Characterization of the oligomerization and ligand-binding properties of recombinant rat lipocalin 11

Biochimica et Biophysica Acta 1834 (2013) 1–7

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Characterization of the oligomerization and ligand-binding properties of recombinant rat lipocalin 11 Yina Gu a, b, Qiang Liu c, Peiyan Chen a, Chenyun Guo a, Yan Liu a, Yufen Zhao a, Yonglian Zhang c, Donghai Lin a, b, d,⁎ a

The key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China Shanghai Key Laboratory for Molecular Andrology, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China d Laboratory of Biomolecular NMR, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China b c

a r t i c l e

i n f o

Article history: Received 6 May 2012 Received in revised form 17 August 2012 Accepted 20 August 2012 Available online 28 August 2012 Keywords: Lipocalin Lcn11 Dimer Disulfide bond Ligand-binding β-barrel

a b s t r a c t Lipocalin 11 (Lcn11), a recently identified member of the lipocalin family, potentially plays crucial physiological roles in male reproduction. In this present work, we cloned, expressed and purified the rat Lcn11 (rLcn11) protein in Escherichia coli. A C59A/C156A substitution was introduced to ameliorate the misfolding and aggregation problem associated with the wild-type protein. From circular dichroism and non-reducing SDS–PAGE, we characterized the conformational properties of rLcn11 as a typical lipocalin scaffold with the conserved disulfide bridge. The results obtained from size-exclusion chromatography, cross-linking experiment and dynamic light scattering analysis indicate that the recombinant rLcn11 protein forms dimer in neutral solution. By using fluorescent probe 8-anilino-1-naphtahlene sulfonic acid (ANS), we found rLcn11 might contain multiple hydrophobic binding sites for ligand binding. Similarly to the odorant-binding protein, rLcn11 processes a moderate affinity for binding 1-aminoanthracene (AMA), implying that Lcn11 might work as a dimeric chemoreception protein in male reproductive system. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Lipocalins constitute a phylogenetically conserved protein family which functions in binding and transport of a variety of physiologically important ligands [1]. Despite low levels of sequence similarities (usually lower than 30% of amino acid identities), this family holds one to three short conserved sequence motifs and a similar folding pattern: an eight-strands β-barrel flanked by an α-helix at the C-terminus [2]. The β-barrel in the lipocalin encloses a central apolar cavity that usually serves for diverse binding of small hydrophobic molecules such as retinol [3], odorant molecules [4], and pheromones [5,6]. Some lipocalins physiologically function in reproductive processes. For example, L-PGDS is the crucial enzyme responsible for synthesizing

Abbreviations: rLcn11, rat lipocalin 11; L-PGDS, lipocalin-type prostaglandin D synthase; OBP, odorant-binding protein; MUP, major urine protein; PBP, pheromone-binding proteins; WT, wild-type; LB, Luria-Bertani; IPTG, isopropyl β-D-thiogalactoside; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; DLS, dynamic light scattering; EGS, Ethylene glycolbis (succinimidylsuccinate); CD, circular dichroism ⁎ Corresponding author at: The Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China. Tel./fax: +86 592 2186078. E-mail address: [email protected] (D. Lin). 1570-9639/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbapap.2012.08.018

prostaglandin D2 [7], rodent major urinary proteins (MUPs) act as pheromone carriers mediating a range of reproductive effects like accelerated puberty [8], behavioral responses [6] and pregnancy block [9]. Meanwhile, several epididymis-specific lipocalins have been identified and characterized, including Lcn5 known as an epididymal retinoicacid-binding protein [10] and Lcn6 associated with sperm maturation [11]. The lipocalin 11 (Lcn11) gene has been recently identified in the epididymal gene cluster at 25.9 Mb from centromere on the locus of mouse chromosome 2 [12,13]. Lcn5, Lcn8, Lcn9, Lcn10 and Lcn12 genes on the same cluster are expressed only in the epididymis, whereas Lcn11 is expressed not only in the epididymis but also in other male reproductive tracts including testis, vas deferens, and prostate [12]. These observations imply that the Lcn11 protein might play specific roles in male reproduction pathway. However, both detailed physiological functions and molecular mechanisms of Lcn11 remain to be addressed. In the present work, we successfully cloned, expressed and purified rat Lcn11 (rLcn11) protein in Escherichia coli. We assessed the oligomerization of rLcn11 in solution using size-exclusion chromatography, cross-linking experiment and dynamic light scattering (DLS) experiment. The physicochemical properties of rLcn11 were characterized by a combination of mass, circular dichroism (CD), non-reducing SDS– PAGE and homology modeling approach. Moreover, ligand-binding

