Dipetalogastin, a potent thrombin inhibitor from the blood-sucking insect Dipetalogaster maximus -: cDNA cloning, expression and characterization

A cDNA coding for the thrombin inhibitor dipetalogastin has been isolated from a stomach library of Dipetalogaster maximus, a blood-sucking insect. The open reading frame of the cloned inhibitor cDNA codes for a Protein of 344 amino-acid residues. Sequence analysis reveals the existence of three repeated homologous main regions, indicating that the inhibitor consists of three domains: Each domain shows a double-headed structure with an internal sequence homology like rhodniin, the thrombin inhibitor from the blood-sucking insect Rhodnius probixus. Peptide sequence comparisons of the deduced amino-acid sequence exhibit a high homology of the domains I and Il to the natural inhibitor dipetalogastin from the stomach content of D. maximus and to rhodniin, respectively. Significant sequence similarities to Kazal-type inhibitors, like the conserved sequence CGXDXXTYXNXC and several cysteine residues, indicate that the thrombin inhibitor from D. maximus is a further blood-sucking insect which belongs to the Kazal-type:family (besides rhodniin). A biologically active recombinant protein corresponding to domain II of the dipetalogastin cDNA was expressed in Escherichia coli. The isolated recombinant dipetalogastin with a molecular mass of,12.91 kDa has proved to be a specific thrombin inhibitor similar to its natural counterpart as well as rhodniin and hirudin. The K-i value of the recombinant dipetalogastin was determined to be 49.3 +/- 22.28 fM.