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
= 7.1
10
M
s
) but also
limulus clotting enzyme (k
= 4.3
10
M
s
).
LICI-2 inhibits mammalian serine proteases, including
-thrombin,
salivary kallikrein, plasmin, and tissue plasminogen activator. The
inactivation of plasmin is the most rapid (k
= 1.2
10
M
s
). The purified LICI-2 is a single chain
glycoprotein with an apparent M
= 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.
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)(
)(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) -
-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
-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
-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. [
-
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
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
) for the inhibition
of LICI-2 toward factor C, clotting enzyme, human
-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
) 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
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
[
-
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
. 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
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
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
and exposed to x-ray film for 1 h.
Protein Concentrations
Concentrations of human
-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
= 58,000)(16) , 21.3 for factor C (M
= 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
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
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
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
= 42,000), under reducing conditions (Fig. 1D). However, under nonreducing conditions, two
bands at M
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
= 1.2
10
M
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
-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
for LICI-2 (without
carbohydrates) was 44,675, a value somewhat larger than that of the
purified protein estimated on SDS-PAGE (M
=
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
-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

-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
. 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
= 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
= 4.3
10
M
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
= 1.2
10
M
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, 
-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
-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
-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.