Proteomic strategy for identifying mollusc shell proteins using mild chemical degradation and trypsin digestion of insoluble organic shell matrix: a pilot study on Haliotis tuberculata.

A successful strategy for the identification of shell proteins is based on proteomic analyses where soluble and insoluble fractions isolated from organic shell matrix are digested with trypsin with the aim of generating peptides, which are used to identify novel shell proteins contained in databases. However, using trypsin as a sole degradative agent is limited by the enzyme's cleavage specificity and is dependent upon the occurrence of lysine and arginine in the shell protein sequence. To bypass this limitation, we investigated the ability of trifluoroacetic acid (TFA), a low-specificity chemical degradative agent, to generate clusters of analyzable peptides from organic shell matrix, suitable for database annotation. Acetic acid-insoluble fractions from Haliotis tuberculata shell were processed by trypsin followed by TFA digestion. The hydrolysates were used to annotate an expressed sequence tag library constructed from the mantle tissue of Haliotis asinina, a tropical abalone species. The characterization of sequences with repeat motifs featured in some of the shell matrix proteins benefited from TFA-induced serial cutting, which can result in peptide ladder series. Using the degradative specificities of TFA and trypsin, we were able to identify five novel shell proteins. This pilot study indicates that a mild chemical digestion of organic shell matrix combined with trypsin generates peptides suitable for proteomic analysis for better characterization of mollusc shell matrix proteins.

Coupling proteomics and transcriptomics for the identification of novel and variant forms of mollusk shell proteins: a study with P. margaritifera.

Shell matrix proteins from Pinctada margaritifera were characterized by combining proteomics analysis of shell organic extracts and transcript sequences, both obtained from the shell-forming cell by using the suppression subtractive hybridization method (SSH) and from an expressed sequence tag (EST) database available from Pinctada maxima mantle tissue. Some of the identified proteins were homologues to proteins reported in other mollusk shells, namely lysine-rich matrix proteins (KRMPs), shematrins and molluscan prismatic and nacreous layer 88 kDa (MPN88). Sequence comparison within and among Pinctada species pointed to intra- and interspecies variations relevant to polymorphism and to evolutionary distance, respectively. In addition, a novel shell matrix protein, linkine was identified. BLAST analysis of the peptide sequences obtained from the shell of P. margaritifera against the EST database revealed the presence of additional proteins: two proteins similar to the Pif97 protein that was identified in the shell of P. fucata, a chitinase-like protein previously identified in Crassostrea gigas, two chitin-binding proteins, and two incomplete sequences of proteins unknown so far in mollusk shells. Combining proteomics and transcriptomics analysis we demonstrate that all these proteins, including linkine, are addressed to the shell. Retrieval of motif-forming sequences, such as chitin-binding, with functional annotation from several peptides nested in the shell could indicate protein involvement in shell patterning.

Proteomic analysis of the organic matrix of the abalone Haliotis asinina calcified shell.

BACKGROUND: The formation of the molluscan shell is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called "calcifying matrix" is a complex mixture of proteins and glycoproteins that is assembled and occluded within the mineral phase during the calcification process. While the importance of the calcifying matrix to shell formation has long been appreciated, most of its protein components remain uncharacterised. RESULTS: Recent expressed sequence tag (EST) investigations of the mantle tissue from the tropical abalone (Haliotis asinina) provide an opportunity to further characterise the proteins in the shell by a proteomic approach. In this study, we have identified a total of 14 proteins from distinct calcified layers of the shell. Only two of these proteins have been previously characterised from abalone shells. Among the novel proteins are several glutamine- and methionine-rich motifs and hydrophobic glycine-, alanine- and acidic aspartate-rich domains. In addition, two of the new proteins contained Kunitz-like and WAP (whey acidic protein) protease inhibitor domains. CONCLUSION: This is one of the first comprehensive proteomic study of a molluscan shell, and should provide a platform for further characterization of matrix protein functions and interactions.

Ultrastructure, chemistry and mineralogy of the growing shell of the European abalone Haliotis tuberculata.

