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Angiotensin-converting enzyme is a GPI-anchored protein releasing factor crucial for fertilization

Nature Medicine volume 11, pages 160–166 (2005)Cite this article

Abstract

The angiotensin-converting enzyme (ACE) is a key regulator of blood pressure. It is known to cleave small peptides, such as angiotensin I and bradykinin and changes their biological activities, leading to upregulation of blood pressure. Here we describe a new activity for ACE: a glycosylphosphatidylinositol (GPI)-anchored protein releasing activity (GPIase activity). Unlike its peptidase activity, GPIase activity is weakly inhibited by the tightly binding ACE inhibitor and not inactivated by substitutions of core amino acid residues for the peptidase activity, suggesting that the active site elements for GPIase differ from those for peptidase activity. ACE shed various GPI-anchored proteins from the cell surface, and the process was accelerated by the lipid raft disruptor filipin. The released products carried portions of the GPI anchor, indicating cleavage within the GPI moiety. Further analysis by high-performance liquid chromatography–mass spectrometry predicted the cleavage site at the mannose-mannose linkage. GPI-anchored proteins such as TESP5 and PH-20 were released from the sperm membrane of wild-type mice but not in Ace knockout sperm in vivo. Moreover, peptidase-inactivated E414D mutant ACE and also PI-PLC rescued the egg-binding deficiency of Ace knockout sperms, implying that ACE plays a crucial role in fertilization through this activity.

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Figure 1: ACE acts as a GPI anchored protein–releasing factor.
Figure 2: Differences in GPIase and peptidase activities of ACE.
Figure 3: Shedding activity of ACE on the cell surface.
Figure 4: Characteristics of GPI cleavage by ACE.
Figure 5: Involvement of ACE GPIase activity in sperm-egg binding.

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References

  1. Kinoshita, T., Ohishi, K. & Takeda, J. GPI-anchor synthesis in mammalian cells: genes, their products, and a deficiency. J. Biochem. 122, 251–257 (1997).

    Article  CAS  PubMed  Google Scholar 

  2. Ikezawa, H. Glycosylphosphatidylinositol (GPI)-anchored proteins. Biol. Pharm. Bull. 25, 409–417 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Nozaki, M. et al. Developmental abnormalities of glycosylphosphatidylinositol-anchor-deficient embryos revealed by Cre/loxP system. Lab. Invest. 79, 293–299 (1999).

    CAS  PubMed  Google Scholar 

  4. Tarutani, M. et al. Tissue-specific knockout of the mouse Pig-a gene reveals important roles for GPI-anchored proteins in skin development. Proc. Natl. Acad. Sci. USA 94, 7400–7405 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Alfieri, J. A. et al. Infertility in female mice with an oocyte-specific knock of GPI-anchored proteins. J. Cell Sci. 116, 2149–2155 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Miyata, T. et al. The cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis. Science 259, 1318–1320 (1993).

    Article  CAS  PubMed  Google Scholar 

  7. Takeda, J. et al. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell 73, 703–711 (1993).

    Article  CAS  PubMed  Google Scholar 

  8. Prusiner, S. B. Prions. Proc. Natl. Acad. Sci. USA 95, 13363–13383 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Aguzzi, A. & Polymenidou, M. Mammalian prion biology: one century of evolving concepts. Cell 116, 313–327 (2004).

    Article  CAS  PubMed  Google Scholar 

  10. Marella, M., Lehmann, S., Grassi, J. & Chabry, J. Filipin prevents pathological prion protein accumulation by reducing endocytosis and inducing cellular PrP release. J. Biol. Chem. 277, 25457–25464 (2002).

    Article  CAS  PubMed  Google Scholar 

  11. Vincent, B. et al. Phorbol ester-regulated cleavage of normal prion protein in HEK293 human cells and murine neurons J. Biol. Chem. 275, 35612–35616 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Kondoh, G. et al. Tissue-inherent fate of GPI revealed by GPI-anchored GFP transgenesis. FEBS Lett. 458, 299–303 (1999).

    Article  CAS  PubMed  Google Scholar 

  13. Mayor, S., Sabharanjak, S. & Maxfield, F. R. Cholesterol-dependent retention of GPI-anchored proteins in endosomes. EMBO J. 17, 4626–4638 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sabharanjak, S., Sharma, P., Parton, R. G. & Mayor, S. GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway. Dev. Cell 2, 411–423 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. Hooper, N. M. Angiotensin converting enzyme: implications from molecular biology for its physiological functions. Int. J. Biochem. 23, 641–647 (1991).

