Sara López-Gomollón

Emma Sevilla

M Teresa Bes

M Luisa Peleato

María F Fillat
email: fillat@unizar.es

New insights into the role of Fur proteins: FurB (All2473) from Anabaena protects DNA and increases cell survival under oxidative stress

Keywords: ferric uptake regulator, heme, cyanobacteria, DNA protection, oxidative stress Fur, ferric uptake regulator, Dps, DNA protection during starvation, PSI, photosystem I, PerR, peroxide-sensing repressor, bp, base pair(s), DTT, 1,4-dithiothreitol, LB, Luria-Bertani, ORF, open reading frame, ROS, reactive oxygen species, IPTG, isopropyl-β-Dthiogalactopyranoside, EMSA, electrophoretic mobility shift assays, MV, methyl viologen (paraquat)

The ferric uptake regulator (Fur) is a prokaryotic transcriptional regulator that controls a large number of genes mainly related to iron metabolism. Frequently, several Fur homologues with different physiological roles are found in the same organism. The genome of the filamentous cyanobacterium Anabaena (Nostoc) sp. PCC 7120 codes for three different fur genes. FurA is an essential protein involved in iron homeostasis that also modulates dinitrogen fixation. FurA interacts with heme, impairing its DNA-binding ability. To explore functional differences between Fur homologues in Anabaena, factors affecting their regulation, as well as some biochemical characteristics have been investigated. While incubation of FurB with heme severely hinders it ability to interact with DNA, binding of heme to FurC could not be detected. Oxidative stress enhances the transcription of the three fur genes, especially of furB and furC. In addition, overexpression of FurA and FurB in E. coli increases survival when the cells are challenged with hydrogen peroxide or methyl viologen, a superoxide anion-generating reagent. When present in saturating concentrations, FurB exhibits unspecific DNA-binding activity and protects DNA from cleavage produced by hydroxyl radicals or DNaseI. Based on these results, it can be suggested that whereas at low concentration FurB would act as a member of the Fur family, at saturating concentrations FurB protects DNA, showing a Dps (DNA protection during starvation)-like behaviour.

INTRODUCTION

Iron participates in numerous metabolic processes and is essential for most living organisms [1]. However, the low solubility of iron in oxygenated waters limits its bioavailability in aquatic environments. This fact, besides the close relationship between iron homeostasis and oxidative stress, make necessary a tight regulation of iron uptake and storage. The ferric uptake regulator (Fur) protein is one of the most important switches for the regulation of iron metabolism in prokaryotes. Under iron-rich conditions, a dimer of Fur binds to specific DNA sequences ("iron-boxes") located in the promoters of iron responsive genes using Fe (II) as co-repressor and avoiding their transcription [2]. Apart from the main group of iron-dependent Fur proteins, the ability of some members of this family to sense other metal ions permits differentiate several subclasses, namely sensors of zinc (Zur) [3], manganese (Mur) [4] or nickel (Nur) [5]. Moreover, some of those Fur-like proteins respond to signals other than metal ions. In fact, using metal-catalyzed protein oxidation some of them regulate DNA-binding activity and thereby respond to the presence of peroxide (PerR) [6] or heme and oxygen (Irr) [7]. The crystal structures of Fur from P. aeruginosa, PerR from B. subtilis and Zur from M. tuberculosis unravel that though each Fur subclass responds to different signals, the overall fold of the N-terminal DNA binding domain and the C-terminal dimerization domain is similar [8][9][10]. Linkage of Fur to the oxidative stress response has been demonstrated in several microorganisms, like Staphylococcus aureus [11] or Mycobacterium tuberculosis [12]. In Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803, many iron inducible genes are also influenced by short-term peroxide or methyl viologen stresses [13,14]. Reactive oxygen species (ROS) are generated during aerobic growth, with the potential to damage almost all cellular components. The formation of these dangerous species is closely related to iron metabolism, since an excess of iron raises the production of ROS through the Fenton reaction [15]. Furthermore, in cyanobacteria iron starvation has shown to be a cause of oxidative stress [16]. Thus, it is not surprising that the cellular responses developed by the organisms to cope with iron starvation and oxidative damage frequently display common elements.

In the filamentous nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 (also known as Nostoc sp.) three different Fur proteins have been identified: FurA (all1691), FurB (all2473) and FurC (alr0957). In vitro assays suggest that these proteins might be cross-regulated [17]. FurA, as well as FurB, bind to the promoter sequences of the three fur genes, whereas FurC has been proposed to modulate the DNA-binding activity of FurA and FurB. FurA is a constitutive and relatively abundant protein that seems to be essential in Anabaena sp. [START_REF] Hernández | Interaction of FurA from Anabaena sp. PCC 7120 with DNA: A reducing environment and the presence of Mn2+ are positive effectors in the binding to isiB and furA promoters[END_REF]. Northern blot analysis shows that under standard growth conditions, mRNAs of furB and furC are less abundant than furA transcripts (our unpublished results). Regulation of FurA takes place at both transcriptional and post-translational levels. Besides a moderate autoregulation, common among fur genes, transcription of Anabaena furA is modulated by the nitrogen regulator NtcA [START_REF] López-Gomollón | Expression of furA is modulated by NtcA and strongly enhanced in heterocysts of Anabaena sp[END_REF] and likely by FurB and FurC [17]. At the post-transcriptional level, an antisense mRNA interferes with the furA transcript [20]. Moreover, post-translational regulation of Fur operated by heme has been reported, since heme binds tightly to FurA impairing its DNA-binding activity [START_REF] Hernández | Heme binds to and inhibits the DNA-binding activity of the global regulator FurA from Anabaena sp. PCC 7120[END_REF]22]. Even though gene regulation is a complex biological process that can happen at several levels, one of the most effective ways for a cell to cope with a change in its environment consists of altering its gene expression. In this work, we show that the expression of the three fur paralogues is strongly affected by oxidative stress. We have used an in vivo approach to demonstrate that, in E. coli, overexpression of FurA and FurB from an inducible promoter prior to oxidative challenge increases cell survival. Moreover, in a purified in vitro system, at saturating concentrations FurB binds to DNA in a sequence-independent fashion and protects DNA from cleavage produced by hydroxyl radicals or DNaseI. Those results unravel a new function for a Fur protein and suggest that in Anabaena, FurA and FurB connect iron regulation to oxidative stress defence.

EXPERIMENTAL PROCEDURES

Strains and culture conditions. The present study was carried out with the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 grown in BG-11 medium at 30ºC [23]. RNAs used in RT-PCR assays were obtained from cultures grown until early-log phase (OD 600 = 0.4) and then challenged with 250 μM H 2 O 2 or 1 mM methyl viologen for 0.5, 1, 2 and 4 hours. All the cultures were bubbled vigorously with filtered air.

Overexpression and purification of FurA, FurB and FurC. Chromosomal DNA from Anabaena sp. was isolated as described previously [24] and used as template for amplification of the furA (all1691), furB (all2473) and furC (alr0957) genes. The PCR products were sequenced and cloned between NcoI and HindIII sites of pET 28a (+). These constructs were used to transform E. coli BL21(DE3) Gold cells to overexpress each Fur protein [17,[START_REF] Bes | Cloning, overexpression and interaction of recombinant Fur from the cyanobacterium Anabaena PCC 7119 with isiB and its own promoter[END_REF]. Recombinant FurA was purified by chromatography through a heparin-sepharose 6 Fast-Flow column (Amersham-Pharmacia) [START_REF] Hernández | Biochemical analysis of the recombinant Fur (ferric uptake regulator) protein from Anabaena PCC 7119: factors affecting its oligomerization state[END_REF]. Recombinant FurB was purified using a heparin-sepharose 6 Fast Flow column (Amersham-Pharmacia) followed by MonoS FPLC column (Pharmacia). Recombinant FurC was purified by heparin-sepharose 6 Fast Flow chromatography followed by MonoQ FPLC column (Pharmacia) as described before [17]. Fur proteins were quantified using the bicinchoninic acid method (BCA™ Protein Assay Reagent Kit, Pierce). FurA was also quantified spectrophotometrically A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. using an extinction coefficient of 13760 M -1 •cm -1 at 276 nm [START_REF] Hernández | Biochemical analysis of the recombinant Fur (ferric uptake regulator) protein from Anabaena PCC 7119: factors affecting its oligomerization state[END_REF]. Quantification was confirmed by densitometric measures in gel using FurA as standard. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS/PAGE) was performed as described by Laemmli [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] using 17% (w/v) polyacrylamide [30:0.8 (w/w) acrylamide/bisacrylamide] gels.

Heme-staining assay.

According to [START_REF] Granath | Identification of hemeproteins in the hemolymph of Biomphalaria glabrata (Gastropoda) separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis[END_REF], a heme-staining procedure was applied to Fur proteins incubated with heme and separated by SDS/PAGE. Heme solution was prepared dissolving heme (hemin, SIGMA) in 0.1 N NaOH. The mixture was diluted in 1 mM sodium phosphate (pH 7.4) to approximately 9.6 M. Aliquots of 5 μM FurA, FurB and FurC were incubated with 1 M heme for 5 minutes. Since Anabaena FurA binds heme [START_REF] Hernández | Heme binds to and inhibits the DNA-binding activity of the global regulator FurA from Anabaena sp. PCC 7120[END_REF], this reaction was used as positive control of the assay. Lysozyme was used as negative control. Samples were analyzed by SDS/PAGE on a 17% (w/v) polyacrilamide gel. The procedure involves staining for heme, distaining and restaining with Coomassie Brilliant Blue for proteins.

Electrophoretic mobility shift assays (EMSA).

