a fixed CDNB concentration of 2 mM. Kinetic parameters were calculated by the method of Lineweaver-Burk. Inhibitory effects on enzyme activity were measured by pre-incubating the enzymes 5 min in presence of GSH (1 mM final concentration) and several quantities of inhibitor (from 0.05 µM to 2 mM final concentration). Reactions were initiated by addition of 1-chloro-2,4-dinitrobenzene (CDNB; final concentration of 1 mM). The concentration of each inhibitor giving 50 % inhibition (IC 50 ) was determined graphically. Journal Immediate Publication. Published on 04 Mar 2008 as manuscript BJ20071464 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071464 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  In the red alga C. crispus, treatments with H 2 O 2 , the auxin analogue 2,4-D and the herbicide glyphosate showed a slight increase of accumulation in GST transcripts (Figure4A). Incubation in presence of the herbicides metolachlor and atrazine, and the insecticide dichlorvos strongly induced expression of the three GST genes. The major effects were observed for CcGST1 with 23-fold induction by metolachlor and 18-fold induction by dichlorvos. Heavy metals caused a modest repression effect on GST gene transcription, with a maximum of a 2-fold decrease. The most marked effects were observed after incubation in the presence of fatty acid derivatives. MeJA treatment induced a 6-to 43-fold change in transcript accumulation. Similar ranges of induction were observed with oxylipins, stronger effects being obtained with 13(S)-HpODE on CcGST2 (12-fold) for the C18 derivatives, and with 15(S)-HETE on CcGST3 (80-fold)

  Among the GST genes isolated from the red algae, CcGST3 represents an intriguing sequence. It exhibits only low similarity with other GSTs from an algal origin. No intron was found in its gene sequence. Despite the fact that we cannot rule out a bacterial origin for this GST, several observations point towards CcGST3 being an endogenous algal sequence: a polyA tail is present at the end of the corresponding mRNA and the likely active sites residues of the encoded protein may be different from 19 Biochemical Journal Immediate Publication. Published on 04 Mar 2008 as manuscript BJ20071464 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071464 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Societythe ones encountered in prokaryotic GSTs. Indeed, GSH-binding is known to involve a conserved cystein residue in the N-terminal portion of the bacterial GSTs. While no such residue is found in the putative GSH-binding domain of CcGST3 (see Supplementary FigureS1), two tyrosine residues are encountered in its N-terminal portion which may play this role. In addition, and as discussed below, CcGST3 follow a transcription pattern similar to the others CcGSTs and sometimes being the most affected during the treatments.

genes during treatment with CuSO 4 .

 4 Incubation of L. digitata sporophytes with oligoguluronates resulted in a strong induction of its GST genes. These oligosaccharides 20 Biochemical Journal Immediate Publication. Published on 04 Mar 2008 as manuscript BJ20071464 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071464 S t a g e 2 ( a ) P O S T -P R I N T are recognized as endogenous elicitors by the alga, inducing oxidative burst production as part of the active defense system [28]. Copper has also been shown to activate transient massive release of reactive oxygen species in this alga (data not shown).Therefore the accumulation of LdGST transcripts may be mediated through induced H 2 O 2 production. This hypothesis is not relevant for C. crispus since application of an oxidative stress seems to have almost no effect on the accumulation of CcGST transcripts.

  These results are however in agreement with published Km GSH values for other GST (Konishi et al., 2005, Park et al., 2005, Urade et al., 1987, Zeng et al., 2005).

  prostaglandin D-synthases (PGDS), catalysing the isomerisation of 22 Biochemical Journal Immediate Publication. Published on 04 Mar 2008 as manuscript BJ20071464 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071464 S t a g e 2 ( a ) P O S T -P R I N T

Figure S1

 S1 Figure S1 Alignment of the predicted amino acid sequence of CcGST3 with

5 6

 5 Results are expressed as means ± SD of four determinations.32Biochemical Journal Immediate Publication. Published on 04 Mar 2008 as manuscript BJ20071464 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071464 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

23

  Results are expressed as the mean of four determinations. 33 Biochemical Journal Immediate Publication. Published on 04 Mar 2008 as manuscript BJ20071464 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071464 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

FIGURE LEGENDS Figure 1

 LEGENDS1 FIGURE LEGENDS

Figure 2

 2 Figure 2 Exon/intron structure of genes encoding GSTs in C. crispus and L. digitata

Figure 3

 3 Figure 3 Unrooted neighbor-joining phylogenetic tree showing the relationship

Figure 4

 4 Figure 4 Real-time PCR analysis of C. crispus and L. digitata GST genes after

37 Biochemical

 37 Figure 2

3 38 4 39

 34 Biochemical Journal Immediate Publication. Published on 04 Mar 2008 as manuscript BJ20071464 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071464 S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 04 Mar 2008 as manuscript BJ20071464 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071464 S t a g e 2 ( a ) P O S T -P R I N T

Table S1 List of oligonucleotide primers used in this study Table S2 Concentrations of substrates used for the determination of specific transferase and peroxidase activities receptor

 S1 -regulated gene expression by arsenite, cadmium, and chromium.

	Toxicology 202, 249-269

Table 1 Substrate specificities of the recombinant CcGST2 and LdGST3 1

 1

	2
	3
		Specific activity
	Substrate	(µmol.min -1 .mg -1 of protein)
		CcGST2	LdGST3
	Aryl halides
	1-chloro-2,4-dinitrobenzene (CDNB)	1.56 ± 0.08	0.98 ± 0.10
	1-bromo-2,4-dinitrobenzene (BDNB)	1.56 ± 0.08	3.34 ± 0.04
	1-fluoro-2,4-dinitrobenzene (FDNB)	20.34 ± 0.59	7.44 ± 0.66
	1-iodo-2,4-dinitrobenzene (IDNB)	3.84 ± 0.12	1.29 ± 0.11
	para-nitrobenzoyl chloride	not detected	not detected
	α,β-unsaturated carbonyls
	Ethacrynic acid	not detected	not detected
	trans-4-phenyl-3-buten-2-one	not detected	not detected
	4-hydroxynonenal (4-HNE)	0.20 ± 0.02	0.69± 0.01
	trans-2-nonenal	not detected	not detected
	Miscellaneous
	para-nitrophenyl acetate	not detected	not detected
	Organic hydroperoxides
	Cumene hydroperoxide	0.15 ± 0.03	0.12 ± 0.01
	t-butyl hydroperoxide	0.04 ± 0.01	0.61 ± 0.12
	13-HpODE	0.23 ± 0.01	0.06 ± 0.01
	13-HpOTE	0.05 ± 0.01	0.08 ± 0.01
	4

Table 2 Inhibitor sensitivities of recombinant CcGST2 and LdGST3 1

 2

	IC 50 (µM)

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 04 Mar 2008 as manuscript BJ20071464 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071464 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society


José Canales

Ascensión Fernández

João Meireles Ribeiro

Alicia Cabezas

Joaquim Rui Rodrigues

José Carlos Cameselle
email: camselle@unex.es

María Jesús Costas
email: macostas@unex.es

Mn 2+ -dependent ADP-ribose/CDP-alcohol pyrophosphatase: a novel metallophosphoesterase family preferentially expressed in rodent immune cells*

Keywords: ADP-ribose, CDP-choline, metallophosphoesterase, Nudix, immune system, signalling

