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Three low molecular weight cysteine proteinase inhibitors of human seminal fluid: Purification and enzyme kinetic properties
Biochimie 95 (2013) 1552e1559
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Research paper
Three low molecular weight cysteine proteinase inhibitors of human seminal fluid: Purification and enzyme kinetic properties Vikash Kumar Yadav a, Nirmal Chhikara a, Kamaldeep Gill a, Sharmistha Dey a, Sarman Singh b, Savita Yadav a, * a b
Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India Department of Lab Medicine, All India Institute of Medical Sciences, New Delhi 110029, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 December 2012 Accepted 9 April 2013 Available online 22 April 2013
The cystatins form a superfamily of structurally related proteins with highly conserved structural folds. They are all potent, reversible, competitive inhibitors of cysteine proteinases (CPs). Proteins from this group present differences in proteinase inhibition despite their high level of structural similarities. In this study, three cysteine proteinase inhibitors (CPIs) of low molecular weight were isolated from human seminal fluid (HSF) by affinity chromatography on carboxymethyl (CM)-papaineSepharose column, purified using various chromatographic procedures and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Matrix-assisted laser desorption-ionization-time-of flight-mass spectrometry (MALDI-TOFMS) identified these proteins as cystatin 9, cystatin SN, and SAP-1 (an N-terminal truncated form of cystatin S). All three CPIs suppressed the activity of papain potentially and showed remarkable heat stability. Interestingly SAP-1 also inhibits the activity of trypsin, chymotrypsin, pepsin, and PSA (prostate specific antigen) and acts as a cross-class protease inhibitor in in vitro studies. Using Surface Plasmon Resonance, we have also observed that SAP-1 shows a significant binding with all these proteases. These studies suggest that SAP-1 is a cross-class inhibitor that may regulate activity of various classes of proteases within the reproductive systems. To our knowledge, this is the first report about purification of CPIs from HSF; the identification of such proteins could provide better insights into the physiological processes and offer intimation for further research. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Chromatography Cross-class inhibition Cystatins Kinetics Papain Surface plasmon resonance
1. Introduction Cysteine proteases are accountable for a lot of biological processes occurring in human body [1]. The main physiological role of CPs is intracellular catabolism of peptides and proteins [2], malfunctioning has been implicated in the development and progression of many diseases [3e6]. The activity of CPs is regulated by their specific natural protein inhibitors called cystatins. Cystatins have been isolated and characterized from different human tissues and body fluids [7e14]. Their inhibitory profiles, as well as their affinities for target enzymes, vary with different CPs. Appropriate steadiness between free CPs and their complexes with inhibitors is critical for the regulation of the proteolytic activity under
Abbreviations: CM, carboxymethyl; CPs, cysteine proteinases; CPIs, cysteine proteinase inhibitors; CRES, cystatin-related epididymal spermatogenic; HSF, human seminal fluid; LMW, low molecular weight; PCI, protein C inhibitor; PSA, prostate specific antigen. * Corresponding author. Tel.: þ91 11 2653201; fax: þ91 11 26588641. E-mail address: [email protected] (S. Yadav). 0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2013.04.007
normal physiological conditions [15e18]. Based on the molecule complexity, cystatins have been categorized into three families [19,20]: Family 1 cystatins (stefins) are found mainly intracellularly and have molecular weights of 12 kDa, family 2 cystatins (S, SA, SN, C) are essentially found extracellularly and have a molecular weight of 14 kDa, and family 3 cystatins are the high molecular weight kininogens. All of the characterized cystatins exhibit sequence homologies. The presence of the different cystatins and their exact functions in the male accessory sex glands is largely still unknown. Cystatin A has been demonstrated to be present in the basal cells, in all cases of benign prostatic hyperplasias (BPH), low-grade prostatic intraepithelial neoplasias (PIN), and high-grade PIN and can aid in the diagnosis of prostatic adenocarcinoma [21]. Cystatin C is highly expressed and widely distributed throughout the male genital tract, suggesting that cystatin C is an important regulator for normal and pathological proteolysis in the male reproductive system [22]. Moreover, the presence of cystatin-related epididymal spermatogenic (CRES) proteins in the sperm acrosome, suggests their role in sperm maturation and fertilization [23]. Seminal
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plasma also contains cystatin S, D, and B in relatively high amounts; however their roles remain to be established. CPIs are the dominant inhibitors in seminal plasma [13,22] therefore, may also have some important, although as yet unknown, roles in fertilization. Further studies are needed to explore their role with special attention to fertility and other functions in semen. In the present study, we have purified and identified three LMW cystatins of HSF using various chromatographic steps in a sequential manner and subsequent characterization of these proteins may promote our knowledge of biochemical mechanisms involved in human fertilization in future. We have also determined the enzymatic properties of the inhibitors to be able to assess their potential capacity as physiologically important inhibitors of CPs of human reproductive tract and/or in HSF. 2. Material and methods 2.1. Sample collection Freshly ejaculated normal human semen was collected and pooled from the Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi. Ethical permission was taken from the same institute for the study (Ref. No.IESC/T-154/ 2010). Proteases (Papain, trypsin, chymotrypsin, pepsin, and
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proteinase K), hemoglobin, and trichloroacetic acid were purchased from SigmaeAldrich (St Louis, MO, USA). Molecular weight marker (Unstained Protein Markers) was from Fermentas. All other chemical and reagents were of analytical grade and obtained from a local supplier. 2.2. Isolation and purification of LMW cystatins To obtain seminal plasma, 20 ml of semen was centrifuged at 1300 g for 15 min at 4 C. The seminal plasma was further clarified by centrifugation at 10,000 g for 15 min. The supernatant was then dialyzed against 50 mM sodium phosphate buffer (pH 6.5, containing 0.2 M NaCl, 2 mM EDTA, and 10 mM sodium azide) and was chromatographed at 4 C on CM-papaineSepharose column, prepared as described in Ref. [24]. After thorough washing with the buffer, proteins with affinity for carboxymethylated papain were eluted with 0.2 M trisodium phosphate, pH 12, containing NaCl, EDTA, and sodium azide, as in the binding buffer above. Eluted sample was neutralized with 2 M sodium phosphate buffer, pH 6.0, and concentrated at 4 C using ultrafiltration (Millipore, Billerica, MA). The concentrated eluate from affinity column was further applied on DEAEeSephacel (10 2.6 cm) column using 50 mM sodium phosphate, pH 6.0. After extensive washing, bound proteins were eluted with linear gradient of NaCl (0e0.5 M) in same buffer (Fig. 1A).
Fig. 1. Purification of HSF cystatins using affinity, ion exchange and gel filtration chromatography. (A) Elution profile on anion exchanger DEAEeSephacel. The fraction with affinity for carboxymethylated papain was applied to DEAEeSephacel column in 50 mM sodium phosphate, pH 6.0. Elution was carried out with a linear gradient of 0e0.5 M NaCl. (B) Purification of SAP-1 on Sephadex G-50. The fraction (peak 2) containing inhibitory activity was pooled and concentrated. (C) Purification of cystatin SN and cystatin 9 on stronganion exchanger column (Resource Q). The flow-through fractions of the DEAEeSephacel column was used as starting material. Cystatin SN was eluted at 0.05 M NaCl and cystatin 9 at 0.1 M (w/v) NaCl. (D) SDS-PAGE pattern of purified cystatins of HSF under reducing condition and stained with Coomassie blue. Lane 1, molecular weight markers (116 kDa: bgalactosidase b, 66 kDa: Bovine Serum Albumin, 45 kDa: Ovalbumin, 35 kDa: Lactate dehydrogenase, 25 kDa: REase Bsp 981, 18.8 kDa: b-Lactoglobulin and Lysozyme: 14.4 kDa); Lane 2, affinity eluent of CM-papaineSepharose; Lane 3, SAP-1; Lane 4, cystatin SN; Lane 5, cystatin 9.