DNA fragments of 389-pb containing the upstream sequence of furA were obtained by PCR and further purified using the GFX DNA purification kit (Amersham Biosciences) as described previously [START_REF] López-Gomollón | Expression of furA is modulated by NtcA and strongly enhanced in heterocysts of Anabaena sp[END_REF]. Electrophoretic mobility shift assays were performed as described [START_REF] Bes | Cloning, overexpression and interaction of recombinant Fur from the cyanobacterium Anabaena PCC 7119 with isiB and its own promoter[END_REF] in a final volume of 20 μl using a core binding buffer which contained 10 mM BisTris-HCl (pH 7.5), 40 mM KCl, 0.1 mg/ml BSA, 1 mM DTT, 5% (v/v) glycerol and 100 μM MnCl 2 . Samples were supplemented with heme at different concentrations. Complexes were separated using 4.8% polyacrilamide gels. Heme solution was prepared by dissolving heme in a (1:9 v/v) 0.1 N NaOH/ethanol solution and filtered through a 0.2 μm filter, as described [START_REF] Hernández | Heme binds to and inhibits the DNA-binding activity of the global regulator FurA from Anabaena sp. PCC 7120[END_REF]. Binding assays were carried out in the presence of a 228-bp control DNA fragment from the human apoE gene to demonstrate the specificity of the DNA-binding activity of FurB. Results were processed with a Gel Doc 2000 Image Analyser from Bio-Rad.

RT-PCR analysis. Anabaena RNA was prepared as described [29], using 40 ml of cells containing 5 μg of chlorophyll a•ml -1 . Anabaena fur genes were reverse transcribed using the Superscript II Reverse Transcriptase (Invitrogen) and the specific reverse oligonucleotides shown in table 1. The RT-PCR mixture contained 1 μg of RNA and 20 pmol of each primer in the annealing buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA and 150 mM KCl). The total reaction volume was 10 μl. Samples were incubated 10 min at 85ºC to denature RNA followed by 60 min at 65ºC to allow annealing of the primer to RNA. After that, 5 pmol of each deoxynucleoside triphosphate (dNTP), 0.2 μmol DTT and 200 U Superscript II Reverse Transcriptase (Invitrogen) were added into the RT-PCR buffer (50mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2 , 20 mM DTT) to a final volume of 20 μl. Samples were reverse transcribed at 47°C for 60 min followed by 15 min incubation at 75°C to inactivate the reverse transcriptase. The products were amplified by PCR using 3 μl of each cDNA as template. The cycling conditions for the reactions were 3 min at 95ºC, followed by 25 cycles of 93°C for 30 s, 50°C for 1 min and 72°C for 1 min, followed by extension at 72°C for 10 min. Positive control reactions using chromosomic Anabaena sp. DNA as template were run in parallel. PCR products were analyzed in 1% agarose gel.

In vitro DNA protection assay. Protection of DNA from damage was performed using pUC19 plasmid DNA (100 ng) in 60 μl final volume of 10 mM BisTris-HCl (pH 7.5), 40 mM KCl, 0.1 mM MgCl 2 and 5% glycerol (v/v) incubated for 10 minutes with FurB (40 pmol or 80 pmol) or BSA (40 pmol or 80 pmol) as negative control. When indicated, 100 μM FeCl 3 , 400 μM EDTA, 150 μM Fe(NH 4 ) 2 (SO 4 ) 2 or 100 mM H 2 O 2 were added to the reaction mixtures and the reaction allowed to evolve for 20 minutes at room temperature. For protection against DNaseI-mediated cleavage, reactions were assembled as above and 10 -5 units DNaseI-FPLC pure (Amersham Biosciences) added in a buffer containing 0.25 mM CaCl 2 , 0.25 mM MgCl 2 and 12.5% glycerol. Samples were incubated for 20 minutes at room temperature. A c c e p t e d M a n u s c r i p t Each reaction mixture was divided in two 30 μl-aliquots, one of them was used as control, while FurB and BSA were removed from the other fraction using a GFX DNA purification kit (Amersham Biosciences). Samples were resolved by electrophoresis in 0.8% agarose gel and visualized by ethidium bromide staining.

Exposure of E. coli to oxidative stress and disk diffusion assays.

Survival of E. coli cells overexpressing FurA, FurB or FurC was analyzed using methyl viologen (MV) or H 2 O 2 disk diffusion assays by a modified version of the method described elsewhere [30]. E. coli BL21 (DE3) Gold bearing pET-28a (+) was used as negative control. Cells were grown in LB (Luria-Bertani) broth at 37ºC with shaking. When OD 600 of the cultures reached a value of 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mM and the cultures were grown for another 2 hours. Cells from induced cultures (200 μl) were spread onto LB agar containing 1 mM IPTG. Then, aliquots (2 μl) of increasing concentrations of H 2 O 2 (Panreac) or methyl viologen (SIGMA) were spotted on top of Whatman paper disks (0.5-cm diameter) that were placed over agar plates. Cells were allowed to grow for 24 h at 37 °C, before measuring the inhibition area around the paper disks.

RESULTS

Analysis of fur expression by RT-PCR.

Regulation of fur genes under different conditions (iron deficiency, nitrogen-fixing conditions, osmotic stress, saline stress and oxidative stress) has been investigated. When using semiquantitative RT-PCR in those studies, it should be considered the existence of an α-furA antisense RNA [START_REF] Hernandez | Identification of a furA cis antisense RNA in the cyanobacterium Anabaena sp. PCC 7120[END_REF] that can interfere with the results of this assay. To avoid this problem, a specific cDNA for each fur gene has been prepared. Transcription of furA slightly increases under iron starvation as well as in nitrogen-fixing conditions, as reported before [START_REF] López-Gomollón | Expression of furA is modulated by NtcA and strongly enhanced in heterocysts of Anabaena sp[END_REF][START_REF] Hernandez | Identification of a furA cis antisense RNA in the cyanobacterium Anabaena sp. PCC 7120[END_REF]. However, these situations do not seem to affect the transcriptional levels of furB and furC. When the cultures were grown under osmotic stress (200 mM sucrose) or salt stress (100 mM NaCl) the amounts of furA, furB and furC mRNAs remain unchanged (data not shown). Under oxidative stress conditions, transcriptional levels of the three fur genes are affected, being the effect much stronger on furB and furC m-RNAs. In all cases, challenge with H 2 O 2 induced a faster response than MV (Figure 1).

FurB protects DNA from cleavage produced by hydroxyl radicals or DNaseI .

The dramatic increase of furB expression under oxidative challenge, its high isoelectric point (pI 8.7) and the fact that at high concentrations FurB binds to DNA in a sequence-independent fashion (our unpublished results), led us to examine whether purified FurB can afford protection against DNA cleavage. Two situations were tested. On the one hand, DNA was challenged with hydroxyl radicals generated via iron-mediated Fenton reactions, on the other DNA was treated with DNaseI. In those experiments, the combination of Fe (III)/EDTA or Fe (II) and H 2 O 2 or addition of DNaseI results in the introduction of single-strand breaks that changes supercoiled DNA mobility in agarose gels (figure 2, lanes 3, 6 and 9). Addition of 40 pmol and 80 pmol of FurB to the reaction mixture containing DNA shows a progressive increase of FurB-DNA complexes, followed by formation of large aggregates that are retained in the wells (figure 2.A.I lanes 4, 5, 7, 8, 10 and 11). FurB binds to supercoiled or linear DNA, suggesting no apparent predilection for either topological form. Removal of FurB before electrophoresis permits to visualize the degree of damage suffered by DNA that was bound to FurB (figure 2.A.II lanes 4, 5, 7, 8, 10 y 11). It can be observed that the presence of increasing amounts of FurB results in a progressive decrease in DNA damage. In order to check the specificity of the DNA protection by FurB, similar experiments were performed in the presence of FurA (not shown) or BSA (figure2.B) instead of FurB. In those cases DNA is nicked by hydroxyl radicals or DNaseI despite the presence of FurA or BSA. Those results show that FurB binds to and protects DNA from cleavage produced by reactive oxygen species or by DNaseI. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

FurB binds heme.

Free heme is a potential cause of oxidative stress inside the cell [START_REF] Gutteridge | Antioxidant protection by haemopexin of haemstimulated lipid peroxidation[END_REF]. It damages lipids, proteins and DNA through the generation of ROS. In particular, redox-active iron released after degradation of heme plays an important role for heme toxicity [START_REF] Kumar | Free heme toxicity and its detoxification systems in humans[END_REF]. In this way, heme detoxification constitutes a way of ensuring cell survival in the redox environment produced by heme. The ability of Fur and other proteins involved in iron homeostasis to bind heme has been reported previously [START_REF] Hernández | Heme binds to and inhibits the DNA-binding activity of the global regulator FurA from Anabaena sp. PCC 7120[END_REF]22,[START_REF] Qi | Heme is an effector molecule for iron-dependent degradation of the bacterial iron-response regulator (Irr) protein[END_REF]. In the case of Anabaena FurA, it has been proposed that heme acts as a potential regulator impairing its DNA-binding activity. With the aim to investigate whether FurB and FurC were able to bind heme, a heme-staining procedure was performed as described previously [START_REF] Hernández | Heme binds to and inhibits the DNA-binding activity of the global regulator FurA from Anabaena sp. PCC 7120[END_REF]. FurA, used as positive control, FurB, FurC and lysozyme, the latter as negative control, were incubated with heme and the complexes resolved by SDS/PAGE. As shown in figure 3.A, FurB binds heme whereas FurC does not seem to interact with it. This result is in good agreement with the existence of the CP (Cys-Pro) regulatory motif in the sequence of FurA and the CPV (Cys-Pro-Val) one in the sequence of FurB (supplementary material figure S1).

Influence of heme in the DNA-binding activity of FurB. Protein-DNA interactions occur between transcription factors and DNA control sequences that lead to gene regulation. They are often the result of extracellular stimuli. The interaction of heme with FurB suggests the possibility that, as in the case of FurA, its DNA-binding activity could be affected. EMSA assays were carried out to assess the influence of heme in the interaction of FurB with the furA promoter. In the absence of heme, FurB binds to P furA , as previously reported [17]. Addition of heme to the binding assay prior to the incubation with P furA inhibits completely the interaction of FurB with DNA, as shown in figure 4.

FurA and FurB increase cell survival under oxidative stress in vivo.

