Abstract
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.
Highlights
= 48,000 estimated by SDS-polyacrylamide gel electro- important forthe host defensein engulfing invading microbes, e, phoresis. It blocks the amidolytic activities of Limulus lipopolysaccharide-sensitiveserine protease, factor by forming a covale1n:lt complex withthe proteas? The second-orderrate constantfor inhibitionof factor C was 2.5 x los M - ~ s-l at 57 "C
In our ongoing studies on the molecular mechanism of hemolymph coagulation in Limulus, we established a serine protease cascade system, much like the mammalian plasma clotting cascade. This cascade is composed of three serine protease zymogens, factor C (41, factor B (51,the proclotting enzyme [6],and
Znhibition of Other Serine Proteases by LZCZ-lb determine whether LICI inhibitsother Limulus serine proteases and mammalian serine proteases, a 10molar excess of the inhibitor was incubated with each protease at 37 "C for 1 h and the remaining amidolytic activity was assayed, as described under
Summary
Times (15 s to 15 min), 50-pl aliquots were removed, and the reaction was terminated by the addition of 950 pl of the buffer containing each substrate (0.6nm).The concentrations of residual enzymes were calcu-. Factor as described under "Experimental Procedures" (Fig. Isolation of LZCZ-derivedPeptide, Sequencing,and Amino AcidAnal- lA).The inhibitor- (LICI)containing fractions werepooled and ysis-The purified inhibitor (700 pg) was reduced, S-pyridylethylated, and digested with lysyl endopeptidase [18].Peptides were separated by reversed-phase HPLC using a pBondasphere CB 300A column concentrated by ultrafiltration and thenapplied to a Sephacryl S-200column (4x 142 cm), equilibrated with 50 m sodium (Nihon Waters Ltd., Tokyo). Znhibition of Other Serine Proteases by LZCZ-lb determine whether LICI inhibitsother Limulus serine proteases and mammalian serine proteases, a 10molar excess of the inhibitor was incubated with each protease at 37 "C for 1 h and the remaining amidolytic activity was assayed, as described under "Experimental Procedures." The inhibitory activity toward.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
