Chun Kim

Zoya Slavinskaya

A Rod Merrill

Stefan H E Kaufmann
email: kaufmann@mpiib-berlin.mpg.de

Human α α α α-Defensins Neutralize Toxins of the Mono-ADP-Ribosyltransferase Family Running title: Neutralization of Bacterial Toxins as Prominent Defense Mechanism of α-Defensins

Keywords: Human neutrophil protein 1, Diphtheria toxin, Pseudomonas exotoxin A

Various bacterial pathogens secrete toxins, which are not only responsible for fatal pathogenesis of disease, but also facilitate evasion from host defenses. One of the best known bacterial toxin groups is the mono-ADP-ribosyltransferase family. Here, we demonstrate that human neutrophil α α α α-defensins are potent inhibitors of the bacterial enzymes, particularly against diphtheria toxin (DT) and Pseudomonas exotoxin A (ETA). Human neutrophil protein 1 (HNP1) inhibited DT-or ETA-mediated ADP-ribosylation of eukaryotic elongation factor 2 (eEF2) and protected HELA cells against DT-or ETA-induced cell death. Kinetic analysis revealed that inhibition of DT and ETA by HNP1 was competitive with respect to eEF2 and uncompetitive against NAD + substrates. Our data reveal that toxin neutralization represents a novel biological function of HNPs in host defense.

INTRODUCTION

Mono-ADP-ribosylation, a covalent chemical modification process, is not only a component of host cell intracellular signaling but is also a widely used mechanism of bacterial pathogenesis [START_REF] Corda | Mono-ADP-ribosylation: a tool for modulating immune response and cell signaling[END_REF]. This reaction is catalyzed by mono-ADP-ribosyltransferases (ART), which transfer the ADP-ribose moiety of NAD + to a target protein with nicotinamide release [START_REF] Okazaki | Mono-ADP-ribosylation: a reversible posttranslational modification of proteins[END_REF]. This enzyme family is structurally classified into two groups: the cholera toxin (CT) group and the diphtheria toxin (DT) group [START_REF] Domenighini | Three conserved consensus sequences identify the NAD-binding site of ADP-ribosylating enzymes, expressed by eukaryotes, bacteria and Teven bacteriophages[END_REF][START_REF] Han | The ARTT motif and a unified structural understanding of substrate recognition in ADP-ribosylating bacterial toxins and eukaryotic ADPribosyltransferases[END_REF].

Although these two groups do not share any significant sequence homology, they show conserved secondary and tertiary structural elements. The CT group bears an NAD + binding pocket characterized by the catalytic Glu, a β/α structural element, and an Arg residue. The majority of bacterial ARTs and mammalian mono-ARTs belong to this group. On the other hand, the DT group including DT and ETA comprises a conserved Glu residue, the β/α motif and a His residue instead of Arg. It is well established that DT and ETA cause cell death by inactivating eEF2 via ADP-ribosylation [START_REF] Collier | Understanding the mode of action of diphtheria toxin: a perspective on progress during the 20th century[END_REF].

The NAD + -dependent and eEF2-dependent ART activities of the toxins were measured as reported previously [START_REF] Armstrong | Application of a fluorometric assay for characterization of the catalytic competency of a domain III fragment of Pseudomonas aeruginosa exotoxin A[END_REF][START_REF] Yates | Elucidation of eukaryotic elongation factor-2 contact sites within the catalytic domain of Pseudomonas aeruginosa exotoxin A[END_REF]. In brief, the NAD + -dependent ART assay was performed with the eEF2 concentration in the assay medium held to 14 µM, while the concentration of ε-NAD + varied between 0 and 500 µM in the presence of HNP1 at 0, 5, 10, 15, and 20 µM. The reactions were initiated with 20 nM (final concentration) ETAc or DTA. The eEF2-dependent ART assay was performed at 500 µM ε-NAD + while varying eEF2 substrate (0-15 µM) in the presence of HNP1 at 0, 5, 7.5, and 10 µM.

The data were analyzed by linear regression analysis of both the Hanes-Woolf and the Lineweaver-Burk (LB) plots. The K i values were determined using Dixon plots, as well as from secondary plots of the slope of the LB plots versus inhibitor concentration.

Infection assay

One day before the assay, HELA cells were seeded in 96-well plates at a density of 2 × 10 4 cells per well in 100 µl RPMI 1640 medium containing 10% fetal calf serum (FCS). For the assay, 4 × 10 5

Corynebacterium diphtheriae cells (ATCC #13812) were added per well and the described amounts of HNP-1 were then added to the cells in serum-free RPMI 1640. Six hours after infection, bacteria were killed by Penicillin and Streptomycin and cells were further incubated for 24h. Cytotoxicity was determined by the CytoTox 96® cytotoxicity assay (Promega).

RESULTS and DISCUSSION

HNP1 is not a substrate of bacterial ARTs

It has been shown recently that HNP1 is a substrate of mammalian ART-1 [START_REF] Paone | ADP ribosylation of human neutrophil peptide-1 regulates its biological properties[END_REF]. Therefore, we first examined whether bacterial ARTs were also able to transfer ADP-ribose to HNP1. CT and pertussis toxin (PT) were tested as representatives of the CT group, and DT and ETA as those of the DT group.

Consistent with previous findings [START_REF] Paone | ADP ribosylation of human neutrophil peptide-1 regulates its biological properties[END_REF], we found distinct absorbance peaks in HNP1 incubated with mouse ART-1 corresponding to the ADP-ribose HNP-1 product(s) (Figure 1, denoted by arrows). In contrast, HNP1 treated with bacterial ARTs showed the same profile as the control (HNP1 without treatment), i.e., no ADP-ribose product was observed (Figure 1). We conclude that HNP1 does not serve as a substrate for these bacterial ARTs.

HNPs protect cells from DT-or ETA-mediated cell death

Next, we determined whether HNPs had any effect on bacterial ART-treated cells. HELA cells treated with DT or ETA succumbed to these toxins within 24 h. In marked contrast, the addition of HNP1 completely abolished cytotoxicity in a dose-dependent manner (Figures 2A and2B). Purified natural HNP1-3 mixtures from human leukocytes showed similar protective capabilities (data not shown) whereas LL-37, another neutrophil cationic peptide with similar size and net charge as HNP1-3, did not display any significant effect (Figures 2A, and2B).

We also examined the effect of HNPs on CT-or PT-treated Chinese hamster ovary (CHO) cells. However, we did not detect any significant difference of cAMP levels between toxin only treated groups and toxin and HNPs treated groups, indicating HNPs do not prevent CT-or PT-mediated intoxication in CHO cells (data not shown).

HNP1 inhibits ADP-ribosylation of eEF2

To determine whether HNP1 directly inhibits ART activity of DT and ETA, we examined the transfer of radiolabelled ADP-ribose from NAD + to eEF2 in vitro. The eEF2 in HELA cell extracts was efficiently ADP ribosylated after incubation with activated DT or ETA. In contrast, ADP-ribosylation of eEF2 was inhibited in the presence of HNP1 in a dose-dependent manner. LL-37 had no significant effect on this reaction suggesting that the inhibition by HNP1 was specific (Figure 3). Surprisingly, linearized HNP1 also showed comparable inhibition activity with intact HNP1. HNP1 contains three intramolecular disulfide linkages to maintain its stable and compact conformation. Our previous study suggested that the unique structure of HNP1 determined by disulfide bonds is a crucial requirement To elucidate the mode of action, we characterized the kinetics of inhibition of HNP1 against both DTA and ETAc. The initial rates of enzyme reaction in the presence of various concentrations of HNP1 provided an IC 50 value of 11.1 ± 2.7 µM against DTA and 19.2 ± 6.5 µM against ETAc (Figures 5A and5B). In competition assays, when the concentration of eEF2 was held constant and that of NAD + was varied, HNP1 was identified as an uncompetitive inhibitor against both toxins, invoking a change in both K M and V max (Figures 6A and6B). On the other hand, when the concentration of NAD + was fixed and that of eEF2 was varied, HNP1 acted as a competitive inhibitor (Figure 6C and inhibition data against DTA not shown). Kinetic and thermodynamic parameters for the interaction of HNP1 with both DTA and ETAc in this study are summarized in Table 1. These data show that HNP1 is a more effective inhibitor against DTA than ETAc (IC 50 values: 11.1 ± 2.6 and 19.2 ± 6.5 µM, respectively; Ki values with variable NAD + : 7.3 ± 1.7 and 13.3 ± 0.9 µM, respectively), which correlates well with the data from the HELA cell protection assay (Figure 2).

Our kinetic studies indicate that HNP1 is a competitive inhibitor against both DT and ETA with respect to eEF2. Mammalian ART-1 has been shown to transfer ADP-ribose to HNP1 [START_REF] Paone | ADP ribosylation of human neutrophil peptide-1 regulates its biological properties[END_REF]. As a corollary, HNP1 would be expected to recognize and bind to the eEF2 docking site within both DT and ETA. It is, therefore, not surprising that HNP1 is a competitive inhibitor against the eEF2 Copyright 2006 Biochemical Society substrate for the toxins. On the other hand, HNP1 acted as an uncompetitive inhibitor for NAD + substrates for the toxins. An uncompetitive inhibitor is defined as an inhibitor binding to the enzyme-substrate (ES) complex but not to the free enzyme (E). Although HNP1 bound to DT and ETA in the absence of NAD + (Figures 4A andB), it was identified as an uncompetitive inhibitor with respect to NAD + in our assays. The following explanation is a plausible rationalization of this observation. It is possible that HNP1 binds to the free E in a unique manner without affecting the binding of NAD + . Yet, once NAD + is accommodated in the E-binding pocket, HNP1 may bind to the ES complex in an inhibitory mode, which results in a kinetically impaired E (lower V max and higher K M ). It remains to be elucidated whether unique HNP1-3 binding pockets exist in such functionally distinct bacterial toxins as LF [START_REF] Kim | Human alpha-defensins neutralize anthrax lethal toxin and protect against its fatal consequences[END_REF], DT, and ETA.

HNP1 Rescues HELA cells from C. diphtheriae-induced cell death.

Having identified DT neutralization as a biological function of HNP1, we decided to examine whether HNP1 neutralizes DT that has been secreted by bacteria. HELA cells were infected with C. diphtheriae and were treated immediately with the indicated amounts of HNP1. As shown in Figure 7, we confirmed that HNP1 protects cells from C. diphtheriae-induced cell death in a dose-dependent manner.

Recently, we identified a new biological function of HNPs, namely neutralization of a secreted bacterial enzyme, the LF of B. anthracis. [START_REF] Kim | Human alpha-defensins neutralize anthrax lethal toxin and protect against its fatal consequences[END_REF]. Consistent with, and in extension of our previous findings, here we show that HNP1-3 act as inhibitors of two additional bacterial toxins, DT and ETA.

