©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Limulus Intracellular Coagulation Inhibitor Type 2
PURIFICATION, CHARACTERIZATION, cDNA CLONING, AND TISSUE LOCALIZATION (*)

(Received for publication, July 28, 1994; and in revised form, October 27, 1994 )

Yoshiki Miura Shun-ichiro Kawabata Yukako Wakamiya Takanori Nakamura (§) Sadaaki Iwanaga (¶)

From the Department of Biology, Faculty of Science, Kyushu University 33, Fukuoka 812, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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 (k(1) = 7.1 times 10^4M s) but also limulus clotting enzyme (k(1) = 4.3 times 10^5M s). LICI-2 inhibits mammalian serine proteases, including alpha-thrombin, salivary kallikrein, plasmin, and tissue plasminogen activator. The inactivation of plasmin is the most rapid (k(1) = 1.2 times 10^6M s). 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.


INTRODUCTION

The hemolymph circulating in horseshoe crabs contains two types of cells, granular hemocytes and cyanocytes(1) . Based on cell morphology, there appears to be only one type of hemocyte in the systemic circulation of the adult intermolt animal, the so called granulocyte or amebocyte(1, 2) . This cell contains an intracellular clotting system present in the large granules and which is triggered by bacterial endotoxins (lipopolysaccharide, LPS)(^1)(3, 4) . The clotting system is composed of at least four serine protease zymogens, factors C(5, 6) , B(7) , G(8) , and proclotting enzyme (9) and a clottable protein, coagulogen(10) . Like mammalian clotting factors, all these limulus factors which participate in the LPS- and (1, 3) -beta-D-glucan-mediated clotting cascades are typical serine protease zymogens related to the trypsin family(11, 12) . In our ongoing studies on the molecular mechanism of the 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 (LICI), specifically inhibits LPS-sensitive serine protease, factor C(6) , and is functionally and structurally related to protease inhibitors of the mammalian plasma serpin superfamily(13) . We have now found a second serpin, designated LICI type 2 (LICI-2) with a unique inhibitory spectrum. We describe herein the purification, characterization, cDNA cloning, and tissue localization of this serpin.


EXPERIMENTAL PROCEDURES

Materials

Factor C(14) , factor B(15) , and the clotting enzyme (16) were purified from T. tridentatus amebocytes. Human alpha-thrombin(17) , rat salivary kallikrein(18) , and bovine plasmin (19) were prepared as previously reported. Tissue plasminogen activator from a human melanoma cell line and human high molecular weight urokinase were kindly provided by Dr. P. Wallen, Umeå University, Umeå, Sweden and Mochida Pharmaceutical Co., Ltd., Tokyo, respectively. The single chain form of tissue plasminogen activator was converted to the two chains form with plasmin, by the method of Heussen et al.(20) . Sephacryl S-200, CM-Sepharose CL-6B, Mono S, and an electrophoresis calibration kit were obtained from Pharmacia LKB Biotechnology, Uppsala, Sweden. Boc-Val-Pro-Arg-pNA, Boc-Leu-Gly-Arg-pNA, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and heparan sulfate were kind gifts from Dr. K. Yoshida (Seikagaku Kogyo, Co., Ltd., Tokyo). Other fluorogenic peptide substrates used were from the Protein Research Foundation, Minoh, Osaka, Japan. Heparin, porcine elastase, and papain were obtained from Sigma, trypsin and alpha-chymotrypsin from Worthington Biochemical Co., Freehold, NJ, and lysyl endopeptidase from Wako Pure Chemical Industries Ltd., Tokyo. Horseradish peroxidase-conjugated goat anti-rabbit IgG and 4-chloro-1-naphthol were obtained from Bio-Rad. [alpha-P]dCTP was purchased from Amersham Japan, Tokyo and nylon membrane from Pall Biosupport Co., Tokyo.

