The shell matrix of the european thorny oyster, Spondylus gaederopus: microstructural and molecular characterization

Abstract

Molluscs, the largest marine phylum, display extraordinary shell diversity and sophisticated biomineral architectures. However, mineral-associated biomolecules involved in biomineralization are still poorly characterized. We report the first comprehensive structural and biomolecular study of Spondylus gaederopus, a pectinoid bivalve with a peculiar shell texture. Having been used since prehistoric times, this is the best-known shell of Europe's cultural heritage. We find that Spondylus microstructure is very poor in organics, which are mostly intercrystalline and concentrated at the interface between structural layers. Using highresolution liquid chromatography tandem mass spectrometry (LC-MS/MS) we characterized several shell protein fractions, isolated by different bleaching treatments. Six shell proteins were identified, which displayed features and domains typically found in biomineralized tissues, including the prevalence of intrinsically disordered regions. However, most of the reconstructed peptide sequences could not be matched to any known shell proteins and probably represent lineage-specific sequences. This work sheds light onto the shell matrix involved in the biomineralization in spondylids. The proteomics data suggests that Spondylus has evolved a shell-forming toolkit, distinct from that of other better studied pectinoids-fine-tuned to produce shell structures with high mechanical properties, while limited in organic content. This study therefore represents an important milestone for future studies on biomineralized skeletons and provides the first reference dataset for forthcoming molecular studies of Spondylus archaeological artifacts.

Shell palaeoproteomics: First application of peptide mass fingerprinting for the rapid identification of mollusc shells in archaeology

“…Remarkably, for Spondylus, the intensity and number of potential marker peaks is considerably higher in the intracrystalline fraction compared to the spectra obtained after just 4 h of bleaching, for which the PMFs were barely detectable. This effect has also been observed in other shells [60], including a study of the Spondylus proteome by tandem mass spectrometry [45], and may be due to the difficulty of breaking down complex networks of proteins with other shell matrix macromolecules, such as chitin. It is likely that the presence of glycosylated proteins, lipoproteins, phospholipids, or proteins with repetitive low complexity domains (RLCD), could influence signal detection (or suppress it completely) [51,61].…”

“…All of the shell samples were already available as fine-grained powders (particle size: 200-500 μm) as they had been used for previous studies [14,16,39,44,45]. The powders represent the bulk fraction of the shell, i.e.…”

“…However, as the future scope of this project is to create a large library of "intracrystalline PMFs", in this work, we give an "operational" definition of the "intracrystalline" proteins as the fraction which can be isolated via a 48-h bleaching step using concentrated NaOCl (~12%). Previous studies have shown that this treatment to be effective for all shell taxa tested thus far, regardless of their age (modern vs fossil) or provenance [14,[36][37][38][39][40]45,57].…”

“…could easily be distinguished from other taxa, while both Spondylus and Ostrea yielded a more uncertain classification. Our hypothesis was that, by retrieving the peptide mass fingerprints (PMFs) of the intracrystalline shell proteins, more secure taxonomic identification could be achieved, especially as new data on Spondylus showed that this mollusc shell has a very distinct protein makeup [45]. This encompasses the second objective of this work, i.e.…”

“…Spondylus, Glycymeris, Cardiidae, Strombus and some foliated shells, e.g. Pecten, Crassostrea, the shell matrix content may be as low as~0.004% by weight [42][43][44][45]. Therefore, considering that sample size is usually a limiting factor for the application of palaeoproteomics to unique archaeological artefacts, sample preparation protocols commonly employed on small samples (typically, < 20 mg) of other biomineralised tissues, e.g.…”