Mark A Wells

Graham S Jackson

Samantha Jones

Laszlo L P Hosszu

C Jeremy Craven

Anthony R Clarke

John Collinge

Jonathan P Waltho
email: j.waltho@sheffield.ac.uk

A Reassessment of Copper (II) Binding in the Full-Length Prion Protein

Keywords: Prion, PrP, Copper (II), Metal binding, NMR, Fluorescence

It has been shown previously that the unfolded N-terminal domain of the prion protein can bind up to six Cu 2+ ions in vitro. This domain contains four tandem repeats of the octapeptide sequence PHGGGWGQ, which alongside the two histidine residues at positions 96 and 111, contribute to its Cu 2+ binding properties. At maximum metal-ion occupancy each Cu 2+ is coordinated by a single imidazole and deprotonated backbone amide groups. However two recent studies of peptides representing the octapeptide repeat region of the protein, have shown that at low Cu 2+ availability an alternative mode of coordination occurs where the metal ion is bound by multiple histidine imidazoles. Both modes of binding are readily populated at pH 7.4, while mild acidification to pH 5.5 selects in favour of the low occupancy, multiple imidazole binding mode. We have used NMR to resolve how Cu 2+ binds to the full-length prion protein under mildly acidic conditions where multiple histidine coordination is dominant. We show that at pH 5.5 the protein binds two Cu 2+ ions and that all six histidines of the unfolded N-terminal domain and the N terminal amine act as ligands. These two sites are of sufficient affinity to be maintained in the presence of millimolar concentrations of competing exogenous histidine. A previously unknown interaction between the N-terminal domain and a site on the C-terminal domain becomes apparent when the protein is loaded with Cu 2+ . Furthermore, the data reveal that sub-stoichiometric quantities of Cu 2+ will cause selfassociation of the prion protein in vitro suggesting that Cu 2+ may play a role in controlling oligomerisation in vivo.

Introduction

Prion diseases are a group of fatal neurodegenerative disorders that includes Creutzfeldt-Jakob disease in humans, scrapie in sheep and bovine spongiform encephalopathy in cattle. In humans such diseases can arise sporadically, be inherited, or occur through infection with prion-contaminated material [START_REF] Collinge | Prion diseases of humans and animals: Their causes and molecular basis[END_REF]2]. The protein only hypothesis states that the particles that cause infection, termed prions, are composed principally or entirely of a misfolded isoform of a host-encoded protein, termed the prion protein (PrP) [START_REF] Griffith | Self-replication and scrapie[END_REF][START_REF] Prusiner | Novel Proteinaceous Infectious Particles Cause Scrapie[END_REF][START_REF] Oesch | A Cellular Gene Encodes Scrapie Prp 27-30[END_REF], with prion propagation occurring by conformational conversion of the normal isoform, PrP C , to the disease related isoform PrP Sc . Mammalian PrP C is a glycolipid anchored cell surface glycoprotein found at high levels in the central nervous system, lymphatic tissue, activated lymphocytes and at neuromuscular junctions [START_REF] Stahl | Scrapie Prion Protein Contains a Phosphatidylinositol Glycolipid[END_REF]. The physiological function of PrP remains unclear; ablation of the PrP gene, Prn-P, in mice results in viable animals without gross developmental or behavioral abnormalities [START_REF] Bueler | Normal Development and Behavior of Mice Lacking the Neuronal Cell-Surface Prp Protein[END_REF], although more detailed studies revealed some phenotypic effects, including altered synaptic physiology [START_REF] Collinge | Prion Protein Is Necessary for Normal Synaptic Function[END_REF].

Biophysical and biochemical studies have demonstrated that PrP specifically binds Cu 2+ and this has led to the proposal that PrP is a metalloprotein in vivo [START_REF] Brown | The cellular prion protein binds copper in vivo[END_REF][10][START_REF] Hornshaw | Copper-Binding to the N-Terminal Tandem Repeat Region of Mammalian and Avian Prion Protein -Structural Studies Using Synthetic Peptides[END_REF][12][START_REF] Miura | Raman spectroscopic study on the copper(II) binding mode of prion octapeptide and its pH dependence[END_REF][START_REF] Viles | Copper binding to the prion protein: Structural implications of four identical cooperative binding sites[END_REF][START_REF] Whittal | Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry[END_REF][START_REF] Burns | Molecular features of the copper binding sites in the octarepeat domain of the prion protein[END_REF][START_REF] Garnett | Copper binding to the octarepeats of the prion protein -Affinity, specificity, folding, and cooperativity: Insights from circular dichroism[END_REF][START_REF] Aronoff-Spencer | Identification of the Cu2+ binding sites in the N-terminal domain of the prion protein by EPR and CD spectroscopy[END_REF][START_REF] Burns | Copper coordination in the fulllength, recombinant prion protein[END_REF][START_REF] Stockel | Prion protein selectively binds copper(II) ions[END_REF][START_REF] Kramer | Prion protein binds copper within the physiological concentration range[END_REF].

PrP C comprises a flexibly disordered N-terminal domain (residues [22] and a globular, mainly α-helical, C-terminal domain (residues 126-231) [START_REF] Riek | NMR structure of the mouse prion protein domain PrP(121-321)[END_REF]. Residues 60-91 of the protein comprise four tandem repeats of the sequence PHGGGWGQ. It is this region, known as the octapeptide repeats, which was first discovered to bind Cu 2+ [10,[START_REF] Hornshaw | Copper-Binding to the N-Terminal Tandem Repeat Region of Mammalian and Avian Prion Protein -Structural Studies Using Synthetic Peptides[END_REF] and a considerable body of work has since been devoted to identifying the number of binding sites, their affinities and the nature of coordination, in both the octapeptide repeats and the full length prion protein, PrP 23-231 ‡ [START_REF] Brown | The cellular prion protein binds copper in vivo[END_REF][12][START_REF] Miura | Raman spectroscopic study on the copper(II) binding mode of prion octapeptide and its pH dependence[END_REF][START_REF] Viles | Copper binding to the prion protein: Structural implications of four identical cooperative binding sites[END_REF][START_REF] Whittal | Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry[END_REF][START_REF] Burns | Molecular features of the copper binding sites in the octarepeat domain of the prion protein[END_REF][START_REF] Garnett | Copper binding to the octarepeats of the prion protein -Affinity, specificity, folding, and cooperativity: Insights from circular dichroism[END_REF][START_REF] Aronoff-Spencer | Identification of the Cu2+ binding sites in the N-terminal domain of the prion protein by EPR and CD spectroscopy[END_REF][START_REF] Burns | Copper coordination in the fulllength, recombinant prion protein[END_REF][START_REF] Stockel | Prion protein selectively binds copper(II) ions[END_REF][START_REF] Kramer | Prion protein binds copper within the physiological concentration range[END_REF]. Until recently, the accepted model of Cu 2+ binding to the full length prion protein, at pH 7.4, is that it binds six Cu 2+ ions at separate sites, one in each of the four octapeptide repeats and two more at sites involving H96 and H111, and each with coordination from a single histidine imidazole and amide nitrogens from nearby glycines [START_REF] Burns | Copper coordination in the fulllength, recombinant prion protein[END_REF].

Two recent studies have shown that the model described above provides an incomplete picture of Cu 2+ binding in the prion protein, by demonstrating that a peptide representing the four octapeptide repeats displays multiple modes of Cu 2+ binding at pH 7.4 [START_REF] Chattopadhyay | The octarepeat domain of the prion protein binds Cu(II) with three distinct coordination modes at pH 7.4[END_REF][START_REF] Valensin | The dimeric and tetrameric octarepeat fragments of prion protein behave differently to its monomeric unit[END_REF].

The peptide can bind a single Cu 2+ ion through multiple histidine coordination but, in the presence of more Cu 2+ , binding of up to four Cu 2+ ions occurs, with all of these coordinated in identical sites -each involving deprotonated backbone amide nitrogen atoms and a single histidine. Perhaps counter-intuitively, these four Cu 2+ binding sites replace a single site of higher affinity. Peptides representing the octapeptide repeats cannot, however, be considered a complete model for Cu 2+ binding to the prion protein.

Cu 2+ binding sites outside the octapeptide repeats have also been reported, involving histidines 96 and 111 [12,[START_REF] Burns | Copper coordination in the fulllength, recombinant prion protein[END_REF][START_REF] Jones | Probing copper2+ binding to the prion protein using diamagnetic nickel2+ and 1H NMR: the unstructured N terminus facilitates the coordination of six copper2+ ions at physiological concentrations[END_REF][START_REF] Jones | Preferential Cu2+ coordination by His96 and His111 induces beta-sheet formation in the unstructured amyloidogenic region of the prion protein[END_REF]. It is thus clear that for a full understanding of Cu 2+ binding to the prion protein, binding to the full-length protein needs to be investigated, and in particular the involvement of multiple histidine Cu 2+ coordination. Given the low availability of Cu 2+ in the extracellular mileu (examined in the discussion section), this higher affinity binding mode is more likely to be substantially populated in vivo.

Fortunately, although the full-length prion protein is poorly soluble above pH 6 [22], it can be studied at lower pH values where multiple histidine Cu 2+ coordination should be favoured. In aqueous solution H + ions will compete for the basic coordinating groups that form a metal binding site, and thus the pK a of the coordinating groups and the pH of the solution are both determinants of the stability of a metal complex. The pK a of the histidine imidazole group is around 6, which is substantially lower than that of a backbone amide nitrogen, and thus coordination by multiple histidines will be maintained at lower pH compared with coordination modes involving deprotonated backbone amide groups. We find that binding of two Cu 2+ ions is maintained in the full-length prion protein at pH 5.5, and have investigated the nature of the coordination in this complex.

Here we report the locations of these binding sites and the identity of the coordinating groups. We demonstrate that these binding sites are of sufficient affinity to be maintained in the presence of substantial concentrations of exogenous histidine, a substance known to bind copper in vivo [START_REF] Linder | Extracellular Copper Substituents and Mammalian Copper Transport[END_REF]. Finally, we show how Cu 2+ binding is altered in a truncated form of PrP, comprising residues 91 to 231 -PrP 91-231 , which has no octapeptide repeat regions, but which can still transmit infection.

Experimental

Expression, purification and metal stripping of PrP 91-231 Human PrP 91-231 , with methionine at position 129, was expressed and purified as described previously [START_REF] Hosszu | The residue 129 polymorphism in human prion protein does not confer susceptibility to CJD by altering the structure or global stability of PrPC[END_REF][START_REF] Jackson | Multiple folding pathways for heterologously expressed human prion protein[END_REF]. The PrP 23-231 and PrP 91-231 constructs used here both have the additional three residue sequence SFR present at the N-terminus which forms part of the thrombin cleavage site. To ensure the protein was free of metal ions, the protein was repeatedly buffer exchanged with 8 M urea, 20 mM EDTA, 25 mM TRIS/acetate, pH 8.0 and then refolded in 5 mM MES, pH 5.5 in an Amicon ultrafiltration unit (3 kDa molecular weight cut off). The protein concentration for refolding was less than 1 mg/ml. All buffers used during and after the metal stripping step were prepared using AnalR grade water and, wherever possible, Aristar grade reagents, and were treated with Chelex resin (BioRAD).

