Significance of tertiary conformation of otoconial matrix proteins - clinical implications.

CONCLUSION: Co-option of the enzyme secretory phospholipase A2 (sPLA2) and adoption of tertiary conformation are essential factors in the multifunctionality of otoconin 90 (OC90) and homologous modulators. OBJECTIVE: To present results of in vitro studies of recombinant otoconial proteins for the understanding of current concepts of biomolecular mechanisms controlling otoconial mineralization. METHODS: In vitro characterization of recombinant otoconial proteins with respect to crystal growth parameters and solution state behavior. Evaluation by HR-SEM, micro-Raman, circular dichroism, in combination with molecular modeling of individual domains and whole OC90. RESULTS: Polymorph selection: recombinant otoconin 22 (rOC22) in vitro selects calcite rather than aragonite, expression of which requires association with an insoluble scaffold most likely provided by Otolin. Alternate folding of rOC22 results in formation of vaterite, the polymorph of primitive fish otoconia and of diseased human otoconia (e.g. Potter's syndrome). Molecular models of OC90 exhibit a surface of uniform negative electrostatic potential, enabling localized supersaturation. We propose that OC90 interacts with Otolin in formation of iso-oriented columns of nano-crystallites, which should ultimately result in assembly of the complex mosaic of native otoconia.

In vitro effects of recombinant otoconin 90 upon calcite crystal growth. Significance of tertiary structure.

Otoconia are biomineral particles of microscopic size essential for perception of gravity and maintenance of balance. Millions of older Americans are affected in their mobility, quality of life and in their health by progressive demineralization of otoconia. Currently, no effective means to prevent or counteract this process are available. Because of prohibitive anatomical and biological constraints, otoconial research is lagging far behind other systems such as bone and teeth. We have overcome these obstacles by generating otoconial matrix proteins by recombinant techniques. In the present study, we evaluated the effects of recombinant Otoconin 90 (OC90), the principal soluble matrix protein upon calcite crystal growth patterns in vitro. Our findings highlight multiple effects, including facilitation of nucleation, and inhibition of crystal growth in a concentration-dependent manner. Moreover, OC90 induces morphologic changes characteristic of native otoconia. OC90 is considerably less acidic than the prototypical invertebrate CaCO(3) -associated protein, but is nevertheless an effective modulator of calcite crystal growth. Based on homology modeling of the sPLA2-like domains of OC90, we propose that the lower density of acidic residues of the primary sequence is compensated by formation of major anionic surface clusters upon folding into tertiary conformation.

Inner ear proteomics of mouse models for deafness, a discovery strategy.

Inner ear dysfunction is often associated with defective hair cells. Therefore, hair cells are the focus of study in many of the mouse mutants showing auditory and vestibular deficits. However, harvesting sufficient numbers of hair cells from the tiny bony mouse inner ear for proteomic analysis is challenging. New approaches that would take advantage of mouse mutants and avoid processing steps, such as decalcification or microdissetion, would be more suitable for proteomic analysis. Here, we propose a novel approach called SSUMM-Subtractive Strategy Using Mouse Mutants. SSUMM takes advantage of the differences between control and affected or mutant samples. We predict that SSUMM would be a useful method in proteomics, especially in those cases in which the investigator must work with small numbers of diverse cell types from a tiny organ. Here, we discuss the potential utility of SSUMM to unravel the protein expression profiles of hair cells using the Pou4f3 mouse mutant as an example. Pou4f3 mutant mice exhibit a total loss of inner and outer hair cells, but supporting cells remain relatively intact in the cochlea, thus providing an excellent model for identifying proteins and transcripts that are specific to the hair cell at all life stages. SSUMM would maximize the sensitivity of the analyses while obviating the need for tedious sessions of microdissection and collection of hair cells. By comparing the mutant to control ears at specific time points, it is possible to identify direct targets of a gene product of interest. Further, SSUMM could be used to identify and analyze inner ear development markers and other known genes/proteins that are coexpressed in the ear. In this short technical report, we also discuss protein-profiling approaches suitable for SSUMM and briefly discuss other approaches used in the field of proteomics.

Microscale analysis of proteins in inner ear tissues and fluids with emphasis on endolymphatic sac, otoconia, and organ of Corti.

