effect of calprismin in vitro, its localization in the shell by immunogold, and its
expression in mantle tissues.
The second protein, caspartin, has been studied in vitro more in detail
(Fig. 13.5), although its sequence is still far from being complete (Marin et al.
2005, 2007a, b). Similarly to calprismin, caspartin is an intracrystalline acidic
protein, which is abundant in the acid-soluble extract of the prisms. Caspartin has
a molecular weight of about 17 kDa. It is extremely enriched in aspartate residues
(more than 60%). Caspartin exhibits better calcium-binding ability than calprismin,
but its affinity for calcium is also weak, i.e., the fixation of calcium is reversible,
and calcium can be easily removed by gentle rinsing of the membrane where
caspartin has been blotted. Caspartin does not seem to be glycosylated, but we do
not exclude the possibility that it may be phosphorylated. An interesting finding
was that caspartin self-agregates in solution: in non-denaturing gels, we were able
to show that caspartin forms di-, tri-, tetra-, penta-, hexa-, and heptamers. On 2D
gels, caspartin migrates as a big spot at pI below 3. However, three tenuous minor
spots of the same molecular weight, one in the acidic domain, and two, basic, are
also observed, which may indicate that caspartin may not be absolutely pure, or that
it exhibits isoforms. In vitro, caspartin is a powerful inhibitor of the precipitation of
calcium carbonate, since it induces an inhibitory effect for concentrations inferior
to 1 mg/ml. Caspartin has also drastic effects on the morphology of calcite crystals
grown in vitro: effects are already observed around 0.2 mg/ml, and they increase
with the concentration of the protein in solution. Between 1 and 3 mg/ml, caspartin
induces the formation of polycrystalline aggregates, together with the formation of
“single crystals,” which exhibit a foliation. When the concentration of caspartin is
increased, the size of the crystals decreases, and above a threshold, caspartin acts as
an inhibitor of calcification and no crystals are produced. A polyclonal antibody,
specific of caspartin, indicated that caspartin, or a similar 17-kDa protein, is also
present in the nacreous layer of Pinna nobilis, but in much lesser amounts (6–8
times less). This antibody was used to localize caspartin directly in the shell of
Pinna nobilis (Marin et al. 2007b). In cross sections, perpendicular to the prisms
axis, we observe that caspartin has two locations: first, it is distributed at the surface
of the prisms (¼ within the prisms), a predictable finding. We do not observe a
particular pattern, but rather, a uniform distribution of the signal at the prisms
surface. Second, more surprisingly, caspartin occupies also an intercristalline
location: it forms a continuous film at the interface between the prisms themselves
and the interprismatic insoluble walls. In other words, the insoluble hydrophobic
interprismatic wall is sandwiched between two caspartin-containing layers. In
longitudinal sections, caspartin is located discontinuously along the prisms. Its
distribution in that dimension is not strictly correlated with the growth lines
observed for each prism, although we observed that caspartin may be more
concentrated on some growth plans (Marin et al. 2007b).
Following the first biochemical characterization of caspartin (Marin et al. 2005),
de novo sequencing of caspartin extracts was performed and different short peptides
were obtained, by using trypsin, pepsin, or AspN digests (Marin et al. 2007b).
However, so far, by using different couples of degenerated oligo primers, we did
376
F. Marin et al.
expression in mantle tissues.
The second protein, caspartin, has been studied in vitro more in detail
(Fig. 13.5), although its sequence is still far from being complete (Marin et al.
2005, 2007a, b). Similarly to calprismin, caspartin is an intracrystalline acidic
protein, which is abundant in the acid-soluble extract of the prisms. Caspartin has
a molecular weight of about 17 kDa. It is extremely enriched in aspartate residues
(more than 60%). Caspartin exhibits better calcium-binding ability than calprismin,
but its affinity for calcium is also weak, i.e., the fixation of calcium is reversible,
and calcium can be easily removed by gentle rinsing of the membrane where
caspartin has been blotted. Caspartin does not seem to be glycosylated, but we do
not exclude the possibility that it may be phosphorylated. An interesting finding
was that caspartin self-agregates in solution: in non-denaturing gels, we were able
to show that caspartin forms di-, tri-, tetra-, penta-, hexa-, and heptamers. On 2D
gels, caspartin migrates as a big spot at pI below 3. However, three tenuous minor
spots of the same molecular weight, one in the acidic domain, and two, basic, are
also observed, which may indicate that caspartin may not be absolutely pure, or that
it exhibits isoforms. In vitro, caspartin is a powerful inhibitor of the precipitation of
calcium carbonate, since it induces an inhibitory effect for concentrations inferior
to 1 mg/ml. Caspartin has also drastic effects on the morphology of calcite crystals
grown in vitro: effects are already observed around 0.2 mg/ml, and they increase
with the concentration of the protein in solution. Between 1 and 3 mg/ml, caspartin
induces the formation of polycrystalline aggregates, together with the formation of
“single crystals,” which exhibit a foliation. When the concentration of caspartin is
increased, the size of the crystals decreases, and above a threshold, caspartin acts as
an inhibitor of calcification and no crystals are produced. A polyclonal antibody,
specific of caspartin, indicated that caspartin, or a similar 17-kDa protein, is also
present in the nacreous layer of Pinna nobilis, but in much lesser amounts (6–8
times less). This antibody was used to localize caspartin directly in the shell of
Pinna nobilis (Marin et al. 2007b). In cross sections, perpendicular to the prisms
axis, we observe that caspartin has two locations: first, it is distributed at the surface
of the prisms (¼ within the prisms), a predictable finding. We do not observe a
particular pattern, but rather, a uniform distribution of the signal at the prisms
surface. Second, more surprisingly, caspartin occupies also an intercristalline
location: it forms a continuous film at the interface between the prisms themselves
and the interprismatic insoluble walls. In other words, the insoluble hydrophobic
interprismatic wall is sandwiched between two caspartin-containing layers. In
longitudinal sections, caspartin is located discontinuously along the prisms. Its
distribution in that dimension is not strictly correlated with the growth lines
observed for each prism, although we observed that caspartin may be more
concentrated on some growth plans (Marin et al. 2007b).
Following the first biochemical characterization of caspartin (Marin et al. 2005),
de novo sequencing of caspartin extracts was performed and different short peptides
were obtained, by using trypsin, pepsin, or AspN digests (Marin et al. 2007b).
However, so far, by using different couples of degenerated oligo primers, we did
376
F. Marin et al.