Expression, purification and metal stripping of full length prion protein, PrP 23-231 Full length human prion protein, with methionine at position 129, was expressed with a N-terminal his-tag containing a thrombin cleavage site, using standard molecular biology protocols. Expression of uniformly 15 N, 13 C labeled protein and the initial purification by Ni-NTA chromatography was carried out using the same procedures described previously for PrP 91-231 [START_REF] Hosszu | The residue 129 polymorphism in human prion protein does not confer susceptibility to CJD by altering the structure or global stability of PrPC[END_REF][START_REF] Jackson | Multiple folding pathways for heterologously expressed human prion protein[END_REF]. Oxidation of the disulfide bond was checked by HPLC [START_REF] Jackson | Multiple folding pathways for heterologously expressed human prion protein[END_REF] and was complete after 24 hours, without the aid of a catalyst. The protein was diluted to less than 1 mg/ml in 6 M guanidine HCl, 50 mM TRIS/HCl, pH 8.0 and then refolded by dialysis against 150 mM acetate/TRIS, 0.02% w/v NaN 3 , pH 3.5. In preparation for the thrombin cleavage step, the pH was gradually increased by successive dialysis steps against 10 mM TRIS/acetate, 0.02 % w/v NaN 3 at pH 4.5, pH 5.5, pH 6.5 and pH 7.0.

The fusion protein was cleaved by adding 0.6 units of thrombin (Novagen) per milligram of protein and CaCl 2 , to a concentration of 2.5 mM. The reaction was incubated at 37 ºC for 24 hours. Cleavage was halted by the addition of the protease inhibitor cocktail Protease Complete (Roche). For the final purification step a higher pH was required and this had to be carried out under denaturing conditions for the protein to be soluble. Urea was thus added to the reaction mixture to give a final concentration of 8 M and the pH was adjusted to 7.5. This material was then loaded onto an SP-Sepharose Fast Flow column (Amersham), pre-equilibrated with 8 M urea, 150 mM NaCl, 50 mM TRIS/HCl pH 7.5. The column was washed extensively with the equilibration buffer. A salt gradient from 150 mM to 600 mM NaCl in 8 M urea, 50 mM TRIS/HCl, pH 7.5 was run over 8 column volumes and the protein was eluted at approximately 300-400 mM NaCl. To remove any metal ions, the purified protein was repeatedly buffer exchanged against 8 M urea, 25 mM EDTA, 20 mM TRIS/acetate, pH 8.0 and then refolded in 150 mM acetate/TRIS, pH 3.5, in an Amicon ultra-filtration unit (3 kDa molecular weight cut off).

The protein concentration for refolding was less than 1 mg/ml. The buffer was exchanged to 20 mM acetate/TRIS pH 4.5 and then finally 10 mM MES pH 5.5. All buffers used during and after the metal stripping step were prepared using AnalR grade water and, wherever possible, Aristar grade reagents, and treated with Chelex resin (BioRAD).

NMR sample preparation

For backbone and sidechain assignment experiments, metal-free full length prion protein in 20 mM MES, pH 5.5 was concentrated to approximately 1 mM protein. NaN 3 (to a final concentration of 1 mM), EDTA free Protease Complete (Roche) and 10% D 2 O were added, and the pH readjusted to 5.5 by addition of MES. The final MES concentration was approximately 30 mM. The sample was spun for 5 minutes at 13,000 rpm in a microcentrifuge, to remove any precipitated material. For titrations with Cu 2+ the samples were prepared likewise, but only concentrated to approximately 0.5 mM or 0.1 mM protein and no NaN 3 was added. An absorbance measurement at 280 nm was taken on an aliquot of the sample diluted into 6 M guanidine HCl, 50 mM Tris/HCl, pH 8.0. The protein concentration was determined based on a calculated extinction coefficient [31].

ICP-MS analysis showed that no significant quantities of transition metals were present in the NMR sample (transition metals above 1 µM were: Cd 5.5 µM, Cu 4.7 µM, Zn 3.6 µM, Fe 1.8 µM). For titration with Cu 2+ a sample of 1.2 mM PrP 91-231 was prepared in 5 mM MES, pH 5.5, with 10% D 2 O. Measurement of the protein concentration was carried out by the same method. Nitric acid washed NMR tubes were used for metal titration samples.

NMR resonance assignment experiments for the full length prion protein -see supplementary online materials and methods

Cu 2+ titrations monitored by NMR

The PrP 91-231 sample was titrated with 4 aliquots of 4 mM CuSO 4 , 10 mM glycine, 5 mM MES pH 5.5 to give mole fractions of Cu 2+ relative to protein of 0.25, 0.50, 0.75 and 1.00. NMR spectra were recorded at 303 K on a Bruker Avance DRX-600 spectrometer.

At each point in the titration a 1 H 15 N HSQC and aromatic and aliphatic 1 H 13 C HSQCs were recorded. The full length prion protein sample was titrated with 13 aliquots of 4 mM CuSO 4 , 10 mM glycine, 5 mM MES pH 5.5, to give mole fractions of Cu 2+ relative to protein of 0.05, 0.10, 0.20 and through to 2.20, in steps of 0.20. NMR spectra were recorded at 303K on a Bruker Avance AV-800 spectrometer. Again at each point in the titration a 1 H 15 N HSQC and aromatic and aliphatic 1 H 13 C HSQCs were recorded.

Determination of dissociation constants

Samples of 3.5 µM full length prion protein in 5 mM MES, pH 5.5 were prepared containing either 10 mM histidine or 100 mM histidine. CuSO 4 was added as a glycine complex in 5 mM MES at pH 5.5, to give the appropriate final Cu 2+ concentrations.

Fluorescence measurements were made at 30 ˚C with an excitation wavelength of 291 nm and an emission wavelength of 345 nm, using a Jasco FP-750 spectrofluorimeter. The contribution of collisional quenching effects was evaluated by titrating samples of 20 µM N-acetyltryptophanamide (matching the concentration of tryptophan residues in the prion protein sample) with Cu 2+ under the same conditions. The prion protein data was then corrected for the measured collisional quenching effect.

Analysis of the data and least squares curve fitting was carried out using the program Grafit (Erithacus Software). To obtain the apparent dissociation constant for the second Cu 2+ binding event, the fluorescence data recorded in the presence of 10 mM histidine were fitted to a discontinuous function describing two phases of metal binding (equation 1), where F 0 is the initial fluorescence and F M is the fluorescence at a given metal concentration, A 1 and A 2 are the amplitudes of the fluorescence changes associated with Copyright 2006 Biochemical Society each event, M is the total metal concentration, P is the total protein concentration and K d2 is the apparent dissociation constant of the second binding event. A linear function describes the first event where the apparent K d value is much less than the concentration of sites, whilst the second event, for which the apparent K d value is similar to the concentration of sites, is described by the quadratically-derived ligand binding equation.

P M ≤       + = P A M F F M 1 0 P M > ( ) ( )         - - + - + + + = P P P M K M K M A A F F d d M 2 4 2 2 2 2 1 0

Equation 1

The data acquired in the presence of 100 mM histidine were fitted to equation 2 which describes metal binding to a single site, with a dissociation constant of K d1 . The apparent K d value of the second site, under these conditions, will be too large to make a significant contribution to the observed fluorescence quenching and hence the contribution of this site was ignored in the fitting procedure.

( )

        - + + - + + + = P MP K M P K M P A F F d d M 2 4 2 1 1 1 0

Equation 2

The true K d values of first and second Cu 2+ binding events at pH 5.5 were calculated using equation 3, where K d(app) , is the measured apparent dissociation constant, K d 1 (His) and K d 2 (His) are the step wise dissociation constants for formation of a Cu(histidine) 2 complex at pH 5.5, (1.2 µM and 160 µM respectively [START_REF] Jackson | Multiple folding pathways for heterologously expressed human prion protein[END_REF]) and the concentration of

Results

NMR-detected effects of Cu 2+ addition

Observation of NMR signals as a protein is titrated with Cu 2+ provides a site specific probe for the identification of nuclei close to the metal ion in a Cu 2+ protein complex.

Proximity to the paramagnetic Cu 2+ ion causes acceleration of the T 2 relaxation of nuclei, and hence broadens NMR signals, leading to a loss of peak height [32]. Nuclei close to the metal binding site can thus be identified by a reduction in the height of their NMR signals as Cu 2+ is titrated into the protein. Backbone and sidechain signals were monitored on addition of Cu 2+ bis-glycinate to the full length prion protein, PrP 23-231 , using 1 H, 15 N and 1 H, 13 C heteronuclear single quantum coherence (HSQC) NMR spectra.

The presence of glycine allows the Cu 2+ to be added as a pH buffered solution, by preventing the precipitation of Cu 2+ as copper hydroxide, which would otherwise occur at

The full length prion protein binds two Cu 2+ ions at pH 5.5

Numerous backbone [N,H N ] § signals, for example those of T191 (Fig. 1A), show very limited reduction in height throughout the titration, indicating that these residues are remote from any metal binding site. In contrast, there are a groups of signals from residues such as S231 (Fig. 1A) for which, initially, the backbone [N,H N ] signal peak heights are largely unaffected by Cu 2+ addition but which rapidly lose height after ca. two molar equivalents of Cu 2+ have been added. This behaviour occurs at multiple sites in the vicinity of residues that have a functional group with a propensity for metal binding (e.g. H140, D144, D178, E207, S231). Similarly, in terms of chemical shift, some [N,H N ] signals exhibit changes only after ca. two molar equivalents of Cu 2+ have been added, including those of S143, D144, Y145 and S231 (Fig. 1B). The affected residues are positioned at several distinct sites on the surface of the protein, so the effects can not be due to a single specific Cu 2+ binding site. Instead they are the result of several different transient interactions with Cu 2+ ions, at separate locations on the protein's surface. The most noteworthy feature of these observations is that the NMR signals from residues involved in these Cu 2+ binding sites are completely unaffected by the first two mole equivalents of Cu 2+ added to the sample. The only explanation for this is that the protein completely sequesters two mole equivalents of Cu 2+ in binding sites of substantially higher affinity. This shows that higher affinity Cu 2+ binding in the full-length prion protein saturates at an apparent Cu:PrP stoichiometry of 1.8 ± 0.2, which is compatible with a stoichiometry of 2:1.

Identification of the Cu 2+ coordinating groups in the PrP(Cu 2+ ) 1 and PrP(Cu 2+ ) 2 complexes

The behaviour of the third category of NMR signals, of which M112 is typical (Fig. 1A), reveals the identity of the coordinating groups that form the two higher affinity Cu 2+ binding sites. This group of [N,H N ] signals show a substantial loss of peak height on addition of the first aliquots of Cu 2+ to the sample, with complete loss of the signal occurring prior to the addition of two molar equivalents of Cu 2+ . The largest reductions in peak heights are in signals from the six histidines residues of the N-terminal domain and from residues nearby in the sequence. This can be seen in Fig. 2A, which shows the reductions in peak heights at a Cu:PrP stoichiometry of 1.8:1. At this stoichiometry, any reductions in peak heights due to the the weaker binding sites, described above, can be eliminated. The binding up to a Cu:PrP stoichiometry approaching 2:1, allows us to rule out the possibility that the effects we attribute to formation of a Cu 2+ protein complex are instead due to non-specific interactions with free Cu 2+ ions.

