Abstract
Background : The shell of the pearl-producing bivalvePinctada margaritiferais composed of an organic cell-freematrix that plays a key role in the dynamic process of biologically-controlled biomineralization. In order to increasegenomic resources and identify shell matrix proteins implicated in biomineralization inP. margaritifera, high-throughput Expressed Sequence Tag (EST) pyrosequencing was undertaken on the calcifying mantle, combinedwith a proteomic analysis of the shell. Results : We report the functional analysis of 276 738 sequences, leading to the constitution of an unprecedentedcatalog of 82P. margaritiferabiomineralization-related mantle protein sequences. Components of the current“chitin-silk fibroin gel-acidic macromolecule”model of biomineralization processes were found, in particular ahomolog of a biomineralization protein (Pif-177) recently discovered inP. fucata. Among these sequences, wecould show the localization of two other biomineralization protein transcripts, pmarg-aspeinandpmarg-pearlin,intwo 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 theP. margaritiferashell organic matrix, we demonstrated the presence of 30 sequences containing almost all of theshell proteins that have been previously described from shell matrix protein analyses of the Pinctadagenus. Theintegration of these two methods allowed the global composition of biomineralizing tissue and calcified structuresto be examined in tandem for the first time. Conclusions : This EST study made on the calcifying tissue ofP. margaritiferais the first description ofpyrosequencing on a pearl-producing bivalve species. Our results provide direct evidence that our EST data setcovers most of the diversity of the matrix protein ofP. margaritiferashell, but also that the mantle transcriptsencode proteins present inP. margaritiferashell, hence demonstrating their implication in shell formation. Combining transcriptomic and proteomic approaches is therefore a powerful way to identify proteins involved inbiomineralization. Data generated in this study supply the most comprehensive list of biomineralization-relatedsequences presently available among protostomian species, and represent a major breakthrough in the field ofmolluskan biomineralization.