Piotr Stefanowicz

Inga Petry-Podgorska

Karolina Kowalewska

Lukasz Jaremko

Mariusz Jaremko

Zbigniew Szewczuk

Electrospray Mass Spectrometry as a Method for Studying the High Pressure Denaturation of Proteins

Keywords: electrospray, high pressure denaturation, hydrogen-deuterium exchange, proteins, secondary structure, ECD; Bioscience

High pressure denaturation of proteins can provide important information concerning their folding and function. These studies require expensive and complicated equipment. In this paper we present a new convenient method for studying high-pressure denaturation of proteins combining deuterium-hydrogen exchange and electrospray mass spectrometry. Application of various values of pressure causes different degrees of protein unfolding resulting in molecules with a different number of protons available for exchange with deuterons. After decompression a protein refolds and a certain number of deuterons is trapped within the hydrophobic core of a refolded protein. Redissolving the deuterated protein in an aqueous buffer initiates the D/H exchange of amides located on the protein surface only, which can be monitored under atmospheric pressure by mass spectrometry. Depending on the degree of deuteration after high pressure treatment, the D/H exchange kinetics are different and indicate how many deuterons were trapped in the protein after refolding. The dependence of this number on pressure gives information on the denaturation state of a protein. The distribution of deuterium along the sequence of a high-pressure-denatured protein was studied the electron capture dissociation (ECD) on a Fourier-transform mass spectrometer, enabling the monitoring of high pressure denaturation with single amino acid resolution.

Introduction

The thermodynamic stability of proteins can provide important biological information concerning their folding and function. High pressure has been long known to change the protein's conformation and, therefore, alter the rate and the equilibrium constants of biological reactions. It exerts an influence on the properties of proteins by the rearrangement and/or destruction of noncovalent bonds, such as hydrogen bonds and hydrophobic and electrostatic interactions, which normally stabilize the tertiary structure of proteins [START_REF] Mozhaev | High pressure effects on protein structure and function[END_REF].Only the native and denaturated structures are well known. There are cases, however, when the intermediate state (a molten globule) is detectable [START_REF] Jonas | High-resolution nuclear magnetic resonance studies of proteins[END_REF]. It has been found that the residual secondary structure of the pressure-assisted, cold-denatured states is very similar to the structure of the early folding intermediates [START_REF] Jonas | High-resolution, high-pressure NMR studies of proteins[END_REF].

High pressure treatment of food or pharmaceutical agents (pascalization) is a new method of sterilization. Some reports suggest that high pressure is not only efficient for the inactivation of bacterial, viral, and fungal pathogens, but may also decrease the infectivity of prions [START_REF] Balny | What lies in the future of high-pressure bioscience?[END_REF]. There are evidences that high pressure can induce the reversible denaturation of some globular proteins [START_REF] Mohana-Borges | Folding of a pressure-denatured model protein[END_REF][START_REF] Nash | Hydrogen-exchange kinetics in the cold denatured state of ribonuclease A[END_REF][START_REF] Zipp | Pressure denaturation of metmyoglobin[END_REF] Pressure is an ideal perturbant in the studies on folding equilibria as it gives valuable thermodynamic information without the need of altering the chemical composition of a solution. High-pressure techniques have been applied in the studies on protein folding and more recently, in the studies on the transition states of that process. Such studies require high-pressure techniques integrated with FTIR, NMR, small-angle X-ray scattering, densitometry, fluorescence, and 4th derivative UV absorbance spectra [START_REF] Yamaguchi | Thermodynamics of unfolding of ribonuclease A under high pressure. A study by proton NMR[END_REF][START_REF] Hoffman | Hydrogen exchange reactions in supercritical media monitored by in situ NMR[END_REF][10][START_REF] Ruan | Unusual effect of high hydrostatic pressure on basic phospholipase A2 from venom of agkistrodon halys pallas[END_REF][START_REF] Vidugiris | Determination of the volume changes for pressure-induced transitions of apomyoglobin between the native, molten-globule, and unfolded states[END_REF]. However, these methods need custom build instruments, specially designed for measurements under high pressure conditions. Such complicated and expensive equipment may limit the availability of those methods.

As the electrospray mass spectrometry (ESI-MS) has become an important tool for protein analysis due to its high specificity and sensitivity, [START_REF] Kaltashov | Mass Spectrometry in Biophysics. Conformation and Dynamics of Biomolecules[END_REF] we decided to apply it in studies on high-pressure denaturation of proteins. A combination of isotopic exchange and mass spectrometry gives additional insight into the protein stability and conformation in a solution [START_REF] Kaltashov | Mass Spectrometry in Biophysics. Conformation and Dynamics of Biomolecules[END_REF]. The hydrogen-deuterium exchange (HDX) is a technique that became one of the major experimental tools to probe both structural and dynamic features of proteins. The replacement of each labile hydrogen atom with deuterium (or vice versa) changes the mass of a protein by one Da.

Since deuteration increases the molecular mass of a protein, mass spectrometry is often used to monitor HDX. In each case the high sensitivity of a contemporary mass spectrometer allows monitoring subtle changes in a very small number of molecules therefore kinetic studies can be carried out. In contrast to other analytical methods, HDX-ESI-MS experiments can be performed successfully even on a mixture of proteins [14,[START_REF] Petry | Conformational stability of six truncated cHMG1a proteins studied in their mixture by H/D exchange and electrospray ionization mass spectrometry[END_REF]. Therefore, the method is very useful for a comprehensive study of compounds with different molecular masses which are difficult to separate by other techniques.

Herein we present a new convenient method for studying high-pressure denaturation of proteins by ESI-MS. We used the method to study the HDX in selected model proteins known to undergo a reversible high-pressure denaturation. As the electrospray mass spectrometric analysis can be performed only under atmospheric pressure, we developed a two-step approach for our purpose (Figure 1). The HDX rate in a protein is closely related to its local environment. In general, a protein in a tightly folded conformation has fewer protons available for deuteration, than the same unfolded protein. Application of various pressures causes different degrees of unfolding and consequently different amounts of protons are available for exchange with deuterons.

After decompression the protein refolds and a certain number of deuterons is trapped within the hydrophobic core of the refolded protein. Redissolving deuterated proteins in an aqueous buffer initiates the D/H exchange (DHX) of deuterons. The exchange rates of the peptide amides' deuterons located on a protein surface are in the range that can be monitored under atmospheric pressure by ESI-MS (Step II in Figure 1). Depending on the degree of deuteration after the first step, the DHX kinetics are different and reveal a number of deuterons trapped in the protein during its refolding. Although the use of the proposed method is limited to proteins that reversibly unfold under high pressure, it provides benefits of study of unpurified proteins or even complex mixtures of proteins, unlike other traditional approaches based on NMR, IR or fluorescence spectroscopy. Therefore, the proposed method may be very useful in the analysis of the folding and the unfolding of proteins in their natural environment.

The measurement of the molecular mass of a deuterated protein provides information on the total number of exchanged amide protons only. The localization of the incorporated deuterons may be achieved by enzymatic cleavage with pepsin followed by chromatographic separation and MS detection of obtained peptides [START_REF] Zhang | Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation[END_REF]. Recently, another method of localization of deuterium atoms in a peptide molecule based on the fragmentation of a protein molecule by electron capture dissociation (ECD) was proposed and discussed [START_REF] Rand | Electron capture dissociation proceeds with a low degree of intramolecular migration of peptide amide hydrogens[END_REF]. Unlike collision induced dissociation (CID), [START_REF] Ferguson | Nonuniform isotope patterns produced by collision-induced dissociation of homogeneously labeled ubiquitin: implications for spatially resolved hydrogen/deuterium exchange ESI-MS studies[END_REF] ECD proceeds with a relatively low degree of hydrogen scrambling, what makes it a method of choice for localization of deuterated amides within apeptide chain. A recent report suggests that this method may be extended to spatially resolved hydrogen exchange experiment on the intact molecule of a protein. This measurement was performed on a ubiquitin molecule by Konermann's group [START_REF] Pan | Electron capture dissociation of electrosprayed protein ions for spatially resolved hydrogen exchange measurments[END_REF]. Herein we performed ECD experiments to reconstruct the distribution of deuterated and undeuterated amide protons along the protein sequence. This paper confirms Konermann's results and is the first one presenting the deuterium distribution within the whole sequence of a protein with a well defined conformation in a solution.

