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Matrix recruitment and calcium sequestration for spatial specific otoconia development.

Yang H, Zhao X, Xu Y, Wang L, He Q, Lundberg YW - PLoS ONE (2011)

Bottom Line: In vivo, the presence of Oc90 in wildtype (wt) mice leads to an enrichment of Ca(2+) in the luminal matrices of the utricle and saccule, whereas absence of Oc90 in the mice leads to drastically reduced matrix-Ca(2+).Molecular modeling and sequence analysis predict structural features that may underlie the interaction and Ca(2+)-sequestering ability of these proteins.Together, the data provide a mechanism for the otoconial matrix assembly and the role of this matrix in accumulating micro-environmental Ca(2+) for efficient CaCO(3) crystallization, thus uncover a critical process governing spatial specific otoconia formation.

View Article: PubMed Central - PubMed

Affiliation: Vestibular Neurogenetics Laboratory, Boys Town National Research Hospital, Omaha, Nebraska, United States of America.

ABSTRACT
Otoconia are bio-crystals anchored to the macular sensory epithelium of the utricle and saccule in the inner ear for motion sensing and bodily balance. Otoconia dislocation, degeneration and ectopic calcification can have detrimental effects on balance and vertigo/dizziness, yet the mechanism underlying otoconia formation is not fully understood. In this study, we show that selected matrix components are recruited to form the crystal matrix and sequester Ca(2+) for spatial specific formation of otoconia. Specifically, otoconin-90 (Oc90) binds otolin through both domains (TH and C1q) of otolin, but full-length otolin shows the strongest interaction. These proteins have much higher expression levels in the utricle and saccule than other inner ear epithelial tissues in mice. In vivo, the presence of Oc90 in wildtype (wt) mice leads to an enrichment of Ca(2+) in the luminal matrices of the utricle and saccule, whereas absence of Oc90 in the mice leads to drastically reduced matrix-Ca(2+). In vitro, either Oc90 or otolin can increase the propensity of extracellular matrix to calcify in cell culture, and co-expression has a synergistic effect on calcification. Molecular modeling and sequence analysis predict structural features that may underlie the interaction and Ca(2+)-sequestering ability of these proteins. Together, the data provide a mechanism for the otoconial matrix assembly and the role of this matrix in accumulating micro-environmental Ca(2+) for efficient CaCO(3) crystallization, thus uncover a critical process governing spatial specific otoconia formation.

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Co-immunoprecipitation (co-IP) of Oc90 with otolin or its domains.HEK293 cells were transfected with full-length Oc90-FLAG, otolin and domains of otolin. Shown in (B, C) are Oc90-FLAG and otolin detected by immunostaining. In (A), protein products were co-immunoprecipitated using anti-FLAG agarose beads. The lanes are co-IP with: 1) empty vectors, 2) full-length (FL) otolin, 3) otolin-TH domain, and 4) otolin-C1q domain.
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pone-0020498-g001: Co-immunoprecipitation (co-IP) of Oc90 with otolin or its domains.HEK293 cells were transfected with full-length Oc90-FLAG, otolin and domains of otolin. Shown in (B, C) are Oc90-FLAG and otolin detected by immunostaining. In (A), protein products were co-immunoprecipitated using anti-FLAG agarose beads. The lanes are co-IP with: 1) empty vectors, 2) full-length (FL) otolin, 3) otolin-TH domain, and 4) otolin-C1q domain.

Mentions: To test whether Oc90 interacts with otolin to form the organic matrix of otoconia and to identify the interacting domains, we performed co-immunoprecipitation (IP). Transfection of HEK293 cells with expression constructs containing full-length Oc90 (Oc90-FLAG), full-length and individual domains of otolin (TH and C1q) yielded high expression efficiency (Figure 1B, 1C). Protein products were co-precipitated using anti-FLAG agarose beads and washed under stringent conditions as described in Materials and Methods. Constituents eluted from the beads were analyzed by Western blotting after separation by 4–15% SDS-PAGE (Figure 1A). Antibodies against the N-terminal regions of Oc90 and otolin [10] were used for detection. Oc90 interacts strongly with full-length otolin (lane 2 in Figure 1A), and both domains of otolin participate synergistically in this interaction (lanes 3 and 4 in Figure 1A). The requirement of both otolin domains is consistent with the finding that the C1q domain of other related proteins is responsible for partner recognition and the TH domain for strengthening the interaction [22]. Both the TH and C1q domains have a high affinity for proteoglycans and Ca2+ as well [39]. As is the case with the sPLA2-like domains in Oc90 [8]; [9], the functional domains of otolin (TH and C1q) are extremely conserved throughout evolution [32].