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Fractions were analyzed for optical density at 280 nm and papain inhibitory activity. Fractions showing protease inhibitory activity were pooled, desalted, and lyophilized. In the next stage of purification, the sample was dissolved and applied to size exclusion chromatography on Sephadex G-50 (120 1.6 cm) in 50 mM sodium phosphate, pH 7.5 (Fig. 1B). Fractions were collected and analyzed for papain inhibitory activity. The flow-through fractions of the DEAEeSephacel column was dialyzed against 20 mM sodium phosphate, pH 7.5, concentrated and applied on Resource Q column (GE-Healthcare), eluted with linear gradient of NaCl (0e0.2 M). cystatin SN was eluted at 0.05 M NaCl and cystatin 9 at 0.1 M NaCl (Fig. 1C). 2.3. Determination of protease inhibitory activity Inhibitory activity was measured with the modified method described by Lee and Lin [25]. 125 mL of sample was preincubated with the same volume of papain (3 mM) dissolved in 50 mM sodium phosphate buffer (pH 6.2), pepsin dissolved in sodium acetate buffer (pH 4.5), and chymotrypsin and trypsin dissolved in TriseHCl buffer (pH 8.2), at 37 C, for 20 min. After preincubation, 250 mL of 1% solution of hemoglobin dissolved in the same buffer was added and the mixture was incubated for 40 min. Then the reaction was stopped by the addition of 1 ml of 5% trichloroacetic acid. Samples were centrifuged at 15,000g for 10 min, and the absorbance of the supernatant was measured at 280 nm. For enzyme standard assay sample was replaced by distilled water and for control assay enzyme solution was replaced by distilled water. The percentage of inhibition was calculated as follows: [(A280 of enzyme standard þ A280 of control) (A280 of sample)]/(A280 of enzyme standard) 100%, where A280 was the absorbance at 280 nm. 2.4. Gel electrophoresis SDS-PAGE was carried out on 12% using 1.5 mm thick slab gels under reducing and non-reducing conditions. Relative molecular masses of separated proteins were estimated using protein standards run in parallel. 2.5. Mass spectrometry Analysis was performed using a Bruker Autoflex MALDI-TOF mass spectrometer equipped with a pulsed 337 nm N2 laser. Operating conditions are as follows: ion source 1 ¼ 19.00 kV, ion source 2 ¼ 16.50 kV, lens voltage ¼ 8.80 kV, reflector voltage ¼ 20.00 kV, optimized pulsed ion extraction time ¼ 80 ns, matrix suppression ¼ 400 Da, and positive reflection mode. Spectra were calibrated externally using the calibrant peptide mixture. Peptides from mass spectra of in-gel digest samples were matched against databases such as Swiss-prot, NCBInr, and MSDB using Mascot search engine (Matrix sciences) for peptide-massfingerprinting. 2.6. Thermal stability of purified inhibitors After purification protease inhibitors were incubated at temperatures ranging from 20 C to 100 C in a water bath for 30 min. The samples were cooled and inhibitory activities against papain were determined. 2.7. SPR-binding studies The binding studies were carried out using the BIAcore 2000 apparatus (Pharmacia BIAcore AB, Uppsala, Sweden). The BIAcore apparatus is a biosensor-based system for real-time specific
interaction analysis [26]. The protein sensor chip was prepared by immobilization of SAP-1 on a research grade CM5 chip (Biosensor AB, Uppsala, Sweden) according to the manufacturer’s recommendations. 60 ml of SAP-1 (30 mg/ml) in 10 mM sodium acetate buffer (pH 5.0) was injected to the flow cell for 4 min at the rate of 5 ml/min and washed for 60 min at the rate of 20 ml/min. The analytes (different proteases) at different concentrations were injected for 4 min at a rate of 10 ml/min. The protein samples were diluted in sodium acetate buffer pH 5.0. Typically, 90 ml of different dilutions of protein were injected at a flow rate of 30 ml/min. The same buffer was passed over the sensor surface at the end of sample injection, to allow dissociation. After a 3 min dissociation phase, the sensor surface was regenerated by 30 ml of 50 mM NaOH and 1 M NaCl. The binding of analytes can be studied by monitoring the change in the resonance unit (RU) values of the sensorgram, where the progress of the interaction was plotted against time, revealing the binding characteristics. Kinetic constants of different proteases were calculated from the association and dissociation phases with BIA evaluation software version 3.0. 3. Results 3.1. Purification of three LMW cystatins from HSF In order to purify cystatins from HSF, the three-step chromatographic procedure was used. The first stage of the process was affinity chromatography on CM-papain affinity column. The affinityeluted protein fractions served as an initial source of cystatins purification. In second stage, affinity-eluted fraction was applied onto a DEAEeSephacel column. As illustrated in Fig. 1A, a single peak of inhibitory activity was detected in eluted fractions of DEAEeSephacel column. The fraction containing protease inhibitory activity was further purified to homogeneity using size exclusion chromatography (Fig. 1B). The cystatin obtained was Nterminal truncated form of cystatin S (SAP-1) and molecular mass corresponds to 14 kDa on SDS-PAGE under reducing conditions (Fig. 1D). In the third stage, two more protease inhibitors were purified and separated through anion-exchange chromatography. The flowthrough of DEAEeSephacel served as the starting material and loaded on strong-anion exchanger Resource Q column at pH 7.5. Two peaks of inhibitory activity were obtained at 0.05 and 0.1 M NaCl (Fig. 1C). These peaks correspond to purified cystatin SN of molecular mass 16 kDa and cystatin 9 of 20 kDa respectively. A Coomassie stained gel of the purified cystatins is shown in Fig. 1D. The protein profile of the CM-papaineSepharose purified preparation was also investigated by SDS-PAGE (Fig. 1D). 3.2. Sequence analysis of purified cystatins Multiple sequence alignments (MSA) of LMW purified cystatins with other members of family 2 cystatins were performed using ClustalW2 on EBI web server (Fig. 2A). Cystatin SN and SAP-1 are member of family 2 cystatins and cystatin 9 also shows maximum homology to this family. Members of family 2 cystatins are about 120 amino acids, have two intra-chain disulfide bonds, and most of them found both in tissues and body fluids. Cystatin 9 shows 18e 27% homology to other members of family 2 cystatins and contains conserved residues which form the inhibitory sites of cystatins, such as the GlyeGly and the ProeTrp in the N-terminus and Cterminus respectively. A close examination of alignment reveals cystatin 9 also have a cysteine rich internal sequence beside the four conserved Cys residues in the cystatins, required for the formation of two disulfide bridges, suggesting that the binding
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Fig. 2. Sequence analysis of family 2 cystatins. (A) Alignment of amino acid sequences of cystatin 9, CRES proteins and members of family 2 cystatins. Purified cystatins are compared with cystatin 8 (accession no. O60676), cystatin 11 (accession no. Q9H112), cystatin S (accession no. P01036), cystatin D (accession no. P28325), and cystatin C (accession no. P01034). Amino acids forming the inhibitory wedge in family 2 cystatins, and the corresponding amino acids in the cystatin 9 are shown in red color. The four cysteines build 2 disulfide linkages in cystatins, are in blue color. The cysteine rich internal sequence is shown in green color in cystatin 9. (B) The branching diagram representing a hierarchy of categories based on degree of similarity or number of shared characteristics.
specificity of cystatin 9 to its target may be different from those of other cystatins. Furthermore, family 2 cystatins also contain a conserved internal sequence glutamineevalineeglycine (QXVXG), which is also completely missing in cystatin 9 (Fig. 2A). 3.3. Kinetic analysis 3.3.1. Antiprotease activity of cystatins Protease inhibition activity of cystatins was tested according to the method of Lee and Lin [25]. Hemoglobin was used as a substrate and percentage inhibition was calculated as described in material and methods. The activity of papain is potentially inhibited by all the purified protease inhibitors but SAP-1 also observed to inhibit the activity of other proteases. We examine the activity of trypsin, chymotrypsin, pepsin, PSA, and proteinase K and found SAP-1 inhibits all these proteases except proteinase K (Fig. 3). SPR results also support the broad spectrum inhibitory property of SAP-1 as it shows significant binding with all proteases. At the inhibitor concentration of 3 mM papain, pepsin, PSA shows almost complete inhibition while in case of trypsin and chymotrypsin 70e80% inhibition is achieved. Negligible inhibition observed in case of proteinase K.
