Benjamin Marie PhD thesis

Evolution of the biomineralizations in molluscs nacreous layers: molecular characterization of calcifying matrix in the cephalopod Nautilus macromphalus and the bivalve Unio pictorum

Defended the 14 may 2008



In metazoan, molluscan shells are one of the most fascinating examples for the investigation of the calcium carbonate biomineralization phenomenon. These exoskeletons are secreted by the outer epithelium of the calcifying mantle. They are composed of 95% of mineral – calcite and/or aragonite -, the rest of the shells being constituted by a mixture of proteins, glycoproteins and polysaccharides, called the organic matrix. This calcifying matrix is directly involved in biomineralization.

This PhD work is focussed on the characterization of organic components associated to the nacreous layer. In molluscs, the nacre is observed in shells of extent species of bivalves, gastropods, cephalopods and also monoplacophorans. Most of the published data on macromolecular compounds of the nacre matrices are based on few species belonging to the genera Pinctada and Pinna for bivalves, and to the genus Haliotis for gastropods. In this study, we investigated the characterization of these compounds in two new models, the freshwater mussel Unio pictorum and the cephalopod Nautilus macromphalus. We think that this comparative approach highlights the molecular mechanisms of nacre formation and their evolutionary story within nacreous shell molluscs.

Unio pictorum is a freshwater bivalve, very common in Burgundy, which exhibits a nacro-prismatic shell exclusively composed of aragonite. Its nacre-associated organic matrix exhibits a remarkable carbonic anhydrase activity, a key enzyme in calcification processes, which had already been detected in the shell of Pinctada sp. SDS-PAGE run under denaturing conditions shows that the acido-soluble nacre matrix is mainly composed of 5 discrete proteins of 95, 50, 29, 16 and 12 kDa apparent molecular weight. A glycosylation study shows that the three heaviest components are substantially glycosylated proteins bearing acidic residues, which are directly involved in mineralization processes. Notably, the 95-kDa protein, which is specific of the nacreous layer, carries sulfated sugars implicated in calcium-binding activity and in the capacity to interact with in vitro CaCO3 crystal precipitation. Partial internal sequences of the nacre-associated proteins were described with mass spectrometry approach.

The nacro-prismatic shell of the cephalopod Nautilus macromphalus is fully aragonitic. SDS-PAGE shows that the acido-soluble matrix is composed of high molecular weight polysaccharides, 63- and 57-kDa glycoproteins and 3-4 calcium-binding proteins migrating between 20 and 10 kDa. On 2-DE, the different components of the nacre matrix migrate either at very acidic or at very basic pI. Partial sequences were obtained by mass spectrometry analysis from purified proteins and complete acido-soluble and acido-insoluble matrix after tryptic digestion. The new sequences exhibit partial similarities with bivalvian nacre proteins, but no homology was detected with gastropod nacre proteins.

Furthermore, we performed a preliminary study of acido-soluble matrices from Rhynchonelliformean brachiopod shells. This work shows that in this phylum the mechanisms of shell formation involve the production of a shell-associated calcifying matrix, which is composed of discrete macromolecular compounds exhibiting various biochemical properties.



Biomineralization, mollusk, nacre, organic matrix, evolution, biochemistry, glycosylation, mass spectrometry, Unio pictorum, Nautilus macromphalus, brachiopodes