Matrix recruitment and calcium sequestration for spatial specific otoconia development.

Yang H, Zhao X, Xu Y, Wang L, He Q, Lundberg YW - PLoS ONE (2011)

Co-immunoprecipitation (co-IP) of Oc90 with otolin or its domains.HEK293 cells were transfected with full-length Oc90-FLAG, otolin and domains of otolin. Shown in (B, C) are Oc90-FLAG and otolin detected by immunostaining. In (A), protein products were co-immunoprecipitated using anti-FLAG agarose beads. The lanes are co-IP with: 1) empty vectors, 2) full-length (FL) otolin, 3) otolin-TH domain, and 4) otolin-C1q domain.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105080&req=5

pone-0020498-g001: Co-immunoprecipitation (co-IP) of Oc90 with otolin or its domains.HEK293 cells were transfected with full-length Oc90-FLAG, otolin and domains of otolin. Shown in (B, C) are Oc90-FLAG and otolin detected by immunostaining. In (A), protein products were co-immunoprecipitated using anti-FLAG agarose beads. The lanes are co-IP with: 1) empty vectors, 2) full-length (FL) otolin, 3) otolin-TH domain, and 4) otolin-C1q domain.
Mentions: To test whether Oc90 interacts with otolin to form the organic matrix of otoconia and to identify the interacting domains, we performed co-immunoprecipitation (IP). Transfection of HEK293 cells with expression constructs containing full-length Oc90 (Oc90-FLAG), full-length and individual domains of otolin (TH and C1q) yielded high expression efficiency (Figure 1B, 1C). Protein products were co-precipitated using anti-FLAG agarose beads and washed under stringent conditions as described in Materials and Methods. Constituents eluted from the beads were analyzed by Western blotting after separation by 4–15% SDS-PAGE (Figure 1A). Antibodies against the N-terminal regions of Oc90 and otolin [10] were used for detection. Oc90 interacts strongly with full-length otolin (lane 2 in Figure 1A), and both domains of otolin participate synergistically in this interaction (lanes 3 and 4 in Figure 1A). The requirement of both otolin domains is consistent with the finding that the C1q domain of other related proteins is responsible for partner recognition and the TH domain for strengthening the interaction [22]. Both the TH and C1q domains have a high affinity for proteoglycans and Ca2+ as well [39]. As is the case with the sPLA2-like domains in Oc90 [8]; [9], the functional domains of otolin (TH and C1q) are extremely conserved throughout evolution [32].

Bottom Line: In vivo, the presence of Oc90 in wildtype (wt) mice leads to an enrichment of Ca(2+) in the luminal matrices of the utricle and saccule, whereas absence of Oc90 in the mice leads to drastically reduced matrix-Ca(2+).Molecular modeling and sequence analysis predict structural features that may underlie the interaction and Ca(2+)-sequestering ability of these proteins.Together, the data provide a mechanism for the otoconial matrix assembly and the role of this matrix in accumulating micro-environmental Ca(2+) for efficient CaCO(3) crystallization, thus uncover a critical process governing spatial specific otoconia formation.

View Article: PubMed Central - PubMed

Affiliation: Vestibular Neurogenetics Laboratory, Boys Town National Research Hospital, Omaha, Nebraska, United States of America.

ABSTRACT
Otoconia are bio-crystals anchored to the macular sensory epithelium of the utricle and saccule in the inner ear for motion sensing and bodily balance. Otoconia dislocation, degeneration and ectopic calcification can have detrimental effects on balance and vertigo/dizziness, yet the mechanism underlying otoconia formation is not fully understood. In this study, we show that selected matrix components are recruited to form the crystal matrix and sequester Ca(2+) for spatial specific formation of otoconia. Specifically, otoconin-90 (Oc90) binds otolin through both domains (TH and C1q) of otolin, but full-length otolin shows the strongest interaction. These proteins have much higher expression levels in the utricle and saccule than other inner ear epithelial tissues in mice. In vivo, the presence of Oc90 in wildtype (wt) mice leads to an enrichment of Ca(2+) in the luminal matrices of the utricle and saccule, whereas absence of Oc90 in the mice leads to drastically reduced matrix-Ca(2+). In vitro, either Oc90 or otolin can increase the propensity of extracellular matrix to calcify in cell culture, and co-expression has a synergistic effect on calcification. Molecular modeling and sequence analysis predict structural features that may underlie the interaction and Ca(2+)-sequestering ability of these proteins. Together, the data provide a mechanism for the otoconial matrix assembly and the role of this matrix in accumulating micro-environmental Ca(2+) for efficient CaCO(3) crystallization, thus uncover a critical process governing spatial specific otoconia formation.

Show MeSH
Related in: MedlinePlus