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A limulus intracellular coagulation inhibitor type 2. Purification, characterization, cDNA cloning, and tissue
Y Miura1, S Kawabata, Y Wakamiya
1Department of Biology, Faculty of Science, Kyushu University 33, Fukuoka, Japan.
Insights
Horseshoe crab hemocytes contain a new serine protease inhibitor, LICI-2, which targets both horseshoe crab and mammalian proteases. This regulated secretory serpin is stored in granules and released upon stimulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Previous identification of limulus intracellular coagulation inhibitor (LICI), a serine protease inhibitor in horseshoe crab hemocytes.
- LICI specifically inhibits limulus lipopolysaccharide-sensitive serine protease, factor C.
Purpose of the Study:
- To identify and characterize a novel serine protease inhibitor, designated LICI type-2 (LICI-2), from horseshoe crab hemocytes.
- To investigate the inhibitory activity, protein structure, gene expression, and localization of LICI-2.
Main Methods:
- Purification of LICI-2 from horseshoe crab hemocytes.
- Determination of inhibitory kinetics against various serine proteases.
- Isolation and sequencing of LICI-2 cDNA.
- Northern blotting for mRNA expression analysis.
- Immunoblotting to determine LICI-2 localization within hemocyte granules.
Main Results:
- LICI-2 inhibits horseshoe crab factor C and limulus clotting enzyme, as well as mammalian serine proteases like alpha-thrombin, kallikrein, plasmin, and tissue plasminogen activator.
- Purified LICI-2 is a 42 kDa glycoprotein; its cDNA codes for a 386 amino acid mature protein with a unique Lys-Ser reactive site.
- LICI-2 mRNA is exclusively expressed in hemocytes; the protein is stored in large granules and released upon stimulation.
Conclusions:
- LICI-2 represents a distinct intracellular serpin with broad inhibitory activity against both invertebrate and mammalian serine proteases.
- The unique reactive site and regulated secretory nature suggest a specialized role for LICI-2 in the horseshoe crab immune response.
- Proposed classification of LICIs into a new subfamily of regulated secretory intracellular serpins.
Abstract:
We described in a foregoing report findings on serpin, a serine protease inhibitor, newly identified in horseshoe crab (Tachypleus tridentatus) hemocytes and we name it limulus intracellular coagulation inhibitor, LICI (Miura, Y., Kawabata, S., and Iwanaga, S. (1994) J. Biol. Chem. 269, 542-547). This serpin specifically inhibits limulus lipopolysaccharide-sensitive serine protease, factor C. In ongoing studies on limulus serpin, we have found another inhibitor, LICI type-2 (LICI-2), which inhibits not only factor C (k1 = 7.1 x 10(4) M-1 S-1) but also limulus clotting enzyme (k1 = 4.3 x 10(5) M-1 S-1). LICI-2 inhibits mammalian serine proteases, including alpha-thrombin, salivary kallikrein, plasmin, and tissue plasminogen activator. The inactivation of plasmin is the most rapid (k1 = 1.2 x 10(6) M-1 S-1). The purified LICI-2 is a single chain glycoprotein with an apparent M(r) = 42,000. A cDNA for LICI-2 was isolated and the open reading frame coded for a mature protein of 386 amino acids, of which 160 residues were confirmed by peptide sequencing. Although LICI-2 shows significant sequence similarity to the previous limulus serpin, LICI-1 (42% identity), LICI-2 contains a unique putative reactive site, -Lys-Ser-, distinct from that of LICI-1 (-Arg-Ser-). Northern blotting revealed expression of LICI-2 mRNA only in hemocytes, and not in heart, brain, stomach, intestine, coxal gland, and skeletal muscle. The immunoblot of large and small granule components with antiserum against purified LICI-2 suggests that LICI-2 is stored specifically in large granules, as in the case of LICI-1, and is released in response to external stimuli. We propose that the LICIs be classified into a new subfamily of intracellular serpins, regulated secretory serpins.
