Jump to ContentJump to Main Navigation
Show Summary Details
More options …

Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred


IMPACT FACTOR 2018: 3.014
5-year IMPACT FACTOR: 3.162

CiteScore 2018: 3.09

SCImago Journal Rank (SJR) 2018: 1.482
Source Normalized Impact per Paper (SNIP) 2018: 0.820

Online
ISSN
1437-4315
See all formats and pricing
More options …
Volume 394, Issue 9

Issues

The active form of goat insulin-like peptide 3 (INSL3) is a single-chain structure comprising three domains B-C-A, constitutively expressed and secreted by testicular Leydig cells

Siqin
  • Laboratory of Animal Reproduction and Physiology, Faculty of Agriculture, Department of Applied Biological Chemistry, Shizuoka University, Suruga-ku, Shizuoka 422-8529, Japan
  • Division of Animal Resource Production, The United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Itaru Minagawa
  • Laboratory of Animal Reproduction and Physiology, Faculty of Agriculture, Department of Applied Biological Chemistry, Shizuoka University, Suruga-ku, Shizuoka 422-8529, Japan
  • Division of Animal Resource Production, The United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Mitsutoshi Okuno / Kimihiko Yamada / Yasushi Sugawara / Yoshio Nagura
  • Livestock Breeding Division, National Livestock Breeding Center Nagano Station, Saku, Nagano 385-0007, Japan
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Koh-Ichi Hamano
  • Education and Research Center of Alpine Field Science, Faculty of Agriculture, Shinshu University, Ninami-Minowa, Nagano 399-4598, Japan
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Enoch Y. Park
  • Laboratory of Biotechnology, Graduate School of Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Hiroshi Sasada
  • Laboratory of Animal Reproduction, Kitasato University School of Veterinary Medicine, Towada 034-8628, Japan
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Tetsuya Kohsaka
  • Corresponding author
  • Laboratory of Animal Reproduction and Physiology, Faculty of Agriculture, Department of Applied Biological Chemistry, Shizuoka University, Suruga-ku, Shizuoka 422-8529, Japan
  • Division of Animal Resource Production, The United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2013-05-16 | DOI: https://doi.org/10.1515/hsz-2012-0357

Abstract

Relaxin-like factor (RLF), also called insulin-like peptide 3 (INSL3), is a member of the insulin/relaxin gene family and is produced by testicular Leydig cells. While the understanding of its effects is growing, very little is known about the structural and functional properties of native INSL3. Here, we demonstrate that native INSL3 isolated from goat testes is a single-chain structure with full biological activity, and is constitutively expressed and secreted by Leydig cells. Using a series of chromatography steps, native INSL3 was highly purified as a single 12-kDa peak as revealed by SDS-PAGE. MS/MS analysis provided 81% sequence coverage and revealed a distinct single-chain structure consisting of the B-, C-, and A-domains deduced previously from the INSL3 cDNA sequence. Moreover, the N-terminal peptide was six amino acid residues longer than predicted. Native INSL3 exhibited full bioactivity in HEK-293 cells expressing the receptor for INSL3. Immunoelectron microscopy and Western blot analysis revealed that INSL3 was secreted by Leydig cells through the constitutive pathway into blood and body fluids. We conclude, therefore, that goat INSL3 is constitutively secreted from Leydig cells as a B-C-A single-chain structure with full biological activity.

Keywords: bioactivity; INSL3; MS/MS; native conformation; purification; subcellular localization

References

  • Adham, I.M., Burkhardt, E., Benahmed, M., and Engel, W. (1993). Cloning of a cDNA for a novel insulin-like peptide of the testicular Leydig cells. J. Biol. Chem. 268, 26668–26672.Google Scholar

  • Anand-Ivell, R.J., Relan, V., Balvers. M., Coiffec-Dorval, I., Fritsch, M., Bathgate, R.A, and Ivell, R. (2006). Expression of the insulin-like peptide 3 (INSL3) hormone-receptor system in the testis. Biol. Reprod. 74, 945–953.Google Scholar

  • Anand-Ivell, R., Heng, K., Hafen, B., Setchell, B., and Ivell, R. (2009). Dynamics of INSL3 peptide expression in the rodent testis. Biol. Reprod. 81, 480–487.Web of ScienceGoogle Scholar

  • Balvers, M., Spiess, A.N., Domagalski, R., Hunt, N., Kilic, E., Mukhopadhyay, A.K., Hanks, E., Charlton, H.M., and Ivell, R. (1998). Relaxin-like factor expression as a marker of differentiation in the mouse testis and ovary. Endocrinology 139, 2960–2970.Google Scholar

