Spine and test skeletal matrices of the Mediterranean sea urchin Arbacia lixula--a comparative characterization of their sugar signature.

Calcified structures of sea urchins are biocomposite materials that comprise a minor fraction of organic macromolecules, such as proteins, glycoproteins and polysaccharides. These macromolecules are thought to collectively regulate mineral deposition during the process of calcification. When occluded, they modify the properties of the mineral. In the present study, the organic matrices (both soluble and insoluble in acetic acid) of spines and tests from the Mediterranean black sea urchin Arbacia lixula were extracted and characterized, in order to determine whether they exhibit similar biochemical signatures. Bulk characterizations were performed by mono-dimensional SDS/PAGE, FT-IR spectroscopy, and an in vitro crystallization assay. We concentrated our efforts on characterization of the sugar moieties. To this end, we determined the monosaccharide content of the soluble and insoluble organic matrices of A. lixula spines and tests by HPAE-PAD, together with their respective lectin-binding profiles via enzyme-linked lectin assay. Finally, we performed in situ localization of N-acetyl glucosamine-containing saccharides on spines and tests using gold-conjugated wheatgerm agglutinin. Our data show that the test and spine matrices exhibit different biochemical signatures with regard to their saccharidic fraction, suggesting that future studies should analyse the regulation of mineral deposition by the matrix in these two mineralized structures in detail. This study re-emphasizes the importance of non-protein moieties, i.e. sugars, in calcium carbonate systems, and highlights the need to clearly identify their function in the biomineralization process.

The test skeletal matrix of the black sea urchin Arbacia lixula.

In the field of biomineralization, the past decade has been marked by the increasing use of high throughput techniques, i.e. proteomics, for identifying in one shot the protein content of complex macromolecular mixtures extracted from mineralized tissues. Although crowned with success, this approach has been restricted so far to a limited set of key-organisms, such as the purple sea urchin Strongylocentrotus purpuratus, the pearl oyster or the abalone, leaving in the shadow non-model organisms. As a consequence, it is still unknown to what extent the calcifying repertoire varies, from group to group, at high (phylum, class), median (order, family) or low (genus, species) taxonomic rank. The present paper shows the first biochemical and proteomic characterization of the test matrix of the Mediterranean black sea urchin Arbacia lixula (Arbacioida). Our work suggests that the skeletal repertoire of A. lixula exhibits some similarities but also several differences with that of the few sea urchin species (S. purpuratus, Paracentrotus lividus), for which molecular data are already available. The differences may be attributable to the taxonomic position of the species considered: A. lixula belongs to an order - Arbacioida - that diverged more than one hundred million years ago from the Camarodonta, which includes the two species S. purpuratus and P. lividus. For the echinoid class, we suggest that large-scale proteomic screening should be performed in order to understand which molecular functions related to calcification are conserved and which ones have been co-opted for biomineralization in particular lineages.

The skeleton of the staghorn coral Acropora millepora: molecular and structural characterization.

The scleractinian coral Acropora millepora is one of the most studied species from the Great Barrier Reef. This species has been used to understand evolutionary, immune and developmental processes in cnidarians. It has also been subject of several ecological studies in order to elucidate reef responses to environmental changes such as temperature rise and ocean acidification (OA). In these contexts, several nucleic acid resources were made available. When combined to a recent proteomic analysis of the coral skeletal organic matrix (SOM), they enabled the identification of several skeletal matrix proteins, making A. millepora into an emerging model for biomineralization studies. Here we describe the skeletal microstructure of A. millepora skeleton, together with a functional and biochemical characterization of its occluded SOM that focuses on the protein and saccharidic moieties. The skeletal matrix proteins show a large range of isoelectric points, compositional patterns and signatures. Besides secreted proteins, there are a significant number of proteins with membrane attachment sites such as transmembrane domains and GPI anchors as well as proteins with integrin binding sites. These features show that the skeletal proteins must have strong adhesion properties in order to function in the calcifying space. Moreover this data suggest a molecular connection between the calcifying epithelium and the skeletal tissue during biocalcification. In terms of sugar moieties, the enrichment of the SOM in arabinose is striking, and the monosaccharide composition exhibits the same signature as that of mucus of acroporid corals. Finally, we observe that the interaction of the acetic acid soluble SOM on the morphology of in vitro grown CaCO3 crystals is very pronounced when compared with the calcifying matrices of some mollusks. In light of these results, we wish to commend Acropora millepora as a model for biocalcification studies in scleractinians, from molecular and structural viewpoints.