Fig. 3. Antiprotease activity of SAP-1against different class of proteases. Hemoglobin was used as the substrate to determine the inhibition activity.
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Further thermal stability of purified cystatins was experienced. The proteins were incubated at different temperatures, ranging from 20 C to 100 C, for 30 min. The maximum inhibitory activity of cystatins was at around 40 C. The inhibitory activity toward papain remained relatively stable up to 80 C (Fig. 4). Only at 100 C the loss in the inhibitory activity (around 20e40%) was observed suggesting a highly stable tertiary structure resistant to heat. SAP-1 was most heat stable inhibitor among purified ones. 3.3.2. Evaluation of binding potential and binding constants using SPR Trypsin, chymotrypsin, pepsin, papain, PSA and, proteinase K were employed for the experiment. SAP-1 interaction with these proteases was analyzed with surface plasmon resonance (SPR) and kinetics constants were calculated. SAP-1 was immobilized on research grade CM5 chip using EDC/NHS chemistry as described in Methods. Proteases were flowed over the chip in different concentrations. Sensograms for the binding of SAP-1 to the proteases are presented in (Fig. 5 & Supplementary file (Fig. S1)). These sensograms were prepared by subtracting the responses from the control cell of the same chip. The profiles of the SPR signal for each of the protease were distinctive. Sensograms of pepsin showed a rapid increase in SPR signal during the association phase corresponding to a fast on-rate (kon), and a gradual reduction in SPR signal during dissociation phase corresponding to a slow off-rate (koff). On the other hand a slower kon with faster koff observed in the sensogram of PSA (Fig. S1). Kinetic analysis of the interactions yielded KA and KD values that are listed in Table 1. The binding kinetics shows that pepsin binds to SAP-1 with dissociation constant of 9.91 108 M, and that PSA binds to SAP-1 with a dissociation constant of 2.69 106 M. The dissociation constant for chymotrypsin, papain and trypsin are 1.17 107 M, 1.38 107 M and 5.63 107 M respectively. There was no interaction observed in case of proteinase K. 4. Discussion Several proteases have been identified in the reproductive tract of mammals [27]. Although their precise functions in reproduction are still unknown. Knowledge of how the activity of proteases is regulated by their inhibitors is important in understanding their role in reproduction. Numerous inhibitors of CPIs have been described and isolated from human. CPIs are present in human
Fig. 4. Thermal stability of purified cystatins. The inhibitor samples were incubated at different temperatures ranging from 20 C to 100 C for 30 min, and inhibitory activity was determined.
semen in much higher concentrations than trypsin inhibitors, which are known to influence fertilization process [28e30]. The presence of cystatins in such a high concentration strongly indicates an efficient need of cysteine proteinase inhibition in reproductive tract. The quantity of CPs in semen is much lower than that of the CPIs; however, vaginal tissue contains large amounts of CPs [31]. Seminal cystatins not only effects proteases present in semen, but they may also protect the sperm from proteases present in secretions of female reproductive tract and also have some other important roles in fertilization, although as yet unknown. It can be proposed that CPIs may inhibit other class of proteases besides CPs in human seminal plasma. There is little existing information on cystatins of seminal fluid, and the present work describes for the first time the purification and enzymatic properties of human seminal cystatins. The results in this study showed that the cystatins can be separated and purified by anion-exchange chromatography, after getting CM-papain affinity eluent. SAP-1 can be separated from other cystatins by anion-exchange (DEAE) chromatography. The protein eluting first in anion-exchange chromatography (Res Q) is cystatin SN which has a pI near 7.0, while the second protein is cystatin 9, which has a pI near 6.0. These three purified proteases are closely related and belong to the family 2 cystatins of the cystatin superfamily of CPIs. Multiple sequence alignments of LMW cystatins reveal that cystatin SN and SAP-1 contains the three conserved regions, thought to be critical for binding to a CP of the papain family. Cystatin 9 also contain the N- and C-terminal conserved regions but the middle region (QXVXG) is missing in cystatin 9 (Fig. 2A). Moreover cystatin 9 also have a cysteine rich internal sequence. These observations suggest that cystatin 9 may have a different mechanism of action as compared to other members of family 2 cystatins. The purified cystatins showed remarkable heat stability. The optimum temperature observed for the activity of cystatins is 30e40 C. Purified inhibitors remained relatively stable up to 80 C. It is known that many of protease inhibitors are thermostable but heat stability is a feature seldom precisely described. High stabilities of the purified inhibitors in broad temperature range are consistent with other cystatins [32,33]. The high heat stability suggests a highly stable tertiary structure of seminal cystatins. The physiological functions of the described proteins are largely unknown. Further experiments are necessary to describe their thermostability and to establish their precise physiological function in fertilization process. Presumably, results of this work will update the already existing catalogs of human seminal CPIs. In order to test the specificity, inhibitory activities against various commercially available proteases (papain, trypsin, chymotrypsin, pepsin, and proteinase K) were compared (Fig. 3). Cystatin SN and cystatin 9 were active only against papain, whereas the third inhibitor SAP-1 potentially inhibits all class of proteases except proteinase K. Fig. 3 shows the protease inhibition assay results starting at 0.1 mM concentration of SAP-1 up to 3 mM, and at 3 mM concentration, it inhibits almost completely all the tested proteases. Binding studies of SAP-1 with proteases also support our data. SPR measures binding interactions on the surface of a biosensor chip and SPR biosensors have been successfully used for quantitative modeling of proteineligand interactions [26]. SAP-1 interacts with the proteases (trypsin, chymotrypsin, papain, pepsin and PSA) with KD values ranging from 106 to 108 M. Among all the proteases tested, pepsin has the lowest dissociation constant (KD) and highest association constant (KA) which indeed corresponds to the highest affinity. The other three proteases, chymotrypsin, papain and trypsin showed comparable binding affinities (Table 1). Though PSA had the lowest affinity towards SAP1among the proteases tested, still the affinity was significant
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Fig. 5. (A) Immobilization profile of SAP-1 on CM5 sensor chip. Each X on the response represents a report point, which provides a value of the response at a given point; (a) During the preconcentration test, the ligand is injected over the unmodified sensor chip surface to determine if the desired immobilization level can be achieved. (b) During the activation step, the carboxymethyl groups on the dextran are converted into reactive esters followed by (c) a washing step of ethanolamine and, then (d) the ligand is injected in short pulses until the desired immobilization level is achieved. (e) During the blocking step, uncrosslinked reactive esters are blocked with ethanolamine. The immobilization level or ligand density is the final response difference from baseline. (B) SPR sensograms of SAP-1 interaction with papain. SAP-1 immobilized on CM5 chip as described in Methods. Concentration of SAP-1 was fixed at 10 mM. Concentration of papain was in decreasing order from top to bottom.
