Peter Stanley

Louise C Serpell

Penelope E Stein

Polymerization of human angiotensinogen: insights into its structural mechanism and functional significance

Keywords: angiotensinogen, serpin, polymers, renin, noninhibitory, hypertension

We have studied the in vitro polymerization of human plasma angiotensinogen (AGT), a noninhibitory member of the SERine Protease INhibitor (serpin) family. Polymerization of AGT is thought to contribute to a high molecular weight form of the protein in plasma that is increased in pregnancy and pregnancy-associated hypertension. Our results show that the polymerization of AGT occurs through a novel mechanism which is primarily dependent on noncovalent linkages, while additional disulphide linkages formed after prolonged incubation are not essential for either formation or stability of polymers. We present the first analyses of AGT polymers by electron microscopy, CD spectroscopy, stability assays and sensitivity to proteinases and we conclude that their structure differs from the "loop-sheet" polymers typical of inhibitory serpins. Histidines within the unique N-terminal extension of AGT appear to influence polymer formation, although it can still take place after their removal by renin. At a functional level, we show that AGT polymers are not substrates for renin, so polymerization of AGT in plasma would predictably lead to decreased formation of AngI with blood pressure lowering. Polymerization may be therefore an appropriate response to hypertension. The ability of AGT to protect its renin cleavage site through polymerization may explain why the AngI decapeptide has remained linked to the large and apparently inactive serpin body through evolution.

INTRODUCTION

Angiotensinogen (AGT) is a 452 amino acid glycoprotein which is a member of the SERine Protease INhibitor (serpin) family of proteins [START_REF] Doolittle | Angiotensinogen is related to the antitrypsin-antithrombin-ovalbumin family[END_REF][START_REF] Irving | Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function[END_REF], showing sequence identities of 25% or less with other plasma serpins and containing a unique 50 residue N-terminal extension.

AGT appears to have evolved a noninhibitory function and has not been found to inhibit any proteinase. In common with at least three other noninhibitory serpins (ovalbumin, pigment epithelial-derived factor and maspin) [START_REF] Stein | Ovalbumin and angiotensinogen lack serpin S R conformational change[END_REF][START_REF] Becerra | Pigment epithelium-derived factor behaves like a noninhibitory serpin. Neurotrophic activity does not require the serpin reactive loop[END_REF][START_REF] Pemberton | The tumor suppressor maspin does not undergo the stressed to relaxed transition or inhibit trypsinlike serine proteases. Evidence that maspin is not a protease inhibitory serpin[END_REF] AGT does not undergo the stressedrelaxed conformational change that characterizes inhibitory members of the family [START_REF] Stein | Ovalbumin and angiotensinogen lack serpin S R conformational change[END_REF].

AGT has a unique role among serpins acting as a precursor for angiotensin peptides. In the initial rate-limiting step of the renin-angiotensin system (RAS), the aspartyl proteinase renin cleaves AGT at its N-terminus, generating the inactive decapeptide angiotensin I (AngI) and leaving the remaining 98% of the molecule as a much larger fragment called des(AngI)AGT. AngI is further hydrolyzed by angiotensin converting enzyme to give the active octapeptide angiotensin II, which regulates blood pressure, salt and water homeostasis, and vascular tone [START_REF] Gould | Kinetics of the human renin and human substrate reaction[END_REF][START_REF] Skeggs | The biochemistry of the reninangiotensin system and its role in hypertension[END_REF]. Linkage studies strongly implicate the AGT gene in the pathogenesis of essential hypertension [START_REF] Lalouel | Angiotensinogen in essential hypertension: from genetics to nephrology[END_REF].

Human plasma AGT is a heterogeneous protein which migrates on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as two bands of approximately 60 kDa. Glycosylation accounts for up to 14% of its molecular mass and plays a predominant role in its heterogeneity [START_REF] Tewksbury | Characterization of human angiotensinogen[END_REF][START_REF] Campbell | The rapid purification and partial characterization of human serum angiotensinogen[END_REF]. In addition to monomeric AGT, a small portion of the circulating AGT has a higher mass and is referred to as high molecular weight AGT (HMrA) [START_REF] Gordon | Chromatographic separation of multiple renin substrates in women: effect of pregnancy and oral contraceptives[END_REF].

Around 3% of the AGT in plasma from normotensive, nonpregnant individuals comprises HMrA. However, the first half of pregnancy is associated with a 4-fold increase in total AGT and 20-fold increase in HMrA in plasma. The proportion of HMrA can reach as high as 28% in pregnancy-associated hypertension [START_REF] Tewksbury | High molecular weight angiotensinogen levels in hypertensive pregnant women[END_REF][START_REF] Tetlow | Changing renin substrates in human pregnancy[END_REF]. HMrA is the major form of AGT in amniotic fluid and in the placenta [START_REF] Lenz | Regional distribution of the angiotensinogens in human placentae[END_REF] where it can comprise up to 80% of the total AGT. One component of HMrA is composed of disulphide-linked complexes with the proform of eosinophil major basic protein [START_REF] Christiansen | Quantification and characterization of pregnancy-associated complexes of angiotensinogen and the proform of eosinophil major basic protein in serum and amniotic fluid[END_REF]. However, these complexes differ from the major form of HMrA in plasma and placental tissue which is composed of polymers of monomeric AGT held together by noncovalent and disulphide bonds [START_REF] Tewksbury | Immunochemical comparison of high molecular weight angiotensinogen from amniotic fluid, plasma of men, and plasma of pregnant women[END_REF][START_REF] Tewksbury | Quantitation of five forms of high molecular weight angiotensinogen from human placenta[END_REF][START_REF] Ramaha | Characterization of different high molecular weight angiotensinogen forms[END_REF]. In addition, most of the Copyright 2006 Biochemical Society placental HMrA is cleaved in the part of the sequence homologous to the reactive centre loop (RCL) of an inhibitory serpin [START_REF] Tewksbury | Quantitation of the five forms of plasma high molecular weight angiotensinogen in women with pregnancy-induced hypertension[END_REF]20].

Two particular areas of contention in the current literature are addressed in this work. The first concerns the nature of the bonds that hold AGT polymers together. Many inhibitory members of the serpin family can form polymers through a "loop-sheet" interaction between the RCL of one molecule and the main -sheet of another, and this noncovalent mechanism has been proposed to explain the formation of HMrA [START_REF] Ramaha | Characterization of different high molecular weight angiotensinogen forms[END_REF]20], while another theory suggests that disulphide linkages play the predominant role in the formation of HMrA [START_REF] Celerier | Characterization of a human angiotensinogen cleaved in its reactive center loop by a proteolytic activity from Chinese hamster ovary cells[END_REF]. The second area of contention concerns the ability of AGT polymers to act as a substrate for renin. Early work on HMrA isolated from plasma concludes that it is recognized by renin [START_REF] Shionoiri | Differences in the kinetic rate constants of normal and high molecular weight renin substrate from term pregnancy human plasma[END_REF][START_REF] Tryon | Kinetic analysis of the reaction of human renin with human Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060444 Copyright 2006 Biochemical Society high and low molecular weight angiotensinogen[END_REF]. However, later work with purified recombinant protein suggests that AGT aggregates are not recognised by renin, possibly due to the presence of intermolecular disulphide bridges [START_REF] Celerier | Characterization of a human angiotensinogen cleaved in its reactive center loop by a proteolytic activity from Chinese hamster ovary cells[END_REF].

Here we describe a study of the polymerization of AGT using an established in vitro system which can replicate the size distribution, nature of the polymer linkages, and state of the RCL found in ex vivo polymers isolated from plasma and placental tissue. We find that the properties of AGT polymers fit neither of the previously reported models and present an alternative mechanism for polymerization. Our findings of the structural nature of AGT polymers serve as a starting point towards understanding their physiological significance.

EXPERIMENTAL

Materials

Unless otherwise stated all reagents were purchased from Sigma (Dorset, UK). Human recombinant renin was a gift from D. Thibeault (Roche, Canada), and 1-antitrypsin was from Athens Research and Technology (Athens, GA). Chromatography media were from Amersham Biosciences (Blue Sepharose 6 FF, DEAE Sepharose FF, Superdex S200 and Superose 12), BioRad (ceramic hydroxyapatite) and Applied Biosystems (POROS 20 PI).

Purification of human AGT

Native AGT was purified from fresh human plasma by 1.4 M and 2.4 M ammonium sulphate fractionation followed by the four chromatography steps described by Tewksbury [START_REF] Tewksbury | Purification of human angiotensinogen[END_REF]: 1. Blue Sepharose 6 FF; 2. DEAE Sepharose FF; 3. Ceramic Hydroxyapatite; and 4. POROS 20 PI. A final gel filtration step on Sephacryl S200 separated the monomeric form of AGT from ex vivo polymers. AGT was stored at 4 o C. Its concentration was determined by UV absorbance at 280 nm using an extinction coefficient (1 mg/ml) of 0.92. Typically, 2.5 mg of monomeric AGT could be purified from 250 ml plasma with a yield of more than 15%. AGT was identified by its immunoreactivity against AGT antibody and its purity

Polymerization of AGT

Native AGT in buffer (0.07 -5.0 mg/ml) was incubated between 37 to 55 o C for durations up to 70 days. Polymerization was assessed on nondenaturing and SDS-PAGE gels and the proteins were visualized by staining with silver (Bio-Rad Silver Stain Plus) or with Coomassie Blue. Residual monomeric protein was quantified by densitometry of stained gels using Quantity One software (Bio-Rad).

To determine the pH dependence of AGT polymerization, samples of AGT were buffer exchanged to 10 mM sodium phosphate, 150 mM NaCl, 1.0 mM EDTA, pH 7.4 and concentrated to 2.0 mg/ml in a 30k cut-off spin concentrator (Vivascience). 2 µl aliquots were diluted 10-fold into 50 mM sodium phosphate, 150 mM NaCl, 0.5 mM EDTA at pH 4.0 to 9.0 and incubated at room temperature for 1 h. Samples (0.2 mg/ml) were then heated at 55 o C for 1 h and run on nondenaturing gels to assess the extent of polymerization.

Polyacrylamide gel electrophoresis and immunoblotting

The discontinuous system of Schagger and von Jagow [START_REF] Schagger | Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa[END_REF] was used for SDS-PAGE. For nondenaturing polyacrylamide gel electrophoresis, gels were cast with 0 to 8 M urea and run with 70 mM Tris, 190 mM glycine, pH 8.8. AGT was electrotransferred to Immobilin-P PVDF (Millipore) and the membranes blocked in 10 % (w/v) dried milk [START_REF] Towbin | Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications[END_REF]. AGT was detected with rabbit antiserum against AGT (at 1/ 15 000 dilution) and revealed with goat anti-rabbit IgG conjugated to horseradish peroxidase on the addition of ECL substrate (Amersham Biosciences). For sequence analysis, AGT was transferred to P SQ PVDF membranes and stained with 0.1 % (w/v) Amido black/ 20% (v/v) methanol.

Circular dichroism

Circular dichroism (CD) spectra were recorded between 190 and 250 nm on a Jasco J-810 spectropolarimeter with 0.3 mg/ml protein in 10 mM sodium phosphate, 50 mM NaCl buffer (pH 7.4) using a 0. Copyright 2006 Biochemical Society followed by monitoring the CD signal at 222 nm between 25 and 95 o C using a heating rate of 1 o C /min. Melting points (Tm) were calculated using an expression for a two state transition as described previously [START_REF] Dafforn | A kinetic mechanism for the polymerization of 1-antitrypsin[END_REF]. The results are the average of three experiments.

