Hydrophilic Shell Matrix Proteins of Nautilus pompilius and the Identification of a Core Set of Conchiferan Domains

Despite being a member of the shelled mollusks (Conchiferans), most members of extant cephalopods have lost their external biomineralized shells, except for the basally diverging Nautilids. Here, we report the result of our study to identify major Shell Matrix Proteins and their domains in the Nautilid Nautilus pompilius, in order to gain a general insight into the evolution of Conchiferan Shell Matrix Proteins. In order to do so, we performed a multiomics study on the shell of N. pompilius, by conducting transcriptomics of its mantle tissue and proteomics of its shell matrix. Analyses of obtained data identified 61 distinct shell-specific sequences. Of the successfully annotated 27 sequences, protein domains were predicted in 19. Comparative analysis of Nautilus sequences with four Conchiferans for which Shell Matrix Protein data were available (the pacific oyster, the pearl oyster, the limpet and the Euhadra snail) revealed that three proteins and six protein domains were conserved in all Conchiferans. Interestingly, when the terrestrial Euhadra snail was excluded, another five proteins and six protein domains were found to be shared among the four marine Conchiferans. Phylogenetic analyses indicated that most of these proteins and domains were probably present in the ancestral Conchiferan, but employed in shell formation later and independently in most clades. Even though further studies utilizing deeper sequencing techniques to obtain genome and full-length sequences, and functional analyses, must be carried out in the future, our results here provide important pieces of information for the elucidation of the evolution of Conchiferan shells at the molecular level.

Independent adoptions of a set of proteins found in the matrix of the mineralized shell-like eggcase of Argonaut octopuses

The Argonaut octopus, commonly called the paper nautilus, has a spiral-coiled shell-like eggcase. As the main characteristics, the eggcase has no internal septum, is composed entirely of calcite with chitosan being the main polycarbonate and is reportedly formed by organic materials secreted from the membranes of the arms. Meanwhile, the biomineralized external "true" shells of the Mollusks, which includes the Cephalopods, are secreted from the mantle tissue. Therefore, the histological origin of the two shells is completely different. The question of how the Argonauts, which phylogenetically diverged from the completely shell-less octopuses, could form a converging shell-like external structure has thus intrigued biologists for a long time. To answer this question, we performed a multi-omics analysis of the transcriptome and proteome of the two congeneric Argonaut species, Argonauta argo and A. hians. Our result indicates that the shell-like eggcase is not a homolog of the shell, even at the protein level, because the Argonauts apparently recruited a different set of protein repertoires to as eggcase matrix proteins (EcMPs). However, we also found the homologs of three shell matrix proteins (SMPs) of the Conchiferan Mollusks, Pif-like, SOD, and TRX, in the eggcase matrix. The proteins were also found in the only surviving shelled Cephalopods, the nautiloid Nautilus pompilius. Phylogenetic analysis revealed that homologous genes of the Conchiferan SMPs and EcMPs were found in the draft genome of shell-less octopuses. Our result reported here thus suggests that the SMP-coding genes are conserved in both shelled and shell-less Cephalopods. Meanwhile, the Argonauts adopted some of the SMP-coding genes and other non-SMP-coding genes, to form a convergent, non-homologous biomineralized external structure, the eggcase, which is autapomorphic to the group.

The ‘Shellome’ of the Crocus Clam Tridacna crocea Emphasizes Essential Components of Mollusk Shell Biomineralization

Molluscan shells are among the most fascinating research objects because of their diverse morphologies and textures. The formation of these delicate biomineralized structures is a matrix-mediated process. A question that arises is what are the essential components required to build these exoskeletons. In order to understand the molecular mechanisms of molluscan shell formation, it is crucial to identify organic macromolecules in different shells from diverse taxa. In the case of bivalves, however, taxon sampling in previous shell proteomics studies are focused predominantly on representatives of the class Pteriomorphia such as pearl oysters, edible oysters and mussels. In this study, we have characterized the shell organic matrix from the crocus clam, Tridacna crocea, (Heterodonta) using various biochemical techniques, including SDS-PAGE, FT-IR, monosaccharide analysis, and enzyme-linked lectin assay (ELLA). Furthermore, we have identified a number of shell matrix proteins (SMPs) using a comprehensive proteomics approach combined to RNA-seq. The biochemical studies confirmed the presence of proteins, polysaccharides, and sulfates in the T. crocea shell organic matrix. Proteomics analysis revealed that the majority of the T. crocea SMPs are novel and dissimilar to known SMPs identified from the other bivalve species. Meanwhile, the SMP repertoire of the crocus clam also includes proteins with conserved functional domains such as chitin-binding domain, VWA domain, and protease inhibitor domain. We also identified BMSP (Blue Mussel Shell Protein, originally reported from Mytilus), which is widely distributed among molluscan shell matrix proteins. Tridacna SMPs also include low-complexity regions (LCRs) that are absent in the other molluscan genomes, indicating that these genes may have evolved in specific lineage. These results highlight the diversity of the organic molecules – in particular proteins – that are essential for molluscan shell formation.

