Félix Rodríguez-Juárez

Enara Aguirre

Susana Cadenas
email: scadenas@cnic.es

Dr E Cano

Dr J Sánchez-Prieto

Dr M Torres

Relative sensitivity of soluble guanylate cyclase and mitochondrial respiration to endogenous nitric oxide at physiological oxygen concentration

Keywords: AMPK, AMP-activated protein kinase, BAY 41-2272, cytochrome c oxidase, high-resolution respirometry, mitochondrial respiration, nitric oxide (NO), oxygen consumption, soluble guanylate cyclase

5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4

INTRODUCTION

Nitric oxide (NO) is produced in cells by the enzyme nitric oxide synthase (NOS) from L- arginine, oxygen (O 2 ) and NADPH. Three isoforms of NOS are well characterized and referred to as neuronal NOS (nNOS, NOS-1), inducible NOS (iNOS, NOS-2) and endothelial NOS (eNOS, NOS-3). A mitochondrial isoform (mtNOS) has also been described, although its nature is controversial [START_REF] Brookes | Mitochondrial nitric oxide synthase[END_REF]. NO exerts a broad spectrum of functions in several systems including the cardiovascular system, the central and peripheral nervous systems and the immune system. Two main cellular targets of NO have been clearly established. NO binds to the heme prosthetic group of soluble guanylate cyclase (sGC) stimulating the production of cGMP from GTP [START_REF] Waldman | Cyclic GMP synthesis and function[END_REF][START_REF] Ignarro | Signal transduction mechanisms involving nitric oxide[END_REF][START_REF] Russwurm | Guanylyl cyclase: NO hits its target[END_REF][START_REF] Russwurm | NO activation of guanylyl cyclase[END_REF]. Through the binding to sGC NO mediates many physiological effects such as smooth muscle relaxation, inhibition of platelet aggregation and neurotransmission [START_REF] Moncada | Nitric oxide: physiology, pathophysiology, and pharmacology[END_REF]. More recently, NO has also been shown to bind to and inhibit cytochrome c oxidase (COX) [START_REF] Brown | Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase[END_REF][START_REF] Cleeter | Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases[END_REF][START_REF] Boveris | Regulation of mitochondrial respiration by oxygen and nitric oxide[END_REF][START_REF] Brookes | Control of mitochondrial respiration by NO, effects of low oxygen and respiration state[END_REF][START_REF] Brunori | Nitric oxide and the respiratory enzyme[END_REF], the terminal enzyme of the mitochondrial electron transport chain, which is responsible of almost all cellular O 2 consumption. The inhibition is reversible and occurs in competition with O 2 . NO-mediated inhibition of O 2 consumption has been reported in isolated COX [START_REF] Brown | Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase[END_REF], mitochondria [START_REF] Cleeter | Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases[END_REF][START_REF] Borutaite | Mitochondria rapidly reduce nitric oxide, and nitric oxide reversibly reduces mitochondrial respiration[END_REF][START_REF] Koivisto | Kinetics of the inhibition of mitochondrial respiration by NO[END_REF], brain nerve terminals [START_REF] Brown | Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase[END_REF] and cultured cells [START_REF] Clementi | On the mechanism by which vascular endothelial cells regulate their oxygen consumption[END_REF][START_REF] Brown | Nitric oxide produced by activated astrocytes rapidly and reversibly inhibits cellular respiration[END_REF]. High concentrations of NO have been shown to cause irreversible decreases in the affinity of COX for O 2 [START_REF] Cooper | Nitric oxide and peroxynitrite cause irreversible increases in the K m for oxygen of mitochondrial cytochrome oxidase: in vitro and in vivo studies[END_REF]. The inhibition of respiration by NO could have pathological repercussions by limiting ATP production, but may also have a physiological function in controlling the rate of cellular O 2 consumption and modulating the production of reactive O 2 species by the respiratory chain [17].

The relative potency of NO towards these targets is a matter of debate. The concentration of NO that causes half-maximal activation of sGC (EC 50 ) has been determined at 250 nM [START_REF] Stone | Spectral and kinetic studies on the activation of soluble guanylate cyclase by nitric oxide[END_REF], 690 nM [START_REF] Russwurm | Functional properties of a naturally occurring isoform of soluble guanylyl cyclase[END_REF] or 1.6 µM [START_REF] Artz | In vitro activation of soluble guanylyl cyclase and nitric oxide release: a comparison of NO donors and NO mimetics[END_REF] for the purified enzyme and 47 nM [START_REF] Bellamy | Sub-second kinetics of the nitric oxide receptor, soluble guanylyl cyclase, in intact cerebellar cells[END_REF], 20 nM [START_REF] Bellamy | Rapid desensitization of the nitric oxide receptor, soluble guanylyl cyclase, underlies diversity of cellular cGMP responses[END_REF] or 2 nM [START_REF] Griffiths | The shaping of nitric oxide signals by a cellular sink[END_REF] in cerebellar cells. The NO concentration that causes half-maximal inhibition of respiration (IC 50 ) at physiological O 2 concentration has been reported as 60 nM in brain synaptosomes [START_REF] Brown | Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase[END_REF] and 11 nM in isolated brown adipose tissue mitochondria [START_REF] Koivisto | Kinetics of the inhibition of mitochondrial respiration by NO[END_REF]. A major limitation of this kind of study is the difficulty in maintaining a constant NO Biochemical Journal Immediate Publication. Published on 18 Apr 2007 as manuscript BJ2007003 © 2007 Biochemical Society supply for extended periods. Moreover, NO donors such as DETA-NO or SNAP are usually used as the NO source despite their known limitations. More recently, it has been reported that sGC is significantly more sensitive to being activated by NO than COX is to being inhibited [START_REF] Bellamy | Differential sensitivity of guanylyl cyclase and mitochondrial respiration to nitric oxide measured using clamped concentrations[END_REF]. In that study, the EC 50 of NO for sGC was 3.9 nM as determined in the purified enzyme, whereas the IC 50 of NO for cellular respiration was determined as 120 nM in cerebellar cells at 30 µM O 2 . The source of NO used was DETA-NO and its concentration was kept constant by an ingenious system in which NO was continuously delivered by the donor and consumed by oxyhaemoglobin (oxyHb). However, it is not clear which enzyme is more sensitive to NO at physiological O 2 tension when NO is produced inside the cells.

The purpose of the present study was to determine the relative sensitivity of the two main targets of NO in cells, sGC and mitochondrial respiration, to endogenous NO at physiological O 2 tension (30 µM). To overcome difficulties associated with the use of NO donors to generate NO, we used a HEK 293 cell line transfected with the iNOS gene under a tetracycline-inducible promoter [START_REF] Mateo | Regulation of hypoxia inducible factor-1α by nitric oxide through mitochondriadependent and -independent pathways[END_REF]. By adding tetracycline to the incubation medium we were able to induce iNOS expression to different levels and thus to produce NO at controlled levels for extended periods. or a monoclonal anti-actin antibody (1:10000, Sigma-Aldrich, St. Louis, MO, U.S.A.) in blocking solution at 4 ºC. Protein bands were detected by incubation for 1 h with horseradish peroxidase-coupled goat anti-rabbit IgG (1:5000, Vector Laboratories, Burlingame, CA, U.S.A.) or goat anti-mouse IgG (1:2500, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) in blocking solution at room temperature, followed by enhanced chemiluminescence (ECL ® ; Amersham Biosciences, Buckinghamshire, U.K.). chambers for cells treated with the same amount of tetracycline; NO production was initiated in one chamber by the addition of L-arginine (1 mM). Because light has been reported to reverse the inhibition of respiration induced by NO [START_REF] Borutaite | Reversal of nitric oxide-, pe3roxynitrite-and S-nitrosothiol-induced inhibition of mitochondrial respiration or complex I activity by light and thiols[END_REF], all experiments were performed in the absence of direct light. NO was quenched by the addition of oxyHb to study the reversibility of respiration inhibition.

Quantification of nitric oxide

Measurement of cGMP

For the measurement of cGMP cells were plated in 6-well plates at 1.5 x 10 6 cells/well.

After reaching confluence, cells were incubated with tetracycline overnight to induce iNOS expression as described above. Plates were transferred to hypoxic chambers (Coy Laboratory Products Inc., Grass Lake, MI, U.S.A.) at 37 ºC and set at an Western blotting (Figure 1a). The addition to the cells of the iNOS substrate L-arginine (1 mM) at 30 µM O 2 immediately triggered NO production (Figure 1b). The quantity of NO produced was dependent on the amount of iNOS expressed and remained constant for at least 10 min. The highest NO concentration obtained, at the greatest expression of iNOS, was 1.2 ± 0.1 µM (n = 3). NO concentration returned to basal after the addition of oxyHb (Figure 5d). The relationship between tetracycline concentration and the maximum value of endogenous NO production at peak time is shown in Figure 1c. The NO concentrations produced by our cells are within the reported physiological range, which appears to be between 1 nM and 1 µM, depending on the tissue and conditions [START_REF] Brown | Nitric oxide regulates mitochondrial respiration and cell functions by inhibiting cytochrome oxidase[END_REF]. Moreover, many cell types will express iNOS and produce high levels of NO when activated by inflammatory mediators, for example endotoxin and interferon γ. Indeed, activated astrocytes accumulate up to 1 µM NO in the medium, with cellular O 2 consumption about half that of control cells [START_REF] Brown | Nitric oxide produced by activated astrocytes rapidly and reversibly inhibits cellular respiration[END_REF]. In the absence of L-arginine, iNOS is able to produce superoxide mainly at the flavin-binding sites of the reductase domain [START_REF] Xia | Inducible nitric-oxide synthase generates superoxide from the reductase domain[END_REF], although at lower levels than the neuronal isoenzyme [START_REF] Weaver | A comparative study of neuronal and inducible nitric oxide synthases: generation of nitric oxide, superoxide, and hydrogen peroxide[END_REF].

Sensitivity of sGC to endogenous NO sGC is a key NO receptor protein involved in NO-mediated biological and pathological reactions. NO activates sGC, leading to increased production of the intracellular messenger cGMP, whose biological effects are mediated by cGMP-dependent protein kinases, cGMP-regulated ion channels and cGMP-dependent phosphodiesterases [START_REF] Schlossmann | cGMP-dependent kinases in drug discovery[END_REF]. To examine the kinetics of NO-induced sGC activation in our system, cells were incubated with tetracycline (5 ng/ml) overnight and then induced with L-arginine (1 mM) to produce NO for periods up to 5 min. We chose 5 ng/ml tetracycline for the time-course experiment, despite the fact that at this concentration NO production was below the µM, which correspond to 21, 3 and 1.5 % O 2 , respectively (Figure 2). cGMP began to accumulate immediately after L-arginine addition, reaching a maximum at 3 min and decreasing slightly at 5 min (Figure 2a). The NO produced upon addition of L-arginine (Figure 1b) activates sGC to generate cGMP from GTP. The amounts of cGMP observed reflect a balance between cGMP production and degradation. cGMP in cells is degraded by phosphodiesterases, which also determine the shape of the cGMP signal [START_REF] Mullershausen | Rapid nitric oxide-induced desensitization of the cGMP response is caused by increased activity of phosphodiesterase type 5 paralleled by phosphorylation of the enzyme[END_REF]. The activity of endogenous phosphodiesterases is different in different cell types, for example phosphodiesterase activity is lower in cerebellar cells than striatal cells [START_REF] Wykes | Kinetics of nitric oxide-cyclic GMP signalling in CNS cells and its possible regulation by cyclic GMP[END_REF]. It has been reported that exposure of cells to DEA NONOate for 2 min desensitises sGC and that the degree of desensitisation is related to the cGMP concentration [START_REF] Bellamy | Rapid desensitization of the nitric oxide receptor, soluble guanylyl cyclase, underlies diversity of cellular cGMP responses[END_REF][START_REF] Wykes | Kinetics of nitric oxide-cyclic GMP signalling in CNS cells and its possible regulation by cyclic GMP[END_REF]. This could explain the decrease in cGMP levels observed at 5 min, particularly considering that our standard assay medium used to determine cGMP contains no phosphodiesterase inhibitor that could lead to artificially high cGMP levels. Addition of 0.5 mM IBMX, an inhibitor of endogenous phosphodiesterases, increased the maximum levels of cGMP in the cell extracts saturating our assay from 3 min onwards, and completely preventing the decrease in cGMP at prolonged incubations (Figure 2b). © 2007 Biochemical Society arginine. We therefore, believe that the slight decrease in cGMP production at low O 2 tension is not related to decreased expression levels of the protein but could instead reflect the fact that NOS activity is O 2 -dependent and that the K m of NOS for O 2 is high [START_REF] Santolini | Differences in three kinetic parameters underpin the unique catalytic profiles of nitric-oxide synthases I, II and III[END_REF]. Supporting this view is the fact that cGMP accumulation at 3 and 1.5 % O 2 was similar in Tet-iNOS 293 cells treated with DEA NONOate (1 µM), from which the release of NO is O 2 -independent (data not shown).

