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Novel Proteins from the Calcifying Shell Matrix of the Pacific Oyster Crassostrea gigas
Abstract
The shell of the Pacific oyster Crassostrea gigas is composed of more than 99% CaCO₃ and of around 0.5% of occluded organic matrix. According to classical views, this matrix is supposed to regulate the shell mineral deposition. In this study, we developed one of the first proteomic approaches applied to mollusk shell in order to characterise the calcifying matrix proteins. The insoluble organic matrix, purified after demineralisation of the shell powder, was digested with trypsin enzyme, and separated on nano-LC, prior to nanospray quadrupole/time-of-flight analysis. MS/MS spectra were searched against the above 220,000 EST sequences available in the public database for Crassostrea. Using this approach, we were able to identify partial or full-length sequence transcripts that encode eight novel shell matrix proteins.
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... DEGs encoding secreted proteins were further investigated whether they (i) were likely to encode shell matrix proteins (SMPs) based upon similarity to proteins that had previously been identified from C. gigas shells, or they had transcriptional regulatory or signaling activity roles. Similarity to SMPs was performed by BLASTP searchers (Camacho et al., 2009) against an in-house database of published proteins that had previously been identified from C. gigas larval and adult shells (Marie et al., 2011;Zhang et al., 2012;Zhao et al., 2018), using an e-value of 1e -100 . As many SMPs contain repetitive low-complexity domains (RLCDs) McDougall et al., 2013;Aguilera et al., 2017), BLASTP searches were conducted without filtering for low-complexity regions and without compositional adjustment. ...
... However, the structure of the periostracal groove is somehow different in that two cell types are distinguishable in C. gigas, columnar cells to the base and planar cells to the tip of the periostracal groove, which are not observed in pearl oysters (Jabbour-Zahab et al., 1992;Parvizi et al., 2017). Given the morphological similarities in the mantle tissue between C. gigas and pearl oysters, it is likely that the differences observed, in terms of the shape and microstructure of their shells (Farre et al., 2011;Lee et al., 2011;Marie et al., 2011;Marie et al., 2012;Mouchi et al., 2016;Checa et al., 2018;Deng et al., 2022), are the product of different shell matrix proteins secreted by the mantle in a species-specific manner (Aguilera et al., 2017), rather than as a product of mantle morphology. Inside the mantle tissue, there are contractile muscle fibers possibly involved in mantle retraction and adipose tissue containing high glycogen content (Berthelin et al., 2000;Álvarez Nogal and Molist Garcıá, 2015;Parvizi et al., 2017). ...
... The mineralogical composition of adult oyster shell is mostly calcite and a small portion of aragonite in the myostracum layer, which is located at the shell inner surface in intimate connection with the adductor muscle (Lee et al., 2011;Marie et al., 2011;Mouchi et al., 2016;Checa et al., 2018). In contrast to most bivalves, C. gigas adult shell textures correspond to a shell-like foliae layer and lenses of chalk (Marie et al., 2011;Checa et al., 2018), a highly porous and poorly organized microstructure that is only found in Ostreidae (Mouchi et al., 2016;Checa et al., 2018). ...
Since the Industrial Revolution, the concentration of atmospheric carbon dioxide (CO 2 ) due to anthropogenic activities has increased at unprecedented rates. One-third of the atmospheric anthropogenic CO 2 emissions are dissolved in the oceans affecting the chemical equilibrium of seawater, which in turn leads to a decrease in pH and carbonate ion (CO 3 ²⁻ ) concentration, a phenomenon known as ocean acidification (OA). This chemical disequilibrium can be detrimental to marine organisms (e.g., mollusks) that fabricate mineralized structures based on calcium carbonate (CaCO 3 ). Most studies on the effect of reduced pH in seawater have been conducted on the early developmental stages of shell-building invertebrates, given less attention to how adult individuals face OA stress. Here, we evaluate histological, secretory, and transcriptional changes in the mantle of adult oysters ( Crassostrea gigas ) exposure to ambient (8.0 ± 0.2) and reduced (7.6 ± 0.2) pH during 20 days. Most histological observations did not show differences in terms of mantle cell morphology. However, Alcian Blue/PAS staining revealed significant differences in the number of Alcian Blue positive cells in the mantle edge, suggesting a decrease in the secretory activity in this morphogenetic zone. Transcriptomic analysis revealed 172 differentially expressed genes (DEGs) between mantle tissues from adult oysters kept in normal and reduced pH conditions. Almost 18% of the DEGs encode secreted proteins that are likely to be contributing to shell fabrication and patterning. 17 of 31 DEGs encoding secreted proteins correspond to oyster-specific genes, highlighting the fact that molluscan shell formation is underpinned by a rapidly evolving secretome. The GO analysis of DEGs encoding secreted proteins showed that they are involved in the cellular response to stimulus, response to stress, protein binding, and ion binding, suggesting these biological processes and molecular functions are altered by OA. This study demonstrates that histology and gene expression profiling can advance our understanding of the cellular and molecular mechanisms underlying adult oyster tolerance to low pH conditions.
... Pacific oysters, like other oyster species, act as foundation species by creating habitats for other estuarine species and providing key ecosystem services to human communities around the world [3][4][5]. Pacific oysters have been widely used as a model species for studying molecular genetics [1,2,6], developmental biology [7][8][9][10], biomineralization [11,12], and adaptation to coastal environments under climate change [13]. Beck et al. [14] have reported that oyster populations have significantly declined on a global scale, with an estimated 85% of oyster reefs gone, far outnumbering the projected loss of coral reefs. ...
... Shells of C. gigas are made up of 99% calcium carbonate and around 0.5% occluded organic matrix [11]. This organic matrix is composed of a combination of proteins, glycoproteins, and polysaccharides that self-assemble and govern the shell's calcium carbonate polymorph (calcite, aragonite), crystallite size, shape, and texture [11]. ...
... Shells of C. gigas are made up of 99% calcium carbonate and around 0.5% occluded organic matrix [11]. This organic matrix is composed of a combination of proteins, glycoproteins, and polysaccharides that self-assemble and govern the shell's calcium carbonate polymorph (calcite, aragonite), crystallite size, shape, and texture [11]. Several molluscan shell matrix proteins (SMPs) have been recently identified in shells, including that of C. gigas, but much of the focus has been on their biomineralization roles [41,42]. ...
The global decline of natural oyster populations emphasizes the need to improve our understanding of their biology. Understanding the role of chemical cues from conspecifics on how oysters occupy appropriate substrata is crucial to learning about their evolution, population dynamics, and chemical communication. Here, a novel role of a macromolecular assembly of shell matrix proteins which act as Crassostrea gigas Settlement Pheromone Protein Components in adult shells is demonstrated as the biological cue responsible for gregarious settlement on conspecifics. A bioassay-guided fractionation approach aided by biochemical and molecular analyses reveals that Gigasin-6 isoform X1 and/or X2 isolated from adult shells is the major inducing cue for larval settlement and may also play a role in postlarva–larva settlement interactions. Other isolated Stains-all-stainable acidic proteins may function as a co-factor and a scaffold/structural framework for other matrix proteins to anchor within this assembly and provide protection. Notably, conspecific cue-mediated larval settlement induction in C. gigas presents a complex system that requires an interplay of different glycans, disulfide bonds, amino acid groups, and phosphorylation crosstalk for recognition. These results may find application in the development of oyster aquacultures which could help recover declining marine species and as targets of anti-fouling agents.
... Folia has not been reported in shells of Pteriidae 40 but is extensively highlighted in shells of Crassostrea spp., representing up to 60-70% of the global shell volume 88,89 . The foliated structure is characterised by an assembly of thin sheets made of laterally joined calcitic blade-like laths (100-400 nm thick, 2-5 μm wide 90,91,51 ) with arrow-point tips, as presented in Fig. 8j 47,91,40,13 . The sheets are stacked Chapter I. Oyster shell recycling: applications and future prospects perpendicular to the inner shell surface, forming a microstructure with a thickness > 100 μm in shells of C. gigas 88,45,91 (Fig. 8k). ...
... Folia has not been reported in shells of Pteriidae 40 but is extensively highlighted in shells of Crassostrea spp., representing up to 60-70% of the global shell volume 88,89 . The foliated structure is characterised by an assembly of thin sheets made of laterally joined calcitic blade-like laths (100-400 nm thick, 2-5 μm wide 90,91,51 ) with arrow-point tips, as presented in Fig. 8j 47,91,40,13 . The sheets are stacked Chapter I. Oyster shell recycling: applications and future prospects perpendicular to the inner shell surface, forming a microstructure with a thickness > 100 μm in shells of C. gigas 88,45,91 (Fig. 8k). ...
... The foliated structure is characterised by an assembly of thin sheets made of laterally joined calcitic blade-like laths (100-400 nm thick, 2-5 μm wide 90,91,51 ) with arrow-point tips, as presented in Fig. 8j 47,91,40,13 . The sheets are stacked Chapter I. Oyster shell recycling: applications and future prospects perpendicular to the inner shell surface, forming a microstructure with a thickness > 100 μm in shells of C. gigas 88,45,91 (Fig. 8k). This highly ordered lamellar structure has a well-defined crystallographic orientation, as reported by Checa et al. (2007) 47 . ...
This thesis program was carried out in a partnership between the oyster farming company Tarbouriech-Médithau, located on the edge of the Thau lagoon (Marseillan), and two institutes of The Balard Chemistry Centre of The University of Montpellier: The Institute of Biomolecules Max Mousseron and The Institute Charles Gerhart Montpellier. The main objective was the identification of valuable compounds from the shells of the oyster Crassostrea gigas Tarbouriech-Médithau with singular morphological and organoleptic properties. This study was positioned within the more general framework of the recycling of the shell of this edible oyster as a source of natural materials, highlighting the advantages and challenges related to its recycling as well as the need to participate to the investigation particular fundamental aspects not described to date.Thus, the pink-purple colour of the shell of the oyster Crassostrea gigas was investigated in order to extract and identify the biomolecules contributing to this colour. The extraction required the development of original and reliable methods, applicable to the extraction of acid-soluble pigments. The structural investigation of these compounds conducted by reverse phase liquid chromatography combined with high-resolution mass spectrometry, by fluorescence spectroscopy and by nuclear magnetic resonance, made possible the newly identification of heterocyclic biomolecules such as porphyrins. The extraction and the identification of these compounds open the way to applications and allow to consider the development of innovative recycling processes of shells of the oyster Crassostrea gigas.
... Pau_g16754.t1 containing protein-like (SVEP1-like) by a BLASTX search (table 1). SVEP is a common SMP in Mollusca (Marie et al. 2010(Marie et al. , 2011(Marie et al. , 2012Mann et al. 2012;Zhang et al. 2012;Gao et al. 2015;Arivalagan et al. 2016) and has VWA (von Willebrand factor type A), EGF domain, and CCP (complement control protein) modules. However, it is difficult to annotate these proteins (Equ15222-15223 and Equ21247) as SVEP1, because they have a Sushi domain but lack other three domains (VWA, EGF, and CCP). ...
... last accessed January 25, 2019) and found 18 domains from 24 matrix proteins (table 1). We then compared them with the domains that were found in 12 previously studied molluscan species (grove snail, Cepaea nemoralis; abalone, H. asinina; limpet, L. gigantea; oyster, Crassostrea gigas; pearl oyster, P. margaritifera and P. maxima; mussel, Mytilus galloprovincialis and M. edulis; clam, Mya truncata; king scallop, P. maximus; fresh water mussels, Elliptio complanata and V. lienosa) (Marie et al. 2011(Marie et al. , 2013(Marie et al. , 2017Mann et al. 2012;Zhang et al. 2012;Mann and Jackson 2014;Gao et al. 2015;Arivalagan et al. 2016Arivalagan et al. , 2017 (supplementary table S7, Supplementary Material online). We showed the results of the domain comparisons among four gastropods E. quaesita, C. nemoralis, H. asinina, and L. gigantea in figure 6. ...
... Several kinds of protease inhibitors (e.g., Kunitz-like, WAP, macroglobulin) have already been found as SMPs in molluscs (Marie et al. 2011(Marie et al. , 2017Zhang et al. 2012;Arivalagan et al. 2017). Protease inhibitors could be involved in the protection of SMPs against several kinds of protease or in the regulation of an immune response pathway (Arivalagan et al. 2016). ...
Over the past decade, many skeletal matrix proteins that are possibly related to calcification have been reported in various calcifying animals. Molluscs are among the most diverse calcifying animals and some gastropods have adapted to terrestrial ecological niches. Although many shell matrix proteins have already been reported in molluscs, most reports have focused on marine molluscs, and the shell matrix proteins (SMPs) of terrestrial snails remain unclear. In addition, some terrestrial stylommatophoran snails have evolved an additional unique calcified character, called a "love dart", used for mating behavior. We identified 54 SMPs in the terrestrial snail Euhadra quaesita, and found that they contain specific domains that are widely conserved in molluscan SMPs. However, our results also suggest that some of them possibly have evolved independently by domain shuffling, domain recruitment, or gene co-option. We then identified four dart matrix proteins (DMPs), and found that two of them are the same proteins as those identified as SMPs. Our results suggest that some DMPs possibly have evolved by independent gene co-option from SMPs during dart evolution events. These results provide a new perspective on the evolution of SMPs and "love darts" in land snails.
... In particular, a 1/10 ratio between ASM and AIM quantified in this study is frequently found for the matrices of several nacro-prismatic bivalves (Marie et al. 2007). Our ASM:AIM values are also comparable to that of Marie et al. (2011) who extracted the equivalent of 0.4% of AIM and 0.05% of ASM from the same species. When fractionated using electrophoresis, the matrix-both ASM and LS-AIM-is constituted of a mixture of polydisperse ('smear') and few discrete macromolecules, i.e. proteins, and the two fractions exhibit similar electrophoresis patterns. ...
