Polypeptide composition of the mammalian tectorial membrane

doi:10.1016/0378-5955(87)90078-5

Hearing Research

Volume 25, Issue 1, 1987, Pages 45-60

https://doi.org/10.1016/0378-5955(87)90078-5 Get rights and content

Abstract

The effects of the enzymes collagenase, pepsin, chondroitinase ABC and keratanase on the polypeptide composition of the mammalian tectorial membrane have been analysed using one dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After reduction at least ten polypeptides can be consistently and clearly recognized in SDS gels with molecular weights relative to globular protein standards of 245, 235,190,165, 155,145,100, 93, 60–73 and 35–49 kDa. With the exception of the 60–73 and 35–49 kDa bands all these polypeptides are sensitive to digestion with bacterial collagenase. The 235, 165, 155, 145 and 93 kDa bands also resist degradation by cold, acidic pepsin. Amino acid analysis of whole tectorial membranes demonstrates that glycine accounts for nearly 25% of the total amino acid content, that proline, hydroxyproline and hydroxylysine are present and that amine sugars can be detected in fairly high concentrations. Estimates based on hydroxyproline content suggest that collagens account for 25–50% of the total tectorial membrane protein. Immunoblotting techniques demonstrate the presence of polypeptides cross reacting with antisera to Type II collagen, Type IX collagen and Type V collagen. Results from immunohistochemical studies confirm that these polypeptides are present in the tectorial membrane and are not contaminants of the isolation procedure. Collagenase treatment of tectorial membranes reveals the presence of an additional non-collagenous polypeptide with an apparent molecular weight of 173 kDa on 7.5% polyacrylamide gels, and polydisperse high molecular weight material spreading over a broad range at the top of the gels. This high molecular weight material and the 173, 60–73 and 35–49 kDa non-collagenous polypeptides are pepsin sensitive and all bind wheat germ agglutinin (WGA) suggesting that they contain N-acetyl glucosamine. The 173 kDa band also binds soybean agglutinin (SBA) suggesting the presence of N-acetyl galactosamine. In the absence of reducing agent the 173 and 60–73 kDa bands are no longer observed and high molecular weight material forming a broad band at the top of the separating gel is seen. The electrophoretic behaviour of this non-collagenous, glycosylated, disulphide bonded, high molecular weight material is altered by treatment with keratanase but not by chondroitinase ABC, The results of this study indicate the tectorial membrane contains at least three different collagen types and, in addition to these collagenous proteins, several non-collagenous, glycosylated polypeptides that may account for as much as 50% of the total tectorial membrane protein.

