Otolith Protein Research Articles

Qualitative Shotgun Proteomics Strategy for Protein Expression Profiling of Fish Otoliths

Despite decades of research on fish otoliths and their capacity to serve as biochronological recorders, much remains unknown about their protein composition, the mechanisms by which proteins are incorporated into the otolith matrix, or the potential for using otolith proteins to provide insight into aspects of fish life history. We examined the protein composition of Atlantic cod (Gadus morhua) otoliths using a state-of-the-art shotgun proteomics approach with liquid chromatography coupled to an electrospray ionization-orbitrap tandem mass spectrometer. In addition to previously known otolith matrix proteins, we discovered over 2000 proteins not previously identified in cod otoliths and more than 1500 proteins not previously identified in any fish otoliths. These included three novel proteins (Somatolactin, F-actin-capping protein subunit beta, Annexin) primarily involved in binding calcium ions and likely mediating crystal nucleation. However, most of the otolith proteins were not necessarily related to otolith formation but rather to other aspects of fish physiology. For example, we identified sex-related biomarkers for males (SPATA6 protein) and females (Vitellogenin-2-like protein). We highlight some noteworthy classes of proteins having diverse functions; however, the primary goal here is not to discuss each protein separately. The number and diverse roles of the proteins discovered in the otoliths suggest that proteomics could reveal critical life history information from archived otolith collections that could be invaluable for understanding aspects of fish biology and population ecology. This proof-of-concept methodology paper provides a novel methodology whereby otolith proteomics can be further explored.

Downregulation of VDR in benign paroxysmal positional vertigo patients inhibits otolith‑associated protein expression levels.

Benign paroxysmal positional vertigo (BPPV) is the most common peripheral vertigo‑associated disease. Vitamin D (VD) helps maintain normal otolith function and may be associated with BPPV. VD exerts its biological functions primarily via the VD receptor (VDR). The present study demonstrated that serum VD levels were significantly decreased in patients with BPPV compared with in controls. VDR, otolith‑associated protein otoconin‑90 (OC90) and NADPH oxidase 3 (NOX3) expression levels were also significantly decreased in patients with BPPV compared with in controls. Furthermore, a positive correlation was observed between VD levels and VDR expression. Receiver operating characteristic curve analysis identified VDR expression levels as a potential diagnostic marker for BPPV. OC90 and NOX3 expression levels were notably lower in the inner ear tissue of VDR knockout mice compared with in those of wild‑type mice. In mice overexpressing VDR, OC90 and NOX3 were also overexpressed. Following intravenous injection of VD in VDR knockout mice, expression levels of OC90 and NOX3 were not significantly different from those in VDR knockout mice injected with saline. This indicated that VDR may be underexpressed in patients with BPPV and was associated with the expression levels of otolith‑associated proteins. Moreover, VDR mediated VD activation, leading to otolith protein formation. The present study provided a novel theoretical basis for BPPV onset that may facilitate the development of more effective diagnostic and treatment options.

In vivo and in vitro analysis of starmaker activity in zebrafish otolith biomineralization.

Otoliths are one of the biominerals whose formation is highly controlled by proteins. The first protein discovered to be involved in otolith biomineralization in zebrafish was starmaker (Stm). Previously, Stm was shown to be responsible for the preferential formation of aragonite, a polymorph of calcium carbonate, in otoliths. In this work, proteomic analysis of adult zebrafish otoliths was performed. Stm is the only highly phosphorylated protein found in our studies. Besides previously studied otolith proteins, we discovered several dozens of unknown proteins that reveal the likely mechanism of biomineralization. A comparison of aragonite and vaterite otoliths showed similarities in protein composition. We observed the presence of Stm in both types of otoliths. In vitro studies of 2 characteristic Stm fragments indicated that the DS-rich region has a special biomineralization activity, especially after phosphorylation.-Kalka, M., Markiewicz, N., Ptak, M., Sone, E. D., Ożyhar, A., Dobryszycki, P., Wojtas, M. In vivo and in vitro analysis of starmaker activity in zebrafish otolith biomineralization.