An integrated study of shell formation was initiated covering the entire life cycle of the marine gastropod Haliotis tuberculata. Shell microstructure, chemistry and mineralogy were investigated by polarized microscopy, scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDX) and infra-red (IR) spectroscopy. SEM images of trochophore and veliger larvae showed the different stages of shell growth from the initial shell field to the late calcified protoconch. Cross-sections revealed the microstructural arrangement of biominerals, showing the progressive mineralization of the organic protoconch prior to metamorphosis. To gain more information on mineralogical composition, EDX analyses and IR spectroscopy were performed along the development stages. The results demonstrated that early protoconch was mostly composed of amorphous calcium carbonate, while veliger stages showed a gradually crystallization under the form of aragonite. Post-metamorphic shell contained two distinct parts, the original protoconch supporting the new juvenile shell characterized by a marked sculptural pattern. The shells from post-larval and juvenile abalones were essentially made of aragonite.

Evolution of nacre: biochemistry and proteomics of the shell organic matrix of the cephalopod Nautilus macromphalus.

In mollusks, one of the most widely studied shell textures is nacre, the lustrous aragonitic layer that constitutes the internal components of the shells of several bivalves, a few gastropods,and one cephalopod: the nautilus. Nacre contains a minor organic fraction, which displays a wide range of functions in relation to the biomineralization process. Here, we have biochemically characterized the nacre matrix of the cephalopod Nautilus macromphalus. The acid-soluble matrix contains a mixture of polydisperse and discrete proteins and glycoproteins, which interact with the formation of calcite crystals. In addition, a few bind calcium ions. Furthermore, we have used a proteomic approach,which was applied to the acetic acid-soluble and -insoluble shell matrices, as well as to spots obtained after 2D gel electrophoresis. Our data demonstrate that the insoluble and soluble matrices, although different in their bulk monosaccharide and amino acid compositions, contain numerous shared peptides. Strikingly, most of the obtained partial sequences are entirely new. A few only partly match with bivalvian nacre proteins.Our findings have implications for knowledge of the long-term evolution of molluskan nacre matrices.

Nacre calcification in the freshwater mussel Unio pictorum: carbonic anhydrase activity and purification of a 95 kDa calcium-binding glycoprotein.

The formation of the molluscan shell is finely tuned by macromolecules of the shell organic matrix. Previous results have shown that the acid-soluble fraction of the nacre matrix of the freshwater paleoheterodont bivalve Unio pictorum shell displays a number of remarkable properties, such as calcium-binding activity, the presence of extensive glycosylations and the capacity to interfere at low concentration with in vitro calcium carbonate precipitation. Here we have found that the nacre-soluble matrix exhibits a carbonic anhydrase activity, an important function in calcification processes. This matrix is composed of three main proteinaceous discrete fractions. The one with the highest apparent molecular weight is a 95 kDa glycoprotein that is specific to the nacreous layer. P95, as it is provisionally named, is enriched in Gly, Glx and Asx and exhibits an apparent pI value of approximately 4, or approximately 7 when chemically deglycosylated. Furthermore, its glycosyl moiety, consisting of sulfated polysaccharides, is involved in calcium binding. Purified fractions of the three main proteins were digested with trypsin, and the resulting peptides were analysed by mass spectrometry. Our results suggest that identical peptides are constitutive domains of the different proteins. Partial primary structures were obtained by de novo sequencing and compared with known sequences from other mollusc shell proteins. Our results are discussed from an evolutionary viewpoint.

Low molecular weight molecules of oyster nacre induce mineralization of the MC3T3-E1 cells.

The nacre layer from the pearl oyster shell is considered as a promising osteoinductive biomaterial. Nacre contains one or more signal molecules capable of stimulating bone formation. The identity and the mode of action of these molecules on the osteoblast differentiation were analyzed. Water-soluble molecules from nacre were fractionated according to dialysis, solvent extraction, and reversed-phase HPLC. The activity of a fraction composed of low molecular weight molecules in the mineralization of the MC3T3-E1 extracellular matrix was investigated. Mineralization of the preosteoblast cells was monitored according to alizarin red staining, Raman spectroscopy, scanning electron microscopy, and quantitative RT-PCR. Molecules isolated from nacre, ranging from 50 to 235 Da, induced a red alizarin staining of the preosteoblasts extracellular matrix after 16 days of culture. Raman spectroscopy demonstrated the presence of hydroxyapatite (HA) in samples treated with these molecules. Scanning electron microscopy pictures showed at the surface of the treated cells the occurrence of clusters of spherical particles resembling to HA. The treatment of cells with nacre molecules accelerated expression of collagen I and increased the mRNA expression of Runx2 and osteopontin. This study indicated that the nacre molecules efficient in bone cell differentiation are certainly different from proteins, and could be useful for in vivo bone repair.