(KD ¼ 2.69 106 M & KA ¼ 3.38 105 M-1) which suggest that SAP1 might be involved in regulation of kallikrein activity cascades. Protein C inhibitor (PCI), a-2 macroglobulin (A2M) and a1-anti chymotrypsin (ACT) are three main inhibitors of PSA reported in HSF. Concentrations of these inhibitors in seminal fluid are low compared to PSA [34e36] suggesting there must be other inhibitors, which may also serve as its inhibitors. SAP-1 seems to be one such inhibitor. Taken together, our studies suggest that the N-terminal truncated form of cystatin S functions as a novel cross-class inhibitor with a structural relationship to the cystatins, but different functional relationship. Although there are several examples of crossclass inhibition between families of cysteine and serine proteases, for example, SQN-5, a serpin, was shown to exhibit dual mechanistic class inhibition by inhibiting both serine and cysteine
Table 1 Affinity constants for SAP-1eproteases interactions. Proteases
KA [1/M]
Papain Chymotrypsin Trypsin Pepsin PSA
7.18 8.13 1.64 1.04 3.38
0.01 0.01 0.30 0.04 0.30
KD [M]
106 106 106 107 105
1.38 1.17 5.63 9.91 2.69
0.01 0.01 0.30 0.04 0.30
107 107 107 108 106
proteases [37]. Another example of cross-class inhibition is cystatin-related epididymal spermatogenic (CRES) protein, related to the family 2 cystatins. CRES inhibited the serine protease prohormone convertase 2 (PC2), a protease involved in prohormone processing in the neuroendocrine system [38]. Moreover, in a recent finding Human Epididymis Protein-4, a serine protease inhibitor, has been shown to inhibit proteases from other classes [39]. Finally, a synthetic peptide representing a domain of cystatin SA that contains the PW motif conserved in CRES and CRES-related proteins was shown to possess inhibitory activity against both cysteine and serine proteases [40], suggesting that, like serpins, some cystatins may also interact with other families of proteases. Indeed, our observation that SAP-1 inhibits various classes of proteases rather than only cysteine protease, is one of the first examples of a cystatin that has acquired new protease inhibitory functions and exhibits cross-class inhibition. N-terminal processing is a common feature of type 2 cystatins that differentially affects the inhibitory property of cystatins [19,41e44]. In general, truncated forms either isolated from natural source or produced by cleavage with enzymes, or by recombinant techniques, show differences in inhibitory activity toward their target protease as compared with the full-length proteins [44e47]. Most cystatins are single-domain proteins composed of 100e120 residues with a characteristic fold presenting the N-terminal segment and two b-hairpin loops for complementation of the
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active site cleft of the target enzyme. They show high affinity for their target enzymes, with equilibrium constants for dissociation of the formed complexes typically in the range of 109e1012 M but here we report SAP-1 binds to its target proteases with relatively low affinity. Collectively, these studies suggest that the N-terminal processing of the cystatins make changes in the specificity and strength of binding to their target proteases. Although cystatin S is reported to inhibit only CPs, this can be presumed that SAP-1 in seminal plasma may inhibit other classes of proteases and thus may influence fertilization. Various classes of protease inhibitors are present in multiple molecular forms in sex glands secretions. They showed some differences in amino acid composition, sequence, isoelectric point and immunological reactivity. These differences might reflect the differences in function of the inhibitors despite their similar inhibitory power against CPs in vitro. Fig. 2B shows the clustering relationships within the cystatin 2 family. Horizontal branch lengths in the dendrogram indicate proportional differences between sequences. It is interesting to speculate on the physiological role of these LMW proteinase inhibitors in human reproduction process. It seems likely that these inhibitors would at least serve a protective function against inappropriate proteolysis both in male and female reproductive tract. An interesting finding in the present study is that SAP-1 inhibits various classes of proteases. Although in vitro studies have clearly established that the SAP-1 is a cross-class protease inhibitor, the in vivo function of this property remains to be established. In summary, here we first time report the purification and characterization of three LMW cystatins of HSF. Purified inhibitors are neutral proteins with remarkable heat stability. Our results showed that SAP-1 is a cross-class protease inhibitor, inhibits different proteases. However, further studies are further required to establish their potential role and elucidate the functions in human reproductive physiology. Hopefully the results of this study and further experiments will shed more light on these interesting and potentially clinically important class of proteins. Acknowledgments We thank the Indian Council of Medical Research (ICMR) and Council of Scientific and Industrial Research (CSIR), New Delhi for the funds, and fellowship granted to VKY and NC. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biochi.2013.04.007. References [1] H.A. Chapman, J.R. Riese, G.P. Shi, Emerging roles for cysteine proteases in human biology, Annu. Rev. Physiol. 59 (1997) 63e88. [2] Z. Grzonka, E. Jankowska, F. Kasprzykowski, R. Kasprzykowska, L. Lankiewicz, W. Wiczk, E. Wieczerzak, J. Ciarkowski, P. Drabik, R. Janowski, M. Kozak, M. Jaskólski, A. Grubb, Structural studies of cysteine proteases and their inhibitors, Acta Biochim. Pol. 48 (2001) 1e20. [3] M. Mussap, M. Plebani, Biochemistry and clinical role of human cystatin C, Crit. Rev. Clin. Lab. Sci. 41 (2004) 467e550. [4] V. Turk, V. Stoka, D. Turk, Cystatins: biochemical and structural properties, and medical relevance, Front. Biosci. 13 (2008) 5406e5420. [5] R. Lertnawapan, A. Bian, Y.H. Rho, V.K. Kawai, P. Raggi, A. Oeser, J.F. Solus, T. Gebretsadik, A. Shintani, C.M. Stein, Cystatin C, renal function, and atherosclerosis in rheumatoid arthritis, J. Rheumatol. 38 (2011) 2297e2300. [6] E. Zerovnik, The emerging role of cystatins in Alzheimer’s disease, Bioessays 31 (2009) 597e599. [7] N. Ramasubbu, M.S. Reddy, E.J. Bergey, G.G. Haraszthy, S.D. Soni, M.J. Levine, Large-scale purification and characterization of the major phosphoproteins and mucins of human submandibular-sublingual saliva, Biochem. J. 280 (1991) 341e352.
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Zhang, P. Tang, S. Zhai, W. Guo, N. Yu, L. Zhao, M. Liu, S. Yang, Expression and clinical significance of cathepsin B and stefin A in laryngeal cancer, Oncol. Rep. 26 (2011) 869e875. [19] V. Turk, W. Bode, The cystatins: protein inhibitors of cysteine proteinases, FEBS Lett. 285 (1991) 213e219. [20] M. Abrahamson, M. Alvarez-Fernandez, C.M. Nathanson, Cystatins, Biochem. Soc. Symp. 70 (2003) 179e199. [21] T. Mirtti, K. Alanen, M. Kallajoki, A. Rinne, K.O. Söderström, Expression of cystatins, high molecular weight cytokeratin, and proliferation markers in prostaticadenocarcinoma and hyperplasia, Prostate 54 (2003) 290e298. [22] T. Jiborn, M. Abrahamson, H. Wallin, J. Malm, A. Lundwall, V. Gadaleanu, P.A. Abrahamsson, A. Bjartell, Cystatin C is highly expressed in the human male reproductive system, J. Androl. 25 (2004) 564e572. [23] G.A. Cornwall, H.H. Von Horsten, S. Whelly, Cystatin-related epididymal spermatogenic aggregates in the epididymis, J. Androl. 32 (2011) 679e685. [24] A. Anastasi, M.A. Brown, A.A. Kembhavi, M.J.H. Nicklin, C.A. Sayers, D.C. Sunter, A.J. Barrett, Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum, Biochem. J. 211 (1983) 129e138. [25] T.M. Lee, Y.H. Lin, Trypsin inhibitor and trypsin-like protease activity in air- or submergence-grown rice (Oryza sativa L.) coleoptiles, Plant Sci. 106 (1995) 43e54. [26] M. Ritzefeld, N. Sewald, Real-time analysis of specific proteineDNA interactions with surface plasmon resonance, J. Amino Acids (2012) 816032. [27] S.L. Lee, Y.H. Wei, The involvement of extracellular proteinases and proteinase inhibitors in mammalian fertilization, Biotechnol. Appl. Biochem. 19 (1994) 31e40. [28] D.Y. Liu, H.W. Baker, Inhibition of acrosin activity with a trypsin inhibitor blocks human sperm penetration of the zona pellucid, Biol. Reprod. 48 (1993) 340e348. [29] M. Deppe, J. Risopatrón, R. Sánchez, Trypsin and chymotrypsin are involved in the progesterone-induced acrosome reaction in canine spermatozoa, Reprod. Domest. Anim. 45 (2010) 453e457. [30] M. Deppe, P. Morales, R. Sánchez, Effect of protease inhibitors on the acrosome reaction and sperm-zona pellucida binding in bovine sperm, Reprod. Domest. Anim. 43 (2008) 713e719. [31] Y. Wu, L. Zhang, H. Jin, J. Zhou, Z. Xie, The role of calpainecalpastatin system in the development of stress urinary incontinence, Int. Urogynecol. J. 21 (2010) 63e68. [32] M. Tarasuk, S. Vichasri Grams, V. Viyanant, R. Grams, Type I cystatin (stefin) is a major component of Fasciola gigantica excretion/secretion product, Mol. Biochem. Parasitol. 167 (2009) 60e71. [33] M. Priyadarshini, B. Bano, Cystatin like thiol proteinase inhibitor from pancreas of Capra hircus: purification and detailed biochemical characterization, Amino Acids 38 (2010) 1001e1110. [34] S.P. Eisenberg, K.K. Hale, P. Heimdal, R.C. Thompson, Location of the proteaseinhibitory region of secretory leukocyte protease inhibitor, J. Biol. Chem. 265 (1990) 7976e7981. [35] W.B. Schill, Quantitative determination of high molecular weight serum proteinase inhibitors in human semen, Andrologia 8 (1976) 359e364. [36] G. Birkenmeier, E. Usbeck, A. Schäfer, A. Otto, H.J. Glander, Prostate specific antigen triggers transformation of seminal alpha2-macroglobulin(alpha2-M) and its binding to alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2-M-R/LRP) on human spermatozoa, Prostate 36 (1998) 219e225. [37] M. Al-Khunaizi, C.J. Luke, Y.S. Askew, S.C. Pak, D.J. Askew, S. Cataltepe, D. Miller, D.R. Mills, C. Tsu, D. Bromme, J.A. Irving, J.C. Whisstock,