To confirm that all six histidines of the N-terminal domain are the coordinating residues, and to determine if any additional coordinating groups are involved, the behavior of histidines, but not those of other histidine residues such as H140 (Fig. 3). Attenuation of signals in this way is expected for nuclei that are close to Cu 2+ in a protein complex, where the exchange rate of Cu 2+ between protein molecules is fast on a NMR timescale (i.e. where the dissociation rate exceeds the chemical shift change induced by binding in Hz). Thus all six histidines (i.e. four from the octapeptide repeats plus H96 and H111) must coordinate Cu 2+ in the PrP(Cu 2+ ) 1 complex, and exchange on a 10 ms or faster timescale. The only other signals reduced in the same fashion were the [Cα,Hα] and [Cβ,Hβ] signals from the N-terminal residue, S20, which is the first of the three residues added to the N-terminus of the full length prion protein sequence to form a thrombin cleavage site. The N-terminal amine is thus also a coordinating group. The hydroxyl group of S20 may be an additional ligand, though the affinity of this group for Cu 2+ is predicted to be orders of magnitude weaker than that of the amine [START_REF] Shriver | Inorganic Chemistry[END_REF]35].

In summary, the data indicate that at least seven functional groups -the six histidines of the N-terminal domain and the N-terminal amine -contribute to coordinating a single Cu 2+ ion in the PrP(Cu 2+ ) 1 complex. Cu 2+ is generally either 3, 4 or 5 coordinate in proteins [36] and hence these seven coordinating groups can not all be simultaneously involved in a single Cu 2+ complex. The higher affinity Cu 2+ binding in the full length prion protein must instead be derived from a rapidly exchanging ensemble of different coordination geometries, all of comparable stability, involving different combinations of the seven coordinating groups.

On increasing the Cu:PrP stoichiometry beyond 1:1, no new protein signals become substantially broadened, until the second binding site is saturated. Therefore, the second binding site must be formed from a subset of the coordinating groups which take part in the ensemble of binding modes which collectively form the highest affinity site.

Self-association of the PrP(Cu 2+ ) 1 complex

In the fourth group of NMR signals another distinct behaviour is observed -peak height is initially reduced by the addition of Cu 2+ but the signals recover towards their initial height as a Cu:PrP stoichiometry of 2:1 is approached (e.g. those of residue K27 in Fig. 1A). This group of [N,H N ] signals includes those from residues in several stretches of the N-terminal domain, namely: 24-48, 100-108, 125-126. When the experiment was repeated with the protein concentration reduced from 440 µM to around 100 µM (K27 Fig. 1A), signals in this group were not dramatically affected by addition of Cu 2+ , indicating that their behaviour at the higher protein concentration must be caused by an intermolecular process. Thus at sub-saturating levels of Cu 2+ and 440 µM protein concentration, a self-associated form of the prion protein must be at least transiently present. Concentration dependent effects are confined to the disordered N-terminal domain and thus show that it is this section of the protein which self-associates. The most probable cause of this self-association is that metal ions are readily coordinated by histidine sidechains from more than one protein molecule, since the concentration of the protein and the effective concentration of histidine residues in the same chain are likely to be similar. This self-associated protein species causes an acceleration of the relaxation of the nuclei in residues such as K27, either by placing them in closer proximity to the Cu 2+ ion or through localized increases in the correlation time. At a Cu:PrP stoichiometry of 1.8:1, where these signals have fully recovered, we can infer that no significant amount of the self-associated species remains. At this point in the titration the effects on signals that arise solely from proximity to Cu 2+ in the monomer can therefore be clearly distinguished and are illustrated in Fig. 2A.

Chemical shift changes in the PrP(Cu 2+ ) 1 and PrP(Cu 2+ ) 2 complexes

Large chemical shift changes were not observed on formation of either PrP(Cu 2+ ) 1 or PrP(Cu 2+ ) 2 complexes, indicating that the electronic environment for the paramagnetic ion must be close to isotropic in both cases [START_REF] Bertini | Metal centred point-dipole approximation[END_REF]. However, smaller chemical shift changes of less than 0.1 ppm in the 15 N dimension and less than 0.02 ppm in the 1 H dimension are present, confirming the rapid exchange of Cu 2+ between prion protein molecules. The distribution of 15 N chemical shift changes for the PrP(Cu 2+ ) 2 complex is plotted in Fig. 2B. Certain signals change in chemical shift up to a Cu:PrP stoichiometry of ca. 2:1 and then stop. This saturable behaviour shows that these chemical shift changes must be associated with the formation of the PrP(Cu 2+ ) 1 and PrP(Cu 2+ ) 2 complexes. The largest shift changes in this category are seen in signals from residues in the region between H96 and H111 (for example N100, K101 and K110 in Fig. 1B), plus some from residues in the first helix (namely H155 and R156) and at the N terminus (namely K24, R25 and K27). Similar but less substantial changes are seen in signals from other residues close to the N-terminus and a few residues nearby H155 and R156 in the C-terminal domain. The locations of these chemical shift changes do not correlate with the changes in peak height, for example the height of signals like K101 and N100 are not substantially affected by Cu 2+ binding, so these changes are unlikely to be a direct result of proximity to Cu 2+ . The shifts observed in the region between H96 and H111 (Fig. 2B) are most likely reporting a conformational redistribution associated with the formation of a partially ordered loop between these residues. However the lack of substantial 1 H chemical shift dispersion in the PrP(Cu 2+ ) 1 and the PrP(Cu 2+ ) 2 complexes indicates Cu 2+ binding does not bring about a highly ordered, sidechain-immobilized structure in the Nterminal domain of the full length prion protein. Instead, Cu 2+ binding at this second site uses the excess metal coordinating residues present in the ensemble of PrP(Cu 2+ ) 1 complexes to produce a poorly ordered structure.

Interaction of the Cu 2+ binding site with the C-terminal domain

Although the C-terminal domain does not contribute directly to Cu 2+ coordination at the higher affinity site, the heights of certain [N,H N ] NMR signals from the C-terminal domain are substantially reduced in the PrP(Cu 2+ ) 2 complex. The largest of these reductions map to a region of the protein surface formed by residues close to the Cterminal end of helix1, (H155 and N159) and the nearby loop between the first beta strand and helix 1 (S135 and R136), (Fig. 4A). The reductions can be explained if this area of the C-terminal domain surface comes in close proximity to the section of the Nterminal domain that coordinates Cu 2+ . An alternative explanation, where these reductions in peak height were due to direct weak Cu 2+ binding at a site on the C-terminal domain can be eliminated -In this scenario the reductions in signal height would be expected to become more dramatic after the higher affinity Cu 2+ binding was saturated, in the same way as is seen at the S231 [N,H N ] signal (Fig. 1A). This does not happen and instead the changes stop at the 2:1 point of the titration showing that they must be related to the saturable higher affinity binding. Chemical shift changes are also observed for the same section of helix 1 (Fig. 2B), arising either from the interaction between domains forming in a Cu 2+ dependent fashion, or from Cu 2+ binding to a region of the N-terminus which is already interacting with the C-terminal domain. Strikingly, the changes observed for full length prion protein, PrP 23- 231 , bound to two Cu 2+ ions are very similar to the changes we observe for the truncated construct PrP 91-231 bound to a single Cu 2+ in the same conditions (Fig. 4B, experiments on this construct are discussed fully in the final results section). Since signals for residues 115 to 134 are not greatly affected by Cu 2+ binding, the sequence responsible for interacting with the C-terminal domain must lie within residues 91-114.

Measurement of Cu 2+ binding affinity

There are two highly specific Cu 2+ binding events in the full-length prion protein that are apparent from the NMR observations. These events can be monitored at much lower protein concentration by measuring the quenching of the intrinsic tryptophan fluorescence, revealing that the two sites have significantly different affinities. In these experiments, the apparent affinities of the two metal binding events are reduced to measurable values by the inclusion of a competitive chelator which competes for free metal ions. With knowledge of the affinity of the competition reagent for the metal, a true dissociation constant for the protein can be calculated from the measured apparent dissociation constant. The affinities of both sites were measured using competition from the amino acid histidine, which forms a Cu 2+ bis-histidinate complex. The affinity of the second of the two Cu 2+ binding sites was measured in the presence of 10 mM histidine.

Under these conditions, fluorescence quenching was linear to a stoichiometry of 1:1, indicating that the first binding event was at the strong binding limit (Throughout, we use "strong binding" to describe a regime where the dissociation constant is much less than the concentration of binding sites and "weak binding" for where it is similar to, or greater than, the concentration of sites. Weak binding is a condition necessary for measuring a dissociation constant in a titration experiment.). In contrast the second binding event had a measurable apparent dissociation constant of 6 µM (Fig. 5A), showing that the affinities of the two binding events are not equal, as might be predicted from the similarity of the coordinating groups in each. To reduce the apparent affinity of the first site to a measurable value it was necessary to use competition with 100 mM histidine. Under these conditions an apparent dissociation constant of 4 µM was measured, with no initial strong binding phase (Fig. 5B). In combination these fluorescence data also show that the first and second binding events are not cooperative, since the data shown in Figures 1A and1B are not compatible with a single dissociation constant process.

The absolute affinity of histidine for Cu 2+ can be used to calculate the affinity at pH 5.5 [START_REF] Jackson | Multiple folding pathways for heterologously expressed human prion protein[END_REF] and using this information we can make estimates of the true dissociation constants for the two binding events in the full-length prion protein at this pH. The interpretation of these competition experiments is ambiguous, however, because it is not known if the protein is interacting with a Cu 2+ ion or a Cu 2+ mono-histidinate complex. If the second binding event in the protein involves a Cu 2+ mono-histidinate complex, log 10 (K d /M) for the Cu 2+ (histidine):protein complex would be -7.0 ± 0.4, whilst if the protein is binding a free Cu 2+ ion then log 10 (K d /M) = -10.8 ± 0.4 (The K d values calculated here are K d values at pH 5.5. To calculate intrinsic K d values it would be necessary to take into account the protonation state of the metal binding site of the protein.). For the first binding event, the apparent dissociation constant translates to a true K d value of log 10 (K d /M) = -8.2 ± 0.5 for binding of a Cu 2+ mono-histidinate complex, or to log 10 (K d /M) = -13.1 ± 0.5 for binding of a free Cu 2+ ion. Despite the ambiguities, it can be concluded that the affinities of the two Cu 2+ binding events at pH 5.5 are both in the sub-micromolar range and are at least an order of magnitude apart.

Identification of Cu 2+ coordinating groups in PrP 91-231

The integrated nature with which all of the histidine sidechains of the N-terminal domain are involved in Cu 2+ coordination in the full-length prion protein, is somewhat surprising given that a truncated form of the protein without the octapeptide repeat regions (PrP 91- 231 ) is also capable of sub-micromolar affinity Cu 2+ binding [12]. In PrP 91-231 , the Cu 2+ binding site was proposed to involve H96 and H111 on the basis of broadening of NMR signals from backbone NH moieties and X-ray absorption data [12,[START_REF] Hasnain | XAFS study of the high-affinity copper-binding site of human PrP91-231 and its low-resolution structure in solution[END_REF]. To investigate the relationship between the two Cu 2+ binding modes, we have carried out further characterization of the PrP 91-231 (Cu 2+ ) 1 complex under equivalent conditions to our investigation of the full-length protein.

