Báo cáo khoa học: Odorant binding protein has the biochemical properties of a scavenger for 4-hydroxy-2-nonenal in mammalian nasal mucosa doc

Odorant binding protein has the biochemical properties of a scavenger for 4-hydroxy-2-nonenal in mammalian nasal mucosa Stefano Grolli, Elisa Merli, Virna Conti, Erika Scaltriti and Roberto Ramoni Dipartimento di Produzioni Animali, Biotecnologie Veterinarie, Qualita ` e Sicurezza degli Alimenti, Universita ` degli Studi di Parma, Italy Reactive oxygen species (ROS) are short-lived radical intermediates generated as a consequence of oxidative metabolism. They can react with virtually all classes of biological molecules and are responsible for most of the cellular damage caused by oxidative stress. In the case of reactions with membrane polyunsaturated fatty acids (PUFA), ROS initiate a lipid peroxidation pro- cess that gives rise to a large number of toxic low molecular mass aldehydes as end products, including the 4-hydroxyalkenals [1,2]. These molecules may be responsible for significant loss of biological activity in proteins and nucleic acids by reacting in a Michael- type addition with the nucleophilic groups (-SH, -NH 2 and imidazole) of amino acids and nucleotides [1,2]. Inactivation of 4-hydroxyalkenals in vivo is achieved by several enzymatic systems [3], with a predominant role played by glutathione S-transferases (GSTs) which catalyse a Michael addition of glutathione to the alde- hyde double bond [4]. Furthermore, 4-hydroxyalkenal cytotoxicity is possible if it is exported via the Keywords 4-hydroxy-2-nonenal; lipocalins; odorant binding protein; oxidative stress; reactive oxygen species Correspondence R. Ramoni, Dipartimento di Produzioni Animali, Biotecnologie Veterinarie, Qualita ` e Sicurezza degli Alimenti, Universita ` di Parma, Via del Taglio 8, 43100 Parma, Italy Fax: +39 052 190 2770 Tel: +39 052 190 2767 E-mail: vetbioc@unipr.it (Received 7 June 2006, revised 18 August 2006, accepted 21 September 2006) doi:10.1111/j.1742-4658.2006.05510.x Odorant binding proteins (OBP) are soluble lipocalins produced in large amounts in the nasal mucosa of several mammalian species. Although OBPs can bind a large variety of odorous compounds, direct and exclusive involvement of these proteins in olfactory perception has not been clearly demonstrated. This study investigated the binding properties and chemical resistance of OBP to the chemically reactive lipid peroxidation end-product 4-hydroxy-2-nonenal (HNE), in an attempt to establish a functional rela- tionship between this protein and the molecular mechanisms combating free radical cellular damage. Experiments were carried out on recombinant porcine and bovine OBPs and results showed that both forms were able to bind HNE with affinities comparable with those of typical OBP ligands (K d ¼ 4.9 and 9.0 lm for porcine and bovine OBP, respectively). Further- more, OBP functionality, as determined by measuring the binding of the fluorescent ligand 1-aminoanthracene, was partially lost only when incuba- ting HNE levels and exposure time to HNE exceeded physiological values in nasal mucosa. Finally, preliminary experiments in a simplified model resembling nasal epithelium showed that extracellular OBP can preserve the viability of an epithelial cell line derived from bovine turbinates exposed to toxic amounts of the aldehyde. These results suggest that OBP, which is expressed at millimolar levels, might reduce HNE toxicity by removing from the nasal mucus a significant fraction of the aldehyde that is produced as a consequence of direct exposure to the oxygen present in inhaled air. Abbreviations AMA, 1-aminoanthracene; BT, bovine turbinate cells; GST, glutathione S-transferase; HNE, 4-hydroxy-2-nonenal; OBP, odorant binding protein; PUFA, polyunsaturated fatty acids; ROS, reactive oxygen species; TTBS, 20 m M Tris ⁄ HCl buffer, pH 7.8 containing 150 mM NaCl and 0.01% (W ⁄ V) Tween 20. FEBS Journal 273 (2006) 5131–5142 ª 2006 The Authors Journal compilation ª 2006 FEBS 5131 bloodstream to molecular targets distributed virtually anywhere in the organism [1,4]. This is a consequence of the small dimensions and moderate hydrophilicity of these compounds, which, in contrast to their lipid precursors, can be solubilized at millimolar levels in the aqueous matrix of biological fluids. Therefore, a protein scavenger that could trap, and eventually deli- ver, 4-hydroxyalkenals to appropriate degradative pathways, might aid other inactivating mechanisms and prevent chemical modification by these molecules in tissues where large-scale lipid peroxidation occurs. The nasal mucosa is constantly exposed to the high oxygen levels present in inhaled air and it has recently been proposed that toxic aldehydes derived from lipid peroxidation might be scavenged by odorant binding proteins (OBPs) [5]. OBPs are soluble proteins present in large amounts (mm levels) on the surface of the nasal mucosa [6]. They can bind a broad spectrum of odorous and nonodorous compounds with good affinities (K d in the lm range), including some toxic 8– 11 carbon aldehydes [7–9] derived from the peroxida- tion of PUFA. OBPs belong to the