It is well known that these toxins are critical for the pathogenesis caused by Corynebacterium diphtheriae and P. aeruginosa [START_REF] Yates | Stealth and mimicry by deadly bacterial toxins[END_REF]. These two toxins use molecular mimicry of an essential part of the eukaryotic host cell's rRNA in order to minimize the risk that the host could develop resistance against them. This is because mutations that might confer resistance to the host cell would also likely Copyright 2006 Biochemical Society interfere with translation efficiency and fidelity in the host [START_REF] Jorgensen | Exotoxin A-eEF2 complex structure indicates ADP ribosylation by ribosome mimicry[END_REF]. As an alternate defense strategy, the host has developed the capacity to neutralize these toxins by binding their enzyme domains with the α-defensins, HNP1-3. The HNP1-3 can be internalized into host cells [START_REF] Nassar | Human alpha-defensin regulates smooth muscle cell contraction: a role for low-density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor[END_REF] and the K d of HNP1 for DT and ETA indicate that it can compete efficiently with eEF2 for association with these toxins inside host cells. HNP1-3 have several advantages over known NAD + analogue inhibitors. First, HNP1-3 can prevent toxin binding to ribosomes by blocking the toxin's association with eEF2. It has been suggested that ART toxins can complex with eEF2 on the ribosomes [START_REF] Jorgensen | Exotoxin A-eEF2 complex structure indicates ADP ribosylation by ribosome mimicry[END_REF][START_REF] Spahn | Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation[END_REF]. Since NAD + analogues fail to prevent toxins from binding to eEF2, the toxin may still be able to interfere with eEF2 on translational complexes. Second, HNP1-3 are more specific for the toxins because NAD + analogues will potentially inhibit various host metabolic enzymes such as dehydrogenases eventually becoming toxic to host cells.

Although HNPs are well known as antimicrobial peptides [27], emerging evidence suggests additional biological functions [28]. Many bacterial pathogens secrete enzymes into the host environment to avoid destruction by the immune system. Because recruited human neutrophils release abundant HNP1-3 locally in the inflammatory regions, we assume that HNP1-3 achieve efficient neutralization of bacterial enzymes in vivo. Hence we propose that neutralization of a group of bacterial toxins constitutes a major function of HNP1-3 in antimicrobial host defense. HELA cell lysates were incubated with DT (A) or ETA (B) in the presence of the indicated amounts of HNP1 or LL-37. The samples were analyzed by SDS-PAGE followed by autoradiography. In the presence of HNP1 at 0.1, 0.5, and 2.5 µM, DT activity decreased to 25, 8, and 0.5% of control DT activity and ETA activity declined to 37, 12, and 1% of control ETA activity, respectively. The binding isotherm for HNP1 with DTA (A) or ETAc (B) was determined from the quenching of the intrinsic protein fluorescence. The raw fluorescence quenching data were converted to fractional saturation values [START_REF] Armstrong | Characterization of competitive inhibitors for the transferase activity of Pseudomonas aeruginosa exotoxin A[END_REF] and plotted against the HNP1 concentration.

FIGURE LEGENDS AND TABLE

Biochemical

  Figures 4A and 4B show the fluorescence quench curves for the intrinsic tryptophan fluorescence of

Biochemical

  Journal Immediate Publication. Published on 3 Jul 2006 as manuscript BJ20060425

Figure 1

 1 Figure 1 Separation of reaction products of HNP1 after incubation with mono-ARTs.

Figure 2

 2 Figure 2 Effect of HNP1 on DT-or ETA-treated cells.

Figure 3

 3 Figure 3 Effect of HNP1 on DT or ETA-mediated ADP-ribosylation of eEF2.

Figure 4

 4 Figure 4 Binding properties of HNP1 to DT and ETA.

Figure 5

 5 Figure 5 Inhibitory properties of HNP1 against DTA and ETAc.

Biochemical Journal Immediate Publication. Published on 3 Jul 2006 as manuscript BJ20060425

Ganz, T. (2004) Defensins: antimicrobial peptides of vertebrates. C. R. Biol. 327, 539-549

ACKOWLEDGEMENT

We thank Dr. Friedrich Nolte for murine ART-1 expression vector and helpful recommendations for purification. We also thank Dr. Hubert Kalbacher for providing LL-37. We are grateful to Robert Hurwitz and Gerry Prentice for expert technical support. S.H.E.K. acknowledges financial support from EU-FP6 (Anthrax Euronet). A.R.M. received support from the Canadian Institutes of Health Research (CIHR) and the Canadian Cystic Fibrosis Research Foundation (CCFF).

were used for examining the inhibitory properties of HNP1 against DTA (A) and ETAc (B). The concentration of the inhibitor that reduces the activity of the enzyme by 50%, the IC 50 value, was determined by nonlinear regression curve fitting using Origin 6.1 (OriginLab, Northhampton, MA).

The data were fit to the exponential first-order decay function.


Peter Prehm
email: prehm@uni-muenster.de

Biosynthesis of Hyaluronan Direction of Chain Elongation

Keywords: Hyaluronan biosynthesis, chain elongation, chain size, recombinant synthase, mechanism, membranes

The mechanism of hyaluronan biosynthesis in vertebrates had been proposed to occur at the reducing end of growing chains. This mechanism was questioned, because a recombinant synthase appeared to add new monosaccharides to the non-reducing end. I reinvestigated this problem with membranes from the eucaryotic B6 cell line. The membranes were incubated with UDP-[ 3 H]GlcNAc and UDP-[ 14 C]GlcA to yield differentially labelled reducing terminal and non-reducing terminal domains. Digestion of the product with a mixture of the exoglycosidases ß-glucuronidase and ß-N-acetylglucosaminidase truncated the hyaluronan chain strictly from the non-reducing end. The change in 3 H/ 14 C ratio of the remaining hyaluronan fraction, during the course of exoglycosidase digestion, confirmed the original results that the native eucaryotic synthase extended hyaluronan at the reducing end. This mechanism demands that the UDP-hyaluronan terminus was bound to the active site within the synthase and should compete with the substrates for binding. Accordingly, increasing substrate concentrations enhanced hyaluronan release from the synthase. A model is proposed that explains the direction of chain elongation at the reducing end by the native synthase and at the non-reducing end by the recombinant synthase based on a loss of binding affinity of the synthase towards the growing UDP-hyaluronan chain.

INTRODUCTION

Hyaluronan is synthesized, without any primer requirement, by alternate addition of Dglucuronic acid and N-acetyl-D-glucosamine units from the corresponding UDP-sugar donors (UDP-GlcA and UDP-GlcNAc) [1;2]. The growing chains are exported from streptococci and from eucaryotic cells by ABC transporters. Transport can be inhibited by inhibitors of multidrug resistance transporters [START_REF] Prehm | Hyaluronate is synthesized at plasma membranes[END_REF][START_REF] Ouskova | Hyaluronan release from Streptococcus pyogenes: Export by an ABC transporter[END_REF][START_REF] Prehm | Inhibition of hyaluronan export from human fibroblasts by inhibitors of multidrug resistance transporters[END_REF][START_REF] Prehm | Inhibitors of hyaluronan export prevent proteoglycan loss from osteoarthritic cartilage[END_REF]. Hyaluronan synthases have been tentatively classified based on primary structure, molecular size, predicted membrane topology, and enzymological characteristics [START_REF] Deangelis | Hyaluronan synthases: fascinating glycosyltransferases from vertebrates, bacterial pathogens, and algal viruses[END_REF]. Class I thus includes the streptococcal, the vertebrate and the viral enzymes, whereas Class II contains a single member, the Pasteurella multocida enzyme.

In 1983 I proposed that hyaluronan chains are elongated at the reducing end, because the digestion kinetics of pulse chase labelled chains with exoglycosidases immediately reduced the incorporated radioactivity that was introduced into the chains during the first pulse reaction [START_REF] Prehm | Synthesis of hyaluronate in differentiated teratocarcinoma cells. Mechanism of chain growth[END_REF]. These experiments were confirmed by Asplund et al. [START_REF] Asplund | Characterization of hyaluronan synthase from a human glioma cell line[END_REF] with a human glioma cell line, and by Tlapak-Simmons et al. [START_REF] Tlapak-Simmons | Hyaluronan biosynthesis by class I streptococcal hyaluronan synthases occurs at the reducing end[END_REF] with streptococci. Both of these native enzyme systems failed to further extend preformed, exogenously added hyaluronan oligosaccharides.

In contrast, the structurally unrelated synthase from Pasteurella multocida synthesizes hyaluronan at the non-reducing end [START_REF] Deangelis | Molecular directionality of polysaccharide polymerization by the Pasteurella multocida hyaluronan synthase[END_REF]. Uncertainty about the direction of hyaluronan synthesis in eucaryotic cells arose, because a recombinant hyaluronan synthase from Xenopus laevis expressed in yeast added new sugar residues to the non-reducing end [START_REF] Bodevin-Authelet | Biosynthesis of Hyaluronan: Direction of chain elongation[END_REF]. In addition, a recombinant synthase was able to elongate hyaluronan oligosaccharides and this should not occur, if synthesis proceeded at the reducing end [START_REF] Hoshi | An engineered hyaluronan synthase: characterization for recombinant human hyaluronan synthase 2 Escherichia coli[END_REF]. Because of these contradictory results, I decided to reinvestigate the directionality of hyaluronan chain elongation catalysed by native eucaryotic hyaluronan synthase applying the double labelling method of Bodevin-Authelet et al. [START_REF] Bodevin-Authelet | Biosynthesis of Hyaluronan: Direction of chain elongation[END_REF]. The results confirmed that the native eucaryotic synthase added new sugars at the reducing end.

The results also shed light on the factors that determine chain length. I previously demonstrated that hyaluronan is liberated from the synthase by dissociation [13]. If nascent hyaluronan is degraded, the synthase elongates it to a size that is determined by thermodynamic factors [14;15]. It is an unexplained observation that the three eucaryotic hyaluronan synthases produce hyaluronan with different chain lengths [START_REF] Itano | Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties[END_REF]. My results

EXPERIMENTAL

Materials

Radiolabelled UDP-[ 14 C]GlcA (specific activity 0.196 Ci/mMole) was obtained from Amersham Biosciences and UDP-[ 3 H]GlcNAc (specific activity 38.5 Ci/mMole) from PerkinElmer Life Sciences. For preparation of the substrate solution, 50 :Ci of UDP-[ 3 H]GlcNAc was mixed with 132 :g of unlabelled UDP-GlcNAc and diluted to 20 ml for the substrate solution to obtain a final concentration of 10 :M and a specific activity of 0.1 Ci/mMole. Additional chemicals were from Sigma-Aldrich Chemical Corporation.