The identity of the ligands was determined by following the titration of Cu 2+ bisglycinate into a sample of 15 N, 13 C labeled PrP 91-231 using heteronuclear NMR methods, as above. After addition of 0.25 molar equivalents of Cu 2+ to PrP 91-231 , a specific group of In previous work the affinity of Cu 2+ binding in the PrP 91-231 construct was overestimated. The affinities reported for the interaction of the truncated prion protein constructs PrP 91-231 (and PrP 52-98 ) with Cu 2+ [12] were calculated from apparent dissociation constants measured in the presence of glycine at pH 8, but without taking into account the extent of protonation of the glycine amino group at this pH, which reduces the stability of the glycinate complex and weakens the competition for Cu 2+ . The stepwise dissociation constants for the Cu 2+ bis-glycinate complex at pH 8 are 0.3 and 3 µM, 40 times larger than the absolute dissociation constants for the formation of this complex from fully deprotonated glycine, which were used in the original calculations.

When this is considered, the dissociation constant for free Cu 2+ binding to PrP 91-231 is in the picomolar range, with a log (K d /M) of -10.1. The equivalent value for PrP 52-98 is -10.5.

Discussion

A model of Cu 2+ binding to the full-length prion protein

A model of Cu 2+ binding to the full length prion protein is illustrated in Fig. 7A. The PrP(Cu 2+ ) 1 complex is an ensemble of species in which Cu 2+ coordination is shared between the six histidines of the N-terminal domain and the N-terminal amine, with rapid exchange of ligands at the metal centre. Some fraction of the PrP(Cu 2+ ) 1 species exists as an oligomer, which is likely to simply comprise Cu 2+ ions coordinated by histidine residues more than one protein molecule. The PrP(Cu 2+ ) 2 complex coordinates Cu 2+ through total occupancy of the ligands used in the PrP(Cu 2+ ) 1 ensemble of complexes.

With the present approach it can not be determined which ligands coordinate each Cu 2+ ion, and it is possible that PrP(Cu 2+ ) 2 is also a rapidly exchanging ensemble of species with different coordination modes. Although the formation of two distinct sites would be a simpler model, this can not account for the broadening of NMR signals from all seven coordinating groups by the first addition of Cu 2+ and the observation that no further signals are broadened after the 1:1 point of the titration. In the wild type protein, lacking the three residue addition in the construct used here, coordination by the S20 amine would almost certainly be replaced by the K23 backbone amine, since the basicity of the backbone amines and the effective molarity of the two amino acids are virtually the same.

The backbone amine group in lysine has a pK a of 9.18 and the dissociation constant for formation of a 1:1 Cu 2+ complex is 10 -7.6 M, whilst for serine these values are 9.60 and 10 -7.9 M respectively [START_REF] Jackson | Multiple folding pathways for heterologously expressed human prion protein[END_REF]. The pK a of the sidechain amine in lysine is 10.72, indicating it is far less likely to be involved in metal binding.

Here we have shown that at pH 5.5 the full length prion protein is capable of binding two Cu 2+ ions with coordination from multiple histidine imidazole groups (Fig. 7A). At first sight, this may appear to contradict some other studies that have reported that the protein can bind five or six Cu 2+ ions at pH 7.4 with a coordination involving deprotonated backbone amides [START_REF] Burns | Copper coordination in the fulllength, recombinant prion protein[END_REF] (Fig. 7B), but the crucial difference between the experiments is the pH. The mode of Cu 2+ coordination that occurs at neutral pH, with maximum Cu 2+ occupancy, has been extensively studied. The octapeptide repeats can bind four Cu 2+ ions, with one ion in each repeat unit, and with coordination of each ion by a histidine Copyright 2006 Biochemical Society imidazole Nδ1, two deprotonated backbone amides from adjacent glycine residues, and a carbonyl oxygen [START_REF] Burns | Molecular features of the copper binding sites in the octarepeat domain of the prion protein[END_REF]. Cu 2+ ions can also bind at two additional and independent sites, involving the residues histidine 96 and 111, again with coordination from the histidine imidazole and nearby backbone amides [START_REF] Burns | Copper coordination in the fulllength, recombinant prion protein[END_REF][START_REF] Jones | Probing copper2+ binding to the prion protein using diamagnetic nickel2+ and 1H NMR: the unstructured N terminus facilitates the coordination of six copper2+ ions at physiological concentrations[END_REF]. This brings the total number of sites to six.

However recent studies of peptides, which comprise the four repeats of the octapeptide sequence, have shown that if the peptide binds only a single Cu 2+ ion, then coordination involves multiple histidine imidazole groups [START_REF] Chattopadhyay | The octarepeat domain of the prion protein binds Cu(II) with three distinct coordination modes at pH 7.4[END_REF][START_REF] Valensin | The dimeric and tetrameric octarepeat fragments of prion protein behave differently to its monomeric unit[END_REF]. When Cu 2+ binding in the peptide is saturated, this form of binding is replaced by the four identical sites involving backbone amides. The latter binding sites are very sensitive to reduction in pH because each involves two deprotonated backbone amide groups and the pK a of this group is well above 7.4. In contrast, multiple histidine coordination would be expected to be maintained at lower pH values because the pK a of the histidine group is around 6, and this is indeed what we observe in the full-length protein at pH 5.5.

The peptide studies of Valensin et al. and Chattopadhyay et al., and our own unpublished data, suggest that the multiple histidine Cu 2+ coordination mode we observe in the fulllength protein at pH 5.5 will be similar to the binding mode which occurs at neutral pH at low Cu 2+ occupancy, even though at maximum Cu 2+ occupancy coordination will be very different (Fig. 7B.). Consistent with this proposal EPR investigations also report that, in the presence of one or two molar equivalents of Cu 2+ at pH 7.4, Cu 2+ coordination is different to that which occurs in the Cu 2+ saturated species [START_REF] Burns | Copper coordination in the fulllength, recombinant prion protein[END_REF]. It is further supported by an X-ray absorption study which found evidence that single and multiple imidazole Cu 2+ coordination might both be accessible in the full length protein at neutral pH [START_REF] Morante | Inter-and intra-octarepeat Cu(II) site geometries in the prion protein -Implications in Cu(II) binding cooperativity and Cu(II)mediated assemblies[END_REF].

The affinity of the prion protein for Cu 2+ and the availability of Cu 2+ are the key determinants of whether the prion protein binds Cu 2+ in vivo. The high capacity, backbone amide coordinated form of Cu 2+ binding, which is accessible at pH 7.4, produces a substantial circular dichroism signal, and this has been used to determine that the affinity of these sites is in the low micromolar range [START_REF] Viles | Copper binding to the prion protein: Structural implications of four identical cooperative binding sites[END_REF]. In vivo, virtually all extracellular Cu 2+ is sequestered in complexes with affinities significantly higher than micromolar, for example with amino acids such as histidine or with proteins such as serum albumin, which have affinities in the low nanomolar and low picomolar range, respectively [START_REF] Linder | Extracellular Copper Substituents and Mammalian Copper Transport[END_REF][START_REF] Masuoka | Intrinsic Stoichiometric Equilibrium-Constants for the Binding of Zinc(Ii) and Copper(Ii) to the High-Affinity Site of Serum-Albumin[END_REF]. A micromolar affinity therefore suggests that the cellular prion protein does not bind Cu 2+ in vivo. However, the preference of the prion protein for multiple histidine Cu 2+ coordination at low Cu 2+ concentrations and at lower pH values, shows that this is likely to be the highest affinity form of binding in the protein. The affinity measurements carried out here indicate that this form of coordination has an affinity at least in the nanomolar range and potentially higher. While not conclusively showing that the prion protein is an authentic cuproprotein, it is clear that the multiple histidine mode of Cu 2+ binding is the most likely to be of biological relevance. In addition, the capability for Cu 2+ binding at pH 5.5 indicates that Cu 2+ binding could be maintained during the cycling of the prion protein through acidic endosomal compartments [START_REF] Shyng | A Prion Protein Cycles between the Cell-Surface and an Endocytic Compartment in Cultured Neuroblastoma-Cells[END_REF], which have been postulated to be the site of PrP Sc formation [START_REF] Mayer | Conversion of Alpha-Helices into Beta-Sheets Features in the Formation of the Scrapie Prion Proteins[END_REF].

It should be emphasized that the Cu 2+ dependent oligomerisation we observe is a distinct process from the oligomerisation involved in formation of either PrP Sc or PrP fibrils. We observe association of the disordered N-terminal domain with no eveidence for any structural changes in the globular C-terminal domain. In contrast formation of PrP Sc or PrP fibrils from monomeric PrP C involves a substantial conformational change within residues 90-231, giving an increase in β-sheet type structure [43,[START_REF] Baskakov | The peculiar nature of unfolding of the human prion protein[END_REF] and does not require residues 23-89 [45], which contain the octapeptide repeats. Nevertheless, our work suggests that metal binding may play a role in controlling self-association of PrP in vivo which could influence the rate of formation of other PrP oligomers such as PrP Sc .

Although the Cu 2+ dependent self-association is relatively weak at pH 5.5, requiring protein concentrations in the order of 0.5 mM, at pH 7.4 self-association is likely to be stronger. At this pH we have found that Cu 2+ binding to a peptide comprising the four tandem octapeptide repeats causes self-association with peptide concentrations in the submicromolar range (Wells, M. A. and Jackson, G. S., unpublished data). Also, in vivo, effective concentrations of the prion protein may be vastly elevated above the true concentration because the protein is anchored to the cell membrane and is further Copyright 2006 Biochemical Society localized in cholesterol rich lipid rafts [START_REF] Vey | Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains[END_REF], which may be sufficient to allow Cu 2+ binding to trigger self-association.

The involvement of H96 and H111 in Cu 2+ binding in the full-length protein shows that metal ion occupancy must play a role in determining the conformation of this section of the protein in the PrP C isoform, as has also been shown for PrP Sc [START_REF] Wadsworth | Strain-specific prion-protein conformation determined by metal ions[END_REF]. Given the known importance of this region of the protein for prion propagation, the role of metal ions in the conversion process should be considered. The formation of a single Cu 2+ binding site by histidine residues from throughout the unstructured N-terminal domain, also has possible implications for understanding the mechanism by which a unique group of pathogenic mutations, the octapeptide repeat insertion mutations, cause disease. Most of the pathogenic mutations seen in inherited prion disease occur within residues 89-231 [START_REF] Collinge | Prion diseases of humans and animals: Their causes and molecular basis[END_REF], which form the relatively protease resistant core of PrP Sc and are sufficient to support prion replication and the development of pathology [45,48]. Insertional mutations comprising addition of integral numbers of the octapeptide repeat sequence to the four repeats of the wild type protein are the exception [START_REF] Collinge | Prion diseases of humans and animals: Their causes and molecular basis[END_REF]. The mechanism by which the octapeptide repeat region influences the formation of PrP Sc is unclear, especially since this section of the protein is unfolded and highly mobile in PrP C , and is rapidly digested by proteinase K in PrP Sc [START_REF] Prusiner | Purification and Structural Studies of a Major Scrapie Prion Protein[END_REF]. However the Cu 2+ mediated interaction demonstrated here between the octapeptide repeats and H96 and H111, illustrates that the insertions would cause alterations in metal coordination which have the potential to modulate PrP Sc formation by causing conformational changes within residues 91-231. shows apo-PrP (top), the PrP(Cu 2+ ) 1 ensemble (middle) and PrP(Cu 2+ ) 2 (bottom), which may also be an ensemble of different coordinations involving the same ligands. Note that the assignment of particular combinations of ligands to any one complex or metal centre is arbitrary. The histidine Nε coordination shown is based on an infra-red study of multiple histidine coordination in the octapeptide repeats [START_REF] Miura | Raman spectroscopic study on the copper(II) binding mode of prion octapeptide and its pH dependence[END_REF]. At pH 7.4, with saturating concentrations of copper, the full-length prion protein can bind at least five Cu 2+ ions, each coordinated by a single imidazole and nearby backbone amides (B).