Assay for LICI-2

The LICI-2 activity was expressed as an inhibitory activity against the limulus purified clotting enzyme. The amidase activity of clotting enzyme was routinely assayed, using the chromogenic substrate, Boc-Leu-Gly-Arg-pNA. Limulus clotting enzyme, 0.01 unit(16) , was preincubated with LICI-2 in 200 µl of 0.1 M Tris-HCl buffer, pH 8.0, containing 0.05% bovine serum albumin and 0.1 M NaCl at 37 °C for 30 min, and 50 µl of 2 mM substrate was then added. The reaction mixture was incubated at 37 °C for 5 min and terminated by the addition of 750 µl of 0.6 M acetic acid. The resulting chromophore was measured at 405 nm. One unit of the inhibitor was defined as the amount of protein that inhibited one unit of clotting enzyme.

Effects of LICI-2 on Other Proteases

Trypsin or elastase (10 nM each) was preincubated, respectively, with 100 nM LICI-2 in 200 µl of 0.1 M Tris-HCl, pH 8.0, containing 2% polyethylene glycol 6000, 0.15 M NaCl, and 20 mM CaCl(2) at 37 °C for 30 min. The conditions used for plasmin, urokinase, and salivary kallikrein were the same as those described above. A fluorogenic substrate, Boc-Val-Pro-Arg-MCA (trypsin), Boc-Ala-Pro-Ala-MCA (elastase), Boc-Val-Leu-Lys-MCA (plasmin), <Glu-Gly-Arg-MCA (urokinase), or carbobenzoxy-Phe-Arg-MCA (kallikrein) was added, respectively, to the mixtures at a final concentration of 0.45 mM. The reaction mixture was further incubated at 37 °C for 10 min, and the reaction was terminated by the addition of 780 µl of 0.6 M acetic acid. The resulting fluorescence was measured with excitation at 380 nm and emission at 440 nm.

Determination of Second-order Rate Constants

The second-order rate constants (k(1)) for the inhibition of LICI-2 toward factor C, clotting enzyme, human alpha-thrombin, bovine plasmin, human urokinase, recombinant tissue type plasminogen activator, and rat salivary kallikrein were determined using the method of Ehrlich et al. (21) as follows; each enzyme (final concentration, 5 nM) was incubated with LICI-2 (final concentration, 25 nM) in 1 ml of 0.02 M Tris-HCl buffer, pH 8.0, containing 0.05% human serum albumin and 0.1 M NaCl at 37 °C. At appropriate times (15 s to 15 min), 50-µl aliquots were removed, and the reaction was terminated by the addition of 950 µl of the buffer containing each substrate (0.6 mM). The concentrations of residual enzymes were calculated from their specific activities. The second-order rate constant (k(1)) was calculated using the standard equation for a second-order reaction(21) .

Isolation of LICI-2-derived Peptides, Sequencing, and Amino Acid Analysis

The purified inhibitor (100 µg) was digested with lysyl endopeptidase(22) . Peptides were separated by reversed-phase high performance liquid chromatography using a µBondasphere C(8) 300A column (Nihon Waters Ltd., Tokyo). Amino acid sequence analysis of purified peptides was performed using a gas-phase sequencer model 473A (Applied Biosystems) with the chemicals and program supplied by the manufacturer. For amino acid analysis, samples were hydrolyzed in 6 M HCl in evacuated and sealed tubes at 110 °C for 24, 48, and 72 h. Cysteines was determined as cysteic acid after performic acid oxidation or as S-carboxymethylcysteine after iodoacetic acid treatment, with or without dithiothreitol. The hydrolyzates were analyzed using a Hitachi L-8500 amino acid analyzer with the chemicals and program supplied by the manufacturer.

