A carboxypeptidase inhibitor from the tick Rhipicephalus bursa: isolation, cDNA cloning, recombinant expression, and characterization.
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.
Arolas
Joan L
JL
Institut de Biotecnologia i de Biomedicina and Departament de Sanitat i d'Anatomia Animals, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain.
Lorenzo
Julia
J
Rovira
Ana
A
Castellà
Joaquim
J
Aviles
Francesc X
FX
Sommerhoff
Christian P
CP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001777:Blood Coagulation / Q000187:drug effects; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002942:Circular Dichroism; D005342:Fibrinolysis / Q000187:drug effects*; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D048494:Rhipicephalus / Q000378:metabolism*; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; D002268:Carboxypeptidases