Adrien Rossary

Khelifa Arab

Dr Jean-Paul Steghens
email: jean-paul.steghens@chu-lyon.fr

Polyunsaturated fatty acids modulate NOX 4 anion superoxide production in human fibroblasts

Keywords: Docosahexaenoic acid, polyunsaturated fatty acids, NADPH oxidase, heme oxygenase, fibroblasts, reactive oxygen species reactive oxygen species, NOX: NADPH oxidase, DUOX: dual oxidase, PUFA: polyunsaturated fatty acid, DPI: diphenyl iodonium, SOD: super oxide dismutase, XO

The strong reactive oxygen species (ROS) production, part of an antioxidant response of human fibroblasts triggered by docosahexaenoic acid, served as a model for deciphering the relative contribution of NADPH oxidase (NOX) to ROS production, as the role of this enzymatic system remains controversial. Using hydroxyethidium fluorescence for fibroblast ROS production, RT-PCR for NOX 4 mRNA quantification, and mRNA silencing, we show that ROS production evolves in parallel with the catalytic activity of NOX and is suppressed by siNOX 4 silencing. Apocynin and plumbagin, specific inhibitors of NOX, prevent ROS production in this cellular model and confirm the role of NOX 4 for this production. Furthermore we show that, in cell lysates, NOX 4 activity can be modulated by polyunsaturated fatty acids (PUFAs) at the micro molar level in the presence of calcium: NOX 4 activity is increased by arachidonic acid (around 175% of control), and conjugated linoleic acid is a potent inhibitor (50% of control).

Unexpectedly, intracellular superoxide dismutase (SOD) does not participate in the modulation of this ROS production and the opposite effects of some PUFA, described in our experiments, could suggest another way of regulating NOX activity.

Introduction

Several novel enzymes, homologs of gp91 phox , the catalytic unit of the protein complex responsible for the oxidative burst in polymorphonuclear cells, have been identified in mammals [START_REF] Meier | The cytochrome b-558 molecules involved in the fibroblast and polymorphonuclear leucocyte superoxide-generating NADPH oxidase systems are structurally and genetically distinct[END_REF][START_REF] Cheng | Homologs of gp91phox: cloning and tissue expression of NOX 3, NOX 4, and NOX 5[END_REF][START_REF] Rao | Expression of nonphagocytic NADPH oxidase system in the ocular lens[END_REF][START_REF] Shiose | A novel superoxide-producing NAD(P)H oxidase in kidney[END_REF]. The identification of new members of this family of enzymes has been an important step in understanding the role of reactive oxygen species (ROS) in the regulation of many biological processes, although their detailed involvement has not yet been fully defined. They are able to produce O 2 .-, which more and more seems implicated in cell signal transduction and communications [START_REF] Lambeth | NOX enzymes and the biology of reactive oxygen[END_REF]. For instance, NADPH oxidases (NOXs) may serve as an oxygen sensor [START_REF] Jones | NADPH oxidase : a universal oxygen sensor ?[END_REF], may be required for cell proliferation [START_REF] Arnold | Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase NOX 1[END_REF][START_REF] Abid | NADPH oxidase activity is requierd for endothelial cell proliferation and migration[END_REF], may participate in vascular reactivity or smooth muscle contractility [START_REF] Souza | Vascular NAD(P)H oxidase is distinct from the phagocytic enzyme and modulates vascular reactivity control[END_REF][START_REF] Thabut | Tumor necrosis factor-α increases airway smooth muscle oxidants production through a NADPH oxidase-like system to enhance myosin light chain phosphorylation and contractility[END_REF], regulate the expression of some proteins [START_REF] Rupin | Role of NADPH oxidase-mediated superoxide production in the regulation of E-selectin expression by endothelial cells subjected to anoxia/reoxygenation[END_REF][START_REF] Fan | Role of neutrophil NADPH oxidase in the mechanisim of tumor necrosis factor-α-induced NF-κB activation and intracellular adhesion molecule-1 expression in endothelial cells[END_REF], and play a role in signal transduction [START_REF] Mahadev | The NAD(P)H oxidase homolog NOX 4 modulates insulin-stimulated generation of H2O2 and plays an integral role in insulin signal transduction[END_REF]. NOXs and Dual oxidases (DUOX) generate ROS in a regulated manner in response to different signals such as growth factors, cytokines, calcium, etc. Surprisingly, overproduction of ROS due to NOX dysfunction is now suggested to be associated with several diseases, leading to increased bioavailability of ROS and subsequent oxidative damage [START_REF] Meyer | A central role for the endothelial NADPH oxidase in atherosclerosis[END_REF][START_REF] Kaminski | Oxidative stress and neutrophil activation -the two keystones of ischemia/reperfusion injury[END_REF][START_REF] Park | NAD(P)H oxidase inhibitor prevents blood pressure elevation and cardiovascular hypertrophy in aldosterone-infused rats[END_REF][START_REF] Pedruzzi | NAD(P)H oxidase NOX 4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells[END_REF].

Recently we described the induction of glutathione synthesis by human fibroblasts triggered with docosahexaenoic methyl ester (DHA met) [START_REF] Arab | Docosahexaenoic acid enhances the antioxidant response of human fibroblasts by upregulating g-glutamyl-cysteinyl ligase and glutathione reductase[END_REF]. The antioxidant response enhanced by human fibroblasts triggered with DHA met was associated with strong production of ROS. While fatty acids are frequently described as potential NOX activators in endothelial cells or neutrophils [START_REF] Cui | Arachidonic acid activates c-jun N-terminal kinase through NADPH oxidase in rabbit proximal tubular epithelial cells[END_REF][START_REF] Zafari | Arachidonic acid metabolites mediate angiotensin IIinduced NADH/NADPH oxidase activity and hypertrophy in vascular smooth muscle cells[END_REF][START_REF] Cherny | Activation of NADPH oxidase-related proton and electron currents in human eosinophils by arachidonic acid[END_REF][START_REF] Shao | Lipid rafts determine efficiency of NADPH oxidase activation in neutrophils[END_REF][START_REF] Rouhanizadeh | Oxidized -1-palmitoyl-2-arachidonoyl -sn -glycero -3phosphorylcholine induces vascular endothelial superoxide production: implication of NADPH oxidase[END_REF], and more and more data report on NOX activity in fibroblasts [START_REF] Dhaunsi | Very long chain fatty acids activate NADPH oxidase in human dermal fibroblasts[END_REF][START_REF] Colston | H2O2 activates NOX 4 through PLA2-dependent arachidonic acid production in adult cardiac fibroblasts[END_REF][START_REF] Dhaunsi | NADPH oxidase in human lung fibroblasts[END_REF][START_REF] Cucoranu | NAD(P)H oxidase 4 mediates transforming growth factorβ1-induced differentiation of cardiac fibroblasts into myofibroblasts[END_REF], few results confirm the effect of fatty acids on fibroblasts and the type of NOX homolog involved in this ROS production. We were therefore interested in deciphering the relative contribution of NOX to ROS production in our cellular model triggered by polyunsaturated fatty acids (PUFAs).

Materials and methods

Chemicals

Cell culture

Human dermal fibroblasts, a gift from Dr Damour, were grown in DMEM containing 10% fetal calf serum (Gibco, Pontoise, 95613, France), fungizone2.5 µg/ml, penicillin 98 U/ml and streptomycin 98 µg/ml (Cambrex, Emerainville, 77184, France). Cultures were maintained at 37°C in a humidified atmosphere with 5% CO2. Cells were harvested after trypsinisation and three phosphate buffer saline (PBS) washes. The total cell lysates were obtained by two successive thawing-freezing cycles in Tris HCl 25 mM buffer (buffer A) pH 7.4 containing Tween 20 0.1%, with 15-second periods in an ultra-sound bath. The total cell lysates were stored at -80°C until analysis.

Protein measurement

Proteins were quantified by the bicinchonic acid method (BCA from Pierce, Interchim, Montluçon, 03103, France) according to the manufacturer's instructions.

HPLC analysis of hydroxyethidium (E_OH + )

Hydroethidine (HE), ethidium (E + ) and hydroxyethidium (E_OH + ), the oxidation product of HE by O 2 .-, were separated by HPLC according to a method modified from Zhao et al. [START_REF] Zhao | Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence[END_REF]. The HPLC system (Agilent HP 1100, Agilent technologies, Massy, 91745, France) was equipped with a fluorescence (Kontron SFM25, Paris 75012, France), or UV (Thermo Spectra system UV 6000 LP, Thermo Electron Corporation, Cergy-Pontoise, 95615, France), or mass spectrometer (Agilent LC/MSD SL) single quadrupole detector.

The mobile phase was ammonium acetate 15 mM, adjusted to pH 4 with acetic acid and 35% (v/v) of methanol. The stationary phase was a C18 reverse-phase column (Atlantis tm , 2.1X150 mm, dC18, 5µm, Waters SAS, Guyancourt, 78280, France).

Samples were positive controls of superoxide anion production obtained by incubation at 37°C of xanthine (500 µM) and xanthine oxidase (0.2 UI/ml) or cells and cell culture medium. One sample volume was mixed with 2 volumes of methanol chilled on ice and then centrifuged before injection. HE, E + , and E_OH + were separated by gradient analysis with a linear increase in methanol concentration from 35% to 90% over 13 min at a flow rate of 0.2 ml/min, with a 5 µl injection volume. In a first step, the elution was monitored by a variable UV detector at 284 and 360 nm and a fluorescence detector with excitation and emission wavelengths at 465 nm and 575 nm, respectively. For the mass spectrometer detector, detection was optimized in a positive electrospray ionization mode (ESI + ) with E_OH + and set at 350°C (drying gas), fragmentor at 175 V and capillary voltage at 3 kV. Chromatograms were recorded as total ion current (TIC) and in single ion monitoring mode at m/z + = 314 for ethidium, 330 for E_OH + , and 301 for the main fragment obtained after cleavage of E_OH + [START_REF] Zhao | Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence[END_REF] as a control. HPLC peak areas were normalized to protein concentration.

Cell culture and measurements on fibroblasts grown with DHA methyl ester or other

PUFAs

Fibroblasts were grown either for 4, 8, 24 or 48 hours in DMEM with 15 µM of DHA methyl ester (final concentration) or for 4 hours with AA, CLA and EPA at the same concentration.

