A Limulus intracellular coagulation inhibitor with characteristics of the serpin superfamily. Purification, characterization, and cDNA cloning.
A Limulus intracellular coagulation inhibitor, designated LICI, was isolated from hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus), using three steps of chromatography, including dextran sulfate-Sepharose CL-6B, Sephacryl S-200, and Mono S. LICI is a single-chain glycoprotein with an apparent M(r) = 48,000 estimated by SDS-polyacrylamide gel electrophoresis. It blocks the amidolytic activities of Limulus lipopolysaccharide-sensitive serine protease, factor C, by forming a covalent 1:1 complex with the protease. The second-order rate constant for inhibition of factor C was 2.5 x 10(6) M-1 s-1 at 37 degrees C. LICI also inhibited human alpha-thrombin, rat salivary kallikrein, bovine plasmin, and trypsin but not Limulus clotting enzyme, Limulus factor B, bovine factor Xa, human factor XIa, human tissue plasminogen activator, human urokinase, chymotrypsin, elastase, and papain. Glycosaminoglycans such as heparin and heparan sulfate had no effect on the inhibitory activity. A cDNA coding for LICI was isolated from a hemocyte cDNA library. The open reading frame of the 1,257-base pair cDNA codes for the mature protein of 394 amino acids, of which 223 residues were confirmed by amino acid sequence analysis. LICI shows significant sequence identities to members of the serpin superfamily, such as human plasminogen activator inhibitor type 2 (40%) and human monocyte/neutrophil elastase inhibitor (39%). LICI contains a putative reactive site, -Arg-Ser-, at the corresponding position present in several inhibitors of the serpin superfamily. The subcellular localization, determined using an anti-LICI polyclonal antibody, indicated that LICI colocates with the Limulus serine protease zymogens in large granules in the hemocyte.
Miura
Y
Y
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
Kawabata
S
S
Iwanaga
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology; D060829:Arthropod Proteins; D001483:Base Sequence; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis* / Q000235:genetics / Q000494:pharmacology; D006025:Glycosaminoglycans / Q000494:pharmacology; D006737:Horseshoe Crabs / Q000737:chemistry*; D006801:Humans; D007447:Invertebrate Hormones / Q000032:analysis* / Q000235:genetics / Q000494:pharmacology; D008969:Molecular Sequence Data; D051381:Rats; D017386:Sequence Homology, Amino Acid; D015843:Serpins / Q000737:chemistry*; D013347:Subcellular Fractions / Q000378:metabolism
D000925:Anticoagulants; D060829:Arthropod Proteins; D018076:DNA, Complementary; D006023:Glycoproteins; D006025:Glycosaminoglycans; D007447:Invertebrate Hormones; C085186:Lici-1 protein, Tachypleus tridentatus; D015843:Serpins