Materials and Methods

Reagents

Deuterium oxide (99.9%D), acetic acid (HPLC grade), formic acid (HPLC grade), acetonitrile (LC-MS grade), ammonium formate (LC-MS grade), hen lysozyme, bovine α-lactalbumin and ubiquitin were purchased from Aldrich.

High Pressure Equipment

The high-pressure denaturation experiments were carried out using a custom-made cylinderpiston type apparatus. The stoppered polypropylene syringe, containing the protein sample was placed in a high-pressure vessel filled with hexane as a transmission medium and compressed (0.1 MPa to 1400 MPa).

High Pressure Experiment Protocol I

The solutions of 1 mg of a protein dissolved in 1 ml of 10 mM ammonium formate in D 2 O (pD 6.9) were incubated at given pressures from 0.1 MPa to 1400 MPa for 30 min at room temperature. The compression and decompression time was approximately 2 min. The samples were frozen in liquid nitrogen immediately after decompression and dried overnight in a freeze-dryer. The DHX experiment under atmospheric pressure was initiated by dissolving dried samples in a 10 mM solution of ammonium formate (pH 6.6) in water. The final concentration of a protein was 1 mg/ml. After a specified DHX time [START_REF] Jonas | High-resolution nuclear magnetic resonance studies of proteins[END_REF][START_REF] Balny | What lies in the future of high-pressure bioscience?[END_REF][START_REF] Yamaguchi | Thermodynamics of unfolding of ribonuclease A under high pressure. A study by proton NMR[END_REF][START_REF] Zhang | Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation[END_REF]30,60,120,240,360, and 480 min) 10 µl of a protein solution was taken and isotopic exchange was quenched with 10 µl of 5% acetic acid that was cooled to approximatelly 0 °C. The sample was immediately analyzed by ESI-MS. Protocol I was applied to lysozyme and αlactalbumin.

High Pressure Experiment Protocol II

The solutions of 0.5 mg of a protein in 0.1 ml of 10 mM ammonium formate in D 2 O were incubated at a given pressure (0.1 MPa to 1400 MPa) for 30 min at room temperature. After decompression samples were immediately diluted with 500 µl of 10 mM ammonium formate (pH 6.6) in water to initiate the DHX under atmospheric pressure. The final concentration of a protein was 0.5 mg/ml. The obtained solution was directly infused into the ion source. Protocol II was applied to lysozyme and ubiquitin.

ESI-MS measurement

The mass spectra were obtained on a Bruker MicrOTOF-Q spectrometer (Bruker Daltonik, Bremen, Germany), equipped with Apollo II electrospray ionization source with ion funnel. The mass spectrometer was operated in the positive ion mode. Mass resolution was 15.000 FWHM. The instrumental parameters were as follows: scan range m/z 400-2300, dry gasnitrogen, temperature 200 ºC, reflector voltage 1300 V, detector voltage 1920 V. The sample in the solution described in High Pressure Experiment Protocol I and II was infused at a flow rate of 3 µL/min. Before each run the instrument was calibrated externally with the Tunemix™ mixture (Bruker Daltonics, Germany) in the quadratic regression mode. The mass accuracy for the calibration was better than 5 ppm. The ECD experiments were performed using an Apex-Qe 7T instrument (Bruker) equipped with the ESI source and a heated hollow cathode dispenser, using standard Bruker ECD settings. The cathode dispenser was heated gradually to 1.7 A. The ECD pulse length was set at 7 ms., and ECD bias was 0.7 V. Analysis and deconvolution of the obtained spectra were carried out with a Biotools (Bruker) software.

Calculation of the extent of deuterium incorporation

The deuterium content of deuterated proteins was determined by the difference in the average mass between deuterated and nondeuterated species. D(r)=M D -M 0 (where D(r) is the number of remaining deuterons, M D is the observed average molecular mass of a deuterated protein sample; M 0 is the observed average molecular mass of a nondeuterated protein sample). D(r) was plotted (Figure 2) as a function of time and the data was fited to the following equation:

D(r)=D 1 exp(-k 1 t)+D 2 exp(-k 2 t)+D 3

(where D 1 -deuterons exchanged with a fast kinetic constant k 1 , D 2 -deuterons exchanged with a lower kinetic constant k 2 , D 3 -deuterons fully protected from the exchange).

Mapping of the exchanged protons in the ubiquitin molecule

Ubiquitin incubated in D 2 O under high pressure was diluted with the 100-fold excess of the ammonium formate (10 mM) aqueous solution. After 60 min of the back exchange, the solution of ubiquitin was brought to pH 3.5 with formic acid, diluted with acetonitrile (1:1) and the ECD spectrum was acquired immediately. The average molecular masses of the corresponding deuterated and undeuterated fragments of ubiquitin (M av ) were calculated using the formula:

M av = Σ(M i A i )/Σ A i
(where M i are the molecular masses for particular isotopic peaks in the isotopic envelope and A i are the abundances of corresponding isotopic peaks). The number of deuterium atoms (D n ) in all of the identified fragments of the c n and (z+1) n type were calculated from the difference in the average mass between nondeuterated and deuterated fragments of ubiquitin.

Results and discussion

A new method of studying high pressure protein denaturation by mass spectrometry was tested using three model proteins -lysozyme, α-lactalbumin, and ubiquitin -that unfold reversibly under high pressure. The CD spectra of those proteins dissolved in ammonium formate buffer measured before and after a high pressure (1400 MPa) treatment (HPT) are exactly the same (not shown in the experimental part). Additionally, the reversibility of high pressure denaturation of proteins is proven by a similarity of the charge state distribution of a protein before and after HPT [START_REF] Babu | The methanol-induced conformational transitions of βlactoglobulin, cytochrome c, and ubiquitin at low pH: a study by electrospray ionization mass spectrometry[END_REF].

To characterize the denaturation degree as a function of pressure, we denatured model proteins in a deuterated solvent (10 mM ammonium formate in deuterium oxide, pD 6.9) by HPT at room temperature. Then the high pressure was released causing the renaturation of a protein. We observed very low temperature changes (∆T less than 2 ºC) during the compression or decompression, suggesting that the thermal effect itself should not influence the protein's stability. The deuterated protein samples were freeze-dried and redissolved in the aqueous buffer at pH 6.6 (protocol I) or, alternatively, diluted 10 times with the aqueous buffer (protocol II). Monitoring of the protein's molecular weight changes enabled us to observe the dependence of the deuteration degree versus pressure (Figure 2). The mass spectrometry analysis of a protein incubated in the deuterated buffer under higher pressure reveals significant changes in the molecular mass of the studied proteins.

