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Shedding of gACE from residual body membrane of rat sperm
Legal Medicine 11 (2009) S309–S310
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Shedding of gACE from residual body membrane of rat sperm Keisuke Takeuchi *, Tomohisa Sakaue, Yoshio Yamamoto, Katsuji Nishi, Iwao Ohkubo Department of Medical Biochemistry, Shiga University of Medical Science, Seta Tsukinowr-cho, Otsu, Shiga 520-2192, Japan
a r t i c l e
i n f o
Article history: Received 16 January 2009 Accepted 2 February 2009 Available online 17 April 2009 Keywords: gACE Sheddase
a b s t r a c t Germinal angiotensin I-converting enzyme (gACE) is expressed only in the testis and is uniquely present in developing spermatids and sperm. We previously purified soluble gACE from porcine seminal plasma, and reported that gACE was secreted from residual body on spermatozoa by the other peptidase(s), namely Sheddase. Using Nma/DNP substrate, it was observed that the shedding activity in testicular fluid was stronger than in the sperm membrane and epididymal fluid. The shedding activity was inhibited by AEBSF and antipain, and not by EDTA and E-64. Accordingly, it is thought that Sheddase is an endo-type of serine peptidase. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Angiotensin I-converting enzyme (ACE; EC 3.4.1.15.1) releases C-terminal dipeptides from angiotensin I and generates the vasoconstrictor peptide angiotensin II [1]. ACE also mediates degradation of the vasodilator bradykinin [1]. Accordingly, it is thought that ACE plays a critical role in the regulation of blood pressure and water and salt metabolism. There are two types of ACE isoforms in mammals, somatic ACE (sACE) and germinal ACE (gACE). ACEs are transcribed from the same gene using alternative promoters [1]. gACE is only expressed in the testis and is uniquely present in developing spermatids and sperm. The enzyme plays an important role in the release of PGI-anchored proteins such as TESP5 and PH-20 [2]. In ACE knockout sperm, GPI-anchored proteins were not released from the sperm membrane. Fertilization does not occur in ACE knockout mice [2]. We previously purified soluble gACE from porcine seminal plasma and reported that gACE in seminal plasma was originated from the residual body on spermatozoa in the testis and epididymis [3]. It is suspected that gACE in seminal plasma was released by other peptidases from the residual body. Here, we report the shedding of rat gACE from the membrane of developing spermatids and mature sperm. 2. Methods 2.1. Release of gACE from rat sperm Rat sperms were obtained from the testis. The sperms were washed three times with PBS, resuspended in 50 mM phosphate * Corresponding author. Tel.: +81 77 548 2161; fax: +81 77 548 2164. E-mail address: [email protected] (K. Takeuchi). 1344-6223/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2009.02.074
buffer, pH 7.0 (or sodium citrate buffer, pH 5.0) with several protease inhibitors, and incubated at 37 °C. After incubation for 12 h, the sperm solution was centrifuged at 1400g for 15 min. The pellet and supernatant were separately obtained. The pellet was then sonicated. Each sample was applied on 12.5% SDS–PAGE and blotted onto a PVDF membrane. gACE was then detected by the method of immunoblot analysis. 2.2. Determination of shedding activity For estimation of ACE-shedding activity, we newly synthesized a specific substrate, Nma-NSARLEK(DNP)-NH2, that contains the cleavage site of rat gACE. Rat testis was cut in half and put into PBS for 60 min. The solution containing sperm was centrifuged at 1500g for 15 min. Using the above Nma-DNP substrate, the sheddase activity was estimated by fluorometric determination (Ex, 340 nm; Em, 440 nm). 3. Results and discussion ACE exists in both membrane-bound and soluble forms. However, little is known about what kinds of peptidases are able to release ACE from the somatic or germinal cell membrane. Oppong and Hooper [4] reported that secretase (sheddase) to somatic ACE exists as the microvilli membrane protein in pig kidney and has characteristics as metalloprotease to be sensitive to EDTA and 1,10-phenanthroline. On the other hand, Thimon et al. [5] recently reported that gACE cleavage from sperm was strongly increased by the presence of epididymal fluid and that shedding activity was inhibited by AEBSF as a serine protease inhibitor. As shown in Fig. 1A, rat gACE was released from testicular sperm in 50 mM phosphate buffer (pH 7.0) but not in 50 mM sodium citrate buffer (pH 5.0). Furthermore, the releasing of gACE from the sperm was strongly inhibited
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K. Takeuchi et al. / Legal Medicine 11 (2009) S309–S310
Fig. 1. Effects of various peptidase inhibitors on shedding of rat testicular sperm gACE. Lane 1: Control (50 mM phosphate buffer, pH 7.0), lane 2: 5 mM EDTA in the same buffer, lane 3: 1 mM AEBSF in the same buffer, lane 4: 30 lM E-64 in the same buffer, lane 5: 100 lM antipain in the same buffer, lane 6: 100 lM leupeptin in the same buffer.
Fig. 2. Detection of ACE-shedding activity for the specific substrate in testicular fluid, sperm and epididymis.
