Human Genetics

, Volume 113, Issue 5, pp 406–416

Mutations in COCH that result in non-syndromic autosomal dominant deafness (DFNA9) affect matrix deposition of cochlin


  • Robert Grabski
    • Department of Cell BiologyUniversity of Alabama at Birmingham
  • Tomasz Szul
    • Department of Cell BiologyUniversity of Alabama at Birmingham
  • Takako Sasaki
    • Max Planck Institute for BiochemistryDepartment of Molecular Medicine
  • Rupert Timpl
    • Max Planck Institute for BiochemistryDepartment of Molecular Medicine
  • Richard Mayne
    • Department of Cell BiologyUniversity of Alabama at Birmingham
  • Barrett Hicks
    • Department of Cell BiologyUniversity of Alabama at Birmingham
    • Department of Cell BiologyUniversity of Alabama at Birmingham
Original Investigation

DOI: 10.1007/s00439-003-0992-7

Cite this article as:
Grabski, R., Szul, T., Sasaki, T. et al. Hum Genet (2003) 113: 406. doi:10.1007/s00439-003-0992-7


The COCH gene mutated in autosomal dominant sensorineural deafness (DFNA9) encodes cochlin, a major constituent of the inner ear extracellular matrix. Sequence analysis of cochlin from DFNA9 patients identified five distinct single-amino-acid mutations within a conserved region (the LCCL domain) of cochlin. To define the molecular basis of DFNA9, we have generated myc-tagged wild-type and mutant cochlins and explored their behavior in transient transfection systems. Western blotting of cell lysates and culture media indicates that wild-type and mutant cochlins are synthesized and secreted in similar amounts. Immunofluorescent staining confirms that all are detected within the endoplasmic reticulum and the Golgi complex of transfected cells. Our findings suggest that COCH mutations are unlikely to cause abnormalities in secretion and suggest that extracellular events might cause DFNA9 pathology. In agreement, we show that wild-type cochlin accumulates in extracellular deposits that closely parallel the matrix component fibronectin, whereas mutant cochlins vary in the amount and pattern of extracellular material. Whereas some mutants exhibit an almost normal deposition pattern, some show complete lack of deposition. Our results suggest that DFNA9 results from gene products that fail to integrate correctly into the extracellular matrix. The partial or complete penetrance of integration defects suggests that DFNA9 pathology may be caused by multiple molecular mechanisms, including compromised ability of cochlin to self-assemble or to form appropriate complexes with other matrix components.


Hearing impairment is the most common human communication disorder, and a familial component is known to be involved in its etiology. Rapid advances in molecular and genetic technologies have led to the identification of an ever-growing number of genes whose products are essential for hearing. Mutations in the COCH (coagulation factor C homology) gene have been shown to correlate with DFNA9, a late-onset autosomal dominant non-syndromic hearing disorder (Verhagen et al. 2000, 2001; Kamarinos et al. 2001; Verstreken et al. 2001; Fransen et al. 1999; de Kok et al. 1999; Robertson et al. 1998; Manolis et al. 1996). Hearing loss begins in the 4th or 5th decade of life and initially involves the high frequencies (Versreken et al. 2001). Deafness is progressive and is usually complete by the 6th decade. In addition to cochlear involvement, DFNA9 patients also exhibit a spectrum of clinical vestibular dysfunctions. Penetrance of the vestibular symptoms is often incomplete, and some patients are minimally affected, whereas others suffer from repeated episodes of vertigo and severe balance disturbances (Verhagen et al. 2001).

