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J Proteomics 2016 Mar 16;136:133-44. doi: 10.1016/j.jprot.2016.01.001.
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A minimal molecular toolkit for mineral deposition? Biochemistry and proteomics of the test matrix of adult specimens of the sea urchin Paracentrotus lividus.

Karakostis K , Zanella-Cléon I , Immel F , Guichard N , Dru P , Lepage T , Plasseraud L , Matranga V , Marin F .

UNLABELLED: The sea urchin endoskeleton consists of a magnesium-rich biocalcite comprising a small amount of occluded organic macromolecules. This structure constitutes a key-model for understanding the mineral--organics interplay, and for conceiving in vitro bio-inspired materials with tailored properties. Here we employed a deep-clean technique to purify the occluded proteins from adult Paracentrotus lividus tests. We characterized them by 1- and 2D-electrophoreses, ELISA and immunoblotting, and using liquid chromatography coupled with Mass Spectrometry (nanoLC-MS/MS), we identified two metalloenzymes (carbonic anhydrase and MMP), a set of MSP130 family members, several C-type lectins (SM29, SM41, PM27) and cytoskeletal proteins. We demonstrate the effect of the protein extract on the crystals, with an in vitro crystallization assay. We suggest that this small set of biomineralization proteins may represent a ''minimal molecular crystallization toolkit''. SIGNIFICANCE: Biominerals often exhibit superior chemical properties, when compared to their inorganic counterparts. This is due pro parte to the proteins that are occluded in the mineral. However, the limited available studies on biomineralization have not yet succeeded in identifying a minimal set of proteins directly involved in the formation of the biomineral in vivo and sufficiently required for in vitro precipitation. Indeed, the high number of proteins identified by high-throughput screening in the recent years does not encourage the possibility of recreating or tailoring the mineral in vitro. Thus, the identification of biomineralization proteins involved in protein-mineral interactions is highly awaited. In the present study, we used the sea urchin, Paracentrotus lividus (P. lividus), to identify the native proteins directly taking part in protein-mineral interactions. We employed an improved deep-clean technique to extract and purify the native occluded skeletal matrix proteins from the test and identified them by the highly sensitive technique of nanoLC-MS/MS. We show that this minimal set of proteins has a shaping effect on the formation of biocalcite in vitro. This work gives insights on the biomineralization of the sea urchin, while it paves the way for the identification of biomineralization proteins in other biomineralizing systems. Understanding the ''biologically controlled mineralization'' will facilitate the in vitro formation and tailoring of biominerals in mild conditions for applications in medicine and materials science.

PubMed ID: 26778142
Article link: J Proteomics

Genes referenced: LOC100887844 LOC115924597 LOC577690 LOC583082 mmp7 msp130 pm27