Cloning and expression of a cDNA encoding a matrix peptide associated with calcification in the exoskeleton of the crayfish.
Calcification-associated peptide (CAP)-1 isolated from the exoskeleton of the crayfish, Procambarus clarkii, has anti-calcification activity and chitin-binding ability and is, therefore, considered to be associated with calcification. In this study, a cDNA encoding CAP-1 was cloned and characterized. An open reading frame encoded a pre-propeptide of 99 amino acid residues, which was composed of a signal peptide, a CAP-1 precursor and two-basic amino acid residues at the C-terminus. The dibasic residues were not observed in the natural CAP-1. Expression analyses using Northern blot and RT-PCR revealed that the mRNA encoding CAP-1 was strongly expressed in the epidermal tissue during the postmolt stage, where and when the calcification takes place. These results support that CAP-1 may play an important role in the calcification of the exoskeleton. Based on the nucleotide sequence of the cDNA encoding CAP-1, a recombinant CAP-1 and that carrying the basic residues at the C-terminus were expressed in Escherichia coli. Anti-calcification assay showed that these recombinant peptides were less active than natural CAP-1, indicating that the phosphate group at the 70th residue, Ser, in natural CAP-1 is important for inhibitory activity and that the paired basic residues have some contribution to the elevation of inhibitory activity.
Inoue
Hirotaka
H
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.
Ohira
Tsuyoshi
T
Ozaki
Noriaki
N
Nagasawa
Hiromichi
H
D000595:Amino Acid Sequence; D000818:Animals; D003400:Astacoidea / Q000235:genetics* / Q000502:physiology*; D001483:Base Sequence; D002113:Calcification, Physiologic / Q000502:physiology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D016326:Extracellular Matrix Proteins; D010455:Peptides; D011994:Recombinant Proteins; C448763:calcification-associated peptide-1, crayfish