To further correlate the release of NO with cGMP effects, we conducted a time-course experiment of cGMP production triggered by DEA NONOate, which is a fast-release NO donor (2.5 min half-life at neutral pH and 37 ºC; Figure 4a). The NO concentration released by 1 µM DEA NONOate in medium containing 1 x 10 7 cells/ml at 37 ºC was 268 ± 56 nM (n = 6). At 3 % O 2 , the pattern of cGMP production after the addition of 1 µM DEA NONOate was similar to that found with endogenous NO produced by tetracycline-induced iNOS: a time-dependent increase in cGMP accumulation which was highest at 3 min and slightly decreased at 5 min (Figure 4a). Again, in the presence of 0.5 mM IBMX cGMP concentration increased significantly, saturating our assay at 3 and 5 min (Figure 4a).

To study the sensitivity of sGC to endogenous NO, cells were incubated overnight with a range of tetracycline concentrations (2-50 ng/ml) and then induced with L-arginine (1 mM) to produce NO for 3 min at physiological O 2 tension (30 µM; Figure 3a). The incubation time of 3 min was selected because this coincided with peak cGMP accumulation in the previous experiment (Figure 2a). cGMP production increased in a dose-dependent manner up to a maximum value of 380 pmol/mg protein at 10 ng tetracycline/ml and then decreased to approximately 230 pmol/mg protein regardless of the tetracycline concentration used. Interestingly, at high tetracycline doses (> 50 ng/ml) NO activated sGC and cGMP production, even in the absence of exogenously added L- arginine (data not shown). The activity of endogenous iNOS normally requires extracellular arginine, in spite of the fact that intracellular arginine concentrations are sufficient to saturate the enzyme. The explanation reported for this is that arginine uptake is required to derepress translation of iNOS [START_REF] Lee | Translational control of inducible nitric oxide synthase expression by arginine can explain the arginine paradox[END_REF]. In our system, however, the Biochemical Journal Immediate Publication. Published on 18 Apr 2007 as manuscript BJ2007003 © 2007 Biochemical Society tetracycline-induced iNOS expression is not subject to this regulation, and low concentrations of intracellular arginine are therefore able to stimulate NO synthesis when iNOS expression is high and this, in turn, activates sGC to produce cGMP.

The accumulation of cGMP observed after overnight incubation of the cells with tetracycline and induction with L-arginine (1 mM) was partially prevented by the addition to the incubation medium (3 min incubation) of inhibitors of sGC (50 µM ODQ; Figures 3a andb) and totally prevented by iNOS inhibitors (500 µM S-EITU; Figure 3b). ODQ completely inhibited cGMP production at low doses of tetracycline but this effect was only partial at high tetracycline doses (Figure 3a) which is consistent with the competitive nature of the inhibition [START_REF] Zhao | Inhibition of soluble guanylate cyclase by ODQ[END_REF]. These experiments show that the observed increase in cGMP can be attributed to the specific activation of sGC by NO. We used HEK 293 cells transfected with empty vector (not containing the iNOS gene) as a negative control. In these cells, overnight treatment with tetracycline and addition of L- arginine did not produce any detectable accumulation of cGMP (data not shown). As a positive control, BAY 41-2272 (10 µM), an NO-independent but haem-dependent sGC activator [START_REF] Stasch | NO-independent regulatory site on soluble guanylate cyclase[END_REF], stimulated cGMP production to levels that saturated our assay (> 500 pmol/mg protein; Figure 3b). Figure 3b also shows that the activation of sGC by BAY 41-2272 was partially prevented by ODQ (50 µM). Figure 6 shows the relationship between the concentration of NO produced endogenously and the amount of cGMP produced by sGC at 3 min, 37 ºC and 30 µM O 2 . The NO concentration that induced halfmaximal activation of the enzyme (EC 50 ) was estimated as 2.9 nM.

To our surprise, concentrations of tetracycline above 10 ng/ml did not yield additional increases in cGMP. To try to explain this result, we performed a dose-response experiment of cGMP production in cells incubated for 3 min with DEA NONOate with or without IBMX (Figure 4b). Again, widely differing DEA NONOate concentrations (2, 20 and 200 µM), which produce very different amounts of NO in medium containing 1 x 10 7 cells/ml at 37 ºC (918 ± 79, 9217 ± 928 nM, n = 5, and > 9 µM, respectively) did not yield significantly different values of cGMP accumulation at 3 min (Figure 4b). The In light of the DEA NONOate experiments it seems likely that, regardless of the NO source, NO concentrations above a certain threshold do not produce additional increases in cGMP accumulation. In other words, sGC becomes insensitive to NO above a particular level or time of exposure. This is supported by the reported desensitisation of sGC to DEA NONOate and the fact that the degree of desensitisation is related to the cGMP concentration [START_REF] Bellamy | Rapid desensitization of the nitric oxide receptor, soluble guanylyl cyclase, underlies diversity of cellular cGMP responses[END_REF][START_REF] Wykes | Kinetics of nitric oxide-cyclic GMP signalling in CNS cells and its possible regulation by cyclic GMP[END_REF]. The combination of phosphodiesterase activity and sGC desensitisation could account for the plateau observed at tetracycline concentrations higher than 10 ng/ml.

Sensitivity of mitochondrial respiration to endogenous NO

The mean capillary O 2 concentration is approximately 30 µM and the typical value inside cells averages 3 µM [START_REF] Gnaiger | Mitochondrial respiration at low levels of oxygen and cytochrome c[END_REF]. These low tissue O 2 tensions do not limit mitochondrial respiration, since the affinity of COX for O 2 is very high, and so there is a very large safety margin to sustain mitochondrial oxidative phosphorylation [START_REF] Gnaiger | Mitochondrial oxygen affinity, respiratory flux control and excess capacity of cytochrome c oxidase[END_REF][START_REF] Rossignol | Mitochondrial threshold effects[END_REF]. Nevertheless, at these O 2 tensions NO might compete significantly for COX binding, and so could For example, in cells stimulated with the highest tetracycline concentration used in these experiments (50 ng/ml), the inhibition of respiration from the control value was 49 % at 50 µM O 2 and 67 % at 30 µM O 2 . The addition of oxyHb to completely quench NO, fully reactivated cellular respiration (Figure 5d), which shows that the inhibitory effect is reversible. The lowest tetracycline concentration that had an effect on respiration at low O 2 was 20 ng/ml (Figure 5b). At physiological O 2 tension (30 µM) the concentration of NO that caused a half-maximal inhibition (IC 50 ) of cellular respiration was 141 nM (Figure 6).

We performed additional measurements of respiration and NO in cells treated with low tetracycline concentrations (2, 5 and 10 ng/ml). For respiration measurement L-arginine was added at 30 µM O 2 instead of 60 µM; NO was not detectable in any case. We measured the respiration at 15 µM O 2 (1.5 % O 2 ). The lack of effect of NO produced by cells treated with 2 ng/ml tetracycline on respiration is shown in Figure 5c. Table 2 shows the values of respiration at 15 µM O 2 as a percentage of the value at 40 µM (before arginine addition) in cells treated with 2, 5 and 10 ng/ml tetracycline. The results indicate that there are no significant differences in any case.

The rate of cellular O 2 consumption is controlled by a number of factors [START_REF] Brand | Control of electron flux through the respiratory chain in mitochondria and cells[END_REF][START_REF] Brown | Control of respiration and ATP synthesis in mammalian mitochondria and cells[END_REF]. The excess capacity of COX explains the low control coefficient of the enzyme over respiration. Therefore, a fall in COX activity will not have a proportional effect on the cellular O 2 consumption rate. In other words, COX activity may be partially inhibited without any effect on mitochondrial respiration, so although low NO concentrations had no effect on the respiration rate of our cells, an actual decrease in COX activity cannot be ruled out. However, there is a threshold value beyond which respiration will be inhibited.

In this respect, it is important to point out a recent study in respiring cells showing that, as the O 2 concentration decreases, there is an early reduction of cytochromes aa

Consequences of NO effects for cell signalling

To further study the relative sensitivities of the two main cellular targets of NO, and considering the fact that COX activity can be partially inhibited without an effect on respiration, we examined the effects of NO on cell signalling through cGMP-dependent protein kinase (PKG) and AMP-activated protein kinase (AMPK) (Figure 7).

PKG is a serine/threonine protein kinase and is one of the major intracellular receptors for cGMP. Activated PKG phosphorylates many intracellular proteins and regulates important physiological functions such as relaxation of vascular smooth muscle, inhibition of cell differentiation and proliferation and inhibition of platelet aggregation and apoptosis [START_REF] Schlossmann | cGMP-dependent kinases in drug discovery[END_REF]. We measured the phosphorylation state of the PKG substrate vasodilator-stimulated phosphoprotein (VASP), which is expressed in most mammalian tissues. Phosphorylation of VASP at Ser157 causes a mobility shift on SDS gel electrophoresis from 46 to 50 kDa, providing a convenient marker of PKG activity. The antibody that we used in this study recognises Ser157-phosphorylated VASP and the nonphosphorylated form. VASP phosphorylation has been established as a marker of the activity of the NO-cGMP pathway in various models [START_REF] Deguchi | Vasodilator-stimulated phosphoprotein (VASP) phosphorylation provides a biomarker for the action of exisulind and related agents that activate protein kinase G[END_REF], including HEK 293 cells [START_REF] Andre | Inducible nitric oxide synthasedependent stimulation of PKGI and phosphorylation of VASP in human embryonic kidney cells[END_REF].

AMPK plays a key role in the regulation of energy homeostasis and is activated by an elevated AMP/ATP ratio produced in response to cellular and environmental stresses, such as heat shock, hypoxia and ischemia [START_REF] Kemp | AMP-activated protein kinase, super metabolic regulator[END_REF]. AMPK is itself phosphorylated by the upstream component of the protein kinase cascade AMP-activated protein kinase kinase with lower values at higher concentrations (Figure 7a). 8-Br-cGMP, a membranepermeable cGMP derivative, induced VASP phosphorylation in the absence of NO, and ODQ, an inhibitor of sGC, confirmed that VASP phosphorylation is dependent on sGC activation (Figure 7b). The increase in AMPK phosphorylation was maximal at 20 ng/ml tetracycline and was maintained at higher NO concentrations (Figure 7c). These experiments agree with the results presented earlier in this study in indicating that the NO-cGMP-dependent protein kinase signalling pathway is activated at lower NO concentrations than the NO-AMP-activated protein kinase pathway.

