RESUMO
A novel peptide named calcification-associated peptide (CAP)-2 was isolated from the exoskeleton of the crayfish, Procambarus clarkii. CAP-2 consists of 65 amino acid residues and has a 44% sequence identity with CAP-1 characterized previously. It has a chitin-binding domain observed in many arthropod cuticle proteins. CAP-2 showed inhibitory activity on calcium carbonate precipitation and chitin-binding ability. A CAP-2 cDNA was cloned using RT-PCR and RACE and the open reading frame encoded a precursor peptide consisting of a signal peptide and CAP-2. RT-PCR revealed that CAP-2 mRNA was exclusively expressed in the epidermal tissue during the postmolt stage, the site and stage being associated with calcification. Calcium-binding assay using recombinant CAP-2 revealed that this peptide had affinity for calcium ions with a Kd value of about 1 mM. All these results suggest that CAP-2 serves as a nucleator or a regulator in the calcification of the exoskeleton.
Assuntos
Astacoidea/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Sequência de Aminoácidos , Animais , Astacoidea/anatomia & histologia , Astacoidea/fisiologia , Carbonato de Cálcio/química , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/farmacologia , Quitina/metabolismo , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Dados de Sequência Molecular , Muda/fisiologia , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Distribuição TecidualRESUMO
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.