Heparin binding carboxypeptidase E protein exhibits antibacterial activity in human semen

International Journal of Biological Macromolecules 64 (2014) 319–327

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Heparin binding carboxypeptidase E protein exhibits antibacterial activity in human semen Sanjay Kumar a , Anil Kumar Tomar a , Sudhuman Singh b , Kamaldeep Gill a , Sharmistha Dey a , Sarman Singh c , Savita Yadav a,∗ a

Department of Biophysics, All India Institute of Medical Sciences (AIIMS), New Delhi, India School of Life Sciences, Jawaharlal Nehru University (JNU), New Delhi, India c Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India b

a r t i c l e

i n f o

Article history: Received 31 July 2013 Received in revised form 13 December 2013 Accepted 15 December 2013 Available online 21 December 2013 Keywords: Semen CPE Heparin Antibacterial

a b s t r a c t Carboxypeptidase E (CPE) cleaves basic amino acid residues at the C-terminal end and involves in the biosynthesis of numerous peptide hormones and neurotransmitters. It was purified from human seminal plasma by ion exchange, heparin affinity and gel filtration chromatography followed by identification through SDS-PAGE and MALDI-TOF/MS analysis, which was further confirmed by western blotting. CPE was characterized as glycoprotein by Periodic Acid Schiff (PAS) staining and treating with deglycosylating enzyme N-glycosidase F. The interaction of CPE with heparin was illustrated by surface plasmon resonance (SPR) and in silico interaction analysis. The association constant (KA ) and dissociation constant (KD ) of CPE with heparin was determined by SPR and found to be 1.06 × 105 M and 9.46 × 10−6 M, respectively. It was detected in human spermatozoa also by western blotting using mouse anti-CPE primary antibody. 20–100 ␮g/ml concentration of CPE was observed as highly effective in killing Escherichia coli by colony forming unit (CFU) assay. We suggest that CPE might act not only in the innate immunity of male reproductive tract but also regulate sperm fertilization process by interacting heparin. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Human semen is complex body fluid consisting of proteins, carbohydrates, lipids and ions. These components involve in different steps of fertilization. The protein macromolecules include different classes of peptidases and proteases. The proteases like kallikrein, seminine and collagenase were well characterized in human seminal plasma [1–6]. Kallikrein cleaves a substrate, known to enhance the motility of spermatozoa [7]. The peptidases comprise of elastase (metallopeptidase), enkephalinase (neutral endopeptidase) and angiotensin convertase (kininase-II) has been implicated previously in seminal plasma [8–10]. The kininase-II hydrolyzes the peptide substrate bradykinin and may involve in reproductive physiology of spermatozoa. Since bradykinin was found in seminal plasma and enhance the sperm motility [11]. Carboxypeptidase was detected in human seminal plasma, which catalyses C-terminal basic amino acid residues from polypeptides and may be a part of proteolytic degradation of semen during the liquefaction process [12] but this study lacks in categorization of carboxypeptidase.

∗ Corresponding author at: Department of Biophysics, All India Institute of Medical sciences (AIIMS), New Delhi 110029, India. Tel.: +91 126546445; fax: +91 126588641. E-mail address: [email protected] (S. Yadav). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.12.020

CPE designed as an enkephalin convertase or carboxypeptidase H, one of the key enzymes responsible for the biosynthesis of peptide hormones and neuropeptides [13,14]. It was found as a membrane bound and soluble forms in secretory granules differing in molecular mass of 2–3 kDa with a region specific relative distribution in the brain, pancreatic islets of Langerhans, rat heart, testis, ovary and lungs [15–18]. The soluble form of CPE is released by hypo-osmotic lysis process under neutral pH, while membrane bound form by a process of detergent extraction or alkaline lysis under the high salt concentration [14]. CPE is different from other forms of carboxypeptidases like CPN and CPB, in that it is active within an optimal acidic pH [19]. Zinc is required as a coordinating metal in the active sites of CPE for hydrolysis of peptide substrate [20]. Met-enkephalin, substance P, endorphin and calcitonin are present in significant amount in human seminal plasma and it has been evidenced that these peptides regulate sperm motility process [21–23]. Thus, CPE may use these peptide hormones as substrate and regulate the reproductive physiology of spermatozoa. The current investigation was employed for the first time to purify and characterize an antibacterial and heparin binding CPE protein from human semen. This protein exhibited a potent antibacterial activity against Escherichia coli and binding ability with heparin. Our results suggest that CPE might act not only in the innate immunity but also in sperm fertilization process by binding heparin.