In conclusion, our results show that the IC 50 for respiratory inhibition by NO is around 50-fold higher than the EC 50 for activation of sGC by NO at tissue O 2 levels. Our data suggest that it is unlikely that NO concentrations that maximally activate sGC could simultaneously inhibit cellular respiration. This agrees with the greater sensitivity of isolated sGC to exogenous NO (DETA-NO) reported for the isolated enzyme in comparison with mitochondrial respiration [START_REF] Bellamy | Differential sensitivity of guanylyl cyclase and mitochondrial respiration to nitric oxide measured using clamped concentrations[END_REF]. Moreover, the NO concentrations that activate the NO-cGMP signalling pathway are lower than those required to activate AMPK. However, given that intracellular O 2 concentrations are an order of magnitude lower than tissue O 2 levels, it is possible that in this lower range of O 2 concentrations, NO regulates cell respiration as well as cGMP production during normal physiology or under pathological conditions of ischemia and hypoxia. The NO concentration that induces half-maximal activation of sGC was estimated as 2.9

© 2007 Biochemical Society 5 EXPERIMENTAL

 5 Using this system we have monitored NO generation with electrochemical NO electrodes (ISO-NOP, World Precision Instruments) and the cGMP production by immunoassay inside hypoxic chambers (Coy Laboratory Products Inc.) to maintain a constant O 2 tension of 30 µM. We have also measured NOmediated inhibition of respiration by high-resolution respirometry (Oroboros Oxygraph-2k). Because the inhibition of COX by NO depends on the O 2 tension, we measured respiration inhibition at physiological O 2 concentration (30 µM). Finally, we have compared the sensitivities to NO of the cGMP-dependent protein kinase (PKG) and AMP-activated protein kinase (AMPK) signalling transduction pathways. Our resultsshow that the concentration of NO that causes half-maximal activation of sGC is around 50-times lower than that which causes half-maximal inhibition of cellular respiration at 30 µM O 2 . We also show that the NO-cGMP signalling pathway is activated at lower NO concentrations than the AMPK pathway. We conclude that sGC is a more sensitive target of endogenous NO than cellular respiration at physiological O 2 concentration. Biochemical Journal Immediate Publication. Published on 18 Apr 2007 as manuscript BJ2007003 Cell culture and reagents Tetracycline-inducible HEK 293 cells stably expressing human iNOS (Tet-iNOS 293) were generated as described elsewhere [25]. Cells were cultured in Dulbecco's modified Eagle's medium (Gibco Invitrogen, Barcelona, Spain) containing 4.5 g/l D-glucose, 10 % (v/v) foetal-calf serum, 200 µg/ml hygromycin B and 15 µg/ml blasticidine, in the dark at 37 ºC in a humidified atmosphere with 5 % CO 2 . S-ethylisothiourea hydrobromide (S-EITU), L-arginine, 8-bromoguanosine-3',5'-cyclomonophosphate sodium salt (8-Br-cGMP), 1H-[1,2,4]oxadiazolo[4,3-a]-6-bromoquinoxazin-1-one (ODQ), 3-isobutyl-1methylxanthine (IBMX) and diethylamine NONOate diethylammonium salt (DEA NONOate) were from Sigma-Aldrich (St. Louis, MO, U.S.A.). Hygromycin B and blasticidin were from Invitrogen. Tetracycline was from Calbiochem (Darmstadt, Germany). Induction of endogenous NO production iNOS expression was induced by overnight incubation of cells in complete growth medium (without selection antibiotics) containing tetracycline (2-1000 ng/ml) and 500 µM S-EITU, a potent inhibitor of iNOS activity. Cells were washed with L-arginine-free Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 1 % foetal-calf serum dialysed to avoid any traces of L-arginine that could trigger undesired NO production. Cells were incubated in L-arginine-free medium for 1 h to completely eliminate S-EITU and tetracycline without activating NO production. Cells were trypsinized and resuspended at 1 x10 7 cells/ml in Hanks balanced salt solution containing 25 mM Hepes. Endogenous NO production was triggered by adding 1 mM L-arginine (see below). Immunoblot analysis of iNOS, VASP and phospho-AMPK Total protein extracts were prepared as follows: for iNOS analysis, attached cells were harvested by trypsinization and resuspended in Hanks balanced salt solution at 1 x 10 7 cells/ml. For VASP and phospho-AMPK analysis, attached cells were scrapped off in 1.5 Biochemical Journal Immediate Publication. Published on 18 Apr 2007 as manuscript BJ2007003 © 2007 Biochemical Society ml ice-cold PBS freshly supplemented with phosphatase inhibitors [6 mM NaF, 10 mM β-glycerophosphate, 10 mM PNPP (p-nitrophenyl phosphate, 1 mM NaVO 3 ] and a protease-inhibitor cocktail (Roche Diagnostics, Barcelona, Spain). Cells were centrifuged at 300 g for 5 min at 4 ºC and the pellet was resuspended in 60 µl ice-cold lysis buffer (20 mM Hepes, pH 7.5, 400 mM NaCl, 20 % (v/v) glycerol, 0.1 mM EDTA, 10 mM NaF, 10 µM Na 2 MoO 4 , 1 mM NaVO 3 , 10 mM PNPP (p-nitrophenyl phosphate), 10 mM βglycerophosphate) supplemented with 1 mM dithiothreitol, 1 mM pefablock, pH 7.4, and a tablet of protease-inhibitor cocktail (Roche Diagnostics, Barcelona, Spain). Cells were lysed on ice for 15 min, and cleared whole-cell extracts were obtained by recovering the supernatant after centrifugation (16000 g for 15 min at 4 ºC). After protein determination (see below), an equal amount of each protein sample (200 µg for iNOS and 120 µg for VASP and phospho-AMPK) was resolved by SDS/7.5 %-(w/v)-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, U.K.) using standard procedures [26]. Thereafter, membranes were blocked with 5 % (w/v) non-fat dry milk in TBS-T (20 mM Tris/HCl, pH 7.2, 150 mM NaCl, 0.1 % Tween 20) and incubated overnight with a polyclonal antibody against iNOS (1:2000, Transduction Laboratories, BD Biosciences, Erembodegem, Belgium), a polyclonal antibody against human VASP (1:1000, Calbiochem, San Diego, CA, U.S.A.), a polyclonal anti-phospho-AMPKα (Thr172) antibody (1:1000, Cell Signaling Technology, Beverly, MA, U.S.A.)

  Endogenous NO production was determined in cells suspended at 1 x 10 7 cells/ml in Hanks balanced salt solution containing 25 mM Hepes. Measurements were taken with an NO electrode (ISO-NOP; World Precision Instruments, Stevenage, Herts., U.K.) in 1 ml of cell suspension in gas-tight chambers gently agitated and kept at 37 ºC. The NO Biochemical Journal Immediate Publication. Published on 18 Apr 2007 as manuscript BJ2007003 © 2007 Biochemical Society electrode was calibrated by addition of known concentrations of NaNO 2 under reducing conditions (KI/H 2 SO 4 ) at 37 ºC. Calibrations were performed at low O 2 tension (30 µM) to take account of the impact of the autooxidation reaction of NO. To initiate NO production, 1 mM L-arginine was added to the chamber at 30 µM O 2 measured with an O 2 electrode (Hansatech, King's Lynn, Norfolk, U.K.). The O 2 electrode was calibrated with air-saturated incubation medium at 37 ºC, assuming an atmospheric O 2 concentration of 200 µM. NO production was monitored for at least 10 min after the addition of L-arginine. Measurement of oxygen consumption Oxygen consumption at physiological tissue O 2 concentration (30 µM) was determined by high-resolution respirometry (Oroboros Oxygraph-2k, Innsbruck, Austria). Cells were trypsinized after overnight treatment with tetracycline and resuspended at 1 x 10 7 cells/ml in Hanks balanced salt solution containing 25 mM Hepes. The instrumental background flux was calculated as a linear function of O 2 concentration and the experimental data were corrected for the whole range of O 2 concentrations using DatLab software (Oroboros Instruments, Innsbruck, Austria). The O 2 concentration in culture medium at air saturation at 37 ºC and local barometric pressure (92.6 kPa) was 175.7 µM (O 2 solubility factor 0.92). Measurements were taken at 37 ºC in parallel Oxygraph-2k

  O 2 concentration of 30 or 15 µM. The medium was replaced with Hanks balanced salt solution Biochemical Journal Immediate Publication. Published on 18 Apr 2007 as manuscript BJ2007003 © 2007 Biochemical Society 8 supplemented with 1 mM L-arginine to initiate NO production, and incubations were carried out for different periods up to 5 min. Afterwards, 0.1 M HCl was added for 30 min to stop endogenous phosphodiesterase activity and to lyse the cells. Lysates were scraped off the plates and centrifuged at 1200 g for 5 min at room temperature, and the supernatant was used directly in the assay. cGMP was measured by immunoassay using the cGMP EIA Kit for Cell and Tissue Lysates (Biomol International, Exeter, U.K.) according to the manufacturer's instructions. Protein concentration was determined in the same supernatants used to quantify cGMP. Protein measurement Protein concentration was measured with the BCA (bichichoninic acid) protein assay kit (Pierce; from Perbio Science, Tattenhall, Chester, Cheshire, U.K.) using BSA as standard. Biochemical Journal Immediate Publication. Published on 18 Apr 2007 as manuscript BJ2007003 © 2007 Biochemical Society 9 RESULTS AND DISCUSSION Production of endogenous NO by Tet-iNOS 293 cells Overnight incubation of cells with a range of concentrations of tetracycline (2-1000 ng/ml) led to a concentration-dependent expression of iNOS protein as determined by

Biochemical

  Journal Immediate Publication. Published on 18 Apr 2007 as manuscript BJ2007003 © 2007 Biochemical Society 10 detection limit of our electrode, because this tetracycline concentration yielded a clear increase in cGMP production with time. cGMP accumulation was determined by immunoassay in hypoxic chambers at three different O 2 concentrations: 200, 30 and 15

  To study the relationship between O 2 concentration and sGC activity we performed the experiments at three different O 2 concentrations. The accumulation of cGMP was essentially independent of the O 2 tension, although at very low tension (1.5 % O 2 ) a slight decrease in cGMP levels was observed (Figure 2a). Effects of O 2 tension on the expression of NOS have been extensively studied in vitro [34,35] and in vivo [36-38]. Moreover, it has been reported that sGC function is impaired in pulmonary arteries of rats exposed to chronic hypoxia [39] and that hypoxia decreases sGC expression in pulmonary artery smooth muscle cells [40]; however, it had been previously shown that sGC expression increases in lungs from rats exposed to hypoxia [41]. In our experiments, cells were exposed to hypoxia only after overnight incubation with tetracycline, during the 1 h incubation in L-arginine-free medium and the subsequent 3 min treatment with L- Biochemical Journal Immediate Publication. Published on 18 Apr 2007 as manuscript BJ2007003

13 addition

 13 of IBMX (0.5 mM) increased cGMP to almost saturate our assay at DEA NONOate concentrations of 2 µM and above.

  inhibit respiration. NO and other inhibitors that raise the enzyme's K m , will decrease the affinity of COX for O 2 and thereby induce O 2 limitation under apparently normoxic conditions. Moreover, biochemical and physiological studies suggest that haemoglobin possesses an allosterically regulated nitrite reductase activity that reduces nitrite to NO along the physiological O 2 gradient [49]. To study the sensitivity of mitochondrial respiration to endogenous NO we determined O 2 concentration and O 2 flux by highresolution respirometry (Oroboros Oxygraph-2k) in Tet-iNOS 293 cells treated with tetracycline (2-50 ng/ml; Table 1). High-resolution respirometry provides a means to accurately determine respiration at low O 2 tension [50]. In these experiments, L-arginine (1 mM) was added to cells at 60 µM O 2 . As shown in Figure 5, in cells treated overnight with a high tetracycline dose (50 ng/ml), cellular O 2 consumption was significantly inhibited immediately after the addition of L-arginine (Figure 5a). The control value in the absence of NO was 14 ± 0.8 pmol O 2 .s -1 .10 -6 cells (not shown). Since the inhibition of Biochemical Journal Immediate Publication. Published on 18 Apr 2007 as manuscript BJ2007003 © 2007 Biochemical Society 14 COX by NO is competitive with O 2 , NO is a more effective inhibitor at low O 2 tensions and hence the degree of inhibition of respiration is dependent on the O 2 concentration.

