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. 2001 Aug 1;357(Pt 3):647–659. doi: 10.1042/0264-6021:3570647

Molecular modelling and experimental studies of mutation and cell-adhesion sites in the fibronectin type III and whey acidic protein domains of human anosmin-1.

A Robertson 1, G S MacColl 1, J A Nash 1, M K Boehm 1, S J Perkins 1, P M Bouloux 1
PMCID: PMC1221995  PMID: 11463336

Abstract

Anosmin-1, the gene product of the KAL gene, is implicated in the pathogenesis of X-linked Kallmann's syndrome. Anosmin-1 protein expression is restricted to the basement membrane and interstitial matrix of tissues affected in this syndrome during development. The anosmin-1 sequence indicates an N-terminal cysteine-rich domain, a whey acidic protein (WAP) domain, four fibronectin type III (FnIII) domains and a C-terminal histidine-rich region, and shows similarity with cell-adhesion molecules, such as neural cell-adhesion molecule, TAG-1 and L1. We investigated the structural and functional significance of three loss-of-function missense mutations of anosmin-1 using comparative modelling of the four FnIII and the WAP domains based on known NMR and crystal structures. Three missense mutation-encoded amino acid substitutions, N267K, E514K and F517L, were mapped to structurally defined positions on the GFCC' beta-sheet face of the first and third FnIII domains. Electrostatic maps demonstrated large basic surfaces containing clusters of conserved predicted heparan sulphate-binding residues adjacent to these mutation sites. To examine these modelling results anosmin-1 was expressed in insect cells. The incorporation of the three mutations into recombinant anosmin-1 had no effect on its secretion. The removal of two dibasic motifs that may constitute potential physiological cleavage sites for anosmin-1 had no effect on cleavage. Peptides based on the anosmin-1 sequences R254--K285 and P504--K527 were then synthesized in order to assess the effect of the three mutations on cellular adhesion, using cell lines that represented potential functional targets of anosmin-1. Peptides (10 microg/ml) incorporating the N267K and E514K substitutions promoted enhanced adhesion to 13.S.1.24 rat olfactory epithelial cells and canine MDCK1 kidney epithelial cells (P<0.01) compared with the wild-type peptides. This result was attributed to the introduction of a lysine residue adjacent to the large basic surfaces. We predict that two of the three missense mutants increase the binding of anosmin-1 to an extracellular target, possibly by enhancing heparan sulphate binding, and that this critically affects the function of anosmin-1.

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Selected References

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  1. Banner D. W., D'Arcy A., Chène C., Winkler F. K., Guha A., Konigsberg W. H., Nemerson Y., Kirchhofer D. The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor. Nature. 1996 Mar 7;380(6569):41–46. doi: 10.1038/380041a0. [DOI] [PubMed] [Google Scholar]
  2. Bateman A., Jouet M., MacFarlane J., Du J. S., Kenwrick S., Chothia C. Outline structure of the human L1 cell adhesion molecule and the sites where mutations cause neurological disorders. EMBO J. 1996 Nov 15;15(22):6050–6059. [PMC free article] [PubMed] [Google Scholar]
  3. Bauvois B., Rouillard D., Sanceau J., Wietzerbin J. IFN-gamma and transforming growth factor-beta 1 differently regulate fibronectin and laminin receptors of human differentiating monocytic cells. J Immunol. 1992 Jun 15;148(12):3912–3919. [PubMed] [Google Scholar]
  4. Bernfield M., Götte M., Park P. W., Reizes O., Fitzgerald M. L., Lincecum J., Zako M. Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem. 1999;68:729–777. doi: 10.1146/annurev.biochem.68.1.729. [DOI] [PubMed] [Google Scholar]