LICI-2-specific DNA Probes

The degenerate nucleotide sequences of the primers used for PCR were based on amino acid sequences of peptides K63 (-NAVFKG-) and K9 (-NHPFMFL-), since the location and spatial relationship of these peptides could be deduced from their sequence similarities to the conserved regions of the serpin superfamily. Sense and antisense nucleotides were synthesized with an EcoRI site at the 5` end, using a DNA synthesizer model 380B and the chemicals and program supplied by the manufacturer (Applied Biosystems). Reactions for PCR contained the cDNA template (corresponding to 0.1 µg of poly(A) RNA) and 200 pmol of each primer and were carried out in a Perkin-Elmer thermal cycler. The PCR products were treated with EcoRI and fractionated on low melting point agarose (Life Technologies, Inc.). Fragments of interest were then ligated into pBluescript II phagemid (Stratagene) for sequence analysis, as described by Sambrook et al.(23) . One clone with 0.6 kb contained the sequence of LICI-2-derived peptides and was used as a probe for screening the gt10 cDNA library.

Screening of cDNA Library

A gt10 cDNA library was constructed from poly(A) RNA, as reported previously(8) . The PCR fragment, labeled with [alpha-P]dCTP using a DNA labeling kit (Pharmacia) served as a probe to screen the gt10 library. After tertiary screening, the cDNA insert was released with EcoRI from the plaque-purified positive clone and subjected to agarose gel electrophoresis. The cDNA insert was then ligated into pBluescript II SK. Subcloning was done using sequential exonuclease digestion using a Deletion Kit (Takara Shuzo Co., Kyoto).

Computer-assisted Analysis of Sequence Data

The LICI-2 sequence was compared with all entries in the SWISS-PROT data base (release 38, February, 1994) using the GeneWorks software package (Ver.2.3 IntelliGenetics, CA). Construction of a phylogenetic tree was done using the unweighted pair group method with arithmetic mean(24) .

Preparation of Antiserum against LICI-2

An antiserum against LICI-2 was raised in rabbits as described(26) . LICI-2 (50 µg) was emulsified in synthetic adjuvant, Titer Max (Vaxal, Inc., GA) and given intradermally. After 4 weeks, a booster with 50 µg of LICI-2 in the same adjuvant was given. Blood sample were taken 1 week after the third injection and the serum was stored at -80 °C.

SDS-PAGE and Immunoblotting

SDS-PAGE was performed in 12.5% slab gels according to the method of Laemmli(25) . The gels were stained with Coomassie Brilliant Blue R-250. For immunoblotting, proteins were transferred to nitrocellulose membranes, using a TRANS-BLOT SD (Bio-Rad) at 15 V for 30 min. The membranes were then treated with the LICI-2 antiserum and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG, as described (26) . Immunoreactive proteins attached to membranes were visualized after horseradish peroxidase reduction of 4-chloro-1-naphthol.

Exocytosis of Limulus Hemocytes

Freshly collected hemocytes were washed and suspended in endotoxin-free 56 mM citrate buffer, pH 4.6, containing 0.5 M NaCl, 10 mM EDTA, and 0.1 M glucose(51) . Hemocytes were then washed with endotoxin-free 0.5 M NaCl and suspended with endotoxin-free 0.5 M NaCl containing 10 mM CaCl(2). Exocytosis was initiated by adding ionophore, A23187 (Sigma) to give a final concentration of 10 µM, and the sample was incubated at 30 °C for 30 min and centrifuged to separate cell aggregation from exocytosis fluid(52) . The supernatant was used for enzyme-linked immunosorbent assay as described(14) .

Northern Blot Analysis of LICI-2

Organs and tissues used for Northern blot analysis were obtained from an adult male Japanese horseshoe crab (T. tridentatus). Immediately after dissection, the hepatopancreas, heart, stomach, intestine, muscles, brain, and coxal gland were excised, washed in sterile water, and placed in liquid nitrogen. The RNAs were prepared from each tissue, using the acid guanidium/thiocyanate/phenol/chloroform method(27) . Then poly(A) RNA of each tissue was prepared using oligotex(TM)-dT30 <super> (Takara Shuzo Co., Kyoto) and electrophoretically separated on a 1.0% agarose gel containing 1.9% formamide and transferred to nylon membrane. A digoxigenin-containing cRNA probe of LICI-2 was generated by transcription by T7 RNA polymerase (Life Technologies, Inc.), using digoxigenin-11-UTP (Boehringer Mannheim) from the LICI-2 clone inserted in pBluescript II. The filter was hybridized with digoxigenin-containing cRNA in hybridization buffer 5 times SSC, 50% formamide, 0.1% N-lauroylsarcosine, 0.02% SDS, and 2% (w/v) blocking reagent (Boehringer Mannheim) at 65 °C for 12 h. The filter was washed with 0.2 times SSC containing 0.1% SDS at 45 °C and then incubated with anti-digoxigenin alkaline phosphatase conjugated Fab fragments in 0.1 M maleic acid, pH 7.5, 0.15 M NaCl, and 1% (w/v) blocking reagent. The filter was washed and incubated with Limugen(TM) PPD (Boehringer Mannheim) in 0.1 M Tris-HCl (pH 9.5), 0.1 M NaCl, and 50 mM MgCl(2) and exposed to x-ray film for 1 h.

