Hitoshi Sawada

Title: Professor Institution: Nagoya University Department: Graduate School of Science Address: Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University 429-93 Sugashima, Toba, Mie 517-0004, Japan Phone: +81-599-34-2217 (Sugashima MBL) or +81-52-789-2514 (Nagoya) FAX: +81-599-34-2456 (Sugashima) or +81-52-789-4204 (Nagoya) E-mail: [email protected]

Education Background: 1. Bachelor of Pharmaceutical Sciences (B.Pharm.Sc.) Degree in Pharmaceutical Sciences from Hokkaido University, Japan, received in 1977. 2. Master of Pharmaceutical Sciences (M.Pharm.Sci.) Degree in Pharmaceutical Sciences from Hokkaido University, Japan, received in 1979. 3. Doctor of Pharmaceutical Sciences (Ph.D.) Degree in Pharmaceutical Sciences from Hokkaido University, Japan, received in 1982.

Professional Career: 1. Assistant Professor, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Japan, 1982-1984. 2. Assistant Professor, Department of Physiology, Wakayama Medical College, Japan, 1984-1986. 3. Assistant Professor, Department of Biology, Tokyo Institute of Technology, Japan, 1986-1988. 4. Associate Professor, Department of Bioscience, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Japan, 1988-1991. 5. Associate Professor, Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Japan, 1991-2002. 6. Full Professor, Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, Japan, 2002-Present (2019).

Editorial Board Members in Scientific Journals: 1. Editorial Board Member of “Zygote” 2. Advisory Board Member of “Molecular Reproduction and Development” 3. Editorial Board Member of “Zoological Letters”

Participation in Professional Organization 1. Member of Japanese Biochemical Society 2. Member of Molecular Biological Society of Japan 3. Member of Zoological Society of Japan 4. Member of Japanese Society of Developmental Biologists

Awards: 1. Japanese Zoological Society Award (September, 2014)

Organized International Symposia: 1. The First International Symposium on the Biology of Ascidians (International Tunicate Meeting), Sapporo (June 26-30, 2000) 2. The Fourth International Symposium on the Molecular and Cell Biology of Egg- and Embryo-

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Coats (MCBEEC or Egg Coat Meeting), Shima (November 8-13, 2004) 3. International Symposium “Intercellular Recognition and Allogeneic Authentication; Perspectives of Reproductive Mechanisms Shared by Animals and Plants”, Nagoya (January 14, 2010) 4. International Symposium on the Mechanisms of Sexual Reproduction in Animals and Plants (Joint Meeting of the Second Allo-Authentication Meeting and the Fifth Egg Coat Meeting), Nagoya (November, 12-16, 2012)

Edited Books: 1. The Biology of Ascidians. ISBN 4-431-70296-2, Springer, 2001 2. Sexual Reproduction in Animals and Plants. ISBN 978-431-54588-0, ISBN 978-4-431-54589- 7 (eBook), Springer, 2014. 3. Fertilization in Animals and Plants (in Japanese), Kagakudojin, 2014.

