Screening molluscan cDNA expression libraries with anti-shell matrix antibodies

In a previous paper [Marin et al., Protein Expr. Purif. 23 (2001) 175], we showed that polyclonal antibodies raised against molluscan shell matrices could be useful tools for visualizing shell proteins after a preparative fractionation of the shell matrix. In this paper, we have used the same antibodies for screening a cDNA library constructed from mantle tissues of the nacro-prismatic bivalve Pinna nobilis. The immunoscreening led to the identification of a new protein, mucoperlin [Marin et al., J. Biol. Chem. 275 (2000) 20667], which was subsequently overexpressed. A polyclonal antibody was obtained from the recombinant mucoperlin. In a control assay, we unambiguously demonstrated that this antibody and one of the sera used for the initial screening hybridize with the same clones. We assess that screening cDNA libraries with antibodies elicited against unfractionated calcifying matrices is a good alternative to oligonucleotide screening techniques, particularly in the field of molluscan biomineralization where only few gene sequences are known.     

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

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

Proteomic analysis of the organic matrix of the abalone <Emphasis Type="Italic">Haliotis asinina</Emphasis> calcified shell

Background  

The formation of the molluscan shell is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called "calcifying matrix" is a complex mixture of proteins and glycoproteins that is assembled and occluded within the mineral phase during the calcification process. While the importance of the calcifying matrix to shell formation has long been appreciated, most of its protein components remain uncharacterised.     

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

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

The shell matrix of the pulmonate land snail Helix aspersa maxima

In mollusks, the shell mineralization process is controlled by an array of proteins, glycoproteins and polysaccharides that collectively constitute the shell matrix. In spite of numerous researches, the shell protein content of a limited number of model species has been investigated. This paper presents biochemical data on the common edible land snail Helix aspersa maxima, a model organism for ecotoxicological purposes, which has however been poorly investigated from a biomineralization viewpoint. The shell matrix of this species was extracted and analyzed biochemically for functional in vitro inhibition assay, for amino acid and monosaccharides compositions. The matrix was further analyzed on 1 and 2D gels and short partial protein sequences were obtained from 2D gel spots. Serological comparisons were established with a set of heterologous antibodies, two of which were subsequently used for subsequent immunogold localization of matrix components. Our data suggest that the shell matrix of Helix aspersa maxima may differ widely from the shell secretory repertoire of the marine mollusks studied so far, such as the gastropod Haliotis or the pearl oyster Pinctada. In particular, most of the biochemical properties generally attributed to soluble shell matrices, such as calcium-binding capability, or the capacity to interfere in vitro with the precipitation of calcium carbonate or to inhibit the precipitation of calcium carbonate, were not recorded with this matrix. This drastic change in the biochemical properties of the landsnail shell matrix puts into question the existence of a unique molecular model for molluscan shell formation, and may be related to terrestrialisation.     

Molecular mechanism of the nacreous layer formation in Pinctada maxima

We have cloned the cDNAs that encode two kinds of molluscan shell matrix proteins, namely N66 and N14, in the nacreous layer of Pinctada maxima. N66 is composed of carbonic anhydrase-like and repeat domains, as described for nacrein (1) in the pearls of P. fucata. N14 is homologous to N16, recently found in the nacreous layer of P. fucata (2) and is characterized by high proportions of Gly, Tyr, and Asn together with NG repeat sequences. The molecular weights of these proteins were estimated as 59,814 and 13,734 Da, respectively. Structural differences were clearly indicated in the alignment and length of the repeat sequences of the sets of the homogeneous proteins (N66/nacrein and N14/N16). The longer repeat sequences of N66 and N14 may be responsible for P. maxima's excellent property of calcification. The in vitro crystallization experiments revealed that the mixture of N66 and N14 could induce platy aragonite layers highly similar to the nacreous layer, once adsorbed onto the membrane of the water-insoluble matrix.     

Distribution and Function of the Nacrein-Related Proteins Inferred from Structural Analysis

Nacrein is the first identified molluscan organic matrix (OM) component considered to be specifically involved in nacreous layer formation (Miyamoto et al. in Proc Natl Acad Sci USA 93:9657–9660, 1996); however, its localization in shell microstructures and phylogeny of molluscs and function still remain unclear. Therefore, to elucidate the functions of the nacrein-related proteins, we set up three experiments focused on (1) the primary structure of the nacrein-related proteins, (2) the tertiary structure of nacrein, and (3) in vitro crystallization of the proteins. In regard to the first experiment, our Western blot analysis and cDNA cloning clearly indicated for the first time the common occurrence of nacrein-related proteins both biochemically and genetically, independent of molluscan phylogeny and shell microstructures. Together with the data reported so far, we classified nacrein-related proteins into four types. Second, we determined the overall structure of nacrein via small-angle x-ray scattering via the program DAMMIN. This kind of research has never yet been attempted for the molluscan OM proteins. Our results inferred the structure of nacrein to be N-shaped based on the low-resolution solution dummy atom model structures that could be derived from the presence of the NG-repeat domain that was intercalated into two CA domains. Third, the result of the crystallization experiment revealed inhibitory activity of crystal formation for nacrein-related proteins when present in free state but the same molecule, when attached to the ISM, may regulate the form and size of aragonite crystal. These results demonstrate the fundamentally important function of nacrein-related proteins in molluscan shell formation. The nucleotide sequences reported in this article have been submitted to GenBank^TK/DDBJ with accession numbers AB252479 to AB252484.     