Here we describe preparatory techniques adapted for the study of proteins of inner ear tissues and fluids that have allowed us to apply state-of-the-art analytical techniques in spite of the minute size and anatomical complexities of this organ. Illustrative examples address unresolved issues of functional and clinical significance. First, we demonstrate how quick-freezing and freeze drying prevents artifacts that arise from sampling endolymphatic sac (ES) content in the liquid state. This set the stage for the generation of the first protein profile of the ES. Identification of crucial proteins will help elucidate mechanisms of endolymph volume regulation and pathogenesis of Meniere's disease. Second, we show how a unique situation allowed identification of otoconial proteins by mass spectrometric analysis without prior separation and we discuss possible roles for these minor otoconins in otoconial development and prevention of degenerative diseases that affect balance. Finally, we demonstrate techniques for the precise dissection of organ of Corti and its substructures, while preserving their near normal chemical state. We extended an earlier study in which we identified a novel calcium-binding protein by IEF, oncomodulin, localized in the outer hair cells and show here the applicability of prefractionation for the screening of calcium-binding proteins of organ of Corti. These studies demonstrate how advanced preparatory and analytical techniques can be applied to studies of the inner ear.

Inner ear proteomics: a fad or hear to stay.

Proteomics, the large-scale analysis of the structure and function of proteins, as well as of protein-protein interactions, has evolved into a major component of 'systems analysis'. This requires the integration of information from different sources and at multiple levels, and involves two distinct parameters, (1) high-throughput protein separation, identification, and characterization, and (2) the extension of the obtained analytical data for the determination of the physiological function. The inner ear poses exceptional challenges to the study of proteomics because of its minute size, poor accessibility, association with complex fluid spaces, and diversity of cell types. Various approaches to the study of proteomics of the inner ear are presented, and success stories, noteworthy failures and what lies ahead, will be discussed.

Mixing model systems: using zebrafish and mouse inner ear mutants and other organ systems to unravel the mystery of otoconial development.

Human vestibular dysfunction is an increasing clinical problem. Degeneration or displacement of otoconia is a significant etiology of age-related balance disorders and Benign Positional Vertigo (BPV). In addition, commonly used antibiotics, such as aminoglycoside antibiotics, can lead to disruption of otoconial structure and function. Despite such clinical significance, relatively little information has been compiled about the development and maintenance of otoconia in humans. Recent studies in model organisms and other mammalian organ systems have revealed some of the proteins and processes required for the normal biomineralization of otoconia and otoliths in the inner ear of vertebrates. Orchestration of extracellular biomineralization requires bringing together ionic and proteinaceous components in time and space. Coordination of these events requires the normal formation of the otocyst and sensory maculae, specific secretion and localization of extracellular matrix proteins, as well as tight regulation of the endolymph ionic environment. Disruption of any of these processes can lead to the formation of abnormally shaped, or ectopic, otoconia, or otoconial agenesis. We propose that normal generation of otoconia requires a complex temporal and spatial control of developmental and biochemical events. In this review, we suggest a new hypothetical model for normal otoconial and otolith formation based on matrix vesicle mineralization in bone which we believe to be supported by information from existing mutants, morphants, and biochemical studies.

Molecular mechanisms underlying ectopic otoconia-like particles in the endolymphatic sac of embryonic mice.

Otoconin-90, the principal otoconial matrix protein, provided a tool to investigate the molecular mechanism of otoconial morphogenesis. The endolymphatic sac of the embryonic chick and guinea pig contain otoconia. Here, we show that the embryonic mouse transiently expresses ectopic otoconia in the endolymphatic sac. Massive precipitate of otoconin-90-positive material is detectable in the lumen of the endolymphatic sac between embryonic day 14.5 and 17.5 with frequent accretion into more heavily staining otoconia-like particles. Otoconin-90 was also localized at the surface and the interior of epithelial cells lining the endolymphatic sac as well as incorporated into free floating cells. In contrast, in situ hybridization failed to detect mRNA in the endolymphatic duct and sac, even though the adjacent nonsensory vestibular structures are heavily stained. Because of ample expression of otoconin-90 protein in the absence of the corresponding mRNA, we conclude that the luminal otoconin-90 is imported via longitudinal flow from the vestibular compartments, where both mRNA and protein are strongly expressed. Because of absence of mRNA, the expression of the corresponding protein by the epithelia lining the endolymphatic sac can only be explained by a resorptive process, as previously proposed on the basis of the movement of luminal macromolecules. The data do not support the previous hypothesis that the transient expression of otoconia-like particles of the endolymphatic sac represents a vestigial phenomenon from the amphibian stage, since amphibia express ample mRNA encoding otoconin-22 in the endolymphatic sac system.

The cochlear F-box protein OCP1 associates with OCP2 and connexin 26.