3 and cc 1 ,

 1 part of the COX enzyme, relative to the decrease in the rate of O 2 consumption [53], a Biochemical Journal Immediate Publication. Published on 18 Apr 2007 as manuscript BJ2007003 © 2007 Biochemical Society mechanism in which NO might be involved and that has important signalling consequences.

(

  AMPKK) and this phosphorylation is required for AMPK activation [57]. The phospho-AMPKα (Thr172) antibody (Cell Signaling) used in this study detects endogenous AMPKα only when phosphorylated at threonine 172, and thus acts as a marker of AMPK activity. Biochemical Journal Immediate Publication. Published on 18 Apr 2007 as manuscript BJ2007003 © 2007 Biochemical Society 16 Time-course experiments indicated that NO-induced phosphorylation was maximal at 1 min for VASP and 15 min for AMPK, as determined by immunoblot (not shown). The increase in VASP phosphorylation was transient (decreasing after 1 min) whereas AMPK phosphorylation was maintained for up to 1 h. The dose-response experiment for VASP phosphorylation by NO produced a maximal P-VASP/VASP ratio at 2 ng/ml tetracycline,

Invest. 96 ,

 96 2661-2666 35. McQuillan, L.P., Leung, G.K., Marsden, P.A., Kostyk, S.K. and Kourembanas, S. (1994) Hypoxia inhibits expression of eNOS via transcriptional and posttranscriptional mechanisms. Am. J. Physiol. 267, H1921-1927 36. Xue, C., Rengasamy, A., Le Cras, T.D., Koberna, P.A., Dailey, G.C. and Johns, R.A. (1994) Distribution of NOS in normoxic vs. hypoxic rat lung: upregulation of NOS by chronic hypoxia. Am. J. Physiol. 267, L667-L678 37. Le Cras, T.D., Xue, C., Rengasamy, A. and Johns, R.A. (1996) Chronic hypoxia upregulates endothelial and inducible NO synthase gene and protein expression in rat lung. Am. J. Physiol. 270, L164-170 38. Xue, C. and Johns, R.A. (1996) Upregulation of nitric oxide synthase correlates temporally with onset of pulmonary vascular remodelling in the hypoxic rat. Hypertension 28, 743-753 39. Crawley, D.E., Zhao, L., Giembycz, M.A., Liu, S., Barnes, P.J., Winter, R.J. and Evans, T.W. (1992) Chronic hypoxia impairs soluble guanylyl cyclase-mediated pulmonary arterial relaxation in the rat. Am. J. Physiol. 263, L325-332 40. Hassoun, P.M., Filippov, G., Fogel, M., Donaldson, C., Kayyali, U.S., Shimoda, L.A. and Bloch, K.D. (2004) Hypoxia decreases expression of soluble guanylate cyclase in cultured rat pulmonary artery smooth muscle cells. Am. J. Respir. Cell Mol. Biol. 30, 908-913 41. Li, D., Zhou, N. and Johns, R.A. (1999) Soluble guanylate cyclase gene expression and localization in rat lung after exposure to hypoxia. Am. J. Physiol. 277, L841-847 Biochemical Journal Immediate Publication. Published on 18 Apr 2007 as manuscript BJ2007003 © 2007 Biochemical Society

Figure 1

 1 Figure 1 Expression of iNOS and production of endogenous NO (a) Immunoblot analysis showing the expression of iNOS after overnight induction of cells with a range of tetracycline concentrations. Immunoblot with anti-actin antibody confirmed equal protein loading (b) Online electrochemical detection of NO gas production in cells treated with tetracycline. NO production was detected immediately after the addition of L-arginine (1 mM) to the chamber of an O 2 electrode containing 1 x 10 7 cells in suspension at 37 ºC. Addition of L-arginine was performed at 30 µM O 2 and NO was monitored for at least 10 min (c) Relationship between the tetracycline concentration used to induce iNOS expression overnight and maximal NO production after L-arginine addition at 30 µM O 2 . The data presented in (a) and (b) are representative of results obtained in three separate experiments. Data in (c) are the means and S.D. of at least three independent experiments.

Figure 2

 2 Figure 2 Time-course of endogenous NO-induced cGMP production at three O 2 concentrations (a) Cells were treated overnight with tetracycline (5 ng/ml), and a time-course of NOactivated cGMP production measured inside hypoxic chambers at three O 2 concentrations (21, 3 and 1.5 % O 2 ). Maximal cGMP accumulation was detected 3 min after L-arginine (1 mM) administration (b) Time-course as in (a) but in the presence of IBMX (0.5 mM), an inhibitor of endogenous phosphodiesterases. Values are the means and S.D. of at least three independent experiments.

Figure 3 Endogenous NO-induced cGMP production at 30 µ µ µ µM O 2 (© 2007 Biochemical Society 29 (

 3229 Figure 3 Endogenous NO-induced cGMP production at 30 µ µ µ µM O 2 (a) Cells were treated overnight with a range of tetracycline concentrations (2-50 ng/ml), and NO-activated cGMP production was determined 3 min after the addition of L- arginine (1 mM) inside hypoxic chambers at 30 µM O 2 in the absence and presence of ODQ (50 µM), a potent and selective inhibitor of NO-sensitive sGC (b) cGMP production in 10 ng/ml tetracycline-treated cells exposed for 3 min to L-arginine (1 mM) or to L-arginine plus the activator BAY 41-2272 (10 µM) at 30 µM O 2 . Addition of ODQ

Figure 4 DEA NONOate-induced cGMP production at 30 µ µ µ µM O 2 (

 42 Figure 4 DEA NONOate-induced cGMP production at 30 µ µ µ µM O 2 (a) Time-course of cGMP production in cells treated with DEA NONOate (1 µM) in the absence and presence of IBMX (0.5 mM) at 30 µM O 2 . Maximal cGMP accumulation was detected at 3 min of incubation. IBMX significantly increased the cGMP accumulation at 3 and 5 min, saturating our assay (b) Dose-response of cGMP production induced by treatment of cells with DEA NONOate (0.02-200 µM) for 3 min at 30 µM O 2 in the absence and presence of IBMX (0.5 mM). Values are the means and S.D. of three independent experiments.

Figure 5

 5 Figure 5 Inhibition of respiration induced by endogenous NO Online recording of O 2 concentration (black line) and O 2 flux (grey line) determined by high-resolution respirometry (Oroboros Oxygraph-2k) in cells pre-treated overnight with (a) 50, (b) 20 or (c) 2 ng/ml tetracycline and then stimulated by addition of L-arginine (1 mM) at an O 2 concentration of 60 µM (a and b) or 30 µM (c). The low range of O 2 concentrations -≤ 40 µM (a and b) and ≤ 25 µM (c) -and the corresponding O 2 flux are shown as a function of time (d) Online simultaneous recording of O 2 concentration (black line) and NO gas production (grey line) in cells treated with tetracycline (1000 ng/ml). NO production started right after L-arginine addition (1 mM) and immediately inhibited respiration. Addition of oxyHb quenched NO and restored the control respiration rate. The data presented in each panel are representative of results obtained in at least three separate experiments.

Figure 6 NO

 6 Figure 6 NO concentration-response curves of cGMP production and inhibition of respiration at 30 µ µ µ µM O 2

  nM. The IC 50 value of NO for cellular respiration was determined to be 141 nM. Values are the means and S.D. of at least three independent experiments. Biochemical Journal Immediate Publication. Published on 18 Apr 2007 as manuscript BJ2007003 © 2007 Biochemical Society

Figure 7

 7 Figure 7 Phosphorylation of VASP and AMPK induced by endogenous NO (a) Immunoblot analysis of non-phosphorylated VASP and VASP phosphorylated at Ser157 (P-VASP) after overnight induction of the cells with a range of tetracycline concentrations. NO production was induced by addition of L-arginine (1 mM) 1 min before cell lysis. The anti-VASP antibody detects two bands, the upper band corresponding to P-VASP. Immunoblot densitometric data are presented as the mean and range of P-VASP/VASP ratio (b) Exposure for 1 min to 8-Br-cGMP (100 µM) induced VASP phosphorylation in control cells (not treated with tetracycline) whereas the addition of ODQ (50 µM) to cells incubated with tetracycline (5 ng/ml) plus 1 min exposure to L-arginine (1 mM) prevented NO-induced VASP phosphorylation (c) Immunoblot analysis showing the expression of phosphorylated AMPK (P-AMPK) after overnight induction of the cells with a range of tetracycline concentrations. NO production was induced by addition of L-arginine (1 mM) 15 min before cell lysis. The data presented in (a-c) are representative of results obtained in two separate experiments.Immunoblots with anti-actin antibody confirmed equal protein loading.

  Figure 3

  36

  Figure 6

TABLE 1

 1 Respiration rate at physiological O 2 (30 µ µ µ µM) and increasing NO concentrations Respiration rate and NO production at 30 µM O 2 in cells treated overnight with tetracycline (2-50 ng/ml) and activated by addition of L-arginine (1 mM). Measurements of O 2 concentration and O 2 flux in control and NO-producing cells (1 x 10 7 cells/ml) were taken in parallel chambers of a high-resolution respirometer (Oroboros Oxygraph-2k) at 37 ºC as explained in Experimental. NO production was initiated by addition of L- arginine at 60 µM O 2 . Values are the means and S.D. of three independent experiments.

	Tetracycline	NO concentration	Respiration at 30 µ µ µ µM O 2
	(ng/ml)	(nM)	(pmol O 2 ⋅s -1 ⋅10 -6 cells)
	2	N.D. 1	14.2 ± 0.17
	5	N.D.	14.7 ± 0.53
	10	N.D.	13.9 ± 0.38
	15	14.5 ± 9.08	15.1 ± 0.50
	20	52.4 ± 9.34	10.9 ± 1.80
	30	318 ± 128	3.69 ± 0.05
	40	576 ± 59.8	3.58 ± 0.45
	50	739 ± 96.9	4.62 ± 0.16

TABLE 2

 2 Respiration rate at low O 2 (15 µ µ µ µM) and low NO concentrations Respiration rate at 15 µM O 2 expressed as a percentage of respiration at 40 µM O 2 (before L-arginine addition) in cells treated with low doses of tetracycline (2, 5 and 10 ng/ml). Measurements of O 2 concentration and O 2 flux in control and NO-producing cells (1 x 10 7 cells/ml) were taken in parallel chambers of a high-resolution respirometer (Oroboros Oxygraph-2k) at 37 ºC as explained in Experimental. NO production was initiated by addition of L-arginine (1 mM) at 30 µM O 2 . Values are the means and S.D. of

	three independent experiments.
	Tetracycline	L-arginine	Respiration at 15 µ µ µ µM
	(ng/ml)	(1 mM)	(% resp. at 40 µM)
	2	+	98.4 ± 1.7
		-	99.8 ± 1.2
		+	96.6 ± 3.0
	5	-	99.7 ± 0.5
		+	97.1 ± 0.8
	10	-	98.2 ± 1.4

Biochemical Journal Immediate Publication. Published on 18 Apr 2007 as manuscript BJ2007003 © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 18 Apr 2007 as manuscript BJ2007003 © 2007 Biochemical Society

N.D. No detectable Biochemical Journal Immediate Publication. Published on 18 Apr

as manuscript BJ2007003 © 2007 Biochemical Society

Acknowledgements

The authors are grateful to Dr. S. Moncada (Wolfson Institute for Biomedical Research, London, U.K.) for helpful comments on the manuscript and for kindly supplying BAY

Dr. J. Mateo (CNIC) for Tet-iNOS 293 cells used in this study, and Dr. S. Bartlett (CNIC) for careful reading of the manuscript. This work was supported by the Spanish Ministry of Science grant BFI2003-03493 awarded to S.C. Predoctoral fellowships from the Spanish Ministry of Science were received by F.R.-J. (F.P.I.) and E.A. S.C. is funded by the "Ramón y Cajal" programme.


