Coupling Proteomics and Transcriptomics for the Identification of Novel and Variant Forms of Mollusk Shell Proteins: A Study with P. margaritifera

Abstract

Shell matrix proteins from Pinctada margaritifera were characterized by combining proteomics analysis of shell organic extracts and transcript sequences, both obtained from the shell-forming cell by using the suppression subtractive hybridization method (SSH) and from an expressed sequence tag (EST) database available from Pinctada maxima mantle tissue. Some of the identified proteins were homologues to proteins reported in other mollusk shells, namely lysine-rich matrix proteins (KRMPs), shematrins and molluscan prismatic and nacreous layer 88 kDa (MPN88). Sequence comparison within and among Pinctada species pointed to intra- and interspecies variations relevant to polymorphism and to evolutionary distance, respectively. In addition, a novel shell matrix protein, linkine was identified. BLAST analysis of the peptide sequences obtained from the shell of P. margaritifera against the EST database revealed the presence of additional proteins: two proteins similar to the Pif97 protein that was identified in the shell of P. fucata, a chitinase-like protein previously identified in Crassostrea gigas, two chitin-binding proteins, and two incomplete sequences of proteins unknown so far in mollusk shells. Combining proteomics and transcriptomics analysis we demonstrate that all these proteins, including linkine, are addressed to the shell. Retrieval of motif-forming sequences, such as chitin-binding, with functional annotation from several peptides nested in the shell could indicate protein involvement in shell patterning.

Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida Elliptio complanata and Villosa lienosa

“…A very recent proteomic investigation [38] has shown that several proteins constituting the byssal acellular structures (tyrosinase, A2M, EGF and VWA containing proteins) are similar to those of the shell layers, demonstrating similarities in the formation and the evolution of these two structures. In addition, mollusc matrix proteins rich in Q have also been found in bivalves: for example, MPN88 was previously characterized from the pearl oyster Pinctada margaritifera [39]. In the shell of the gastropod Lottia, at least four different matrix proteins present similar features [29].…”

Identification and Characterisation of a Calcium Carbonate‐Binding Protein, Blue Mussel Shell Protein (BMSP), from the Nacreous Layer

“…Here, we explored the possibility that other molluscs also have a Pif-related protein that plays a role in nacreous layer formation. Although a Pif homologue from the Pinctada genus has been identified from the expressed sequence tag (EST) database, [26,27] the diversity of Pif molecules among various molluscan species is still unknown. Thus, it remains unclear if the role of Pif represents a common mechanism underlying nacreous layer formation.…”

Proteomic Strategy for Identifying Mollusc Shell Proteins Using Mild Chemical Degradation and Trypsin Digestion of Insoluble Organic Shell Matrix: A Pilot Study on Haliotis tuberculata

“…Again, this in silico annotation strategy is not efficient at identifying new shell proteins with an unexpected composition. In this context, it is now necessary to bridge the gap between high throughput identification of putative shell proteins via EST construction and guaranteed shell targeting (Berland et al 2011).…”