The shell organic matrix of the crossed lamellar queen conch shell (Strombus gigas).

In molluscs, the shell organic matrix comprises a large set of biomineral-occluded proteins, glycoproteins and polysaccharides that are secreted by the calcifying mantle epithelium, and are supposed to display several functions related to the synthesis of the shell. In the present paper, we have characterized biochemically the shell matrix associated to the crossed-lamellar structure of the giant queen conch Strombus gigas. The acid-soluble (ASM) and acid-insoluble (AIM) matrices represent an extremely minor fraction of the shell. Both are constituted of polydisperse and of few discrete proteins among which three fractions, obtained by preparative SDS-PAGE and named 1P3, 2P3 and 3P3, are dominant and were further characterized. Compared to other matrices, the acid-soluble matrix is weakly glycosylated (3%) and among the discrete components, only 3P3 seems noticeably glycosylated. The monosaccharide composition of the ASM shows that mannose represents the main monosaccharide. To our knowledge, this is the first report of a high ratio of this sugar in a skeletal matrix. Furthermore, the ASM interacts with the in vitro crystallization of calcium carbonate, but this interaction is moderate. It differs from that of the isolated 1P3 fraction but is similar to that of the 2P3 and 3P3 fractions. At last, antibodies developed from the 3P3 fraction were used to localize this fraction within the shell by immunogold. This study is the first one aiming at characterizing the organic matrix associated to the crossed-lamellar structure of the queen conch shell.

The Medicago truncatula sucrose transporter family: characterization and implication of key members in carbon partitioning towards arbuscular mycorrhizal fungi.

We identified de novo sucrose transporter (SUT) genes involved in long-distance transport of sucrose from photosynthetic source leaves towards sink organs in the model leguminous species Medicago truncatula. The identification and functional analysis of sugar transporters provide key information on mechanisms that underlie carbon partitioning in plant-microorganism interactions. In that way, full-length sequences of the M. truncatula SUT (MtSUT) family were retrieved and biochemical characterization of MtSUT members was performed by heterologous expression in yeast. The MtSUT family now comprises six genes which distribute among Dicotyledonous clades. MtSUT1-1 and MtSUT4-1 are key members in regard to their expression profiles in source leaves and sink roots and were characterized as functional H(+)/sucrose transporters. Physiological and molecular responses to phosphorus supply and inoculation by the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was studied by gene expression and sugar quantification analyses. Sucrose represents the main sugar transport form in M. truncatula and the expression profiles of MtSUT1-1, MtSUT2, and MtSUT4-1 highlight a fine-tuning regulation for beneficial sugar fluxes towards the fungal symbiont. Taken together, these results suggest distinct functions for proteins from the SUT1, SUT2, and SUT4 clades in plant and in biotrophic interactions.

Novel molluskan biomineralization proteins retrieved from proteomics: a case study with Upsalin.

The formation of the molluskan shell is regulated by an array of extracellular proteins secreted by the calcifying epithelial cells of the mantle. These proteins remain occluded within the recently formed biominerals. To date, many shell proteins have been retrieved, but only a few of them, such as nacreins, have clearly identified functions. In this particular case, by combining molecular biology and biochemical approaches, we performed the molecular characterization of a novel protein that we named Upsalin, associated with the nacreous shell of the freshwater mussel Unio pictorum. The full sequence of the upsalin transcript was obtained by RT-PCR and 5'/3' RACE, and the expression pattern of the transcript was studied by PCR and qPCR. Upsalin is a 12 kDa protein with a basic theoretical pI. The presence of Upsalin in the shell was demonstrated by extraction of the acetic-acid-soluble nacre matrix, purification of a shell protein fraction by mono-dimensional preparative SDS-PAGE, and by submitting this fraction, after trypsic digestion, to nano-LC-MS/MS. In vitro experiments with the purified protein showed that it interferes poorly with the precipitation of calcium carbonate. Homology searches also could not affiliate Upsalin to any other protein of known function, leaving open the question of its exact role in shell formation. An antibody raised against an immunogenic peptide of Upsalin was found to be specific to this protein and was subsequently assayed for immunogold localization of the target protein in the shell, revealing the ubiquitous presence of Upsalin in the nacreous and prismatic layers. Recently, with the application of high-throughput proteomic studies to shells, the number of candidate proteins without clear functions has been increasing exponentially. The Upsalin example highlights the crucial need, for the scientific community dealing with biomineralization in general, to dedicate the coming years to designing experimental approaches, such as gene silencing, that focus on the functions of mineral-associated proteins.