COCH maps to the long arm of human chromosome 14 in bands q11.2–13 (Robertson et al. 1997). The genomic structure of COCH has been determined (de Kok et al. 1999; Robertson et al. 1998): COCH has 12 exons with the initiator ATG start codon being located in exon 2 and the reading frame ending in exon 12. The exon-intron borders correlate with the modular structure of cochlin. Cochlin contains an N-terminal signal peptide (SP), an LCCL domain highly homologous to a domain found in the clotting factor C of the invertebrate Limulus (Japanese horseshoe crab; Trexler et al. 2000), in the late gestation lung protein Lgl1, and in the eye protein vitrin (Mayne et al. 1999), and two von Willebrand factor A (vWFA) domains (Fig. 1A). The LCCL domain is encoded by exons 4 and 5, vWFA1 by exons 8–10, and vWFA2 by exons 11 and 12 (Robertson et al. 1998). The presence of a signal peptide (characteristic for secretory proteins) and vWFA domains (characteristic for collagen-binding matrix proteins) is consistent with the finding that cochlin comprises the major non-collagen component of the extracellular matrix (ECM) of the inner ear (Ikezono et al. 2000). Two human cochlin isoforms (~63kDa and ~40kDa; Robertson et al. 2001) and three bovine cochlin isoforms (~63kDa, ~44kDa, and ~40kDa), which exhibit significant molecular heterogeneity (Ikezono et al. 2000), have been detected within the inner ear in vivo. Sequence analysis of the smaller isoforms indicates that both lack the LCCL domain (Ikezono et al. 2000). The size heterogeneity may be attributable to alternative mRNA splicing (three mRNA species of 2.0, 2.3, and 2.9 kb are present in the human cochlea; Robertson et al. 1997), exon skipping, or post-translational proteolytic processing. Cochlin mRNA and protein are highly expressed in the fibrocytes of the spiral ligament and the spiral limbus in the cochlea, and in the stroma underlying the sensory epithelium of the crest ampullaris in the vestibular labyrinth (Robertson et al. 2001). Significantly, in patients afflicted with DFNA9, there is a marked decrease in the density of cells that normally express cochlin and an increase in the accumulation of mucopolysaccharide deposits that obstruct the cochlear and vestibular nerve channels (Robertson et al. 1998; Merchant et al. 2000; Khetarpal 2000.
Fig. 1A, B.

Cochlin mutants used in this study. A Schematic diagram of cochlin domain structure. A 26 amino acids signal peptide (SP, yellow) is followed by the LCCL domain (amino acids 34–133, blue), two vWFA domains (vWFA1 amino acids 142–321, VWFA2 amino acids 362–535, both orange). To facilitate detection, an myc/his tag (Myc/His) has been inserted at the C-terminus (gray, black). Red arrows Mutated amino acids within LCCL. Cysteine residues (C) are marked in green. Sequence alignments of LCCL domains in human, mouse, and chicken cochlin, Limulus factor C (LCF_TACTR), chicken cocoacrisp protein (COCOACRISP), and human vitrin (VITRIN) are shown below. Identical residues are shown in blue. DFNA9 mutations are indicated in red. Cysteines are marked in green. B Ribbon diagram of the LCCL domain showing mutant amino acids in red and di-sulfide bonds in black

Analysis of the COCH gene in families afflicted with DFNA9 has identified five different mutations in the LCCL domain, the only region showing alterations (de Kok et al. 1999; Fransen et al. 1999; Kamarinos et al. 2001; Verhagen et al. 2001; Verstreken et al. 2001; Robertson et al. 1998). All are single-base-pair substitutions that result in an amino acid change in residues that are identical in human, cow, mouse, and chicken (except for a conserved substitution of valine 66 to isoleucine in the chicken sequence). In vivo, the mutations influence only the larger isoform of cochlin, since the smaller isoforms do not contain the LCCL domain (Ikezono et al. 2001). Structural analysis of bacterially expressed LCCL fragment of wild-type and mutant cochlins indicates that four of the five described mutations (excluding the W117R mutation) disrupt the normal structure and lead to misfolding of the domain (Liepinsh et al. 2001). The consequences of such misfolding and the way in which they result in DFNA9 pathology have not been explored.

