A C. elegans Zona Pellucida domain protein functions via its ZPc domain


Autoři: Jennifer D. Cohen aff001;  Jessica G. Bermudez aff002;  Matthew C. Good aff002;  Meera V. Sundaram aff001
Působiště autorů: Department of Genetics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States of America aff001;  Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America aff002;  Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, United States of America aff003
Vyšlo v časopise: A C. elegans Zona Pellucida domain protein functions via its ZPc domain. PLoS Genet 16(11): e1009188. doi:10.1371/journal.pgen.1009188
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1009188

Souhrn

Zona Pellucida domain (ZP) proteins are critical components of the body’s external-most protective layers, apical extracellular matrices (aECMs). Although their loss or dysfunction is associated with many diseases, it remains unclear how ZP proteins assemble in aECMs. Current models suggest that ZP proteins polymerize via their ZPn subdomains, while ZPc subdomains modulate ZPn behavior. Using the model organism C. elegans, we investigated the aECM assembly of one ZP protein, LET-653, which shapes several tubes. Contrary to prevailing models, we find that LET-653 localizes and functions via its ZPc domain. Furthermore, we show that ZPc domain function requires cleavage at the LET-653 C-terminus, likely in part to relieve inhibition of the ZPc by the ZPn domain, but also to promote some other aspect of ZPc domain function. In vitro, the ZPc, but not ZPn, domain bound crystalline aggregates. These data offer a new model for ZP function whereby the ZPc domain is primarily responsible for matrix incorporation and tissue shaping.

Klíčová slova:

Caenorhabditis elegans – Extracellular matrix – Extracellular matrix proteins – Fluorescence imaging – Fluorescence recovery after photobleaching – Polymerization – Transfection – Vulva


Zdroje

1. Luschnig S, Uv A. Luminal matrices: an inside view on organ morphogenesis. Exp Cell Res. 2014;321(1):64–70. doi: 10.1016/j.yexcr.2013.09.010 24075963

2. Gaudette S, Hughes D, Boller M. The endothelial glycocalyx: Structure and function in health and critical illness. J Vet Emerg Crit Care (San Antonio). 2020. doi: 10.1111/vec.12925 32067360

3. Halliday HL. The fascinating story of surfactant. J Paediatr Child Health. 2017;53(4):327–332. doi: 10.1111/jpc.13500 28271629

4. Hansson GC. Mucus and mucins in diseases of the intestinal and respiratory tracts. J Intern Med. 2019;285(5):479–490. doi: 10.1111/joim.12910 30963635

5. Gordts P, Esko JD. The heparan sulfate proteoglycan grip on hyperlipidemia and atherosclerosis. Matrix Biol. 2018;71–72, 262–282. doi: 10.1016/j.matbio.2018.05.010 29803939

6. Husain N, Pellikka M, Hong H, Klimentova T, Choe KM, Clandinin TR, Tepass U. The agrin/perlecan-related protein eyes shut is essential for epithelial lumen formation in the Drosophila retina. Dev Cell. 2006;11(4):483–493. doi: 10.1016/j.devcel.2006.08.012 17011488

7. Hwang HY, Olson SK, Esko JD, Horvitz HR. Caenorhabditis elegans early embryogenesis and vulval morphogenesis require chondroitin biosynthesis. Nature. 2003;423(6938):439–443. doi: 10.1038/nature01634 12761549

8. Jovine L, Qi H, Williams Z, Litscher E, Wassarman PM. The ZP domain is a conserved module for polymerization of extracellular proteins. Nat Cell Biol. 2002;4(6):457–461. doi: 10.1038/ncb802 12021773

9. Porter KR, Tamm I. Direct visualization of a mucoprotein component of urine. J Biol Chem. 1955;212(1):135–140. 13233216

10. Lane MC, Koehl MA, Wilt F, Keller R. A role for regulated secretion of apical extracellular matrix during epithelial invagination in the sea urchin. Development. 1993;117(3), 1049–1060. 8325234