Both protocols provide similar conclusions on the tested proteins, although 10% of protein deuterons remain unexchanged in the case of protocol II. The high pressure experiment for this protein was performed according to protocol I. The time course of DHX depends on the HPT of the sample. The sample of the protein incubated under high pressure presents a higher content of deuterium (more than 60 deuterons for the sample incubated under pressure of 1400 MPa), than the protein incubated in the deuterated buffer at the atmospheric pressure (20 deuterons only). Deuterium entrapped inside the molecule of the protein is exchanged relatively slowly (t½ ~ 10h at room temperature, pH 6.6). Changes in the content of deuterium plotted as a function of pressure provide information on the denaturation of the protein under high pressure. The dependences of the number of deuterons trapped in the hydrophobic core of the proteins after the high pressure treatment, followed by the 1 hour backexchange in the aqueous buffer, as a function of the applied pressure (denaturation curve) are presented in Figures 2. b. The curves were fitted to the sigmoidal Boltzmann function (the general form of the equation is: y = (A 1 -A 2 )/[1+exp((x-x 0 )/B) +A 2 ). A similar equation was used by Powell and Fitzgerald [START_REF] Powell | Accuracy and precision of a new H/D exchange and mass spectrometry based technique for measuring the thermodynamic properties of protein -peptide complexes[END_REF] to describe the HDX under denaturing conditions. For lysozyme data were analysed separately in two sets corresponding to the range 0.1-700 and 600-1400 MPa. Pressure below 400 MPa did not influence the content of deuterium in the protein sample. The renaturation of αlactalbumin previously denaturated at the pressure of 600 MPa entraps approximately 20 deuterons more than the protein denaturated at 400 MPa. A further increase of pressure had very little effect on the number of additional deuterons trapped in the hydrophobic core of the protein. The transition midpoint of α-lactalbumin is close to 480 MPa. This value is in a good agreement with the data reported previously by Jonas [START_REF] Jonas | High-resolution nuclear magnetic resonance studies of proteins[END_REF]. The application of a combination of advanced high-resolution nuclear magnetic resonance techniques and high-pressure measurements, evidences that α-lactalbumin is completely unfolded under the pressure of 500 MPa, whereas it forms a molten globule state below 400 MPa.

The corresponding experiment for lysozyme was performed using both protocol I and II. In both cases the denaturation curves are similar and the denaturation occurs in two steps. The changes in the yield of ultraviolet fluorescence emission of lysozyme with pressure permitted the detection of two distinct steps of its denaturation, at 400-800 MPa and 800-1100 MPa (transition 1 and 2, respectively) [START_REF] Li | Plurality of pressure-denatured forms in chymotrypsinogen and lysozyme[END_REF].

Lysozyme contains 129 amino acid residues that fold into two structural domains: the α-domain consists of four α-helices and a 3 10 helix; the β-domain includes a long triplestranded β-sheet, a short double stranded β-sheet, a 3 10 helix, and a long loop [START_REF] Blake | Structure of Hen Egg-White Lysozyme: A Three-dimensional Fourier Synthesis at 2 Å Resolution[END_REF].The dependence of a number of deuterons trapped within the protein molecule as a function of pressure suggests the existence of two stages of denaturation: the first in the range of 0.1-600 MPa and the second in the range of 600-1400 MPa. The presented plot (Figure 2.II.b and Figure 2.III.b) suggests a two-stage HPT unfolding of lysozyme that reflects transition stages 1 and 2, as suggested by Li [START_REF] Li | Plurality of pressure-denatured forms in chymotrypsinogen and lysozyme[END_REF]. Two transition midpoints were observed, at about 400 and 850 MPa, for the experiment performed according either to protocol I or II.

The pressure values causing appearance of transition stages 1 and 2 of lysozyme are in a good agreement for protocols both I and II. The ∆M 1 and ∆M 2 values (marked in Figure 2.II.b and Figure 2.III.b) represent the number of amide protons exchanged during the first and the second stage. The protons must be involved in the formation of the internal hydrogen bonds in the native structure and they are exposed toward the solvent in the transition stages 1 and 2. The ∆M 1 and ∆M 2 values are 11 and 42 respectively and the difference between them is 31. These results may lead to the conclusion that 11 amino acid residues in the protein interior are exposed to the solvent in the intermediate state, whereas additional 31 residues are exposed at a pressure higher than 1000 MPa. On the basis of the above results we can not point out the amino acid residues that undergo the isotopic exchange in the transition stages 1 and 2. However, Samarasinghe et al. [START_REF] Samarasinghe | High-resolution NMR study of the pressure-induced unfolding of lysozyme[END_REF] suggested that the α-domain undergoes denaturation at a lower pressure, whereas denaturation of the β-domain requires a higher pressure (above 500 MPa).

The pressure denaturation of ubiquitin was performed using protocol II. Ubiquitin shows a very simple denaturation curve with the midpoint at approx 500 MPa. (Figure 2.IV.b) The data obtained by Herberhold and Winter, [START_REF] Herberhold | Temperature-and pressure-induced unfolding and refolding of ubiquitin: A static and kinetic fourier transform infrared spectroscopy study[END_REF] using high pressure FT-IR are in a good agreement with our results. The pressure dependence of the FT-IR spectra of deuteriumexchanged ubiquitin at pD 7.0 and 21 °C revealed drastic changes in the spectral shape of the amide I' band at 540 MPa [START_REF] Herberhold | Temperature-and pressure-induced unfolding and refolding of ubiquitin: A static and kinetic fourier transform infrared spectroscopy study[END_REF]. The total number of protons exchanged during the denaturation process is approx 20. Only one inflection point on the sigmoidal plot in Figure 2.IV.b suggests that unfolding of ubiquitin occurs in one cooperative step. This result is in an agreement with the results obtained by Jonas [START_REF] Jonas | High-resolution, high-pressure NMR studies of proteins[END_REF].

During HPT, the unfolded structure of ubiquitin exchanges all the amide protons present in the core of the protein fast. The incubation of ubiquitin in the aqueous buffer (performed after the renaturation caused by the decompression) results in afast backexchange of the amide protons located in the unstructured fragments of the protein backbone. The deuterons which are involved in the formation of the hydrogen bonds stabilizing the α or β structures are protected from DHX.

We applied the ECD method to determine the distribution of deuterons along the sequence of ubiquitin. The molecule of ubiquitin is the most relevant model for such a study because the protein does not contain disulphide bonds, what makes ECD fragmentation clearer. Additionally, a moderate number of amino acid moieties makes the results obtained for ubiquitin easy to interpretate.

The solution of ubiquitin was exposed to the pressure of 800 MPa for 30 min. We observed that a further increase in pressure (up to 1400 MPa) did not influence the number of exchanged protons what indicates that a whole protein molecule is denaturated at 800 MPa. After the decompression, the solution of the protein was immediately diluted with the aqueous buffer (10 mM ammonium formate in H 2 O) and incubated for over 60 min. That time was sufficient for the exchange of the majority of labile protons located on the protein surface. Finally, the sample was diluted twice with acetonitrile containing formic acid (0.4%) and immediately subjected to the ECD experiment. The obtained spectrum of ubiquitin reveals the presence of 18 deuterium atoms, which proves the retention of the majority of deuterons in the protein molecule at the experimental conditions. The ECD fragmentation was performed for the ion at m/z in the range of 714-718 which corresponds to deuterated protein molecules with the charge of +12. Figure 3 presents the crude fragmentation data.