Here, we have explored whether mutations in cochlin affect its synthesis, secretion, and post-secretion behavior. Our data suggest a DFNA9 mechanism in which mutant cochlins do not correctly integrate into the ECM, leading to structural changes in the supportive matrix of the cochlear and vestibular systems. Such ECM changes are likely to have pronounced effects on the sensory function of both organs.

Materials and methods

Reagents and antibodies

Restriction enzymes and molecular reagents were from Promega (Madison, Wis.), New England BioLabs (Beverly, Mass.), or Qiagen (Chatsworth, Calif.). SuperSignal West Pico Chemiluminescence Substrate was from Pierce (Rockford, Ill.). Rabbit polyclonal antibodies against p115 and mouse polyclonal antibodies against GM130 have been described previously (Barroso et al. 1995; Nelson et al. 1998). Rabbit polyclonal anti-myc antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.), and monoclonal anti-myc antibodies were obtained from Invitrogen (Carlsbad, Calif.). Goat anti-rat and anti-mouse antibodies conjugated with Oregon Green or Texas Red-X and Alexa Fluor 594 Phalloidin were purchased from Molecular Probes (Eugene, Ore.). Horseradish peroxidase (HRP)-labeled sheep anti-rabbit IgG antibody, HRP-labeled donkey anti-mouse IgG antibody, and HRP-labeled rabbit anti-goat IgG antibody were from Amersham Life Science (Buckinghamshire, England). Antibodies against calreticulin were from Affinity BioReagents (Golden, Colo.). Antibodies against fibronectin, actinin, paxilin, and vinculin were kindly provided by Dr. Anne Woods (UAB).


Gene mutation nomenclature used in this article follows the recommendations of den Dunnen and Antonarakis (2001). Gene symbols used in this article follow the recommendations of the HUGO Gene Nomenclature Committee (Povey et al. 2001).The authors have made every attempt to perform the study in accordance with the recommendations made by Cooper et al. (2002).

Generation of constructs

Full-length human cochlin cDNA was provided by Dr. Rupert Timpl and subcloned first into pcDNA3.1(+) and then into pcDNA4/TO/Myc-His, both from Invitrogen (Carlsbad, Calif.). Point mutations were introduced with Quick Change Site-Directed Mutagenesis Kit, according to the manufacturer's protocol (Stratagene, La Jolla, Calif.). All mutated cochlin sequences were verified by sequencing.

In vitro transcription/translation

Cochlin was generated in vitro by using the TNT T7 Coupled Reticulocyte Lysate System from Promega, according to the manufacturer's directions. An aliquot of 2 μg cochlin-pBS cDNA was used in a total reaction volume of 100 μl. Trans 35S Label from ICN Pharmaceuticals was used as the source of 35S-methionine and 35S-cysteine. The reaction was carried out at 30°C for 4 h.

Cell culture

HeLa cells were grown in Dulbecco's modified Eagle's medium with glucose and glutamine (Mediatech, Comprehensive Cancer Center of the University of Alabama at Birmingham), supplemented with 10% fetal bovine serum (FBS, Life Technologies, Grand Island, N.Y.), 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies).

