Skip to main content
PMC home page
Journal of Medical Genetics logoLink to Journal of Medical Genetics
. 2003 Jul;40(7):479–486. doi: 10.1136/jmg.40.7.479

Subcellular localisation, secretion, and post-translational processing of normal cochlin, and of mutants causing the sensorineural deafness and vestibular disorder, DFNA9

N Robertson 1, S Hamaker 1, V Patriub 1, J Aster 1, C Morton 1
PMCID: PMC1735525  PMID: 12843317

Abstract

Five missense mutations in the FCH/LCCL domain of the COCH gene, encoding the protein cochlin, are pathogenic for the autosomal dominant hearing loss and vestibular dysfunction disorder, DFNA9. To date, the function of cochlin and the mechanism of pathogenesis of the mutations are unknown. We have used the biological system of transient transfections of the entire protein coding region of COCH into several mammalian cell lines, to investigate various functional properties of cochlin. By western blot analysis of lysates prepared from transfected cells, we show that cochlin is a secreted protein. Immunocytochemistry shows concentrated localisation of cochlin in perinuclear structures consistent with the Golgi apparatus and endoplasmic reticulum, showing intracellular passage through these secretory compartments. We detected that cochlin is proteolytically cleaved between the FCH/LCCL domain and the downstream vWFA domains, resulting in a smaller cochlin isoform of ~50 kDa. Interestingly, this isoform lacks the entire mutation bearing FCH/LCCL domain. We have also shown that cochlin is N-glycosylated in its mature secreted form. Previous studies of the FCH/LCCL domain alone, expressed in bacteria, have demonstrated that three of four DFNA9 mutations cause misfolding of this domain. Characteristic eosinophilic deposits in DFNA9 affected inner ear structures could be the result of aberrant folding, secretion, or solubility of mutated cochlins, as in certain other pathological states in which misfolded proteins accumulate and aggregate causing toxicity. To examine the biological consequences of cochlin misfolding, we made separate constructs with three of the DFNA9 mutations and performed parallel studies of the mutated and wild type cochlins. We detected that mutated cochlins are not retained intracellularly, and are able to be secreted adequately by the cells, through the Golgi/ER secretory pathway, and also undergo proteolytic cleavage and glycosylation. These results suggest that DFNA9 mutations may manifest deleterious effects beyond the point of secretion, in the unique environment of the extracellular matrix of the inner ear by disrupting cochlin function or interfering with protein-protein interactions involving the FCH/LCCL domain. It is also possible that the mutations may result in aggregation of cochlin in vivo over a longer time course, as supported by the late onset and progressive nature of this disorder.

