Characterization of otoconin-95, the major protein of murine otoconia, provides insights into the formation of these inner ear biominerals

Abstract

During the course of a study aimed at identifying inner ear-specific transcripts, a 1,906-bp murine cDNA predicted to encode a secreted 469-aa protein with two domains of homology with the secreted phospholipases A 2 was isolated. This transcript is specifically expressed in the inner ear from embryonic day 9.5. The encoded 95-kDa glycoprotein is the major protein of the utricular and saccular otoconia and thus was named otoconin-95. By immunohistof luorescence, otoconin-95 also was detected in the cupulae of the semicircular canals and in previously undescribed transient granular structures of the cochlea. Otoconin-95 was found to be synthesized by various nonsensory cell types, but not by the supporting cells of the sensory epithelia, which produce the otoconial precursor vesicles. In addition, multiple isoforms generated by differential splicing were observed in different combinations during development. Based on the present results, we propose a model for the formation of the otoconia.The mammalian inner ear is composed of two sensory organs: the cochlea, which is the auditory component, and the vestibule, responsible for the control of balance. The latter consists of the saccule and the utricle, which detect linear acceleration, and the three semicircular canals sensitive to angular acceleration. Each sensory epithelium is covered by an acellular gelatinous membrane, namely the tectorial membrane in the cochlea, the cupulae in the ampullae of the semicircular canals, and the otoconial membranes in the saccule and the utricle. The displacement of the acellular membrane relative to the sensory epithelia leads to the deflection of the stereocilia of sensory hair cells, which in turn opens the mechanotransduction channels, leading to cell depolarization.The otoconial membranes of the saccule and utricle are overlayed with dense biominerals made of a filamentous organic matrix and calcium carbonate (CaCO 3 ). These biominerals are found as large deposits (otoliths) in most fish and as numerous small crystals (otoconia) in all other vertebrates. Otoliths and otoconia have been classified in three groups according to the crystalline form of CaCO 3 , i.e., vaterite, aragonite, or calcite. In mammals, otoconia are calcitic, whereas in amphibians they are calcitic in the utricle and aragonitic in the saccule (1-4). Each type of otoconia is characterized by a distinct set of proteins collectively named otoconins (3). The major protein of aragonitic otoconia has been purified from Xenopus laevis saccule and sequenced (5). In contrast, none of the otoconins of the calcitic otoconia have been characterized so far. In mice, the rate of otoconia production is highest between embryonic days 15 and 17 (E15-E17), although the biominerals continue to grow until postnatal day 7 (P7) (6, 7). Different hypotheses for the genesis of otoconia have been proposed (reviewed in ref. 8). It is now generally accepted that otoconia are produced from protrusions of the saccular and utricular supporting cells that form vesicular str...

Osteopontin is not Critical for Otoconia Formation or Balance Function

“…Interestingly, OPN and Oc90 are co-expressed in vestibular dark cells and the endolymphatic sac, with the onset of Oc90 expression being much earlier than that for OPN (Verpy et al 1999;Sakagami 2000;Ignatova et al 2004). Given the negligible role of OPN in otoconia formation, such cellular co-localization may be coincidental or may merely reflect cellular activities in secretion and/or ion fluxes, two processes that may provide some components for otoconia formation or maintain homeostasis of the endolymph.…”

“…Both types of matrices contain sulfated glycosaminoglycans (as GAGs or proteoglycans), osteopontin (OPN), fetuin-A, and members of the collagen X family (collagen X in bone and otolin in otoconia; Lundberg et al 2006;Zhao et al 2007). The fourth known otoconia component protein, Oc90, is the predominant otoconial matrix protein (Wang et al 1998;Verpy et al 1999) whose role in bone formation has yet to be studied.…”

Molecular Genetics of Vestibular Organ Development

“…Given the anatomical differences between the maculae and cristae, it is surprising that, besides NT-3, no other genes have been reported to be differentially expressed in the maculae and not the cristae. Even otoconin-95, a major component of the otoconia, is not restricted to the utricle and saccule but rather broadly expressed in the nonsensory regions of the inner ear (Verpy et al 1999). However, several genes, although not exclusively expressed in the utricle or saccule, such as Otx1, Otx2, Hmx2 and Hmx3, and Ngn1, when knocked out resulted in an incomplete separation of the utricle and saccule that often affected the development of the two maculae (Wang et al 1998;Cantos et al 2000;Ma et al 2000).…”

Interfacial Kinetic and Binding Properties of the Complete Set of Human and Mouse Groups I, II, V, X, and XII Secreted Phospholipases A2

“…Many of these sPLA 2 s were identified in the past few years, and studies are under way to determine their physiological functions. A catalytically inactive sPLA 2 -like protein called otoconin-95 was also identified in mice and humans (7,8). The different sPLA 2 paralogs are not closely related isoforms because the amino acid identity between any two is in the range of Ͻ15-50%.…”