... Gigasin-6 is a 34 kDa (302 AA residue long), leucine-rich protein with a basic pI, which exhibits a C beta-lactamase-like domain, a domain that catalyses the opening and hydrolysis of the beta-lactamine ring of this class of antibiotics, which include penicillins and cephalosporins. Interestingly, Gigasin-6 was one of the eight proteins that were identified by us in a former study on the shell constituents of C. gigas (Marie et al. 2011). Its function in calcification remains unknown. ...
... Only specific areas of the chalky deposits, standing out from the rest of these structures, are stained by SER324 different function than that related to apoptosis. Note that this protein was not identified in our former proteomic study (Marie et al. 2011) on the whole matrix: we observed indeed that performing proteomics on electrophoresis fractions improve proteomics signals and allows the identification of rare proteins that are currently overshadowed by abundant ones in the mixture of the skeletal matrix macromolecules (Kanold et al. 2015). One intriguing aspect of our proteomic analysis is that the two nacrein-like proteins exhibit a molecular weight higher than that expected from the electrophoresis fraction (26-30 kDa), while that of the cell death abnormality protein 1-like and of Gigasin-6 fit approximately into this molecular weight range. ...
Mollusc shells are organic–inorganic biocomposites, arranged in a limited number of superimposed calcified
layers that generally exhibit very different organization of their crystallites. Because of their attractive mechanical and crystallographic properties, these shell layers have been the focus of several physical and biochemical characterizations. In particular, recent proteomic data obtained from individual layers suggest that their protein contents are different. However, the direct visual evidence that some macromolecular
components are layer-specific is rather tenuous. This paper is based on a non-conventional immunogold labelling approach to localize proteins in the shell of the edible oyster Crassostrea gigas. The shell microstructure of this model organism is predominantly composed of foliated calcite, interspersed by discontinuous pockets of ‘chalky layers’, a porous microstructure typical of bivalves of the ostreid family. By developing a polyclonal antibody (in two rats) elicited against a proteinaceous shell fraction, we obtained differential staining of the two microstructures. We assert that our labelling is microstructure discriminant. The difference in labelling of the two shell microstructures suggests either that they are formed by a variation
of the secretory repertoire of the shell-forming cells of the calcifying mantle epithelium or that the chalky layer may be formed via a completely different mechanism. Our results allow a first glimpse on the subtle regulatory mechanisms that drive the process of chalky and foliated layers deposition.
... Although this method does not discriminate between genes involved in biomineralization and those that are not, in silico predictions of secreted proteins have been used to identify and compare putative shell matrix proteins (SMPs) in several taxa [63,72,78,79]. Further, proteomic studies have revealed proteins secreted by the mantle that are actually incorporated into the shell [45,64,[79][80][81][82][83][84][85][86][87][88], narrowing the gap in our understanding of gene expression in the mantle epithelium and the final destination of proteins in mineralized structures. Despite advances in our understanding in this area, relatively few comparative studies have been performed and taxon sampling has been limited to just two of the eight of the major lineages of Mollusca (Bivalvia and Gastropoda). ...
... Many of the novel genes comprising the mantle secretome include RLCD-containing proteins [63,78,82]. These domains can either be in completely novel domain configurations or be combined with more ancient domains, such as observed in carbonic anhydrases [92]. ...
... Despite the unclear origin and evolution of RLCDcontaining proteins, their prevalence suggests that proteins containing these domains are important components of the mantle secretome [63,78,82]. The apparent high rate of evolution of RLCD-containing proteins may contributeat least in partto the high levels of gene novelty found in all mantle secretomes examined to date. ...
An external skeleton is an essential part of the body plan of many animals and is thought to be one of the key factors that enabled the great expansion in animal diversity and disparity during the Cambrian explosion. Molluscs are considered ideal to study the evolution of biomineralization because of their diversity of highly complex, robust and patterned shells. The molluscan shell forms externally at the interface of animal and environment, and involves controlled deposition of calcium carbonate within a framework of macromolecules that are secreted from the dorsal mantle epithelium. Despite its deep conservation within Mollusca, the mantle is capable of producing an incredible diversity of shell patterns, and macro- and micro-architectures. Here we review recent developments within the field of molluscan biomineralization, focusing on the genes expressed in the mantle that encode secreted proteins. The so-called mantle secretome appears to regulate shell deposition and patterning and in some cases becomes part of the shell matrix. Recent transcriptomic and proteomic studies have revealed marked differences in the mantle secretomes of even closely-related molluscs; these typically exceed expected differences based on characteristics of the external shell. All mantle secretomes surveyed to date include novel genes encoding lineage-restricted proteins and unique combinations of co-opted ancient genes. A surprisingly large proportion of both ancient and novel secreted proteins containing simple repetitive motifs or domains that are often modular in construction. These repetitive low complexity domains (RLCDs) appear to further promote the evolvability of the mantle secretome, resulting in domain shuffling, expansion and loss. RLCD families further evolve via slippage and other mechanisms associated with repetitive sequences. As analogous types of secreted proteins are expressed in biomineralizing tissues in other animals, insights into the evolution of the genes underlying molluscan shell formation may be applied more broadly to understanding the evolution of metazoan biomineralization.
... Willebrand factor D and EGF domain-containing protein, VWA domain-containing protein 7 (Vwa), collagen alpha-1(X) chain and perlucin-like protein were downregulated. EGF domain-containing proteins were found to be involved in shell formation in Crassostrea gigas, exhibiting sequence similarities with proteins extracted from prismatic layer of pearl oysters (Marie et al., 2011). Thus, the downregulation of expression of EGF domain-containing protein coding genes could potentially make the prismatic layer more soluble under OA. ...
... The reduction in organic content of the shell is thought to be mainly due to the loss of periostracal proteins and prismatic layer proteins as no microstructural impairment was observed in folia. If there was less expression of SMPs from the folia layer, the microstructure will also be affected, as proteins are important in the shell microstructure formation (Borbas et al., 1991;Checa, 2018;Marie et al., 2011). This loss of periostracum organic layer leaves the shell further prone to dissolution as periostracum acts as a protective layer (Peck et al., 2016). ...
Biomineralisation is one of the key processes that is notably affected in marine calcifiers such as oysters under ocean acidification (OA). Understanding molecular changes in the biomineralisation process under OA and its heritability, therefore, is key to developing conservation strategies for protecting ecologically and economically important oyster species. To do this, in this study we have explicitly chosen the tissue involved in biomineralisation (mantle) of an estuarine commercial oyster species, Crassostrea hongkongensis. The primary aim of this study is to understand the influence of DNA methylation over gene expression of mantle tissue under decreased ~ pH 7.4, a proxy of OA, and to extrapolate if these molecular changes can be observed in the product of biomineralisation – the shell. We grew early juvenile C. hongkongensis, under decreased ~ pH 7.4 and control ~ pH 8.0 over 4.5 months and studied OA‐induced DNA methylation and gene expression patterns along with shell properties such as microstructure, crystal orientation and hardness. The population of oysters used in this study was found to be moderately resilient to OA at the end of the experiment. The expression of key biomineralisation related genes such as carbonic anhydrase and alkaline phosphatase remained unaffected, thus, mechanical properties of the shell (shell growth rate, hardness and crystal orientation) were also maintained without any significant difference between control and OA conditions with signs of severe dissolution. In addition, this study makes three major conclusions: 1) higher expression of Ca2+ binding/signalling related genes in the mantle play a key role in maintaining biomineralisation under OA, 2) DNA methylation changes occur in response to OA, however, these methylation changes do not directly control gene expression, 3) OA would be more of a ‘dissolution problem’ rather than a ‘biomineralisation problem’ for resilient species that maintain calcification rate with normal shell growth and mechanical properties.
... The outer calcified part is composed of calcite prisms of 20-200 lm in height and 5-40 lm in width, while the middle and inner shell layers include usually a combination of three types of microstructures: regularly foliated, crossed foliated and chalky deposits (Carter 1990). The foliated structure consists of arrow point ending tabular laths measuring 2-5 lm wide and 200-250 lm thick (Checa et al. 2007;Marie et al. 2011b). The chalky structures are composed of platy calcite petals of variable sizes. ...
... Such a finding might suggest that the inducing factors could be similar, or at least, that functional domains responsible for the effect could be similar, whatever the shell proteins that exhibit these functional domains. Recently, the identification of some shell matrix proteins from the shell of Crassostrea gigas has been performed (Marie et al. 2011b;Oliveira et al. 2012). Among the putative protein candidates to investigate further, gigasin-2 contains two conserved epidermal growth factor (EGF) domains. ...
Mollusc shells are composed of more than 95% calcium carbonate and less than 5% organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. In this study, we investigated the effects of matrix macromolecular components extracted from the shells of two edible molluscs of economic interest, i.e., the blue mussel Mytilus edulis and the Pacific oyster Crassostrea gigas. The potential biological activities of these organic molecules were analysed on human dermal fibroblasts in primary culture. Our results demonstrate that shell extracts of the two studied molluscs modulate the metabolic activities of the cells. In addition, the extracts caused a decrease of type I collagen and a concomitant increase of active MMP-1, both at the mRNA and the protein levels. Therefore, our results suggest that shell extracts from M. edulis and C. gigas contain molecules that promote the catabolic pathway of human dermal fibroblasts. This work emphasises the potential use of these shell matrices in the context of anti-fibrotic strategies, particularly against scleroderma. More generally, it stresses the usefulness to valorise bivalve shells that are coproducts of shellfish farming activity.
... The presence of these various VWA and chitin-binding domains suggests that these proteins might participate in structuring the chitin/protein nacre scaffold. In addition, we identified other ECM-related proteins, such as DMP-like, MSP130-like, mytilin3-like, ANF-receptor containing or Ca-binding proteins, for which homologues have previously been described from other proteins associated with biomineralization [30][31][32] . The involvement of these proteins in biomineralization processes is discussed below. ...
... So far, no other function than those related to biomineralization has been described for this protein. Interestingly, during our previous investigation of the shell proteins of the Pacific oyster Crassostrea gigas, we detected a protein, called gigasin-3, whose partial sequence exhibits a noticeable similarity to S. purpuratus MSP-130 [31]. Even more recently, the msp130 gene was also identified in a calcified tube-forming polychaete [65]. ...
The formation of the molluscan shell nacre 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, glycoproteins and polysaccharides that is assembled and occluded
within the mineral phase during the calcification process. Better molecularlevel
characterization of the substances that regulate nacre formation is still
required. Notable advances in expressed tag sequencing of freshwater mussels,
such as Elliptio complanata and Villosa lienosa, provide a pre-requisite to
further characterize bivalve nacre proteins by a proteomic approach. In this
study, we have identified a total of 48 different proteins from the insoluble
matrices of the nacre, 31 of which are common to both E. complanata and
V. lienosa. A few of these proteins, such as PIF, MSI60, CA, shematrin-like,
Kunitz-like, LamG, chitin-binding-containing proteins, together with A-,
D-, G-, M- and Q-rich proteins, appear to be analogues, if not true homologues,
of proteins previously described from the pearl oyster or the edible
mussel nacre matrices, thus forming a remarkable list of deeply conserved
nacre proteins. This work constitutes a comprehensive nacre proteomic
study of non-pteriomorphid bivalves that has enabled us to describe the
molecular basis of a deeply conserved biomineralization toolkit among nacreous
shell-bearing bivalves, with regard to proteins associated with other
shell microstructures, with those of other mollusc classes (gastropods,
cephalopods) and, finally, with other lophotrochozoans (brachiopods).
... Such a novel shell microstructure proves particularly beneficial for the sessile lifestyle of oysters, providing them with a robust and flexible attachment mechanism. Previously, various SMPs have been identified and characterized in the whole shell of Pacific oyster Crassostrea gigas, using proteomic approaches [28][29][30][31]. These studies have significantly expanded our understanding of the complex protein composition of the oyster shell. ...
Background
Molluscan shell, composed of a diverse range of architectures and microstructures, is a classic model system to study the relationships between molecular evolution and biomineralized structure formation. The shells of oysters differ from those of other molluscs by possessing a novel microstructure, chalky calcite, which facilitates adaptation to the sessile lifestyle. However, the genetic basis and evolutionary origin of this adaptive innovation remain largely unexplored.
Results
We report the first whole-genome assembly and shell proteomes of the Iwagaki oyster Crassostrea nippona. Multi-omic integrative analyses revealed that independently expanded and co-opted tyrosinase, peroxidase, TIMP genes may contribute to the chalky layer formation in oysters. Comparisons with other molluscan shell proteomes imply that von Willebrand factor type A and chitin-binding domains are basic members of molluscan biomineralization toolkit. Genome-wide identification and analyses of these two domains in 19 metazoans enabled us to propose that the well-known Pif may share a common origin in the last common ancestor of Bilateria. Furthermore, Pif and LamG3 genes acquire new genetic function for shell mineralization in bivalves and the chalky calcite formation in oysters likely through a combination of gene duplication and domain reorganization.
Conclusions
The spatial expression of SMP genes in the mantle and molecular evolution of Pif are potentially involved in regulation of the chalky calcite deposition, thereby shaping the high plasticity of the oyster shell to adapt to a sessile lifestyle. This study further highlights neo-functionalization as a crucial mechanism for the diversification of shell mineralization and microstructures in molluscs, which may be applied more widely for studies on the evolution of metazoan biomineralization.
... Shell matrix proteins have been recovered from and studied in different marine calcifying taxa, including sponges, mollusks, corals, brachiopods, and echinoderms [18][19][20][21][22]. From these studies, certain conserved functional proteins such as carbonic anhydrase have been recovered from the shell making tissues or direct from the soluble organic shell matrix [23]. ...
