Ulrike Böer

Julia Eglins

Doris Krause

Susanne Schnell

Christof Schöfl

Willhart Knepel

Enhancement by lithium of cAMP-induced CRE/CREB-directed gene transcription conferred by TORC to the CREB basic region leucine zipper domain

Keywords: lithium, cAMP-responsive element binding protein (CREB), basic leucine zipper region (bZip), transducer of regulated CREB (TORC), coactivator, reporter gene assays CRE, cAMP-responsive element, CREB, cAMP-responsive element binding protein, bZip, basic leucine zipper domain, TORC, transducer of regulated CREB, GSK3 , glycogen synthase kinase 3, IMP, inositol monophosphatase, EMSA, electromobility shift assay

The molecular mechanism of action of lithium salts in the treatment of bipolar disorder is not well understood. As their therapeutic action requires chronic treatment, adaptive neuronal processes are suggested to be involved. The molecular basis of this are changes in gene expression regulated by transcription factors like the cAMP-responsive element (CRE) binding protein (CREB). CREB contains a transactivation domain, in which Ser-119 is phosphorylated upon activation, and a basic leucine zipper region (bZip). The bZip serves for CREB dimerization and DNA-binding but also contributes to CREB transactivation by recruiting the coactivator TORC (transducer of regulated CREB). Here, the effect of lithium on CRE/CREB-directed gene transcription was investigated. Electrically excitable cells were transfected with CRE/CREB-driven luciferase reporter genes. LiCl (6 mM or higher) induced an up to 4.7-fold increase in 8BrcAMP-stimulated CRE/CREB-directed transcription. This increase was not due to enhanced Ser-119-phosphorylation or DNA-binding of CREB. Also the known targets inositol-monophosphatase and glycogen-synthase-kinase 3 were not involved as specific GSK3 -inhibitors and inositol replenishment did not mimic and abolish, respectively, the effect of lithium. However, lithium no longer enhanced CREB activity when the CREB-bZip was deleted or the TORC binding site inside the CREB-bZip was specifically mutated (CREB-R300A). Otherwise, TORC overexpression conferred lithium responsiveness to CREB-bZip or the CRE-containing truncated rat somatostatin promoter. This indicates that lithium enhances cAMP-induced CRE/CREB-directed transcription, conferred by TORC to the CREB-bZip. We thus support the hypothesis that lithium salts modulate CRE/CREBdependent gene transcription and suggest the CREB coactivator TORC as new molecular target of lithium.

INTRODUCTION

Lithium salts are widely used in the therapy of bipolar disorder [START_REF] Cade | John Frederick Joseph Cade: family memories on the occasion of the 50th anniversary of his discovery of the use of lithium in mania[END_REF]. A hallmark of the therapeutic concept of lithium treatment is the chronic administration. To achieve the moodstabilizing and phase prophylactic action, treatment with lithium for several months has been shown to be necessary. Though the effectiveness of lithium is indisputable its mechanism of action is still poorly understood. Some molecular targets for lithium have been identified.

Among those are enzymes like the adenylyl cyclase, the glycogen synthase kinase 3 and inositol monophosphatase [START_REF] Quiroz | MOLECULAR EFFECTS of lithium[END_REF]. As monovalent cation lithium is able to inhibit these enzymes and thereby subsequently induces changes in second messenger levels and signalling cascades which are thought to contribute to the therapeutic action of lithium. However, these changes occur immediately and therefore are not sufficient to explain the extremely delayed onset of the therapeutic action. Thus, subsequent to early changes in enzyme activities particularly long lasting adaptive neuronal processes are suggested to be involved. Indeed, lithium-induced plasticity could be shown in hippocampal neurons both in vitro and in vivo after chronic treatment [START_REF] Kim | Lithium selectively increases neuronal differentiation of hippocampal neural progenitor cells both in vitro and in vivo[END_REF]. The molecular basis for these adaptive changes is the expression of genes regulated by transcription factors. Thus, transcription factors could be a target of lithium, initiating changes in gene expression, which can be summarized as neuronal adaptation. One important transcription factor in the brain is CREB (cAMP-responsive element binding protein). CREB is involved in adaptive processes like learning, memory [START_REF] Silva | CREB and memory[END_REF] and opiod-induced addiction [START_REF] Nestler | Molecular neurobiology of addiction[END_REF]. Among the CREB-regulated genes there are several involved in neurotransmission and signalling [START_REF] Impey | Defining the CREB regulon: a genome-wide analysis of transcription factor regulatory regions[END_REF].

CREB belongs to the family of basic leucine zipper transcription factors which contain Cterminally a leucine zipper dimerization domain and a basic region, by which the cAMPresponsive element (CRE) is bound [START_REF] Shaywitz | CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals[END_REF]. CREB can be activated by various signalling pathways, which converge in the phosphorylation of Ser-119 located in the transactivation domain. Transactivation of transcription is accomplished by the recruitment of coactivators, which interact with the basal transcription machinery. In addition to the long-known coactivator CBP (CREB binding protein) [START_REF] Shaywitz | CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals[END_REF] which binds to the phosphorylated transactivation domain, more recently another coactivator has been discovered. TORC (transducer of regulated CREB activity), an 80 kDa protein which exists in three isoforms [START_REF] Conkright | TORCs: transducers of regulated CREB activity[END_REF], binds to the leucine zipper domain and contributes to the transcriptional activity of CREB.

There are few studies on the action of lithium on CREB. The majority of them focus on the phosphorylation of CREB [START_REF] Tardito | Reduced CREB phosphorylation after chronic lithium treatment is associated with downregulation of CaM kinase IV in rat hippocampus[END_REF][10][START_REF] Kopnisky | Chronic lithium treatment antagonizes glutamate-induced decrease of phosphorylated CREB in neurons via reducing protein phosphatase 1 and increasing MEK activities[END_REF] and its DNA binding [START_REF] Grimes | CREB DNA binding activity is inhibited by glycogen synthase kinase-3 beta and facilitated by lithium[END_REF]13]. However, the DNA binding of CREB plays a minor role in the regulation of its activity, as also non-phosphorylated CREB is able to bind the CRE [START_REF] Shaywitz | CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals[END_REF]. Furthermore it has been shown before [START_REF] Schwaninger | Involvement of the Ca(2+)-dependent phosphatase calcineurin in gene transcription that is stimulated by cAMP through cAMP response elements[END_REF][START_REF] Pham | CRE-mediated gene transcription in neocortical neuronal plasticity during the developmental critical period[END_REF][16], that phosphorylation is a necessary prerequisite for the transcriptional activation of CREB but not sufficient. Therefore, CREB activity cannot be determined by measuring its phosphorylation or DNA binding alone but rather by determining the expression of a CRE/CREB-driven gene. Thus, in the present study we focus on the effect of lithium on the transcriptional activity of CREB as revealed by CRE-or Gal4-system-driven luciferase reporter gene constructs. The expression of the reporter gene records the complete process including DNA binding, phosphorylation, and also the recruitment of coactivators.

The present study shows that lithium strongly enhances the cAMP-stimulated CRE-mediated transcription and CREB activity. This effect depends on the basic leucine zipper region. containing the luciferase gene under control of four repeats of the CRE from the somatostatin promoter and a truncated thymidine kinase promoter (Fig. 1), -65SMSLuc, containing the luciferase gene under control of the truncated rat somatostatin promoter (-65 to +54) [START_REF] Schwaninger | Inhibition of cAMP-responsive element-mediated gene transcription by cyclosporin A and FK506 after membrane depolarization[END_REF] and 5xGal4E1B-Luc [START_REF] Kruger | Inhibition of CREB-and cAMP response element-mediated gene transcription by the immunosuppressive drugs cyclosporin A and FK506 in T cells[END_REF] which contains the luciferase gene under control of 5 repeats of the responsive element for the yeast transcription factor GAL4. Expression vectors for the overexpression of GAL4-CREB fusion proteins were GAL4-CREB 327 [20], encoding full length CREB, GAL4-CREB 261 [21], which encodes a truncated CREB lacking the basic region and leucine zipper domain, and GAL4-CREB-R300A. The GAL4-CREB-R300A mutant was generated by primerless PCR using the following primers (mutation underlined) 5' tgt tta gaa aac gca gtg gca gtg ctt 3' (forward), 5' aag cac tgc cac tgc gtt ttc taa aca 3' (reverse), flanking primers and GAL4-CREB327 as template. For PCR cloning of GAL4-bZip encoding the basic leucine zipper domain (CREB 262-327 ), the primers 5' cgcggatcctagcacgaaagagagaggtcc 3' (forward) (BamHI site in italics, coding sequence underlined), 5' cgc tct aga tta tta atc tga ttt gtg gca gta aag gtc 3' (reverse) (XbaI site in italics, stop codons bold, coding region underlined), and GAL4-CREB 327 as template were used followed by cloning into the GAL4 domain-coding vector pSG424. TORC1-coding sequence was kindly provided by Marc Labow, (Novartis Pharma AG, Basel, Switzerland) and subcloned into pcDNA3.

Cell culture, transfection and stimulation

Hamster insulinoma tumor cells (HIT-T15) [START_REF] Schwaninger | Inhibition of cAMP-responsive element-mediated gene transcription by cyclosporin A and FK506 after membrane depolarization[END_REF] were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 5% horse serum, 100 units/ml penicillin and 100 µg/ml streptomycin. The cells were transiently transfected by the DEAE-dextran method [START_REF] Schwaninger | Inhibition of cAMP-responsive element-mediated gene transcription by cyclosporin A and FK506 after membrane depolarization[END_REF] with 2 µg of the indicated plasmid per 6-cm dish.

Cytomegalovirus green fluorescent protein expression vectors (1 µg per 6-cm dish) were added as second reporters to control for transfection efficiency. Cells were treated with 1 mM myoinositol 8 h prior to harvest, with 20 mM LiCl or the GSK3 inhibitors SB215763 (30 µM) or SB415286 (50 µM) 7 h prior to harvest, and with 1 or 2 mM 8BrcAMP, 40 mM KCl or 10 µM forskolin 6 h prior to harvest. Cell extracts were prepared 48 h after transfection. The luciferase assay was performed as previously described [START_REF] Schwaninger | Inhibition of cAMP-responsive element-mediated gene transcription by cyclosporin A and FK506 after membrane depolarization[END_REF]. Fluorescence of the green fluorescent protein was measured in a Packard FluoroCount with excitation wave length at 485 nm and emission wave length at 530 nm. For the determination of the intracellular lithium concentration, cells were lysed by freeze-thaw cycles and the lithium concentration in supernatants was measured by ion-selective potentiometry using an Easylyte Na/K/Li analyzer (Bedford, USA).

