Structural determination and functional analysis of a soluble matrix protein associated with calcification of the exoskeleton of the crayfish, Procambarus clarkii.
A novel matrix protein named calcification-associated soluble protein-2 (Casp-2) was isolated from the acetic acid-soluble fraction of the exoskeleton of the crayfish Procambarus clarkii, and its primary structure was determined by a combination of peptide sequencing, mass spectral analysis, and cDNA cloning. Casp-2 consists of 117 amino acid residues and has a chitin-binding consensus sequence, the so-called Rebers-Riddiford (R-R) consensus sequence. Casp-2 exhibited an inhibitory activity on calcium carbonate precipitation from its supersaturated solution in vitro, suggesting association with calcification of the exoskeleton. Reverse transcription PCR and Northern blot analyses indicated that the Casp-2 gene was expressed only at the epidermis throughout the molting stages, and most strongly at the late pre-molt stage. Recombinant Casp-2 showed weak affinity to chitin in spite of having the R-R consensus sequence. These results indicate that Casp-2 interacts loosely with chitin fibrils and regulates calcification in the cuticle.
Inoue
Hirotaka
H
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Yuasa-Hashimoto
Naomi
N
Suzuki
Michio
M
Nagasawa
Hiromichi
H
D000595:Amino Acid Sequence; D000818:Animals; D003400:Astacoidea / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002113:Calcification, Physiologic; D002686:Chitin / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D029941:Fish Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009928:Organ Specificity; D011485:Protein Binding; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012995:Solubility
D018076:DNA, Complementary; D029941:Fish Proteins; D002686:Chitin