Effects of temperature on tissue–diet isotopic spacing of nitrogen and carbon in otolith organic matter

Reconstruction of the trophic position of a fish can be performed by analysing stable nitrogen and carbon isotopes in otolith protein. However, ambient temperature may affect the tissue–diet isotopic spacing of stable isotopes from diet to predator tissue and bias estimates of trophic position. To test this, otolith protein, heart and muscle tissue from a rearing experiment with juvenile cod held at different temperatures (4, 7, 10 and 14°C) were analysed. There was no significant effect of temperature on otolith δ15N, whereas muscle and heart exhibited a slight decrease in δ15N values with increasing temperature corresponding to maximum of 0.6‰ over the 10°C range. By contrast, the otolith protein δ13C values at 4 and 7°C were significantly higher than for 10 and 14°C, suggesting an approximate 1‰ increased tissue–diet enrichment at the lower temperatures. Temperature had no significant effect on muscle and heart δ13C values. Considering the annual mean variation in ocean temperatures, our results indicate that the trophic signals recorded in the otoliths will reflect changes in diet isotope values with little bias from the ambient temperature experienced by the fish.

Not All Inner Ears are the Same: Otolith Matrix Proteins in the Inner Ear of Sub-Adult Cichlid Fish, Oreochromis Mossambicus, Reveal Insights Into the Biomineralization Process.

The fish ear stones (otoliths) consist mainly of calcium carbonate and have lower amounts of a proteinous matrix. This matrix consists of macromolecules, which directly control the biomineralization process. We analyzed the composition of this proteinous matrix by mass spectrometry in a shotgun approach. For this purpose, an enhanced protein purification technique was developed that excludes any potential contamination of proteins from body fluids. Using this method we identified eight proteins in the inner ear of Oreochromis mossambicus. These include the common otolith matrix proteins (OMP-1, otolin-1, neuroserpin, SPARC and otoconin), and three proteins (alpha tectorin, otogelin and transferrin) not previously localized to the otoliths. Moreover, we were able to exclude the occurrence of two matrix proteins (starmaker and pre-cerebellin-like protein) known from other fish species. In further analyses, we show that the absence of the OMP starmaker corresponds to calcitic otoliths and that pre-cerebellin-like protein is not present at any stage during the development of the otoliths of the inner ear. This study shows O. mossambicus does not have all of the known otolith proteins indicating that the matrix proteins in the inner ear of fish are not the same across species. Further functional studies of the novel proteins we identified during otolith development are required.

Spatial Expression of Otolith Matrix Protein-1 and Otolin-1 in Normally and Kinetotically Swimming Fish.

Kinetosis (motion sickness) has been repeatedly shown to affect some fish of a given clutch following the transition from 1g to microgravity or from hypergravity to 1g. This susceptibility to kinetosis may be correlated with irregular inner ear otolith growth. Otoliths are mainly composed of calcium carbonate and matrix proteins, which play an important role in the process of otolith mineralization. Here, we examine the morphology of otoliths and the expression pattern of the major otolith proteins OMP-1 and otolin-1 in a series of hypergravity experiments. In the utricle, OMP-1 is present in centripetal (medial) and centrifugal (lateral) regions of the meshwork area. In the saccule, OMP-1 was expressed within a dorsal and a ventral narrow band of the meshwork area opposite to the periphery of the sulcus acusticus. In normal animals, the spatial expression pattern of OMP-1 reaches more posteriorly in the centrifugal aspect and is considerably broader in the centripetal portion of the utricle compared to kinetotic animals. However, otolin-1 was not expressed in the utricule. In the saccule, no differences were observed for either gene when comparing normal and kinetotically behaving fish. The difference in the utricular OMP-1 expression pattern between normally and kinetotically swimming fish indicates a different otolith morphology and thus a different geometry of the otoliths resting on the corresponding sensory maculae. As the utricle is the endorgan responsible for sensing gravity, the aberrant morphology of the utricular otoliths, based on OMP-1 expression, likely leads to the observed kinetotic behavior.

The Role of the C1q Domain of Zebrafish Otolin 1a in Otolith Morphogenesis

Otolin1 is an extracellular matrix protein of otoliths (“ear stones” of fishes), and otoconia (“ear dust” of higher vertebrates). These acellular biominerals are essential for the sense of balance; dislodging results in the most common human balance disorder, benign paroxysmal positional vertigo. Otolin1 comprises of a collagen and a C1q domain, similar to atypical collagens VIII and X. We propose that Otolin1 forms a scaffold to which other otolith proteins, Ca2+ and CO32– ions bind during otolith morphogenesis, with C1q trimers as hubs and collagen triple-helices as spokes. To test this model, we investigated whether the Otolin1 C1q domain is necessary and sufficient to form trimers and higher complexes in vitro. The zebrafish Otolin1a C1q domain was expressed in bacteria either alone or fused to thioredoxin. Multimerization of affinity purified recombinant proteins was assessed by size-exclusion chromatography and gel electrophoresis. Under denaturing conditions, recombinant C1q proteins formed monomers and trimers, but not dimers or higher-order complexes. Under native conditions, they formed only higher-order complexes. In contrast, thioredoxin alone only appeared as a monomer. Our results are consistent with the proposed ability of the Otolin C1q domain to form trimers that, in turn, assemble into higher-order complexes. Experiments are underway to characterize the various complexes in more detail and to test whether the C1q domain is necessary to trimerize of full-length Otolin1a. (Supported by NIH R01DC006962)