Western blot

For CREB/ phospho-CREB determination, HIT-T15 cells were treated 3 h prior to harvest with 20 mM LiCl and 2 h prior to harvest with 2 mM 8BrcAMP. For measurement of -catenin accumulation, the cells were treated 6 h prior to harvest with 20 mM LiCl or the GSK3 inhibitors SB215763 (30 µM) or SB415286 (50 µM). Proteins in the cell lysates were separated by 10% SDS-PAGE and transferred to nitrocellulose membrane by semi dry blotting. CREB, phospho-CREB or -catenin was immunostained with specific antibodies and respective peroxidase-labeled secondary antibodies. Immunoreactivity was detected using the ECL reaction. Specific bands were quantified densitometrically and the ratio between intensity of CREB and phospho-CREB from the same lysate was calculated. For -catenin blots identical loading was assured by GAPDH staining.

Electrophoretic mobility shift assay (EMSA)

For EMSA, HIT-T15 cells were treated with 20 mM LiCl 2 h prior to harvest and with 2 mM 8BrcAMP 1 h prior to harvest. Nuclear extracts were prepared according to Schreiber [START_REF] Schreiber | Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells[END_REF].

The synthetic complementary oligonucleotides SCE [17] with 5'-GATC overhangs were annealed and labelled by a fill-in reaction using [ 32 P]dCTP and Klenow enzyme. Using 11 µg protein from the nuclear extracts, the EMSA was performed as described [23]. Briefly, nuclear extracts were incubated in 20 µl (total volume) containing 20 mM HEPES pH 7.9, 1 mM EDTA, 0.5 mM dithiothreitol, 5 mM MgCl 2 , 70 mM KCl, 10% glycerol, 0.2 µg poly(dI-dC) as non-specific competitor and 16 pmol of unlabeled wildtype or mutant SCE as specific competitor, if indicated, for 10 min on ice. Then, 20,000 cpm of the labelled probe were added and incubated 15 min on ice. Proteins were separated on 5% non-denaturing polyacrylamide gel electrophoresis and bound probes detected by autoradiography.

Measurement of intracellular calcium [Ca 2+ ] i

HIT-T15 cells were subcultured on coverslips at 37° C in medium containing 130 mM NaCl, 4.7 mM KCl, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 1.5 mM CaCl 2 , 6 mM glucose, 20 mM HEPES and 2% BSA (w/v), aerated with 100% O 2 (v/v), pH 7.4. Where indicated, cells were pretreated with 20 mM LiCl for 1 h before loading with 5 µM fura-2/AM, in the presence of Pluronic F-127 0.1% (w/v) at 37 °C for 30 min. After loading, the coverslips were washed, mounted in a temperature-controlled superfusion chamber (37°C), and placed on the platform of a Carl Zeiss Axiovert IM 135 equipped with a 40x Achrostigmat oil immersion objective (Carl Zeiss, Jena, Germany). The chamber was superfused with the same buffer as above with 0.1% BSA (w/v), without 0.1% Pluronic F-127 (w/v) and the respective test agents at a flow rate of 0.75 ml/min. Ca 2+ measurements were taken on cells of average size and healthy appearance (round in shape, no membrane blebs). Fura-2 fluorescence from single cells was recorded with a dual excitation spectrofluorometer system (Deltascan 4000, Photon Technology Instruments, Wedel, Germany). [Ca 2+ ] i was calculated according to the formula [START_REF] Grynkiewicz | A new generation of Ca2+ indicators with greatly improved fluorescence properties[END_REF], R max , R min and B are constants that were determined in the superfusion chamber from solutions containing fura-2 free acid (1 µM) and various concentrations of free Ca 2+ (data not shown). All records have been corrected for autofluorescence of unloaded cells at each wavelength before the ratio was used.

[Ca 2+ ] i = K d x B x (R -R min )/(R max -R), where K d = 225 nM

Materials

All reagents, unless otherwise indicated, are from Sigma-Aldrich, München, Germany. Stock solutions were prepared in water, except for forskolin, SB-215763 and SB-415286 (DMSO).

Controls received the solvent only. [ 32 P]dCTP and ECL system was obtained from GE Healthcare (München, Germany). All enzymes were purchased from Fermentas GmbH, St.

Leon-Roth, Germany. SB-215763 and SB-415286 were obtained from GlaxoSmithKline, München, Germany. Phospho-CREB antibody was from NewEngland Biolabs, Frankfurt a.M., Germany and -catenin antibody was from BD biosciences, Heidelberg, Germany.

Statistics

RESULTS

To investigate the effect of lithium salts on CRE-directed gene transcription, a transient transfection assay was used. The electrically excitable hamster insulinoma tumor cell line HIT was transfected with a DNA construct in which the expression of a reporter gene is controlled by the CRE from the rat somatostatin promoter. This CRE contains the consensus sequence TGACGTCA and is well known as a high affinity CREB binding site that mediates responsiveness to cAMP and Ca 2± [17]. The construct (Fig. 1) containing four copies of the CRE in front of the truncated viral thymidine kinase promoter and the luciferase gene, has been characterized before in HIT cells [START_REF] Schwaninger | Inhibition by antidepressant drugs of cyclic AMP response element-binding protein/cyclic AMP response element-directed gene transcription[END_REF]. The CRE conferred negligible low basal transcriptional activity (Fig. 1). Stimulation with the adenylyl cyclase activator forskolin resulted in a 27.5-fold increase of CRE-mediated transcription, as described before [START_REF] Schwaninger | Inhibition by antidepressant drugs of cyclic AMP response element-binding protein/cyclic AMP response element-directed gene transcription[END_REF].

For lithium treatment, a concentration of 20 mM was chosen. This concentration resulted in an intracellular lithium concentration of 3.2 ± 0.2 mM. Treatment with 20 mM LiCl only slightly increased basal CRE activity (not significant) (Fig. 1). In contrast, lithium treatment resulted decreased of forskolin-induced CRE activity by 41% (Fig. 1). This decrease is most likely due to the inhibition of the adenylyl cyclase activity by lithium [START_REF] Mork | The effects of lithium in vitro and ex vivo on adenylate cyclase in brain are exerted by distinct mechanisms[END_REF] as the measurement of intracellular cAMP levels indicated markedly reduced levels after lithium treatment (data not shown).

Enhancement by lithium of cAMP-induced transcription

To test, whether, in addition to its effect on cAMP formation, lithium can influence cAMPinduced transcription; HIT cells were stimulated with 8BrcAMP, an analogue for cAMP with a higher stability and increased membrane permeability. Stimulation with 1 mM 8BrcAMP resulted in a 4.7-fold increase in CRE-mediated transcription (Fig. 2, left panel). In contrast to the effect on forskolin stimulation, lithium in combination with 8BrcAMP exerted a robust enhancing effect on CREB activity by 22.3-fold. Relative to the 8BrcAMP-induced stimulation, lithium treatment resulted in a 4.7-fold increase (Fig. 2).

As HIT cells are electrically excitable [START_REF] Schwaninger | Inhibition by antidepressant drugs of cyclic AMP response element-binding protein/cyclic AMP response element-directed gene transcription[END_REF], we asked whether lithium could influence calciuminduced CRE-mediated transcription [START_REF] Schwaninger | Inhibition by antidepressant drugs of cyclic AMP response element-binding protein/cyclic AMP response element-directed gene transcription[END_REF]. HIT cells were treated with 40 mM KCl to induce membrane depolarization and subsequent calcium influx. Membrane depolarization led to a 6.5-fold increase of CRE-mediated transcription (Fig. 2, left panel). Lithium treatment increased depolarization-induced CRE activity only 1.39-fold, which was not significant. In contrast, the combined stimulus of 8BrcAMP plus depolarization, which resulted in a 140-fold increase of CRE-mediated gene transcription, was further enhanced by lithium by 1.8fold (Fig. 2, right panel) which was highly significant. Since lithium enhanced transcription mot only after stimulation by cAMP but also after stimulation by cAMP plus KCl, lithium may affect transcription not through an increase in cytosolic calcium concentrations. For further Pretreatment with lithium resulted in a slightly decreased calcium rise (38 ± 11 nM), which was not significant (Student's t-test). Stimulation with both, 8BrcAMP and KCl, increased Ca 2+ levels by 226 ± 25 nM over basal. Pretreatment with lithium had no effect (218 ± 37 nM). The results support the notion that the enhancing effect of lithium on CRE-mediated transcription is not caused by elevated intracellular calcium levels.

To examine the concentration-response relationship of the effect of lithium on 8BrcAMPstimulated CRE activation was dose-dependent, increasing concentrations of lithium were employed. A significant enhancing effect of lithium was detectable at 6 mM (2.3-fold increase of 8BrcAMP-induced activity) (Fig. 3). Higher concentrations (20 and 50 mM LiCl) enhanced 8BrcAMP-induced activity about 4-fold (Fig. 3) and 11.4-fold, respectively (data not shown).

The dose-response curve did not show a sigmoid characteristic as it reached no maximum. Thus, a half maximal effective dose could not be calculated.

Role of the known lithium targets GSK3ß and inositol-monophosphatase

Lithium salts are known to inhibit glycogen-synthase-kinase 3 (GSK3 ) and inositolmonophosphatase (IMP) [START_REF] Quiroz | MOLECULAR EFFECTS of lithium[END_REF]. Therefore the question was addressed whether these two enzymes are involved in the effect of lithium on CRE-mediated gene transcription.

Two different GSK3 inhibitors were used. Both, SB216763 and SB415286 did not mimic the effect of lithium (Fig. 4a). SB216763, SB415286 and lithium induced -catenin accumulation indicating efficient inhibition of GSK3 (data not shown).

Lithium inhibits IMP and thus depletes inositol in the cell [START_REF] Quiroz | MOLECULAR EFFECTS of lithium[END_REF]. Therefore, cells were treated concurrently with inositol to replenish a presumed depletion in inositol storage. The addition of inositol did not change the effect of lithium on 8BrcAMP-induced CRE-mediated transcription (Fig. 4b). Thus, these well known biochemical targets of lithium most likely were not responsible for the stimulation of CRE-mediated transcription by lithium.

