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⁄
Trang 1of 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, -NH2 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 (Kd¼ 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 m M NaCl and 0.01% (W ⁄ V) Tween 20.
Trang 2bloodstream 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 (Kdin 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
Kd 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
Trang 3reversible 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
0
20
40
60
80
100
120
140
160
180
0 20 40 60 80 100 120 140 160 180
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0.0 0.2 0.4 0.6 0.8 1.0 1.2
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.
Trang 4of 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 Kd 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 90C)
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 37C) 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] μ M / [OBP] μ M × 2 0 2 10 20 50 0 2 10 20 50
A
0.0
0.2
0.4
0.6
0.8
1.0
1.2
B
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.
Trang 5in 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
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] µ M / [pOBP] µ M [HNE] µ M / [bOBP] µ M × 2
0
2
4
6
8
10
0 2 4 6 8 10 12 14
[HNE] µ M / [pOBP] µ M
0.0
0.2
0.4
0.6
0.8
1.0
1.2
[HNE] µ M / [bOBP] µ M × 2
0.0 0.2 0.4 0.6 0.8 1.0 1.2
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 reacincuba-ting residues was plotted versus the ratios between incubaincuba-ting 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.
Trang 6resistance of OBP to chemical attack by the aldehyde,
especially under experimental conditions in which
incu-bation times are closer to physiological values found
in vivoin 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 20C 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 phase-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 Kd 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
Trang 7the 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
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 l M HNE in the presence of 40 l M 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.
Trang 8OBP 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, nastracheobronchi-al 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
Trang 9The concentration was controlled on the spectrophotometer
(e223¼ 13750 m)1Æcm)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)1Æcm)1for 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 4C 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
plot5.0 (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 4C, 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 (excitaexcita-tion 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 Kd 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 Kd values were then calculated from Eqn (1) [17]:
Ktrued ¼ Kappd 1
1þ ð1=KAMA
where KAMA
d is the dissociation constant of the AMA–OBP complex
Competitive binding tests with heat-denatured OBPs (incubated overnight at 90C) were performed to determine the specificity of the interaction between the protein and HNE
Trang 10Immunoblotting 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 37C 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 4C 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 bindbind-ing
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 4C
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 4C 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 lL in
20 mm Tris⁄ HCl buffer pH 7.8) were incubated for 20 min while the temperature of 25C 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)
A 6· 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
37C, 5% CO2in 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