Transcriptome and proteome analysis of Pinctada margaritifera calcifying mantle and shell: focus on biomineralization.
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.
Ifremer - Laboratoire de Biotechnologie et de Qualité de Perle, Centre Océanologique du Pacifique - BP 7004, 98719 Taravao, French Polynesia.
Caroline
C
Joubert
David
D
Piquemal
Benjamin
B
Marie
Laurent
L
Manchon
Fabien
F
Pierrat
Isabelle
I
Zanella-Cléon
Nathalie
N
Cochennec-Laureau
Yannick
Y
Gueguen
Caroline
C
Montagnani
D000595:Amino Acid Sequence; D000825:Animal Structures / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002113:Calcification, Physiologic / Q000235:genetics*; D020451:Contig Mapping; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D017403:In Situ Hybridization; D008903:Minerals / Q000378:metabolism*; D008958:Models, Molecular; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D049911:Pinctada / Q000033:anatomy & histology* / Q000235:genetics*; D020543:Proteome / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D008903:Minerals; D020543:Proteome; D012333:RNA, Messenger