    Article  CAS  PubMed  Google Scholar 

  16. Turner, A. J. & Hooper, N. M. The angiotensin-converting enzyme gene family: genomics and pharmacology. Trends Pharmacol. Sci. 23, 177–183 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Hooper, N. M. Families of zinc metalloproteases. FEBS Lett. 354, 1–6 (1994).

    Article  CAS  PubMed  Google Scholar 

  18. Wei, L., Alhenc-Gelas, F., Corvol, P. & Clauser, E. The two homologous domains of human angiotensin I-converting enzyme are both catalytically active. J. Biol. Chem. 266, 9002–9008 (1991).

    CAS  PubMed  Google Scholar 

  19. Natesh, R., Schwager, S. L. U., Sturrock, E. D. & Acharya, K. R. Crystal structure of the human angiotensin-converting enzyme-lisinopril complex. Nature 421, 551–554 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Crackower, M. A., et al. Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature 417, 822–828 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Li, W. et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450–454 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Scallon, B. J. et al. Primary structure and functional activity of a phosphatidylinositol-glycan-specific phospholipase D. Science 252, 446–448 (1991).

    Article  CAS  PubMed  Google Scholar 

  23. Tujioka, H., Misumi, Y., Takami, N. and Ikehara, Y. Posttranslational modification of glycosylphosphatidylinositol (GPI)-specific phospholipase D and its activity in cleavage of GPI anchors. Biochem. Biophys. Res. Commun. 251, 737–743 (1998).

    Article  CAS  PubMed  Google Scholar 

  24. Watanabe, T., Wada, N., Kim, E. E., Wyckoff, H. W. & Chou, J. Y. Mutations of a single amino acid converts germ cell alkaline phosphatase to placental alkaline phosphatase. J. Biol. Chem. 266, 21174–21178 (1991).

    CAS  PubMed  Google Scholar 

  25. Carvajal, N. et al. Non-chelating inhibition of the H10N variant of human liver arginase by EDTA. J. Inorg. Biochem. 98, 1465–1469 (2004).

    Article  CAS  PubMed  Google Scholar 

  26. Nyborg, J. K. & Peersen, O. B. That zincing feeling: the effect of EDTA on the behaviour of zinc-binding transcriptional regulators. Biochem. J. 381, e3–e4 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Nicholas, B-J. et al. Rapid cycling of lipid raft markers between the cell surface and Golgi complex. J. Cell Biol. 153, 529–541 (2001).

    Article  Google Scholar 

  28. Simons, K. & Ikonen, E. Functional rafts in cell membranes. Nature 387, 569–572 (1997).

    Article  CAS  PubMed  Google Scholar 

  29. Brown, D. A. & London, E. Functions of lipid rafts in biological membranes. Annu. Rev. Cell Dev. Biol. 14, 111–136 (1998).

    Article  CAS  PubMed  Google Scholar 

  30. Varma, R. & Mayor, S. GPI-anchored proteins are organized in submicron domains at the cell surface. Nature 394, 798–801 (1998).

    Article  CAS  PubMed  Google Scholar 

  31. Hanada, K., Izawa, K., Nishijima, M. & Akamatsu, Y. Sphingolipid deficiency induces hypersensitivity of CD14, a glycosylphosphatidylinositol-anchored protein, to phosphatidylinositol-specific phospholipase C. J. Biol. Chem. 268, 13820–13823 (1993).

    CAS  PubMed  Google Scholar 

  32. Chen, R. et al. Mammalian glycosylphosphatidylinositol anchor transfer to proteins and posttransfer deacylation. Proc. Natl. Acad. Sci. USA 95, 9512–9517 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Krege, J. H. et al. Male-female differences in fertility and blood pressure in ACE-deficient mice. Nature 375, 146–148 (1995).

    Article  CAS  PubMed  Google Scholar 

  34. Hagaman, J. R., et al. Angiotensin-converting enzyme and male fertility. Proc. Natl. Acad. Sci. USA 95, 2552–2557 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Honda, A., Yamagata, K., Sugiura, S., Watanabe, K. & Baba, T. A mouse serine protease TESP5 is selectively included into lipid rafts of sperm membrane presumably as a glycosylphosphatidylinositol-anchored protein. J. Biol. Chem. 277, 16976–16984 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Lin, Y., Mahan, K., Lathrop, W. F., Myles, D. G. & Primakoff, P. A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg. J. Cell Biol. 125, 1157–1163 (1994).

    Article  CAS  PubMed  Google Scholar 

  37. Roberts, W. L., Myher, J. J., Kuksis, A., Low, M. G. & Rosenberry, T. L. Lipid analysis of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase. Palmitoylation of inositol results in resistance to phosphatidylinositol-specific phospholipase C. J. Biol. Chem. 263, 18766–18775 (1988).