In sight of the results shown to this point, we sought to investigate whether the Fur family is involved in protecting cells from oxidative damage in vivo. If that was the case, the presence of high amounts of Fur in the cells induced by H 2 O 2 or methyl viologen should increase cell survival. Fur proteins from Anabaena were overexpressed in E. coli from the IPTG-inducible promoter T7 of pET-28a (+) [17]. Those E. coli cells were allow to grow on LB-agar plates supplemented with IPTG and Whatman paper disks on top containing increasing concentrations of H 2 O 2 or MV. Figure 5 shows that when the cells are challenged with H 2 O 2 , overexpression of FurA increases significantly cell survival, as shown by a smaller zone of growth inhibition. Cells overexpressing FurB are also less sensitive to H 2 O 2 damage. In the MV assays, similar results were observed (figure 6). In both cases, the effect is more evident at high concentrations of H 2 O 2 or MV. Overexpression of FurC seems not to influence bacterial survival in any condition tested.

DISCUSSION

The existence of different Fur homologues in distantly related organisms suggests an evolutionary specialization. Although all Fur proteins show common characteristics, there are substantial differences among the signals affecting their affinity for DNA, the sets of target genes or the elements that modulate their transcription. To get a better understanding of the roles of fur paralogues in Anabaena, we have investigated the regulation of those genes under different situations, as well as the behaviour of their protein products in face of agents that could induce DNA damage. While osmotic and saline stresses did not affect the transcription of fur genes, oxidative challenge lead to increased levels of the three fur transcripts, particularly of furB and furC. This indicates that the raise of the transcription is not a general response to any stress and are consistent with previous reports linking iron-responsive gene expression and oxidative stress response [13,14,16,[START_REF] Kobayashi | Response to oxidative stress involves a novel peroxiredoxin gene in the unicellular cyanobacterium Synechocystis sp. PCC 6803[END_REF]. In many microorganisms, Fur proteins regulate the expression of genes involved in the concerted defence against oxidative stress, such as superoxide dismutase or catalase. In the unicellular cyanobacterium Synechocystis sp. PCC 6803, the Fur-like Slr1738 plays a regulatory role in the induction of the potent antioxidant gene sll1621(ahpC) [14,[START_REF] Kobayashi | Response to oxidative stress involves a novel peroxiredoxin gene in the unicellular cyanobacterium Synechocystis sp. PCC 6803[END_REF]. Both genes are strongly induced by methyl viologen or peroxide treatment and are located contiguously in the genome. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

In addition to ROS generated by the Fenton´s reaction and by the adventitious delivery of electrons from the photosynthetic and respiratory pathways to oxygen, the potential of heme to catalyse free radical formation has to be considered. Histidine residues and sometimes cysteine-proline (CP) [36] or cysteine-proline-valine (CPV) [START_REF] Zhang | Heme binds to a short sequence that serves a regulatory function in diverse proteins[END_REF] sequences, have been proposed to be involved in the interaction between heme and proteins. A common feature of the Fur family is the presence of a histidine-rich motif that is involved in metal binding. Moreover, FurA and FurB from Anabaena contain in their sequences CP or CPV motifs, respectively, located in their carboxyl ends (figure S1), which accounts for the ability to bind metal and dimerise [8,[START_REF] Coy | Structural dynamics and functional domains of the fur protein[END_REF]. The CP or CPV sequences are thought to be regulatory elements for non-covalent binding of heme to proteins [START_REF] Zhang | Heme binds to a short sequence that serves a regulatory function in diverse proteins[END_REF]. Several functions are proposed for these sequences. They could act, for instance, as a secondary heme binding sites or contribute to the complex formation by increasing the concentration of heme around them [START_REF] Hernández | Heme binds to and inhibits the DNA-binding activity of the global regulator FurA from Anabaena sp. PCC 7120[END_REF]. The presence of the CP motif in the sequence of FurA and CPV one in the sequence of FurB is in good agreement with the ability of these proteins to bind heme, while FurC, that lacks any of these signatures, does not interact with it. In Rhizobiales, heme regulates the degradation of the Fur-related response regulator Irr, whose stability depends on the iron status [START_REF] Qi | Heme is an effector molecule for iron-dependent degradation of the bacterial iron-response regulator (Irr) protein[END_REF]. In the case of FurA and FurB, degradation products are not detected after incubation with heme, suggesting that heme plays a different role in Irr than in Fur proteins. Different mechanisms of redox control of gene expression have evolved to adapt rapidly to redox changes within bacteria. The regulator FixL utilizes heme as co-repressor to elicit a conformational change in response to oxygen [START_REF] Bauer | Mechanisms for redox control of gene expression[END_REF]. Similarly, the interaction of heme with FurA and FurB could produce a conformational change providing a mechanism of post-translational regulation. This model is in good agreement with the fact that heme impairs the DNA binding activity of FurB and, as previously reported, of FurA [START_REF] Hernández | Heme binds to and inhibits the DNA-binding activity of the global regulator FurA from Anabaena sp. PCC 7120[END_REF]. Fur regulation seems to be a complex process which involves not only the existence of Fe(II) and reducing conditions [START_REF] Hernández | Interaction of FurA from Anabaena sp. PCC 7120 with DNA: A reducing environment and the presence of Mn2+ are positive effectors in the binding to isiB and furA promoters[END_REF]. The DNA-binding ability of Fur, and consequently, its functionality, is impaired also by the dimerisation rate and by heme binding. The contexts of the fur homologues in Anabaena are rather different that those of the corresponding ortologues in Synechocystis sp. PCC 6803. While the peroxide-dependent slr1738 is contiguos to ahpC, the orthologue to furB, sll1937, lies divergently next to zntC, that codes for a component of an ABC type zinc uptake transporter [40]. Analysis of the sequences flanking furB and furC in Anabaena (Cyanobase: http://bacteria.kazusa.or.jp/cyano/) shows that the ORFs contiguous to those fur genes encode hypothetical or unknown proteins. On the other hand, the closest homologues to ahpC and zntC, the all1541 and all0833 genes, are located next to other ORFs unrelated to the fur family. Our results demonstrate that FurB protects DNA from cleavage produced by hydroxyl radicals or DNaseI. In addition, overexpression of FurA and FurB in E. coli increases survival when the cells are stressed with hydrogen peroxide or methyl viologen. Although overexpression of FurC does not seem to influence cell survival, it could be hypothesized that FurC might play a role in the regulation of cyanobacterial metabolic processes that are absent in E. coli, such as those related to photosynthesis. In fact, expression of furC is enhanced specifically by addition of methyl viologen, a superoxide anion-generating reagent. In photosynthetic organisms, the most important production of superoxide anion takes place in the photosynthetic electron transport chain, when molecular oxygen adventitiously oxidizes PSI [41]. Thus, FurC might be involved in the protection of the photosynthetic proteins from oxidative damage. In the case of FurA, it might increase cell survival modulating a set of genes involved in the detoxification of ROS. Indeed, FurA recognises the regulatory regions of several key genes related to redox homeostasis, such as gor and a number of thioredoxins [START_REF] López-Gomollón | Cross-talk between iron and nitrogen regulatory networks in Anabaena (Nostoc) sp. PCC 7120: identification of overlapping genes in FurA and NtcA regulons[END_REF] suggesting that FurA could coordinate the cellular response to oxidative stress. Overexpression of FurB possibly increases cell survival under oxidative challenge avoiding DNA damage by unspecific binding. The cationic isoelectric point of FurB (theoretical pI = 8.7), together with its high affinity to DNA, its ability to bind heme and its DNA-protective activity suggest that FurB could act as a Dps-like protein [43]. In addition to Dps, there are other bacterial, relatively nonspecific DNA-binding proteins that exert different functions depending on the level of expression. H-NS is one of the most abundant chromatin-associated proteins [START_REF] Tupper | The chromatin-associated protein H-NS alters DNA topology in vitro[END_REF]. At sub-saturating concentrations, H-NS exhibits a preferred DNA-binding site modulating transcription of the proU operon. However, when present in saturating concentrations, H-NS binds to DNA in a sequence-independent fashion. This could be the case of furB, whose transcription is triggered by reactive oxygen species. Under those conditions, the level of FurB would be high enough to coat DNA unspecifically and prevent oxidative damage. To our knowledge, this is the first evidence that a Fur protein protects DNA from cleavage by direct binding. A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. (A) 5 μM of each protein were incubated 5 minutes with heme. FurA (MW= 17.2 KDa) was used as positive control or the experiment. Lysozyme (MW= 14.3 KDa) was used as a control of non-specific binding of heme to protein. Samples were analyzed in SDS/PAGE and a specific heme-staining assay was performed [START_REF] Granath | Identification of hemeproteins in the hemolymph of Biomphalaria glabrata (Gastropoda) separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis[END_REF]. (B) After that, proteins were detected with Coomassie blue staining. The sizes of the molecular mass markers are indicated on the left side. Note that the samples are loaded using a non-reducing loading buffer, thus several oligomeric forms of FurA, FurB and FurC could be detected. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 22 Oct 2008 as manuscript BJ20081066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081066

Biochemical

  Journal Immediate Publication. Published on 22 Oct 2008 as manuscript BJ20081066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081066

  Biochemical Journal Immediate Publication. Published on 22 Oct 2008 as manuscript BJ20081066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081066

Biochemical

  Journal Immediate Publication. Published on 22 Oct 2008 as manuscript BJ20081066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081066

Biochemical

  Journal Immediate Publication. Published on 22 Oct 2008 as manuscript BJ20081066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081066 A c c e p t e d M a n u s c r i p t

Figure 1 .

 1 Figure 1. RT-PCR analysis of the fur family transcription under oxidative stress conditions. Expression of furA, furB and furC under oxidative stress conditions produced by 250 μM H 2 O 2 and 1 mM methyl viologen. Transcripts were analyzed at different times by RT-PCR. PCR reactions with the corresponding oligonucleotides using chromosomic Anabaena sp. DNA as template were carried out as positive controls.

Figure 2 .

 2 Figure 2. In vitro DNA protection assay.The introduction of a single-stranded break in the plasmid DNA converts it to a nicked form changing its mobility in an agarose gel. As the substrate was used 100 ng of pUC plasmid. When indicated, Fe (III)/EDTA or Fe (II) and H 2 O 2 or DNaseI were added to the samples as denoted below the panels. FurB (40 pmol and 80 pmol) as is shown in panel A, and BSA (40 pmol and 80 pmol) as negative control, in panel B, were added to the samples. After incubation for 20 minutes at room temperature, the reactions were stopped and proteins were removed from half of each sample. DNA was visualized by electrophoresis in 0.8% agarose gel. N: nicked DNA, L: linear DNA, SC: supercoiled DNA.