The Mn 2+ -dependent ADP-ribose/CDP-alcohol pyrophosphatase (ADPRibase-Mn) is isolated from rat liver supernatants after separation from Mg 2+ -activated ADP-ribose pyrophosphatases devoid of CDP-alcohol pyrophosphatase activity (ADPRibase-I and ADPRibase-II). The latter are putative Nudix hydrolases, while the molecular identity of ADPRibase-Mn is unknown. MALDI mass spectrometry data from rat ADPRibase-Mn pointed to a hypothetical protein that was cloned and expressed, and showed the expected specificity. It is encoded by the RGD1309906 rat gene, so far annotated just as 'hydrolase'. ADPRibase-Mn is not a Nudix hydrolase, but it shows the sequence and structural features typical of the metallophosphoesterase superfamily. It may constitute a protein family of their own, which appears to be specific to vertebrates, plants and algae. ADP-ribose was successfully docked to a model of rat ADPRibase-Mn revealing its putative active centre. Microarray data from the GEO database indicated the mouse gen 2310004I24Rik, orthologous of RGD1309906, is preferentially expressed in immune cells. This was confirmed by northern and activity assay of ADPRibase-Mn in rat tissues. A possible role of ADPRibase-Mn in immune cell signaling is suggested by the second messenger role of ADP-ribose, that activates TRPM2 ion channels as a mediator of oxidative/nitrosative stress, and by the signaling function assigned to many of the microarray profile neighbours of 2310004I24Rik. Furthermore, an influence of ADPRibase-Mn in the CDP-choline or CDP-ethanolamine pathways of phospholipid biosynthesis cannot be discarded.

INTRODUCTION

ADP-ribose is part of the NAD(P)-derived network of regulators. It is a primary product of NAD, formed by hydrolysis of the N-glycosidic linkage between nicotinamide and ribose, and also a secondary product formed by turnover of NAD primary metabolites [START_REF] Hilz | ADP-ribose. A historical overview[END_REF][START_REF] Berger | The new life of a centenarian: signalling functions of NAD(P)[END_REF]. Its reducing ribose moiety is prone to react nonenzymically, which explains its protein-glycating character and its reputation of being cytotoxic [START_REF] Jacobson | ADP-ribose in glycation and glycoxidation reactions[END_REF][START_REF] Just | Cysteine-specific ADP-ribosylation of actin[END_REF][START_REF] Wondrak | Histone carbonylation in vivo and in vitro[END_REF]. On the other hand, intracellular ADP-ribose is also a regulator of ion channels. Very interesting is the role of ADP-ribose as an activator of transient receptor potential melastatin channel-2 (TRPM2) that participates in Ca 2+ -mediated cell death. The channel opens in response to ADP-ribose as a mediator of oxidative/nitrosative stress in the immune system, a major site for TRPM2 expression [START_REF] Perraud | ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology[END_REF][START_REF] Sano | Immunocyte Ca 2+ influx system mediated by LTRPC2[END_REF][START_REF] Gerth | Extracellular NAD + regulates intracellular free calcium concentration in human monocytes[END_REF][START_REF] Kühn | TRPM2: a calcium influx pathway regulated by oxidative stress and the novel second messenger ADPribose[END_REF][START_REF] Perraud | Accumulation of free ADP-ribose from mitochondria mediates oxidative stress-induced gating of TRPM2 cation channels[END_REF][START_REF] Gasser | Activation of T cell calcium influx by the second messenger ADPribose[END_REF].

ADP-ribose levels in mammals are likely controlled by a low-K m , Mg 2+dependent, specific ADP-ribose pyrophosphatase [START_REF] Perraud | ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology[END_REF][START_REF] Miró | A specific, low K m ADP-ribose pyrophosphatase from rat liver[END_REF][START_REF] Lin | Cloning, expression and characterisation of a human Nudix hydrolase specific for adenosine 5'-diphosphoribose (ADP-ribose)[END_REF][START_REF] Perraud | NUDT9, a member of the Nudix hydrolase family, is an evolutionarily conserved mitochondrial ADP-ribose pyrophosphatase[END_REF][START_REF] Carloto | The specific, submicromolar K m ADP-ribose pyrophosphatase purified from human placenta is enzymically indistinguishable from recombinant NUDT9 protein, including a selectivity for Mn 2+ as activating cation and increase in K m for ADP-ribose, both elicited by H 2 O 2[END_REF], that belongs to the Nudix superfamily of NDP-X hydrolases [16,[START_REF] Mclennan | The Nudix hydrolase superfamily[END_REF]. However, there are other mammalian ADPribose hydrolases with diverse preferences for NDP-X substrates and for Mg 2+ or Mn 2+ as activators. In rat liver, ADP-ribose can be hydrolysed by the broad-specificity nucleotide pyrophosphatase or phosphodiesterase I ectoenzymes [START_REF] Cameselle | Two low K m hydrolytic activities on dinucleoside 5',5"'-P 1 ,P 4 -tetraphosphates in rat liver. Characterization as the specific dinucleoside tetraphosphatase and a phosphodiesterase I-like enzyme[END_REF] and four intracellular hydrolases [19,[START_REF] Bernet | Rat liver mitochondrial ADP-ribose pyrophosphatase in the matrix space with low K m for free ADP-ribose[END_REF]. ADPRibase-I (cytosolic) and ADPRibase-m (mitochondrial) are specific ADP-ribose pyrophosphatases that hydrolyse only ADPribose and the non-physiological IDP-ribose in the presence of Mg 2+ . Their specificity is less strict in the presence of Mn 2+ , but Mg 2+ is likely to be the physiological activator. ADPRibase-II (cytosolic) is a Mg 2+ -dependent ADP-sugar pyrophosphatase, also less strict in its NDP-X specificity when assayed with Mn 2+ . Finally, ADPRibase-Mn is not activated by Mg 2+ , but by low micromolar Mn 2+ . Its specificity is unusual, as it hydrolyses ADP-ribose, IDP-ribose, CDP-glycerol, CDP-choline and CDPethanolamine, but not other non-reducing ADP-sugars or CDP-glucose [19].

None of these rat ADP-ribose hydrolases have been cloned, but ADPRibase-I and ADPRibase-m are near identical to human Nudix enzymes NUDT9 and NUDT9 [START_REF] Perraud | ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology[END_REF][START_REF] Lin | Cloning, expression and characterisation of a human Nudix hydrolase specific for adenosine 5'-diphosphoribose (ADP-ribose)[END_REF][START_REF] Perraud | NUDT9, a member of the Nudix hydrolase family, is an evolutionarily conserved mitochondrial ADP-ribose pyrophosphatase[END_REF][START_REF] Carloto | The specific, submicromolar K m ADP-ribose pyrophosphatase purified from human placenta is enzymically indistinguishable from recombinant NUDT9 protein, including a selectivity for Mn 2+ as activating cation and increase in K m for ADP-ribose, both elicited by H 2 O 2[END_REF][19][START_REF] Bernet | Rat liver mitochondrial ADP-ribose pyrophosphatase in the matrix space with low K m for free ADP-ribose[END_REF][START_REF] Ribeiro | Human placenta hydrolases active on free ADP-ribose: an ADP-sugar pyrophosphatase and a specific ADP-ribose pyrophosphatase[END_REF], both encoded by the NUDT9 gene, while ADPRibase-II is related to human NUDT5 [START_REF] Ribeiro | Human placenta hydrolases active on free ADP-ribose: an ADP-sugar pyrophosphatase and a specific ADP-ribose pyrophosphatase[END_REF]22], also a Nudix enzyme encoded by the NUDT5 gene. In contrast, ADPRibase-Mn is not molecularly identified. Here, we are showing it forms a novel metallophosphoesterase family preferentially expressed in rodent immune tissues and cells. EXPERIMENTAL Chemicals and biochemicals 1,10-Phenanthroline monohydrate, and sodium salts of ADP-ribose, ADP-glucose, CDP-choline, CDP-ethanolamine, CDP-glycerol, CDP-glucose, UDP-glucose, CDP, AMP and CMP were from Sigma. ADP (potassium salt), Tris base (2-amino-2-[hydroxymethyl]-1,3-propanediol) and alkaline phosphatase (grade I from calf intestine) were from Roche. MnCl 2 , MgCl 2 , KCl, NaCl, and EDTA (Titriplex TM III) were highpurity preparations from Merck. Purification of rat liver ADPRibase-Mn and MALDI mass spectrometry of trypsin digests Rat ADPRibase-Mn was purified from 100,000 g liver supernatants by a six-step chromatographic procedure. The first five steps were as previously described [19] and, for the final step, the enzyme obtained from 30 g of liver was adsorbed to a 2.5 cm x 1.5 cm Reactive Red 120-agarose column (Sigma) equilibrated in 20 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, washed with 5 ml of KCl 0.5 M in the same buffer, followed by 10 ml of equilibrating buffer. The enzyme (3.5 ml) was recovered with a wash with water and concentrated to 0.1 ml in a centrifugal filter with a 3K-cutoff polyethersulfone membrane (Filtron Technology Corp, Northborough, MA). This preparation contained a single protein band of 38 kDa. After staining for 1 h with Coomassie blue G-250 (1 g/l in 50% methanol) and destaining in 40% methanol, the band was cut out and processed for trypsin digestion and MALDI mass spectrometry as described [START_REF] Cabezas | Identification of human and rat FAD-AMP lyase (cyclic FMN forming) as ATPdependent dihydroxyacetone kinases[END_REF] (Servicio de Proteómica, UAM, Madrid, Spain). The peptide-mass fingerprint (MS mode), and the ion spectra (MS/MS mode) of two fragments of 1746.88 Da and 2126.98 Da were used for database querying.