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[43] K. Shibuya, H. Kaji, T. Itoh, Y. Ohyama, A. Tsujikami, S. Tate, A. Takeda, I. Kumagai, I. Hirao, K. Miura, et al., Human cystatin A is inactivated by engineered truncation. The NH2-terminal region of the cysteine proteinase inhibitor is essential for expression of its inhibitory activity, Biochemistry 34 (1995) 12185e12192. [44] A. Pavlova, I. Björk, Grafting of features of cystatins C or B into the N-terminal region or second binding loop of cystatin A (stefin A) substantially enhances inhibition of cysteine proteinases, Biochemistry 42 (2003) 11326e11333. [45] A. Baron, A. DeCarlo, J. Featherstone, Functional aspects of the human salivary cystatins in the oral environment, Oral Dis. 5 (1999) 234e240. [46] P. Lindahl, M. Nycander, K. Ylinenjärvi, E. Pol, I. Björk, Characterization by rapid-kinetic and equilibrium methods of the interaction between N-terminally truncated forms of chicken cystatin and the cysteine proteinases papain and actinidin, Biochem. J. 286 (1992) 165e171. [47] E. Saitoh, K. Minaguchi, O. Ishibashi, Production and characterization of two variants of human cystatin SA encoded by two alleles at the CST2 locus of the type 2 cystatin gene family, Arch. Biochem. Biophys. 352 (1998) 199e206.
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Research paper
Three low molecular weight cysteine proteinase inhibitors of human seminal fluid: Purification and enzyme kinetic properties Vikash Kumar Yadav a, Nirmal Chhikara a, Kamaldeep Gill a, Sharmistha Dey a, Sarman Singh b, Savita Yadav a, * a b
Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India Department of Lab Medicine, All India Institute of Medical Sciences, New Delhi 110029, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 December 2012 Accepted 9 April 2013 Available online 22 April 2013
The cystatins form a superfamily of structurally related proteins with highly conserved structural folds. They are all potent, reversible, competitive inhibitors of cysteine proteinases (CPs). Proteins from this group present differences in proteinase inhibition despite their high level of structural similarities. In this study, three cysteine proteinase inhibitors (CPIs) of low molecular weight were isolated from human seminal fluid (HSF) by affinity chromatography on carboxymethyl (CM)-papaineSepharose column, purified using various chromatographic procedures and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Matrix-assisted laser desorption-ionization-time-of flight-mass spectrometry (MALDI-TOFMS) identified these proteins as cystatin 9, cystatin SN, and SAP-1 (an N-terminal truncated form of cystatin S). All three CPIs suppressed the activity of papain potentially and showed remarkable heat stability. Interestingly SAP-1 also inhibits the activity of trypsin, chymotrypsin, pepsin, and PSA (prostate specific antigen) and acts as a cross-class protease inhibitor in in vitro studies. Using Surface Plasmon Resonance, we have also observed that SAP-1 shows a significant binding with all these proteases. These studies suggest that SAP-1 is a cross-class inhibitor that may regulate activity of various classes of proteases within the reproductive systems. To our knowledge, this is the first report about purification of CPIs from HSF; the identification of such proteins could provide better insights into the physiological processes and offer intimation for further research. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Chromatography Cross-class inhibition Cystatins Kinetics Papain Surface plasmon resonance
1. Introduction Cysteine proteases are accountable for a lot of biological processes occurring in human body [1]. The main physiological role of CPs is intracellular catabolism of peptides and proteins [2], malfunctioning has been implicated in the development and progression of many diseases [3e6]. The activity of CPs is regulated by their specific natural protein inhibitors called cystatins. Cystatins have been isolated and characterized from different human tissues and body fluids [7e14]. Their inhibitory profiles, as well as their affinities for target enzymes, vary with different CPs. Appropriate steadiness between free CPs and their complexes with inhibitors is critical for the regulation of the proteolytic activity under
Abbreviations: CM, carboxymethyl; CPs, cysteine proteinases; CPIs, cysteine proteinase inhibitors; CRES, cystatin-related epididymal spermatogenic; HSF, human seminal fluid; LMW, low molecular weight; PCI, protein C inhibitor; PSA, prostate specific antigen. * Corresponding author. Tel.: þ91 11 2653201; fax: þ91 11 26588641. E-mail address: [email protected] (S. Yadav). 0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2013.04.007
normal physiological conditions [15e18]. Based on the molecule complexity, cystatins have been categorized into three families [19,20]: Family 1 cystatins (stefins) are found mainly intracellularly and have molecular weights of 12 kDa, family 2 cystatins (S, SA, SN, C) are essentially found extracellularly and have a molecular weight of 14 kDa, and family 3 cystatins are the high molecular weight kininogens. All of the characterized cystatins exhibit sequence homologies. The presence of the different cystatins and their exact functions in the male accessory sex glands is largely still unknown. Cystatin A has been demonstrated to be present in the basal cells, in all cases of benign prostatic hyperplasias (BPH), low-grade prostatic intraepithelial neoplasias (PIN), and high-grade PIN and can aid in the diagnosis of prostatic adenocarcinoma [21]. Cystatin C is highly expressed and widely distributed throughout the male genital tract, suggesting that cystatin C is an important regulator for normal and pathological proteolysis in the male reproductive system [22]. Moreover, the presence of cystatin-related epididymal spermatogenic (CRES) proteins in the sperm acrosome, suggests their role in sperm maturation and fertilization [23]. Seminal
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plasma also contains cystatin S, D, and B in relatively high amounts; however their roles remain to be established. CPIs are the dominant inhibitors in seminal plasma [13,22] therefore, may also have some important, although as yet unknown, roles in fertilization. Further studies are needed to explore their role with special attention to fertility and other functions in semen. In the present study, we have purified and identified three LMW cystatins of HSF using various chromatographic steps in a sequential manner and subsequent characterization of these proteins may promote our knowledge of biochemical mechanisms involved in human fertilization in future. We have also determined the enzymatic properties of the inhibitors to be able to assess their potential capacity as physiologically important inhibitors of CPs of human reproductive tract and/or in HSF. 2. Material and methods 2.1. Sample collection Freshly ejaculated normal human semen was collected and pooled from the Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi. Ethical permission was taken from the same institute for the study (Ref. No.IESC/T-154/ 2010). Proteases (Papain, trypsin, chymotrypsin, pepsin, and
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proteinase K), hemoglobin, and trichloroacetic acid were purchased from SigmaeAldrich (St Louis, MO, USA). Molecular weight marker (Unstained Protein Markers) was from Fermentas. All other chemical and reagents were of analytical grade and obtained from a local supplier. 2.2. Isolation and purification of LMW cystatins To obtain seminal plasma, 20 ml of semen was centrifuged at 1300 g for 15 min at 4 C. The seminal plasma was further clarified by centrifugation at 10,000 g for 15 min. The supernatant was then dialyzed against 50 mM sodium phosphate buffer (pH 6.5, containing 0.2 M NaCl, 2 mM EDTA, and 10 mM sodium azide) and was chromatographed at 4 C on CM-papaineSepharose column, prepared as described in Ref. [24]. After thorough washing with the buffer, proteins with affinity for carboxymethylated papain were eluted with 0.2 M trisodium phosphate, pH 12, containing NaCl, EDTA, and sodium azide, as in the binding buffer above. Eluted sample was neutralized with 2 M sodium phosphate buffer, pH 6.0, and concentrated at 4 C using ultrafiltration (Millipore, Billerica, MA). The concentrated eluate from affinity column was further applied on DEAEeSephacel (10 2.6 cm) column using 50 mM sodium phosphate, pH 6.0. After extensive washing, bound proteins were eluted with linear gradient of NaCl (0e0.5 M) in same buffer (Fig. 1A).