Transfections and immunofluorescence microscopy

Cells were transfected with the Calcium Phosphate Transfection System (Life Technologies) or with TransIT Polyamine Transfection Reagents (Mirus Corporation, Madison, Wis.), according to the manufacturer's protocols. Some 18–48 h after transfection, cells were fixed in 3% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature. Paraformaldehyde was quenched with 10 mM ammonium chloride, and cells were permeabilized with PBS, 0.1% Triton X-100, for 7 min at room temperature. In some experiments, cells were not permeabilized. The coverslips were washed (3×2 min) with PBS, then blocked in PBS, 0.4% fish skin gelatin, 0.2% Tween 20 for 5 min, followed by blocking in PBS, 2.5% goat serum, 0.2% Tween 20 for 5 min. Cells were incubated with primary antibody diluted in PBS, 0.4% fish skin gelatin, 0.2% Tween 20 for 45 min at 37°C. Coverslips were washed (5×5 min) with PBS, 0.2% Tween 20. Secondary antibodies coupled to Oregon Green or Texas red-X were diluted in 2.5% goat serum and incubated on coverslips for 30 min at 37°C. Coverslips were washed as above and mounted on slides in 9:1 glycerol:PBS with 0.1% q-phenylenediamine. Fluorescence patterns were visualized with a Leitz Orthoplan epifluorescence microscope (Wetzlar, Germany). Optical sections were captured with a charge-coupled-device high-resolution camera equipped with a camera/computer interface. Images were analyzed with a power Mac by using IPLab Spectrum software (Scanalytics, Fairfax, Va.). Actin filaments were visualized by labeling with Alexa Fluor 594 Phalloidin (Molecular Probes) diluted with 0.05% Tween 20 in PBS. Nuclei were counterstained with Hoechst 33258.

Immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and immunoblotting

HeLa cells were solubilized in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM TRIS, pH 8.0) supplemented with Complete Protease Inhibitors (Roche) 24 h after transfection. After incubation at 4°C for 30 min, samples were centrifuged at 16,000 g for 15 min. Supernatants were incubated at 4°C with 2 μg anti-myc antibodies for 2 h, followed by 20 μl 50% (v/v) protein A-Sepharose 4FF for 1 h. Beads were recovered by centrifugation and then washed four times with RIPA buffer containing inhibitors. Precipitates were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Following SDS-PAGE, proteins were transferred to NitroPure nitrocellulose membrane (Micron Separations, Westborough, Mass.), and the membrane subjected to immunoblotting as previously described (Gao et al. 1998).

Protein N-glycosidase treatment

HeLa cells were lysed in RIPA buffer with protease inhibitors for 15 min on ice 24 h after transfection. Cell lysates were supplemented with 5 μl 10% NP-40, 5 μl 10× G7 buffer (NEB), and 1.5 μl protein N-glycosidase F (PNGase F), and incubated for 45 min at 37°C in 50 μl total volume. Samples were then supplemented with an equal volume of 2× Laemmli buffer, analyzed by SDS-PAGE, and immunoblotted with anti-myc antibodies.


Generation of cochlin constructs analogous to mutants that cause DFNA9

A full length clone encoding wild-type human cochlin was used as a template in polymerase chain reactions (PCRs) that introduced single-amino-acid substitutions into the coding region. The wild-type and mutant cochlins were then subcloned into a mammalian expression vector that added a myc/his-tag to the C-terminus of each protein (Fig. 1A).

Expression and secretion of wild-type and mutant cochlins

Human HeLa cells were transfected with myc/his-tagged wild-type and mutant cochlins. Cochlin expression was examined 24 h later by Western immunoblotting with anti-myc antibodies. As shown in Fig. 2A, a band of ~63kDa is observed in each lysate, indicating efficient expression of recombinant cochlins. The level of expression is roughly equivalent between the various constructs. The band of ~63kDa represents cochlin, since it is not present in cells mock-transfected with an empty plasmid (control lane). Cellular cochlin is glycosylated since the band of ~63kDa collapses to bands of ~61kDa and 60kDa when samples are treated with PNGase (Fig. 2C). Cochlin contains two NXS/T consensus sites for N-linked glycosylation (at positions 100 and 221), and the presence of two bands suggests that both sites accept oligosaccharide chains in vivo. Our data supports the hypothesis that glycosylation is responsible for the charge heterogeneity observed in cochlins isolated from bovine inner ear (Ikezano et al. 2001).
Fig. 2A–C.