PCR cloning and sequencing of rat ADPRibase-Mn cDNA

The 1014-nt ORF coding for rat ADPRibase-Mn (mRNA accession number BC088174) was amplified by PCR from a rat liver Marathon Ready cDNA preparation with the Advantage TM cDNA polymerase mix (both from Clontech). The forward primer GTCTGGAATTCCATGGCTGATAAACCGGAC contained an EcoRI site (underlined) followed by C and the 18 first nucleotides of the ORF, with a designed G12A substitution; the reverse primer GCTCCGTCGACTTATTCCCTTTTGTAATTC contained a SalI site (underlined) followed by the reverse complement of the 19 last nucleotides of the ORF. A product of the expected size was obtained, cut with EcoRI and SalI, and inserted into the corresponding sites of the pGEX-6P-3 plasmid (Amersham) to obtain plasmid pGEX-6P-3-rADPRMn. Both strands of the insert were sequenced in the Servicio de Secuenciación Automática (IIB, CSIC-UAM, Madrid, Spain).

Recombinant ADPRibase-Mn expression and purification

One hundred-ml cultures of BL21 cells transformed with pGEX-6P-3-rADPRMn, were grown at 30°C and induced with IPTG. The GST/ADPRibase-Mn fusion recovered in post-sonication supernatants (in 10 ml of 20 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 mM DTT, 50 mM KCl, containing one dissolved tablet of protease inhibitor cocktail Complete TM Mini; from Roche) was adsorbed to a GSH-Sepharose column (1.5 ml; Amersham) equilibrated in the same buffer without protease inhibitors. The unbound proteins were washed out with 15 ml of 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 150 mM NaCl. ADPRibase-Mn was separated from the GST tag by in-column proteolysis with PreScission TM protease followed by elution with wash buffer. The eluted enzyme (1.8 ml) was frozen at -80ºC in 50-µl aliquots. This was a high-purity enzyme preparation (Figure S1, lane 6). Upon thawing, the enzyme was stable at least for one week. The expression and purification procedure was carried out three times with the same results. The elutions were performed with equilibrating buffer at 1 ml/min and the chromatographic profiles were recorded at 280 nm. For ADPRibase-Mn activity assay, 0.25 ml fractions were collected.

Enzyme activity assays

The hydrolytic activities of ADPRibase-Mn on NDP-X substrates were assayed at 37˚C by coupling to alkaline phosphatase and measuring colorimetrically the amount of P i formed with a dodecyl sulphate-ascorbate-molybdate reagent [START_REF] Ribeiro | Human placenta hydrolases active on free ADP-ribose: an ADP-sugar pyrophosphatase and a specific ADP-ribose pyrophosphatase[END_REF]. The assay mixtures contained, except when otherwise stated, 50 mM Tris-HCl (pH 7.5 at 25ºC), 500 µM NDP-X, 100 µM MnCl 2 , 1.5 µg/ml (5.5 units/ml) alkaline phosphatase, and the appropriate amount of enzyme sample. For the assay of the chromatographic profiles of ADPRibase-I, ADPRibase-Mn and ADPRibase-II in fractionated rat tissue extracts, 1 mg bovine serum albumin/ml was included in the reaction mixtures. Whenever the substrate was NDP or NMP, alkaline phosphatase was omitted. For the pH-activity profiles, different buffers were used, MnCl 2 was increased to 1 mM, and the concentration of alkaline phosphatase was doubled. In every case, the reaction was stopped and colour developed by addition of either 0.7 ml of standard P i reagent to 0.05-0.1 ml or, when higher sensitivity was needed, 0.5 ml of a concentrated reagent to 0.4 ml of reaction mixture. For studies of enzyme saturation, initial rates were measured at diverse substrate concentrations and the data were adjusted by non-linear regression to the Michaelis-Menten equation [START_REF] Cleland | The statistical analysis of enzyme kinetic data[END_REF]. For inactivation studies with o-phenanthroline, the activity was assayed in standard reaction mixtures without alkaline phosphatase, by measuring the conversion of ADP-ribose to AMP by HPLC after 15 min of reaction. Enzyme activities were linear with time and amount of enzyme. Blanks without enzyme and/or substrate were run in parallel. One unit enzyme activity is defined as 1 µmol substrate hydrolysed/min.

HPLC detection of NMP as reaction product of ADPRibase-Mn

Reaction mixtures with all the substrates hydrolysed by ADPRibase-Mn were analysed by ion-pair reverse-phase HPLC in a 15 cm x 0.4 cm octadecylsilica column (Kromasil 100, Teknokroma, Spain) with a 1 cm x 0.4 cm pre-column of the same material, using a Hewlett-Packard HP1100 chromatograph. Two elution buffers were used: A, 5 mM sodium phosphate, pH 7.0, 20 mM tetrabutylammonium and 10 % (vol/vol) methanol; B, same as A with 100 mM sodium phosphate. Before each run, the column was equilibrated in buffer A. Samples of 20 µl (reaction mixture) were injected, and the elution was accomplished at 1 ml/min with a linear, 10-min 5-100 mM phosphate gradient, followed by a 10-min isocratic wash with buffer B. S2; Sigma), was used. The blot was probed with a digoxigenin-labeled cDNA containing the full ORF of rat ADPRibase-Mn. The labelling was performed with the DIG High Prime DNA Labeling kit (Roche) according to manufacturer instructions. Hybridisation and detection were performed by the optimized protocol described elsewhere [25] except that probe concentration was 10 ng cDNA/ml, the hybridisation steps were carried out at 55ºC, and ready-to-use CSPD was the substrate for the chemiluminescent reaction catalysed by alkaline phosphataselabeled anti-digoxigenin (Roche). The results were recorded first by luminescent quantitative detection (Kodak Image Station 2000R) under conditions in which signal accumulation was linear with time and with the amount of labelled probe and, second, by exposure of X-ray film to the chemiluminescent blot.

