Protein Chemistry and Structure
A Limulus Intracellular Coagulation Inhibitor Type 2: PURIFICATION, CHARACTERIZATION, cDNA CLONING, AND TISSUE LOCALIZATION (∗)

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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 × 104M -1 s -1 ) but also limulus clotting enzyme (k1 = 4.3 × 105M -1 s -1 ). LICI-2 inhibits mammalian serine proteases, including α-thrombin, salivary kallikrein, plasmin, and tissue plasminogen activator. The inactivation of plasmin is the most rapid (k1 = 1.2 × 106M -1 s -1 ). The purified LICI-2 is a single chain glycoprotein with an apparent Mr = 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.

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This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and by the Sasakawa Scientific Research Grant from the Japan Science Society (to Y. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) D32211.

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Present address: Institute for Enzyme Research, University of Tokushima, Tokushima 770, Japan.