Skeletal Organic Matrices in Molluscs: Origin, Evolution, Diagenesis
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The mollusc shell comprises a small amount of organic macromolecules, mostly proteins and polysaccharides, which, all together, constitute the skeletal organic matrix (SOM). In the recent years, the study of the SOM of about two dozens of mollusc species via transcriptomics and/or proteomics has led to the identification of hundreds of shell-associated proteins. This rapidly growing set of data allows several comparisons, shedding light on similarities and differences at the primary structure level and on some peculiar evolutionary mechanisms that may have affected SOM proteins. In addition, it constitutes a prerequisite for investigating the SOM repertoires of sub-fossils or fossil specimens, closely related to known extant species, in order to revisit diagenetic processes, i.e. how SOM proteins degrade during fossilization. These two aspects are briefly exemplified here: on the one hand, Aplysia californica, the sea hare, exhibits a vestigial internal shell that has kept a proteomic signature similar to that found in fully functional external shells. On the other hand, subfossil specimens of the giant clam Tridacna, collected in French Polynesia, precisely dated and analysed by proteomics for their SOM content, comprise several preserved proteins that can still be identified by their peptide signature, in spite of information losses likely due to diagenetic transformations.
KeywordsMollusc Shell Proteomics Protein Sequences Functional domains Diagenesis
The mollusc shell is a remarkable composite material made of calcium carbonate at 99% and of a minor organic fraction, the skeletal organic matrix (SOM). During the mineral deposition process, the SOM is secreted by the shell-forming organ, the mantle, and remains occluded. It is considered to be the main regulator of crystallization. In addition, it exhibits an interesting potential for preservation in fossil or subfossil samples (Hare et al. 1980). Classical biochemical characterizations indicate that the SOM consists in a mixture of proteins and polysaccharides (Marin et al. 2012). For decades, the SOM was considered as a ‘black box’ and analysed biochemically in bulk. Nowadays, high-throughput screening of SOMs, via the combined use of proteomics and transcriptomics, has allowed the identification of hundreds of shell proteins that are putatively involved in shell biosynthesis, in about two dozens of model mollusc genera comprising mostly bivalves, gastropods, and, in a lesser extent, cephalopods (Marin et al. 2016). This wealth of molecular data has considerably blurred the outlines of the SOM. However, taken individually, each of these ‘shell repertoires’ represents a key component of the calcifying machinery for making a shell, and shell repertoires can be compared to each other, shedding light on the macroevolution of calcifying matrices (Kocot et al. 2016; Marie et al. 2017). In addition to giving information on the calcification process and its macroevolution, molecular data collected from shells of extant molluscs is a prerequisite for obtaining – whenever possible – similar proteins in archaeological or fossil shell samples, an emerging field defined as paleoproteomics (Demarchi et al. 2016; Wallace and Schiffbauer 2016).
In the present paper, we briefly describe two unpublished examples on the use of proteomics to identify shell proteins: the first example relates to the Californian sea hare, Aplysia californica, a heterobranch gastropod that belong to a family, the Aplysiidae, characterized by an internal atrophied shell which is weakly calcified. The second example relates to subfossil specimens of the giant clam, Tridacna sp., collected in French Polynesia and precisely dated. In both cases, proteomics was performed from their extracted SOM.
34.2 Materials and Methods
Fresh shells of the Californian sea hare Aplysia californica were obtained from RSMAS at the University of Miami (Ph. Gillette) after sacrifice of the living animals according to ethical rules. Six shells of the giant clam Tridacna sp., including fresh, recent and subfossil specimens, were collected by one of us (E. S) on two sites of French Polynesia: Motu Piti Aau, Motu Mute, Bora Bora (Society Islands) and Motu Tepapuri, Mangareva (Gambier archipelago). The fresh Tridacna specimens were used as reference material.
34.2.2 Structural and Geochemical Characterizations
Series of structural characterizations of the shells were performed by SEM observations on polished sections or on freshly broken shell pieces that were slightly etched (EDTA 1% wt/vol, 3–5 min.). Minute fragments were sampled and powdered and the powder analysed by FT-IR spectroscopy, in order to check the mineralogy (aragonite). For Tridacna samples, thick sections were made for cathodoluminescence, epifluorescence and XRD analyses. In addition, the subfossil samples were dated via 14C measurements (Beta Analytic, Miami, FL, USA).