Figure 3 .

 3 Figure 3. Heme binding and staining assay.(A) 5 μM of each protein were incubated 5 minutes with heme. FurA (MW= 17.2 KDa) was used as positive control or the experiment. Lysozyme (MW= 14.3 KDa) was used as a control of non-specific binding of heme to protein. Samples were analyzed in SDS/PAGE and a specific heme-staining assay was performed[START_REF] Granath | Identification of hemeproteins in the hemolymph of Biomphalaria glabrata (Gastropoda) separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis[END_REF]. (B) After that, proteins were detected with Coomassie blue staining. The sizes of the molecular mass markers are indicated on the left side. Note that the samples are loaded using a non-reducing loading buffer, thus several oligomeric forms of FurA, FurB and FurC could be detected.

Figure 4 .

 4 Figure 4. Inhibition of in vitro DNA binding activity of FurB by heme.Electrophoretic mobility shift assay of FurB to a 389-bp DNA fragment containing the Anabaena sp. furA promoter. A fragment of exon IV from the human apoE gene was used as non-specific competitor DNA. Lane 1: free DNA. Lane 2: 500 nM FurB. Lane 3: Reaction mixture in the presence of 1:5 FurB /heme concentration ratio (500 nM FurB and 2.5 μM heme). Lane 4: Reaction mixture in the presence of 1:8 FurB /heme concentration ratio (500 nM FurB and 4 μM heme).

Figure 5 .

 5 Figure 5. H 2 O 2 disk assay.In the assay, LB-agar plates were inoculated from liquid cultures of E. coli strains overexpressing FurA, FurB and FurC in the presence of 1 mM IPTG. E. coli containing pET-28a(+) was used as negative control. Sensitivity to H 2 O 2 was measured by the zone of growth inhibition (diameter). (A) Effect of H 2 O 2 on growth of E. coli bearing (a) pET-28a(+) plasmid (negative control), (b) furA cloned into pET-28a(+), (c) furB cloned into pET-28a(+), (d) furC cloned into pET-28a(+).H 2 O 2 concentration assayed: (1) 9.7 M, (2) 4.85 M, (3) 1.94 M, (4) 0.97 M and (5) 0.48 M. (B) The zone of inhibition at each concentration of H 2 O 2 of negative control (black bars) and furA (light grey bars), furB (white bars), furC (dark grey bars) cloned into pET-28a(+) was measured five times, and the mean values were represented. The error bars are the standard errors of the mean value.

Figure 6 .

 6 Figure 6. Methyl viologen disk assay. In the assay, LB-agar plates were inoculated from liquid cultures of E. coli strains overexpressing FurA, FurB and FurC in the presence of 1mM IPTG. Sensitivity to MV was measured by the zone of growth inhibition (diameter). (A) Effect of methyl viologen on growth of E. coli bearing (a) pET-28a(+) plasmid (negative control), (b) furA cloned into pET-28a(+), (c) furB cloned into pET-28a(+), (d) furC cloned into pET-28a(+). MV concentration assayed: (1) 1000 mM, (2) 500 mM, (3) 100 mM, (4) 75 mM, (5) 50 mM, (6) 25 mM and (7) 10 mM. (B) The zone of inhibition at each concentration of MV of negative control (black bars) and furA (light grey bars), furB (white bars), furC (dark grey bars) cloned into pET-28a(+)was measured five times, and the mean values were represented. The error bars are the standard errors of the mean value.

Figure 1 .

 1 Figure 1. RT-PCR analysis of fur family expression under oxidative stress conditions

Figure 2 .Figure 3 .

 23 Figure 2. In vitro DNA protection assay

Figure 4 .

 4 Figure 4. Inhibition of in vitro DNA binding activity of FurB by heme

Figure 5 .

 5 Figure 5. H 2 O 2 disk assay

Figure 6 .

 6 Figure 6. Methyl viologen disk assay

Table 1 .

 1 Oligonucleotides used in this work

	Biochemical Journal Immediate Publication. Published on 22 Oct 2008 as manuscript BJ20081066
	Primer	Sequence (5´_ 3´)	Description
	FurN	ATTACCATGGCTGTCTACAC	RT-PCR of
	FurC	CTTGTAAGCTTACCAGGTGC	furA
	all2473-N	ATAGTCCATGGGAGCCATAC	RT-PCR of
	all2473-C CAGTAGTTTAAGCTTTTGACTA	furB
	alr0957-N GACATAACCATGGAGCAACAG	RT-PCR of
	alr0957-C	CTACTCCCAAGCTTTCTACTC	furC

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

ACKNOWLEDGEMENTS

The authors thank Dr Silvia Pellicer for helpful discussions. This work was supported by grant BFU2006-03454/BMC (Programa nacional de I+D, Ministerio de Educación y Ciencia, Spain).


Majd B Protty

Nicholas A Watkins

Dario Colombo

Steven G Thomas

Victoria L Heath

John Herbert

Roy Bicknell

Yotis A Senis

Leonie K Ashman

Fedor Berditchevski

Fedor BERDITCHEVSKI § Willem H Ouwehand †oe

Steve P Watson

Michael G Tomlinson
email: m.g.tomlinson@bham.ac.uk

Identification of

Keywords: platelet, megakaryocyte, tetraspanin, Tspan9, membrane microdomain, GPVI

INTRODUCTION

Platelets play an essential role in preventing excessive blood loss at sites of vascular injury by plugging holes in damaged blood vessels and promoting the clotting cascade. Platelets are also important in inflammation through recruitment of leukocytes, and in blood vessel repair through the release of growth factors. However, platelets also play a deleterious role in causing heart attack and stroke when activated in diseased vessels [START_REF] Langer | Platelet-vessel wall interactions in atherosclerotic disease[END_REF]. Critical to these events are a variety of well-characterised surface receptor proteins which combine to induce powerful platelet activation when they encounter exposed sub-endothelial matrix proteins in the damaged vessel wall [START_REF] Varga-Szabo | Cell adhesion mechanisms in platelets[END_REF]. It is becoming increasingly clear that such cell surface proteins and their associated signaling proteins are not randomly distributed in the plasma membrane, but are instead compartmentalized into membrane microdomains [START_REF] Jacobson | Lipid rafts: at a crossroad between cell biology and physics[END_REF].

The tetraspanins are a large family of four-transmembrane glycoproteins that are thought to function as 'molecular organizers' by forming a type of membrane microdomain that is distinct from lipid rafts [START_REF] Hemler | Tetraspanin functions and associated microdomains[END_REF][START_REF] Le Naour | Membrane microdomains and proteomics: lessons from tetraspanin microdomains and comparison with lipid rafts[END_REF][START_REF] Levy | The tetraspanin web modulates immune-signalling complexes[END_REF]. This is achieved through tetraspanin-tetraspanin associations and specific tetraspanin interactions with so-called partner proteins, which include integrins, immunoglobulin superfamily proteins and G protein-coupled receptors. Such a fundamental organizing role is consistent with tetraspanin expression throughout the fungi, plant and animal kingdoms and their essential roles in processes such as cell adhesion, motility and signal transduction [START_REF] Hemler | Tetraspanin functions and associated microdomains[END_REF].

An essential role for tetraspanins in platelets was demonstrated by mild bleeding phenotypes in mice deficient for either of the tetraspanins CD151 or Tspan32 (previously named TSSC6 or PHEMX) [START_REF] Lau | The tetraspanin superfamily member CD151 regulates outsidein integrin {alpha}IIb{beta}3 signaling and platelet function[END_REF][START_REF] Goschnick | Impaired "outside-in" integrin alphaIIbbeta3 signaling and thrombus stability in TSSC6-deficient mice[END_REF]. These similar phenotypes appear to be due to impaired signaling by the major platelet integrin DIIbE3, but their weak nature suggests functional redundancy between tetraspanins. However, only two other tetraspanins, CD9 and CD63, have been identified on platelets using antibodies [START_REF] Goschnick | Tetraspanins-structural and signalling scaffolds that regulate platelet function[END_REF]. Identification of other platelet tetraspanins is impaired by a lack of antibodies to most of the 33 mammalian tetraspanins. Additionally, the anucleate platelet has minimal mRNA levels [10], which makes reverse transcriptase-polymerase chain reaction (RT-PCR) challenging due to the issue of contamination with mRNA from other blood cells.

The present study aims to identify a novel platelet tetraspanin through genomic analyses of megakaryocytes, the platelet progenitors, and subsequent antibody generation to confirm expression on platelets.

MATERIALS AND METHODS

Genomics

The serial analysis of gene expression (SAGE) library from primary mouse megakaryocytes was generated using the I-SAGE Long Kit (Invitrogen) as described previously [11]. SAGE tags were identified using SAGE2000 4.5 Analysis Software (Invitrogen) and each tetraspanin tag was confirmed manually by referring to GenBank entries in the National Center for Biotechnology Information public database. The HaemAtlas microarray data was generated, as described [12], from monocytes (CD14 + ), B cells (CD19 + ), helper T cells (CD4 + ), natural killer cells (CD56 + ), granulocytes (CD66b + ) and cytotoxic T cells (CD8 + ) isolated from seven human volunteers, and erythroblasts and megakaryocytes differentiated from CD34 + haematopoietic stem cells.

Cell culture

The HEK 293T human embryonic kidney cell line was cultured in DMEM supplemented with 10% foetal bovine serum, penicillin, streptomycin, and glutamine. The DG75 and Raji human B cell lines, the HPB-ALL and Jurkat human T cell lines, the DAMI and MEG-01 human megakaryocyte-like cell lines, and the HEL human erythroleukaemia line, were all cultured in RPMI supplemented with 10% foetal bovine serum, penicillin, streptomycin, and glutamine.