Protein Concentrations

Concentrations of human alpha-thrombin, bovine factor Xa, bovine plasmin, and rat salivary kallikrein were determined by the active site titration with p-nitrophenyl p`-guanidinobenzoate(28) . Active site concentrations of limulus clotting enzyme and factor C were titrated with human antithrombin III by the method of Lawrence et al.(29) . Concentrations for other proteins were calculated from their extinction coefficients of 1% solution at 280 nm as follows: 7.6 for the clotting enzyme (M(r) = 58,000)(16) , 21.3 for factor C (M(r) = 123,000)(5) . For limulus factor G (8) and urokinase, the concentrations were estimated by assuming extinction coefficient = 10. The concentration of the purified LICI-2 was calculated from the data on amino acid analysis.


RESULTS

Purification of LICI-2

The lysate prepared from 43 g (wet weight) of hemocytes was first fractionated on a dextran sulfate-Sepharose CL-6B column (5 times 23 cm)(13) , and fractions were assayed for inhibitory activity toward clotting enzyme, as described under ``Experimental Procedures.'' The 0.5 M NaCl fractions containing inhibitor (LICI-2) were pooled and concentrated by ultrafiltration and then applied to a Sephacryl S-200 column (4 times 142 cm), equilibrated with 20 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl (Fig. 1A). LICI-2 fractions were pooled and dialyzed against 20 mM Tris-HCl, pH 8.0, containing 0.05 M NaCl and applied to CM-Sepharose CL-6B (2 times 16 cm) equilibrated with 20 mM Tris-HCl, pH 8.0, containing 50 mM NaCl (Fig. 1B). The proteins were eluted with a liner gradient of 0.05-0.35 M NaCl. Active fractions were pooled and dialyzed against 50 mM sodium phosphate buffer, pH 6.0. The dialyzed sample was then applied to a Mono S column equilibrated with the same buffer, and the protein was eluted with a liner gradient of 0-0.5 M NaCl (Fig. 1C). Purification procedures were performed at 4 °C, except for Mono S chromatography which was done at room temperature. The purified LICI-2 gave a single protein band on SDS-PAGE (M(r) = 42,000), under reducing conditions (Fig. 1D). However, under nonreducing conditions, two bands at M(r) 42,000 and 40,000 were seen, the former of which corresponded to that of the reduced sample, suggesting that a disulfide bond in LICI-2 is unstable to boiling with SDS at pH 7.5 (20 mM Tris-HCl). About 2 mg of LICI-2 was reproducibly isolated from 43 g of hemocytes.


Figure 1: Elution profiles for LICI-2 from various columns and SDS-PAGE of LICI-2. Experimental details are presented under ``Results.'' A, Sephacryl S-200. B, CM-Sepharose CL-6B. C, Mono S. D, SDS-PAGE of purified LICI-2. Three µg of LICI-2 was subjected to SDS-PAGE, under nonreducing (lane 1) and reducing conditions (lane 2). The open circles and solid lines indicate the remaining clotting enzyme activity and the absorbance of the eluate at 280 nm, respectively.