The data obtained for the HPT protein were compared to those obtained for unmodified ubiquitin. The analysis of the ECD spectra was carried out with a Biotools program and is presented in the Table 1. Fragments detected in the ECD spectrum belong to class c n (total number of identified c fragments is 37) and (z+1) n (19 identified fragments). The identified c n and (z+1) n fragments cover 49% and 25%, respectively, of the ubiquitin sequence which allows us to map the distribution of deuterium atoms in the sequence of ubiquitin. Masses of deuterium containing fragments of the deuterated protein are shifted to higher m/z values as compared to the fragments of nondeuterated ubiquitin. The differences between these masses reflect the number of deuterons in the fragments. Figure 4 shows the content of deuterium D n in the fragment ions generated upon ECD fragmentation. The number of deuterons for particular ions is plotted versus the residue number (n) separately for the c and (z+1) series. This plot shows regions of high and low slopes of the D n number dependence on n. The high slopes correspond to the structured fragments. In contrast the low slopes correspond to the unstructured fragments of the protein chain characterized by a low deuterium content. The removal of consecutive non-deuterated amino acid residues from the low slope regionsdoes not influence the number of deuterons trapped in the molecule. On the other hand, the removal of the amino acid residues containing deuteron in their amide bonds reduces the number of remaining deuterons and suggests that the given amide bond is involved in the formation of a hydrogen bond stabilizing the secondary structure of the protein molecule. Recently, the localization of deuterons situated within short peptides has been analyzed by Rand et al [START_REF] Rand | Electron capture dissociation proceeds with a low degree of intramolecular migration of peptide amide hydrogens[END_REF] by ECD. Our results show that the localization of deuterons by ECD may also be successfully performed on an intact deuterated protein.

The ECD data indicate that deuterium atoms remain in the ordered fragments of ubiquitin, mainly in β-strands 1-7 and 11-15 and in α-helical fragments. There is a difference between the deuterium content in the C-and the N-terminal part of that protein. The observed distribution of deuterium corresponds well to the structure of a protein molecule with a long and flexible C-terminal motif which is responsible for its interaction with other proteins.

Using the NMR technique, Pan and Briggs [START_REF] Pan | Hydrogen exchange in native and alcohol forms of ubiquitin[END_REF] and Bougault et al.

[27] investigated the accessibility and the structural stability of residues along the ubiquitin polypeptide chain by monitoring the changes in the cross peaks' intensities in a series of 2D 15 N-1 H HSQC experiments. The kinetic data concerning the exchange rate of particular protons measured by NMR are depicted in Figure 5. There is a good correlation between the data obtained by ECD and NMR. Both analyses reveal that the amide hydrogens from the unstructured regions undergo a fast isotopic exchange, whereas the hydrogens involved in the stabilization of βstrands and α-helices are exchanged significantly slower. This correlation may further confirm that ubiquitin under the pressure of 800 MPa undergoes a complete denaturation and after decompression the protein refolds into its native state. Our results demonstrate that the ECD based procedure provides data that allow to evaluate the retention of deuterium in particular segments of the protein chain. This approach seems to be more convenient and faster as compared to the previous methods based on a fast peptic hydrolysis of a deuterated protein combined with the LC-MS detection of obtained fragments [START_REF] Zhang | Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation[END_REF].

Conclusions

The application of the H/D exchange combined with ESI-MS methods for the analysis of a protein's high-pressure denaturation has been shown. Although the effects of highpressure on a protein's stability and conformation were not analyzed directly, the proposed technique enables the observation of the two-stage unfolding process of lysozyme as well as the detrmination of the pressure values that totally unfold the studied proteins. The obtained values of critical pressure for the denaturation of all analyzed model proteins are consistent with those reported previously in literature, what confirms the usefulness of the proposed method. The deuteration pattern of a protein with a single amino acid resolution was established from the deuteration levels of the fragment ions generated by ECD. The results obtained suggest that a combination of the H/D exchange and ICR-electrospray mass spectrometry is a fast and reliable method of analysis of the reversibly denatured proteins.

Figure captions

Figure 1 A schematic representation of the isotopic exchange of a protein under high pressure measured by ESI-MS. The first step is the incubation of a protein in the deuterated buffer at the defined pressure for 30 minutes. Under high pressure (P 2 ) more protons are exchanged with deuterons due to unfolding of the protein, as compared to the experiment at the atmospheric pressure (P 1 ). The decompression causes refolding of the protein and trapping a number of deuterons in its interior. The second step involves redissolving a deuterated protein in the aqueous buffer and ESI-MS monitoring of changes of the molecular mass with time.

The list of abbreviations

Figure 2 .

 2 I.a presents an example of the D/H exchange kinetics of α-lactalbumin monitored by ESI-MS.

Figure 2

 2 Figure 2 High pressure denaturation of: I: α-lactalbumin (protocol I); II: lysozyme (protocol I); III: lysozyme (protocol II); IV: ubiquitin (protocol II). a: DHX kinetics of proteins treated with D 2 O at different pressures; b: Graph of the number of deuterons trapped in the hydrophobic core of a protein after the high pressure treatment followed by the 1 hour backexchange in the aqueous buffer as a function of the applied pressure. Legend: -0.1 MPa, -200 MPa, -300 MPa, -400 MPa, -450 MPa, -550 MPa, -600 MPa, -800 MPa, -1000 MPa, -1200 MPa, -1400 MPa.

Figure 3

 3 Figure 3 Crude ECD spectrum of the (+12) ubiquitin ion that allows sequencing of 60% of the protein chain. The spectrum is divided into two parts presented in panel A and B. The values of m/z and z for identified c-and (z+1)-ions are given in the Table 1.

Figure 4

 4 Figure 4 Deuteration levels of the c-and (z+1)-fragment ions (line a and b, respectively) generated from ECD. The deuteration pattern is readily recognized from the deuterium levels of the fragment ions generated by ECD. Representation of the secondary structure motifs along the sequence is adapted from the ubiquitin NMR structure in the Protein Data Bank (PDB id 1d3z).

Figure 5

 5 Figure 5 NMR H/D exchange data published previously by Bougault [27] for ubiquitin dissolved in phosphate buffer. Fast exchange protons are depicted by lower pascals. Protons with a low isotopic exchange rate (represented by higher pascals) are involved in the stabilization of the secondary structure of the protein. Representation of the secondary structure motifs along the sequence is adapted from the ubiquitin NMR structure in the Protein Data Bank (PDB id 1d3z).

A c c e p t e d M a n u s c r i p t

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Table 1 Identified c-and (z+1) -fragment ions of deuterated and nondeuterated ubiquitin obtained by ECD-MS

 1 . m/z corresponds to the average values calculated from the isotopic patterns of particular ions.

	fragment charge m/z	m/z	fragment charge m/z	m/z
	ion	state	nondeuterated deuterated	ion	state	nondeuterated deuterated
	C4	1	537.59	539.82	C60	7	966.97	968.86
	C5	1	636.70	639.05	C61	8	860.36	862.12
	C7	2	433.49	435.01	C63	9	793.36	794.97
	C8	1	979.15	982.52	C64	9	807.71	809.27
	C10	1	1136.92	1140.43	C68	9	856.43	858.13
	C11	2	633.23	635.03	C70	9	880.02	881.75
	C16	2	912.08	914.97	C71	9	892.59	894.39
	C17	2	961.66	964.57	C75	10	851.69	853.22
	C20	2	1118.28	1121.13	(Z+1)8	2	434.54	434.95
	C21	2	1175.86	1178.85	(Z+1)10	2	559.69	560.12
	C23	2	1282.94	1286.23	(Z+1)11	2	610.24	610.85
	C24	3	898.69	901.15	(Z+1)12	2	653.78	654.85
								8

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  Bioscience Reports Immediate Publication. Published on 13 Mar 2009 as manuscript BSR20090015 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090015 A c c e p t e d M a n u s c r i p t

	ECD	Electron capture dissociation
	FTIR	Fourier transform infrared spectroscopy
	NMR	Nuclear magnetic resonance
	UV spectra Ultraviolet spectra
	ESI-MS	Electrospray mass spectrometry
	HDX	Hydrogen -deuterium exchange
	DHX	Deuterium -hydrogen exchange
	CID	Collision induced dissociation
	LC-MS	Liquid chromatography-mass spectrometry
	HPLC	High performance liquid chromatography
	CD	Circular dichroism
	HPT	High pressure treatment
	ICR	Ion cyclotron resonance
		11

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Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgement

This work was supported by a grant No. N N401 222734 from the Ministry of Science and Higher Education of Poland.