lipocalins, a family of structurally related soluble proteins that bind differ- ent types of small hydrophobic molecules [10,11]. In general, OBPs are monomeric proteins with a molecu- lar mass of 19 kDa. A nine-strand beta-barrel defines the ligand-binding site, connected by a short linker (hinge sequence) to a C-terminal a helix [12,13] of unknown function. Dimeric OBPs have also been des- cribed, although less frequently. For example, bovine OBP is peculiar in that it is a dimer with domain swapping [14,15]. There is currently a lack of informa- tion on the in vivo binding properties of OBP, the low binding specificity, however, suggests several hypothe- ses for its role as a versatile carrier ⁄ scavenger involved in different molecular mechanisms within the nasal mucosa: (a) OBP might be involved in olfactory perireceptor events behaving either as carrier of odorous compounds to their receptors or as a scavenger of excess odours; (b) OBP, at least in ruminants, might have a protective prophylactic role towards parasitosis and infectious diseases carried by insects; (c) OBP might protect against oxidative stress by removing toxic compounds locally produced by lipid peroxidation or inhalation. Experimental evidence in favour of the first hypothe- sis include the binding capacity of OBPs for odorous compounds and the abundance of OBPs in nasal tissue [6], and the recent report of an in vitro interaction between porcine OBP and an olfactory receptor [16]. The second hypothesis is supported by the identifica- tion of the natural ligand of bovine OBP, the insect attractant 1-octen-3-ol, a component of bovine breath that is produced by rumenal microflora [17]. OBP may render animals less attractive for insects by trapping 1-octen-3-ol from expired breath as it passes through nasal turbinates, resulting in a general decrease in bites. This, in turn, would cause a reduction in vector- mediated parasitosis and infectious diseases. Evaluation of the third hypothesis, the molecular basis of which is described above, is the aim of this study. Here, we report the binding properties, chemical modification and chemical resistance of recombinant bovine and porcine OBPs with respect to 4-hydroxy- 2-nonenal (HNE), the most abundant and extensively characterized toxic 4-hydroxyalkenal derived from per- oxidation of x-6 PUFA [1,2]. Our results show that OBP can bind HNE in a reversible equilibrium and retain a relevant fraction of its binding capacity after chemical modifications induced by the aldehyde. Fur- thermore, preliminary experiments indicate that extra- cellular OBP can prevent HNE-induced cytotoxicity in an epithelial cell line derived from bovine nasal turbin- ates. Taken together, the data suggest that, in vivo, OBP may trap compounds derived from peroxidation of PUFA and lead them from the mucus present on nasal epithelia to the digestive tract for their chemical inactivation. Results Protein purification and functional characterization SDS ⁄ PAGE of the purified forms of recombinant por- cine and bovine OBP gave two single bands at the expected molecular masses. Binding capacity was tes- ted using the fluorescent ligand 1-aminoanthracene (AMA). Hyperbolic titration curves (Fig. 1A,B) giving K d values of 1.5 and 0.5 lm, and saturation levels of 0.9 and 1.83 for porcine and bovine OBP, respectively were in agreement with functional preparations of native and recombinant OBP [18]. Direct binding test to detect HNE–OBP reversible binding complexes The experiment, showing the formation of reversible HNE–OBP binding complexes, was based on the assumption that affinity (dissociation constants in the lm range) and binding stoichiometry (1 mole of lig- and ⁄ OBP equivalent) for HNE are similar to those of the typical OBP ligands. In addition, the spectrophoto- metric binding assay used here allowed us to discrimin- ate, in the same test, between HNE molecules forming OBP as a scavenger for HNE S. Grolli et al. 5132 FEBS Journal 273 (2006) 5131–5142 ª 2006 The Authors Journal compilation ª 2006 FEBS reversible complexes with OBP and those irreversibly bound as HNE–OBP covalent adducts. The experiment, in which 90 lm HNE was incuba- ted with a slight molar excess of OBP, showed that all the aldehyde was reversibly complexed to the protein- binding site, independent of incubation time. In fact, HNE could be quantitatively displaced by a large excess of undecanal, an OBP ligand whose binding complexes with the protein have been resolved at the structural level using X-ray crystallography [5,19]. Undecanal is known to bind within the barrel of OBP, thus results from the displacement experiments indica- ted that HNE was bound entirely within the barrel of OBP. The immediate and quantitative displacement of HNE by undecanal indicates that, as expected in the case of binding equilibria, the formation of reversible HNE–OBP complexes is faster than the covalent reaction between the aldehyde and protein nucleophilic amino acids. The different rates of these two reactions further indicate (see below) the potential efficacy of HNE scavenging by OBP, preventing formation of covalent adducts between the aldehyde and its