Fig. 3. Detection of gACE-shedding from the sperm membrane during epididymal transit by immunofluorescence microscopy using gACE antibody.
by 1 mM AEBSF and 100 lM antipain but was not inhibited by EDTA, E-64 and leupeptin (Fig. 1B). These results suggest that the gACEshedding enzyme is a serine peptidase reacting at neutral pH. Furthermore, we tried to determine the localization of the gACEshedding enzyme (sheddase) in the rat genital tract using a newly synthesized specific substrate. As shown in Fig. 2, the sheddase activity was detected in testicular fluid, testicular sperm and epididymis. The activity in testicular fluid was 4-fold stronger than the activities in testicular sperm and epididymis. The activity in rat testicular fluid was inhibited by AEBSF and DFP (data not shown). In addition, we tried to observe the localization of gACE-shedding in the porcine genital tract. As shown in Fig. 3, gACE in the membrane of sperm was released during epididymis transit. Most of gACE localized at the membrane surface of testicular sperm was released in the cauda region of the epididymis. These result agree with results reported by Thimon et al. [5]. Thus, solublized gACE is thought to work for release of PGI-anchored proteins such as TESP5 and PH20 and to play an important role in fertilization.
Conflict of interest None. References [1] Corvol P, Eyries M, Soubrier F. Peptidyl-dipeptidase A/angiotensin I-converting enzyme. In: Barrett A, Rowlings ND, Woessner JF, editors. Handbook of porteolytic enzymes. Amsterdam: Elsevier Academic Press; 2004. p. 332–46. [2] Kondoh G, Tojo H, Nakatani Y, Komazawa N, Murata C, Yamagata K, et al. Angiotensin-converting enzyme is GPI-anchored protein releasing factor crucial for fertilization. Nat Med 2005;11:160–6. [3] Takeuchi K, Araki H, Sakaue T, Yamamoto Y, Fujiwara M, Nishi K, et al. Porcine germinal angiotensin I-converting enzyme: Isolation, characterization and molecular cloning. Comp Biochem Biophys B Biochem Mol Biol 2007;146:215–26. [4] Oppong SY, Hooper NM. Characterization of a secretase activity which releases angiotensin-converting enzyme from the membrane. Biochem J 1993;292: 597–603. [5] Thimon V, Metayer S, Belghazi M, Dacheux F, Dacheux JL, Gatti JL. Shedding of the germinal angiotensin I-converting enzyme (gACE) involves a serine protease and is activated by epididymal fluid. Biol Reprod 2005;73:881–90.
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Shedding of gACE from residual body membrane of rat sperm Keisuke Takeuchi *, Tomohisa Sakaue, Yoshio Yamamoto, Katsuji Nishi, Iwao Ohkubo Department of Medical Biochemistry, Shiga University of Medical Science, Seta Tsukinowr-cho, Otsu, Shiga 520-2192, Japan
a r t i c l e
i n f o
Article history: Received 16 January 2009 Accepted 2 February 2009 Available online 17 April 2009 Keywords: gACE Sheddase
a b s t r a c t Germinal angiotensin I-converting enzyme (gACE) is expressed only in the testis and is uniquely present in developing spermatids and sperm. We previously purified soluble gACE from porcine seminal plasma, and reported that gACE was secreted from residual body on spermatozoa by the other peptidase(s), namely Sheddase. Using Nma/DNP substrate, it was observed that the shedding activity in testicular fluid was stronger than in the sperm membrane and epididymal fluid. The shedding activity was inhibited by AEBSF and antipain, and not by EDTA and E-64. Accordingly, it is thought that Sheddase is an endo-type of serine peptidase. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Angiotensin I-converting enzyme (ACE; EC 3.4.1.15.1) releases C-terminal dipeptides from angiotensin I and generates the vasoconstrictor peptide angiotensin II [1]. ACE also mediates degradation of the vasodilator bradykinin [1]. Accordingly, it is thought that ACE plays a critical role in the regulation of blood pressure and water and salt metabolism. There are two types of ACE isoforms in mammals, somatic ACE (sACE) and germinal ACE (gACE). ACEs are transcribed from the same gene using alternative promoters [1]. gACE is only expressed in the testis and is uniquely present in developing spermatids and sperm. The enzyme plays an important role in the release of PGI-anchored proteins such as TESP5 and PH-20 [2]. In ACE knockout sperm, GPI-anchored proteins were not released from the sperm membrane. Fertilization does not occur in ACE knockout mice [2]. We previously purified soluble gACE from porcine seminal plasma and reported that gACE in seminal plasma was originated from the residual body on spermatozoa in the testis and epididymis [3]. It is suspected that gACE in seminal plasma was released by other peptidases from the residual body. Here, we report the shedding of rat gACE from the membrane of developing spermatids and mature sperm. 2. Methods 2.1. Release of gACE from rat sperm Rat sperms were obtained from the testis. The sperms were washed three times with PBS, resuspended in 50 mM phosphate * Corresponding author. Tel.: +81 77 548 2161; fax: +81 77 548 2164. E-mail address: [email protected] (K. Takeuchi). 1344-6223/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2009.02.074
