function is poorly understood. Application of molecular biological techniques such
as RNAi and anti-sense RNA (S€ ollner et al. 2003; Murayama et al. 2005) will help
better understanding of the function of matrix proteins.
Orchestia cavimana, the terrestrial crustacean, stores calcium carbonate in the
posterior ceca of the midgut during the pre-molt stage to keep calcium carbonate for
recalcification of the new cuticle at the next molting stage. A phosphoprotein with
an apparent molecular mass of 23 kDa on a gel of SDS-PAGE was purified from the
EDTA-soluble fraction of the posterior ceca, and named Orchestin (Luquet et al.
1996; Testenie `re et al. 2002; Hecker et al. 2003, 2004), because it has no sequence
homology with known proteins. Orchestin can bind calcium, and this ability
depends on phosphorylation of Ser residues. The molecular mass calculated from
the amino acid sequence deduced from the nucleotide sequence of a cDNA was
12.5 kD. The discrepancy may be due to the strong acidity of this protein with
a high proportion of acidic amino acid residues (30%) and phosphorylated serine
residues. In situ hybridization, Northern blot analysis, and immunohistochemistry
showed that Orchestin was synthesized specifically not only during the pre-molt
stage but also during the post-molt stage as a component of the organic matrix
of calcium carbonate precipitates. Thus, Orchestin is probably a key molecule in
calcium carbonate precipitation process in this species.
11.4 Identification of Matrix Peptides and Proteins
in Exoskeleton
The main calcification site in crustaceans is the cuticle of exoskeleton. By using
almost the same method as that used for identification of GAMP in gastroliths,
organic matrices seemingly responsible for calcification were searched in the
extract of crayfish exoskeleton. The difference in the searching strategy from
that for identification of GAMP was that we aimed at searching compounds with
calcification inhibitory activity. For this purpose, we modified the method for
assessing calcification inhibitory activity developed previously (Wheeler et al.
1981) to miniaturize the scale of experiment to about a tenth for purification of
inhibitory compounds (Fig. 11.6). In this case, we followed the increase in the
turbidity of the solution caused by precipitation instead of following the decrease
of the pH value (Inoue et al. 2001).
Extraction with an SDS/dithiothreitol solution from insoluble carapace exoskeleton after decalcification with dilute acetic acid, followed by three steps of HPLC
purification afforded two calcification inhibitory peptides, named calcificationassociated peptide (CAP)-1 and -2 (Inoue et al. 2001, 2004). The yields of CAP-1
and -2 were 7 and 12 mg, respectively, from 1 g of dried exoskeleton, each being
far less than that of GAMP. Sequence analyses of CAP-1 and -2 clarified that they
consisted of 78 and 65 amino acid residues (Fig. 11.7). These peptides are rich
in acidic amino acid residues and share about 60% sequence similarity. They both
11 Structure and Function of Matrix Proteins and Peptides
321
as RNAi and anti-sense RNA (S€ ollner et al. 2003; Murayama et al. 2005) will help
better understanding of the function of matrix proteins.
Orchestia cavimana, the terrestrial crustacean, stores calcium carbonate in the
posterior ceca of the midgut during the pre-molt stage to keep calcium carbonate for
recalcification of the new cuticle at the next molting stage. A phosphoprotein with
an apparent molecular mass of 23 kDa on a gel of SDS-PAGE was purified from the
EDTA-soluble fraction of the posterior ceca, and named Orchestin (Luquet et al.
1996; Testenie `re et al. 2002; Hecker et al. 2003, 2004), because it has no sequence
homology with known proteins. Orchestin can bind calcium, and this ability
depends on phosphorylation of Ser residues. The molecular mass calculated from
the amino acid sequence deduced from the nucleotide sequence of a cDNA was
12.5 kD. The discrepancy may be due to the strong acidity of this protein with
a high proportion of acidic amino acid residues (30%) and phosphorylated serine
residues. In situ hybridization, Northern blot analysis, and immunohistochemistry
showed that Orchestin was synthesized specifically not only during the pre-molt
stage but also during the post-molt stage as a component of the organic matrix
of calcium carbonate precipitates. Thus, Orchestin is probably a key molecule in
calcium carbonate precipitation process in this species.
11.4 Identification of Matrix Peptides and Proteins
in Exoskeleton
The main calcification site in crustaceans is the cuticle of exoskeleton. By using
almost the same method as that used for identification of GAMP in gastroliths,
organic matrices seemingly responsible for calcification were searched in the
extract of crayfish exoskeleton. The difference in the searching strategy from
that for identification of GAMP was that we aimed at searching compounds with
calcification inhibitory activity. For this purpose, we modified the method for
assessing calcification inhibitory activity developed previously (Wheeler et al.
1981) to miniaturize the scale of experiment to about a tenth for purification of
inhibitory compounds (Fig. 11.6). In this case, we followed the increase in the
turbidity of the solution caused by precipitation instead of following the decrease
of the pH value (Inoue et al. 2001).
Extraction with an SDS/dithiothreitol solution from insoluble carapace exoskeleton after decalcification with dilute acetic acid, followed by three steps of HPLC
purification afforded two calcification inhibitory peptides, named calcificationassociated peptide (CAP)-1 and -2 (Inoue et al. 2001, 2004). The yields of CAP-1
and -2 were 7 and 12 mg, respectively, from 1 g of dried exoskeleton, each being
far less than that of GAMP. Sequence analyses of CAP-1 and -2 clarified that they
consisted of 78 and 65 amino acid residues (Fig. 11.7). These peptides are rich
in acidic amino acid residues and share about 60% sequence similarity. They both
11 Structure and Function of Matrix Proteins and Peptides
321