Abstract
Insulin-like 3 (INSL3) is a novel circulating peptide hormone that is produced by testicular Leydig cells and ovarian thecal and luteal cells. In males, INSL3 is responsible for testicular descent during foetal life and suppresses germ cell apoptosis in adult males, whereas in females, it causes oocyte maturation. Antagonists of INSL3 thus have significant potential clinical application as contraceptives in both males and females. Previous work has shown that the INSL3 receptor binding region is largely confined to the B-chain central α-helix of the hormone and a conformationally constrained analogue of this has modest receptor binding and INSL3 antagonist activity. In the present study, we have employed and evaluated several approaches for increasing the α-helicity of this peptide in order to better present the key receptor binding residues and increase its affinity for the receptor. Analogues of INSL3 with higher α-helicity generally had higher receptor binding affinity although other structural considerations limit their effectiveness.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Adham IM, Burkhardt E, Benahmed M et al (1993) Cloning of a cDNA for a novel insulin-like peptide of the testicular Leydig cells. J Biol Chem 268:26668–26672
Andrade MA, Chacon P, Merelo JJ et al (1993) Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng 6:383–390. doi:10.1093/protein/6.4.383
Avbelj F (2000) Amino acid conformational preferences and solvation of polar backbone atoms in peptides and proteins. J Mol Biol 300:1335–1359. doi:10.1006/jmbi.2000.3901
Avbelj F, Fele L (1998) Role of main-chain electrostatics, hydrophobic effect and side-chain conformational entropy in determining the secondary structure of proteins. J Mol Biol 279:665–684. doi:10.1006/jmbi.1998.1792
Bachelot T, Ray-Coquard I, Catimel G et al (2000) Multivariable analysis of prognostic factors for toxicity and survival for patients enrolled in phase I clinical trials. Ann Oncol 11:151–4773. doi:10.1023/A:1008368319526
Bai Y, Englander SW (1994) Hydrogen bond strength and beta-sheet propensities: the role of a side chain blocking effect. Proteins 18:262–266. doi:10.1002/prot.340180307
Bai Y, Milne JS, Mayne L et al (1993) Primary structure effects on peptide group hydrogen exchange. Proteins 17:75–86. doi:10.1002/prot.340170110
Barazza A, Wittelsberger A, Fiori N et al (2005) Bioactive N-terminal undecapeptides derived from parathyroid hormone: the role of alpha-helicity. J Pept Res 65:23–35. doi:10.1111/j.1399-3011.2005.00207.x
Barlow DJ, Thornton JM (1988) Helix geometry in proteins. J Mol Biol 201:601–619. doi:10.1016/0022-2836(88)90641-9
Beck-Sickinger AG, Jung G (1995) Structure-activity relationships of neuropeptide Y analogues with respect to Y1 and Y2 receptors. Biopolymers 37:123–142. doi:10.1002/bip.360370207
Blaber M, Zhang XJ, Matthews BW (1993) Structural basis of amino acid alpha helix propensity. Science 260:1637–1640. doi:10.1126/science.8503008
Blaber M, Zhang XJ, Lindstrom JD et al (1994) Determination of alpha-helix propensity within the context of a folded protein. Sites 44 and 131 in bacteriophage T4 lysozyme. J Mol Biol 235:600–624. doi:10.1006/jmbi.1994.1016
Bogatcheva NV, Truong A, Feng S et al (2003) GREAT/LGR8 is the only receptor for insulin-like 3 peptide. Mol Endocrinol 17:2639–2646. doi:10.1210/me.2003-0096
Büllesbach EE, Schwabe C (1995) A novel Leydig cell cDNA-derived protein is a relaxin-like factor. J Biol Chem 270:16011–16015. doi:10.1074/jbc.270.27.16011