Research activity: 1) Ascidian sperm lysin: Fertilization is a precisely controlled process involving many gamete molecules in sperm binding to and penetration through the extracellular matrix of the egg. After sperm bind to the extracellular matrix (vitelline coat), they undergo the acrosome reaction which exposes and partially releases a lytic agent called “lysin” to digest the vitelline coat for the sperm penetration. The vitelline coat sperm lysin is generally a protease in deuterostomes. The molecular mechanism of the actual degradation of the vitelline coat, however, remains poorly understood. In order to understand the lysin system, we have been studying the fertilization mechanism in ascidians (Urochordata) because we can obtain large quantities of gametes which are readily fertilized in the laboratory. Whereas ascidians are hermaphrodites, which release sperm and eggs simultaneously, many ascidians, including Halocynthia roretzi, are strictly self-sterile. Therefore, after sperm recognize the vitelline coat as nonself, the sperm lysin system is thought to be activated. We revealed that two sperm -like proteases, and spermosin, the latter of which is a novel sperm protease with -like substrate specificity, are essential for fertilization in H. roretzi. These proteases contain motifs involved in binding to the vitelline coat. We found that the proteasome rather than trypsin-like proteases has a direct lytic activity toward the vitelline coat. The target for the ascidian lysin was found to be a 70-kDa vitelline coat component called HrVC70, which is made up of 12 EGF-like repeats. In addition to the proteasome system, the ubiquitination system toward the HrVC70 was found to be necessary for ascidian fertilization. HrVC70 on the vitelline coat is ubiquitinated by insemination, and the fertilization is inhibited by anti-multi-Ub mAb (FK2). Biochemical studies on the structures and roles in fertilization of the two trypsin-like proteases, acrosin and spermosin, and also on the novel extracellular ubiquitin-proteasome system, which plays an essential role in the degradation of the ascidian vitelline coat, are now in progress in my laboratory. 2) Mechanisms of self-sterility (self/nonself-recognition) in Halocynthia roretzi: Halocynthia roretzi is strictly self-sterile. How nonself cells (gametes) can be recognized by ascidians without such acquired immune system as MHCs and antibodies. The vitelline coat protein HrVC70 is considered to be a sperm receptor at fertilization. We are getting the evidence that HrVC70 is a self/nonself-recognition protein. It was also found that several amino acids in HrVC70 are substituted among individuals. The binding partners of HrVC70 in sperm surface were identified as HrUrabin, a GPI-anchored cysteine-rich secretory protein, and HrTTSP-1, a type II transmembrane , on the sperm surface. These proteins are able to bind to HrVC70, but it is still unknown whether these proteins are capable of recognizing the allogeneic difference of HrVC70. 3) Mechanisms of self-sterility (self/nonself-recognition) in Ciona intestinalis (C. robusta): In Ciona intestinalis, we recently identified the gamete proteins responsible for self/nonself- 2

recognition during fertilization by positional cloning. Genetic analysis revealed that two genetic loci, which reside in chromosome 2 (A-locus) and 7 (B-locus), are responsible for self- incompatibility. In both loci, highly polymorphic sperm PKDREJ-like proteins, which are referred to as s-Themis-A and s-Themis-B, and the highly polymorphic fibrinogen-like proteins on the vitelline coat, called v-Themis-A and v-Themis-B, appear to play a key role in the allorecognition system. Interestingly, v-Themis genes locate in the first intron of s-Themis genes, respectively. From the genetic analysis, we found that s-Themis-A and s-Themis-B recognize v-Themis-A and v-Themis-B, respectively. In the case that s-Themis-A and s- Themis-B recognize v-Themis-A and v-Themis-B as self-proteins, respectively, sperm must recognize the vitelline coat as self and detach the vitelline coat. We noticed that the mechanisms of self-incompatibility in ascidians are very similar to those in flowering plants. These results imply the occurrence of the common mechanism of sexual reproduction shared with animals and plants.

Selected Publications:

1) *Sawada, H., Yokosawa, H., and Ishii, S. (1984). Purification and characterization of two types of trypsin-like from sperm of the ascidian (Prochordata) Halocynthia roretzi. Evidence for the presence of spermosin, a novel acrosin-like . J. Biol. Chem. 259, 2900-2904. 2) *Sawada, H., Yokosawa, H., Someno, T., Saino, T., and Ishii, S. (1984). Evidence for the participation of two sperm proteases, spermosin and acrosin, in fertilization of the ascidian, Halocynthia roretzi: Inhibitory effects of leupeptin analogs on enzyme activities and fertilization. Dev. Biol. 105, 246-249. 3) *Sawada, H., Kawahigashi, M., Yokosawa, H., and Ishii, S. (1985). Trypsin-like enzyme from eggs of the ascidian (Protochordate), Halocynthia roretzi. Purification, properties, and physiological role. J. Biol. Chem. 260, 15694-15698. 4) *Sawada, H., Tsuji, S., Kusumoto, S., Doi, Y., and Matsushita, H. (1986). Preclinical increase in activity of muscle microsomal trypsin-like protease in murine muscular dystrophy, C57BL/10-mdx. FEBS Lett. 199, 193-197. 5) Takagi Sawada, M., Someno, T., Hoshi, M., and Sawada, H. (1989). Inhibition of starfish oocyte maturation by leupeptin analogs, potent trypsin inhibitors. Dev. Biol. 133, 609- 612. 6) *Takagi Sawada, M., Someno, T., Hoshi, M., and Sawada, H. (1992). Participation of 650-kDa protease (20S proteasome) in starfish oocyte maturation. Dev. Biol. 150, 414- 418. 7) Matsuura, K., *Sawada, H., and Yokosawa, H. (1993). Purification and properties of N- acetylglucosaminidase from eggs of the ascidian, Halocynthia roretzi. Eur. J. Biochem. 218, 535-541. 8) *Sawada, H., Muto, K., Fujimuro, M., Akaishi, T., Takagi Sawada, M., Yokosawa, H., and Goldberg, A. L. (1993). Different ratios in 20 S proteasomes and regulatory subunit complexes in two forms of the 26 S proteasome purified from rabbit skeletal muscle. FEBS Lett. 335, 207-212. 9) Yamazaki, K., Suzuki, R., Hojo, E., Kondo, S., Kato, Y., Kamioka, K., Hoshi, M., and *Sawada, H. (1994). Trypsin-like hatching enzyme of mouse blastocysts: Evidence for its participation in hatching process before zona shedding of embryos. Develop. Growth Differ. 36, 149-154. 10) *Fujimuro, M., Sawada, H., and Yokosawa, H. (1994). Production and characterization of monoclonal antibodies specific to multi-ubiquitin chains of polyubiquitinated proteins. FEBS Lett. 349, 173-180.

