Molecular Human Reproduction, Vol. 8, No. 1, 32-36,
January 2002
© 2002 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Matrix metalloproteinase (MMP)-2 and MMP-9 activities in human seminal plasma
1 Department of Functional Bioanalysis, 2 Department of Chemistry of Hygiene, Meiji Pharmaceutical University, 2–522–1 Noshio, Kiyose, Tokyo 204-8588, 3 Department of Obstetrics and Gynecology, Tachikawa Kyosai Hospital, Tokyo and 4 Department of Urology, Ichikawa General Hospital and Tokyo Dental College, Chiba, Japan
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Abstract |
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We report on the existence of two kinds of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, in human seminal plasma. Partial purification of the proteinases was achieved by two steps, consisting of chromatography on a gel-filtration column and then on a gelatin affinity column. Proteinase activities in the chromatography extracts were shown to hydrolyse a fluorescent substrate specific to MMPs (Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2). The proteinases were detected using gelatin-zymography, but were not detected using casein-zymography, and were also inhibited by EDTA, EGTA and o-phenanthroline. Molecular weights of the proteinases were determined by SDS–PAGE, gelatin-zymography and Western blot to be ~92, 84, 72, 67, 52 and 45 kDa. Gelatin-zymography showed three major bands of activity at 72, 67 and 52 kDa and minor bands at 92, 84 and 45 kDa. Apart from the two smallest bands, these proteinases were all recognized by the polyclonal antibodies for MMP-2 or MMP-9. These results indicate that two kinds of pro-form and active-form matrix metalloproteinases, MMP-2 and MMP-9, and their degradation products, are present in human seminal plasma.
human seminal plasma/matrix metalloproteinase/MMP/proteinase/zymography
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Seminal fluid contains many kinds of proteinases which are secreted from the prostate gland. The existence of many proteinase enzymes such as collagenase-like peptidase (Lukac and Koren, 1979
), gelatinolytic proteinases (Yin et al., 1990; Wilson et al., 1993), plasminogen activator (Astedt et al., 1976), pepsinogen II (Reese et al., 1986), Pz-peptidase (Lessley and Garner, 1985) and prostatic specific antigen (McGee and Herr, 1988) in human seminal plasma is known. However, the identity and function of metalloproteinases, which contain collagenase-like peptidase and gelatinolytic proteinase activities, is mostly unknown.
The matrix metalloproteinase (MMP) family consists of at least 20 structurally related zinc metalloendopeptidases capable of degrading the extracellular matrix components. These enzymes participate in embryonic development, morphogenesis, blastocyst implantation, angiogenesis and tissue resorption, and in diseases such as arthritis, cancer cell invasion and metastasis (Woessner, 1994; Nagase, 1996). Lymphocytes, macrophages and neutrophils are also known to release MMP-2 and MMP-9 in a cell-specific manner, following specific stimulation for MMP induction and regulation (Welgus et al., 1990; Miltenburg et al., 1995).
Gelatinase A (MMP-2) and gelatinase B (MMP-9) are also known as 72 kDa gelatinase/type IV collagenase and 92 kDa gelatinase/type IV collagenase respectively. MMP-2 has the ability to hydrolyse gelatins, type I, IV, V, VII and XI collagens, fibronectin, laminin, large tenascin-C, aggrecans and elastin. MMP-9 has the ability to hydrolyse activity of gelatins, type III, IV, V and XIV collagens, aggrecans, elastin and entactin. Both gelatinases have three repeat domains that are homologous to the type II domain of fibronectin. These domains are responsible for the ability of MMP-2 and MMP-9 to bind to gelatin, collagen I and IV, and laminin (Nagase, 1996).
In this paper, we report the existence of these two kinds of matrix metalloproteinases (MMP-2 and MMP-9) in human seminal plasma.