Effect of lithium on CREB phosphorylation and CRE binding

An important step in the activation of CREB is its phosphorylation on Ser-119. Therefore, the ratio of phosphorylated to non-phosphorylated CREB was determined by western blot analysis. Treatment with lithium alone did not change the ratio of P-CREB to CREB (Fig. 5). 8BrcAMP induced a slight 1.3-fold increase in CREB Phosphorylation. This Phosphorylation was not detectably changed by lithium (Fig. 5). However, as the values only approached The DNA binding of CREB was not changed by any treatment (Fig. 6b) (p=0.9, effect of treatment, two-way-ANOVA). Thus, the enhancement of CRE-mediated gene transcription was not related to an effect of lithium on the binding of CREB to its responsive element as revealed by EMSA.

Lithium responsiveness depends on the CREB-bZip

To further investigate the mechanism of the observed action of lithium on CREB-mediated transcription the Gal4 system was employed. Expression vectors encoding the DNA-binding domain of the yeast transcription factor GAL4 fused to the transcription factor of interest were cotransfected with a GAL4 binding site-driven luciferase reporter gene. This allows the investigation of the transactivational potential of the transcription factor of interest, independent from its DNA binding and endogenous background. The GAL4 domain itself did not show any responsiveness to the stimuli used (data not shown).

When full length CREB was fused to the GAL4 DNA-binding domain (GAL4-CREB) the treatment with 8BrcAMP and lithium mimicked the effect observed when using the CREdriven promoter (Fig. 7). 20 mM LiCl alone had no effect on gene transcription. The combination of lithium with the cAMP stimulus resulted in a robust enhancement of gene transcription (2.9-fold over 8BrcAMP alone (Fig. 7). To test whether the lithium effect depends on the bZip domain of CREB, a truncated mutant of CREB (aa1-261) was fused to GAL4 (GAL4-CREB 261 ). This construct contains the transactivation domain of CREB (including Ser-119) but lacks the basic leucine zipper domain of CREB. With GAL4-CREB 261 , the lithium effect was completely abolished (Fig. 7). Stimulation with 20 mM LiCl increased transcription only slightly, 1 mM 8BrcAMP 1.6-fold, and the combination of both stimuli 2.43fold, which did not reach significance (two-way ANOVA) (Fig. 7). Thus, the basic leucine zipper domain seemed to be crucial to confer the lithium effect on CREB.

The coactivator TORC confers lithium responsiveness

As the coactivator TORC binds to the bZip [START_REF] Conkright | TORCs: transducers of regulated CREB activity[END_REF], the involvement of TORC in the lithium effect was tested. A CREB mutant with an exchange of R300 to A in the bZip has been reported to be deficient in TORC binding [START_REF] Screaton | The CREB coactivator TORC2 functions as a calcium-and cAMP-sensitive coincidence detector[END_REF]. Thus, a GAL4-CREB-R300A mutant was employed (Fig. 8a). As reported before, this mutant responded only poorly to the cAMP stimulus [START_REF] Screaton | The CREB coactivator TORC2 functions as a calcium-and cAMP-sensitive coincidence detector[END_REF] and also lithium failed to induce a significant enhancement (Fig. 8a). To confirm the importance of the bZip and TORC for the lithium effect, a construct containing the Gal4-DNA-binding domain fused to the isolated CREB-bZip was employed (GAL4-bZip). Without cotransfection, the bZip showed no activation by cAMP and lithium (Fig. 8a). However, when TORC1 was overexpressed, the bZip was activated by 8BrcAMP and more importantly, also the lithium effect was restored completely (2.4-fold increase, relative to the cAMP stimulus) (Fig. 8a).

GAL4 and GAL4-bZip-R300A were not activated by TORC overexpression (data not shown).

Finally, we tested whether TORC could confer the lithium effect also on a native promoter.

Therefore, a promoter (-65SMSLuc) containing one CRE in its homologous context was employed. The truncated promoter showed only poor response to either stimulus (Fig. 8b). In contrast, after overexpression of TORC1, lithium was able to enhance the cAMP-stimulated promoter activity 1.94-fold (Fig. 8b). Thus, as shown with the Gal4-system and a native promoter, TORC is able to confer the lithium-induced enhancement of cAMP-stimulated CREB activity.

DISCUSSION

Lithium salts have been established for more than 50 years as the most effective treatment for bipolar disorder [START_REF] Cade | John Frederick Joseph Cade: family memories on the occasion of the 50th anniversary of his discovery of the use of lithium in mania[END_REF]. Lithium salts have an acute silencing action, making them beneficial for the treatment not only of manic phases of bipolar disorder but also of psychotic manic states. More important, they are the only treatment that prevents the cycling between manic and depressive states and also reduces the suicide rate [28]. The requirement of chronic treatment of up to several months implicates the involvement of changes in gene expression based on the modulation of transcription factors like CREB.

CREB has been implicated in several important brain functions. In addition to physiological functions like learning and memory [START_REF] Silva | CREB and memory[END_REF] there is evidence that CREB also plays a role in the development of mental diseases [START_REF] Nestler | Neurobiology of depression[END_REF]. Major depression and bipolar disorder both are thought to be related to changes in the cAMP/PKA/CREB system and treatment with antidepressants and mood stabilizers are suggested to correct for deficits in neuronal signalling [START_REF] Stewart | Abnormalities in the cAMP signaling pathway in post-mortem brain tissue from the Stanley Neuropathology Consortium[END_REF].

In this study, the action of the clinically well-established mood stabilizer lithium on CRE/CREB-driven gene transcription was investigated. The major findings are i) lithium induces a robust increase in cAMP-stimulated CRE-driven gene transcription and enhances CREB activity; ii) this effect of lithium does not appear to be related to an enhanced CREB-Ser-119 phosphorylation, an increased CREB-DNA binding or the inhibition of GSK3 and IMP; iii) the enhancement by lithium of CREB activity seems to be conferred by the coactivator TORC binding to the CREB basic leucine zipper region.

To study the effect of lithium on CRE/CRB-directed gene transcription, a luciferase reporter gene construct was employed where reporter gene expression is regulated by four repeats of the somatostatin CRE. This CRE contains the palindromic core binding site with the consensus sequence 5'-TGACGTCA-3'. In combination with the flanking regions, the somatostatin CRE forms a binding site with a notably high affinity for CREB [START_REF] Shaywitz | CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals[END_REF]. The 4xSomCRET81Luc construct has been thoroughly characterized in the pancreatic -cell line HIT-T15 [17,[START_REF] Schwaninger | Inhibition by antidepressant drugs of cyclic AMP response element-binding protein/cyclic AMP response element-directed gene transcription[END_REF] as well as in other cells including neuronal cell lines [START_REF] Kruger | Inhibition of CREB-and cAMP response element-mediated gene transcription by the immunosuppressive drugs cyclosporin A and FK506 in T cells[END_REF]. HIT-cells resemble neuronal cells in important aspects. They are electric excitability, express calcium and potassium channels and also have neuron-specific transcription factors.

Lithium induced a robust increase in 8BrcAMP-induced CRE/CREB-directed gene transcription. The dose-response curve showed a significant effect with 6 mM lithium. The measurement of the cytosolic lithium concentration revealed that the intracellular level of lithium was about 6-fold lower than in the cell culture medium. As plasma levels of 1 mM lithium have been proven to be effective in patients, we conclude that lithium at therapeutic concentrations is able to increase CRE/CREB transcriptional activity. A simple explanation for the effect of lithium could be an induction of calcium influx, since the combination of calcium and cAMP stimuli has been shown to be super additive in the . However, the combined stimulus, 8BrcAMP plus KCl, was also enhanced by lithium. Moreover, the determination of intracellular calcium concentrations revealed, if anything, rather decreased calcium levels by lithium after cAMP-stimulation. This could be due to an enhanced efflux of calcium caused by a replacement of sodium by lithium in a sodium/calcium exchanger which has been reported previously [START_REF] Palty | Lithium-calcium exchange is mediated by a distinct potassium-independent sodium-calcium exchanger[END_REF].

Two well-known targets of lithium are IMP and GSK3 [START_REF] Quiroz | MOLECULAR EFFECTS of lithium[END_REF] Both do not appear to be involved in the observed effect of lithium on CRE-driven gene transcription. IMP inhibition and subsequent inositol depletion have been suggested to be the common mechanism of action of three mood stabilizing agents [START_REF] Williams | A common mechanism of action for three mood-stabilizing drugs[END_REF] as lithium, valproate and carbamazepine stabilized neuronal growth cones in an inositol-reversible manner. However, the action of lithium on CRE/CREB-directed transcription was not affected by the addition of inositol, which should prevent the assumed inositol depletion by lithium. GSK3 has been shown to be inhibited by lithium at concentrations of about 1-2 mM [START_REF] Klein | A molecular mechanism for the effect of lithium on development[END_REF] and is discussed to mediate the therapeutic effects of lithium [START_REF] Jope | Mood stabilizers, glycogen synthase kinase-3beta and cell survival[END_REF]. As CREB is phosphorylated by GSK3 on Ser-115 [START_REF] Fiol | A secondary phosphorylation of CREB341 at Ser129 is required for the cAMP-mediated control of gene expression. A role for glycogen synthase kinase-3 in the control of gene expression[END_REF], lithium may influence CREB activity via GSK3 . It has been shown that the phosphorylation of CREB by GSK3 reduces the DNA binding of CREB and thus attenuated CREB activity [START_REF] Grimes | CREB DNA binding activity is inhibited by glycogen synthase kinase-3 beta and facilitated by lithium[END_REF]. However, in the present study, two specific GSK-inhibitors did not mimic the effect of lithium on CRE/CREB-directed gene transcription. Moreover, the EMSA revealed no effect of lithium on CREB DNA-binding. Noteworthy, lithium and the two GSK3 inhibitors induced an accumulation of -catenin indicating that GSK3 was active and the inhibitors were effective. Thus, inhibition of GSK3 most likely does not contribute to the effect of lithium on cAMPinduced CRE/CREB-directed gene transcription.

