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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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Molecular modeling of the C1q domain of otolin.(A) The tertiary structures of Col10a1-C1q domain (green) and otolin-C1q domain (magenta) are nearly super-imposable. The modeled structure is consistent with the crystal structure of human COL10A1. Ca2+-binding residues are labeled and projected as sticks. (B) The surface of the otolin-C1q domain with temperature factors denoted in different colors. (C) Clustering of residues and the cationic surface of otolin-C1q domain. Residues are colored according to their electrical charges and hydrophobic nature. The nine key cationic residues are labeled and projected as sticks. (D) The protein backbone RMSD during 1ns simulation. After 400ps, the structure is stable. The 400–1000 ps range was used for computing the final structure. (E) The temperature factor (RMS fluctuation) of each residue. The residues with higher RMS fluctuation usually are exposed on molecule surface and may construct active sites. The Ca2+-binding residues in (A) are labeled.
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pone-0020498-g007: Molecular modeling of the C1q domain of otolin.(A) The tertiary structures of Col10a1-C1q domain (green) and otolin-C1q domain (magenta) are nearly super-imposable. The modeled structure is consistent with the crystal structure of human COL10A1. Ca2+-binding residues are labeled and projected as sticks. (B) The surface of the otolin-C1q domain with temperature factors denoted in different colors. (C) Clustering of residues and the cationic surface of otolin-C1q domain. Residues are colored according to their electrical charges and hydrophobic nature. The nine key cationic residues are labeled and projected as sticks. (D) The protein backbone RMSD during 1ns simulation. After 400ps, the structure is stable. The 400–1000 ps range was used for computing the final structure. (E) The temperature factor (RMS fluctuation) of each residue. The residues with higher RMS fluctuation usually are exposed on molecule surface and may construct active sites. The Ca2+-binding residues in (A) are labeled.

Mentions: Both otolin and Col10a1 have a highly conserved C-terminal globular C1q (gC1q) domain, sharing 68% amino acid sequence similarity to each other, with 55 identical (*), 20 conservative (:) and 18 semi-conservative (.) residues among a total of 136aa (Figure 6). The inter-subunit contacts in the trimer are almost entirely hydrophobic with key contact residues highlighted in yellow in Figure 6. Thirteen out of 20 residues in Col10a1 are conserved in otolin. Based on molecular modeling results, the tertiary structures of murine otolin and Col10a1 are nearly super-imposable (Figure 7A). Most importantly, the two critical aspartic acids (D, highlighted in Figures 6 & 7) that provide the ligands for a cluster of four Ca2+ ions in Col10a1 are conserved in otolin (E422 binds Ca2+ equally well). With other residues (R421 and Y425), they form a pocket with high temperature factors for Ca2+ binding (Figure 7B, E). Additionally, a stretch of nine positively charged residues is present nearby the pocket and surrounded by several hydrogen-bonding residues (Figure 7C), which constitutes a large cationic and hydrogen-bonding surface and should represent a putative functional site/region for Oc90 binding. Possible electrostatic interactions and hydrogen bonding between otolin and Oc90 is further supported by the clustering of anionic and hydrogen-bonding residues on the surface of a modeled Oc90 structure [44]. At the physiological pH, otolin has a net positive charge of 11.5 with a predicted pI of 9.2, and Oc90 has a negative charge of −23.0 with a predicted pI of 4.5. The non-polar residues may also facilitate the interaction of otolin with Oc90. Clustering of non-polar residues was also found on the surface of murine Oc90 [45] and bullfrog Oc22 [44], a homolog of Oc90 but with only one sPLA2-like domain.


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)

Molecular modeling of the C1q domain of otolin.(A) The tertiary structures of Col10a1-C1q domain (green) and otolin-C1q domain (magenta) are nearly super-imposable. The modeled structure is consistent with the crystal structure of human COL10A1. Ca2+-binding residues are labeled and projected as sticks. (B) The surface of the otolin-C1q domain with temperature factors denoted in different colors. (C) Clustering of residues and the cationic surface of otolin-C1q domain. Residues are colored according to their electrical charges and hydrophobic nature. The nine key cationic residues are labeled and projected as sticks. (D) The protein backbone RMSD during 1ns simulation. After 400ps, the structure is stable. The 400–1000 ps range was used for computing the final structure. (E) The temperature factor (RMS fluctuation) of each residue. The residues with higher RMS fluctuation usually are exposed on molecule surface and may construct active sites. The Ca2+-binding residues in (A) are labeled.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105080&req=5

pone-0020498-g007: Molecular modeling of the C1q domain of otolin.(A) The tertiary structures of Col10a1-C1q domain (green) and otolin-C1q domain (magenta) are nearly super-imposable. The modeled structure is consistent with the crystal structure of human COL10A1. Ca2+-binding residues are labeled and projected as sticks. (B) The surface of the otolin-C1q domain with temperature factors denoted in different colors. (C) Clustering of residues and the cationic surface of otolin-C1q domain. Residues are colored according to their electrical charges and hydrophobic nature. The nine key cationic residues are labeled and projected as sticks. (D) The protein backbone RMSD during 1ns simulation. After 400ps, the structure is stable. The 400–1000 ps range was used for computing the final structure. (E) The temperature factor (RMS fluctuation) of each residue. The residues with higher RMS fluctuation usually are exposed on molecule surface and may construct active sites. The Ca2+-binding residues in (A) are labeled.
Mentions: Both otolin and Col10a1 have a highly conserved C-terminal globular C1q (gC1q) domain, sharing 68% amino acid sequence similarity to each other, with 55 identical (*), 20 conservative (:) and 18 semi-conservative (.) residues among a total of 136aa (Figure 6). The inter-subunit contacts in the trimer are almost entirely hydrophobic with key contact residues highlighted in yellow in Figure 6. Thirteen out of 20 residues in Col10a1 are conserved in otolin. Based on molecular modeling results, the tertiary structures of murine otolin and Col10a1 are nearly super-imposable (Figure 7A). Most importantly, the two critical aspartic acids (D, highlighted in Figures 6 & 7) that provide the ligands for a cluster of four Ca2+ ions in Col10a1 are conserved in otolin (E422 binds Ca2+ equally well). With other residues (R421 and Y425), they form a pocket with high temperature factors for Ca2+ binding (Figure 7B, E). Additionally, a stretch of nine positively charged residues is present nearby the pocket and surrounded by several hydrogen-bonding residues (Figure 7C), which constitutes a large cationic and hydrogen-bonding surface and should represent a putative functional site/region for Oc90 binding. Possible electrostatic interactions and hydrogen bonding between otolin and Oc90 is further supported by the clustering of anionic and hydrogen-bonding residues on the surface of a modeled Oc90 structure [44]. At the physiological pH, otolin has a net positive charge of 11.5 with a predicted pI of 9.2, and Oc90 has a negative charge of −23.0 with a predicted pI of 4.5. The non-polar residues may also facilitate the interaction of otolin with Oc90. Clustering of non-polar residues was also found on the surface of murine Oc90 [45] and bullfrog Oc22 [44], a homolog of Oc90 but with only one sPLA2-like domain.

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