Tables and Figures

pH 5 . 5 [

 55 33]. The overlap in chemical shift of the signals from the octapeptide repeat region meant that these residues were represented by a single set of resonances, each of which was a combination of four or five signals, one from each repeating unit and from a pseudo octapeptide repeat sequence at positions 52-59. The effects of titration with Cu 2+ on the height of the protein's NMR signals fall into four broad categories, of which representative examples are shown in Fig. 1A. Certain signals are almost completely unaffected; others are initially unaffected but are attenuated after ca. 2 molar equivalents of Cu 2+ have been added; a third group show substantial attenuation after addition of sub-stoichiometric quantities of Cu 2+ ; the final group behave similarly to these but begin to recover after ca. 1.4 molar equivalents of copper have been added.

  1 H 15 N HSQC data thus point to all six histidines of the N-terminal domain being involved in the formation of the higher affinity complexes. Importantly, no large changes in behaviour are observed for any of the NMR signals on passing a Cu:PrP stoichiometry of 1:1, suggesting the first and second binding sites are not distinct from Biochemical Journal Immediate Publication. Published on 6 Jul 2006 as manuscript BJ20060458 Copyright 2006 Biochemical Society each other -a point which is discussed in more detail below. The behaviour of signals such as S231 [N,H N ] (Fig. 1A), which are largely protected from attenuation by Cu 2+

  [C,H] NMR signals on titration with Cu 2+ was also measured. Signals completely attenuated by the addition of sub-stoichiometric amounts of Cu 2+ included the [Cβ,Hβ] , [Cδ2,Hδ2] and [Cε1,Hε1] signals of H96, H111 and all of the octapeptide repeat

[

  C,H] signals were completely attenuated, whilst the majority of signals, such as V122, were unaffected by Cu 2+ addition (Fig. 6). The attenuated signals included the [Cβ,Hβ], [Cδ2,Hδ2] and [Cε1,Hε1] resonances of H96 and H111. In addition, the [Cα,Hα] and [Cβ,Hβ] signals of the N-terminal serine, S88, also became unobservable. As in the case of our PrP 23-231 construct, S88 was one of three residues inserted on the N-terminal side of the PrP 91-231 sequence to form a thrombin cleavage site. The total quenching of these signals at sub-stoichiometric Cu 2+ concentrations again shows that exchange of Cu 2+ between PrP 91-231 molecules occurs rapidly within the timescale of the NMR experiment. None of the sidechain signals for other potential ligands (namely Q91, T95, S97, Q98, N100, S103, T107, N108, M109 and M112) or signals for backbone amides exhibited this complete quenching at the first addition of Cu 2+ , thus demonstrating that at pH 5.5 PrP 91-231 coordinates Cu 2+ through the two histidine imidazoles of the N-terminal region and the N-terminal amine.

Figure 1 .

 1 Figure 1. Titration of full length human prion protein with Cu 2+ bis-glycinate monitored by H 15 N HSQC. (A) NMR signal heights for residues K27, M112, T191 and S231. (B) 15 N chemical shift changes for residues N100, K101, K110 and S231. All data is from a titration of 440 µM protein, except the data shown by triangles in the plot for K27, which is from a titration of 110 µM protein.

Figure 2 .

 2 Figure 2. Changes in the backbone [N,H N ] NMR signals induced by Cu 2+ binding to full human prion protein plotted against residue number. The degenerate octapeptide repeat signals are plotted in the order (GG)WGQ(P)HG and no data is presented for the bracketed glycines residues due to signal overlap. Signal height changes are shown, as a fraction of the starting value, for the addition of 1.8 mole equivalents of Cu 2+ bisglycinate (A). The large reductions at residues 24-25, 95-96, 110-112 and the octapeptide repeat HG sequence arise from paramagnetic quenching due to the proximity of these residues to Cu 2+ in the PrP(Cu 2+ ) 2 complex. (B) The 15 N chemical shift changes after addition of 1.8 mole equivalents of Cu 2+ bis-glycinate in ppm.

Figure 3 .

 3 Figure 3. Reductions in the heights of histidine sidechain [Cδ,Hδ] NMR signals induced by proximity to Cu 2+ in PrP23-231 . Plots show the signal height as a fraction of its starting value during titration with Cu 2+ for H111 (A), part of the higher affinity binding site, and H140 (B), which only binds Cu 2+ after higher affinity binding is saturated.

Figure 4 .

 4 Figure 4. The Cu 2+ -bound N-terminal domain contacts the C-terminal domain. The reductions in the heights of [N,H N ] signals in the C-terminal domain of PrP, induced by proximity to the Cu 2+ bound to the N-terminal domain, are illustrated on the backbone structure by a color scale, ranging from dark blue to cyan (0-30% peak height reduction) and cyan to green (30-60% reduction). Data is shown for the full length prion protein loaded with two Cu 2+ ions (A) and the truncated PrP 91-231 construct, loaded with one Cu 2+ ion (B). Residues for which there are no data are coloured grey. Signals from the area of the C-terminal domain that contacts the unfolded N-terminal domain see the largest reductions (coloured green) because they are closer to the Cu 2+ ion bound to the unfolded domain. The unfolded domain of the protein and the Cu 2+ ion bound to it are omitted for clarity.

Figure 5 .Figure 7 .

 57 Figure 5. Cu 2+ binding to full length human prion protein monitored by tryptophan fluorescence quenching. The graphs the tryptophan fluorescence, as a percentage of the starting fluorescence, as Cu 2+ bis-glycinate is titrated into samples of 3.5 µM prion protein at pH 5.5 in the presence of two different competition systems, (A) 10 mM histidine and (B) 100 mM histidine. The point at which one mole equivalent of Cu 2+ hasbeen added to the protein, and the highest affinity site is therefore saturated, is labeled 1:1 in (A). The lines of best fit to the binding equations (described in the materials and methods section) are shown.

Biochemical Journal Immediate Publication. Published on 6 Jul 2006 as manuscript BJ20060458

Biochemical Journal Immediate Publication. Published on 6 Jul 2006 as manuscript BJ20060458

Biochemical Journal Immediate Publication. Published on 6 Jul 2006 as manuscript BJ20060458

Biochemical Journal Immediate Publication. Published on 6 Jul 2006 as manuscript BJ20060458

Biochemical Journal Immediate Publication. Published on 6 Jul 2006 as manuscript BJ20060458 Copyright 2006 Biochemical Society

Gill, S. C. and Vonhippel, P. H. (1989) Calculation of Protein Extinction Biochemical Journal Immediate Publication. Published on 6 Jul 2006 as manuscript BJ20060458

Coefficients from Amino-Acid Sequence Data. Analytical Biochemistry. 182, 319-326

Processing in Lysosomes -the New Therapeutic Target in Neurodegenerative Disease.Lancet. 340, 156-159

Acknowledgments

This work was funded by the U.K. Medical Research Council and the U.K. Biotechnology and Biological Sciences Research Council. The authors thank Clare Trevitt for assistance with protein expression and purification, Matt Cliff and Lilia Milanesi for critical reading of the manuscript, Cameron McLeod and Neil Bramall for trace metal analysis of NMR samples, and Accelrys for the provision of Felix.

Abbreviations

HSQC, heteronuclear single quantum coherence; K d , dissociation constant; PrP, prion protein; PrP C , cellular PrP Sc , pathogenic (scrapie) isoform; Prion protein constructs will be referred to as PrP x-y with the residues they are composed of given as a superscript, e.g. PrP 23-231 for full length prion protein; PrP(Cu 2+ ) 1 and PrP(Cu 2+ ) 2 , complexes of the full-length prion protein with one and two Cu 2+ ions respectively; 2D 1 H 15 N HSQC and 2D 1 H 13 C HSQC crosspeaks will be described by giving the two coupled nuclei which produce the crosspeak in brackets, e.


Silvia Belem-Gonçalves

Pascale Tsan

Jean-Marc Lancelin

Tito L M Alves

Vera M Salim

Françoise Besson
email: f-besson@univ-lyon1.fr

Interfacial Behaviour of Bovine Testis Hyaluronidase

Keywords: Bovine testis hyaluronidase, hyaluronic acid, Langmuir film, FTIR spectroscopy FTIR, Fourier Transform Infrared, GPI, glycosylphosphatidylinositol, Ks, surface elasticity modulus

SYNOPSIS

The interfacial properties of bovine testicular hyaluronidase were suggested by demonstrating the association of hyaluronidase activity with membranes prepared from bovine testis. Protein adsorption to the air/water interface was investigated using surface pressure-area isotherms. Whatever the way to obtain interfacial films (protein injection or deposition), the hyaluronidase exhibited a significant affinity for the air/water interface. The isotherm obtained 180 min after protein injection into a pH 5.3 subphase was similar to the isotherm obtained after spreading the same amount of protein on the same subphase, indicating that bovine testicular hyaluronidase molecules adopted similar arrangement and/or conformation at the interface. Increasing the subphase pH from 5.3 to 8 resulted in changes of the protein isotherms. These modifications, which could correspond to the small pH-induced conformational changes observed by FTIR spectroscopy, were discussed in relation with the pH influence on the hyaluronidase activity. Adding hyaluronic acid, the enzyme substrate, to the subphase tested the stability of the hyaluronidase interfacial properties. The presence of hyaluronic acid in the subphase did not modify the protein adsorption and allowed the substrate binding to a preformed film of hyaluronidase at pH 5.3, the optimal pH for the enzyme activity. Such effects of hyaluronic acid were not observed when the subphase was constituted of pure water, medium where the enzyme activity was negligible. These influences of hyaluronic acid were discussed in relation with the modelled structure of bovine testis hyaluronidase where a hydrophobic region was supposed to be at the opposite of the catalytic site.