Christelle Bonod-Bidaud

Mickaël Beraud

Elisabeth Vaganay

Frédéric Delacoux

Bernard Font

David Js Hulmes

Dr Florence Ruggiero
email: f.ruggiero@ibcp.fr

David J S Hulmes

Enzymatic cleavage specificity of the Proalpha1(V) chain processing analysed by site-directed mutagenesis

Keywords: proteolytic processing, metalloproteinases, procollagens, extracellular matrix, transient cell transfection, site-directed mutagenesis

INTRODUCTION

Most extracellular matrix proteins are synthesized by cells as precursors that undergo subsequent proteolytic processing into mature and functional molecules. Among these proteins are members of the collagen superfamily. Collagens undergo a great variety of proteolytic modifications involved in biosynthesis, fibrillogenesis, functional activation, shedding of transmembrane collagens and production of functional collagen-derived fragments also called matricryptins. The fate and functions of the released fragments derived from collagens, i.e. propeptides, collagen ectodomains and other cryptic non collagenous domains, are still under intensive investigation [START_REF] Hulmes | The collagen superfamily: from the extracellular matrix to the cell membrane[END_REF]. A large repertoire of proteinases is responsible for these processing interactions. Included among such enzymes are the ADAMTS and the BMP-1/tolloid families of metalloproteinases, and more recently the furinlike proprotein convertases [START_REF] Trackman | Diverse biological functions of extracellular collagen processing enzymes[END_REF]. Tolloid metalloproteinases play key roles in the control and orchestration of extracellular matrix deposition, cohesion, mineralization and cross-linking through proteolytic processing of the various extracellular matrix proteins. Their enzymatic activities include processing of fibrillar procollagens [START_REF] Prockop | Procollagen N-proteinase and procollagen C-proteinase. Two unusual metalloproteinases that are essential for procollagen processing probably have important roles in development and cell signalling[END_REF], laminin 5 and collagen VII [START_REF] Sasaki | Short arm region of laminin-5 gamma2 chain: structure, mechanism of processing and binding to heparin and proteins[END_REF][START_REF] Colombo | Procollagen VII self-assembly depends on site-specific interactions and is promoted by cleavage of the NC2 domain with procollagen C-proteinase[END_REF], maturation of small leucine-rich proteoglycans [START_REF] Scott | Bone morphogenetic protein-1 processes probiglycan[END_REF][START_REF] Ge | Bone morphogenetic protein-1/tolloid-related metalloproteinases process osteoglycin and enhance its ability to regulate collagen fibrillogenesis[END_REF], proteolytic activation of lysyl oxidase [START_REF] Panchenko | Metalloproteinase activity secreted by fibrogenic cells in the processing of prolysyl oxidase. Potential role of procollagen C-proteinase[END_REF] and the release of bioactive domains from SIBLING proteins (dentin sialophosphoprotein and dentin matrix protein-1) [START_REF] Steiglitz | Bone morphogenetic protein-1/Tolloid-like proteinases process dentin matrix protein-1[END_REF] and perlecan [START_REF] Gonzalez | BMP-1/Tolloid-like metalloproteases process endorepellin, the angiostatic C-terminal fragment of perlecan[END_REF].

The proteolytic processing of procollagen V is complex and depends on the activity of several enzymes. Collagen V is a minor fibrillar collagen with a broad distribution in tissues such as dermis, tendons, bones, blood vessels and cornea in which it can assemble in three different known stoichiometries. The heterotrimer 1(V) 2 2(V) is the most abundant and is widely distributed, whereas the heterotrimer 1(V) 2(V) 3(V) and the homotrimer 1(V) 3 show more restricted distributions [START_REF] Fichard | Another look at collagen V and XI molecules[END_REF][START_REF] Fichard | Human recombinant alpha1(V) collagen chain. Homotrimeric assembly and subsequent processing[END_REF]. The physiological relevance of these three molecular forms is unclear. However, a wealth of evidence favors the role of the heterotrimer 1(V) 2 2(V) in the control of fibril assembly [START_REF] Birk | Type V collagen: heterotypic type I/V collagen interactions in the regulation of fibril assembly[END_REF], a function that is not shared with by the homotrimer 1(V) 3 [START_REF] Chanut-Delalande | Control of heterotypic fibril formation by collagen V is determined by chain stoichiometry[END_REF]. A key step in collagen fibril formation is the proteolytic removal of the non collagenous N-and C-propeptides. BMP-1 cleaves pro 1(V) at both N-and Ctermini. These processing cleavages release a part of the N-propeptide at the N-terminus, the TSPN-1 domain, and the C-propeptide at the C-terminus [START_REF] Imamura | Bone morphogenetic protein-1 processes the NH2-terminal propeptide and furin-like proprotein convertase processes the COOH-terminal propeptide of pro-1(V) collagen[END_REF][START_REF] Kessler | Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V homotrimer[END_REF]. ADAMTS-2 was recently shown to cleave the heterotrimer pro 1(V) 2 2(V) extracted form skin and the recombinant homotrimer pro 1(V) 3 [START_REF] Colige | Domains and maturation processes that regulate the activity of ADAMTS-2, a metalloproteinase cleaving the aminopropeptide of fibrillar procollagens types I-III and V[END_REF]. From the determination of the cleavage site, it was deduced that the released fragment includes the TSPN-1 domain and the variable region [START_REF] Colige | Domains and maturation processes that regulate the activity of ADAMTS-2, a metalloproteinase cleaving the aminopropeptide of fibrillar procollagens types I-III and V[END_REF]. The C-propeptide of the recombinant homotrimer pro 1(V) 3 was shown to be cleaved rapidly in the conditioned medium of transfected HEK-293 cells [START_REF] Fichard | Human recombinant alpha1(V) collagen chain. Homotrimeric assembly and subsequent processing[END_REF]. It was further shown that an endogenous furin-like proprotein convertase was responsible for this cleavage [START_REF] Imamura | Bone morphogenetic protein-1 processes the NH2-terminal propeptide and furin-like proprotein convertase processes the COOH-terminal propeptide of pro-1(V) collagen[END_REF].

Interestingly, not all of these processing interactions could have been predicted by analyzing the presence of conserved consensus sequences in the N-and C-terminal regions of the pro 1(V) chain or based on our knowledge about the processing of the fibrillar procollagens.

Here, the proteolytic processing specificity of the pro 1(V) chain by BMP-1 and by furin-like proprotein convertases was analysed in details by site directed mutagenesis. In vitro digestion assays are commonly used to investigate matrix protein processing and give, in many cases, satisfactory results. However, fastidious extraction and purification steps are often necessary to obtain limited amounts of unprocessed proteins and active enzymes since most of them are present in trace amounts in tissues. These problems have been partly solved by expressing recombinant matrix proteins and enzymes, though this alternative method does not avoid purification procedures. Therefore, in the present study, the proteolytic processing of procollagen V was approached by using transient cell transfection for allowing rapid and straightforward analysis of processing interactions. Using this method, we provide new and reliable informations on the enzymatic cleavage specificity of procollagen V.

MATERIALS AND METHODS

Plasmids

Plasmids pCEP4-pro 1V containing the full-length cDNA of the human pro 1(V) chain, and pCEP4-BMP-1 were already described [START_REF] Fichard | Human recombinant alpha1(V) collagen chain. Homotrimeric assembly and subsequent processing[END_REF][START_REF] Moali | Substrate-specific modulation of a multisubstrate proteinase. C-terminal processing of fibrillar procollagens is the only BMP-1dependent activity to be enhanced by PCPE-1[END_REF]. pCEP4-N 1(V) construct was obtained from pCEP4-pro 1V, after a KpnI/XhoI digestion (from nt 1 to nt 1783) and a subcloning in pCEP4 previously digested with the same enzymes. N 1(V) is a small domain of 64 kDa containing the NC3, COL2 and NC2 domains and eleven triplets from COL1 (Fig. 1A).

Mutagenesis

For mutagenesis, N 1(V) and pro 1(V) had to be subcloned into the pcDNA3 plasmid (Invitrogen). After KpnI/XhoI digestion of N 1(V) from PCEP4-N 1(V), and KpnI/KpnI digestion of pro 1(V) from pCEP4-pro 1V, fragments were introduced into pcDNA3 linearized with KpnI/XhoI or KpnI/KpnI digestions, respectively. Mutation of the putative cleavage site of the 1(V) C-propeptide by furin (Fig. 1B) was done on pcDNA3-pro 1(V), and different simple, double or triple mutations of the BMP-1 cleavage site of N 1(V) (Fig. 1B) were done on pcDNA3-N 1(V). All mutations were done using the Quick-change II XL site-directed mutagenesis kit (Stratagen) according to the manufacturers' instructions, using

Cell culture

Human embryonic kidney (HEK) 293 EBNA cells were grown in DMEM medium supplemented with 10% fetal calf serum and penicillin-streptomycin cocktail (all from Sigma Aldrich, USA) at 37°C in a 5% CO 2 incubator. Stable cell lines were selected and grown in the same medium supplemented with 200 µg/ml hygromycin B (Calbiochem,USA).

Production and purification of recombinant proteins

pCEP4-BMP-1 and pCEP4-N 1(V) were transfected in HEK 293-EBNA cells by electroporation and the transfected cells were selected by adding hygromycin B (200 µg/ml) during 7-10 days to DMEM culture medium supplemented with 10% fetal calf serum [START_REF] Fichard | Human recombinant alpha1(V) collagen chain. Homotrimeric assembly and subsequent processing[END_REF].

Resistant 293-EBNA cell media were tested for expression of the recombinant proteins by 6% SDS-PAGE followed by Coomassie blue staining. Large amounts of serum-free medium from transfected HEK-293 EBNA cells were collected every 48 h and stored at -20°C until used.

The purified BMP-1 was obtained as previously described [START_REF] Moali | Substrate-specific modulation of a multisubstrate proteinase. C-terminal processing of fibrillar procollagens is the only BMP-1dependent activity to be enhanced by PCPE-1[END_REF]. The N 1(V) fragment was purified from conditioned media by 2 steps of ion exchange chromatography. Conditioned medium (360 ml) was dialyzed against 50 mM Tris/HCl pH 7.6, 100 mM NaCl, 2 M Urea.

After centrifugation, the supernatant was passed over a DEAE column (DE 52, Whatman) and subsequently eluted with a linear 0-0.6 M NaCl gradient. Pools containing purified recombinant N 1(V) fragment were recovered and dialyzed against 50 mM Tris/HCl pH 7.6, 100 mM NaCl. Samples were then subjected to a HitrapQ column (Pharmacia, Biotech) and eluted by a NaCl gradient. Recombinant protein-containing fractions were analyzed by SDS-PAGE on a 6% gel and dialyzed against a 50 mM Tris/HCl pH 7.6, 100 mM NaCl. Purified N 1(V) fragment was stored at -20°C until used.

Polyclonal and monoclonal antibodies

Polyclonal antibodies to BMP-1 were kindly provided by Dr. Efrat Kessler [START_REF] Parsons | Mechanical load enhances procollagen processing in dermal fibroblasts by regulating levels of bone morphogenetic protein-1 (BMP-1)/Procollagen C-Proteinase Exp[END_REF]. For production of monoclonal antibody against the N propeptide of the pro 1(V), mice were immunized with the purified recombinant N 1(V) fragment. Among the tested hybridomas, three clones produced an antibody able to recognize N 1(V) both by ELISA and Western blotting. Using the various recombinant N-propeptide domains, it was determined that the clones named 6A7 and 18G5 were specific for the variable region and to for the NC2 domain, respectively. Neither of them recognized the TSPN-1 domain (Fig. 2C).