  • Bogatcheva, N.V., Truong, A., Feng, S., Engel, W., Adham, I.M., and Agoulnik, A.I. (2003). GREAT/LGR8 is the only receptor for insulin-like 3 peptide. Mol. Endocrinol. 17, 2639–2646.Google Scholar

  • Büllesbach, E.E. and Schwabe, C. (1995). A novel Leydig cell cDNA-derived protein is a relaxin-like factor. J. Biol. Chem. 270, 16011–16015.Google Scholar

  • Büllesbach, E.E. and Schwabe, C. (2002). The primary structure and the disulfide links of the bovine relaxin-like factor (RLF). Biochemistry 41, 274–281.Google Scholar

  • Deveson, S., Forsyth, I.A., and Arendt, J. (1992). Retardation of pubertal development by prenatal long days in goat kids born in autumn. J. Reprod. Fertil. 95, 629–637.Google Scholar

  • Feng, S., Bogatcheva, N.V., Truong, A., Korchin, B., Bishop, C.E., Klonisch, T., Agoulnik, I.U., and Agoulnik, A.I. (2007). Developmental expression and gene regulation of insulin-like 3 receptor RXFP2 in mouse male reproductive organs. Biol. Reprod. 77, 671–680.Google Scholar

  • Ferlin, A. and Foresta, C. (2005). Insulin-like factor 3: a novel circulating hormone of testicular origin in humans. Ann. N. Y. Acad. Sci. 1041, 497–505.Google Scholar

  • Filonzi, M., Cardoso, L.C., Pimenta, M.T., Queiróz, D.B., Avellar, M.C., Porto, C.S., and Lazari, M.F. (2007). Relaxin family peptide receptors Rxfp1 and Rxfp2: mapping of the mRNA and protein distribution in the reproductive tract of the male rat. Reprod. Biol. Endocrinol. 5, 29 (12 pages).Web of ScienceGoogle Scholar

  • Gorlov, I.P., Kamat, A., Bogatcheva, N.V., Jones, E., Lamb, D.J., Truong, A., Bishop, C.E., McElreavey, K., and Agoulnik, A.I. (2002). Mutations of the GREAT gene cause cryptorchidism. Hum. Mol. Genet. 11, 2309–2318.Google Scholar

  • Gustafsson, K., Sultana, T., Zetterström, C.K., Setchell, B.P., Siddiqui, A., Weber, G., and Söder, O. (2002). Production and secretion of interleukin-1alpha proteins by rat testis. Biochem. Biophys. Res. Commun. 297, 492–497.Google Scholar

  • Halban, P.A. and Irminger, J.C. (1994). Sorting and processing of secretory proteins. Biochem. J. 299, 1–18.Google Scholar

  • Hess, H.H., Lees, M.B., and Derr, J.E. (1978). A linear Lowry-Folin assay for both water-soluble and sodium dodecyl sulfate-solubilized proteins. Anal. Biochem. 85, 295–300.Google Scholar

  • Hombach-Klonisch, S., Tetens, F., Kauffold, J., Steger, K., Fischer, B., and Klonisch, T. (1999). Molecular cloning and localization of caprine relaxin-like factor (RLF) mRNA within the goat testis. Mol. Reprod. Dev. 53, 135–141.Google Scholar

  • Hombach-Klonisch, S., Buchmann, J., Sarun, S., Fischer, B., and Klonisch, T. (2000). Relaxin-like factor (RLF) is differentially expressed in the normal and neoplastic human mammary gland. Cancer 89, 2161–2168.Google Scholar

  • Hombach-Klonisch, S., Schön, J., Kehlen, A., Blottner, S., and Klonisch, T. (2004). Seasonal expression of INSL3 and Lgr8/Insl3 receptor transcripts indicates variable differentiation of Leydig cells in the roe deer testis. Biol. Reprod. 71, 1079–1087.Google Scholar

  • Hsu, S.Y., Nakabayashi, K., Nishi, S., Kumagai, J., Kudo, M., Sherwood, O.D., and Hsueh, A.J. (2002). Activation of orphan receptors by the hormone relaxin. Science 295, 671–674.Google Scholar

  • Ivell, R. and Anand-Ivell, R. (2009). Biology of insulin-like factor 3 in human reproduction. Human Reprod. Update 15, 463–476.Web of ScienceGoogle Scholar

  • Ivell, R. and Bathgate, R.A. (2002). Reproductive biology of the relaxin-like factor (RLF/INSL3). Biol. Reprod. 67, 699–705.Google Scholar

  • Ivell, R., Kotula-Balak, M., Glynn, D., Heng, K., and Anand-Ivell, R. (2011). Relaxin family peptides in the male reproductive system– a critical appraisal. Mol. Hum. Reprod. 17, 71–84.Web of ScienceGoogle Scholar