Isolation of plasma membranes

B6 cells, a hybrid cell line derived from mouse mammary carcinoma and a Chinese hamster lung cell line which produced large amounts of hyaluronan [17;18] were grown in suspension culture in Dulbecco's modified Eagles medium supplemented with streptomycin/penicillin (100 units of each/ml), kanamycin (100 units/ml) and 10 % foetal calf serum. A suspension culture was grown to a density of 2x10 5 /ml in 4 culture flasks (180 cm 2 surface area). Four hours before cell harvest, hyaluronan synthesis was stimulated by supplementation of additional foetal calf serum (5 %). The cells were sedimented at 1500 g for 5 min, washed with phosphate buffered saline (PBS) and suspended in 30 ml of ice-cold PBS [START_REF] Prehm | Hyaluronate is synthesized at plasma membranes[END_REF]. The cells were transferred into a Parr-bomb, exposed to a nitrogen pressure of 900 psi for 15 min and disrupted by nitrogen cavitation [START_REF] Klempner | Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation[END_REF]. Nuclei were sedimented at 2000 g for 10 min and the particulate fraction was obtained by centrifugation at 20.000 g for 20 min.

Hyaluronan Synthesis and Isolation

For synthesis of [ 3 H]hyaluronan, the membrane fraction was suspended in 1 ml of prewarmed substrate for hyaluronan synthesis that contained 8 :M UDP-GlcA, 10 :M UDP-[ 3 H]GlcNAc, 4 mM dithiothreitol, 20 mM MgCl 2 in 50 mM TRIS-malonate pH 7.0 and incubated again at 37/C for 30 min. The membranes were sedimented by centrifugation for 5 min at 14.000 g. An aliquot (1/3) was solubilized in 1 % SDS and heating to 90/C for 5 min.

Undissolved material was sedimented by centrifugation for 5 min at 14.000 g and the supernatant was applied to gel filtration on Sephacryl S-1000 (1.4x 64 cm) with 10 mM NH 4 HCO 3 as eluant at a flow rate of 14 ml/h. Fractions of 2 ml were collected and the radioactivity of 100 :l was determined. For synthesis of dual labelled hyaluronan, the rest of the membrane fraction (2/3) was sedimented and suspended in 1 ml of prewarmed substrate for hyaluronan synthesis that contained 8 :M UDP-[ 14 C]GlcA, 150 :M UDP-GlcNAc, 4 mM dithiothreitol, 20 mM MgCl 2 in 50 mM TRIS-malonate pH 7.0 and incubated at 37/C for 15 min. The membrane fraction was again sedimented, solubilized in SDS and applied to gel filtration on Sephacryl S-1000 as described above.

Enzymatic Degradation of Hyaluronan

The dual labelled hyaluronan fractions from the Sephacryl S-1000 column (fractions 25 to 35) were collected, lyophylized to dryness and dissolved in 4 ml of 0.1 M sodium acetate pH 5.2. The enzymes ß-N-acetylglucosaminidase (0.8 U) and ß-glucuronidase (10000 U) were added and incubated in four aliquots in dialysis bags at 37/C and simultaneously dialyzed against 5 l of 10 mM sodium acetate pH 5.2. After different time periods, the radioactivity of the remaining [ 3 H/ 14 C]hyaluronan in the dialysis bags was determined.

Biochemical Journal Immediate Publication. Published on 23 May 2006 as manuscript BJ20060431

Copyright 2006 Biochemical Society

RESULTS

Synthesis of dual labelled hyaluronan

The eukaryotic hyaluronan synthase has a rather high K M value for UDP-GlcNAc (about 60 :M) and thus high substrate concentrations are required to obtain sufficient chain elongation.

On the other hand, large amounts of UDP-[ 3 H]GlcNAc with high specific radioactivity impede the purification of [ 3 H/ 14 C]hyaluronan. Therefore a series of preliminary experiments were performed to evaluate the optimal experimental conditions to produce dual labelled hyaluronan chains by incubation of membranes from the eukaryotic B6 cell line with different substrate concentrations, specific radioactivities, and synthesis periods. A compromise was found that coped with both a sufficient chain elongation rate and a measurable [ 3 H]hyaluronan radioactivity. Hyaluronan was first labelled by incubation of membranes with UDP-[ 3 H]GlcNAc at a concentration of 10 :M und unlabelled 8 :M UDP-GlcA for 30 min. The [ 3 H]hyaluronan was analysed by gel filtration using Sephacryl S-1000. Fig. 1a shows that most of the [ 3 H]hyaluronan was included in the middle of the column eluate. In a second experiment, the membranes were incubated with a substrate solution containing 8 :M UDP-[ 14 C]GlcA and unlabelled 150 :M UDP-GlcNAc for 15 min and subjected again to gelfiltration. Fig. 1b shows that this labelling condition yielded a size of hyaluronan that was also included in the column eluate. In the third experiment, the two incubation conditions were utilized sequentially and the size of [ 3 H/ 14 C]hyaluronan was again analysed by gelfiltration. Fig. 1c shows that [ 3 H]hyaluronan was further elongated by [ 14 C]hyaluronan. For the subsequent exoglycosidase digestion, the [ 3 H/ 14 C]hyaluronan containing fractions were isolated.

Exoglycosidase digestion of dual labelled hyaluronan

The dual labelled hyaluronan of high molecular weight was subjected to graded exoglycosidase (ß-N-acetylglucosaminidase and ß-glucuronidase) digestion as described in the Methods section. The digestion was performed with simultaneous dialysis. Aliquots in the dialysis bags were withdrawn at various time periods to determine the residual radioactivity.

The results are shown in Table 1. The radioactivity of the dual labelled hyaluronan decreased continuously. The ratio of 3

Effect of substrate concentration on hyaluronan release from the synthase

If the terminal UDP-hyaluronan is bound to the synthase during elongation at the sites where the substrates also bind, the concentration of the substrates should influence not only the rate of elongation, but also the rate of chain release. Therefore membranes from B6 cells were incubated with different concentrations of substrates and the release of hyaluronan from the membranes into the soluble fractions was determined. Fig. 2a shows that hyaluronan was first incorporated into the membrane fraction and then released. The proportion of soluble hyaluronan increased with the substrate concentrations (Fig. 2b). Simular results were obtained, when only the concentration of substrate was altered (data not shown). Thus the substrates appeared to compete with UDP-hyaluronan for binding to the synthase.

DISCUSSION

It would be desirable to obtain a homogeneous preparation of double labelled hyaluronan for an experimental proof of the direction of hyaluronan chain elongation by exoglycosidase digestion. However, I have shown previously that continuous new chain initiation occurred during hyaluronan synthesis [START_REF] Prehm | Synthesis of hyaluronate in differentiated teratocarcinoma cells. Characterization of the synthase[END_REF] and thus conclusions can only be derived from enriched hyaluronan preparations in our study on the native enzyme as well as on the recombinant enzyme by Bodevin-Authelet et al. [START_REF] Bodevin-Authelet | Biosynthesis of Hyaluronan: Direction of chain elongation[END_REF]. Therefore, the double labelled hyaluronan preparation used for exoglycosidase digestion was heterogeneous and its size overlapped with the single labelled hyaluronan. But most of the first labelled [ 3 H]hyaluronan increased in molecular weight during the second incubation and coeluted with the second [ 14 C]labelled hyaluronan, indicating that it had been further elongated to some extent. This heterogeneity is reflected in the simultaneous release of both labels during exoglycosidase digestion, and conclusions are only based on differences of release rates. In contrast to the study of Bodevin-Authelet et al. [START_REF] Bodevin-Authelet | Biosynthesis of Hyaluronan: Direction of chain elongation[END_REF] on recombinant hyaluronan synthase, I found that hyaluronan was elongated at the reducing end by the native synthase confirming the original mechanism

[1;2].
It is surprising that a recombinant eucaryotic hyaluronan synthase expressed in yeast synthesizes hyaluronan at the non-reducing end [START_REF] Bodevin-Authelet | Biosynthesis of Hyaluronan: Direction of chain elongation[END_REF], because the structural homology between streptococcal and vertebrate synthases should reflect similar mechanisms, as opposed to the synthase from Pasteurella. This discrepancy calls for an explanation. It can be found in a detailed consideration of the experimental conditions and the probable mechanism of enzymatic reactions.

It is obvious that the two substrates UDP-GlcA and UDP-GlcNAc must bind to two active sites within the enzyme. Since no other molecule is required for chain initiation and elongation, the growing UDP-hyaluronan shuttles from one substrate to the other during elongation as illustrated in Fig. 3. Polymerisation at the reducing end is therefore dependent on a high affinity of the growing UDP-hyaluronan to both active sites. It is likely that the binding affinities of UDP-hyaluronan to these sites is proportional to those for the two substrates. The binding affinity of UDP-GlcA and UDP-GlcNAc is reflected by the K Mvalues which have been measured for both the native and the recombinant synthases. Copyright 2006 Biochemical Society demonstrates that the recombinant synthase has dramatically reduced affinities for both substrates. Even detergent solubilization appeared to decrease binding affinity. It is thus likely that UDP-hyaluronan that binds to the same sites has also reduced affinity to the synthase. An additional component that holds the growing hyaluronan chain close to the synthase in the native membrane is the hyaluronan exporter, because it has been demonstrated that hyaluronan export and synthesis are closely associated in eucaryotic plasma membranes [START_REF] Prehm | Inhibition of hyaluronan export from human fibroblasts by inhibitors of multidrug resistance transporters[END_REF] and that growing hyaluronan chains exert feedback inhibition on the synthase [14;15]. In addition, the exporter is also absent from the recombinant enzyme preparations.

The possible scenario for synthesis at the non-reducing end by the recombinant synthase is illustrated in Fig. 3. At high substrate concentrations that were used for assessment of growth direction of the recombinant synthase (150 :M UDP-GlcNAc and 50 :M UDP-GlcA) [START_REF] Bodevin-Authelet | Biosynthesis of Hyaluronan: Direction of chain elongation[END_REF],

the active sites will be constantly occupied by substrates. Any initially formed UDPhyaluronan will be ousted from the synthase. However, the enzymatic activities for the transferase reactions remain the same or are even increased, as indicated by the increased v max values. Thus the synthase tries to find targets for transfer of monosaccharides from the nucleotide sugars. The only abundant targets are soluble nucleotides sugars. It is thus likely that the synthase transfers the monosaccharides to soluble substrate, instead of transferring bound UDP-hyaluronan. In fact, Bodevin-Authelet et al. only analysed soluble hyaluronan after precipitation of membrane proteins with trichloroacetic acid and unfortunately, did not indicate whether there existed any membrane bound hyaluronan [START_REF] Bodevin-Authelet | Biosynthesis of Hyaluronan: Direction of chain elongation[END_REF]. Such a trichloroacetic acid precipitation has previously been shown to precipitate most of the newly synthesized hyaluronan [START_REF] Sugahara | Biosynthesis of hyaluronic acid by Streptococcus[END_REF], especially after the short periods of chain growth that were used for determination of direction of elongation .