buffer, pH 7.0 (or sodium citrate buffer, pH 5.0) with several protease inhibitors, and incubated at 37 °C. After incubation for 12 h, the sperm solution was centrifuged at 1400g for 15 min. The pellet and supernatant were separately obtained. The pellet was then sonicated. Each sample was applied on 12.5% SDS–PAGE and blotted onto a PVDF membrane. gACE was then detected by the method of immunoblot analysis. 2.2. Determination of shedding activity For estimation of ACE-shedding activity, we newly synthesized a specific substrate, Nma-NSARLEK(DNP)-NH2, that contains the cleavage site of rat gACE. Rat testis was cut in half and put into PBS for 60 min. The solution containing sperm was centrifuged at 1500g for 15 min. Using the above Nma-DNP substrate, the sheddase activity was estimated by fluorometric determination (Ex, 340 nm; Em, 440 nm). 3. Results and discussion ACE exists in both membrane-bound and soluble forms. However, little is known about what kinds of peptidases are able to release ACE from the somatic or germinal cell membrane. Oppong and Hooper [4] reported that secretase (sheddase) to somatic ACE exists as the microvilli membrane protein in pig kidney and has characteristics as metalloprotease to be sensitive to EDTA and 1,10-phenanthroline. On the other hand, Thimon et al. [5] recently reported that gACE cleavage from sperm was strongly increased by the presence of epididymal fluid and that shedding activity was inhibited by AEBSF as a serine protease inhibitor. As shown in Fig. 1A, rat gACE was released from testicular sperm in 50 mM phosphate buffer (pH 7.0) but not in 50 mM sodium citrate buffer (pH 5.0). Furthermore, the releasing of gACE from the sperm was strongly inhibited
S310
K. Takeuchi et al. / Legal Medicine 11 (2009) S309–S310
Fig. 1. Effects of various peptidase inhibitors on shedding of rat testicular sperm gACE. Lane 1: Control (50 mM phosphate buffer, pH 7.0), lane 2: 5 mM EDTA in the same buffer, lane 3: 1 mM AEBSF in the same buffer, lane 4: 30 lM E-64 in the same buffer, lane 5: 100 lM antipain in the same buffer, lane 6: 100 lM leupeptin in the same buffer.
Fig. 2. Detection of ACE-shedding activity for the specific substrate in testicular fluid, sperm and epididymis.
Fig. 3. Detection of gACE-shedding from the sperm membrane during epididymal transit by immunofluorescence microscopy using gACE antibody.
by 1 mM AEBSF and 100 lM antipain but was not inhibited by EDTA, E-64 and leupeptin (Fig. 1B). These results suggest that the gACEshedding enzyme is a serine peptidase reacting at neutral pH. Furthermore, we tried to determine the localization of the gACEshedding enzyme (sheddase) in the rat genital tract using a newly synthesized specific substrate. As shown in Fig. 2, the sheddase activity was detected in testicular fluid, testicular sperm and epididymis. The activity in testicular fluid was 4-fold stronger than the activities in testicular sperm and epididymis. The activity in rat testicular fluid was inhibited by AEBSF and DFP (data not shown). In addition, we tried to observe the localization of gACE-shedding in the porcine genital tract. As shown in Fig. 3, gACE in the membrane of sperm was released during epididymis transit. Most of gACE localized at the membrane surface of testicular sperm was released in the cauda region of the epididymis. These result agree with results reported by Thimon et al. [5]. Thus, solublized gACE is thought to work for release of PGI-anchored proteins such as TESP5 and PH20 and to play an important role in fertilization.
Conflict of interest None. References [1] Corvol P, Eyries M, Soubrier F. Peptidyl-dipeptidase A/angiotensin I-converting enzyme. In: Barrett A, Rowlings ND, Woessner JF, editors. Handbook of porteolytic enzymes. Amsterdam: Elsevier Academic Press; 2004. p. 332–46. [2] Kondoh G, Tojo H, Nakatani Y, Komazawa N, Murata C, Yamagata K, et al. Angiotensin-converting enzyme is GPI-anchored protein releasing factor crucial for fertilization. Nat Med 2005;11:160–6. [3] Takeuchi K, Araki H, Sakaue T, Yamamoto Y, Fujiwara M, Nishi K, et al. Porcine germinal angiotensin I-converting enzyme: Isolation, characterization and molecular cloning. Comp Biochem Biophys B Biochem Mol Biol 2007;146:215–26. [4] Oppong SY, Hooper NM. Characterization of a secretase activity which releases angiotensin-converting enzyme from the membrane. Biochem J 1993;292: 597–603. [5] Thimon V, Metayer S, Belghazi M, Dacheux F, Dacheux JL, Gatti JL. Shedding of the germinal angiotensin I-converting enzyme (gACE) involves a serine protease and is activated by epididymal fluid. Biol Reprod 2005;73:881–90.