As the two best known lithium targets seemed not to be involved we focused on the effect of lithium on CREB activity. Western blot analyses revealed that the robust enhancement of cAMP-induced CRE/CREB-directed gene transcription was not accompanied by an increased phosphorylation of CREB. Unchanged CREB phosphorylation after lithium treatment of an osteoblastic cell line [36] or in cerebellar granule cells of acutely treated rats [START_REF] Kopnisky | Chronic lithium treatment antagonizes glutamate-induced decrease of phosphorylated CREB in neurons via reducing protein phosphatase 1 and increasing MEK activities[END_REF] has been shown before. Moreover, in addition to the lacking impact on CREB phosphorylation it was shown that the lithium effect was not dependent on the Ser-119 containing transactivation domain but required the existence of the CREB-bZip. We here confirm a discrepancy between CRE/CREB-mediated transcription and phosphorylation, which has been reported before in HIT cells after cyclosporine A treatment [START_REF] Schwaninger | Involvement of the Ca(2+)-dependent phosphatase calcineurin in gene transcription that is stimulated by cAMP through cAMP response elements[END_REF] or in cortical [START_REF] Pham | CRE-mediated gene transcription in neocortical neuronal plasticity during the developmental critical period[END_REF] and hippocampal neurons [16]. Until recently, CREB transcription activity was generally explained by the classic transactivation pathway of CREB e.g. phosphorylation of Ser-119 and recruitment of CBP, though the involvement of additional factors has also been assumed Nowadays, a new coactivator has been discovered; the transducer of regulated CREB (TORC) [START_REF] Conkright | TORCs: transducers of regulated CREB activity[END_REF]. TORC confers CREB transactivation by binding to the CREB-bZip independent from phosphorylation at Ser-119. TORC exists in three isoforms and has been shown to be localized under basal conditions in the cytosol. Upon stimulation with cAMP it translocates to the nucleus and binds to the leucine zipper of CREB where it contributes to transcriptional activity. As the effect of lithium on CREB activity and the action of TORC converge at the CREB-bZip, the involvement of TORC into the lithium action was shown. Three approaches were used. First, arginine 300 which has been shown to be crucial for the CREB-TORC interaction was mutated to alanine. Here, lithium failed to induce an enhancement of CREB activity. Second, the isolated CREB-bZip showed activation neither by cAMP nor by lithium; however, overexpression of recombinant TORC completely restored the lithium effect. As the isolated CREB-bZip was not sufficient to mediate the lithium effect, one could assume that the recruitment of endogenous TORC was less stable or occurred with lower affinity. The conformation of the CREB-bZip or its position related to the DNA is presumably suboptimal in the GAL4-bZip construct used here, suggesting that the CREB-TORC interaction is stabilized by other CREB regions than the bZip. Overexpression results in higher concentration of TORC in the nucleus which could overcome these deficits. Third, the somatostatin CRE in its homologous context was employed. The promoter contains the CRE which was used to create the oligomerized promoter in 4xSomCRET81Luc. Overexpression of TORC conferred the responsiveness of the native promoter to lithium, thus indicating that also in the homologous context TORC was mediating the lithium effect.

What could be the consequences of these findings? Generally, lithium could be able to modulate the expression of CRE/CREB-regulated genes [START_REF] Impey | Defining the CREB regulon: a genome-wide analysis of transcription factor regulatory regions[END_REF]. The brain derived neurotrophic factor (BDNF) gene and the antiapoptotic Bcl-2 gene are regulated by CREB which has been implicated in neuroprotective and neuroplasticity processes. It was shown that lithium treatment increased expression of BDNF and Bcl-2 in rat hippocampus and cortex [10], which has been considered to contribute to the neuroprotective action of lithium. Furthermore, in the hippocampus it has been shown, that TORC1 was essential for CREBinduced long-term potentiation, a form of synaptic plasticity involved in long-term-memory formation [16]. The hippocampus plays a pivotal role in the control of mood and emotion. In addition, hippocampal volume reduction was associated with bipolar disorder [38]. Thus, one could assume that the mood-stabilizing action of lithium involves CRE/CREB-TORCmediated gene expression particularly in this brain region. We here for the first time provide evidence that lithium plays a direct role in the CRE/CREBdirected gene transcription. We showed that independent from interfering with signalling cascades lithium affects directly CREB activity and suggest the coactivator TORC as a new target for its molecular mechanism of action. This will not only deepen the understanding of

Furthermore, 4 Biochemical

 4 TORC was shown to confer lithium responsiveness to CREB-bZip. Thus, the present study, for the first time, identifies the CREB-bZip in combination with the coactivator TORC as a new molecular target of lithium. Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070796 © 2007 The Authors Journal compilation © 2007 Biochemical Society EXPERIMENTAL Plasmids: Reporter gene constructs used for transfection were 4xSomCRET81Luc [17]

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  Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070796 © 2007 The Authors Journal compilation © 2007 Biochemical Society

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  Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070796 © 2007 The Authors Journal compilation © 2007 Biochemical Society

7 Biochemical

 7 All analyses (two-way ANOVA with Bonferroni post hoc test, Student's t-test) as indicated in the legends to the figures were performed using the soft ware STATISTICA 7.1, statsoft, Tulsa, USA. Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070796 © 2007 The Authors Journal compilation © 2007 Biochemical Society

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  Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070796 © 2007 The Authors Journal compilation © 2007 Biochemical Society significance (p=0,065, one-way ANOVA), probably because of the low 8BrcAMP concentration, these are only tendencies which, nevertheless, most likely do not account for the observed effect of lithium on CRE-mediated transcription.Another possibility for the action of lithium on CRE-mediated gene transcription could be the modulation of the DNA binding of CREB. Therefore, EMSAs with nuclear extracts from lithium treated cells and the somatostatin CRE were performed. Nuclear CREB binding to the CRE was clearly detectable as shown before [17] (Fig.6a). The specificity of the band was confirmed in competition experiments. Adding an excess of unlabelled CRE markedly decreased the intensity of the band to 8.7 % of control, whereas or a similar excess of a mutant CRE was almost ineffective (reduction to 76.1% of control).

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  Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070796 © 2007 The Authors Journal compilation © 2007 Biochemical Society

11 Biochemical

 11 Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070796 © 2007 The Authors Journal compilation © 2007 Biochemical Society

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 12 Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070796 © 2007 The Authors Journal compilation © 2007 Biochemical Society activation of CRE/CREB-directed gene transcription [17]

14 Biochemical 15 Biochemical

 1415 Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070796 © 2007 The Authors Journal compilation © 2007 Biochemical Society the therapeutic action of lithium salts. It also provides new insight into the regulation of gene expression whereas the interplay between transcription factors and their coactivators can be modulated by ions like lithium. Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070796 © 2007 The Authors Journal compilation © 2007 Biochemical Society Tyson, D. R., Swarthout, J. T., Jefcoat, S. C. and Partridge, N. C. (2002) PTH induction of transcriptional activity of the cAMP response element-binding protein requires the serine 129 site and glycogen synthase kinase-3 activity, but not casein kinase II sites. Endocrinology 143, 674-682 37 Mayr, B. and Montminy, M. (2001) Transcriptional regulation by the phosphorylationdependent factor CREB. Nat Rev Mol Cell Biol

Fig. 4

 4 Fig. 4 Effect of GSK3ß inhibitors (a) and inositol (b) on cAMP-induced CRE/CREB-directed gene transcription. HIT-T15 cells were transfected with the reporter gene construct 4xSomCRET81Luc. a) 7 h prior to harvest cells were treated with 20 mM LiCl or the GSK3 inhibitors SB215763 (30 µM) or SB415286 (50 µM) and 6 h prior to harvest with 1 mM 8BrcAMP. b) 8 h prior to harvest 1 mM myoinositol was added, 7 h prior to harvest cells were treated with 20 mM LiCl and 6 h prior to harvest with 1 mM 8BrcAMP. 48 h after

Fig. 5

 5 Fig. 5 Effect of lithium on Ser119 phosphorylation of CREB. HIT-T15 cells were treated 3 h prior to harvest with 20 mM LiCl and 2 h prior to harvest with 2 mM 8BrcAMP. Cell lysates were subjected to western blot analysis and CREB and phospho-CREB immunostained with specific antibodies. a) Immunoblot from a typical experiment. b) Densitrometric quantification of the bands. Specific bands of P-CREB and CREB from the same lysate were quantified and the ratio between p-CRE and CREB was calculated. Values are means ± SEM of 3 independent experiments.

Fig. 6

 6 Fig. 6Effect of lithium on the binding of CREB to the somatostatin CRE as revealed by the EMSA.HIT-T15 cells were treated with 20 mMLiCl 2 h prior to harvest and with 2 mM 8BrcAMP 1 h prior to harvest. Nuclear extracts were incubated with labelled oligodeoxynucleotides containing the somatostatin CRE (SCE). Unlabelled wildtype or mutant oligonucleotides as competitors were added at a 450-fold molar excess. Protein complexes were separated on a non-denaturing SDS gel and probes visualized by autoradiography. a) autoradiography of a typical experiment. The arrow indicates the mutation sensitive complexes b) Densitometric quantification of specific bands. Values are means ± SEM from 6 independent experiments.

Fig. 7 22 BiochemicalFig. 8

 7228 Fig. 7Effect of lithium on cAMP-induced transcriptional activity of CREB wildtype and CREB mutant lacking the basic leucine zipper region, using the heterologous GAL4-system.HIT-T15 cells were transfected with the Gal4-driven reporter gene construct G5E1B-Luc and GAL4-fusion proteins containing full length CREB or CREB1-261. 7 h prior to harvest cells were treated with 20 mM LiCl and 6 h prior to harvest with 2 mM 8BrcAMP. 48 h after transfection luciferase activity was determined. Values are expressed as % of the respective untreated control. Values are means of 3 independent experiments ± SEM each done in duplicate. ***:p<0.001, two-way ANOVA, Bonferroni post-hoc test.

8 Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070796 © 2007 The Authors Journal compilation © 2007 Biochemical Society investigation

  , the intracellular calcium levels of HIT cells after different stimuli were recorded. 20 mM LiCl did not change the basal intracellular Ca 2+ level (180 ± 3 nM (control) vs. 176 ± 5 nM (lithium)). Stimulation with 2 mM 8BrcAMP increased intracellular Ca 2+ by 84 ± 16 nM.

Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070796 © 2007 The Authors Journal compilation © 2007 Biochemical Society LEGENDS TO THE FIGURES Fig. 1

  Effect of lithium on forskolin-stimulated CRE/CREB-directed gene transcription.

