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From the Departments of * Functional Bioanalysis
and Chemistry of Hygiene, Meiji Pharmaceutical
University, Kiyose, Tokyo, Japan; and the
Department of Obstetrics and Gynecology,
Tachikawa Kyosai Hospital, Tachikawa, Tokyo, Japan.
| Correspondence to: Dr K. Shimokawa, Department of Functional Bioanalysis, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan. |
| Received for publication June 3, 2002; accepted for publication September 12, 2002. |
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Abstract |
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Key words: Human seminal plasma, matrix metalloproteinase
The existence of four kinds of TIMPs (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) has been confirmed, and these TIMPs have about 37% sequence identity to each other including 12 conserved half-cysteines. Human TIMP-1 and TIMP-3 are glycoproteins of about 29 kd and 27 kd, respectively. Human TIMP-2 and TIMP-4 are nonglycoproteins of about 21 kd and 22 kd, respectively (Sternlicht et al, 2001). All TIMPs inhibit MMPs by forming a 1:1 molecular complex. The construction of human TIMPs indicates that the molecule consists of the N-terminal domain possesses with inhibitory activity and the C-terminal domain of TIMPs appears to influence the interaction with MMPs (Nagase, 1996). Gelatinases (MMP-9 and MMP-2) often form complexes with TIMP-1 and TIMP-2, respectively. These specific complexes are formed through interaction of the C-terminal domains of MMPs and TIMPs, and the N-terminal domains of TIMPs in the MMP-9/TIMP-1 and MMP-2/TIMP-2 complexes are unoccupied.
In this paper we report the existence of two kinds of TIMPs (TIMP-1 and TIMP-2) and their complexes with gelatinases in human seminal plasma.
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Materials and Methods |
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Human Seminal Plasma
Human semen was collected from volunteers who visited Tachikawa Kyosai
Hospital 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 x
g for 30 minutes 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 Complexes
Partial purification of complexes were previously described by Shimokawa et
al (2002). The presence of
MMPs in all chromatography fractions was monitored by gelatin-zymography. The
homogeneity of the MMPs following the final chromatography step was examined
by SDS-PAGE and Western blot analysis.
SDS-PAGE
SDS-PAGE was performed using 12% total acrylamide under reducing conditions
as previously described (Laemmli,
1970). Proteins were stained with Coomassie brilliant blue (CBB)
R-250 or silver nitrate.
Gelatin-Zymography
The chromatography extracts were mixed with nonreducing SDS gel sample
buffer and applied without boiling to a 12% polyacrylamide gel containing 0.1%
SDS and 1 mg/mL gelatin 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
CaCl2, 5 µmol/L ZnCl, 0.02% NaN3, and 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 24 hours. Proteins
were stained with CBB R-250 solution.
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, and 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 abilities to digest
synthetic fluorogenic substrates,
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 hours, 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 Company, Japan).
Western Blot Analysis
For Western blot analysis, samples electrophoresed by 12% SDS-PAGE were
electroblotted onto PVDF membranes as previously described
(Burnette, 1981). Nonspecific
binding of immunoglobulin (Ig) G was blocked by a 3% skim milk solution. The
membranes were incubated with the primary antibodies at a 1:5000 dilution for
1 hour. The two primary polyclonal antibodies of sheep anti-human MMP-2 or
sheep anti-human MMP-9, and the four primary synthetic peptide antibodies to
rabbit anti-human TIMP-1, TIMP-2, TIMP-3, or TIMP-4 were used in all
experiments. After the membranes received an extensive washing they were
incubated with peroxidase-conjugated goat anti-rabbit IgG at a 1:50 000
dilution for 1 hour at room temperature. Protein bands were detected using the
ECL Plus Western blotting detection system with subsequent exposure to x-ray
film.
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Results |
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The fractions in tubes 66-93 produced many bands on SDS-PAGE (Figure 2A) and gelatin hydrolyzed activities indicated by gelatin-zymography (Figure 2B). The bands of proMMP-9 (92 kd), proMMP-2 (72 kd), and MMP-2 (67 kd) were detected on fractions 66-84, and these bands corresponded with standard proMMP-9, proMMP-2, and MMP-2 mixture solutions.
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Western blot analysis using the antibodies of anti-human MMP-2 and MMP-9 revealed two bands of proMMP-2 and MMP-2 on the membrane against anti-human MMP-2 antibody (Figure 2C), but the bands were not detected against anti-human MMP-9 antibody (data not shown).
Figure 2 (D and E) shows two bands on each membrane against anti-human TIMP-1 and TIMP-2 antibodies by Western blot analysis, but the bands were not detected against anti-human TIMP-3 and TIMP-4 antibodies (data not shown). The antibodies to TIMP-1 and TIMP-2 recognized proteins of 29 and 23 kd (Figure 2D), and 21 and 18 kd, respectively (Figure 2E). The bands of standard recombinant full-length TIMPs (TIMP-1 and TIMP-2) were detected against anti-human TIMP-1 and TIMP-2 antibodies (Figure 2, D and E). These bands were consistent with standard recombinant full-length TIMP-1 and TIMP-2 proteins. The major band was full-length TIMP-1 (29 kd), and the minor band was low-molecular-weight TIMP-1 (23 kd; Figure 2D). Similarly, the major band was full-length TIMP-2 (21 kd), and the minor band was low molecular-weight TIMP-2 (18 kd; Figure 2E).
