Pendola M et al. (2018), Secrets of the Sea Urchin Spicule Revealed: Pro...

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ACS Omega 2018 Sep 30;39:11823-11830. doi: 10.1021/acsomega.8b01697.

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Secrets of the Sea Urchin Spicule Revealed: Protein Cooperativity Is Responsible for ACC Transformation, Intracrystalline Incorporation, and Guided Mineral Particle Assembly in Biocomposite Material Formation.

Pendola M , Jain G , Huang YC , Gebauer D , Evans JS .

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The formation of the sea urchin spicule involves the stabilization and transformation of amorphous calcium carbonate (ACC) and assembly of ACC nanoparticle precursors into a mesoscale single crystal of fracture-resistant calcite. This process of particle assembly or attachment is under the control of a family of proteins known as the spicule matrix [Strongylocentrotus purpuratus (SpSM)] proteome. Recently, two members of this proteome, SpSM50 and the glycoprotein SpSM30B/C-G (in recombinant forms), were found to interact together via SpSM30B/C-G oligosaccharide-SpSM50 protein interactions to form hybrid protein hydrogels with unique physical properties. In this study, we investigate the mineralization properties of this hybrid hydrogel alongside the hydrogels formed by SpSM50 and SpSM30B/C-G individually. We find that the SpSM50 + SpSM30B/C-G hybrid hydrogel is synergistic with regard to surface modifications and intracrystalline inclusions of existing calcite crystals, the inhibition of ACC formation, and the kinetic destabilization of ACC to form a crystalline phase. Most importantly, the hybrid hydrogel phase assembles and organizes mineral particles into discrete clusters or domains within in vitro mineralization environments. Thus, the interactions of SpSM50 and SpSM30B/C-G, mediated by carbohydrate-protein binding, reflect the need for protein cooperativity for the ACC-to-crystalline transformation, intracrystalline void formation, and guided mineral particle assembly processes that are instrumental in spicule formation.

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Genes referenced: LOC100887844 LOC590371

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	Figure 1. SEM images of mineral deposits retrieved from micromineralization assays. 1:1 refers to the equimolar rSpSM30B/C-G–rSpSM50 mixture. Mineralized protein deposits are indicated by white arrows. Protein-free control image is shown as an inset to the rSpSM30B/C-G image (where scale bar = 2 μm). Note that vaterite crystals are also observed in both the rSpSM30B/C-G and rSpSM50 mineralization assays (Figure S1, Supporting Information).
	Figure 2. SEM images of (A) representative Ir-coated 1:1 rSpSM30B/C-G–rSpSM50 calcite crystal and background-mineralized hydrogel deposits; (B) FIB sectioning of the same crystal, revealing the intracrystalline nanoporosities that are distributed throughout the crystal interior. Scale bar = 200 nm. Enlargement of the image in (B) can be found in the Supporting Information (Figure S2). In contrast, the calcite generated in protein (−) conditions features few, if any, intracrystalline nanoporosities.25,29,38−40
	Figure 3. (A) Amount of free Ca(II) ions and (B) ion product of calcium carbonate in the absence and presence of 500 nM rSpSM30B/C-G, rSpSM50, and the 1:1 protein mixture at pH 8.5 as a function of time. The dashed black line signifies the amount of added Ca(II) ions during titration. The curves represent the average amounts of three individual reference experiments and two protein experiments. The error bars signify ±1-σ standard deviation (see Table 1).
	Figure 4. (Top row) μCT images (XY-planes along the z-axis) of representative control [(−) protein] and rSpSM50 and rSpSM30B/C-G mineralization assay vials. Scale bars = 1 mm. Numbers refer to a mineral particle number quantitated in each sample volume. (Bottom row) μCT XZ-sagittal plane volumetric sections (4 mm × 4 mm × 1.9 mm) of microvial mineralization samples. Note the presence of suspended mineral particles above the bottom of the vials. For rSpSM50 and rSpSM30B/C-G, note the presence of mineral particle layering. Scale bar = 1 mm; height of each image = 1.9 mm. Orientation axes shown. Note in all images the hemispherical region, which is the remnant plastic sprue stub that remains from the vial manufacturing process.26
	Figure 5. (Top row) μCT image (along z-axis) of a representative 1:1 rSpSM30B/C-G–rSpSM50 mineralization assay vial. Scale bars = 1 mm. Numbers refer to the mineral particle number quantitated in sample volume. (Bottom row) μCT XZ-sagittal plane volumetric section (4 mm × 4 mm × 1.9 mm) of the same sample. In both images, note the unique distribution of mineral particles, which is compared to the distributions as seen in Figure 4. Arrows point to representative dense mineral clusters. Scale bar = 1 mm; height of each image = 1.9 mm. Orientation axes shown.

References [+] :

Berman, Intercalation of sea urchin proteins in calcite: study of a crystalline composite material. 1990, Pubmed, Echinobase