A carboxypeptidase inhibitor from the tick Rhipicephalus bursa: Isolation, cDNA cloning, recombinant expression, and characterization

Joan L. Arolas, Julia Lorenzo, Ana Rovira, Joaquim Castellà, Francesc X. Aviles, Christian P. Sommerhoff

Research output: Contribution to journalArticleResearchpeer-review

62 Citations (Scopus)

Abstract

A novel proteinaceous metallo-carboxypeptidase inhibitor, named tick carboxypeptidase inhibitor (TCI), was isolated from the ixodid tick Rhipicephalus bursa and N-terminally sequenced. The complete cDNA encoding this protein was cloned from tick mRNA by reverse transcription-PCR and rapid amplification of cDNA ends techniques. The full-length TCI cDNA contains an open reading frame coding for a precursor protein of 97 amino acid residues that consists of a predicted signal peptide of 22 residues and of mature TCI, a 75-residue cysteine-rich protein (12 Cys). The deduced amino acid sequence shows no homology to other known proteins; the C terminus, however, resembles those of other protein metallo-carboxypeptidase inhibitors, suggesting a common mechanism of inhibition. Recombinant TCI expressed in Escherichia coli is fully functional and inhibits carboxypeptidases of the A/B subfamily with equilibrium dissociation constants in the nanomolar range. Structural analyses by circular dichroism and nuclear magnetic resonance indicate that TCI is a protein strongly constrained by disulfide bonds, unusually stable over a wide pH range and highly resistant to denaturing conditions. As a tight binding inhibitor of plasma carboxypeptidase B, also known as thrombin-activatable fibrinolysis inhibitor, recombinant TCI stimulates fibrinolysis in vitro and thus may have potential for applications to prevent or treat thrombotic disorders.
Original languageEnglish
Pages (from-to)3441-3448
JournalJournal of Biological Chemistry
Volume280
Issue number5
DOIs
Publication statusPublished - 4 Feb 2005

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