Full Text

The Full Text of this article is available as a PDF (266.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bruijn L. I., Houseweart M. K., Kato S., Anderson K. L., Anderson S. D., Ohama E., Reaume A. G., Scott R. W., Cleveland D. W. Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1. Science. 1998 Sep 18;281(5384):1851–1854. doi: 10.1126/science.281.5384.1851. [DOI] [PubMed] [Google Scholar]
  2. Davies S. W., Turmaine M., Cozens B. A., DiFiglia M., Sharp A. H., Ross C. A., Scherzinger E., Wanker E. E., Mangiarini L., Bates G. P. Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell. 1997 Aug 8;90(3):537–548. doi: 10.1016/s0092-8674(00)80513-9. [DOI] [PubMed] [Google Scholar]
  3. Delrieu Isabelle, Waller Cecilia C., Mota Maria M., Grainger Munira, Langhorne Jean, Holder Anthony A. PSLAP, a protein with multiple adhesive motifs, is expressed in Plasmodium falciparum gametocytes. Mol Biochem Parasitol. 2002 Apr 30;121(1):11–20. doi: 10.1016/s0166-6851(02)00016-6. [DOI] [PubMed] [Google Scholar]
  4. Durham H. D., Roy J., Dong L., Figlewicz D. A. Aggregation of mutant Cu/Zn superoxide dismutase proteins in a culture model of ALS. J Neuropathol Exp Neurol. 1997 May;56(5):523–530. doi: 10.1097/00005072-199705000-00008. [DOI] [PubMed] [Google Scholar]
  5. Fransen E., Verstreken M., Verhagen W. I., Wuyts F. L., Huygen P. L., D'Haese P., Robertson N. G., Morton C. C., McGuirt W. T., Smith R. J. High prevalence of symptoms of Menière's disease in three families with a mutation in the COCH gene. Hum Mol Genet. 1999 Aug;8(8):1425–1429. doi: 10.1093/hmg/8.8.1425. [DOI] [PubMed] [Google Scholar]
  6. Garcia-Mata Rafael, Gao Ya-Sheng, Sztul Elizabeth. Hassles with taking out the garbage: aggravating aggresomes. Traffic. 2002 Jun;3(6):388–396. doi: 10.1034/j.1600-0854.2002.30602.x. [DOI] [PubMed] [Google Scholar]
  7. Ikezono T., Omori A., Ichinose S., Pawankar R., Watanabe A., Yagi T. Identification of the protein product of the Coch gene (hereditary deafness gene) as the major component of bovine inner ear protein. Biochim Biophys Acta. 2001 Mar 26;1535(3):258–265. doi: 10.1016/s0925-4439(00)00101-0. [DOI] [PubMed] [Google Scholar]
  8. Kamarinos M., McGill J., Lynch M., Dahl H. Identification of a novel COCH mutation, I109N, highlights the similar clinical features observed in DFNA9 families. Hum Mutat. 2001 Apr;17(4):351–351. doi: 10.1002/humu.37. [DOI] [PubMed] [Google Scholar]
  9. Khetarpal U. Autosomal dominant sensorineural hearing loss. Further temporal bone findings. Arch Otolaryngol Head Neck Surg. 1993 Jan;119(1):106–108. doi: 10.1001/archotol.1993.01880130108016. [DOI] [PubMed] [Google Scholar]
  10. Khetarpal U. DFNA9 is a progressive audiovestibular dysfunction with a microfibrillar deposit in the inner ear. Laryngoscope. 2000 Aug;110(8):1379–1384. doi: 10.1097/00005537-200008000-00030. [DOI] [PubMed] [Google Scholar]
  11. Liepinsh E., Trexler M., Kaikkonen A., Weigelt J., Bányai L., Patthy L., Otting G. NMR structure of the LCCL domain and implications for DFNA9 deafness disorder. EMBO J. 2001 Oct 1;20(19):5347–5353. doi: 10.1093/emboj/20.19.5347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Maley F., Trimble R. B., Tarentino A. L., Plummer T. H., Jr Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal Biochem. 1989 Aug 1;180(2):195–204. doi: 10.1016/0003-2697(89)90115-2. [DOI] [PubMed] [Google Scholar]
  13. Masliah E., Rockenstein E., Veinbergs I., Mallory M., Hashimoto M., Takeda A., Sagara Y., Sisk A., Mucke L. Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders. Science. 2000 Feb 18;287(5456):1265–1269. doi: 10.1126/science.287.5456.1265. [DOI] [PubMed] [Google Scholar]
  14. Merchant S. N., Linthicum F. H., Nadol J. B., Jr Histopathology of the inner ear in DFNA9. Adv Otorhinolaryngol. 2000;56:212–217. doi: 10.1159/000059105. [DOI] [PubMed] [Google Scholar]
  15. Riordan J. R. Cystic fibrosis as a disease of misprocessing of the cystic fibrosis transmembrane conductance regulator glycoprotein. Am J Hum Genet. 1999 Jun;64(6):1499–1504. doi: 10.1086/302429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Robertson N. G., Khetarpal U., Gutiérrez-Espeleta G. A., Bieber F. R., Morton C. C. Isolation of novel and known genes from a human fetal cochlear cDNA library using subtractive hybridization and differential screening. Genomics. 1994 Sep 1;23(1):42–50. doi: 10.1006/geno.1994.1457. [DOI] [PubMed] [Google Scholar]
  17. Robertson N. G., Lu L., Heller S., Merchant S. N., Eavey R. D., McKenna M., Nadol J. B., Jr, Miyamoto R. T., Linthicum F. H., Jr, Lubianca Neto J. F. Mutations in a novel cochlear gene cause DFNA9, a human nonsyndromic deafness with vestibular dysfunction. Nat Genet. 1998 Nov;20(3):299–303. doi: 10.1038/3118. [DOI] [PubMed] [Google Scholar]
  18. Robertson N. G., Resendes B. L., Lin J. S., Lee C., Aster J. C., Adams J. C., Morton C. C. Inner ear localization of mRNA and protein products of COCH, mutated in the sensorineural deafness and vestibular disorder, DFNA9. Hum Mol Genet. 2001 Oct 15;10(22):2493–2500. doi: 10.1093/hmg/10.22.2493. [DOI] [PubMed] [Google Scholar]
  19. Robertson N. G., Skvorak A. B., Yin Y., Weremowicz S., Johnson K. R., Kovatch K. A., Battey J. F., Bieber F. R., Morton C. C. Mapping and characterization of a novel cochlear gene in human and in mouse: a positional candidate gene for a deafness disorder, DFNA9. Genomics. 1997 Dec 15;46(3):345–354. doi: 10.1006/geno.1997.5067. [DOI] [PubMed] [Google Scholar]
  20. Roth Jürgen. Protein N-glycosylation along the secretory pathway: relationship to organelle topography and function, protein quality control, and cell interactions. Chem Rev. 2002 Feb;102(2):285–303. doi: 10.1021/cr000423j. [DOI] [PubMed] [Google Scholar]
  21. Takahashi R. H., Nam E. E., Edgar M., Gouras G. K. Alzheimer beta-amyloid peptides: normal and abnormal localization. Histol Histopathol. 2002 Jan;17(1):239–246. doi: 10.14670/HH-17.239. [DOI] [PubMed] [Google Scholar]
  22. Trexler M., Bányai L., Patthy L. The LCCL module. Eur J Biochem. 2000 Sep;267(18):5751–5757. doi: 10.1046/j.1432-1327.2000.01641.x. [DOI] [PubMed] [Google Scholar]
  23. Waelter S., Boeddrich A., Lurz R., Scherzinger E., Lueder G., Lehrach H., Wanker E. E. Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation. Mol Biol Cell. 2001 May;12(5):1393–1407. doi: 10.1091/mbc.12.5.1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Zoghbi H. Y., Orr H. T. Glutamine repeats and neurodegeneration. Annu Rev Neurosci. 2000;23:217–247. doi: 10.1146/annurev.neuro.23.1.217. [DOI] [PubMed] [Google Scholar]
  25. de Kok Y. J., Bom S. J., Brunt T. M., Kemperman M. H., van Beusekom E., van der Velde-Visser S. D., Robertson N. G., Morton C. C., Huygen P. L., Verhagen W. I. A Pro51Ser mutation in the COCH gene is associated with late onset autosomal dominant progressive sensorineural hearing loss with vestibular defects. Hum Mol Genet. 1999 Feb;8(2):361–366. doi: 10.1093/hmg/8.2.361. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Medical Genetics are provided here courtesy of BMJ Publishing Group

RESOURCES