As described later, phosphorylation enhances calcification inhibitory activity and
therefore seems to be important in calcification of the cuticle. The phosphorylation
of CAP-1 influenced the shape of crystals, when in vitro calcium carbonate precipitation was performed (Sugawara et al. 2006; Yamamoto et al. 2008).
Cloning of cDNAs encoding CAP-1 and -2 were attained by the conventional
method (Inoue et al. 2003, 2004). The precursor peptide of CAP-1 consists of
a signal peptide, CAP-1 and two basic amino acid residues at the C-terminus.
Therefore, the mature CAP-1 is produced through posttranslational processing
of cleavage of a signal peptide and removal of the C-terminal dibasic residues
and phosphorylation of Ser
70 . On the other hand, the precursor peptide of CAP-2 is
simple and consists of only a signal peptide and CAP-2. RT-PCR analyses showed
that the CAP-1 and -2 genes were expressed only in the epidermis during the time
from the end of the pre-molt stage to the early post-molt stage, indicating their
production precedent to and concomitant with calcification of new cuticle.
Using these cDNAs, recombinant peptides of CAP-1 and -2 were prepared using
an Escherichia coli expression system and used for their functional analyses (Inoue
et al. 2007). The chitin-binding ability of these peptides was demonstrated by the
experiment of incubation with commercially available chitin. This ability seems
to be consistent with the fact that CAP-1 and -2 were not easily extracted with dilute
acetic acid but extracted with an SDS-containing solution at high temperature. This
also means that these peptides exist as a complex with chitin in the cuticle. The
calcium-binding ability of recombinant CAP-1 (dephosphorylated CAP-1 with an
additional Ala at the N-terminus) was verified by incubation with
45
CaCl 2 . Further
analyses indicated that the calcium binding comprised high-affinity and low-affinity
bindings. Considering that CAP-1 shows calcification inhibitory activity and has
abilities of chitin binding and calcium binding, CAP-1 may serve as a bifunctional
molecule to bind to chitin for formation of scaffold for calcification and to regulate
calcification including the initiation of calcium carbonate deposition.
11.5 Structure-Activity Relationship of a Cuticle Matrix
Peptide
Structure-function relationship study has rarely been performed in the research
of biomineralization, perhaps partly because the function of organic matrices still
remains obscure and partly because peptide chemists are not aware of the importance of peptides and proteins in biomineralization. Recently, detailed studies on
the relationship between peptide sequence and calcification-inhibitory activity of
a matrix peptide in the cuticle were conducted. The following is an example of
structure-function relationship study on CAP-1.
As mentioned previously, CAP-1 is a 78-amino acid residue peptide and has
a chitin-binding sequence (the R-R consensus sequence) well conserved among
11 Structure and Function of Matrix Proteins and Peptides
323
therefore seems to be important in calcification of the cuticle. The phosphorylation
of CAP-1 influenced the shape of crystals, when in vitro calcium carbonate precipitation was performed (Sugawara et al. 2006; Yamamoto et al. 2008).
Cloning of cDNAs encoding CAP-1 and -2 were attained by the conventional
method (Inoue et al. 2003, 2004). The precursor peptide of CAP-1 consists of
a signal peptide, CAP-1 and two basic amino acid residues at the C-terminus.
Therefore, the mature CAP-1 is produced through posttranslational processing
of cleavage of a signal peptide and removal of the C-terminal dibasic residues
and phosphorylation of Ser
70 . On the other hand, the precursor peptide of CAP-2 is
simple and consists of only a signal peptide and CAP-2. RT-PCR analyses showed
that the CAP-1 and -2 genes were expressed only in the epidermis during the time
from the end of the pre-molt stage to the early post-molt stage, indicating their
production precedent to and concomitant with calcification of new cuticle.
Using these cDNAs, recombinant peptides of CAP-1 and -2 were prepared using
an Escherichia coli expression system and used for their functional analyses (Inoue
et al. 2007). The chitin-binding ability of these peptides was demonstrated by the
experiment of incubation with commercially available chitin. This ability seems
to be consistent with the fact that CAP-1 and -2 were not easily extracted with dilute
acetic acid but extracted with an SDS-containing solution at high temperature. This
also means that these peptides exist as a complex with chitin in the cuticle. The
calcium-binding ability of recombinant CAP-1 (dephosphorylated CAP-1 with an
additional Ala at the N-terminus) was verified by incubation with
45
CaCl 2 . Further
analyses indicated that the calcium binding comprised high-affinity and low-affinity
bindings. Considering that CAP-1 shows calcification inhibitory activity and has
abilities of chitin binding and calcium binding, CAP-1 may serve as a bifunctional
molecule to bind to chitin for formation of scaffold for calcification and to regulate
calcification including the initiation of calcium carbonate deposition.
11.5 Structure-Activity Relationship of a Cuticle Matrix
Peptide
Structure-function relationship study has rarely been performed in the research
of biomineralization, perhaps partly because the function of organic matrices still
remains obscure and partly because peptide chemists are not aware of the importance of peptides and proteins in biomineralization. Recently, detailed studies on
the relationship between peptide sequence and calcification-inhibitory activity of
a matrix peptide in the cuticle were conducted. The following is an example of
structure-function relationship study on CAP-1.
As mentioned previously, CAP-1 is a 78-amino acid residue peptide and has
a chitin-binding sequence (the R-R consensus sequence) well conserved among
11 Structure and Function of Matrix Proteins and Peptides
323