interest that calcification and decalcification occur concurrently at the different
tissues in an individual; at the pre-molt stage, decalcification and calcification
occur in the cuticle and stomach, respectively, while at the post-molt stage they
do in a completely converse manner (Shechter et al. 2008a). The former process
is triggered by the molting hormone, but it is not clear whether the latter process is
under endocrine control or not.
11.3 Identification of Matrix Proteins in the Tissues
for Temporary Storage of Calcium Carbonate
Gastroliths are formed only prior to molting as a temporary storage of calcium
carbonate and do not contain any cells. A gastrolith of the crayfish, Procambarus
clarkii, was decalcified with 1 M acetic acid, and insoluble materials remained
with almost the same shape as the original gastrolith like a sponge rubber. The
insoluble material was then treated with a solution containing 1% SDS and 10 mM
dithiothreitol in a boiling water bath for 10 min. The extract was applied to reversephase HPLC, which gave almost one peak on the chromatogram. The peak material
was a protein, and the yield was about 460 mg/g gastrolith. This protein was a novel
protein and was named gastrolith matrix protein (GAMP). The time-of-flight
mass spectrum of GAMP showed that the molecular mass was around 55 kDa. The
N-terminal sequencing failed to identify any sequence, indicating that the N-terminus
was blocked. So, GAMP was digested with some proteolytic enzymes. The resulting
fragment peptides were separated by reverse-phase HPLC, and their amino acid
sequences were determined (Ishii et al. 1998). By combining the sequences
overlapped, five fragments with a total of 225 amino acid residues were obtained.
The residue after SDS extraction was dried, dissolved in [
2 H]-formic acid,
and subjected to
1 H NMR measurement. The spectrum was almost identical with
authentic chitin commercially available, indicating that the residual material was
chitin (Ishii et al. 1996). Thus, GAMP was tightly bound to chitin in the gastrolith
and was not released with dilute acetic acid but extracted with a hot SDS solution,
although we could not find any known chitin-binding domains in the amino acid
sequence of GAMP. Therefore, GAMP may have a novel chitin-binding domain.
Based on the partial amino acid sequences thus determined, a cDNA encoding
GAMP was cloned by combination of RT-PCR with 5
0 and 3
0 RACEs (Tsutsui et al.
1999). An open reading frame of 1,515 bp encoded a precursor protein consisting
of a signal peptide (18 amino acid residues) and GAMP (487 amino acid residues)
(Fig. 11.5). MS/MS analyses of the N-terminal 10-residue peptide obtained by
digestion with endoproteinase Asp-N demonstrated that the N-terminus was blocked
by a pyroglutamic acid residue. The deduced amino acid sequence of GAMP
indicated that it contained two kinds of tandem repeated sequences; 17 repeats of
10 amino acid residues near the N-terminus and 15 repeats of 5 amino acid residues
near the C-terminus. Although repeated sequences were found in many other matrix
11 Structure and Function of Matrix Proteins and Peptides
319
tissues in an individual; at the pre-molt stage, decalcification and calcification
occur in the cuticle and stomach, respectively, while at the post-molt stage they
do in a completely converse manner (Shechter et al. 2008a). The former process
is triggered by the molting hormone, but it is not clear whether the latter process is
under endocrine control or not.
11.3 Identification of Matrix Proteins in the Tissues
for Temporary Storage of Calcium Carbonate
Gastroliths are formed only prior to molting as a temporary storage of calcium
carbonate and do not contain any cells. A gastrolith of the crayfish, Procambarus
clarkii, was decalcified with 1 M acetic acid, and insoluble materials remained
with almost the same shape as the original gastrolith like a sponge rubber. The
insoluble material was then treated with a solution containing 1% SDS and 10 mM
dithiothreitol in a boiling water bath for 10 min. The extract was applied to reversephase HPLC, which gave almost one peak on the chromatogram. The peak material
was a protein, and the yield was about 460 mg/g gastrolith. This protein was a novel
protein and was named gastrolith matrix protein (GAMP). The time-of-flight
mass spectrum of GAMP showed that the molecular mass was around 55 kDa. The
N-terminal sequencing failed to identify any sequence, indicating that the N-terminus
was blocked. So, GAMP was digested with some proteolytic enzymes. The resulting
fragment peptides were separated by reverse-phase HPLC, and their amino acid
sequences were determined (Ishii et al. 1998). By combining the sequences
overlapped, five fragments with a total of 225 amino acid residues were obtained.
The residue after SDS extraction was dried, dissolved in [
2 H]-formic acid,
and subjected to
1 H NMR measurement. The spectrum was almost identical with
authentic chitin commercially available, indicating that the residual material was
chitin (Ishii et al. 1996). Thus, GAMP was tightly bound to chitin in the gastrolith
and was not released with dilute acetic acid but extracted with a hot SDS solution,
although we could not find any known chitin-binding domains in the amino acid
sequence of GAMP. Therefore, GAMP may have a novel chitin-binding domain.
Based on the partial amino acid sequences thus determined, a cDNA encoding
GAMP was cloned by combination of RT-PCR with 5
0 and 3
0 RACEs (Tsutsui et al.
1999). An open reading frame of 1,515 bp encoded a precursor protein consisting
of a signal peptide (18 amino acid residues) and GAMP (487 amino acid residues)
(Fig. 11.5). MS/MS analyses of the N-terminal 10-residue peptide obtained by
digestion with endoproteinase Asp-N demonstrated that the N-terminus was blocked
by a pyroglutamic acid residue. The deduced amino acid sequence of GAMP
indicated that it contained two kinds of tandem repeated sequences; 17 repeats of
10 amino acid residues near the N-terminus and 15 repeats of 5 amino acid residues
near the C-terminus. Although repeated sequences were found in many other matrix
11 Structure and Function of Matrix Proteins and Peptides
319