Rat tissue or cell extract fractionation to assay ADPRibase-I, ADPRibase-Mn and ADPRibase-II Female Wistar rats weighing 200-220 g, fed ad libitum, were provided by the Servicio de Animalario of the Universidad de Extremadura, Badajoz. The animals were killed by rapid decapitation and the organs of interest (liver, skeletal muscle, spleen and thymus) were immediately dissected. After washing in cold saline, the tissue was blotted dry and weighed. Depending on the organ, 0.35-1.3 g of tissue were processed. The protocol to obtain rat splenocytes and to remove red blood cells was as described [START_REF] Kruisbeek | Isolation and fractionation of mononuclear cell populations[END_REF], except that spleens were crushed in 10 ml of cold saline, the red cell lysing buffer was purchased from Roche, and the final washes of splenocytes were performed with 14.5 mM Tris, pH 7.6, 5 µM CaCl 2 , 98 µM MgCl 2 , 0.54 mM KCl, 126 mM NaCl, and 0.1 g/l of glucose.

The separation of ADPRibase-I, ADPRibase-Mn and ADPRibase-II was carried out, with some simplification, as described [19]. The tissue samples were homogenised on ice, in buffer containing 50 mM Tris, pH 7.5, 0.5 mM EDTA, 1 mM PMSF, using 6 ml/g tissue (liver and muscle), 9 ml/g tissue (spleen and thymus), or 4.5 ml per washed splenocyte pellet obtained from one spleen. A hand-driven Potter type was used, except for muscle that was homogenized first in a motor-driven blendor and then finished in the Potter. With every tissue, 4-5 ml of homogenate was centrifuged for 60 min at 4ºC and 33,000 rpm in a Beckman SW45 rotor. The supernatant was chromatographed in a Sephadex G-100 column (1 cm x 90 cm) equilibrated and eluted with 20 mM Tris, pH 7.5, 0.5 mM EDTA at 20 ml/h. Fractions of about 2.5 ml were collected and the Mg 2+and the Mn 2+ -dependent ADP-ribose hydrolase activities were assayed. A small peak of activity eluting in the void volume was discarded, and the fractions of a large, included peak of ADP-ribose hydrolase, active with both Mg 2+ or Mn 2+ , were pooled. This peak contained ADPRibase-I, ADPRibase-Mn and ADPRibase-II, and it was applied to a DEAE-cellulose column (1.5 cm x 12 cm) equilibrated in 20 mM Tris, pH 7.5, and washed with 2-3 volumes of 20 mM Tris, pH 7.5, 0.5 mM EDTA, 50 mM KCl, followed by a 130-ml linear gradient of 50-400 mM KCl in the same buffer. The three enzymes were resolved within the gradient, allowing their individual quantitation. A careful accounting was kept such that activities quantitated after the DEAE-cellulose step could be extrapolated back and referred to total tissue protein measured in the homogenate with bovine serum albumin as a standard [27]. The theoretical coordinates of rat ADPRibase-Mn were taken from the SwissProt Repository [START_REF] Kopp | UniProt AC Q5M886, model for similar to RIKEN cDNA 2310004I24 gene from Rattus norvegicus (rat)[END_REF]. This homology model is based on the crystal structure of the zebrafish protein (pdb entry: 2NXF), which contains two Zn 2+ ions in a typical metallophosphoesterase dinuclear centre. Since there was an excellent structural similarity between the zebrafish and the rat proteins concerning the residues located within 5 Å of the Zn 2+ ions (RMSD 0.082 Å), the metal coordinates of the crystal structure were used to place them in the homology model. The protonation state of His side chains was assigned with the program Reduce [START_REF] Word | Asparagine and glutamine: using hydrogen atom contacts in the choice of sidechain amide orientation[END_REF]. Atom types and Kollman partial charges were assigned with AutoDockTools (ADT). The zinc ions were modelled with charge 2+ and default AutoDock 4 radius and well depth. ADP-ribose was prepared with Marvin 4.1.13, 2007, ChemAxon (http://www.chemaxon.com), its Gasteiger charges were calculated with ADT (total charge: 2-), and all its torsionable groups were made flexible except 6-NH 2 , and 2´, 3´, 2´´and 3´´-OH. Docking of ADPribose was run with AutoDock 4 [START_REF] Morris | Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function[END_REF] within a scoring grid (sized 33.8 29.2 29.2 Å with a resolution of 0.375 Å) centred around the putative active-site. AutoDock was run with the Lamarckian genetic algorithm with default parameters except that: 100 independent runs were performed; the maximum number of energy evaluations per run was set to 5 10 7 ; and the step sizes were adjusted to 0.2 Å for translations and 5º for orientations and torsions.

RESULTS

Tandem-mass spectrometry of rat liver ADPRibase-Mn pointed to a hypothetical rat protein that was cloned and expressed

For identification, a peptide-mass fingerprint of the 38-kDa band of rat liver ADPRibase-Mn was obtained. In a Mascot search [START_REF] Perkins | Probability-based protein identification by searching sequence databases using mass spectrometry data[END_REF] against the NCBInr database, it did not give any significant candidate. Therefore, two peptides of 1746.88 Da and 2126.98 Da were analysed by tandem mass spectrometry. Searches with these data pointed to two tryptic peptides formed by amino acids 47-61 (HSLVHLQGAIEDWNK) and 102-118 (VPVHHTWGNHEFYNFSR) of a 337 amino-acid hypothetical rat protein (accession No. AAH88174). Its theoretical mass of 38710 Da agreed with the 38-kDa protein band. It was recorded as a product of the rat gene RGD1309906 (GeneID 287406) which was annotated just as 'hydrolase'. The coding sequence (accession No. BC088174) of the hypothetical protein was used to design the PCR cloning described under Experimental. Plasmid pGEX-6P-3-rADPRMn contained, in frame with a GST affinity label, the PCR-amplified, 1014-nt ORF coding for ADPRibase-Mn. Relative to the ORF used as reference, it displayed 3 nucleotide changes: G12A (due to the design of the forward PCR primer), A131G and A636G, only one causing an amino acid change (Y44C). This sequence was deposited in GenBank (accession No. EU037900).

The GST/ADPRibase-Mn fusion of theoretical 65080 Da was expressed from pGEX-6P-3-rADPRMn as a 65-kDa protein (Figure S1, lane 2), and it was adsorbed to GSH-Sepharose. The ADPRibase-Mn moiety was recovered by in-column proteolysis as a 40-kDa protein (Figure S1, lane 6). This agrees with an expected mass of 39360 Da, sum of ADPRibase-Mn and the N-terminal extension (GPLGSPNS) left by proteolysis. Characterisation of the recombinant protein as a Mn 2+ -dependent ADP-ribose/CDPalcohol pyrophosphatase identical to ADPRibase-Mn Like rat liver ADPRibase-Mn [19], the recombinant enzyme hydrolysed ADP-ribose and CDP-alcohols. At 500 µM substrate and 100 µM MnCl 2 , CDP-choline gave the highest rate, followed by CDP-glycerol, CDP-ethanolamine, ADP-ribose and ADP (Table 1). However, kinetic parameters derived from hyperbolic saturation curves (Figure S3) showed ADP-ribose as the best substrate (k cat /K m = 328000 M -1 s -1 ), whereas CDP-alcohols and ADP were hydrolysed with 3.5-15 fold lower efficiencies (Table 1). ADP-glucose, UDP-glucose, CDP-glucose, CDP, CMP and AMP were not hydrolysed.

All the reactions involved the hydrolysis of the phosphoanhydride linkage, as: (i) P i was formed by alkaline phosphatase from the NDP-X reaction products but not from the substrates; (ii) P i was directly formed from ADP; (iii) AMP or CMP were identified as products by HPLC (results not shown).

ADPRibase-Mn was inactive without added Mn 2+ and showed high affinity for it, since saturation was observed at low micromolar Mn 2+ , well below the 500 µM substrate concentration (Figure S3). The concentration of MnCl 2 giving the halfmaximal rate was 1-4 µM. In no case, Mg 2+ substituted for Mn 2+ .