    CAS  PubMed  Google Scholar 

  38. McLeskey, S. B., Dowds, C., Carballada, R., White, R. R. & Saling, P. M. Molecules involved in mammalian sperm-egg interaction. Int. Rev. Cytol. 177, 57–113 (1998).

    Article  CAS  PubMed  Google Scholar 

  39. Doan, T. N., Gletsu, N., Cole, J. & Bernstein, K. E. Genetic manipulation of the rennin-angiotensin system. Curr. Opin. Nephrol. Hypertens. 10, 483–491 (2001).

    Article  CAS  PubMed  Google Scholar 

  40. Bergaya, S., et al. Decreased flow-dependent dilation in carotid arteries of tissue kallikrein-knockout mice. Circ. Res. 88, 593–599 (2001).

    Article  CAS  PubMed  Google Scholar 

  41. Meier, P., et al. Soluble dimeric prion protein binds PrPSc in vivo and antagonizes prion disease. Cell 113, 49–60 (2003).

    Article  CAS  PubMed  Google Scholar 

  42. Tojo, H. Properties of an electrospray emitter coated with material of low surface energy. J. Chromatogr. A 1056, 223–228 (2004).

    Article  CAS  PubMed  Google Scholar 

  43. Kasahara, Y. & Ashihara, Y. Colorimetry of angiotensin-I converting enzyme activity in serum. Clin. Chem. 27, 1922–1925 (1981).

    CAS  PubMed  Google Scholar 

  44. Kinoshita, T., Medof, M. E., Silber, R. & Nussenzweig, V. Distribution of decay-accelerating factor in the peripheral blood of normal individuals and patients with paroxysmal nocturnal hemoglobinuria. J. Exp. Med. 162, 75–92 (1985).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank K. Ohishi and Y. Tashima for technical assistance, T. Baba for providing anti-TESP5 antibody and P. Primakoff for providing anti-PH-20 antibody. We are also grateful to V.W. Keng and D.G. Myles for the critical reading of the manuscript. This work was supported by grants from the Osaka Medical Research Foundation for Incurable Diseases and the Ministry of Education, Science, Sports, and Culture of Japan.

Author information

Author notes

  1. Gen Kondoh

    Present address: Laboratory of Animal Experiments for Regeneration, Institute for Frontier Medical Sciences, Kyoto University, Japan

  2. Gen Kondoh, Taroh Kinoshita & Ryo Taguchi

    Present address: CREST, Japan Science and Technology Society, 53 Syogoin-Kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan

  3. Kazuo Yamagata

    Present address: Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba Science City, Ibaraki, 305-8572, Japan

Authors and Affiliations

  1. Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Suita, 2-2 Yamadaoka, Osaka, 565-0871, Japan

    Gen Kondoh, Yuka Nakatani, Nobuyasu Komazawa & Junji Takeda

  2. Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Osaka University, Suita, 2-2 Yamadaoka, Osaka, 565-0871, Japan

    Hiromasa Tojo

  3. Department of Metabolome, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Chie Murata & Ryo Taguchi

  4. Genome Information Research Center, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Kazuo Yamagata & Masaru Okabe

  5. Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Yusuke Maeda & Taroh Kinoshita

  6. Center for Advanced Science and Innovation, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Junji Takeda

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Supplementary information

Supplementary Fig. 1 (download PDF )

Detection of GPIase activity in testicular germ cells. (PDF 56 kb)

Supplementary Fig. 2 (download PDF )

Lack of Gpi-pld expression in the mouse testicular germ cell. (PDF 104 kb)

Supplementary Fig. 3 (download PDF )

Proteomics analysis of the purified 100-kDa protein. (PDF 37 kb)

Supplementary Fig. 4 (download PDF )

The testicular isoform of wild-type (ACE-WT) and peptidase-inactivated mutants (ACE-E414D and ACE-H413K, H417K) were produced and purified as described in Methods. (PDF 48 kb)

Supplementary Fig. 5 (download PDF )

PLAP substrate produced and purified as described in Methods. (PDF 30 kb)

Supplementary Fig. 6 (download PDF )

ACE-S results in shedding of DAF from K562 cell surface. K562 cells were treated with filipin and then with 1.0 μM ACE-S as described in the Methods. (PDF 53 kb)

Supplementary Fig. 7 (download PDF )

Pups born from knockout sperm pretreated with wild-type Ace-t. (PDF 75 kb)

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Kondoh, G., Tojo, H., Nakatani, Y. et al. Angiotensin-converting enzyme is a GPI-anchored protein releasing factor crucial for fertilization. Nat Med 11, 160–166 (2005). https://doi.org/10.1038/nm1179

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