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2. Materials and methods 2.1. Collection and processing of semen samples Human semen samples were collected after 3 days of sexual abstinence in Department of Laboratory Medicine, All India Institute of Medical Sciences (AIIMS), New Delhi, India. These samples were kept for liquefaction at room temperature, cocktail of protease inhibitors (Sigma–Aldrich, USA) added and were evaluated for various parameters such as semen volume, sperm concentration and sperm motility according to WHO 2009 guidelines. Normozoospermic semen samples having a sperm-count more than 20 millions/ml and sperm motility more than 60% were chosen in this study. The samples were pooled and centrifuged at 10,000 × g for 20 min at 4 ◦ C. The supernatant was clarified further by centrifuging at 10,000 × g for 20 min at 4 ◦ C. The supernatant was finally stored at −20 ◦ C as seminal plasma for further use. This study was approved by the Institute Ethics Committee, AIIMS, New Delhi and informed consent was obtained from each of the individuals. 2.2. Purification of CPE from seminal plasma using chromatography The pooled crude seminal plasma (10 ml) was diluted in ammonium acetate buffer (50 mM, pH 6.0) and loaded into the pre-equilibrated CM-sephadex C-50 cation exchanger column. The unbound proteins were collected and concentrated using ultrafiltration unit (Millipore, MD) having 3 kDa cut off membrane. It was further applied to heparin-Sepharose CL-6B affinity column, which was equilibrated with ammonium acetate buffer. The unbound proteins were washed out and bound proteins were eluted by stepwise salt gradient 0.1 M, 0.2 M and 0.5 M NaCl in ammonium acetate buffer (50 mM, pH 6.0). The optical density of each fraction was measured at 280 nm. The protein fractions of each peak (0.1 M, 0.2 M and 0.5 M) were pooled separately, desalted and concentrated. 10% SDS-PAGE was used to resolve the proteins of each peak under reducing conditions. Twenty milligrams of lyophilized heparin bound proteins eluted by 0.1 M NaCl were dissolved in 500 ␮l of ammonium acetate buffer (50 mM, pH 6.0) and loaded to sephadex G-75 (1.6 cm × 125 cm) gel filtration column. The column was pre-equilibrated with 50 mM ammonium acetate buffer (pH 6.0) containing 0.15 M NaCl. One ml volume of each protein fraction was collected at the flow rate of 6 ml/h and monitored at 280 nm. The purity and molecular weight of proteins of each peak were analyzed on 10% SDS-PAGE under reducing and nonreducing conditions as previously described protocol [24]. The gel was stained with Coomassie Brilliant Blue G-250. The peak II protein solution of gel filtration was also visualized by silver staining to rule out the other protein contaminants. In brief, SDS-PAGE gel was kept overnight in a fixing solution (40% ethanol, 10% glacial acetic acid and 50% distilled water) and washed with 30% ethanol for 20 min followed by washing with milli-Q water for 20 min (4 times). The gel was then sensitized in 0.02% Na2 S2 O3 solution for 1 min and incubated in silver stain (0.2% AgNO3 and 0.02% formaldehyde) for 30 min in dark. The gel was kept in solution containing 3% Na2 CO3 , 0.0005% Na2 S2 O3 and 0.05% formaldehyde for 5 min by continuous shaking till the spots appeared. Finally, the gel was stopped by 11% glycine solution (Fig. 1). 2.3. Mass spectrometric analysis and protein identification The intact molecular mass of protein solution (second peak of gel filtration) was analyzed by MALDI-TOF/MS. 1 mL of protein (1 mg/mL) was added to 1 mL of sinapinic acid matrix solution (dissolved in acetonitrile/water (1:1) at a concentration of 5 mg/mL),