In order to confirm the implication of NOX in ROS production, fibroblasts were grown in the presence of apocynin 2.5 µM or plumbagin 1 µM or DPI [START_REF] Shiose | A novel superoxide-producing NAD(P)H oxidase in kidney[END_REF]

Silencing of NOX 4

To confirm the involvement of NOX expression in DHA treatment, we inhibited NOX 4 expression with small interfering RNA (siRNA). Twenty-one nucleotide doublestranded siRNAs (from Qiagen HP genome wide siRNA databank, cat number : SI00117663 www.qiagen.com; 5 nM), targeting the NOX 4 sequence, were used. Nonsilencing fluorescein-labeled control (5 nM) was used as the negative siRNA control (scrambled siRNA).

SiRNA transfection to fibroblasts was performed according to the manufacturer's instructions and was monitored with the control siRNA labeled with fluorescein (scrambled siRNA). Quantitative RT-PCR was performed to compare NOX 4 mRNA degradation in the presence and absence of siNOX 4.

After 36 hours silencing, fibroblasts were triggered by DHA for 4 hours and cellular ROS production on whole cells was performed by flow cytometry.

Evaluation of cellular ROS production by flow cytometry

In order to assess the production of ROS and superoxide anion, fibroblasts were loaded with hydroethidine (HE). After 0, 4, 8, 24, and 48 hours of culture, cells were harvested and washed twice with PBS, incubated with 2 µM of HE in PBS at 37°C for 30 min.

Then the cell suspension was submitted to fluorescent flow cytometry analysis (FACScan flow cytometer, BD Biosciences) on a log scale for 10,000 events (cell counts). The oxidation of HE was measured, with the excitation set at 488 nm, as an increase in fluorescence at 585.

Statistical analysis

Statistical

Results

Effects of inhibitors and fatty acids on whole cell fluorescence at 4 hours

HPLC analysis of hydroxyethidium (E_OH + )

As described by Zhao et al. [START_REF] Zhao | Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence[END_REF], hydroethidine (HE) is known to produce a specific oxidation product, named hydroxyethidium, in the presence of superoxide anion. In

Silencing of NOX 4

To confirm the involvement of NOX 4 expression in response to DHA treatment, we inhibited NOX 4 expression with small interfering RNA (siRNA). reported the effect of only very long-chain fatty acids on NOX 2 activity [START_REF] Dhaunsi | Very long chain fatty acids activate NADPH oxidase in human dermal fibroblasts[END_REF]. In our model, with fibroblasts grown with DHA met (22:6 n-3), an increase in ROS production at 4 hours was observed [START_REF] Arab | Docosahexaenoic acid enhances the antioxidant response of human fibroblasts by upregulating g-glutamyl-cysteinyl ligase and glutathione reductase[END_REF]. This model therefore seemed appropriate for deciphering the relative contribution of NOX isoenzymes to this ROS production and for investigating the potential modulation of this catalytic activity by fatty acids.

Preliminary results showed that ROS production obtained with cell lysates incubated with NADPH was totally inhibited by DPI (a well known NOX inhibitor acting on FAD coenzyme) and with added SOD, the enzyme responsible for physiological O 2

.-

degradation. As demonstrated by nitrogen bubbling, this signal was also dependent on O 2 . The latter inhibition was within a very close range of those obtained with DPI or SOD, suggesting that a unique catalytic activity, dependent on a FAD coenzyme, required NADPH and O 2 for O 2 .

-production, as these ROS were SOD-sensitive.

Moreover, inhibition was in the same range for specific NOX inhibitors (plumbagin [START_REF] Ding | Inhibition of NOX 4 activity by plumbagin, a plant-derived bioactive naphtoquinone[END_REF] and apocynin) as well as for non specific inhibitors (DPI or SOD, specific for O 2 .-but not for NOX). These results strongly suggested that this ROS production was due essentially to NOX activity.

To confirm this hypothesis, we investigated the action of NOX inhibitors such as apocynin, plumbagin or DPI on whole cells triggered by DHA. As shown in figure 1A, apocynin, a specific NOX inhibitor which prevents NOX subunit assembly, totally inhibited ROS production under DHA, thus confirming the participation of NOX in the production of ROS at 4 hours. Moreover, plumbagin, described as a NOX 4 inhibitor [START_REF] Ding | Inhibition of NOX 4 activity by plumbagin, a plant-derived bioactive naphtoquinone[END_REF], also totally inhibited ROS production under DHA, thus defining NOX 4 as the NOX homolog of our fibroblasts.

Unlike apocynin, DPI acts by covalent binding to FAD; in this manner, it is a general inhibitor of all enzymes requiring FAD as a coenzyme. As described in the literature [START_REF] Riganti | Diphenyleneiodonium inhibits the cell redox metabolism and induces oxidatives stress[END_REF][START_REF] Li | DPI induces mitochondrial superoxide-mediated apoptosis[END_REF], DPI induces oxidative stress and apoptosis via mitochondrial O 2 .-production. Our results confirmed these observations, and also that DPI, active on many enzymes ex vivo, Lipids in general, and AA in particular, are known to interact with NOX in phagocytic and nonphagocytic cells [START_REF] Cui | Arachidonic acid activates c-jun N-terminal kinase through NADPH oxidase in rabbit proximal tubular epithelial cells[END_REF][START_REF] Zafari | Arachidonic acid metabolites mediate angiotensin IIinduced NADH/NADPH oxidase activity and hypertrophy in vascular smooth muscle cells[END_REF][START_REF] Cherny | Activation of NADPH oxidase-related proton and electron currents in human eosinophils by arachidonic acid[END_REF][START_REF] Shao | Lipid rafts determine efficiency of NADPH oxidase activation in neutrophils[END_REF][START_REF] Rouhanizadeh | Oxidized -1-palmitoyl-2-arachidonoyl -sn -glycero -3phosphorylcholine induces vascular endothelial superoxide production: implication of NADPH oxidase[END_REF][START_REF] Dhaunsi | Very long chain fatty acids activate NADPH oxidase in human dermal fibroblasts[END_REF], and a recent publication has implicated very long-chain fatty acids in the activation of NOX in some peroxisomal disorders of human dermal fibroblasts [START_REF] Dhaunsi | Very long chain fatty acids activate NADPH oxidase in human dermal fibroblasts[END_REF]. In a previous publication [START_REF] Rossary | Régulation de l'activité des NADPH oxydases en présence d'acides gras[END_REF], we investigated the effect of DHA, free or as a methyl ester, and of arachidonic acid (AA) on fibroblast extracts: in our experiments, neither calcium, described as a NOX 5 activator [START_REF] Banfi | A Ca 2+ -activated NADPH oxidase in testi, spleen, and lymph nodes[END_REF], nor arachidonic acid (AA) alone, known as a gp91 phox activator [START_REF] Cherny | Activation of NADPH oxidase-related proton and electron currents in human eosinophils by arachidonic acid[END_REF][START_REF] Shao | Lipid rafts determine efficiency of NADPH oxidase activation in neutrophils[END_REF], had any effect on NOX catalytic activity of fibroblast lysates. However AA associated with calcium strongly increased NOX activity (175% of control); this result recalls the work of Xiao-Lan Cui et al [START_REF] Cui | Arachidonic acid activates c-jun N-terminal kinase through NADPH oxidase in rabbit proximal tubular epithelial cells[END_REF].

is
According to these authors, AA activates the c-jun N-terminal kinase through NOX in rabbit proximal tubular epithelial cells. In our model, NOX catalytic activity was due to NOX 4, as demonstrated by RT-PCR. In agreement with previous results on Renox, the first name of NOX 4 [START_REF] Cui | Arachidonic acid activates c-jun N-terminal kinase through NADPH oxidase in rabbit proximal tubular epithelial cells[END_REF][START_REF] Cherny | Activation of NADPH oxidase-related proton and electron currents in human eosinophils by arachidonic acid[END_REF], NOX 4 was responsive to arachidonic acid and required Ca 2+ mobilization.

Unexpectedly, neither DHA (free or as a methyl ester) nor EPA, both with calcium, activated NOX on cell lysates [START_REF] Rossary | Régulation de l'activité des NADPH oxydases en présence d'acides gras[END_REF], whereas they strongly induced NOX activity on whole fibroblasts triggered for 4 hours (fig 1B). Very recently we showed that, in fibroblasts triggered by DHA met, DHA increased threefold and induced a profound change in total cell lipid composition [START_REF] Arab | Docosahexaenoic acid enhances the antioxidant response of human fibroblasts by upregulating g-glutamyl-cysteinyl ligase and glutathione reductase[END_REF] with a low cellular AA increase. These results strongly suggest that, when PUFA induce a huge O 2 .

-production, it could be due to release of AA by membranes, with subsequent NOX activation. Thus, this increase in

O 2 .
-production in the first 4 hours could play the role of an intracellular messenger, a role already suggested in the model by Xia-Lan Cui et al. in 1997 [START_REF] Cui | Arachidonic acid activates c-jun N-terminal kinase through NADPH oxidase in rabbit proximal tubular epithelial cells[END_REF] after AA activation and more recently by Colston et al. in 2005 [25]. A general mechanism involving AA and calcium should be in agreement with the results of Bouzidi et al. [START_REF] Bouzidi | Binding of arachidonic acid to myeloidrelated proteins (S100A8/A9) enhances phagocytic NADPH oxidase activation[END_REF],

who reported this association as a NOX activator with an effect mediated by the myeloid-related proteins (S100A8/A9) which bind both calcium and AA. Furthermore, the hypothesis that AA is released from membranes could explain the similar activation of NOX obtained with many lipids: according to Rouhanizadeh et al. [START_REF] Rouhanizadeh | Oxidized -1-palmitoyl-2-arachidonoyl -sn -glycero -3phosphorylcholine induces vascular endothelial superoxide production: implication of NADPH oxidase[END_REF], "the specific mechanism(s) by which ox-PAPC induces NADPH oxidase activity and O 2 .

-production remain to be defined and the effect may be similar to that reported for lysophosphatidylcholine (LPS) which stimulates monocyte chemoattractant protein-1 (MCP-1) expression and O 2 .

