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 f rom a stomach library of Dipetalogaster maximus, a blood-sucking insect. Th e open reading frame of the cloned inhibitor cDNA codes for a Protein of 34 4 amino-acid residues. Sequence analysis reveals the existence of three rep eated homologous main regions, indicating that the inhibitor consists of th ree domains: Each domain shows a double-headed structure with an internal s equence homology like rhodniin, the thrombin inhibitor from the blood-sucki ng insect Rhodnius probixus. Peptide sequence comparisons of the deduced am ino-acid sequence exhibit a high homology of the domains I and Il to the na tural inhibitor dipetalogastin from the stomach content of D. maximus and t o rhodniin, respectively. Significant sequence similarities to Kazal-type i nhibitors, like the conserved sequence CGXDXXTYXNXC and several cysteine re sidues, indicate that the thrombin inhibitor from D. maximus is a further b lood-sucking insect which belongs to the Kazal-type:family (besides rhodnii n). A biologically active recombinant protein corresponding to domain II of the dipetalogastin cDNA was expressed in Escherichia coli. The isolated re combinant dipetalogastin with a molecular mass of,12.91 kDa has proved to b e a specific thrombin inhibitor similar to its natural counterpart as well as rhodniin and hirudin. The K-i value of the recombinant dipetalogastin wa s determined to be 49.3 +/- 22.28 fM.