Anna Kaminski

Hannah J Welters

Edward R Kaminski

Prof Noel G Morgan
email: noel.morgan@pms.ac.uk

Human and rodent pancreatic β-cells express IL-4 receptors and IL-4 protects against β-cell apoptosis by activation of the PI-3-Kinase and Jak/STAT pathways

Keywords: Interleukin-4, Interleukin-4 receptors, islet cells, pancreatic beta cells, cell death, cytokines, apoptosis

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Human and rodent pancreatic β-cells express IL-4 receptors and IL-4 protects against β-cell apoptosis by activation of the PI-3-Kinase and Jak/STAT pathways

INTRODUCTION

Autoimmune destruction of pancreatic beta-cells in Type I diabetes (T1D) is thought to occur as a result of the disruption of normal pancreatic immunoregulation which may, in part, reflect a functional polarisation of the T helper (Th) subset of CD4+ lymphocytes [START_REF] Rabinovitch | Role of cytokines in the pathogenesis and therapy of diabetes Type I[END_REF]. Secretion of proinflammatory cytokines, such as interleukin (IL)-1 IL-6, interferon (IFN)- and tumour necrosis factor (TNF)- by Th1 lymphocytes and macrophages in the pancreatic environment are associated with increasing loss of insulin producing β-cells in the pancreatic islets [START_REF] Mandrup-Poulsen | The role of IL-1 in the pathogenesis of IDDM[END_REF][START_REF] Rabinovitch | Un update on cytokines in the pathogenesis of insulin-dependent diabetes mellitus[END_REF][START_REF] Eizirik | A choice of death -the signal transduction of immune-mediated beta-cell apoptosis[END_REF]. This is exacerbated by a concomitant reduction of secretion of anti-inflammatory cytokines, such as IL-4, by Th2 lymphocytes [START_REF] Berman | Decreased IL-4 production in new onset of tyoe 1 inulin dependent diabetes mellitus[END_REF][START_REF] Szelakowska | Decreased in vitro IL-4 and IL-10 production by peripheral blood in first degree relatives at high risk of diabetes type I[END_REF][START_REF] Rapoport | Decreased secretion of Th2 cytokines precedes up-regulated and delayed secretion of Th1 cytokines in activated peripheral blood mononuclear cells from patients with insulin-dependent diabetes mellitus[END_REF]. In animal models, IL-4 has been proposed to play a protective role in the development of diabetes [START_REF] Rapoport | Interleukin-4 reverses T cell proliferative unresponsiveness and prevents the onset of diabetes in nonobese diabetec mice[END_REF][START_REF] Cameron | Immunotherapy of spontaneous type 1 diabetes in nonobese diabetec mice by systemic interleukin-4 treatment employing adenovirus vector-mediated gene transfer[END_REF], while patients with elevated levels of IL-4 may be resistant to the development of diabetes [START_REF] Wilson | Extreme Th1 bias in invariant Valpha24JalphaQ T cells in type 1 diabetes[END_REF].

IL-4 is a multifunctional cytokine that plays a critical role in the regulation of immune responses. Its effects depend upon binding to and signalling through a receptor complex consisting of the IL-4 receptor alpha (IL-4Rα) chain and either the cytokine receptor common gamma chain (γc) (in type I IL-4 receptors) or the α1-chain of IL-13 receptors (in type II IL-4 receptors), resulting in a series of phosphorylation events mediated by receptor-associated kinases [START_REF] Nelms | The IL-4 receptor: signalling mechanism and biologic function[END_REF]. In turn, these signal to promote cell growth, resistance to apoptosis, and/or gene activation and differentiation. In other cell types, heterodimerisation of the IL-4Rα chain with the γc (common-γ) chain results in tyrosine phosphorylation of the Jak family of protein kinases in response to IL-4 [START_REF] Nelms | The IL-4 receptor: signalling mechanism and biologic function[END_REF][START_REF] Keegan | An IL-4 receptor region containing an insulin receptor motif is important for IL-4-mediated IRS-1 phosphorylation and cell growth[END_REF]. The activation of Jak kinases culminates in the phosphorylation of the transcription factor STAT6, to propagate the signal arising from binding of IL-4. In addition, IRS-1 and IRS-2 can also become phosphorylated as a result of interaction of IL-4 with the IL-4Rα [START_REF] Xiao | Specificity of interleukin-2 receptor gamma chain superfamily cytokines is mediated by insulin receptor substrate-dependent pathway[END_REF][START_REF] Wang | A role of insulin-interleukin (IL)-4 receptor motif of the IL-4 receptor alpha-chain in regulating activation of insulin receptor substrate 2 and signal transducer and activator of transcription 6 pathways. Analysis by mutagenesis[END_REF]. This response promotes their high affinity association with the p85 regulatory subunit of PI-3-kinase leading, in turn, to activation of PI-3-kinase and downstream kinases such as Akt [START_REF] Kennedy | The PI-3-kinase/Akt signalling pathway delivers an anti-apoptotic signal[END_REF].

In a previous study we confirmed, at both the RNA and protein level, the presence of each component of the Type 1 and Type II IL-4 receptors in rat pancreatic β-cells [START_REF] Kaminski | Pre-incubation with IL-4 mediates protective effect against the loss of pancreatic beta-cell viability induced by pro-inflammatory cytokines[END_REF]. In addition we showed that IL-4 had the capacity to attenuate the cytotoxic effects of IL-1β/IFNγ on pancreatic β-cells [START_REF] Kaminski | Pre-incubation with IL-4 mediates protective effect against the loss of pancreatic beta-cell viability induced by pro-inflammatory cytokines[END_REF]. However it was not established which signaling pathway is responsible for the anti-apoptotic effects of IL-4 in β-cells. Therefore, the aim of the present study was to confirm that IL-4R are found on human pancreatic β-cells and to investigate the signaling pathways by which IL-4 exerts its effects in β-cells.

EXPERIMENTAL

Materials

RPMI 1640, trypsin/EDTA, phosphate-buffered saline (PBS), penicillin and streptomycin were purchased from Invitrogen, UK. Anti-human IL-4 receptor antibody was purchased from R&D Systems, UK. DNase free ribonuclease was from Roche, Lewes, UK. Rabbit anti-mouse HRP, DakoREAL Envision/HRP rabbit anti-mouse antibody, REAL DAB chromogen and DAKO

Growth and incubation of rodent clonal β-cells

BRIN-BD11 and INS-1 cells were used in the studies. The cells were cultured in RPMI-1640 medium supplemented with 10% foetal calf serum (FCS), 2mM L-glutamine, 400 U/ml penicillin and 200 µg/ml streptomycin. INS-1E Flp-In-TRex cells were established and grown as described by Welters et al [START_REF] Welters | Conditional expression of hepatocyte nuclear factor-1beta, the maturity-onset diabetes of the young-5 gene product, influences the viability and functional competence of pancreatic beta-cells[END_REF]. Cells inducibly expressing the tyrosine phosphatase PTP-BL were generated using Flp recombinase [START_REF] Welters | Conditional expression of hepatocyte nuclear factor-1beta, the maturity-onset diabetes of the young-5 gene product, influences the viability and functional competence of pancreatic beta-cells[END_REF][START_REF] Welters | The protein tyrosine phosphatase, PTP-BL, modulates pancreatic β-cell proliferation by interaction with the Wnt signalling pathway[END_REF] and were grown in the presence of hygromycin to maintain selection pressure. When required, over-expression of PTP-BL was achieved by culture in the presence of tetracycline for 24h.