Inhibition of Limulus Clotting Enzyme by LICI-2

LICI-2 (25 nM) was allowed to react with limulus clotting enzyme (5.0 nM), and the remaining amidolytic activity was assayed at different times. The inhibition was time-dependent and at least 10-min incubation was required for complete inhibition (inset in Fig. 2). Fig. 2also shows the stoichiometry for inhibition of LICI-2 to limulus clotting enzyme. The clotting enzyme was incubated with increasing amounts of LICI-2 for 15 min and there was a parallel decrease in the amidolytic activity of the enzyme. The intersection of the extrapolated line occurred at an inhibitor: enzyme molar ratio of 1.0.


Figure 2: Reaction stoichiometry in the inhibition of limulus clotting enzyme with LICI-2. Limulus clotting enzyme (1 pmol/ml) was mixed with different concentrations of LICI-2 (0-10 pmol/ml), and the mixtures were incubated at 37 °C for 15 min. The remaining amidolytic activity was measured. In inset, the mixture of limulus clotting enzyme (5.0 pmol/ml) and LICI-2 (25 pmol/ml) was incubated at 37 °C and at intervals, aliquots of the mixture were taken for assay of the limulus clotting enzyme activity. Experimental details are presented under ``Experimental Procedures.''



Inhibitions of Other Serine Proteases by LICI-2

To determine whether LICI-2 inhibits other limulus clotting factors and mammalian serine proteases, a 10 M excess of the inhibitor was incubated with each protease at 37 °C for 30 min, and the remaining amidolytic activity was assayed, as described under ``Experimental Procedures.'' Limulus factor C and factor G (53) activities, in addition to that of the clotting enzyme, were inhibited by LICI-2. Furthermore, LICI-2 inhibited the activities of bovine plasmin, human thrombin, urokinase, tissue type plasminogen activator, rat salivary kallikrein, and trypsin (Table 1). Among them, the inactivation of plasmin was the most rapid (k(1) = 1.2 times 10^6M s). A thiol protease such as papain was not inhibited by LICI-2 (data not shown). LICI-2 formed a stable one-to-one complex with each protease which did not dissociate in SDS sampling buffer. For example, the interaction of LICI-2 (42 kDa) with the limulus factor C protease domain (B-chain, 34 kDa) (6) yielded a 77-kDa complex, under reducing conditions (Fig. 3, lane 2). Furthermore, each of the complexes formed between LICI-2 and limulus clotting enzyme-H chain (lane 3, 74 kDa), or thrombin (lane 4, 74 kDa), was also detectable.




Figure 3: Complex formation of purified LICI-2 with serine proteases. Three pmol of LICI-2 was incubated with excess proteases (30 pmol) for 60 min at 37 °C and subjected to SDS-PAGE, under reducing conditions. The complex between LICI-2 and proteases was identified by immunoblotting, using anti-LICI-2 antiserum. Lane 1, LICI-2; lane 2, factor C; lanes 3, limulus clotting enzyme; and lane 4, thrombin.



Effects of Various Glycosaminoglycans on LICI-2 Activity

Glycosaminoglycans potentiate the anticoagulant activities of antithrombin III and heparin cofactor II(30, 31) . Therefore, effects of glycosaminoglycans (50 and 500 µg/ml) on the LICI-2 activity were tested. Glycosaminoglycans including heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate had little or no effect on the inhibitory activity of LICI-2, respectively (data not shown).

Peptide Sequencing

Intact LICI-2 (100 pmol) was subjected to amino acid sequence analysis and the partial amino-terminal sequence of ELHFYKEKADRSHENLK- was determined. The 12 peptides derived from LICI-2 were then isolated and sequenced, the results of which provided the sequences of 160 amino acids (underlined amino acids in Fig. 4).


Figure 4: Nucleotide sequence and deduced amino acid sequence of LICI-2. Nucleotides and amino acid residues are numbered on the right. Underlines represent sequences determined by amino acid sequence analysis of isolated peptides. Outlined letters represent a putative reactive site. A closed diamond indicates an attachment site for N-linked sugar chain.