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Materials and methods |
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Materials
GCL-2000-sf-cellulofine and Gelatin-cellulofine for chromatography columns were purchased from Seikagaku Kogyo Co., Japan. Fluorescent substrate (Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2), sheep anti-human MMP-2 and sheep anti-human MMP-9 polyclonal antibodies were from Calbiochem Co. Standard proMMP-9, proMMP-2 and MMP-2 mixture solutions were from Yagai Research Center Co., Japan. Dimethyl sulphoxide (DMSO), EDTA, EGTA and o-phenanthroline were from Nakalai Tesque Co. Ltd, Japan. Polyvinylidene difluoride (PVDF) membrane and protein standards for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) were from Bio-Rad Laboratories. Brij 35 was from Sigma Chemical Co. Block Ace (dry milk) solution was from Dainippon Pharmacy Co., Japan. All reagents used, including substrates, antibodies and proteinase inhibitors were of analytical grade or were medically used products.
Human seminal plasma
Human semen was collected from volunteers who visited several hospitals in Tokyo. Informed consent was obtained from all volunteers and ethical approval was obtained from a Committee of Ethics associated with Tachikawa Kyosai Hospital, Tokyo. Azoospermic samples were excluded from the study. After liquefaction, the semen was centrifuged at 14 000 rpm for 30 min to separate the seminal plasma, which was then passed through a 0.45 µm filter. The seminal plasma preparations were frozen at –40°C until use.
Partial purification of MMPs
All purifications were performed at 4°C. Seminal plasma supernatant was centrifuged (6 ml) and loaded onto a GCL-2000-sf-cellulofine gel-filtration chromatography column (2.5x90 cm), equilibrated with 50 mmol/l Tris-HCl buffer, pH 7.5, 0.15 mol/l NaCl, 10 mmol/l CaCl2, 0.02% NaN3 (TNC buffer). The column was eluted at a rate of 10 ml/h and 3.5 ml fractions were collected and assessed by fluorescent substrate hydrolysis. The fractions containing MMP activities were collected, and then applied to a gelatin-cellulofine chromatography column (2.5x8 cm), equilibrated in TNC buffer. The column was washed with TNC buffer containing 1 mol/l NaCl, and then eluted with the same buffer containing 5% DMSO. The fractions were collected at 5.0 ml/tube and assessed by fluorescent substrate hydrolysis. The presence of MMPs in all the chromatography fractions was also monitored by gelatin-zymography. The homogeneity of the MMPs following the final chromatography step was examined by SDS–PAGE, gelatin-zymography and Western blot analysis.
SDS–PAGE electrophoresis
SDS–PAGE electrophoresis was performed using 10% total acrylamide under reducing or non-reducing conditions, as previously described (Laemmli, 1970). Proteins were stained with Coomassie Brilliant Blue R-250 or silver nitrate. Standard proteins included phosphorylase B (101 kDa), bovine serum albumin (79 kDa), ovalbumin (50.1 kDa), carbonic anhydrase (34.7 kDa), soybean trypsin inhibitor (28.4 kDa) and lysozyme (20.8 kDa).
Gelatin- and casein-zymography
The chromatography extracts were mixed with non-reducing SDS gel sample buffer and applied without boiling to a 10% polyacrylamide gel containing 0.1% SDS and 1 mg/ml gelatin or casein solution (Wilson et al., 1993). After electrophoresis, the gels were washed in 50 mmol/l Tris-HCl (pH 7.5) containing 0.15 mol/l NaCl, 5 mmol/l CaC12, 5 µmol/l ZnCl, 0.02% NaN3, 0.25% Triton X-100 (three changes) at room temperature, and then incubated in the same buffer without Triton X-100 (two changes) at 37°C for 20 h. Proteins were stained by Coomassie Brilliant Blue R-250 solution. To examine the inhibition of enzyme activity, samples at a final concentration of 10 mmol/l EDTA, EGTA or o-phenanthroline were incubated at room temperature for 1 h, mixed with non-reducing SDS gel sample buffer [1:1 (v/v)], then applied without boiling to a 10% polyacrylamide gel containing 0.1% SDS and 1 mg/ml gelatin. After electrophoresis, the gel was washed, incubated in the zymography buffer with each inhibitor (at a final concentration of 10 mmol/l) at 37°C for 20 h, and stained with Coomassie Brilliant Blue R-250.