cellular targets. In fact, when OBP is in molar excess with respect to HNE and its concentration is at least one order of magnitude higher than the dissociation con- stant of the reversible binding complex, it can be expected that the amount of free aldehyde might be negligible. Determination of the dissociation constant of HNE–OBP complexes Affinities for HNE were determined by measuring the progressive chasing of saturating amounts of AMA bound to OBP in response to increasing concentrations [AMA] µ M 02468 Fluorescence intensity 0 20 40 60 80 100 120 140 160 180 [AMA] µ M 02468 Fluorescence intensity 0 20 40 60 80 100 120 140 160 180 [HNE] µ M 0 10203040 Relative fluorescence intensity 0.0 0.2 0.4 0.6 0.8 1.0 1.2 [HNE] µ M 0204060 Relative fluorescence intensity 0.0 0.2 0.4 0.6 0.8 1.0 1.2 A B C D Fig. 1. Binding curves of the fluorescent ligand AMA to porcine (A) and bovine (B) OBP. The curves report the emission fluorescence inten- sity at 480 nm of the AMA–OBP complex, upon excitation at 380 nm, versus the concentration of AMA. Competitions between HNE and AMA are shown for porcine (C) and bovine (D) OBP. Protein samples were incubated with a fixed saturating amount of AMA and increasing HNE. Each point on the y-axis shows the concentration of AMA still bound per OBP monomer relative to the initial value, on a scale of 0–1, versus the concentration of HNE. S. Grolli et al. OBP as a scavenger for HNE FEBS Journal 273 (2006) 5131–5142 ª 2006 The Authors Journal compilation ª 2006 FEBS 5133 of the aldehyde (Fig. 1C,D). Competition curves, matching a two-parameter hyperbolic decay model, gave apparent dissociation constants of 11.0 and 21.0 lm for porcine and bovine OBP, respectively. These values, given in Eqn (1) (see Experimental pro- cedures), resulted in true K d values for HNE of 4.9 and 9.0 lm, which are comparable with those of other typical OBP ligands determined under the same experi- mental conditions [19]. Competitive titrations with heat-denatured OBPs (overnight incubation at 90 °C) were performed as negative controls, and showed that formation of the binding complex between HNE and OBP is strictly dependent on the structural integrity of the protein (not shown). Western blotting and ligand-binding assay of HNE-modified OBP The immunoblot analysis reported in Fig. 2A shows the progressive formation of HNE–OBP adducts after incubation (5 h at 37 °C) of a fixed amount of OBP (26 and 52 lm for bovine and porcine OBP, respect- ively) with increasing aldehyde concentrations (0.1– 2.5 mm). Molar HNE levels exceeded those of OBP and covered the range reported in the literature for this aldehyde in the case of lipid peroxidation in vivo [1]. The labelling pattern showed that bovine OBP can be chemically modified by lower amounts of HNE than the porcine form. This is probably because of the higher number of possibly HNE-reacting amino acid side chains (His and Lys) in bovine OBP (13.2% of the amino acidic residues) compared with the porcine form (8.3%) [12], and because of their spatial arrange- ment in the 3D structure of the proteins (see below). The functionality of these HNE chemically modified OBPs was determined by measuring residual binding for the fluorescent ligand AMA. The plots in Fig. 2B,C show that, even in the case of very high incubating [HNE] ⁄ [OBP] molar ratios (50 : 1), both forms lost only a fraction of their initial AMA-binding capability. Titration of HNE covalent adducts in OBP These titration curves showed that formation of HNE– OBP adducts was definitely time-dependent, passing from 20 min, i.e. the time necessary for clearance of substances dissolved in nasal mucus [20], to the arbi- trary value of 16 h (Fig. 3A,B). This is particularly evi- dent for porcine OBP, but similar behaviour was seen [HNE] m M 00.1 2.51.00.5 0 0.1 2.51.00.5 [HNE] μ M / [OBP] μ M × 2 0 2 10 20 50 0 2 10 20 50 porcine OBP bovine OBP A [HNE] µM / [pOBP] µM 0 102030405060 Residual AMA binding 0.0 0.2 0.4 0.6 0.8 1.0 1.2 B [HNE] µ M / [pOBP] µ M × 2 0 102030405060 Residual AMA binding 0.0 0.2 0.4 0.6 0.8 1.0 1.2 C Fig. 2. (A) Immunoblotting of porcine and bovine OBP HNE-reacting groups. The immunostaining of OBP samples (1 mgÆmL )1 ), treated with increasing amounts of HNE (0.1–2.5 m M), was visualized after incubation with a rabbit serum raised against HNE–protein adducts. Ligand- binding tests showing the functionality of the same HNE-treated porcine (B) and bovine (C) OBPs as in the immunostaining. The plots show the residual AMA-binding capacity versus the ratios between incubating HNE and OBP equivalents. Single data points on the y-axes are reported relative to the functionalities of OBP samples that had not been treated with HNE. OBP as a scavenger for HNE S. Grolli et al. 5134 FEBS Journal 273 (2006) 5131–5142 ª 2006 The Authors Journal compilation ª 2006 FEBS in the case of the bovine form. The divergence between the different incubation times, occurring immediately for porcine OBP and at molar [HNE] ⁄ [OBP] ratios > 10 for bovine OBP, led us to hypothesize that, in vivo, only a small fraction of the putative HNE- reacting protein residues might be present as HNE- covalent adducts. In