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11) Satoh, K., Nishikawa, T., Yokosawa, H., and *Sawada, H. (1995). Phosphorylation of proteasome substrate by a protein kinase associated with the 26 S proteasome is linked to the ATP-dependent proteolysis of the 26 S proteasome. Biochem. Biophys. Res. Commun. 213, 7-14. 12) Matsuura, K., *Sawada, H., and Yokosawa, H. (1995). N-Acetylglucosaminidase inhibitor isolated from the vitelline coat of ascidian eggs is a candidate for sperm receptor. Biochem. Biophys. Res. Commun. 213, 311-316. 13) Akaishi, T., Yokosawa, H., and *Sawada, H. (1995). Regulatory subunit complex dissociated from 26 S proteasome: Isolation and characterization. Biochim. Biophys. Acta 1245, 331-338. 14) Akaishi, T., Shiomi, T., *Sawada, H., and Yokosawa, H. (1996). Purification and properties of the 26 S proteasome from the rat brain: Evidence for its degradation of myelin basic protein in a ubiquitin-dependent manner. Brain Res. 727, 139-144. 15) *Takagi Sawada, M., Kyozuka, K., Izumi, K., and Sawada, H. (1997). The proteasome is an essential mediator of the activation of pre-MPF during starfish oocyte maturation. Biochem. Biophys. Res. Commun. 236, 40-43. 16) *Sawada, H., Akaishi, T., Katsu, M., and Yokosawa, H. (1997). Difference between PA700-like proteasome activator complex and the regulatory complex dissociated from the 26S proteasome implies the involvement of modulating factors in the 26S proteasome assembly. FEBS Lett. 412, 521-525. 17) *Sawada, H., Pinto, M. R., and De Santis, R. (1998). Participation of sperm proteasome in fertilization of the Phlebobranch ascidian, Ciona intestinalis. Mol. Reprod. Dev. 50, 493-498. 18) Satoh, K., Sasajima, H., Nyoumura, K., Yokosawa, H., and *Sawada, H. (2001). Assembly of the 26S proteasome is regulated by phosphorylation of the p45/Rpt6 ATPase subunit. Biochemistry 40, 314-319. 19) Kodama, E., Baba, T., Yokosawa, H., and *Sawada, H. (2001). cDNA cloning and functional analysis of ascidian sperm proacrosin. J. Biol. Chem. 276, 24594-24600. 20) *Sawada, H., Sakai, N., Abe, Y., Tanaka, E., Takahashi, Y., Fujino, J., Kodama, E., Takizawa, S., and Yokosawa, H. (2002). Extracellular ubiquitination and proteasome- mediated degradation of the ascidian sperm receptor. Proc. Natl. Acad. Sci. U.S.A. 99, 1223-1228. 21) Kodama, E., Baba, T., Kohno, N., Satoh, S., Yokosawa, H., and *Sawada, H. (2002). Spermosin, a trypsin-like protease from ascidian sperm: cDNA cloning, protein structures and functional analysis. Eur. J. Biochem. 269, 657-663. 22) *Sawada, H., Takahashi, Y., Fujino, J., Flores, S. Y., and Yokosawa, H. (2002). Localization and roles in fertilization of sperm proteasomes in the ascidian Halocynthia roretzi. Mol. Reprod. Dev. 62, 271-276. 23) *Sawada, H. (2002). Ascidian sperm lysin system. Zool. Sci. 19, 139-151. 24) Sakai, N., *Sawada, H., and Yokosawa, H. (2003). Extracellular ubiquitin system implicated in fertilization of the ascidian, Halocynthia roretzi: Isolation and characterization. Dev. Biol. 264, 299-307. 25) *Sawada, H., Tanaka, E., Ban, E., Yamasaki, C., Fujino, J., Ooura, K., Abe, Y., Matsumoto, K., and Yokosawa, H. (2004). Self/nonself recognition in ascidian fertilization: vitelline coat protein HrVC70 is a candidate allorecognition molecule. Proc. Natl. Acad. Sci. U.S.A. 101, 15615-15620. 26) Ban, S., Harada, Y., Yokosawa, H., and *Sawada, H. (2005). Highly polymorphic vitelline-coat protein HaVC80 from the ascidian, Halocynthia aurantium: Structural analysis and involvement in self/nonself recognition during fertilization. Dev. Biol. 286, 440-451.