Immunocytochemical staining for IL-4 receptor expression

Paraffin embedded human pancreatic sections obtained at autopsy from non-diabetic individuals. They were kindly provided by Dr AK Foulis (Glasgow) and were used with full ethical permission (North Glasgow NHS Trust). The sections were de-waxed in xylene (2 x 4 minutes). Slides were then rehydrated through 100% ethanol for 1 min, 90% ethanol for 1 min, 70% ethanol for 1 min, 100% methanol for 1 min and H 2 O for 5 min. Slides were blocked with 0.5ml normal goat serum for 5 minutes and primary mouse anti-human IL-4 receptor antibody, isotypespecific control IgG or anti-insulin serum applied and incubated overnight at 4C o . After washing in Tris-buffered saline pH 7.4, secondary antibody conjugated to horseradish peroxidise was applied and the slides were incubated for further 1hr. After washing in Tris-buffered saline DAB substrate was applied for 10 minutes. After further washing, slides were stained with haematoxylin for 5 minutes. Dehydration of the slides was performed by immersion in 50% ethanol (5 min) 70% ethanol (5 min) 90% ethanol (5 min) and finally 100% ethanol (2 x 5 min). Slides were cleared in xylene (2 x 5 min) and mounted in DPX medium for examination.

Flow cytometric analysis of cell death

β-cell death was analysed using flow cytometry. Following incubation with test reagents, the culture medium containing detached cells was harvested. Adherent cells were trypsinised and both the detached and adherent cells were collected by centrifugation (300g; 5min). They were resuspended in 200 µl of phosphate-buffered saline (PBS) and fixed with ice-cold ethanol: PBS (70 : 30 v:v) for 30 min on ice. After fixation the cells were centrifuged for 5 min at 300g and resuspended in 820 µl of PBS. 80µl of propidium iodide (0.5 mg/ml) and 5µl of DNase free ribonuclease were added and incubation continued for 30 min at 37 o C. Cellular DNA was analysed using a flow cytometer and the extent of DNA fragmentation determined as an index of loss of viability. Experiments were performed in triplicate and were repeated on at least three occasions.

Western blotting analysis

β-cells were incubated with appropriate reagents and homogenised in lysis buffer (20mM Tris, 150mM NaCl, 1 mM EDTA, 1% Triton X and protease inhibitors; pH 7.4) for protein extraction. The homogenate was then centrifuged at 2000g for 10 min at 4 o C and the supernatant was collected, aliquoted and stored at -80 o C until use. The protein concentration was determined using bovine serum albumin (BSA) as standard. Immunoblotting was carried out using 70µg of protein loaded into BioRad Criterion TM Precast 12% gels. Separated proteins were transferred into PVDF membranes. Membranes were then blocked in Tris buffered saline 0.1% Tween-20 (TBS-T, pH 7.5) containing 2% BSA and 5% non-fat dried milk for 1h at room temperature before adding primary antibody. After overnight incubation at 4 o C, the membranes were washed three times in TBS-T and secondary goat anti-rabbit IgG HRP conjugated antibody added for 1 h. The membranes were washed 3 times for 5 min in TBS-T. Protein bands were detected using CDP Star chemiluminescent Substrate.

MTS assay

β-cells were seeded in 96 well plates at 10 4 cells/well and, after 24 h incubation, IL-4 added with or without relevant inhibitors (wortmannin (500nM), Jak3 (78nM)). After a further 24 h incubation, MTS assay was performed according to manufacturer's instructions. Briefly, 20ul of MTS reagent was added to each well and the mixture incubated for 1h at 37 o C. Absorbance was read at 490nm.

Statistical analysis

Statistical analysis was performed using the Student's t-test or by analysis of variance, and results were considered significant when P<0.05

RESULTS

Expression of IL-4 receptors in human pancreas

In a previous study we demonstrated that rat pancreatic β-cell lines express receptors for IL-4 at both the RNA and protein level [START_REF] Kaminski | Pre-incubation with IL-4 mediates protective effect against the loss of pancreatic beta-cell viability induced by pro-inflammatory cytokines[END_REF]. However, it has not been established whether primary βcells also express the receptor and, therefore, immunohistochemical analysis was undertaken in sections of normal adult human pancreas to verify this (Fig. 1 upper panel). Intensely positive staining with anti-IL-4Rα antibodies was seen in the majority of islet endocrine cells, consistent with the presence of IL-4 receptors on human β-cells. Similar data were obtained in pancreas samples from 4 separate individuals and, in each case, the majority of islets were stained with anti-IL4 receptor antibody. Immunostaining of the adjacent section for insulin (Fig. 1; middle panel) suggested that fewer cells stained positively for the hormone than for IL-4 receptor implying that the receptors may be present on both β-cells and on other islet endocrine cell types. In addition to the labelling of islet endocrine cells, immunopositivity for IL-4 receptor was also detected in the surrounding exocrine tissue, although this was less intense than on islet cells. The staining intensity was lost when the primary antibody was omitted (not shown) or replaced with isotype control mouse IgG (Fig. 1-lower panel).

Activation of the PI-3-kinase pathway in beta-cells treated with IL-4

Previous studies by our own and other groups have established that the cytotoxic actions of proinflammatory cytokines in β-cells are attenuated by pre-incubation with IL-4 [START_REF] Kaminski | Pre-incubation with IL-4 mediates protective effect against the loss of pancreatic beta-cell viability induced by pro-inflammatory cytokines[END_REF][START_REF] Souza | Interaction between pro-inflammatory and anti-inflammatory cytokines in insulin-producing cells[END_REF]. This was confirmed in the present work (Fig. 2) where the extent of cell loss caused by incubation of βcells with IL-1β & IFNγ was attenuated by approximately 65% in the presence of 20ng/ml IL-4. This effect was prevented by the presence of the PI-3-kinase inhibitor wortmannin (Fig. 2) suggesting that activation of the PI-3-kinase pathway is involved. In support of this, Western blotting revealed that incubation of cells with IL-4 resulted in increased phosphorylation of Akt, a downstream target of PI-3-kinase (Fig. 3). The extent of this increase was less marked than that induced in parallel studies with 10nM IGF-1 (Fig. 3) but, nevertheless, the response to IL-4 resulted in a clear increase in Akt phosphorylation.

Activation of Jak/STAT signalling in beta-cells treated with IL-4

In immune cells, the activation of IL-4 receptors leads to alterations in gene transcription as well as to increases in PI-3-kinase activity and it is thought that these effects may reflect the activation of different signalling pathways by IL-4 receptors [START_REF] Nelms | The IL-4 receptor: signalling mechanism and biologic function[END_REF]. In particular, IL-4R activation is frequently associated with changes in the phosphorylation status of STAT proteins, especially STAT6. Therefore we next examined whether the Jak-STAT pathway may also play a role in mediating the response to IL-4 in β-cells. Initially, the effects of an inhibitor of Jak-3 kinase (WHI-P154) were investigated and it was observed that this significantly reduced the cytoprotective response to IL-4 in beta-cells exposed to pro-inflammatory cytokines (Fig. 4).