  • Jutras, I., Seidah, N.G., Reudelhuber, T.L., and Brechler, V. (1997). Two activation states of the prohormone convertase PC1 in the secretory pathway. J. Biol. Chem. 272, 15184–15188.Google Scholar

  • Kato, S., Siqin, Minagawa, I., Aoshima, T., Sagata, D., Konishi, H., Yogo, K., Kawarasaki, T., Sasada, H., Tomogane, H., and Kohsaka, T. (2010). Evidence for expression of relaxin hormone-receptor system in the boar testis. J. Endocrinol. 207, 135–149.Google Scholar

  • Kawamura, K., Kumagai, J., Sudo, S., Chun, S., Pisarska, M., Morita, H., Toppari, J., Fu, P., Wade, J.D., Bathgate, R.A., et al. (2004). Paracrine regulation of mammalian oocyte maturation and male germ cell survival. Proc. Natl. Acad. Sci. USA 101, 7323–7328.Google Scholar

  • Kelly, R.B. (1985). Pathways of protein secretion in eukaryotes. Science 230, 25–32.Google Scholar

  • Klonisch, T., Steger, K., Kehlen, A., Allen, W.R., Froehlich, C., Kauffold, J., Bergmann, M., and Hombach-Klonisch, S. (2003). INSL3 ligand-receptor system in the equine testis. Biol. Reprod. 68, 1975–1981.Google Scholar

  • Koeva, Y.A., Bakalska, M.V., Atanassova, N.N., and Davidoff, M.S. (2008). INSLF3-LGR8 ligand-receptor system in testes of mature rats after exposure to ethane dimethanesulphonate. Folia Med. (Plovdiv) 50, 37–42.Google Scholar

  • Kohsaka, T., Sasada, H., and Masaki, J. (1992). Subcellular localization of the antigenic sites of relaxin in the luteal cells of the pregnant rat using an improved immunocytochemical technique. Anim. Reprod. Sci. 29, 123–132.Google Scholar

  • Kohsaka, T., Sasada, H., and Masaki, J. (1993a). Subcellular localization of the maturation process of relaxin in rat luteal cells during pregnancy as revealed by immunogold labeling. Anim. Reprod. Sci. 34, 159–166.Google Scholar

  • Kohsaka, T., Takahara, H., Sugawara, K., and Tagami, S. (1993b). Endogenous heterogeneity of relaxin and sequence of the major form in pregnant sow ovaries. Biol. Chem. Hoppe Seyler 374, 203–210.Google Scholar

  • Kumagai, J., Hsu, S.Y., Matsumi, H., Roh, J.S., Fu, P., Wade, J.D., Bathgate, R.A., and Hsueh, A.J. (2002). INSL3/Leydig insulin-like peptide activates the LGR8 receptor important in testis descent. J. Biol. Chem. 277, 31283–31286.Google Scholar

  • LeRoith, D. and Roberts, C.T. (2003). The insulin-like growth factor system and cancer. Cancer Lett. 195, 127–137.Google Scholar

  • Lunstra, D.D., Ford, J.J., Christenson, R.K., and Allrich, R.D. (1986). Changes in Leydig cell ultrastructure and function during pubertal development in the boar. Biol. Reprod. 34, 145–158.Google Scholar

  • Marriott, D., Gillece-Castro, B., and Gorman, C.M. (1992). Prohormone convertase-1 will process prorelaxin, a member of the insulin family of hormones. Mol. Endocrinol. 6, 1441–1450.PubMedGoogle Scholar

  • McKinnell, C., Sharpe, R.M., Mahood, K., Hallmark, N., Scott, H., Ivell, R., Staub, C., Jégou, B., Haag, F., Koch-Nolte, F., et al. (2005). Expression of insulin-like factor 3 protein in the rat testis during fetal and postnatal development and in relation to cryptorchidism induced by in utero exposure to di (n-Butyl) phthalate. Endocrinology 146, 4536–4544.Google Scholar

  • Minagawa, I., Fukuda, M., Kohriki, H., Shibata, M., Park, E.Y., Kawarasaki, T., and Kohsaka, T. (2012). Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B-C-A with full biological activity in boars. Biochem. J. 441, 265–273.Web of ScienceGoogle Scholar

  • Nakayama, K. (1997). Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Biochem. J. 327, 625–635.Google Scholar

  • Nef, S. and Parada, L.F. (1999). Cryptorchidism in mice mutant for Insl3. Nature Genetics 22, 295–299.Google Scholar

  • Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1–6.Google Scholar

  • Ogine, T., Kohsaka, T., and Taya, K. (1999). Time-resolved fluoroimmunoassay (TR-FIA) of porcine relaxin. Exp. Clin. Endocrinol. Diabetes 107, 276–280.Google Scholar