Incubation with 2.5 mM EDTA did not affect ADPRibase-Mn activity. This was in agreement with the stability of the enzyme frozen for months in a buffer with 1 mM EDTA. However, o-phenanthroline caused near full inactivation in 1.5 h (Figure 1).

The effect of pH on ADPRibase-Mn activities was studied in the range of pH 6-10. The profile of ADP-ribose hydrolase showed an optimum at pH 8.0-8.5, whereas the activities on ADP and CDP-alcohols were highest at pH 6-8, and decreased abruptly at higher pH (Figure 2).

ADPRibase-Mn behaved as a monomer, with an apparent mass of 30 kDa by gel filtration (Figure S4), in good agreement with the native mass of 32 kDa observed for the enzyme purified from rat liver [19].

Characterisation of ADPRibase-Mn as a protein belonging to the metallophosphoesterase, not to the Nudix superfamily In contrast to other ADP-ribose pyrophophatases, ADPRibase-Mn lacked the amino acid signature of the Nudix superfamily. Searches in the InterPro site (http://www.ebi.ac.uk/InterProScan) and Superfamily database (http://supfam.org/SUPERFAMILY) identified a metallophosphoesterase domain (IPR004843), indicating that ADPRibase-Mn belongs to the Pfam metallophos family (accession ID PF00149), and is a member of the Scop metallo-dependent phosphatase superfamily (accession ID SSF56300). These proteins contain (1) a dimetal centre with diverse ion pairs or combinations, (2) a secondary structure signature within (3) a four-layered fold with two -sheets flanked by -helices ( / / / fold), and (4) a disperse sequence signature that includes, in five conserved regions, the amino acids coordinated with the metal ions: DX [START_REF] Lohse | Insights derived from the structures of the Ser/Thr phosphatases calcineurin and protein phosphatase 1[END_REF][33][START_REF] Richter | 5' Cyclic nucleotide phosphodiesterases class III: members, structure, and catalytic mechanism[END_REF][START_REF] Shenoy | Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis[END_REF]. The aligment of ADPRibase-Mn orthologues found by BlastP searches (Table 2) revealed these five regions (Figure 3, I-V).

[H/X]-(X)n-GDXX[D/X]-(X)n-GNH[D/E]-(X)n- [G/X]H-(X)n-GHX[H/X]
The crystal structure of the zebrafish (Danio rerio) orthologue complexed with P i and two Zn 2+ ions [36], and a homology model of the rat protein [START_REF] Kopp | UniProt AC Q5M886, model for similar to RIKEN cDNA 2310004I24 gene from Rattus norvegicus (rat)[END_REF] were found in public databases. They display the / / / metallophosphoesterase fold, with the five regions of the sequence signature brought together each at the C-end of a differentstrand: regions I-III belong to one of the -sheets, and regions IV-V to the other. Seven of the conserved amino acids, which show an excellent structural similarity between the zebrafish and the rat proteins, form a dimetal centre located at the bottom of a groove. The gene encoding ADPRibase-Mn in Rattus norvegicus and mammalian orthologues A BlastN query to the R. norvegicus genome using the ORF sequence of rat ADPRibase-Mn revealed that the gene RGD1309906 (GeneID 287406; also known as MDS006 and MGC108803) is the only significantly related locus. It is a predicted gene located in rat chromosome 10 (10q24) that contains five exons, of which only the three last ones are translated and were hit by the BlastN search. The ADPRibase-Mn ORF spans from the 5´-end of exon 3 to within exon 5 (Figure 5). The reference rat genome contains the coding sequence of hypothetical protein LOC287406 (accession No. BC088174), identical to that coding for recombinant ADPRibase-Mn except for the nucleotide differences mentioned above. Figure 5 shows also the organisation of the mouse and human genes and the ORFs coding for ADPRibase-Mn orthologues in these organisms. TBlastN and BlastN searches pointed to an inverse 427-pb repetition including most of exon 5 and located 130 kb downstream of the rat gene, a feature not observed in the mouse or human genomes.

In human, mouse and rat, in addition to ADPRibase-Mn orthologues of 337-342 amino acids (Figure 3), BlastP searches found shorter protein variants due to an alternative 5´-splice site of the intron following the first translated exon. They were either conceptual translations from alternatively spliced transcripts (e.g. accession Nos BC001294 and BC070155 for human, and AK009142 for mouse mRNAs) or genomic predictions (accession number EDL10423 for mouse and EDM04778 for rat proteins). The alternative 5´-splice leaves 86-nt of the intron 5´-end attached to the preceding exon after splicing (Figure 5). The alternative transcription pattern contains an in-frame stop codon either within the retained intron segment (human and mouse) or in the next exon (rat). By the time of submitting this article, some databases point to the shorter version of the protein as the gene product.

Restricted taxonomic distribution of ADPRibase-Mn orthologues Orthologous genes of RGD1309906 are recorded only among vertebrates, higher plants and algae. As this seemed an important phylogenetic feature, we run BlastP and TBlastN searches that found ADPRibase-Mn orthologous in those phyla, but not in invertebrates and fungi (Table 2). In the latter cases, TBlastN searches of complete genomes, confirmed that the absence of orthologous proteins was not due just to their not being recorded yet.

ADPRibase-Mn gene expression: differential expression in immune and non-immune rodent tissues

Upon inspection of the Gene Expression Omnibus (GEO) database for ADPRibase-Mnrelated genes, a genomics-based investigation aimed to identify mouse immune-genes came to our attention (GEO dataset GDS2068 [START_REF] Hutton | Microarray and comparative genomics-based identification of genes and gene regulatory regions of the mouse immune system[END_REF]). Out of the 8734 genomic features explored in this microarray experiment, 360 show preferential expression in thymus, spleen, peripheral blood mononuclear cells, lymph nodes (unstimulated or stimulated), or in vitro activated T-cells. These features were linked to 333 unique genes, one of them being 2310004I24Rik, the orthologue of ADPRibase-Mn. The probe representing this gene in the microarray is a cDNA of the alternatively spliced mRNA (AK009142) that encodes the short protein variant (see above and Figure 5). It is likely to hybridize with both types of transcripts as they differ only in a 86-nt insert within a length of 1500 nt. The expression profile of 2310004I24Rik is shown in Figure S5, together with the heat plot of its profile neighbours (listed in Table 3 with their assigned functions [START_REF] Hutton | Microarray and comparative genomics-based identification of genes and gene regulatory regions of the mouse immune system[END_REF]). To confirm the preferential expression of ADPRibase-Mn in rodent immune tissues, the UniGene database was inspected, and a northern blot analysis and a fractionation study of the ADP-ribose pyrophosphatases in soluble extracts of selected rat tissues were run.

Using the UniGene's EST Profile Viewer to analyse mouse ESTs, one can see that ADPRibase-Mn is most strongly expressed in thymus (Table S1). Expression in spleen is also observed but not as prominently as in the microarray.

The northern blot was hybridised with a rat cDNA probe that revealed a major transcript of 1.4 kb in all tissues analysed (Figure 6), in agreement with the structure of RGD1309906 gene (Figure 5). Longer transcripts of 3 kb and 5 kb were also observed in some tissues. Quantitation of the RNA signals indicated higher expression in thymus and spleen than in the other rat tissues.

