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 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.
Marie
Benjamin
B
UMR CNRS 5561 Biogéosciences, Université de Bourgogne, Dijon, France. benjamin.marie@u-bourgogne.fr
Luquet
Gilles
G
Bédouet
Laurent
L
Milet
Christian
C
Guichard
Nathalie
N
Medakovic
Davorin
D
Marin
Frédéric
F
D000595:Amino Acid Sequence; D000818:Animals; D049872:Bivalvia / Q000033:anatomy & histology* / Q000145:classification / Q000201:enzymology / Q000378:metabolism*; D002113:Calcification, Physiologic; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002254:Carbonates; D002256:Carbonic Anhydrases / Q000378:metabolism*; D004789:Enzyme Activation; D005618:Fresh Water; D005782:Gels; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry; D008855:Microscopy, Electron, Scanning; D008970:Molecular Weight; D040901:Proteomics; D017421:Sequence Analysis; D012995:Solubility
D002135:Calcium-Binding Proteins; D002254:Carbonates; D005782:Gels; D006023:Glycoproteins; D002256:Carbonic Anhydrases; D002118:Calcium