The ability to determine the complete amino acid sequence of proteins resulted in the development of molecular phylogeny. The technique was first applied to proteins such as cytochrome c and hemoglobin. With the subsequent development of molecular biology techniques, the approach could be extended to protein-coding DNA sequences. These techniques have thus far been applied primarily to extant species. However, conceptually they could just as easily be applied to the preserved proteins of fossil species. Soft tissue preservation of proteins is rare or absent in invertebrate fossils and thus far has not yielded suitable starting material. However, biomineralized tissue, such as the carbonate shells of molluscs, is a potential source. Here, crystallization of the calcium carbonate inorganic phase has occurred in and directed by an organic matrix consisting of various proteins, glycoproteins, and polysaccharides.
In fact, these matrix proteins are easily isolated from modern mollusc shells by dissolution of the carbonate phase (e.g. with weak acid or the chelating agent EDTA), yielding soluble and insoluble (in water) fractions of proteins. Initial studies of these proteins, primarily from bivalves, were hindered by the complexity of the mixtures, in part due to multiple protein species, and their possible post-translational modification (e.g. by phosphorylation and glycosylation). Improved purification techniques have allowed separation and sequencing of individual protein species. In addition, even partial amino acid sequences, with the application of the techniques of molecular biology, have allowed determination of the DNA sequences (and thus the amino acid sequences) corresponding to these proteins. Marin et al. (2008) have recently reviewed these proteins.
The question remains, can the mollusc shell proteins contribute to molecular phylogeny studies of fossil molluscs? More specifically, do these shell proteins survive fossilization intact or at least in large discrete fragments that can be isolated from fossils and sequenced? Weiner et al. (1976) described the isolation of glycoproteins from the late Cretaceous (80 Ma old) bivalve Scabrotrigonia thoracica shells . Several discrete polypeptide bands were separated on polyacrylamide gel electrophoresis, and amino acid analyses (but not sequence studies) carried out. More recently, isolation of the insoluble fraction of shell proteins from the calcitic outer layer of the Middle Miocene (8-18 Ma) gastropod Ecphora has been reported (Nance et al., 2015). While further fractionation of the proteins in this fraction has not yet been carried out, the physical character of the residue and NMR and amino acid analysis studies suggest the possibility of large or intact polypeptides that can be sequenced. The insoluble protein fraction also contains the red-brown pigment (presumably a carotenoid) that marks Ecphora in fossil form, and likely in life.
To restate the question, can data be extracted from the sequences of fossil shell proteins that will allow us to construct a molecular phylogeny of the molluscs? Given the advances in the resolution of complex protein mixtures and the increased sensitivity of our analytical techniques, the answer at this point is, pending further studies, a hopeful “maybe”.
Nance, J.R., Armstrong, J.T., Cody, G.D., Fogel, M.L., and Hazen, R.M. (2015) Preserved macroscopic polymeric sheets of shell-binding protein in the Middle Miocene (8 to 18 Ma)gastropod Ecphora, Geochemical Perspectives Letters 1, 1-9.
Marin, F., Luquet, G., Marie, B., and Medakovic, D. (2008) Molluscan shell proteins: Primary structure, origin, and evolution. Current Topics in Developmental Biology 80, 209-276.
Weiner, S., Lowenstam, H.A., and Hood, L. (1976) Characterization of 80-million-year-old mollusc shell proteins. Proc. Natl. Acad. Sci. USA 73(8), 2541-2545.