Expression of wild-type (WT) and mutant cochlins. HeLa cells were transfected with wild-type or mutant cochlins. A Cell lysates were prepared 24 h later and analyzed by immunoblotting with anti-myc antibodies. A band of ~63kDa is detected in cells transfected with wild-type and mutant cochlins but not in mock-transfected (control) cells. B Culture media were collected 24 h later, immunoprecipitated with anti-myc antibodies, and the immunoprecipitates immunoblotted with anti-myc antibodies. A band of ~69kDa is detected in media of cells transfected with wild-type and mutant cochlins but not in mock-transfected (control) media. C Lysate from cells transfected with wild-type cochlin was mock-treated () or treated (+) with PNGase and analyzed by immunoblotting with anti-myc antibodies. Two faster migrating proteins (61kD, 60kD) indicate two glycosylation sites in cochlin

To determine whether mutant cochlins can be secreted from cells or are misfolded and removed through the endoplasmic-reticulum-associated degradation (ERAD) pathway, culture media were analyzed for cochlin. As shown in Fig. 2B, a band of ~69kDa is detected in culture media from cells expressing the wild-type or mutant cochlins, suggesting that the mutant proteins traverse the secretory pathway and are not extensively degraded. Only the I109 N mutant shows slightly reduced levels of secretion, possibly because of ERAD degradation or intracellular retention. The increase in size between the secreted and cellular cochlins (~69kDa versus ~63kDa) is attributable to processing of the N-linked oligosaccharide chains (data not shown).

Intracellular and extracellular localization of wild-type cochlin

To localize wild-type cochlin, cells either were not permeabilized to visualize exclusively extracellular cochlin or were permeabilized to allow the visualization of extracellular and intracellular cochlin. As shown in Fig. 3A, cochlin is found in a punctate extracellular pattern (open arrowheads) in non-permeabilized cells. In permeabilized cells (Fig. 3B), cochlin is present in a diffuse reticular pattern and is concentrated in a peri-nuclear region, in addition to the peripheral punctate pattern (open arrowheads). The identity of the intracellular compartments containing cochlin was explored by double label immunofluorescence with a Golgi (p115) and an ER (calreticulin) marker protein. As shown in Fig. 3C, cochlin extensively co-localized with p115 within the peri-nuclear Golgi complex (arrows). Cochlin also co-localized with calreticulin in a thin reticular ER network (Fig. 3D, arrowheads). Cochlin therefore appears to traverse the major compartments of the secretory pathway.
Fig. 3A–D.

Localization of wild-type cochlin. HeLa cells transfected with wild-type cochlin were not permeabilized (A) or permeabilized (B–D) and processed for immunofluorescence with anti-myc antibodies (A–D), and either anti-p115 (C), or anti-calreticulin antibodies (D). Wild-type cochlin is found in extracellular punctate deposits (open arrowheads in A), in the Golgi complex (arrows in C), and in the ER (arrowheads in D)

The relationship between the extracellular deposits of cochlin and the major ECM component fibronectin is shown in Fig. 4A. The punctate cochlin pattern parallels that of fibronectin, and cochlin and fibronectin often co-localize (open arrowheads). Extracellular cochlin deposits are not at focal adhesion sites, since they do not co-localize with the known focal adhesion markers actinin (Fig. 4B), vinculin (Fig. 4C), or paxillin (data not shown).
Fig. 4A–C.

Extracellular deposition of wild-type cochlin. HeLa cells transfected with wild-type cochlin were permeabilized and processed for immunofluorescence with anti-myc antibodies and either anti-fibronectin (A), anti-actinin (B), or anti-vinculin antibodies (C). Extracellular cochlin co-distributes with fibronectin (open arrowheads in A), but does not localize to focal adhesion sites (open arrowheads in B, C)

Intracellular localization of mutant cochlins

The intracellular localization of mutant cochlins was explored in permeabilized cells. As shown in Fig. 5, all mutants co-localized with calreticulin in a diffuse reticular pattern characteristic of the ER. Similarly, all mutants co-localized with p115 in the peri-nuclear Golgi region (Fig. 6). The W117R and P51S mutants showed significant staining within the Golgi complex (arrows), similar to wild-type cochlin (see Fig. 3C). The V66G, G88E, and I109 N mutants appeared more reticular and may have been less concentrated in the Golgi region.
Fig. 5.