FLAG-tagged tetraspanin constructs

Tetraspanin cDNAs were generated by PCR from the following sources: DAMI human megakaryocyte-like cell line cDNA for CD9 and CD151; specific cDNA for CD63 [13]; and mouse primary megakaryocyte cDNA for Tspan9 [11]. These were cloned into the pEF6/Myc-His vector (Invitrogen) which had been modified to include an in-frame, upstream sequence for the FLAG epitope tag. The endogenous stop codons were retained for each tetraspanin construct to prevent translation of the Myc and His epitope tags.

Antibodies

The mouse negative control monoclonal antibody (MOPC 21) and the rabbit anti-FLAG antibody were from Sigma, while the normal rabbit control IgG was from Upstate. The mouse monoclonal antibodies to human tetraspanins CD9 (C9-BB and 1AA2) [START_REF] Berditchevski | Characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains (TM4 proteins)[END_REF][START_REF] Sincock | Localization of the transmembrane 4 superfamily (TM4SF) member PETA-3 (CD151) in normal human tissues: comparison with CD9, CD63, and alpha5beta1 integrin[END_REF], CD63 (6H1 and 12F12) [16,[START_REF] Zannettino | A powerful new technique for isolating genes encoding cell surface antigens using retroviral expression cloning[END_REF] and CD151 (11B1) [START_REF] Sincock | Localization of the transmembrane 4 superfamily (TM4SF) member PETA-3 (CD151) in normal human tissues: comparison with CD9, CD63, and alpha5beta1 integrin[END_REF], and to human GPVI (204-11) [18] and GPIbD (WM23) [19], were as described, while those to integrins DIIb (SZ22), D2 (Gi9) and D6 (4F10) were from Beckman Coulter, Immunotech and Serotec, respectively.

Generation of Tspan9 polyclonal antibodies

A peptide of sequence QHIHRTGKKYDA, corresponding to the COOH terminus of human and mouse Tspan9, conjugated to keyhole limpet haemocyanin via an additional NH 2 -terminal cysteine residue, was used to immunise one rabbit and one chicken by Eurogentec. IgY was purified from chicken yolks using the Pierce Eggcellent Chicken IgY Purification Kit (Perbio). Rabbit and chicken anti-Tspan9 antibodies were then peptide-purified using the Pierce SulfoLink Kit according to the manufacturer's protocol (Perbio).

Confocal microscopy

Foetal liver mouse megakaryocytes were prepared and cultured as described [20], and mature megakaryocytes were collected after five days by passing the culture over a bovine serum albumin step gradient. Megakaryocytes were allowed to spread for two hours on coverslips coated with 100 Pg/ml fibrinogen. HEK 293T cells were allowed to adhere to glass coverslips prior to calcium phosphate transfection with FLAG-tagged Tspan9, and incubated for 24 hours. Both megakaryocytes and HEK 293T cells were fixed and prepared for fluorescence microscopy as described previously [START_REF] Thomas | Studies on the actin-binding protein HS1 in platelets[END_REF]. The rabbit anti-Tspan9 antibody was used at a concentration of 2 Pg/ml. Images were acquired using a DMIRE2 Leica confocal microscope (Leica) with a 63x oil immersion 1.40 NA plan-apochromat oil immersion lens. Images were captured using Leica Confocal Software and offline analysis was performed with Adobe Photoshop.

Quantitation of tetraspanins in human platelets

Tetraspanins were quantitated using six human donors: three males of Caucasian, Chinese and Indian origin, and three females of Arabic, Caucasian and Japanese origin. Washed platelets were prepared as previously described [22], but with the modification that platelets were taken from the top third of the platelet-rich plasma to ensure that they were essentially free of other blood cells.

The number of copies of CD9 per platelet cell surface was determined using the anti-CD9 monoclonal antibody 1AA2 [START_REF] Sincock | Localization of the transmembrane 4 superfamily (TM4SF) member PETA-3 (CD151) in normal human tissues: comparison with CD9, CD63, and alpha5beta1 integrin[END_REF] and the Platelet Calibrator Kit (Biocytex), according to the manufacturer's instructions.

The relative expression levels of Tspan9 versus CD9, CD63 and CD151 in platelets were determined using a quantitative western blotting method that we have used previously to quantitate protein tyrosine kinases in lymphocytes [START_REF] Tomlinson | Reconstitution of Btk signaling by the atypical Tec family tyrosine kinases Bmx and Txk[END_REF][START_REF] Tomlinson | Expression and function of Tec, Itk, and Btk in lymphocytes: evidence for a unique role for Tec[END_REF]. Reference samples of FLAG-tagged forms of each tetraspanin were first generated by transiently transfecting each construct into HEK 293T cells using the calcium phosphate protocol, lysing the cells two days later with 1% NP40 and 1% dodecylmaltoside lysis buffer (the latter to fully solubilise lipid rafts), immunoprecipitating with mouse anti-FLAG (M2) agarose beads (Sigma), washing with stringent RIPA buffer to remove associated proteins, western blotting under non-reducing conditions with rabbit anti-FLAG (Sigma), and quantitating the bands using ECL chemiluminescence in combination with the GeneGnome quantitative western blotting system (Syngene). Specific FLAG-tetraspanin reference samples and human platelet lysates, lysed as for the HEK 293T cells, were then western blotted under non-reducing conditions using tetraspanin-specific antibodies (C9-BB for CD9, 6H1 for CD63, 11B1 for CD151 and rabbit anti-Tspan9), and the bands quantitated using the GeneGnome. Finally, the relative expression levels of each tetraspanin were calculated by comparing the anti-FLAG and anti-tetraspanin quantitation data, with the expression of CD9 arbitrarily set to 100. Washed human platelets were suspended at a concentration of 5 x 10 8 per ml in phosphatebuffered saline (PBS) containing 1 mg/ml sulfo-NHS-SS-biotin (Perbio) and gently rotated for 30 minutes at room temperature. The biotinylation reaction was quenched with 100 mM glycine in PBS, and the platelets pelleted and washed once more with PBS. Platelets (5 x 10 8 ) were lysed on ice in 1 ml of either 1% Brij97 (containing 1 mM CaCl 2 and 1 mM MgCl 2 ) or 1% Triton X-100 lysis buffer (containing 1 mM EDTA); both lysis buffers otherwise contained 10 mM Tris pH 7.5, 150 mM NaCl, 0.01% sodium azide, 200 Pg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 10 Pg/ml aprotinin, 10 Pg/ml leupeptin, 1 Pg/ml pepstatin A, 10 PM NaF and 2 mM sodium orthovanadate. Lysates were pre-cleared at 4 ż C for 30 minutes each with 10 Pl protein G sepharose (Sigma) followed by 10 Pl protein G sepharose pre-coupled with 1 Pg of either MOPC mouse or normal rabbit control Ig. Pre-cleared lysates were immunoprecipitated at 4 ż C for 90 minutes with specific antibodies (1AA2 for CD9, 11B1 for CD151, rabbit anti-Tspan9, 4F10 for D6 and Gi9 for D2), pre-coupled to 10 Pl protein G sepharose, and washed four times with lysis buffer on ice. Non-reducing samples were separated on 4-12% gradient gels (Invitrogen) for blotting with IRDye 800CW-conjugated streptavidin (LI-COR). The blots were visualised using the Odyssey Infrared Imaging System (LI-COR).

Platelet biotinylation and biochemical analyses

Co-immunoprecipitations using non-biotinylated platelets were performed as described above, with the exception that western blots were visualised using Western Lightning chemiluminescence reagents (Perkin Elmer) and Hyperfilm (Amersham Biosciences), which was developed using a Compact X4 film processor (Xograph Imaging Systems).

Cleavage of N-linked oligosaccharides from Tspan9 in vitro was performed using Nglycosidase F (PNGase F) according to the manufacturer's protocol (New England BioLabs).

Results

Primary mouse megakaryocytes express mRNA for 19 tetraspanins A total of 33 tetraspanins have been identified in the human and mouse genomes, but only four have been identified as platelet-expressed using specific antibodies, namely CD9, CD63, CD151 and Tspan32 [START_REF] Goschnick | Tetraspanins-structural and signalling scaffolds that regulate platelet function[END_REF]. This is primarily due to a lack of antibodies and to the technical difficulties in performing genomics in the platelet [10]. To identify tetraspanins that are potentially expressed in platelets, we examined mRNA expression in their nucleated precursor cell, the megakaryocyte, through re-analysis of our previously reported serial analysis of gene expression (SAGE) library [11]. SAGE provides a quantitative measure of relative mRNA levels by essentially converting each mRNA into a short piece of DNA, termed a SAGE tag, which can be ligated with other tags and readily sequenced [START_REF] Saha | Using the transcriptome to annotate the genome[END_REF]. SAGE identified 16 tetraspanins in primary mouse megakaryocytes (Figure 1). The expression of each was confirmed by RT-PCR, which also identified an additional three tetraspanins (CD82, Tspan5 and Tspan18) that fell below the threshold of detection in our SAGE library (data not shown). The most abundant tetraspanin was CD63, followed by CD9 and CD151, each of which is known to be expressed in platelets. The other platelet tetraspanin, Tspan32, was also relatively highly expressed in megakaryocytes. Of the other 15 tetraspanins identified, most have not been reported as megakaryocyte-expressed and remain unstudied at the protein level.