Comparative ultrastructure and carbohydrate composition of gastroliths from astacidae, cambaridae and parastacidae freshwater crayfish (crustacea, decapoda).

Crustaceans have to cyclically replace their rigid exoskeleton in order to grow. Most of them harden this skeleton by a calcification process. Some decapods (land crabs, lobsters and crayfish) elaborate calcium storage structures as a reservoir of calcium ions in their stomach wall, as so-called gastroliths. For a better understanding of the cyclic elaboration of these calcium deposits, we studied the ultrastructure of gastroliths from freshwater crayfish by using a combination of microscopic and physical techniques. Because sugars are also molecules putatively involved in the elaboration process of these biomineralizations, we also determined their carbohydrate composition. This study was performed in a comparative perspective on crayfish species belonging to the infra-order Astacidea (Decapoda, Malacostraca): three species from the Astacoidea superfamily and one species from the Parastacoidea superfamily. We observed that all the gastroliths exhibit a similar dense network of protein-chitin fibers, from macro- to nanoscale, within which calcium is precipitated as amorphous calcium carbonate. Nevertheless, they are not very similar at the molecular level, notably as regards their carbohydrate composition. Besides glucosamine, the basic carbohydrate component of chitin, we evidenced the presence of other sugars, some of which are species-specific like rhamnose and galacturonic acid whereas xylose and mannose could be linked to proteoglycan components.

Nautilin-63, a novel acidic glycoprotein from the shell nacre of Nautilus macromphalus.

UNLABELLED: In molluscs, and more generally in metazoan organisms, the production of a calcified skeleton is a complex molecular process that is regulated by the secretion of an extracellular organic matrix. This matrix constitutes a cohesive and functional macromolecular assemblage, containing mainly proteins, glycoproteins and polysaccharides that, together, control the biomineral formation. These macromolecules interact with the extruded precursor mineral ions, mainly calcium and bicarbonate, to form complex organo-mineral composites of well-defined microstructures. For several reasons related to its remarkable mechanical properties and to its high value in jewelry, nacre is by far the most studied molluscan shell microstructure and constitutes a key model in biomineralization research. To understand the molecular mechanism that controls the formation of the shell nacreous layer, we have investigated the biochemistry of Nautilin-63, one of the main nacre matrix proteins of the cephalopod Nautilus macromphalus. After purification of Nautilin-63 by preparative electrophoresis, we demonstrate that this soluble protein is glycine-aspartate-rich, that it is highly glycosylated, that its sugar moieties are acidic, and that it is able to bind chitin in vitro. Interestingly, Nautilin-63 strongly interacts with the morphology of CaCO(3) crystals precipitated in vitro but, unexpectedly, it exhibits an extremely weak ability to inhibit in vitro the precipitation of CaCO(3) . The partial resolution of its amino acid sequence by de novo sequencing of its tryptic peptides indicates that Nautilin-63 exhibits short collagenous-like domains. Owing to specific polyclonal antibodies raised against the purified protein, Nautilin-63 was immunolocalized mainly in the intertabular nacre matrix. In conclusion, Nautilin-63 exhibits 'hybrid' biochemical properties that are found both in the soluble and insoluble proteins, rendering it difficult to classify according to the standard view on nacre proteins. DATABASE: The protein sequences of N63 appear on the UniProt Knowledgebase under accession number P86702.