34.2.3 Extraction of the Aplysia and Tridacna SOMs
All Tridacna shells were scrupulously abraded and cleaned (two or three extended bleaching treatments with sodium hypochlorite) in order to remove putative contaminants. The clean powders were decalcified overnight with acetic acid, and the soluble and insoluble fractions were fractionated by centrifugation. All the subsequent steps leading to freeze-dried matrices were performed as previously described (Ramos-Silva et al. 2014). For Aplysia californica, the thinness of shells required adapting the cleaning/extraction protocol, which consisted first in protein desorption in successive bathes of TBS buffer containing Tween 20 (0.1%), NaN3 (0.001%), pH 9.2 for 1 week. After drying and reduction into powder, the samples were decalcified and centrifuged, leading to the fractionation of the soluble and insoluble matrices. The soluble fraction was desalted by several centrifugations/resuspension in water, in Vivaspin 20 cells (3 kDa cutoff), while the insoluble was rinsed with water. Both fractions were freeze-dried.
34.2.4 Proteomics on SOMs
All lyophilized samples were submitted to proteomic analysis (3P5 platform, Institut Cochin, Université Paris Descartes, Paris), after trypsic digestion, as previously described (Immel et al. 2016). For Aplysia californica, assigning identified peptides to known proteins was performed by using Mascot program (version 2.5, MatrixScience, London, UK) against the nonredundant NCBInr database. The search was restricted to ‘Other Metazoa’ dataset, which comprises a large collection of transcriptomic and genomic sequences from Aplysia californica, publicly accessible at NCBI (www.ncbi.nlm.nih.gov/). For Tridacna sp., an unpublished EST database provided by one of us (Dr. T. Takeuchi) from mantle tissues of the coral reef-associated crocus giant clam Tridacna crocea was used for protein identification.
34.3.1 Proteomics on Aplysia californica Shell Matrices
34.3.2 Proteomics on Subfossil Tridacna Samples from French Polynesia
The subfossil samples of the giant clam Tridacna sp. were carefully checked for their preservation state, taking the fresh shells as reference. In particular, micro-samplings made across the thickness of the shell to identify the mineralogy via FT-IR spectroscopy showed that all shells were fully aragonitic and not recrystallized (data not shown). All of them exhibited the classical crossed-lamellar microstructure. In the subfossil shells, we however saw important alterations and perforations in their outermost and innermost layers (which were subsequently discarded) while the core layer was intact.
This paper illustrates how proteomics contributes to answer questions related to the functions, evolution and diagenesis of SOMs in mollusc shell. In the first case, we explored the protein composition of the internal shell of the Californian sea hare, Aplysia californica. Aplysiidae are usually considered as a very derived gastropod family that has emerged only 25 million years ago (Klussmann-Kolb 2004) during the Oligocene epoch. This family is characterized by the presence of an atrophied and weakly calcified internal shell, which has completely lost its primary function, the protection of the soft tissues. In spite of this regressive evolution, it is remarkable to observe that the shell of A. californica has conserved a protein repertoire that exhibits a similar signature as the ones from fully functional external shells, in terms of diversity of protein families present in the matrix.
The second example deals with the diagenetic processes that affect organic matrices associated to calcium carbonate biominerals. In a previous paper, we showed that artificial diagenesis experiments performed on fresh nacre powder samples resulted in two phenomena recorded by proteomic analyses: a decrease of the number of identified proteins correlated to harsh diagenetic conditions and, in parallel, a decrease of the number of peptides identified per protein (Parker et al. 2015). Our analyses performed on the SOMs of subfossil Tridacna tend to correlate this finding. This example gives interesting perspectives for the coming time, i.e. the possibility to track the diagenetic pathway of each shell protein, taken individually, in sub-fossil/fossil of increasing age.
This work was supported by NEWFELPRO programme (Dr. D. Medakovic), by funds from OIST (T. Takeuchi), by EC2CO project (I. Bundeleva, F. Marin) and by annual funds from UMR Biogeosciences (F. Marin).
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