A second index of improved viability was also measured in these experiments, MTS reduction, which provides information on the functional status of mitochondrial dehydrogenases. It was observed that exposure of beta-cells to IL-4 resulted in a marked increase in MTS reduction, consistent with an enhanced metabolic propensity of the cells in the presence of the antiinflammatory cytokine (Fig. 5). Importantly, this response was progressively reduced in cells over-expressing the tyrosine phosphatase, PTP-BL, which is active against phospho-STAT proteins (Fig. 5). In order to confirm this more directly, Western blotting of cell extracts was undertaken using an antiserum raised against tyrosine phosphorylated STAT6. The results revealed a marked increase in phopsho-STAT6 formation in β-cells exposed to IL-4 (Fig. 6). Strikingly, as in the case of IL-4-induced MTS reduction (Fig. 5), this response was entirely lost in cells over-expressing the tyrosine-specific phosphatase, PTP-BL (Fig. 6). . Since the majority of islet cells were stained by the anti-IL-4 receptor antibodies and, in serial sections, by anti-insulin serum (Fig. 1 middle panel), this suggests that human β-cells express the receptor. As such, these results provide support to our earlier conclusion that IL-4 receptors are expressed on clonal pancreatic β-cells derived from rat [START_REF] Kaminski | Pre-incubation with IL-4 mediates protective effect against the loss of pancreatic beta-cell viability induced by pro-inflammatory cytokines[END_REF] and they show that expression of these molecules is not restricted to rodent islet cells. However, the pattern of immunohistochemical staining implies that, at least in man, other islet endocrine cells may also express IL-4 receptors since positive immunostaining for these molecules did not appear to be restricted only to β-cells. Nevertheless, the presence of IL-4 receptors on normal human pancreatic β-cells is significant since it implies that, as in rat [START_REF] Kaminski | Pre-incubation with IL-4 mediates protective effect against the loss of pancreatic beta-cell viability induced by pro-inflammatory cytokines[END_REF] these receptors may play a protective role against cell damage mediated by pro-inflammatory cytokines. As such, it is clear that the balance between the influence of pro-and antiinflammatory cytokines (including IL-4) on β-cells might be an important determinant of their ultimate fate during the progression towards type 1 diabetes. In this context, it is interesting to note that a relative lack of IL-4 production has been reported in patients with type 1 diabetes in a number of studies. For example, peripheral blood mononuclear cells (PBMC) from human diabetic patients were shown to produce a decreased level of IL-4 when challenged with polyclonal activators such as anti-CD3 antibody or phytohemagglutinin [START_REF] Berman | Decreased IL-4 production in new onset of tyoe 1 inulin dependent diabetes mellitus[END_REF]. Moreover, others have reported that elevated IFNγ/IL-4 ratios correlate with the presence of destructive insulitis and impaired Th2 cell cytokine production from cells that infiltrate the pancreas of diabetic patients [START_REF] Kolb | Benign versus destructive insulitis[END_REF][START_REF] Farilla | Presence of interleukin 4 or interleukin 10, but not both cytokines in pancreatic tissue of two patients with recently diagnosed diabetes mellitus type I[END_REF][START_REF] Vrabelova | Protein microarray analysis as a tool for monitoring cellular autoreactivity in type 1 diabetes patients and their relatives[END_REF].

In view of these considerations, it is important to determine the intracellular signalling pathways by which IL-4 may mediate its effects in β-cells. Accordingly, we have employed clonal rat β-cell lines for functional analysis in the present work since we showed previously that recombinant IL-4 significantly reduced apoptotic β-cell death induced by pro-inflammatory cytokines in these cells [START_REF] Kaminski | Pre-incubation with IL-4 mediates protective effect against the loss of pancreatic beta-cell viability induced by pro-inflammatory cytokines[END_REF]. In addition, another group has recently demonstrated a protective effect of IL-4, IL-10 and IL-13 in blocking pro-inflammatory cytokine-induced activation of caspase-3 [START_REF] Souza | Interaction between pro-inflammatory and anti-inflammatory cytokines in insulin-producing cells[END_REF]. Previous work in other cells, has established that different signalling pathways can be activated upon addition of IL-4 [START_REF] Xiao | Specificity of interleukin-2 receptor gamma chain superfamily cytokines is mediated by insulin receptor substrate-dependent pathway[END_REF][START_REF] Wang | A role of insulin-interleukin (IL)-4 receptor motif of the IL-4 receptor alpha-chain in regulating activation of insulin receptor substrate 2 and signal transducer and activator of transcription 6 pathways. Analysis by mutagenesis[END_REF] and, in particular, it has been proposed that IL-4 mediated heterodimerisation of the IL-4Rα chain with the γc (common-γ) chain results in tyrosine phosphorylation of the Jak family of protein kinases, particularly Jak1 and Jak3. This appears to reflect the binding of Jak1 to the IL-4Rα chain, while Jak3 associates with the γc chain [START_REF] Nelms | The IL-4 receptor: signalling mechanism and biologic function[END_REF][START_REF] Keegan | An IL-4 receptor region containing an insulin receptor motif is important for IL-4-mediated IRS-1 phosphorylation and cell growth[END_REF]. The activation of Jak kinases culminates in the phosphorylation of the transcription factor STAT6, to propagate the signal arising from binding of IL-4. However, in addition to the Jak kinases, a second group of molecules that often become phosphorylated as a result of interaction of IL-4 with the IL-4Rα chain are the insulin receptor substrates, particularly IRS-1 and IRS-2 [START_REF] Xiao | Specificity of interleukin-2 receptor gamma chain superfamily cytokines is mediated by insulin receptor substrate-dependent pathway[END_REF][START_REF] Wang | A role of insulin-interleukin (IL)-4 receptor motif of the IL-4 receptor alpha-chain in regulating activation of insulin receptor substrate 2 and signal transducer and activator of transcription 6 pathways. Analysis by mutagenesis[END_REF]. This response leads to the tyrosine phosphorylation of sites located within both IRS1 and IRS2, thereby promoting their high affinity association with the p85 regulatory subunit of PI-3-kinase. This in turn leads to activation of PI-3-kinase and thence to activation of downstream kinases such as Akt that are important in mediating cytoprotection and cell proliferation [START_REF] Kennedy | The PI-3-kinase/Akt signalling pathway delivers an anti-apoptotic signal[END_REF]. In the present work we report that the ability of IL-4 to attenuate β-cell death induced by pro-inflammatory cytokines can be ameliorated by the addition of the PI-3-kinase inhibitor, wortmannin (Fig. 2). This suggests strongly that the PI-3-kinase pathway is activated in rat βcells exposed to IL-4 and that it can contribute to cytoprotection. In support of this, Western blotting analysis revealed that incubation of β-cells with IL-4 results in increased phosphorylation of Akt (Fig. 3). These results imply that functional activation of IL-4Rα complexes occurs during exposure of β-cells to IL-4 and that subsequent activation of the PI-3kinase pathway plays a role in mediating the effects of the cytokine. However, it is clear that activation of a second signalling pathway involving Jak kinases is also associated with IL-4R activation. Thus, flow cytometry was used to measure the percentage of cells undergoing apoptosis after exposure to pro-inflammatory cytokines and it was observed that a selective inhibitor of Jak3 significantly reduced the cytoprotective response to IL-4 (Fig. 4). Evidence for the involvement of Jak3/STAT6 was reinforced by the observation that IL-4 directly promotes STAT6 phosphorylation in β-cells, as measured by Western blotting (Fig. 6). Thus, we conclude that both PI-3-kinase and Jak3/STAT6 are activated by IL-4 in β-cells and that both signalling pathways may contribute to cytoprotection. Interestingly, this conclusion differs from data obtained with mouse T-cells where these pathways are also activated but appear to be dispensable for cytoprotection [START_REF] Wurster | Stat6 and IRS-2 cooperate in interleukin 4 (IL-4)-induced proliferation and differentiation but are dispensable for IL-4 dependent rescue from apoptosis[END_REF]. Thus, the primary signalling mechanism(s) mediating the protective effects of IL-4 may differ between pancreatic β-cells and cells of the immune system.