Electron microscopy

Samples for electron microscopy were prepared by incubating native AGT (0.5 mg/ml) in 10 mM sodium phosphate, 50 mM NaCl, pH 7.4 at 55 o C for 24 h. A 4 µl drop was placed onto a carbon-coated grid for 2 min. The grid was blotted, washed with water, negatively stained with 2 % (w/v) uranyl acetate, and air-dried. The grid was examined in a Hitachi 7100 transmission electron microscope at 80 kV. Images were recorded at magnifications of 3-60 k with a Gatan Multi-Scan 794 CCD camera using DigitalMicrograph 3.4.4 software.

Image processing was done with Adobe PhotoShop 6.0 software. Individual molecules were visualized at 57,000-fold magnification with photographic enlargement.

RESULTS

Formation and properties of angiotensinogen polymers

Incubation of monomeric AGT at physiological temperature and in physiological buffer at concentrations comparable to those in plasma for periods up to 10 weeks produced discrete high molecular mass ladders of dimers, trimers and higher order polymers (Figure 1(i)).

This process was assisted by mild denaturation either by incubation with urea at low temperature (Figure 1(ii)) or by higher temperatures (Figure 1 (iii)).

Monomeric AGT incubated at 55 o C rapidly formed polymers which co-migrated with unheated ex vivo AGT polymers isolated from plasma (Figure 1(iii)). Nondenaturing gels showed polymer formation after 5 min, with the order of polymers increasing after longer incubation times, and almost no monomer remaining by 6 h (Figure 2A(i)). The initial polymers dissociated back to the monomer totally on nonreducing SDS-PAGE (Figure 2A(ii)) but after 2 h polymers were visible on nonreducing SDS-PAGE which only dissociated to the monomer after reduction (not shown). After 54 h incubation, AGT formed higher order polymers that were stacked to the top of both types of gel.

Formation of polymers on heating RCL-cleaved AGT

Out of a panel of proteinases, only thermolysin was found to cleave the RCL of AGT at the P6-P5 (412Q, 413L) position, matching the cleavage site found in HMrA isolated from

Electron microscopy

Polymers were examined by electron microscopy to confirm that the multimers seen on polyacrylamide gels were ordered polymers and not disordered aggregates. This revealed the presence of mostly linear species, similar to those observed with 1-antitrypsin (Figure 2C(i)). The majority of the filaments were less than 50 nm in length; however, some longer filaments extended to over 100 nm (Figure 2C(ii)).

Kinetic analysis of AGT polymerization

AGT formed polymers when incubated at 48 o C for 22 h at concentrations between 0.07 and 0.7 mg/ml with loss of monomeric AGT (Figure 3A). The extent of polymerization was assessed by quantification of the monomeric AGT on nondenaturing gels. The decreases in monomer were linear when fitted to a reciprocal plot against time indicating that this was a second order reaction. The overall second order rate constant from a double logarithmic plot of initial velocity against AGT concentration was 3.25 + 0.63 L mole -1 sec -1 .

The effect of thiol group modifications on the ability of AGT to polymerize

Comparison of the amounts of monomeric AGT remaining on nondenaturing and SDS-PAGE gels after heat induced polymerization provides an indication of the relative contributions of noncovalent and covalent linkages (Figure 3B). Covalent linkages were not significant until after 6 h when total polymerization was virtually complete, implying that nearly all polymerization before 6 h was due to noncovalent linkages. Copyright 2006 Biochemical Society

Free cysteine residues of AGT were modified with NEM or DTT prior to heat-induced polymerization to investigate the contribution of disulphide bonding. Nondenaturing PAGE analysis of the products showed a ladder of multimer species totally independent of the state of the cysteine residues (Figure 3C(i)) while nonreducing SDS-PAGE showed complete absence of the normal ladder of disulphide-linked polymers after cysteine reduction or alkylation (Figure 3C(ii)). When pre-polymerized AGT was reduced with DTT, the modified protein still appeared as a polymer on nondenaturing PAGE. Moreover, the size distribution of the polymers was unaltered suggesting that the disulphide bridges linked adjacent monomers in the same strand and not separate polymers (not shown).

Circular dichroism signal changes during the polymerization of AGT

Circular dichroism (CD) was used to study structural changes during AGT polymerization (Figure 4). The far-UV CD spectrum of native AGT monomers indicated the usual presence of both -helix and -sheet in a folded protein. However, the presence of a double minimum at 208 nm and 220 nm (Figure 4A) differed from the CD spectra of typical inhibitory serpins at pH 7.4 [START_REF] Bruch | Plasma serine proteinase inhibitors (serpins) exhibit major conformational changes and a large increase in conformational stability upon cleavage at their reactive sites[END_REF]. The far-UV CD spectrum of cleaved AGT monomers was identical to that of intact AGT (Figure 4A) confirming that RCL cleavage was not accompanied by significant conformational change.

The effect of polymerization on the far-UV CD spectrum of AGT was identical for intact and cleaved AGT. Figure 4B shows an overall 20% reduction in the negative CD signal between 200-240 nm on heating. In addition, the overall shape of the CD spectrum after polymerization differed from that obtained for monomers. Instead of the broad level minimum between 208 and 220 nm there was a single minimum at 215 nm. This pattern differs from the CD spectra of typical inhibitory serpin polymers.The stability of monomeric and polymeric AGT was assessed by monitoring the change in CD signal at 222 nm while increasing the temperature. Figure 5A shows the melting curve for monomeric intact AGT with a calculated Tm of 60.6 (± 0. Copyright 2006 Biochemical Society

Characterisation of the unfolding of AGT on urea gels

The stabilities of monomeric and polymeric AGT were compared on nondenaturing gels containing 0 to 8 M urea. Monomeric AGT unfolded with a profile similar to that of inhibitory serpins with an unfolding transition at ~4 M urea (Figure 5B). The monomer was stable below 2 M urea and fully unfolded above 6 M urea with a dynamic region in between. AGT polymers unfolded in an identical manner to the monomer with a transition at ~4 M urea and becoming fully unfolded and running as a tight band in 8 M urea. Faint higher molecular weight bands were visible in 8 M urea and presumably represented the small proportion of covalently linked monomers.

Proteolytic cleavage of the reactive centre loop in premade AGT polymers

Susceptibility of the RCL of native and polymerized AGT to cleavage by thermolysin was compared. Cleavage of native AGT yields a 4 kDa peptide and major fragments of 56 and 52 kDa (Figure 6(i)). Cleavage of polymerized AGT showed an identical pattern on SDS-PAGE (Figure 6(i)), confirming that the sites of interaction with thermolysin do not become hidden or disrupted in the polymers. Furthermore, cleavage of the RCL in AGT polymers was not associated with dissociation of the polymeric structure (Figure 6(ii)).

The effect of pH on AGT polymerization

Polymerization of AGT was assessed after incubation at pH 4 to 9. The formation of polymers was insignificant below pH 6.0 and most marked above pH 7.0 (Figure 7A), suggesting that polymerization might depend on the protonation of a histidine residue.

Treatment of monomeric AGT with DEPC allowed substantial polymerization under neutral and acidic conditions, with no monomer remaining at any pH value tested (Figure 7B).

There are three histidine residues between the AGT N-terminus and the disulphide linkage which connects Cys18 to Cys138 on the serpin body [START_REF] Streatfeild-James | Angiotensinogen cleavage by renin: importance of a structurally constrained N-terminus[END_REF]. The effect of breaking this disulphide bridge was tested. AGT that had been reduced with DTT prior to heating was found to polymerize with the formation of noncovalent linkages at pH 7.4, in exactly the same way as intact AGT, although covalent linkages were totally absent (Figure 3B).

Moreover, reduced AGT polymerized under neutral conditions similarly to DEPC modified AGT (Figure 7C). The influence of AngI, which includes two histidine residues (His6 and His9), on polymerization was assessed by heating des(AngI)AGT. Renin cleavage of AGT had no effect on polymerization at physiological pH: des(AngI)AGT formed polymers Copyright 2006 Biochemical Society through noncovalent and disulphide linkages, exactly as intact AGT (not shown). However, des(AngI)AGT also polymerized at pH 7.0 and below (Figure 7D).

Effect of AGT polymerization on Angiotensin I generation

The effect of polymerization on the ability of renin to cleave the N-terminus of AGT was investigated. Monomeric AGT was polymerized at 55 o C for periods up to 18 h. Aliquots were withdrawn, hydrolysed with renin, and run on reducing SDS-PAGE and immunoblotted. The release of the N-terminal AngI decapeptide was followed by loss of reactivity against an -AngI antibody, with total AGT assessed with an -AGT antibody.

Prior polymerization of AGT proportionally reduced its ability to be cleaved by renin (Figure 8A). This loss in substrate behaviour was essentially complete after 2 h (Figure 2A).

However, even after incubation at 55 o C for 54 h, AGT polymers never became fully resistant to renin and a small loss of AngI remained detectable (not shown).

Monomeric intact AGT and thermolysin cleaved AGT were treated at 55 o C for 2 h to produce polymers without significant disulphide bridging prior to incubation with renin for up to 8 h. RCL cleavage was found to make no difference to the response of monomeric or polymeric AGT to renin (Figure 8B). The monomeric AGT samples released AngI at equivalent rates over 8 h. However, after an initial loss of AngI, both intact and RCLcleaved polymers were totally resistant to further renin hydrolysis.

DISCUSSION

Under physiological conditions, typical inhibitory serpins exist in a metastable state and require partial unfolding by chemical denaturants or heating just below their melting temperatures to induce polymerization. AGT, like other noninhibitory serpins, does not have a metastable native conformation, but we show here that it can polymerize under conditions similar to those which induce polymerization of inhibitory serpins (Figure 1).

These in vitro AGT polymers were found to be similar to the AGT multimers in HMrA isolated from human plasma and placental tissue which have been previously characterized with masses ranging from 200-550 kDa [START_REF] Tewksbury | Quantitation of five forms of high molecular weight angiotensinogen from human placenta[END_REF][START_REF] Ramaha | Characterization of different high molecular weight angiotensinogen forms[END_REF]20]. In addition, electron microscopy of in vitro AGT polymers revealed bead-like, linear chains of molecules (Figure 2C) similar to polymers formed in vivo by inhibitory serpins [START_REF] Lomas | 1-antitrypsin Siiyama (Ser53 Phe). Further evidence for intracellular loop-sheet polymerization[END_REF][START_REF] Davis | Familial dementia caused by polymerization of mutant neuroserpin[END_REF], as well as polymers formed in vitro at elevated temperatures [START_REF] Lomas | The mechanism of Z 1-antitrypsin accumulation in the liver[END_REF][START_REF] Mahadeva | Heteropolymerization of S, I, and Z 1-antitrypsin and liver cirrhosis[END_REF]. This suggests that the increase in mass of AGT was due to Copyright 2006 Biochemical Society the formation of dimers, trimers and higher order polymers rather than nonspecific aggregation.