spotted onto a stainless steel MALDI target plate and dried at room temperature before analysis. Mass spectra were obtained with an average of ten shots of the laser beam in a Bruker Daltonics Autoflex MALDI-TOF/MS instrument operating in linear and positive ion mode. To analyze the protein, mass spectra were acquired using the instrument parameters: pulsed ion extraction delay of 30 ns, ion source voltage one, 20 kV, ion source voltage two, 18.65 kV and ion source lens voltage 7.1 kV. The single protein band was also manually excised from the SDS-PAGE gel. The dye was completely removed by washing the bands thrice in acetonitrile: H2 O (50%, v/v) containing ammonium bicarbonate (25 mM). The in-gel tryptic digestion was carried out as previously described method [25]. Four microliters of reconstituted sample was mixed in 1 mL of sinapinic acid matrix solution (Bruker Daltonik, Germany) and spotted onto a MALDI target plate. The analysis was carried out using a Bruker Autoflex MALDI-TOF mass spectrometer. Peptides from mass spectra were matched against protein NCBI human databases using the mascot search engine (Matrix Sciences, London, UK) for peptide-mass-fingerprinting (PMF). The top mascot score was used for the selection of significant protein. 2.4. Western blotting To further validate the mass spectrometry results, western blotting experiments were performed using primary-antibody against CPE. The second peak of gel filtration fractions was analyzed by 10% SDS-PAGE. The proteins were transferred to nitrocellulose membrane (Millipore, MA) and blocked with 50 mM phosphate buffer saline (pH 7.4, 200 mM NaCl) containing 5% skimmed milk for 3 h at room temperature. The membrane was then probed with mouse anti-CPE primary antibody (BD Bioscience) diluted in 1:2000 in PBS. Then the blot was washed with PBS containing 0.1% Tween-20 followed by incubating it with secondary antibodies (goat antimouse Ig G, 1:2000) labeled with horseradish peroxidase. Further it was developed with enhanced chemiluminenscence (ECL) substrate and exposed to X-ray film. 2.5. Identification of CPE in spermatozoa Human spermatozoa were obtained by centrifuging the liquefied semen at 2000 rpm for 15 min. The spermatozoa pellet was washed with PBS containing cocktail of protease inhibitors by repeated centrifugations (2000 rpm for 15 min) to remove residual soluble components. Only spermatozoa suspensions whose final wash does not show immunoreactivity with anti-CPE antibody was used for the experiments. Then, it was sonicated for four times with 10 s on and off pulse at 4 ◦ C in 150 ␮l of lysis buffer consisting of 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 1 mM EDTA and a cocktail of protease inhibitors. The mixtures were dialyzed against PBS for 24 h. After dialysis, it was mixed in sample buffer and analyzed on SDS-PAGE under reducing condition. The location of CPE band was detected by western blotting with mouse anti-CPE primary antibody as described above. 2.6. Glycosylation analysis Periodic Acid Schiff (PAS) staining was conducted to characterize CPE as glycoprotein. The purified CPE was analyzed on 10% SDS-PAGE. After electrophoresis, the gel was submerged in 12.5% (w/v) trichloroacetic acid (TCA) for 30 min followed by extensive washing with distilled water. Then, it was incubated with 1% periodic acid (prepared in 3% acetic acid) for 50 min at room temperature and washed thrice with distilled water. The gel was stained with fuschin-sulphite in dark for 1 h and was washed thrice

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Fig. 1. Purification of CPE from human seminal plasma using ion exchange, affinity and gel filtration chromatography. (A) Elution profile of heparin-Sepharose CL-6B affinity column. The peaks formed as a function of X-axis as an elution volume in ml and Y-axis is absorbance at 280 nm. (B) SDS-PAGE pattern of peaks obtained from heparinSepharose CL-6B affinity column. M – protein molecular weight marker. Lane I – elution profile of peak I (0.1 M elution). Lanes II and III – elution profile of peak II (0.2 M elution), and peak III (0.5 M elution), respectively. (C) Elution profile of Sephadex G-75. The peaks were obtained as a function of X-axis as an elution volume in ml and Y-axis is absorbance at 280 nm. (D) The standard curve for G-75 and a line drawn from the position of CPE on the standard curve. (E) and (F) 10% SDS-PAGE was applied to analyze the second peak of Sephadex G-75 in reducing and non-reducing conditions, respectively. Lane I – protein molecular weight marker. Lane II – single band of CPE in second peak in reducing condition (E) and non-reducing conditions (F).