  5. Boehm M. K., Mayans M. O., Thornton J. D., Begent R. H., Keep P. A., Perkins S. J. Extended glycoprotein structure of the seven domains in human carcinoembryonic antigen by X-ray and neutron solution scattering and an automated curve fitting procedure: implications for cellular adhesion. J Mol Biol. 1996 Jun 21;259(4):718–736. doi: 10.1006/jmbi.1996.0353. [DOI] [PubMed] [Google Scholar]
  6. Bunch T. A., Grinblat Y., Goldstein L. S. Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res. 1988 Feb 11;16(3):1043–1061. doi: 10.1093/nar/16.3.1043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burgoon M. P., Grumet M., Mauro V., Edelman G. M., Cunningham B. A. Structure of the chicken neuron-glia cell adhesion molecule, Ng-CAM: origin of the polypeptides and relation to the Ig superfamily. J Cell Biol. 1991 Mar;112(5):1017–1029. doi: 10.1083/jcb.112.5.1017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Busby T. F., Argraves W. S., Brew S. A., Pechik I., Gilliland G. L., Ingham K. C. Heparin binding by fibronectin module III-13 involves six discontinuous basic residues brought together to form a cationic cradle. J Biol Chem. 1995 Aug 4;270(31):18558–18562. doi: 10.1074/jbc.270.31.18558. [DOI] [PubMed] [Google Scholar]
  9. Chothia C., Jones E. Y. The molecular structure of cell adhesion molecules. Annu Rev Biochem. 1997;66:823–862. doi: 10.1146/annurev.biochem.66.1.823. [DOI] [PubMed] [Google Scholar]
  10. Coronas V., Féron F., Hen R., Sicard G., Jourdan F., Moyse E. In vitro induction of apoptosis or differentiation by dopamine in an immortalized olfactory neuronal cell line. J Neurochem. 1997 Nov;69(5):1870–1881. doi: 10.1046/j.1471-4159.1997.69051870.x. [DOI] [PubMed] [Google Scholar]
  11. Dandekar A. M., Robinson E. A., Appella E., Qasba P. K. Complete sequence analysis of cDNA clones encoding rat whey phosphoprotein: homology to a protease inhibitor. Proc Natl Acad Sci U S A. 1982 Jul;79(13):3987–3991. doi: 10.1073/pnas.79.13.3987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dickinson C. D., Veerapandian B., Dai X. P., Hamlin R. C., Xuong N. H., Ruoslahti E., Ely K. R. Crystal structure of the tenth type III cell adhesion module of human fibronectin. J Mol Biol. 1994 Mar 4;236(4):1079–1092. doi: 10.1016/0022-2836(94)90013-2. [DOI] [PubMed] [Google Scholar]
  13. Duke V. M., Winyard P. J., Thorogood P., Soothill P., Bouloux P. M., Woolf A. S. KAL, a gene mutated in Kallmann's syndrome, is expressed in the first trimester of human development. Mol Cell Endocrinol. 1995 Apr 28;110(1-2):73–79. doi: 10.1016/0303-7207(95)03518-c. [DOI] [PubMed] [Google Scholar]
  14. Duke V., Quinton R., Gordon I., Bouloux P. M., Woolf A. S. Proteinuria, hypertension and chronic renal failure in X-linked Kallmann's syndrome, a defined genetic cause of solitary functioning kidney. Nephrol Dial Transplant. 1998 Aug;13(8):1998–2003. doi: 10.1093/ndt/13.8.1998. [DOI] [PubMed] [Google Scholar]
  15. Edelman G. M., Crossin K. L. Cell adhesion molecules: implications for a molecular histology. Annu Rev Biochem. 1991;60:155–190. doi: 10.1146/annurev.bi.60.070191.001103. [DOI] [PubMed] [Google Scholar]
  16. Farrell P. J., Behie L. A., Iatrou K. Transformed Lepidopteran insect cells: new sources of recombinant human tissue plasminogen activator. Biotechnol Bioeng. 1999 Aug 20;64(4):426–433. [PubMed] [Google Scholar]
  17. Francart C., Dauchez M., Alix A. J., Lippens G. Solution structure of R-elafin, a specific inhibitor of elastase. J Mol Biol. 1997 May 9;268(3):666–677. doi: 10.1006/jmbi.1997.0983. [DOI] [PubMed] [Google Scholar]
  18. Franco B., Guioli S., Pragliola A., Incerti B., Bardoni B., Tonlorenzi R., Carrozzo R., Maestrini E., Pieretti M., Taillon-Miller P. A gene deleted in Kallmann's syndrome shares homology with neural cell adhesion and axonal path-finding molecules. Nature. 1991 Oct 10;353(6344):529–536. doi: 10.1038/353529a0. [DOI] [PubMed] [Google Scholar]