Loop-sheet polymerization of serpins through s4A and s7A linkages proceeds through two steps: a fast unimolecular phase independent of protein concentration consistent with a conformational change within the monomer, and a slow bimolecular phase dependent on protein concentration representing the formation of polymers. Both phases have been described by first order rate constants [START_REF] Dafforn | A kinetic mechanism for the polymerization of 1-antitrypsin[END_REF][START_REF] Crowther | Nucleation of 1antichymotrypsin polymerization[END_REF]. Our experiments at 48 o C with nondenaturing gels measured the rate-limiting production of polymer and did not report on the fast phase (Figure 3A). Velocities derived from the loss of monomer were dependent on protein concentration and defined polymerization as a second order reaction. The second order rate constant of 3.25 L mole -1 sec -1 expressed as a range of first order constants (0.5 -5.0 x 10 -5 sec -1 ) showed that the rates of AGT polymerization were comparable to those for the slow phases of other wildtype serpins [START_REF] Dafforn | A kinetic mechanism for the polymerization of 1-antitrypsin[END_REF][START_REF] Crowther | Nucleation of 1antichymotrypsin polymerization[END_REF][START_REF] Zhou | Polymerization of plasminogen activator inhibitor-1[END_REF].

The polymerization of AGT was slower at physiological temperature but detectable over several weeks (Fig

1(i))
, a rate that was nevertheless probably sufficient to explain the low levels of polymerized AGT found in the circulating plasma of normotensive, nonpregnant individuals. Within the range 0.07 -0.7 mg /ml AGT polymerization was dependent on the square of the concentration, so that a four-fold increase in AGT would increase the velocity of polymerization 16-fold. Interestingly, a four-fold increase in total AGT over the same concentration range in the first half of pregnancy is associated with a 20-fold increase in HMrA [START_REF] Tewksbury | High molecular weight angiotensinogen levels in hypertensive pregnant women[END_REF][START_REF] Tetlow | Changing renin substrates in human pregnancy[END_REF].

Ex vivo HMrA is held together by both noncovalent bonds and disulphide bonds [START_REF] Ramaha | Characterization of different high molecular weight angiotensinogen forms[END_REF][START_REF] Tewksbury | Quantitation of the five forms of plasma high molecular weight angiotensinogen in women with pregnancy-induced hypertension[END_REF][20] and our results suggest that in vitro AGT polymers were stabilized in the same way (Figures 2A andB). We found that the formation of the SDS-sensitive noncovalent linkages and the intermolecular disulphide linkages were temporally separated during polymerization (Figure 3B), with noncovalent bonds forming first as the most important linkage, followed by later disulphide linkages. Indeed, polymerization still occurred in the absence of disulphide linkage (Figure 3C). Furthermore, if pre-made polymers were reduced with DTT, there was no difference in the size distribution of the polymers (not shown). Copyright 2006 Biochemical Society Presumably, disulphide linkages were formed between adjacent monomers on the same strand rather than between separate polymer strands.

We considered the possibility that AGT polymers possessed the same type of noncovalent linkage as polymers of inhibitory serpins. Polymerization of inhibitory serpins typically involves the intact RCL from one monomer inserting into the A--sheet of another monomer in the strand 4A position that it would normally occupy during proteinase inhibition [START_REF] Sivasothy | Pathogenic 1-antitrypsin polymers are formed by reactive loop--sheet A linkage[END_REF]. AGT polymerization was unlikely to involve this A-sheet mechanism since there is good evidence that the RCL of AGT cannot insert into -sheet A due, firstly, to bulky side chains in the hinge region (P10-P12) of the RCL that would prevent loop insertion [START_REF] Stein | Ovalbumin and angiotensinogen lack serpin S R conformational change[END_REF], and secondly, to sequence features in the A-sheet that would prevent sheet opening [START_REF] Stein | Serpin tertiary structure transformation[END_REF]. In addition, in this work we have shown that both RCL-cleaved and intact AGT polymerized in the same way on heating (Figure 2A,B), suggesting that polymerization did not involve the RCL. Furthermore, AGT polymers displayed a far-UV CD minimum at 215 nm (Figure 4B) while typical A-sheet polymers retain the single 222 nm trough of the monomer [START_REF] Dafforn | A kinetic mechanism for the polymerization of 1-antitrypsin[END_REF]. We also found that AGT polymers did not have the enhanced stability characteristic of typical serpin A-sheet polymers (Figure 5B) [START_REF] Lomas | Preparation and characterization of latent 1-antitrypsin[END_REF].

In contrast to A-sheet polymers where the inserted P8-P3 sequence of the RCL is inaccessible [START_REF] Sivasothy | Pathogenic 1-antitrypsin polymers are formed by reactive loop--sheet A linkage[END_REF][START_REF] Elliott | Inhibitory conformation of the reactive loop of 1-antitrypsin[END_REF], the P6-P5 position of the RCL of AGT remained exposed after polymerization (Figure 6A(i)). Moreover, cleavage of the RCL of polymers did not disrupt their structure (Figure 6A(ii). Accessibility of the RCL was also inconsistent with other polymer models which involve linkages of the RCL with -sheet C or the edge of sheet A [START_REF] Zhou | Polymerization of plasminogen activator inhibitor-1[END_REF][START_REF] Bruce | Biochemical Society thermolabile conformational changes of antithrombin Rouen-VI (187 Asn Asp)[END_REF][START_REF] Carrell | Biological implications of a 3A structure of dimeric antithrombin[END_REF][START_REF] Lomas | 1-Antitrypsin Mmalton (Phe52 ) forms loop-sheet polymers in vivo. Evidence for the C sheet mechanism of polymerization[END_REF][START_REF] Sharp | The active conformation of plasminogen activator inhibitor 1, a target for drugs to control fibrinolysis and cell adhesion[END_REF]. The formation of a stable binary complex between a serpin and an exogenous peptide corresponding to its RCL has been used as a marker of the accessibility of its A-sheet [START_REF] Lomas | The mechanism of Z 1-antitrypsin accumulation in the liver[END_REF][START_REF] Schulze | Structural transition of 1-antitrypsin by a peptide sequentially similar to -strand s4A[END_REF]. A peptide corresponding to the wildtype sequence of the AGT RCL fails to insert in the AGT A--sheet [START_REF] Ramaha | Characterization of different high molecular weight angiotensinogen forms[END_REF], suggesting that its A-sheet cannot open. We confirmed this finding using a peptide corresponding to the RCL of antithrombin which has been found to insert in the A-sheet of many different serpins (not shown).

An unexpected finding was that AGT polymerization was dependent on pH (Figure 7A).

Involvement of histidine residues (pKa 6.0) was confirmed by DEPC modification which allowed polymerization under acidic conditions (Figure 7B). Furthermore, demodification Copyright 2006 Biochemical Society by hydroxylamine led to nearly complete recovery of the inhibition of polymerization at pH 6.0 (not shown). Involvement of N-terminal histidine residues was supported by our findings that AGT could be induced to polymerize under acidic conditions by either reducing the disulphide bridge to allow the N-terminus to move from its original position (Figure 7C), or by removal of the AngI peptide with renin (Figure 7D). However, the presence of the N-terminus was not essential for polymerization since des(AngI)AGT produced polymers at physiological pH in the same way as intact AGT (not shown). A mechanism to distinguish between active AGT and inactive des(AngI)AGT would be a desirable component of the RAS. While active AGT does not polymerize significantly below physiological pH, des(AngI) AGT can form polymers at pH 7.0 (Figure 7A andD).

This might allow discrimination of the two forms under conditions of mild acidosis, for instance at sites of inflammation [START_REF] Ramaha | Release and degradation of angiotensin I and angiotensin II from angiotensinogen by neutrophil serine proteinases[END_REF] or within intracellular compartments [START_REF] Tewksbury | Detection of a receptor for angiotensinogen on placental cells[END_REF].

The effect of AGT polymerization on its role as a renin substrate was investigated under physiological conditions of pH, ionic strength, temperature and AGT concentration, following the reaction by directly assaying the loss of AngI from the body of the serpin.

Our results showed progressive inaccessibility of the N-terminus of AGT to renin with associated failure of the polymer to act as an efficient renin substrate (Figure 8A).

Resistance to renin was proportional to the formation of noncovalent polymers and not disulphide linkages. Recognition of monomeric AGT by renin was unaffected by the state of its RCL (Figure 8B) consistent with the lack of any significant structural changes associated with RCL cleavage. RCL-cleaved and RCL-intact polymers were equally unable to act as renin substrates although this was not clear until the later stages of the reaction when the polymers became resistant to further loss of AngI (Figure 8B). The initial 15% loss in AngI was too great to be explained by residual monomer and may reflect the hydrolysis of the N-termini of AGT molecules at the ends of polymer chains. Copyright 2006 Biochemical Society that of native PAI-1 [START_REF] Zhou | Polymerization of plasminogen activator inhibitor-1[END_REF][START_REF] Sharp | The active conformation of plasminogen activator inhibitor 1, a target for drugs to control fibrinolysis and cell adhesion[END_REF] (Figure 9). The increase in overall -sheet structure characterized by an aberrant -strand linkage is consistent with the 215 nm minimum of the AGT polymer far-UV spectrum (Figure 4). AGT polymers lacked the extreme stability typical of serpin polymers formed through an A-sheet -strand insertion (Figure 5B). This lower stability is compatible with an additional edge strand forming hydrogen bonds on only one side. The stable closure of the A-sheet seen in AGT would be a necessity for the formation of this s7A linkage. Other features of this model in keeping with our findings are (i) the reactive centre loop remains accessible while the N-terminus is hidden and inaccessible to renin (Figures 6 and8), (ii) the Cys18-Cys138 disulphide linkage is close enough to Cys232 for a thiol exchange to link adjacent monomers and further inactivate AGT (Figure 2A,B; Figure 3B,C) [START_REF] Streatfeild-James | Angiotensinogen cleavage by renin: importance of a structurally constrained N-terminus[END_REF], and (iii) the proximity of N-terminal histidine residues to positively charged residues in Helix F may be prevented by their protonation at acidic pH (Figure 7).

A proportion of ex vivo HMrA consists of monomers in which the RCL has been cleaved at the P6-P5 position. Our experiments provide two alternative explanations for the presence of cleaved polymers in vivo: either cleaved AGT monomers polymerize or intact AGT monomers polymerize and undergo subsequent proteinase cleavage. Monomeric cleaved AGT is not detectable in human plasma [START_REF] Celerier | Characterization of a human angiotensinogen cleaved in its reactive center loop by a proteolytic activity from Chinese hamster ovary cells[END_REF] and yet cleaved AGT polymers can be the predominant form [20] which makes the latter the more plausible route.

We have shown that polymerization of AGT can be induced in vitro at temperatures above 45 o C, but obviously this mechanism is not relevant in vivo. The presence of destabilizing mutations might enhance polymerization of AGT under physiological conditions, as with other serpins [START_REF] Carrell | The biostructural pathology of the serpins: critical function of sheet opening mechanism[END_REF]. However, this cannot explain the increased polymerization associated with pregnancy because the primary structure of this protein is unaltered. A microenvironment or a protein partner specific to pregnancy (possibly within the placenta) might reduce the stability of the monomer enough to accelerate polymerization at 37 o C.

Another possibility is that changes in the highly heterogeneous glycosylation pattern of AGT during pregnancy may affect its behaviour. Alternatively, polymerization may occur intracellularly with de novo protein in a partially unfolded state. Short chain polymers entering the circulation could then seed AGT polymerization at other sites [START_REF] Crowther | Nucleation of 1antichymotrypsin polymerization[END_REF]. Copyright 2006 Biochemical Society

The cleavage of AGT by renin is the rate-limiting step in the renin-angiotensin system [START_REF] Gould | Kinetics of the human renin and human substrate reaction[END_REF][START_REF] Skeggs | The biochemistry of the reninangiotensin system and its role in hypertension[END_REF].

