Dataset Information

In-depth, high-accuracy proteomics of sea urchin tooth organic matrix.

ABSTRACT: BACKGROUND:The organic matrix contained in biominerals plays an important role in regulating mineralization and in determining biomineral properties. However, most components of biomineral matrices remain unknown at present. In sea urchin tooth, which is an important model for developmental biology and biomineralization, only few matrix components have been identified. The recent publication of the Strongylocentrotus purpuratus genome sequence rendered possible not only the identification of genes potentially coding for matrix proteins, but also the direct identification of proteins contained in matrices of skeletal elements by in-depth, high-accuracy proteomic analysis. RESULTS:We identified 138 proteins in the matrix of tooth powder. Only 56 of these proteins were previously identified in the matrices of test (shell) and spine. Among the novel components was an interesting group of five proteins containing alanine- and proline-rich neutral or basic motifs separated by acidic glycine-rich motifs. In addition, four of the five proteins contained either one or two predicted Kazal protease inhibitor domains. The major components of tooth matrix were however largely identical to the set of spicule matrix proteins and MSP130-related proteins identified in test (shell) and spine matrix. Comparison of the matrices of crushed teeth to intact teeth revealed a marked dilution of known intracrystalline matrix proteins and a concomitant increase in some intracellular proteins. CONCLUSION:This report presents the most comprehensive list of sea urchin tooth matrix proteins available at present. The complex mixture of proteins identified may reflect many different aspects of the mineralization process. A comparison between intact tooth matrix, presumably containing odontoblast remnants, and crushed tooth matrix served to differentiate between matrix components and possible contributions of cellular remnants. Because LC-MS/MS-based methods directly measures peptides our results validate many predicted genes and confirm the existence of the corresponding proteins. Knowledge of the components of this model system may stimulate further experiments aiming at the elucidation of structure, function, and interaction of biomineral matrix components.

SUBMITTER: Mann K

PROVIDER: S-EPMC2614417 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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In-depth, high-accuracy proteomics of sea urchin tooth organic matrix.

Mann Karlheinz K Poustka Albert J AJ Mann Matthias M

Proteome science 20081209

<h4>Background</h4>The organic matrix contained in biominerals plays an important role in regulating mineralization and in determining biomineral properties. However, most components of biomineral matrices remain unknown at present. In sea urchin tooth, which is an important model for developmental biology and biomineralization, only few matrix components have been identified. The recent publication of the Strongylocentrotus purpuratus genome sequence rendered possible not only the identificatio ...[more]

PMID: 19068105

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Project description:BackgroundSea urchin is a major model organism for developmental biology and biomineralization research. However, identification of proteins involved in larval skeleton formation and mineralization processes in the embryo and adult, and the molecular characterization of such proteins, has just gained momentum with the sequencing of the Strongylocentrotus purpuratus genome and the introduction of high-throughput proteomics into the field.ResultsThe present report contains the determination of test (shell) and tooth organic matrix phosphoproteomes. Altogether 34 phosphoproteins were identified in the biomineral organic matrices. Most phosphoproteins were specific for one compartment, only two were identified in both matrices. The sea urchin phosphoproteomes contained several obvious orthologs of mammalian proteins, such as a Src family tyrosine kinase, protein kinase C-delta 1, Dickkopf-1 and other signal transduction components, or nucleobindin. In most cases phosphorylation sites were conserved between sea urchin and mammalian proteins. However, the majority of phosphoproteins had no mammalian counterpart. The most interesting of the sea urchin-specific phosphoproteins, from the perspective of biomineralization research, was an abundant highly phosphorylated and very acidic tooth matrix protein composed of 35 very similar short sequence repeats, a predicted N-terminal secretion signal sequence, and an Asp-rich C-terminal motif, contained in [Glean3:18919].ConclusionsThe 64 phosphorylation sites determined represent the most comprehensive list of experimentally identified sea urchin protein phosphorylation sites at present and are an important addition to the recently analyzed Strongylocentrotus purpuratus shell and tooth proteomes. The identified phosphoproteins included a major, highly phosphorylated protein, [Glean3:18919], for which we suggest the name phosphodontin. Although not sequence-related to such highly phosphorylated acidic mammalian dental phosphoproteins as phosphoryn or dentin matrix protein-1, phosphodontin may perform similar functions in the sea urchin tooth. More than half of the detected proteins were not previously identified at the protein level, thus confirming the existence of proteins only known as genomic sequences previously.