  19. Georgopoulos N. A., Pralong F. P., Seidman C. E., Seidman J. G., Crowley W. F., Jr, Vallejo M. Genetic heterogeneity evidenced by low incidence of KAL-1 gene mutations in sporadic cases of gonadotropin-releasing hormone deficiency. J Clin Endocrinol Metab. 1997 Jan;82(1):213–217. doi: 10.1210/jcem.82.1.3692. [DOI] [PubMed] [Google Scholar]
  20. Gong Q., Shipley M. T. Evidence that pioneer olfactory axons regulate telencephalon cell cycle kinetics to induce the formation of the olfactory bulb. Neuron. 1995 Jan;14(1):91–101. doi: 10.1016/0896-6273(95)90243-0. [DOI] [PubMed] [Google Scholar]
  21. Gu W. X., Colquhoun-Kerr J. S., Kopp P., Bode H. H., Jameson J. L. A novel aminoterminal mutation in the KAL-1 gene in a large pedigree with X-linked Kallmann syndrome. Mol Genet Metab. 1998 Sep;65(1):59–61. doi: 10.1006/mgme.1998.2732. [DOI] [PubMed] [Google Scholar]
  22. Hardelin J. P., Julliard A. K., Moniot B., Soussi-Yanicostas N., Verney C., Schwanzel-Fukuda M., Ayer-Le Lievre C., Petit C. Anosmin-1 is a regionally restricted component of basement membranes and interstitial matrices during organogenesis: implications for the developmental anomalies of X chromosome-linked Kallmann syndrome. Dev Dyn. 1999 May;215(1):26–44. doi: 10.1002/(SICI)1097-0177(199905)215:1<26::AID-DVDY4>3.0.CO;2-D. [DOI] [PubMed] [Google Scholar]
  23. Hardelin J. P., Levilliers J., Blanchard S., Carel J. C., Leutenegger M., Pinard-Bertelletto J. P., Bouloux P., Petit C. Heterogeneity in the mutations responsible for X chromosome-linked Kallmann syndrome. Hum Mol Genet. 1993 Apr;2(4):373–377. doi: 10.1093/hmg/2.4.373. [DOI] [PubMed] [Google Scholar]
  24. Hardelin J. P., Levilliers J., del Castillo I., Cohen-Salmon M., Legouis R., Blanchard S., Compain S., Bouloux P., Kirk J., Moraine C. X chromosome-linked Kallmann syndrome: stop mutations validate the candidate gene. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8190–8194. doi: 10.1073/pnas.89.17.8190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Harlos K., Martin D. M., O'Brien D. P., Jones E. Y., Stuart D. I., Polikarpov I., Miller A., Tuddenham E. G., Boys C. W. Crystal structure of the extracellular region of human tissue factor. Nature. 1994 Aug 25;370(6491):662–666. doi: 10.1038/370662a0. [DOI] [PubMed] [Google Scholar]
  26. Hennighausen L. G., Sippel A. E. Mouse whey acidic protein is a novel member of the family of 'four-disulfide core' proteins. Nucleic Acids Res. 1982 Apr 24;10(8):2677–2684. doi: 10.1093/nar/10.8.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hileman R. E., Fromm J. R., Weiler J. M., Linhardt R. J. Glycosaminoglycan-protein interactions: definition of consensus sites in glycosaminoglycan binding proteins. Bioessays. 1998 Feb;20(2):156–167. doi: 10.1002/(SICI)1521-1878(199802)20:2<156::AID-BIES8>3.0.CO;2-R. [DOI] [PubMed] [Google Scholar]
  28. Huber A. H., Wang Y. M., Bieber A. J., Bjorkman P. J. Crystal structure of tandem type III fibronectin domains from Drosophila neuroglian at 2.0 A. Neuron. 1994 Apr;12(4):717–731. doi: 10.1016/0896-6273(94)90326-3. [DOI] [PubMed] [Google Scholar]
  29. Jenkins P. V., Pasi K. J., Perkins S. J. Molecular modeling of ligand and mutation sites of the type A domains of human von Willebrand factor and their relevance to von Willebrand's disease. Blood. 1998 Mar 15;91(6):2032–2044. [PubMed] [Google Scholar]
  30. Kabsch W., Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577–2637. doi: 10.1002/bip.360221211. [DOI] [PubMed] [Google Scholar]
  31. Kayyem J. F., Roman J. M., de la Rosa E. J., Schwarz U., Dreyer W. J. Bravo/Nr-CAM is closely related to the cell adhesion molecules L1 and Ng-CAM and has a similar heterodimer structure. J Cell Biol. 1992 Sep;118(5):1259–1270. doi: 10.1083/jcb.118.5.1259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kirk J. M., Grant D. B., Besser G. M., Shalet S., Quinton R., Smith C. S., White M., Edwards O., Bouloux P. M. Unilateral renal aplasia in X-linked Kallmann's syndrome. Clin Genet. 1994 Sep;46(3):260–262. doi: 10.1111/j.1399-0004.1994.tb04238.x. [DOI] [PubMed] [Google Scholar]