the case of bovine OBP, titrations did not reach the putative end-point of 21 residues assumed from the amino acid sequence. Indeed, titration could be almost completed (9 HNE-covalent adducts ⁄ 13 putative HNE targets) only with porcine OBP after 16 h in the pres- ence of a very large excess of aldheyde. The curves, which were steeper for [HNE] ⁄ [OBP] molar ratios < 20, indicated facilitated reactivity of groups of nucleophilic amino acids probably located on the protein surfaces, which, on the basis of their 3D struc- tures, are 5 and 11 for porcine and bovine OBP, respectively [13,14]. The number of HNE-modified residues correlated with a progressive decrease in protein functionality, as shown in Fig. 3C,D which shows the residual AMA- binding capacity versus [HNE] ⁄ [OBP] molar ratios. Loss of protein functionality was more evident for the 16-h incubations, in which at least 50% of residual lig- and-binding capacity was maintained at the highest [HNE] ⁄ [OBP] values. In the case of 20-min incuba- tions, HNE inactivation was even less efficient and the fraction of functional protein remained > 70%. The data suggest that HNE-covalent adducts do not com- pletely disrupt the 3D arrangement of the native proteins. Furthermore, they indicate the relevant HNE reacting groupsResidual AMA binding HNE reacting groups Residual AMA binding [HNE] µ M / [pOBP] µ M [HNE] µ M / [bOBP] µ M × 2 0 10203040506070 0 2 4 6 8 10 0 1020304050 0 2 4 6 8 10 12 14 [HNE] µ M / [pOBP] µ M 0 10203040506070 0.0 0.2 0.4 0.6 0.8 1.0 1.2 [HNE] µ M / [bOBP] µ M × 2 0 1020304050 0.0 0.2 0.4 0.6 0.8 1.0 1.2 A B CD Fig. 3. (A,B) Spectrophotometric titrations of covalent adducts between HNE and porcine (A) and bovine (B) OBP, after 20 min (open sym- bols) and 16 h incubation with increasing amounts of the aldheyde. Samples were concentrated using Centricon filters and the number of HNE–OBP covalent adducts was determined by subtracting the amounts of HNE in the ultrafiltrate from the corresponding levels of incuba- ting HNE. The number of HNE reacting residues was plotted versus the ratios between incubating HNE and OBP equivalents. Ligand-binding tests showing the functionality of the same HNE-treated porcine (C) and bovine (D) OBP samples from the spectrophotometric titration. The plots, showing the residual AMA-binding capacity versus the ratios between incubating HNE and OBP equivalents, are reported as in Fig. 2B,C. S. Grolli et al. OBP as a scavenger for HNE FEBS Journal 273 (2006) 5131–5142 ª 2006 The Authors Journal compilation ª 2006 FEBS 5135 resistance of OBP to chemical attack by the aldehyde, especially under experimental conditions in which incu- bation times are closer to physiological values found in vivo in nasal mucosa. Biochemical characterization of OBP exposed to HNE under conditions simulating oxidative stress in nasal mucosa In this experiment, we incubated HNE and OBP under experimental conditions simulating those of the nasal mucosa. OBP concentrations were those presumed for this tissue (0.5 and 1 mm for bovine and porcine OBP, respectively) [6]; HNE concentrations (2 mm) were in the range reported for acute oxidative stress in vivo. Incubation was carried out at 20 °C for 20 min, i.e. the time necessary for the clearance of substances dis- solved in nasal mucus [20]. Under these conditions, formation of HNE–protein adducts was negligible and changes in binding properties were not detected. The results confirm those of the previous experiment in which, at short incubation times and when the molar ratio of HNE and OBP was < 5, the aldehyde did not form a relevant number of covalent adducts with the protein (Fig. 3A,B). Evaluation of the protective role of OBP against HNE cytotoxicity in a simplified model simulating the nasal epithelium The aim of this experiment was a preliminary evalua- tion of the protective role of OBP against chemical aggression by HNE on living cells. We incubated the epithelial cell line BT, derived from bovine nasal tur- binates [21], with a cytotoxic amount (20 lm) of HNE in the absence and presence of a molar excess of bovine OBP (40 lm). The protein was maintained in the extracellular environment by immobilization to a Ni-NTA affinity chromatography matrix, in order to simulate the conditions in the nasal mucosa. We employed a form of bOBP with an N-terminal 6· His- tag (i-bOBP). The structural and functional properties of this form (in terms of aggregation state and binding with different ligands including AMA, HNE and unde- canal) are analogous to those of the native form (data not shown). The cytotoxic effects of 20 lm HNE on BT cells were clearly shown within few hours. Cells showed changes in morphology and viability. Essentially, cells treated with HNE alone became detached from culture dishes or showed marked changes in cellular morphol- ogy (i.e. shrinking and rounding) evident under phase- contrast microscopy (Fig. 4B). By contrast, no signs of cytotoxicity were present in control cultures or cells treated with HNE in the presence of a molar excess of i-bOBP (Fig. 4A,C). Trypan Blue exclusion test con- firmed that cell viability was preserved in the presence of