As stated in the Introduction, besides ADPRibase-Mn, rat liver supernatants contain other two ADP-ribose pyrophosphatases, named ADRibase-I and ADPRibase-II, which work with either Mg 2+ or Mn 2+ as the activating cation [19]. To distinguish the ADP-ribose pyrophosphatase activity of ADPRibase-Mn from the other hydrolases, fractionation of tissue supernatants was necessary (Figure S6). This allowed the quantitation of the three enzymes in five different rat sources: thymus, spleen, splenocytes, liver and skeletal muscle (Table 4). ADPRibase-Mn activity in thymus and spleen was 2.5-5-fold higher than in liver and muscle. The ratio was even higher when ADPRibase-Mn was quantitated in splenocytes, where the activity was 4-8-fold higher than in the non-immune tissues (Table 4).

DISCUSSION

Our results provide a molecular identity for ADPRibase-Mn and a biochemical basis for the functional annotation of the rat gene RGD1309906, likely extrapolable to other mammalian genes coding for strongly similar hypothetical metallophosphoesterases. This includes the mouse gene 2310004I24Rik and the human gene c17orf48, which respectively encode proteins 95% and 85% identical to rat ADPRibase-Mn.

Not every known NDP-X hydrolase is a member of the Nudix superfamily [38, 39] but, in fact, all those with a clear or relative preference for ADP-ribose [13-15, 22, 40, 41] or CDP-alcohol [START_REF] Tirrell | YZGD from Paenibacillus thiaminolyticus, a pyridoxal phosphatase of the HAD (haloacid dehalogenase) superfamily and a versatile member of the Nudix (nucleoside diphosphate x) hydrolase superfamily[END_REF][START_REF] Xu | The 26 Nudix hydrolases of Bacillus cereus, a close relative of Bacillus anthracis[END_REF] are. It has been pointed out that the Mn 2+ -dependent Nudix hydrolase YZGD, from Paenibacillus thiaminolyticus, displays a specificity similar to ADPRibase-Mn [START_REF] Tirrell | YZGD from Paenibacillus thiaminolyticus, a pyridoxal phosphatase of the HAD (haloacid dehalogenase) superfamily and a versatile member of the Nudix (nucleoside diphosphate x) hydrolase superfamily[END_REF]. Therefore, that the latter is a metallophosphoesterase was relatively unexpected. In the Scop classification, the metallo-dependent phosphatase superfamily is currently divided in several families, including: purple acid phosphatase; DNA double-strand break repair nuclease; 5´-nucleotidase (syn. UDPsugar hydrolase), N-terminal domain; protein serine/threonine phosphatase; and others. Searches in the Superfamily server (http://supfam.org/SUPERFAMILY) failed to asign ADPRibase-Mn to any established Scop family (no E-value < 0.01 was obtained), suggesting that it may constitute a novel protein family that can be provisionally described as ADP-ribose/CDP-alcohol pyrophosphatase.

ADPRibase-Mn is inactive in the absence of added metals, and is activated by low micromolar concentrations of Mn 2+ , but not of Mg 2+ . Activation by Mn 2+ reflects metal binding to the enzyme, not just to the free substrate, as half saturation was obtained at 1-4 µM MnCl 2 , >100 fold lower than the substrate. This is compatible with Mn 2+ being a natural activator of the enzyme, as free Mn 2+ is estimated to be 0.7 µM in hepatocytes from fed rats and 0.25 µM in hepatocytes from fasted rats [START_REF] Ash | Determination of free and bound manganese(II) in hepatocytes from fed and fasted rats[END_REF].

The metal centre of metallophosphoesterases can contain a couple of the same or different metals in varying combinations. However, it is unclear whether activation by Mn 2+ reflects binding to the dinuclear centre or otherwise. In fact, the structure of the zebrafish orthologue of ADPRibase-Mn was determined with two Zn 2+ ions in the dinuclear centre, but it also contained two Zn 2+ ions at other locations [36]. The timedependent inactivation of rat ADPRibase-Mn by phenanthroline, not by EDTA, could indicate that a metal different from Mn 2+ could be part of the enzyme, perhaps Fe or Zn, that form more stable complexes with the former chelator. However, phenanthroline could also be acting as an inhibitor by binding to the active centre. In summary, further evidence is needed to elucidate the nature of the metals bound to ADPRibase-Mn and their structural and/or functional role.

The putative active centre of ADPRibase-Mn is in the groove where P i lies bound to the Zn 2+ ions of the crystal structure of the zebrafish protein [36]. In the homology model of the rat protein, a docked ADP-ribose fits into that groove, with thephosphate of ADP occupying a position similar to P i in the X-ray structure. All the ADP-ribose interactions with the Zn 2+ ions, and most of those established with the protein, involve the pyrophosphate group (Figure 4 and legend), and it is likely that some of them are important for catalysis. For instance, His111 (part of the conserved GNHE region III in Figure 3) interacts with the oxygen of the phosphoanhydride linkage hydrolysed by ADPRibase-Mn, as in other metallophosphoesterases where this interaction is catalytically relevant [44]. On the other hand, the specificity of ADPRibase-Mn for NDP(-X) compounds (Table 1) should be accounted for by interactions of the N and the X portions of the substrate. In the ADP-ribose docking model, while the pyrophosphate group is in a rather closed environment, adenine and the distal ribose protrude towards more open areas of the protein surface (Figure 4B). On the N side of ADP-ribose, a hydrophobic contact of adenine with Leu196 is observed, but not any hydrogen bond. On the X side, two possible interactions of the 1´´-OH group with backbone atoms of His278 and His280, and a favorable contact of the ribose ring with Cys253 are observed. It is unclear whether these interactions alone account for the preferences of ADPRibase-Mn for ADP versus CDP, for NDP-X versus the corresponding NDPs, and for ADP-ribose versus ADP-glucose as summarized in Table 1. Anyhow, it must be stressed that the theoretical structure of rat ADPRibase-Mn is derived by homology to a zebrafish protein complexed with P i , a ligand much smaller than ADP-ribose. In a true enzyme-substrate complex, specific interactions may depend on an induced fit of the enzyme which P i would not possibly evoke.