Background
As a group of sessile crustaceans that were being misclassified as mollusks by Carl Linnaeus, barnacles produce calcareous shell plates which, in most species, are permanently attached to the substratum. As biomineralization has independently evolved in multiple marine invertebrate taxa, a key question is how biomineralization has driven the evolution of genetic toolkits underlying shell formation. Here, we explore the shell proteome of the deep-sea acorn barnacle Bathylasma hirsutum (Hoek, 1883) using an integrated transcriptomic-proteomic approach and compare the properties of barnacle shell proteins with molluscan shell matrix proteins.
Results
We identified 31 B. hirsutum barnacle shell proteins (BSPs), including a series of key biomineralization proteins, such as carbonic anhydrase and C-type lectin. More than half of barnacle specific shell proteins (BSSPs) exhibit unknown functions. The amino acid composition of these BSSPs were biased toward A, D, E, G, S, P and Q, and were acidic and hydrophilic. Almost all BSSPs were detected with repetitive low complexity domains. Similar to molluscan shell matrix proteins, RLCDs in D-, and E-rich BSSPs constituted up to 50% amino acid of the whole protein. RLCDs in Q-rich proteins also exhibited similarity to a Q-rich abalone shell matrix protein and an insect cuticle protein.
Conclusion
From the B. hirsutum shell proteome, certain proteins such as carbonic anhydrase, C-type lectin, and peroxidase were implicated in shell formation or protein cross-linking across sessile invertebrate taxa. Despite the lack of sequence homology, D- and Q-rich BSSPs share similar features with molluscan shell matrix proteins in sequence redundancy, amino acid bias and thereby protein isoelectric point and hydropathy. Such convergence may reflect that similar selection pressures shape the molecular evolution of biomineralization and shell formation genes in marine invertebrates.
... In non-model organisms such as fish, complete proteome data is not available, except the model organism such as zebrafish. The main areas of research in aquaculture are related to improving health status of fish, reproduction, and developmental biology (Schultz et al. 2013;Karlsen et al. 2011;Talakhun et al. 2014;Klinbunga et al. 2012;Marie et al. 2011) to understand the immunology and physiology for species that represent commercial and wild stock (Provan et al. 2013;Braceland et al. 2013). The first database of fish, FISHPROT (http://www.cifri.ernet.in/Fishprot/index.html), in India was developed by the Central Inland Fisheries Research Institute (CIFRI), Barrackpore, especially for biomarker discovery and evaluation. ...
Fish is a crucial source of nutritious food all over the world. Aquatic ecosystems are highly exposed to anthropogenic contaminants, leading to questions about the availability of high-quality nutritional sources that are safe. It is important to identify pathways in the reproduction and development of fish due to ecosystem stressors in order to enhance well-being and monitoring programs for fish. Xenobiotics accumulate in the organism’s reproductive and developmental systems and cause toxicity. An advanced technique such as proteomic approach can solve the present issue in commercially important species in the wild as well as aquaculture to evaluate developmental biology, physiological state, and the impact of environmental toxicity in fish. The recent enhancement of the mass spectrometry approach of proteomic methods can lead to the future in the field of biology. Identified proteins could map crucial possible mechanisms of toxicity of xenobiotics in ecosystems. In this chapter, we surveyed scientific reports that were published related to proteomics and advanced tool of proteomics to classify recent developments in the xenobiotic sector and for extensive interpretations of biomarkers in fish physiology for toxicity.KeywordsApplicationsBiomarkerProteomicsQuantificationXenobiotics
... In non-model organisms such as fish, complete proteome data is not available, except the model organism such as zebrafish. The main areas of research in aquaculture are related to improving health status of fish, reproduction, and developmental biology Karlsen et al. 2011;Talakhun et al. 2014;Klinbunga et al. 2012;Marie et al. 2011) to understand the immunology and physiology for species that represent commercial and wild stock (Provan et al. 2013;Braceland et al. 2013). The first database of fish, FISHPROT (http://www.cifri.ernet.in/Fishprot/index.html), in India was developed by the Central Inland Fisheries Research Institute (CIFRI), Barrackpore, especially for biomarker discovery and evaluation. ...
Among the major concerns associated globally with aquaculture or fish farming, top-notch and prime one is the treatment and management of xenobiotics. Aquatic ecosystems are heavily and negatively impacted by these chemicals. Xenobiotics impact aquaculture industry in a number of ways, be it the decline in the production or remaining in the system for so long that it hampers treatment and management. Because of its presence for a large time in waterbodies, these compounds can build up in food chains via the consumption of aquaculture products. There are good reports where these chemicals build-up in human body leading to cancers. One of the main agendas of xenobiotic management is its degradation. Degradation involves bioremediation (bioattenuation, biostimulation, and bioaugmentation). Burkholderia, Bacillus, Pseudomonas, Sphingomonas, Kocuria, Chromohalobacter, and Achromobacter have been reported to play a critical role in the degradation of these compounds. Bioremediation process can be improved with advancements in molecular biological techniques like genome editing, which allows the modification of microbial strains with an increased capacity for digesting several xenobiotics simultaneously and/or at a quick rate the bioremediation process can be improved.KeywordsXenobioticDegradationBioremediationGenome editing
... Classic models of oyster or bivalve biomineralization proposed by biologists focus on the secreted SMPs that are incorporated in specific microstructures of the shell [6][7][8][9][10][11]; whereas the directional solidification model on molluscan biomineralization proposed by material scientists describes biomineralization as a directional process where the crystallization starts from the top periostracum template (organic coating on shell) as random nano granules and reaches steady state towards the mantle epithelium (shell architecture (figure 1b) and directional fabrication model (figure 1c) [12][13][14]. The directional solidification model considers the biochemical control exerted by the organism through SMPs as boundary conditions that guide the 'self-assembly' of the shell [14]. ...
Biomineralization is one of the key biochemical processes in calcifying bivalve species such as oysters that is affected by ocean acidification (OA). Larval life stages of oysters are made of aragonite crystals whereas the adults are made of calcite and/or aragonite. Though both calcite and aragonite are crystal polymorphs of calcium carbonate, they have different mechanical properties and hence it is important to study the micro and nano structure of different life stages of oyster shells under OA to understand the mechanisms by which OA affects biomineralization ontogeny. Here, we have studied the larval and juvenile life stages of an economically and ecologically important estuarine oyster species, Crassostrea hongkongensis, under OA with focus over shell fabrication under OA (pHNBS 7.4). We also look at the effect of parental exposure to OA on larvae and juvenile microstructure. The micro and nanostructure characterization reveals directional fabrication of oyster shells, with more organized structure as biomineralization progresses. Under OA, both the larval and juvenile stages show directional dissolution, i.e. the earlier formed shell layers undergo dissolution at first, owing to longer exposure time. Despite dissolution, the micro and nanostructure of the shell remains unaffected under OA, irrespective of parental exposure history.
... The organic matrix is the major source of macromolecules including glycoproteins, chitin, and acidic polysaccharides that play important roles in crystal nucleation and growth (Marin et al., 2005;Addadi et al., 2006;Suzuki et al., 2009). Shell matrix proteins (SMPs) have been identified from a variety of mollusk species using genomic, transcriptomic, and proteomic approaches in Crassostrea gigas (Marie et al., 2011;Zhang et al., 2012), Hyriopsis cumingii (Berland et al., 2013), Mytilus edulis (Liao et al., 2015), Haliotis asinine (Marie et al., 2010) and ...
Shell formation is a dynamic process involving organic matrix secretion and calcification. In this study, we characterized shell morphogenesis during larval development in Crassostrea gigas. Using scanning electron microscopy (SEM) and fluorescence staining, we demonstrated that shell field, the first morphologically distinguishable shell-forming tissue, became visible soon after enlargement of the blastopore at the anterior end of the trochophore. Shell organic matrix namely protein polysaccharides and calcified structure appeared as a slit at the dorsal side of the embryo. The early shell field began to extend along the dorsal side of the trochophore larvae, and became a saddle shaped shell field that gave rise to the prodissoconch I embryonic shell in the early D-shaped larvae. Subsequently, prodissoconch II shell was formed in the late D-shaped larvae with a characteristic appearance of growth lines. To identify gene expression markers for studying shell formation, we isolated three potential larval shell formation genes CgPOU2F1, CgSox5, and CgPax6 and analyzed their expression during shell morphogenesis. The three potential shell formation genes possessed a similar pattern of expression. Their expression was detected in the shell gland and shell field regions in early D-shaped larvae, hereafter, their expression was detected at the larval mantle edge in the calcified shell stages. Together, these studies provide knowledge of shell morphogenesis in pacific oyster and molecular markers for studying the molecular regulation of biomineralization and shell formation.
... Interestingly, gigasin-6 isoforms X1 and X2, as well as dentin sialophosphoprotein-like, were upregulated in the conspecific cue-induced PL batch. Gigasin-6 and its isoforms have been reported as a component in the adult shell matrix in C. gigas [60,61], while our sequence homology search on dentin sialophosphoproteinlike showed close homology to folian-cv1 in C. viriginica [62]. Others were related to signal regulation processes such as calmodulin A (LOC105327998), Wnt inhibitory factor 1/EGF-like domain containing protein 2 (Wif-1) (LOC105335286), tyrosine-protein kinase receptor Tie (LOC105338560), beta-2 adrenergic receptor (LOC105336903), nuclear receptors (LOC105338842, LOC105345246), and FMFRamide receptor (LOC105343238). ...
The molecular mechanisms underlying the conspecific cue-mediated larval settlement in Crassostrea gigas is not yet fully understood. In this study, we described and compared the transcriptomes of competent pediveligers (Pedi) and conspecific cue-induced postlarvae (PL). A total of 2383 candidate transcripts were identified: 740 upregulated and 1643 downregulated transcripts, after settlement. Gene Ontology analysis revealed active chitin binding, calcium ion binding, and extracellular region processes in both stages. Results showed that the differential expression trend of six candidate transcripts were consistent between the quantitative real-time PCR and transcriptome data. The differential transcript expression related to shell formation showed closely linked dynamics with a gene regulatory network that may involve the interplay of various hormone receptors, neurotransmitters, and neuropeptide receptors working together in a concerted way in the Pedi and PL stages. Our results highlight the transcriptome dynamics underlying the settlement of oysters on conspecific adult shells and demonstrate the potential use of this cue as an attractant for wild and hatchery-grown oyster larval attachment on artificial substrates. It also suggests the possible involvement of an ecdysone signal pathway that may be linked to a neuroendocrine-biomineralization crosstalk in C. gigas settlement.
... The epidermal growth factor (EGF)-like domain is a common domain in molluscan SMPs (Aguilera et al. 2017). In the Pacific oyster C. gigas, Gigasin-2, which contains a signal peptide and two EGF-like domains in tandem, was first identified by LC-MS/MS analysis (Marie, Zanella-Cléon, Guichard, et al. 2011). Later, similar EGF-like proteins (EGFL proteins) were found as SMPs from a number of bivalves Zhang et al. 2012;Gao et al. 2015;Liao et al. 2015;Iwamoto et al. 2020). ...
Several types of shell matrix proteins (SMPs) have been identified in molluscan shells. Their diversity is the consequence of various molecular processes, including domain shuffling and gene duplication. However, the evolutionary origin of most SMPs remains unclear. In this study, we investigated the evolutionary process EGF-like and Zona Pellucida (ZP) domains containing SMPs. Two types of the proteins (EGFL and EGFZP) were found in the pearl oyster, Pinctada fucata. In contrast, only EGFZP was identified in the gastropods. Phylogenetic analysis and genomic arrangement studies showed that EGFL and EGFZP formed a clade in bivalves, and their encoding genes were localized in tandem repeats on the same scaffold. In P. fucata, EGFL genes were expressed in the outer part of mantle epithelial cells are related to the calcitic shell formation. However, in both P. fucata and the limpet Nipponacmea fuscoviridis, EGFZP genes were expressed in the inner part of the mantle epithelial cells are related to aragonitic shell formation. Furthermore, our analysis showed that in P. fucata, the ZP domain interact with eight SMPs that have various functions in the nacreous shell mineralization. The data suggest that the ZP domain can interact with other SMPs, and EGFL evolution in pterimorph bivalves represents an example of neo-functionalization that involves the acquisition of a novel protein through gene duplication.
... (Livingston et al., 2006;Mann et al., 2008;Mann et al., 2010;Mann and Edsinger, 2014). Such an organic matrix is formed prior to secretion of the mineralized part of the shell and is thus of crucial importance for conchiferan mollusks, but the respective factors involved are also present in other metazoans that lack a shell (Supplementary Table S2) (Marie et al., 2010;Marie et al., 2011a;Marie et al., 2011b;Marie et al., 2012;Marin et al., 2014). Altogether, our data point towards a shared "molecular biomineralization toolbox" across Metazoa, but a broader taxon sampling especially from key invertebrate phyla are required for deeper evolutionary insights. ...
Mollusks are known for their highly diverse repertoire of body plans that often includes external armor in form of mineralized hardparts. Representatives of the Conchifera, one of the two major lineages that comprises taxa which originated from a uni-shelled ancestor (Monoplacophora, Gastropoda, Cephalopoda, Scaphopoda, Bivalvia), are particularly relevant regarding the evolution of mollusk shells. Previous studies have found that the shell matrix of the adult shell (teleoconch) is rapidly evolving and that the gene set involved in shell formation is highly taxon-specific. However, detailed annotation of genes expressed in tissues involved in the formation of the embryonic shell (protoconch I) or the larval shell (protoconch II) are currently lacking. Here, we analyzed the genetic toolbox involved in embryonic and larval shell formation in the quagga mussel Dreissena rostriformis using single cell RNA sequencing. We found significant differences in genes expressed during embryonic and larval shell secretion, calling into question ontogenetic homology of these transitory bivalve shell types. Further ortholog comparisons throughout Metazoa indicates that a common genetic biomineralization toolbox, that was secondarily co-opted into molluscan shell formation, was already present in the last common metazoan ancestor. Genes included are engrailed, carbonic anhydrase, and tyrosinase homologs. However, we found that 25% of the genes expressed in the embryonic shell field of D. rostriformis lack an ortholog match with any other metazoan. This indicates that not only adult but also embryonic mollusk shells may be fast-evolving structures. We raise the question as to what degree, and on which taxonomic level, the gene complement involved in conchiferan protoconch formation may be lineage-specific or conserved across taxa.