Isolation of a cDNA Clone and Nucleotide Sequence of LICI-2

The LICI-2-specific probe with 0.6 kb was identified with oligonucleotides corresponding to peptides derived from LICI-2, using PCR and DNA sequence analysis. When the probe was used to screen a hemocyte cDNA library (500,000 recombinant phages), one positive clone with a 1.4-kb insert was found and was subjected to restriction mapping followed by sequence determination of both strands, by sequential exonuclease digestion. The nucleotide and deduced amino acid sequences are shown in Fig. 4. The cDNA included 1,341 nucleotides with an open reading frame of 1,257 nucleotides. A stop codon TAA (nucleotide position 55) was followed by an initiation Met beginning at position 67. The open reading frame for the LICI-2 cDNA encoded for a mature protein of 386 amino acid residues and a signal sequence of 22 residues with a typical hydrophobic core. Cleavage at the Gln-Glu bond with a typical motif for the recognition of signal peptidase (32) yielded a mature protein with an NH(2)-terminal sequence identical with that of the purified LICI-2. Amino acid sequences of the isolated peptides corresponded exactly to the protein sequence deduced from the cDNA sequence, thereby clearly indicating that the isolated cDNA clone codes for LICI-2.

The amino acid analysis of LICI-2 agreed well with the amino acid composition deduced from the cDNA (Table 2). While LICI-1 previously characterized contained both hexosamines, only glucosamine was detected in LICI-2 (Table 2). There is a potential N-linked glycosylation site at Asn and amino acid sequence analysis of the peptides revealed no phenylthiohydantoin-Asn at that position (data not shown). Therefore, Asn is probably modified by the N-linked sugar chain. The calculated M(r) for LICI-2 (without carbohydrates) was 44,675, a value somewhat larger than that of the purified protein estimated on SDS-PAGE (M(r) = 42,000). Compared with findings of no cysteines in LICI-1, LICI-2 was predicted to contain 2 cysteines from the cDNA sequence, and 2.1 mol of cysteic acids were actually detected after performic acid oxidation. These cysteines probably form a disulfide bridge, since carboxymethylcysteines were detected only in the hydrolysate of the sample S-alkylated after reduction (data not shown).



Sequence Similarity to Other Serpins

A search of SWISS-PROT showed the striking similarity of LICI-2 to members of the serpin superfamily, as shown in Fig. 5. LICI-2 was most closely related to LICI-1 (42%) and more to mammalian intracellular serpins with human plasminogen activator inhibitor type 2 (36%) (33) and human monocyte/neutrophil elastase inhibitor (35%) (34) than to insect serpins, such as an elastase inhibitor from Manduca sexta (28%)(35) , antitrypsin (29%)(36) , and antichymotrypsin 1 (26%) (37) from Bombyx mori. It also shared a 34% sequence similarity with human antithrombin III. Comparing LICI-2 with other serpins (Fig. 5), there was a putative reactive site, -Lys-Ser-, in the COOH-terminal region, as is the case with the serpin superfamily. Furthermore, Ala at the NH(2)-terminal side of the reactive center loop of serpins, referred to as the P12 site (which is known as a critical determinant of inhibitor status of serpins), was conserved in LICI-2 as well as LICI-1(13) . As LICI-1 also contained the consensus sequence FLF(F/L)I in the corresponding region (which is known as a clearance signal, for instance, FVFLM for alpha(1)-antitrypsin, exposed after formation of the serpin-enzyme complex), a similar clearance mechanism may function in the horseshoe crab.


Figure 5: Alignment of the amino acid sequence of LICI-2 with those of LICI-1(13) , human monocyte/neutrophil elastase (LEI) (34) and human plasminogen activator inhibitor type 2 (PAI-2)(33) . Manual alignment and position numbers are based on the sequence of LICI-2, with appropriate gaps. Residues identical to LICI-2 are boxed.



Expression of LICI-2 mRNA in Various Tissues

To determine the size of the mRNA and to investigate tissue specific expression of LICI-2, Northern blot analysis was carried out using poly(A) RNAs of hemocytes, heart, hepatopancreas, brain, stomach, intestine, skeletal muscle, and coxal gland (Fig. 6). Using the LICI-2 cRNA probe of about 850 bases, the expression of LICI-2 was only detected in hemocytes but not in other tissues. The LICI-2 transcript had approximately 1.8 kb.