The chemical reaction is virtually identical to the initial step of polymerisation that results in the first disaccharide unit for elongation at the reducing end. But now elongation proceeds at the non-reducing end. The only difference is the transferred moiety: UDP-hyaluronan to the nucleotide sugars for the synthesis at the reducing end and the nucleotide sugars to hyaluronan for the synthesis at the non-reducing end. This may also explain the failure of Bodevin-Authelet et al. [START_REF] Bodevin-Authelet | Biosynthesis of Hyaluronan: Direction of chain elongation[END_REF] to chase a pulse labelled hyaluronan chain. In contrast, a chase Copyright 2006 Biochemical Society was demonstrated previously for the natural eucaryotic synthase [START_REF] Prehm | Inhibition of hyaluronate synthesis[END_REF] and again in this paper (Fig. 1).

In accord with hyaluronan chain elongation at the reducing end were results that nucleotide sugars should compete with UDP-hyaluronan for binding to the enzyme and high concentrations increased shedding and reduced the chain length. Thus intracellular concentrations of the nucleotide sugars and the affinity of the growing UDP-hyaluronan chain to the enzyme determine the dissociation rate. ") or 1:3 (•, ª) with buffer. At the times indicated the membranes were sedimented for 3 min at 14.000 g, dissolved in 1% SDS and applied to descending paper chromatography (solid symbols). The supernatants (open symbols) were applied directly [START_REF] Prehm | Synthesis of hyaluronate in differentiated teratocarcinoma cells. Characterization of the synthase[END_REF]. The origins containing hyaluronan were excised and the radioactivities were determined (A). From these data the ratios of released and membrane bound hyaluronan were calculated (B).

LEGENDS

  H/ 14 C decreased during digestion. This result was verified by two independent experiments. It indicated that the [ 3 H]hyaluronan introduced first into the growing chain was digested faster. Consequently, most of the hyaluronan chains must have Biochemical Journal Immediate Publication. Published on 23 May 2006 as manuscript BJ20060431 Copyright 2006 Biochemical Society been elongated at the reducing end.

Biochemical

  Journal Immediate Publication. Published on 23 May 2006 as manuscript BJ20060431

Fig. 1

 1 Fig.1 Synthesis of dual labelled hyaluronan (A) A membrane fraction of B6 cells was incubated with UDP-[ 3 H]GlcNAc and unlabelled UDP-GlcA for 30 min. The membranes were solubilized in 1% SDS and subjected to gel filtration on a Sephacryl S-1000 column (1.4 x 64 cm). Fractions of 2 ml were collected and analysed for 3 H (~) radioactivity. The scale of the fractions from 51 to 65 containing the substrate was reduced by the factor of 10. (B) The membranes were incubated with UDP-[ 14 C]GlcA and unlabelled UDP-GlcNAc for 15 min and analysed as above for 14 C () radioactivity. (C) The membranes were first incubated with UDP-[ 3 H]GlcNAc and unlabelled UDP-GlcA for 30 min and then with UDP-[ 14 C]GlcA and unlabelled UDP-GlcNAc for 15 min and analysed as above for 3 H (~) and 14 C () radioactivity. Fractions 23 to 36 were combined for subsequent digestion.

Fig. 2

 2 Fig. 2 Release of hyaluronan from the membrane bound synthaseHyaluronan was synthesized by B6 membranes in the presence of substrate containing 8 :M UDP-[ 14 C]GlcA and unlabelled 166 :M UDP-GlcNAc (, G), or at substrate diluted 1:2 (!,

Fig. 3

 3 Fig. 3 Transfer reactions for hyaluronan synthesis at the reducing and non-reducing endAfter dissaccharide formation, high affinity binding of UDP-hyaluronan to the active sites of the native enzyme E ensures that elongation proceeds at the reducing end. Reduced binding affinity to the recombinant enzyme E leads to transfer of monosaccharides to soluble substrates at the non-reducing end.

E

  UD P-G lcA -G lcN ac UD P-G lcN ac E UD P-G lcN ac-Glc -Gl cN ac

Table 2 Biochemical Journal Immediate Publication. Published on 23 May 2006 as manuscript BJ20060431

 2

Table 1 Digestion dual labelled hyaluronan with exoglycosidases

 1

	Experiment 1
	Digestion	3 H (cpm) 14 C (cpm) 3 H/ 14 C ratio
	period (h)
	0	7162	14216	0,50
	3	4281	10157	0,42
	9	2076	6498	0,32
	27	1606	5780	0,28
	Experiment 2
	Digestion	3 H (cpm) 14 C (cpm) 3 H/ 14 C ratio
	period (h)
	0	3725	10560	0,35
	3	2799	9512	0,29
	8	1434	5264	0,27
	24	1461	5616	0,26

Biochemical Journal Immediate Publication. Published on 23 May 2006 as manuscript BJ20060431 Copyright 2006 Biochemical Society

TABLE 2 Comparison of published Michaelis Menten constants for native and recombinant eucaryotic hyaluronan synthases

 2

		Km (:M) for	K m (:M) for	V max (nmol/h/mg)	V max (nmol/h/mg)	Reference
		UDP-GlcA	UDP-GlcNAc	for UDP-GlcA	for UDP-GlcNAc
	native chick fibroblasts	20	50	10	13	[22]
	native human	3	21	70	70	[23]
	fibroblasts
	native human	1	12	6	7	[24]
	fibroblasts
	native mouse	10.8	58.4	7	1	[25]
	fibroblasts
	detergent-solubilized,	50	100			[26]
	native mouse
	oligodendroglioma
	recombinant xenopus	60	235			[27]
	DG42
	recombinant mouse	60	950	2772	3090	[28]
	HAS1

Biochemical Journal Immediate Publication. Published on 23 May 2006 as manuscript BJ20060431 Copyright 2006 Biochemical Society Prehm, Fig. 1

ACKNOWLEDGEMENTS

The author thanks Drs. R. Pohlmann and R. Stern for critical review of the manuscript and U.

Rasmussen and R. Schulz for excellent technical assistance.


Michèle Brillard-Bourdet

Ahmed Hamdaoui

Eric Hajjar

Christian Boudier

Nathalie Reuter

Laurence Ehret-Sabatier

Joseph G Bieth

Francis Gauthier
email: gauthier@univ-tours.fr

A novel locust (Schistocerca gregaria) serine protease inhibitor with a high affinity for neutrophil elastase

Keywords: PPE, porcine pancreatic elastase, HNE, human neutrophil elastase, Abz, O-aminobenzoyl, serine protease, kinetics, kazal inhibitor, neutrophil elastase, insect

h e n y l ) e t h y l e n e d i a m i n e ;

I g e p a l C A -6 3 0 , (octylphenoxy)polyethoxyethanol;; OMTKY-III, turkey ovomucoid trypsin inhibitor third

INTRODUCTION

Neutrophil elastase is thought to be the major protease involved in tissue destruction during inflammation. This is especially true for lung diseases such as cystic fibrosis, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS), which are accompanied by considerable recruitment of neutrophils. Anti-inflammatory therapy using protease inhibitors may be an efficient means of reducing the unopposed proteolytic activity (e.g HNE activity) that occurs when neutrophils invade inflammatory sites (reviewed in [START_REF] Tremblay | Anti-inflammatory activity of neutrophil elastase inhibitors[END_REF]). A number of studies using natural and recombinant inhibitors have had mixed results due to ineffective administration, or the inactivation of the inhibitor by mechanical or physicochemical constraints in the local environment [START_REF] Tremblay | Anti-inflammatory activity of neutrophil elastase inhibitors[END_REF]. There is thus a need for molecular structures that possess the physicochemical and mechanical properties required for use in vivo.

At least 2500 sequences homologous to those of known protease inhibitors can be retrieved from data banks, and assigned to one of 48 families [START_REF] Rawlings | Evolutionary families of peptidase inhibitors[END_REF]. The classical inhibitors of HNE and other neutrophil proteases are members of families I1 (Kazal), I2, I3, (Kunitz), I4 (serpin), I17 (elafin).

The most prominent physiological inhibitor of HNE is α 1 -Pi (I4 family), the recombinant form of which has been given by inhalation or intravenously to treat cystic fibrosis and α1-Pi deficiency.

Several other inhibitors, including MNEI, SLPI and elafin/pre-elafin, have also been given as aerosols or systemically to inhibit neutrophil elastase. Most of these natural inhibitors are sensitive to oxidation and/or proteolytic degradation by other proteases that are present at inflammatory sites [START_REF] Tremblay | Anti-inflammatory activity of neutrophil elastase inhibitors[END_REF]. Recombinant protease inhibitors based on the sequences of natural endogenous inhibitors have been developed to overcome some of these drawbacks and a few have proved to have beneficial anti-inflammatory properties [START_REF] Honore | Beneficial effect of an inhibitor of leukocyte elastase (EPI-hNE-4) in presence of repeated lung injuries[END_REF]. Synthetic low Mr elastase inhibitors have also been developed, but their side effects and limited efficacy are barriers to further work on them [START_REF] Tremblay | Anti-inflammatory activity of neutrophil elastase inhibitors[END_REF].

Insects have many more serine proteases than do humans, but they have relatively few serine protease inhibitors [4] [START_REF] Simonet | Genomics, evolution and biological functions of the pacifastin peptide family: a conserved serine protease inhibitor family in arthropods[END_REF]. A new family of low Mr protease inhibitors, the pacifastins, was found recently in arthropods [START_REF] Simonet | Structural and functional properties of a novel serine protease inhibiting peptide family in arthropods[END_REF].They all have 6 cysteine residues arranged in a conserved disulfide pattern [START_REF] Simonet | Structural and functional properties of a novel serine protease inhibiting peptide family in arthropods[END_REF]. Most of them preferentially inhibit chymotrypsin and trypsin. Five pacifastin inhibitors have been purified from the ovary of the desert locust Schistocerca gregaria [START_REF] Hamdaoui | Purification and characterization of a group of five novel peptide serine protease inhibitors from ovaries of the desert locust, Schistocerca gregaria[END_REF]. We have now isolated and purified to homogeneity two forms of a novel inhibitor of higher Mr, greglin, from the same material. The protein is a powerful neutrophil elastase inhibitor that also inhibits pancreatic elastase and chymotrypsin-like proteases.

We used computational tools to look for sequence homologies with other proteinaceous inhibitors and to predict the secondary and tertiary structures of greglin.

EXPERIMENTAL

Materials

Schistocerca gregaria ovaries were obtained as previously described [START_REF] Hamdaoui | Purification and characterization of a group of five novel peptide serine protease inhibitors from ovaries of the desert locust, Schistocerca gregaria[END_REF][START_REF] Hamdaoui | Purification of a novel, heat-stable serine protease inhibitor protein from ovaries of the desert locust, Schistocerca gregaria[END_REF]. Porcine pancreatic elastase (PPE; EC 3.4. 21.36), human neutrophil elastase (HNE; EC 3.4. 21.31), protease 3 (EC 3.4.21.76), cathepsin G (EC 3.4. 21.20) and human α 1 -Pi were purified [START_REF] Martodam | A rapid procedure for the large scale purification of elastase and cathepsin G from human sputum[END_REF]10] or obtained from Athens Research and Technology (Athens, GA, USA). Porcine pancreatic trypsin (EC 3.4. 21.4) and subtilisin Carlsberg (EC 3.4.21.62) were obtained from Sigma (St Quentin Fallavier, France).