11. Syed ZA, Bougé AL, Byri S, Chavoshi TM, Tång E, Bouhin H, et al. A luminal glycoprotein drives dose-dependent diameter expansion of the Drosophila melanogaster hindgut tube. PLoS Genet. 2012;8(8):e1002850. doi: 10.1371/journal.pgen.1002850 22876194

12. Chappell D, Jacob M, Paul O, Rehm M, Welsch U, Stoeckelhuber M. et al. The glycocalyx of the human umbilical vein endothelial cell: an impressive structure ex vivo but not in culture. Circ Res. 2009;104(11):1313–1317. doi: 10.1161/CIRCRESAHA.108.187831 19423849

13. Johansson ME, Sjovall H, Hansson GC. The gastrointestinal mucus system in health and disease. Nat Rev Gastroenterol Hepatol. 2013;10(6):352–361. doi: 10.1038/nrgastro.2013.35 23478383

14. Plaza S, Chanut-Delalande H, Fernandes I, Wassarman PM, Payre F. From A to Z: apical structures and zona pellucida-domain proteins. Trends Cell Biol. 2010;20(9):524–532. doi: 10.1016/j.tcb.2010.06.002 20598543

15. Gupta SK. The Human Egg’s Zona Pellucida. Curr Top Dev Biol. 2018;130:379–411. doi: 10.1016/bs.ctdb.2018.01.001 29853184

16. Yan B, Zhang ZZ, Huang LY, Shen HL, Han ZG. OIT3 deficiency impairs uric acid reabsorption in renal tubule. FEBS Lett. 2012;586(6):760–765. doi: 10.1016/j.febslet.2012.01.038 22306318

17. Legan PK, Rau A, Keen JN, Richardson GP. The mouse tectorins. Modular matrix proteins of the inner ear homologous to components of the sperm-egg adhesion system. J Biol Chem. 1997;272(13):8791–8801. doi: 10.1074/jbc.272.13.8791 9079715

18. Op De Beeck K, Vermeire S., Rutgeerts P, Bossuyt X. Antibodies to GP2, the major zymogen granule membrane glycoprotein, in inflammatory bowel diseases. Gut. 2012;61(1):162–164; author reply 164–165. doi: 10.1136/gut.2010.233148 21193445

19. Renner M, Bergmann G, Krebs I, End C, Lyer S, Hilberg F, et al. DMBT1 confers mucosal protection in vivo and a deletion variant is associated with Crohn’s disease. Gastroenterology. 2007;133(5):1499–1509. doi: 10.1053/j.gastro.2007.08.007 17983803

20. McAllister KA, Grogg KM, Johnson DW, Gallione CJ, Baldwin MA, Jackson CE, et al. Endoglin, a TGF-beta binding protein of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1. Nat Genet. 1994;8(4):345–351. doi: 10.1038/ng1294-345 7894484

21. Wong SH, Hamel L, Chevalier S, Philip A. Endoglin expression on human microvascular endothelial cells association with betaglycan and formation of higher order complexes with TGF-beta signalling receptors. Eur J Biochem. 2000;267(17):5550–5560. doi: 10.1046/j.1432-1327.2000.01621.x 10951214

22. Cummings D, Cruise M, Lopez R, Roggenbuck D, Jairath V, Wang Y, et al. Loss of tolerance to glycoprotein 2 isoforms 1 and 4 is associated with Crohn’s disease of the pouch. Aliment Pharmacol Ther. 2018;48(11–12):1251–1259. doi: 10.1111/apt.15034 30411391

23. Mapes J, Li Q, Kannan A, Anandan L, Laws M, Lydon JP, et al. CUZD1 is a critical mediator of the JAK/STAT5 signaling pathway that controls mammary gland development during pregnancy. PLoS Genet. 2017;13(3):e1006654. doi: 10.1371/journal.pgen.1006654 28278176

24. Wheeler E, Thomas S. Diagnosis and Long-term Management of Uromodulin Kidney Disease. Cureus. 2019;11(3):e4270. doi: 10.7759/cureus.4270 31157132

