bands in the acetic acid-soluble prisms extract, below 10 kDa, and above 60 kDa, on
1D electrophoretic profiles (Marin et al. 2007b). However, sequence information
on these bands is missing, so far. When the matrix of the complete prismatic
layer – and not only the sodium hypochlorite-isolated prisms – is extracted
(P. Narayanappa, unpublished, 2010), a huge set of additional protein bands are
visualized on gel, probably comprising proteins which are weakly linked to the
mineral phase. Similarly, in the nacre extracts, several proteins bands have been
detected in the acetic acid-soluble and in the Laemmli-soluble fractions (Marin
et al. 2000). For none of these proteins, we possess sequence information.
Other putative shell proteins have been retrieved by one of us (P. Narayanappa
un published, 2010) by screening our cDNA expression library of the mantle of P.
nobilis with anti-caspartin and anti-calprismin polyclonal antibodies. This operation yielded additional clones encoding putative proteins that have a “shell” signature: short hydrophobic or acidic motifs, predominance of few amino acids, such as
glutamic acid, serine or leucine in the overall composition of the protein. Among
them, the translated product of clone CLP2T7 is enriched in glutamic acid (18%), in
leucine (10%), and in serine (10.3%), while the one of clone CSP3 is characterized
by an extremely acidic C-terminus (with a poly-D domain). Interestingly, in parallel
to the immunoscreening, a proteomic analysis of the acetic acid-soluble prisms
matrix of P. nobilis yielded different peptides. One of them belongs to CSP3
translated sequence (Narayanappa et al., in preparation), proving that this putative
protein is truly a shell matrix protein of the prismatic layer. Our results are being
compiled for their subsequent publication.
13.4.4 Effect of the Acidic Proteins of P. nobilis
on “Calcification,” Sensu Lato
13.4.4.1 “Classical” Biochemical Effects
As described in Sect. 4.3, the identified shell proteins of P. nobilis exert different
biochemical effects in relation with calcification. These effects, such as the calcium-binding effect, the inhibition effect, or the interference with CaCO 3 crystallization, can be quantified in a “test tube.” Although they give us precious
information on the putative functions of the tested protein, we are fully aware
that, by no means, they substitute to the “function.” In the present paragraph, we
clearly dissociate the effects – which are known – from the true functions – which
are still elusive.
The calcium-binding property of the shell proteins of P. nobilis is an interesting
property, prone to discussion. In our hands, we always observed that the calciumbinding ability of caspartin, of calprismin, and of the nacre and prisms matrices in
general, was labile. We observed that the level of calcium-binding was better for the
whole matrices than for its separate constituents, like caspartin or calprismin, when
13 Acidic Shell Proteins of the Mediterranean Fan Mussel Pinna nobilis
379
1D electrophoretic profiles (Marin et al. 2007b). However, sequence information
on these bands is missing, so far. When the matrix of the complete prismatic
layer – and not only the sodium hypochlorite-isolated prisms – is extracted
(P. Narayanappa, unpublished, 2010), a huge set of additional protein bands are
visualized on gel, probably comprising proteins which are weakly linked to the
mineral phase. Similarly, in the nacre extracts, several proteins bands have been
detected in the acetic acid-soluble and in the Laemmli-soluble fractions (Marin
et al. 2000). For none of these proteins, we possess sequence information.
Other putative shell proteins have been retrieved by one of us (P. Narayanappa
un published, 2010) by screening our cDNA expression library of the mantle of P.
nobilis with anti-caspartin and anti-calprismin polyclonal antibodies. This operation yielded additional clones encoding putative proteins that have a “shell” signature: short hydrophobic or acidic motifs, predominance of few amino acids, such as
glutamic acid, serine or leucine in the overall composition of the protein. Among
them, the translated product of clone CLP2T7 is enriched in glutamic acid (18%), in
leucine (10%), and in serine (10.3%), while the one of clone CSP3 is characterized
by an extremely acidic C-terminus (with a poly-D domain). Interestingly, in parallel
to the immunoscreening, a proteomic analysis of the acetic acid-soluble prisms
matrix of P. nobilis yielded different peptides. One of them belongs to CSP3
translated sequence (Narayanappa et al., in preparation), proving that this putative
protein is truly a shell matrix protein of the prismatic layer. Our results are being
compiled for their subsequent publication.
13.4.4 Effect of the Acidic Proteins of P. nobilis
on “Calcification,” Sensu Lato
13.4.4.1 “Classical” Biochemical Effects
As described in Sect. 4.3, the identified shell proteins of P. nobilis exert different
biochemical effects in relation with calcification. These effects, such as the calcium-binding effect, the inhibition effect, or the interference with CaCO 3 crystallization, can be quantified in a “test tube.” Although they give us precious
information on the putative functions of the tested protein, we are fully aware
that, by no means, they substitute to the “function.” In the present paragraph, we
clearly dissociate the effects – which are known – from the true functions – which
are still elusive.
The calcium-binding property of the shell proteins of P. nobilis is an interesting
property, prone to discussion. In our hands, we always observed that the calciumbinding ability of caspartin, of calprismin, and of the nacre and prisms matrices in
general, was labile. We observed that the level of calcium-binding was better for the
whole matrices than for its separate constituents, like caspartin or calprismin, when
13 Acidic Shell Proteins of the Mediterranean Fan Mussel Pinna nobilis
379