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Intrinsically disordered and pliable Starmaker-like protein from medaka (Oryzias latipes) controls the formation of calcium carbonate crystals.

Różycka M, Wojtas M, Jakób M, Stigloher C, Grzeszkowiak M, Mazur M, Ożyhar A - PLoS ONE (2014)

Bottom Line: Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths.An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals.The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wrocław, Poland.

ABSTRACT
Fish otoliths, biominerals composed of calcium carbonate with a small amount of organic matrix, are involved in the functioning of the inner ear. Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths. Recently, a gene was identified encoding the Starmaker-like (Stm-l) protein from medaka (Oryzias latipes), a putative homologue of Stm and human dentine sialophosphoprotein. Although there is no sequence similarity between Stm-l and Stm, Stm-l was suggested to be involved in the biomineralization of otoliths, as had been observed for Stm even before. The molecular properties and functioning of Stm-l as a putative regulatory protein in otolith formation have not been characterized yet. A comprehensive biochemical and biophysical analysis of recombinant Stm-l, along with in silico examinations, indicated that Stm-l exhibits properties of a coil-like intrinsically disordered protein. Stm-l possesses an elongated and pliable structure that is able to adopt a more ordered and rigid conformation under the influence of different factors. An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals. The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed.

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The effect of Stm-l on calcium carbonate mineralization.(A) SEM images of calcium carbonate crystals grown 48 h. Crystals grown in the absence of any protein (a, h, o), in the presence of trypsin at a concentration of 100 µg/mL (b, i, p), and in the presence of Stm-l at the following concentrations: 1 µg/mL (c, j, q), 5 µg/mL (d, k, r), 10 µg/mL (e, l, s), 20 µg/mL (f, m, t), 50 µg/mL (g, n, u). Concentrations of calcium ions were 5 mM (a–g), 10 mM (h–n), or 20 mM (o–u). Asterisks indicate additional, less thermodynamically stable, large spherical vaterite crystals which were present at every calcium ion and protein concentration and in the controls. The scale bar on the upper left corner of each panel represents a 200 µm-distance. (B) 10× magnification of the representative crystals shown on panel (A). The scale bar on the upper left corner of each panel represents a 20 µm-distance. Other details as in (A).
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pone-0114308-g006: The effect of Stm-l on calcium carbonate mineralization.(A) SEM images of calcium carbonate crystals grown 48 h. Crystals grown in the absence of any protein (a, h, o), in the presence of trypsin at a concentration of 100 µg/mL (b, i, p), and in the presence of Stm-l at the following concentrations: 1 µg/mL (c, j, q), 5 µg/mL (d, k, r), 10 µg/mL (e, l, s), 20 µg/mL (f, m, t), 50 µg/mL (g, n, u). Concentrations of calcium ions were 5 mM (a–g), 10 mM (h–n), or 20 mM (o–u). Asterisks indicate additional, less thermodynamically stable, large spherical vaterite crystals which were present at every calcium ion and protein concentration and in the controls. The scale bar on the upper left corner of each panel represents a 200 µm-distance. (B) 10× magnification of the representative crystals shown on panel (A). The scale bar on the upper left corner of each panel represents a 20 µm-distance. Other details as in (A).

Mentions: Fig. 6 shows the SEM morphologies of crystals grown in the presence of Stm-l in various concentrations, or in the presence of trypsin (as a control protein not involved in biomineralization), or without any protein during mineralization period of 48 h. Calcium carbonate crystals grown in the presence of Stm-l (Fig. 6A and B, images c–g, j–n, q–u) differed significantly in shape and size from the crystals obtained without any protein (Fig. 6A and B, images a, h, o), or in the presence of trypsin (Fig. 6A and B, images b, i, p). Noteworthy is the fact, that all the control crystals were prismatic. It was previously shown that synthetic calcite usually grows as an almost isotropic rhombohedron in the hexagonal lattice in which the calcium and carbonate ions are closely packed to obtain the most thermodynamic stability [75]. In contrast, crystals with Stm-l had a significantly different growth pattern which changed with increasing protein concentrations. The impact of Stm-l was already visible at a concentration of 1 µg/mL, when all the crystals became rounded at the edges (Fig. 6A and B, images c, j, q). However, a higher concentration of 5 µg/mL revealed crystals with noticeably rounded edges and characteristic stair-like structures (Fig. 6A and B, images d, k, r). At a protein concentration of 10 µg/mL – independent of the calcium ion concentration – we observed a heterogenic, two-sized population of crystals with significantly different dimensions (Fig. 6A and B, images e, l, s). Crystals were the most modified at a concentration of 20 µg/mL, when the edges and some of the faces of the crystals completely disappeared and the stair-like structures that had formed on the surface of the crystals were distinctly noticeable (Fig. 6A and B, images f, m, t). This effect was enhanced at the highest protein concentration of 50 µg/mL (Fig. 6A and B, images g, n, u). A higher protein concentration of 100 µg/mL was also tested, but the effect on the calcium carbonate mineralization was similar that for 50 µg/mL (data not shown). Micro-Raman analysis showed that crystals obtained in the presence of Stm-l even at the highest concentration of protein are calcite (Fig. 7). The same results was obtained for crystals obtained without any proteins or in the presence or trypsin (data not shown).