Project description:BackgroundThe sea urchin embryo has been an important model organism in developmental biology for more than a century. This is due to its relatively simple construction, translucent appearance, and the possibility to follow the fate of individual cells as development to the pluteus larva proceeds. Because the larvae contain tiny calcitic skeletal elements, the spicules, they are also important model organisms for biomineralization research. Similar to other biominerals the spicule contains an organic matrix, which is thought to play an important role in its formation. However, only few spicule matrix proteins were identified previously.ResultsUsing mass spectrometry-based methods we have identified 231 proteins in the matrix of the S. purpuratus spicule matrix. Approximately two thirds of the identified proteins are either known or predicted to be extracellular proteins or transmembrane proteins with large ectodomains. The ectodomains may have been solubilized by partial proteolysis and subsequently integrated into the growing spicule. The most abundant protein of the spicule matrix is SM50. SM50-related proteins, SM30-related proteins, MSP130 and related proteins, matrix metalloproteases and carbonic anhydrase are among the most abundant components.ConclusionsThe spicule matrix is a relatively complex mixture of proteins not only containing matrix-specific proteins with a function in matrix assembly or mineralization, but also: 1) proteins possibly important for the formation of the continuous membrane delineating the mineralization space; 2) proteins for secretory processes delivering proteinaceous or non-proteinaceous precursors; 3) or proteins reflecting signaling events at the cell/matrix interface. Comparison of the proteomes of different skeletal matrices allows prediction of proteins of general importance for mineralization in sea urchins, such as SM50, SM30-E, SM29 or MSP130. The comparisons also help point out putative tissue-specific proteins, such as tooth phosphodontin or specific spicule matrix metalloproteases of the MMP18/19 group. Furthermore, the direct sequence analysis of peptides by MS/MS validates many predicted genes and confirms the existence of the corresponding proteins.

Project description:The sea urchin Paracentrotus lividus (Lamarck, 1816) is a keystone herbivore in the Mediterranean Sea due to its ability to transform macroalgal-dominated communities into barren areas characterized by increased cover of bare substrates and encrusting coralline algae, reduced biodiversity and altered ecosystem functions. P. lividus is also an excellent animal model for toxicology, physiology and biology investigations having been used for more than a century as a model for embryological studies with synchronously developing embryos which are easy to manipulate and analyze for morphological aberrations. Despite its importance for the scientific community, the complete genome is still not fully annotated. To date, only a few molecular tools are available and a few Next Generation Sequencing (NGS) studies have been performed. Here we aimed at setting-up an RNA extraction method to obtain high quality and sufficient quantity of RNA for NGS from P. lividus embryos at the pluteus stage. We compared five different RNA extraction protocols from four different pools of plutei (500, 1000, 2500 and 5000 embryos): TRIzol®, and four widely-used Silica Membrane kits, GenElute™ Mammalian Total RNA Miniprep Kit, RNAqueous® Micro Kit, RNeasy® Micro Kit and Aurum™ Total RNA Mini Kit. The quantity of RNA isolated was evaluated using NanoDrop. The quality, considering the purity, was measured as A260/A280 and A260/230 ratios. The integrity was measured by RNA Integrity Number (RIN). Our results demonstrated that the most efficient procedures were GenElute, RNeasy and Aurum, producing a sufficient quantity of RNA for NGS. The Bioanalyzer profiles and RIN values revealed that the most efficient methods guaranteeing for RNA integrity were RNeasy and Aurum combined with an initial preservation in RNAlater. This research represents the first attempt to standardize a method for high-quality RNA extraction from sea urchin embryos at the pluteus stage, providing a new resource for this established model marine organism.

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In-depth, high-accuracy proteomics of sea urchin tooth organic matrix.

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In-depth, high-accuracy proteomics of sea urchin tooth organic matrix.

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