  33. Lander A. D. Understanding the molecules of neural cell contacts: emerging patterns of structure and function. Trends Neurosci. 1989 May;12(5):189–195. doi: 10.1016/0166-2236(89)90070-2. [DOI] [PubMed] [Google Scholar]
  34. Leahy D. J., Aukhil I., Erickson H. P. 2.0 A crystal structure of a four-domain segment of human fibronectin encompassing the RGD loop and synergy region. Cell. 1996 Jan 12;84(1):155–164. doi: 10.1016/s0092-8674(00)81002-8. [DOI] [PubMed] [Google Scholar]
  35. Leahy D. J., Hendrickson W. A., Aukhil I., Erickson H. P. Structure of a fibronectin type III domain from tenascin phased by MAD analysis of the selenomethionyl protein. Science. 1992 Nov 6;258(5084):987–991. doi: 10.1126/science.1279805. [DOI] [PubMed] [Google Scholar]
  36. Lee B., Richards F. M. The interpretation of protein structures: estimation of static accessibility. J Mol Biol. 1971 Feb 14;55(3):379–400. doi: 10.1016/0022-2836(71)90324-x. [DOI] [PubMed] [Google Scholar]
  37. Legouis R., Cohen-Salmon M., del Castillo I., Levilliers J., Capy L., Mornow J. P., Petit C. Characterization of the chicken and quail homologues of the human gene responsible for the X-linked Kallmann syndrome. Genomics. 1993 Aug;17(2):516–518. doi: 10.1006/geno.1993.1360. [DOI] [PubMed] [Google Scholar]
  38. Legouis R., Hardelin J. P., Levilliers J., Claverie J. M., Compain S., Wunderle V., Millasseau P., Le Paslier D., Cohen D., Caterina D. The candidate gene for the X-linked Kallmann syndrome encodes a protein related to adhesion molecules. Cell. 1991 Oct 18;67(2):423–435. doi: 10.1016/0092-8674(91)90193-3. [DOI] [PubMed] [Google Scholar]
  39. Livnah O., Stura E. A., Johnson D. L., Middleton S. A., Mulcahy L. S., Wrighton N. C., Dower W. J., Jolliffe L. K., Wilson I. A. Functional mimicry of a protein hormone by a peptide agonist: the EPO receptor complex at 2.8 A. Science. 1996 Jul 26;273(5274):464–471. doi: 10.1126/science.273.5274.464. [DOI] [PubMed] [Google Scholar]
  40. Lutz B., Rugarli E. I., Eichele G., Ballabio A. X-linked Kallmann syndrome. A neuronal targeting defect in the olfactory system? FEBS Lett. 1993 Jun 28;325(1-2):128–134. doi: 10.1016/0014-5793(93)81428-3. [DOI] [PubMed] [Google Scholar]
  41. Main A. L., Harvey T. S., Baron M., Boyd J., Campbell I. D. The three-dimensional structure of the tenth type III module of fibronectin: an insight into RGD-mediated interactions. Cell. 1992 Nov 13;71(4):671–678. doi: 10.1016/0092-8674(92)90600-h. [DOI] [PubMed] [Google Scholar]
  42. Margalit H., Fischer N., Ben-Sasson S. A. Comparative analysis of structurally defined heparin binding sequences reveals a distinct spatial distribution of basic residues. J Biol Chem. 1993 Sep 15;268(26):19228–19231. [PubMed] [Google Scholar]
  43. Maya-Nuñez G., Zenteno J. C., Ulloa-Aguirre A., Kofman-Alfaro S., Mendez J. P. A recurrent missense mutation in the KAL gene in patients with X-linked Kallmann's syndrome. J Clin Endocrinol Metab. 1998 May;83(5):1650–1653. doi: 10.1210/jcem.83.5.4817. [DOI] [PubMed] [Google Scholar]
  44. Maya-Núez G., Cuevas-Covarrubias S., Zenteno J. C., Ulloa-Aguirre A., Kofman-Alfaro S., Méndez J. P. Contiguous gene syndrome due to deletion of the first three exons of the Kallmann gene and complete deletion of the steroid sulphatase gene. Clin Endocrinol (Oxf) 1998 Jun;48(6):713–718. doi: 10.1046/j.1365-2265.1998.00406.x. [DOI] [PubMed] [Google Scholar]
  45. Meitinger T., Heye B., Petit C., Levilliers J., Golla A., Moraine C., Dalla Piccola B., Sippell W. G., Murken J., Ballabio A. Definitive localization of X-linked Kallman syndrome (hypogonadotropic hypogonadism and anosmia) to Xp22.3: close linkage to the hypervariable repeat sequence CRI-S232. Am J Hum Genet. 1990 Oct;47(4):664–669. [PMC free article] [PubMed] [Google Scholar]
  46. Muller Y. A., Ultsch M. H., de Vos A. M. The crystal structure of the extracellular domain of human tissue factor refined to 1.7 A resolution. J Mol Biol. 1996 Feb 16;256(1):144–159. doi: 10.1006/jmbi.1996.0073. [DOI] [PubMed] [Google Scholar]