The effect of molecules in mother-of-pearl on the decrease in bone resorption through the inhibition of osteoclast cathepsin K.

This study evaluates the effect of the mother-of-pearl (nacre) organic matrix on mammalian osteoclast activity and on cathepsin K protease. Rabbit osteoclasts were cultured on bovine cortical bone slices in the presence of water-soluble molecules extracted from nacre of the pearl oyster Pinctada margaritifera. Osteoclast resorption activity was determined by quantification of the resorption surface area on bovine bone slices. Papain and cathepsin K, B and L inhibition tests were performed in the presence of the nacre water-soluble extracts. The active crude extract was fractionated by dialysis and reversed-phase high-performance liquid chromatography before electrospray mass spectrometry analysis of inhibitory fractions. The water-soluble molecules extracted from nacre decreased bone resorption without jeopardizing osteoclast survival. The hydrolytic activity of cysteine proteinases was reduced when the enzymes were incubated with the nacre water-soluble molecules. Trending towards characterization of the molecules involved, it appears that cathepsin K inhibitors remain in different nacre water-soluble organic matrix subfractions, composed of low molecular weight molecules. Mollusk shell nacre contains molecules capable of reducing osteoclast bone resorption activity by inhibiting cathepsin K, giving a new facet of the bioactivity of nacre as bone biomaterial.

Proteomics analysis of the nacre soluble and insoluble proteins from the oyster Pinctada margaritifera.

Shell nacre is laid upon an organic cell-free matrix, part of which, paradoxically, is water soluble and displays biological activities. Proteins in the native shell also constitute an insoluble network and offer a model for studying supramolecular organization as a means of self-ordering. Consequently, difficulties are encountered in extraction and purification strategies for protein characterization. In this work, water-soluble proteins and the insoluble conhiolin residue of the nacre of Pinctada margaritifera matrix were analyzed via a proteomics approach. Two sequences homologous to nacre matrix proteins of other Pinctada species were identified in the water-soluble extract. One of them is known as a fundamental component of the insoluble organic matrix of nacre. In the conchiolin, the insoluble residue, four homologs of Pinctada nacre matrix proteins were found. Two of them were the same as the molecules characterized in the water-soluble extract. Results established that soluble and insoluble proteins of the nacre organic matrix share constitutive material. Surprisingly, a peptide in the conchiolin residue was found homologous to a prismatic matrix protein of Pinctada fucata, suggesting that prismatic and nacre matrices may share common proteins. The insoluble properties of shell matrix proteins appear to arise from structural organization via multimerization. The oxidative activity, found in the water-soluble fraction of the nacre matrix, is proposed as a leading process in the transformation of transient soluble proteins into the insoluble network of conchiolin during nacre growth.

Heterogeneity of proteinase inhibitors in the water-soluble organic matrix from the oyster nacre.

We extracted proteinase inhibitors from the nacre of the oyster Pinctada margaritifera with water. Mixing the nacre powder with water for 20 h led to a water-soluble fraction [0.24% (wt/wt) of nacre]. After dialysis of the water-soluble matrix through 6- to 8-kDa and 0.5-kDa membranes, the proteinase inhibitors were divided into low and high molecular weight fractions that contained inhibitors of papain, bovine cathepsin B, and human cathepsin L. We studied the heterogeneity of the inhibitors after separating the low molecular weight fraction according to charge and hydrophobicity. After multistep purification, mass spectrometry analysis revealed that a potent inhibitory fraction contained several molecules. This observation demonstrates the difficulties encountered in attempting to isolate individual metabolites from the complex mixture of molecules present in nacre matrix. Interestingly, the low molecular weight fraction contained specific inhibitors that could discern between cathepsin B and cathepsin L. The nacre organic inhibitors were active against several cysteine proteinases, yet they were more specific in relation to serine proteinases, because only proteinase K was inhibited. These results demonstrate, for the first time, the presence of active proteinase inhibitors in the mollusc shell, and it is possible that these inhibitors may play a role in either protection of proteins involved in shell formation or in defense against parasites, or both.

Zona localization of shell matrix proteins in mantle of Haliotis tuberculata (Mollusca, Gastropoda).