To obtain antibodies able to recognize specifically the TSPN-1 region, rabbits were immunized with the recombinant TSPN-1 domain produced in E. Coli. The TSPN-1 cDNA (nt 258-723) was generated by polymerase chain reaction (PCR) using the N 1 clone as template. Two oligonucleotides flanking the desired sequence were designed, forward: 5'-TATGAATTCCCACCAAGCAGCTGTACCCTG-3' and reverse: 5'-TATCTGCAGGCTGTAGTGCTCACAGTAATC-3', the underlined sequences introducing a EcoRI site at the 5' end and a PstI site at the 3' end. The resulting PCR product was subcloned in the EcoRI and PstI sites of a pT7/7-6His expression vector [START_REF] Nègre | Definition of a consensus DNA-binding site for the Escherichia coli pleiotropic regulatory protein[END_REF]. The resulting plasmid, named pTSPN1-6His, encoded the TSPN-1 domain of the human pro 1(V) chain, under the control of the E. coli phage T7 promoter. E. coli host strain BL21 (DE3) was transformed with pTSPN1-6His and cells were harvested after 2 hours of IPTG induction as previously described [START_REF] Delacoux | Molecular features of the collagen V heparin binding site[END_REF]. Purified TSPN-1 domain was obtained by subjecting cell lysates to a Ni 2+ -NTA agarose column (Qiagen). The recombinant protein was analyzed by 15% SDS-PAGE followed by Coomassie blue staining. Polyclonal antibodies obtained after immunization with the recombinant protein were purified on a CNBr-activated Sepharose 4B column coupled with the purified recombinant N 1(V) fragment as indicated by the manufacturer (Pharmacia Biotech). The purified antibodies were able to recognize N 1(V) (Fig. 2C lane 1) and the recombinant TSPN-1 domain (data not shown) both by ELISA and Western blotting. Rabbit polyclonal antibodies to human pepsinized collagen V were obtained from Novotec (Lyon, France).

Transient cell transfection

For transient transfection experiments, wild-type HEK-293 EBNA cells or HEK-293 EBNA cells stably transfected with pCEP4-BMP-1 were plated in 100 mm Petri dishes with 1.5 x 10 6 cells and transfected the following day with the different plasmids using the calcium phosphate transfection kit from Invitrogen. 24 hours after transfection, cells were washed three times with PBS and placed in serum-free medium supplemented with 50 µg/ml sodium ascorbate. Media were collected 48 hours after transfection for protein analysis. Efficiency of transfection was analyzed by transfecting the cells with a plasmid that expresses green fluorescent protein (GFP). The percentage of transfection was calculated by counting green cells 48 hours after transfection with an inversed microscope equipped with fluorescence (Nikon). The values varied from 50% to 70%.

Protein analysis

Proteins were recovered from transfected cell media by addition of 1% Triton X-100 followed by precipitation with 10% trichloroacetic acid (TCA). After centrifugation, pellets were washed twice with ethanol and acetone, resuspended in Laemmli buffer and analyzed by 12%, 10% or 6% SDS-PAGE as indicated, under reducing conditions. Proteins were electrotransferred onto polyvinylidene difluoride membranes (Immobilon-P, Millipore, France) overnight in 10 mM CAPS pH 11, 5% methanol. Then, membranes were saturated for 2 hours in PBS supplemented with 10% non-fat milk. For N 1 digestion by BMP-1, double immunolabelling using two different colorimetric detection kits was performed to discriminate the BMP-1 band from the N 1 bands. Membranes were first probed with polyclonal primary antibodies against the TSPN-1 domain followed by incubation with alkaline phosphatase coupled goat anti-rabbit secondary antibodies (BioRad) and detection using an alkaline phosphatase colorimetric kit (AP Color kit, BioRad). Then membranes were probed with polyclonal primary antibodies to BMP-1 followed by incubation with peroxidase coupled goat anti-rabbit secondary antibodies (DAKO A/S, Denmark) and the signal was detected using a peroxidase colorimetric kit (Sigma Aldrich). For the furin site mutants, membranes were probed with the monoclonal 6A7 antibody followed by alkaline phosphatase coupled goat anti-mouse secondary antibodies incubation (BioRad) and detected using an alkaline phosphatase colorimetric kit. For the BMP-1 mutants, N 1 and TSPN fragments were detected with polyclonal primary antibodies against the TSPN-1 domain, followed by incubation with horseradish peroxidase coupled goat anti-rabbit secondary antibodies (DAKO A/S, Denmark) and signals were detected using an ECL reagent (Amersham Biosciences) and X-ray film exposure. When indicated, 18G5 antibody was used to detect the N 1 fragment after BMP-1 cleavage. Membranes were then de-hybridized according to the manufacturer's instructions and probed with antibodies to BMP-1 and treated as described above.

Analytical and electron microscopy methods

Amino acid sequence analysis was performed by automated Edman degradation using an Applied Biosystems 473A protein sequencer. For N-terminal sequencing of the TSPN-1 released fragment, the membrane, after immunotransfer, was treated with pyroglutamate aminopeptidase (Boehringer-Mannheim) to remove the pyroglutamic acid blocking groups before performing automated Edman degradation.

For rotary shadowing, purified N 1 fragment was diluted to 5-10 µg/ml with 0.1 M ammonium acetate. Samples were mixed with glycerol (1:1), sprayed onto freshly cleaved mica sheets and immediately placed on the holder of a MED 010 evaporator (Balzers). Rotary shadowing was carried out as previously described [START_REF] Ruggiero | Human corneal fibrillogenesis. Collagen V structural analysis and fibrillar assembly by stromal fibroblasts in culture[END_REF]. Observations of replicas were performed with a Philips CM120 microscope at the "Centre Technique des Microstructures" (Université Lyon 1, Villeurbanne, France).

The possible triple helix folding of N 1 was ascertained by pepsin digestion experiments and circular dichroïsm. Pepsin digestion experiments were performed on the N 1 fragment and the recombinant homotrimeric 1(V) as positive control [START_REF] Fichard | Human recombinant alpha1(V) collagen chain. Homotrimeric assembly and subsequent processing[END_REF]. Samples were treated with pepsin for 2 hrs at room temperature at an enzyme/substrate ratio of 1:10 in acetic acid 0.5 M.

Digestion products were analyzed by 5-20% gradient (for N 1) or 6 % (for the procollagen V homotrimer) SDS-PAGE, under reducing conditions, followed by Coomassie blue staining.

The N 1 fragment was analysed by circular dichroïsm before and after pepsin treatment. The pepsinized recombinant homotrimer collagen V, referred to as 1TH [START_REF] Fichard | Human recombinant alpha1(V) collagen chain. Homotrimeric assembly and subsequent processing[END_REF], was analyzed as control. Spectra were recorded at 20°C in 50 mM acetic acid (for 1TH) or 20 mM sodium phosphate pH 7.0 (for N 1) on a Chirascan apparatus (Applied photophysics). Measurements were performed with either 0.5 mm or 0.2 mm path length cells. Spectra were collected at 0.5 nm intervals over the wavelength range from 260 to 180 nm.

RESULTS

Expression and characterization of recombinant N 1(V) fragment

The Npro 1(V) fragment, referred to as N 1, was designed to encompass the complete Npropeptide of the human 1(V) chain, including the NC3, COL2 and NC2 domains plus eleven triplets of COL1 (Fig. 1A). The fragment contains the BMP-1 cleavage site, which is located between the TSPN-1 domain and the variable region (Fig. 1A). The theoretical molecular weight of the corresponding fragment is 64 kDa but electrophoretic analysis of serum-free medium from Npro 1(V) transfected 293-EBNA cells demonstrated a protein band at 116 kDa (Fig. 2A, lane 1), which was absent in non-transfected cell medium (data not shown). The identity of the recombinant protein was confirmed by N-terminal sequencing after electrotransfer. The determined sequence, Ala-Gln-Pro-Ala, starts with the first aminoacid of the pro 1(V) chain after the peptide signal cleavage site [START_REF] Fichard | Human recombinant alpha1(V) collagen chain. Homotrimeric assembly and subsequent processing[END_REF]. This indicates that the recombinant protein, even though it migrated much slower than expected, corresponds to the Npro 1(V) fragment. This is in agreement with previous results on the recombinant production of the entire pro 1(V) chain. Collagenase digestion of the recombinant pro 1(V) chains revealed a 86 kDa fragment corresponding to the entire N-propeptide [START_REF] Fichard | Human recombinant alpha1(V) collagen chain. Homotrimeric assembly and subsequent processing[END_REF]. The difference in migration (116 kDa vs 86 kDa) matches correlates with the presence in the construct of an additional sequence encoding the complete COL2 and NC2 domains and the 11 triplets of the COL1 domain (Fig. 1A). Two steps of anion exchange chromatography were performed to purify the recombinant fragment (Fig. 2A). Rotary-shadowing of the purified N 1 fragment revealed an homogenous preparation of 50 nm long molecules with a globular domain at one extremity, the TSPN-1 domain, and an extended tail corresponding to the variable region (Fig. 2B). This observation attests for the proper folding of the NC3 domain.

The COL2 and COL1 domains were not visible in our preparations indicating that these domains do not fold into stable triple helices. Moreover, formation of a trimer should be visualized as a fish-spear shape with 3 TSPN globular domains at one end of the molecule.

We definitively excluded the possibility that N 1 folds into a triple helix from pepsin digestion experiments and circular dichroïsm analysis. As shown in Figure 2C, N 1 was completely digested by pepsin (Fig. 2C, lane 2) whereas pepsin digestion of the 1(V) homotrimer in the same conditions showed a pepsin-resistant band (Fig. 2C, lane 4) corresponding to the triple helix domain referred to as 1TH [START_REF] Fichard | Human recombinant alpha1(V) collagen chain. Homotrimeric assembly and subsequent processing[END_REF]. CD spectra were monitored for N 1 before and after pepsin digestion and compared to the spectrum of 1TH which was typical of a triple helix conformation (a negative minimum peak at 197 nm and a positive peak around 222 nm) (Fig. 2D). The spectrum obtained for N (1) before pepsinization corresponded to signals from the different sub-domains of the fragment, particularly the non collagenous domains, TSPN and the variable region (Fig. 2D). After pepsin digestion, no particular signal was monitored for N 1 (Fig. 2D) corroborating the results obtained with SDS-PAGE analysis (Fig. 2C).

The purified fragment N (1) was used to obtain monoclonal antibodies to different regions of the N-propeptide. None of the hybridomas obtained recognized the TSPN-1 domain. The monoclonal antibody to the N-propeptide, called 6A7, was selected for this study, whose epitope was located in the variable region. It was shown to recognize the complete pro 1(V) chain and the N 1 fragment (Fig. 3, lanes 4 and 5) but not the released BMP-1 fragment, the TSPN-1 domain (data not shown). Polyclonal antibodies to the TSPN-1 domain were obtained by immunizing rabbits with the purified recombinant domain produced in bacteria.

These antibodies, pAbTSPN, recognized the complete pro 1(V) chain, the N 1 fragment (Fig. 3, lanes 1 and 2) and the recombinant TSPN-1 domain (data not shown).

N 1 is processed by BMP-1

HEK 293 cells were transiently transfected with both N 1 and BMP-1 constructs, in order to visualize digestion products immediately after SDS-PAGE analysis or immunoblotting. We chose the HEK 293 cell line for several reasons. These cells have been successfully used in the past for production of both matrix proteins and enzymes because correct protein folding and high expression level are achieved. In addition, they express endogenous furin [START_REF] Tillet | Recombinant analysis of human alpha 1 (XVI) collagen. Evidence for processing of the N-terminal globular domain[END_REF], a proprotein convertase enzyme involved in functional processing of metalloproteinases, notably BMP-1 and ADAMTS. BMP-1 processing of the C-propeptide of fibrillar collagens is known to be independent of the triple helical conformation [START_REF] Kessler | Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V homotrimer[END_REF]. To test whether the Npropeptide cleavage by BMP-1 is also conformation independent, we chose to perform experiments using the monomeric N-propeptide recombinant fragment as substrate.