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properties of rLcn11 were addressed by fluorescence titration experiments using 8-anilino-1-naphtahlene sulfonic acid (ANS) and 1-aminoanthracene (AMA). Our results suggest that Lcn11 might behave as a dimeric chemoreception protein in male reproductive process. 2. Materials and methods 2.1. Materials The pET-22b vector was from Novagen. The in-fusion cloning kit and KOD-Plus-Mutagenesis-Kit were purchased from Clone-tech and TOYOBO companies, respectively. Ni-NTA superflow resin was obtained from Qiagen. The BCA protein assay kit was purchased from Pierce. Ethylene glycolbis [succinimidyl succinate] (EGS), fluorescent probes ANS and AMA were brought from Sigma. 2.2. Sequence alignment and homology modeling Amino acid sequences of homologous lipocalins from the GenBank database (http://www.ncbi.nlm.nih.gov) were aligned by Clustal W [14]. The three-dimensional structure of the dimeric rLcn11 protein was modeled using the 3Djigsaw program [15] and the HADDOCK [16] web service. The crystal structure of A chain in dimeric trichosurin protein was selected as template (PDB ID: 2R73_A), which shares a sequence similarity of 63% with the rLcn11 protein. The key residues on the dimer interface of trichosurin were matched to rLcn11 and inputted as active residues on the HADDOCK web server. Ten modeling structure clusters of the dimeric rLcn11 were built. The structure with the best docking score has been chosen and displayed. The modeling structure was visualized and inspected with the MOLMOL software [17]. 2.3. Rat Lcn11 cloning A pair of primers carrying Nde I and Xhol I restriction sites were designed (5′-AAGGAGATATACATATGCTTCAGGATCAAACCAATGTT-3′, 5′-GGTGGTGGTGCTCGAGGAGTGTGTCACACTCACGAGAGAA-3′). PCR experiments were performed using the plasmid pGEM-T-rLcn11 as template. The amplification products were one-step cloned into the pET-22b expression vector using the in-fusion enzyme. The double cysteine mutations (C59A/C156A) were performed by KOD-PlusMutagenesis-Kit. The generated mutant was referred to C59A/C156A rLcn11. All the constructions were sequenced and analyzed.

Tris/HCl, 50 mM NaCl, 100 mM sucrose, 1 mM EDTA, 1 mM GSH, 0.1 mM GSSG, pH 8.2). After 24 h the buffer was changed to 1 L of buffer D (50 mM Tris/HCl, 50 mM NaCl, pH 8.2). 2.5. Size exclusion chromatography The proteins were further purified and analyzed by fast protein liquid chromatography (FPLC) on a Superdex-75 column (GE Healthcare, Piscataway, NJ) with buffer E (50 mM NaH2PO4, pH 6.8) at a flow rate of 0.6 mL/min. The column was calibrated with bovine serum albumin (66.2 kDa), ovalbumin (44.0 kDa) and L-PGDS (20.0 kDa) in buffer E. Buffer E was used in all the following experiments for characterization of the rLcn11 protein. The presence of putative disulfide bridge of C59A/C156A rLcn1l was confirmed by non-reducing SDS–PAGE. The protein concentrations were determined using a BCA protein assay kit. 2.6. Protein cross-linking experiment The cross-linking agent EGS was dissolved in dimethyl sulfoxide (DMSO), and then added a 40-fold molar excess to the rLcn11 protein solution in buffer E. After incubation at 4 °C for 30 min, the reaction was terminated by addition of the denaturing buffer and analyzed by SDS–PAGE. 2.7. Dynamic light scattering experiment Dynamic light scattering experiment was performed at 4 °C with a DynaPro NanoStar™ instrument (Wyatt Technology, Europe GmbH). A sample of purified C59A/C156A rLcn11 in buffer E (10 μL) was centrifuged at 16,000 × g for 10 min followed by injection into a sample cuvette. Thirty parallel measurements were conducted, then the molecular weight of the protein was estimated from the hydrodynamic diameter using globular conformation model by the Dynamics V7 software (Wyatt Technology Europe GmbH). 2.8. Mass spectrometry Matrix-assisted laser desorption ionization time-of-flight (MALDITOF) mass experiments were performed on a Bruker microFlex MALDITOF-MS spectrometer equipped with a nitrogen laser of 337 nm wavelength. The protein sample was applied to a pre-spotted thin-layer matrix sinapinic acid and operated in the positive ion/linear mode with an accelerating voltage of 20 kV.