-production by NADPH oxidase. Similar activation is also reported for other lysolipids such as phosphatidic acid and platelet-activating factors, which induce mitogenic signaling pathways."

We already showed that fibroblasts triggered by DHA [START_REF] Arab | Docosahexaenoic acid enhances the antioxidant response of human fibroblasts by upregulating g-glutamyl-cysteinyl ligase and glutathione reductase[END_REF] and other PUFAs, except CLA [START_REF] Arab | Conjugated linoleic acid, unlike other unsaturated fatty acids, strongly induces glutathione synthesis withot any lipoperoxidation[END_REF], produce superoxide anion and activate an antioxidant response. Now we demonstrate that this ROS production is due to NOX 4 activation. During this antioxidant response, we were unable to detect any change either in mRNA SOD1 expression or total cellular catalytic SOD activity. For this reason, regulation of ROS production by cellular SOD seems to be excluded, at least during the relatively short time periods of our experiments. However, during the antioxidant response triggered by different PUFAs [START_REF] Arab | Docosahexaenoic acid enhances the antioxidant response of human fibroblasts by upregulating g-glutamyl-cysteinyl ligase and glutathione reductase[END_REF][START_REF] Arab | Conjugated linoleic acid, unlike other unsaturated fatty acids, strongly induces glutathione synthesis withot any lipoperoxidation[END_REF], we demonstrate the induction of HO-1, a typical enzyme of the antioxidant response as it contains some antioxidant responsive elements in its genepromoter region. HO-1 produces CO through heme degradation and its induction strongly suggests that, apart from regulation at the transcriptional level [START_REF] Martyn | Functional analysis of NOX 4 reveals unique characteristics compared to other NADPH oxidases[END_REF] confirmed by a decrease of NOX 4 and p22 phox mRNA expression, a further factor of regulation for NOX activity, as suggested recently [START_REF] Rossary | Régulation de l'activité des NADPH oxydases en présence d'acides gras[END_REF], could be through CO inhibition [START_REF] Kim | CO as a cellular signaling molecule[END_REF] or heme degradation [START_REF] Taillé | Induction of heme oxygenase-1 inhibits NAD(P)H oxidase activity by down-regulating cytochrome b558 expression via the reduction of heme availability[END_REF].

As CLA is the only PUFA able to induce an antioxidant response (testified by glutathione synthesis up-regulation) without NOX 4 activation, our cellular model may be also useful to explore the different facets and steps leading to a "physiological" defense mechanism, the antioxidant response, and question the exact role of ROS for its signaling.

2 [

 2 Chemicals were obtained as follows: diphenyl iodonium (DPI), super oxide dismutase (SOD), xanthine, plumbagin and apocynin from Sigma-Aldrich, (Sigma-Aldrich, Saint Quentin Fallavier, 38297, France); arachidonic acid (AA, 20:4 n-6), docosahexaenoic acid (DHA, 22:6 n-3) and methyl ester (DHA met), conjugated linoleic acid (CLA, 18:9Z, 11E]), eicosapentaenoic acid (EPA, 20:5n-3), from Cayman Chemical (Interchim, Montluçon, 03103, France); xanthine oxidase (XO), NADPH from Roche (Roche, Meylan, 38242, France); hydroethidine (HE) from Fluoprobe (Interchim, Montluçon, 03103, France). All other reagents were of analytical grade. All concentrations are final concentrations in incubation or culture medium.

  We investigated the fluorescence of ROS production of fibroblasts triggered for 4 hours with DPI (data not shown), apocynin, plumbagin and/or with fatty acids. The vehicle controls -DMSO for DPI, ethanol for apocynin, plumbagin and DHA met -had no significant effect on ROS production. DPI increased production of ROS at 4 hours to 143 ± 4% of control, and this increase was cumulative with that due to DHA, reaching 185 ± 1%.On the other hand, E_OH + fluorescence induced by DHA alone was 165 ± 18 % of control and that of the inhibitors, alone or with DHA, was between 104 and 112 % of control (fig 1A). The effects of fatty acids on fluorescence produced by whole cells (through NOX activity) are shown on fig 1B: both DHA and EPA significatively increased cell fluorescence (148 ± 23% and 143 ± 16%) and only AA increased signal up to 202 ± 10%. Fluorescence of fibroblasts triggered by CLA for 4 hours was lower that than of control, but not significatively.

2 and 4 )

 24 order to demonstrate the production of superoxide by fibroblasts triggered by fatty acids, we performed LC/MS analysis for hydroxyethidium on different samples including culture media and cells. As a positive control for superoxide anion production, we used a xanthine-xanthine oxidase assay for which typical chromatograms are shown in figure 2A. The TIC chromatogram is shown on the upper graph, and specific ion current chromatograms are displayed for m/z + 314 = E+ (fig 2A2), 330 = E_OH+ (fig 2A3), and 301, the main fragment of 330 (fig 2A4). Obviously m/z + 330 and 301 have the same retention time (RT) which is different of that both of HE (RT = 11.04) and E + (RT = 8.85) . Figure 2B shows similar chromatograms obtained for fibroblasts triggered for 2 hours with AA 15 µM and HE 5 µM. Figure 2C displays the results (mean of triplicate) for culture media, in the same conditions, with a 2.4-fold increase in E_OH+ production and a production similar to ethanol control if SOD (15UI/ml) is added. These experiments are a further confirmation of superoxide anion production by NOX of fibroblasts triggered by different PUFAs. Time course analysis of cell culture with DHA methyl ester We grew human fibroblasts with 15 µM of DHA methyl ester (DHA met) up to 48 hours. Fluorescent measurements of O 2 .-production were performed at 0, 4, 8, 24, and 48 hours. At the same time points, different mRNAs were quantified by quantitative RT-PCR. During this time period, neither SOD 1 mRNA expression nor total SOD catalytic activity were significatively different from control (data not shown). O 2 .-production measured by fluorescence of E_OH + (fig 3A) peaked at 4 hours (174 ± 28%) and then decreased until 48 hours. Cell culture controls (fibroblasts at time 0 and fibroblasts grown with ethanol for 48 hours) were not significatively different from each other. Quantification of mRNA for NOX 4 and p22 phox First, we investigated the mRNA expression of the different NOX homologs (NOX 1, in human fibroblasts by agarose gel electrophoresis and RT-PCR. Amplification was detected only with NOX 4 and p22 phox primers on fibroblasts whereas NOX 2 primers were able to detect mRNA expression in white blood cells as a control. Second, the time course of mRNA expression for NOX 4 showed a significant decrease at 48 hours (fig 3B). During the same time period, p22 phox mRNA expression decreased gradually (fig 3C), and the absence of mRNA expression for NOX 1 and 2 was confirmed.

  After 36 hours silencing, quantitative RT-PCRs were performed to compare NOX 4 mRNA degradation in the presence and absence of siNOX 4 (fig 4A). Expression remained only 6% of basal level. Fibroblasts treated by siNOX 4 were triggered by DHA for 4 hours. Neither NOX catalytic activity on total cell lysates (data not shown) nor fluorescent ROS production performed on whole cells (fig 4B) showed any activation under DHA and siNOX 4. Discussion NOXs, cellular sources of reactive oxygen species, use O 2 and NADPH as substrates and FAD as coenzyme. As sources of ROS, they are detectable by lucigenin luminescence, a fast tool for screening of NOX catalytic activity. However lucigenin specificity is often questioned and we were strongly advised to use hydroxyethidium analysis either by fluorescence or LC/MS for O 2 .-production. NOXs, particularly NOX 1 and 2, areclaimed to be activated by some fatty acids[START_REF] Cui | Arachidonic acid activates c-jun N-terminal kinase through NADPH oxidase in rabbit proximal tubular epithelial cells[END_REF][START_REF] Zafari | Arachidonic acid metabolites mediate angiotensin IIinduced NADH/NADPH oxidase activity and hypertrophy in vascular smooth muscle cells[END_REF][START_REF] Cherny | Activation of NADPH oxidase-related proton and electron currents in human eosinophils by arachidonic acid[END_REF][START_REF] Shao | Lipid rafts determine efficiency of NADPH oxidase activation in neutrophils[END_REF][START_REF] Rouhanizadeh | Oxidized -1-palmitoyl-2-arachidonoyl -sn -glycero -3phosphorylcholine induces vascular endothelial superoxide production: implication of NADPH oxidase[END_REF]. In human fibroblasts, Dhaunsi et al.

  not relevant for the investigation of NOX activity in intact cells. Quantification by RT-PCR of mRNA for NOXs confirmed that mRNA for NOX 4 was the only NOX homolog mRNA present to explain ROS production. After 36 hours silencing with siRNA for NOX 4, quantitative RT-PCR showed NOX 4 mRNA degradation (fig 4A). Furthermore fibroblasts silenced by siNOX 4 (for 36 hours) and triggered by DHA for 4 hours displayed no cellular ROS production (fig 4B) unlikefibroblasts silenced by the scrambled siRNA. Associated with the fact that NOX catalytic activity measurement[START_REF] Rossary | Régulation de l'activité des NADPH oxydases en présence d'acides gras[END_REF] and E_OH+ fluorescence on whole cells show a similar pattern, these results strongly confirm the role of NOX 4 as the only NOX isoenzyme for ROS production in human fibroblasts and are in agreement with very recent publications on fibroblasts[25,27;33].

5 'Figure 1 A

 51 Figure 1

Figure 4 A

 4 Figure 4

Figure 1 :

 1 Figure 1: Effects of specific inhibitors (A) and fatty acids (B) on whole cells (fibroblasts grown for 4 hours). Each bar corresponds to the mean of at least three assays (*p<0.05, **p<0.01, ***p<0.001). A: Apocynin or plumbagin inhibition of the fluorescence emitted by fibroblasts triggered by DHA. Controls correspond to fibroblasts grown for 4 hours with ethanol (negative), DHA (positive) or inhibitor alone. B: Enhancement of fluorescence of fibroblasts triggered with 15µM of AA, DHA or EPA, for 4 hours. Unlike other PUFAs, CLA decreases the fluorescence. Control corresponds to fibroblasts triggered with ethanol for 4 hours.