	HIT-T15 cells were transfected with the indicated reporter gene construct
	2, 599-609 4xSomCRET81Luc. 7 h prior to harvest cells were treated with 20 mM LiCl and 6 h prior to
	38 harvest with 10 µM forskolin. 48 h after transfection luciferase activity was determined. Blumberg, H. P., Kaufman, J., Martin, A., Whiteman, R., Zhang, J. H., Gore, J. C.,
	Charney, D. S., Krystal, J. H. and Peterson, B. S. (2003) Amygdala and hippocampal Values are expressed as % of the controls that received solvent only. Values are means of 4
	volumes in adolescents and adults with bipolar disorder. Arch Gen Psychiatry 60, independent experiments ± SEM each done in duplicate. FSK: forskolin. TK: thymidine
	1201-1208 kinase promoter. Luciferase: coding sequence of the firefly luciferase gene. ***:p<0.001, two-
	way ANOVA, Bonferroni post-hoc test.
	Fig. 2
	Effect of lithium on cAMP-and membrane depolarization-induced CRE/CREB-directed gene
	transcription. HIT-T15 cells were transfected with the reporter gene construct
	Fig. 3
	Concentration-response curve for the effect on cAMP-induced CRE/CREB-directed gene
	transcription. HIT-T15 cells were transfected with the reporter gene construct
	4xSomCRET81Luc. 7 h prior to harvest cells were treated with increasing concentrations of
	LiCl (20 µM-20 mM) and 6 h prior to harvest with 1 mM 8BrcAMP. 48 h after transfection
	cells luciferase activity was determined. Values of 8BrcAMP-treated cells that received no
	LiCl were set to 100%. Values are means of 3 independent experiments ± SEM each done in
	duplicate. *: p<0.05, Student's t-test; **: p<0.01, Student's t-test.
	20

4xSomCRET81Luc.7 h prior to harvest cells were treated with 20 mM LiCl and 6 h prior to harvest with 2 mM 8BrcAMP or 45 mM KCl. 48 h after transfection luciferase activity was determined. Values are expressed as % of control (no 8BrcAMP, no KCl). Values are means of 3 independent experiments ± SEM each done in duplicate. ***:p<0.001, two-way ANOVA, Bonferroni post-hoc test.

Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070796 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 14 Aug 2007 as manuscript BJ20070796 © 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGMENTS

We thank Elke Oetjen for her scientific advice and reading the manuscript.

The work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 402 (A3) to W.K.).


Anna Ohradanova

Daniela Vullo

Juraj Kopacek

Claudia Temperini

Tatiana Betakova

Silvia Pastorekova

Jaromir Pastorek

Claudiu T Supuran
email: claudiu.supuran@unifi.it

Reconstitution of carbonic anhydrase activity of the cell surface binding protein of vaccinia virus

Keywords: carbonic anhydrase, vaccinia virus, in vitro mutagenesis hCA, human carbonic anhydrase, vaccCA, vaccinia virus CA-like protein, vaccinia virus cell surface-binding protein, GST, glutathione S-transferase, IPTG, isopropyl β-D-thiogalactoside, PBS, phosphate-buffered saline, E-I complex, enzyme-inhibitor complex, RT-PCR, reverse-

The N-terminal region of a 32-kDa cell surface-binding protein, encoded by D8L gene of vaccinia virus, shows sequence homology to carbonic anhydrases (CAs, EC 4.2.1.1). The active CAs catalyze the reversible hydration of CO 2 to bicarbonate participating in many physiological processes. The CA-like domain of vaccinia protein (vaccCA) contains one of the three conserved histidines required for coordination to the catalytic zinc ion and for enzyme activity. Here we report the engineering of catalytically active vaccCA mutants by introduction of the missing histidines into the wild type protein. The wild type vaccCA was inactive as a catalyst and does not bind sulfonamide CA inhibitors. Its position on a phylogram with other human CAs shows a relationship with the acatalytic isoforms CA X and XI, suggesting that the corresponding viral gene was acquired from the human genome by horizontal gene transfer. The single mutants (vaccCA N92H/Y69H) showed low enzyme activity and low affinity for acetazolamide, a classical sulfonamide CA inhibitor. The activity of the double mutant, vaccCA N92H/Y69H, was much higher, of the same order of magnitude as that of some human isoforms, namely CA VA and CA XII.

Moreover, its affinity to acetazolamide was high, comparable to that of the most efficient human isoenzyme, CA II (in the low nanomolar range). Multiplication of vaccinia virus in HeLa cells transfected with the vaccCA N92H/Y69H double mutant was almost twice more efficient than in wild type vaccCA transfectants, suggesting that the reconstitution of the enzyme activity improved the virus life cycle.

Introduction

Poxviruses constitute a family of viruses characterized by a large, linear dsDNA genome, a cytoplasmic site of replication and complex virion morphology [START_REF] Condit | In a nutshell: structure and assembly of the vaccinia virion[END_REF][START_REF] Moss | Poxvirus entry and membrane fusion[END_REF]. The most notorious member of the family is the variola virus, the causative agent of smallpox, whereas the laboratory prototype virus used for the study of poxviruses is vaccinia, the virus used as a live, naturally attenuated vaccine for the eradication of smallpox [START_REF] Condit | In a nutshell: structure and assembly of the vaccinia virion[END_REF][START_REF] Moss | Poxvirus entry and membrane fusion[END_REF][START_REF] Tatsis | Vaccinia virus as a tool for immunologic studies[END_REF]. The poxviruses have a long history of causing disease, and their biological effects in humans and other mammals have been extensively studied [START_REF] Essajee | Poxvirus vaccines for cancer and HIV therapy[END_REF]. In the 80s and 90s, genetic engineering techniques were applied to vaccinia in order to create replicating recombinant vectors that could express inserted genes encoding other proteins, which were able to elicit a specific adaptive immune response to foreign antigens in animal models, creating the basis of gene delivery approaches for the treatment of diseases (mainly viral infections and tumors) [START_REF] Essajee | Poxvirus vaccines for cancer and HIV therapy[END_REF][START_REF] Moroziewicz | Gene therapy with poxvirus vectors[END_REF]. Although intensively studied, much is still to be learned about vaccinia virus, considering the fact that its approximately 200 kbp long genome encodes for more than 200 different proteins, some of which have not been much investigated up to now [START_REF] Condit | In a nutshell: structure and assembly of the vaccinia virion[END_REF][START_REF] Resch | Protein composition of the vaccinia virus mature virion[END_REF].

In 1990, Maa et al [START_REF] Maa | Structural and functional characterization of a cell surface binding protein of vaccinia virus[END_REF] identified a 32-kDa protein of vaccinia, which was shown to be a part of the virus envelope binding machinery to cells. The gene encoding this 32-kDa viral protein was then mapped and sequenced, and was found to encode a 35,426-Da protein with a large N-terminal domain, showing sequence homology to α-carbonic anhydrases (CAs, EC 4.2.1.1), a family of zinc enzymes widespread all over the phylogenetic tree [START_REF] Lai | The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surface[END_REF][START_REF] Rodriguez | Insertional inactivation of the vaccinia virus 32-kilodalton gene is associated with attenuation in mice and reduction of viral gene expression in polarized epithelial cells[END_REF][START_REF] Supuran | Carbonic Anhydrase -Its Inhibitors and Activators[END_REF], and a C-terminal domain with sequence similarity to the attachment glycoprotein VP7 of rotavirus and some other transmembrane proteins [START_REF] Maa | Structural and functional characterization of a cell surface binding protein of vaccinia virus[END_REF]. The 32-kDa protein was basic, with a pI around 8.7, and was probably dimeric due to a disulfide bridge involving the unique cysteine residue at amino acid position 262 [START_REF] Maa | Structural and functional characterization of a cell surface binding protein of vaccinia virus[END_REF]. It was also shown to be a vaccinia virus antigen, with predicted antigenic sites located near amino acids 108-110 (carbonic anhydrase domain) and 298-299 (transmembrane domain). This protein was found to bind to cell surface chondroitin sulphate of cultured cells and participate in efficient virus replication in both cultured cells and in infected animals [START_REF] Maa | Structural and functional characterization of a cell surface binding protein of vaccinia virus[END_REF][START_REF] Lai | The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surface[END_REF][START_REF] Rodriguez | Insertional inactivation of the vaccinia virus 32-kilodalton gene is associated with attenuation in mice and reduction of viral gene expression in polarized epithelial cells[END_REF][START_REF] Hsiao | Vaccinia virus envelope D8L protein binds to cell surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells[END_REF]. As there are no other known viral CAs or CA-like proteins [START_REF] Supuran | Carbonic Anhydrase -Its Inhibitors and Activators[END_REF][START_REF] Pastorekova | Carbonic anhydrases: current state of the art, therapeutic applications and future prospects[END_REF][START_REF] Sly | Human carbonic anhydrases and carbonic anhydrase defficiencies[END_REF][14], we decided to investigate this 32 kDa protein discovered by Maa et al. [START_REF] Maa | Structural and functional characterization of a cell surface binding protein of vaccinia virus[END_REF]. In this paper we report the cloning of vaccinia CA-like proteins (vaccCAs) in which we have mutated two amino acids belonging to the putative enzyme active site in such a way as to bind zinc ions, the metal cofactor essential for Biochemical Journal Immediate Publication. Published on 6 Jul 2007 as manuscript BJ20070816 © 2007 The Authors Journal compilation © 2007 Biochemical Society catalytic activity in all α-CAs [START_REF] Supuran | Carbonic Anhydrase -Its Inhibitors and Activators[END_REF][START_REF] Pastorekova | Carbonic anhydrases: current state of the art, therapeutic applications and future prospects[END_REF][START_REF] Sly | Human carbonic anhydrases and carbonic anhydrase defficiencies[END_REF]. We were able to engineer a variant of vaccCA possessing enzyme activity for the physiologic reaction catalyzed by CAs, i.e., CO 2 hydration to bicarbonate, of the same order of magnitude as exhibited by some human CAs (hCAs). Furthermore, this active vaccCA was also inhibited by sulfonamide CA inhibitors (some of which are clinically used drugs [START_REF] Supuran | Carbonic anhydrase inhibitors[END_REF][START_REF] Scozzafava | Modulation of carbonic anhydrase activity and its applications in therapy[END_REF]) in the low nanomolar range, indicating that the engineered protein is indeed a viral CA.

Experimental methods

Cell culture, viral and bacterial strains

HeLa cells derived from human cervical carcinoma were cultivated in DMEM supplemented with 10% FCS (BioWhittaker, Verviers, Belgium), 100 IU/ml penicillin, 100 µg/ml streptomycin sulfate and 0.25 µg/ml Amphotericin B (BioWhittaker) in a humidified atmosphere with 5% CO 2 at 37 °C.