Sparc Protein Is Required for Normal Growth of Zebrafish Otoliths

Otoliths and the homologous otoconia in the inner ear are essential for balance. Their morphogenesis is less understood than that of other biominerals, such as bone, and only a small number of their constituent proteins have been characterized. As a novel approach to identify unknown otolith proteins, we employed shotgun proteomics to analyze crude extracts from trout and catfish otoliths. We found three proteins that had not been associated previously with otolith or otoconia formation: 'Secreted acidic cysteine rich glycoprotein' (Sparc), an important bone protein that binds collagen and Ca(2+); precerebellin-like protein, which contains a C1q domain and may associate with the collagenous otolin-1 during its assembly into a framework; and neuroserpin, a serine protease inhibitor that may regulate local protease activity during framework assembly. We then used the zebrafish to investigate whether Sparc plays a role in otolith morphogenesis. Immunodetection demonstrated that Sparc is a true constituent of otoliths. Knockdown of Sparc expression in morphant zebrafish resulted in four principal types of defective otoliths: smaller, extra and ectopic, missing and fused, or completely absent. Smaller size was the predominant phenotype and independent of the severity of otic-vesicle defects. These results suggested that Sparc is directly required for normal otolith growth.

Otolith accretion rates: Does size really matter?

This study examined the relationship between otolith size and growth in juvenile cod ( Gadus morhua L.). Two groups of juvenile cod were reared under different food ration and temperature regimes to obtain fish of similar somatic size but with different sized otoliths. The two groups were subjected to alternating temperature regimes and intermediate ration levels. Large otoliths grew significantly faster than the small ones and variation between individuals was extensive. The ratio of otolith growth during cold and warm temperature exposure did not differ between groups, and the observed growth pattern is therefore not attributable to differential growth within individual temperature periods. The ratio decreased with otolith size, presumably as a result of ontogenetic decrease in otolith protein composition. These results suggest that processes coupled to the metabolic rate of the endolymphatic epithelium are the key driver behind otolith growth.

Transition metal binding to cod otolith proteins

Otolith microchemistry can be very useful in identifying fish populations and reconstructing fish movements. Recent attempts have been made to evaluate otoliths as proxies of ambient levels of transition metals, but findings have been inconsistent. Some of the difficulty with obtaining a definitive answer stems from an incomplete understanding of the biological control of transition metal speciation in otoliths. Metals may be incorporated as part of the calcium carbonate phase, trapped in interstitial spaces within the crystal, or associated with the protein matrix. Metal binding to the protein phase may be inferred from its structural and biochemical properties but has not been observed previously. Inherent difficulties with the extraction of metal-binding proteins in their native state from the calcium carbonate phase make them extraordinarily difficult to measure. We have developed a method that facilitates the extraction of otolith proteins without total disruption of transition metal binding. Chelating agents such as EDTA, used in the decalcification of otoliths, can demetallate the proteins if allowed to reach equilibrium; however, if the reaction is halted prior to equilibration, intact metal–protein complexes can be obtained. Using such an approach, we have confirmed the presence of copper and zinc in the soluble portion of the protein matrix of cod otoliths, and we have established that between 70% and 100% of copper and 40% to 60% of zinc found in whole otoliths are associated with the soluble part of the protein matrix. Manganese was not observed to be associated with the protein, indicating that it is either weakly bound or that no binding is present. Our results, combined with an understanding of the biological control of these metals, suggest that otoliths are not likely to be reliable indicators of copper and zinc exposure, but they may provide useful insight into fish growth and physiological development.

Analysis of the soluble matrix of vaterite otoliths of juvenile herring ( Clupea harengus): do crystalline otoliths have less protein?