2.4. Recombinant rLcn11 expression and refolding The pET22b–rLcn11 plasmid was transformed into E. coli BL21 (DE3) cells. The classical system was designed for the expression of rLcn11 with a minimal N-terminal His6-tag. Transformed cells were inoculated in LB (Luria-Bertani) medium at 37 °C with 100 μg/mL ampicillin. When OD600 reached 0.6–0.8, the rLcn11 expression was induced with 0.5 mM IPTG (isopropyl β-D-thiogalactoside). After 5 h, cells were harvested by centrifugation at 6000 × g for 10 min at 4 °C, then frozen at −80 °C. Frozen cell pellets were thawed and resuspended in buffer A (50 mM Na2HPO4, 500 mM NaCl, 5% glycerol, pH 8.0), then lysed by ultrasonic breaking at 400 W for 30 min. The cell lysate was centrifuged at 10,000 × g for 30 min to pellet insoluble inclusion bodies. The inclusion bodies were washed twice by buffered 0.5% (v/v) Triton X-100, 2 M NaCl sequentially. For each buffer, the inclusion bodies were resuspended, incubated on ice for 30 min, centrifuged (10,000 × g at 4 °C for 10 min) and then suspended again in the next buffer. Thereafter, inclusion bodies were treated with 30 mL of buffer B (8 M urea, 50 mM Tris/HCl, pH 8.2), and incubated for 2 h on ice to denature and solubilize inclusion bodies. The denatured proteins were purified by Ni-NTA affinity chromatography and then refolded by dialysis at 4 °C against 1 L of buffer C (50 mM

2.9. Circular dichroism spectroscopy Circular dichroism spectra were recorded on a JASCO-810 spectropolarimeter (Jasco, Tokyo, Japan) with a quartz cell of 0.1 cm path length. The measurements were conducted on WT and C59A/ C156A rLcn11 (0.01 mM) in buffer E at 25 °C. Spectra were recorded from 190 to 260 nm by accumulating three consecutive scans with a bandwidth of 1.0 nm and a response time constant of 1.0 s. The spectra were processed by first subtracting a blank spectrum followed by baseline correction, and then normalized for the mean residue weight according to Eq. (1) [18]:

½θMRW ¼

½θobs ⋅MR c⋅d⋅N A

ð1Þ

where [θ]obs denotes the measured ellipticity, MR the molecular mass of rLcn11 [g/mol], c the protein concentration [mg/mL], d the path length of the quartz cuvette [cm], and NA the number of amino acid residues.

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2.10. Fluorescence binding assay

3. Results and discussion

Ligand binding experiments were performed at 25 °C using a Hitachi F-4500 spectrofluorimeter with slits set at 5 nm bandwidth. In the case of AMA binding to rLcn11, fluorescence was excited at 295 nm and detected at 504 nm. The titration experiments were performed on different samples of rLcn11 (2 μM) incubated at 25 °C for 2 h with various amount of AMA ranging from 0.3 to 15 μM. The slight volume increase of the ligand stock solution in the titration process was neglected. Three repeated measurements were performed, and each one was corrected by a subtraction of the blank value. The titration data were analyzed by the Origin software according to Eq. (2).