Fig. 1. Purification of HSF cystatins using affinity, ion exchange and gel filtration chromatography. (A) Elution profile on anion exchanger DEAEeSephacel. The fraction with affinity for carboxymethylated papain was applied to DEAEeSephacel column in 50 mM sodium phosphate, pH 6.0. Elution was carried out with a linear gradient of 0e0.5 M NaCl. (B) Purification of SAP-1 on Sephadex G-50. The fraction (peak 2) containing inhibitory activity was pooled and concentrated. (C) Purification of cystatin SN and cystatin 9 on stronganion exchanger column (Resource Q). The flow-through fractions of the DEAEeSephacel column was used as starting material. Cystatin SN was eluted at 0.05 M NaCl and cystatin 9 at 0.1 M (w/v) NaCl. (D) SDS-PAGE pattern of purified cystatins of HSF under reducing condition and stained with Coomassie blue. Lane 1, molecular weight markers (116 kDa: bgalactosidase b, 66 kDa: Bovine Serum Albumin, 45 kDa: Ovalbumin, 35 kDa: Lactate dehydrogenase, 25 kDa: REase Bsp 981, 18.8 kDa: b-Lactoglobulin and Lysozyme: 14.4 kDa); Lane 2, affinity eluent of CM-papaineSepharose; Lane 3, SAP-1; Lane 4, cystatin SN; Lane 5, cystatin 9.
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Fractions were analyzed for optical density at 280 nm and papain inhibitory activity. Fractions showing protease inhibitory activity were pooled, desalted, and lyophilized. In the next stage of purification, the sample was dissolved and applied to size exclusion chromatography on Sephadex G-50 (120 1.6 cm) in 50 mM sodium phosphate, pH 7.5 (Fig. 1B). Fractions were collected and analyzed for papain inhibitory activity. The flow-through fractions of the DEAEeSephacel column was dialyzed against 20 mM sodium phosphate, pH 7.5, concentrated and applied on Resource Q column (GE-Healthcare), eluted with linear gradient of NaCl (0e0.2 M). cystatin SN was eluted at 0.05 M NaCl and cystatin 9 at 0.1 M NaCl (Fig. 1C). 2.3. Determination of protease inhibitory activity Inhibitory activity was measured with the modified method described by Lee and Lin [25]. 125 mL of sample was preincubated with the same volume of papain (3 mM) dissolved in 50 mM sodium phosphate buffer (pH 6.2), pepsin dissolved in sodium acetate buffer (pH 4.5), and chymotrypsin and trypsin dissolved in TriseHCl buffer (pH 8.2), at 37 C, for 20 min. After preincubation, 250 mL of 1% solution of hemoglobin dissolved in the same buffer was added and the mixture was incubated for 40 min. Then the reaction was stopped by the addition of 1 ml of 5% trichloroacetic acid. Samples were centrifuged at 15,000g for 10 min, and the absorbance of the supernatant was measured at 280 nm. For enzyme standard assay sample was replaced by distilled water and for control assay enzyme solution was replaced by distilled water. The percentage of inhibition was calculated as follows: [(A280 of enzyme standard þ A280 of control) (A280 of sample)]/(A280 of enzyme standard) 100%, where A280 was the absorbance at 280 nm. 2.4. Gel electrophoresis SDS-PAGE was carried out on 12% using 1.5 mm thick slab gels under reducing and non-reducing conditions. Relative molecular masses of separated proteins were estimated using protein standards run in parallel. 2.5. Mass spectrometry Analysis was performed using a Bruker Autoflex MALDI-TOF mass spectrometer equipped with a pulsed 337 nm N2 laser. Operating conditions are as follows: ion source 1 ¼ 19.00 kV, ion source 2 ¼ 16.50 kV, lens voltage ¼ 8.80 kV, reflector voltage ¼ 20.00 kV, optimized pulsed ion extraction time ¼ 80 ns, matrix suppression ¼ 400 Da, and positive reflection mode. Spectra were calibrated externally using the calibrant peptide mixture. Peptides from mass spectra of in-gel digest samples were matched against databases such as Swiss-prot, NCBInr, and MSDB using Mascot search engine (Matrix sciences) for peptide-massfingerprinting. 2.6. Thermal stability of purified inhibitors After purification protease inhibitors were incubated at temperatures ranging from 20 C to 100 C in a water bath for 30 min. The samples were cooled and inhibitory activities against papain were determined. 2.7. SPR-binding studies The binding studies were carried out using the BIAcore 2000 apparatus (Pharmacia BIAcore AB, Uppsala, Sweden). The BIAcore apparatus is a biosensor-based system for real-time specific
interaction analysis [26]. The protein sensor chip was prepared by immobilization of SAP-1 on a research grade CM5 chip (Biosensor AB, Uppsala, Sweden) according to the manufacturer’s recommendations. 60 ml of SAP-1 (30 mg/ml) in 10 mM sodium acetate buffer (pH 5.0) was injected to the flow cell for 4 min at the rate of 5 ml/min and washed for 60 min at the rate of 20 ml/min. The analytes (different proteases) at different concentrations were injected for 4 min at a rate of 10 ml/min. The protein samples were diluted in sodium acetate buffer pH 5.0. Typically, 90 ml of different dilutions of protein were injected at a flow rate of 30 ml/min. The same buffer was passed over the sensor surface at the end of sample injection, to allow dissociation. After a 3 min dissociation phase, the sensor surface was regenerated by 30 ml of 50 mM NaOH and 1 M NaCl. The binding of analytes can be studied by monitoring the change in the resonance unit (RU) values of the sensorgram, where the progress of the interaction was plotted against time, revealing the binding characteristics. Kinetic constants of different proteases were calculated from the association and dissociation phases with BIA evaluation software version 3.0. 3. Results 3.1. Purification of three LMW cystatins from HSF In order to purify cystatins from HSF, the three-step chromatographic procedure was used. The first stage of the process was affinity chromatography on CM-papain affinity column. The affinityeluted protein fractions served as an initial source of cystatins purification. In second stage, affinity-eluted fraction was applied onto a DEAEeSephacel column. As illustrated in Fig. 1A, a single peak of inhibitory activity was detected in eluted fractions of DEAEeSephacel column. The fraction containing protease inhibitory activity was further purified to homogeneity using size exclusion chromatography (Fig. 1B). The cystatin obtained was Nterminal truncated form of cystatin S (SAP-1) and molecular mass corresponds to 14 kDa on SDS-PAGE under reducing conditions (Fig. 1D). In the third stage, two more protease inhibitors were purified and separated through anion-exchange chromatography. The flowthrough of DEAEeSephacel served as the starting material and loaded on strong-anion exchanger Resource Q column at pH 7.5. Two peaks of inhibitory activity were obtained at 0.05 and 0.1 M NaCl (Fig. 1C). These peaks correspond to purified cystatin SN of molecular mass 16 kDa and cystatin 9 of 20 kDa respectively. A Coomassie stained gel of the purified cystatins is shown in Fig. 1D. The protein profile of the CM-papaineSepharose purified preparation was also investigated by SDS-PAGE (Fig. 1D). 3.2. Sequence analysis of purified cystatins Multiple sequence alignments (MSA) of LMW purified cystatins with other members of family 2 cystatins were performed using ClustalW2 on EBI web server (Fig. 2A). Cystatin SN and SAP-1 are member of family 2 cystatins and cystatin 9 also shows maximum homology to this family. Members of family 2 cystatins are about 120 amino acids, have two intra-chain disulfide bonds, and most of them found both in tissues and body fluids. Cystatin 9 shows 18e 27% homology to other members of family 2 cystatins and contains conserved residues which form the inhibitory sites of cystatins, such as the GlyeGly and the ProeTrp in the N-terminus and Cterminus respectively. A close examination of alignment reveals cystatin 9 also have a cysteine rich internal sequence beside the four conserved Cys residues in the cystatins, required for the formation of two disulfide bridges, suggesting that the binding
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Fig. 2. Sequence analysis of family 2 cystatins. (A) Alignment of amino acid sequences of cystatin 9, CRES proteins and members of family 2 cystatins. Purified cystatins are compared with cystatin 8 (accession no. O60676), cystatin 11 (accession no. Q9H112), cystatin S (accession no. P01036), cystatin D (accession no. P28325), and cystatin C (accession no. P01034). Amino acids forming the inhibitory wedge in family 2 cystatins, and the corresponding amino acids in the cystatin 9 are shown in red color. The four cysteines build 2 disulfide linkages in cystatins, are in blue color. The cysteine rich internal sequence is shown in green color in cystatin 9. (B) The branching diagram representing a hierarchy of categories based on degree of similarity or number of shared characteristics.