INTRODUCTION

Hyaluronidases are present in many tissues and organs (such as kidney, liver, skin, testes and uterus), as well as in venom (bees, wasps or cobra) [1]. These enzymes are capable of hydrolysing randomly hyaluronic acid [START_REF] Tranchepain | A complete set of hyaluronan fragments obtained from hydrolysis catalyzed by hyaluronidase: Application to studies of hyaluronan mass distribution by simple HPLC devices[END_REF], one of the most abundant constituents of the extracellular matrix [START_REF] Toole | Hyaluronan: from extracellular glue to pericellular cue[END_REF][START_REF] Laurent | Hyaluronan[END_REF]. They also act on chondroitin or chondroitin sulphates [START_REF] Csoka | Hyaluronidases in tissue invasion[END_REF][START_REF] Saitoh | Enzymatic reconstruction of glycosaminoglycan oligosaccharide chains using the transglycosylation reaction of bovine testicular hyaluronidase[END_REF][START_REF] Takagaki | Enzymatic reconstruction of a hybrid glycosaminoglycan containing 6-sulfated, 4-sulfated, and unsulfated N-acetylgalactosamine[END_REF][START_REF] Takagaki | Enzymatic reconstruction of dermatan sulfate[END_REF]. Hyaluronidases are implicated in many biological functions, including allergy [START_REF] Kakegawa | Inhibitory effects of hydrangenol derivatives on the activation of hyaluronidase and their antiallergic activities[END_REF], inflammation [START_REF] Szary | Indomethacin as inhibitor of hyaluronidase[END_REF][START_REF] Feinberg | Hyaluronate in vasculogenesis[END_REF][START_REF] West | The effect of hyaluronate and its oligosaccharides on endothelial cell proliferation and monolayer integrity[END_REF][START_REF] Horton | Hyaluronan fragments synergize with interferon-gamma to induce the C-X-C chemokines mig and interferoninducible protein-10 in mouse macrophages[END_REF], migration of cancer cells [START_REF] Cameron | Ascorbic acid and cancer: a review[END_REF] and microvascular permeability [START_REF] Sunnergren | The effects of hyaluronidase on interstitial hydration, plasma protein exclusion, and microvascular permeability in the isolated perfused rat heart[END_REF].

Six human hyaluronidases have been to date cloned [START_REF] Csoka | The six hyaluronidase-like genes in the human and mouse genomes[END_REF], and three of them have been expressed in different cell lines, but, due to difficult isolation and/or purification, the enzymes have not been produced in enough amounts for enzymatic characterisation and structural determination. Moreover bovine testicular hyaluronidase, which is commercially avaible, has been chosen as a model for several studies. Thus, the hyaluronan hydrolysis catalysed by this enzyme has been studied by investigating the hydrolysis kinetics at pH 5 and 37 °C [START_REF] Asteriou | Inhibition of hyaluronan hydrolysis catalysed by hyaluronidase at high substrate concentration and low ionic strength[END_REF]. The authors demonstrated that the hyaluronan hydrolysis by hyaluronidase was inhibited at high substrate concentration and low ionic strength and suggested the formation of non-specific complexes between hyaluronan and hyaluronidase based on electrostatic interactions. Furthermore Monte Carlo simulation of the mechanism of bovine testicular hyaluronidase reaction showed that condensation reaction is much weaker than the transglycosylation reaction but contributes to product distribution at the final stage of the reaction, preventing complete hydrolysis of the substrates [18].

Even if bovine testicular hyaluronidase and the distantly related bee venom hyaluronidase are well known representatives of the eukaryotic enzymes, structural information is only available for the bee venom hyaluronidase [START_REF] Markovic-Housley | Crystal structure of hyaluronidase, a major allergen of bee venom[END_REF]. This is the reason why we were interested in the characterisation of bovine testicular hyaluronidase. In this work, we looked for interfacial properties of the bovine testicular hyaluronidase because we demonstrated that, in a bovine testis homogenate, a significant amount of the hyaluronidase activity could be recovered associated with membranes. We then modelled the bovine testicular hyaluronidase by using crystal structure of the bee venom hyaluronidase in order to localize the hydrophobic amino acids on the surface of the molecule. Thus the suspected interfacial properties of the protein were studied by using the commercial bovine testicular hyaluronidase and the ability of this protein to form films at the air-water interface in different conditions were investigated. The pHsensitive interfacial properties of the enzyme were compared with the pH-sensitive enzymatic activity of the protein on hyaluronic acid as well as with the pH-induced conformational changes evidenced by FTIR spectroscopy. Finally the influence of hyaluronic acid on the interfacial properties of the bovine testicular hyaluronidase was studied.

MATERIALS and METHODS

Materials

Hyaluronic acid from human umbilical cord and hyaluronidase from bovine testis (type IV-S) were purchased from Sigma-Aldrich and deuterated water (²H 2 O) from Merck.

All organic solvents were analytical grade.

Isolation of bovine testis membranes

Total membranes were prepared as previously described for bovine kidney [START_REF] Bonnin | Protein and lipid analysis of detergent-resistant membranes isolated from bovine kidney[END_REF] with minor modifications. Bovine testis tissue was cut into small pieces and homogenized in 10 mM Tris buffer, pH 7.4 containing 150 mM NaCl (Tris buffer) and 0.25 M sucrose with pepstatin and leupeptin (0.25 mM each). The homogenate was centrifuged at 150,000 g for 30 min at 4°C. The pellets containing total membranes were further washed three times at 4°C with the same buffer and centrifuged at 150,000 g as above.

Protein and enzyme assays

Protein concentrations were determined by the method of Bradford [START_REF] Bradford | A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] using bovine serum albumin as standard. Hyaluronidase activity was measured in a 100 mM phosphate buffer pH 5.3 containing 150 mM NaCl as previously described [START_REF] Borders | Purification and partial characterization of testicular hyaluronidase[END_REF]. One enzyme unit hydrolyzes half of the hyaluronic acid present in the assay during the 10 min reaction time. Alkaline phosphatase activity was measured at 37°C in a 25 mM glycine buffer pH 11 containing p-nitrophenyl phosphate as substrate. Release of pnitrophenolate was followed at 420 nm. One enzyme unit hydrolyzes one µmole pnitrophenyl phosphate /min [START_REF] Bonnin | Protein and lipid analysis of detergent-resistant membranes isolated from bovine kidney[END_REF].

Monolayer technique

All experiments were performed at a constant temperature of 21.0 ± 0.1 °C. The film balance was built by R&K (Riegler & Kirstein, Wiesbaden, Germany) and equipped with a Wilhelmy-type surface-pressure measuring system as previously described [START_REF] El Kirat | Role of calcium and membrane organization on phospholipase D localization and activity. Competition between a soluble substrate and an insoluble substrate[END_REF].

The subphase buffer used was a 100 mM phosphate buffer containing or not 150 mM NaCl at different pH. In all experiments, the subphase was continuously stirred with a magnetic stirrer spinning at 100 rpm. In order to measure the hyaluronidase adsorption at constant surface area, the experiments were performed on a small Teflon dish (diameter, 3 cm) with a subphase volume of 7 mL. The enzyme was injected in the subphase at a final concentration of 1.4 µg/mL. Hyaluronidase adsorption at the airwater interface, measured by tensiometry, was followed as an increase in surface pressure. The π-A isotherms of the protein were measured either after deposition or after injection of a known quantity of hyaluronidase either on or into the subphase buffer of the Langmuir trough (dimensions: 165 cm² and 120 mL subphase). After 15 min, the surface was compressed at a velocity of 6 cm²/min. The adsorption times indicated on the compression-decompression-recompression π-A isotherms correspond to the cumulated adsorption time at zero surface pressure. Indeed, we consider that the protein is weakly adsorbed at the interface during the decompression. Surface elasticity moduli were calculated from the pressure-area data obtained from the monolayer compressions using the following equation: Ks = -Adπ/dA, where A is the molecular area at the indicated surface pressure π.

FTIR spectroscopy measurements

Infrared data were acquired, as previously described [START_REF] Bonnin | A FTIR spectroscopy evidence of the interactions between wheat germ agglutinin and N-acetylglucosamine residues[END_REF], with a Nicolet 510M FTIR spectrometer continuously purged with dry filtered air (Balston regenerating desiccant dryer). Spectra were recorded at 25°C with 128 interferograms and Fourier transformed.

The resolution was 4 cm -1 . 20 µl of a hyaluronidase solution at 10 mg/mL, freshly prepared in a 100 mM phosphate 2 H 2 O buffer containing 150 mM NaCl at different pH, were deposited into a CaF 2 cell. The final spectrum was fitted by the mean of a curve fitting program computing the Lorentzian band shape components in the amide I region.

The wavenumber position of each component band was fixed while the width and the height varied in order to obtain the best fit. The wavenumber position and the number of component bands were determined by using the second derivative spectra. After decomposition, the percentage of each component band was calculated as 100 (Ax/At), where Ax is a component band area and At is the amide band area. IR difference spectra were obtained by using the intensity of the characteristic Tyr band at 1515 cm -1 as reference for the hyaluronidase concentration.

Modelling of the three-dimensional structure of bovine testicular hyaluronidase

Bovine testicular hyaluronidase (sequence accession code Q7YS45; [START_REF] Chowpongpang | Cloning and characterization of the bovine testicular PH-20 hyaluronidase[END_REF]) were modelled with version 4.0 of the MODELLER program [START_REF] Sali | Comparative protein modelling by satisfaction of spatial restraints[END_REF] using the hyaluronidase from bee venom, the closest three-dimensional structure available (1FCU Protein Data Bank accession code; [START_REF] Markovic-Housley | Crystal structure of hyaluronidase, a major allergen of bee venom[END_REF]) and the sequence alignment shown in Fig. 1.

RESULTS

Hyaluronidase from bovine testis is membrane-bound

During assays for preparation of hyaluronidase from bovine testis, an unexpected result was obtained. Indeed a testicular homogenate was prepared and separated by classical centrifugation at 150,000 g to obtain a soluble protein supernatant and a total membrane pellet. Only about 50% of the hyaluronidase activity of the homogenate were recovered in the soluble protein fraction. To test the efficiency of the membrane preparation, the activity of alkaline phosphatase, a GPI-anchored membrane marker [START_REF] Bonnin | Protein and lipid analysis of detergent-resistant membranes isolated from bovine kidney[END_REF], was titrated in parallel with the activity of hyaluronidase. About 80% of the alkaline phosphatase activity of the homogenate were recovered associated with membranes, showing a good separation of the membranes. Furthermore three washings of total membranes with buffer did not induce a release of both enzymes. Since it had been demonstrated that the bovine testicular hyaluronidase does not possess a lipidic modification [START_REF] Meyer | The soluble hyaluronidase from bull testes is a fragment of the membrane-bound PH-20 enzyme[END_REF], its membrane association could be due to amphiphilic properties.

Hyaluronidase modelling

We then looked for the repartition of hydrophobic amino acids (potent candidates to induce amphiphilic properties of proteins) on the surface of the bovine testicular hyaluronidase molecule after constructing the 3D model of this enzyme on the base of the known crystal structure of bee venom hyaluronidase [START_REF] Markovic-Housley | Crystal structure of hyaluronidase, a major allergen of bee venom[END_REF]. The sequence alignment used is given in Fig. 1 showing that there are an N-terminal region (corresponding to 12 amino acids) and a C-terminal region (corresponding to about 100 amino acids) which were not be modelled. Fig. 1 also shows the presence of several groups of hydrophobic amino acids along the sequence and Fig. 2 indicates the repartition of the hydrophobic amino acid residues on the surface of the modelled molecule of testicular hyaluronidase.

The top view shows the presence of a hydrophobic patch around the N-and C-terminal regions of the testicular enzyme and the lateral view shows that this hydrophobic regions are located more or less at the opposite of the catalytic site containing two conserved negatively charged amino acids.