Polymerization of AGT into HMrA would lead to reduced generation of AngI and AngII, decreased vasoconstriction and a reduction in blood pressure. Polymerization of AGT could therefore occur as a pathological effect, contributing to the low blood pressure and "shock" associated with many severe illnesses. However, since the polymerization of AGT would lead to a reduction in blood pressure, elevated HMrA is also actually an appropriate response to hypertension. Such appropriate polymerization would be unique among serpins to date. Polymerization of many inhibitory serpins has been described, but in all cases it results in a deficiency of the serpin because the polymerized protein is inactive [START_REF] Davis | Familial dementia caused by polymerization of mutant neuroserpin[END_REF][START_REF] Bruce | Biochemical Society thermolabile conformational changes of antithrombin Rouen-VI (187 Asn Asp)[END_REF][START_REF] Lomas | Serpinopathies and the conformational dementias[END_REF][START_REF] Stein | What do dysfunctional serpins tell us about molecular mobility and disease?[END_REF]. The ability of AGT to modulate its activity as a renin substrate through polymerization could explain the puzzling evolutionary conservation of a serpin framework in this large protein that apparently functions simply as a peptide donor.

FIGURES

  Polyacrylamide gel protein markers were from Bio-Rad. Polyclonal antibody to AGT was a gift from D. Tewksbury (Marshfield Medical Research Foundation, USA) and the AngI polyclonal antibody was purchased from Cambio (Cambridge, UK). A synthetic peptide Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060444 Copyright 2006 Biochemical Society corresponding to the P14-P3 sequence of antithrombin (acetyl-SEAAASTAVVIA) was supplied by MWB (Cambridge, UK).

  was assessed by SDS-and nondenaturing-PAGE. Monomeric protein migrated as a doublet band on both types of gel. Both bands were confirmed to be intact AGT by N-terminal sequencing (1DRVYI) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) of derived tryptic peptides. N-terminal sequencing was performed by the Protein and Nucleic Acid Chemistry facility of the Department of Biochemistry, University of Cambridge, and MALDI-TOF by the Proteomics Facility of the MRC Dunn Human Nutrition Unit, Cambridge.

  2 mm path length quartz cuvette thermostatically controlled at 25 o C. Each spectrum represents the accumulation of ten scans. Thermal denaturation was Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060444

Biochemical

  Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060444 Copyright 2006 Biochemical Society placental tissue. Cleavage with thermolysin reduced the size of both 60 and 56 kDa AGT bands with generation of a 4 kDa peptide (Figure 2B(i)). The observed mass of the peptide (m/z = 4414.0) matched the calculated mass (Mr = 4414.1) of the C-terminal sequence Leu413 to Ala 452, the last residue of mature AGT. N-terminal sequencing of the peptide confirmed the primary cleavage site at P6-P5 within the RCL. Native AGT was easily distinguished from the RCL-cleaved protein on SDS-PAGE but not on nondenaturing gels (Figure 2B(ii)). The effect of RCL cleavage on the polymerization of AGT was determined by heating thermolysin cleaved AGT at 55 o C for up to 54 h (Figure 2B(ii) and (iii)). RCLcleaved AGT retained the ability to polymerize with identical kinetics to those of intact AGT (Figure 2A).

  3) o C, similar to typical inhibitory serpins. Thermolysin-cleaved AGT showed an almost identical heat stability profile (Tm = 59.8 (± 0.4) o C (not shown)). For polymeric AGT, the change in the 222 nm signal with temperature was far less marked, not decreasing at all below 80 o C (Figure 5A). Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060444

  Our results strongly exclude the involvement of the reactive centre loop and an open Asheet in the formation of AGT polymers so we have looked for alternative -strand donors and acceptors. A potential -strand donor would be the amino terminus of AGT which is extended by 50 residues compared to other serpins. A possible model for the AGT polymer is shown in Figure9in which the N-terminal extension of one molecule anneals as a dissociable strand on the edge of the A-sheet of another to give an s7A linkage similar to Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060444

Figure 1

 1 Figure 1 Polymerization of AGT under physiological conditionsAGT was incubated at 37 o C and 0.07 mg/ml in 1 x PBS (i) for 0 to 70 days, (ii) for 24 h containing 0, 2, 4 or 6 M urea, and (iii) for[START_REF] Tewksbury | Purification of human angiotensinogen[END_REF] h at 40, 45, 50 or 55 o C, as indicated. Aliquots were analyzed by 7.5% nondenaturing PAGE and silver staining. Lanes contained 1.4 µg of protein. The positions of monomers (m), dimers (d), trimers (tr) and tetramers (te) are marked with arrows. Ex vivo AGT polymers isolated from human plasma are shown (E).

Figure 2 Figure 4

 24 Figure 2 Analysis of AGT polymers by gel electrophoresis and electron microscopy(A) Polymerization of intact AGT. AGT was incubated at 55 o C and 0.2 mg/ml in 30 mM sodium phosphate, 150 mM NaCl, 0.5 mM EDTA, pH 7.4 (referred to as buffer). Aliquots taken over time (0 to 45 min and 2 to 54 h) were analyzed by (i) 7.5% nondenaturing PAGE and (ii) 10% nonreducing SDS-PAGE. Lanes contained (i) 4 µg and (ii) 2 µg of protein.

Figure 5

 5 Figure 5 Stability of AGT monomers and polymers (A) Thermal stability of AGT monomers and polymers. CD signal at 222 nm for monomeric (solid line) and polymeric (dashed line) samples of AGT while increasing temperature at 1 o C /min. Polymers were formed by incubating AGT in buffer at 1.5 mg/ml at 55 o C for 24 h. Before recording, samples were diluted to 0.3 mg/ml in 10 mM sodium phosphate, 50 mM NaCl, pH7.4. (B) The urea-induced unfolding of AGT monomers and polymers. Composite gel of 7.5% nondenaturing gel electrophoresis in the presence of 0 M, 2 M, 4 M, 6 M and 8 M urea of AGT monomers (M) and polymers (P) (4 µg protein each lane). AGT polymers formed by heating AGT (0.2 mg /ml) in buffer at 55 o C for 3 h.

Figure 6

 6 Figure 6 Absence of reactive centre loop-sheet interactions in AGT polymersCleavage of monomeric and polymeric AGT with thermolysin. Monomeric and polymeric AGT were incubated in reaction buffer at 37 o C for 1 h before loading on (i) 10% SDS-PAGE (2 µg protein / lane) and (ii) 7.5% non-denaturing gel (4 µg protein / lane). The ratios of protein:proteinase were (0) no thermolysin, (1) 900:1, (2) 300:1 based on w/w.

Figure 7 Figure 8

 78 Figure 7 pH-dependence of the polymerization of AGT Following (A) no treatment, (B) histidine modification with 1.0 mM DEPC at 25 o C for 15 min in 20 mM sodium phosphate, 75 mM NaCl, 1.0 mM EDTA, pH 6.0, (C) reduction with 10 mM dithiothreitol (DTT) at 25 o C for 2 h in 20 mM sodium phosphate, 75 mM NaCl, 1.0 mM EDTA, pH 7.0, and (D) cleavage by 3.0 nM renin at 37 o C for 72 h in 30 mM sodium phosphate, 150 mM NaCl, 1.0 mM EDTA, pH 6.0, buffers were adjusted to 150 mM NaCl and pH from 4 to 9 as indicated, and AGT heated at 55 o C for 1 h. Samples were loaded on 7.5% nondenaturing gels (4 µg protein / lane). (A) to (D), Lane C contains unheated sample of each treated AGT.

Figure 9

 9 Figure 9 Proposed model of an AGT s7A loop-sheet dimer Our dimer model was built from a model of the AGT monomer [29] based on sequence alignment with ovalbumin. This model does not include the 69 N-terminal amino acids of AGT, which are illustrated by a dotted line. The disulphide bond between Cys18 and Cys138 and the position of the reactive centre loop (RCL) are indicated. A. Side view of the proposed AGT dimer showing how the N-terminus of AGT would be hidden and inaccessible to renin. The proximity of Cys138 and Cys232 is evident. B. View rotated 90 o showing how the N-terminal extension of an AGT monomer could be positioned to form a new strand against the edge of -sheet A of a second monomer. The position of Cys232 adjacent to Helix F is indicated ( ).

Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060444

Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060444 Copyright 2006 Biochemical Society Chemical modification of AGT

	Stock solutions of dithiothreitol (DTT) in water and N-ethylmaleimide (NEM) in absolute
	ethanol were freshly prepared. Native AGT (1.0 mg/ml) in 20 mM sodium phosphate, 75
	mM NaCl, 1.0 mM EDTA, pH 7.0 was incubated with 10 mM DTT or NEM in a volume of
	50 µl at 25 o C for 2 h. The reduced and alkylated forms of AGT were then diluted 5-fold in
	buffer and incubated for 54 h at 4 o C or 55 o C.
	Stock solutions of DEPC were freshly prepared in absolute ethanol. Native AGT (1.0
	mg/ml) in 20 mM sodium phosphate, 75 mM NaCl, 1.0 mM EDTA, pH 6.0 was incubated
	with 1.0 mM DEPC in a volume of 50 µl at 25 o C for 15 min. The reaction was quenched
	with 5.0 mM imidazole. As a control, an equivalent volume of absolute ethanol (minus
	DEPC) was added to a parallel mixture and treated identically.

Preparation of reactive centre -cleaved and N-terminal -cleaved forms of AGT

	To prepare reactive centre loop-cleaved AGT, native AGT (0.5 mg/ml) was incubated with
	thermolysin (50 µg/ml) at a final protein: proteinase ratio of 200:1 (w/w) in 20 mM Tris-
	Cl, 150 mM NaCl, 2 mM CaCl 2 , pH 7.0 at 37 o C for 60 min and the reaction stopped by the
	addition of EDTA to 10 mM. Cleavage was confirmed by SDS-PAGE, mass spectroscopy,
	and N-terminal sequencing of the products. Cleaved AGT was buffer exchanged and
	purified by gel filtration on Superose 12.
	Large scale N-terminal cleavage of AGT (200 µg; 0.2 mg/ml) by recombinant human renin
	(3.0 nM) was performed at 37 o C in 30 mM sodium phosphate, 150 mM NaCl, 1 mM
	EDTA, pH 6.0. After 72 h, completion was confirmed by the lack of immunoreactivity
	against an -AngI antibody. Des(AngI)AGT was buffer exchanged to pH 7.4, separated
	from AngI and other products by gel filtration on Superose 12, and concentrated in a spin
	concentrator.

Accessibility of the reactive centre loop in AGT polymers

	and polymer forms were incubated with thermolysin at protein: proteinase ratios up to
	250:1 (w/w) at 37 o C for 60 min. Reactions were stopped by placing on ice and adding
	EDTA to 10 mM.
	Native AGT (0.4 mg/ml) was either heated at 55 o C for 54 h to form polymers or stored at 4
	o C as the monomeric form. The presence of polymers in the heated sample was confirmed
	by nondenaturing PAGE, followed by addition of CaCl 2 to 2.5 mM. 5 µg aliquots of native

Renin cleavage of monomeric and polymeric AGT

	Native AGT (0.15 mg/ml) in buffer was heated at 55 o C for up to 18 h. Aliquots were
	removed at intervals and run on gels to confirm the extent of polymerization. Further
	aliquots (2 µg) were removed, diluted with an equal volume of 3.0 nM renin in
	physiological buffer (pH 7.4) and incubated at 37 o C for 1 h. Reactions were stopped by the
	addition of reducing SDS-PAGE loading buffer and snap-frozen in liquid nitrogen.
	Native AGT and RCL cleaved AGT (0.3 mg/ml) were heated at 55 o C for 2 h or stored at 4
	o C. Aliquots were removed and run on gels to confirm the extent of polymerization. Further

aliquots (10 µg) were removed, diluted with an equal volume of 1.0 nM renin in physiological buffer (pH 7.4), and incubated at 37 o C for intervals up to 8 h. The reactions were stopped as above. Aliquots were run on duplicate SDS-PAGE gels, and transferred to PVDF membrane for Western blot analysis with -AGT and -AngI antibodies.

Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060444

Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060444

Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060444

Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060444

Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060444

Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060444

Biochemical Journal Immediate Publication. Published on 27 Jul 2006 as manuscript BJ20060444 Copyright 2006 Biochemical Society

ACKNOWLEDGEMENTS

We wish to thank Ms. Diane Thibeault (BioMega Research Division of Boehringer Ingelheim (Canada) Ltd.) for the gift of recombinant renin, Dr. Duane Tewksbury (Marshfield Medical Research Foundation, USA) for the gift of polyclonal antibody to AGT, Dr. Aiwu Zhou (CIMR, Cambridge) for providing RCL peptides, Dr.

Michael Harbour for performing mass spectrometry (Proteomics Facility, MRC Dunn Human Nutrition Unit, Cambridge), and Dr Rosa Streatfeild-James for the angiotensinogen model. We are grateful to Dr. Didier Belorgey and Dr. Aiwu Zhou for helpful discussion in the preparation of this manuscript.

This study was supported by the Wellcome Trust (Grant 054518/Z/98/Z).


Dimitris Platis

Brian J Smith

Trevor Huyton

Nikolaos E Labrou
email: lambrou@aua.gr

Structure-guided design of a

Keywords: neuraminidase inhibitors, influenza, Structure-guided design, Cibacron Blue 3GA CB3GA, Cibacron Blue 3GA, HPLC, high-performance liquid chromatography, NA, neuraminidase, SBS, sulphate binding subsite

  L'archive ouverte pluridisciplinaire

INTRODUCTION

Influenza is a highly contagious, acute viral respiratory disease which occurs seasonally in most parts of the world. Epidemics with high attack rates, a short incubation period, and rapid progression of the disease through the population are characteristic [START_REF] Nicholls | Pandemic influenza: the inside story[END_REF]. The hallmark of influenza is this epidemicity. The public health impact of influenza is dramatic. Greater than 10,000 deaths occurred in each of 7 U.S. epidemics in the period 1977-1988, and greater than 40,000 deaths occurred during two of these epidemics. The 1918 "Spanish Flu" killed more than 500,000 people in the United States, and 20-50 million people worldwide [START_REF] Palese | What can we learn from reconstructing the extinct 1918 pandemic influenza virus?[END_REF]. Vaccination remains the primary method for prevention of influenza, but vaccine strains must be continually updated and their protective efficacy is limited in patients beyond 65 years of age, the major target group [3]. An alternative lies in antiviral drugs. Influenza neuraminidase (NA) has been proven as a valid therapeutic target for antiviral drugs due to its essential role in the viral replication cycle [START_REF] Colman | Influenza virus neuraminidase: structure, antibodies and inhibitors[END_REF]. NA is thought to enhance viral mobility via hydrolysis of the - [START_REF] Palese | What can we learn from reconstructing the extinct 1918 pandemic influenza virus?[END_REF]3) or - [START_REF] Palese | What can we learn from reconstructing the extinct 1918 pandemic influenza virus?[END_REF][START_REF] Palese | Characterization of temperature sensitive influenza virus mutants defective in neuraminidase[END_REF] glycosidic linkage between a terminal sialic acid (Neu5Ac) residue and its adjacent carbohydrate moiety on the host receptor.

These molecules with terminal Neu5Ac are also the target receptors for the viral hemagglutinin (HA) [START_REF] Ward | Structure of the influenza virus hemagglutinin[END_REF], the major surface glycoprotein on the viral particle surface.

NA destroys these HA receptors, allowing progeny virus particles, budding from infected cell surfaces, to be released [START_REF] Palese | Characterization of temperature sensitive influenza virus mutants defective in neuraminidase[END_REF][START_REF] Murti | Distribution of hemagglutinin and neuraminidase on influenza virions as revealed by immunoelectron microscopy[END_REF].

Neuraminidase is a tetramer of identical, 60 kDa, glycosylated subunits. X-ray diffraction studies revealed a polypeptide fold of sheets, six in total, arranged like the blades of a propeller. Each sheet is composed of four antiparallel strands and has the topology of the letter 'W'. The active site is centrally located on the neuraminidase subunit and lies on the propeller axis at the N-terminal ends of the first strand of each sheet [START_REF] Colman | Influenza virus neuraminidase: structure, antibodies and inhibitors[END_REF].

The discovery of inhibitors of NA for the treatment of influenza infection has been an active area of research [START_REF] Chand | Design and synthesis of benzoic acid derivatives as influenza neuraminidase inhibitors using structure-based drug design[END_REF][START_REF] Smith | Structural studies of the resistance of influenza virus neuraminidase to inhibitors[END_REF][START_REF] Smith | Dihydropyrancarboxamides Related to Zanamivir: A New Series of Inhibitors of Influenza Virus Sialidases. 1. Discovery, synthesis, biological activity, and structure-activity relationships of 4-Guanidino-and 4-Amino-4H-pyran-6carboxamides[END_REF][START_REF] Maring | Structure-based characterization and optimization of novel hydrophobic binding interactions in a series of pyrrolidine influenza neuraminidase inhibitors[END_REF][START_REF] Macdonald | Dimeric zanamivir conjugates with various linking groups are potent, long-lasting inhibitors of influenza neuraminidase including H5N1 avian influenza[END_REF][START_REF] Wang | Design, synthesis, and structural analysis of inhibitors of influenza neuraminidase containing a 2,3-disubstituted tetrahydrofuran-5-carboxylic acid core[END_REF]. NA inhibitors are a class of anti-influenza drugs used for both prophylaxis and treatment of influenza virus infections. Modern approaches for finding new leads for therapeutic targets are increasingly based on threedimensional information about receptors [START_REF] Blundell | Structure-based drug design[END_REF][START_REF] Hardy | The impact of structure-guided drug design on clinical agents[END_REF]. The ready availability of crystal structures of inhibitor/NA complexes has enabled a detailed analysis of the structural basis for potent inhibition [START_REF] Varghese | Three-dimensional structure of theneuraminidase of influenza virus A/Tokyo/3/67 at 2.2 resolution[END_REF][START_REF] Von Itzstein | Rational design of potent sialidase-based inhibitors of influenza virus replication[END_REF]. For example, with the aid of the X-ray crystal structure of NA complexed with sialic acid or 2,3-dehydro-sialic acid, the nanomolar inhibitors zanamivir and oseltamivir were designed as enzyme substrate mimics [START_REF] Von Itzstein | Rational design of potent sialidase-based inhibitors of influenza virus replication[END_REF][START_REF] Colman | Zanamivir: an influenza virus neuraminidase inhibitor[END_REF][START_REF] Varghese | Development of neuraminidase inhibitors as anti-influenza virus drugs[END_REF]. These compounds display high potency and specificity both in vitro and in vivo and are effective prophylactics. Both drugs were introduced into clinical practice in various parts of the world during 1999-2002, and share similar potency and specificity for all subtypes of influenza A and B virus NA [START_REF] Colman | Zanamivir: an influenza virus neuraminidase inhibitor[END_REF][START_REF] Varghese | Development of neuraminidase inhibitors as anti-influenza virus drugs[END_REF].

The development of neuraminidase inhibitor-resistant influenza virus strains is a serious concern and there is an urgent need for research on newer anti-neuraminidase agents [START_REF] Nicholls | Pandemic influenza: the inside story[END_REF][START_REF] Palese | What can we learn from reconstructing the extinct 1918 pandemic influenza virus?[END_REF][START_REF] Smith | Structural studies of the resistance of influenza virus neuraminidase to inhibitors[END_REF]. In the present report we describe a structure-guided approach to the design of a new class of non-carbohydrate benzyl-sulphonate inhibitors, and examine their binding features in the active-site pocket of influenza NA. Non-carbohydrate inhibitors give the advantages of high chemical and metabolic stability

MATERIALS AND METHODS

Materials.

Cyanuric chloride (1,3,5-sym-trichlorotriazine), Cibacron Blue 3GA (CB3GA), 1amino-4-bromo-2-methyl-4a,9a-dihydroanthracene-9,10-dione and all other reagents and chemicals were of analytical grade and obtained from Sigma-Aldrich Co (USA).

Crystalline bovine serum albumin (fraction V) was obtained from Boehringer Mannheim, Germany.

Expression and purification of NA.

Expression of

/Beijing/262/95 H1N1 influenza NA was accomplished in expressSF+ insect cells, using the Baculovirus Expression Vector system [START_REF] Dalakouras | Development of recombinant protein-based influenza vaccine: expression and purification of 1 1 influenza virus neuraminidase[END_REF].

Purification of NA was carried out as described in [START_REF] Dalakouras | Development of recombinant protein-based influenza vaccine: expression and purification of 1 1 influenza virus neuraminidase[END_REF].

Synthesis and purification of CB3GA and other analogues.

Purification of CB3GA.

Solid commercial dye (purity 61.3 %, w/w) was purified to homogeneity in two stages according the procedure of Labrou & Clonis [START_REF] Labrou | The interaction of Candida boidinii formate dehydrogenase with a new family of chimeric biomimetic dye-ligands[END_REF], as follows: 100 mg dye was dissolved in deionised water (20 ml) by stirring at room temperature. The solution was extracted twice with diethyl ether (2 x 20 ml), the aqueous phase concentrated (approx. threefold) on a rotary evaporator and the dye precipitated by addition of cold acetone (60 ml). The precipitate was filtered through Whatman filter paper and dried overnight under reduced pressure. Dried dye was dissolved in water/methanol (5 ml, 50/50 v/v) and filtered through a 0.45-ìm-pore-size cellulose membrane filter (Millipore). The dye solution was applied on a lipophilic Sephadex LH-20 column (2.5 x 30 cm) previously equilibrated in water/methanol (50/50 v/v). The column was developed isocratically at a flow rate of 0.1 ml min -1 cm -1 . Fractions (5 ml) were collected and analyzed by TLC and those with the pure dye were pooled, concentrated by 60 % on a rotary evaporator under reduced pressure, before the product was lyophilized and stored desiccated at 4°C.

Analysis of pure dye was performed by TLC and HPLC, whereas dye concentration was determined spectrophotometrically at 620 nm using a molar absorption coefficient of 12.6 l mmol -1 cm -1 [START_REF] Labrou | The interaction of Candida boidinii formate dehydrogenase with a new family of chimeric biomimetic dye-ligands[END_REF]. Ascending TLC was performed on precoated plastic sheets with silica gel 60 (0.2 mm; Merck) using solvent system butanol-1/propanol-2/ethylacetate/water (2/4/1/3). HPLC analysis was carried out on a C18 reverse phase S5 ODS2 Spherisorb silica column (250 mm x 4.6 mm I.D.). The starting solvent system composed of 80% (v/v) methanol and 20% (v/v) water containing 0.1% (w/v) N-acetyltrimethylammonium bromide.