Tspan9 mRNA is relatively specific to human megakaryocytes versus other haematopoietic cells

Since SAGE is quantitative, different SAGE libraries can be compared with each other to identify cell type-specific genes. We have previously performed such comparisons to show that the most megakaryocyte-specific genes tend to be important for platelet function [11]. Similar analyses of tetraspanins, however, revealed that none were specific to megakaryocytes (data not shown), although the fact that nine of the tetraspanins had only five tags or less made it difficult to determine statistically whether any of these were over-represented in megakaryocytes versus other cell types. To address this issue, we used our blood cell expression atlas data [12] to compare tetraspanin mRNA expression in megakaryocytes versus seven other haematopoietic cell types. The Illumina HumanWG-6 v2 Expression BeadChips, used in this study, is a recently developed bead-based DNA microarray that largely overcomes the problems of intra-and interarray reproducibility associated with earlier microarray types [26]. Interestingly, the tetraspanins that were more highly expressed in megakaryocytes than other cell types were the known platelet tetraspanins CD9, CD63, CD151 and Tspan32, as well as Tspan4 and Tspan9 (Supplementary Figure 1). The most megakaryocyte-specific of these was Tspan9 (Figure 2), a functionally unstudied tetraspanin that was originally named NET-5 [START_REF] Serru | Sequence and expression of seven new tetraspans[END_REF], and which we and others had identified as a candidate platelet tetraspanin using proteomic analyses of the human platelet surface [11, 28] and DNA microarray analyses of human megakaryocytes [29]. Moreover, Tspan9 is remarkably highly conserved during evolution, with amino acid identities of 97% for human versus mouse and 90% for human versus zebrafish, suggesting that this tetraspanin may be functionally important in megakaryocyte and platelet biology. To determine whether Tspan9 is indeed a new platelet tetraspanin, we generated the first antibodies to this protein in the form of rabbit and chicken polyclonal antisera to the COOHterminal cytoplasmic tail of Tspan9, a region that is identical in amino acid sequence between human and mouse. The antisera were peptide-purified and their efficacy confirmed by western blotting lysates of HEK 293T cells transiently transfected with mouse Tspan9 (Figure 3A). The multiple bands detected in the western blot of transfected Tspan9 are likely to represent immature glycoforms that may be relatively abundant in an over-expression system. Indeed, Tspan9 has a single predicted N-linked glycosylation site, and treatment of Tspan9 immunoprecipitates with an N-glycosidase reduced the banding pattern to a single species (Figure 3B). Moreover, N-glycosidase treatment of Tspan9 immunoprecipitates from human platelet lysates confirmed that Tspan9 is a glycoprotein that is expressed in this cell type (Figure 3B). Confocal microscopy analysis of Tspan9-transfected cells, in which not all of the cells were positive due to the transient nature of the transfection, suggested a predominantly plasma membrane localisation for Tspan9 (Figure 3C).

The Tspan9 antibody was used to western blot whole cell lysates of human platelets and a panel of haematopoietic cell lines which had been normalised for protein content (Figure 3D). Tspan9 was readily detected in platelets but not in the other cell types, including the megakaryocyte-like cell lines DAMI and MEG-01, suggesting that Tspan9 is predominantly restricted to primary cells of this lineage (Figure 3D). Tspan9 was similarly detected in mouse platelet lysates, as well as in lung, with relatively weak expression in brain, kidney, liver and spleen (Figure 3E). Bone marrow, a major site for megakaryocytopoiesis, was positive upon longer exposure (data not shown), but its weak signal was likely due to the relatively low numbers of megakaryocytes in this tissue. Indeed, expression of Tspan9 protein in primary mouse megakaryocytes (Figure 3F), and the proplatelet structures that they form (Figure 3G), was confirmed by fluorescence microscopy, a technique that was used because of difficulties in obtaining enough mature megakaryocytes for western blotting. Tspan9 staining throughout the megakaryocyte suggests localisation to the demarcation membrane system, which is an extensive network of membrane channels that is connected to, and is thought to originate from, the plasma membrane [START_REF] Battinelli | Delivering new insight into the biology of megakaryopoiesis and thrombopoiesis[END_REF].

Quantitation of tetraspanins in human platelets

Quantitation of Tspan9 expression levels on platelets may provide clues as to potential binding partners, which may be expressed at similar levels. The human platelet cell surface is relatively well characterised with respect to copy numbers of the major glycoproteins, but amongst tetraspanins only CD9 has been quantitated. Three different groups, using iodinated CD9 antibodies, reported 65,000 [31], 46,000 [START_REF] Hato | Exposure of platelet fibrinogen receptors by a monoclonal antibody to CD9 antigen[END_REF] and 38,000 [33] copies per platelet. To determine whether these discrepancies were likely due to donor variation, we quantitated CD9 levels on the platelets of six donors, of diverse ethnic backgrounds, using a flow cytometry-based method that we have previously used to quantify other platelet glycoproteins [START_REF] Best | GPVI levels in platelets: relationship to platelet function at high shear[END_REF]. CD9 levels were similar between individuals, with a mean of 49,000 copies per platelet and standard deviation of 3,560 (Figure 4). This suggests that the 65,000 to 38,000 range in previously reported copy numbers [31-33] reflects differences in antibodies and methodology. Nevertheless, a copy number of approximately 50,000 ranks CD9 as a major component of the platelet cell surface, compared to the two other major glycoproteins, integrin DIIbE3 (80,000 copies) [START_REF] Wagner | Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets[END_REF] and GPIb-IX-V (25,000) [START_REF] Du | Glycoprotein Ib and glycoprotein IX are fully complexed in the intact platelet membrane[END_REF].

Quantitation of Tspan9 by the flow cytometry-based method is not readily applicable to intracellular epitopes, such as the COOH-terminal cytoplasmic tail of Tspan9 to which our antibody was raised. Therefore we quantitated Tspan9 in human platelets relative to CD9, and other tetraspanins, using a western blotting protocol with epitope-tagged standards, an approach we used previously to quantitate protein tyrosine kinases in lymphocytes [START_REF] Tomlinson | Reconstitution of Btk signaling by the atypical Tec family tyrosine kinases Bmx and Txk[END_REF][START_REF] Tomlinson | Expression and function of Tec, Itk, and Btk in lymphocytes: evidence for a unique role for Tec[END_REF]. The platelet lysates were made in a lysis buffer that included dodecylmaltoside to fully solubilise lipid rafts, although Tspan9 and other platelet tetraspanins appear to be largely excluded from lipid rafts (data not shown and [START_REF] Israels | Platelet tetraspanin complexes and their association with lipid rafts[END_REF]). Using the same six donors as for the previous CD9 quantitation (Figure 4), we found Tspan9 to be expressed at 6% of the level of CD9, with similar expression between donors (Figure 5). In comparison, CD151 was expressed at a 2.3-fold greater level than Tspan9, whereas CD63 was 3-fold lower than Tspan9 (Figure 5). These data suggest that the Tspan9 expression level, like that of other tetraspanins, is tightly regulated in human platelets, and can be estimated at approximately 2,800 surface copies with a standard deviation of 600, assuming the same cell surface to intracellular localisation ratio for CD9 and Tspan9.

Tspan9 is a component of tetraspanin microdomains on human platelets

To determine whether Tspan9 is a component of tetraspanin microdomains, Tspan9-associated cell surface proteins were analysed following Tspan9 immunoprecipitation in two different detergents, Brij97 and Triton X-100. In the presence of calcium and magnesium ions, 1% Brij97 is a relatively weak detergent that is known to largely maintain tetraspanin-tetraspanin interactions. In contrast, the more stringent detergent 1% Triton X-100 disrupts tetraspanintetraspanin interactions but maintains certain tetraspanin-partner protein interactions [START_REF] Le Naour | Membrane microdomains and proteomics: lessons from tetraspanin microdomains and comparison with lipid rafts[END_REF][START_REF] Hemler | Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain[END_REF].

As shown in Figure 6A (left panel), Tspan9 immunoprecipitation from Brij97 lysates of surface biotinylated platelets contained several proteins. Similar patterns of immunoprecipitated proteins were observed in immunoprecipitations for CD9, CD151 and the tetraspanin-associated laminin-binding integrin D6E1 [START_REF] Hemler | Tetraspanin functions and associated microdomains[END_REF] (Figure 6A, left panel). Consistent with its relatively high expression level, CD9 immunoprecipitation yielded more intense bands. As a control, the collagen-binding D2E1 integrin, which is not thought to be tetraspanin-associated [START_REF] Hemler | Tetraspanin functions and associated microdomains[END_REF], was predominantly immunoprecipitated as a doublet corresponding to the molecular weights of the integrin D2 and E1 chains (Figure 6A, left panel). For each immunoprecipitation in Triton X-100 (Figure 6A, upper right panel), the major bands were the immunoprecipitated proteins themselves, with some weaker bands, presumably representing relatively tightly associated proteins, detected on a longer exposure (Figure 6A, lower right panel). For Tspan9, the most prominent of these was a 43 kD protein (p43) that was also present in CD151, but not CD9, immunoprecipitates (Figure 6A, lower right panel). A 60 kD protein (p60) was also identified in Tspan9 and D6 immunoprecipitates (Figure 6A, lower right panel), which corresponded to a major component of tetraspanin microdomains observed in Brij97 immunoprecipitates (Figure 6A, left panel). These two proteins may represent novel binding partners for Tspan9.

Since Tspan9 and other tetraspanins co-immunoprecipitate a similar pattern of surface biotinylated proteins in Brij97, this suggests that Tspan9 is a component of tetraspanin microdomains. To provide further supporting evidence for this idea, immunoprecipitations from non-biotinylated platelets were blotted for Tspan9 (Figure 6B, upper panels). In Brij97, Tspan9 was detected in CD9, CD151 and D6 immunoprecipitates, but not in those for control antibody or D2, suggesting that Tspan9 is tetraspanin-associated. These interactions were not observed in more stringent Triton X-100 lysis conditions that are thought to disrupt tetraspanin-tetraspanin interactions [START_REF] Le Naour | Membrane microdomains and proteomics: lessons from tetraspanin microdomains and comparison with lipid rafts[END_REF][START_REF] Hemler | Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain[END_REF]. Interestingly, Tspan9 was immunoprecipitated more efficiently in Triton X-100 than in Brij97, despite similar solubilities in the two detergents as shown by western blotting of whole cell lysates (Figure 6B, upper panels). This result is consistent with Figure 6A, in which a biotinylated 27 kD protein band corresponding to Tspan9 was immunoprecipitated more efficiently in Triton X-100. These findings suggest that the epitope for the Tspan9 antibody, which is 12 amino acids long, COOH-terminal and membrane-proximal, is less accessible to an immunoprecipitating antibody in the context of intact tetraspanin microdomains in Brij97, perhaps due to steric hindrance by other tetraspanins or associated proteins.