with 0.5% metabisulphite. Finally, it was washed with running tap water for 2 min to remove excessive stain. CPE was deglycosylated with N-glycosidase F (Sigma–Aldrich) for 24 h at 37 ◦ C. Each deglycosylation reaction was composed of CPE (20 ␮g), 1 ␮l of glycoprotein reaction buffer (containing 5 mM DTT and 0.5 M NaCl in 20 mM Na2 HPO4 ), 2 ␮l of deglycosylation enzyme (N-glycosidase F) and H2 O to make a 10 ␮l total reaction volume. A parallel reaction (without N-glycosidase F) for CPE was carried out as a control. The both reactions were stopped by boiling in SDS sample buffer at 100 ◦ C for 5 min and analyzed through western blotting after separation of proteins on 10% SDS-PAGE. Expasy proteomics NetNGlyc 1.0 server was used to predict the N-glycosylation sites available in CPE (Fig. 2). 2.7. Interactions of CPE with heparin 2.7.1. Surface plasmon resonance (SPR) studies The binding interactions between CPE and heparin were studied by SPR analysis with an automatic instrument BIAcore 2000

(Biosensor AB, Uppsala, Sweden). Heparin (30 mg) was dissolved in 3 mL of 50 mM sodium bicarbonate (pH 8.3) and incubated with 50 mM Sulfo-N-hydroxy succinimide-long chain biotin (SNHS-LCbiotin) for 2 h at 4 ◦ C. It was dialyzed against 50 mM sodium bicarbonate buffer (pH 8.3) to remove free biotin followed by lyophilization and stored at −20 ◦ C. It was dissolved in 10 mM HBS-EP buffer (150 mM Sodium Chloride, 3 mM EDTA, 0.005% Polysorbate 20) up to 0.1 mg/ml. 5 ␮l of biotinylated heparin was injected over flow cell 2 of the CM5 sensor chip at a flow rate of 5 ␮l/min and was immobilized. The unreacted groups were blocked by 20 ␮l of 1 M ethanolamine (pH 8.5). 30 ␮l of saturated biotin was used as a background control in flow cell 1 on the chip. The different concentrations (75, 150 and 300 nM) of purified CPE suspended in 10 mM sodium acetate buffer were injected into different flow cells (one with immobilized heparin and other without heparin as a reference cell) at a constant flow rate of 5 ␮l/min through microflow system for 3 min. The dissociations between them were followed by 10 mM sodium acetate buffer (pH 5.0) for 2 min and the regeneration of the heparin bound with purified CPE was carried out

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Fig. 2. (A) MALDI-TOF/MS spectra of whole protein CPE. The peaks obtained as a function of percentage abundance (Y) against m/z (mass/charge) ratio (X axis). The molecular mass protein is shown. (B) Immunoblotting of CPE using mouse anti-CPE primary antibody. Lane I – purified CPE in peak II of Sephadex G-75 gel filtration, Lane II – CPE in crude seminal plasma. (C) It does not show band, when it was incubated in secondary antibody only (without primary antibody) and used as negative control. (D) 10% SDS-PAGE patterns of human spermatozoa lysates. Lane I – protein molecular weight, Lane II – spermatozoa lysates. (E) A single band of CPE detected by western blotting in spermatozoa lysates. (F) It does not show band when it was incubated in secondary antibody only (without primary antibody) and used as negative control. (G) PAS staining shows a single pink band of CPE as a glycoprotein in SDS-PAGE. (H) Western blot analysis showing deglycosylation of CPE. CPE protein band shifted in lower position (Lane II) after treatment with N-glycosidase F as compared to CPE (Lane I) not treated with N-glycosidase F.

using 0.1 mM NaOH. The all the experiments were repeated in triplicates to reduce the errors. The equilibrium association constant (KA ) and dissociation constant (KD ) were determined by fitting the primary sensorgram data into 1:1 Langmuir binding model using BIA evaluation software 3.0 (GE Healthcare). 2.7.2. In silico structure analysis Amino acid sequence of CPE was retrieved from Protein Knowledgebase, UniProtKB (P16870) and its structure was modeled by homology modeling method using Modeler 9v7. The 3-D structure model was further refined by KoBaMIN and structure quality was checked using Structural Analysis and Verification Server (SAVS) (http://nihserver.mbi.ucla.edu/SAVES) by analyzing residue-by-residue geometry and overall structure geometry. Structure co-ordinates for heparin disaccharide and heptasaccharide were obtained from complex structures 4DY0 and 1SR5, respectively, from Protein Data Bank (www.rcsb.org). In silico docking analysis of CPE with heparin was performed by Hex program [26] based on shape and electrostatics correlations. UCSF Chimera v1.5.3 was used as a visualization tool and for generating highresolution 3D structure figures [27] (Fig. 3).