The hydrolyzed analogue of 1-Amino-4-[3-(4,6-dichlorotriazin-2-ylamino)-4sulphophenylamino]anthraquinone-2-sulphonic acid was prepared by incubating in 0.1 M Na 2 CO 3 at 25 C for 1h as previously described [START_REF] Labrou | Molecular modeling for the design of dye-ligands and their interaction with mitochondrial malate dehydrogenase[END_REF]. Purification of the analogue was achieved by preparative TLC on silica gel 60 plates, using the solvent system butanol-1/propanol-2/ethylacetate/water (2/4/1/3). The blue band was scraped from the plate, and the product was eluted from silica with water. 1-amino-4-[(4amino-3-methylphenyl)amino]-2-methyl-4a,9a-dihydroanthracene-9,10-dione was synthesized as described previously [START_REF] Labrou | Molecular modeling for the design of dye-ligands and their interaction with mitochondrial malate dehydrogenase[END_REF]. All dyes were found to be at least 98 % pure.

Synthesis of azido-CB3GA.

Azido-CB3GA was synthesised by diazotization of the anthraquinone amino group of CB3GA using the NaNO 2 /HCl system following by nucleophilic displacement of the diazotized intermediate with NaN 3 , as following: to a cooled and stirred solution of CB3GA (20 mg) in 1.5 ml of HCl (3M), a solution of sodium nitrite (10 mg in 0.2 ml of water) was added drop wise. The reaction mixture was kept cold by adding crushed ice and was continuously stirred. Immediately following, a solution of sodium azide (12 mg in 0.5 ml water) was gradually added to the vigorously stirred mixture.

Stirring and cooling of the solution in an ice bath was continued for 2 h. Purification of azido-CB3GA was achieved by preparative TLC on silica gel 60 plates using the solvent system propanol/AcOH/H 2 O (4/1/1). The red band was scraped from the plate, and the product was eluted from silica with water. The purity of the product was confirmed by HPLC. HPLC analysis was effected on a C18 reverse phase S5 ODS2

Spherisorb silica column (250 mm x 4.6 mm I.D.). The starting solvent system composed of 80% (v/v) methanol and 20% (v/v) water containing 0.1% (w/v) Nacetyltrimethylammonium bromide. Azido-CB3GA was found to be at least 98.4% pure.

Assay of enzyme activity and protein.

Enzyme assays were performed at pH 5.9 at 37°C according to the published method [START_REF] Schauer | Sialidase (neuraminidase)[END_REF] using sialyl-lactose as substrate. Observed reaction velocities were corrected for spontaneous reaction rates where necessary. All initial velocities were determined in triplicate in buffers equilibrated at constant temperature. Turnover numbers were calculated on the basis of one active site per subunit. Protein concentration was determined by the method of Bradford [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] using bovine serum albumin (fraction V) as standard. Kinetic inhibition studies were carried out using sialyl-lactose as substrate at pH 5.9 at 37°C. No significant interference was observed from the inhibitors. Kinetic constants were deduced from Dixon's plot using the computer program GraFit (Erithacus Software Ltd., Leatherbarrow, 1998).

Difference spectroscopy.

Difference spectral titrations were performed on a Perkin-Elmer Lamda16 double beam double monochromator UV-VIS spectrophotometer at 37 C. Enzyme solution (1 ml; 10.5 M in 20 mM potassium phosphate, pH 5.9) and enzyme solvent (1 ml; 20 mM potassium phosphate, pH 5.9) were placed in the sample and reference black-wall silica cuvettes (10 mm path length), respectively, and the baseline difference spectrum was recorded in the range 700-500 nm. Identical volumes (2-5 l) of dye solutions (0.5 mM) were added to both cuvettes and the difference spectra recorded after each addition. The difference absorption at max was measured relative to a zeroabsorbance reference area at 750 nm. The data were analyzed using the following equation [START_REF] Axarli | Characterization of the ligandin site of maize glutathione S-transferase I[END_REF]:

] [ ] [ A = A max ligand K ligand D equation 1
where A is the difference absorption at max after each addition of dye-ligand, and A max is the maximum difference absorption at max at saturated concentration of dye.

The data were fitted to equation 1 using non-linear fitting using the computer program GraFit (Erithacus Software Ltd., Leatherbarrow, 1998).

Synthesis of triazine-based sulphonate analogues.

The synthesis was carried out as described in [START_REF] Morrill | Rational combinatorial chemistry-based selection, synthesis and evaluation of an affinity adsorbent for recombinant human clotting factor VII[END_REF][START_REF] Palanisamy | Synthesis and evaluation of affinity adsorbents for glycoproteins: an artificial lectin[END_REF] with the following modifications: cyanuric chloride (1 mmol) was dissolved in 20 ml of ice-cold acetone:water (2:1) and added slowly to a solution of o-aminobenzenesulphonic acid (1 mmol, 20 ml ice-cold acetone:water, 2:1) over a period of 20 min under constant stirring. During the course of the reaction the pH was maintained at 6-7 by addition of 0.5 M NaHCO 3 and the temperature at 0-4 C. The progress of the reaction was monitored by thin layer chromatography (silica, chloroform/methanol/acetic acid, 8/4/0.5, v/v) until no cyanuric acid was detectable (after about 2 hours). On completion, 1mmol of o-aminobenzenesulphonic acid was added to the reaction and the mixture was heated to 40-50 C for 24 hours. The reaction was monitored by thin layer chromatography (silica, chloroform/methanol/acetic acid, 8/4/0.5, v/v). During the course of the reaction the pH was maintained at 6-7 by addition of 0.5 M NaHCO 3 . On completion, the reaction was run on silica gel plates (20 x 20 cm) (solvent: chloroform/methanol/acetic acid, 8/4/0.5, v/v). The band corresponding to the bis-substituted triazine product was identified under UV and excised. The bissubstituted product was eluted from silica by subsequent resuspension in a mixture of Copyright 2006 Biochemical Society acetone:water (1:1) (5 ml x 3 times), the acetone removed by evaporation and the remaining liquid by lyophilization. The pure product (purity >95%) was subjected to MS analysis. The same procedure was followed for the synthesis of other bissubstituted triazine derivatives.

Reaction of azido-CB3GA with NA.

Recombinant NA (1 unit) was incubated at 4 C in the dark with azido-CB3GA (20 M) in 20 mM potassium phosphate buffer, pH 5.9. The solution (200 l) was placed 2 cm in front of a UV lamp and irradiated. After various times of irradiation, an aliquot was diluted 10-fold with buffer and residual enzyme activity was determined.

When Neu5Ac (1 mM) was tested for its ability to protect against inactivation, it was preincubated with the enzyme for 10 min prior to the addition of azido-CB3GA.

Stoichiometry of azido-CB3GA binding to NA.

Recombinant NA in 0.1 M potassium phosphate buffer, pH 6, was inactivated with azido-CB3GA at 4 C. The dye-inactivated enzyme was separated from the free dye by ultrafiltration (in an Amicon stirred cell 8050 carrying a Diaflo YM10 ultrafiltration membrane; cut-off 10 kDa) after extensive washing with distilled water.

Further separation was achieved by gel-filtration chromatography by applying the inactive dye-enzyme complex to a Sephadex G-25 column (9 cm x 1.6 cm) equilibrated with water, and collecting fractions (0.5 ml) at a flow rate of 10 ml/h. The solution with dye-inactivated NA was then lyophilized and stored at -20 ºC. The lyophilized enzyme covalent complex was dissolved in 8 M urea, and the absorbance was determined spectrophotometrically at 520 nm using a molar extinction coefficient of 9.5 L/cm . mmol which was determined in 8 M urea. The protein concentration was determined by the method of Lowry [START_REF] Palanisamy | Synthesis and evaluation of affinity adsorbents for glycoproteins: an artificial lectin[END_REF]; no dye interference was observed in protein determinations.

Chymotryptic digestion of the azido-CB3GA covalent complex and peptide purification using high performance liquid chromatography.

Lyophilized neraminidase-azido-CB3GA covalent complex (100 µg) was dissolved in Hepes/NaOH buffer, (0.1 M, pH 7.0, 1 ml) and denatured by the addition of solid urea to yield 8 M solution. The denatured enzyme was treated by DTT (10 mM, 25 C, 1 h) Copyright 2006 Biochemical Society and N-ethyl-maleimide was added to a final concentration of 15 mM, and the solution incubated for 30 min at room temperature. The enzyme was then dialyzed against 0.1 M ammonium bicarbonate buffer, pH 8.3. The enzyme was digested by addition of 10 µg of chymotrypsin. The digestion was allowed to continue overnight at 30 ºC before the mixture was lyophilized and stored dry at -20 ºC. Separation of the resulting peptides was achieved on a C18 reverse phase S5 ODS2 Spherisorb silica column (250 mm x 4.6 mm I.D.). Analysis was effected with a H 2 O/acetonitrile linear gradient containing 0.1% trifluoroacetic acid (0-80% acetonitrile during 80 min) at a flow rate 0.5 ml/min. Fractions of 0.5 ml were collected. The eluents were monitored at both 220 nm and 520 nm.

Molecular docking

The complex of NA with sulphate ions (PDB code 2B8H) was crystallized from buffer with ammonium sulphate [START_REF] Smith | X-ray Crystal Structure of a Calcium Deficient Form of Influenza Virus Neuraminidase: Implications for Substrate Binding[END_REF]. This structure was prepared for docking by removing all crystallographic water molecules, solvent molecules and ions, and adding hydrogen atoms to fulfil unsatisfied valencies using the Sybyl program [30].

The positions of the hydrogen atoms was adjusted manually to ensure correct hydrogen-bonding patterns, then energy minimized until the maximum gradient was less than 0.2 kJ mol -1 Å -1 (with protein atoms held fixed). All-atom Kollman charges were assigned to protein atoms. Ionizable amino acids were modelled in their ionized state, with the exception of D151 that was neutral. The ligand was constructed manually, minimized, and Gasteiger charges assigned using the Sybyl program [30].

The DOCK [START_REF] Ewing | DOCK 4.0: search strategies for automated molecular docking of flexible molecule databases[END_REF] suite of programs was employed for predicting the geometry of the ligand bound to the protein. Electrostatic and van der Waals energies were evaluated on a grid [START_REF] Meng | Automated docking with gridbased energy evaluation[END_REF] with a 0.3 Å spacing in the region spanning all residues within 12 Å of the active-site aspartate D151. A distance-dependent dielectric ( = 4r) and an energy cut-off distance of 10 Å were used in evaluating the interaction energy between protein and ligand. The position of the sulphate-sulphur atoms in the X-ray crystal structure was used to define the docking spheres; matching of sphere centres with ligand sulphur atoms used a maximum distance tolerance of 0.5 Å. Ligand conformational flexibility was explored through torsion angle sampling and minimization. The highest-ranked docked conformation was subjected to energy minimization in Sybyl [30] with all protein atoms held fixed.

RESULTS & DISCUSSION

Structure-guided lead ligand design.

Analysis of the crystal structure of whale N9 NA (PDB code 2B8H) [START_REF] Smith | X-ray Crystal Structure of a Calcium Deficient Form of Influenza Virus Neuraminidase: Implications for Substrate Binding[END_REF], determined in sulphate buffer, revealed that four sulphate ions are located at well defined positions with sulphur atoms 6.0 -7.6 Å apart (Figure 1A). One of them is located (sulphate binding subsite 1, SBS1) in the position usually occupied by the substrate's carboxylate moiety and forms ionic interactions with the side chain guanidinium groups of the arginine residues 119, 294 and 372 (numbering according to Figure 1B).

