Proteomic Identification of Novel Proteins from the Calcifying Shell Matrix of the Manila Clam Venerupis Philippinarum

Marie, Benjamin; Trinkler, Nolwenn; Zanella-Cléon, Isabelle; Guichard, Nathalie; Becchi, Michel; Paillard, Christine; Marin, Frédéric

doi:10.1007/s10126-010-9357-0

Cited by 43 publications

(31 citation statements)

References 29 publications

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“…Upsalin has a predicted signal peptide with a cleavage site between the positions 16 and 17 and a putative transmembrane region (positions [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23]. The mature form of the protein consists of 109 aa residues and has a theoretical molecular weight of 12.3 kDa.…”

Section: Primary Structure and Molecular Features Of Upsalinmentioning

confidence: 99%

“…The generated EST datasets were recently combined with proteomic data from shell extracts, [17] enabling the identification of biomineralization-related proteins at a record rate. So far, this methodology has been applied to a number of molluskan groups: clams, [18] mussels, [19] oysters, [17,20] and abalone. [21] Thanks to proteomics, it has been possible to identify new homologous proteins belonging to the set of sequences that exhibit conserved domains such as N66 and nacrein-like homologous sequences with carbonic anhydrase domains.…”

Section: Introductionmentioning

confidence: 99%

“…Examples include MUSPs-2 and -3 (edible mussel), [19] MRNP34 (pearl oyster), [25] and IMSPs-1, -2, and -3 (manila clam). [18] The growing number of these "orphan shell proteins", without known functions, highlights the need for their complete characterization, in order to understand their roles in biomineralization. In this context, we report the characterization-both at transcriptional and protein level-of a novel biomineralization protein that does not exhibit any homology with previous known shell proteins.…”

Section: Introductionmentioning

confidence: 99%

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Novel Molluskan Biomineralization Proteins Retrieved from Proteomics: A Case Study with Upsalin

et al. 2012

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The formation of the molluskan shell is regulated by an array of extracellular proteins secreted by the calcifying epithelial cells of the mantle. These proteins remain occluded within the recently formed biominerals. To date, many shell proteins have been retrieved, but only a few of them, such as nacreins, have clearly identified functions. In this particular case, by combining molecular biology and biochemical approaches, we performed the molecular characterization of a novel protein that we named Upsalin, associated with the nacreous shell of the freshwater mussel Unio pictorum. The full sequence of the upsalin transcript was obtained by RT‐PCR and 5′/3′ RACE, and the expression pattern of the transcript was studied by PCR and qPCR. Upsalin is a 12 kDa protein with a basic theoretical pI. The presence of Upsalin in the shell was demonstrated by extraction of the acetic‐acid‐soluble nacre matrix, purification of a shell protein fraction by mono‐dimensional preparative SDS‐PAGE, and by submitting this fraction, after trypsic digestion, to nano‐LC‐MS/MS. In vitro experiments with the purified protein showed that it interferes poorly with the precipitation of calcium carbonate. Homology searches also could not affiliate Upsalin to any other protein of known function, leaving open the question of its exact role in shell formation. An antibody raised against an immunogenic peptide of Upsalin was found to be specific to this protein and was subsequently assayed for immunogold localization of the target protein in the shell, revealing the ubiquitous presence of Upsalin in the nacreous and prismatic layers. Recently, with the application of high‐throughput proteomic studies to shells, the number of candidate proteins without clear functions has been increasing exponentially. The Upsalin example highlights the crucial need, for the scientific community dealing with biomineralization in general, to dedicate the coming years to designing experimental approaches, such as gene silencing, that focus on the functions of mineral‐associated proteins.

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Section: Primary Structure and Molecular Features Of Upsalinmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Novel Molluskan Biomineralization Proteins Retrieved from Proteomics: A Case Study with Upsalin

et al. 2012

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“…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.…”

Section: Introductionmentioning

confidence: 99%

Studies on the Chemical Structures of Organic Matrices and Their Functions in the Biomineralization Processes of Molluscan Shells

Suzuki¹,

Kogure²,

Nagasawa³

2017

AGri-Biosci. Monogr. (AGBM)

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Keywordscalcium carbonate and an organic matrix. In the course of shell formation, the periostracum, which is not mineralized and covers the external surface of the shell, is formed first; subsequently, the prismatic layer is formed on the periostracum. Finally, the nacreous layer is formed on the prismatic layer (Checa 2000). The nacreous layer is made of tablets of aragonite single crystals. The aragonite crystal compartment in the nacreous layer is sandwiched between sheets of organic matrix. In contrast, the prismatic layer is composed of columnar calcite surrounded by the organic matrix. The calcite crystals, surrounded by the organic framework, are oriented on the c-axis perpendicular to the shell surface.The microstructure of the shell of the limpet, Lottia kogamogai, consists of five distinct layers stacked in Studies on the Chemical Structures of Organic Matrices and Their Functions in the Biomineralization Processes of Molluscan Shells AbstractMolluscan shells protect the soft body from predators and the external environment and consist of calcium carbonate in an organic matrix. The interaction between calcium carbonate and the organic matrix forms the microstructure of the molluscan shell. In this review, we discuss several organic molecules that may be important in the formation of the shell microstructure. The iridescent color of pearls is attributed to the characteristic nacreous microstructure of molluscan shells. The Japanese pearl oyster, Pinctada fucata, is used in pearl aquaculture in Japan. The shell of P. fucata consists of two layers, prismatic and nacreous. Prismalin-14 in the prismatic layer interacts with calcium carbonate and binds to chitin. Pif in the nacreous layer interacts with aragonite crystals and plays important roles in forming the organic framework in a compartment-like structure. In contrast, limpets have a crossed lamellar microstructure in their shells. The organic matrices of limpet shells induce the formation of spindle-like aragonite crystals. Recent studies have increased our understanding of the calcification process of molluscan shells, and the findings can be applied to increase yields of high-quality pearls, lowering the cost and energy of pearl aquaculture.

