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Nacre calcification in the freshwater mussel Unio pictorum : carbonic anhydrase activity and purification of a 95-kDa calcium-binding glycoprotein

The formation of the molluscan shell is finely tuned by macromolecules of the shell organic matrix. Previous results have shown that the acido-soluble fraction of the nacre matrix of the freshwater paleoheterodont bivalve Unio pictorum shell displays a number of remarkable properties, such as calcium-binding activity, presence of extensive glycosylations, capacity to interfere at low concentration with the in vitro calcium carbonate precipitation. In the present paper, we found that the nacre soluble matrix exhibits a carbonic anhydrase activity, an important function in calcification processes. This matrix is composed of 3 main proteinaceous discrete fractions. The one with the highest apparent molecular weight is a 95- kDa glycoprotein, which is specific of the nacreous layer. P95, as it is provisionally named, is enriched in Gly, Glx and Asx and exhibits an apparent pI value around 4, and close to 7 when chemically deglycosylated. Furthermore, its glycosyl moiety, constituted of sulfated polysaccharides, is involved in calcium binding. Purified fractions of the 3 main proteins were digested with trypsin and the resulting peptides were analysed by mass spectrometry. Our results suggest that identical peptides are constitutive domains of the different proteins. Partial primary structures were obtained by de novo sequencing, and compared with known sequences from other mollusc shell proteins. Our results are discussed in an evolutionary viewpoint.

biomineralization; mollusc shell nacre; carbonic anhydrase; 2-DE; two-dimensional electrophoresis; organic matrix

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