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Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell: focus on biomineralization.

Joubert C, Piquemal D, Marie B, Manchon L, Pierrat F, Zanella-Cléon I, Cochennec-Laureau N, Gueguen Y, Montagnani C - BMC Genomics (2010)

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ifremer - Laboratoire de Biotechnologie et de Qualité de Perle, Centre Océanologique du Pacifique - BP 7004, 98719 Taravao, French Polynesia.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

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Gene Ontology annotation of annotated unique sequences (singletons and contigs) from P. margaritifera. Distribution (%) of gene ontology terms among the annotated unique sequences. GO ontologies are represented as general function categories. Among the 10 004 unique sequences with GO assignation, 5976 (59.7%) are involved in biological processes (A), 6855 (68.5%) have molecular functions (B) and 5737 (57.3%) are cellular components (C). Specialized terms of the binding category repartition (D) shows 17% of sequences implicated in ion binding.
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Figure 1: Gene Ontology annotation of annotated unique sequences (singletons and contigs) from P. margaritifera. Distribution (%) of gene ontology terms among the annotated unique sequences. GO ontologies are represented as general function categories. Among the 10 004 unique sequences with GO assignation, 5976 (59.7%) are involved in biological processes (A), 6855 (68.5%) have molecular functions (B) and 5737 (57.3%) are cellular components (C). Specialized terms of the binding category repartition (D) shows 17% of sequences implicated in ion binding.

Mentions: Gene Ontology (GO) assignment was carried out on unique sequences in order to categorize transcripts from P. margaritifera mantle by putative function. The GO project provides a structured and controlled vocabulary of terms (ontologies) for describing gene product characteristics and gene product annotation data [43]. In our study, 10 004 unique sequences (13.0%) were successfully assigned to one or more GO terms. Among these, following the functional classification with the three unrelated GO ontologies, 5976 (59.7%) are involved in biological processes, 6855 (68.5%) have molecular functions and 5737 (57.3%) are cellular components. For each of these three ontologies, annotated sequences are mainly distributed among two or three of the general term categories. Within the 5976 unique sequences involved in biological processes, 5006 (83.8%) and 4191 (70.1%) are dedicated to cellular processes and metabolic processes, respectively (Figure 1A). Similarly, in the molecular functions sub-ontology, 5208 (76.0%) and 3704 (54.0%) of the 6855 unique sequences have binding and catalytic activity, respectively (Figure 1B). Finally, of the 5737 unique sequences predicted to be cellular components, 5656 (98.6%) and 3868 (67.4%) are related to cell and organelle components, respectively (Figure 1C). These results constitute common features among EST databases available from marine organisms, and in particular mollusks [40,44].


Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell: focus on biomineralization.

Joubert C, Piquemal D, Marie B, Manchon L, Pierrat F, Zanella-Cléon I, Cochennec-Laureau N, Gueguen Y, Montagnani C - BMC Genomics (2010)

Gene Ontology annotation of annotated unique sequences (singletons and contigs) from P. margaritifera. Distribution (%) of gene ontology terms among the annotated unique sequences. GO ontologies are represented as general function categories. Among the 10 004 unique sequences with GO assignation, 5976 (59.7%) are involved in biological processes (A), 6855 (68.5%) have molecular functions (B) and 5737 (57.3%) are cellular components (C). Specialized terms of the binding category repartition (D) shows 17% of sequences implicated in ion binding.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3091754&req=5

Figure 1: Gene Ontology annotation of annotated unique sequences (singletons and contigs) from P. margaritifera. Distribution (%) of gene ontology terms among the annotated unique sequences. GO ontologies are represented as general function categories. Among the 10 004 unique sequences with GO assignation, 5976 (59.7%) are involved in biological processes (A), 6855 (68.5%) have molecular functions (B) and 5737 (57.3%) are cellular components (C). Specialized terms of the binding category repartition (D) shows 17% of sequences implicated in ion binding.
Mentions: Gene Ontology (GO) assignment was carried out on unique sequences in order to categorize transcripts from P. margaritifera mantle by putative function. The GO project provides a structured and controlled vocabulary of terms (ontologies) for describing gene product characteristics and gene product annotation data [43]. In our study, 10 004 unique sequences (13.0%) were successfully assigned to one or more GO terms. Among these, following the functional classification with the three unrelated GO ontologies, 5976 (59.7%) are involved in biological processes, 6855 (68.5%) have molecular functions and 5737 (57.3%) are cellular components. For each of these three ontologies, annotated sequences are mainly distributed among two or three of the general term categories. Within the 5976 unique sequences involved in biological processes, 5006 (83.8%) and 4191 (70.1%) are dedicated to cellular processes and metabolic processes, respectively (Figure 1A). Similarly, in the molecular functions sub-ontology, 5208 (76.0%) and 3704 (54.0%) of the 6855 unique sequences have binding and catalytic activity, respectively (Figure 1B). Finally, of the 5737 unique sequences predicted to be cellular components, 5656 (98.6%) and 3868 (67.4%) are related to cell and organelle components, respectively (Figure 1C). These results constitute common features among EST databases available from marine organisms, and in particular mollusks [40,44].

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ifremer - Laboratoire de Biotechnologie et de Qualité de Perle, Centre Océanologique du Pacifique - BP 7004, 98719 Taravao, French Polynesia.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus