(Received for publication, July 20, 1994)
From the
Horseshoe crab hemocyte lysate responds to
(13)-
-D-glucans, initiating an enzymatic cascade,
which culminates in clot formation.We have purified to homogeneity the
serine protease zymogen factor G, which is directly activated by
(1
3)-
-D-glucans and which initiates the hemolymph
clotting cascade. Factor G is a heterodimeric protein composed of two
noncovalently associated subunits
(72 kDa) and
(37 kDa). In
the presence of (1
3)-
-D-glucans such as curdlan and
paramylon, factor G is autocatalytically activated to an active serine
protease named factor G. This activation is accompanied by limited
proteolysis of both subunits: the 72-kDa subunit
is cleaved to
55-kDa and 17-kDa fragments, and the 37-kDa subunit
is shortened
to 34 kDa. Longer incubations with (1
3)-
-D-glucans
result in cleavage of the 55-kDa fragment to 46 kDa and the 34-kDa
fragment to 32 kDa, with concomitant loss of amidase activity.
Reconstitution experiments using purified proteins participating in the
hemolymph clotting cascade demonstrate that factor G is capable of
activating proclotting enzyme directly, resulting in the conversion of
coagulogen to coagulin gel. Thus, purified factor G is shown to be the
primary initiator of the (1
3)-
-D-glucan-sensitive
coagulation pathway in the horseshoe crab hemocyte lysate.
The evolution of an effective system for microbial defense is
central to the survival and perpetuity of higher organisms.
Invertebrates, which lack typical immune systems, have developed unique
modalities to detect and respond to microbial surface antigens, such as
lipopolysaccharide (LPS), ()peptideglycan, and
(1
3)-
-D-glucan. Because both invertebrates and
vertebrate animals respond to these substances, it is likely that a
system recognizing these epitopes emerged at a common stage in the
evolution of these animals.
(13)-
-D-glucan and
its derivatives, integral components of the cell wall of fungi and
plants, can stimulate the host defense systems of many vertebrate and
invertebrate animals(1, 2) . In mammals,
(1
3)-
-D-glucans have been shown to evoke antitumor
activity caused by the stimulation of the reticuloendothelial
system(3) . Arthropods also exhibit several well characterized
(1
3)-
-D-glucan-sensitive systems. One of the better
known examples is the prophenoloxidase cascade system found mainly in
the hemolymph of insects. In this system,
(1
3)-
-D-glucans activate prophenoloxidase via a
serine protease, leading to melanin formation(4, 5) ;
the formed melanin then encapsulates invading foreign organisms and
immobilizes them.
Among other arthropods, horseshoe crab (or limulus) hemolymph is known to be very sensitive to bacterial LPS (reviewed in (6) and (7) ). A trace amount of LPS activates the hemocytes to degranulate and release LPS-sensitive coagulation factors and antimicrobial substances. Among the proteins released from the cells, an LPS-sensitive serine protease zymogen, factor C, is autocatalytically converted to its active form, factor C, in the presence of LPS(8, 9) . The active factor C activates zymogen factor B to factor B(10, 11) , which then activates proclotting enzyme to clotting enzyme(12, 13) . The resulting clotting enzyme catalyzes the activation of coagulogen, resulting in the formation of an insoluble coagulin gel(14, 15) . Thus, Gram-negative bacteria invading the horseshoe crab hemolymph are engulfed in the coagulin gel and are subsequently killed by antimicrobial substances, such as anti-LPS factor and tachyplesins (16, 17, 18) .
Because of its high
sensitivity, this LPS-sensitive clotting reaction is utilized to
quantitate a trace amount of endotoxin (the limulus test or limulus
amebocyte lysate test). During the diagnostic application of the
limulus test, it was pointed out that positive reactions were observed
with plasma of some patients even in the absence of LPS(19) .
Since some of those patients suffered from fungal infection or were
undergoing hemodialysis with cellulose dialyzers, this pseudopositive
reaction had been suggested to be at least partly caused by
(13)-
-D-glucans. In 1981, we and others reported
the presence of a (1
3)-
-D-glucan-sensitive protease
zymogen in hemocyte lysate, which was distinct from any of the
components of the LPS-mediated coagulation
pathway(20, 21) . We tentatively called the zymogen
factor G and showed that the activation of factor G caused the gelation
of the lysate through the activation of proclotting enzyme.
Previous
efforts at the purification of factor G have been unsuccessful because
of its instability during isolation procedures, and it has therefore
not been definitively determined if active factor G directly activates
proclotting enzyme or whether other factors are necessary (20, 21) . In this report, we describe the
purification and characterization of factor G. The purified protein
consisted of two subunits, which was consistent with the deduced amino
acid sequence based on cDNA cloning(22) . The purified factor G
when activated can itself directly activate proclotting enzyme;
moreover, the three cascade components, factor G, proclotting enzyme,
and coagulogen, constitute the minimal elements that are both necessary
and sufficient for reconstituting the
(13)-
-D-glucan-mediated cascade.
Figure 1:
Purification of Factor G. A,
dextran sulfate-Sepharose CL-6B chromatography. Proteins were eluted
with 0.05 M, 0.15 M, 0.25 M, 0.5 M,
and 2.0 M NaCl containing 20 mM Tris-HCl (pH 8.0). B, ConA-Sepharose chromatography. The column was washed with
0.5 M NaCl containing 20 mM Tris-HCl (pH 8.0) and
then eluted with 0.5 M methyl--D-glucoside (
-MG) containing 20 mM Tris-HCl (pH 8.0) and 0.5 M NaCl. C, Sephacryl S-200 HR chromatography.
