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 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 Ki value of the recombinant dipetalogastin was determined to be 49.3 +/- 22.28 fM.
Mende
K
K
Research Unit Pharmacological Hemostaseology, Medical Faculty, Friedrich Schiller University Jena, Germany. kat@bug.mpg.uni-jena.de
Petoukhova
O
O
Koulitchkova
V
V
Schaub
G A
GA
Lange
U
U
Kaufmann
R
R
Nowak
G
G
D000595:Amino Acid Sequence; D000818:Animals; D000991:Antithrombins / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015723:Gene Library; D019476:Insect Proteins / Q000494:pharmacology*; D007313:Insecta; D007700:Kinetics; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry; D016366:Open Reading Frames; D011480:Protease Inhibitors / Q000737:chemistry; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013917:Thrombin / Q000008:administration & dosage / Q000037:antagonists & inhibitors*
D000991:Antithrombins; D018076:DNA, Complementary; D019476:Insect Proteins; D009711:Nucleotides; D011480:Protease Inhibitors; D020033:Protein Isoforms; C407584:dipetalogastin protein, Dipetalogaster maximus; D013917:Thrombin; D003545:Cysteine