3.1. Cloning, expression, refolding and purification

½RL ¼

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð½R þ ½L þ K d Þ− ð½R þ ½L þ K d Þ2 −4½R½L 2

ð2Þ

where [R] and [L] are total concentrations of the protein and the ligand, respectively. [RL] is the concentration of the complex, and Kd is the apparent dissociation constant. For ANS, fluorescence was excited at 370 nm and detected at 499 nm. The stock solutions of the ligand were added stepwise in 0.4–2 μL of aliquots to 2 mL of the purified rLcn11 protein solution (5 μM) in buffer E. After each titration, the protein sample was mixed and incubated in the dark for 2 min before fluorescence measurements. Scatchard analysis was carried out to determine binding parameters for ANS–rLcn11 interaction. An experimentally accessible parameter r (Eq. (3)), defined as the average number of bound ANS molecules per monomeric rLcn11 molecule, is introduced in the case of n moles of ligands bind to one protein, assuming n sites are equal and independent.

n½Lcn11−ANS r¼ Lcn11−ANS þ ½Lcn11free

ð3Þ

Eq. (3) can be rearranged into a suitable form for graphical treatment (Eq. (4)). r n r ¼ − ½ANSfree K d K d

ð4Þ

Thus a plot of r/[ANS]free against r gives a straight line with a slope of − 1/Kd and an intercept of n/Kd on the x axis.

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The open reading frame of rLcn11 encodes 177 amino acid residues with a highly hydrophobic transmembrane domain of 16 residues at the N-terminus identified as signal peptide. The coding sequence of the mature rLcn11 protein (17–177) was cloned into the pET-22b vector with a His6-tag at the C-terminus. The amino acid sequence of rLcn11 contains four cysteines (Cys59, Cys84, Cys156 and Cys174). Sequence alignment illustrates that rLcn11 holds two conserved cysteines Cys84 and Cys174, which may form a typical intramolecular disulfide bridge of the lipocalin family [19] (Fig. 1). No extra disulfide bond would be formed between the nonconservative cysteines, Cys59 and Cys156. Therefore, they might be involved in the wrong formation of either intramolecular or intermolecular disulfide bonds, which would potentially trigger protein misfolding and aggregation. Previous studies demonstrated that mutation of the nonconservative cysteines usually does not significantly change tertiary structures and functions of lipocalins, but might benefit the expression and purification of the recombinant proteins [20,21]. Thus, we introduced the C59A/C156A substitution for the wild-type (WT) rLcn11 protein to ameliorate the misfolding and aggregation problem. Both WT rLcn11 and C59A/C156A mutant were mostly expressed in inclusion bodies with a maximal amount when the expression was conducted at 37 °C for 5 h in the present of 0.5 mM IPTG. The supernatant of cell lysis was abandoned which contained most of extraneous proteins but little target proteins (Fig. S1, lane 3). After the inclusion bodies were washed by two steps and purified by a Ni-NTA affinity column, the eluted fraction was obtained with a purity of approximately 90% (Fig. S1, lane 4). Precipitated impurities were further removed during the slow refolding process by dialysis against buffers. Both the WT and C59A/C156A proteins were obtained with high purity (>95%) after refolding (Fig. S1, lanes 5 and 6). The C59A/C156A proteins were obtained with a yield of approximately 17 mg for 1 L of LB culture by collecting the dimer peak in the size-exclusion chromatogram (Fig. 2a), which is much higher than that of the WT proteins (3 mg/L culture). The mass spectrometry analysis confirms the primary structure of the recombinant C59A/C156A rLcn11 protein, which is associated with the peak of 19,452.6 Da (Fig. S2). This value is in accordance with the theoretical molecular weight of recombinant C59A/C156A rLcn11. 3.2. Evidence for protein dimerization Size-exclusion chromatograms of both WT and C59A/C156A rLcn11 exhibit two major peaks at positions of about 8.0 and 11.2 mL, suggesting that refolded rLcn11 proteins possess at least two oligomeric

Fig. 1. The structure-based sequence alignment of rLcn11 and four homologous lipocalins. The primary sequences of rLcn11 (Gene ID: 100169711), trichosurin (PDB ID: 2R73_A), bovine odorant-binding protein (PDB ID: 2HLV), major horse allergen Equ c 1 (PDB ID: 1EW3_A) and bovine β-lactoglobulin (PDB ID: 1DV9) were aligned by Clustal W. The conserved amino acids are colored in red. Schematic diagram of rLcn11 secondary structure was displayed by ESPrint.