... We assumed dry shell weight was 100% CaCO 3 and that other components of shell (e.g., protein matrix, periostracum) were negligible (Marie et al., 2011). Because these oysters are harvested and then largely exported to other watersheds, this net growth represents net calcification within the growing area. ...
Many studies have examined the vulnerability of calcifying organisms, such as the eastern oyster (Crassostrea virginica), to externally forced ocean acidification, but the opposite interaction whereby oysters alter their local carbonate conditions has received far less attention. We present an exploratory model for isolating the impact that net calcification and respiration of aquacultured eastern oysters can have on calcite and aragonite saturation states, in the context of varying temperature, ocean-estuary mixing, and air-sea gas exchange. We apply the model to the Damariscotta River Estuary in Maine which has experienced rapid expansion of oyster aquaculture in the last decade. Our model uses oyster shell growth over the summer season and a previously derived relationship between net calcification and respiration to quantify impacts of net oyster calcification and gross metabolism on carbonate saturation states in open tidal waters. Under 2018 industry size and climate conditions, we estimate that oysters can lower carbonate saturation states by up to 5% (i.e., 0.17 and 0.11 units on calcite and aragonite saturation states, respectively) per day in late summer, with an average of 3% over the growing season. Perturbations from temperature and air-sea exchange are similar in magnitude. Under 2050 climate conditions and 2018 industry size, calcite saturation state will decrease by up to an additional 0.54 units. If the industry expands 3-fold by 2050, the calcite and aragonite saturation states may decrease by 0.73 and 0.47 units, respectively, on average for the latter half of the growing season when compared to 2018 climate conditions and industry size. Collectively, our results indicate that dense aggregations of oysters can have a significant role on estuarine carbonate chemistry.
... Perlucin regulates shell formation by accelerating CaCO 3 precipitation and biomineralization (Lin et al. 2013), and perlucin-like proteins are involved in shell growth in both Haliotis discus hannai and P. fucata (Shi and He 2014;Choi et al. 2015). Fibronectin-related proteins have five type-III domains for integrin binding and cell adhesion and may interact with other extracellular matrix proteins in bivalve shells (Marie et al. 2011;Zhang et al. 2012). Collagen, elastin, and keratin are the most common fibrous proteins in eukaryotes found in connective tissues (including bones and cartilages), skin, and epithelial appendages (nails and hair) (Tatham and Shewry 2000;Fratzl 2008;Langel 2009). ...
Growth of the eastern oyster Crassostrea virginica, a major aquaculture species in the USA, is highly variable and not well understood at molecular levels. As growth of mollusks is confined in shells constructed by the mantle, mantle transcriptomes of large (fast-growing) and small (slow-growing) eastern oysters were sequenced and compared in this study. Transcription was observed for 31,186 genes, among which 104 genes were differentially expressed between the large and small oysters, including 48 upregulated and 56 downregulated in large oysters. Differentially expressed genes (DEGs) included genes from diverse pathways highlighting the complexity of shell formation and growth regulations. Seventeen of the 48 upregulated DEGs were related to shell matrix formation, most of which were upregulated in large oysters, indicating that large oysters are more active in biomineralization and shell formation. Genomic and transcriptomic analyses identified 22 genes encoding novel polyalanine containing proteins (Pacps) with characteristic motifs for matrix function that are tandemly duplicated on one chromosome, all specifically expressed in mantle and at higher levels in large oysters, suggesting that these expanded Pacps play important roles in shell formation and growth. Analysis of sequence variation identified 244,964 SNPs with 328 associated with growth. This study provides novel candidate genes and markers for shell formation and growth, and suggests that genes related to shell formation are important for the complex regulation of growth in the eastern oyster and possibly other bivalve mollusks. Results of this study show that both transcriptional modulation and functional polymorphism are important in determining growth.
... Although previous studies have demonstrated the potential osteogenic activity of matrix proteins by proteomics [11], there is no direct evidence of a synergistic effect between matrix proteins and bone remodeling. Therefore, this study mainly examines the effects of the matrix proteins in oyster shells on osteoblasts in vitro and vivo. ...
Matrix protein is secreted by the membrane of bivalve shellfish to and used to regulate shell biomineralization. In this study, we extracted water-soluble matrix protein (WSMP) from oyster shells to investigate its effects on osteogenic differentiation and mineralization of MC3T3-E1 cells and osteoporosis rats. Our results suggested that WSMP was an acidic glycoprotein by amino acid analysis and secondary structure analysis. In vitro, WSMP could promote osteoblastic proliferation. Moreover, alkaline phosphatase (ALP) and osteocalcin (OCN) were increased, mineralized nodules were increased, and BMP-2 expression was up-regulated. Additionally, in vivo, tartrate-resistant acid phosphatase (TRAP) and Bone alkaline phosphatase (BALP) expressions in the medium-dose and high-dose groups were significantly decreased compared with the model group, while OCN expression was significantly increased. Bone mineral density (BMD) and bone mineral content (BMC) of bone recovered significantly. In summary, WSMP can promote the proliferation, differentiation and mineralization of osteoblasts in vitro and in vivo.
... The SMPs together with sugars, polysaccharides, lipids and pigments, form an extracellular organic matrix that is secreted to the extrapallial space, at the interface between the shell and the mantle tissue, and directly induces crystal nucleation, growth, morphology and deposition (Addadi and Weiner, 1985;Mann, 1988;Albeck et al., 1996;Belcher et al., 1996;Samata et al., 1999;Kono et al., 2000). The process results in a stable and well-packed organo-mineral assemblage with 96-99% of mineral phase and up to 4% of organic matrix, depending on the mollusc species (Marie et al., 2009(Marie et al., , 2011Ramos-Silva et al., 2012). The exceptional mechanical properties of the shell biomaterials are due to: (1) the three-dimensional arrangement of crystal aggregates, i.e. shell microstructures, and (2) the occluded organic matrix (Wainwright et al., 1982;Weiner and Addadi, 1997). ...
Shelled pteropods, known as ‘sea butterflies’, are a group of small gastropods that spend their entire lives swimming and drifting in the open ocean. They build thin shells of aragonite, a metastable polymorph of calcium carbonate. Pteropod shells have been shown to experience dissolution and reduced thickness with a decrease in pH and therefore represent valuable bioindicators to monitor the impacts of ocean acidification. Over the past decades, several studies have highlighted the striking diversity of shell microstructures in pteropods, with exceptional mechanical properties, but their evolution and future in acidified waters remains uncertain. Here, we revisit the body-of-work on pteropod biomineralization, focusing on shell microstructures and their evolution. The evolutionary history of pteropods was recently resolved, and thus it is timely to examine their shell microstructures in such context. We analyse new images of shells from fossils and recent species providing a comprehensive overview of their structural diversity. Pteropod shells are made of the crossed lamellar and prismatic microstructures common in molluscs, but also of curved nanofibers which are proposed to form a helical three-dimensional structure. Our analyses suggest that the curved fibres emerged before the split between coiled and uncoiled pteropods and that they form incomplete to multiple helical turns. The curved fibres are seen as an important trait in the adaptation to a planktonic lifestyle, giving maximum strength and flexibility to the pteropod thin and lightweight shells. Finally, we also elucidate on the candidate biomineralization genes underpinning the shell diversity in these important indicators of ocean health.
... These data agree with previous data reported for oyster shells. 21 We also verified that there was no change in the mineral phase during the milling process. In fact, the X-ray diffraction pattern and the FTIR spectrum revealed that only magnesium calcite was present after the grinding ( Figure SI3). ...
Seashells are a calcium-carbonate-based material that can be converted into valuable advanced functional materials. Seashells are also a waste material from aquaculture. They are produced in millions of tonnes per year and represent an environmental issue. They uniquely contain an intraskeletal organic matrix rich in carboxylate groups that so far has not been exploited or has been even removed, when they were used as calcium carbonate substitutes. The intraskeletal organic matrix allows for a so far never reported covalent functionalization. Such a process strengthens the surface functionalization with respect to adsorption and, most importantly, opens up the possibility for the functionalization of the biogenic calcium carbonate with a wide variety of molecules by means of organic chemistry reactions. As a proof of concept, powdered waste oyster shells were covalently functionalized with a fluorescent probe. The impact of this research can be terrific in the valorization of CaCO3 from biogenic wastes providing advanced functional products tailored for individual applications. Moreover, its consequences on the environment and society will epitomize a perfect example of a circular economy.
... Cette protéine possède toutefois des activités anti-Listeria monocytogenes et anti-Salmonella enterica sv. Enteritidis, dont elle limite la croissance, grâce à son domaine de liaison à l'héparine (Rehault-Godbert et al, 2013).4.3 Caractérisation des protéines identifiées dans les matrices organiques d'invertébrés biominéralisantsDe nombreuses analyses protéomiques et transcriptomiques ont permis d'étudier la composition des matrices organiques associées aux biominéraux chez un certain nombre d'invertébrés biominéralisants, tant chez les mollusques(Bedouet et al, 2007b, Joubert et al, 2010, Marie et al, 2010a, Kinoshita et al, 2011, Marie et al, 2011a, Marie et al, 2011b, Marie et al, 2011c que chez les coraux(Drake et al, 2013, Ramos-Silva et al, 2013 ou les oursins. Différentes protéines ont également été isolées et leurs fonctions ont pu être caractérisées. ...
... oysters and clams) has been the most widely investigated. Key bivalve species include pearl oysters of the Pinctada genus and edible oysters such as the Pacific oyster Crassostrea gigas [213][214][215][216][217][218][219][220][221][222][223][224][225][226][227][228][229][230][231]. These studies set the foundation for genome-wide surveys of shell-forming genes and combined with gene knockdown experiments have guided selective breeding programs towards the development of high quality pearl farming [210,214,229,[232][233][234][235][236][237]. ...
Gastropods are the largest and most diverse class of mollusc and include species that are well studied within the areas of taxonomy, aquaculture, biomineralization, ecology, microbiome and health. Gastropod research has been expanding since the mid-2000s, largely due to large-scale data integration from next-generation sequencing and mass spectrometry in which transcripts, proteins and metabolites can be readily explored systematically. Correspondingly, the huge data added a great deal of complexity for data organization, visualization and interpretation. Here, we reviewed the recent advances involving gastropod omics ('gastropodomics') research from hundreds of publications and online genomics databases. By summarizing the current publicly available data, we present an insight for the design of useful data integrating tools and strategies for comparative omics studies in the future. Additionally, we discuss the future of omics applications in aquaculture, natural pharmaceutical biodiscovery and pest management, as well as to monitor the impact of environmental stressors.
... Bulk analysis of decalcified oyster shells shows that the organic component contains proteins [13][14][15][16], which are often enriched in acidic residues and which can be post-translationally modified, as well as other components like polysaccharides. Furthermore, in situ techniques [17][18][19][20] have localized proteins to sheath-like structures that cover individual CaCO 3 crystallites. ...
Infrared (IR) vibrational scattering scanning near-field optical microscopy ( s -SNOM) has advanced to become a powerful nanoimaging and spectroscopy technique with applications ranging from biological to quantum materials. However, full spatiospectral s -SNOM continues to be challenged by long measurement times and drift during the acquisition of large associated datasets. Here, we demonstrate a novel approach of computational spatiospectral s -SNOM by transforming the basis from the stationary frame into the rotating frame of the IR carrier frequency. We demonstrate an acceleration of IR s -SNOM data collection by a factor of 10 or more in combination with prior knowledge of the electronic or vibrational resonances to be probed, the IR source excitation spectrum, and other general sample characteristics. As an example, we apply rotating-frame s -SNOM (R-sSNOM) to chemical nanoimaging of ultrathin protein sheets in a mollusk shell. R-sSNOM enables high-voxel-density imaging of sparsely distributed molecules in an extended matrix. It is generally applicable to many multiscale material systems with sparse features and can be extended to other spectroscopic nanoimaging modalities.
... [27,33]). Several of the differentially expressed transcripts are from genes known to code for parts of the shell matrix, such as nacrein [42], papilin (also a metalloprotease inhibitor; [40]), chitin-binding protein [43] and a protein with a beta-lactamase domain that is known to be part of the shell matrix, but with a currently unknown function [44]. Nacrein has a carbonic anhydrase domain [42], and has previously been shown to be strongly expressed prior to the initiation of shell formation in blue mussels [45]. ...
Background:
Despite recent work to characterize gene expression changes associated with larval development in oysters, the mechanism by which the larval shell is first formed is still largely unknown. In Crassostrea gigas, this shell forms within the first 24 h post fertilization, and it has been demonstrated that changes in water chemistry can cause delays in shell formation, shell deformations and higher mortality rates. In this study, we use the delay in shell formation associated with exposure to CO2-acidified seawater to identify genes correlated with initial shell deposition.
Results:
By fitting linear models to gene expression data in ambient and low aragonite saturation treatments, we are able to isolate 37 annotated genes correlated with initial larval shell formation, which can be categorized into 1) ion transporters, 2) shell matrix proteins and 3) protease inhibitors. Clustering of the gene expression data into co-expression networks further supports the result of the linear models, and also implies an important role of dynein motor proteins as transporters of cellular components during the initial shell formation process.
Conclusions:
Using an RNA-Seq approach with high temporal resolution allows us to identify a conceptual model for how oyster larval calcification is initiated. This work provides a foundation for further studies on how genetic variation in these identified genes could affect fitness of oyster populations subjected to future environmental changes, such as ocean acidification.