Hyaluronidase adsorption at the air-water interface

The hyaluronidase amphiphilic properties suggested by its membrane association and its modelling led us to investigate possible interfacial properties of the protein. This was first studied by determining the adsorption of protein at the air-water interface. The same amount of protein (12 µg) was injected into different pH subphases, giving a final enzyme concentration of 1.4 µg/mL. The adsorption was monitored by measuring the surface pressure at constant area as a function of time. When injected into the subphase at pH 5.3 (Fig. 3), a lag time of about 20 min was necessary to detect the protein at the interface. Then the surface pressure increased gradually until a maximum was reached after about 70 min, indicating that a stable monolayer was formed at the interface.

Varying the pH subphase did not significantly modify the kinetic of the protein adsorption (Fig. 3).

If our data are compared with the air-water interface adsorption of the amyloid precursor protein (APP), which has been shown to exhibit interfacial properties [START_REF] Lahdo | The amyloid precursor protein interacts with neutral lipids[END_REF], it appears that, at similar concentrations, hyaluronidase and APP show similar adsorption kinetic. This indicates that hyaluronidase effectively exhibits interfacial properties.

Hyaluronidase isotherms

Then the adsorption of the protein at the air-water interface was followed by measuring successive π-A isotherms after injection of 12 µg of hyaluronidase into the trough subphase buffer to reach a final concentration of 0.1 µg/mL. The trough surface was compressed after various delays to obtain π-A isotherms presented in Fig. 4A. The molecular area at each surface pressure increased with increasing time, indicating that the protein was adsorbed at the air-water interface [29][START_REF] Gicquaud | Adsorption of actin at the air-water interface: a monolayer study[END_REF]. The protein adsorption was not measured over 180 min since the isotherm obtained after that time was similar to that obtained when the 12 µg of protein was deposited onto the subphase buffer, indicating that, after a 180 min delay, roughly all the protein had reached the interface (Fig. 4A).

The surface elasticity modulus (Ks) of the last π-A isotherm was represented against the surface pressure in Fig. 4B. The variation of Ks during the compression exhibits three phases. During the first phase, the Ks increased linearly until the surface pressure reached 5 mN/m. For pressures between 8 and 12 mN/m, the Ks increase was lower and stabilised and for pressure higher than12 mN/m the Ks decreased.

Influence of the buffer composition on the hyaluronidase isotherm

We were then interested in the influence of pH on the interfacial properties of the hyaluronidase. The protein was dissolved in the corresponding subphase buffer and spread at the air-water interface. Measuring π-A isotherms were after spreading the protein on different buffered subphases tested the influence of pH on the interfacial properties of hyaluronidase (Fig. 5). The hyaluronidase isotherms were shifted to lower molecular area at pH 5.3 and pH 6.2 as compared with pH 7.2 and pH 8. These shifts decreased during the compression of the hyaluronidase monolayer and at the highest pressures, all the curves tend to the same value. Similar experiments were performed with hyaluronidase solubilized in water with a water subphase (Fig. 5). In these conditions the shift toward higher molecular area is observed all along the isotherm.

Influence of hyaluronic acid on the hyaluronidase adsorption

The absence of interfacial properties of hyaluronic acid was first tested. Hyaluronic acid was injected at 8.3 µg/mL into pH 5.3 buffer or water subphases. Thirty minutes after the injection and at several 30-min intervals, the interface was compressed and the pressure was measured during the compression. No variation of the pressure was observed whatever the subphase or the waiting time, indicating the absence of interfacial property for the hyaluronidase substrate.

The influence of hyaluronic acid on the protein ability to reach the air-water interface was then tested. Successive π-A isotherms were measured after injecting 0.1 µg/mL hyaluronidase into pH 5.3 buffer subphase containing 3.3 µg/mL hyaluronic acid and were compared with those obtained in the absence of enzyme substrate. For each adsorption delay, isotherms obtained with or without hyaluronic acid were pratically superposed. Increasing hyaluronidase concentration up to 0.2 µg/mL allowed the detection of small differences in the adsorption kinetics as seen in (Fig. 6).

Similar experiments were performed with hyaluronidase solubilized in water and injected at 0.2 µg/mL in a water subphase containing, or not, 3.3 µg/mL hyaluronic acid. Successive π-A isotherms were measured during the protein adsorption and the pressures induced by the protein adsorption at an area of 20 cm² were plotted versus the adsorption delays (Fig. 6). In the absence of hyaluronic acid, the protein in water subphase reached the interface more slowly than it did at the same concentration in pH 5.3 subphase. In the presence of hyaluronic acid, the protein in water induced no significant pressure during interface compression, indicating that the protein remained in the subphase.

Adsorption of hyaluronic acid on the hyaluronidase monolayer

We then tested if the interaction between the enzyme and its soluble substrate would destabilise hyaluronidase films at the air-water interface. So the hyaluronidase was spread at the surface of the pH 5.3 buffer subphase containing different concentrations of hyaluronic acid (1.7 or 3.3 µg/mL) and the isotherms were measured 30 min after the protein deposition. For the two hyaluronic acid concentrations, the isotherms measured in the presence of hyaluronic acid were compared to the isotherm measured in its absence (Fig. 7A). In all the cases, the isotherms measured with hyaluronic acid shifted toward higher molecular area as compared with the isotherm without hyaluronic acid.

These shifts increased with increasing the hyaluronic acid concentration. Furthermore, when the isotherms were measured 60 min after the injection of hyaluronic acid no significant changes were observed as compared with the isotherms obtained 30 min after the injection (data not shown). The surface elasticity moduli (Ks) of the π-A isotherm before and after hyaluronic acid injection were represented against the surface pressure in Fig. 7B. For the lowest surface pressures (until about 7 mN/m), Ks were increased in the presence, confirming that the interaction between the hyaluronidase and its substrate.

The influence of the subphase on the interaction between hyaluronic acid and hyaluronidase was then tested. When the pH of the buffer subphase was increased from 5.3 to 8 and the protein was dissolved in the corresponding buffer, the isotherm shifts toward higher molecular area were still observed in the presence of hyaluronic acid (data not shown). On the contrary, such a shift of the isotherm toward higher molecular area was not observed when the protein dissolved in water was spread on a water subphase (Fig. 7A).

FTIR spectroscopy studies of the secondary structure of hyaluronidase

The differences observed in the interfacial properties of the enzyme with the wide variations of pH, as well as interaction with hyaluronic acid, may influence the conformation of the bovine hyaluronidase. Indeed some amino acid residues may change their local charges and potentially flexible hydrophobic segments buried in the molecule may move to the surface of molecule during its movement to the interface. This is the reason why FTIR spectroscopy was used to look for pH-induced changes in the conformation of hyaluronidase.

The ²H 2 O buffer-subtracted FTIR spectrum of hyaluronidase at pH 5.3 (p²H 5.5) and its deconvolution are shown in Fig. 8. Amide I bands, at 1700-1610 cm -1 , are principally C=O stretching vibrations with some N-H bending and C-H stretching vibrations while amide II bands, at 1575-1480 cm -1 , are principally N-H bending vibrations with some CN stretching vibrations. The remaining intense amide II component band at 1545 cm -1 pointed out an incomplete NH/N²H exchange. This incomplete deuteration corresponds to N-H residues buried in the hydrophobic regions of the protein since increasing the incubation time in the ²H 2 O buffer did not modify the amide II band intensity. The deconvolution of the amide I bande gives four component bands at 1634, 1650, 1657 and 1675 cm -1 . The 1634 cm -1 band associated with the 1675 cm -1 band corresponds to intramolecular β-sheets, although overlap from β-turns can also contribute to this last band. The 1657 cm -1 band in ²H 2 O is usually produced by α-helices but it cannot be excluded to originate from turns or 3 10 -helices. The 1650 cm -1 band in ²H 2 O corresponds to unordered structures. Besides, it is well known that some amino acid side chain groups absorb in amide I and II regions [START_REF] Barth | The infrared absorption of amino acid side chains[END_REF]. The 1515 cm -1 band, corresponding to Tyr residues, was easily recognized while the bands at 1585 and 1565 cm -1 might correspond to Asp and Glu COO -residues. The four amide I component bands at 1675, 1657, 1650 and 1634 cm -1 , represent respectively 6, 20, 33 and 41% of the original band.

Then, in order to explain the pH-sensitivity of the hyaluronidase, we looked for hypothetical pH-induced conformational changes of this protein. ²H 2 O buffer-subtracted FTIR spectra of hyaluronidase with increasing pH show a slight decrease in the maximum of the amide I band (from 1643 cm -1 at pH 5.3, to 1642 cm -1 at pH 6.2, 1641 cm -1 at pH 7.2 and 1640 cm -1 at pH 8) and a decrease in intensity of the amide II band, pointing out a NH/N²H exchange increase (Fig. 9A). The pH-induced conformational changes of hyaluronidase could be better seen on the IR difference spectra, obtained by subtracting the FTIR spectrum of hyaluronidase at pH 5.3 to the FTIR spectrum at another pH (Fig. 9B). When pH varied from 6.2 to 8, the negative bands of the IR difference spectra at 1657 cm -1 , corresponding to α-helices, decreased in intensity while the positive bands at 1625 cm -1 , corresponding to intermolecular β-sheets, increased. This indicates that intermolecular β-sheets appeared at the expense of α-helices. At pH 8, the positive peak of the IR difference spectrum at 1625 cm -1 involved 2-3 % of the amino acids of the protein, meaning that about 11 amino acids were implicated in the formation of intermolecular β-sheets. Furthermore the FTIR spectrum of hyaluronidase measured in non-buffered ²H 2 O was compared with the spectrum measured at pH 5.3 and the resulting IR difference spectrum was similar to that obtained by subtracting the FTIR spectrum at pH 5.3 to the FTIR spectrum at pH 6.2 (data not shown).

DISCUSSION

Sperm and testicular hyaluronidases have the same amino acid sequence but differ by their post-translational modification [START_REF] Meyer | The soluble hyaluronidase from bull testes is a fragment of the membrane-bound PH-20 enzyme[END_REF]. Indeed the sperm hyaluronidase possesses a GPI anchor while the testicular hyaluronidase does not. In this work, we showed that the total membranes, obtained by classical centrifugation at 150,000 g of a bovine testis homogenate, contained hyaluronidase and that three washings of total membranes with buffer did not induce a release of the enzyme. This unexpected membrane association could be explained if bovine testicular hyaluronidase possesses amphiphilic properties.

In order to test this hypothesis, the presence of surface hydrophobic areas at the surface of the bovine testicular hyaluronidase molecule was estimated after modelling the protein with the crystal structure of the bee hyaluronidase. A hydrophobic region, more or less at the opposite of the catalytic site, was evidenced at the surface of the bovine testicular hyaluronidase molecule. This region could be responsible for the amphiphilic properties of the protein but it must be remembered that the modelling did not involved all the amino acids of the protein. Indeed the non-modelled C-terminal peptide part, which is not significantly enriched in hydrophobic amino acids, could mask the hydrophobic region.