Bovine pancreatic chymotrypsin (EC 3.4.21.1) was purchased from Worthington (Lakewood, NJ, USA) and papain (EC 3.4.22.2) from Roche Diagnostics (Meylan, France). The active sites of all the above proteases were titrated using published methods [11 , 12-14] Approval was obtained from the Ethics Committee of our Institution.

Purification of greglin

The detailed purification procedure is reported in the "Supplementary material section". Briefly, an homogenate of 150 S gregaria ovaries was precipitated between 50-80% solid ammonium sulphate and the resulting pellet suspended and fractionated by gel filtration. The collected fractions were checked for their anti-elastase activity using Suc-(Ala) 3 -p-NA as a substrate. The elastase-inhibiting fractions were pooled and chromatographed on an anion exchange column.

The bound material was eluted with 0 to 300 mM gradient of NaCl and the fractions containing inhibitor detected as above and further chromatographed on a Mono Q HR 5/5 column. The two major peaks were further purified by chromatography on the same column and their purity checked by SDS gel (12% ) electrophoresis and rp-h.p.l.c. using a C4 Brownlee cartridge.

Kinetic measurements

Experimental details related to the measurements of enzymes activities on chromogenic and fluorogenic substrates are given in the "Supplementary material" section.

Determination of active inhibitor concentrations

The active concentration of each inhibitor was determined with active site-titrated HNE.

Increasing quantities of inhibitor were incubated with 0.38 µM enzyme in 990 µl reaction mixtures for 10 min. The remaining enzyme activities were measured by adding 10 µl 50 mM Suc-(Ala) 3 -p-NA. Under these conditions the decrease in HNE activity as a function of both inhibitor concentrations was linear up to about 90 % inhibition. We used the linear portion of the inhibition curve to deduce the active concentration of both inhibitory species assuming a 1 : 1 binding stoichiometry.

Determination of K i, the equilibrium dissociation constant for the complexes formed between greglin and various proteases

Constant amounts of a given protease were reacted with increasing amounts of inhibitor in 990 µL buffered reaction mixtures for 20 min. The remaining enzymic activities were measured by adding 10 µL of appropriate substrate solution [START_REF] Bieth | Theoretical and practical aspects of proteinase inhibition kinetics[END_REF]. Further details are given in the "Supplementary material" section.

Determination of k ass and k diss , the association and dissociation rate constants for the interaction of HNE with greglin

The association and dissociation kinetics for the reaction of HNE with greglin 1 and 2 were investigated by the progress curve method [START_REF] Bieth | Theoretical and practical aspects of proteinase inhibition kinetics[END_REF] using a stopped-flow apparatus (SFM3, Bio-Logic, Claix, France) [START_REF] Bieth | Theoretical and practical aspects of proteinase inhibition kinetics[END_REF][START_REF] Kellenberger | Serine protease inhibition by insect peptides containing a cysteine knot and a triplestranded beta-sheet[END_REF]. Data acquisition and processing were done with the manufacturer's BioKine software.

Assessment of the residual greglin inhibitory activity after incubation with non-target proteolytic enzymes

2.8 nM HNE was incubated with 14 nM native or protease-treated inhibitor for 10 min at 37 ° C in 212 µL buffer. The residual enzyme activity was measured by adding 2 µl 0.2 mM Abz-APEEIMRRQ-EDDnp.

Inhibition by greglin of the HNE activity in cystic fibrosis sputum supernatant

The sputum samples collected from adult cystic fibrosis patients were homogenized in 10 mM phosphate-buffered saline (PBS), pH 7.4 (3 mL/g sputum) and centrifuged at 10,000 x g for 10 min at 4°C. The concentrations of active HNE in these samples were deduced by comparing their enzyme activities with that of pure, active-site-titrated HNE using Abz-APEEIMRRQ-EDDnp (1.33 µM final). Buffered reaction mixtures containing greglin (3 to 36 nM final) were incubated with samples of sputum supernatant containing HNE (30 nM final) for 10 min. at 37°C. Residual HNE activity was measured spectrofluorometrically using Abz-APEEIMRRQ-EDDnp as substrate.

Determination of the primary structure of greglin

The complete amino acid sequences of the two reduced and alkylated greglin isoforms were determined by automated N-terminal sequencing of the purified inhibitors and the enzymatically and chemically cleaved forms using an Applied Biosystems Procise pulsed liquid sequencer with the chemicals and program recommended by the manufacturer. Inhibitor (1 nanomol) was reduced with dithiothreitol and alkylated with 2 µL 4-vinyl pyridine (see Supplementary Materials). Samples were desalted by rp-h.p.l.c. and incubated (final concentration: 4.6 nM) with trypsin (2.0 nM final) or chymotrypsin (1.5 nM final) in appropriate buffers. The products were separated by rp-h.p.l.c, freeze-dried and sequenced. . The reduced, alkylated inhibitors (1.5 nM final) were also incubated in the dark at 20°C for 18 hours with 75 mg/mL cyanogen bromide in 70 % formic acid. The products were separated by rph.p.l.c, freeze-dried and sequenced. C-terminal sequences were determined by mass spectrometry using a Bruker BIFLEX III TM mass spectrometer (Bremen, Germany) in linear positive ion mode. Samples were prepared by the sandwich method [START_REF] Kussmann | Matrix-assisted laser desorption/ionization mass spectrometric peptide mapping of the neural cell adhesion protein neurolin purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis or acidic precipitation[END_REF]. Purified peptides were incubated at 25 °C for 3 hours with 10 ng/µL carboxypeptidase P (Sigma) in 10 µL 50 mM sodium citrate buffer , pH 4.0.

Prediction of the secondary and tertiary structures of greglin

Sequence analysis tools

The BLAST (http://www.ebi.ac.uk/blastall/) and MEROPS (http://merops.sanger.ac.uk/) suites of programs were used to look for homologies in the sequence databases. We used T-coffee (http://igs-server.cnrs-mrs.fr/Tcoffee/tcoffee_cgi/index.cgi) to generate multiple sequence alignments, and NetPhos (http://www.cbs.dtu.dk/services/NetPhos/) to predict phosphorylation sites.

Secondary structure prediction

Seconday structural elements were predicted using sspro [START_REF] Baldi | Exploiting the past and the future in protein secondary structure prediction[END_REF], nnpredict [START_REF] Kneller | Improvements in protein secondary structure prediction by an enhanced neural network[END_REF], psipred, sam, jufo through the Robetta server [START_REF] Kim | Protein structure prediction and analysis using the Robetta server[END_REF], sable and profsec through the GeneSilico metaserver (http://genesilico.pl/meta). These programs were selected to cover the whole range of methods (neural network, hidden Markov chain, position specific and profile matrices, etc.) and parameters (solvent representation, amino acid properties, etc.) that are currently available.

Tertiary structure prediction

To predict the tertiary structure of greglin, we used the automated GeneSilico metaserver (http://genesilico.pl/meta) that uses results from the inbgu, 3dpssm, ffas, mgenthreader, sam, sparks, fugues, and 3dpssm servers. The Robetta server was used to build the structure of greglin starting from its primary sequence alone [START_REF] Kim | Protein structure prediction and analysis using the Robetta server[END_REF].

SwissPdbViewer (http://au.expasy.org/spdbv/) was used to generate multiple structural alignments and to superimpose themodels obtained. Copyright 2006 Biochemical Society oxidant was removed by rp-h.p.l.c. on a C4 column and oxidation was checked by mass spectrometry using a M@LDI L/R Waters micromass apparatus. The anti-PPE activity of the desalted inhibitor was assayed as above.

Greglin physicochemical properties

Effect of pH and temperature on inhibitor activity

Greglin was incubated for 20 hours at 37°C in 0.5 M glycine-HCl buffer pH 2.5; 50mM hepes buffer pH 7.4; and in 0.5 M Tris-base pH 11.0, and its residual inhibitory activity assayed in the hepes buffer. The effect of temperature was investigated by incubating greglin (3.85 µM) in hepes buffer pH 7.4 for one hour at temperatures from 37°C to 90°C and then measuring its residual inhibitory activity at 37° C.

Reaction of greglin with PPE and HNE under acidic conditions

The greglin reactive bond (P1-P1') was tentatively identified by incubating the inhibitor with target enzymes at low pH and looking for putative cleavage sites by N-terminal sequencing, as described for soya-bean trypsin inhibitor [START_REF] Sealock | Enzymatic replacement of the arginyl by a lysyl residue in the reactive site of soybean trypsin inhibitor[END_REF]. Experimental details are provided in the "Supplementary Material" section.

Susceptibility of greglin activity to proteolytic enzymes

The susceptibility of greglin anti-elastase activity was investigated by incubating the inhibitor with serine (trypsin, endoprotease Glu-C), cysteine (papain) and metallo (pseudolysin) proteases.

The mixtures were then separated by rp-h.p.l.c. and the inhibitory properties of the eluted peaks analysed (see Supplementary Materials).

RESULTS

Purification of two greglin isoforms from a crude homogenate of S gregaria ovaries:

We isolated two forms of an anti-PPE protein referred to as "greglin" from the Schistocerca gregaria ovaries using a combination of salt precipitation, size-exclusion and anion exchange chromatographies. The inhibitory activity of greglin was used to monitor its presence along the purification procedure. The protein was eluted from the Mono Q column as two close peaks that were further purified by rp-h.p.l.c on a Brownlee C4 column for sequence analysis, and gave a single band when run on a SDS gel (Figure 1A). A homogenate of 150 ovaries gave about 4 mg of purified greglin. The sequence of each greglin isoform was obtained by sequencing overlapping peptides produced by cleaving the reduced alkylated rp-h.p.l.c.-purified proteins with cyanogen bromide (Met 14 and Met 68), trypsin (Lys 52, Lys 70 and Lys 81) and chymotrypsin (Leu 27, Tyr42 and Tyr 6) (Figure 1B). Both sequences contained 83 identical amino-acid residues except that one sequence had no identifiable residues at positions 8, 11 and 15, whereas the other had seryl residues at these positions. This strongly suggests that the seryl residues in the first sequence are glycosylated or phosphorylated. The molecular mass calculated from the greglin sequence was 9,229 Da, while mass spectrometry gave a minimal mass of 9,600 Da for and a maximal mass of 9,844 Da, consistent with a triphosphorylation. Intermediate masses consistent with mono and diphosphorylation were also recorded. The Net Phos program of the Expasy server predicted the phosphorylation of the Ser residues at positions 6, 8, 11 and 15 and 37 in agreement with sequence data that found no seryl residues at positions 8, 11 and 15 in one isoform, i.e. those with the best predictive scores. But there may well be additional post translational modifications that could explain the differences between the mass deduced from amino acid analysis and those obtained experimentally. We identified 6 Cys residues unambiguously by N-terminal sequencing after 4-vinyl-pyridine alkylation. The C-terminal sequence was confirmed by carboxypeptidase P digestion plus mass spectrometric analysis of the products. The data indicated a 303 Da decrease in the molecular mass, consistent with the KSS C-terminal sequence. In addition, cleavage of the native inhibitor with trypsin gave a molecule of lower mass (-175 Da), in agreement with the release of the two C-terminal seryl residues. Incubation of the trypsin-reacted products with carboxypeptidase P reduced the molecular mass by a further 128 Da, confirming the presence of a lysine residue at position 81.