... The rapid development of molecular biology since the mid-twentieth century has assisted in determining the entire sequence of matrix proteins found in the mollusc shell. Exhaustive analyses (e.g., proteome, transcriptome, and whole genome analyses) of mollusc shell formation have only recently begun (Joubert et al. 2010;Berland et al. 2011;Marie et al. 2010Marie et al. , 2011aMarie et al. , 2011bMarie et al. , 2011c. To reveal the mechanism of formation of the shell microstructure, we identified key organic molecules using functional analysis. ...
... Epidermal growth factor (EGF) domain-containing SMPs were first identified from C. gigas 44 and named as Cgigas-IMSP-2.They were subsequently discovered in P. maxima, P. margaritifera, and L. gigantea 12,14 . Generally, the SMP consists of both EGF-like domain and one zona pellucida (ZP) domain. ...
The calcifying shell is an excellent model for studying biomineralization and evolution. However, the molecular mechanisms of shell formation are only beginning to be elucidated in Mollusca. It is known that shell matrix proteins (SMPs) play important roles in shell formation. With increasing data of shell matrix proteomes from various species, we carried out a BLASTp bioinformatics analysis using the shell matrix proteome from Crassostrea gigas against 443 SMPs from nine other species. The highly conserved tyrosinase and chitin related proteins were identified in bivalve. In addition, the relatively conserved proteins containing domains of carbonic anhydrase, Sushi, Von Willebrand factor type A, and chitin binding, were identified from all the ten species. Moreover, 25 genes encoding SMPs were annotated and characterized that are involved in CaCO3 crystallization and represent chitin related or ECM related proteins. Together, data from these analyses provide new knowledge underlying the molecular mechanism of shell formation in C.gigas, supporting a refined shell formation model including chitin and ECM-related proteins.
... Interest at the level of gene and protein function will provide a more comprehensive understanding of a species' biology and its response/adaptation to the environment. The main areas of research that have adhered to this technology have been aquaculture for improving fish health and nutrition, welfare assessment and stress reduction, diseases and the use of antibiotics and vaccines, aquatic toxicology, and the identification of major environmental threats and the mode of action (MOA) of water contaminants [52][53][54]; developmental biology and reproduction [55][56][57]; and physiology and immune mechanisms in selected wildlife and commercial species [58][59][60][61]. The conventional proteomic techniques such as quantitative 2DE and 2D-DIGE in combination with MS have been the most used to address specific proteomic questions raised in aquatic sciences. ...
Proteomics is a field of growing importance in animal and aquatic sciences. Similar to other proteomic approaches, top-down proteomics is slowly making its way within the vast array of proteomic approaches that researchers have access to. This opinion and mini-review article is dedicated to top-down proteomics and how its use can be of importance to animal and aquatic sciences. Herein, we include an overview of the principles of top-down proteomics and how it differs regarding other more commonly used proteomic methods, especially bottom-up proteomics. In addition, we provide relevant sections on how the approach was or can be used as a research tool and conclude with our opinions of future use in animal and aquatic sciences.
... New opportunities for high throughput approaches (mass spectrometry-based proteomics) and deep proteome coverage in aquaculture species were created due to the complementary information available at the level of the genome and transcriptome [12][13][14][15]. The OMICs technologies have been employed in many different research topics from nutrition [16,17], to the disclosure of immune mechanisms and response to infection [12,18,19], characterization of the reproductive systems [15,[20][21][22][23], understanding of the effects of climate change [24,25], characterization of neuropeptides and hormone like elements in crustaceans [26,27], growth and development [28], risk assessment and toxicity of environmental contaminants [29][30][31][32][33], and mechanisms of detoxification of contaminants [34]. ...
Aquaculture is one of the fastest growing world industries due to the increased demand of fishery products
for human consumption and capture restrictions as a result of aquatic ecosystems
exploitation. Aquaculture is therefore an extremely competitive business with major challenges to keep a high quality farmed fish through a sustainable production system. These challenges imposed quite important changes in this more traditional market, namely at the level of integrating scientific knowledge and research. Proteomics presents itself as a powerful tool not only for a better understanding of the marine
organisms biology but also to provide solutions to deal with changes and the increasing demand in the system’s production line to ensure the required supply. In this book chapter we will give an overview of aquaculture nowadays, its challenges and describe relevant proteomics
studies in several areas of this industry. A brief description of the proteomics technical approaches applied to aquaculture will also be addressed.
... We first tested a set of nacreous molluscs: the abalone Haliotis asinina (Marie et al., 2010b), the edible mussel Mytilus (Marie et al., 2011b), the pearl oyster from French Polynesia Pinctada margaritifera (Marie et al., 2011c, submitted). In addition, some non-nacreous molluscs were similarly analyzed: the Manila clam Venerupis philipinarum (Marie et al., 2011d), the edible oyster Crassostrea gigas (Marie et al., 2011e), and the giant owl limpet Lottia gigantea (Marie et al., submitted). This combined approach allows dramatically increasing the quantity of information on shell proteins in different models. ...
Since the mid-seventies, there has been a considerable emphasis on
the key-role played by extracellular organic macromolecules associated to
mollusc shell biomineralization. In particular, the proteins occluded within the
shell are supposed to fulfill several distinct functions, listed as follows: to
provide a gel-like or colloidal micro-environment where crystallization can
occur, to compartmentalize this environment in relation to the future
microstructure, to promote nucleation and favour crystal growth in privileged
crystallographic axes, to stop crystal growth when necessary. Beside these
functions related to the interaction with the mineral, some specific shell
proteins function as enzymes, while others exert signalling activities towards
the calcifying mantle epithelium.
The topographic models of shell mineralization, which emerged a decade
ago for describing nacre ultrastructure, translate very imperfectly the complexity
of the calcification process at the molecular level. For a few years, we have
undertaken to obtain and compare the “shellomes”—the full shell protein
contents of several mollusc models—by combining a proteomic approach to
available EST data sets. Surprisingly, our findings suggest that, from model to
model (pearl oyster versus mussel, for example), the nacre protein contents
may exhibit very few similarities. In this review paper, functional and
evolutionary implications of our data are briefly discussed.
... Although the process of shell development is a wellstudied subject in molluscs (Kniprath, 1980;Eyster, 1983;Medaković, 2000;Hohagen and Jackson, 2013), understanding of the calcium transport mechanisms is not comprehensive. The general view of bivalve shell formation suggests that biomineralisation occurs via precipitation of CaCO 3 crystals in the organic matrix from a supersaturated solution of CaCO 3 in the extrapallial space i.e. the compartment between the outer mantle epithelium (OME) and the shell (Wheeler and Sikes, 1984;Levi-Kalisman et al., 2001;Joubert et al., 2010;Marie et al., 2011;Marin et al., 2012). This hypothesis has been questioned by models suggesting that too large volumes of such solutions would be required for this to be feasible (Addadi et al., 2006) and that the extrapallial fluid is undersaturated with regard to CaCO 3 (Misogianes and Chasteen, 1979;Heinemann et al., 2012). ...
Shell growth of oysters requires calcium uptake from the environment and transport to the area of shell formation. A shell regeneration assay in combination with radiolabelled calcium was used to investigate uptake and distribution of calcium to different tissues and hemolymph fractions in Pacific oysters, Crassostrea gigas (Bivalvia, Ostreoida). Oysters were notched at the shell margin and subsequently sampled for hemolymph and grading of shell regeneration during a two week experimental period. Half of the oysters were additionally exposed to 45Ca and sampled for hemolymph and tissues. Total plasma calcium concentrations increased in notched oysters compared to controls on 1, 2 and 7 days after notching. A decrease in plasma calcium levels was apparent on day 4, for both total and ionic calcium. The shell regeneration assay in the notched oysters resulted in a visible deposition of CaCO3 onto the regenerate from day 7 onwards. This was coinciding with an increased uptake of total calcium on days 11 and 14 as well as free, i.e. ionic and ligand-bound calcium, on day 14. At day 1, notching also increased calcium uptake into the mantle tissues, in areas above the notch and near the hinge. During the experiment, both the total hemocyte count and the number of granulocytes increased in notched compared to control oysters. The present study suggests that induced shell damage results in a dynamic regulation of the calcium uptake from the environment and the distribution of calcium within the body, starting directly after notching. Increases in both total calcium concentrations and uptake rates coincided with the visible depositions of CaCO3 on the regenerate shell. C. gigas was found to transport calcium mainly in the ionic form in the hemolymph, with only minor parts being bound to proteins or smaller ligands. Hemolymph measurement also revealed that C. gigas is able to regulate the extracellular concentrations of calcium and potassium. The changes in plasma calcium concentrations and speciation, concomitant with increases in granulocytes indicate that multiple calcium transport processes are activated after induced shell damage.
The fine structure and chemical composition of the shell growth margin were compared in diploid and triploid oysters, Crassostrea gigas (Thunberg 1793), reared to commercial size in a Crimean marine farm. The diploid oysters were deposited from plankton, whereas the triploid ones were obtained from an Atlantic coast nursery. An electron scanning microscope SEM Hitachi U 3500 with built-in software Oxford Ultin Max 65 for microanalysis was employed in the study. The shell growth margin is shown to consist of two layers: periostracum and prismatic. The periostracum in diploid oysters is smooth and porous, whereas the periostracum of triploid oysters is volumetric and shows longitudinal folds. The prismatic layer of both right and left shell valves consists of prisms surrounded by organic membranes. In contrast to diploid oysters, triploid ones have longer prism facets, their calcite filling is significantly lower than normal, their interprismatic organic membranes are discontinuous and contain calcium carbonate. The proportion of organic matter in diploid oyster shells is significantly higher than that in triploid ones. The factors affecting the fine structure of oyster shells differing in ploidy are discussed.
Acrasids are large, fast-moving, omnivorous amoebae. However, under certain conditions, they can also cooperate to form multicellular fruiting bodies in a process known as aggregative multicellularity (AGM). This makes acrasids the only known example of multicellularity among the earliest branches of eukaryotes (formerly superkingdom Excavata) and thus the outgroup to all other known multicellular eukaryotes. We have sequenced the genome of Acrasis kona , along with transcriptomes from cells in pre-, mid- and post-development. We find the A. kona genome to be rich in novelty, genes acquired by horizontal transfer and, especially, multigene families. The latter include nearly half of the amoeba’s protein coding capacity, and many of these families show differential expression among life cycle stages. Development in A. kona appears to be molecularly simple, requiring substantial upregulation of only 449 genes compared to 2762 in the only other AGM model, Dictyostelium discoideum. However, unlike the dictyostelid, developing A. kona also does not appear to be starving, being instead very metabolically active and inducing neither autophagy nor increasing ubiquitin-tagged proteolysis. Thus, contrary to current expectations, starvation does not appear to be essential for AGM development. Moreover, despite the ~ 2 billion years of evolution separating the two amoebae, their development appears to employ remarkably similar pathways for signaling, motility and construction of an extracellular matrix surrounding the developing cell mass. In addition, much of this similarity is shared with the clonal multicellularity of animals. This makes the acrasid something of a “bare bones” developmental model and suggests that much of the basic tool kit for multicellular development arose very early in eukaryotic evolution.
Since the Industrial Revolution, the concentration of atmospheric carbon dioxide (CO2) due to anthropogenic activities has increased at unprecedented rates. One-third of the atmospheric anthropogenic CO2 emissions are dissolved in the oceans affecting the chemical equilibrium of seawater, which in turn leads to a decrease in pH and carbonate ion (CO32-) concentration, a phenomenon known as ocean acidification (OA). This chemical disequilibrium can be detrimental to marine organisms (e.g., mollusks) that fabricate mineralized structures based on calcium carbonate (CaCO3). Most studies on the effect of reduced pH in seawater have been conducted on the early developmental stages of shell-building invertebrates, neglecting how adult individuals face OA stress. Here, we evaluate histological, secretory, and transcriptional changes in the mantle of adult oysters (Crassostrea gigas) exposure to ambient (8.0 +- 0.2) and reduced (7.6 +- 0.2) pH during 20 days. Most histological observations did not show differences in terms of mantle cell morphology. However, Alcian Blue/PAS staining revealed significant differences in the number of Alcian Blue positive cells in the mantle edge, suggesting a decrease in the secretory activity in this morphogenetic zone. Transcriptomic analysis revealed 172 differentially expressed genes (DEGs) between mantle tissues from adult oysters kept in normal and reduced pH conditions. Almost 18% of the DEGs encode secreted proteins that are likely to be contributing to shell fabrication and patterning. 17 of 31 DEGs encoding secreted proteins correspond to oyster-specific genes, highlighting the fact that molluscan shell formation is underpinned by a rapidly evolving secretome. The GO analysis of DEGs encoding secreted proteins showed that they are involved in the cellular response to stimulus, response to stress, protein binding, and ion binding, suggesting these biological processes and molecular functions are altered by OA. This study demonstrates that histology and gene expression profiling can advance our understanding of the cellular and molecular mechanisms underlying adult oyster tolerance to low pH conditions.
The echinoderms are an ideal group to understand evolution from a holistic, interdisciplinary framework. The genetic regulatory networks underpinning development in echinoderms are some of the best known for any model group. Additionally, the echinoderms have an excellent fossil record, elucidating in in detail the evolutionary changes underpinning morphological evolution. In this Element, the echinoderms are discussed as a model group for molecular palaeobiological studies, integrating what is known of their development, genomes, and fossil record. Together, these insights shed light on the molecular and morphological evolution underpinning the vast biodiversity of echinoderms, and the animal kingdom more generally.
The ASM extracted from the shells of P. placenta can stabilize ACC and inhibit secondary nucleation for 10 hours, and an explosive secondary nucleation and quick crystal growth from 50 nm to 10 μm can be finished on the shell surface in one hour.
