Nacre Calcification in the Freshwater Mussel Unio pictorum: Carbonic Anhydrase Activity and Purification of a 95 kDa Calcium‐Binding Glycoprotein

Abstract

The formation of the molluscan shell is finely tuned by macromolecules of the shell organic matrix. Previous results have shown that the acid-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, the presence of extensive glycosylations and the capacity to interfere at low concentration with in vitro calcium carbonate precipitation. Here we have found that the nacre-soluble matrix exhibits a carbonic anhydrase activity, an important function in calcification processes. This matrix is composed of three main proteinaceous discrete fractions. The one with the highest apparent molecular weight is a 95 kDa glycoprotein that is specific to the nacreous layer. P95, as it is provisionally named, is enriched in Gly, Glx and Asx and exhibits an apparent pI value of approximately 4, or approximately 7 when chemically deglycosylated. Furthermore, its glycosyl moiety, consisting of sulfated polysaccharides, is involved in calcium binding. Purified fractions of the three 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 from an evolutionary viewpoint.

Evolution of Nacre: Biochemistry and Proteomics of the Shell Organic Matrix of the Cephalopod Nautilus macromphalus

“…Some of our recent data suggest that these modules may serve as "functional blocks" in different proteins, soluble or not. [52,53] Finally, we cannot exclude a mechanism opposite to polymerization: protein cleavage. Some proteins of the AIM may indeed maturate and be cleaved into short peptides, which would be easily solubilized.…”

Novel Molluskan Biomineralization Proteins Retrieved from Proteomics: A Case Study with Upsalin

“…Furthermore, Upsalin is only weakly glycosylated, and it is very unlikely that its sugar moieties exert a role in concentrating calcium ions in the vicinity of the nucleation sites [39] as has been proposed for other proteins. [11,32] Previous calcium-binding studies on the ASM of Unio pictorum, by staining with Stains All and by autoradiography with 45 Ca, suggest weak calcium binding ability at molecular weights below 14 kDa. [7] Consistently with this, Upsalin does not interfere in vitro with the precipitation of calcium carbonate.…”

“…Upsalin has a predicted signal peptide with a cleavage site between the positions 16 and 17 and a putative transmembrane region (positions [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23]. The mature form of the protein consists of 109 aa residues and has a theoretical molecular weight of 12.3 kDa.…”

“…[1,10] Proteins of the soluble matrix can be further fractionated by electrophoresis or HPLC, and subjected to sequence analysis through mass spectrometry [11] or Edman degradation. [12] In order to obtain the full-length primary structures, degenerate primers are designed from partial protein sequences for amplification of shell protein-encoding transcripts, extracted from mantle tissues.…”

“…Some proteins are true shell proteins, but because they have been sequenced directly, information at the transcript level is not available. [11,12] Conversely, some proteins have been characterized only at the transcript level, with demonstration of their presence in the shell remaining to be achieved. [15] Lately, the introduction of high-throughput approaches to mollusks is changing this picture.…”