One of the oxygen atoms of this sulphate anion also comes in close contact (~3.2 Å) with a carboxylate oxygen atom of D152. Outside the substrate-binding site, but close to D152 is a second sulphate anion that forms a hydrogen bond with the N 1 atom of H151 (SBS2). Extending further from the binding site there are two more sulphates (SBS3 and SBS4), the first interacting with the side chain atoms of R153 and N201, and the second forms a hydrogen bond with the N of W458 from a neighbouring monomer. These SBSs represent novel binding sites which may be explored to generate chemical feature-based pharmacophore models of the binding site of this enzyme since the SBSs are located at generally conserved positions, overlapping the active site (Figure 1B).

In an effort to discover novel, non-carbohydrate inhibitors of influenza virus neuraminidase we hypothesized that compounds which contain sulphonate groups in appropriate positions, able to occupy the SBSs, might be bound tightly by the enzyme.

A well-known polysulphonate molecule that acts as an inhibitor for several enzymes and proteins is Cibacron Blue 3GA (CB3GA, compound 4, Figure 2). CB3GA has been engaged in biochemical and enzymological [START_REF] Lyon | Novel class of bivalent glutathione S-transferase inhibitors[END_REF] studies, and in protein purification [START_REF] Labrou | Design and selection of affinity ligands for affinity chromatography[END_REF]. It has been well established that CB3GA tends to bind preferentially to the active-site regions of globular proteins and mimic the binding of the naturally occurring anionic substrates and coenzymes such as NADH, ATP, coenzyme A, Copyright 2006 Biochemical Society flavins, and folate [START_REF] Labrou | The interaction of Candida boidinii formate dehydrogenase with a new family of chimeric biomimetic dye-ligands[END_REF][START_REF] Labrou | Molecular modeling for the design of dye-ligands and their interaction with mitochondrial malate dehydrogenase[END_REF][START_REF] Axarli | Characterization of the ligandin site of maize glutathione S-transferase I[END_REF][START_REF] Subramanian | Spectral changes induced in Cibacron Blue F3GA by salts, organic solvents, and polypeptides: implications for blue dye interaction with proteins[END_REF]. The specific interaction of sulphonic groups with protonated amino acid residues (Arg, His, Lys) on the biomolecules has been recently investigated by Friess and Zenobi [START_REF] Friess | Protein Structure Information from Mass Spectrometry? Selective Titration of Arginine Residues by Sulfonates[END_REF], who have shown that the charged part of CB3GA (sulphonic groups) binds preferentially to protonated amino acid residues (Arg, His, Lys) on the biomolecules to form noncovalent complexes, whereas other sulphonates of simpler structure, such as naphthalene-sulphonic acid derivatives and 1-anilino-naphthalene-8-sulphonic acid, were found to bind preferentially to Arg. The present study was undertaken to determine whether sulphonate molecules [(e.g. CB3GA and its fragments (compounds 1-5, Figure 2)] are able to occupy the SBSs and inhibit NA, with the potential that it may lead to a new family of sulphonatesubstituted affinity ligands.

Investigation of the interaction of NA with CB3GA and its fragments by difference spectroscopy and kinetic inhibition studies.

The interaction of CB3GA (compound 4, Figure 2) with NA was assessed by difference spectroscopy in the 750-500 nm region by studying the perturbation of the absorption spectrum of this compound. In the presence of NA the absorption spectrum of the dye undergoes a red shift, producing difference spectra consisting of positive maximum at the 675 nm region. Figure 3A depicts original difference spectra. CB3GA displays two broad peaks (a 675 nm positive and a 590 nm negative) and an isosbestic point at 630 nm, following a shift from its original absorbance maximum (612 nm).

The shape and the wavelengths that correspond to maximum and minimum of the dye spectrum remained unchanged during titration experiments; furthermore, no timedependent changes of absorbance were observed. These findings indicate no irreversible binding of dye to NA, and formation of a single type of complex. The increase in the extinction at positive maximum after each addition of the dye exhibits a hyperbolic dependence on the concentration of dye, indicating the formation of dye . NA complex (Figure 3B). This phenomenon was useful for the calculation of K D of the dye . NA complexes. The results showed that NA interacts with CB3GA with high affinity, 2.1 0.2 M (Table 1).

Subramanian [START_REF] Subramanian | Spectral changes induced in Cibacron Blue F3GA by salts, organic solvents, and polypeptides: implications for blue dye interaction with proteins[END_REF] has shown that the shape of the spectrum describing the dyeenzyme complex is characteristic of the nature of interaction. The difference spectra of the CB3GA, in a high salt environment, is characterized by positive maximum (690 nm) and negative double minima (630 and 585 nm), whereas the difference spectrum of the dye in binary aqueous solvents displayed a positive peak and a positive shoulder at approximately 655 and 610 nm, respectively, with a small negative contribution below 550 nm. From Figure 3A it therefore appears that the anthraquinone chromogen of CB3GA is probably located in a rather hydrophobic environment. This observation is further supported by the molecular modelling studies (see below).

To analyze which fragment of the CB3GA molecule is responsible or necessary for strong binding to NA the ability of structural fragments of CB3GA (compounds 1-3,5, Figure 2) to bind NA was investigated by kinetic inhibition analysis, and the results listed in Table 1. The results showed that each of the CB3GA fragments exhibits a different affinity for NA compared to the parent CB3GA. In general, all analogues are able to bind NA, however, the relative affinity of analogues differs by approximately 220-fold. The data presented on Table 2 points to the conclusion that smaller inhibitors (e.g. compound 5, the terminal fragment derived from the parent CB3GA) exhibits highest affinity for NA. The anthraquinone moiety (compound 1) seems not to contribute significantly to the binding affinity.

Localization of CB3GA binding site by photoaffinity labelling experiments.

Inactivation of NA by azido-CB3GA.

To characterize and locate precisely the CB3GA-binding site of NA affinity labelling experiments were performed. Affinity labelling is a useful tool for the identification and probing of specific, catalytic and regulatory sites in purified enzymes and proteins [START_REF] Pettigrew | 3-Methyleneoxindole: an affinity label of glutathione S-transferase pi which targets tryptophan 38[END_REF][START_REF] Wang | Probing subunit interactions in alpha class rat liver glutathione S-transferase with the photoaffinity label glutathionyl S-[4 (succinimidyl)benzophenone][END_REF]. In the present study a new photoactive derivative of CB3GA (azido-CB3GA, Figure 4A) was synthesized and evaluated for its ability to react covalently with NA.

NA in the presence of azido-CB3GA is inactivated by irradiation. Figure 4B illustrates the decrease in the enzyme activity as a function of time of irradiation. As controls, incubation of enzyme with azido-CB3GA in the dark, or irradiation of the enzyme under the same conditions but without the reagent, caused no significant loss of enzyme activity. These results show that loss of enzyme activity was due to the reaction of enzyme and photoactivated reagent. The rate of inactivation under these conditions was determined to be 0.017 0.002 min -1 . The inactivation of NA by azido-CB3GA was irreversible and enzyme activity could not be recovered by extensive dialysis nor after gel-filtration chromatography on Sephadex G-25 column in the presence or absence of 8M urea.

To determine the stoichiometry of dye binding to NA, NA was completely inactivated by the dye and the dye-enzyme covalent complex was resolved from free dye by gel filtration on Sephadex G-25 and ultrafiltration. The molar ratio of [Dye]/[NA active site] was determined by measuring the dye spectrophotometrically in urea solution, and the protein by the method of Lowry et al. [START_REF] Lowry | Protein Measurement with the Folin Phenol Reagent[END_REF]. The molar ratio of dye to NA active site was 0.94 0.15, using a subunit molecular weight 51.7 kDa, showing that the dye reacts stoichiometrically with the enzyme and therefore indicates a specific interaction between dye and protein.

The ability of specific ligands (e.g. substrates and inhibitors) to prevent enzyme inactivation by an irreversible inhibitor is the usual criterion used in arguing for binding site-directed modification [START_REF] Labrou | The interaction of Candida boidinii formate dehydrogenase with a new family of chimeric biomimetic dye-ligands[END_REF][START_REF] Labrou | Molecular modeling for the design of dye-ligands and their interaction with mitochondrial malate dehydrogenase[END_REF][START_REF] Axarli | Characterization of the ligandin site of maize glutathione S-transferase I[END_REF][START_REF] Pettigrew | 3-Methyleneoxindole: an affinity label of glutathione S-transferase pi which targets tryptophan 38[END_REF][START_REF] Wang | Probing subunit interactions in alpha class rat liver glutathione S-transferase with the photoaffinity label glutathionyl S-[4 (succinimidyl)benzophenone][END_REF]. Figure 4B shows that the rate of enzyme inactivation by azido-CB3GA decreased in the presence of 1 mM Neu5Ac, indicating that the dye interacts with the enzyme at the substrate-binding site.

Isolation and analysis of peptides from NA modified by azido-CB3GA.

Modified NA was subjected to chymotrypsin digestion followed by fractionation on a reverse-phase HPLC column. Essentially, a single red peak (azido-CB3GA-labelled peptide) was eluted from the column. The red peak was freeze-dried and subjected to amino acid analysis and sequencing. The overall recovery of modified peptide, based on the initial amount of modified enzyme was 36%. Edman sequence analysis of the labelled peptide gave the sequence EECSCYPDTGTVMCVCXDNW, where X indicates that no phenylthiohydantoin derivative was detected in the cycle. By comparison with the amino acid sequence of NA, the X in the peptide was identified as R294, indicating that the side chain of R294 is the group responsible for reacting with the azido group of the dye.

Mapping of the CB3GA binding site by molecular modelling studies.

Molecular modelling studies were employed to provide a detailed picture of CB3GA interaction with NA. CB3GA was docked to the active site of NA with its three sulphate groups located in SBSs 1, 2 and 3. In addition to the interactions of the sulphate groups with the protein mentioned above for the individual sulphate groups, the anthraquinone moiety makes hydrophobic contacts with the aliphatic atoms of the side chains of A248, T249 and N348. The final geometry of the ligand bound is presented in Figure 5. The data listed in Table 1 and the results from molecular modelling studies suggests that the binding of CB3GA and its fragments to NA may be primarily achieved by the sulphonate moieties that provide the driving force for positioning and recognition of the analogues. This conclusion is also supported by the crystal structures of CB3GA-horse liver alcohol dehydrogenase [START_REF] Biellmann | X-ray studies of the binding of Cibacron blue F3GA to liver alcohol dehydrogenase[END_REF] and CB3GAglutathione S-transferase complexes [START_REF] Oakley | The ligandin (non-substrate) binding site of human Pi class glutathione transferase is located in the electrophile binding site (H-site)[END_REF]. The interaction of sulphonate groups of CB3GA with an arginine has been observed by Biellmann et al. [START_REF] Biellmann | X-ray studies of the binding of Cibacron blue F3GA to liver alcohol dehydrogenase[END_REF]. Lowe et al. have used these data to investigate the interaction of CB3GA and ADH in more detail [START_REF] Lowe | Design and application of bio-mimetic dyes in biotechnology[END_REF]: they found that different parts in the molecular structure of CB3GA exhibited completely different reactivities, except for two of the sulphonate groups. The terminal sulphonate group as well as the sulphonate group in the linking diaminobenzene-unit were found to always interact with two arginine residues of ADH. Later, Burton and coworkers [START_REF] Burton | Design and applications of biomimetic anthraquinone dyes. III. Anthraquinone-immobilised C.I. reactive blue 2[END_REF] considered the role of the third sulphonate group bound to the anthraquinone-system. Too close proximity to the amino group inhibits a strong sulphonate dye-protein interaction. Burton also demonstrated the strong affinity of sulphonates to positively charged guanidino groups; almost no affinity between dye and protein remained after substitution of the guanidino-with a trimethylammonium-group. In the case of the CB3GA-glutathione S-transferase complex the sulphonic group linked on the anthraquinone ring interacts with the guanidyl group of R13 [START_REF] Oakley | The ligandin (non-substrate) binding site of human Pi class glutathione transferase is located in the electrophile binding site (H-site)[END_REF]. In the model of CB3GA docked to NA the distance between the anthraquinone amine and R294 is 3.4 Å. Thus, replacement of this amine with an azide would place the azide in close proximity to R294, in agreement with the photoaffinity labelling experiments that identified R294 as the reactive species.