ER localization of mutant cochlins. HeLa cells transfected with mutant cochlins were permeabilized and processed for immunofluorescence with anti-myc and anti-calreticulin antibodies. All cochlins are detected within the reticular ER network

Fig. 6.

Golgi localization of mutant cochlins. HeLa cells transfected with mutant cochlins were permeabilized and processed for immunofluorescence with anti-myc and anti-p115 antibodies. All mutant forms show partial localization in the Golgi, and the W117R and P51S mutant forms appear more concentrated in the Golgi (arrows)

Extracellular localization of mutant cochlins

To determine whether mutant cochlins are deposited extracellularly, non-permeabilized cells were processed for immunofluorescence. As shown in Fig. 7, the W117R mutant and, to a lesser extent, the P51S mutant showed extracellular deposition that paralleled or co-localized with fibronectin (open arrowheads). The overall distributions of W117R and P51S appeared analogous to that of wild-type cochlin (see Fig. 4A). Significantly, the V66G, G88E, and I109 N mutants did not show extracellular deposits. The low level of cochlin labeling was attributed to the partial permeabilization of intracellular compartments during cell fixation.
Fig. 7.

Extracellular localization of mutant cochlins. HeLa cells transfected with mutant cochlins were not permeabilized. Cells were processed for immunofluorescence with anti-myc and anti-fibronectin antibodies. The W117R and P51S mutants show a deposition pattern that is co-localized with fibronectin (open arrowheads). The V66G, G88E, and I109 N mutants lack extracellular deposits. The intracellular signal is attributable to partial permeabilization during cell fixation


The last decade has witnessed rapid progress in the discovery and characterization of genes involved in hereditary deafness (Petersen 2002; Steel and Bussoli 2001). COCH is responsible for post-lingual progressive autosomal-dominant hearing loss, DFNA9. Pathology of DFNA9 manifests as a sensorineural hearing loss starting in high frequencies and gradually leads to anacusis. DFNA9 is associated with significant histopathological changes in which acidophilic deposits are found in the peripheral auditory and vestibular systems. In patients with DFNA9 caused by the V66G, the G88E, and the W117R mutations, such accumulations of mucopolysaccharides have been found to obstruct the cochlear and vestibular nerve channels (Robertson et al. 1998; Khetarpal 2000; Merchant et al. 2000). Patients also show a significant decrease in the number of cells that normally express cochlin in the spiral lamina, spiral limbus, and spiral ligament, suggesting that the accumulation of deposits may be related to cell death.

The COCH gene product cochlin is a secretory protein that constitutes the major non-collagen component of the ECM of the inner ear (Ikezono et al. 2001). Cochlin contains two vWFA domains that are characteristic for proteins interacting with fibrillar collagens, suggesting a possible role in linking ECM components. Cochlin also contains a rare LCCL domain consisting of a central α-helix surrounded by six short β-sheets and stabilized by two di-sulfide bonds (Liepinsh et al. 2001; Fig. 1B). DFNA9 patients contain five distinct single-amino-acid substitutions, all located within the LCCL domain. All mutations, except the W117R substitution, result in misfolding of the LCCL domain. Interestingly, the W117R mutation causes the same histopathology as the "misfolded" V66G and G88E mutations, raising the possibility that different molecular mechanisms may lead to DFNA9 pathology.

To characterize the molecular basis of DFNA9 deafness, we generated mutant cochlins analogous to those found in DFNA9 patients and characterized their behavior in a transient transfection system.