Tetraspanin microdomains on human platelets contain GPVI but not GPIbD or DIIbE3

The identities of most of the tetraspanin-associated platelet proteins, observed in tetraspanin immunoprecipitations from Brij97 lysates (Figure 6A, left panel), are not known, but their molecular weights suggest as candidates the major platelet glycoproteins GPIbD (the von Willebrand receptor), DIIbE3 (the major platelet integrin) and GPVI (the main signalling receptor for collagen). However, GPIbD and DIIb were not detected in western blots of tetraspanin immunoprecipitates (Figure 6B, middle and lower panels), suggesting that these proteins are not tetraspanin-associated. A similar experiment proved difficult for GPVI because in western blots this protein was obscured by the immunoglobulin heavy chain from the immunoprecipitating antibodies (data not shown).

To overcome this problem, GPVI immunoprecipitates were analysed for tetraspanin-associated proteins (Figure 6C) and tetraspanins themselves (Figure 6D). GPVI immunoprecipitation from Brij97 lysates of biotinylated platelets yielded a strikingly similar pattern of biotinylated proteins to CD151 immunoprecipitation (Figure 6C). In Triton X-100, most of these interactions were lost, leaving a major 62 kD band in GPVI immunoprecipitates and a major 27 kD band in CD151 immunoprecipitates (Figure 6C), corresponding to the molecular weights of GPVI and CD151, respectively.

To confirm that GPVI was associated with tetraspanins in these immunoprecipitation experiments, specific CD9 and CD151 blots were performed (Figure 6D). In Brij97 lysates, both tetraspanins were detected in GPVI immunoprecipitates, but not in those for control or D2 antibodies. These interactions were lost in Triton X-100 lysates. The GPVItetraspanin interaction appeared to be less robust than the positive control D6E1-tetraspanin interaction, the latter of which was even detected in more stringent Triton X-100 lysates (Figure 6D), consistent with the direct and relatively strong nature of the CD151 interaction with this integrin [START_REF] Hemler | Tetraspanin functions and associated microdomains[END_REF]. Together these data suggest that GPVI is a novel tetraspanin-associated protein.

Discussion

In the present study, we found 19 tetraspanins to be expressed in megakaryocytes and raised the first antibodies to the previously uncharacterized tetraspanin Tspan9, which was originally named NET-5 [START_REF] Serru | Sequence and expression of seven new tetraspans[END_REF]. Tspan9 protein expression was relatively specific to platelets when compared to other blood cell types. Moreover, biochemical analyses demonstrated that Tspan9 was a novel component of tetraspanin microdomains on the platelet surface. This is the first characterisation of the tetraspanin Tspan9 at the protein level through the generation of antibodies. Of the 33 tetraspanins in the human genome, antibodies have been reported to less than half. As such, we do not know the full complement of tetraspanins that are expressed in a given cell type and which other proteins are recruited to tetraspanin microdomains. The probable reasons for the lack of antibody reagents are firstly the relatively small size of tetraspanins and their association with larger proteins in tetraspanin microdomains [START_REF] Min | Structural basis for tetraspanin functions as revealed by the cryo-EM structure of uroplakin complexes at 6-A resolution[END_REF]. This may render them virtually inaccessible to antibody recognition in the context of intact cells, which are the predominant immunogens used to date for the generation of tetraspanin monoclonal antibodies. Secondly, tetraspanin protein sequences are highly conserved during evolution [40], so the number of tetraspanin non-self epitopes is very low. However, we managed to overcome these problems by raising antibodies in rabbit and chicken to the intracellular COOH-terminal tail of human Tspan9. Although this approach was successful for Tspan9, it is not applicable to all tetraspanins, since some have cytoplasmic tails that are predicted to contain as few as four amino acids and are therefore too short to be used as immunogens. Moreover, we have failed to raise good antibodies to Tspan3, Tspan15, Tspan17, Tspan32 and Tspan33 using this method, although an antibody that recognised both Tspan13 and Tspan31 was successfully generated (MP and MGT, unpublished data).

In order to identify platelet tetraspanins in the absence of antibodies, we measured mRNA levels in megakaryocytes, the platelet progenitor cells, since platelets have extremely low levels of mRNA [10]. To do this, we took advantage of a mouse megakaryocyte SAGE library that we had generated previously [11], and more recent DNA microarray analyses of human megakaryocytes and seven other haematopoietic cell types [12]. SAGE, which detects essentially all expressed genes in a quantitative manner, identified the four platelet tetraspanins, CD63, CD9, CD151 and Tspan32, amongst the six most highly expressed mouse megakaryocyte tetraspanins. We identified an additional 15 tetraspanins, at generally lower expression levels, most of which have not been reported on megakaryocytes or platelets [START_REF] Goschnick | Tetraspanins-structural and signalling scaffolds that regulate platelet function[END_REF], but which nevertheless may have important roles in, for example, the regulation of G protein-coupled receptors that are expressed at relatively low levels. A total of 19 tetraspanins in megakaryocytes may be typical for other cell types. For example, a figure of 20 tetraspanins has been estimated for leukocytes [41], and we have found 22 for endothelial cells upon analyses of 11 publicly-available SAGE libraries (JH, RB and MGT, unpublished data). Unlike SAGE, the microarray data is not a reliable indicator of relative tetraspanin expression in megakaryocytes, due to the potential for differential hybridisation efficiencies for different genes. However, the microarrays do enable a comparison of the expression levels of individual tetraspanins between different cell types. Those tetraspanins that have been reported to be expressed in human platelets using antibodies (CD9, CD63, CD151 and Tspan32) [START_REF] Goschnick | Tetraspanins-structural and signalling scaffolds that regulate platelet function[END_REF], or by mass spectrometry peptide sequencing (Tspan9, Tspan15 and Tspan33) [11, 28], were relatively highly represented in human megakaryocytes. In contrast, a number of tetraspanins were not detected by either SAGE or DNA microarray, for example the CD9-related Tspan2 and the CD151-related Tspan11, suggesting that they are not expressed by the megakaryocyte/platelet lineage and thus do not compensate for CD9 and CD151 when these genes are knocked out in platelets. Finally, CD37, CD81 and Tspan4 were identified at the mRNA level in megakaryocytes but are absent at the protein level from human platelets (data not shown). It is possible that these tetraspanins are also not expressed at the protein level in megakaryocytes. Alternatively they may be megakaryocyte-expressed but actively excluded from platelets during platelet formation.

Genomic approaches, such as SAGE and DNA microarrays, do not prove expression at the protein level. However, using our new antibodies to Tspan9, we confirmed its expression on megakaryocytes and platelets by immunofluorescence microscopy, western blotting and immunoprecipitation from surface-biotinylated cells. Consistent with its relative mRNA specificity for megakaryocytes within the haematopoietic lineage, Tspan9 was not readily detected in human B and T cell lines, or mouse thymus, and only weakly in spleen. Tspan9 was only detected in primary megakaryocytes and proplatelets, and not human megakaryocyte-like cell lines, highlighting that these cells are not good models for primary cells. Tspan9 is not entirely restricted to megakaryocytes and platelets, though, since expression was also detected in mouse lung, brain and kidney. In human platelets, we found that Tspan9 expression was tightly regulated between donors at a level of 6% of CD9, 43% of CD151 and three times the level of CD63 (this relatively low protein expression level for the latter in platelets contrasts with its high mRNA expression in megakaryocytes, for reasons that are currently unknown). Since we also showed that CD9 is the major platelet tetraspanin at 49,000 cell surface copies per cell, we can estimate a Tspan9 copy number of approximately 2,800 on the platelet surface, assuming that the plasma membrane to intracellular ratio is the same for each protein. Therefore it seems unlikely that the major platelet glycoproteins such as integrin DIIbE3 (80,000 copies) [START_REF] Wagner | Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets[END_REF] or GPIb-IX-V (25,000 copies) [START_REF] Du | Glycoprotein Ib and glycoprotein IX are fully complexed in the intact platelet membrane[END_REF] would be direct binding partners for Tspan9. We did detect 43 and 60 kD proteins in relatively stringent co-immunoprecipitations with Tspan9, and we are working to identify these proteins using proteomics.

Immunoprecipitation of tetraspanins CD9, CD151 and Tspan9, and the CD151-associated integrin, D6E1, from surface biotinylated platelets revealed a distinct pattern of proteins in the relatively weak detergent Brij97, in which tetraspanin microdomains are thought to remain largely intact [START_REF] Le Naour | Membrane microdomains and proteomics: lessons from tetraspanin microdomains and comparison with lipid rafts[END_REF][START_REF] Hemler | Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain[END_REF]. These proteins are likely to represent the major components of tetraspanin microdomains on the platelet cell surface. Using specific antibodies, we found that these tetraspanin-associated proteins do not include three of the major platelet glycoproteins, namely GPIb-IX-V, D2E1 or DIIbE3. The latter result was somewhat surprising, since DIIbE3 was reported to co-immunoprecipitate with CD151 in HEL cells lysed in the mild detergent CHAPS [START_REF] Fitter | Transmembrane 4 superfamily protein CD151 (PETA-3) associates with beta 1 and alpha IIb beta 3 integrins in haemopoietic cell lines and modulates cell-cell adhesion[END_REF], and with CD151 and Tspan32 in mouse platelets lysed in Triton X-100 [START_REF] Lau | The tetraspanin superfamily member CD151 regulates outsidein integrin {alpha}IIb{beta}3 signaling and platelet function[END_REF][START_REF] Goschnick | Impaired "outside-in" integrin alphaIIbbeta3 signaling and thrombus stability in TSSC6-deficient mice[END_REF]. However, we did identify GPVI as a novel component of tetraspanin microdomains using coimmunoprecipitation experiments with CD9 and CD151 in Brij97. GPVI interaction with CD9 and CD151 was lost in more stringent Triton X-100 detergent, which does not preserve tetraspanin-tetraspanin interactions and hence microdomain integrity [START_REF] Le Naour | Membrane microdomains and proteomics: lessons from tetraspanin microdomains and comparison with lipid rafts[END_REF][START_REF] Hemler | Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain[END_REF]. This suggests that either the GPVI-tetraspanin interaction is relatively weak or that GPVI interacts more strongly with an unidentified tetraspanin. If the latter is true, Tspan9 is unlikely to be the direct binding partner for GPVI because we could not detect Tspan9 in GPVI immunoprecipitates (data not shown). The functional consequences of GPVI-tetraspanin association are currently unknown. Interestingly, the antigen receptors on B and T lymphocytes, which signal via an immunoreceptor tyrosine-based activation motif (ITAM)-based mechanism in common with the GPVI/FcRJ complex [43], are dependent on tetraspanins for their normal function [START_REF] Cherukuri | B cell signaling is regulated by induced palmitoylation of CD81[END_REF][START_REF] Cherukuri | The tetraspanin CD81 is necessary for partitioning of coligated CD19/CD21-B cell antigen receptor complexes into signaling-active lipid rafts[END_REF][46][START_REF] Van Spriel | A regulatory role for CD37 in T cell proliferation[END_REF][START_REF] Wright | Characterization of mice lacking the tetraspanin superfamily member CD151[END_REF]. Particularly well characterised is the role of the tetraspanin CD81 on B cells, where it interacts with the CD19 glycoprotein. CD19 is a signalling molecule which forms a complex with the complement receptor CD21, and is recruited to the B cell receptor complex upon its engagement by complement-opsonized antigen. In the absence of CD81, the activated B cell receptor fails to localise correctly to lipid raft microdomains and fails to induce sustained signalling [START_REF] Cherukuri | B cell signaling is regulated by induced palmitoylation of CD81[END_REF][START_REF] Cherukuri | The tetraspanin CD81 is necessary for partitioning of coligated CD19/CD21-B cell antigen receptor complexes into signaling-active lipid rafts[END_REF]. Moreover, CD19 biosynthetic maturation and trafficking to the plasma membrane are impaired [49]. Our future studies will aim to determine whether a specific platelet tetraspanin can similarly regulate GPVI maturation, trafficking and/or signal transduction.