Figure 6: Northern blot analysis of mRNA isolated from various tissues of horseshoe crab. Lane 1, hemocyte; lane 2, heart; lane 3, hepatopancreas; lane 4, stomach; lane 5, intestine; lane 6, coxal gland; lane 7, brain; and lane 8, skeletal muscle. Details are presented under ``Experimental Procedures.''



Subcellular Localization and Release of LICI-2 from Hemocytes

Antiserum raised against purified LICI-2 was used to identify localization of this coagulation inhibitor in hemocytes. The isolated large and small granules from the hemocytes (50) were first treated with 1% SDS at 100 °C for 2 min and subjected to SDS-PAGE, under reducing conditions for immunoblotting. The anti-LICI-2 antiserum recognized the 42-kDa LICI-2 in the extract of large granules (Fig. 7). However, we found no immunoreactive materials in the extract of small granules with this antiserum, indicating that LICI-2 is located in the large granule. We examined whether LICI-2 could be secreted from hemocytes by an external stimulation. When hemocytes were treated with calcium ionophore A23187, which has been shown to induce exocytosis of limulus hemocytes(51, 52) , LICI-2 was released into the extracellular fluid as detected by immunoblotting and enzyme-linked immunosorbent assay (Fig. 8). Under the conditions used, hemocytes were not lysed, since lactate dehydrogenase activity, a cytosolic marker enzyme, was negligible (data not shown) (51, 52) .


Figure 7: Localization of LICI-2. Ten µg each of large and small granule samples was subjected to SDS-PAGE under reducing conditions(50) . LICI-2 was identified by immunoblotting. Lane 1, LICI-2; lane 2, large granule; and lane 3, small granule. Samples were separated by a sucrose gradient centrifuge method(50) .




Figure 8: Release of LICI-2 from hemocytes. Fresh hemocytes were incubated with 10 µM ionophore A23187, and the exocytosis fluid was diluted with a solution containing 0.5 M NaCl and 10 mM CaCl(2). The diluted sample was subjected to enzyme-linked immunosorbent assay and LICI-2 antigen was detected by anti-LICI-2 antiserum(14) . Open and closed circles represent presence and absence of the ionophore A23187, respectively. The inset shows Western blot analysis using anti-LICI-2 antiserum.




DISCUSSION

In the present study, a second intracellular protease inhibitor type 2 (LICI-2) from limulus hemocytes was purified and characterized and the entire sequence of a cDNA coding for LICI-2 was determined. LICI-2 is a single chain glycoprotein consisting of 386 amino acids with an apparent M(r) = 42,000, under reducing conditions. LICI-1 previously reported (13) mainly inhibits factor C in the limulus coagulation cascade(12) . On the other hand, LICI-2 inhibits not only factor C but also clotting enzyme (k(1) = 4.3 times 10^5M s) by forming a covalent 1:1 complex, probably through the putative reactive site, -Lys-Ser-. In serpins, the amino acid at the P1 site generally reflects the substrate specificity of target proteases. The selectivity of the Lys at position of P1 site seems to be in good agreement with the kinetic study, since the second-order rate constant against plasmin (k(1) = 1.2 times 10^6M s) was highest between proteases tested. Therefore, LICI-2 could be classified as a Lys-serpin, such as rat kallikrein-binding protein (38) and silkworm antitrypsin(36) .

The expression of mRNA for LICI-2 was detected only in hemocytes, and the same result was also obtained for LICI-1 previously reported(13) . Although mammalian plasma serpins are mainly expressed in liver, mRNAs for LICIs were not detected in hepatopancreas with functions analogous to the liver and pancreas of vertebrates. Furthermore, both LICI-1 (13) and LICI-2 are located in the large granules in hemocytes. Therefore, in horseshoe crabs, all of the serine protease zymogens for coagulation and their specific serpins so far identified are co-localized in the same granules, thereby indicating a more effective coagulation and regulation at local lesions.