25. Verhoeven K, Van Laer L, Kirschhofer K, Legan PK, Hughes DC, Schatteman I, et al. Mutations in the human alpha-tectorin gene cause autosomal dominant non-syndromic hearing impairment. Nat Genet. 1998;19(1):60–62. doi: 10.1038/ng0598-60 9590290

26. Roggenbuck D, Hausdorf G, Martinez-Gamboa L, Reinhold D, Buttner T, Jungblut PR, et al. Identification of GP2, the major zymogen granule membrane glycoprotein, as the autoantigen of pancreatic antibodies in Crohn’s disease. Gut. 2009;58(12):1620–1628. doi: 10.1136/gut.2008.162495 19549613

27. Mustapha M, Weil D, Chardenoux S, Elias S, El-Zir E, Beckmann JS, et al. An alpha-tectorin gene defect causes a newly identified autosomal recessive form of sensorineural pre-lingual non-syndromic deafness, DFNB21. Hum Mol Genet. 1999;8(3):409–412. doi: 10.1093/hmg/8.3.409 9949200

28. Lin SJ, Hu Y, Zhu J, Woodruff TK, Jardetzky TS. Structure of betaglycan zona pellucida (ZP)-C domain provides insights into ZP-mediated protein polymerization and TGF-beta binding. Proc Natl Acad Sci U S A. 2011;108(13):5232–5236. doi: 10.1073/pnas.1010689108 21402931

29. Saito T, Bokhove M, Croci R, Zamora-Caballero S, Han L, Letarte M, et al. Structural Basis of the Human Endoglin-BMP9 Interaction: Insights into BMP Signaling and HHT1. Cell Rep. 2017;19(9):1917–1928. doi: 10.1016/j.celrep.2017.05.011 28564608

30. Bokhove M., & Jovine L. (2018). Structure of Zona Pellucida Module Proteins. Curr Top Dev Biol, 130, 413–442. doi: 10.1016/bs.ctdb.2018.02.007 29853186

31. Shen HL, Xu ZG, Huang LY, Liu D, Lin DH, Cao JB, et al. Liver-specific ZP domain-containing protein (LZP) as a new partner of Tamm-Horsfall protein harbors on renal tubules. Mol Cell Biochem. 2009;321(1–2):73–83. doi: 10.1007/s11010-008-9921-3 18830570

32. Wilburn DB, Swanson WJ. The "ZP domain" is not one, but likely two independent domains. Mol Reprod Dev. 2017;84(4):284–285. doi: 10.1002/mrd.22781 28176401

33. Han L, Monne M, Okumura H, Schwend T, Cherry AL, Flot D, et al. Insights into egg coat assembly and egg-sperm interaction from the X-ray structure of full-length ZP3. Cell. 2010;143(3):404–415. doi: 10.1016/j.cell.2010.09.041 20970175

34. Cohen JD, Flatt KM, Schroeder NE, Sundaram MV. Epithelial Shaping by Diverse Apical Extracellular Matrices Requires the Nidogen Domain Protein DEX-1 in Caenorhabditis elegans. Genetics. 2019;211(1):185–200. doi: 10.1534/genetics.118.301752 30409789

35. Bokhove M, Nishimura K, Brunati M, Han L, de Sanctis D, Rampoldi L, et al. A structured interdomain linker directs self-polymerization of human uromodulin. Proc Natl Acad Sci U S A. 2016;113(6):1552–1557. doi: 10.1073/pnas.1519803113 26811476

36. Jovine L, Janssen WG, Litscher ES, Wassarman PM. The PLAC1-homology region of the ZP domain is sufficient for protein polymerisation. BMC Biochem. 2006;7:11. doi: 10.1186/1471-2091-7-11 16600035

37. Louros NN, Chrysina ED, Baltatzis GE, Patsouris ES, Hamodrakas SJ, Iconomidou VA. A common ‘aggregation-prone’ interface possibly participates in the self-assembly of human zona pellucida proteins. FEBS Lett. 2016;590(5):619–630. doi: 10.1002/1873-3468.12099 26879157

