Shell Matrix Research Articles

Cloning of Ovocalyxin-36, a Novel Chicken Eggshell Protein Related to Lipopolysaccharide-binding Proteins, Bactericidal Permeability-increasing Proteins, and Plunc Family Proteins

The avian eggshell is a composite biomaterial composed of noncalcifying eggshell membranes and the overlying calcified shell matrix. The shell is deposited in a uterine fluid where the concentration of different protein species varies at different stages of its formation. The role of avian eggshell proteins during shell formation remains poorly understood, and we have sought to identify and characterize the individual components in order to gain insight into their function during elaboration of the eggshell. In this study, we have used direct sequencing, immunochemistry, expression screening, and EST data base mining to clone and characterize a 1995-bp full-length cDNA sequence corresponding to a novel chicken eggshell protein that we have named Ovocalyxin-36 (OCX-36). Ovocalyxin-36 protein was only detected in the regions of the oviduct where egg-shell formation takes place; uterine OCX-36 message was strongly up-regulated during eggshell calcification. OCX-36 localized to the calcified eggshell predominantly in the inner part of the shell, and to the shell membranes. BlastN data base searching indicates that there is no mammalian version of OCX-36; however, the protein sequence is 20-25% homologous to proteins associated with the innate immune response as follows: lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and Plunc family proteins. Moreover, the genomic organization of these proteins and OCX-36 appears to be highly conserved. These observations suggest that OCX-36 is a novel and specific chicken eggshell protein related to the superfamily of lipopolysaccharide-binding proteins/bactericidal permeability-increasing proteins and Plunc proteins. OCX-36 may therefore participate in natural defense mechanisms that keep the egg free of pathogens.

The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z.

BackgroundChitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain.ResultsEnzymatic mollusc chitin synthesis was investigated in vivo by using the small-molecule drug NikkomycinZ, a structural analogue to the sugar donor substrate UDP-N-acetyl-D-glucosamine (UDP-GlcNAc). The impact on mollusc shell formation was analyzed by binocular microscopy, polarized light video microscopy in vivo, and scanning electron microscopy data obtained from shell material formed in the presence of NikkomycinZ. The partial inhibition of chitin synthesis in vivo during larval development by NikkomycinZ (5 μM – 10 μM) dramatically alters the structure and thus the functionality of the larval shell at various growth fronts, such as the bivalve hinge and the shell's edges.ConclusionProvided that NikkomycinZ mainly affects chitin synthesis in molluscs, the presented data suggest that the mollusc chitin synthase fulfils an important enzymatic role in the coordinated formation of larval bivalve shells. It can be speculated that chitin synthesis bears the potential to contribute via signal transduction pathways to the implementation of hierarchical patterns into chitin mineral-composites such as prismatic, nacre, and crossed-lamellar shell types.

Culture of mantle epithelial cells expressing shell matrix proteins from scallop Patinopecten yessoensis

In molluscs, mantle epithelial cells secrete organic matrix proteins to form shells. In this study, we established a culture of mantle epithelial cells by using the mantle pallial layer of scallops. We aimed to identify the mantle epithelial cells expressing scallop shell matrix proteins and establish a culture system of epithelial cells. After the mantle pallial layer was carefully isolated from the mantle tissue, explant culture was performed at 4°C. Most cells that migrated from the explant tissue were round cells. Most of the adhered cells retained round morphology, while some of the cells adhered to the dish and showed morphology similar to that of epithelial-like and fibroblast-like cells. When the cultured cells were immuno stained with a polyclonal antibody against the shell matrix protein, the antibody recognized many of the adhered cells. An estimation of the number of epithelial cells revealed that approximately 70% of the adhered cells were epithelial cells. This is the first report to describe epithelial cells in cultured mantle cells, which express shell matrix proteins. This culture system may be a useful method for characterization of the mantle epithelial cells.