Figure 2 :

 2 Figure 2: LC/MS analysis of hydroxyethidium produced from O 2 .-with hydroethidine. A and B: chromatograms of a xanthine-xanthine oxidase assay and of a total cell lysate, respectively. Total ion current chromatograms are shown in A1 and B1. Specific chromatograms are displayed at m/z + = 314 for ethidium (at RT = 8.8) in A2 and B2, at m/z + = 330 for hydroxyethidium (at RT = 9.6) in A3 and B3 and at m/z + = 301 for the main fragment of hydroxyethidium (at RT = 9.6) in A4 and B4. C: hydroxyethidium production in cell culture medium by fibroblasts triggered for 2 hours by arachidonic acid at 15 µM, with or without SOD at 15 UI/ml. Each bar is the mean of triplicates (*p<0.05, ***p<0.001).

Figure 3 :

 3 Figure 3: Time course of ROS production (A) and mRNA expression (B,C) in fibroblasts grown with DHA methyl ester 15 µM, for 48 hours. Ethanol is the vehicle control at 48 hours. All data are expressed as a ratio with the reference at time 0. (*p<0.05, **p<0.01). A: Evaluation of Total ROS production by hydroxyethidium fluorescence. B: NOX 4 mRNA expression. C: p22 phox mRNA expression.

Figure 4 :

 4 Figure 4: Silencing of NOX 4 in human fibroblasts. A: Quantitative RT-PCR of NOX 4 mRNA after 36 hours silencing in presence and absence of siNOX 4 on fibroblasts. B: ROS production measured by hydroxyethidium on whole cells triggered by DHA for 4 hours, after 36 hours silencing in presence and absence of siNOX 4. Nonsilencing control (5 nM) was used as the negative siRNA control (scrambled siRNA). (*p<0.05, **p<0.01).

  analysis was carried out by GraphPad analysis (version 2.1). All data are expressed as means ± SE. Comparisons among groups were performed by the Student's t-test or one-way ANOVA, with Dunnett's multiple range tests. The significance level was 0.05.

Table 1

 1

	Name	Accession number	Primer sequences	Fragment lentgh (pb)
	β actin NM_001101	Forward Reverse

Biochemical Journal Immediate Publication. Published on 2 May 2007 as manuscript BJ20061009 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements

We thank Sarah Somerville (IARC, Lyon France) for careful English editing. Biochemical Journal Immediate Publication. Published on 2 May 2007 as manuscript BJ20061009


Karim Hnia

Dora Zouiten

Sonia Cantel

Delphine Chazalette

Gérald Hugon

Jean- Alain Fehrentz

Ahmed Masmoudi

Ann Diment

Janice Bramham

Dominique Mornet

Steve J Winder
email: s.winder@sheffield.ac.uk

ZZ DOMAIN OF DYSTROPHIN AND UTROPHIN: TOPOLOGY AND MAPPING OF A β-DYSTROGLYCAN INTERACTION SITE

Keywords: cysteine-rich region, Duchenne muscular dystrophy, dystroglycan, dystrophin, utrophin, zinc binding, AAS atomic absorption spectroscopy

Dystrophin forms part of a vital link between actin cytoskeleton and extracellular

matrix via the transmembrane adhesion receptor dystroglycan. Dystrophin and its autosomal homologue utrophin, interact with β -dystroglycan via their highly conserved carboxyterminal cysteine-rich regions, comprising WW, EF hand and ZZ domains. The EF hand region stabilises the WW domain providing the main interaction site between dystrophin or utrophin and dystroglycan. The ZZ domain, containing a predicted zinc finger motif, stabilises the WW and EF hand domains and strengthens the overall interaction between dystrophin or utrophin and β-dystroglycan. Using bacterially expressed ZZ domain we demonstrate a conformational effect of zinc binding to the ZZ domain, and identify two zinc binding regions within the ZZ domain by SPOTs overlay assays. Epitope-mapping of the dystrophin ZZ domain was carried out with new monoclonal antibodies by ELISA, overlay assay and immunohistochemistry. One monoclonal antibody defined a discrete region of the ZZ domain that interacts with β-dystroglycan. The epitope was localised to the conformationaly sensitive second zinc binding site in the ZZ domain. Our results suggest that residues 3326-3332 of dystrophin, forms a crucial part of the contact region between dystrophin and β-dystroglycan and provides new insight into ZZ domain organisation and function.

INTRODUCTION

Dystrophin loss leads to the progressive muscle wasting disease Duchenne muscular dystrophy (DMD). Following the identification of the DMD gene by Kunkel and colleagues by positional cloning in the late 1980's [START_REF] Koenig | Complete cloning of the Duchenne muscular-dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals[END_REF] it was realised that the protein product dystrophin was a large cytoskeletal protein with homology to the spectrin family of proteins [START_REF] Hoffman | Dystrophin: the protein product of the Duchenne muscular dystrophy locus[END_REF][START_REF] Koenig | The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein[END_REF][START_REF] Davison | Alpha-actinins and the DMD protein contain spectrin-like repeats[END_REF]. Like spectrin and α-actinin, dystrophin and utrophin can provide a link between the F-actin cytoskeleton and transmembrane proteins, and in the specific case of dystrophin through the transmembrane adhesion receptor protein dystroglycan, and so to the extracellular matrix [START_REF] Ervasti | A role for the dystrophin glycoprotein complex as a transmembrane linker between laminin and actin[END_REF]. The much rarer and often milder allelic form of DMD known as Becker muscular dystrophy (BMD) allows the expression of a variable amount of altered dystrophin protein [START_REF] Koenig | The molecular-basis for Duchenne versus Becker muscular-dystrophy -correlation of severity with type of deletion[END_REF]. Analysis of the genotype/phenotype relationship for deletion mutations in these patients has revealed valuable information about the functional importance of different regions of the dystrophin protein [START_REF] Beggs | Exploring the Molecular-Basis For Variability Among Patients With Becker Muscular-Dystrophy -Dystrophin Gene and Protein Studies[END_REF].

Dystrophin and its autosomal homologue utrophin [START_REF] Tinsley | Primary structure of dystrophin-related protein[END_REF] comprise 4 main regions. From N to C terminus: an actin-binding domain, a series of spectrin-like repeats, a cysteine-rich region, and a dimeric coiled coil region. The actin-binding domain and cysteine-rich region show highest overall sequence similarity between the two proteins, and the mutational spectrum from analysis of BMD patients points to a greater functional importance for these regions in dystrophin.

Mutations in the actin binding region give rise to severe BMD, whilst despite the presence of some dystrophin protein product, mutations in the cysteine-rich region give rise to an DMD phenotype [START_REF] Beggs | Exploring the Molecular-Basis For Variability Among Patients With Becker Muscular-Dystrophy -Dystrophin Gene and Protein Studies[END_REF]. These findings from human subjects were recapitulated in an elegant series of experiments using transgenic mice expressing dystrophin constructs containing a similar spectrum of deletion and mutations, reviewed in [START_REF] Chamberlain | The structure and function of dystrophin[END_REF]. The human and mouse data demonstrate that from a functional point of view the physical links between dystrophin and the actin cytoskeleton via the actin-binding domain, and the link between cysteine-rich region and dystroglycan are crucial to dystrophin function. The spectrin repeats are to a large extent redundant, and only mild phenotypes are associated with loss of the C-terminal coiled coil repeats. Within the cysteine-rich region, arguably the most important region of dystrophin, are three distinct domains that contribute to the interaction between dystrophin and the c terminus of dystroglycan: the WW, EF-hand and ZZ domains [10][START_REF] Bork | The WW domain: a signalling site in dystrophin?[END_REF][START_REF] Ponting | ZZ and TAZ: new putative zinc fingers in dystrophin and other proteins[END_REF]. The WW domain is the primary site of interaction between dystrophin (or utrophin) and the last c-terminal 15 amino acids of β-dystroglycan [13,[START_REF] Jung | Identification and characterization of the dystrophin anchoring site on β-dystroglycan[END_REF]. The stability of the WW domain and its affinity for β-dystroglycan is greatly increased by the EF

MATERIALS AND METHODS

Peptides and constructs -All peptides used were obtained as previously described [START_REF] Pons | Utrophin localisation in normal and dystrophin-deficient heart[END_REF].

Overlapping 7 residue synthetic peptides were produced to span the dystrophin or utrophin ZZ domains, D3311-3342 and U3068-3099 respectively as represented in Fig. 1. Dystrophin and utrophin differ by only two isoleucines in this region I3322 and I3336 (dystrophin numbering) replaced by two valine residues in utrophin sequences (V3079 and V3093). A ZZ domain construct comprising residues 3049-3114 of human utrophin was generated by PCR including Nde1 and Sal1 restriction sites and cloned into the modified pET vector pSJW1 [START_REF] Winder | Protein production in 3 different expression vectors from a single PCR product[END_REF]. Point mutations in cysteine residues were generated by overlap extension mutagenesis. The non-fusion protein was expressed in E. coli BL21(DE3) and purified from inclusion bodies using CM Sephadex and Gel filtration chromatography in the presence of 6M urea. Urea was removed by step-wise dialysis into 10mM PIPES pH 7.0, 1mM ascorbate with either 1mM ZnCl 2 or 1mM phenanthroline. The cytoplasmic domain only of mouse β-dystroglycan, residues 775-895, was expressed in E. coli BL21(DE3) and purified as described previously [START_REF] Chen | Direct interaction of β-dystroglycan with F-actin[END_REF].

Antibodies -The dystrophin ZZ domain sequence (3311-3342) was linked to keyhole limpet haemocyanin (KLH) and injected into mice according to a previously described protocol [20].

Spleen cells were fused to myeloma to obtain specific hybridomas and antibodies were selected by ELISA using the antigenic peptide. The dystrophin and utrophin C-terminal polyclonal antibodies (H4 and K7 respectively) were raised in New Zealand rabbits by repeated intradermal injections. Peptides of the last 16 and 11 C-terminal amino acids of dystrophin or utrophin respectively were conjugated via a cysteine residue to KLH and used as antigen. Antibodies were purified and characterised as previously described [21]. Tissues -All tissues; muscle, lung and sciatic nerve, were dissected immediately after death and rapidly frozen in 2-methylbutane, cooled in liquid nitrogen and stored at -80°C until use. Crude muscle membrane homogenate was prepared using freshly dissected muscle according to a previous protocol [START_REF] Fabbrizio | Re-evaluation of the distributions of dystrophin and utrophin in sciatic nerve[END_REF].

