elicited against the recombinant mucoperlin reacted only with nacre extracts, but
not at all with prisms extracts, on Western blots, on dot blots or on ELISA
(Fig. 13.6d). This clearly suggested that mucoperlin is specific of the nacreous
layer of Pinna nobilis. To our knowledge, this was the first convincing demonstration of the existence of a nacre-specific protein among molluscs. Our finding was
confirmed by screening mucoperlin clones with anti-prisms matrix and anti-nacre
matrix antibodies, separately (Marin et al. 2003a), and by histological staining:
using conventional staining of shell cross sections, we showed that mucoperlin was
localized in the nacre layer exclusively, and that it was particularly concentrated in
the intertabular zones, i.e., the short lateral sides of the tablets, also defined as the
zone that is comprised between adjacent tablets (Fig. 13.6e, f). In addition, a
staining was also observed in the interlamellar matrix, but this staining was much
weaker. Later, we repeated the immunohistological experiments by using the more
precise immunogold technique (Marin et al. 2007b). We then observed a
completely superimposed staining pattern, a concentration of white spots localizing
mucoperlin in the intertabular zones (F. Marin, unpublished data, 2006;
Fig. 13.6g–i; Marin 2009).
The identification and molecular characterization of mucoperlin, an acidic
mucin-like protein, in a shell was a “premie `re” and established a bridge between
molecular physiology, especially that of vertebrates, and biomineralization in general. Mucins are indeed involved in several functions, such as lubrication, protection
of epithelial tissues against chemical or microbial aggressors. They are also involved
in cell signaling (Bafna et al. 2008). Interestingly, in vertebrates, mucins are also
associated with fluids that calcify or that prevent calcification. In the buccal cavity,
the saliva is a supersaturated fluid, which – fortunately for us – never calcifies.
The main reason is that saliva contains a cortege of proteins that prevent the
deposition of calcium salts. Salivary mucins, when free in solution, do not allow
calcium salts crystals to grow further (Tabak et al. 1985; Nieuw-Amerongen et al.
1989; Tabak 1995). Furthermore, these mucins can adhere on tooth hydroxyapatite
with a strong affinity, and prevent their demineralization (Meyer-Lueckel et al.
2006). Similarly, in the bladder, urine comprises a set of proteins, among which
the Tamm-Horsfall Protein (THP) and urinary mucins, the function of which is to
prevent the precipitation of calcium oxalates or of uric acid (Grases and Llobera
1998). Finally, the gallbladder is also an organ in contact with supersaturated fluids
(Afdhal et al. 1995). In pathological situations (lithiasis), gallbladder mucins are
frequently involved in the deposition of mineralized concretions (Lechene de la
Porte et al. 1996), an observation which suggests that they may be also involved
in nucleation processes. Clearly, mucins in general seem to play important –
although probably underestimated – functions associated with calcification.
13.4.3.3 Other Shell Proteins of P. nobilis
Although caspartin and calprismin represent the two dominant intracrystalline
proteins of the isolated prisms of P. nobilis, we detected about eight minor protein
378
F. Marin et al.
not at all with prisms extracts, on Western blots, on dot blots or on ELISA
(Fig. 13.6d). This clearly suggested that mucoperlin is specific of the nacreous
layer of Pinna nobilis. To our knowledge, this was the first convincing demonstration of the existence of a nacre-specific protein among molluscs. Our finding was
confirmed by screening mucoperlin clones with anti-prisms matrix and anti-nacre
matrix antibodies, separately (Marin et al. 2003a), and by histological staining:
using conventional staining of shell cross sections, we showed that mucoperlin was
localized in the nacre layer exclusively, and that it was particularly concentrated in
the intertabular zones, i.e., the short lateral sides of the tablets, also defined as the
zone that is comprised between adjacent tablets (Fig. 13.6e, f). In addition, a
staining was also observed in the interlamellar matrix, but this staining was much
weaker. Later, we repeated the immunohistological experiments by using the more
precise immunogold technique (Marin et al. 2007b). We then observed a
completely superimposed staining pattern, a concentration of white spots localizing
mucoperlin in the intertabular zones (F. Marin, unpublished data, 2006;
Fig. 13.6g–i; Marin 2009).
The identification and molecular characterization of mucoperlin, an acidic
mucin-like protein, in a shell was a “premie `re” and established a bridge between
molecular physiology, especially that of vertebrates, and biomineralization in general. Mucins are indeed involved in several functions, such as lubrication, protection
of epithelial tissues against chemical or microbial aggressors. They are also involved
in cell signaling (Bafna et al. 2008). Interestingly, in vertebrates, mucins are also
associated with fluids that calcify or that prevent calcification. In the buccal cavity,
the saliva is a supersaturated fluid, which – fortunately for us – never calcifies.
The main reason is that saliva contains a cortege of proteins that prevent the
deposition of calcium salts. Salivary mucins, when free in solution, do not allow
calcium salts crystals to grow further (Tabak et al. 1985; Nieuw-Amerongen et al.
1989; Tabak 1995). Furthermore, these mucins can adhere on tooth hydroxyapatite
with a strong affinity, and prevent their demineralization (Meyer-Lueckel et al.
2006). Similarly, in the bladder, urine comprises a set of proteins, among which
the Tamm-Horsfall Protein (THP) and urinary mucins, the function of which is to
prevent the precipitation of calcium oxalates or of uric acid (Grases and Llobera
1998). Finally, the gallbladder is also an organ in contact with supersaturated fluids
(Afdhal et al. 1995). In pathological situations (lithiasis), gallbladder mucins are
frequently involved in the deposition of mineralized concretions (Lechene de la
Porte et al. 1996), an observation which suggests that they may be also involved
in nucleation processes. Clearly, mucins in general seem to play important –
although probably underestimated – functions associated with calcification.
13.4.3.3 Other Shell Proteins of P. nobilis
Although caspartin and calprismin represent the two dominant intracrystalline
proteins of the isolated prisms of P. nobilis, we detected about eight minor protein
378
F. Marin et al.