We found no significant relationship between the whole sequence of greglin and any of the protease inhibitors searching the MEROPS database [START_REF] Rawlings | MEROPS: the peptidase database[END_REF]. However using only part of the sequence (residues P26 to C69), the program retrieved two putative inhibitors from Ciona intestinalis (O97362) and from Drosophila melanogaster (Q9VE57) which share 38% and 36% homology, respectively, with this part of the greglin sequence.

We tentatively aligned the greglin sequence with those of these two peptides and with ovomucoid domain 3 (OMTKY-III), a typical Kazal inhibitor [START_REF] Laskowski | Protein inhibitors of proteinases[END_REF], and with the Kazal-related peptide PEC-60 [START_REF] Liepinsh | Solution structure and dynamics of PEC-60, a protein of the Kazal type inhibitor family, determined by nuclear magnetic resonance spectroscopy[END_REF], identified by fold recognition methods as the closest structural homologue to greglin (see Copyright 2006 Biochemical Society next paragraph). The best scores were obtained with the C intestinalis and D melanogaster peptides; these were the only two that gave significant E-values (Table I). However, only three of six Cys residues in greglin aligned with those in the Kazal-related molecules (Figure 1C). This suggests a different disulfide bridge pattern, but we failed to identify this pattern due to the resistance of greglin to proteolysis. We then used computer-based structural studies to tentatively assign greglin to a family of peptidase inhibitors.

Secondary and tertiary structure predictions:

The secondary structure elements of the full-length greglin sequence (83 amino acids) were predicted using 7 different programs (Fig. 2A). All the programs except SABLE agreed on the presence of three main beta strands in greglin at positions 27-35, 41-45 and 59-64. They also predicted an alpha helix starting at R66 up to L76. The characteristic scaffold of the Kazal family of inhibitors consists of a central alpha helix, an adjacent three-stranded beta-sheet and a proteinase-binding loop cross-connected through disulfide bridges 1-5, 2-4 and 3-6, whereas inhibitors of the pacifastin family have beta strands only as secondary structure elements [START_REF] Gaspari | A simple fold with variations: the pacifastin inhibitor family[END_REF].

The predicted secondary structure of greglin is thus more closely related to that of Kazal inhibitors than to that of pacifastin inhibitors.

Because few sequence homologies were found by searching sequence databases (max 38%), we used protein fold recognition methods via the "GeneSilico" metaserver. The folding of greglin is predicted to contain alpha helices and beta sheets ("a+b" group of the SCOP classification; http://scop.mrc-lmb.cam.ac.uk/scop/). Most of the fold-recognition programs identified PEC-60 (PDB ID: 1PCE) as the closest structural homologue of greglin. Intermediate models (those not containing the variable loops) that correspond to the best hits of the fold recognition searches were aligned structurally to PEC-60 using the tool "Domain fit" of "SwissPdbViewer" (Fig 2B).

The cores are readily superimposed, but there are deviations in the loops and the N-terminal region of 1PCE could only be aligned with one model (Figure 2C). We manually improved the structural alignment of greglin using insertions and deletions (Figure 2B). However, only one disulfide bridge was generated by this approach while there are 3 disulfide bridges in the folds of the Kazal-type inhibitors. Therefore, greglin appears to be only distantly related to the Kazal-type inhibitors and it could well have a novel folding pattern.

Inhibition of serine proteases by greglin

The inhibitory activity of greglin was assayed toward a series of serine protease to delineate its specificity. In addition to PPE used to monitor its purification, those included the related human neutrophil proteases Cat G and protease 3, porcine trypin, bovine pancreatic chymotrypsin and subtilisin Carlsberg. Preliminary experiments showed that both greglin isoforms reacted similarly with the enzymes tested, so that both were equally used in the following kinetic experiments.

Preliminary experiments also showed that HNE forms a much tighter complex with greglin than does PPE. We used this property to titrate the inhibitor. Increasing quantities of the inhibitor were reacted with 0.38 µM HNE as describe in the experimental section. The residual enzyme activity decreased linearly with the greglin concentration up to about 90 % inhibition, indicating that the inhibitor binds the enzyme tightly under the experimental conditions used (Figure 3), and suggesting a value of K i at least two orders of magnitude lower than the HNE concentration used, in accordance with theory [START_REF] Kneller | Improvements in protein secondary structure prediction by an enhanced neural network[END_REF]. This experiment could not be used to determine K i but allowed accurate titration of the inhibitor by fitting the data of the linear portion of the curve to the best theoretical straight line, whose intercept with the x axis was used to calculate the concentration of the active greglin (Figure 3A). Since the present titration data could not be used to measure K i , the equilibrium dissociation constant for the greglin : HNE complex, this parameter was obtained from k ass and k diss , the association and dissociation rate constants. These parameters were measured using the progress curve method. Enzyme and inhibitor were reacted under pseudo-first order conditions ([I] 0 ≥ 10 x [E] 0 ) in the presence of 0.2 mM MeOsuc-(Ala) 2 -Pro-Ala-TBE with stopped flow mixing and detection. The rate of HNE inhibition was investigated at various inhibitor concentrations (Figure 3B).

The inset of Figure 3B shows a typical progress curve used to follow the time course of HNE inhibition. In all cases a pre-steady-state of product accumulation preceded its release at a constant rate, indicating a reversible interaction between enzyme and inhibitor. Non-linear regression analysis of the progress curves (Figure 3B) yielded k, the apparent pseudo-first order rate constant for the approach to equilibrium [START_REF] Kneller | Improvements in protein secondary structure prediction by an enhanced neural network[END_REF]. Copyright 2006 Biochemical Society mechanism: the second order rate constant k ass was calculated from the slope of the straight line on Figure 3B, its intercept with the ordinate yielding k diss . We found k ass = 1.23 ± 0.16 x 10 7 M - 1 .s -1 and k diss = 0.043 ± 0.010 s -1 . K i = 3.49 ± 1.25 x 10 -9 M was deduced from K i = k diss /k ass .. The K i for the complexes between greglin and the other enzymes were obtained from equilibrium titration experiments with experimental conditions compatible with concave inhibition curves. The effects of increasing greglin concentrations on PPE activity (18 nM final) are illustrated in Figure 4. The residual enzyme activity was assessed by adding the substrate (Suc-(Ala) 3 -p-NA) to the equilibrium reaction mixtures. The rate of product release accelerated for about 30 s and reached a steady state. This time course of substrate breakdown indicates three things: first, that the enzyme partially dissociates from a reversible complex with inhibitor as a result of disturbance by substrate of the initial equilibrium; second, that substrate and inhibitor compete for binding to PPE; and third, that a new equilibrium between PPE, greglin, substrate and their complexes is reached. The plot of the steady state rate of substrate hydrolysis versus the inhibitor concentration was analyzed [START_REF] Bieth | Theoretical and practical aspects of proteinase inhibition kinetics[END_REF] to obtain K i(app) , the substrate-dependant equilibrium dissociation constant for the greglin : PPE complex (Figure 4). The true K i value was obtained from K i = K i(app) /(1+[S] 0 /K m ), where [S] 0 is the initial substrate concentration and K m the Michaelis constant obtained by classical means.

This procedure was used to study the interaction of greglin with bovine chymotrypsin, subtilisin Carlsberg and the human cathepsin G. The K i for all enzyme : inhibitor pairs are shown in Table II. Porcine trypsin and protease 3 were not inhibited under these conditions.

Greglin physicochemical properties

Resistance to proteolysis: Greglin was incubated with HNE and PPE at low pH to locate the enzyme-binding site through a peptide bond cleavage [START_REF] Sealock | Enzymatic replacement of the arginyl by a lysyl residue in the reactive site of soybean trypsin inhibitor[END_REF]. Sequence analysis of the rp-h.p.l.c.fractionated mixtures revealed that greglin had been N-terminally truncated at E10, Q12 and L27 by PPE and at M14 by HNE. The N-terminally cleaved inhibitor (minus 26 residues) retained full inhibitory activity towards PPE or HNE at pH 7.4. The observation that P1-P26 N-terminal segment is not involved in inhibition agrees with the total lack of homology between this segment and the sequences of other protease inhibitors.

Native greglin was also incubated with other proteases, including serine (trypsin and Glu-C), cysteine (papain) and metallo (pseudolysin) proteases, at various molar ratio. The resulting mixtures were fractionated by rp-h.p.l.c. and the main peaks sequenced and analysed for their HNE-inhibitory properties. Again the cleavage sites were located within the N-terminal segment (P1-P26), except that trypsin cleaved at K81 before the penultimate C-terminal residue of the protein. Greglin remained a potent HNE inhibitor even after prolonged incubation and fractionation by rp-h.p.l.c., whatever the protease used. We conclude that N-terminal (minus 26) and C-terminal (minus 3) truncations of greglin do not alter its inhibitory properties.

The resistance of greglin anti-HNE activity to pseudolysin was also compared to that of α 1 -P i , the major physiological inhibitor of HNE. Figure 6A shows the effect of incubating greglin and α 1 -PI with various amounts of pseudolysin for 15 min on their anti-HNE activity. Greglin retained most of its inhibitory capacity even at the highest enzyme concentration used, whereas α 1 -Pi activity was reduced by 80 % at E = 50 nM or completely lost at E = 500 nM (Figure 5A).

pH and temperature stability: The recovery of inhibitory activity after rp-h.p.l.c. demonstratesd that the two forms of greglin resist denaturation by acid and acetonitrile. We investigated the resistance of the inhibitor to low and high pH by incubating it overnight at pH 2.5 and at pH 11.0. The inhibitory properties were not significantly altered (Figure 5B). Similarly, we found no significant loss of activity when the inhibitor was incubated in hepes buffer for one hour at temperatures from 37 to 90°C (data not shown).

Sensitivity to oxidation and reduction : Chemical and physiological oxidizing system can alter the biological activity of many proteins by converting their exposed methionyl residues to methionine sulfoxide. This can dramatically lower the activity of protein protease inhibitors, especially those having a methionyl residue at position P1 or P1' of their reactive site [27][START_REF] Nobar | Oxidized elafin and trappin poorly inhibit the elastolytic activity of neutrophil elastase and proteinase 3[END_REF][START_REF] Vogelmeier | Anti-neutrophil elastase defense of the normal human respiratory epithelial surface provided by the secretory leukoprotease inhibitor[END_REF].