Analysis of NA inhibition by benzyl-sulphonate analogues.

To analyze the contribution of the sulphonic group orientation (e.g. ortho-, meta-, para) in compound 5, two additional analogues were synthesized and their inhibitory activity was evaluated for NA using kinetic analysis. The inhibition constants (K i ) 1. All analogues exhibited a competitive-type of inhibition with respect to substrate, indicating that the analogues bind at the substratebinding site of NA. Substitution on the triazine ring for 3-aminobenzyl-sulphonic acid led to compound 6 (Figure 2) which exhibits higher affinity compared to compound 5.

On the other hand, the isomer bearing bis-substituted 4-aminobenzyl-sulphonic acid (compound 7) clearly displayed a higher K i value corresponding to about 132-fold reduction in affinity compared to compound 6. The importance of the presence of sulphonic group in the analogues was demonstrated by the compounds 8 and 9. For example, compound 8 with a single sulphonic group displays reduced affinity for NA compared to 6, whereas compound 9 lacking both sulphonic groups did not show any appreciable binding to NA.

Molecular modelling studies were also employed to provide a detailed picture of the interaction of compounds 5, 6 and 7 with NA. The analogues were docked with the sulphonic groups occupying SBSs 1 and 2 (Figure 6), consistent with their ability to inhibit competitively. This mode of binding differs from that predicted for CB3GA (Figure 5) in which the sulphonic groups of the bis-benzylsulphonate-triazine moiety bind SBSs 2 and 3. The chlorine of compound 7, although largely solvent-exposed, interacts with the hydrophobic pocket occupied by the anthraquionone of CB3GA.

The chlorines of compounds 5 and 6, however, occupy a hydrophobic region that does not directly participate in binding substrate. The results from the inhibition studies (Table 1) in combination with the molecular modelling studies allow us to speculate on the origins of binding of sulphonic acid analogues to NA. The positioning of the analogues with NA may be primarily achieved by ionic interactions of the sulphonate moiety with the side chain guanidinium groups of the arginine residues 119, 294 and 372 (subsite AS1). These interactions may provide the driving force for positioning and recognition of the analogues. This is in line with the observation that compound 8, with a single sulphonic group, displays good affinity for NA, whereas compound 9, lacking both sulphonic groups, does not show any appreciable binding to NA.

Compounds possessing two sulphonate groups will have a greater affinity than those with one or no sulphonate. Further support may be obtained from the recent work of Friess and Zenobi [START_REF] Friess | Protein Structure Information from Mass Spectrometry? Selective Titration of Arginine Residues by Sulfonates[END_REF], who employed Matrix Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry, to demonstrate that sulphonic acid derivatives bind to arginine with higher selectivity compared to other basic sites (e.g. lysine and Copyright 2006 Biochemical Society histidine) of proteins. The high selectivity of sulphonate compounds for arginine is not fully understood. The principal arginine-sulphonate interaction is electrostatic in nature (a salt bridge), enhanced by ionic-hydrogen bonds that are especially favourable because of the near-perfect shape complementary of the two bidentate binding partners [START_REF] Friess | Protein Structure Information from Mass Spectrometry? Selective Titration of Arginine Residues by Sulfonates[END_REF].

CONCLUSIONS

The polysulphonate molecule CB3GA and several of it structural components were shown to bind to NA. Difference spectroscopy analysis indicated that these compounds bound reversibly to the substrate binding site. CB3GA binds NA with high affinity, 2.1 M; a derivative lacking the anthraquinone moiety binds with K i of 0.13 M. Docking studies of CB3GA to NA confirmed high structural complementarity between the sulphate binding sites observed in the X-ray crystal structure and the sulphonates of the inhibitor. The anthraquinone component of the molecule was predicted to contact several hydrophobic residues on the rim of the active-site pocket, which was supported by the difference spectroscopy analysis. An azido derivative of CB3GA was shown to specifically covalently bind an active-site arginine residue in excellent agreement with the predicted conformation of CB3GA with NA.

The concept of selective staining by dyes of animal tissue and microbial organisms by Ehrlich lay the foundations of present-day chemotherapy. During the 1960's, CIBA introduced the Cibacron range of dyes, which have found utility not only as dyes but also in biomedical research. Although CB3GA and its structural components exhibit wide specificity [START_REF] Labrou | The interaction of Candida boidinii formate dehydrogenase with a new family of chimeric biomimetic dye-ligands[END_REF][START_REF] Subramanian | Spectral changes induced in Cibacron Blue F3GA by salts, organic solvents, and polypeptides: implications for blue dye interaction with proteins[END_REF][START_REF] Biellmann | X-ray studies of the binding of Cibacron blue F3GA to liver alcohol dehydrogenase[END_REF], they provide access to lead structures towards the development of non-carbohydrate inhibitors to influenza NA. analogues and their interaction with horse liver alcohol dehydrogenase and other adenine nucleotide-binding proteins. J. Chromatogr. 508, 109-125.

Figure 2 .

 2 Figure 2. The structure of CB3GA analogues used in the present study. 1: 1-amino-4bromo-9,10-dioxo-4a,9,9a,10-tetrahydroanthracene-2-sulphonic acid; 2: 1-amino-4-[(4-amino-3-sulphophenyl)amino]-9,10-dioxo-4a,9,9a,10-tetrahydroanthracene-2sulphonic acid; 3: hydrolized 1-Amino-4-[3-(4,6-dichlorotriazin-2-ylamino)-4sulphophenylamino]anthraquinone-2-sulphonic acid, 4: Cibacron Blue 3GA; 5: 2-({4chloro-6-[(2-sulphophenyl)amino]-1,3,5-triazin-2-yl}amino)benzenesulphonic acid; 6: 3-({4-chloro-6-[(3-sulphinophenyl)amino]-1,3,5-triazin-2-yl}amino)benzenesulphonic acid; 7: 4-({4-chloro-6-[(4-sulphophenyl)amino]-1,3,5triazin-2-yl}amino)benzenesulphonic acid; 8: 3-[(4-anilino-6-chloro-1,3,5-triazin-2yl)amino]benzenesulphonic acid; 9: 6-chloro-N,N'-diphenyl-1,3,5-triazine-2,4diamine.

Figure 3 .

 3 Figure 3. Difference spectra titrations. A. Difference spectra of CB3GA (from top to bottom: 15, 5, 2, µM CB3GA) in the presence of recombinant neuraminidase at pH 5.9 and 37°C. B. The difference absorbance at 675 nm as a function of the total CB3GA concentration (2-20 µM) for NA.

Figure 4 .A.

 4 Figure 4. A. The structure of azido-CB3GA. B. Photoaffinity labelling of neuraminidase by azido-CB3GA at pH 5.9 and 4 o C. Recombinant neuraminidase was incubated in the absence (filled rhomb) or in the presence of azido-CB3GA at concentration of 20 µM (filled square). At the times indicated, aliquots were withdrawn diluted and assayed for enzymatic activity. Enzyme was also incubated in the presence of 20 µM azido-CB3GA and Neu5Ac (1 mM, open square).

Figure 5 .

 5 Figure 5.Interaction of CB3GA with NA from docking calculations. The solvent accessible surface of the sulphate binding sites is colored blue (SBS1), red (SBS2) and green (SBS3). The sulphate ions identified in the X-ray analysis are displayed as thin rods.Side-chains of specific residues contributing to the SBSs and interacting with the ligand are presented as heavy rods beneath the transparent surface. Atoms are coloured white (H), cyan (C), blue (N), red (O), yellow (S) and grey (Cl). The sulphur atoms of the ligand coincide with the sulphate sulphur atoms. The figure was prepared using DINO (http://www.dino3d.org).

Figure 6 . 9 Figure 2 .

 692 Figure 6. Modelling of benzyl-sulphonate analogues in the active site of NA. 2aminobenzyl-sulphonate -compound 5 (left panel); 3-aminobenzyl-sulphonatecompound 6 (middle panel); 4-aminobenzyl-sulphonate -compound 7 (right panel).Colours of the surface of NA and atoms are the same as detailed in Figure5. The sulphate ions observed in the X-ray structure are presented as thin rods.

Biochemical Journal Immediate Publication. Published on 16 Jun 2006 as manuscript BJ20060447

Biochemical Journal Immediate Publication. Published on 16 Jun 2006 as manuscript BJ20060447

Biochemical Journal Immediate Publication. Published on 16 Jun 2006 as manuscript BJ20060447

Biochemical Journal Immediate Publication. Published on 16 Jun 2006 as manuscript BJ20060447 Copyright 2006 Biochemical Society obtained

  are summarized in Table

Biochemical Journal Immediate Publication. Published on 16 Jun 2006 as manuscript BJ20060447

Biochemical Journal Immediate Publication. Published on 16 Jun 2006 as manuscript BJ20060447 Copyright 2006 Biochemical SocietyTable 1 .

 1 Binding characteristics of CB3GA and its fragments to recombinant neuraminidase. The structures of analogues are depicted in Figure2. Inhibition constants (K i ) were determined by kinetic inhibition studies.

	Compound	K i ( M)]
	1	56.5 4
	2	12.5 2
	3	12.8 1.9
	4	2.9 0.2*
	5	0.28 0.04
	6	0.13 0.05
	7	17.1 1.9
	8	4.3 0.7
	9	No detectable binding
	*The value of 2.1 0.2 for compound 4 was determined by difference spectra titrations.

Biochemical Journal

Immediate Publication. Published on 16 Jun 2006 as manuscript BJ20060447 Copyright 2006 Biochemical Society FIGURE LEGENTS Figure 1. A. The sulphate binding sites SBS1-4 of NA as observed from the 2.2 Å crystal structure (PDB code 2B8H, [29]).

  Sulphate anions are presented as thin tubes, and the side-chains of binding residues are displayed as heavy tubes.

	B. The sulphate binding residues of influenza A neuraminidases of different
	subtypes (N1 to N9). Amino acids that form the sulphate binding sites SBS1-4 are
	marked with *. The partial alignments were produced using ESPript. Abbreviations
	for neuraminidases are in brackets: /Beijing/262/95 (N1)
	A/Chicken/California/9420/2001(N2); A/turkey/California/6878/79(N3);
	A/mink/Sweden/E12665/84(N4); A/shearwater/Australia/1/72 (N5);
	A/sanderling/Delaware/1258/86(N6); avian/FPV/Weybridge (N7);
	A/Turkey/Canada/63 (N8); A/whale/Maine/1/84(N9).