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Sequence alignment of rLcn11 and four homologous lipocalins illustrates that rLcn11 possesses the typical primary structure characteristic of the lipocalin family in the present of several conserved amino acid residues (Fig. 1). Most of the homologous lipocalins sharing higher similarities with rLcn11 could form dimers, including trichosurin (63% similarities) [22], bovine odorant-binding protein (bOBP, 49% similarities) [23], major horse allergen Equ c 1 (45% similarities) [24] and bovine β-lactoglobulin (bBLG, 40% similarities) [25]. As a metatherian lipocalin from the milk whey, trichosurin dimerizes in solution at pH 4.6 and pH 8.2. At the core of the dimerization interface, aromatic interactions, salt bridges and numerous intermolecular hydrogen bonds contribute to the stability of the trichosurin dimer [22]. Furthermore, bovine OBP also exists as dimer in neutral solution in which each monomer has a α-helix protruding out of β-barrel and crossing the dimer interface called “domain-swapped” protein structure [26,27]. Besides, allergen Equ c1 crystallizes and also behaves as a dimeric protein in solution at pH 7.0, as indicated by gel filtration analysis [24,28]. On the other hand, previous works showed that both acidic pH environment and low protein concentration could usually favor pH-induced dissociation of dimeric lipocalins [22,28,29]. Our work demonstrates that the recombinant rLcn11 dimer could also be partly dissociated when pH was decreased to 2.6 and protein concentration was reduced to 5 μM (data not shown). While there is a small peak (~ 10 mL) between the peaks of oligomer and dimer on the size-exclusion chromatogram, it could be assigned as the misfolding protein instead of another oligomeric rLcn11 protein like trimer. Since there is no observable trimer band at 66 kDa on the SDS–PAGE diagram after cross-linking for this collection of the protein peak. Besides, from dynamic light scattering analysis, we did not observe an average molecular weight consistent with the rLcn11 trimer. In general, we confirm that the recombinant rLcn11 protein could form a dimer in neutral solution through a series of size-exclusion chromatography, cross-linking experiment and DLS analysis. 3.3. Conformational characterization

Fig. 2. (a) Size-exclusion chromatography of C59A/C156A rLcn11 (solid line) and WT rLcn11 (dotted line) at pH 6.8. The elution was monitored by absorbance at A280. (b) SDS–PAGE analysis of the cross-linking experiment: lane 1, C59A/C156A rLcn11 before cross-linking reaction; lane 2, C59A/C156A rLcn11 after crosslinking reaction with EGS; lane 3, lysozyme (negative control) before crosslinking reaction; lane 4, lysozyme after cross-linking reaction. The band of dimeric rLcn11 is marked by an arrow. No band of lysozyme dimer was observed.

statuses in buffer E (Fig. 2a). The first peak (8.0 mL) corresponds to a protein with a molecular weight larger than 66.2 kDa, indicating an oligomeric pattern higher than trimer. The polymer formation might result from the highly hydrophobic property of rLcn11 and/or wrong disulfide bridges formed by solvent accessible cysteines [21]. The second peak (11.2 mL) is related to a protein with a molecular weight less than 44 kDa. Taking account of the theoretical molecular weight of dimeric rLcn11 (39 kDa), the second peak could be assigned as dimeric rLcn11 protein. This peak was collected for the following cross-linking and DLS experiments with the same protein concentration (0.03 mM) in buffer E. We performed cross-linking experiments to confirm the dimeric status of C59A/C156A rLcn11. We observed a band between 35 and 45 kDa in the SDS–PAGE diagram (Fig. 2b), which was associated with dimeric rLcn11. This result confirms again that the second peak in the size-exclusion chromatogram (Fig. 2a) is related to dimeric rLcn11. Furthermore, DLS experiments measured the hydrodynamic diameter of the putative dimeric rLcn11 protein (Fig. S3). Based on the measured diameter of 2.88 nm, the average molecular weight of the rLcn11 protein was estimated to be 40 kDa, indicative of the dimeric status of C59A/C156A rLcn11 in neutral solution.

Far-UV CD spectra of dimeric WT and C59A/C156A rLcn11 display a minimum near 215 nm and a positive peak below 200 nm (Fig. 3), which obviously reveals the typical secondary structure of a predominantly β-sheet protein. Both WT and C59A/C156A rLcn11 contain abundant β-sheet structures (>60%), indicating that dimeric rLcn11 is a well-folded β-rich protein. To confirm the formation of the intramolecular disulfide bond between Cys84 and Cys174, we compared the reducing and nonreducing SDS–PAGE diagrams of C59A/C156A rLcn11 (Fig. S4, with and without DTT). A non-reduced band close to the molecular weight

Fig. 3. Far-UV CD spectrum of WT rLcn11 (solid line) and C59A/C156A rLcn11 (dotted line) in 50 mM NaH2PO4 buffer (pH 6.8) at 25 °C.