Büllesbach EE, Schwabe C (1999) Tryptophan B27 in the relaxin-like factor (RLF) is crucial for RLF receptor-binding. Biochemistry 38:3073–3078. doi:10.1021/bi982687u
Büllesbach EE, Schwabe C (2005) LGR8 signal activation by the relaxin-like factor. J Biol Chem 280:14586–14590. doi:10.1074/jbc.M414443200
Büllesbach EE, Schwabe C (2007) Structure of the transmembrane signal initiation site of the relaxin-like factor (RLF/INSL3). Biochemistry 46:9722–9727. doi:10.1021/bi700708s
Burkhardt E, Adham IM, Hobohm U et al (1994) A human cDNA coding for the Leydig insulin-like peptide (Ley I-L). Hum Genet 94:91–94. doi:10.1007/BF02272850
Compton LA, Johnson WC Jr (1986) Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication. Anal Biochem 155:155–167. doi:10.1016/0003-2697(86)90241-1
Connelly GP, Bai Y, Jeng MF et al (1993) Isotope effects in peptide group hydrogen exchange. Proteins 17:87–92. doi:10.1002/prot.340170111
Del Borgo MP, Hughes RA, Bathgate RA et al (2006) Analogs of insulin-like peptide 3 (INSL3) B-chain are LGR8 antagonists in vitro and in vivo. J Biol Chem 281:13068–13074. doi:10.1074/jbc.M600472200
Feng S, Cortessis VK, Hwang A et al (2004) Mutation analysis of INSL3 and GREAT/LGR8 genes in familial cryptorchidism. Urology 64:1032–1036. doi:10.1016/j.urology.2004.06.051
Ferlin A, Pepe A, Gianesello L et al (2008) Mutations in the insulin-like factor 3 receptor are associated with osteoporosis. J Bone Miner Res 23:683–693. doi:10.1359/jbmr.080204
Foresta C, Ferlin A (2004) Role of INSL3 and LGR8 in cryptorchidism and testicular functions. Reprod Biomed Online 9:294–298
Hossain MA, Rosengren KJ, Haugaard-Jonsson LM et al (2008) The A-chain of human relaxin family peptides has distinct roles in the binding and activation of the different relaxin family peptide receptors. J Biol Chem 283:17287–17297. doi:10.1074/jbc.M801911200
Houston ME Jr, Gannon CL, Kay CM et al (1995) Lactam bridge stabilization of alpha-helical peptides: ring size, orientation and positional effects. J Pept Sci 1:274–282. doi:10.1002/psc.310010408
Hsu SY (2003) New insights into the evolution of the relaxin-LGR signaling system. Trends Endocrinol Metab 14:303–309. doi:10.1016/S1043-2760(03)00106-1
Ivell R, Einspanier A (2002) Relaxin peptides are new global players. Trends Endocrinol Metab 13:343–348. doi:10.1016/S1043-2760(02)00664-1
Kawamura K, Kumagai J, Sudo S et al (2004) Paracrine regulation of mammalian oocyte maturation and male germ cell survival. Proc Natl Acad Sci USA 101:7323–7328. doi:10.1073/pnas.0307061101
Klonisch T, Mustafa T, Bialek J et al (2005) Human medullary thyroid carcinoma: a source and potential target for relaxin-like hormones. Ann N Y Acad Sci 1041:449–461. doi:10.1196/annals.1282.069
Lees JG, Miles AJ, Wien F et al (2006) A reference database for circular dichroism spectroscopy covering fold and secondary structure space. Bioinformatics 22:1955–1962. doi:10.1093/bioinformatics/btl327
Lobley A, Wallace BA (2001) DICHROWEB: a website for the analysis of protein secondary structure from circular dichroism spectra. Biophys J 80:373
Lobley A, Whitmore L, Wallace BA (2002) DICHROWEB: an interactive website for the analysis of protein secondary structure from circular dichroism spectra. Bioinformatics 18:211–212. doi:10.1093/bioinformatics/18.1.211