Skeletal matrix proteins of invertebrate animals: Comparative analysis of their amino acid sequences

The genetic bases of skeletogenesis are expected to shed light on the origins of metazoan biomineralization. Here we review aspects of genetic machineries of invertebrate skeletogenesis, including regulatory genes involved in biomineralization as well, and with an enumerative reference to the genes encoding skeletal matrix proteins. The complete primary structure has been determined for a total of 77 skeletal matrix proteins in invertebrates representing five animal phyla. Presence of repeated sequences and prevalence of acidic proteins stand as common features among those proteins. Similarities are interpreted as convergence because these proteins are not similar at the primary structure level. C-type lectin-like domains are shared by the calcium carbonate skeletal matrix proteins of molluscs and deuterostomes. However, the important sites for carbohydrate binding are not conserved between these two groups. Several arthropod skeletal matrix proteins have the Rebers-Riddiford consensus sequence which is characteristic of non-calcified cuticular proteins of arthropods, indicating that these skeletal matrix proteins were recruited from the non-calcified cuticular proteins after arthropods diverged from other metazoan groups. Dermatopontin, a molluscan shell matrix protein, is also inferred to represent a cooption for biomineralization after molluscs diverged from other metazoan groups based on the molecular phylogemetic analysis. Those findings support the premise that the genetic machineries of biomineralization evolved independently many times after the divergence of metazoan phyla, and that some common genes that served for other functions have been coopted for biomineralization in various lineages.

A novel matrix protein family participating in the prismatic layer framework formation of pearl oyster, Pinctada fucata

Understanding the molecular composition and the formation mechanism of shell matrix framework is of great interest for biomineralization in mollusk shell. The cDNAs encoding a novel matrix protein family (KRMP) were cloned from the mantle of pearl oyster, Pinctada fucata. Analysis of the deduced amino acid sequences revealed that KRMP have a high proportion of lysine, glycine, and tyrosine, and their predict isoelectric points are higher than any other identified shell matrix protein to our knowledge. The deduced amino acid sequences of KRMP can be divided into three regions, including an N-terminal signal peptide, a lysine-rich basic region interacting with acidic proteins or CO 3 2−, and a Gly/Tyr-rich region involved in the protein cross-link via quinone-tanning process. RT-PCR and in situ hybridization demonstrated that KRMP mRNA was specifically expressed in the mantle edge, involved in the prismatic layer formation. Taken together, it seems that KRMP is a matrix protein family participating in the framework formation of prismatic layer.

Molecular Evolution and Functionally Important Structures of Molluscan Dermatopontin: Implications for the Origins of Molluscan Shell Matrix Proteins

A major shell matrix protein originally obtained from a freshwater snail is a molluscan homologue of Dermatopontins, a group of Metazoan proteins also called TRAMP (tyrosine-rich acidic matrix protein). We sequenced and identified 14 molluscan homologues of Dermatopontin from eight snail species belonging to the order Basommatophora and Stylommatophora. The bassommatophoran Dermatopontins fell into three types, one is suggested to be a shell matrix protein and the others are proteins having more general functions based on gene expression analyses. N-glycosylation is inferred to be important for the function involved in shell calcification, because potential N-glycosylation sites were found exclusively in the Dermatopontins considered as shell matrix proteins. The stylommatophoran Dermatopontins fell into two types, also suggested to comprise a shell matrix protein and a protein having a more general function. Phylogenetic analyses using maximum likelihood and Bayesian methods revealed that gene duplication events occurred independently in both basommatophoran and stylommatophoran lineages. These results suggest that the dermatopontin genes were co-opted for molluscan calcification at least twice independently after the divergence of basommatophoran and stylommatophoran lineages, or more recently than we have expected.

Development of Near Zero-Order Release Dosage Forms Using Three-Dimensional Printing (3-DP™) Technology

ABSTRACTThree near zero-order controlled-release pseudoephedrine hydrochloride (PEH) formulations demonstrating proportional release rates were developed using 3-Dimensional Printing (3-DP™) technology. Mixtures of Kollidon SR and hydroxypropylmethyl cellulose (HPMC) were used as drug carriers. The release rates were adjusted by varying the Kollidon SR-HPMC ratio while keeping fabrication parameters constant. The dosage forms were composed of an immediate release core and a release rate regulating shell, fabricated with an aqueous PEH and an ethanolic triethyl citrate (TEC) binder, respectively. The dosage form design called for the drug to be released via diffusional pathways formed by HPMC in the shell matrix. The release rate was shown to increase correspondingly with the fraction of HPMC contained in the polymer blend. The designed formulations resulted in dosage forms that were insensitive to changes in pH of the dissolution medium, paddle stirring rate, and the presence/absence of a sinker. The near zero-order release properties were unchanged regardless of the dissolution test being performed on either single cubes or on a group of eight cubes encased within a gelatin capsule shell. The chemical and dissolution properties of the three formulations remained unchanged following 1 month's exposure to 25°C/60% RH or 40°C/75% RH environment under open container condition. The in vivo performance of the three formulations was evaluated using a single-dose, randomized, open-label, four-way crossover clinical study composed of 10 fasted healthy volunteers. The pharmacokinetic parameters were analyzed using a noncompartmental model. Qualitative rank order linear correlations between in vivo absorption profiles and in vitro dissolution parameters (with slope and intercept close to unity and origin, respectively) were obtained for all three formulations, indicating good support for a Level A in vivo/in vitro correlation.