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Fig. 4. Front view of the three-dimensional structure of dimeric rLcn11 constructed by homology modeling and HADDOCK software. Eight cysteine residues and two conserved disulfide bridges are displayed as red sticks.

marker of 18.4 kDa is clearly distinguished from the reduced band located between 18.4 and 25.0 kDa. The protein sample without DTT migrates faster than that with DTT, suggesting that the non-reduced protein might adopt a more compact shape due to the presence of the intramolecular disulfide bond. In addition, non-reduced protein does not display a band associated with dimeric C59A/C156A rLcn11 (about 40 kDa), implying that the dimerization of rLcn11 is not due to the wrong formation of intermolecular disulfide bridge. The three-dimensional structure of dimeric rLcn11 was modeled using the crystal structure of A chain in dimeric trichosurin as templet. The rLcn11 protein adopts a typical scaffold of the lipocalin family: eight-stranded antiparallel β-sheet composing the β-barrel framework with one α-helix near the C-terminus (Fig. 4). A highly-conserved disulfide bond is formed between two conservative cysteines (Cys84 and Cys174) in each chain, which ties the C-terminus to the β4 strand. Moreover, two nonconservative cysteines (Cys59, Cys156) appear on the protein surface, which have been replaced with alanines in the C59A/C156A mutant. The intermolecular interactions among four antiparallel β-strands (β5–β8) and their connecting loops contribute to the formation of rLcn11 putative dimer interface. Further structural investigation will be expected to explain the details of rLcn11 protein dimerization. 3.4. Ligand binding assay Lipocalins exhibit diverse functions mostly related to ligand binding abilities. We performed ligand binding assays with fluorescent probes ANS and AMA to access the molecular mechanism of ligand transportation function for rLcn11. We used ANS as a probe to evaluate the binding affinity of rLcn11 for hydrophobic ligands. From fluorescence titration curves (Fig. 5a) and Scatchard plot (Fig. 5b), we determined two binding parameters, dissociate constant Kd and stoichiometric proportion n. As indicated by Eq. (4), a plot of r/[ANS]free against r gives a straight line with a slope of − 1/Kd . The Kd value was determined to be 4.9 ± 0.3 μM,indicative of a moderate affinity between C59A/C156A rLcn11 and ANS. We thereby suggest that rLcn11 contains the hydrophobic patch for transporting hydrophobic ligands, similarly to other members of the lipocalin family. In addition, the fluorescence titration curve of WT rLcn11 does not show

significant difference from that of the C59A/C156A mutant (Fig. 5a), implying WT and C59A/C156A rLcn11 possess almost identical binding affinity for ANS. Since the C59A/C156A mutation does not significantly change the conformation and hydrophobic ligand-binding affinity of rLcn11, we suggest that Cys59, Cys156 and the nearby residues in the protein might not participate in binding hydrophobic ligands. Evidences from cysteine mutations in other lipocalins, such as C176A rLcn12 mutant [21] and C116S ApoD mutant [20], also confirmed that minor chemical differences between WT lipocalins and the cysteine mutants did not distinctly alter affinities for binding ligands. Furthermore, Fig. 5b indicates that one monomeric rLcn11 molecule binds three ANS molecules. As the intracavity space of rLcn11 is not large enough to accommodate three ANS molecules, we suggest that a hydrophobic patch potentially exists outside the β-barrel which provides an extra binding site for ANS. Previous works have reported internal and external ANS binding sites on tear lipocalin [30] and betalactoglobulin [31]. Electrostatic interactions between ANS derivatives and positively charged side chains in lipocalins, together with van der Waals interactions, account for higher affinities of the external binding sites on lipocalins for binding ANS [32]. Moreover, a hydrophobic patch on the surface of bovine beta-lactoglobulin was detected and defined as an external binding site [33]. Thus, the rLcn11 protein might also possess similar external binding site for hydrophobic ligands. Based on the assumption of equivalent and independent sites, the measured Kd is thereby a macroscopic apparent dissociation constant describing the identical affinity for all bound isomers in all binding steps. AMA is another fluorescent probe that is widely used to detect hydrophobic cavities in lipocalins. Different from the probe ANS, titration of AMA to the rLcn11 solution induced a decrease in the protein tryptophan fluorescence emission at 340 nm accompanied with an increase of the emission intensity at 504 nm corresponding to AMA fluorescence (Fig. 6a). The decrease of tryptophan fluorescence intensity might result from both ligand-binding inducing quenching and resonance energy transfer between tryptophan residues and AMA [34]. The rLcn11 protein contains two tryptophan residues, Trp38 and Trp148. The modeling structure of rLcn11 shows that Trp38 is located in a hydrophobic environment, while Trp148 is situated on the external hydrophilic surface. Since AMA shows a weak fluorescence emission

