Different secretory repertoires control the biomineralization processes of prism and nacre deposition of the pearl oyster shell

Abstract

Mollusca evolutionary success can be attributed partly to their efficiency to sustain and protect their soft body with an external biomineralized structure, the shell. Current knowledge of the protein set responsible for the formation of the shell microstructural polymorphism and unique properties remains largely patchy. In Pinctada margaritifera and Pinctada maxima, we identified 80 shell matrix proteins, among which 66 are entirely unique. This is the only description of the whole "biomineralization toolkit" of the matrices that, at least in part, is thought to regulate the formation of the prismatic and nacreous shell layers in the pearl oysters. We unambiguously demonstrate that prisms and nacre are assembled from very different protein repertoires. This suggests that these layers do not derive from each other.

Fig. 1.

P. margaritifera prism and nacre SMPs. The nacre (A) and prism (B) AIM proteins were digested with trypsin, and the resulting peptides were analyzed by mass spectrometry (MS/MS mode). P. margaritifera prism (C) and nacre (D) SMPs that presented at least two matching peptides, or for which identification was further confirmed in P. maxima by homolog protein detection, are listed. Raw MS/MS data were directly interrogated against the assembled mantle EST data set (17). An asterisk indicates protein sequences discovered in this analysis. Predicted signal peptide can be retrieved from all EST-translated products that match shell proteomic data, indicating that these proteins are secreted. Full-length sequences of 52 unique SMPs (17 nacre and 35 prism proteins) were deposited on National Center for Biotechnology Information database (Datasets S1 and S2). No additional proteins were identified from the P. margaritifera prism and nacre acid-soluble matrices (Table S1). N: nacre; P: prism; Pfu: Pinctada fucata; Cgig: Crassostrea gigas. “>” indicates that mass spectrometry identification scores are higher in one of the two layers when detected in both shell AIMs. Shematrin8 was then considered as prism SMP, and Nacrein and NUSP-18 as nacre SMPs, accordingly. (Scale bars, 5 and 50 µm for A and B, respectively.)

Fig. 2.

Comparison of prism and nacre SMPs of P. margaritifera and P. maxima. Prisms and nacre proteins identified in both species by MS/MS analyses are circled in blue/green or red/orange, respectively. In P. margaritifera, 48 proteins were detected in prisms, and 33 in nacre. Forty-five proteins are prism-specific and 30 are nacre-specific. Only three proteins are common to the two layers. In P. maxima, 28 proteins were detected in prisms (with 26 prism-specific), 17 in nacre (with 15 nacre-specific), and only 2 in common proteins. Twenty-four proteins are common to P. margaritifera and P. maxima prism AIMs, and 15 are common to both nacres. From the 43 SMPs detected in P. maxima, 41 homologs can be retrieved in P. margaritifera and present high sequence similarities (above 85–95% sequence identity), giving a congruent picture with previous phylogenetic data for these species (20). From the 80 different Pinctada SMPs identified here, 77 can be specifically detected in prisms and nacre. Three proteins only, Shematrin8, Nacrein, and NUSP-18, are found in both shell layers.

Fig. 3.

Immunolocalization of nacre SMPs on shell and mantle of P. margaritifera. A polyclonal antibody raised against a solubilized fraction of nacre AIM was used to identify nacre proteins (A) on Western blot (N, nacre; P, prisms), (B) in nacre cross-section by immunogold (scale bar, 1 μm), and (C) in mantle epithelia by immunofluorescence. mf: middle fold; of: outer fold; pg: periostracal groove; oe: outer epithelium. We note that the fact that more gold particles are observed on the upper interlamellar side of nacre tablets, rather than on the down side, is mainly due to the microtopography of nacre fractures and to the angle of observation.

Fig. 4.

Prism and nacre SMP gene expression in mantle edge and pallium of P. margaritifera estimated by high-throughput qRT-PCR (Fluidigm). Comparison of In(mantle edge/mantle pallium) [ln(ME/MP)] expression ratio (fold/fold) of prism and nacre SMPs. Protein names are indicated in blue and red colors for prism and nacre SMPs, respectively. The three SMPs detected in both prism and nacre layers are indicated with an asterisk.

Fig. 5.

Localization of prism and nacre transcripts in P. margaritifera mantle by in situ hybridization. (A) MP10, Clp-1, and Fibronectin-1 transcripts are expressed in the outer epithelium of the mantle edge. (B) NUSP-1, Pearlin, and MNRP34 transcripts are expressed in the outer epithelium of the mantle pallium. Paraffin-embedded sections of oyster tissues were hybridized with antisense or sense single-stranded cDNA probes labeled with digoxigenin. Positive cells are stained in dark blue. Sense probes showed no hybridization (Fig. S4). Black arrows symbolize the epithelial cell limits of prism and nacre transcript expression. (Scale bars, 1 mm in large view and 50 μm on stained-cell enlargements.) mf: middle fold; of: outer fold; pg: periostracal groove; oe: outer epithelium.