Intrinsically disordered and pliable Starmaker-like protein from medaka (Oryzias latipes) controls the formation of calcium carbonate crystals.

Różycka M, Wojtas M, Jakób M, Stigloher C, Grzeszkowiak M, Mazur M, Ożyhar A - PLoS ONE (2014)

The effect of Stm-l on calcium carbonate mineralization.(A) SEM images of calcium carbonate crystals grown 48 h. Crystals grown in the absence of any protein (a, h, o), in the presence of trypsin at a concentration of 100 µg/mL (b, i, p), and in the presence of Stm-l at the following concentrations: 1 µg/mL (c, j, q), 5 µg/mL (d, k, r), 10 µg/mL (e, l, s), 20 µg/mL (f, m, t), 50 µg/mL (g, n, u). Concentrations of calcium ions were 5 mM (a–g), 10 mM (h–n), or 20 mM (o–u). Asterisks indicate additional, less thermodynamically stable, large spherical vaterite crystals which were present at every calcium ion and protein concentration and in the controls. The scale bar on the upper left corner of each panel represents a 200 µm-distance. (B) 10× magnification of the representative crystals shown on panel (A). The scale bar on the upper left corner of each panel represents a 20 µm-distance. Other details as in (A).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260845&req=5

pone-0114308-g006: The effect of Stm-l on calcium carbonate mineralization.(A) SEM images of calcium carbonate crystals grown 48 h. Crystals grown in the absence of any protein (a, h, o), in the presence of trypsin at a concentration of 100 µg/mL (b, i, p), and in the presence of Stm-l at the following concentrations: 1 µg/mL (c, j, q), 5 µg/mL (d, k, r), 10 µg/mL (e, l, s), 20 µg/mL (f, m, t), 50 µg/mL (g, n, u). Concentrations of calcium ions were 5 mM (a–g), 10 mM (h–n), or 20 mM (o–u). Asterisks indicate additional, less thermodynamically stable, large spherical vaterite crystals which were present at every calcium ion and protein concentration and in the controls. The scale bar on the upper left corner of each panel represents a 200 µm-distance. (B) 10× magnification of the representative crystals shown on panel (A). The scale bar on the upper left corner of each panel represents a 20 µm-distance. Other details as in (A).
Mentions: Fig. 6 shows the SEM morphologies of crystals grown in the presence of Stm-l in various concentrations, or in the presence of trypsin (as a control protein not involved in biomineralization), or without any protein during mineralization period of 48 h. Calcium carbonate crystals grown in the presence of Stm-l (Fig. 6A and B, images c–g, j–n, q–u) differed significantly in shape and size from the crystals obtained without any protein (Fig. 6A and B, images a, h, o), or in the presence of trypsin (Fig. 6A and B, images b, i, p). Noteworthy is the fact, that all the control crystals were prismatic. It was previously shown that synthetic calcite usually grows as an almost isotropic rhombohedron in the hexagonal lattice in which the calcium and carbonate ions are closely packed to obtain the most thermodynamic stability [75]. In contrast, crystals with Stm-l had a significantly different growth pattern which changed with increasing protein concentrations. The impact of Stm-l was already visible at a concentration of 1 µg/mL, when all the crystals became rounded at the edges (Fig. 6A and B, images c, j, q). However, a higher concentration of 5 µg/mL revealed crystals with noticeably rounded edges and characteristic stair-like structures (Fig. 6A and B, images d, k, r). At a protein concentration of 10 µg/mL – independent of the calcium ion concentration – we observed a heterogenic, two-sized population of crystals with significantly different dimensions (Fig. 6A and B, images e, l, s). Crystals were the most modified at a concentration of 20 µg/mL, when the edges and some of the faces of the crystals completely disappeared and the stair-like structures that had formed on the surface of the crystals were distinctly noticeable (Fig. 6A and B, images f, m, t). This effect was enhanced at the highest protein concentration of 50 µg/mL (Fig. 6A and B, images g, n, u). A higher protein concentration of 100 µg/mL was also tested, but the effect on the calcium carbonate mineralization was similar that for 50 µg/mL (data not shown). Micro-Raman analysis showed that crystals obtained in the presence of Stm-l even at the highest concentration of protein are calcite (Fig. 7). The same results was obtained for crystals obtained without any proteins or in the presence or trypsin (data not shown).

Bottom Line: Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths.An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals.The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wrocław, Poland.

ABSTRACT
Fish otoliths, biominerals composed of calcium carbonate with a small amount of organic matrix, are involved in the functioning of the inner ear. Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths. Recently, a gene was identified encoding the Starmaker-like (Stm-l) protein from medaka (Oryzias latipes), a putative homologue of Stm and human dentine sialophosphoprotein. Although there is no sequence similarity between Stm-l and Stm, Stm-l was suggested to be involved in the biomineralization of otoliths, as had been observed for Stm even before. The molecular properties and functioning of Stm-l as a putative regulatory protein in otolith formation have not been characterized yet. A comprehensive biochemical and biophysical analysis of recombinant Stm-l, along with in silico examinations, indicated that Stm-l exhibits properties of a coil-like intrinsically disordered protein. Stm-l possesses an elongated and pliable structure that is able to adopt a more ordered and rigid conformation under the influence of different factors. An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals. The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed.

Show MeSH
Related in: MedlinePlus