ADPRibase-Mn may have a function in vertebrate immune systems. Mining of expression databases identified a mouse microarray experiment in which gene 2310004I24Rik, that codes for the mouse orthologue of ADPRibase-Mn, is labeled as an 'immune gene', preferentially expressed in immune versus non-immune cells and tissues [START_REF] Hutton | Microarray and comparative genomics-based identification of genes and gene regulatory regions of the mouse immune system[END_REF]. As a complement to this, rat ADPRibase-Mn mRNAs were found more abundant (Figure 6) and enzyme activity was higher (Table 4) in thymus and spleen than in nonimmune rat tissues. ADPRibase-Mn activity was even higher in splenocytes, that include spleen immunocytes (Table 4). Such a pattern was not observed for the Nudixtype ADPRibase-I (Nudt9) and ADPRibase-II (Nudt5), either when assaying enzyme activities in rat tissues (Table 4) or in the RNA expression of the mouse orthologue of the rat Nudt5 gene that codes for ADPRibase-II (a probe for the Nudt9 orthologue, that codes for ADPRibase-I, was not included in the microarray [START_REF] Hutton | Microarray and comparative genomics-based identification of genes and gene regulatory regions of the mouse immune system[END_REF]). Also in agreement with a role in the vertebrate immune system, a restriction was found in the taxonomic distribution of ADPRibase-Mn orthologues. BlastP and TblastN searches revealed they occur in all vertebrates with the possible exception of birds, but not in invertebrates and 2). This contrasts, e.g., with genes orthologous to Nudt9 (coding for ADPRibase-I) which, besides in vertebrates, is represented in Drosophila and C. elegans [START_REF] Perraud | NUDT9, a member of the Nudix hydrolase family, is an evolutionarily conserved mitochondrial ADP-ribose pyrophosphatase[END_REF], or to Nudt5 (coding for ADPRibase-II), which is represented in C. elegans and Saccharomyces [22]. The immune role of ADPRibase-Mn is unknown. In the above-mentioned microarray study [START_REF] Hutton | Microarray and comparative genomics-based identification of genes and gene regulatory regions of the mouse immune system[END_REF], a cellular function was assigned to 298 of the 333 mouse immune genes identified, grouped in about 20 functional sets labeled as e.g. defense, apoptosis, immune, signaling, metabolism, etc (see e.g. Table 3). Immunegene 2310004I24Rik, here shown to code for the mouse orthologue of ADPRibase-Mn, was annotated just as metallophosphoesterase, and could not be assigned to either of those functional sets [START_REF] Hutton | Microarray and comparative genomics-based identification of genes and gene regulatory regions of the mouse immune system[END_REF]. The role of ADP-ribose as a second messenger of stress in immune cells [START_REF] Kühn | TRPM2: a calcium influx pathway regulated by oxidative stress and the novel second messenger ADPribose[END_REF][START_REF] Perraud | Accumulation of free ADP-ribose from mitochondria mediates oxidative stress-induced gating of TRPM2 cation channels[END_REF][START_REF] Vu | Intramolecular ADP-ribose transfer reactions and calcium signalling. Potential role of 2'-phospho-cyclic ADP-ribose in oxidative stress[END_REF][START_REF] Fonfria | TRPM2 channel opening in response to oxidative stress is dependent on activation of poly(ADP-ribose) polymerase[END_REF][START_REF] Naziroglu | New molecular mechanisms on the activation of TRPM2 channels by oxidative stress and ADP-Ribose[END_REF] points to a signaling role for ADPRibase-Mn. This agrees with the roles of the immune genes that are profile neighbours of 2310004I24Rik in the microarray study. While 16% of the 298 immune genes with a known role are marked 'signaling' [START_REF] Hutton | Microarray and comparative genomics-based identification of genes and gene regulatory regions of the mouse immune system[END_REF], this increases to 43% among the profile neighbours of 2310004I24Rik (Table 3). ADPRibase-Mn in the immune system could be related to ADP-ribose turnover and to the termination of its effects on TRPM2 channels. Since the immunegene microarray does not contain a TRPM2 probe, to compare 2310004I24Rik and TRPM2 expression in mouse tissues, we inspected the UniGene database using the EST Profile Viewer. Both genes are expressed at low levels (e.g. as compared with -actin), what makes comparisons difficult in terms of numbers of ESTs counted. Anyhow, considering only body sites for which a high number of total ESTs is on record (> 40000), one can see that TRPM2 expression is accompannied by ADPRibase-Mn. This represents only a partial correlation, because several sites express ADPRibase-Mn but not TRPM2 (Table S1). CDP-choline and CDP-ethanolamine are key intermediates in biosynthetic pathways of, respectively, phosphatidylcholine and phosphatidylethanolamine [START_REF] Vance | Phospholipid biosynthesis in mammalian cells[END_REF]49]. The formation of CDP-choline by CTP-phosphocholine cytidylyltransferase is ratelimiting and important for the regulation of phosphatidylcholine biosynthesis. ADPRibase-Mn is the only mammalian NDP-X hydrolase showing a relative preference for CDP-choline (Table 1). Working simultaneously with the cytidylyltransferase, ADPRibase-Mn would form a futile substrate cycle that could contribute also to regulation:

(cytidylyltransferase) CTP + phosphocholine CDP-choline + PP i (ADPRibase-Mn) CDP-choline + H 2 O CMP + phosphocholine A similar reasoning can be applied to the CDP-etanolamine pathway. In conclusion, a role of ADPRibase-Mn related to its CDP-alcohol substrates, cannot be discarded. The list was found by automatically seaching for 2310004I24Rik gene profile neighbours in the GEO dataset GDS2068 within the GEO Profiles database at the NCBI site. Out of the 31 records found, only those classified as 'immune genes' are listed together with their assigned functions [START_REF] Hutton | Microarray and comparative genomics-based identification of genes and gene regulatory regions of the mouse immune system[END_REF]. Expression profiles are shown in Figure S5. The activities were quantitated after separation of the three ADPRibases by gelfiltration of tissue supernatants followed by ion-exchange chromatography (Figure S6). The results are referred to an amount of tissue containing 1 g protein (assayed in the crude homogenates), and are means ± S.D. of three independent experiments, each corresponding to one animal. Figure 2 pH profiles of the activities of recombinant ADPRibase-Mn Initial rates of hydrolysis were measured with the indicated substrate, in the presence of 1 mM MnCl 2 . The buffers used were: 100 mM Tris-acetate (pH 6-7), 100 mM Tris-HCl (pH 7-9) and 100 mM CAPS/NaOH (pH 9-10.1). Reaction mixtures of 0.4 ml were prepared and, at the end of the incubation period, 0.1 ml was used for the assay of activity, and 0.3 ml for pH mesurement with a glass electrode. sequentially conserved (Figure 3) and superimposable to those of the zebrafish protein (not shown), except Leu196 that is conserved but not superimposable in both proteins.

ADPRibase

Figure 5 The rat ADPRibase-Mn gene and orthologues in mouse and human genomes

The exon(black boxes)-intron(thin lines) structures are shown as they currently appear in the NCBI Gene database, including the ORFs coding for rat ADPRibase-Mn and its mouse and human orthologues after splicing. The exons of the rat gene are currently supported by a 1.3 kb mRNA (BC088174) which agrees with the size of the most abundant transcript seen in a northern blot (Figure 6). The white prolongations of the first translated exon show how the mRNAs would remain after operation of the alternative 5´-splice in the next intron (see the text). The alternative splicing would give rise to proteins shorter than ADPRibase-Mn and its orthologues, due to an in-frame stop codon either in the portion of intron retained as exon (mouse and human) or in the next exon (rat).

Human c17orf48 gene

Biochemical

  Journal Immediate Publication. Published on 19 Mar 2008 as manuscript BJ20071471 THIS IS NOT THE FINAL VERSION -see doi:10. 1042/ BJ20071471 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 19 Mar 2008 as manuscript BJ20071471 THIS IS NOT THE FINAL VERSION -see doi:10. 1042/ BJ20071471 S t a g e 2 ( a ) P O S T -P R I N T Native molecular weight of ADPRibase-Mn by gel filtration chromatography Samples of ADPRibase-Mn and molecular weight markers were chromatographed in a Protein-Pak 125 column (7.8 mm x 300 mm) from Waters, equilibrated in 50 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl, and connected to a Hewlett-Packard HP1090 chromatograph.

Biochemical

  Journal Immediate Publication. Published on 19 Mar 2008 as manuscript BJ20071471 THIS IS NOT THE FINAL VERSION -see doi:10. 1042/ BJ20071471 S t a g e 2 ( a ) P O S T -P R I N T Docking of ADP-ribose to the homology model of rat ADPRibase-Mn

  Biochemical Journal Immediate Publication. Published on 19 Mar 2008 as manuscript BJ20071471 THIS IS NOT THE FINAL VERSION -see doi:10. 1042/ BJ20071471 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 19 Mar 2008 as manuscript BJ20071471 THIS IS NOT THE FINAL VERSION -see doi:10. 1042/ BJ20071471 S t a g e 2 ( a ) P O S T -P R I N T ADP-ribose was succesfully docked to the rat protein model into this site, indicating that it can be the active centre (Figure 4A,B). The amino acids that form the dimetallic centre and/or might interact with the substrate are shown in Figure 4C.