38. Louros NN, Iconomidou VA, Giannelou P, Hamodrakas SJ. Structural analysis of peptide-analogues of human Zona Pellucida ZP1 protein with amyloidogenic properties: insights into mammalian Zona Pellucida formation. PLoS One. 2013;8(9):e73258. doi: 10.1371/journal.pone.0073258 24069181

39. Darie CC, Janssen WG, Litscher ES, Wassarman PM. Purified trout egg vitelline envelope proteins VEbeta and VEgamma polymerize into homomeric fibrils from dimers in vitro. Biochim Biophys Acta. 2008;1784(2):385–392. doi: 10.1016/j.bbapap.2007.10.011 18067874

40. Litscher ES, Janssen WG, Darie CC, Wassarman PM. Purified mouse egg zona pellucida glycoproteins polymerize into homomeric fibrils under non-denaturing conditions. J Cell Physiol. 2008;214(1):153–157. doi: 10.1002/jcp.21174 17559063

41. Monne M, Han L, Schwend T, Burendahl S, Jovine L. Crystal structure of the ZP-N domain of ZP3 reveals the core fold of animal egg coats. Nature. 2008;456(7222):653–657. doi: 10.1038/nature07599 19052627

42. Williams Z, Wassarman PM. Secretion of mouse ZP3, the sperm receptor, requires cleavage of its polypeptide at a consensus furin cleavage-site. Biochemistry. 2001;40(4):929–937. doi: 10.1021/bi002275x 11170414

43. Jimenez-Movilla M, Dean J. ZP2 and ZP3 cytoplasmic tails prevent premature interactions and ensure incorporation into the zona pellucida. J Cell Sci. 2011;124(Pt 6):940–950. doi: 10.1242/jcs.079988 21378311

44. Jovine L, Qi H, Williams Z, Litscher ES, Wassarman PM. A duplicated motif controls assembly of zona pellucida domain proteins. Proc Natl Acad Sci U S A. 2004;101(16):5922–5927. doi: 10.1073/pnas.0401600101 15079052

45. Boja ES, Hoodbhoy T, Fales HM, Dean J. Structural characterization of native mouse zona pellucida proteins using mass spectrometry. J Biol Chem. 2003;278(36):34189–34202. doi: 10.1074/jbc.M304026200 12799386

46. Litscher ES, Qi H, Wassarman PM. Mouse zona pellucida glycoproteins mZP2 and mZP3 undergo carboxy-terminal proteolytic processing in growing oocytes. Biochemistry. 1999;38(38):12280–12287. doi: 10.1021/bi991154y 10493795

47. Zhao M, Gold L, Dorward H, Liang LF, Hoodbhoy T, Boja E, et al. Mutation of a conserved hydrophobic patch prevents incorporation of ZP3 into the zona pellucida surrounding mouse eggs. Mol Cell Biol. 2003;23(24):8982–8991. doi: 10.1128/mcb.23.24.8982-8991.2003 14645511

48. Brunati M, Perucca S, Han L, Cattaneo A, Consolato F, Andolfo A, et al. The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin. Elife. 2015;4:e08887. doi: 10.7554/eLife.08887 26673890

49. Fahrenkamp E, Algarra B, Jovine L. Mammalian egg coat modifications and the block to polyspermy. Mol Reprod Dev. 2020. doi: 10.1002/mrd.23320 32003503

50. Fernandes I, Chanut-Delalande H, Ferrer P, Latapie Y, Waltzer L, Affolter M, et al. Zona pellucida domain proteins remodel the apical compartment for localized cell shape changes. Dev Cell. 2010;18(1):64–76. doi: 10.1016/j.devcel.2009.11.009 20152178

51. Forman-Rubinsky R, Cohen JD, Sundaram MV. Lipocalins Are Required for Apical Extracellular Matrix Organization and Remodeling in Caenorhabditis elegans. Genetics. 2017;207(2):625–642. doi: 10.1534/genetics.117.300207 28842397