The phylogenic tree for 12 serpins is shown in Fig. 9. The positions of the LICIs in the tree are far from mammalian plasma serpins. Even invertebrate serpins, antitrypsin (36) and antichymotrypsin 1 (37) from the larval hemolymph of B. mori, are also located far apart among LICIs. LICIs could well occupy the position of ovalbumin and an intracellular serpin branch rather than the insect serpins.


Figure 9: Phylogenetic tree of 12 serpin sequences, including LICI-1 and -2. Abbreviations for proteins are; A1AT-HUMAN, alpha(1)-antitrypsin human(47) ; A1AT-BOMMO, antitrypsin Bombyx mori(36) ; PAI1-HUMAN, PAI-1 human(48) ; ACH1-BOMMO, antichymotrypsin 1, B.(37) ; LEI-HUMAN(34) , HORSE(39) ; and porcine (40) leukocyte elastase inhibitor; PTI, placental thrombin inhibitor(41) ; PAI-2(30) ; OVAL-CHICK, ovalbumin chicken(49) .



Of intracellular serpins, human leukocyte/monocyte elastase inhibitor (34) , equine elastase inhibitor(39) , porcine leukocyte neutral protease inhibitor(40) , placental thrombin inhibitor(41, 42) , and maspin (43) have been reported. The key features of these intracellular serpins are the lack of signal sequences destined for the endoplasmic reticulum and the presence of an oxidation-sensitive residue, Met, in proximity to their reactive sites(41) . LICI-2 also contains a Met residue at the P3 site, has a higher sensitivity to N-chlorosuccinimide, and oxidation inactivates the LICI-2 activity (data not shown). Although the physiological importance of the labile Met residue remains to be defined, these intracellular inhibitors may be involved in the regulation of intracellular protein degradation(39) , cell apoptosis(44) , and tumor suppressor(41) . Despite the structural similarity between LICIs and these intracellular serpins, both LICI-1 and -2, have typical NH(2)-terminal signal peptides destined for the endoplasmic reticulum. LICIs are stored in large granules, probably through a regulated secretory pathway in limulus hemocytes and may be co-released with several coagulation factors and bactericidal peptides(12) , in response to external LPS stimulation. By contrast, mammalian intracellular serpins are localized in the cytosolic fraction and are not secreted into plasma or culture media(39, 42) , except for PAI-2(45) . A part of PAI-2 enters the endoplasmic reticulum through the NH(2)-terminal hydrophobic region of the molecule and is constitutively secreted into the blood circulation(46) . Therefore, from the standpoint of localization, LICIs could be classified into a new subfamily of intracellular serpins, a regulated secretory serpin.


FOOTNOTES

*
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(TM)/EMBL Data Bank with accession number(s) D32211[GenBank].

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

To whom correspondence should be addressed: Dept. of Biology, Faculty of Science, Kyushu University 33, Higashi-ku, Hakozaki, Fukuoka 812, Japan. Tel.: 81-92-632-2742; Fax: 81-92-632-2742.

(^1)
The abbreviations used are: LPS, lipopolysaccharide; LICI, limulus intracellular coagulation inhibitor; serpin, serine protease inhibitor; PAI, plasminogen activator inhibitor; Boc, N-tert-butoxycarbonyl; MCA, 4-methylcoumaryl-7-amide; pNA, p-nitroanilide; PAGE, polyacrylamide gel electrophoresis; kb, kilobases; K, lysyl endopeptidasedigested peptide.


ACKNOWLEDGEMENTS

We are grateful to Dr. F. Shishikura for guidance in organ dissection and identification of the horseshoe crab. We also thank H. Hashimoto and T. Yano for expert technical assistance with peptide sequencing and amino acid analysis, S. Yukawa for her assistance in preparing the anti-LICI-2 antiserum, Y. Kuroki for her assistance in immunoblotting, Dr. T. Muta for valuable suggestions and helpful discussions, and M. Ohara for helpful comments on the manuscript.


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