  47. O'Neill M. J., Tridjaja B., Smith M. J., Bell K. M., Warne G. L., Sinclair A. H. Familial Kallmann syndrome: a novel splice acceptor mutation in the KAL gene. Hum Mutat. 1998;11(4):340–342. [PubMed] [Google Scholar]
  48. Rugarli E. I., Ghezzi C., Valsecchi V., Ballabio A. The Kallmann syndrome gene product expressed in COS cells is cleaved on the cell surface to yield a diffusible component. Hum Mol Genet. 1996 Aug;5(8):1109–1115. doi: 10.1093/hmg/5.8.1109. [DOI] [PubMed] [Google Scholar]
  49. Schlessinger J., Lax I., Lemmon M. Regulation of growth factor activation by proteoglycans: what is the role of the low affinity receptors? Cell. 1995 Nov 3;83(3):357–360. doi: 10.1016/0092-8674(95)90112-4. [DOI] [PubMed] [Google Scholar]
  50. Schneider I. Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol. 1972 Apr;27(2):353–365. [PubMed] [Google Scholar]
  51. Soussi-Yanicostas N., Faivre-Sarrailh C., Hardelin J. P., Levilliers J., Rougon G., Petit C. Anosmin-1 underlying the X chromosome-linked Kallmann syndrome is an adhesion molecule that can modulate neurite growth in a cell-type specific manner. J Cell Sci. 1998 Oct;111(Pt 19):2953–2965. doi: 10.1242/jcs.111.19.2953. [DOI] [PubMed] [Google Scholar]
  52. Soussi-Yanicostas N., Hardelin J. P., Arroyo-Jimenez M. M., Ardouin O., Legouis R., Levilliers J., Traincard F., Betton J. M., Cabanié L., Petit C. Initial characterization of anosmin-1, a putative extracellular matrix protein synthesized by definite neuronal cell populations in the central nervous system. J Cell Sci. 1996 Jul;109(Pt 7):1749–1757. doi: 10.1242/jcs.109.7.1749. [DOI] [PubMed] [Google Scholar]
  53. Streuli M., Krueger N. X., Ariniello P. D., Tang M., Munro J. M., Blattler W. A., Adler D. A., Disteche C. M., Saito H. Expression of the receptor-linked protein tyrosine phosphatase LAR: proteolytic cleavage and shedding of the CAM-like extracellular region. EMBO J. 1992 Mar;11(3):897–907. doi: 10.1002/j.1460-2075.1992.tb05128.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Tsunemi M., Matsuura Y., Sakakibara S., Katsube Y. Crystal structure of an elastase-specific inhibitor elafin complexed with porcine pancreatic elastase determined at 1.9 A resolution. Biochemistry. 1996 Sep 10;35(36):11570–11576. doi: 10.1021/bi960900l. [DOI] [PubMed] [Google Scholar]
  55. Volkmer H., Hassel B., Wolff J. M., Frank R., Rathjen F. G. Structure of the axonal surface recognition molecule neurofascin and its relationship to a neural subgroup of the immunoglobulin superfamily. J Cell Biol. 1992 Jul;118(1):149–161. doi: 10.1083/jcb.118.1.149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yamasaki K., Naito S., Anaguchi H., Ohkubo T., Ota Y. Solution structure of an extracellular domain containing the WSxWS motif of the granulocyte colony-stimulating factor receptor and its interaction with ligand. Nat Struct Biol. 1997 Jun;4(6):498–504. doi: 10.1038/nsb0697-498. [DOI] [PubMed] [Google Scholar]
  57. Yokosaki Y., Matsuura N., Higashiyama S., Murakami I., Obara M., Yamakido M., Shigeto N., Chen J., Sheppard D. Identification of the ligand binding site for the integrin alpha9 beta1 in the third fibronectin type III repeat of tenascin-C. J Biol Chem. 1998 May 8;273(19):11423–11428. doi: 10.1074/jbc.273.19.11423. [DOI] [PubMed] [Google Scholar]
  58. de Vos A. M., Ultsch M., Kossiakoff A. A. Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science. 1992 Jan 17;255(5042):306–312. doi: 10.1126/science.1549776. [DOI] [PubMed] [Google Scholar]
  59. del Castillo I., Cohen-Salmon M., Blanchard S., Lutfalla G., Petit C. Structure of the X-linked Kallmann syndrome gene and its homologous pseudogene on the Y chromosome. Nat Genet. 1992 Dec;2(4):305–310. doi: 10.1038/ng1292-305. [DOI] [PubMed] [Google Scholar]

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