proteins identified, the sequences were different from one another and the significance of the repeated sequences was unclear. Gene expression analyses showed
that GAMP mRNA was present only during pre-molt stage at the gastrolith disk.
The expression was significantly enhanced by 20-hydroxyecdysone (molting
hormone) in in vitro incubation of the gastrolith disk (Tsutsui et al. 1999).
Immunohistochemistry using an anti-GAMP antiserum showed that GAMP was
present in gastrolith disk epithelial cells and uniformly in the gastrolith, indicating
that GAMP is produced by and secreted from the gastrolith disk cells constantly
during the formation of gastrolith (Takagi et al. 2000). GAMP inhibited calcium
carbonate precipitation from its supersaturated solution at more than 5 Â 10
À8 M.
All these results strongly indicate that GAMP plays an important role in gastrolith
formation.
An EDTA-soluble matrix protein in the gastroliths was also studied using
a different species of the crayfish, Cherax quadricarinatus (Shechter et al.
2008b). A major component in the EDTA-soluble fraction was detected on an
SDS-PAGE gel as a band stained with Coomassie brilliant blue at an apparent
molecular mass of 65 kDa and named GAP 65 because of a novel protein. The
deduced amino acid sequence of GAP 65 from the nucleotide sequence of a cDNA
encoding GAP 65 revealed that it consists of 528 amino acid residues and contains
three known domains, chitin-binding domain, a low-density lipoprotein receptor
class A domain, and a polysaccharide deacetylase domain. It is not clear whether
the latter two domains are related to calcification or not. The GAP 65 gene was
expressed at the gastrolith disk and subepidermal tissue, but not at stomach wall.
The expression was greatly enhanced only at the gastrolith disk during the pre-molt
stage, suggesting the association of GAP 65 with gastrolith formation and the
induction of gene expression of GAP 65 by molting hormone. The RNAi knockdown experiment on GAP 65 indicated that it induced an abnormal ultrastructure of
gastrolith. Although many matrix proteins have been identified chemically, their
AAA
33 bp
1515 bp
2871 bp
SP
GAMP
394
467
62
231
N
C
Q V A Q E Q A Q E G
G S X S F
A
G
1
17
1
15
Fig. 11.5 Schematic representation of the structure of GAMP cDNA and protein.Two kinds of
characteristic repeated sequences are present in GAMP
320
H. Nagasawa
that GAMP mRNA was present only during pre-molt stage at the gastrolith disk.
The expression was significantly enhanced by 20-hydroxyecdysone (molting
hormone) in in vitro incubation of the gastrolith disk (Tsutsui et al. 1999).
Immunohistochemistry using an anti-GAMP antiserum showed that GAMP was
present in gastrolith disk epithelial cells and uniformly in the gastrolith, indicating
that GAMP is produced by and secreted from the gastrolith disk cells constantly
during the formation of gastrolith (Takagi et al. 2000). GAMP inhibited calcium
carbonate precipitation from its supersaturated solution at more than 5 Â 10
À8 M.
All these results strongly indicate that GAMP plays an important role in gastrolith
formation.
An EDTA-soluble matrix protein in the gastroliths was also studied using
a different species of the crayfish, Cherax quadricarinatus (Shechter et al.
2008b). A major component in the EDTA-soluble fraction was detected on an
SDS-PAGE gel as a band stained with Coomassie brilliant blue at an apparent
molecular mass of 65 kDa and named GAP 65 because of a novel protein. The
deduced amino acid sequence of GAP 65 from the nucleotide sequence of a cDNA
encoding GAP 65 revealed that it consists of 528 amino acid residues and contains
three known domains, chitin-binding domain, a low-density lipoprotein receptor
class A domain, and a polysaccharide deacetylase domain. It is not clear whether
the latter two domains are related to calcification or not. The GAP 65 gene was
expressed at the gastrolith disk and subepidermal tissue, but not at stomach wall.
The expression was greatly enhanced only at the gastrolith disk during the pre-molt
stage, suggesting the association of GAP 65 with gastrolith formation and the
induction of gene expression of GAP 65 by molting hormone. The RNAi knockdown experiment on GAP 65 indicated that it induced an abnormal ultrastructure of
gastrolith. Although many matrix proteins have been identified chemically, their
AAA
33 bp
1515 bp
2871 bp
SP
GAMP
394
467
62
231
N
C
Q V A Q E Q A Q E G
G S X S F
A
G
1
17
1
15
Fig. 11.5 Schematic representation of the structure of GAMP cDNA and protein.Two kinds of
characteristic repeated sequences are present in GAMP
320
H. Nagasawa