New data on molluscs and their shell microstructures from the Middle Cambrian Gowers Formation, Australia

Abstract: Numerous new cases of preserved shell microstructure were discovered in molluscs from the Middle Cambrian Gowers Formation (Ptychagnostus atavus/Peronopsis opimus Zone, Floran Stage) in the Georgina Basin, Australia. The new data provide further evidence that, by the Middle Cambrian, molluscan shell microstructures were diverse, and many molluscs had a complex shell with multiple types of shell microstructure. In addition, many new occurrences of laminar microstructures are described herein. For many, the nature of these laminar microstructures is not known, but in three species the microstructure is foliated calcite, and in at least two the microstructure is more likely to have been calcitic semi‐nacre, a type of microstructure known in brachiopods and bryozoans but unknown in modern molluscs. This commonality among these three closely related lophotrochozoans underscores a similar mechanism of biomineralization. Moreover, these observations suggest a prevalence of calcite‐shelled lineages among molluscs from the Middle Cambrian, a time of calcite seas. In addition, the broad occurrence of laminar, nacre‐like microstructures in many of these fossils reveals how widespread these strong (fracture‐resistant) microstructures were in Middle Cambrian molluscs. Additionally, a few specimens of Yochelcionella preserve imprints of a bilaterally symmetrical pair of muscle scars. New taxa described here include Corystos thorntoniensis gen. et sp. nov., Yochelcionella snorkorum sp. nov., Yochelcionella saginata sp. nov., and Anhuiconus? agrenon sp. nov.     

A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats

The nucleotide sequences of partial cDNA clones coding for the core protein of a human polymorphic epithelial mucin were determined, and a large domain was found to consist of a 60-base pair tandem repeat sequence. The cDNA clones were originally selected (Gendler, S. J., Burchell, J. M., Duhig, T., Lamport, D., White, R., Parker, M., and Taylor-Papadimitriou, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6060-6064) using three monoclonal antibodies which show differential reactivity with the mucin produced by normal and malignant breast. Two of the epitopes are exposed in the normally processed and cancer-associated mucin, while one epitope is unmasked only in the cancer-associated mucin (Burchell, J. M., Durbin, H., and Taylor-Papadimitriou, J. (1983) J. Immunol. 131, 508-513; Burchell, J., Gendler, S., Taylor-Papadimitriou, J., Girling, A., Lewis, A., Millis, R., and Lamport, D. (1987) Cancer Res. 47, 5476-5482). We show here that all three antibodies react with a synthetic peptide with an amino acid sequence corresponding to that predicted by the tandem repeat. Identification of the epitopes preferentially expressed on the cancer-associated mucin should allow a directed approach to the development of tumor-specific antibodies using synthetic peptides as immunogens.     

A Bivalve Biomineralization Toolbox

Mollusc shells are a result of the deposition of crystalline and amorphous calcite catalyzed by enzymes and shell matrix proteins (SMP). 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 SMPs 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 the 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 shell matrix of the freshwater mussel Unio pictorum (Paleoheterodonta, Unionoida). Involvement of acidic polysaccharides from glycoproteins in nacre mineralization

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.     

Large-scale fractionation of molluscan shell matrix

The numerous proteins occluded within the molluscan shell play a key role in the control of the mineralization process. Although extensively studied, these proteins are still poorly known, mainly because they are difficult to fractionate. In the present paper, we present, for the first time, a simple combined strategy for separating successfully large amounts of molluscan shell proteins. Since shell proteins do not absorb at 280 nm, our approach is based on the "blind" separation of these proteins by a preparative denaturing electrophoresis. They are subsequently detected on dot-blot with polyclonal antibodies raised against the unfractionated soluble matrix. In the present case, this approach allows one to collect enough purified proteins to obtain amino-acid composition as well as N-terminal sequences, and to perform in vitro tests and glycosylation studies. Furthermore, this method permits one to raise polyclonal antibodies against the isolated proteins.     