Synthesis and secretion

We show that wild-type and mutant cochlins are synthesized as glycoproteins containing 2 N-linked carbohydrate chains in HeLa cells adapted to culture. Cochlin contains two asparagines that fit the NXS/T consensus sequence for glycosylation. Asp100 (NYS) lies within the LCCL domain, whereas Asp221 (NFT) is contained within the vWFA 1 region. Significantly, glycosylation of Asp100 appears unperturbed in all mutant cochlins, suggesting that the mutations do not fundamentally disrupt the global structure of the LCCL domain in vivo. Glycosylation of Asp221 is unlikely to be affected by mutations within the LCCL, since the LCCL and vWFA domains appear to fold autonomously (Liepinsh et al. 2001). That mutations within the LCCL domain do not cause global misfolding is supported by the finding that all mutant cochlins are secreted from cells. Misfolded secretory proteins are usually removed from the ER through retro-translocation and proteasome-mediated degradation (Hampton 2002). Some of the mutations may affect transit through the secretory pathway, since wild-type W117R and P51S show higher concentration within the Golgi than do the V66G, G88E, and I109 N mutants.

ECM deposition

Our results show that DFNA9 is unlikely to be caused by significant defects in cochlin synthesis, glycosylation, or secretion and suggest that the acidophilic deposits and loss of cellularity may be linked to extracellular matrix-related events. In support, we document differences in the matrix association of wild-type and mutant cochlins.

The most drastic effects are observed with the V66G, G88E, and I109 N mutants. Despite being secreted from cells (Fig. 2B), these mutants proteins do not localize to the ECM; they might integrate into ECM in situ, by binding to fibrillar collagens or other ECM molecules through their vWFA domains. However, the use of HeLa cells that do not express collagen or other cochlea-specific matrix proteins has allowed the visualization of the deposition defect. The finding that LCCL domains containing the V66G or G88E mutations misfold and aggregate when expressed in bacteria (Liepinsh et al. 2001) has led to the suggestion that the accumulation of acidophilic deposits in DFNA9 patients might results from the intracellular or extracellular deposition of misfolded cochlin mutants. Our data do not support such a model: mutant cochlins were not found to aggregate intracellularly and were not detected in extracellular deposits. It is likely that the difference in misfolding lies in the specialized redox environment that is present within the mammalian ER and that supports di-sulfide bonds formation and protein folding (Fassio and Sitia 2002). Mutant cochlins may achieve a more native folding state in mammalian cells. Analogous conditions would be present in DFNA9 patients, suggesting that acute misfolding and aggregation of mutant cochlins are unlikely to account for DFNA9 histopathology.

This is also strongly suggested by the behavior of the W117R and P51S mutants. These mutant proteins form extracellular deposits that are indistinguishable from wild-type cochlin and yet cause the same histopathology as the V66G and G88E mutants. It is likely that, in situ, they are unable to interact with their cognate ECM components. Together, the data suggest that the LCCL domain may have multiple functions and that multiple protein-protein interactions may underlie cochlin localization and function in vivo. First, the LCCL domain appears to mediate cochlin deposition within the ECM, whereas the V66G, G88E, and I109 N mutations inhibit this process. Deposition is possibly linked to LCCL-mediated homo-oligomerization of cochlin or association with specific ECM molecules. Second, the LCCL domain is likely to mediate interactions with additional ECM components, with the W117R and P51S mutations inhibiting such events. These interactions may involve ECM molecules specific to the cochlea and the vestibular system. The data are consistent with cochlin acting as a master regulator that organizes and gate-keeps the specific architecture of ECM in the cochlear and vestibular systems. Alterations in cochlin ability to integrate into the ECM or to interact with specific ECM components may represent two molecular mechanisms that lead to DFNA9 deafness.


We thank Dr. Cynthia Morton and Nancy Robertson for helpful discussion and sharing of results prior to publication. We are grateful to Dr. Anne Woods for providing antibodies and explanations and to Dr. Ben Sha for modeling the LCCL domain.

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© Springer-Verlag 2003