52. Gill HK, Cohen JD, Ayala-Figueroa J, Forman-Rubinsky R, Poggioli C, Bickard K, et al. Integrity of Narrow Epithelial Tubes in the C. elegans Excretory System Requires a Transient Luminal Matrix. PLoS Genet. 2016;12(8):e1006205. doi: 10.1371/journal.pgen.1006205 27482894

53. Kelley M, Yochem J, Krieg M, Calixto A, Heiman MG, Kuzmanov A, et al. FBN-1, a fibrillin-related protein, is required for resistance of the epidermis to mechanical deformation during C. elegans embryogenesis. Elife. 2015;4. doi: 10.7554/eLife.06565 25798732

54. Low IIC, Williams CR, Chong MK, McLachlan IG, Wierbowski BM, Kolotuev I, Heiman M. G. Morphogenesis of neurons and glia within an epithelium. Development. 2019;146(4). doi: 10.1242/dev.171124 30683663

55. Muriel JM, Brannan M, Taylor K, Johnstone IL, Lithgow GJ, Tuckwell D. M142.2 (cut-6):a novel Caenorhabditis elegans matrix gene important for dauer body shape. Dev Biol. 2003;260(2):339–351. doi: 10.1016/s0012-1606(03)00237-9 12921736

56. Sapio MR, Hilliard MA, Cermola M, Favre R, Bazzicalupo P. The Zona Pellucida domain containing proteins, CUT-1, CUT-3 and CUT-5, play essential roles in the development of the larval alae in Caenorhabditis elegans. Dev Biol. 2005;282(1):231–245. doi: 10.1016/j.ydbio.2005.03.011 15936343

57. Sebastiano M, Lassandro F, Bazzicalupo P. cut-1 a Caenorhabditis elegans gene coding for a dauer-specific noncollagenous component of the cuticle. Dev Biol. 1991;146(2):519–530. doi: 10.1016/0012-1606(91)90253-y 1864469

58. Vuong-Brender TTK, Suman SK, Labouesse M. The apical ECM preserves embryonic integrity and distributes mechanical stress during morphogenesis. Development. 2017;144(23):4336–4349. doi: 10.1242/dev.150383 28526752

59. Yu RY, Nguyen CQ, Hall DH, Chow KL. Expression of ram-5 in the structural cell is required for sensory ray morphogenesis in Caenorhabditis elegans male tail. Embo J. 2000;19(14):3542–3555. doi: 10.1093/emboj/19.14.3542 10899109

60. Corsi AK, Wightman B, Chalfie M. A Transparent window into biology: A primer on Caenorhabditis elegans. WormBook. 2015;1–31. doi: 10.1895/wormbook.1.177.1 26088431

61. Cohen JD, Sparacio AP, Belfi AC, Forman-Rubinsky R, Hall DH, Maul-Newby H, et al. A multi-layered and dynamic apical extracellular matrix shapes the vulva lumen in Caenorhabditis elegans. Elife. 2020;9:e57874. doi: 10.7554/eLife.57874 32975517

62. Buechner M, Hall DH, Bhatt H, Hedgecock EM. Cystic canal mutants in Caenorhabditis elegans are defective in the apical membrane domain of the renal (excretory) cell. Dev Biol. 1999;214(1):227–241. doi: 10.1006/dbio.1999.9398 10491271

63. Jones SJ, Baillie DL. Characterization of the let-653 gene in Caenorhabditis elegans. Mol Gen Genet. 1995;248(6):719–726. doi: 10.1007/BF02191712 7476875

64. Pedelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS. Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol. 2006 24(1):79–88. doi: 10.1038/nbt1172 16369541

65. Greenspan P, Mayer EP, Fowler SD. Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol. 1985;100(3);965–973. doi: 10.1083/jcb.100.3.965 3972906