Processing experiments using cell factory showed that a released 34 kDa fragment is detected in conditioned medium from cells co-transfected with N 1 and BMP-1 constructs using pAbTSPN (Fig. 4A, lane 2). This band was not detected when cells were transfected with N 1 construct alone (Fig. 4A, lane 1). The production of active BMP-1 by cells after cotransfection was assessed by probing the same membrane with BMP-1 antibodies as described in Materials and Methods (Fig. 4A lane 2). In contrast to the control (Fig. 4A lane 1), a clear band migrating at the expected position was detected in cells co-transfected with both constructs (Fig. 4A lane 2). N-terminal sequence of the released fragment confirmed that it corresponds to the N-terminal portion of the N 1 fragment. The processed N 1 fragment was identified by probing the membrane with the 18G5 antibody (not shown) and the N-terminal sequence, X-Asp-Pro-Asn-Pro, was determined after deblocking with pyroglutamate endopeptidase. It corresponds to the sequence previously described by others [START_REF] Imamura | Bone morphogenetic protein-1 processes the NH2-terminal propeptide and furin-like proprotein convertase processes the COOH-terminal propeptide of pro-1(V) collagen[END_REF]. In vitro experiments were carried out in parallel with the purified recombinant fragment and enzyme.

Western blot analysis of samples after overnight incubation at 37°C of the purified N 1 and BMP-1 also showed the appearance of a 34 kDa fragment recognized by pAbTSPN. The same blot was probed with BMP-1 antibodies to reveal the presence of the enzyme band in the assay contrary to the control in which BMP-1 was omitted (Fig. 4A, lanes 3 and 4). However, contrary to the processing assays in cells, considerable protein breakdown was observed after overnight incubation. A similar experiment was performed using cells stably transfected with the BMP-1 construct (Fig. 4B, lane 1). Analysis of conditioned media after single transfection with the N 1 construct revealed the presence of a dense band corresponding to the released TSPN-1 domain (Fig. 4B, lane 2). Our results indicate that, as for C-propeptide processing, triple helical conformation is not necessary for BMP-1 cleavage of collagen V N-propeptide.

BMP-1cleavage specificity of N 1 processing

With the exception of procollagens V and XI N-terminal processing, all the previously identified BMP-1 cleavage sites contain an aspartic acid residue at the P1' position that appears to be indispensable for BMP-1 activity [START_REF] Kessler | Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V homotrimer[END_REF]. The BMP-1 cleavage site in the Npropeptide of the pro 1(V) chain is different from the consensus site described for other matrix proteins [START_REF] Ge | Developmental roles of the BMP1/TLD metalloproteinases Birth Defects[END_REF] and corresponds to the Ser-254/Gln-255 peptide bond between the TSPN-1 domain and the variable region (Fig. 5A). The P1' residue indispensable for the cleavage is thus not an aspartic acid residue but a glutamine. To investigate which residue is essential for BMP-1 activity, six different mutants were generated from the N 1 wild type construct (Fig. 1B): three single mutants S254A, Q255A and D256A, two double mutants S254A/Q255N and Q255A/D256A and the triple mutant S254A/Q255A/D256A. Mutation to Ala of Gln-255 at the P1' position of the cleavage site might introduce a known cleavage site for BMP-1, Ala-Asp [START_REF] Kessler | Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V homotrimer[END_REF], with regard to the presence of an Asp residue at the P2' position (Fig. 1B). To address this possibility, Gln-255 was mutated to Asn in the double mutant S254A/Q255N. The D267A mutant located downstream to the cleavage site was also generated as a control (Fig. 1B). A screening for BMP-1 cleavage efficiency was assayed by transfection of mutant and wild type constructs into BMP1-293 EBNA cells (Fig. 5C) for which BMP-1 expression level was checked using antibodies against BMP-1 (Fig. 5B).

Alternatively, co-transfection of both N 1 and BMP-1 constructs in HEK-293 EBNA cells was performed to confirm the results (not shown).

Processing of the procollagen V C-propeptide by BMP-1

BMP-1 and furin are both capable of in vitro processing of the pro 1(V) C-propeptide chain [START_REF] Kessler | Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V homotrimer[END_REF]. Proteolytic C-propeptide removal by furin occurred between Arg-1585 and Asn-1586 (Fig. 1B), a cleavage site located upstream to the previously identified cleavage site of BMP-1 (Asp-1594/Asp-1595) [START_REF] Imamura | Bone morphogenetic protein-1 processes the NH2-terminal propeptide and furin-like proprotein convertase processes the COOH-terminal propeptide of pro-1(V) collagen[END_REF][START_REF] Kessler | Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V homotrimer[END_REF]. In vitro, processing of the pro 1(V) C-propeptide by furin is more efficient than processing by BMP-1. We thus took advantage of the "in cellulo" method to study 1(V) C-propeptide processing by BMP-1 in the absence of furin cleavage. The use of decanoyl-RVKR-chloromethyl ketone to protect from furin activity in HEK-293 cells only inhibited furin cleavage by about 50-70% [START_REF] Kessler | Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V homotrimer[END_REF]. We thus sought to abolish the furin cleavage site of the pro 1(V) chain by mutating to alanines the two arginines, Arg-1584 and Arg-1585, located at positions P2 and P1 of the cleavage site (Fig. 1B). The furin mutant and wild type constructs were transiently transfected into HEK-293 cells and conditioned media were analyzed by SDS-PAGE. As can be seen (Fig. 6A), the R1584/1585A mutations efficiently protected the C-propeptide cleavage from furin activity (Fig. 4A, lane 2). Only a very faint band migrating at the pN 1(V) position was observed in conditioned medium of mutant cells by Western blotting with the 6A7 antibody (Fig. 6B, lane 3). The pro 1R1584/1585A construct was then transfected into BMP-1 293 cells to analyze C-propeptide cleavage efficiency compared to the wild type (Fig. 6C andD). BMP-1 processing of pro 1R1584/1585A homotrimers yielded two bands recognized by the 6A7 antibody (Fig. 6 C, lane 2). The largest corresponds to the pC 1(V) form that migrated with a mobility faster than that of pN 1(V) (Fig. 6B, lane 3). In the same experimental conditions, processing of the wild type homotrimer by BMP-1 gave a unique band corresponding to the mature 1(V) chain (Fig. 6C, lane 1). In agreement with these results, 12% SDS-PAGE gel analysis (Fig. 6D) revealed the absence of the band corresponding to the furin cleavage in BMP-1 293 cells transiently transfected with pro 1R1584/1585A construct (Fig. 6D, lane 3 compared to lanes 1 and 2). The BMP-1 activity on the N-propeptide in the cell factory assay was attested by the presence of a faster band of same intensity (Fig. 6D, lanes 2 and 3), which corresponds to the N-terminal portion of the pro 1(V) homotrimer released by BMP-1. As can be seen (Fig. 6D), the C-propeptide cleavage by BMP-1 was not enhanced in the absence of furin cleavage.

The corresponding band migrating at about 38 kDa was of similar intensity in wild type and mutant samples (Fig. 6D, lanes 2 and 3). These data confirm in vitro experiments [START_REF] Kessler | Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V homotrimer[END_REF] and indicate that BMP-1 is capable of processing the C-propeptide even though less efficiently than furin.

DISCUSSION

Removal of the N-and C-propeptides of fibrillar collagens is accomplished by procollagen proteinases of the ADAMTS and BMP-1/TLD families respectively. This post-translational event plays a crucial role in fibril assembly [START_REF] Kadler | Assembly of collagen fibrils de novo by cleavage of the type I pC-collagen with procollagen C-proteinase. Assay of critical concentration demonstrates that collagen self-assembly is a classical example of an entropydriven process[END_REF] and proteolytic products of extracellular matrix proteins may have important biological functions [START_REF] Trackman | Diverse biological functions of extracellular collagen processing enzymes[END_REF]. Collagen V shows a partial retention of N-propeptide that inhibits lateral fibril growth [START_REF] Linsenmeyer | Type V collagen: molecular structure and fibrillar organization of the chicken alpha 1(V) NH2-terminal domain, a putative regulator of corneal fibrillogenesis[END_REF]. The excision of the Npropeptide can occur at different sites in a tissue specific manner and be ensured by BMP-1 and/or ADAMTS-2 [START_REF] Imamura | Bone morphogenetic protein-1 processes the NH2-terminal propeptide and furin-like proprotein convertase processes the COOH-terminal propeptide of pro-1(V) collagen[END_REF][START_REF] Colige | Domains and maturation processes that regulate the activity of ADAMTS-2, a metalloproteinase cleaving the aminopropeptide of fibrillar procollagens types I-III and V[END_REF]. Another feature of the minor fibrillar collagen V is that enzyme activities responsible for C-propeptide cleavage (BMP-1 and/or furin) are chain-dependant [START_REF] Kessler | Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V homotrimer[END_REF][START_REF] Unsöld | Biosynthetic processing of the pro-a1(V)pro-a2(V) collagen heterotrimer by bone morphogenetic protein-1 and furin-like proprotein convertases[END_REF].

In order to analyze processing of the procollagen V at the molecular level, we took the advantages of a cell approach based on the transient transfection of cells with plasmids that each encode one of the partners (enzyme and substrate) followed by direct analysis of digestion products. The major advantages of this procedure are the rapid screening of processing interactions, the lack of protein breakdown during purification and enzyme digestion steps and the analysis of enzyme activity in a cellular environment.

Procollagen V can be processed at the C-terminal end by two enzymes, furin and BMP-1. The consensus recognition sequence for furin cleavage is Arg-Xaa-Arg/Lys-Arg (Xaa for any other residue). Excision of the C-propeptide from the pro 1(V) chain by BMP-1 activity occurs at a site (Asp-1594/1595) next to the furin cleavage site (Arg-1585/Asn-1586) [START_REF] Imamura | Bone morphogenetic protein-1 processes the NH2-terminal propeptide and furin-like proprotein convertase processes the COOH-terminal propeptide of pro-1(V) collagen[END_REF].

Kessler et al. [START_REF] Kessler | Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V homotrimer[END_REF] showed that the C-propeptide processing by BMP-1 was much less efficient than furin cleavage and 4-fold less efficient than the cleavage of the N-propeptide by BMP-1.

Partial inhibition of furin activity, using enzyme inhibitors, showed that BMP-1 is capable of cleaving the C-propeptide from the intact molecule [START_REF] Kessler | Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V homotrimer[END_REF]. To accurately analyze the physiological relevance of BMP-1 cleavage, it was important to carry out experiments in a cellular environment and in the complete absence of furin activity. The double mutation (Arg1584/1585Ala) completely abolished furin activity and showed that the C-propeptide fragment released by BMP-1 activity did not result exclusively from further cleavage of the furin-derived fragment. However, a consistent finding over the course of several experiments was the lower efficiency of the C-propeptide cleavage by BMP-1 compared to furin and no enhancement of BMP-1 activity was obtained after mutation of the furin cleavage site. Given the strong conservation of this domain in all the fibrillar procollagens [START_REF] Boot-Handford | A novel and highly conserved collagen (pro(alpha)1(XXVII)) with a unique expression pattern and unusual molecular characteristics establishes a new clade within the vertebrate fibrillar collagen family[END_REF], a common processing mechanism was expected. However, this does not turn out right. It has been shown

that the C-propeptides of the 1 and 3 chains of collagen V, a member of the fibrillar collagen family, are efficiently cleaved by furin activity [START_REF] Imamura | Bone morphogenetic protein-1 processes the NH2-terminal propeptide and furin-like proprotein convertase processes the COOH-terminal propeptide of pro-1(V) collagen[END_REF][START_REF] Unsöld | Biosynthetic processing of the pro-a1(V)pro-a2(V) collagen heterotrimer by bone morphogenetic protein-1 and furin-like proprotein convertases[END_REF]. The procollagen Cproteinase enhancer (PCPE) is a potent stimulator of the BMP-1 processing of the major fibrillar collagens [START_REF] Ge | Developmental roles of the BMP1/TLD metalloproteinases Birth Defects[END_REF]. But the possible role of PCPE as an enhancer of BMP-1 cleavage of the fibrillar collagen V C-propeptides remains an open question. Low amounts of PCPE were detected in the 293 HEK cell media [START_REF] Moali | Substrate-specific modulation of a multisubstrate proteinase. C-terminal processing of fibrillar procollagens is the only BMP-1dependent activity to be enhanced by PCPE-1[END_REF]. The data presented here indicate either that PCPE enhancement of BMP-1 processing is not targeted to all members of the collagen fibrillar family or that the cells produce insufficient amount of PCPE to efficiently increase BMP-1 activity. The later hypothesis is likely since maximum enhancement activity appears to occur at a PCPE:procollagen molar ratio of 1:1 [START_REF] Moschcovich | Folding and activity of recombinant human procollagen C-proteinase enhancer[END_REF].