Vaccinia virus, strain CR19 (wild type), was a kind gift of Dr. Gustav Russ, Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovakia. E. coli strains JM109 and BL21-CodonPlus (DE3)-RIPL (Stratagene, La Jolla, U.S.A.) were used for cloning and protein expression, respectively.

Plasmids, primers and cloning

The gene for vaccinia virus carbonic anhydrase-like protein (vaccinia virus cell surfacebinding protein -the National Center for Biotechnology Information (NCBI) accession number J05190) was amplified from a boiled culture medium containing viral particles by PCR using oligonucleotides VaccCAs and VaccCAa (sequences of all primers used are listed in Table 1; nucleotides introduced for cloning are lowercase; mutations are boldface) using Phusion polymerase (Finnzymes, Espoo, Finland). The PCR product was first Mutants of vaccinia virus CA-like protein designated N92H and Y69H, according to the amino acids mutated, were generated by two-step PCR-directed mutagenesis using the pBKS-vaccCAwt plasmid as a template. One reaction of the first round was performed using sense mutagenic oligonucleotide N92Hs or Y69Hs, respectively, and antisense pBluescript specific external primer R. In the second reaction, antisense mutagenic primers N92Ha or Y69Ha were used with sense pBluescript specific external primer F. For the second round, PCRs were carried out using sense and antisense pBluescript specific external primers for both mutants and the PCR products from the first round were used as templates. Final PCR products of single mutants were gel purified, processed with EcoRI and XhoI and inserted into pGEX-4T-1 vector linearised with EcoRI and XhoI, creating plasmids pGEX-4T-1-N92H and pGEX-4T-1-Y69H. Double mutant N92H/Y69H of vaccinia virus CA-like protein was similarly generated later using plasmid construct pGEX-4T-1-N92H as a template. First PCRs were carried out using sense and antisense Y69H-mutagenic primers and a corresponding pGEX-4T-1 specific external primers pGEXA and pGEXS. Obtained PCR product was inserted into pGEX-4T-1 vector using EcoRI and XhoI yielding a plasmid designated pGEX-4T-1-vaccCAdm.

All PCR reactions were performed using the following amplification program: an initial denaturation at 98 °C for 30 s, then denaturation a t 98 °C for 10 s, annealing at 62-68 °C for 30 s and extension at 72 °C for 20-35 s for a t otal of 30 cycles, and finally 5 min at 72 °C. All pGEX-vaccinia constructs were confirmed by sequencing using ABIPrism BigDye terminator V3.1 sequencing kit for fluorescent detection and ABIPrism 3100 genetic analyser (Applied Biosystems, Foster City, U.S.A.).

For eukaryotic expression, cDNAs of the wild type and the double mutant of vaccinia virus CA-like protein were cloned from the plasmids pGEX-4T-1-vaccCAwt and pGEX-4T-1-vaccCAdm, respectively, into phCMV1 expression vector (Genlantis, San Diego, U.S.A.) using EcoRI and NotI enzymes.

Recombinant protein expression and purification

Verified plasmid constructs pGEX-4T-1-vaccCAwt, -N92H, -Y69H and -vaccCAdm were was induced with 0.4 mM IPTG at 30 °C for 4 hours. Recombinant proteins were purified using Glutathione Sepharose 4B medium and cleaved from their GST-tag overnight at 4 °C with thrombin (Sigma-Aldrich) while still atta ched to Sepharose beads; as reported earlier by us for the purification of several mammalian CA isozymes [START_REF] Pastorekova | Carbonic anhydrase inhibitors: The first selective, membraneimpermeant inhibitors targeting the tumor-associated isozyme IX[END_REF][START_REF] Svastova | Hypoxia activates the capacity of tumor-associated carbonic anhydrase IX to acidify extracellular pH[END_REF][START_REF] Nishimori | Carbonic anhydrase inhibitors. The mitochondrial isozyme VB as a new target for sulfonamide and sulfamate inhibitors[END_REF]. After elution from the beads (which retained the GST part of the fusion protein), the concentration of recombinant proteins was determined by measuring its absorption A 260 as well as by SDS-PAGE followed by Coommassie Brilliant Blue R-250 staining. For the double mutant possessing good CA activity (see later in the text), the obtained bacterial lysate was applied to a prepacked Glutathione Sepharose 4B column (Amersham), the column was extensively washed with ice-cold PBS and then the GST-vaccCAdm fusion protein was eluted with a buffer consisting of 5 mM reduced glutathione in 50 mM Tris-HCl pH 8.0.

Finally the GST part of the fusion protein was cleaved with thrombin and the vaccCAdm was further purified by sulfonamide affinity chromatography [START_REF] Khalifah | Carbon-13 nuclear magnetic resonance probe of active-site ionizations in human carbonic anhydrase B[END_REF], the amount of enzyme being determined by spectrophotometric measurements and its activity by stopped-flow experiments, with CO 2 as substrate [START_REF] Khalifah | The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C[END_REF]. The CO 2 hydrase activity has been measured as described below.

CA activity/inhibition assay

An Applied Photophysics stopped-flow instrument has been used for assaying the CA/vaccCA catalysed CO 2 hydration activity [START_REF] Khalifah | The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C[END_REF]. Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.5) as buffer, 0.1 M Na 2 SO 4 (for maintaining constant the ionic strength), following the CA-catalyzed CO 2 hydration reaction for a period of 10-100 s. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates.

Stock solutions of inhibitor (1 mM) were prepared in distilled-deionized water with 10-20% (v/v) DMSO (which is not inhibitory at these concentrations) and dilutions up to 0.01 nM were done thereafter with distilled-deionized water. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear Biochemical Journal Immediate Publication. Published on 6 Jul 2007 as manuscript BJ20070816 © 2007 The Authors Journal compilation © 2007 Biochemical Society least-squares methods using PRISM 3, from Lineweaver-Burk plots, as reported earlier, and represent the mean from at least three different determinations [START_REF] Khalifah | The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C[END_REF].

Generation of HeLa cells expressing vaccCA proteins

HeLa cells plated in 35 mm Petri dishes were transfected with 2 µg of the phCMV1-vaccCAwt, phCMV1-vaccCAdm plasmids and an empty phCMV1 vector using GenePorter transfection reagent (Genlantis, San Diego, U.S.A.). Two days after transfection, the cells were split (onto seven 60 mm Petri dishes) and subjected to a two-week selection in a medium containing Geneticin G418 (Invitrogen, Carlsbad, U.S.A.) in the concentration of 600 µg/ml. The resistant colonies were pooled and tested for the expression of ectopic genes by RT-PCR using vaccCAs and Y69Ha primers and primers for β-actin that served as internal standard (see the list in Table 1).

Plaque assay

The effect of the wild-type and the double mutant of vaccinia CA-like protein on vaccinia virus replication was compared after the infection of stably transfected HeLa cells where ectopically expressed vaccinia CA-like protein variants could be packed into virus particles. Confluent HeLa cell monolayers grown in 96-well tissue culture dishes were infected with 5-fold serial dilutions of vaccinia virus starting at a multiplicity of infection (MOI) of 1 PFU per cell. After 1 h of adsorption, the monolayers were washed and covered with DMEM containing 0.5 % methylcellulose. After two days, the cells were stained with crystal violet and PFU/ml for the vaccinia virus grown in different tranfectants was determined.

Computer analysis of CA sequences

Amino acid sequence alignments were carried out using the program CLUSTAL W, version 1.83 [START_REF] Higgins | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice[END_REF]. Protein phylogeny tree reconstructions were performed using the MEGA3.1 software [START_REF] Kumar | MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment[END_REF]. Trees were obtained using the distance/neighbor joining method and bootstrap statistical analyses were performed with 1000 replicates.

Results and discussion

By catalyzing the interconversion between carbon dioxide and bicarbonate, with generation of a proton, the CAs operate on three very simple molecules/ions involved in a variety of critical life processes [START_REF] Supuran | Carbonic Anhydrase -Its Inhibitors and Activators[END_REF][START_REF] Pastorekova | Carbonic anhydrases: current state of the art, therapeutic applications and future prospects[END_REF][START_REF] Sly | Human carbonic anhydrases and carbonic anhydrase defficiencies[END_REF][14][START_REF] Supuran | Carbonic anhydrase inhibitors[END_REF]. Among them, the most important ones are pH regulation, respiration, secretion of electrolytes, biosynthesis of some important biomolecules such as urea, glucose, lipids and pyrimidines, excretion of acid and salts, carcinogenesis, signalling, etc. [START_REF] Supuran | Carbonic Anhydrase -Its Inhibitors and Activators[END_REF][START_REF] Pastorekova | Carbonic anhydrases: current state of the art, therapeutic applications and future prospects[END_REF][START_REF] Sly | Human carbonic anhydrases and carbonic anhydrase defficiencies[END_REF][14][START_REF] Supuran | Carbonic anhydrase inhibitors[END_REF]. Different isozymes among the 16 presently known isoforms in mammals participate to such processes. These isoforms display very diverse cellular localization, catalytic activity and susceptibility for inhibition/activation by various endogenous or exogenous regulators of activity. In humans only 15 of the 16 mammalian isoforms are present, due to the fact that CA XV is encoded by a pseudogene in all primates investigated so far [START_REF] Hilvo | Characterization of CA XV, a new GPI-anchored form of carbonic anhydrase[END_REF]. Among these remaining 15 isoforms, 12 possess catalytic activity for the CO 2 hydration reaction, whereas CA VIII, X and XI (also denominated CARPs, for CA-related proteins) are devoid of this activity due to a lack of one or more histidine residues coordinating the catalytically critical Zn(II) ion within the Whereas the physiological function of most catalytically active CAs is rather well understood, the three CARPs (i.e., isozymes CA VIII, X and XI) still represent a biochemical and physiological enigma [26]. The CARPs lack one or more histidine residues required to bind the zinc ion, which is essential for the CO 2 hydration activity of all CAs. The Zn(II) ion within the enzyme active site is tetrahedrically coordinated to the imidazole moieties of three His residues (His94, 96 and 119, CA II numbering [START_REF] Supuran | Carbonic Anhydrase -Its Inhibitors and Activators[END_REF]), whereas the fourth ligand is a water molecule/hydroxide ion which acts as a strong nucleophile on the substrate (CO 2 ) bound within a hydrophobic pocket in its neighborhood [START_REF] Supuran | Carbonic Anhydrase -Its Inhibitors and Activators[END_REF][START_REF] Pastorekova | Carbonic anhydrases: current state of the art, therapeutic applications and future prospects[END_REF][START_REF] Sly | Human carbonic anhydrases and carbonic anhydrase defficiencies[END_REF][14][START_REF] Supuran | Carbonic anhydrase inhibitors[END_REF][START_REF] Scozzafava | Modulation of carbonic anhydrase activity and its applications in therapy[END_REF][START_REF] Pastorekova | Carbonic anhydrase inhibitors: The first selective, membraneimpermeant inhibitors targeting the tumor-associated isozyme IX[END_REF][START_REF] Svastova | Hypoxia activates the capacity of tumor-associated carbonic anhydrase IX to acidify extracellular pH[END_REF]. The CARPs were shown to be consistently expressed in the brain, and CARP VIII was also found to be expressed in some peripheral tissues An inactive CA-like domain has also been identified in the N-terminal regions of two receptor protein-tyrosine phosphatases, RPTPβ and RPTPγ [START_REF] Peles | Multi-ligand interactions with receptor-like protein tyrosine phosphatase β: implications for intercellular signalling[END_REF][START_REF] Barnea | Identification of a carbonic anhydrase-like domain in the extracellular region of RPTP gamma defines a new subfamily of receptor tyrosine phosphatases[END_REF]. RPTPβ is expressed on the surface of glial cells and utilizes the CA domain to bind the cell recognition molecule contactin on neuronal cells, thus supporting its role in neuronal adhesion and neurite outgrowth [START_REF] Peles | The carbonic anhydrase domain of receptor tyrosine phosphatase β is a functional ligand for the axonal cell recognition molecule contactin[END_REF]. RPTPγ is expressed in a variety of tissues. It appears developmentally regulated and is implicated in hematopoetic differentiation [START_REF] Sorio | Receptor protein tyrosine phosphatase gamma, PTPγ, regulates hematopoietic differentiation[END_REF]. Both these phosphatases contain only one of the three His residues that bind zinc and are crucial for CA activity. On this basis it has been proposed that their CA domains function rather as a protein-binding pocket.