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27) *Harada, Y., and Sawada, H. (2006). Proteins interacting with the ascidian vitelline-coat sperm receptor HrVC70 as revealed by yeast two-hybrid screening. Mol. Reprod. Dev. 74, 1478-1487. 28) Yokota, N., and *Sawada, H. (2007). Sperm proteasomes are responsible for the acrosome reaction and sperm penetration of the vitelline envelope during fertilization of the sea urchin Pseudocentrotus depressus. Dev. Biol. 308, 222-231. 29) *Harada, Y., Takagaki, Y., Sunagawa, M., Saito, T., Yamada, L., Taniguchi, H., Shoguchi, E., and *Sawada, H. (2008). Mechanism of self-sterility in a hermaphroditic chordate. Science 320, 548-550. 30) Urayama, S., Harada, Y., Nakagawa, Y., Ban, S., Akasaka, M., Kawasaki, N., and *Sawada, H. (2008). Ascidian sperm glycosylphosphatidylinositol-anchored CRISP-like protein as a binding partner for an allorecognizable sperm receptor on the vitelline coat. J. Biol. Chem. 283, 21725-21733. 31) Harada, Y., and *Sawada, H. (2008). Allorecognition mechanisms during ascidian fertilization. Int. J. Dev. Biol. 52, 637-645. 32) Yamada, L., Saito, T., Taniguchi, H., *Sawada, H., and *Harada, Y. (2009). Comprehensive egg coat proteome of the ascidian Ciona intestinalis reveals gamete recognition molecules involved in self-sterility. J. Biol. Chem. 284, 9402-9410. 33) Yokota, N., Harada, Y., and *Sawada, H. (2010). Identification of testis-specific ubiquitin-conjugating enzyme in the ascidian Ciona intestinalis. Mol. Reprod. Dev. 77, 640-647. 34) Yamaguchi, A., Saito, T., Yamada, L., Taniguchi, H., Harada, Y., and *Sawada, H. (2011). Identification and localization of the sperm CRISP family protein CiUrabin involved in gamete interaction in the ascidian Ciona intestinalis. Mol. Reprod. Dev. 78, 488-497. 35) Yokota, N., Kataoka, Y., Hashii, N., Kawasaki, N., and *Sawada, H. (2011). Sperm- specific C-terminal processing of the proteasome PSMA1/a6 subunit. Biochem. Biophys. Res. Commun. 410, 809-815. 36) Saito, T., Shiba, T., Inaba, K., *Yamada, L., and *Sawada, H. (2012). Self- incompatibility response induced by calcium increase in sperm of the ascidian Ciona intestinalis. Proc. Natl. Acad. Sci. U.S.A. 109, 4158-4162. 37) Otsuka, K., Yamada, L., and *Sawada H. (2013). cDNA cloning, localization and candidate binding partners of acid-extractable vitelline-coat protein Ci-v-Themis-like in the ascidian Ciona intestinalis. Mol. Reprod. Dev. 80, 840-848. 38) Nakazawa, S., Shirae-Kurabayashi, M., Otsuka, K., and *Sawada, H. (2015). Proteomics of ionomycin-induced ascidian sperm reaction: Released and exposed sperm proteins in the ascidian Ciona intestinalis. Proteomics, 15, 4064-4079. 39) Brozovic M, Martin C, Dantec C, Dauga D, Mendez M, Simion P, Percher M, Laporte B, Scornavacca C, Di Gregorio A, Fujiwara S, Gineste M, Lowe EK, Piette J, Racioppi C, Ristoratore F, Sasakura Y, Takatori N, Brown TC, Delsuc F, Douzery E, Gissi C, McDougall A, Nishida H, Sawada H, Swalla BJ, Yasuo H, and *Lemaire P. (2016). ANISEED 2015: a digital framework for the comparative developmental biology of ascidians. Nucleic Acids Res. 44, D808-818. 40) Mino, M., and *Sawada, H. (2016) Follicle cell trypsin-like protease HrOvochymase: its cDNA cloning, localization, and involvement in the late stage of oogenesis in the ascidian Halocynthia roretzi. Mol. Reprod. Dev. 83, 347-58. 41) *Sawada, H., Shirae-Kurabayashi M., and Numakunai, T. (2017). Spawning of the ascidian Halocynthia roretzi. Mol. Reprod. Dev. 84, 93. 42) Seo, T., Sakon, T., Nakazawa, S., Nishioka, A., Watanabe, K., Matsumoto, K., Akasaka, M., Shioi, N., Sawada, H., and *Araki, S. (2017). Haemorrhagic snake

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metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell–cell adhesions in vitro and induces haemorrhage in vivo. FEBS J. 284, 1657-1671. 43) *Nakazawa, S., Shirae-Kurabayashi, M., and *Sawada, H. (2019). The role of metalloproteases in fertilisation in the ascidian Ciona robusta. Scientific Reports 1009. 44) Tanaka, Y., Yamada, S., Connop, S. L., Hashii, N., Sawada, H., Shih, Y., and *Nishida, H. (2019). Vitelline membrane proteins promote left-sided nodal expression after neurula rotation in the ascidian, Halocynthia roretzi. Dev. Biol. 449, 52-61.

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