In summary, we have shown that Tspan9 is relatively highly expressed in the megakaryocyte/platelet lineage and is a component of tetraspanin microdomains on the platelet surface. Tspan9 is remarkably highly conserved during evolution, even relative to most other tetraspanins. A feature of conserved amino acids is their involvement in intra-molecular or intermolecular interactions [START_REF] Kim | Relating three-dimensional structures to protein networks provides evolutionary insights[END_REF]. This raises the possibility that almost the entire surface of Tspan9 is engaged in protein-protein interactions. Indeed, we hypothesise that Tspan9 has multiple simultaneous interacting partners and functions as an interaction 'hub' within tetraspanin microdomains. Tspan9 may therefore be essential for microdomain organisation and for the function of microdomain-associated proteins on the platelet surface. The generation of Tspan9deficient mice is in progress to test this hypothesis. (A) HEK 293T cells were transiently transfected with empty vector control, FLAG-tagged human CD9, CD63, CD151 or mouse Tspan9 (note that the Tspan9 antibody epitope is identical in protein sequence between human and mouse). Mouse anti-FLAG immunoprecipitates were western blotted with a rabbit anti-FLAG antibody and each tetraspanin quantitated, following ECL chemiluminescence, using the GeneGnome quantitative western blotting system from Syngene. These FLAG-tetraspanin samples were then compared to whole cell lysates of washed human platelets by western blotting for (B) CD9, (C) CD63, (D) CD151 and (E) Tspan9 and the relative intensities quantitated as in panel A. (F) The data in panels A-E, for platelets from six human donors of diverse ethnic backgrounds, were used to quantitate the relative expression levels of each tetraspanin in human platelets. The data are presented as mean relative expression levels and the error bars represent the standard deviation. Figure 6. Tspan9 is a component of platelet tetraspanin microdomains that include GPVI and D6E1, but not DIIbE3, D2E1 or GPIbD. (A) Human washed platelets were surface biotinylated, lysed in 1% Brij97 (left panel) or more stringent 1% Triton X-100 (right panels, including a longer exposure), immunoprecipitated with the indicated antibodies and biotinylated proteins detected by western blotting with streptavidin using the Odyssey Infrared Imaging System from LI-COR. The position of certain proteins is indicated by arrows. These data are representative of four experiments using different donors. (B) Immunoprecipitations were performed as described in part A, with the exceptions that non-biotinylated platelets were used, that western blotting was performed with Tspan9, GPIbD and DIIb antibodies, and that blots were visualised using chemiluminescence and film. (C) CD151 and GPVI immunoprecipitations were performed from 1% Brij97 (B) or 1% Triton X-100 (T) lysates of biotinylated platelets. Biotinylated proteins were detected by western blotting with streptavidin and visualised using chemiluminescence and film. (D) GPVI, D2 and D6 immunoprecipitations were western blotted with CD9 and CD151 antibodies as described in part B. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

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Figure 1 .

 1 Figure 1. Tetraspanin expression in primary mouse megakaryocytes. A LongSAGE library was generated from mouse megakaryocytes grown in vitro from bone marrow precursors [11]. The bar chart shows the number of SAGE tags, for those tetraspanins that were identified, out of 53,046 total tags.

Figure 2 .

 2 Figure 2. Tspan9 is relatively specific to human megakaryocytes when compared to other haematopoietic cell types. Whole genome expression data was obtained for eight different human blood cells using Illumina HumanWG-6 v2 Expression BeadChips. Mean normalised intensity values are shown, with error bars representing standard error from either 4 or 7 replicates. The dashed line represents the cut-off for present/absent.

Figure 3 .

 3 Figure3. Generation of a new antibody identifies the Tspan9 glycoprotein as a novel megakaryocyte/platelet tetraspanin. (A) HEK 293T cells were transiently transfected with empty vector (-) or FLAG-tagged mouse Tspan9 (+) and whole cell lysates western blotted with the peptide-purified rabbit anti-Tspan9 antibody. Essentially identical data were generated with the chicken anti-Tspan9 antibody (data not shown). (B) FLAG-tagged mouse Tspan9, transiently transfected into HEK 293T cells, and endogenous Tspan9 from human platelets, were immunoprecipitated with rabbit anti-Tspan9 antibody and subjected to control (-) or Nglycosidase (+) treatment with the enzyme PNGase F. Samples were analysed by western blotting with chicken anti-Tspan9 antibody. (C) HEK 293T cells were transiently transfected with FLAG-tagged mouse Tspan9, fixed, permeabilised and analysed by fluorescence confocal microscopy with the rabbit anti-Tspan9 antibody. The left panel identifies the cells by differential interference contrast (DIC) microscopy and the right panel is a mid-plane confocal section showing Tspan9 expression in a subpopulation of the cells. (D) Whole cell lysates of the human cell lines DG75 and Raji (B cells), HPB-ALL and Jurkat (T cells), DAMI (megakaryocyte-like), HEL (erythroleukaemia) and MEG-01 (megakaryocyte-like), and human washed platelets were normalised for protein content and then western blotted with the rabbit anti-Tspan9 antibody. (E) Whole cell lysates of various mouse tissues and washed mouse platelets were normalised for protein content and then western blotted with the rabbit anti-Tspan9 antibody. In this panel and the previous panel, essentially identical data were generated with the chicken anti-Tspan9 antibody (data not shown). (F) Mouse megakaryocytes and (G) proplatelets were generated from cultured foetal liver cells, allowed to spread on fibrinogen, fixed, permeabilised and analysed by fluorescence confocal microscopy with the rabbit anti-Tspan9 antibody. The left panel identifies the cells by DIC and the right panel shows Tspan9 staining. Control staining with normal rabbit immunoglobulin did not yield a substantial signal (data not shown).

Figure 4 .

 4 Figure 4. CD9 is expressed at 49,000 surface copies per cell on human platelets. The number of surface copies of CD9 per platelet was determined using the mouse IgG1 anti-CD9 monoclonal antibody 1AA2 and the Platelet Calibrator Kit from Biocytex. (A) The calibrator beads, coated with the indicated numbers of mouse IgG1 antibody molecules, were stained with FITC-conjugated anti-mouse antibody and analysed by flow cytometry. (B) Washed human platelets from a representative donor were stained with an IgG1 control or the CD9 monoclonal antibody, followed by FITC-conjugated anti-mouse antibody and analysed by flow cytometry.

Figure 5 .

 5 Figure 5. Quantitation of Tspan9, CD63 and CD151, relative to CD9, in human platelets.(A) HEK 293T cells were transiently transfected with empty vector control, FLAG-tagged human CD9, CD63, CD151 or mouse Tspan9 (note that the Tspan9 antibody epitope is identical in protein sequence between human and mouse). Mouse anti-FLAG immunoprecipitates were western blotted with a rabbit anti-FLAG antibody and each tetraspanin quantitated, following ECL chemiluminescence, using the GeneGnome quantitative western blotting system from Syngene. These FLAG-tetraspanin samples were then compared to whole cell lysates of washed human platelets by western blotting for (B) CD9, (C) CD63, (D) CD151 and (E) Tspan9 and the relative intensities quantitated as in panel A. (F) The data in panels A-E, for platelets from six human donors of diverse ethnic backgrounds, were used to quantitate the relative expression levels of each tetraspanin in human platelets. The data are presented as mean relative expression levels and the error bars represent the standard deviation.

Biochemical 1 Biochemical 2 Biochemical 3

 123 Figure 1

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Acknowledgements

We are grateful to members of the Watson Lab for their advice and comments. We thank Michael Berndt and Masaaki Moroi for kindly providing the anti-GPIbD and GPVI mAbs, respectively, Jon Frampton, John Gordon, Martin Rowe and Arthur Weiss for gifts of cell lines, Kate Fitzpatrick-Ellis for generating the Tspan9 construct, and Alex Mazharian and Sonia Severin for helping to establish the methodology for megakaryocyte culture from foetal liver. M.G. Tomlinson is supported by a MRC New Investigator Award and a Wellcome Trust Value in People Award; D. Colombo holds a BHF Studentship; S.P. Watson holds a BHF Chair; N.A. Watkins and W.H. Ouwehand are funded by grants from the 6 th Framework Program of the European Union (LSHM-CT-2004-503485) and from the National Institute for Health Research (NIHR) to NHS Blood and Transplant.

Disclosure of Conflicts of Interest

The authors state that they have no conflict of interest.