Similarly to CARPs and RPTPβ/γ, the N-terminal CA-like domain of the cell surface binding protein of vaccinia virus identified by Maa et al [START_REF] Maa | Structural and functional characterization of a cell surface binding protein of vaccinia virus[END_REF] shows a high sequence homology with mammalian CAs, but lacks two of the zinc-coordinating histidines present in the active site of these enzymes. More precisely, a Tyr is present at position 69, and an Asn is found at position 92, instead of His at the corresponding positions 96 and 119 in CA II. Figure 1 shows the sequence alignment of the CA domains of all human isozymes (including CARPs), RPTPβ/γ and vaccCA. The sequence homology between the CA domains of these proteins ranges from 22% to 67%. A phylogenetic analysis of these proteins (Figure 2) revealed that vaccCA is evolutionarily similar to CARPs XI and X, whereas it is more distantly related to human CARP VIII, CA XIII, CA VII, CA VA and CA VB. On the other hand, all other CAs are clustered on different branches of the phylogenetic tree, suggesting that they are less evolutionarily related to vaccCA.

In order to investigate the possibility of engineering a CA-like active site in this viral protein, we decided to mutate the vaccCA amino acids Tyr69 and Asn92 -corresponding to positions 96 and 119 in CA I and II -to histidines. Thus, we have generated three mutant vaccCAs, i.e., N92H vaccCA, Y69H vaccCA, and the double mutant, N92H/Y69H vaccCA (see Experimental section for details). GST-fusion proteins were obtained for the wild type and the three mutant vaccCAs that were purified by a procedure previously optimized for the production of high amounts of various CA isozymes for medicinal chemistry purposes. Two-column chromatographic passages were utilized, the first After thrombin cleavage of the chimeric protein, a second column based on aromatic sulfonamide Sepharose allowed the purification of the proteins with CA-like activity (this second column could not be used for the wild type vaccCA which was not retained on sulfonamide affinity columns). The average yield after two purification procedures ranged between 1-3 mg per liter culture. The CO 2 hydrase activity of the obtained proteins has been measured by a stopped flow assay. Their inhibition by the prototypic sulfonamide CA inhibitor acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide, AAZ) was also assessed [START_REF] Supuran | Carbonic Anhydrase -Its Inhibitors and Activators[END_REF].

Data included in Table 2 show that the wild type vaccCA protein is devoid of CO 2 hydrase activity, as expected for a protein unable to bind zinc, being thus similar to CARP VIII; X and XI [26]. On the other hand, the two single mutants N92H and Y69H, and much more the double mutant, vaccCA N92H/Y69H show enzymatic activity typical of a CA-like protein. Nevertheless, the single mutants showed a low activity, with k cat and k cat /K M values similar to those of CA III (the least effective catalyst among the mammalian CAs for the physiologic reaction [START_REF] Supuran | Carbonic Anhydrase -Its Inhibitors and Activators[END_REF]). This may be explained by the presence of only two His residues within the active site leading to a rather low affinity for Zn(II), and as a consequence, probably the active site of these enzymes is only partially occupied by zinc. This hypothesis is also confirmed by the finding that the sulfonamide CA inhibitor acetazolamide binds to these two proteins with a rather low affinity (inhibition constants in the range of 76-89 nM). However, the fact that the sulfonamide does bind represents a clear sign that the active site of the mutated viral proteins can be also formed when only two His residues are present, but the catalytic activity is low. In the case of the double mutant, vaccCA N92H/Y69H, the CO 2 hydrase activity is much higher as compared to the single mutants, with k cat and k cat /K M values similar to those of some catalytically efficient human isoforms, such as CA VA and CA XII. The other enzymes considered in Table 2 are much better catalysts for this reaction, especially CA II, which is in fact one of the best catalysts known in nature (Table 2) [START_REF] Supuran | Carbonic Anhydrase -Its Inhibitors and Activators[END_REF]. Furthermore, acetazolamide binds with very high affinity to the double mutant, showing an inhibition constant of the same order of magnitude as that against CA II. This is clear proof that we were able to engineer the active site of a viral CA isozyme, which similarly to other α-CAs shows activity for the CO 2 hydration reaction and binds the typical CA inhibitors, sulfonamides such as acetazolamide, with nanomolar affinity. How can we explain these findings? Due to the high sequence homology between vaccCA and other α-CAs, most of the amino acid residues needed for the construction of the active site architecture useful for CO 2 hydration are already present in the wild type vaccinia protein. By inserting the amino acid residues (two histidines) in positions equivalent to His96 and His119 in the CA II sequence, these features are further accentuated, leading to a viral CA with a moderate CA activity for the physiologic reaction (in the same range as the activity of the human isozymes CA VA and CA XII), which also binds the sulfonamide CA inhibitors efficiently. In fact, other residues in the neighborhood of the Zn(II) ion, more precisely Thr198 (CA II numbering), which forms a hydrogen bond to the water coordinated to Zn(II) as the fourth ligand in all other α-CAs [START_REF] Supuran | Carbonic Anhydrase -Its Inhibitors and Activators[END_REF][START_REF] Pastorekova | Carbonic anhydrases: current state of the art, therapeutic applications and future prospects[END_REF][START_REF] Sly | Human carbonic anhydrases and carbonic anhydrase defficiencies[END_REF][14][START_REF] Supuran | Carbonic anhydrase inhibitors[END_REF][START_REF] Scozzafava | Modulation of carbonic anhydrase activity and its applications in therapy[END_REF][START_REF] Pastorekova | Carbonic anhydrase inhibitors: The first selective, membraneimpermeant inhibitors targeting the tumor-associated isozyme IX[END_REF] and Thr199, which is involved in the binding of inhibitors, together with Thr199 [START_REF] Supuran | Carbonic Anhydrase -Its Inhibitors and Activators[END_REF][START_REF] Pastorekova | Carbonic anhydrases: current state of the art, therapeutic applications and future prospects[END_REF][START_REF] Sly | Human carbonic anhydrases and carbonic anhydrase defficiencies[END_REF][14][START_REF] Supuran | Carbonic anhydrase inhibitors[END_REF][START_REF] Scozzafava | Modulation of carbonic anhydrase activity and its applications in therapy[END_REF][START_REF] Pastorekova | Carbonic anhydrase inhibitors: The first selective, membraneimpermeant inhibitors targeting the tumor-associated isozyme IX[END_REF], are also present in vaccCA, and this probably helps in stabilizing the tetrahedral geometry of Zn(II) during the catalytic cycle. In the close proximity of another important residue for the catalytic cycle, His64 (in CA II), vaccCA (and CA III) contains Lys (see Figure 1). This residue is involved in the proton shuttling between the active site and the environment, with generation of the zinc hydroxide, catalytically effective species of the enzyme [START_REF] Supuran | Carbonic Anhydrase -Its Inhibitors and Activators[END_REF][START_REF] Pastorekova | Carbonic anhydrases: current state of the art, therapeutic applications and future prospects[END_REF][START_REF] Sly | Human carbonic anhydrases and carbonic anhydrase defficiencies[END_REF][14][START_REF] Supuran | Carbonic anhydrase inhibitors[END_REF][START_REF] Scozzafava | Modulation of carbonic anhydrase activity and its applications in therapy[END_REF][START_REF] Pastorekova | Carbonic anhydrase inhibitors: The first selective, membraneimpermeant inhibitors targeting the tumor-associated isozyme IX[END_REF]. Obviously, the εamino group present in Lys is a less efficient proton acceptor as compared to the imidazole moiety of a His residue, and this may explain the lower catalytic activity of vaccCA (double mutant), which is of the same order of magnitude as that of CA III. However, our double mutant is in fact approximately 83 times more efficient as a catalyst for CO 2 hydration as compared to CA III (Table 2). This is probably due to the fact that CA III has Phe197, a very bulky amino acid which highly restricts its active site, whereas the equivalent amino acid residues in vaccCA all possess smaller side chains, and thus the steric impairment is not present in its active site, allowing for a better catalytic efficiency.