Biphasic and Dually Coordinated Expression of the Genes Encoding Major Shell Matrix Proteins in the Pearl Oyster Pinctada fucata

Regional expression patterns of shell matrix protein genes of Pinctada fucata were investigated using real-time quantitative polymerase chain reaction (PCR) and in situ hybridization. Six shell matrix proteins examined in this study indicated a distinct biphasic pattern of expression, falling into one of the following three groups: (1) expressed only in the more dorsal region of the mantle (MSI60 and N16); (2) expressed only in the more ventral region (MSI31, Prismalin-14, and Aspein); and (3) expressed in both regions (nacrein). The ubiquity of the last protein probably reflects its general role as a carbonate-producing enzyme, while the other groups are interpreted as corresponding to the distinction between the two varieties of shell layers, the aragonitic nacreous layer and the calcitic prismatic layer. In addition, the constituent genes of each of these two groups indicated similar levels of relative expression among different sites even among different individuals, suggesting that the genes of each group share a single upstream regulatory factor, respectively, and that these genes are expressed in a dually coordinated fashion.

The distribution of chitin in larval shells of the bivalve mollusk Mytilus galloprovincialis

The insoluble matrix of larval shells of the marine bivalve mollusk Mytilus galloprovincialis is investigated by confocal laser scanning microscopy using a GFP fusion protein with a chitin-binding domain for labeling of chitinous structures. We show that chitinous material is present in the larval shell, presumably as a chitin–protein complex. We further show that the structure of the chitinous material changes with the development of the larvae. We conclude from the presence of characteristic chitinous structures in certain shell regions that chitin fulfills an important function in the formation and functionality of larval bivalve shells.

CDNA clonings of shell matrix proteins from scallop shell

A cDNA encoding the matrix shell protein from scallop (MSP-2) has been cloned. Comparison of the sequence of MSP-2 with the GenBank database revealed the sequence displays a high degree of homology with MSP-1, which was recently identified in the shell of the scallop Patinopecten yessoensis. Matrix shell protein-2 contains only 323 amino acids, while MSP-1 comprises 824 amino acids. The structural difference between MSP-1 and MSP-2 is mainly the presence or absence of the tandem repeat arrangement. In addition, multiple isoforms of MSP-2 were found. These isoforms share a high degree of sequence similarity to each other, suggesting that the MSP-2, MSP-2 isoforms and MSP-1 may constitute the same family. The MSP-2 isoforms in scallop shell may be responsible for the formation of molluscan shells.

METABOLIC EFFECTS OF ACUTE EXPOSURE TO METHOPRENE IN THE AMERICAN LOBSTER, HOMARUS AMERICANUS

Methoprene was a constituent of the pesticide cocktail applied to the Western Long Island Sound (WLIS) watershed area during the summer of 1999. Subsequently, the seasonal lobster catches from the WLIS have decreased dramatically. We have been engaged in ongoing studies of the effects of methoprene on larval, juvenile and adult lobsters. Most recently, we found that Stage IV larvae exposed to 50 ppb methoprene experience >90% mortality rate after 3 days. Bioaccumulation studies on adult lobsters showed that methoprene concentrated against the gradient of the surrounding seawater (50 ppb) in hepatopancreas (1.55 ppm), gonad (5.18 ppm), epithelial tissue (6.17 ppm) and, most significantly, the eyestalks (28.83 ppm). Exposure to methoprene altered the expression of the stress proteins and the pattern of ubiquitinylation of cytosolic proteins by Day 1 Stage I larvae and by epithelial tissue of postmolt juvenile lobsters. Postmolt juvenile animals also demonstrated an altered pattern of protein phosphorylation in their epithelial tissues following methoprene exposure, indicating that it may interfere with cell signaling pathways. Increasing concentrations of methoprene were associated with increasing chitoproteins in the microsomal fractions of Day 1 Stage I larvae, suggesting that methoprene may compromise the exocytosis of shell matrix precursors from the epithelial cells. Methoprene did not, however, alter the activity of chitin synthase in these larvae. Although it is likely that a combination of harmful events and exposures led to the reduced lobster population in WLIS, methoprene may have contributed to the decline both by direct toxic effects and by disrupting homeostatic processes.