2.8. Antibacterial property of CPE 2.8.1. Colony forming unit assay (CFU) A colony forming unit (CFU) assay was employed to determine the antimicrobial activity of CPE in the presence and absence of its specific inhibitor GEMSA (2-guanidinoethylmercaptosuccinic acid, Santa Cruz, India). E. coli cells were allowed to grow in Luria–Bertani broth. The cells at log phase were washed with ice cold 10 mM sodium phosphate buffer (pH 7.4) and exposed to purified CPE protein diluted (in the absence of GEMSA) in 10 mM sodium phosphate buffer (pH 7.4) with or without NaCl (concentration ranging 0–350 mM) for 3 h at 37 ◦ C. After incubation, the treated cells were diluted in different concentrations (10, 20, 40, 60 and 100 ␮g/mL) in 10 mM sodium phosphate buffer (pH 7.4) and spread on 1 mm thick Luria–Bertani agar plates for overnight at 37 ◦ C. The same experiments were repeated with purified CPE incubated with 1 mM GEMSA. Surviving colonies were counted by hand and the formula: % survival = 100 × (number of colonies surviving after the treatment of E. coli with CPE/total number of colonies survived for E. coli not treated with CPE) was used to calculate the antibacterial activity. All values were the means of results from at least three experiments.

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Fig. 3. (A) SPR sensorgrams shows the interaction of CPE with heparin. Heparin immobilized on CM5 sensor chip as described in Section 2. The concentration of heparin was fixed at 10 mM while the concentrations of CPE varied from top to bottom 300, 150, and 75 nM, respectively. (B) Ramachandran plot analysis showing different regions of modeled CPE protein. (C–F) Docking analysis for CPE interaction with heparin disaccharide and heparin heptasaccharide. (C) Heparin disaccharide (stick model) binding on CPE surface. (D) CPE (ribbon model) interaction with heparin disaccharide (stick model); CPE amino acid residues critical for interaction are labeled and shown as sticks. (E) Heparin heptasaccharide (ball and stick model) binding on CPE surface. (F) CPE (ribbon model) interaction with heparin heptasaccharide (wire model); CPE residues critical for interaction are labeled and shown as sticks.

2.8.2. Scanning electron microscopy (SEM) The effect of CPE on morphology of E. coli cells were observed under SEM as described in the protocol [28]. E. coli bacterial cells grown at log phase were resuspended in 10 mM phosphate buffer saline (PBS) and incubated with 20 ␮l purified CPE protein (1 mg/ml) for 4 h at 37 ◦ C. Bacterial cells were washed thrice at

2000 rpm for 15 min each and kept overnight in 4% glutaraldehyde for fixation at 4 ◦ C. These were filtered onto a 0.1 ␮m polycarbonate membrane with the help of a vacuum pump. The filter membrane was washed with 0.15 M sodium phosphate buffer followed by dehydration with graded ethanol series (30–100%) and stored in dryer for 24 h. The mounting was done on aluminum specimen

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Fig. 4. (A) E. coli were incubated with different concentrations of CPE and antibacterial activity was determined by CFU assay. The assays were performed in triplicate. The values are indicated as mean ± SD and P values < 0.01. (B) E. coli were incubated with 40 ␮g of CPE in varied concentration of NaCl (0–350 mM) in 10 mM phosphate buffer. (C) The scanning electron micrographs illustrate the E. coli cells treated with CPE for 3 h. (D) Normal E. coli cells not treatment with CPE. Note that the treatment of E. coli with CPE displaying roughening and shrinkage of cells surfaces with normal E. coli cells, indicating the membrane killing of bacteria.