specificity of cystatin 9 to its target may be different from those of other cystatins. Furthermore, family 2 cystatins also contain a conserved internal sequence glutamineevalineeglycine (QXVXG), which is also completely missing in cystatin 9 (Fig. 2A). 3.3. Kinetic analysis 3.3.1. Antiprotease activity of cystatins Protease inhibition activity of cystatins was tested according to the method of Lee and Lin [25]. Hemoglobin was used as a substrate and percentage inhibition was calculated as described in material and methods. The activity of papain is potentially inhibited by all the purified protease inhibitors but SAP-1 also observed to inhibit the activity of other proteases. We examine the activity of trypsin, chymotrypsin, pepsin, PSA, and proteinase K and found SAP-1 inhibits all these proteases except proteinase K (Fig. 3). SPR results also support the broad spectrum inhibitory property of SAP-1 as it shows significant binding with all proteases. At the inhibitor concentration of 3 mM papain, pepsin, PSA shows almost complete inhibition while in case of trypsin and chymotrypsin 70e80% inhibition is achieved. Negligible inhibition observed in case of proteinase K.
Fig. 3. Antiprotease activity of SAP-1against different class of proteases. Hemoglobin was used as the substrate to determine the inhibition activity.
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Further thermal stability of purified cystatins was experienced. The proteins were incubated at different temperatures, ranging from 20 C to 100 C, for 30 min. The maximum inhibitory activity of cystatins was at around 40 C. The inhibitory activity toward papain remained relatively stable up to 80 C (Fig. 4). Only at 100 C the loss in the inhibitory activity (around 20e40%) was observed suggesting a highly stable tertiary structure resistant to heat. SAP-1 was most heat stable inhibitor among purified ones. 3.3.2. Evaluation of binding potential and binding constants using SPR Trypsin, chymotrypsin, pepsin, papain, PSA and, proteinase K were employed for the experiment. SAP-1 interaction with these proteases was analyzed with surface plasmon resonance (SPR) and kinetics constants were calculated. SAP-1 was immobilized on research grade CM5 chip using EDC/NHS chemistry as described in Methods. Proteases were flowed over the chip in different concentrations. Sensograms for the binding of SAP-1 to the proteases are presented in (Fig. 5 & Supplementary file (Fig. S1)). These sensograms were prepared by subtracting the responses from the control cell of the same chip. The profiles of the SPR signal for each of the protease were distinctive. Sensograms of pepsin showed a rapid increase in SPR signal during the association phase corresponding to a fast on-rate (kon), and a gradual reduction in SPR signal during dissociation phase corresponding to a slow off-rate (koff). On the other hand a slower kon with faster koff observed in the sensogram of PSA (Fig. S1). Kinetic analysis of the interactions yielded KA and KD values that are listed in Table 1. The binding kinetics shows that pepsin binds to SAP-1 with dissociation constant of 9.91 108 M, and that PSA binds to SAP-1 with a dissociation constant of 2.69 106 M. The dissociation constant for chymotrypsin, papain and trypsin are 1.17 107 M, 1.38 107 M and 5.63 107 M respectively. There was no interaction observed in case of proteinase K. 4. Discussion Several proteases have been identified in the reproductive tract of mammals [27]. Although their precise functions in reproduction are still unknown. Knowledge of how the activity of proteases is regulated by their inhibitors is important in understanding their role in reproduction. Numerous inhibitors of CPIs have been described and isolated from human. CPIs are present in human
Fig. 4. Thermal stability of purified cystatins. The inhibitor samples were incubated at different temperatures ranging from 20 C to 100 C for 30 min, and inhibitory activity was determined.
semen in much higher concentrations than trypsin inhibitors, which are known to influence fertilization process [28e30]. The presence of cystatins in such a high concentration strongly indicates an efficient need of cysteine proteinase inhibition in reproductive tract. The quantity of CPs in semen is much lower than that of the CPIs; however, vaginal tissue contains large amounts of CPs [31]. Seminal cystatins not only effects proteases present in semen, but they may also protect the sperm from proteases present in secretions of female reproductive tract and also have some other important roles in fertilization, although as yet unknown. It can be proposed that CPIs may inhibit other class of proteases besides CPs in human seminal plasma. There is little existing information on cystatins of seminal fluid, and the present work describes for the first time the purification and enzymatic properties of human seminal cystatins. The results in this study showed that the cystatins can be separated and purified by anion-exchange chromatography, after getting CM-papain affinity eluent. SAP-1 can be separated from other cystatins by anion-exchange (DEAE) chromatography. The protein eluting first in anion-exchange chromatography (Res Q) is cystatin SN which has a pI near 7.0, while the second protein is cystatin 9, which has a pI near 6.0. These three purified proteases are closely related and belong to the family 2 cystatins of the cystatin superfamily of CPIs. Multiple sequence alignments of LMW cystatins reveal that cystatin SN and SAP-1 contains the three conserved regions, thought to be critical for binding to a CP of the papain family. Cystatin 9 also contain the N- and C-terminal conserved regions but the middle region (QXVXG) is missing in cystatin 9 (Fig. 2A). Moreover cystatin 9 also have a cysteine rich internal sequence. These observations suggest that cystatin 9 may have a different mechanism of action as compared to other members of family 2 cystatins. The purified cystatins showed remarkable heat stability. The optimum temperature observed for the activity of cystatins is 30e40 C. Purified inhibitors remained relatively stable up to 80 C. It is known that many of protease inhibitors are thermostable but heat stability is a feature seldom precisely described. High stabilities of the purified inhibitors in broad temperature range are consistent with other cystatins [32,33]. The high heat stability suggests a highly stable tertiary structure of seminal cystatins. The physiological functions of the described proteins are largely unknown. Further experiments are necessary to describe their thermostability and to establish their precise physiological function in fertilization process. Presumably, results of this work will update the already existing catalogs of human seminal CPIs. In order to test the specificity, inhibitory activities against various commercially available proteases (papain, trypsin, chymotrypsin, pepsin, and proteinase K) were compared (Fig. 3). Cystatin SN and cystatin 9 were active only against papain, whereas the third inhibitor SAP-1 potentially inhibits all class of proteases except proteinase K. Fig. 3 shows the protease inhibition assay results starting at 0.1 mM concentration of SAP-1 up to 3 mM, and at 3 mM concentration, it inhibits almost completely all the tested proteases. Binding studies of SAP-1 with proteases also support our data. SPR measures binding interactions on the surface of a biosensor chip and SPR biosensors have been successfully used for quantitative modeling of proteineligand interactions [26]. SAP-1 interacts with the proteases (trypsin, chymotrypsin, papain, pepsin and PSA) with KD values ranging from 106 to 108 M. Among all the proteases tested, pepsin has the lowest dissociation constant (KD) and highest association constant (KA) which indeed corresponds to the highest affinity. The other three proteases, chymotrypsin, papain and trypsin showed comparable binding affinities (Table 1). Though PSA had the lowest affinity towards SAP1among the proteases tested, still the affinity was significant
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Fig. 5. (A) Immobilization profile of SAP-1 on CM5 sensor chip. Each X on the response represents a report point, which provides a value of the response at a given point; (a) During the preconcentration test, the ligand is injected over the unmodified sensor chip surface to determine if the desired immobilization level can be achieved. (b) During the activation step, the carboxymethyl groups on the dextran are converted into reactive esters followed by (c) a washing step of ethanolamine and, then (d) the ligand is injected in short pulses until the desired immobilization level is achieved. (e) During the blocking step, uncrosslinked reactive esters are blocked with ethanolamine. The immobilization level or ligand density is the final response difference from baseline. (B) SPR sensograms of SAP-1 interaction with papain. SAP-1 immobilized on CM5 chip as described in Methods. Concentration of SAP-1 was fixed at 10 mM. Concentration of papain was in decreasing order from top to bottom.