To learn whether introduction of the CA catalytic activity into vaccinia virus cell surface binding protein could have any biological effect on infectious properties of the virus, we attempted to produce virions containing ectopically expressed vaccCA protein variants. Three stable tranfectants of HeLa cells had been created that expressed either full length vaccinia virus CA-like protein or its catalytically active counterpart designated here as vaccCAdm. As a control, HeLa cells had been transfected with an empty vector carrying a assay, the titer of recombinant vaccinia virus containing vaccCAdm in its envelope was determined as 4.3 x 10 7 PFU/ml, whereas the titer of the virus grown on HeLa cells expressing the wild type vaccCA was close to the titer of the control (2.4 x 10 7 PFU/ml comparing to 2.7 x 10 7 PFU/ml). The size of plaques was similar in all samples (see Figure 3).

Based on the recent knowledge, it is difficult to anticipate a possible function of the CA domain of vaccCA. This viral cell surface binding protein has been shown to contribute to virus entry, but the CA domain was considered dispensable for its binding capacity [START_REF] Maa | Structural and functional characterization of a cell surface binding protein of vaccinia virus[END_REF]. On the other hand, others have demonstrated that the N-terminal region (amino acids 1-264) of 32-kDa vaccinia virus CA-like protein is important in vaccinia virus adsorption to cells [START_REF] Hsiao | Vaccinia virus envelope D8L protein binds to cell surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells[END_REF]. As a binding partner, a cell surface glycosaminoglycan chondroitin sulphate was presented [START_REF] Hsiao | Vaccinia virus envelope D8L protein binds to cell surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells[END_REF], but the precise region of interaction between the two molecules has not been mapped so far.

To ascertain how the gained CO 2 hydrase function affected overall structure of the protein which may prove important for adsorption properties of the virus, we also derived 3-D models of the CA domain of vaccCA proteins in comparison with that of RPTPβ and CA II, which is well characterized from the structural point of view. In a set of supplementary figures A-E we present the X-ray crystal structure of CA II and the corresponding 3-D model, which was obtained with the program PHYRE. This model well matches the crystal structure (compare Figures A andB). The model of RPTPβ CA domain was also included for comparison (Figure C), since the pocket structure of the catalytically inactive CA domain of this protein was shown to serve as a receptor site that binds contactin and modulates adhesion and differentiation of neurons [START_REF] Peles | Multi-ligand interactions with receptor-like protein tyrosine phosphatase β: implications for intercellular signalling[END_REF]. The vaccCA 3-D models were obtained from the amino acid sequences of the CA domains of the wild type protein (Figure D) and the catalytically active double mutant (Figure E). It may be observed that the globular shapes of CA II, RPTPβ and vaccCA are quite similar. The active site of CA II forms a wide and deep pocket, but also the acatalytic RPTPβ and vaccCA display a pocket-like structure. Interestingly, introduction of two mutations leading to the acquisition of zinc ion-binding capacity did not show any remarkable effect on the tertiary structure of the double mutant, suggesting that the formation of the deep pocket-like structure does not depend on the binding of the zinc ion, but seems to be affected by the amino acid composition of the entire CA domain. This, however, supports our experimental results Genome analyses of poxviruses revealed the existence of numerous genes homologous to eukaryotic genes present in their host organisms. Therefore, it has been suggested that the evolution of poxviruses involved multiple events of gene acquisition [START_REF] Hughes | Poxvirus genome evolution by gene gain and loss[END_REF]. Due to this horizontal gene transfer poxviruses express many proteins that are functionally and structurally similar to host proteins, such as DNA polymerase, thymidine kinase, ribonucleotide reductase, DNA toposiomerase, etc. It is well conceivable that vaccCA has also been acquired by means of a horizontal gene transfer. Its close similarity to CARP X and XI preferably points to an ancestral gene that led to CARPs (in mammals) and in parallel to vaccCA (Figure 2). However, taking into account the results of our mutagenesis study, it cannot also be excluded that the virus originally acquired an evolutionary even older gene, i.e., a common ancestor of the catalytic and acatalytic CA isoforms, which lost its catalytic activity over evolution in the virus, since it was not critical for the vaccinia virus life cycle.

Biochemical

In conclusion, we engineered the active viral CA active site by mutating two amino acid residues in the vaccCA protein. The double mutant vaccCA N92H/Y69H showed a catalytic activity of the same order of magnitude as that of some physiologically relevant human CA isoforms. The double mutant (and to a lesser degree also the two single mutants reported here) also bound sulfonamide CA inhibitors, such as acetazolamide, with a good efficacy. Moreover, when expressed in HeLa cells, the double mutant was able to slightly increase the titer of the virus. However, the biological significance of this finding cannot be judged merely on in vitro data from cell culture, as the carbonic anhydrase activity is particularly relevant at the physiological level in the living organisms.

Acknowledgement

  subcloned into the EcoRV site of pBluescript KS-(Stratagene), creating the plasmid pBKS-vaccCAwt. The target fragment was retrieved by digestion with restriction enzymes EcoRI and XhoI and cloned into pGEX-4T-1 (Amersham Biosciences, Chalfont St Giles, United Kingdom), creating a plasmid designated pGEX-4T-1-vaccCAwt. Biochemical Journal Immediate Publication. Published on 6 Jul 2007 as manuscript BJ20070816 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  transformed into BL21-CodonPlus (DE3)-RIPL cells. An expression of GST-fusion proteins Biochemical Journal Immediate Publication. Published on 6 Jul 2007 as manuscript BJ20070816 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  3-D protein models were obtained with the program PHYRE, a successor of the program 3D-PSSM [24]. Biochemical Journal Immediate Publication. Published on 6 Jul 2007 as manuscript BJ20070816 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  enzyme active site [26]. Indeed, CA I-III, VII and XIII are soluble cytosolic isozymes, CA IV and XV are extracellular membrane-anchored enzymes by means of glycosylphosphatidylinositol (GPI) tails, CA VA and VB are mitochondrial enzymes, CA VI is the only secreted CA isoform, initially identified in sheep saliva and parotid glands, whereas CA IX, XII and XIV are transmembrane proteins with the active site situated outside the cell [10, 15, 25, 26].

  [26]. Similarly to the Biochemical Journal Immediate Publication. Published on 6 Jul 2007 as manuscript BJ20070816 © 2007 The Authors Journal compilation © 2007 Biochemical Society catalytically active transmembrane isoforms CAs IX and XII, CARP VIII has also been reported to be overexpressed in certain types of carcinoma [26].

Biochemical

  Journal Immediate Publication. Published on 6 Jul 2007 as manuscript BJ20070816 © 2007 The Authors Journal compilation © 2007 Biochemical Society involving a Glutathione Sepharose column which retained the GST-vaccCA fusion protein.

Biochemical

  Journal Immediate Publication. Published on 6 Jul 2007 as manuscript BJ20070816 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  neomycin resistance gene. Prior to infection with CR19 strain of vaccinia virus, expression of vaccCAwt and vaccCAdm was confirmed by RT-PCR (data not shown). By plaque Biochemical Journal Immediate Publication. Published on 6 Jul 2007 as manuscript BJ20070816 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  Journal Immediate Publication. Published on 6 Jul 2007 as manuscript BJ20070816 © 2007 The Authors Journal compilation © 2007 Biochemical Society where no dramatic changes in properties of vaccCAwt and vaccCAdm virions were observed.

  This work was supported by the grants from Slovak Government (BITCET SP 337/2003) and from EU (EUROXY project LSHC-CT-2003-502932). Biochemical Journal Immediate Publication. Published on 6 Jul 2007 as manuscript BJ20070816 © 2007 The Authors Journal compilation © 2007 Biochemical SocietyTable.1 List of primers used for cloning and RT-PCR analysis of vaccinia virus carbonic anhydrase-like protein and its mutant variants

Figure 1

 1 Figure 1

Figure 2

 2 Figure 2Unrooted phylogenetic tree of human CA isozymes I-XIV, human RPTP β/γ and vaccinia CA-like protein based on the alignment of carbonic anhydrase domains corresponding to amino acid residues 1-260 of hCA II. The tree was constructed using the distance (neighbor joining) method using MEGA3.1 software. A bootstrap of 1000 replicates was performed and the number on each node indicates the percentage with which each branch topology was supported. The bar shown represents the genetic distance.

Figure 3 A

 3 Figure 3A plaque assay of vaccinia virus, strain CR19, on HeLa cells expressing vaccCA protein variants. Representative wells of HeLa cells stably tranfected with cDNA of vaccCAwt and vaccCAdm and infected with 5-fold serial dilutions vaccinia virus, strain CR19. After 1 h of adsorption, the monolayers were washed and covered with DMEM containing 0.5 %

Figure 2 Biochemical

 2 Figure 2

Table 2

 2 Kinetic parameters for the CO 2 hydration reaction catalysed by the cytosolic α-hCA

	isozymes I-III, the mitochondrial isozymes VA and VB, the secreted isoform hCA VI (full
	length) and the transmembrane isozymes hCA IX (catalytic domain), and hCA XII (catalytic
	domain), at 20 °C and pH 7.5, vaccCA (wt) and the t hree mutants prepared during this
	study, and their inhibition data with acetazolamide AAZ (5-acetamido-1,3,4-thiadiazole-2-
	sulfonamide), a clinically used drug.
	Isozyme	Activity level	k cat *		k cat /K m *	K I (AAZ)*
			( s	-1 )		(M -1 .s	-1 )	(nM)
	hCA I	moderate	2.0x10	5	5.0x10	7	250 ± 13
	hCA II	very high	1.4x10	6	1.5x10	8	12 ± 0.6
	hCA III	very low	1.0x10 4	3.0x10	5	300,000 ± 5000
	hCA VA	low	2.9x10	5	2.9x10	7	63 ± 4
	hCA VB	high	9.5x10	5	9.8x10	7	54 ± 5
	hCA VI	moderate	3.4x10 5	4.9x10 7	11 ± 1
	hCA IX	high	3.8x10 5	5.5x10	7	25 ± 3
	hCA XII	moderate	4.2x10 5	3.5x10	7	5.7 ± 0.4
	vaccCAwt	devoid	-			-		-
	vaccCA N92H	low	7.8x10 4	(3.7±0.2)x10	6	76 ± 6
	vaccCA Y69H	low	8.3x10 4	(2.3±0.1)x10	6	89 ± 7
	vaccCA N92H/Y69H moderate	1.2x10 5	(2.5±0.2)x10	7	12 ± 1
	* Mean ± standard error (from three different determinations). Kinetic data for hCA I -hCA XII are
	from the literature [10,12-17], whereas vaccCA data are reported here for the first time

Biochemical Journal Immediate Publication. Published on 6 Jul 2007 as manuscript BJ20070816 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 6 Jul 2007 as manuscript BJ20070816 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society