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“…By combining transcriptomics and proteomics, it is possible to discriminate true-shell forming proteins from those which have a signal peptide and are predicted to be secreted, but are not incorporated into the shell , Marie et al 2010a, Berland et al 2011, Marie et al 2011a, Marie et al 2011b, Marie et al 2011c, Bédouet et al 2012, Mann et al 2012, Marie et al 2012b, Mann and Edsinger 2014, Mann and Jackson 2014, narrowing the gap between gene expression in the mantle epithelium and the final destination of proteins in mineralised structures. In addition, the genomes of the molluscs Crassostrea gigas, Pinctada fucata and Lottia gigantea (Takeuchi et al 2012, Zhang et al 2012, Simakov et al 2013) and high-throughput proteomics have enabled the direct analysis of SMPs and their protein modifications (Mann et al 2012, Zhang et al 2012, Miyamoto et al 2013, Mann and Edsinger 2014, revealing an unexpected SMP diversity and a high complexity of the shell formation process in molluscs.…”

Section: The Mantle Appears To Use Conserved Developmental Regulatorsmentioning

confidence: 99%

Investigation of gene family evolution and the molecular basis of shell formation in molluscs

Aguilera¹

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Since the emergence of shell-bearing molluscs in the Early Cambrian, diverse shell forms have evolved. Modern molluscs are renowned for a highly complex and robust shell, which is the product of the orchestrated expression of genes and secretion of a large number of proteins and other macromolecules from the epithelium of a specialised organ called the mantle. Molluscan shells display remarkable morphological diversity, structure and ornamentation, however the molecular mechanisms underlying the evolution and formation of the shell remain largely unknown. In this thesis, I seek to investigate the nature of the mantle transcriptome focussing on the timing of origin of genes expressed in the mantle and the evolutionary history of gene families that encode secreted proteins in representatives of bivalve and gastropod species.First, by comparative transcriptomics of the mantle, I determined the origin and evolution of gene families expressed in this organ. Gene origin analyses show that most genes expressed in the mantle are taxon-restricted innovations (i.e. unique to a particular species), although a large number of genes arose along the stems leading to the bilaterian and molluscan last common ancestors. Focussing on gene families that encode secreted proteins that are likely to be embedded within the shell and/or to regulate shell construction, I found that these are comprised of both ancient and lineage-specific gene families. The evolution of these gene families is highly dynamic with prolific gene family gains and losses over evolutionary time. By comparing sequences, I also detected indications of positively selected gene families over molluscan evolution. These gene families show unique patterns of enrichment of protein domains, and presumably molecular functions, that are taxa-specific, suggesting that bivalves and gastropods use almost completely different secretory repertoire to construct their shells.I then focused on an ancient gene family expressed in the molluscan mantles -the tyrosinase gene family -to understand how a conserved gene family has been co-opted into the biomineralisation pathway. I found no evidence of large-scale expansion of tyrosinase genes in gastropods, whereas tyrosinase genes in bivalves have undergone substantial independent gene expansions in at least two lineages (Pinctada spp. and Crassostrea gigas), and that the resulting gene duplicates have been co-opted into the mantle gene regulatory network. Many of the tyrosinase genes are found in clusters within the genomes and are expressed at relatively high levels in the mantle. Detailed 3 comparisons of tyrosinase gene expression in different regions of the mantle in two closely related pearl oysters, P. maxima and P. margaritifera, reveal that recently evolved orthologous genes have unique expression profiles across the mantle with high expression levels at the distal mantle edge, suggesting roles in colouration and/or prismatic shell layer construction. Differences in expression levels are consistent with the rapid evo...

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Proteomic Identification of Novel Proteins from the Calcifying Shell Matrix of the Manila Clam Venerupis Philippinarum

Cited by 43 publications

References 29 publications

Novel Molluskan Biomineralization Proteins Retrieved from Proteomics: A Case Study with Upsalin

Novel Molluskan Biomineralization Proteins Retrieved from Proteomics: A Case Study with Upsalin

Studies on the Chemical Structures of Organic Matrices and Their Functions in the Biomineralization Processes of Molluscan Shells

Investigation of gene family evolution and the molecular basis of shell formation in molluscs

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