Dipetalogastin, a potent thrombin inhibitor from the blood‐sucking insectDipetalogaster maximus

Abstract

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 prolixus. Peptide sequence comparisons of the deduced amino-acid sequence exhibit a high homology of the domains I and II 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.Keywords: thrombin inhibitor; Kazal-type; blood-sucking insect; bug; cDNA cloning.Hematophagous animals are a particularly rich source of anticoagulant substances. In order to prevent the clotting during sucking of host blood and during digestion of fed blood, they have developed various mechanisms to interfere with blood coagulation. Among the highly specific enzymes and inhibitors involved in the coagulation [1], the thrombin inhibitors are the most prominent anticoagulants, having been described and characterized from leeches [2±4], snake venoms [9] and also from insects [5±8,10,11].The trypsin-like serine protease thrombin is a multifunctional key enzyme in the blood coagulation cascade. During coagulation thrombin converts fibrinogen into fibrin, activates other coagulation factors such as factor V, VIII, XIII and protein C [12,13], interacts with different cells, induces platelet aggregation and stimulates platelet secretion [14].While the anticoagulants from the blood-sucking insects Rhodnius prolixus and Triatoma pallidipennis have been investigated intensively [10,11,15±22], only sparse knowledge about anticoagulant activities from a further blood-sucking insect Dipetalogaster maximus [23,24] is available. Recently, we have isolated and characterized biochemically the specific natural thrombin inhibitor dipetalogastin from the stomach content of D. maximus [24]. This inhibitor consists of four isoforms each with a molecular mass of approximately 12 kDa. Natural dipetalogastin forms only 1 : 1 molar complexes with thrombin and acts as a slow, tight-binding inhibitor of throm...

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A Novel Serine Protease Inhibitor Acts as an Immunomodulatory Switch while Maintaining Homeostasis

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