A growth model of the cockle ( Cerastoderma edule L.) tested in the Oosterschelde estuary (The Netherlands)

We present an ecophysiological model of the bivalve Cerastoderma edule that simulates individual growth and reproduction under ambient conditions in temperature and food availability in the Oosterschelde estuary, SW Netherlands. The model contains feedback loops in the uptake and metabolism of food and in the partitioning of carbon to the internal state variables: somatic tissue, storage, organic shell matrix and gametes. The model was calibrated for 24 parameters, based on random distributions of parameter values. This procedure includes an estimate of confidence intervals of the output variables. The simulated growth of shell length and animal wet and dry weight reflected the observed values of growth in the field for the period 1993 – 1997. The model is a tool for the integration of ecophysiological knowledge of this species and also for carrying-capacity studies of shellfish culture and for environmental management of populations in estuarine and coastal areas.

Structural Characterization of the Major Extrapallial Fluid Protein of the MolluscMytilus edulis: Implications for Function

The major protein component of the extrapallial fluid of the mollusc Mytilus edulis has been previously isolated and partially characterized. It was postulated to play a role in shell mineralization because of its intriguing property of Ca(2+)-binding-induced self-assembling. However, it also binds other divalent ions, including Cd(2+), Cu(2+), Mn(2+), and Mg(2+). Herein is the initial report on the characterization of the primary structure of the extrapallial (EP) protein by RT-PCR and cDNA sequencing methods and by de novo peptide sequencing with mass spectrometry. The EP protein is comprised of 213 amino acids postcleavage of a signal peptide of 23 amino acids. The protein is rich in His, Glu, and Asp residues. The site of N-glycosylation, "NHTE", at amino acid positions 54-57 and the intramolecular disulfide bond between Cys 139 and Cys 171 of the protein have been characterized also. Sequence comparisons reveal that the EP protein possesses little homology to any presently known matrix proteins previously isolated from mollusc shells but rather it highly resembles a heavy metal binding protein and a histidine-rich glycoprotein, both from the hemolymph of M. edulis. The predicted domain profile and amino acid composition suggest that its N-terminus may be involved in calcium binding. The abundance of histidine residues of the protein may account for its heavy metal binding properties. Thus, the EP protein perhaps has multiple functions, serving as a Ca(2+)-transport protein, a shell matrix protein, and a heavy metal detoxification protein.

Identifying repaired shell damage and abnormal calcification in the stout razor clam Tagelus plebeius as a tool to investigate its ecological interactions

Analysis of acetate peels of shell sections of the stout razor clam Tagelus plebeius from the Mar Chiquita coastal lagoon (37° 32′S, 57°19′W, Argentina) revealed the presence of a series of repaired shell margin breaks and different types of abnormal calcifications on the inner surface of their valves. Shell damage and subsequent repair was observed in 73% of the specimens analysed (70.3% of them with scars in both valves around the shell margin, 54.5% with the posterior shell area damaged in one or both valves, 30% with more than one damage). There were only few sediment grains incorporated in the shell matrix when scars occurred around the shell margin. A field experiment suggests that this pattern of shell damage results from natural re-burrowing (vertical movement) activities. However, 10% of the individuals showed an inner shell alteration forming a blister full of sediment grains in the area of the shell pallial sinus. These blisters were associated with repaired shell breaks in the posterior part of the valve, which was confirmed with a field experiment. The development of such blisters could be an indirect consequence of sub-lethal predatory attacks by the American oystercatcher Haematopus palliatus, given that this species breaks the posterior part of the shells of stout razor clams when extracting them from the sediments. The third type of shell alteration (94.3% of the shells) was an orange to brown coloration on the inner shell surface concurrently with irregular carbonate deposition that, in some cases, results in the formation of localised calcium carbonate concretions. This pattern is associated with the presence of metacercariae of gymnophallid parasites. In conclusion, Tagelus plebeius is able to repair its shell after damage produced by different agents. These repairs are very distinctive and, thus, they can be used as evidence of interactions between this clam and its environment (e.g. sediment characteristics) and the associated community (e.g., predators, parasites).

