was priorly incubated with antibodies raised against the non-fractionated matrix
than by using silver nitrate or Coomassie Blue staining on the gel. For unknown
reasons, it seems that discrete components of the matrix are more immunogenic
than smearing macromolecules, which results in the improvement of the signal/
background ratio (if we consider that the smearing materials are part of the
“background effect”). We used this property to isolate two prominent prism matrix
proteins as follows: after the isolation of single prisms with dilute sodium hypochlorite, the intraprismatic matrix was extracted by dilute cold acetic acid,
according to our standard procedure (Marin et al. 2001). Then, the matrix was
fractionated by preparative electrophoresis, which allowed the “blind” collection of
numerous fractions. Each fraction was blotted on a PVDF membrane by dot-blot
apparatus, and the membrane was subsequently treated with anti-prisms or antinacre matrix antibodies, according to a classical procedure used in Western blot.
This allowed detecting in which fractions were eluted the proteins of interest.
Proteins were collected, and extensively dialyzed before being freeze-dried. The
technique was described in Marin et al. (2001) and in Marin (2003).
In this manner, we identified two new proteins, that we subsequently named
caspartin and calprismin, respectively (Marin and Luquet 2005; Marin et al. 2005).
Calprismin is an acidic intracrystalline soluble protein of 38 kDa of apparent
molecular weight. It is enriched in Ala, Asx, Thr, and Pro residues, these four
residues constituting 55% of the sequence. The N-terminus of calprismin (61 a
residues, about 18% of the sequence) was sequenced. It is enriched in Glu residues
and presents a pattern containing four cysteine residues that is also observed in
different extracellular matrix (ECM) proteins. A blast search did not allow finding
affiliation of calprismin with other shell proteins, neither with other ECM proteins.
Calprismin is a very weak calcium-binding protein, and this property is extremely
labile. On 2D-gel, calprismin migrates as a thick spot that is localized below pI 3.
Two other diffuse spots, which are slightly less acidic, suggest that calprismin may
have different isoforms/glycoforms. The enzymatic deglycosylation of calprismin
with a cocktail of endoglycosidases led to a loss of few kDa of apparent molecular
weight on a gel. This weight loss is correlated with the disappearance of Alcian blue
staining of the protein after deglycosylation. Calprismin may be rather unstable
since it degrades when performing a chemical deglycosylation with TFMS acid.
The analysis of its N-terminal sequence, and the comparison with other N/Cterminal sequences of prisms-associated proteins allowed obtaining general
features (Evans 2008): abundance of anionic residues, low amount of cationic
residues, variable amounts of hydrogen-bonding donor/acceptor (HBDA). Like
other proteins associated to prisms, calprismin may be intrinsically disordered,
Fig. 13.5 (continued) (lane 4), 20 mg (lane 5). (f–h) Immunogold localization of caspartin. (f)
cross section, low magnification. (g) cross section, high magnification of a triple junction. (h)
longitudinal section of a prism. (i–k) in vitro CaCO 3 crystallization experiment in the presence of
purified caspartin. (i,j) polycrystalline aggregates formed at 2 mg/ml of caspartin. (k) “single”
crystal formed at 3 mg/ml of caspartin
◂
374
F. Marin et al.
than by using silver nitrate or Coomassie Blue staining on the gel. For unknown
reasons, it seems that discrete components of the matrix are more immunogenic
than smearing macromolecules, which results in the improvement of the signal/
background ratio (if we consider that the smearing materials are part of the
“background effect”). We used this property to isolate two prominent prism matrix
proteins as follows: after the isolation of single prisms with dilute sodium hypochlorite, the intraprismatic matrix was extracted by dilute cold acetic acid,
according to our standard procedure (Marin et al. 2001). Then, the matrix was
fractionated by preparative electrophoresis, which allowed the “blind” collection of
numerous fractions. Each fraction was blotted on a PVDF membrane by dot-blot
apparatus, and the membrane was subsequently treated with anti-prisms or antinacre matrix antibodies, according to a classical procedure used in Western blot.
This allowed detecting in which fractions were eluted the proteins of interest.
Proteins were collected, and extensively dialyzed before being freeze-dried. The
technique was described in Marin et al. (2001) and in Marin (2003).
In this manner, we identified two new proteins, that we subsequently named
caspartin and calprismin, respectively (Marin and Luquet 2005; Marin et al. 2005).
Calprismin is an acidic intracrystalline soluble protein of 38 kDa of apparent
molecular weight. It is enriched in Ala, Asx, Thr, and Pro residues, these four
residues constituting 55% of the sequence. The N-terminus of calprismin (61 a
residues, about 18% of the sequence) was sequenced. It is enriched in Glu residues
and presents a pattern containing four cysteine residues that is also observed in
different extracellular matrix (ECM) proteins. A blast search did not allow finding
affiliation of calprismin with other shell proteins, neither with other ECM proteins.
Calprismin is a very weak calcium-binding protein, and this property is extremely
labile. On 2D-gel, calprismin migrates as a thick spot that is localized below pI 3.
Two other diffuse spots, which are slightly less acidic, suggest that calprismin may
have different isoforms/glycoforms. The enzymatic deglycosylation of calprismin
with a cocktail of endoglycosidases led to a loss of few kDa of apparent molecular
weight on a gel. This weight loss is correlated with the disappearance of Alcian blue
staining of the protein after deglycosylation. Calprismin may be rather unstable
since it degrades when performing a chemical deglycosylation with TFMS acid.
The analysis of its N-terminal sequence, and the comparison with other N/Cterminal sequences of prisms-associated proteins allowed obtaining general
features (Evans 2008): abundance of anionic residues, low amount of cationic
residues, variable amounts of hydrogen-bonding donor/acceptor (HBDA). Like
other proteins associated to prisms, calprismin may be intrinsically disordered,
Fig. 13.5 (continued) (lane 4), 20 mg (lane 5). (f–h) Immunogold localization of caspartin. (f)
cross section, low magnification. (g) cross section, high magnification of a triple junction. (h)
longitudinal section of a prism. (i–k) in vitro CaCO 3 crystallization experiment in the presence of
purified caspartin. (i,j) polycrystalline aggregates formed at 2 mg/ml of caspartin. (k) “single”
crystal formed at 3 mg/ml of caspartin
◂
374
F. Marin et al.