ELISA -ZZ domain sequence synthetic peptide (3311-3342) was coated on micro-titre plates at 0.1 mg/ml, and incubated at 4°C overnight in PBS. After washes, the plates were blocked with 0.05% Tween 20 in PBS buffer containing 1% BSA for 20 min at 37°C, then incubated with each monoclonal antibody for 2h at 37°C. Reaction was revealed by adding alkaline phosphatase labelled anti-mouse IgG for 30 min at 37°C and signal was detected at 405 nm using pnitrophenylphosphatase (Sigma) in diethanolamine buffer, pH 9.8. ELISA assay was performed to test epitope mapping for each monoclonal antibody using the 7 residue synthetic peptides corresponding to the overlapping sequences spanning the D3311-3342 or U3068-3099 ZZ domain. Freshly prepared peptide, 7.7 mg/ml in 0.05% Tween 20 PBS buffer, was coated onto micro-titre plates in triplicate then incubated with each monoclonal antibody to test their reactivity. For competitive ELISA experiments micro-titre plates were coated in triplicate with peptide 19. After blocking as above, plates were incubated at 4°C overnight with either crude muscle membrane homogenate (0.1 mg/ml), purified β-dystroglycan (9µg/ml) or with blocking buffer. Monoclonal antibody (13D2) and the commercial β-dystroglycan antibody (1:500, 43DAG/8D5 Novocastra) were added successively. Reactions were revealed as described above.

Immunofluorescence -Cryostat sections (10 µm) of unfixed muscle (Rabbit and Torpedo marmorata) were labelled with the antibodies described above. Immunoreactivity was detected with Cy3-conjugated sheep anti-mouse IgG (Euromedex).

Western blotting -Fresh extracts were prepared from 0.01 g of muscle tissue homogenised in 150 µl of 5% SDS buffer (50 mM Tris/HCl pH 8.0, 10 mM EDTA) supplemented with 1% of trypsin inhibitor and 1% saponin. After centrifugation (10 min at 13000 g), supernatant protein concentrations were estimated using the BCA protein assay (Pierce). Samples were separated in duplicate by SDS-PAGE (3-10% or 5-15%), transferred to nitrocellulose and developed with appropriate antibody essentially as described previously [13] All monoclonal antibodies were tested in competition with an excess of corresponding synthetic peptides (1mg of peptide per µg/ml of monoclonal antibody [START_REF] Khurana | The subcellular distribution of chromosome 6-encoded dystrophin-related protein in the brain[END_REF]) on both cryostat sections or western blots. All monoclonal antibodies were completely blocked by their specific peptides (Fig 4).

Far western blotting overlay -Blotted nitrocellulose sheets (0.2 µm) containing total muscle protein extract were blocked for 1 h at room temperature with 10 mM triethanolamine, pH 7.6, 140 mM NaCl, 1mM CaCl2, and 1mM MgCl2 containing 5% BSA and then pre-incubated with peptide 19 (0.1 mg/ml) in 0.5 % BSA containing 1mM dithiothreitol or 0.5% BSA alone (control) for 24 hours. Crude membrane homogenates were applied to the nitrocellulose membrane overnight at 4°C with gentle agitation. Membranes were blocked in Tris buffer containing 0.1% Tween 20, 3% BSA (w/v). After washing, proteins were revealed with dystrophin C-terminal antibody (H4).

ZZ domain zinc binding -Circular dichroism (CD) was carried out on a Jobin Yvon CD6 spectrophotometer using 0.6 ml cuvettes. ZZ domain was analysed in the presence of 1mM ZnCl 2 or 1mM 1,10-phenanthroline. Aliquots of similar samples were also subjected to SDS-PAGE in the absence or presence of zinc. Atomic absorption spectroscopy (AAS) was performed on a Unicam Solaar 929 spectrometer using ZnCl 2 solutions prepared in the same buffer as the ZZ domain as standards at a wavelength of 213.9 nm with a bandpass of 0.5 nm and taking the average of 3 readings at 1 sec intervals. For metal ion competition assays, 10µg of purified ZZ was spotted onto nitrocellulose and allowed to air dry. Membranes were probed with 65 ZnCl 2 as described previously [START_REF] Schiff | Characterization of a Zinc Blotting Technique: Evidence that a Retroviral Gag Protein Binds Zinc[END_REF] using 200µM 65 ZnCl 2 (20µCi/ml) and an excess of the indicated counter ions. Autoradiographs were quantified by densitometry and data expressed relative to 65 ZnCl 2 alone = 1.0. A SPOTs peptide array comprising 46 peptides of 20 residues each differing by 1 amino acid covering the complete utrophin ZZ domain (3049-3114) was synthesised as described previously [13]. 65 ZnCl 2 binding was performed as described above; a separate control experiment was performed in the presence of excess 10mM ZnCl 2 .

RESULTS

ZZ domain zinc binding:

Like other proteins that bind divalent cations, purified bacterially expressed utrophin ZZ domain showed a small upward mobility-shift on SDS-PAGE when the protein was prepared in the presence of zinc, whereas ZZ domain prepared in the presence of the chelator 1,10-phenanthroline ran slightly faster (Fig 2A inset). This is indicative of a structural change induced by zinc binding altering the SDS-PAGE mobility, a phenomenon commonly seen in cation binding proteins, such as calmodulin when subjected to SDS-PAGE in the presence or absence of calcium, for example see [25]. A conformational change upon zinc binding was also substantiated by CD. The CD spectrum of ZZ domain in the presence of zinc showed a slightly higher propensity toward helical structure compared to ZZ domain with phenanthroline (Fig 2A ).

Determination of divalent cation specificity for the ZZ domain by 65 Zn-overlay revealed most competition for zinc binding by excess zinc or copper (Fig 2B) which is in keeping with the complexing ability of divalent cations of the transition metals which generally follow the Irving-Williams series Mn<Fe<Co<Ni<Cu>Zn which in turn are all greater than the alkaline earth metals, Ca 2+ , Mg 2+ etc [START_REF] Dawson | Data for Biochemical Research[END_REF]. This trend is also evident on quantification of this and two other independent experiments (Fig 2C). These data suggest the ZZ domain is a bona fide zinc binding protein. The original description of the ZZ domain highlighted 4 conserved cysteine residues in the cysteine rich regions of dystrophin and utrophin [START_REF] Ponting | ZZ and TAZ: new putative zinc fingers in dystrophin and other proteins[END_REF] leading to the suggestion that the ZZ domain might adopt a structure equivalent to half of a LIM domain [27]. ZZ domains in most other proteins however, contain 6 conserved cysteine residues in positions that could coordinate zinc with additional histidine and other potential liganding residues (Fig 1B) [START_REF] Ponting | ZZ and TAZ: new putative zinc fingers in dystrophin and other proteins[END_REF]. 65 Zn-overlay assays: We therefore mutated each of the five cysteine residues in the utrophin ZZ domain and performed 65 Zn-overlay assays. No qualitative difference was observed in zinc binding as determined by autoradiography suggesting that the ZZ domain did not adopt a half LIM domain-like conformation, as removal of any one cysteine should prevent zinc coordination.

For the constructs where enough soluble material could be produced, we further examined the ZZ domain zinc binding using AAS. The wild type purified protein, refolded from urea, contained sub-stoichiometric quantities of zinc, 0.65±0.03 mol Zn/mol ZZ suggesting that the protein was not all correctly folded. However the zinc content of the utrophin ZZ domain mutant C3071A appeared stoichiometric at 2.14±0.1 mol Zn/mol ZZ: thus two rather than one zinc ion bound to the ZZ domain. We hypothesise that the mutation of C3071 to alanine removed a 'free' uncoordinated cysteine residue that led to inappropriate zinc coordination, possible disulphide formation and aggregation of the domain leading to insolubility. To confirm the stoichiometry of zinc binding determined by AAS, we used a peptide SPOTs array of 20 amino acid peptides each differing from the previous by one amino acid spanning the 66 residues of the ZZ domain to examine 65 Zn binding. As shown in Figure 3A, two distinct regions of the utrophin ZZ domain bound zinc, in part corroborating the AAS data. Zinc is typically coordinated by Cys and His, but Asp, Glu are also able to act as ligands with water contributing in catalytic situations [START_REF] Vallee | Zinc coordination, function, and structure of zinc enzymes and other proteins[END_REF]. In the first zinc-binding region, peptides 6-16, contained a glutamic acid, a histidine, two of the conserved cysteines and a further cysteine unique to dystrophin and utrophin. Each potential liganding residue was separated by two residues which could theoretically accommodate the coordination of a zinc ion (Fig 3A). The other region, peptides 30-37, contained the second pair of conserved cysteines, a histidine and an aspartic acid (Fig 3A). Rather than a zinc finger, the ZZ domain may therefore contain two zinc 'knuckles' as depicted in Figure 3B, though other conformations are possible. The binding of zinc to the peptides regions described above must rely on some local sequence specificity and not the simple presence of potential coordinating ligands, as peptides [START_REF] Lenk | A cysteine 3340 substitution in the dystroglycan-binding domain of dystrophin associated with Duchenne muscular dystrophy, mental retardation and absence of the ERG b-wave[END_REF][44][45][46], which all contain a pair of histidines and a pair of cysteines in a configuration one might expect to be able bind zinc, showed no labelling with 65 Zn (Fig 3A). The synthesis of SPOTs peptides is not quantitative, therefore strength of binding interaction cannot necessarily be inferred from the intensity in any one SPOT as the quantities of peptide in each SPOT may differ.

However, the presence of radioactivity of broadly similar intensity across several SPOTs is likely to indicate a similar affinity for the ligand over those SPOTs.

