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.
Highlights
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 Iimulus intracellular coagulation inhibitor, LICI (Miura, Y., Kawabata, S., and Iwanaga, S. (1994) J
Purification of LICI type-2 (LICI-2)-The lysate prepared from 43 g of hemocytes was first fractionated on a dextran sulfate-Sepharose CL-6B column (5 X 23 em) [13], and fractions were assayed for inhibitory activity toward clotting enzyme, as described under "Experimental Procedures." The 0.5 M NaCI fractions containing inhibitor (LICI-2) were pooled and concentrated by ultrafiltration and applied to a Sephacryl S-200 column (4 X 142 em), equilibrated with 20 mu Tris-HCI, pH 8.0, containing 0.5 M NaCI (Fig. lA)
About 2 mg ofLICI-2 was reproducibly isolated from 43 g of hemocytes
Summary
(Received for publication, July 28, 1994, and in revised form, October 27, 1994). Yoshiki Miura, Shun-ichiro Kawabata, Yukako Wakamiya, Takanori Nakamuraj, and Sadaaki Iwanaga§. 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 Iimulus intracellular coagulation inhibitor, LICI This serpin inhibits limulus lipopolysaccharide-sensitive serine protease, factor C. In our ongoing studies on the molecular mechanism ofthe limulus clotting pathway, we have obtained evidence for the existence of a typical serine protease inhibitor (serpin) in the Japanese horseshoe crab (Tachypleus tridentatus) hemocytes [13]. This serpin, named limulus intracellular coagulation inhibitor We describe the purification, characterization, cDNA cloning, and tissue localization of this serpin
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