  • Rorsman, P. and Renström, E.I. (2003). Insulinn granule dynamics in pancreatic beta cells. Diabetologia 46, 1029–1045.Google Scholar

  • Sadeghian, H., Anand-Ivell, R., Balvers, M., Relan, V., and Ivell, R. (2005). Constitutive regulation of the Insl3 gene in rat Leydig cells. Mol. Cell. Endocrinol. 241, 10–20.Google Scholar

  • Siqin, Kotani, M., Aoshima, T., Nakai, M., Fuchigami, M., Odanaka, Y., Sugawara, Y., Yogo, K., Nagura, Y., Hamano, K., et al. (2010a). Protein localization of relaxin-like factor in goat testes and its expression pattern during sexual development. Nihon Chikusan Gakkaiho 81, 1–9.Google Scholar

  • Siqin, Nakai, M., Hagi, T., Kato, S., Pittia, A.M, Kotani, M., Odanaka, Y., Sugawara, Y., Hamano, K., Yogo, K., et al. (2010b). Partial cDNA sequence of a relaxin-like factor (RLF) receptor, LGR8 and possible existence of the RLF ligand-receptor ststem in goat testes. Anim. Sci. J. 81, 681–686.Web of ScienceGoogle Scholar

  • Smith, K.J., Wade, J.D., Claasz, A.A., Otvos, L. Jr., Temelcos, C., Kubota, Y., Hutson, J.M., Tregear, G.W., and Bathgate, R.A. (2001). Chemical synthesis and biological activity of rat INSL3. J. Pept. Sci. 7, 495–501.Google Scholar

  • Steiner, D.F. (1998). The proprotein convertases. Curr. Opin. Chem. Biol. 2, 31–39.PubMedCrossrefGoogle Scholar

  • von Heijne, G. (1986). A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683–4690.Google Scholar

  • Zimmermann, S., Steding, G., Emmen, J.M., Brinkmann, A.O., Nayernia, K., Holstein, A.F., Engel, W., and Adham, I.M. (1999). Targeted disruption of the Insl3 gene causes bilateral cryptorchidism. Mol. Endocrinol. 13, 681–691.Google Scholar

About the article

Corresponding author: Tetsuya Kohsaka, Laboratory of Animal Reproduction and Physiology, Faculty of Agriculture, Department of Applied Biological Chemistry, Shizuoka University, Suruga-ku, Shizuoka 422-8529, Japan; and Division of Animal Resource Production, The United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan


Received: 2012-12-27

Accepted: 2013-05-14

Published Online: 2013-05-16

Published in Print: 2013-09-01


Citation Information: Biological Chemistry, Volume 394, Issue 9, Pages 1181–1194, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/hsz-2012-0357.

Export Citation

©2013 by Walter de Gruyter Berlin Boston.Get Permission

Citing Articles

Here you can find all Crossref-listed publications in which this article is cited. If you would like to receive automatic email messages as soon as this article is cited in other publications, simply activate the “Citation Alert” on the top of this page.

[1]
Michelle L. Halls, Ross A. D. Bathgate, Steve W. Sutton, Thomas B. Dschietzig, Roger J. Summers, and Eliot H. Ohlstein
Pharmacological Reviews, 2015, Volume 67, Number 2, Page 389
[2]
I. Minagawa, Y. Murata, K. Terada, M. Shibata, E. Y. Park, H. Sasada, and T. Kohsaka
Andrologia, 2018, Page e13010
[3]
Takatsugu Miyazaki, Masaaki Ishizaki, Hideo Dohra, Sungjo Park, Andre Terzic, Tatsuya Kato, Tetsuya Kohsaka, and Enoch Y. Park
Scientific Reports, 2017, Volume 7, Number 1
[5]
Ali M. Pitia, Kyoko Uchiyama, Hiroaki Sano, Masashi Kinukawa, Yoshiaki Minato, Hiroshi Sasada, and Tetsuya Kohsaka
Animal Science Journal, 2017, Volume 88, Number 4, Page 678
[6]
M.A. Hannan, N. Kawate, Y. Fukami, I.N. Pathirana, E.E. Büllesbach, T. Inaba, and H. Tamada
Theriogenology, 2016, Volume 86, Number 3, Page 749
[7]
Ali Mohammed Pitia, Itaru Minagawa, Naoto Uera, Koh-Ichi Hamano, Yasushi Sugawara, Yoshio Nagura, Yoshihisa Hasegawa, Toshifumi Oyamada, Hiroshi Sasada, and Tetsuya Kohsaka
Cell and Tissue Research, 2015, Volume 362, Number 2, Page 407
[8]
Ravinder Anand-Ivell and Richard Ivell
Molecular and Cellular Endocrinology, 2014, Volume 382, Number 1, Page 472

Comments (0)

Please log in or register to comment.
Log in