and coated with 15 nm thick gold platinum alloy. Ultimately, samples were taken to scanning electron microscope (LEO Electron Microscopy Inc., Thornwood, NY) using an accelerating voltage of 20 kV (Fig. 4). 3. Results 3.1. Purification of native CPE from human seminal plasma The concentrated unbound proteins from CM sephadex C-50 cation exchanger column were fractionated on heparin CL-6B affinity column as three peaks I, II and III (Fig. 1A). These peaks were resolved on SDS PAGE (Fig. 1B) and CPE immunoreactivity was found only in peak I (Supplementary Fig. S1). Six bands were visualized in pooled and concentrated fractions of 0.1 M elution (peak I) on SDS-PAGE (Fig. 1B, Lane I). The peak I was further separated in sephadex G-75 gel permeation column. Four peaks were observed in the gel filtration chromatography, out of which the immunoreactivity was observed only in peak II (Fig. 1C). The standard curve for gel filtration was prepared between elution volume and molecular weight of standards (Fig. 1D). The single band was seen in peak II protein elution, which corresponds to the apparent molecular weight of 50 kDa on SDS-PAGE under

reducing and non-reducing condition in Coomassie stained gel (Fig. 1E and F, Lane II). The silver stain demonstrates that there were no other protein contaminants in SDS-PAGE gel except single band in peak II protein solution of gel filtration (Supplementary Fig. S2). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2013. 12.020. The protein solution of peak II was further processed for their intact molecular mass determination and purity by MALDI-TOF, where molecular mass observed at 50,159.5 Da (Fig. 2A). The single band was also excised manually, in gel digested with trypsin and the spectra were finally identified as CPE by MALDI-TOF/MS analysis (Supplementary Fig. S3). The detailed report is summarized in Table 1 and Supplementary Table S1. Western blot of purified CPE protein (peak II of gel filtration curve) and crude seminal plasma confirmed the presence of CPE using mouse anti-CPE primary antibody (Fig. 2B, Lane I and Lane II). While, no band was observed in negative control (Fig. 2C), when incubated with secondary antibody only. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2013. 12.020.

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Table 1 Identification of single band on SDS-PAGE (Lane II in Fig. 1E) of peak II in gel filtration column (Fig. 1C) by MALDI-TOF/MS. Protein name

Accession number

Number of peptides matched

Sequence coverage (%)

Mowse score

Database search

Carboxypeptidase E

gi|4503009

11

31.7

93

NCBInr

3.2. CPE identified in human spermatozoa Lysates prepared from human spermatozoa were used for this study. To avoid possible individual differences, these lysates were obtained from pooled samples at least four different ejaculates. The western blotting experiment was performed to analyze the CPE in human spermatozoa lysates as discussed in material and method. The lysates were diluted in sample buffer and analyzed on SDSPAGE according to their molecular weight (Fig. 2D, Lane II). Then, a strong protein band was developed in western blotting using CPE primary antibodies (Fig. 2E). While, no band was observed in negative control (Fig. 2F) when incubated with secondary antibody only. 3.3. Glycosylation studies Purified CPE was confirmed as a glycoprotein in the form of pink band on SDS-PAGE by PAS staining (Fig. 2G). The purified CPE was further deglycosylated with N-glycosidase F and compared with CPE protein (untreated with N Glycosidase F) in western blotting. There was shifting of band in a lower position in case of deglycosylated as a comparison to glycosylated form of CPE (Fig. 2H). The predictions for glycosylation sites in CPE protein by NetNGlyc 1.0 servers revealed that protein had two potential glycosylation sites at Asn139 and Asn390 position well above the threshold. 3.4. CPE binds heparin with higher affinity The interactions of CPE with heparin were analyzed by surface plasmon resonance (SPR) and the association constant (KA ) for the formation of molecular complex and dissociation constant (KD ) were determined. The binding of analytes was monitored by changes in RU values of the sensorgram, where the progress of the interaction was plotted against time, indicating the binding characteristics. Three different concentrations of CPE (75, 150 and 300 nM) were passed over the immobilized heparin and RU values were observed as 300, 600 and 960, respectively. RU values were determined using the standard curve of purified CPE. The linear change in RU values with varying concentrations of CPE indicated the change in mass on the heparin immobilized on chip with time and the dissociation constant (KD ) was found to be 9.46 × 10−6 M (Fig. 3A). The 3-D structure of human CPE (P16870) was predicted by Modeler, refined by KoBaMIN and analyzed by SAVS. Procheck Ramachandran plot analysis revealed that structure obtained had 99.4% residues in allowed region and only 0.6% residues (ASP132 and GLU226) in generously allowed regions (Fig. 3B). This structure was passed by Verify 3D (with 89.6% residues having an average 3D-1D score > 0.2) and overall quality factor was 82.8 (Errat). Best CPE and heparin complexes predicted by Hex were analyzed carefully by Chimera. The aim of this analysis was to find an orientation which can provide important information about heparin interacting site on CPE. In silico structure analysis revealed that CPE has high affinity to heparin, as inferred by a large number of interacting residues. A cavity formed by CPE residues, including ASN172, LYS175, MET195, PRO198, SER292, ASN293, ASN348, THR361, ALA363, LYS364 was predicted as a heparin-binding site in case of a heparin disaccharide (Fig. 3C and D). When docking analysis was performed with heparin heptasaccharide, an additional