The pro 1(V) N-propeptide BMP-1 cleavage site, which occurs at the peptide bond Ser-254/Gln-255, differs from those previously described [START_REF] Kessler | Bone morphogenetic protein-1 (BMP-1) mediates C-terminal processing of procollagen V homotrimer[END_REF], most remarkably in the absence of an invariant aspartate at the P1'position. Proteolytic processing of pro 1(XI) by BMP-1 occurs at an analogous cleavage site (Fig. 7) [START_REF] Medeck | BMP-1-mediated proteolytic processing of alternatively spliced isoforms of collagen type XI[END_REF]. The pro 3(V) chain was shown to be cleaved at a previously unreported site [START_REF] Gopalakrishnan | Biosynthetic processing of the pro-a1(V)proa2(V)proa3(V) procollagen heterotrimer[END_REF]. Though the primary sequence of the pro 3(V) chain is highly homologous to pro 1(V), BMP-1 cleavage occurs at a different peptide bond located between Gln-463 and Ala-464 (Fig. 7). In that case, the complete N-propeptide is released. Again, the cleavage sequence lacks an aspartate at the P1' position and residues adjacent to the site showed no homology with the residues flanking any previously characterized cleavage sites of BMP-1 (Fig. 7). Along this line, Colige and collaborators [START_REF] Colige | Domains and maturation processes that regulate the activity of ADAMTS-2, a metalloproteinase cleaving the aminopropeptide of fibrillar procollagens types I-III and V[END_REF] showed that ADAMTS-2 processes the aminopropeptide of pro-1(V) homotrimer at the end of the variable domain at a cleavage site (Pro-Ala) different from the previously described sites (Pro/Ala-Glu) for ADAMTS-2. In generating mutants to the previously identified cleavage site, we demonstrate that the residue crucial for BMP-1 cleavage is the Asp at the P2' position of the cleavage site whereas the P1' Gln does not influence BMP-1 activity. This result was unexpected. First, the P1' Gln is conserved in the pro 1(V) and pro 1(XI) chains of various species [START_REF] Imamura | Bone morphogenetic protein-1 processes the NH2-terminal propeptide and furin-like proprotein convertase processes the COOH-terminal propeptide of pro-1(V) collagen[END_REF] but the mutation Q255A and the double mutation S254A/Q255N BMP-1 only slightly affect the activity of BMP-1. Second, the presence of a conserved Asp residue is known to be important for BMP-1 cleavage but at the P1' position. The presence of a conserved P3' Pro adjacent to the Asp residue can play an important role in BMP-1 activity although prolines are not found at similar positions in all previously identified BMP-1 cleavage sites. The fact that mutation of the P1 Ser has no effect on BMP-1 activity is less surprising since this residue is not conserved in the pro 1(XI) chains [START_REF] Imamura | Bone morphogenetic protein-1 processes the NH2-terminal propeptide and furin-like proprotein convertase processes the COOH-terminal propeptide of pro-1(V) collagen[END_REF].

Fibrillar collagens are the most abundant structural proteins in the extracellular matrix. They undergo N-and C-propeptide processing that implicates specific proteinases. Collagen V is a minor fibrillar collagen that can be distinguished from the others by its capacity to control fibrillogenesis. In addition, this molecule is submitted to a particular processing and is involved in fundamental processes such as development and human connective tissues disorders. Here, in exploring the N-and C-terminal proteolytic processing of pro 1(V) chain using several mutants, our results provide new relevant informations on this complex and poorly understood mechanism essential to the function of the extracellular matrix proteins. A, 6% SDS-PAGE analysis of transfected HEK-293 cell medium with the human pro 1(V) construct (lane 1) and with the mutant construct pro 1R1584/1585A (lane 2). B, Western blot analysis of (lane 1) purified pN 1(V) homotrimer as running position standard, transfected HEK-293 cell medium with the human pro 1(V) construct (lane 2) and with the mutant

  '. The cDNA sequences of the mutants were thoroughly checked.

  Conditioned media were analyzed byWestern blotting using anti-TSPN-1 antibodies. The results showed that N-propeptide cleavage was either unaffected or only slightly affected in cells expressing the single mutants S254A and Q255A and the double mutant S254A/Q255N (Fig.5Clanes 3, 4 and 6) as well as in D267A control (not shown). In striking contrast, the data showed that the P2' residue Asp is crucial for BMP-1 activity (Fig.5C, lane 5). In agreement with this result, BMP-1 cleavage of the double Q255A-D256A mutant was greatly affected (Fig.5C, lane 7). Transfection with the triple mutant construct completely abolished the N-propeptide cleavage by BMP-1(Fig.5C, lane 8). Our results highlight the importance of the Asp residue at the P2' position in the 1(V) N-propeptide cleavage by BMP-1.

Figure 1 :

 1 Figure 1: Constructs and mutants of the human pro 1(V) chain. A, schematic representation of the N-propeptide construct referred to as N 1. It consists of NC3, COL2, NC2 and eleven triplets from COL1. B, Sequences of the C-propeptide encompassing the furin cleavage site and of the R1584/1585A mutant. The two arginines at positions 1584 and 1585 were mutated to alanine. C, Sequences of the N-propeptide encompassing the BMP-1 cleavage site and of the BMP-1 cleavage site mutants. COL, collagenous domain; NC, noncollagenous domain. Mutated residues are underlined. Arrows indicate cleavage sites.

Figure 2 :

 2 Figure 2: Production and characterization of N 1(V) fragment and antibodies. A, 6%SDS-PAGE analysis of recombinant N 1(V) fragment produced in 293-cells. Electrophoretic patterns of serum-free medium from transfected cells (lane 1) and of the purified N 1(V) fragment (lane 2). B, Rotary-shadowing images of recombinant purified N 1(V) fragment revealing the presence of a compact globular domain (TSPN-1 domain) and an extended rodlike domain (variable region). C, 5%-20% gradient SDS-PAGE analysis of N 1(V) (lane 1) and N 1(V) digested with pepsin (lane 2) and 6% SDS-PAGE analysis of 1(V) homotrimeric molecule treated without (lane 3) and with pepsin (lane 4). D, Circular dichroism spectra of 1(V)TH, N 1 and N 1 after pepsin digestion. Buffer baselines were systematically subtracted.

Figure 3 :

 3 Figure 3: Western-blot characterization of the polyclonal antibodies to the TSPN-1 domain (pAbTSPN) and the monoclonal antibody (mAb6A7) directed against the variable region of the N 1(V) fragment. pAbTSPN (lanes 1 and 2) and the monoclonal antibody 6A7 (lanes 4 and 5) recognized the complete pro 1(V) chain (lanes 2 and 5) and the N 1(V) fragment (lanes 1 and 4). Lane 3 shows pro 1(V) chain recognition by polyclonal antibodies directed against the collagen V triple helix (from Novotec). Epitopes of the different antibodies are indicated on the schematic representation of the N 1(V) fragment presented below. Left, running positions of protein standards are indicated in kDa.

Figure 4 :

 4 Figure 4: BMP-1 cleavage of the N-propeptide domain of the pro 1(V) chain. A, Western blot analysis of BMP-1 processing of the N-propeptide of the pro 1(V) chain in 293-EHK cells (lanes 1 and 2) compared to in vitro experiments with purified proteins (lanes 3 and 4). Electrophoretic patterns of 293-HEK cell media transfected with N 1 construct alone (lane 1)

Figure 5 :

 5 Figure 5: BMP-1 cleavage specificity of N 1 processing analysed by site-directed mutagenesis. A, Sequence for the N-propeptide encompassing the BMP-1 cleavage site. The mutated residues S254, Q255, D256 and D267 are in boldface, the BMP-1 cleavage site is underlined. B, Western blot analysis showing the level of BMP-1 expression in HEK-293 transfected with BMP-1. Electrophoretic patterns of cell media transfected with a mock plasmid as negative control (lane 1), and with BMP-1 construct (lanes 2 and 3). A low level of proBMP-1 is observed in wild type HEK-293 cells transfected with a control plasmid, while the high expression level of the mature form of BMP-1 is reproducibly observed in transfected HEK-293 cell media. C, Electrophoretic patterns of cleavage products obtained by processing wild type N 1(V) and mutant constructs using cell factory assays. Western blots were performed to monitor efficiency of BMP-1 cleavage. Membranes were probed with pAbTSPN. Cells expressing BMP-1 (B, lane 2) were transfected with a mock plasmid as negative control (lane 1), wild-type N 1 construct (lane 2) or with the mutant constructs (lane 3) S254A; (lane 4) Q255A, (lane 5) D256A, (lane 6) S254A-Q255N, (lane 7) Q255A-D256A and (lane 8) S254A-Q255A-D256A. Mutants D256A, S254A-Q255A-D256A and Q255A-D256A showed no or weak BMP-1 activity. Right, running positions of the different processed and unprocessed forms.

Figure 6 :

 6 Figure 6: Effect of furin site mutagenesis on the 1(V) C-propeptide cleavage by BMP-1.

  construct pro 1R1584/1585A (lane 3). Membranes were probed with mAb 6A7. Western blot shows that the C-propeptide cleavage is completely abolished in HEK-293 cells transfected with the furin mutant. C, Western blot analysis (mAb 6A7) of the cleavage products in 293-HEK cells expressing BMP-1, transfected with the pro 1(V) construct (lane 1) and with the mutant construct pro 1R1584/1585A (lane 2). Right panel, schematic representation of the different pro 1(V) processed forms and cleavage sites. N, N-propeptide; C, C-propeptide; TH, triple helix domain. D, Electrophoretic patterns of cleavage products obtained by processing pro 1(V) (lanes 1 and 2) and the furin mutant pro 1R1584/1585A (lane 3) in 293-HEK cells expressing recombinant BMP-1 and endogenous furin (lanes 2 and 3) and in wild type HEK-293 cells (lane 1). Samples were run on a 12% SDS-PAGE and stained with Coomassie blue. Right, running positions of the different processed forms.

Figure 7 :

 7 Figure 7: Alignment of sequences corresponding to the BMP-1 cleavage site of the Npropeptide of the human pro 1(V) (A) and pro 3(V) (B) chains with human pro 1(XI). COL, collagenous domain, NC, non collagenous domain. Conserved residues are boldface, arrows indicate known BMP-1 cleavage sites in (A) human pro 1(V) and pro 1(XI) and (B) pro 3(V) (accession numbers are NM000093, NM001854 and NM015719 respectively).

  Figure 5

  Figure 6

  Figure 7

Biochemical Journal Immediate Publication. Published on 4 Apr 2007 as manuscript BJ20070051

Biochemical Journal Immediate Publication. Published on 4 Apr 2007 as manuscript BJ20070051 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements: We thank Efrat Kessler for the generous gift of BMP-1 antibodies and for helpful discussion on BMP-1 specificity, Jean-Claude Cortay for providing the pT7/7-6His expression vector plasmid and Yvette Descollonges for the production of monoclonal antibody to the N-propeptide of the human 1(V) chain. Circular dichroism experiments have been performed with the help of Rolland Montserret at the plateform "Production And Purification of Proteins" from the IFR 128 BioSciences Gerland (Université Lyon 1). This work was supported by grants from the "Association pour la Recherche contre le Cancer" (n°3652), the Emergence Research Program (Région Rhône-Alpes), GIS Maladies Rares INSERM (A04115SP) and the European Commission (Contract NMP2-CT-2003-504017). MB is the recipient of an Emergence Research Program bursary (Région Rhône-Alpes).

The abbreviations used are: BMP-1, bone morphogenetic protein-1; ADAMTS, a disintegrin and metalloproteinase domain with thrombospondin type 1 repeats; N-pro, N-propeptide; Cpro, C-propeptide.