MolluscDB: a genome and transcriptome database for molluscs

As sequencing becomes more accessible and affordable, the analysis of genomic and transcriptomic data has become a cornerstone of many research initiatives. Communities with a focus on particular taxa or ecosystems need solutions capable of aggregating genomic resources and serving them in a standardized and analysis-friendly manner. Taxon-focussed resources can be more flexible in addressing the needs of a research community than can universal or general databases. Here, we present MolluscDB, a genome and transcriptome database for molluscs. MolluscDB offers a rich ecosystem of tools, including an Ensembl browser, a BLAST server for homology searches and an HTTP server from which any dataset present in the database can be downloaded. To demonstrate the utility of the database and verify the quality of its data, we imported data from assembled genomes and transcriptomes of 22 species, estimated the phylogeny of Mollusca using single-copy orthologues, explored patterns of gene family size change and interrogated the data for biomineralization-associated enzymes and shell matrix proteins. MolluscDB provides an easy-to-use and openly accessible data resource for the research community. This article is part of the Theo Murphy meeting issue ‘Molluscan genomics: broad insights and future directions for a neglected phylum’.

PU14, a Novel Matrix Protein, Participates in Pearl Oyster, Pinctada Fucata, Shell Formation

Biomineralization is a widespread biological process, involved in the formation of shells, teeth, and bones. Shell matrix proteins have been widely studied for their importance during shell formation. In 2015, our group identified 72 unique shell matrix proteins in Pinctada fucata, among which PU14 is a matrix protein detected in the soluble fraction that solely exists in the prismatic layer. However, the function of PU14 is still unclear. In this study, the full-length cDNA sequence of PU14 was obtained and functional analyses of PU14 protein during shell formation were performed. The deduced protein has a molecular mass of 77.8 kDa and an isoelectric point of 11.34. The primary protein structure contains Gln-rich and random repeat units, which are typical characteristics of matrix protein and indicate its potential function during shell formation. In vivo and in vitro experiments indicated PU14 has prismatic layer functions during shell formation. The tissue expression patterns showed that PU14 was mainly expressed in the mantle tissue, which is consistent with prismatic layer formation. Notching experiments suggested that PU14 responded to repair and regenerate the injured shell. After inhibiting gene expression by injecting PU14-specific double-stranded RNA, the inner surface of the prismatic layer changed significantly and became rougher. Further, in vitro experiments showed that recombinant protein rPU14 impacted calcite crystal morphology. Taken together, characterization and functional analyses of a novel matrix protein, PU14, provide new insights about basic matrix proteins and their functions during shell formation.

Phylogenetic comparisons reveal mosaic histories of larval and adult shell matrix protein deployment in pteriomorph bivalves

Molluscan shells are organo-mineral composites, in which the dominant calcium carbonate is intimately associated with an organic matrix comprised mainly of proteins and polysaccharides. However, whether the various shell matrix proteins (SMPs) date to the origin of hard skeletons in the Cambrian, or whether they represent later deployment through adaptive evolution, is still debated. In order to address this issue and to better understand the origins and evolution of biomineralization, phylogenetic analyses have been performed on the three SMP families, Von Willebrand factor type A (VWA) and chitin-binding domain-containing protein (VWA-CB dcp), chitobiase, and carbonic anhydrase (CA), which exist in both larval and adult shell proteomes in the bivalves, Crassostrea gigas and Pinctada fucata. In VWA-CB dcp and chitobiase, paralogs for larval and adult SMPs evolved before the divergence of these species. CA-SMPs have been taken as evidence for ancient origins of SMPs by their presumed indispensable function in biomineralization and ubiquitous distribution in molluscs. However, our results indicate gene duplications that gave rise to separate deployments as larval and adult CA-SMPs occurred independently in each lineage after their divergence, which is considerably more recent than hitherto assumed, supporting the “recent heritage and fast evolution” scenario for SMP evolution.

Hydrophilic Shell Matrix Proteins of Nautilus pompilius and The Identification of a Core Set of Conchiferan Domains

Despite being a member of the shelled mollusks (Conchiferans), most members of extant cephalopods have lost their external biomineralized shells, except for the Nautiloids. Here, we report the result of our study to identify major Shell Matrix Proteins and their domains in the Nautiloid Nautilus pompilius, in order to gain a general insight into the evolution of Conchiferan Shell Matrix Proteins. In order to do so, we conducted transcriptomics of the mantle, and proteomics of the shell of N. pompilius simultaneously. Analyses of obtained data identified 61 distinct shell-specific sequences. Of the successfully annotated 27 sequences, protein domains were predicted in 19. Comparative analysis of Nautilus sequences with four Conchiferans for which Shell Matrix Protein data were available (the pacific oyster, the pearl oyster, the limpet, and the Euhadra snail) revealed that three proteins and six domains of the shell proteins are conserved in all Conchiferans. Interestingly, when the terrestrial Euhadra snail was excluded, another five proteins and six domains were found to be shared among the four marine Conchiferans. Phylogenetic analyses indicated that most of these proteins and domains were present in the ancestral Conchiferan, but employed in shell formation later and independently in most clades. Although further studies utilizing deeper sequencing techniques to obtain genome and full-length sequences, and functional analyses, must be done in the future, our results here provide important pieces of information for the elucidation of the evolution of Conchiferan shells at the molecular level.