Organic matrix from molluscan shells has the potential to regulate calcium carbonate deposition and crystallization. Control of crystal growth thus seems to depend on control of matrix protein secretion or activation processes in the mantle cells, about which little is known. Biomineralization is a highly orchestrated biological process. The aim of this work was to provide information about the source of shell matrix macromolecule production, within the external epithelium of the mantle. An in vivo approach was chosen to describe the histologic changes in the outer epithelium and in blood sinus distribution, associated with mantle cells implicated in shell matrix production. Our results characterized a topographic and time-dependent zonation of matrix proteins involved in shell biomineralization in the mantle of Haliotis.

The water-soluble matrix fraction from the nacre of Pinctada maxima produces earlier mineralization of MC3T3-E1 mouse pre-osteoblasts.

Nacre or mother of pearl is a calcified structure that forms the lustrous inner layer of some shells. We studied the biological activity of the water-soluble matrix (WSM) extracted from powdered nacre from the shell of the pearl oyster, Pinctada maxima, on the MC3T3-E1 pre-osteoblast cell line from mouse calvaria. This cell line has the ability to differentiate into osteoblasts and to mineralize in the presence of beta-glycerophosphate and ascorbic acid. Cell proliferation and alkaline phosphatase activity were measured as markers of osteoblast differentiation, and mineralization was analyzed. These studies revealed that WSM stimulates osteoblast differentiation and mineralization by day 6 instead of the 21-day period required for cells grown in normal mineralizing media. We compared the activity of WSM with that of dexamethasone on this cell line. WSM can inhibit alkaline phosphatase (ALP) activity and the activity of dexamethasone on MC3T3-E1 cells. This study shows that nacre WSM could speed up the differentiation and mineralization of this cell line more effectively than dexamethasone.

Soluble silk-like organic matrix in the nacreous layer of the bivalve Pinctada maxima.

Nacre organic matrix has been conventionally classified as both 'water-soluble' and 'water-insoluble', based on its solubility in aqueous solutions after decalcification with acid or EDTA. Some characteristics (aspartic acid-rich, silk-fibroin-like content) were specifically attributed to either one or the other. The comparative study on the technique of extraction (extraction with water alone vs. demineralization with EDTA) presented here, seems to reveal that this generally accepted classification may need to be reconsidered. Actually, the nondecalcified soluble organic matrix, extracted in ultra-pure water, displays many of the characteristics of what until now has been called 'insoluble matrix'. We present the results obtained on this extract and on a conventional EDTA-soluble matrix, with various characterization methods: fractionation by size-exclusion and anion-exchange HPLC, amino acid analysis, glycosaminoglycan and calcium quantification, SDS/PAGE and FTIR spectroscopy. We propose that the model for the interlamellar matrix sheets of nacre given by Nakahara [In: Biomineralization and Biological Metal Accumulation, Westbroek, P. & deJong, E.W., eds, (1983) pp. 225-230. Reidel, Dordrecht, Holland] and Weiner and Traub [Phil. Trans. R. Soc. Lond. B (1984) 304, 425-434] may no longer be valid. The most recent model, proposed by Levi-Kalisman et al. [J. Struct. Biol. (2001) 135, 8-17], seemed to be more in accordance with our findings.

Bioactivity of nacre water-soluble organic matrix from the bivalve mollusk Pinctada maxima in three mammalian cell types: fibroblasts, bone marrow stromal cells and osteoblasts.

In vivo and in vitro studies provide strong evidence of the osteogenic activity of nacre obtained from Pinctada maxima. The in vitro studies indicate that diffusible factors from nacre are involved in cell stimulation. The water-soluble matrix (WSM) was extracted from nacre by a non-decalcifying process, and four fractions (SE(1)-SE(4)) were separated by SE-HPLC. Those fractions were tested in vitro on MRC5 fibroblasts. Alkaline phosphatase (ALP) activity was measured as a marker of osteoblastic differentiation. The anti-apoptotic protein Bcl-2 was also immunodetected in cultured osteoblasts from rat calvaria. WSM and fraction SE(4) increased ALP activity. BMP-2 had the same effect on the cells as WSM and SE(4). WSM greatly increased the amount of Bcl-2 in the cytoplasm and nucleus of osteoblasts. These in vitro studies support our initial hypothesis that nacre organic matrix (WSM) of a bivalve mollusk contains signal-molecules that can stimulate the osteogenic pathway in mammalian cells that are targets for bone induction.