Molecular paleobiology provides a promising avenue to merge data from deep time, molecular biology and genomics, gaining insights into the evolutionary process at multiple levels. The echinoderm skeleton is a model for molecular paleobioloogical studies. I begin with an overview of the skeletogenic process in echinoderms, as well as a discussion of what gene regulatory networks are, and why they are of interest to paleobiologists. I then highlight recent advances in the evolution of the echinoderm skeleton from both paleobiological and molecular/functional genomic perspectives, highlighting examples where diverse approaches provide complementary insight and discussing potential of this field of research.
The exoskeleton outside the soft body in shelled Mollusca defends against physical damage and parasite invasion and frequently undergoes biological and abiotic disturbance in natural and farmed environments, causing shell damage and environmental exposure, thereby threatening survival. To investigate the immune response and biocalcification after shell damage in pearl oyster Pinctada fucata martensii, a semi-sessile bivalve with a well-developed shell structure, we constructed a shell damage repair model and detected the transcriptome of mantle edge. In the first stage (4–12 h post-shell notching) the suppression of the secretion of the shell matrix protein (SMP) and activation of immune signaling pathways suggested the functional transformation of the mantle from biomineralization to immune defense. In the second stage (12–24 h), the enhancement of protein synthesis and processing, protein export-related functions, and energy production-related pathways indicated that the mantle prepared materials and energy for repairing the damaged shell and tissues. In the third stage, the upregulation of 68 SMPs involved in the shell formation indicated the reopening of shell fabrication and the repair of mantle tissue cells from immune defense to biomineralization. Furthermore, some SMPs burdened immune response molecules and the controller of biomineralization, which indicated the crosslinking between immune response and biomineralization. Our research outlines three stages an individual suffers during shell damage and provides insights into immune response and biocalcification crosslinking in pearl oyster and improves understanding of how the mantle tissue can quickly respond to environmental threats.
Matrix protein is a kind of secretory protein that regulates the biomineralization of the bivalve shell. In this study, a water-soluble matrix protein (WSMP) from Pacific oysters (Crassostrea gigs) shell was isolated, and its structure was analyzed in detail, in addition to its anti-osteoporosis activity in vitro and in vivo. Results showed that WSMP was an acidic protein with an apparent molecular mass of 47 and 79 kDa and contained a glycoprotein structure. In vitro, the reduction of Tartrate-resistant acid phosphatase (TRAP) and deoxypyridinoline (DPD) indicated that osteoclast activity was inhibited compared with the model group. Moreover, the increased osteocalcin (OCN) and BMD levels suggested that the high osteoblast activity and bone mineralization was improved. SEM analysis of the femur showed that there were fewer bone pits in experimental groups, which was consistent with the above results. In vivo, WSMP promoted the expression of alkaline phosphatase (ALP) and osteogenic differentiation factor BMP-2 in osteoblasts. In addition, the activity of osteoclasts was inhibited by regulating the process of osteoclast differentiation induced by RANKL. Both in vitro and in vivo studies showed that WSMP could promote osteogenesis and inhibit osteoclast absorption, thus demonstrating their potential applications in osteoporosis.
Mollusc shells are a result of the deposition of crystalline and amorphous calcite catalysed by enzymes and shell matrix proteins. Developing a detailed understanding of bivalve mollusc biomineralization pathways is complicated not only by the multiplicity of shell forms and microstructures in this class, but also by the evolution of associated proteins by domain co-option and domain shuffling. In spite of this, a minimal biomineralization toolbox comprising proteins and protein domains critical for shell production across species has been identified. Using a matched pair design to reduce experimental noise from inter-individual variation, combined with damage-repair experiments and a database of biomineralization shell matrix proteins (SMP) derived from published works, proteins were identified that are likely to be involved in shell calcification. Eighteen new, shared proteins likely to be involved in the processes related to the calcification of shells were identified by analysis of genes expressed during repair in Crassostrea gigas, Mytilus edulis and Pecten maximus. Genes involved in ion transport were also identified as potentially involved in calcification either via the maintenance of cell acid-base balance or transport of critical ions to the extrapallial space, the site of shell assembly. These data expand the number of candidate biomineralization proteins in bivalve molluscs for future functional studies and define a minimal functional protein domain set required to produce solid microstructures from soluble calcium carbonate. This is important for understanding molluscan shell evolution, the likely impacts of environmental change on biomineralization processes, materials science, and biomimicry research.
The shells of the Pacific oyster Crassostrea gigas contain calcite crystals with three types of microstructures: prismatic, chalky, and foliated layers. Many shell matrix proteins were annotated from the shells of C. gigas; however, it is unclear which SMPs play important roles in their shell mineralization. The matrix proteins have never been reported from the chalky layer. In this study, we identified a chalky layer-derived EGF-like domain-containing protein (CgELC) from the chalky layer of C. gigas shells. The gene sequence of the CgELC was encoded under CGI_ 10017544 of the C. gigas genome database. Only peptide fragments in the N-terminal region of CGI_ 10017544 were detected by LC-MS/MS analyses, suggesting that CGI_ 10017544 was digested at the predicted protease digestion dibasic site by post-translational modification to become a mature CgELC protein. We produced three types of CgELC recombinant proteins, namely, the full length CgELC, as well as the N-terminal and C-terminal parts of CgELC (CgELC-N or –C, respectively), for in vitro crystallization experiments. In the presence of these recombinant proteins, the aggregation of polycrystalline calcite was observed. Some fibrous organic components seemed to be incorporated into the calcite crystals in the presence of the r-CgELC protein. We also noted different features in the crystallization between CgELC-N and CgELC-C; some crystals were inhibited crystal plane formation and contained many columnar prisms inside the crystals (CgELC-N) and formed numerous holes on their surfaces (CgELC-C). These results suggest that CgELC is involved in crystal aggregation and incorporated into calcite crystals.
In this study, the SMPs from four commercially important and divergent bivalve species crassostrea gigas (pacific oyster), Mya truncata (soft shell clam), Mytilus edulis (blue mussel) and Pecten maximus (king scallop) were extracted and analysed using standardized extraction protocol and proteomic pipeline. This enables us to identify critical elements of basic biomineralization tool kit for calcification process irrespective of their shell morphology, mineralogy and microstructure. In addition, it enables the identification of SMPs that are specific to calcite and aragonite mineralogies. The signifiant numbers of SMPs found species-specific were hypothesized as adaptation to their modus vivendi. In fact, the latter proteins possess immunity-related functions and fit into specific pathway, phenoloxidase, suggesting their role in defense against pathogen. The comparative study of shell proteome of mussels living in full marine condition, North Sea and the Iow saline Baltic Sea showed the modulation of the SMPs that constitute the basic biomineralization tool kit. Higher modulation of chitin related proteins and non-modulated protein such as carbonic anhydrase, EGF and fibronectin domain containing proteins points out the impaired scaffold and mineral nucleation process in Baltic mussel. The modulation of immunity related proteins denote the influence of biotic components. These investigations show the functional diversity of SMPs and their roles beyond shell formation in the bivalvesand put forth the idea that shell is dynamic, endowed with both biochemical and mechanical protection.
The ivory shell, Babylonia areolata, is a commercially important aquaculture species in the southeast coast of mainland China. The middle veliger stage, later veliger stage, and juvenile stage are distinct larval stages in B. areolata development. In this study, we used label-free quantification proteomics analysis of the three developmental stages of B. areolata. We identified a total of 5,583 proteins, of which 1,419 proteins expression level showed significant differential expression. The results of gene ontology enrichment analysis showed that the number of proteins involved in metabolic and cellular processes were the most abundant. Those proteins mostly had functions such as binding, catalytic activity and transporter activity. The results of Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that the number of proteins involved in the ribosome, carbon metabolism, and lysosome pathways were the most abundant, indicating that protein synthesis and the immune response were active during the three stages of development. This is the first study to use proteomics and real-time PCR to study the early developmental stages of B. areolata, which could provide relevant data on gastropod development. Our results provide insights into the novel aspects of protein function in shell formation, body torsion, changes in feeding habits, attachment and metamorphosis, immune-related activities in B. areolata larvae.
Molluscs fabricate shells of incredible diversity and complexity by localized secretions from the dorsal epithelium of the mantle. Although distantly-related molluscs express remarkably different secreted gene products, it remains unclear if the evolution of shell structure and pattern is underpinned by the differential co-option of conserved genes or the integration of lineage-specific genes into the mantle regulatory program. To address this, we compare the mantle transcriptomes of 11 bivalves and gastropods of varying relatedness. We find that each species, including four Pinctada (pearl oyster) species that diverged within the last 20 million years ago, expresses a unique mantle secretome. Lineage- or species-specific genes comprise a large proportion of each species' mantle secretome. A majority of these secreted proteins have unique domain architectures that include repetitive low complexity domains (RLCDs), which evolve rapidly, and have a proclivity to expand, contract and rearrange in the genome. There are also a large number of secretome genes expressed in the mantle that arose before the origin of gastropods and bivalves. Each species expresses a unique set of these more ancient genes consistent with their independent co-option into these mantle gene regulatory networks. From this analysis, we infer lineage-specific secretomes underlie shell diversity, and include both rapidly-evolving RLCD-containing proteins, and the continual recruitment and loss of both ancient and recently-evolved genes into the periphery of the regulatory network controlling gene expression in the mantle epithelium.
Bivalves have evolved a range of complex shell forming mechanisms that are reflected by their incredible diversity in shell mineralogy and microstructures. A suite of proteins exported to the shell matrix space plays a significant role in controlling these features, in addition to underpinning some of the physical properties of the shell itself. Although, there is a general consensus that a minimum basic protein tool kit is required for shell construction, to date, this remains undefined. In this study the shell matrix proteins (SMPs) of four highly divergent bivalves (The Pacific oyster, Crassostrea gigas; the blue mussel, Mytilus edulis; the clam, Mya truncata and the king scallop, Pecten maximus) were analyzed in an identical fashion using proteomics pipeline. This enabled us to identify the critical elements of a " basic tool kit " for calcification processes, which were conserved across the taxa irrespective of the shell morphology and arrangement of the crystal surfaces. In addition, protein domains controlling the crystal layers specific to aragonite and calcite were also identified. Intriguingly, a significant number of the identified SMPs contained domains related to immune functions. These were often are unique to each species implying their involvement not only in immunity, but also environmental adaptation. This suggests that the SMPs are selectively exported in a complex mix to endow the shell with both mechanical protection and biochemical defense.
Boosted by the development of cutting-edge “omics” technologies, powerful tools have been developed to support traditional fruit crop research. Comparative “omics” studies have been extensively applied to investigate complex biological processes, such as fruit development
and ripening, pointing out unique pathways, genes and proteins involved in these processes. Due to the availability of new technologies, reduced experimental costs, and optimized protein extraction protocols for recalcitrant plant tissues, proteomics is rapidly expanding, reaching fruit species regarded as non-model plant systems.
Olea europaea
can be undoubtedly ranked as a non-model plant species, thus suffering from a dearth of proteomic investigation when compared to other fruit species. In this chapter, we will briefly travel through the proteomic history of olives as an example of a non-model tree crop, characterized by a proteomic investigation still in its infancy but appearing to be promising. We will highlight what has been already done and we will draw the attention of the reader especially on what can be still done.
Bacterial competence, which can be natural or induced, allows the uptake of exogenous double stranded DNA (dsDNA) into a competent bacterium. This process is known as transformation. A multiprotein assembly binds and processes the dsDNA to import one strand and degrade another yet the underlying molecular mechanisms are relatively poorly understood. Here distant relationships of domains in Competence protein EC (ComEC) of Bacillus subtilis (Uniprot: P39695) were characterised. DNA-protein interactions were investigated in silico by analysing models for structural conservation, surface electrostatics and structure-based DNA binding propensity; and by data-driven macromolecular docking of DNA to models. Our findings suggest that the DUF4131 domain contains a cryptic DNA-binding OB fold domain and that the β-lactamase-like domain is the hitherto cryptic competence nuclease. This article is protected by copyright. All rights reserved.
Calcium carbonate skeletal tissues in metazoans comprise a small quantity of occluded organic macromolecules, mostly proteins and polysaccharides that constitute the skeletal matrix. Because its functions in modulating the biomineralization process are well known, the skeletal matrix has been extensively studied, successively via classical biochemical approaches, via molecular biology and, in recent years, via transcriptomics and proteomics. The optimistic view that the deposition of calcium carbonate minerals requires a limited number of macromolecules has been challenged, in the last decade, by high-throughput approaches. Such approaches have made possible the rapid identification of large sets of mineral-associated proteins, i.e., 'skeletal repertoires' or 'skeletomes', in several calcifying animal models, ranging from sponges to echinoderms. One of the consequences of this expanding set of data is that a simple definition of the skeletal matrix is no longer possible. This increase in available data, however, makes it easier to compare skeletal repertoires, shedding light on the fundamental evolutionary mechanisms affecting matrix components.
A mussel (Mytilus galloprovincialis) cDNA encoding Mgfp2, a major component of the adhesive plaque that anchors mussels tightly to underwater surfaces was isolated.
It encoded a protein mainly consisted of epidermal growth factor-like repeats, containing tyrosine residues that will be converted
to 3,4-dihydroxyphenylalanine near C and N termini. Amino acid residues important for cell-cell interaction in other epidermal
growth factor-like proteins were, however, not conserved in the structure of Mgfp2. RNA blot analysis on adult tissues showed
foot-specific expression of this gene, while the analysis on developing larvae showed that the expression starts with formation
of the foot. These results suggest that the function of Mgfp2 has been specialized to form the adhesive plaque.