Otoliths are calcium carbonate concretions laid down in the inner ear of fish and used in fish age estimation. Otoliths precipitate in the form of aragonite but aberrant precipitation may result in vaterite formation instead of aragonite. Vaterite otoliths are more translucent than aragonite. The quantity of HCl-soluble proteins (SP) was measured in the vaterite otoliths and their aragonite pairs of one year old reared herring Clupea harengus to assess the changes induced by the precipitation of vaterite in the amount of soluble proteins in the otolith. Results showed that vaterite otoliths had as much soluble proteins as their aragonite pairs ( p>0.05). Due to the lower density of the vaterite, vaterite otoliths were lighter than their aragonite pairs ( p<0.05) which explained that protein concentrations were significantly higher ( p<0.05) than in aragonite otoliths. These results indicate that the precipitation of vaterite in otoliths did not affect the inclusion of soluble proteins. Furthermore, they suggest that soluble proteins do not affect the translucent or opaque appearance of otoliths. Differences in translucency may instead be caused by the amounts of insoluble proteins or by differences in the physical properties of proteins. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the otolith proteins revealed two bands at 50 and 62 kDa in both aragonite and vaterite otoliths suggesting that the precipitation of vaterite in the otolith is not controlled by either of these two proteins present in the otolith.

Effect of age and temperature on amino acid composition and the content of different protein types of juvenile Atlantic cod (Gadus morhua) otoliths

The purpose of this study was to analyse the amino acid composition of otolith matrix protein, estimate the proportion of the water-soluble protein fraction, and analyse the effect of matrix composition on otolith visual appearance. Juvenile Atlantic cod (Gadus morhua) were reared under constant temperature and feeding conditions and sampled at the beginning and the end of the experiment. The amino acid composition was dominated by asparagine, glutamic acid, leucine, serine, and proline. A change in amino acid composition was observed with increasing temperature and time, caused by changing proportions of the water-soluble and -insoluble protein fractions. Feeding level had no effect. The relative content of water-soluble protein was linearly related to fish dry weight and temperature. Otolith opacity, defined as the percentage of incident light absorbed by an otolith section, did not differ significantly between experimental treatments. The soluble protein fraction had a positive, albeit insignificant, correlation with opacity. Using opacity and otolith volume, deposited total otolith protein content was estimated with an R2 of 0.91, where otolith volume alone explained 83% of the observed variation.

Atlantic cod (Gadus morhua) growth and otolith accretion characteristics modelled in a bioenergetics context

The purpose of this work is to present a conceptual model for fish otolith growth in which somatic growth is related to otolith growth and opacity. The model is based on known mechanisms of CaCO3 and protein incorporation into the otolith. Model parameters were derived from laboratory experiments and from the literature. A sensitivity analysis showed that the model was robust to measurement errors in most input parameters. The most sensitive parameters were the exponents of the otolith length–weight and otolith protein – whole-body protein synthesis relationships and the proportionality constant between metabolism and otolith growth rate. Application to experimental data resulted in good agreement between back-calculated and observed fish sizes. In the growth experiment, the average back-calculated weights were slightly lower than the average observed weights, but the correlation was highly significant. In the starvation experiment, the back-calculated weights were also highly correlated with observed weights, with slightly declining residuals with fish size. Unlike previous back-calculation methods, this model has the ability to detect periods of starvation and estimate growth histories in both growing and starving fish.

Thyroid hormones are necessary for teleostean otolith growth

Experiments were conducted to understand the effects of thyroid hormones and thiourea (TU) on otolith growth during metamorphosis of leptocephali. TU is an anti-thyroid hormone drug that inhibits the production of thyroxine (T 4 ) and triiodothyronine (T 3 ) in the thyroid tissue. Fully grown leptocephali of tarpon Megalops cyprinoides collected at the mouth of the Gong-Shy-Tyan Brook, N Taiwan, were immediately treated with 10 ppb T 3 and 300 ppm TU for the experimental period of 13 d. The newly grown otolith lengths measured from the maximum radius were 77.0 ± 12.0, 91.9 ± 7.2 and 47.9 ± 6.3 pm in the control, T 3 and TU groups, respectively. The newly grown otolith length of the T 3 group was significantly longer than those of the control and TU groups, and the control group had significantly larger length values than the TU group. The T 3 treatment stimulated otolith growth, increasing daily increments from 7 to 13 cm (control) to 9 to 16 pm (T 3 ) during the growth peak of metamorphosis. Maximum daily increments were only 6 to 10 pm in TU-treated tarpons. The otoliths of the fish treated with T 3 and TU had increments deposited daily. Otoliths of T 3 -treated and control fish grew throughout the experimental period, but otoliths of TU-treated fish stopped growth beginning on Day 6 to Day 9. These results suggest that thyroid hormones are essential for otolith growth, probably by regulating the expressions and functions of the otolith protein matrix and ion transporters in the saccular epithelium.