Monoclonal antibody selection and epitope mapping: Monoclonal antibodies directed towards

the ZZ domain of dystrophin (3311-3342) were collected as hybrid supernatants, selected by ELISA using recombinant ZZ domain and named 13D2, 12D7, 14A4 and 4G3. Specificity and epitope mapping was performed for the four selected supernatants by ELISA against the series of 7 residue peptides from the dystrophin ZZ domain (Fig. 1A) including those common to dystrophin and utrophin. A specific response is obtained with peptide 19 (D3326-3332 / U3081-3089) for 13D2 supernatant, with peptide 1 (D3315-3331 / U3072-3072) for 12D7 supernatant and peptide 33 (D3335-3341) for 14A4 supernatant whilst no specific response was obtained with supernatant 4G3 (Fig. 4A). Peptide 33 is unique to dystrophin as it contains one of the isoleucines that is substituted by valine in the utrophin ZZ domain. We therefore re-screened serum 14A4 hybrid supernatant against all peptides that spanned the equivalent region in dystrophin and utrophin, but as shown in Figure 4B, supernatant 14A4 was specific for peptide 33 and did not recognise the equivalent peptide form utrophin with an Ileu to Val substitution (peptide 34). The respective epitopes of the selected antibodies are presented schematically in Fig. 4C taking into account the topological information derived from Figure 3.

Immunodetection of the dystrophin ZZ domain:

The four monoclonal antibodies were tested by immunofluorescence detection using both Torpedo marmorata and rabbit muscle (Fig. 5AB).

Serum 13D2 shows no specific sarcolemmal labelling whereas both 12D7 and 14A4 antibodies produced the expected sarcolemmal labelling typical of dystrophin staining in skeletal muscle. Specific staining was blocked by preincubation of the hybrid supernatant with the respective peptide. The 4G3 antibody, which did not detect any specific dystrophin or utrophin ZZ domain peptide, produced a labelling pattern only in the cytoplasmic compartment of muscle fibres.

These antibodies were further tested by western blot on total protein homogenates from rabbit skeletal muscle (Fig. 5C) or T. marmorata (not shown). In keeping with the immunofluorescence detection pattern in muscle sections, no protein band was detected using the 13D2 antibody while a 400 kDa protein band, corresponding to the expected molecular mass of dystrophin, was obtained by 12D7 and 14A4 antibodies (Fig. 5). Specific staining was again blocked by preincubation of the serum with the respective peptide. Using protein extract from dystrophin deficient mdx mouse muscle (Fig. 5D) 12D7 and 14A4 antibody fail to give a 400 kDa protein band indicating that these antibodies recognize specifically the dystrophin ZZ domain sequence.

The 4G3 monoclonal antibody revealed an unknown protein band with molecular mass of about 170 kDa. Monoclonal supernatant 13D2 specifically recognised peptide 19 common to the dystrophin/utrophin ZZ domain (Fig 4A). It is surprising therefore that it did not recognise dystrophin in tissue sections or on western blots (Fig 5AB). This lack of reactivity might imply that the epitope for 13D2 is masked in tissue sections or is dependent on conformation, the latter might also explain its lack of reactivity in western blots.

ELISA competition assays:

In order to further investigate the reasons behind this lack of reactivity we performed competitive ELISA experiments in micro-titre plates coated with peptide 19. In two separate experiments we examined the competition between purified β-dystroglycan or a skeletal muscle membrane fraction enriched for β-dystroglycan, and the 13D2 ZZ domain antibody or 43DAG/8D5, a monoclonal hybrid supernatant against β-dystroglycan. All experiments, including controls were carried out in the presence of 1% BSA. The two experiments produced qualitatively similar results (Fig. 6AB). 13D2 incubation produced a consistent high signal across all wells (Fig. 6AB), consistent with the specificity of this monoclonal antibody for peptide 19 (Fig 4). The monoclonal antibody 43DAG/8D5 against βdystroglycan however, gave a consistent low signal across all wells, demonstrating that, like the related antiserum MANDAG2, this hybridoma supernatant does not recognise peptide 19 and is specific for the WW domain interaction sequence and PPPYVP epitope at the C-terminus of βdystroglycan (Supplementary Fig 1 .) and as reported previously [13,29]. Increasing concentrations of either purified recombinant β-dystroglycan (Fig. 6A) or dystroglycancontaining membrane fraction (Fig. 6B), competed for 13D2 binding to peptide 19 reducing the signal from successive wells. Conversely the detection of β-dystroglycan with 43DAG/8D5 revealed an increase in β-dystroglycan binding to peptide 19 with increasing β-dystroglycan or dystroglycan-containing membrane fraction competition across successive wells. Thus peptide 19 appears to be able to bind β-dystroglycan which can compete specifically for the 13D2 monoclonal antibody suggesting that the region of the dystrophin and utrophin ZZ domains, corresponding to peptide 19, is an additional binding site for β-dystroglycan on dystrophin and utrophin. Furthermore, the ability of 43DAG/8D5 to still recognise β-dystroglycan bound to peptide 19, suggests that the two binding events are independent in this context.

DISCCUSSION

The ZZ domain: ZZ domains are a highly conserved and widespread zinc binding motif first identified in dystrophin/utrophin and the transcriptional co-activator CBP/p300 [START_REF] Ponting | ZZ and TAZ: new putative zinc fingers in dystrophin and other proteins[END_REF] (Figure 8).

In dystrophin, the first half of the carboxy-terminal domain and ZZ domain-containing cysteinerich region has been implicated in binding to β-dystroglycan, and when mutated gives rise to a severe muscle-wasting phenotype [START_REF] Hoffman | Dystrophin: the protein product of the Duchenne muscular dystrophy locus[END_REF][START_REF] Campbell | Association of dystrophin and an integral membrane glycoprotein[END_REF][START_REF] Suzuki | Glycoprotein-binding site of dystrophin is confined to the cysteine-rich domain and the first half of the carboxyterminal domain[END_REF]. This region contains several modular protein domains: a WW domain, two incomplete but putative calcium-binding sites (EF hands) [START_REF] Chung | WW and EF hand domains of dystrophin-family proteins mediate dystroglycan binding[END_REF] and a ZZ domain (zinc finger domain) [START_REF] Ponting | ZZ and TAZ: new putative zinc fingers in dystrophin and other proteins[END_REF]. An examination of the spectrum of point mutations in the human dystrophin gene in the ZZ domain region (Table 1), reveals that 4 of these mutations, where a full-length protein product may be produced, are in zinc liganding residues implicated in our studies. The missense mutation C3313F produced a DMD phenotype, though no information is available as to whether this individual had any dystrophin protein expression [33]. D3335H on the other hand was identified in an individual with a normal level of correctly localised full length dystrophin and dystrophin associated proteins, but yet a severe DMD phenotype [START_REF] Goldberg | A dystrophin missense mutation showing persistence of dystrophin and dystrophin-associated proteins yet a severe phenotype[END_REF].

From our analysis of 65 Zn peptide SPOTs overlay, D3335 is a potential liganding residue (Fig 3), but mutation of this residue to histidine in biochemical experiments had no effect on βdystroglycan binding [START_REF] Ishikawa-Sakurai | ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to beta-dystroglycan[END_REF]. Although Asp to His is not normally considered a conservative substitution, in this context His could replace Asp and still coordinate zinc, thus preserving protein conformation, and as suggested from the patient phenotype, presumably some function too. It may be that D3335 has another important functional role that so far has not been elucidated and that is not supported by a His substitution, but it is difficult to predict what that might be. Aspartic acid in this position is conserved in the majority of ZZ domain sequences (Figure 8), suggesting a functional importance [START_REF] Ponting | ZZ and TAZ: new putative zinc fingers in dystrophin and other proteins[END_REF].

Zinc binding: An NMR structure of the ZZ domain from CBP (CRE Binding Protein) revealed a cross-brace zinc finger motif [START_REF] Legge | ZZ Domain of CBP: an Unusual Zinc Finger Fold in a Protein Interaction Module[END_REF], where the individual zinc ions are coordinated by non-linear sequence elements, with the protein backbone crossing back on itself to form a zinc binding site (Figure 8B). The SPOTs analysis carried out in Figure 3 sequence [START_REF] Legge | ZZ Domain of CBP: an Unusual Zinc Finger Fold in a Protein Interaction Module[END_REF]. Due to insertions in the dystrophin/utrophin sequences this corresponds to 27 and 28 residues apart, too long to provide an unambiguous interpretation in the peptide array. But as highlighted in Figure 3, it does not appear that just any combination of 4 residues that could coordinate zinc, will bind zinc. The two pairs of cysteine and histidine residues in peptides 43-46 of the SPOTs are appropriately spaced to bind zinc (Fig 3A ) but do not, suggesting a degree of specificity to the other sequences identified by this method that do bind zinc. Moreover, unlike almost all other ZZ domain sequences in the databases, vertebrate dystrophin and utrophin lack three of the conserved residues that form the second zinc binding site in the CBP ZZ domain: the central pair of cysteines and one of the c-terminal histidines (Figure 8B) [START_REF] Ponting | ZZ and TAZ: new putative zinc fingers in dystrophin and other proteins[END_REF][START_REF] Legge | ZZ Domain of CBP: an Unusual Zinc Finger Fold in a Protein Interaction Module[END_REF]. This would suggest that dystrophin and utrophin bind zinc in a different conformation and this conformation may be specific to the ability of these proteins to interact with dystroglycan through multiple modes of interaction as discussed below. Interestingly the vertebrate dystrobrevins and invertebrate dystrophins and dystrobrevins, do have cysteine residues in these positions, suggesting some functional divergence with the emergence of distinct dystrophin and utrophin genes in vertebrates.