(KD ¼ 2.69 106 M & KA ¼ 3.38 105 M-1) which suggest that SAP1 might be involved in regulation of kallikrein activity cascades. Protein C inhibitor (PCI), a-2 macroglobulin (A2M) and a1-anti chymotrypsin (ACT) are three main inhibitors of PSA reported in HSF. Concentrations of these inhibitors in seminal fluid are low compared to PSA [34e36] suggesting there must be other inhibitors, which may also serve as its inhibitors. SAP-1 seems to be one such inhibitor. Taken together, our studies suggest that the N-terminal truncated form of cystatin S functions as a novel cross-class inhibitor with a structural relationship to the cystatins, but different functional relationship. Although there are several examples of crossclass inhibition between families of cysteine and serine proteases, for example, SQN-5, a serpin, was shown to exhibit dual mechanistic class inhibition by inhibiting both serine and cysteine
Table 1 Affinity constants for SAP-1eproteases interactions. Proteases
KA [1/M]
Papain Chymotrypsin Trypsin Pepsin PSA
7.18 8.13 1.64 1.04 3.38
0.01 0.01 0.30 0.04 0.30
KD [M]
106 106 106 107 105
1.38 1.17 5.63 9.91 2.69
0.01 0.01 0.30 0.04 0.30
107 107 107 108 106
proteases [37]. Another example of cross-class inhibition is cystatin-related epididymal spermatogenic (CRES) protein, related to the family 2 cystatins. CRES inhibited the serine protease prohormone convertase 2 (PC2), a protease involved in prohormone processing in the neuroendocrine system [38]. Moreover, in a recent finding Human Epididymis Protein-4, a serine protease inhibitor, has been shown to inhibit proteases from other classes [39]. Finally, a synthetic peptide representing a domain of cystatin SA that contains the PW motif conserved in CRES and CRES-related proteins was shown to possess inhibitory activity against both cysteine and serine proteases [40], suggesting that, like serpins, some cystatins may also interact with other families of proteases. Indeed, our observation that SAP-1 inhibits various classes of proteases rather than only cysteine protease, is one of the first examples of a cystatin that has acquired new protease inhibitory functions and exhibits cross-class inhibition. N-terminal processing is a common feature of type 2 cystatins that differentially affects the inhibitory property of cystatins [19,41e44]. In general, truncated forms either isolated from natural source or produced by cleavage with enzymes, or by recombinant techniques, show differences in inhibitory activity toward their target protease as compared with the full-length proteins [44e47]. Most cystatins are single-domain proteins composed of 100e120 residues with a characteristic fold presenting the N-terminal segment and two b-hairpin loops for complementation of the
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active site cleft of the target enzyme. They show high affinity for their target enzymes, with equilibrium constants for dissociation of the formed complexes typically in the range of 109e1012 M but here we report SAP-1 binds to its target proteases with relatively low affinity. Collectively, these studies suggest that the N-terminal processing of the cystatins make changes in the specificity and strength of binding to their target proteases. Although cystatin S is reported to inhibit only CPs, this can be presumed that SAP-1 in seminal plasma may inhibit other classes of proteases and thus may influence fertilization. Various classes of protease inhibitors are present in multiple molecular forms in sex glands secretions. They showed some differences in amino acid composition, sequence, isoelectric point and immunological reactivity. These differences might reflect the differences in function of the inhibitors despite their similar inhibitory power against CPs in vitro. Fig. 2B shows the clustering relationships within the cystatin 2 family. Horizontal branch lengths in the dendrogram indicate proportional differences between sequences. It is interesting to speculate on the physiological role of these LMW proteinase inhibitors in human reproduction process. It seems likely that these inhibitors would at least serve a protective function against inappropriate proteolysis both in male and female reproductive tract. An interesting finding in the present study is that SAP-1 inhibits various classes of proteases. Although in vitro studies have clearly established that the SAP-1 is a cross-class protease inhibitor, the in vivo function of this property remains to be established. In summary, here we first time report the purification and characterization of three LMW cystatins of HSF. Purified inhibitors are neutral proteins with remarkable heat stability. Our results showed that SAP-1 is a cross-class protease inhibitor, inhibits different proteases. However, further studies are further required to establish their potential role and elucidate the functions in human reproductive physiology. Hopefully the results of this study and further experiments will shed more light on these interesting and potentially clinically important class of proteins. Acknowledgments We thank the Indian Council of Medical Research (ICMR) and Council of Scientific and Industrial Research (CSIR), New Delhi for the funds, and fellowship granted to VKY and NC. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biochi.2013.04.007. References [1] H.A. Chapman, J.R. Riese, G.P. Shi, Emerging roles for cysteine proteases in human biology, Annu. Rev. Physiol. 59 (1997) 63e88. [2] Z. Grzonka, E. Jankowska, F. Kasprzykowski, R. Kasprzykowska, L. Lankiewicz, W. Wiczk, E. Wieczerzak, J. Ciarkowski, P. Drabik, R. Janowski, M. Kozak, M. Jaskólski, A. Grubb, Structural studies of cysteine proteases and their inhibitors, Acta Biochim. Pol. 48 (2001) 1e20. [3] M. Mussap, M. Plebani, Biochemistry and clinical role of human cystatin C, Crit. Rev. Clin. Lab. Sci. 41 (2004) 467e550. [4] V. Turk, V. Stoka, D. Turk, Cystatins: biochemical and structural properties, and medical relevance, Front. Biosci. 13 (2008) 5406e5420. [5] R. Lertnawapan, A. Bian, Y.H. Rho, V.K. Kawai, P. Raggi, A. Oeser, J.F. Solus, T. Gebretsadik, A. Shintani, C.M. Stein, Cystatin C, renal function, and atherosclerosis in rheumatoid arthritis, J. Rheumatol. 38 (2011) 2297e2300. [6] E. Zerovnik, The emerging role of cystatins in Alzheimer’s disease, Bioessays 31 (2009) 597e599. [7] N. Ramasubbu, M.S. Reddy, E.J. Bergey, G.G. Haraszthy, S.D. Soni, M.J. Levine, Large-scale purification and characterization of the major phosphoproteins and mucins of human submandibular-sublingual saliva, Biochem. J. 280 (1991) 341e352.
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