A novel ferritin subunit involved in shell formation from the pearl oyster (Pinctada fucata)

Iron is one of the most important minor elements in the shell of bivalves. This study was designed to investigate the involvement of ferritin, the principal protein for iron storage, in shell formation. A novel ferritin cDNA from the pearl oyster (Pinctada fucata) was isolated and characterized. The ferritin cDNA encodes a 206 amino acid polypeptide, which shares high similarity with snail soma ferritin and the H-chains of mammalian ferritins. Oyster ferritin mRNA shows the highest level of expression in the mantle, the organ for shell formation. In situ hybridization analysis revealed that oyster ferritin mRNA is expressed at the highest level at the mantle fold, a region essential for metal accumulation and contributes to metal incorporation into the shell. Taken together, these results suggest that ferritin is involved in shell formation by iron storage. The identification and characterization of oyster ferritin also helps to further understand the structural and functional properties of molluscan ferritins.     

文蛤SRBI基因克隆及其在不同壳色群体中的差异表达

为了研究SRBI基因的结构、功能以及在贝类壳色形成中的作用, 利用SMART RACE技术克隆得到文蛤(Meretrix meretrix) SRBI (Mm-SRBI)基因的全长序列, 并对其内含子特征及不同组织、不同壳色群体外套膜中的表达差异进行了分析。结果表明: Mm-SRBI基因cDNA全长1676 bp, 开放阅读框1515 bp, 编码504个氨基酸, 结构域预测发现有一个CD36结构域; 氨基酸序列比对发现, 与华贵栉孔扇贝的同源性最高(55%), 与其他物种的相似性在34%—40%, 表明该基因变异较大; 在Mm-SRBI基因中扩增出12个内含子, 均存在于开放阅读框中, 且都遵循GT-AG原则; 荧光定量PCR (qRT-PCR)结果表明, Mm-SRBI在闭壳肌、外套膜、斧足、鳃、内脏团和水管6个组织均有表达, 其中在外套膜中表达量显著高于其他组织(P<0.01), 这可能与外套膜中类胡萝卜素含量较高有关; 不同壳色群体外套膜中基因表达分析表明,Mm-SRBI在黑斑和红壳文蛤中的表达量显著高于白壳文蛤(P<0.05)。实验结果为文蛤壳色形成研究奠定了基础。     

A novel phosphorylated glycoprotein in the shell matrix of the oyster Crassostrea nippona

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.     

Zona Localization of Shell Matrix Proteins in Mantle of <Emphasis Type="Italic">Haliotis tuberculata</Emphasis> (Mollusca,Gastropoda)

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

Purification and characterization of calcium-binding conchiolin shell peptides from the mollusc,Haliotis rufescens,as a function of development

Summary Conchiolin peptides of the molluscan shell are believed to determine structural organization and facilitate calcification during shell formation. Changes in patterns of conchiolin synthesis during development, and the possible contribution of these peptides to shell formation, have been investigated by purification and characterization of the soluble peptides extracted from the shell of the gastropod mollusc,Haliotis rufescens (red abalone), at various stages of development. Shell peptides were purified from young post-larvae, juveniles and adults by gel-filtration column chromatography in aggregation-reducing bicarbonate buffers. Calcium-binding domains were detected spectrophotometrically after reaction with a cationic carbocyanine dye. Juvenile and adult shell peptides were found to be heterogeneous, and rich in aspartic acid and glycine residues; in contrast, post-larval shells were found to contain one major glycine-rich component. The juvenile shell peptide population shares components from each of the other two populations, suggesting that the synthesis of the different shell peptides results from the differential expression of a multi-gene family, in a developmentally controlled progression. Enzymatic analyses suggest that calcium binds to the aspartic acid residues of the peptide core, rather than to satellite groups such as phosphate, sulfate or carbohydrate. The possibility is discussed that the aspartic acid residues found in shell peptides may play an important role in the calcification of the abalone shell matrix. The methods demonstrated here also should prove useful for the purification, characterization, and comparative analysis of calcium-binding proteins of connective tissues, extracellular matrices and support structures in many other systems.Abbreviations Asp aspartic acid - BSA bovine serum albumin - Da daltons - EDTA ethylenediaminetetraacetic acid - GABA -aminobutyric acid - HPLC high-pressure liquid chromatography - ODS octadecylsilane - OPA o-phthaldialdehyde - SDS sodium dodecyl sulfate     

A novel matrix protein participating in the nacre framework formation of pearl oyster,Pinctada fucata

Understanding the molecular composition is of great interest for both nacre formation mechanism and biomineralization in mollusk shell. A cDNA clone encoding an MSI31 relative, termed MSI7 because of its estimated molecular mass of 7.3 kDa, was isolated from the pearl oyster, Pinctada fucata. This novel protein shares similarity with MSI31, a prismatic framework protein of P. fucata. It is peculiar that MSI7 is much shorter in size, harboring only the Gly-rich sequence that has been proposed to be critical for Ca(2+) binding. In situ hybridization result showed that MSI7 mRNA was expressed specifically at the folds and outer epithelia of the mantle, indicating that MSI7 participates in the framework formation of both the nacreous layer and prismatic layer. In vitro experiment on the function of MSI7 suggested that it accelerates the nucleation and precipitation of CaCO(3). Taken together, we have identified a novel matrix protein of the pearl oyster, which may play an important role in determining the texture of nacre.