Since greglin contains 3 methionyl residues, its activity might be altered by oxidants. The inhibitor was incubated with N-chlorosuccinimide and its oxidation checked by mass spectrometry. The observed molecular mass was consistent with the oxidation of the three Met residues but HNE-inhibitory capacity of oxidized greglin was unaltered (Figure 5C). This absence of significant change in the greglin anti-HNE property suggests that no methionyl residue occupies a critical position in the inhibitor reactive site. But greglin lost all its inhibitory capacity once its disulfide bridges had been reduced by 10mM dithiothreitol and alkylated with iodoacetamide. The greglin tertiary structure therefore is essential for inhibition, which suggests that the sequence from the first to the last Cys residues (Cys33 to Cys76) is required for inhibitory activity. Iinhibition of HNE in cystic fibrosis sputum: Purulent sputum is a complex biological medium containing elements (oxidants, proteolytic enzymes) able to depress the activity of local physiological inhibitors [START_REF] Matheson | Enzymatic inactivation of human alpha-1-proteinase inhibitor by neutrophil myeloperoxidase[END_REF]. We reacted increasing amounts of greglin with fixed amounts of cystic fibrosis sputum supernatant to delineate the anti-HNE efficiency of the inhibitor in sputum.

The remaining HNE-activity was measured by adding the fluorescent substrate Abz-APEEIMRRQ-EDDnp to the equilibrium mixtures. The relative enzyme activity decreased curvilinearly with the quantity of inhibitor (Figure 6), as anticipated from the K i value in hepes buffer (Table II), the greglin concentration, and the protease concentration (30nM) that gave a

[HNE]/Ki ≈ 8.5 [START_REF] Bieth | Theoretical and practical aspects of proteinase inhibition kinetics[END_REF]. Since inhibition of HNE by greglin in sputum is best described by the K i measured in this environment, we analyzed the inhibition data as indicated in the legend of figure 4 to determine this parameter. The concave inhibition curve shown in figure 6 was generated using the best estimate of K i(app) . After correction for the substrate (K m = 15 µM, determined by classical methods), we found K i = 3.1 ± 0.43 nM, a value close to that determined by stoppedflow kinetics with the purified enzyme:inhibitor system. Thus the sputum components did not inactivate the anti-HNE function of greglin during the time of the experiment and did not dissociate the enzyme-inhibitor complex.

DISCUSSION

Anti inflammatory therapy using protease inhibitors has long been suggested as an efficient means of controlling the unopposed proteolytic activity that occurs at inflammatory sites.

Neutrophil elastase is one of the main proteases released from activated and dying PMNs and it is involved in tissue destruction and inflammation. A variety of natural, recombinant and synthetic inhibitors have been used as anti-inflammatory drugs to target HNE but the results to date have not been entirely satisfactory [START_REF] Tremblay | Anti-inflammatory activity of neutrophil elastase inhibitors[END_REF]. New elastase inhibitors that have different structural and/or physicochemical properties from those described so far could lead to new, therapeutically useful elastase inhibitors.

Arthropods and other invertebrates are a rich source of protease inhibitors. Several belonging to the Kunitz and serpin families have been isolated, especially from haemolymph [START_REF] Polanowski | Serine proteinase inhibitors from insect hemolymph[END_REF]. Two new families of low molecular weight serine protease inhibitors were discovered recently: one in silkworms [START_REF] Pham | A new family of serine protease inhibitors (Bombyx family) as established from the unique topological relation between the positions of disulphide bridges and reactive site[END_REF] and the other in locusts [6, 7, 17 , 33] and crayfish [START_REF] Liang | Pacifastin, a novel 155-kDa heterodimeric proteinase inhibitor containing a unique transferrin chain[END_REF]. The function of these serine protease inhibitors and their distribution in invertebrate, especially arthropods, remains a matter of debate; but this could be due to the far larger number of serine proteases in insects than in human and other vertebrates [4]. Five serine protease inhibitors, designated SGPI 1-5, have been isolated from the ovaries of the locust (S. gregaria) [START_REF] Hamdaoui | Purification and characterization of a group of five novel peptide serine protease inhibitors from ovaries of the desert locust, Schistocerca gregaria[END_REF]; they are members of the pacifastin family of peptidase inhibitors. These low Mr (about 35 residues) inhibitors are found only in invertebrates and they all have a similar cysteine array [START_REF] Simonet | Genomics, evolution and biological functions of the pacifastin peptide family: a conserved serine protease inhibitor family in arthropods[END_REF]. Aqueous homogenates of mature locust ovaries also contain potent trypsin, chymotrypsin and elastase inhibitory activities. The trypsin and chymotrypsin inhibitor is a 14 kDa, heat stable peptide that has been purified and characterized [START_REF] Hamdaoui | Purification of a novel, heat-stable serine protease inhibitor protein from ovaries of the desert locust, Schistocerca gregaria[END_REF], but the protein(s) responsible for the anti-elastase activity is still unidentified.

The protease inhibitors we have purified from the locust ovary are two forms of a single protein, greglin. They have similar inhibitory behaviors towards all the tested proteases. Greglin reversibly inhibits enzymes of the elastase and chymotrypsin families. It forms tight complexes with subtilisin and HNE, but has no effect on the activities of trypsin or protease 3. To our knowledge, no other potent HNE inhibitor has been purified from locusts. Most of the known locust inhibitors belong to the pacifastin family and preferentially inhibit trypsin and chymotrypsin [START_REF] Hamdaoui | Purification of a novel, heat-stable serine protease inhibitor protein from ovaries of the desert locust, Schistocerca gregaria[END_REF][START_REF] Kellenberger | Serine protease inhibition by insect peptides containing a cysteine knot and a triplestranded beta-sheet[END_REF]. The sequence of greglin is only distantly related to that of any other protease inhibitor. The putative Kazal-type inhibitor of Ciona intestinalis is the most closely related, as the C-terminal region of the sequence is 38% homologous. The function of the greglin N-terminal extension that takes no part in inhibition remains unexplained, but its sensitivity to proteolysis contrasts with that of the inhibitory C-terminal domain. Though this domain has some physicochemical properties in common with members of other low Mr inhibitors, it also has features that can be exploited in drug development.

The stopped-flow kinetics of HNE inhibition by greglin show that the latter is a fast acting inhibitor that binds the enzyme via a simple bimolecular mechanism under the experimental conditions used. The HNE : greglin complex formation is governed by the second order rate constant k ass = 1.23 x 10 7 M -1 s -1 , a value that is similar to, or higher than that reported for the main physiological inhibitors of this enzyme: α 1 -PI (1.3 x 10 7 M -1 s -1 [START_REF] Frommherz | Heparin strongly decreases the rate of inhibition of neutrophil elastase by alpha 1-proteinase inhibitor[END_REF], which irreversibly inhibits its target proteases, and the reversible inhibitors SLPI (6.4 x 10 6 M -1 s -1 [START_REF] Boudier | Mucus proteinase inhibitor: a fast-acting inhibitor of leucocyte elastase[END_REF]) and elafin Copyright 2006 Biochemical Society (3.6 x 10 6 M -1 s -1 [37]). The rate of HNE inhibition by greglin is also similar to that measured for eglin c (k ass = 1.3 x 10 7 M -1 s -1 [START_REF] Braun | Kinetic studies on the interaction of eglin c with human leukocyte elastase and cathepsin G[END_REF]), another elastase inhibitor from the leech. The K i for the HNE : greglin complex is significantly higher than those for the interactions between HNE and the reversible inhibitors SLPI (0.3 nM [START_REF] Boudier | Mucus proteinase inhibitor: a fast-acting inhibitor of leucocyte elastase[END_REF] and elafin (0.2nM [37]). Despite this relatively low affinity, greglin concentrations of about 10 -8 molar efficiently inhibit the elastase activity in purulent sputum.

Greglin also resists oxidation, while α 1 -PI, SLPI and elafin, the major protease inhibitors of the lung, do not. The HNE-inhibitory activity of these three human proteins is reduced by their interaction with the oxidant species produced by the myeloperoxidase -hydrogen peroxide system of triggered neutrophils [START_REF] Carp | Potential mediator of inflammation. Phagocyte-derived oxidants suppress the elastase-inhibitory capacity of alpha 1-proteinase inhibitor in vitro[END_REF], or with N-chlorosuccinimide [27,[START_REF] Nobar | Oxidized elafin and trappin poorly inhibit the elastolytic activity of neutrophil elastase and proteinase 3[END_REF]. Oxidative inactivation of the inhibitors is due to the conversion of a methionyl residue involved in protease binding to methionine sulfoxide. As greglin is not inactivated by incubation with Nchlorosuccinimide, it probably has no methionyl residue in such a critical position (P1 or P'1) in its reactive site. This also suggests that greglin would retain its anti-elastase activity in a chronic inflammation environment with high concentrations of oxidant species and lysosomal proteinases produced by continuously recruited neutrophils.

Pseudolysin, a metalloenzyme that contributes to the virulence of P aeruginosa, can break down many proteins, including α 1 -PI and secretory leukocyte protease inhibitor. Both inhibitors are inactivated by limited cleavage in their reactive site regions [START_REF] Johnson | Inactivation of human bronchial mucosal proteinase inhibitor by Pseudomonas aeruginosa elastase[END_REF][START_REF] Morihara | Purification of human plasma alpha 1-proteinase inhibitor and its inactivation by Pseudomonas aeruginosa elastase[END_REF]. This mechanism, in which inhibitors behave as substrates, may cause a significant loss of active inhibitors and promote the breakdown of lung connective tissues by neutrophil proteases. We find that greglin resists inactivation by several proteolytic enzymes, including high concentrations of trypsin, papain and pseudolysin. This is important for the development of a therapeutic system, since P aeruginosa infection accompanies the inflammation of airways in patients with cystic fibrosis [START_REF] Wretlind | Pseudomonas aeruginosa elastase and its role in pseudomonas infections[END_REF].

The predicted secondary and tertiary structures suggest that greglin is more closely related to the Kazal inhibitors than to pacifastin inhibitors. However the distribution of Cys residues within the greglin sequence does not fit the disulfide pattern found in classical Kazal inhibitors, which all contain three disulfide bridges. Though the inhibitory site of Kazal inhibitors lies within the Cys2-Cys4 disulfide loop, computer-based analysis predicts that the greglin inhibitory site lies in the N-terminal extended strand, part of which is not necessary for inhibition. We therefore believe that greglin is folded in a manner different from that of classical Kazal-type inhibitors. However, non-classical Kazal-type inhibitors with different folding [START_REF] Lauber | Homologous proteins with different folds: the three-dimensional structures of domains 1 and 6 of the multiple Kazal-type inhibitor LEKTI[END_REF] or different positioning of their cysteine residues [START_REF] Hemmi | Structural and functional study of an Anemonia elastase inhibitor, a "nonclassical" Kazal-type inhibitor from Anemonia sulcata[END_REF] have been described. Our results and observations suggest that it is difficult to assign a fold to the greglin sequence using computer-based methods alone. For example, none of the ab initio models constructed by the Robetta server (http://robetta.bakerlab.org/) was folded like any known inhibitor (not shown).