Compared to oxygen isotopes, the carbon isotope composition of biogenic carbonates is less commonly used as proxy for palaeoenvironmental reconstructions because shell δ13C is derived from both dissolved inorganic (seawater) and organic carbon sources (food), and interactions between these two pools make it difficult to unambiguously identify any independent effect of either. The main purpose of this study was to demonstrate any direct impact of variable food supply on bivalve shell δ13C signatures, using low/high rations of a 13C-light mixed algal diet fed to 14-month-old (adult) cultured Japanese Crassostrea gigas under otherwise essentially identical in vitro conditions during 3 summer months (May, June and July 2003, seawater temperature means at 16, 18 and 20 °C respectively) in experimental tanks at the Argenton laboratory along the Brittany Atlantic coast of France. At a daily ration of 12% (versus 4%) oyster dry weight, the newly grown part of the shells (hinge region) showed significantly lower δ13C values, by 3.5‰ (high ration: mean of −5.8 ± 1.1‰, n = 10; low ration: mean of −2.3 ± 0.7‰, n = 6; ANOVA Scheffe’s test, p
The deposition of precise arrays of inorganic crystals in many organisms involves controlled nucleation at interfaces between the crystals and substrate macromolecules. These inorganic-organic molecular recognition processes have potential technological application.
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.
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.
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.
The shell of the pearl-producing bivalve Pinctada margaritifera is composed of an organic cell-free matrix that plays a key role in the dynamic process of biologically-controlled biomineralization. In order to increase genomic resources and identify shell matrix proteins implicated in biomineralization in P. margaritifera, high-throughput Expressed Sequence Tag (EST) pyrosequencing was undertaken on the calcifying mantle, combined with a proteomic analysis of the shell.
We report the functional analysis of 276 738 sequences, leading to the constitution of an unprecedented catalog of 82 P. margaritifera biomineralization-related mantle protein sequences. Components of the current "chitin-silk fibroin gel-acidic macromolecule" model of biomineralization processes were found, in particular a homolog of a biomineralization protein (Pif-177) recently discovered in P. fucata. Among these sequences, we could show the localization of two other biomineralization protein transcripts, pmarg-aspein and pmarg-pearlin, in two distinct areas of the outer mantle epithelium, suggesting their implication in calcite and aragonite formation. Finally, by combining the EST approach with a proteomic mass spectrometry analysis of proteins isolated from the P. margaritifera shell organic matrix, we demonstrated the presence of 30 sequences containing almost all of the shell proteins that have been previously described from shell matrix protein analyses of the Pinctada genus. The integration of these two methods allowed the global composition of biomineralizing tissue and calcified structures to be examined in tandem for the first time.
This EST study made on the calcifying tissue of P. margaritifera is the first description of pyrosequencing on a pearl-producing bivalve species. Our results provide direct evidence that our EST data set covers most of the diversity of the matrix protein of P. margaritifera shell, but also that the mantle transcripts encode proteins present in P. margaritifera shell, hence demonstrating their implication in shell formation. Combining transcriptomic and proteomic approaches is therefore a powerful way to identify proteins involved in biomineralization. Data generated in this study supply the most comprehensive list of biomineralization-related sequences presently available among protostomian species, and represent a major breakthrough in the field of molluskan biomineralization.
The 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.
Using 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.
The 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.
The capacity to biomineralize is closely linked to the rapid expansion of animal life during the early Cambrian, with many skeletonized phyla first appearing in the fossil record at this time. The appearance of disparate molluscan forms during this period leaves open the possibility that shells evolved independently and in parallel in at least some groups. To test this proposition and gain insight into the evolution of structural genes that contribute to shell fabrication, we compared genes expressed in nacre (mother-of-pearl) forming cells in the mantle of the bivalve Pinctada maxima and the gastropod Haliotis asinina. Despite both species having highly lustrous nacre, we find extensive differences in these expressed gene sets. Following the removal of housekeeping genes, less than 10% of all gene clusters are shared between these molluscs, with some being conserved biomineralization genes that are also found in deuterostomes. These differences extend to secreted proteins that may localize to the organic shell matrix, with less than 15% of this secretome being shared. Despite these differences, H. asinina and P. maxima both secrete proteins with repetitive low-complexity domains (RLCDs). Pinctada maxima RLCD proteins-for example, the shematrins-are predominated by silk/fibroin-like domains, which are absent from the H. asinina data set. Comparisons of shematrin genes across three species of Pinctada indicate that this gene family has undergone extensive divergent evolution within pearl oysters. We also detect fundamental bivalve-gastropod differences in extracellular matrix proteins involved in mollusc-shell formation. Pinctada maxima expresses a chitin synthase at high levels and several chitin deacetylation genes, whereas only one protein involved in chitin interactions is present in the H. asinina data set, suggesting that the organic matrix on which calcification proceeds differs fundamentally between these species. Large-scale differences in genes expressed in nacre-forming cells of Pinctada and Haliotis are compatible with the hypothesis that gastropod and bivalve nacre is the result of convergent evolution. The expression of novel biomineralizing RLCD proteins in each of these two molluscs and, interestingly, sea urchins suggests that the evolution of such structural proteins has occurred independently multiple times in the Metazoa.
Although bivalves are among the most-studied marine organisms because of their ecological role and economic importance, very little information is available on the genome sequences of oyster species. This report documents three large-scale cDNA sequencing projects for the Pacific oyster Crassostrea gigas initiated to provide a large number of expressed sequence tags that were subsequently compiled in a publicly accessible database. This resource allowed for the identification of a large number of transcripts and provides valuable information for ongoing investigations of tissue-specific and stimulus-dependant gene expression patterns. These data are crucial for constructing comprehensive DNA microarrays, identifying single nucleotide polymorphisms and microsatellites in coding regions, and for identifying genes when the entire genome sequence of C. gigas becomes available.
In the present paper, we report the production of 40,845 high-quality ESTs that identify 29,745 unique transcribed sequences consisting of 7,940 contigs and 21,805 singletons. All of these new sequences, together with existing public sequence data, have been compiled into a publicly-available Website http://public-contigbrowser.sigenae.org:9090/Crassostrea_gigas/index.html. Approximately 43% of the unique ESTs had significant matches against the SwissProt database and 27% were annotated using Gene Ontology terms. In addition, we identified a total of 208 in silico microsatellites from the ESTs, with 173 having sufficient flanking sequence for primer design. We also identified a total of 7,530 putative in silico, single-nucleotide polymorphisms using existing and newly-generated EST resources for the Pacific oyster.
A publicly-available database has been populated with 29,745 unique sequences for the Pacific oyster Crassostrea gigas. The database provides many tools to search cleaned and assembled ESTs. The user may input and submit several filters, such as protein or nucleotide hits, to select and download relevant elements. This database constitutes one of the most developed genomic resources accessible among Lophotrochozoans, an orphan clade of bilateral animals. These data will accelerate the development of both genomics and genetics in a commercially-important species with the highest annual, commercial production of any aquatic organism.
Background:
Human activities have increased atmospheric concentrations of carbon dioxide by 36% during the past 200 years. One third of all anthropogenic CO(2) has been absorbed by the oceans, reducing pH by about 0.1 of a unit and significantly altering their carbonate chemistry. There is widespread concern that these changes are altering marine habitats severely, but little or no attention has been given to the biota of estuarine and coastal settings, ecosystems that are less pH buffered because of naturally reduced alkalinity.
Methodology/principal findings:
To address CO(2)-induced changes to estuarine calcification, veliger larvae of two oyster species, the Eastern oyster (Crassostrea virginica), and the Suminoe oyster (Crassostrea ariakensis) were grown in estuarine water under four pCO(2) regimes, 280, 380, 560 and 800 microatm, to simulate atmospheric conditions in the pre-industrial era, present, and projected future concentrations in 50 and 100 years respectively. CO(2) manipulations were made using an automated negative feedback control system that allowed continuous and precise control over the pCO(2) in experimental aquaria. Larval growth was measured using image analysis, and calcification was measured by chemical analysis of calcium in their shells. C. virginica experienced a 16% decrease in shell area and a 42% reduction in calcium content when pre-industrial and end of 21(st) century pCO(2) treatments were compared. C. ariakensis showed no change to either growth or calcification. Both species demonstrated net calcification and growth, even when aragonite was undersaturated, a result that runs counter to previous expectations for invertebrate larvae that produce aragonite shells.
Conclusions and significance:
Our results suggest that temperate estuarine and coastal ecosystems are vulnerable to the expected changes in water chemistry due to elevated atmospheric CO(2) and that biological responses to acidification, especially calcifying biota, will be species-specific and therefore much more variable and complex than reported previously.
Matrix evolutions: We have biochemically characterized the nacre matrix of the cephalopod Nautilus macromphalus, in part by a proteomic approach applied to the acetic acid-soluble and -insoluble shell matrices, as well as to spots obtained after 2D gel electrophoresis. Strikingly, most of the obtained partial sequences are entirely new, whereas a few correspond only partly with bivalvian nacre proteins. Our findings shed new light on the macroevolution of nacre matrix proteins.
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.
Among molluscs, the shell biomineralization process is controlled by a set of extracellular macromolecular components secreted by the calcifying mantle. In spite of several studies, these components are mainly known in bivalves from only few members of pteriomorph groups. In the present case, we investigated the biochemical properties of the aragonitic shell of the freshwater bivalve Unio pictorum (Paleoheterodonta, Unionoida). Analysis of the amino acid composition reveals a high amount of glycine, aspartate and alanine in the acid-soluble extract, whereas the acid-insoluble one is rich in alanine and glycine. Monosaccharidic analysis indicates that the insoluble matrix comprises a high amount of glucosamine. Furthermore, a high ratio of the carbohydrates of the soluble matrix is sulfated. Electrophoretic analysis of the acid-soluble matrix revealed discrete bands. Stains-All, Alcian Blue, periodic acid/Schiff and autoradiography with (45)Ca after electrophoretic separation revealed three major polyanionic calcium-binding glycoproteins, which exhibit an apparent molecular mass of 95, 50 and 29 kDa, respectively. Two-dimensional gel electrophoresis shows that these bands, provisionally named P95, P50 and P29, are composed of numerous isoforms, the majority of which have acidic isoelectric points. Chemical deglycosylation of the matrix with trifluoromethanesulfonic acid induces a drastic shift of both the apparent molecular mass and the isoelectric point of these matrix components. This treatment induces also a modification of the shape of CaCO(3) crystals grown in vitro and a loss of the calcium-binding ability of two of the main matrix proteins (P95 and P50). Our findings strongly suggest that post-translational modifications display important functions in mollusc shell calcification.
In the last few years, the field of molluscan biomineralization has known a tremendous mutation, regarding fundamental concepts on biomineralization regulation as well as regarding the methods of investigation. The most recent advances deal more particularly with the structure of shell biominerals at nanoscale and the identification of an increasing number of shell matrix protein components. Although the matrix is quantitatively a minor constituent in the shell of mollusks (less than 5% w/w), it is, however, the major component that controls different aspects of the shell formation processes: synthesis of transient amorphous minerals and evolution to crystalline phases, choice of the calcium carbonate polymorph (calcite vs aragonite), organization of crystallites in complex shell textures (microstructures). Until recently, the classical paradigm in molluscan shell biomineralization was to consider that the control of shell synthesis was performed primarily by two antagonistic mechanisms: crystal nucleation and growth inhibition. New concepts and emerging models try now to translate a more complex reality, which is remarkably illustrated by the wide variety of shell proteins, characterized since the mid-1990s, and described in this chapter. These proteins cover a broad spectrum of pI, from very acidic to very basic. The primary structure of a number of them is composed of different modules, suggesting that these proteins are multifunctional. Some of them exhibit enzymatic activities. Others may be involved in cell signaling. The oldness of shell proteins is discussed, in relation with the Cambrian appearance of the mollusks as a mineralizing phylum and with the Phanerozoic evolution of this group. Nowadays, the extracellular calcifying shell matrix appears as a whole integrated system, which regulates protein-mineral and protein-protein interactions as well as feedback interactions between the biominerals and the calcifying epithelium that synthesized them. Consequently, the molluscan shell matrix may be a source of bioactive molecules that would offer interesting perspectives in biomaterials and biomedical fields.
Many mineralizing organisms selectively form either calcite or aragonite, two polymorphs of calcium carbonate with very similar
crystalline structures. Understanding how these organisms achieve this control has represented a major challenge in the field
of biomineralization. Macromolecules extracted from the aragonitic shell layers of some mollusks induced aragonite formation
in vitro when preadsorbed on a substrate of β-chitin and silk fibroin. Macromolecules from calcitic shell layers induced mainly
calcite formation under the same conditions. The results suggest that these macromolecules are responsible for the precipitation
of either aragonite or calcite in vivo.
Soluble proteins in the scallop (Patinopecten yessoensis) foliated calcite shell layer were characterized using biochemical
and molecular biological techniques. SDS PAGE of these molecules revealed three major protein bands, 97 kD, 72 kD, and 49
kD in molecular weight, when stained with Coomassie Brilliant Blue. Periodic Acid Schiff staining and Stains-All staining
indicated that these proteins are slightly glycosylated and may have cation-binding potential. N-terminal sequencing of the
three proteins revealed that all three share the same amino acid sequence at least for the first 20 residues. A partial amino
acid sequence of 436 amino acids of one of these proteins (MSP-1) was deduced by characterization of the complementary DNA
encoding the protein. The deduced sequence is composed of a high proportion of Ser (31%), Gly (25%), and Asp (20%), typifying
an acidic glycoprotein of mineralized tissues. The protein has a basic domain near the N-terminus and two highly conserved
Asp-rich domains interspersed in three Ser and Gly-rich regions. In contrast with prevalent expectations, (Asp-Gly)n-, (Asp-Ser)n-,
and (Asp-Gly-X-Gly-X-Gly)n-type sequence motifs do not exist in the Asp-rich domains, demanding revision of previous theories
of protein-mineral interactions.