OCP1 and OCP2 are the most abundant proteins in the organ of Corti. Their distributions map identically to the epithelial gap-junction system, which unites the supporting cell population. Sequence data imply that OCP1 and OCP2 are subunits of an SCF E3 ubiquitin ligase. Consistent with that hypothesis, electrophoretic mobility-shift assays and pull-down assays with immobilized OCP1 demonstrate the formation of an OCP1-OCP2 complex. Sedimentation equilibrium data indicate that the complex is heterodimeric. The coincidence of the OCP1-OCP2 distribution and the epithelial gap-junction system suggests that one or more connexin isoforms may be targets of an SCF(OCP1) complex. Significantly, immobilized OCP1 binds (35)S-labeled connexin 26 (Cx26) produced by in vitro transcription-translation. Moreover, Cx26 can be co-immunoprecipitated from extracts of the organ of Corti by immobilized anti-OCP1, implying that OCP1 and Cx26 may associate in vivo. Given that lesions in the Cx26 gene (GJB2) are the most common cause of hereditary deafness, the OCP1-Cx26 interaction has substantial biomedical relevance.

Toward an understanding of cochlear homeostasis: the impact of location and the role of OCP1 and OCP2.

The central role of the supporting cell population, or epithelial support complex (ESC), in cochlear homeostasis has gained general acceptance. That the details of this role may vary markedly with location, however, remains poorly appreciated. For example, the K+ recirculation pathway may well be dictated by position along the cochlear axis: a perilymphatic route near the apex and a transcellular one near the base. The ESC expresses very high levels of OCP1 and OCP2, now known to be components of a novel, organ of Corti (OC)-specific SCF ubiquitin ligase (SCF(OCP1)). In the SCF(OCP1) cnmplex, OCP1 presumably binds selected protein targets, positioning them for ubiquitination. The recent demonstration that recombinant OCP1 interacts non-covalently with Cx26 suggests that the connexins may be target proteins for SCF(OCP1). Although ubiquitination has classically been viewed as a signal for subsequent destruction by the 26S proteasome, the energy-limited state of the OC prompts consideration of alternative fates, e.g. reversible internalization. The ESC also expresses several components of the Wingless/Wnt signaling pathway. Significantly, two of the gap-junction proteins expressed in the OC, Cx43 and Cx30, are known targets of the Wnt pathway. On the basis of these observations, a working hypothesis is proposed wherein the Wnt pathway activates connexin expression, while OCP1 regulates its degradation.

Non-syndromic vestibular disorder with otoconial agenesis in tilted/mergulhador mice caused by mutations in otopetrin 1.

Otoconia are biominerals within the utricle and saccule of the inner ear that are critical for the perception of gravity and linear acceleration. The classical mouse mutant tilted (tlt) and a new allele, mergulhador (mlh), are recessive mutations that affect balance by impairing otoconial morphogenesis without causing collateral deafness. The mechanisms governing otoconial biosynthesis are not known. Here we show that tlt and mlh are mutant alleles of a novel gene (Otopetrin 1, Otop1), encoding a multi-transmembrane domain protein that is expressed in the macula of the developing otocyst. Both mutants carry single point mutations leading to non-conservative amino acid substitutions that affect two putative transmembrane (TM) domains (tlt, Ala(151)-->Glu in TM3; mlh, Leu(408)-->Gln in TM8). Otop1 and Otop1-like paralogues, Otop2 and Otop3, define a new gene family with homology to the C. elegans and D. melanoganster DUF270 genes.

OCP2 immunoreactivity in the human fetal cochlea at weeks 11, 17, 20, and 28, and the human adult cochlea.

The two most abundant proteins of the organ of Corti, OCP1 and OCP2, are acidic, cytosolic, low molecular weight proteins diffusely distributed within the cytoplasm of supporting cells. A recent study by Henzl et al. (2001) found first, that these two proteins co-localize with connexin 26 along the epithelial gap junction system and second, that OCP2 could participate with OCP1 in an organ of Corti-specific SCF complex (Skp1, cul1in, and Fbp), a ubiquitin ligase complex. Previous study has also implicated OCP2 in the recycling and regulation of intracellular K(+) efflux as well as pH homeostatic mechanisms. In the present study, we document the emergence and distribution features of OCP2 through various stages (weeks 11-28) of gestation in human fetal cochleae. Four fetal cochleae, the cochleae of a normal hearing human adult and a mature rat for positive control were fixed in 4% formalin within 2 h post mortem. Immunohistochemical studies were performed using a rabbit polyclonal antibody raised against a synthetic peptide corresponding to amino acids 3-16. Specimens were mounted in paraffin sections. Results show that OCP2 immunoreactivity is evident at a prenatal age of 11 weeks, peaks in expression at the onset of cochlear function at 20 weeks and achieves adult-like patterns of distribution just prior to histological maturation at 28 weeks. Though this protein could be associated with the development, maturation, and electrochemical maintenance of the cochlear gap junction system, the nature of this protein's function in the developing and mature human cochlea remains unclear.