Enzyme activity assays by fluorescent substrate hydrolysis
Enzyme activity assays were performed in 50 mmol/l Tris-HCl buffer, pH 7.5, 0.15 mol/l NaCl, 10 mmol/l CaCl2, 0.02% NaN3 (TNC buffer) containing 0.05% Brij 35 and 50 µmol/l ZnSO4, as previously described (Netzel-Arnett et al., 1991; Bickett et al., 1993). The fractions were tested for their ability to digest a synthetic fluorogenic substrate, Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 (a general MMP substrate). Each fraction was therefore incubated with 1 µmol/l substrate at 37°C for 20 h, and the reaction was stopped by the addition of 3% acetic acid. Fluorescence was measured using wavelengths of 280 nm (excitation) and 360 nm (emission) with a fluorescence reader (F-4010; Hitachi Co., Japan).
Western blot analysis
For Western blot analysis, samples electrophoresed by 10% SDS–PAGE were electroblotted onto PVDF membranes, as previously described (Burnette, 1981). Non-specific binding of immunoglobulin (Ig)G was blocked by a Block Ace solution. The membranes were incubated with the primary antibodies at a 1:5000 dilution for 1 h. The two primary polyclonal antibodies of sheep anti-human MMP-2 or sheep anti-human MMP-9 were used in all the experiments. After extensive washing of the membranes, they were incubated with peroxidase-conjugated goat anti-sheep IgG at a 1:50 000 dilution for 1 h at room temperature. Protein bands were detected using the ECL plus Western blotting detection system (Amersham Pharmacia Biotech. UK Ltd) with subsequent exposure to X-ray film.
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Results |
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The seminal plasma supernatant was loaded on a GCL-2000-sf-cellulofine gel-filtration chromatography column. As shown in Figure 1
, several peaks with MMP fluorescent hydrolytic activity were obtained. The fractions in tubes 65–95 contained proteinase activities assessed by both fluorescent substrate hydrolysis (Figure 1) and gelatin-zymography (Figure 2). No proteinase activites were detected by casein-zymography (data not shown). The fractions in tubes 135–145 had proteinase activities detected by fluorescent substrate hydrolysis (Figure 1), but did not have activities when assessed by gelatin-zymography (data not shown). The fractions in tubes 65–95 containing both types of MMP activities were collected and loaded onto a gelatin-cellulofine chromatography column. The proteinases were eluted with TNC buffer containing 1 mol/l NaCl and 5% DMSO and assessed by gelatin zymography (Figure 3). The peaks (tubes 117–119) were collected and concentrated using an Ultracent-30 concentrator (Tosoh Co. Ltd, Japan). The concentrated sample was then examined by SDS–PAGE, by Western blot against anti-human MMP-2 and MMP-9, and by gelatin- and casein-zymography. As shown in Figure 4, the purity of the final preparation was examined by gel electrophoresis in both reducing and non-reducing conditions (Figure 4a,b).
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The partially purified proteinases produced several bands; the major band was 72 kDa and the minor bands were 92, 67 and 52 kDa on SDS–PAGE (silver stain). On Western blot analysis using the antibodies of anti-human MMP-2 and anti-human MMP-9 (Figure 4c,d
), the two bands were shown on each membrane respectively. The polyclonal antibodies of MMP-2 and MMP-9 recognized proteins of ~72 and 67 kDa (Figure 4c), and ~92 and 84 kDa respectively (Figure 4d). Proteinase activities of a mixture of standard proteinases (proMMP-9, pro MMP-2 and MMP-2) and of the concentrated sample were detected by gelatin-zymography (Figures 4e,f). The three bands of standard MMP-2 and MMP-9 were observed on the gelatin-zymography gel (Figure 4e). The upper band is pro-form MMP-9 (92 kDa), the middle band is pro-form MMP-2 (72 kDa) and the lower band is active-form MMP-2 (67 kDa). In the concentrated sample, three major bands of 72, 67 and 52 kDa were observed, along with other minor bands of 92, 84 and 45 kDa, by gelatin-zymography (Figure 4f). The activity of all these proteinases was inhibited by EDTA (Figure 4g), EGTA and o-phenanthroline (data not shown), as assessed by gelatin zymography. |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Gelatinolytic proteinase activities have been previously reported in human seminal plasma (Yin et al., 1990
; Wilson et al., 1993). These proteinase activities were detected by gelatin-zymography, as three prominent bands of activity at 90, 66 and 60 kDa, as well as nine other bands of lesser activity (158–34 kDa). These proteinases were dependent upon calcium for their optimal activity, and were shown to hydrolyse gelatin but not casein. However, the identity and function of these proteinases is not clear. We report that two kinds of pro-form and active-form metalloproteinases, MMP-2 and MMP-9, and their degradation products are present in human seminal plasma, as detected by gelatin-zymography and Western blot analysis.