interacting cavity formed by ARG33, ILE34, TYR35, and THR36 was found critical. Additionally, two other residues HIS58 and LEU169 provided more stability to the first interactive site (Fig. 3E and F). It is well known that positively charged residues are critical for heparin interaction as it is negatively charged and both of the interacting sites predicted in our study also contain positively charged residues. 3.5. Antibacterial activity The percentage survival rate of E. coli was not reduced significantly with different concentrations of CPE in the presence of inhibitor GEMSA (Supplementary Fig. S4) in CFU assay. Whereas, it demonstrated detectable killing activity against E. coli bacteria in a concentration dependent manner (Fig. 4A) in the absence of GEMSA. Though, CPE (at 10 ␮g/ml concentration) did not produce any bactericidal activity in 3 h of incubation but percentage survival of bacteria was reduced with increasing concentration from 20 to 100 ␮g/ml for the same incubation period. BSA at a concentration of 100 ␮g/ml did not exhibit bacterial killing and was included as a negative control in the assay (data not mentioned). The antimicrobial property of protein and peptides is affected by salt concentrations because monovalent or divalent cations involve electrostatic interactions with bacterial surface [27]. CPE maintained its antibacterial activity in the broad range of NaCl concentration (Fig. 4B). In the absence of NaCl, the percentage survival of bacteria due to CPE was found to be zero. There was an increase in bacterial survival with rise in the NaCl concentration from 50 to 350 mM. This resulted that CPE produce salt tolerant antibacterial property. Supplementary material related to this artican be found, in the online version, at cle http://dx.doi.org/10.1016/j.ijbiomac.2013.12.020. The antibacterial action of proteins undergoes various mechanisms during processing of bacteria. Some proteins involved autolytic processing to stimulate its antibacterial property [29], while cationic proteins undergoes through the membrane permeabilization to kill bacterial cells [30]. SEM experiment was performed to examine the effect of CPE on the morphology of E. coli. The E. coli cells were grown, incubated with CPE and observed under SEM. The untreated E. coli cells appeared smooth and normal surface morphology (Fig. 4D). While, treated E. coli cells with CPE exhibits membrane wrinkling and surface blebbing (Fig. 4C). Such type of changes produced by CPE in E. coli gives evidence of its membrane dependent killing. 4. Discussion The seminal fluids contain a variety of biochemical components that are essential for sperm functions. Male infertility is responsible for the failure of conception in approximately 30% of infertile couples [31] and no definite cause has been reported in 25% of those couples [32]. Spermatozoa along with seminal plasma are distributed into the female reproductive tract in physiological reproduction and seminal plasma proteins play a key role in conception during vitro fertilization [33]. Therefore, in that context we have purified CPE from human seminal plasma by relatively simple three-step procedure of ion exchange, affinity and gel permeation chromatography. It was identified by