The resolution of the greglin 3D structure or the production of recombinant forms with single mutation points at the putative inhibitory site will be needed before we can assign this inhibitor to a family and understand the molecular basis of its physicochemical properties. (C)Alignment of the sequences of greglin (gre) and kazal-related homologues calculated using Tcoffee (http://igs-server.cnrs-mrs.fr/Tcoffee/tcoffee_cgi/index.cgi) [START_REF] Notredame | T-Coffee: A novel method for fast and accurate multiple sequence alignment[END_REF]. (OMTKY-III (turkey ovomucoid domain 3) (ovo), porcine PEC-60 (pec), C intestinalis (cio) and D melanogaster (dro)). The inhibitory site of OMTKY-III (P4 to P3') is underlined. The scores resulting from the the multiple sequence analysis are identified as bad, average and good. (B) Linear dependence of k, the pseudo-first order rate constant for HNE inhibition as a function of the greglin concentration, indicating that I and E interact according to a bimolecular mechanism. k ass and k diss could therefore be obtained from a linear fit of the data to equation 1 [START_REF] Bieth | Theoretical and practical aspects of proteinase inhibition kinetics[END_REF].

€ k = k diss + k ass [I] 0 1+ [S] 0 /K m ( ) (1)
The theoretical straight line shown was generated using the best estimate of k ass and k diss (1.23 ± 0.15 x 10 7 M -1 .s -1 and 4.30 ± 0.97 x 10 -2 s -1 , respectively) with K m = 20.9 µM for the MeOsuc-(Ala) 2 -Pro-Ala-TBE : HNE interaction.

Inset: stopped-flow trace showing product accumulation as a function of time for the reaction of 0.32 µM greglin with 30 nM HNE in the presence of 0.2 mM MeOsuc-(Ala) 2 -Pro-Ala-TBE. The best estimate of the rate constant k was obtained, as published [START_REF] Bieth | Theoretical and practical aspects of proteinase inhibition kinetics[END_REF], by fitting the progress curve data to equation 2:

[ ] (

) ( )

kt s z s e k v v t v P - - - + = 1 / (2)
where [P] stands for the product concentration at any time t and v z and v s are the velocity at t = 0 and the steady state velocity, respectively. The best theoretical curve represented by a smooth line was generated with k = 0.45 ± 0.035 s -1 , v z = 2.98 ± 0.14 µM.s -1 and v s = 0.29 ± 0.013 µM.s -1 .

Figure 4:

Effect of increasing quantities of greglin on the activity of 18 nM PPE. The residual enzyme activity was measured with (Suc-(Ala) 3 -p-NA) as indicated in the text. K i(app), the substratedependant K i , was obtained by fitting the experimental equilibrium titration data to equation 3 [START_REF] Bieth | Theoretical and practical aspects of proteinase inhibition kinetics[END_REF]:

( ) ( ) ( ) ( ) 0 0 0 2 app i 0 0 app i 0 0 ] [ 2 ] [ ] [ 4 ] [ ] [ ] [ ] [ 1 E I E K I E K I E a       - + + - + + - = ( 3
)
where a is the enzyme relative activity as defined in the legend of figure 3.

[E] 0 and [I] 0 are the initial enzyme and inhibitor concentration, respectively. The theoretical curve was generated with K i(app) = 0.102 µM.

  . The concentrations given here refer to active protein concentrations. Pseudolysin (E.C 3.4.24.26), endoprotease Glu-C (EC 3.4.21.19) were from Calbiochem (VWR, Strasbourg, France) and Sigma respectively. Succinyl-Ala-Ala-Ala-p-nitroanilide [Suc-(Ala) 3 -p-NA], methoxysuccinyl-Ala-Ala-Pro-Ala-thiobenzyl ester [MeOsuc-(Ala) 2 -Pro-Ala-TBE], benzoyl-Arg-p-nitroanilide (BAPNA), succinyl-Ala-Ala-Pro-Phe-p-nitroanilide [Suc-(Ala) 2 -Pro-Phe-p-NA] and methoxysuccinyl-Lys(2-picolinoyl)-Ala-Pro-Val-p-nitroanilide [MeOsuc-Lys-(pico)-Ala-Pro-Val-p-NA, the chromogenic substrates of the various proteases were purchased from Bachem (Bubendorf, Switzerland). The HNE fluorogenic substrate Abz-Ala-Pro-Glu-Glu-Ile-Met-Arg-Arg-Gln-EDDnp (Abz-APEEIMRRQ-EDDnp) was used as described [15]. Sputum samples were collected from adult cystic fibrosis patients after informed consent.

  Oxidation by N-chlorosuccinimide Greglin (13 µM final) was incubated at room temperature with N-chlorosuccinimide (125 µM final) in 50mM Hepes buffer pH 7.4, 150 mM NaCl, 0.05% Igepal CA-630 for 20 min. Excess Biochemical Journal Immediate Publication. Published on 13 Jul 2006 as manuscript BJ20060437

k

  increased linearly as a function of the inhibitor concentration (Fig 3B), suggesting that no reaction intermediate accumulates over the range of inhibitor concentrations used. Inhibition was therefore analysed assuming that HNE and greglin associate via a simple bimolecular Biochemical Journal Immediate Publication. Published on 13 Jul 2006 as manuscript BJ20060437

Figure 1 :

 1 Figure 1: Purification of greglin: (A) SDS gel electrophoresis of purified greglin after ion exchange chromatography (B) Complete amino-acid sequence of greglin obtained from Nterminal sequencing of overlapping peptides produced by chemical and proteolytic cleavages followed by rp-h.p.l.c separation of each greglin isoform after reduction and alkylation. Proteolytically-cleaved and chemically-cleaved fragments are underlined.

Figure 2 :

 2 Figure 2: Prediction of the greglin secondary and tertiary structures :(A) The full amino acid sequence of greglin is displayed on the first line. Each of the other 7 lines reports the secondary structure predicted by the different programs used. The residues predicted to be involved in beta-strands are annotated with the letter "E ". The residues that are predicted to be in helical regions are annotated with the letter "H".(B) Fold recognition sequences and fold recognition structural alignments. Sequence alignments were based on the structural alignment between 1PCE and a subset of "intermediate" models generated by the Genesilico server from the best hits of the fold recognition search. The first line is the greglin sequence, which was fitted manually to the template structure (1PCE). The identifier at the beginning of each line refers to the program (ffas, inbgu, sam) and the structure (1PCE, 1BUS, 1OVO, 1ROT) used to build each of the "intermediate" models. SwissPdbViewer (Domain Fit) was used to calculate the structural superimposition and generate the corresponding alignment. Residues in light grey are the residues of the intermediate models that were aligned with those of the template 1PCE by the fitting operations.

Figure 3 :

 3 Figure 3: (A) Effect of increasing quantities of greglin on the activity of 0.38 µM HNE. The enzyme relative activity is the ratio of the steady state rate of Suc-(Ala) 3 -p-NA hydrolysis (see text) measured in the presence of inhibitor to the rate measured in its absence. The straight line drawn through the data is theoretical. It shows that greglin titrates the enzyme under the experimental conditions used. Its intercept with the x axis was used to calculate the concentration of the active inhibitor.

Figure 5 :Figure 6 :

 56 Figure 5 : (A) Effect of pseudolysin on the anti-HNE activities of greglin and α 1 -Pi : Inhibitors (4µM final) were incubated with pseudolysin (0.5 nM-0.5 µM) as indicated in the text. Results show the median and maximal values (n ≥ 2) (B) Resistance of greglin activity to oxidation and acetonitrile: Greglin 2 (77 µM final) was incubated with 125 µM N-chlorosuccinimide. Residual greglin inhibitory activity was then measured and compared with those of the untreated inhibitor and inhibitor after rp-h.p.l.c. The HNE concentration was adjusted so that inhibition by native greglin was incomplete (90%). Results show the median and maximal values (n ≥ 2) (C) Resistance of greglin activity to acid and base conditions: Solutions of purified greglin were incubated at pH 2.5, pH 7.4 and pH 11.0 at 37°C overnight and their capacity to inhibit neutrophil elastase measured. Results show the median and maximal values (n ≥ 2)

Biochemical Journal Immediate Publication. Published on 13 Jul 2006 as manuscript BJ20060437

Biochemical Journal Immediate Publication. Published on 13 Jul 2006 as manuscript BJ20060437

Table I :

 I Sequence alignments of greglin residues P26 to C 69 with Kazal-related inhibitors from Ciona intestinalis (Swiss-Prot ID: 097362), Drosophila melanogaster (Swiss-Prot ID: Q9VE57), pig PEC-60 (Swiss-Prot ID: P37109) and OMTKY3 (Swiss-Prot ID: P68390) (using the LALIGN program from Fasta.E value* : homologous proteins were taken to be those for which the aligned amino acid sequences were related with an E value of 0.001 or less

	sequence comparison	% alignment	E Value
	greglin-O97362	36	0.0005
	greglin-Q9VE57	33	0.00091
	O97362-Q9VE57	27	0.014
	greglin-P37109	34	1.7
	Greglin-P68390	27	1.400

Biochemical Journal Immediate Publication. Published on 13 Jul 2006 as manuscript BJ20060437 Copyright 2006 Biochemical SocietyTable II :

 II Equilibrium dissociation constant (K i ) for the interaction of greglin with the various proteases.

	Enzyme	K i (nM)
	PPE	58.3 ± 12
	HNE	3.6 ± 2.1 a
	PR 3	NI b
	cathepsin G	153.5 ± 10
	chymotrypsin	26.7 ± 10.8
	subtilisin	0.68 ± 0.10
	trypsin	NI b

a calculated from k ass and k diss (see text) b NI, no inhibition

Acknowledgements : We thank « Vaincre la mucoviscidose » for financial support, . Luiz Juliano, Universidade Federal, São Paulo, Brazil for synthesizing the Abz-APEEIMRRQ-EDDnp substrate, Valérie Labas, Department of proteomics, Institut National de la Recherche Agronomique, Tours-Nouzilly for performing mass spectrum analyses, Sylvie Attucci for her help in preparing the manuscript, Jose W. Saldanha and Janusz Bujnicki for fruitful discussions and Owen Parkes for editing the English text. Funding for N.R and E.H. was provided by the National Program for Research in Functional Genomics in Norway (FUGE) in the Research Council of Norway.

SEDDGSVSPESQEMSYLELPLPSISPLIYAPVCVEDSNSDFYLEVNECEVRKCGCEAGFVYTFVPREMCKATTSLCPMQTKSS --------------EEEE--------EEEEEEEEE-----EEEEE---EEEE-------EEEE--HHHHHHHHHH--------PSIPRED (1) ----------H-----E----------EEEEEEEE-----EEEEEE--E---------EEEEEE-HHHH--------------SAM (2) ----------------HH---------EEEEEEEE-----EEEE-----HHH------EEEEEE-HHHHH-------------JUFO (3) --------------EE----------EEE--EEEE-----EEEEE---EE--------EEEEE--HHHHHHH-----------SSPRO (4) -

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