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Analysis of the mouse TM by gel electrophoresis reveals the presence of a number of proteins, many of which are sensitive to digestion with bacterial collagenase (see Fig. 2A). These collagenase-sensitive polypeptides are now known to be derived from type II, type IX, and type XI collagen (Richardson, Russell, Duance, & Bailey, 1987; Thalmann et al., 1987), collagens that are typically found in cartilage. Type V collagen may also be present (Richardson et al., 1987).
The tectorial membrane is an extracellular matrix that lies over the apical surface of the auditory epithelia in the inner ears of reptiles, birds, and mammals. Recent studies have shown it is composed of a small set of proteins, some of which are only produced at high levels in the ear and many of which are the products of genes that, when mutated, cause nonsyndromic forms of human hereditary deafness. Quite how the proteins of the tectorial membrane are assembled within the lumen of the inner ear to form a structure that is precisely regulated in its size and physical properties along the length of a tonotopically organized hearing organ is a question that remains to be fully answered. In this brief review we will summarize what is known thus far about the structure, protein composition, and function of the tectorial membrane in birds and mammals, describe how the tectorial membrane develops, and discuss major events that have occurred during the evolution of this extracellular matrix.
Tectorins crosslink type II collagen fibrils and connect the tectorial membrane to the spiral limbus
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Further investigations would clarify whether the TM performs one or more of these functions. The TM is composed of four types of collagens (II, V, IX and XI), seven types of noncollagenous glycoproteins (α-tectorin, β-tectorin, carcinoembryonic antigen-related cell adhesion molecule 16 – CEACAM16, otogelin, otogelin-like, otoancorin and otolin) and two glycosaminoglycans (uronic acid and keratan sulfate) (Thalmann et al., 1986, 1987, 1993; Richardson et al., 1987; Thalmann, 1993; Zwaenepoel et al., 2002; Deans et al., 2010; Zheng et al., 2011; Kammerer et al., 2012; Oonk et al., 2014). During embryonic development, all these molecules self-assemble into the open fluid environment of the endolymph.
All inner ear organs possess extracellular matrix appendices over the sensory epithelia that are crucial for their proper function. The tectorial membrane (TM) is a gelatinous acellular membrane located above the hearing sensory epithelium and is composed mostly of type II collagen, and α and β tectorins. TM molecules self-assemble in the endolymph fluid environment, interacting medially with the spiral limbus and distally with the outer hair cell stereocilia. Here, we used immunogold labeling in freeze-substituted mouse cochleae to assess the fine localization of both tectorins in distinct TM regions. We observed that the TM adheres to the spiral limbus through a dense thin matrix enriched in α- and β-tectorin, both likely bound to the membranes of interdental cells. Freeze-etching images revealed that type II collagen fibrils were crosslinked by short thin filaments (4 ± 1.5 nm, width), resembling another collagen type protein, or chains of globular elements (15 ± 3.2 nm, diameter). Gold-particles for both tectorins also localized adjacent to the type II collagen fibrils, suggesting that these globules might be composed essentially of α- and β-tectorins. Finally, the presence of gold-particles at the TM lower side suggests that the outer hair cell stereocilia membrane has a molecular partner to tectorins, probably stereocilin, allowing the physical connection between the TM and the organ of Corti.
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The predominantly afferent innervated inner hair cells (IHC) take the role of a sensory receptor whereas predominantly efferent innervated outer hair cells (OHC) play an important role in the cochlear amplifier [2]. The longest row of stereocilia from the OHC is embedded in the underside of the tectorial membrane [3]. OHC‘s influence the excitability of IHC by modifying the interaction between the tectorial membrane and the reticular lamina system [4].
Similar to other zona pellucida mutations in the alpha-tectorin (TECTA) gene, the p.Y1870C alteration in DFNA8/12 causes prelingual, nonsyndromic, autosomal dominant hearing loss. Here we investigated the effect of p.Y1870C on reverse transduction by audiometric studies in the family.
Pure tone audiometry, brainstem evoked response audiometry, the Freiburger test for speech understanding and transient evoked and distortion product otoacoustic emissions were assessed in three available affected members bearing p.Y1870C.
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The solid component of the tectorial membrane (TM) is a porous matrix made up of the radial collagen fibers and the striated sheet matrix. The striated sheet matrix is believed to contribute to shear impedance in both the radial and longitudinal directions, but the molecular mechanisms involved have not been determined. A missense mutation in Tecta, a gene that encodes for the α-tectorin protein in the striated sheet matrix, causes a 60-dB threshold shift in mice with relatively little reduction in outer hair cell amplification. Here, we show that this threshold shift is coupled to changes in shear impedance, response to osmotic pressure, and concentration of fixed charge of the TM. In Tecta^Y^1870C/+ mice, the tectorin content of the TM was reduced, as was the content of glycoconjugates reacting with the lectin wheat germ agglutinin. Charge measurements showed a decrease in fixed charge concentration from $- 6.4 \pm 1.4$ mmol/L in wild-types to $- 2.1 \pm 0.7$ mmol/L in Tecta^Y^1870C/+ TMs. TMs from Tecta^Y^1870C/+ mice showed little volume change in response to osmotic pressure compared to those of wild-type mice. The magnitude of both radial and longitudinal TM shear impedance was reduced by $10 \pm 1.6$ dB in Tecta^Y^1870C/+ mice. However, the phase of shear impedance was unchanged. These changes are consistent with an increase in the porosity of the TM and a corresponding decrease of the solid fraction. Mechanisms by which these changes can affect the coupling between outer and inner hair cells are discussed.
Col11a2 deletion reveals the molecular basis for tectorial membrane mechanical anisotropy
2009, Biophysical Journal
Citation Excerpt :
However, the extent to which these fibers contribute to TM mechanical properties has not been determined experimentally. The radial collagen fibrils of the TM consist primarily of collagen type II covalently linked to lesser amounts of collagen types IX and XI, and possibly V (14–17). A mouse model of genetic hearing loss allows us to investigate how these radial collagen fibrils affect TM stiffness and cochlear sensitivity.
The tectorial membrane (TM) has a significantly larger stiffness in the radial direction than other directions, a prominent mechanical anisotropy that is believed to be critical for the proper functioning of the cochlea. To determine the molecular basis of this anisotropy, we measured material properties of TMs from mice with a targeted deletion of Col11a2, which encodes for collagen XI. In light micrographs, the density of TM radial collagen fibers was lower in Col11a2 –/– mice than wild-types. Tone-evoked distortion product otoacoustic emission and auditory brainstem response measurements in Col11a2 –/– mice were reduced by 30–50 dB independent of frequency as compared with wild-types, showing that the sensitivity loss is cochlear in origin. Stress-strain measurements made using osmotic pressure revealed no significant dependence of TM bulk compressibility on the presence of collagen XI. Charge measurements made by placing the TM as an electrical conduit between two baths revealed no change in the density of charge affixed to the TM matrix in Col11a2 –/– mice. Measurements of mechanical shear impedance revealed a 5.5 ± 0.8 dB decrease in radial shear impedance and a 3.3 ± 0.3 dB decrease in longitudinal shear impedance resulting from the Col11a2 deletion. The ratio of radial to longitudinal shear impedance fell from 1.8 ± 0.7 for TMs from wild-type mice to 1.0 ± 0.1 for those from Col11a2 –/– mice. These results show that the organization of collagen into radial fibrils is responsible for the mechanical anisotropy of the TM. This anisotropy can be attributed to increased mechanical coupling provided by the collagen fibrils. Mechanisms by which changes in TM material properties may contribute to the threshold elevation in Col11a2 –/– mice are discussed.

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Polypeptide composition of the mammalian tectorial membrane

Abstract

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Biosci. Rep.

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Biochem. J.

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Acta Oto-Laryngol.

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