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with a maximum at 560 nm in aqueous buffer, but exhibits an enhanced fluorescence emission with a maximum at 504 nm in a hydrophobic environment (Fig. 6a). A blue shift of AMA fluorescence emission was observed which accounts for the more hydrophobic environmental change of AMA upon binding to rLcn11. Therefore, our data suggest that AMA binds to a hydrophobic patch around Trp38 in rLcn11 protein. Further, we determined the binding affinity of AMA to rLcn11 by fluorescence titration experiments. Fitting the fluorescence titration curve (Fig. 6b) gave an apparent dissociation constant of 1.69 μM for interaction between AMA and C59A/C156A rLcn11, which is similar to the binding affinities previously reported for other lipocalins, such as OBPs [35–37], pheromone-binding proteins (PBPs) [34], α1-glycoprotein [38], and bovine beta-lactoglobulin [39]. In bovine OBP–AMA complexes, AMA binds to the internal pocket of OBP, which was confirmed as the general binding site for other odorant molecules like 1-octen-3-ol [37,40]. Moreover, the previous work demonstrated that Phe35 and Tyr82, locating in the buried cavity of the β-barrel in porcine OBP, were involved in uptake and release of both AMA and the pheromone undecanal [41]. In these experiments, AMA was generally used as a substitute of odorants and pheromones to detect the biological functions of chemoreception proteins. The binding affinity of rLcn11 for AMA is close to those of other homologous proteins, implying that rLcn11 might also contain similar binding sites for pheromones and odorants. However, the competitive binding assay for rLcn11 showed that, bound AMA molecules were not readily to be replaced by other ligand molecules like 1-octen-3-ol and undecanal (data not shown). Overall, rLcn11 displays an analogous characteristic of binding AMA, suggesting

Fig. 6. (a) The fluorescence emission spectra of AMA binding assay show a maximum at 340 nm for C59A/C156A rLcn11 and 504 nm for AMA. (b) Fluorescence titration curve of C59A/C156A rLcn11 with AMA.

it might also act as a chemoreception protein for transporting small chemical ligands such as pheromones and odorants. Taking into account its specific expression and putative physiological function in the male reproductive tracts, future studies would be conducted to explore the potential ligands of rLcn11 in such pathway. 3.5. Conclusions In summary, we have established an efficient approach for expression and purification of the rLcn11 protein. Our results indicate that rLcn11 forms dimer in neutral solution, which shares the typical β-barrel scaffold and a conserved intramolecular disulfide bond with other lipocalins. Furthermore, ligand binding assays implicate that rLcn11 might contain hydrophobic patches both inside and outside the β-barrel. Based on these data, we presume that rLcn11 potentially functions as a chemoreception protein for transporting small hydrophobic ligands in male reproductive process. This work provides insights into the structure–function relationship of Lcn11 and may be helpful for the studies of other dimeric lipocalins. Acknowledgements

Fig. 5. (a) Fluorescence titration curves of WT rLcn11 (dotted line) and C59A/C156A rLcn11 (solid line) with ANS. (b) Scatchard plot for ANS binding to C59A/C156A rLcn11.

We thank Prof. Xu Shen for providing the help with the DLS measurement. This work was financially supported by grants from the Natural Science Foundation of China (nos. 91129713, 31170717, and 30730026), the Program of Shanghai Subject Chief Scientist (nos. 09XD1405100).

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Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.bbapap.2012.08.018.

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