title: A minimal molecular toolkit for mineral deposition? Biochemistry and proteomics of the test matrix of adult specimens of the sea urchin Paracentrotus lividus.
abstract: 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'.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Cell Stress and Environment Research Group, Consiglio Nazionale delle Ricerche, Istituto di Biomedicina e Immunologia Molecolare "Alberto Monroy", Via Ugo La Malfa 153, 90146 Palermo, Italy; INSERM UMR S 1162, Institut de Génétique Moléculaire, Hôpital Saint-Louis, 27 Rue Juliette Dodu, 75010 Paris, France. Electronic address: chem898@yahoo.gr.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Karakostis
authors.affiliation: Protein Science Facility, Institut de Biologie et Chimie des Protéines, 7, passage du Vercors, 69367 Lyon cedex 07, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Zanella-Cléon
authors.affiliation: UMR CNRS 6282 Biogéosciences, Université de Bourgogne Franche-Comté (UBFC), 6 boulevard Gabriel, 21000 Dijon, France.
authors.forename: Françoise
authors.initials: F
authors.lastname: Immel
authors.affiliation: UMR CNRS 6282 Biogéosciences, Université de Bourgogne Franche-Comté (UBFC), 6 boulevard Gabriel, 21000 Dijon, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Guichard
authors.affiliation: UMR 7009 CNRS/UPMC - Laboratoire de Biologie du Développement, Observatoire Océanologique de Villefranche-sur-Mer, BP 28, Quai de la Darse, 06234 Villefranche-sur-Mer cedex, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Dru
authors.affiliation: UMR 7009 CNRS/UPMC - Laboratoire de Biologie du Développement, Observatoire Océanologique de Villefranche-sur-Mer, BP 28, Quai de la Darse, 06234 Villefranche-sur-Mer cedex, France; IBV - Institut de Biologie Valrose, CNRS UMR7277, Inserm U1091, UNS Université Nice Sophia Antipolis, Parc Valrose, 06108 Nice cedex 2, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Lepage
authors.affiliation: ICMUB, UMR CNRS 6302, Université de Bourgogne Franche-Comté (UBFC), Faculté des Sciences Mirande, 9 Avenue A. Savary, F-21000 Dijon, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Plasseraud
authors.affiliation: Cell Stress and Environment Research Group, Consiglio Nazionale delle Ricerche, Istituto di Biomedicina e Immunologia Molecolare "Alberto Monroy", Via Ugo La Malfa 153, 90146 Palermo, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Matranga
authors.affiliation: UMR CNRS 6282 Biogéosciences, Université de Bourgogne Franche-Comté (UBFC), 6 boulevard Gabriel, 21000 Dijon, France. Electronic address: Frederic.marin@u-bourgogne.fr.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Marin
mesh_terms: D000818:Animals; D005109:Extracellular Matrix / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D013058:Mass Spectrometry; D047328:Paracentrotus / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*
chemical_list: D016326:Extracellular Matrix Proteins
keywords: Biomineralization; C-type lectin; Calcifying matrix; Calcite; Carbonic anhydrase; Proteomics; Sea urchin