i-bOBP (> 95% vital cells versus < 5% vital cells in wells treated with HNE alone; data not shown). Discussion We investigated the hypothesis that the lipocalin odor- ant binding protein might represent, in nasal tissue, a carrier and ⁄ or a scavenger for toxic aldehydes derived from the peroxidation of unsaturated fatty acids. This study was realized through characterization of the binding properties and chemical resistance of OBP to HNE, an alken-aldehyde involved in the pathogene- sis of several acute and chronic diseases. The hypothe- sis that OBP might actually protect living cells against HNE cytotoxicity was also evaluated. To verify whe- ther the property of scavenging toxic aldehydes may have general significance in mammals, experiments were carried out using OBPs from bovine and porcine species, two proteins with relevant differences in amino acid sequence and 3D structure. The experimental study was divided into four parts: (a) the detection and characterization of the reversible binding complex between OBP and HNE; (b) investi- gation of the chemical modifications of OBP as a result of incubation with large excesses of HNE and evaluation of their effects on the binding properties of the protein; (c) determination of OBP functionality after incubation with HNE under experimental condi- tions simulating the oxidative stress environment of nasal mucosa; and (d) determination of the protective role of extracellular OBP against HNE toxicity in living cells. Experimental results from the first two parts clearly showed that the biochemical properties of OBP match those of an efficient carrier ⁄ scavenger for HNE, and eventually for other alken-aldehydes that the protein could bind. First, direct and competitive binding tests showed that the formation of reversible OBP–HNE complexes, whose K d values are in the lm range, match those of most OBP ligands. This similarity suggests that the binding of HNE might occur with the same features determined by X-ray crystallography for other OBP ligands of similar structure [5,19]. Hence HNE, like undecanal and 1-octen-3-ol, must likely adapt its con- formation to the shape of the binding site, establishing very few specific interactions with the amino acid side chains, which do not undergo significant motion. Fur- ther structural studies of the OBP–HNE complexes in OBP as a scavenger for HNE S. Grolli et al. 5136 FEBS Journal 273 (2006) 5131–5142 ª 2006 The Authors Journal compilation ª 2006 FEBS the crystal and in solution will elucidate detailed struc- tural and functional aspects of the binding process. The reactivity of HNE towards OBP was investi- gated using two different methodologies: western blotting, which showed progressive formation of HNE–OBP adducts after incubation with increasing amounts of the aldehyde, and a spectrophotometric ti- tration that allowed quantitative determination of the HNE reactive amino acids in OBP. Results from these experiments indicated that the formation of HNE– OBP covalent adducts is largely dependent on the incubation time and the molar ratio between the alde- hyde and protein. In the case of the titrations, it could be shown quantitatively that the number of modified residues increased when incubation of the HNE–OBP mixtures was increased from 20 min to 16 h, and when the aldehyde was present in high molar excess (> 5). A comparison between the molar [HNE] ⁄ [OBP] ratios in these experiments and those presumed to be found in nasal mucosa (< 5) [1,6] suggests that in this tissue 4 hours 24 hours controlHNEHNE + i-bOBP A A B B C C Fig. 4. Prevention of HNE-induced cytotoxicity by bOBP in BT cells. Cells were cultivated in a Costar Transwell plate (lower compartment) for 72 h and then incubated with control medium (A), 20 l M HNE (B) or 20 lM HNE in the presence of 40 lM i-bOBP (C), added to the upper compartment. Phase-contrast photomicrography images, taken after 4 and 24 h, show that i-bOBP prevents HNE-induced cytotoxicity. Original magnification: ·100. S. Grolli et al. OBP as a scavenger for HNE FEBS Journal 273 (2006) 5131–5142 ª 2006 The Authors Journal compilation ª 2006 FEBS 5137 OBP might be substantially free of chemical HNE modifications. This is confirmed by MS mass determi- nations of native porcine and bovine OBP samples [18] (R Ramoni, unpublished results), whose values invaria- bly matched those expected on the basis of the amino acid sequences. The biochemical characterization resulting from the first two parts of this study indicates that this type of protein might represent a general carrier ⁄ scavenger for HNE. However, it must be considered that most of these experiments were conducted under conditions that, in terms of OBP and HNE levels, cannot be real- istically compared with those present in vivo in nasal mucosa. Hence, to further evaluate the credibility of the ‘scavenger’ hypothesis, we designed the last two parts of this study to simulate the physiological condi- tions in this tissue. We evaluated the functional prop- erties of the protein and in particular the chemical resistance and binding capacity with respect to HNE. The incubation time of 20 min is the time required for the