Enhancement of Natural Pigment Extraction Using Bacillus Species Xylanase

Pigment extracts from the root of Lithospermum erythrorhizon are used as natural red dyes, as well as basic drugs due to their numerous pharmacological activities. In recent years, the demand for such natural pigment materials has increased; however, in natural dye production, the pigment yield is strongly affected by the source of cultivation, extracting conditions, and solvents. Accordingly, this study proposes a method of enzymatic pigment production based on the introduction of hydrolytic enzymes prior to the usual extraction to avoid repeated pigment extraction. The matrix destruction in the epidermal layer of the root by the enzymes was found to improve the pigment extractability, that is, the increment of K(L), the mass transfer coefficient, representing the pigment mobility in the epidermal layer. The root tissue maceration by the hydrolytic enzymes was also measured to evaluate the pigment extractability, and a linear relationship was observed between the K(L) values and the tissue maceration up to the addition of 3000 units/g of xylanase, indicating that the enzymatic maceration proportionally increases the interfacial area between the pigment and the solvent. Bacillus sp. DX107 xylanase only served to increase the extractability of the pigment by loosening the root shell matrix, without affecting the contents and color properties of the pigment, as almost no difference was found in the color between the pigments extracted using xylanase and those extracted according to the traditional method.

Asprich: A Novel Aspartic Acid‐Rich Protein Family from the Prismatic Shell Matrix of the Bivalve Atrina rigida

Almost all mineralized tissues contain proteins that are unusually acidic. As they are also often intimately associated with the mineral phase, they are thought to fulfill important functions in controlling mineral formation. Relatively little is known about these important proteins, because their acidic nature causes technical difficulties during purification and characterization procedures. Much effort has been made to overcome these problems, particularly in the study of mollusk-shell formation. To date about 16 proteins from mollusk-shell organic matrices have been sequenced, but only two are unusually rich in aspartic and glutamic acids. Here we screened a cDNA library made from the mRNA of the shell-forming cells of a bivalve, Atrina rigida, using probes for short Asp-containing repeat sequences, and identified ten different proteins. Using more specific probes designed from one subgroup of conserved sequences, we obtained the full sequences of a family of seven aspartic acid-rich proteins, which we named "Asprich"; a subfamily of the unusually acidic shell-matrix proteins. Polyclonal antibodies raised against a synthetic peptide of the conserved acidic1 domain of these proteins reacted specifically with the matrix components of the calcitic prismatic layer, but not with those of the aragonitic nacreous layer. Thus the Asprich proteins are constituents of the prismatic layer shell matrix. We can identify different domains within these sequences, including a signal peptide characteristic of proteins destined for extracellular secretion, a conserved domain rich in aspartic acid that contains a sequence very similar to the calcium-binding domain of Calsequestrin, and another domain rich in aspartic acid, that varies between the seven sequences. We also identified a domain with DEAD repeats that may have Mg-binding capabilities. Although we do not know, as yet, the function of these proteins, their generally conserved sequences do indicate that they might well fulfill basic functions in shell formation.

The life history responses of the abalone pest, Terebrasabella heterouncinata, under natural and aquaculture conditions