By testing with a fluorescent substrate specific to MMPs, several peaks with hydrolytic activity were obtained. One fraction (tube 66) had one prominent band of 92 kDa, and another fraction (tube 74) had two prominent bands of 72 and 67 kDa. Another fraction (tube 84) had several minor bands from 52 to 25 kDa, as assessed by gelatin-zymography. These enzymes had gelatin hydrolytic activities, and were inhibited by EDTA, EGTA and o-phenanthroline (metalloproteinase inhibitor), although a 25 kDa band remained uninhibited (data not shown). We expect that these activities were the metalloproteinases proMMP-9 (92 kDa), proMMP-2 (72 kDa) and MMP-2 (67 kDa), and that their degradation products were present at lower molecular weights. This could be expected because these proteinases, MMP-2 and MMP-9, each have gelatin binding domains in their catalytic domains (Collier et al., 1992; Banyai et al., 1994).
The fractions with hydrolytic activity (tubes 65–95) were loaded onto a gelatin-cellulofine chromatography column. The partially purified proteinases produced several bands by SDS–PAGE; a major band was present at 72 kDa and minor bands were at 92, 67 and 52 kDa. The 92 kDa proMMP-9 band was faint in comparison with the 72 kDa proMMP-2 band. As MMP-9 is a glycoprotein, it did not stain fully by the silver stain reagent. The proteinases recognized by the polyclonal antibodies against MMP-2 and MMP-9 were the 72 and 67 kDa bands representing proMMP-2 and MMP-2, and the 92 and 84 kDa bands representing proMMP-9 and MMP-9 respectively. These bands and two additional bands of 52 and 45 kDa were all detected in the partially purified sample by gelatin-zymography. We expect that the 52 and 45 kDa bands were degradation products of MMP-2 or MMP-9, as they had gelatin binding domains in the molecule (as shown by binding to the gelatin-cellulofine column) and gelatinolytic activity (as demonstrated by gelatin-zymography). However, these proteinases were not recognized by the anti-MMP-2 and anti-MMP-9 antibodies. We expect that these antibodies would recognize the C-terminal domain in MMP, but would not recognize the catalytic domain of MMP-2 or MMP-9. Both the 52 and 45 kDa proteinases would be expected to contain the catalytic domain of MMP-2 or MMP-9 and lack the C-terminal domain. Comparing the standard mixture (proMMP-9, proMMP-2 and MMP-2) with the final partially purified sample using gelatin-zymography, these proteinase activities showed the same positions of proMMP-9 (92 kDa), proMMP-2 (72 kDa) and MMP-2 (67 kDa). The final sample and the standard mixture were not detected by casein-zymography. This result showed that it is not possible to use this method for the detection of MMP-9 and MMP-2. The gelatin-zymography showed that these proteinases were all inhibited by EDTA, EGTA and o-phenanthroline, all of which are inhibitors of metalloproteinases. These data further verified that these enzymes are all metalloproteinases.
Seminal coagulation and liquefaction after ejaculation are important functions for fertilization. In particular, the digestion of seminal proteins by proteinases is important for semen liquefaction. The representative proteinase of semen is prostatic specific antigen (PSA), which can cleave the cross-linked semenogelin, the major gel-forming protein of seminal vesicle secretions (Robert and Gagnon, 1999). PSA is a prominent serine proteinase in prostatic secretions and has a chymotrypsin-like activity. As proMMP-9 is partially activated by chymotrypsin, it may be activated by PSA in seminal plasma. Furthermore, the digestion of cross-linked semenogelin by PSA may occur in association with activated MMPs.
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Acknowledgements |
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This work was supported by a grant for the promotion of the advancement of education and research in graduate schools of Japan.
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Notes |
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5 To whom correspondence should be addressed. E-mail: kshimoka{at}my-pharm.ac.jp
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References |
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Submitted on June 11, 2001; accepted on October 18, 2001.
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