clearance of a molecule from the nasal mucosa [20], while incubating OBP and HNE levels, both in the mm range, were plausible physiological values [1,6]. This exposure to HNE did not give appreciable chem- ical modification of OBP, further confirming that OPB ⁄ HNE binding in vivo should be reversible and should leave OBPs binding properties unmodified. We also evaluated the capacity of OBP to protect living cells from HNE chemical aggression. To better simu- late the nasal mucosa environment, an epithelial cell line derived from bovine nasal turbinates was used. Incubating OBP was kept in the extracellular environ- ment to simulate its tissue localization in the mucus that covers the nasal epithelia. The experimental results, although preliminary and not quantitative, clearly show that OBP can protect living cells from cytotoxic HNE. These results can be considered as a further indica- tion of the role of this protein as a plausible scavenger for HNE in vivo. In particular, because the amount of OBP in nasal tissue is at least two orders of magnitude higher with respect to the dissociation constant of the binding complex with HNE (and eventually other toxic molecules with similar structure), it is possible that a large fraction of the aldehyde produced from lipid peroxidation might be trapped by this protein. Nasal mucus, which flows from the nasal turbinates to the pharynx with a clearance time of $ 20 min, might drive the OBP–HNE complexes into the first tract of the digestive system where they are inactivated. Hence, this mechanism might be considered as an extracellular counterpart of the chemical inactivation of HNE that occurs intracellularly via GST and other enzymes that are abundantly expressed in nasal mucosa [22]. Fur- thermore, it must be stressed that OBP is a secreted protein, and as such, might have particular relevance in the protection of olfactory receptors, which are pro- teins that play a crucial role in the behaviour and sur- vival of most animal species. In fact, their extracellular domains, which bear the binding sites for odorous compounds, are completely embedded in nasal mucus [23,24] and consequently exposed to the reactive mole- cules rising from oxidative stress. Importantly, this study shows that the OBPs from two animal species, with 42% sequence homology and relevant differences in 3D structure, display similar binding modalities and chemical resistance to HNE. This indicates that the protective role with respect to HNE and other alken-aldehydes might be hypothesized for most OBPs and thus might have a general signifi- cance in mammals. The same role that we hypothesize here for OBP has been also proposed for human tear lipocalin (lcn-1), a protein belonging to the same family. It is produced by the lachrymal and lingual salivary glands, and has been found to be expressed by several other secretory tissues such as prostate, mucosal glands of the tracheobronchi- al tree, nasal mucosa and sweat glands [25]. Human tear lipocalin has significant sequence homology with the human forms of OBP and, at least in humans, par- tially shares a similar tissue distribution [26]. This pro- tein can bind HNE, but compared with OBP, has a binding spectrum that is particularly oriented to com- pounds like 8-isoprostane or 7-b-hydroxycholesterol, with higher molecular masses and hydrophobicity. Despite the different binding spectrum, however, OBP and lcn-1 may cooperate as complementary scavengers in the removal of toxic compounds derived from lipid peroxidation in tissues where they are both present. There are several other lipocalins that bind small hydrophobic molecules, but their physiological role has not yet been univocally and unambiguously established [11]. Further investigations, based on binding tests with molecules derived from lipid peroxidation, might give new insights into the ligand specificity and binding affinity of these proteins and, in turn, their function in lipocalin-driven molecular mechanisms for the removal and inactivation of toxic compounds produced as a consequence of oxidative stress in different tissues. Experimental procedures Materials HNE (27 mm), in hexane, was purchased from Alexis Bio- chemicals (Lausanne, Switzerland) and stored at )80 °C. OBP as a scavenger for HNE S. Grolli et al. 5138 FEBS Journal 273 (2006) 5131–5142 ª 2006 The Authors Journal compilation ª 2006 FEBS The concentration was controlled on the spectrophotometer (e 223 ¼ 13750 m )1 Æcm )1 ) after dilution in water. AMA was from Sigma Aldrich (Milan, Italy). All other reagents, purchased from different companies, were of ana- lytical grade. Rabbit serum raised against HNE–protein adducts was from Alpha Diagnostics International (San Antonio, TX), poly(vinylidene difluoride) was from Millipore (Milan, Italy) and the reagents for electrophoresis and western blot- ting were from Sigma Aldrich. Media and reagents for cell cultures were from Gibco (Milan, Italy). Costar Transwell culture plates were from Corning (Schiphol-Rijk, the Netherlands). OBP purification and functionality test with AMA Recombinant forms of porcine and bovine OBPs were purified from BL21-DE3 Escherichia coli