Biochemical

  Journal Immediate Publication. Published on 19 Mar 2008 as manuscript BJ20071471 THIS IS NOT THE FINAL VERSION -see doi:10. 1042/ BJ20071471 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 19 Mar 2008 as manuscript BJ20071471 THIS IS NOT THE FINAL VERSION -see doi:10. 1042/ BJ20071471 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 19 Mar 2008 as manuscript BJ20071471 THIS IS NOT THE FINAL VERSION -see doi:10. 1042/ BJ20071471 S t a g e 2 ( a ) P O S T -P R I N T most lower eukaryots (Table

Biochemical

  Journal Immediate Publication. Published on 19 Mar 2008 as manuscript BJ20071471 THIS IS NOT THE FINAL VERSION -see doi:10. 1042/ BJ20071471 S t a g e 2 ( a ) P O S T -P R I N T Table 2 ADPRibase-Mn orthologues in representative vertebrates and plants, and absence of orthologues in invertebrates and fungi BlastP searches were run in the NCBI site using the conceptual translation of the ORF cloned from rat liver cDNA (GenBank accession EU037900) as the query against the NCBInr protein database. Only representative hits are shown for vertebrates and plants. When the BlastP program failed to find a putative orthologous protein, TBlastN searches were run against the NCBInr nucleotide database and the assembled genomes of relevant taxonomic groups. The results shown were obtained on September 8, 2007. Publication. Published on 19 Mar 2008 as manuscript BJ20071471 THIS IS NOT THE FINAL VERSION -see doi:10. 1042/ BJ20071471 S t a g e 2 ( a ) P O S T -P R I N T

Figure 1

 1 Figure 1 Inactivation of recombinant ADPRibase-Mn by o-phenanthroline Enzyme samples were diluted 1:1 in 5 mM ( ) o-phenanthroline or ( ) EDTA each dissolved in 50 mM Tris, pH 7.5, and were incubated for the indicated lengths at 37ºC. Enzyme aliquots were taken at the end of the incubation, diluted 100-fold in 50 mM Tris-HCl, pH 7.5, and immediately used for ADP-ribose hydrolase activity assay by HPLC.

Figure 3

 3 Figure 3 Protein alignment of rat ADPRibase-Mn orthologues showing the conservation of a metallophosphoesterase sequence signatureThe sequences were aligned using the Clustal-W program. Orthologous proteins (with Genbank accession numbers): Rnor, Rattus norvegicus (conceptual translation of EU037900); Hsap, Homo sapiens (NP_064618); Mmu, Mus musculus (AAH04029); Xlae, Xenopus laevis (NP_001080428); Drer, Danio rerio (NP_956715); Atha, Arabidopsis thaliana (NP_194204); Osat1 and Osat2, Oryza sativa (NP_001060698 and NP_001062482); Oluc, Ostreococcus lucimarinus (green algae) (XP_001419532). The asterisks and the points below the sequences mark residues conserved as identities or similarities, respectively. The white letters on dark background represent conserved amino acids that are part of the metallophosphoesterase sequence signature divided in five regions (I-V): DX[H/X]-(X)n-GDXX[D/X]-(X)n-GNH[D/E]-(X)n-[G/X]H-(X)n-GHX[H/X][START_REF] Lohse | Insights derived from the structures of the Ser/Thr phosphatases calcineurin and protein phosphatase 1[END_REF][33][START_REF] Richter | 5' Cyclic nucleotide phosphodiesterases class III: members, structure, and catalytic mechanism[END_REF][START_REF] Shenoy | Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis[END_REF]. The vertical marks at the top of the sequences are positioned at intervals of ten amino acids relative to the Rattus norvegicus sequence.

Figure 4

 4 Figure 4 Theoretical model of rat ADPRibase-Mn with docked ADP-ribose The figure shows different views of the lowest-energy pose found after docking as described in the Experimental section. The two Zn 2+ ions are shown as yellow spheres and ADP-ribose is shown in blue (adenine), red (pyrophosphate) and green (ribose-1 and ribose-2, respectively proximal and distal to the adenine). Oxygen atoms of thephosphate group interact with Zn1 and Zn2. (A) Full homology model showing the / / / fold, typical of metallophosphoesterases, and the docked ADP-ribose. (B) Molecular surface of the active centre with ADP-ribose, as viewed from above in A. (C) Stereo view of a selection of amino acids that form the dimetallic centre and/or might interact with the substrate, as viewed from the right-hand side in A. Possible interactions of amino acid side chains in C are as follows. (i) Coordination with Zn1: Asp25, Gln27, Asp74 and His280. (ii) Coordination with Zn2: Asp74, Asn110, His241 and His278. (iii) H bridge with O-P : Gln27, His278 and His280. (iv) H bridge with O-P : Arg43 and His111. (v) H bridge with the phosphoanhydride O: Asn110 and His111. (vi) Hydrophobic contact with the adenine rings: Leu196. (vii) Contacts with ribose-1 or ribose-2 rings: Phe210 and Cys253, respectively. All the mentioned amino acids are

Figure 6 S t a g e 2 4 Biochemical

 624 Figure 6 Expression of RGD1309906 transcripts in rat tissuesA multiple rat-tissue northern blot is shown after hybridisation with digoxineninlabelled ADPRibase-Mn cDNA. The figure shows a 3-h exposure of X-ray film to the chemiluminescent membrane, made 19-h after starting the detection reaction. Numbers below the lanes represent the quantitation (as percentages of the thymus signal) of the 1.4-kb band by image analysis during the first 3 h of detection.

Figure 5 Biochemical

 5 Figure 5

Figure 6 Biochemical

 6 Figure 6

Table 1

 1 Substrate specificity and kinetic parameters of ADP-ribose/CDP-alcohol Kinetic parameters are expressed ± S.E. of the pooled data of 2-3 experiments[START_REF] Cleland | The statistical analysis of enzyme kinetic data[END_REF].

	pyrophosphatase
		Enzyme
		purified	Enzyme expressed from cloned DNA
		from rat		(this work)
		liver
		Activity at	Activity at
	Substrate a	500 µM	500 µM	k cat	d	K m	d	k cat / K m
		substrate b	substrate c
		(%)	(%)	s -1		µM	M -1 s -1
	ADP-ribose	100	100 ± 8	12.8 ± 0.2	39 ± 2	328200
	CDP-choline	140	156 ± 23	28.5 ± 0.6	300 ± 22	95000
	CDP-ethanolamine	89	84 ± 18	41.3 ± 1.1 1422 ± 99	29000
	CDP-glycerol	89	103 ± 10	28.1 ± 1.4	304 ± 59	92400
	ADP	27	26 ± 4	4.4 ± 0.2	201 ± 30	21400
	a Neither the purified nor the expressed enzymes displayed significant activity over
	ADP-glucose, UDP-glucose, CDP-glucose, CDP, CMP and AMP (< 3%).
	b Taken from [19].
	c Means ± S.D. of 3 experiments; 100% was 23 µmol/min/mg; protein assayed
	according to [50].

d

Table 3

 3 ADPRibase-Mn profile neighbours of expression among the genes preferentially expressed in mouse immune cells and tissues

Table 4

 4 Preferential expression of ADPRibase-Mn activity in rat immune tissues

Licenced copy . Copy ing is not permitted, ex cept w ith prior permission and as allow ed by law . © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy . Copy ing is not permitted, ex cept w ith prior permission and as allow ed by law . © 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGMENTS

We are grateful to Antonio Sillero and María Antonia Günther for their generous support of this research. JRR is indebted to the Escola Superior de Tecnologia e Gestão, Instituto Politécnico de Leiria, Portugal, for permission to be on leave. Thanks are due to the Ministerio de Educación y Ciencia, Spain, for Grant BFU2006-00510, cofinanced by FEDER, to the Consejería de Infraestructuras y Desarrollo Tecnológico, Junta de Extremadura Spain, for Grants GRU06031 and GRU07066, cofinanced by FSE and FEDER, and to the Vicerrectorado de Investigación, Desarrollo e Innovación, Universidad de Extremadura, for Grant A7-09.