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MALDI-TOF/MS and further confirmed by western blotting using mouse anti CPE primary antibody. The purified seminal plasma CPE shows an apparent molecular weight 50 kDa on SDS-PAGE under reducing and non reducing condition, which illustrated that it is found in monomer form. Although the seminal carboxypeptidase exists as heterodimer of 32 kDa and 26 kDa under reducing conditions [12]. PAS staining has demonstrated the glycosylated form of CPE. The PNGase F treatment with CPE produced a shift of band position on western blot due deglycosylation, which was supported by presence of two N linked glycosylation sites at Asn 139 and 390 Asn position as per prediction by NetNGlyc 1.0 server. This confirmed that CPE in human seminal fluid is N-glycosylated. CPE is synthesized as precursor containing 25 amino acid signal amino acids that directs it to rough endoplasmic reticulum and is then removed followed by its glycosylation through post-translational modification in the Golgi complex to form secondary and tertiary structures [34]. Glycosylation is essential for cellular and biological functions of proteins [35]. CPE was identified in spermatozoa lysates as well as in seminal plasma by western blotting experiment. Some proteins have been reported in both seminal plasma and spermatozoa but little is understood about their distribution in both the specimen. Eppin, lactoferrrin and clusterin were synthesized in the testis and secreted in the seminal fluid indicate that some amount of these come into the seminal plasma while rest of its amount likely to assemble on the spermatozoa during the last step of spermiogenesis [36]. Here, CPE shows the similarities with that of carboxypeptidase M (CPM) in molecular weight but CPM has been reported as bound to cell membrane in placenta, kidney, lung and blood vessels [37], while CPE was found in soluble form. Although, the source of CPE in human semen is still not known. It has been previously hypothesized that CPE may be membrane bound in prostate [12,38]. Therefore, further research is needed to point out the source of secretion of CPE in male reproductive tract. Heparin binding ability of CPE was assessed using heparin sepharose column chromatography, which was further confirmed by SPR studies. In silico interaction analysis performed in present study, also supports our SPR data and suggests that CPE binds to heparin with higher affinity than other proteins of human seminal plasma like lactoferrin (LF) fragment and semenogelin I (SGI) fragment [39]. The some of the seminal plasma proteins interact with acid polysaccharides of the heparin and hyaluronan types. These proteins play important role in the formation of oviduct sperm reservoir, sperm capacitation and sperm–oviduct interaction. Glycosaminoglycans (GAGs) are present in high concentration in the female reproductive tract that modulates capacitation reaction by binding to the proteins of spermatozoa [40]. Heparin is the one of the most potent GAGs tested, which enhances capacitation in bovine and rabbit spermatozoa [41–43]. Heparin does not involve alone in capacitation of epididymal spermatozoa. But, when accessory gland proteins (having the ability to bind heparin) are incubated with epididymal spermatozoa, these spermatozoa became able to undergo capacitation and respond to the zona pellucida during acrosome reaction [44]. Here, CPE was also detected in human spermatozoa lysates, which may have a role in capacitation by interacting heparin present in the female reproductive tract in human and regulate the fertilization process. A number of microorganisms may infect the reproductive tract tissues and semen in humans and other animals, which can alter the reproductive and endocrine functions of the animals. These pathogens may enter the testis through the blood and lymphatic vessels crossed the interstitial compartment of the testis, rete testis and scrotum skin [45]. The dysfunction of the epididymis may be because of the microorganisms present in the blood vessels supplying these organs and may cause infertility [46]. The human seminal

plasma has been known for its antibacterial properties for decades although its active agent had not been detected [47,48]. Some proteins and peptides of human seminal plasma display antibacterial properties. ␤-Defensins involves in the antimicrobial properties by interacting and disruption of the membrane of target microorganism [32,49]. We are reporting here for the first time the antibacterial activity of CPE against E. coli in the absence of GEMSA. It demonstrates that CPE is synthesized in the male reproductive tract where it may contribute specific role in inhibition of bacterial growth and may protect the reproductive organs like prostate, epididymis and testis from damage to occur. Furthermore, the bacterial growth was not influenced by CPE in the presence of specific inhibitor GEMSA, which indicates that enzymatic activity is required for the antibacterial property of CPE. In conclusion, we identified a CPE in both human seminal plasma and spermatozoa. This report highlights the antibacterial activity and heparin binding properties of CPE. Which contributes the new information on the function of CPE in both fertilization and innate immunity in human. Hence, the function of CPE in human semen is unclear; therefore, this research output will widen our knowledge on investigation of CPE related to reproductive functions in future. Acknowledgements We thank to Electron Microscopy Facility, All India Institute of Medical Sciences, New Delhi and Advance Instrumentation Research Facility (AIRF), JNU, New Delhi for SEM experiments. This work is supported by the grants from Indian Council of Medical Research (ICMR), New Delhi. Sanjay Kumar and Anil Kumar Tomar thank ICMR, New Delhi for their fellowships. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]

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