Correlations between structural properties of bivalve shell organic matrix and its proposed functions in the regulation of biomineralization were examined using proteinaceous fractions obtained from the shell of the oyster Crassostrea virginica (Gmelin) following dissolution of the mineral with ethylenediaminetetraacetate (EDTA). Matrix isolated in this way contains a continuous size distribution of proteins ranging from relatively small molecular weight (Mr) soluble matrix (SM) components to insoluble matrix (IM) components. Regulation of mineralization by these components was determined primarily by their reduction of crystal growth rate in an in vitro assay. For all fractions tested, there was an inverse correlation between Mr and regulatory activity. However, matrix properties other than molecular size may be important in regulation of crystal growth in vivo in that the larger but less acidic of two soluble matrix proteins was a more effective inhibitor of spicule formation by sea urchin embryos. Base treatment of IM and high molecular weight SM fractions that had little or no inhibitory activity when untreated, resulted in constituents that had a molecular weight distribution and in vitro inhibitory activity in the same range as whole SM. These results, combined with the finding that a major fraction of IM has an amino acid composition very similar to the highly charged SM fractions, suggest that much of the matrix is made up of similar molecules, and that their function in crystal growth regulation may change as they interact to form units of increasing size. A second class of IM was isolated which contained dihydroxyphenylalanine (dopa) and a preponderance of hydrophobic amino acids. This material may represent the basic structural framework of the matrix.
Atomic-force microscopy of the major class of soluble matrix protein from the oyster Crassostrea virginica revealed that this protein forms a ring structure, perhaps rendering the NH2-terminus unavailable to Edman degradation. Cleavage of these proteins using mild acid hydrolysis and hydroxylamine produced linear peptides that were able to be sequenced. Peptides consisted of a domain of polyserine-phosphoserine and runs of aspartic acid of 4 to 7 residues, with several other amino acids present. Hydrophobic domains were also isolated. Although phylogenetically distant, oyster shell-matrix protein is similar to the phosphophoryn isolated from dentin and, to a lesser extent, proteins isolated from verteorate bone.
The calcified shell layer of the American oyster Crassostrea virginica Gmelin consists of the calcitic prismatic layer and calcitostracum. The EDTA-insoluble matrix of the prismatic layer has a relatively high non-polar amino acid content, a common feature of calcitic prismatic layers of many bivalve species. The proteins of the prismatic layer wall matrix are shown by immunocytochemistry to be different from those of the intercrystalline matrix of the same layer.Immunocytochemical studies of both layers indicate that the insoluble matrix fraction constitutes the major framework of the matrix. The soluble matrix, with its glycosaminoglycans (GAGs), seems to be at the surface of the insoluble matrix and surrounds calcite crystals. Soluble and insoluble matrices appear to be synthesized separately, but packaged into the mucous droplets of the outer shell-side epithelium of the mantle, where they aggregate in groups and are secreted into the extrapallial cavities.
The superimposed layers of the true oyster shell have distinct morphology. The shells are mainly calcitic, comprising an outer prismatic region and inner foliated structure that is frequently interrupted by lenses of chalky calcitic deposits. Aragonite is restricted to the myostracum and ligament. Electron backscatter diffraction (EBSD) analysis has shown that despite the variations in structural morphology, the mineralized layers of the oyster shell maintain a single crystallographic orientation with the crystallographic c-axis orientated perpendicular to outer and inner shell surfaces. Varying crystal morphology, while maintaining crystallographic unity, may be an evolutionary trait that forms a crack-resistant shell with optimum strength and flexibility.
1.1. The organic matrix of the shell of the oyster, Crassostrea virginica, has been isolated and analysed as two fractions. These consist of a protein with a high content of glycine and dicarboxylic amino acids, and a carbohydrate. The infrared spectrum of the carbohydrate fraction indicates that it is a sulphated polysaccharide.2.2. X-ray diffraction of powdered samples of the intact matrix and the polysaccharide fraction give identical results.3.3. Material isolated as organic matrix frequently contains clay contaminants.4.4. The organic matrix from the calcite shell of the oyster has been recalcified from a solution which produces the aragonite polymorph. The matrix shows some ability to induce the formation of calcite under these conditions.5.5. The influence of the organic matrix upon the secretion of the shell under normal circumstances is discussed.
The highly acidic soluble organic matrix (SM) isolated from shells of the Antarctic scallop, Adamussium colbecki, was shown to consist of 1.5% carbohydrate by weight and 12.8% phosphate by weight. Total SM is composed of approximately 31% Asx, 29% Ser, and 18% Gly. Separation of the SM using RP-HPLC yielded a minimum of six protein fractions labeled RP-1 through RP-6 in order of elution off the column. The first fraction, RP-1, was found to be an effective inhibitor of calcium carbonate crystal nucleation in vitro suggesting a role for this protein in the regulation of shell mineralization. A less acidic fraction, RP-3, showed less inhibitory activity and dephosphorylation of RP-1 resulted in almost complete loss of inhibitory activity. Automated Edman degradation was used to sequence peptides generated by chemical cleavage of RP-1. Mild acid hydrolysis yielded peptides with sequences of N-S-G-D-D-D-D-G-G-OH, N-S-G-G-(S,G)-G-OH, and N-S-G-R-G-OH. Cleavage with hydroxylamine yielded peptides of N-D-D-D-D-D-D-D-D-OH, N-L-Y-Y-OH, and N-A-V-G-E-S-D-OH. These data suggest biochemical similarities between this SM and other SM proteins isolated from both calcium carbonate and calcium phosphate biominerals, and presents evidence of a primary domain structure similar to that described for oyster SM and phosphophoryn isolated from rat dentin.
A mussel (Mytilus galloprovincialis) cDNA encoding Mgfp2, a major component of the adhesive plaque that anchors mussels tightly to underwater surfaces was isolated. It encoded a protein mainly consisted of epidermal growth factor-like repeats, containing tyrosine residues that will be converted to 3,4-dihydroxyphenylalanine near C and N termini. Amino acid residues important for cell-cell interaction in other epidermal growth factor-like proteins were, however, not conserved in the structure of Mgfp2. RNA blot analysis on adult tissues showed foot-specific expression of this gene, while the analysis on developing larvae showed that the expression starts with formation of the foot. These results suggest that the function of Mgfp2 has been specialized to form the adhesive plaque.
It is believed that the polymorphism observed in calcium carbonate crystals, such as aragonite and calcite in mollusk shells, is controlled by organic matrix proteins secreted from the mantle epithelia. However, the fine structures of these proteins are still unknown, and to understand the molecular mechanisms of mineralization process, detailed structural analyses of the organic matrix proteins are essential. For this, we have carried out purification, characterization, and cDNA cloning of nacrein, which is a soluble organic matrix protein in the nacreous layer of oyster pearls. Northern blot analysis showed that the nacrein transcript was specifically expressed in mantle pallial. Analysis of the deduced amino acid sequence revealed that the protein contained two functional domains: one was a carbonic anhydrase and another was a Gly-Xaa-Asn (Xaa = Asp, Asn, or Glu) repeat domain; however, the carbonic anhydrase domain was split into two subdomains with insertion of the Gly-Xaa-Asn repeat domain between them. Our findings suggest that nacrein actually functions as a matrix protein whose repeated Gly-Xaa-Asn domain possibly binds calcium and as a carbonic anhydrase that catalyzes the HCO3- formation, thus participating in calcium carbonate crystal formation of the nacreous layer.
Ca2+ ions play crucial roles in many matrix-matrix, cell-matrix and cell-cell contacts. Recent X-ray and NMR structure determinations have revealed an intriguing diversity of Ca(2+)-binding sites in extracellular proteins, ranging from the stabilization of isolated domains to intimate involvement in the superstructure of macromolecular assemblies. The central role of Ca2+ in extracellular proteins is illustrated by the molecular characterization of hereditary connective tissue disorders in humans. Point mutations of Ca(2+)-binding residues in fibrillin and cartilage oligomeric matrix protein are responsible for Marfan syndrome and pseudoachondroplasia, respectively. We also discuss the possibility that structure and function of extracellular proteins may be regulated by physiologically relevant Ca2+ gradients.
An expressed sequence tag program was undertaken to isolate genes involved in defense mechanisms of the Pacific oyster, Crassostrea gigas. Putative function could be assigned to 54% of the 1142 sequenced cDNAs. We built a public database where all EST information are accessible through numerous search profiles (http://www.ifremer.fr/GigasBase). Based on sequence similarities we identified 20 genes that may be implicated in immune function. We investigated the expression of four of these genes during bacterial challenge of oysters. Three of them were induced in response to challenge lending support to their involvement in oyster immunity. Moreover, four other genes were highly homologous to components of the NF-kappa B signaling pathway which is involved in innate immune response in Drosophila and mammals. Altogether, our results open a new way to investigate the immune response in mollusks.
The complete primary structure of MSP-1, a major water-soluble glycoprotein in the foliated calcite shell layer of the scallop Patinopecten yessoensis, is reported. The full-length complementary DNA for MSP-1 isolated by polymerase chain reaction contained a sequence for a signal peptide of 20 amino acids followed by a polypeptide of 820 amino acids with calculated molecular mass of 74.5 kDa. The deduced amino acid sequence of MSP-1 includes a high proportion of Ser (32%), Gly (25%), and Asp (20%), and the predicted isoelectric point is 3.2; in these respects, MSP-1 is a typical acidic glycoprotein of mineralized tissues. A repeated modular structure characterizes MSP-1, with a sequence unit between 158 and 177 amino acids in length being repeated 4 times in tandem in the middle part of the protein. The repeated unit comprises 3 modules (SG, D, and K domains), each having a distinct amino acid composition and sequence. The SG domain is almost exclusively composed of Ser and Gly residues. The D domain is rich in Asp residues, potential N-glycosylation and phosphorylation sites. The K domain is rich in Gly residues and has a core of basic residues. The Asp residues are arranged more or less regularly in the D domains, exhibiting some repeated motifs such as Asp-Gly-Ser-Asp and Asp-Ser-Asp. Further, the 4 D domains indicate remarkable overall sequence similarities to each other. These observations suggest that the regular arrangements of COO(-) groups in the D domain side chains may be important for specific control of crystal growth.
The biological approach to forming crystals is proving to be most surprising. Mollusks build their shells by using a hydrophobic silk gel, very acidic aspartic acid rich proteins, and apparently also an amorphous precursor phase from which the crystals form. All this takes place in a highly structured chitinous framework. Here we present ideas on how these disparate components work together to produce the highly structured pearly nacreous layer of the mollusk shell.
The foliated layer of bivalves is constituted by platy calcite crystals, or laths, surrounded by an organic layer, and which are arranged into sheets (folia). Therefore, the foliated microstructure can be considered the calcitic analogue to nacre. In this paper, the foliated microstructure has been studied in detail using electron and X-ray diffraction techniques, together with SEM observations on naturally decalcified shells, to investigate the crystallographic organization on different length scales and to resolve among previous contradictory results. This layer is highly organized and displays a coherent crystallographic orientation. The surface of the laths of the foliated layer is constituted by calcite crystals oriented with their c-axis tilted opposite to the growth direction of the laths and one of its {101 4} rhombohedral faces looking in the growth direction. These faces are only expressed as the terminal faces of the laths, whereas the main surfaces of laths coincide with {101 8} rhombohedral faces. This arrangement was consistently found in all specimens studied, which leads us to the provisional conclusion that, unlike previous studies, there is only one possible crystallographic arrangement for the foliated layer. Future studies on other species will help to ascertain this assertion.
A calcium-binding soluble protein extracted from oyster shell suppresses calcium carbonate nucleation and decreases the rate of crystal growth in vitro. These findings suggest that soluble matrix may regulate shell growth.
The generation of EST information is an essential step in the genomic characterisation of species. In the context of the European Network Marine Genomics, a common goal was to significantly increase the amount of ESTs in commercial marine mollusk species and more specifically in the less studied but ecologically and commercially important groups, such as mussel and clam genera. Normalized cDNA libraries were constructed for four different relevant bivalves species (Crassostrea gigas, Mytilus edulis, Ruditapes decussatus and Bathymodiolus azoricus), using numerous tissues and physiological conditions. In this paper, we present the analysis of the 13,013 expressed sequence tags (ESTs) generated. Each EST library was independently assembled and 1300-3000 unique sequences were identified in each species. For the different species, functional categories could be assigned to only about 16 to 27% of ESTs using the GO annotation tool. All sequences have been incorporated into a publicly available database and form the basis for subsequent microarray design, SNP detection and polymorphism analysis, and the placement of novel markers on genetic linkage maps.
We found a novel 52 kDa matrix glycoprotein MPP1 in the shell of Crassostrea nippona that was unusually acidic and heavily phosphorylated. Deduced from the nucleotide sequence of 1.9 kb cDNA, which is likely to encode MPP1 with high probability, the primary structure of this protein shows a modular structure characterized by repeat sequences rich in Asp, Ser and Gly. The most remarkable of these is the DE-rich sequence, in which continuous repeats of Asp are interrupted by a single Cys residue. Disulfide-dependent MPP1 polymers occurring in the form of multimeric insoluble gels are estimated to contain repetitive locations of the anionic molecules of phosphates and acidic amino acids, particularly Asp. Thus, MPP1 and its polymers possess characteristic features of a charged molecule for oyster biomineralization, namely accumulation and trapping of Ca2+. In addition, MPP1 is the first organic matrix component considered to be expressed in both the foliated and prismatic layers of the molluscan shell microstructure. In vitro crystallization assays demonstrate the induction of tabular crystals with a completely different morphology from those formed spontaneously, indicating that MPP1 and its polymers are potentially the agent that controls crystal growth and shell microstructure.
Shellfish face uncertain future in high CO 2 world: influence of acidification on oyster larvae calcification and growth in estuaries A carbonic anhydrase from the nacreous layer in oyster pearls
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Parallel evolution of nacre building gene sets in molluscs
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