One of the factors contributing to the affinity of the protein for the interface is the presence of hydrophobic areas on the surface of the protein [START_REF] Wierenga | Protein exposed hydrophobicity reduces the kinetic barrier for adsorption of ovalbumin to the air-water interface[END_REF]. The monolayer technique, which is a powerful method for assaying the surface activities of proteins [START_REF] Graham | Proteins at liquid interfaces[END_REF], was then applied to bovine testicular hyaluronidase. Another important advantage of this method is that its requires only small amounts of proteins. Following the protein adsorption by direct measure of the pressure increase after injection into a small trough shows that bovine testicular hyaluronidase exhibits a significant surface activity.

Measuring successive isotherms after protein injection was the other way chosen to determine the enzyme adsorption at the air/buffer interface in a time-dependent manner.

Furthermore, it was shown that hyaluronidase remains at the interface when spread and the surface pressure developed by the protein after injection, as a consequence of its adsorption at the interface, leads to values similar to those obtained by direct spreading onto the surface. This indicates that, as it has been shown for other proteins [START_REF] Graham | Proteins at liquid interfaces[END_REF][START_REF] Vernoux | Interfacial behaviour of cytoplasmic and mitochondrial creatine kinase oligomeric states[END_REF][START_REF] Angeletti | Surface activity and interaction of StarD7 with phospholipid monolayers[END_REF][START_REF] Carrizo | The amphiphilic character of glycogenin[END_REF], the arrangement adopted by the bovine testicular hyaluronidase at the interface is independent of the method used to form the film. This was confirmed by the similarities in the pressure-dependent variations of surface elasticity modulus (Ks) observed after injection or spreading. Furthermore, the Ks variations suggest that compression induce a marked rearrangement of the organisation and distribution of the protein at the interface. One may consider that the plateau, observed between 8 and 12 mN/m, could be due to equilibrium between the protein desorption and adsorption at the air-water interface. It seems that above 12 mN/m, there was either a protein desorption or a reorganisation of the protein at the air-water interface during the monolayer compression. It is interesting to notice that the maximum value of the surface elasticity modulus (at about 30 mN/m) was obtained at a pressure (10 mN/m) similar to those observed for the phospholipid-hydroperoxide glutathione peroxidase [START_REF] Morandat | Adsorption of a phospholipid-hydroperoxide glutathione peroxidase into phospholipid monolayers at the air-water interface[END_REF] and for the creatine kinase [START_REF] Vernoux | Interfacial behaviour of cytoplasmic and mitochondrial creatine kinase oligomeric states[END_REF].

Several factors (pH, salts and hyaluronic acid concentration as well as the glycosaminoglycan microenvironment of hyaluronidase) are known to affect the enzymatic activity of bovine testicular hyaluronidase [START_REF] Csoka | Hyaluronidases in tissue invasion[END_REF][START_REF] Asteriou | Inhibition of hyaluronan hydrolysis catalysed by hyaluronidase at high substrate concentration and low ionic strength[END_REF][START_REF] Maksimenko | Resistance of dextran-modified hyaluronidase to inhibition by heparin[END_REF]. Increasing the subphase pH from 5.3 to 8 (corresponding to a decrease of the optimal conditions for the enzymatic activity of hyaluronidase) induces small but significant differences in the π-A isotherms. This might reflect pH-induced differences either in the desorption of protein segments or in the short-range intermolecular interactions. Indeed decreasing the pH could decrease the repulsive forces between the protein molecules by neutralising some ionic charges on the protein surface since the pI of the bovine testicular hyaluronidase is about 5 [START_REF] Malmgren | Characteristics of testicular hyaluronidase[END_REF]. Similarly, the larger shift observed in the isotherm measured on water (condition which gives only 7% of the enzymatic activity measured at pH 5.3) could be explained by the absence of salts. Furthermore the variations observed in the isotherms could result from the small structural changes observed by FTIR spectroscopy.

Since hydrophobic interactions on the surface of the hyaluronidase molecule have been previously suggested to play a role during the formation of conjugates between hyaluronidase and glycosaminoglycans [START_REF] Maksimenko | Role of the glycosaminoglycan microenvironment of hyaluronidase in regulation of its endoglycosidase activity[END_REF], we then looked for the surface activity of the protein in the presence of its substrate, the hyaluronic acid. In a first set of experiments, the influence of hyaluronic acid on the hyaluronidase ability to reach the interface was followed by measuring successive π-A isotherms after injection of the protein into a pH 5.3 buffered subphase containing, or not, the enzyme substrate. In these conditions (corresponding to the optimal conditions for the enzymatic activity), hyaluronic acid did not significantly decrease the amount of hyaluronidase detected at the interface. The presence of the catalytic site at the opposite of the hydrophobic region (as suggested by protein modelling) could explain the persistence of the adsorption of the protein in the presence of hyaluronic acid.

In a second set of experiments, injecting the enzyme substrate under a pre-formed film of hyaluronidase on a pH 5.3-buffered subphase followed the influence of hyaluronic acid on the hyaluronidase ability to remain at the air-water interface. Whatever the substrate concentration, the isotherms measured in the presence of hyaluronic acid shifted toward higher molecular area as compared with the isotherm in its absence.

These shifts increased with increasing the hyaluronic acid concentration. This indicates that the presence of the water-soluble hyaluronic acid into the subphase did not induce a delocalisation of the protein from the air-water interface to the bulk subphase (no shift of the isotherm toward lower molecular area). Furthermore, the isotherm shift toward higher molecular area showed that the protein at the interface was still able to interact with the hyaluronic acid, the complex "hyaluronidase-hyaluronic acid" having a higher molecular area than the protein alone. Moreover, the absence of significant changes between the isotherms measured at different times after the hyaluronic acid injection might suggest that the enzyme binding to its substrate occurred faster than the hyaluronic acid hydrolysis by hyaluronidase. The experiment temperature (21°C) which is lower than the physiological temperature could explain this result.

Moreover, the subphase influences the interaction between hyaluronidase and its substrate. When the pH of the buffer subphase varied from 5.3 to 8 (pH range allowing the hydrolysis of hyaluronic acid by the enzyme, even if there is a decrease of enzymatic activity), the presence of hyaluronic acid induces isotherm shifts toward higher molecular area. On the contrary, when the subphase contained only water (condition which did not induce significant hyaluronic acid hydrolysis), such a shift toward higher molecular area was not observed. Furthermore, when hyaluronidase was injected into a water subphase, it reached the interface more slowly than the protein injected into the subphase at pH 5.3, the pI of the protein, and the addition of hyaluronic acid induced the remaining of the protein into the subphase. The hyaluronic acidinduced inhibition of the adsorption of the protein in water could be due either to an increase of the charges at the surface of the molecule or to a partial denaturation of the hyaluronidase, masking the hydrophobic region of the protein.

In conclusion we have shown that bovine testicular hyaluronidase exhibits surface activity depending on the composition of the medium where it was solubilized. Maximum of interfacial properties was observed in conditions corresponding to the maximum of enzymatic activity. At this stage, a deeper knownledge of the influence of the pH as well as a better characterization of the interactions between the protein and hyaluronic acid at the air-water interface, which might influence the 3D structure of the bovine hyaluronidase are necessary. Such studies would need the use of specific interfacial biophysical techniques.

Figure 1 .

 1 Figure 1. Sequence alignment of bovine testis hyaluronidase with bee venom hyaluronidase used for the modelling. HYA_BOVINE refers to bovine testis hyaluronidase (sequence accession code Q7YS45) and HYA_BEE to bee venom hyaluronidase (Q08169). Regions in black boxes correspond to undefined structure in bovine hyaluronidase model (this work) or bee hyaluronidase crystal structure (1FCU Protein Data Bank accession code). Conserved residues of the active site (D112 and E114 in HYA_BOVINE, corresponding to D111 and E113 in HYA_BEE) are shown with bold letters. Hydrophobic amino acids of HYA_BOVINE, identified according to [http://www.inrp.fr/Acces/Biogeo/model3d/chimdata/scripts/globine/aa.htm], are underlined.

Figure 2 .

 2 Figure 2. Hydrophobic residues in the model of bovine testis hyaluronidase (A, B). A: Top view of the N-and C-terminal regions indicating C ter , N ter and hydrophobic amino acids (black area). B: View showing the N-terminal region opposite to the Asp and Glu conserved residues of the active site, as well as hydrophobic amino acids (black area). This modelled structure of bovine testis hyaluronidase did not take account of the amino acids in black boxes for HYA_BOVINE in Fig. 1.

Figure 3 .

 3 Figure 3. Kinetics of surface pressure increases due to hyaluronidase adsorption at the air-water interface at 21°C.

Figure 4 .

 4 Figure 4. Successive π-A isotherms during hyaluronidase adsorption at the air-water interface at 21 °C. A: π-A isotherms measured 15 min (curve a), 35 min (curve b), 65 min (curve c), 125 min (curve d), and 180 min (curve e) after 12 µg-protein injection into pH 5.3 buffer subphase at zero surface pressure or after 12 µg-protein spreading (curve f). B: Monolayer Ks calculated from curve e isotherm and represented as a function of surface pressure.

Figure 5 .

 5 Figure 5. Influence of pH on the hyaluronidase isotherms after 12 µg-protein spreading onto different buffer subphases at 21 °C. Subphases were 100 mM phosphate, 150 mM NaCl buffer at pH 5.3 (full squares), pH 6.2 (open triangles), pH 7.2 (full circles), pH 8 (open diamonds) and pH 5.6 water (full triangles).

Figure 6 .

 6 Figure 6. Kinetic of hyaluronidase adsorption at 21 °C.The pressures induced by the protein adsorption (0.2 µg/mL) at a 20 cm² area (obtained from the π-A isotherms) were plotted versus the adsorption delays. Assays with pH 5.3 buffer (triangles) or pH 5.6 water (circles) subphases containing no (full lines) or 3.3 µg/mL (dotted lines) hyaluronic acid.

Figure 7 .

 7 Figure 7. Influence of hyaluronic acid on preformed hyaluronidase films at 21 °C. A: π-A isotherms of hyaluronidase dissolved in pH 5.3 buffer (full symbols) or in pH 5.6 water (open symbols) and spread onto pH 5.3 buffer or water subphases containing no hyaluronic acid (squares), 1.7 µg/mL (diamonds) or 3.3 µg/mL (triangles) hyaluronic acid. Isotherms were measured 30 min after the protein deposition. B: π-A isotherm-derived Ks of hyaluronidase in pH 5.3 buffer subphases containing no (curve a) or 1.7 µg/mL (curve b) hyaluronic acid.

Figure 8 .Figure 1 Figure 2 A

 812 Figure 8. ²H 2 O buffer-subtracted FTIR spectrum of hyaluronidase at pH 5.3.

Biochemical Journal Immediate Publication. Published on 13 Jun 2006 as manuscript BJ20060485 Copyright 2006 Biochemical Society

Biochemical Journal Immediate Publication. Published on 13 Jun 2006 as manuscript BJ20060485

ACKNOWLEDGEMENTS

S. B. G. was recipient of a Ph.D. fellowship from the Brazilian government (CAPES, Project N° BEX 0801/04-6). We thank A. Kouzayha for helpful discussions.

Original spectrum (full line), best-fitted individual component bands of the amide I and II regions (full lines) and their resulting summation (dotted line). Protein concentration was 10 mg/mL.