strains trans- formed with the expression vector pT7-7 containing the different OBP cDNAs as previously reported [18]. The purity of each OBP preparation was determined by SDS ⁄ PAGE and protein concentrations were calculated based on the absorbance values at 280 nm (13 000 and 48 000 m )1 Æcm )1 for porcine and bovine OBP, respectively). The functionality of the different OBP forms was deter- mined by direct titrations using the fluorescent ligand AMA as reported previously [18]. Briefly, 1 mL samples of 1 lm OBP, in 20 mm Tris ⁄ HCl buffer pH 7.8, were incu- bated overnight at 4 °C in the presence of increasing con- centrations of AMA (0.156–10 lm). Fluorescence emission spectra between 450 and 550 nm were recorded with a Perkin-Elmer LS 50 luminescence spectrometer (excitation and emission slits of 5 nm) at a fixed excitation wavelength of 380 nm and the formation of the AMA–OBP complex was followed as an increase in the fluorescence emission intensity at 480 nm. Dissociation constants for the AMA– OBP complexes were determined from the hyperbolic titra- tion curves using the nonlinear fitting program sigma plot 5.0 (Cambridge Software Corp., Cambridge, MA). The concentration of the AMA–OBP complex was deter- mined on the basis of emission spectra obtained when incubating AMA (0.1–10 lm) with saturating amounts of both OBP forms. Spectrophotometric assay for the detection of HNE–OBP binding complexes Two 0.4 lL aliquots of HNE were taken from a 27 mm stock solution in hexane and dried under a nitrogen stream to remove the organic solvent. The samples were resuspend- ed in 100 lL of different solutions containing, respectively, 120 lm porcine OBP and 60 lm bovine OBP in 20 mm Tris ⁄ HCl buffer pH 7.8. In both samples the concentration of HNE was 90 lm. After two different incubation times, 20 min at room temperature and 16 h at 4 °C, one half (50 lL) of each OBP–HNE sample was concentrated to a final volume of 5 lL using an Ultrafree-MC, 10 000 Da cut-off, Centrifugal Filter (Millipore, Medford, MA). The HNE eventually not bound to OBP was recovered in the ultrafiltrate and quantified spectrophotometrically after dilution in 1 mL of 20 mm Tris ⁄ HCl buffer pH 7.8. The HNE molecules specifically interacting with the binding sites of the two OBP forms were then displaced by the addition of a large excess of the OBP ligand undecanal (730 lL of a 5 mm solution in Tris ⁄ HCl pH 7.8) and separ- ated from the proteins with a second concentration step. Because no other component in the ultrafiltrate had an absorption band in the UV region, the amount of HNE released from OBP could be determined spectrophotome- trically. A 100 lL sample of 90 lm HNE alone in Tris buffer was processed in parallel to evaluate the amount of HNE eventually retained by nonspecific interactions with the membrane of the Ultrafree-MC Centrifugal Filters. Competitive binding test to determine the dissociation constant for HNE–OBP complexes The dissociation constants for the complexes between the different OBP forms and HNE were determined, as des- cribed previously for other ligands [17,19], by competitive binding tests with the fluorescent ligand AMA. Recombin- ant porcine (1 lm) and bovine OBP (0.5 lm), dissolved in 20 mm Tris ⁄ HCl buffer pH 7.8, were preincubated at room temperature for 20 min with AMA (1.5 and 1.0 lm for por- cine and bovine OBP, respectively). Samples (1 mL) of these solutions were then poured into different tubes con- taining increasing amounts of HNE (1.0–70.0 lm) in which the hexane of the HNE stock solution had been previously removed under a nitrogen stream. The samples were incuba- ted for another 30 min at room temperature and the binding of HNE was followed as a decrease of the fluorescence emission of the AMA–OBP complex at 480 nm upon excita- tion at 380 nm (excitation and emission slits 5 nm). Titra- tion in the absence of OBP allowed us to exclude that HNE might lead to changes of the fluorescence intensity of AMA. The apparent K d values for the HNE–OBP complexes were determined from the competition curves analysed as two parameters hyperbolic decays using the nonlinear fit- ting function of sigma plot 5.0 (Cambridge Software Corp.). The true K d values were then calculated from Eqn (1) [17]: K true d ¼ K app d  1 1 þð1=K AMA d ½AMAÞ ð1Þ where K AMA d is the dissociation constant of the AMA–OBP complex. Competitive binding tests with heat-denatured OBPs (incubated overnight at 90 °C) were performed to determine the specificity of the interaction between the protein and HNE. S. Grolli et al. OBP as a scavenger for HNE FEBS Journal 273 (2006) 5131–5142 ª 2006 The Authors Journal compilation ª 2006 FEBS 5139 Immunoblotting of HNE-modified OBP Formation of HNE–OBP covalent adducts was induced by incubating 0.5 mL of 1 mgÆmL )1 porcine and bovine OBP (52 and 26 lm respectively) for 4 h at 37 °C with increasing super-saturating amounts of HNE (0.1–2.5 mm). The high- est levels of HNE were chosen to exceed, in terms of molar ratio, the number of protein amino acid residues (13 and 21 for porcine and bovine OBP, respectively) that could react with the aldehyde. Aliquots (10 lL) were run on SDS ⁄ PAGE [27] while the remaining volume of each sam- ple, after dialysis in 20 mm Tris ⁄ HCl buffer pH 7.8, was stored at 4 °C for a functionality assay with the OBP fluor- escent ligand AMA (see below). After electrophoresis, the protein bands were transferred onto a poly(vinylidene diflu- oride) membrane [28] that was blocked with 5% skimmed dry milk dissolved in 20 mm Tris ⁄ HCl buffer, pH 7.8 con- taining 150 mm NaCl, and 0.01% (w ⁄ v) Tween 20 (TTBS). After washing with TTBS the membrane was incubated with an anti-(HNE–protein adducts) serum raised in rabbit and diluted (·1000) in TTBS ⁄ containing 1% (w ⁄ v) skim- med dry milk. These two steps were repeated with the secondary antibody conjugated to horseradish peroxidase (dilution factor 2000). HNE–chemically modified OBP bands were visualized with diamino-benzidine. The functionality of the OBP samples employed in the immunoblotting experiment was tested with the fluorescent ligand AMA as described above. The residual AMA bind- ing of each sample was normalized to reference binding val- ues obtained with OBP samples which had not been treated with HNE. Titration of HNE-reacting residues in OBP The titration was specifically designed for the quantitative determination of the OBP HNE-reacting residues that had been displayed, at the qualitative level, throughout the immunoblotting experiment described above. Briefly, samples of porcine and bovine OBP (120 and 60 lm, respectively) in 0.2 mL of 20 mm Tris ⁄ HCl pH 7.8 buffer were incubated with increasing amounts of HNE (0.09–5 mm). One half of each sample was stored at 4 °C for an overnight incubation (16 h) while the remaining part, after 20 min, was treated with 0.4 mL of a solution con- taining a large excess (5 mm) of the OBP ligand undecanal [5–19], to displace the aliquot of HNE present in the bind- ing site. The solutions were then concentrated on Ultrafree- MC, 10 000 Da cut-off, centrifugal filters and the amount of HNE not associated with OBP was determined spectro- photometrically in the ultrafiltrates on the basis of the absorbance at 223 nm. The number of HNE covalent adducts ⁄ equivalent of OBP was finally calculated by sub- tracting the values determined in the ultrafiltrates from the initial amounts of HNE. The second aliquot of each sample that had been stored at 4 °C was than treated with the same procedure to determine if the number of covalent adducts increased with time (16 h). Biochemical characterization of OBP exposed to HNE in a test tube assay simulating conditions of oxidative stress in nasal mucosa These experiments were carried out incubating porcine and bovine OBP at their presumed physiological concentrations in nasal mucosa (1 and 0.5 mm, respectively, for pOBP and bOBP) [6], with HNE at the levels reported in case of acute oxidative stress in vivo (2.0 mm) [1]. Samples (50 lLin 20 mm Tris ⁄ HCl buffer pH 7.8) were incubated for 20 min while the temperature of 25 °C is that of inhaled air in favourable environments of most mammalian species [22]. The solutions were then concentrated on Ultrafree-MC, 10 000 Da cut-off, centrifugal filters after the addition of 0.95 mL of 5 mm undecanal in the same Tris buffer. The amount of HNE-reacting groups was determined as repor- ted above. The functionality of the OBP samples was finally determined with AMA direct binding tests. Evaluation of the protective role of OBP against HNE cytotoxicity in a simplified model simulating nasal mucosa Preparation of immobilized bovine OBP (i-bOBP) A6· His affinity tag was placed at the N-terminus of bovine OBP by PCR using a specific primer. The fused cDNA was subcloned in the expression vector pT7-7 and the expression of the protein was realized as reported above for the recombinant forms of porcine and bovine OBP. The purification of the protein was obtained by affinity chroma- tography with a Ni-NTA Agarose (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The aggregation state was determined by gel permeation by a Superdex 70 column (Amersham Pharmacia, Milano Italy) in FPLC, while the binding tests with AMA, HNE and Undecanal were carried out as described above for the recombinant OBPs. Following purification, the protein was immobilized again to the Ni-NTA-Agarose (i-bOBP) and was employed for the tests on living cells described below. Cell culture and treatment BT epithelial cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (v ⁄ v) and 50 lgÆmL )1 gentamycin. Cells were grown at 37 °C, 5% CO 2 in a humidified incubator. Exponentially growing cells were seeded in 2 mL of reduced serum medium (2.5% fetal bovine serum) at a den- sity of 40 000 cellsÆwell )1 in the lower compartment of a six-well Costar Transwell plate. After 72 h, the following treatments were added in the upper compartment of the OBP as a scavenger for HNE S. 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Odorant binding protein has the biochemical properties of a scavenger for 4-hydroxy-2-nonenal in mammalian nasal mucosa Stefano Grolli, Elisa Merli,. proteins (OBP) are soluble lipocalins produced in large amounts in the nasal mucosa of several mammalian species. Although OBPs can bind a large variety of