To explore the importance of the Jak/STAT pathway further, β-cells over-expressing one specific member of the class of protein tyrosine phosphatases (PTP) PTP-BL [START_REF] Welters | The protein tyrosine phosphatase, PTP-BL, modulates pancreatic β-cell proliferation by interaction with the Wnt signalling pathway[END_REF] were employed. PTP-BL has recently been demonstrated to antagonise the phosphorylation of STAT6 mediated by IL-4 [START_REF] Nakahira | Regulation of signal transducer and activator of transcription signalling by tyrosine phosphatise PTP-BL[END_REF] and, consistent with this, the increase in tyrosine phosphorylation of STAT6 mediated by IL-4 was completely prevented in β-cells over-expressing PTP-BL (Fig. 6). Thus, as suggested for immune cells [START_REF] Nakahira | Regulation of signal transducer and activator of transcription signalling by tyrosine phosphatise PTP-BL[END_REF] these data imply that PTP-BL could be involved in the regulation of IL-4 signalling in β-cells. Moreover, the results demonstrate that the Jak3/STAT6 pathway can be functionally manipulated in β-cells and they suggest that it is involved in the regulation of β-cell survival in response to IL-4. Further support for this proposal derives from our observation that the increase in β-cell MTS reduction induced by IL-4 (an index of improved metabolic activity) was significantly attenuated by up-regulation of PTP-BL (Fig. 5).

Therefore, we have provided evidence for the presence of IL-4 receptors on the surface of human pancreatic β-cells. In addition, we reveal that the functional activity of IL-4 involves activation of both PI-3-kinase and Jak3/STAT6 pathways and that these can be opposed by increased expression of the tyrosine phosphatase, PTP-BL.

Taken together, the data imply that targeting of IL-4 receptors may offer a means to attenuate the loss of pancreatic β-cells occurring during autoimmune diabetes. Furthermore, they may have similar implications for therapeutic intervention in type 2 diabetes since recent evidence has also implicated modest islet inflammation [START_REF] Ehses | Increased number of islet-associated macrophages in type 2 diabetes[END_REF][START_REF] Richardson | Islet associated macrophages in type 2 diabetes[END_REF] and increased IL-1β production in mediating β-cell demise [START_REF] Donath | Cytokines and (beta)-cell biology: from concept to clinical translation[END_REF]. Phosphorylation of STAT6 following incubation with IL-4. INS-1 cells inducibly expressing the tyrosine phosphatase PTP-BL were generated using Flp recombinase. When required, over expression of PTP-BL was achieved by culture in the presence of 1µg/ml tetracycline for 24h. Un-induced cells received vehicle. IL-4 (20ng/ml) was included for the final 30 min of culture, as shown. Proteins were extracted, subjected to SDS-PAGE electrophoresis and immunostained with anti-pSTAT6 antibody. β-actin was used as a loading control (lower panel). Data are from a representative experiment which was repeated twice with similar results.

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  Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090021 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090021 A c c e p t e d M a n u s c r i p t 4 REAL substrate buffer were from DAKO, Ely, UK. DPX Mountant was purchased from Fluka, UK. IL-4, IL-1β, IFNγ, goat anti-rabbit Alkaline Phosphatase IgG antibody, protease inhibitors, CDB-Star Chemiluminescent Substrate and Wortmannin were from Sigma, Poole, UK. CellTiter 96 AQ ueous One Solution Cell Proliferation (MTS) Assay was from Promega, Southampton, UK. Rabbit anti-rat pAkt antibody was from Santa Cruz, UK. Jak3 inhibitor II (WHI-P154) was from Calbiochem, Nottingham, UK.

Bioscience

  Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090021 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090021 A c c e p t e d M a n u s c r i p t

  Bioscience Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090021 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090021 A c c e p t e d M a n u s c r i p t 6

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  Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090021 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090021 A c c e p t e d M a n u s c r i p t DISCUSSION The present results demonstrate the presence of IL-4 receptors on human adult pancreatic islet cells by immunohistochemistry (Fig 1)

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  Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090021 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090021 A c c e p t e d M a n u s c r i p t

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  Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090021 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090021 A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t

Figure 1 .

 1 Figure 1. Expression of IL-4 receptors (IL-4R) on human islet cells. Sections of adult human pancreas were stained with anti-IL-4Rα serum to detect expression of the receptor. (Upper panel) A typical islet is shown in which the majority of endocrine cells were stained (brown) consistent with expression of IL-4Rα. (Middle panel) The adjacent section was stained for insulin (brown) to reveal the presence of β-cells. (Lower panel) Sections of pancreas were also exposed to control IgG (isotope specific IgG for the antiserum to IL-4 receptor) and were unstained.

Figure 2 .

 2 Figure 2.Involvement of the PI-3-kinase pathway in mediating the protective effects of IL-4 in βcells. Cells were incubated under control conditions (white bars) or with IL-4 (20ng/ml; grey bars) for 48h. After this time, IL-1β (2ng/ml) plus IFNγ (2ng/ml) was added in the absence or presence of wortmannin (500nM) as shown. Cells were incubated for further 24h then harvested and analysed by flow cytometry. Each point is the mean (± S.E.M.) from at least four separate experiments. **p<0.01 relative to IL-1β/IFNγ in the absence of IL-4.

Figure 3 .

 3 Figure 3. Phosphorylation of Akt in pancreatic β-cells exposed to IL-4. Cells were incubated for 10 min under control conditions or in the presence of IGF-1 (10nM) or IL-4 (20ng/ml) as shown.Proteins were then extracted and fractionated by SDS-PAGE followed by immunoblotting with either anti-phospho-Akt antibody (p-Akt; upper panel) or total Akt antibody (lower panel). Data are from a representative experiment which was repeated twice with similar results.

Figure 4 .

 4 Figure 4. Effects of an inhibitor of Jak3 on the cytoprotective actions of IL4 in β-cells. Cells were incubated under control conditions (white bar) or in the presence of pro-inflammatory cytokines (Cyto -IL-1β (2ng/ml) plus IFNγ (2ng/ml)) IL-1β & IFNγ plus IL-4 (20ng/ml); IL-1β & IFNγ plus the Jak-3 inhibitor WHI-P154 (Jak; 1μM) or IL-1β & IFNγ/IL-4 plus WHI-P154, as shown for 24h. After this time the cells were harvested and their viability determined by flow cytometry. *p<0.001 relative to cells incubated under control conditions; ** p<0.01 relative to cells incubated in the presence of IL-1β & IFNγ alone; ***p<0.001 relative to cells incubated with IL-1β/IFNγ and IL-4 in the absence of Jak-3 inhibitor.

Figure 5 .

 5 Figure 5. MTS reduction in β-cells treated with IL-4. Cells were treated with increasing concentrations of tetracycline (tet; 20ng/ml or 1000ng/ml) for 24h to induce the expression of PTP-BL. IL-4 (20ng/ml) was included during this incubation period, as shown. MTS reagent was then added and the absorbance measured 1h later. Results are expressed relative to control (100%) and are shown as mean values ± SEM from replicates of 8. The experiment was repeated twice with similar results. *p<0.001 relative to the absence of IL-4; **p<0.05 relative to IL-4 alone; ***p<0.001 relative to IL-4 alone.

Figure 6 .

 6 Figure 6.

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  Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090021 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090021 A c c e p t e d M a n u s c r i p t

Figure 1 Bioscience

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Figure 2 Untreated

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Figure 3 ControlBioscience

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Figure 6 Bioscience

 6 Figure 6

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Bioscience Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090021 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090021

ACKNOWLEDGEMENTS

The study was funded by the European Foundation for the Study of Diabetes (EFSD) and Diabetes UK. Thanks are also due to Sarah Richardson for assistance with immunohistochemistry.