Dystroglycan binding, role of the WW domain:

We and others have independently mapped the site for dystrophin and utrophin binding to dystroglycan to the last 15 amino acids at the cterminus of β-dystroglycan [13, [START_REF] Jung | Identification and characterization of the dystrophin anchoring site on β-dystroglycan[END_REF][START_REF] Rentschler | The WW domain of dystrophin requires EF-hands region to interact with betadystroglycan[END_REF][37][START_REF] Rosa | Localisation of the dystrophin binding site at the carboxyl terminus of β-dystroglycan[END_REF][START_REF] Di Vignano | Contribution of the different modules in the utrophin carboxy-terminal region to the formation and regulation of the DAP complex[END_REF]. The major determinant within these 15 residues being the sequence PPPY a consensus for type I WW domain interactions [13,[START_REF] Rentschler | The WW domain of dystrophin requires EF-hands region to interact with betadystroglycan[END_REF]37,[START_REF] Di Vignano | Contribution of the different modules in the utrophin carboxy-terminal region to the formation and regulation of the DAP complex[END_REF]. Despite a necessary requirement for the PPPY motif in dystroglycan for dystrophin/utrophin binding via their WW domains [29], this is not sufficient. Where this requirement has been analysed in more detail, it has been found that constructs corresponding to the WW domain of dystrophin or utrophin alone cannot bind β-dystroglycan, the addition of the EF hand region allows some binding, but full binding potential is not realised until the ZZ domain is included [13,[START_REF] Jung | Identification and characterization of the dystrophin anchoring site on β-dystroglycan[END_REF][START_REF] Rentschler | The WW domain of dystrophin requires EF-hands region to interact with betadystroglycan[END_REF][START_REF] Suzuki | Glycoprotein-binding site of dystrophin is confined to the cysteine-rich domain and the first half of the carboxyterminal domain[END_REF][START_REF] Ishikawa-Sakurai | ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to beta-dystroglycan[END_REF][START_REF] Di Vignano | Contribution of the different modules in the utrophin carboxy-terminal region to the formation and regulation of the DAP complex[END_REF][START_REF] Suzuki | Molecular organisation at the glycoprotein-complex-binding site of dystrophin: three dystrophin-associated proteins bind directly to the carboxy-terminal portion of dystrophin[END_REF]. Unlike most other WW domains which exhibit autonomous binding activity toward their cognate peptide ligands [29], the WW domains of dystrophin and utrophin as a minimum require the support of the EF hand region. The reasons for this became clear on elucidation of a crystal structure of the dystrophin WW-EF region in complex with a βdystroglycan peptide [START_REF] Huang | Structure of a WW domain containing fragment of dystrophin in complex with β-dystroglycan[END_REF]. This revealed that the WW domain was supported extensively by the EF hand region including a contribution to the β-dystroglycan binding interface and confirming earlier elegant biochemical studies by this group [START_REF] Rentschler | The WW domain of dystrophin requires EF-hands region to interact with betadystroglycan[END_REF].

Dystroglycan binding, role of the ZZ domain: Detailed biochemical analyses have pointed to a role for the ZZ domain in further supporting and contributing to this binding interface [START_REF] Rentschler | The WW domain of dystrophin requires EF-hands region to interact with betadystroglycan[END_REF][START_REF] Ishikawa-Sakurai | ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to beta-dystroglycan[END_REF]. Moreover Rentschler et al. even go so far as to suggest that there is an additional binding site between dystroglycan and non-WW domain regions in dystrophin [START_REF] Rentschler | The WW domain of dystrophin requires EF-hands region to interact with betadystroglycan[END_REF], possibly the ZZ domain.

Transgenic mdx mice expressing various dystrophin gene constructs showed that the dystrophic change in mdx muscle can be rescued [START_REF] Rafael | Forced expression of dystrophin deletion constructs reveals structure-function correlations[END_REF]42]. Because the binding of dystrophin to βdystroglycan is required to prevent the dystrophic phenotype, deletion models can be considered to exhibit effective and physiological binding activity in these mice. In this context, the fulllength cDNA with deletion of exons 68-70 (ZZ domain) failed to rescue the phenotype [START_REF] Rafael | Forced expression of dystrophin deletion constructs reveals structure-function correlations[END_REF] despite the presence of the WW-EF region. Another model with deletion of exons 64-67 (EF hands) also failed to rescue the phenotype [42]. This model, which includes the WW domain encoded by exons 62-63 but lacks the subsequent EF1 and EF2, suggests as in the biochemical experiments, that the WW domain alone is not sufficient for effective binding to β-dystroglycan.

On the other hand, a mouse with deletion of exons 71-78 could rescue the phenotype, suggesting that the C-terminal region encoded by these exons is not essential for the binding [42]. All these result are compatible with the fact that the C-terminal region of dystrophin spanning 3311-3342the ZZ domain, is crucial for binding to β-dystroglycan. Our results suggest that we could limit the direct binding activity of this crucial region to the residues D3326-3332 or U3083-3089 of dystrophin or utrophin respectively (peptide 19). Taken together these result are consistent with there being a second dystroglycan-binding site between dystrophin/utrophin ZZ domain and dystroglycan in addition to the WW domain-mediated interaction with the extreme c-terminus of β-dystroglycan. As shown in figure 6, this region is likely to be distinct from the well characterised PPPYVP site due to the inability of 43DAG/8D5 to compete for peptide 19 binding to purified β-dystroglycan. This additional binding site further stabilises the β-dystroglycan -WW domain interaction, which itself is supported by the EF hand region, and explains why the complete WW-EF-ZZ region appears to be required for full binding activity between βdystroglycan and dystrophin or utrophin.

ZZ domain mutations in humans:

The first missense mutation reported in the C-terminal region was C3340Y from a patient with DMD and mental retardation, but with ~20% of normal levels of dystrophin immunoreactivity and some β-dystroglycan staining at the sarcolemma [START_REF] Lenk | A cysteine 3340 substitution in the dystroglycan-binding domain of dystrophin associated with Duchenne muscular dystrophy, mental retardation and absence of the ERG b-wave[END_REF].

From our analyses, C3340 would be predicted to be essential for the formation of the second zinc knuckle (Fig 3), and mutation to tyrosine would ablate the zinc binding site. Biochemical analysis of this mutation, and also an engineered C3340S substitution, in the context of the interaction between dystrophin and β-dystroglycan also demonstrates a loss of function [START_REF] Ishikawa-Sakurai | ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to beta-dystroglycan[END_REF] in support of the patient data and our own analysis of the zinc binding site. From the biochemical analysis of mutations of all the cysteines in the dystrophin ZZ domain, only C3340Y prevents dystroglycan binding. Mutations C3313Y, C3316Y and C3319Y still allow dystroglycan binding [START_REF] Ishikawa-Sakurai | ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to beta-dystroglycan[END_REF], suggesting some plasticity in zinc binding in the first zinc knuckle. This is evident from the SPOTs overlay in Figure 3A, where there are five potential liganding residues, but only four would be required to coordinate zinc. The situation for utrophin appears different however.

Mutation of any of the 3 cysteine residues in the first zinc knuckle did not support dystroglycan binding, whereas mutation of either cysteine in the second zinc knuckle did support dystroglycan binding [START_REF] Ishikawa-Sakurai | ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to beta-dystroglycan[END_REF]. This observation is supported in part by our own data, where antibody 13D2 directed against the second zinc knuckle completely blocked dystroglycan binding to dystrophin but had a reduced ability to block utrophin binding ( Fig 7) suggesting that the second zinc knuckle is less important for the utrophin-dystroglycan interaction.

Summary:

The above data provides a conceptual advance on previously reported findings [START_REF] Rentschler | The WW domain of dystrophin requires EF-hands region to interact with betadystroglycan[END_REF][START_REF] Ishikawa-Sakurai | ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to beta-dystroglycan[END_REF] and defines the ZZ domain in concert with the WW domain as being a crucial structural and functional part of the dystrophin/utrophinβ-dystroglycan interaction that is involved in anchoring these proteins to the cell membrane. The identification of the short dystrophin peptide YRSLKHF defines a second β-dystroglycan binding site in dystrophin and utrophin and raises the possibility of identifying further specific antibodies that could be used to distinguish between dystrophin and utrophin in this highly similar region. Finally biochemical approaches, epitope mapping, ELISA and overlay, provide new evidence for the zinc binding properties and βdystroglycan interaction site on the ZZ domain, and highlights distinct differences in the mode of interaction between β-dystroglycan and the dystrophin or utrophin ZZ domains. Antibodies 12D7 and 14A4 both produced a clear sarcolemmal staining pattern consistent with dystrophin staining (A2/B2,A3/B3), which in both cases was competed by an excess of the specific peptide to which the antibody was raised (A2'/B2',A3'/B3'). Antibody 4G3, which did not detect a specific peptide by ELISA exhibited extensive background staining in both T. marmorata and rabbit muscle (A4/B4). Surprisingly antibody 13D2 produced only a very weak background staining pattern that was not further reduced in the presence of excess peptide, with no clear sarcolemmal staining in either muscle type (A1/A1', B1/B1'). Bar =50 µm. In keeping with the immunohistological observation antibodies 12D7 and 14A4 recognised an ~ 400kDa band in western blots of rabbit muscle extracts, again consistent with dystrophin staining, which was competed away with the specific peptide, whereas antibody 13D2 did not detect anything (C). Antibody 4G3, which did not detect a specific peptide or label the sarcolemma, recognised an unknown 170kDa protein in western blots. The specificity of antibodies 12D7 and 14A4 was further tested on muscle extracts from mdx mice that lack dystrophin (D). A previously characterised antibody specific for the carboxy-termini of dystrophin (H4, far left lane) confirmed the absence of dystrophin, whereas the presence of utrophin was revealed by antibody K7 (near left lane). Antibodies 12D7 and 14A4 detected no bands on western blots of mdx skeletal muscle confirming their specificity for dystrophin (right lanes). The protein bands detected are indicated according molecular mass markers (Biorad). B. A scheme to demonstrate the positions of Zn coordinating residues as proposed for the utrophin ZZ domain and as elucidated structurally for the CBP ZZ domain [START_REF] Legge | ZZ Domain of CBP: an Unusual Zinc Finger Fold in a Protein Interaction Module[END_REF]. Numbers beneath each sequence refer to the positions of the liganding residues coordinating each individual zinc ion. For utrophin these are proposed to be consecutive in the linear sequence, whereas CBP adopts a cross-braced organisation.