66. Hawe A, Sutter M, Jiskoot W. Extrinsic fluorescent dyes as tools for protein characterization. Pharm Res. 2008;25(7):1487–1499. doi: 10.1007/s11095-007-9516-9 18172579

67. Sackett DL, Wolff J. Nile red as a polarity-sensitive fluorescent probe of hydrophobic protein surfaces. Anal Biochem. 1987;167(2):228–234. doi: 10.1016/0003-2697(87)90157-6 3442318

68. Weadick CJ. Molecular evolutionary analysis of nematode Zona Pellucida (ZP) modules reveals disulfide-bond reshuffling and standalone ZP-C domains. Genome Biol Evol. 2020;. doi: 10.1093/gbe/evaa095 32426804

69. Stanisich JJ, Zyla DS, Afanasyev P, Xu J, Kipp A, Olinger E, et al. The cryo-EM structure of the human uromodulin filament core reveals a unique assembly mechanism. Elife. 2020;9.e60265. doi: 10.7554/eLife.60265 32815518

70. Stsiapanava A, Xu C, Brunati M, Zamora-Caballero S, Schaeffer C, Han L, Carroni M, et al. Cryo-EM Structure of Native Human Uromodulin, a Zona Pellucida Module Polymer. BioRxiv. 2020. https://doi.org/10.1101/2020.05.28.119206

71. Diestel U, Resch M, Meinhardt K, Weiler S, Hellmann TV, Mueller TD, et al. Identification of a Novel TGF-beta-Binding Site in the Zona Pellucida C-terminal (ZP-C) Domain of TGF-beta-Receptor-3 (TGFR-3). PLoS One. 2013;8(6):e67214. doi: 10.1371/journal.pone.0067214 23826237

72. Smith SJ, Davidson LA, Rebeiz M. Evolutionary expansion of apical extracellular matrix is required for the elongation of cells in a novel structure. Elife. 2020;9. doi: 10.7554/eLife.55965 32338602

73. Raj I, Sadat Al Hosseini H, Dioguardi E, Nishimura K, Han L, Villa A, et al. Structural Basis of Egg Coat-Sperm Recognition at Fertilization. Cell. 2017:169(7), 1315–1326.e1317. doi: 10.1016/j.cell.2017.05.033 28622512

74. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77(1):71–94. 4366476

75. Dodt HU, Zieglgansberger W. Visualizing unstained neurons in living brain slices by infrared DIC-videomicroscopy. Brain Res. 1990;537(1–2);333–336. doi: 10.1016/0006-8993(90)90380-t 2085783

76. Bidaud-Meynard A, Nicolle O, Heck M, Le Cunff Y, Michaux G. A V0-ATPase-dependent apical trafficking pathway maintains the polarity of the intestinal absorptive membrane. Development. 2019;146(11). doi: 10.1242/dev.174508 31110027

77. Nance J, Frøkjær-Jensen C. The Caenorhabditis elegans Transgenic Toolbox. Genetics. 2019;212(4):959–990. doi: 10.1534/genetics.119.301506 31405997

78. Reits EA, Neefjes JJ. From fixed to FRAP: measuring protein mobility and activity in living cells. Nat Cell Biol. 2001;3(6):145–147. doi: 10.1038/35078615 11389456

79. Yanagawa S, Lee JS, Ishimoto A. Identification and characterization of a novel line of Drosophila Schneider S2 cells that respond to wingless signaling. J Biol Chem. 1998;273(48):32353–32359. doi: 10.1074/jbc.273.48.32353 9822716

80. Stiernagle T. Maintenance of C. elegans. WormBook. 2006;1–11. doi: 10.1895/wormbook.1.101.1 18050451

81. Tordai H, Banyai L, Patthy L. The PAN module: the N-terminal domains of plasminogen and hepatocyte growth factor are homologous with the apple domains of the prekallikrein family and with a novel domain found in numerous nematode proteins. FEBS Lett. 1999;461(1–2):63–67. doi: 10.1016/s0014-5793(99)01416-7 10561497

82. Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0 7108955


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