The sabellid, Terebrasabella heterouncinata, is a small (<5 mm) intratubular brooder that lives in burrows within the host’s shell matrix. It is a semi-continuous breeder and despite producing small numbers of large eggs, infestation by this animal has reached epidemic proportions on local abalone farms. The present study compared the morphometrics and reproductive characteristics of worms from farmed and wild abalone, in the Walker Bay area of the south Western Cape Province of South Africa, to gain insights into why this animal has become so successful under aquaculture conditions. The farms designated farm A and farm B each had one “on-farm” site, and two wild sites, while farm C had two on-farm sites and two wild sites. The wild sites were natural abalone habitats located within 2.5 km of the farms. Our results conclusively showed that environmental conditions prevalent on the farms enhanced the reproductive success of these worms relative to that observed in its natural environment. At farms B and C, worms occurred in significantly higher densities at the on-farm sites than in the corresponding wild samples, but at farm A, density was equally low at the three sites. At all three farms, a greater proportion of the population was reproductively active in the on-farm samples than in the wild samples. Worms on farmed abalone had a higher instantaneous fecundity, brooded more clutches simultaneously and were larger than their conspecifics from the wild. There was a positive correlation between adult size and brood size and the number of clutches brooded simultaneously. Within the three on-farm sites there was a negative correlation between egg volume and brood size, indicating a trade-off between these traits. However, such a trade-off was not apparent between sites, with brood size being higher at the on-farm sites than at the wild sites, irrespective of egg size. This suggests that the stable nutrient-enriched environment on the farm led to an increase in fecundity without compromising the size (and implicitly the quality) of the eggs. Worm density did not have a significant effect on body size or any other reproductive traits at most sites, and the density of T. heterouncinata was unaffected by the density of other shell-infesting polychaetes. The results suggest that the farm environment has selected for larger, more fecund worms that breed rapidly with high recruitment success as a consequence of abundant nutrients, high host density, habitat stability and a possible lack of predation and interspecific competition.

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.

Conservation of signal molecules involved in biomineralisation control in calcifying matrices of bone and shell

Bone and nacre feature highly orchestrated biomineralised structures. They are calcified structures that share an intervening organic matrix laid by specialized cells, bone cells in vertebrates and epithelial mantle cells in molluscs. Organic framework provides a scaffold for crystallisation and directs mineralisation. Bone and shell likely undergo self-reparation when damaged, clues to the presence in these mineralised structures of signal molecules, targeting the cells involved in their renewal. In molluscs, calcium carbonate crystallise under two mineral polymorphs, aragonite or calcite. Sudo et al. [22] reported two exclusive proteins in the shell of a bivalvia molluscan, Pinctada fucata, MSI 60 in aragonite and MSI 31 in calcite. We built up specific antibodies and experimented immunohistochemistry in Haliotis. These tools revealed the presence of Pinctada-like proteins within Haliotis mantle cells, in a zonation consistent with shell matrix mineralisation control involvement. Another facet of matrix-mediated biomineralisation control arises from bone and nacre interactivity. In vivo experiments of nacre implantation in sheep and rabbit established the biocompatibilty of nacre and bone. The nacreous part from the shell of Pinctada, a bivalve mollusc, was implanted as bone device in sheep and showed that nacre passes bone acceptance. Nacre implants were not subjected to intolerance reaction and the recipient bone provided with nacre underwent a sequence of bone regeneration within an osteoprogenitor-rich cell layer. The water-soluble organic matrix was extracted from powdered nacre by a gentle non-decalcifiying process. Three mammalian cell types, fibroblasts (human), bone marrow stromal cells (rat) and pre-osteoblasts (mouse) were used to characterize in vitro the effect of nacre water-soluble matrix on mammal bone cell lineage. In vitro studies provided evidence for the presence, in nacre organic matrix, of signal molecules responsible for the recruitment of mammal cells in the osteogenic pathway and bone cell activation undergoing a complete sequence of mineralisation. Retrieving like-proteins in shell matrix of molluscs from distant taxa and bone nacre interactivity provides convergent data supporting the conservation of molecular signals for biomineralisation control within the organic framework of biomineralised structures. To cite this article: C. Milet et al., C. R. Palevol 3 (2004) .

Molluscan shell proteins

The shell secreted by molluscs is one of the most remarkable examples of a matrix-mediated mineralisation performed outside living tissues. The calcifying matrix is a mixture of proteins, glycoproteins, and polysaccharides that precisely self-assemble and control the CaCO 3 polymorph (calcite, aragonite), the size, the shapes of the crystallites, and finally, the texture of the shell. In spite of several biochemical studies, the molecular aspects of the shell building are far from being understood. The present article makes an overview of the most recent molecular data on the proteinaceous components of the shell matrix. These data put into question the classical models of molluscan mineralisation. Furthermore, they show that shell proteins are diverse and multifunctional and that they may have different origins. To cite this article: F. Marin, G. Luquet, C. R. Palevol 3 (2004) .