Far-western blotting

  of β-dystroglycan with Dystrophin and utrophin ZZ domains: Total mouse muscle extract transferred to nitrocellulose was overlayed with a dystrophin-enriched crude membrane homogenate of mouse skeletal muscle, or with a utrophin-enriched crude membrane homogenate of mdx mouse skeletal muscle or sciatic nerve. The dystrophin in the enriched dystrophin homogenate bound to β-dystroglycan on the nitrocellulose membrane as revealed by blotting for the dystrophin c terminus (Fig 7, panel A3; Pep19-). When the blot was pre-incubated with peptide 19 however, the binding of dystrophin in the membrane homogenate to dystroglycan on the blot was strongly reduced (Fig 7, panel A3; Pep19+). This resultdemonstrates that the peptide 19 can effectively block dystrophin binding to intact βdystroglycan again highlighting the importance of the ZZ domain for the dystrophin-dystroglycan interaction. In a similar experiment overlaying an enriched utrophin homogenate from dystrophin-deficient mdx muscle or sciatic nerve, utrophin detection using the C-terminal utrophin antibody (K7) showed that the fraction of utrophin that bound β-dystroglycan was less compared to that observed with dystrophin, suggesting that utrophin has a lower affinity for βdystroglycan (Fig7, panel B3; Pep19-). However, when peptide 19 was applied there was still a slight reduction in utrophin binding (Fig.7panel B3; Pep19+). The apparent difference in inhibition of binding to β-dystroglycan is not likely to be due to the slightly lower dystrophin+utrophin content seen in the mdx mice samples, compare quantifications in panels A4 and B4. The difference in competition for β-dystroglycan binding probably reflects a real difference in the mode of binding between the dystrophin or utrophin ZZ domain, and βdystroglycan. This may be due to other DGC components assembly differently in the mdx mouse in the presence of utrophin which contributes to a slightly different mode of interaction with dystroglycan. Biochemical Journal Immediate Publication. Published on 29 Sep 2006 as manuscript BJ20061051

  could not reveal coordination of zinc through such a structure as the individual peptides were only 20 amino acids long, and from the CBP ZZ domain structure the liganding residues are 21 and 26 residues apart in the linear Biochemical Journal Immediate Publication. Published on 29 Sep 2006 as manuscript BJ20061051

Figure 1 :

 1 Figure 1: Amino acid alignment of the ZZ domains of dystrophin (Dys) and utrophin (Utr) indicating identical residues (|) and highly conserved residues (:) and delimiting amino acid residue numbers (large bold letters in the centre of the figure. Above and below these sequences are the sequences of the series of overlapping 7 residue peptides used in this study, all are numbered except the first four which could not be synthesised to high enough purity. Peptides above the alignment are to dystrophin including those which are underlined which are common between dystrophin and utrophin, those below the alignment are unique to utrophin.

Figure 2 :

 2 Figure 2: Zinc binding to the utrophin ZZ domain. A. CD spectrum of the utrophin ZZ domain in the presence of 1mM ZnCl 2 (dashed line) or absence of zinc (presence of phenanthroline, solid line) indicating a modest structural change on zinc binding. This is reflected in the slightly altered mobility of the ZZ domain on SDS-PAGE in the presence of ZnCl 2 (inset). B. Autoradiographs of individual 65 ZnCl 2 overlays of purified ZZ domain in the absence (top left square) or presence of an excess of the indicated competing divalent cation. 65 Zn binding is competed in keeping with the Irving-Williams series Mn<Fe<Co<Ni<Cu>Zn>>Ca/Mg. A subset of the data in part B were published previously in brief [44]. C. Densitometric analysis of the data in B and two additional independent experiments, with 65 ZnCl 2 binding in the presence of the indicated competing divalent cations represented relative to 65 ZnCl 2 alone = 1. Mean ± SEM n=3.

Figure 3 :

 3 Figure 3: Utrophin ZZ domain Peptide SPOTs 65 Zn overlay. A. A 46 peptide, 20 residue SPOTs peptide array overlapping by one amino acid and covering the utrophin ZZ domain was overlaid with 65 ZnCl 2 and subjected to autoradiography. Individual peptides are numbered 1-46 with the corresponding sequence shown. Positions 47-50 in the array were blank. Two regions of 65 Zn binding were observed spanning peptides 6-16 and 30-37 respectively, potential residues involved in the coordination of zinc are highlighted in grey and bold underline. Another region with the potential to bind 65 Zn (peptides 43-46: potential coordinating residues highlighted in grey) showed no 65 Zn binding by this approach. The two zinc binding regions are represented schematically in B.

Figure 4 :Figure 3 ,

 43 Figure 4: ZZ domain monoclonal antibody epitope mapping. A. Epitope determination of monoclonal antibodies specificity by ELISA assay using synthetic peptides (number on x axis; see Fig 1). Results are represented as histograms; 13D2, 12D7 and 14A4 antibodies all recognised a single specific dystrophin peptide, whereas antibody 4G3 did not recognise any peptide. 14A4 was reprobed against a series of overlapping dystrophin and utrophin peptides (panel B). Again the 14A4 antibody appeared specific for the dystrophin sequence D3335-3341 sequence, and did not recognise the when I3336 substitution to V3039 in the utrophin sequence (peptide 34). The epitopes are represented on the topological map of the ZZ domain derived from Figure 3, individual epitopes are shaded grey and the non-conserved isoleucine residues (valine in utrophin) are indicated by asterisks.

Figure 5 :

 5 Figure 5: Immunodetection of dystrophin in muscle. Serial cryostat sections of Torpedo marmorata (A) or rabbit muscle (B) were labelled using the four monoclonal antibodies.

BiochemicalFigure 6 :

 6 Figure 6: Competitive ELISA binding for peptide 19. Microtitre plates coated with or without peptide 19 were detected with antibody 13D2 (raised against peptide 19) or 43DAG/8D5 (43DAG; raised against the β-dystroglycan cytoplasmic domain). In panel A, peptide 19 was detected by 13D2 at similar levels in all wells (open squares) and not detected by 43DAG (closed squares). On incubation with increasing concentrations of purified β-dystroglycan cytoplasmic domain (β-DG; 0.5-9µg.ml) 13D2 detection of peptide 19 was specifically reduced (open diamonds) whereas incubation of empty or peptide 19 coated wells with β-DG and detection with 43DAG showed a specific increase in signal. These results demonstrate a β-DG specific competition for 13D2 binding to peptide 19, confirming peptide 19 as a β-dystroglycan binding site. Qualitatively similar results were obtained in competitive ELISA binding for peptide 19 using a dystroglycan-enriched fraction of skeletal muscle (Ht; 1-10µg/ml panel B). Increasing concentrations of homogenate competed for 13D2 detection of peptide 19 (open triangle), whereas the homogenate had no effect on the detection of β-DG coated wells by 43DAG (closed circles), other controls were as panel A.

Figure 7 :

 7 Figure 7:Overlay experiments using electroblotted total crude muscle membrane homogenate (Ht). Panels (A1 and A2) correspond to the native nitrocellulose with total electrotransferred Ht revealed by LG5 and H4 polyclonal antibodies (anti-β-dystroglycan and anti-dystrophin respectively). Membranes were overlayed with enriched dystrophin homogenate comprising dystrophin and its associated proteins. Detection was performed with a dystrophin C-terminal antibody (H4). When peptide 19 was pre-incubated before overlay with the dystrophin enriched homogenate only a 400 kDa band was detected with H4 polyclonal antibody suggesting that the binding site for β-dystroglycan was blocked (A3). Panel B represents the same overlay experiment performed with utrophin enriched homogenate from mdx mouse comprising utrophin and its associated proteins detected by the C-terminal β-dystroglycan and utrophin antibodies (panel B1 and panel B2 respectively). Panel B3 showed that the utrophin binding properties for β-dystroglycan are slightly different from that of dystrophin and were not reduced to the same extent when peptide 19 was applied. Panels A4 and B4 represent densitometric quantification of the amounts of dystrophin (DYS) and utrophin (UTR) present in the two fractions.

Figure 8 .

 8 Figure 8. Alignment of ZZ domain sequences from dystrophin family proteins and other unrelated proteins from different phyla, A. Gene name, organism and residue numbers for the Z domain used in the alignment are shown. The alignment was generated using ClustalW and the 60% consensus sequence derived from the SMART database entry for the ZZ domain is shown at the bottom. All cysteines are coloured yellow, single amino acids that fit the consensus and conserved aromatic and basic residues that form the core of the ZZ domain are also coloured. Capitalised letters represent conserved amino acids (in the full alignment), and the lowercase letters and symbols denote conserved features; positive, +; aromatic, a; small, s; polar, p; big, b and hydrophobic, h. A more extensive alignment of 47 ZZ domain sequences can be found at http://www.expasy.org/cgibin/aligner?psa=PS01357&color=1&maxinsert=10&linelen=0

  a,b These mutations produced frame-shifting stop codons 34 a and 8 b codons respectively downstream of the point mutation. Consequently the DMD phenotypes could be due to the frame-shift and or truncation rather than any specific point mutation. A rare single amino acid deletion of a conserved glutamate 3367 just c-terminal of the ZZ domain resulted in a DMD phenotype despite normal correctly localised protein [47].

Figure 1 .Figure 2 .Figure 3 .Figure 5CD .Figure 6 .Figure 7 .

 1235CD67 Figure 1. Hnia et al.

Biochemical Journal Immediate Publication. Published on 29 Sep 2006 as manuscript BJ20061051

Table 1 .

 1

	Phenotype	Dystrophin ZZ domain	Source
	3311 AKCNICKECPIIGFRYRSLKHFNYDICQSCFF 3343
	DMD	AKFNICKECPIIGFRYRSLKHFNYDICQSCFF	[33]
	BMD	AKCNICKERPIIGFRYRSLKHFNYDICQSCFF	[45]
	DMD	AKCNICKECPIIGFRYRSLKHFNYHICQSCFF	[34]
	DMD	AKCNICKECPIIGFRYRSLKHFNYDIYQSCFF	[43]
	DMD a	AKCNICKECPIIGFRYRSLKHFNYDICLSCFF	[45]
	DMD b	AKCNICKECPIIGFRYRSLKHFNYDICFSCFF	[46]

Table 1 . Missense mutations in the ZZ domain of dystrophin.

 1

Biochemical Journal Immediate Publication. Published on 29 Sep 2006 as manuscript BJ20061051

Figure 4. Hnia et al. Biochemical Journal Immediate Publication. Published on 29 Sep 2006 as manuscript BJ20061051

ACKNOWLEDGEMENTS

KH was supported by the "Association Française contre les Myopathies" (AFM, Fellowship N° 10529), DM by Montpellier1 University and CNRS, and SJW by the BBSRC (ICR07596), MRC (G0000104) and Wellcome Trust (042180). We are extremely grateful to Jürgen Wehland for

synthesis of the ZZ domain peptide SPOT array and to Louise Anderson, who sadly and prematurely passed away in 2005, for generous supplies of 43DAG/8D5.

CONFLICT OF INTEREST

The authors declare they have no competing interests.