Ma S, Bonaventure P, Ferraro T et al (2007) Relaxin-3 in GABA projection neurons of nucleus incertus suggests widespread influence on forebrain circuits via G-protein-coupled receptor-135 in the rat. Neuroscience 144:165–190. doi:10.1016/j.neuroscience.2006.08.072
Marshall GR, Hodgkin EE, Langs DA et al (1990) Factors governing helical preference of peptides containing multiple alpha, alpha-dialkyl amino acids. Proc Natl Acad Sci USA 87:487–491. doi:10.1073/pnas.87.1.487
Mimna R, Tuchscherer G, Mutter M (2007) Toward the design of highly efficient, readily accessible peptide N-caps for the induction of helical conformations. Int J Pept Res Ther 13:237–244. doi:10.1007/s10989-006-9073-9
Nef S, Parada LF (1999) Cryptorchidism in mice mutant for Insl3. Nat Genet 22:295–299. doi:10.1038/10364
Park JI, Semyonov J, Yi W (2008) Regulation of receptor signaling by relaxin A chain motifs: derivation of pan-specific and LGR7-specific human relaxin analogs. J Biol Chem 283(46):32099–32109
Roche PJ, Butkus A, Wintour EM et al (1996) Structure and expression of Leydig insulin-like peptide mRNA in the sheep. Mol Cell Endocrinol 121:171–177. doi:10.1016/0303-7207(96)03861-0
Rosengren KJ, Zhang S, Lin F et al (2006) Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3. J Biol Chem 281:28287–28295. doi:10.1074/jbc.M603829200
Scott DJ, Layfield S, Yan Y et al (2006) Characterization of novel splice variants of LGR7 and LGR8 reveals that receptor signaling is mediated by their unique low density lipoprotein class A modules. J Biol Chem 281:34942–34954. doi:10.1074/jbc.M602728200
Shabanpoor F, Bathgate RA, Hossain MA et al (2007) Design, synthesis and pharmacological evaluation of cyclic mimetics of the insulin-like peptide 3 (INSL3) B-chain. J Pept Sci 13:113–120. doi:10.1002/psc.807
Shabanpoor F, Hughes RA, Bathgate RA et al (2008) Solid-phase synthesis of europium-labeled human INSL3 as a novel probe for the study of ligand-receptor interactions. Bioconjug Chem 19:1456–1463. doi:10.1021/bc800127p
Shepherd NE, Hoang HN, Desai VS et al (2006) Modular alpha-helical mimetics with antiviral activity against respiratory syncitial virus. J Am Chem Soc 128:13284–13289. doi:10.1021/ja064058a
Spiess AN, Balvers M, Tena-Sempere M et al (1999) Structure and expression of the rat relaxin-like factor (RLF) gene. Mol Reprod Dev 54:319–325. doi:10.1002/(SICI)1098-2795(199912)54:4<319::AID-MRD1>3.0.CO;2-Z
Tashima LS, Hieber AD, Greenwood FC et al (1995) The human Leydig insulin-like (hLEY I-L) gene is expressed in the corpus luteum and trophoblast. J Clin Endocrinol Metab 80:707–710. doi:10.1210/jc.80.2.707
Werle M, Bernkop-Schnürch A (2006) Strategies to improve plasma half life time of peptide and protein drugs. Amino Acids 30:351–367. doi:10.1007/s00726-005-0289-3
Whitmore L, Wallace BA (2004) DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res 32:W668–W673
Zimmermann S, Steding G, Emmen JM et al (1999) Targeted disruption of the Insl3 gene causes bilateral cryptorchidism. Mol Endocrinol 13:681–691. doi:10.1210/me.13.5.681
Acknowledgments
We thank Tania Ferraro for help with binding assays. This work was funded by National Health and Medical Research Council of Australia Project grants #350245 and 509048 to JDW, RADB and RAH.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Shabanpoor, F., Hughes, R.A., Zhang, S. et al. Effect of helix-promoting strategies on the biological activity of novel analogues of the B-chain of INSL3. Amino Acids 38, 121–131 (2010). https://doi.org/10.1007/s00726-008-0219-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00726-008-0219-2