The fractions in tubes 65-95 were loaded onto a gelatin-cellulofine column and eluted by 5% dimethyl sulf-oxide solution (Figure 3), and the peaks (tubes 80-84) were collected and concentrated.
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The concentrated sample was examined by SDS-PAGE and Western blot against anti-human MMP-9, MMP-2, TIMP-1, and TIMP-2 (Figure 4). The partially purified proteins produced several bands on SDS-PAGE (Figure 4a). Western blot analysis using the antibodies of anti-human MMP-9, MMP-2, TIMP-1, and TIMP-2 showed bands on the membrane (Figure 4, b—g). The antibodies to MMP-9 and MMP-2 recognized proteins of 92, 72, and 67 kd, and the antibodies to TIMP-1 and TIMP-2 recognized proteins of 29 and 21 kd, respectively. Standard recombinant full-length TIMPs (TIMP-1 and TIMP-2) were observed as prominent bands on each membrane (Figure 4, d and f). In the concentrated sample, the bands of MMP-2 and TIMP-2 were observed as a prominent band (Figure 4, c and g), but the bands of MMP-9 and TIMP-1 were faint on each membrane (Figure 4, b and e).
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Discussion |
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Another group of researchers reported that trypsin, chymotrypsin, and human neutrophil elastase (HNE) cleave TIMP-1 and destroy MMP-inhibitory activity (Okada et al, 1988). Trypsin and chymotrypsin both digest TIMP-1 into several small fragments, whereas HNE cleaves TIMP-1 into two major fragments, suggesting that a single cleavage of the inhibitor leads to its inactivation. These low-molecular-weight TIMPs may be degraded by some proteinase; for example, prostatic specific antigen (PSA) in seminal plasma (Robert and Gagnon, 1999), because PSA is a prominent serine proteinase in prostatic secretions and has a chymotrypsin-like activity.
The fractions in tubes 65-95 were loaded onto a gelatin-cellulofine column (Figure 3). The concentrated sample was examined by Western blotting against anti-human MMP-9, MMP-2, TIMP-1, and TIMP-2 (Figure 4). Western blot analysis using the antibodies detected the band on each membrane (Figure 2, B—G). These antibodies recognized proteins of 92, 72, 67, 29, and 21 kd, respectively. We have already reported the existence of proMMP-9 (92 kd), proMMP-2 (72 kd), and MMP-2 (67 kd) in concentrated samples after gelatin-cellulofine affinity column chromatography (Shimokawa et al, 2002), and these bands were recognized against anti-human MMP-9 and MMP-2 in the concentrated sample (Figure 4, b and c). On the other hand, standard recombinant TIMPs (TIMP-1 and TIMP-2) were observed as one band on each membrane (Figure 4, d and f). In the concentrated sample, these bands were consistent with standard recombinant TIMPs (Figure 4, e and g). Therefore, these results indicate that these TIMPs partially existed as complexes of proMMP-9/TIMP-1, proMMP-2/TIMP-2, and MMP-2/TIMP-2 in human seminal plasma, because proMMP-9, proMMP-2, and MMP-2 were bound to the gelatin affinity column by each having gelatin binding domains in their catalytic domains (Collier et al, 1992; Banyai et al, 1994).
Another group of researchers reported that when TIMP-1 was cleaved of the Val69-Cys70 bond by HNE, its inhibitory activity was destroyed. However, cleavage of this bond by HNE was prevented when TIMP-1 formed a complex with the catalytic domain of MMP-3 (Nagase et al, 1997). Our data show that these TIMPs were not degraded in a concentrated sample (Figure 4, e and g). We expected that cleavage of TIMPs was prevented by the formation of complexes, and that free TIMPs were degraded by some proteinases in human seminal plasma.
Recently, another group reported the presence of heparin-binding protein (HBP) in bovine seminal fluid (McCauley et al, 2001). The molecular mass of purified HBP was 24 kd under reducing conditions. The N-terminal 20-amino acid sequence of HBP shared significant identity (90%) with a bovine TIMP-2, however, the role of HBP in bovine seminal fluid is unknown.
The digestion of seminal proteins by proteinases is important for semen liquefaction. The representative proteinase in semen is PSA, which can cleave the cross-linked semenogelin, the major gel-forming protein of seminal vesicle secretions (Robert and Gagnon, 1999). It may be that MMP-9/TIMP-1 and MMP-2/TIMP-2 complexes, are involved in the regulation of physiological processes in human semen; for example, they might be associated with the digestion of cross-linked semenogelin by PSA. However, the role of human MMPs and TIMPs in human seminal plasma is unknown.
In summary, our data show that proMMP-9/TIMP-1, proMMP-2/TIMP-2, MMP-2/TIMP-2, free TIMP-1, and TIMP-2 exist in human seminal plasma, and that partially free TIMPs were degraded by some proteinases in human seminal plasma.
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Footnotes |
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