Proteins were eluted with 50 mM sodium phosphate (pH 8.0).
Fractions indicated by bars were pooled. Absorbance at 280 nm
is shown in solid lines. Factor G (open circles) and
factor G (closed circles) activities are shown in units per
ml. See ``Experimental Procedures'' for further
details.
SDS-polyacrylamide gel electrophoresis of this fraction revealed two
bands of 72 kDa and 37 kDa under reducing conditions and 72 kDa and 32
kDa under nonreducing conditions (Fig. 2A). Because the
native molecular mass as estimated by Superose 6 gel filtration was
found to be 110 kDa, zymogen factor G is likely a heterodimeric protein
composed of two noncovalently associated subunits. We named these two
subunits (the 72 kDa-subunit) and
(the 37-kDa subunit),
respectively. The amino acid composition of purified factor G (Table 1) was consistent with that deduced from the cDNA
sequences(22) . A summary of the purification of zymogen factor
G is shown in Table 2.
Figure 2: SDS-polyacrylamide gel electrophoresis of the purified zymogen factor G (A) and its native molecular mass on Superose 6 gel filtration (B). A, The purified factor G was subjected to 12.5% SDS-polyacrylamide gel electrophoresis in the presence (+SH) or absence (-SH) of 2-mercaptoethanol. The gel was stained with Coomassie Brilliant Blue. B, The native molecular mass of factor G was determined by fast protein liquid chromatography on a Superose 6 HR 10/30 column. The protein standards (closed circles) were blue dextran 2000 (2,000 kDa), ovalbumin (43 kDa), and chymotrypsinogen (25 kDa). Factor G activity (open circle) was recovered at the position of 110 kDa.
The purified factor G was sensitive to freezing and thawing; approximately 40-80% of the activity was lost after freezing once at -80 °C. 0.5 mg/ml of BSA, 10% polyethylene glycol 4000, 0.1% Triton X-100, or 0.1% Nonidet P-40 enhanced the stability of factor G approximately 4-fold upon storage at 4 °C, with more than 75% of the activity remaining even after the freezing and thawing (data not shown). The factor G amidase activity was increased 1.5-fold in the presence of 0.2-1.0 M NaCl and was inhibited to 40% by 10-160 units/ml of heparin (data not shown).
The
optimal concentration of curdlan was dependent upon the amount of
factor G present (Fig. 3), peaking at a molar ratio of
approximately 10:1. Fig. 4shows the time course (A)
and the structural changes (B) resulting from the activation
of factor G in the presence of curdlan. The amidase activity of factor
G increased gradually and peaked at 20 min, corresponding with the
replacement of the 72-kDa subunit and the 37-kDa subunit
with three new bands of 55 kDa, 34 kDa, and 17 kDa. The amino-terminal
sequence of the 55-kDa fragment was
Glu-Ser-Asn-Thr-Asn-Gly-Ile-X-Tyr-His-Ile-Tyr-Ser-X-Glu-
(data not shown), corresponding to residues 151-165 of the 72-kDa
subunit
(22) ; sequence analysis of the 17-kDa band showed
that it corresponded to the amino terminus of this subunit (data not
shown; (22) ). On the other hand, the 37-kDa subunit
was
converted to a 34-kDa fragment during activation. Under nonreducing
conditions, the activated subunit
migrated slightly slower,
indicating that the cleavage site is in a disulfide loop. The 55-kDa
band was further degraded to a 46-kDa fragment after a 20-min
incubation, which appeared to correlate with the loss of activity. A
small portion of the 34-kDa band was also fragmented to a 32-kDa
polypeptide after 30-40 min.
Figure 3: Dose-dependent curve of curdlan for the activation of factor G. Three different concentrations of factor G were incubated with various concentrations of curdlan, and the amidolytic activity was measured with Boc-E(OBzl)GR-MCA as a substrate. See ``Experimental Procedures'' for further details.
Figure 4: Time course of factor G activation by curdlan. Time course of the factor G amidolytic activity to hydrolyze Boc-E(OBzl)GR-MCA (A) and the structural changes (B) of factor G after the incubation with curdlan are shown. B, SDS-polyacrylamide gel electrophoresis on 12.5% gel in the presence (+SH) or absence (-SH) of 2-mercaptoethanol. See ``Experimental Procedures'' for further details.
Since the activation of factor G
is associated with limited proteolysis of both subunits and
, the three serine proteases involved in the horseshoe crab
coagulation cascade (6) were examined to see whether they were
capable of activating zymogen factor G. Neither factor C, clotting
enzyme, nor partially purified factor B exhibited factor G-activating
activity; nor did human
-thrombin or digestive proteases, such as
trypsin or chymotrypsin (data not shown). This was not because of the
degradation of the protease domain of factor G, because curdlan-induced
factor G activity emerged after incubation of the zymogen factor G with
these proteases (data not shown).
The amidase
activity of factor G was sensitive to diisopropyl fluorophosphate and
leupeptin but not to E-64 or EDTA, indicating that it is a serine
protease (data not shown). Table 5shows the effects of various
protease inhibitors on the activity of factor G. Horseshoe crab
hemocytes are known to contain at least two types of serpin type serine
protease inhibitors, named LICI-1 (23) and LICI-2. ()Whereas LICI-1 had no effect on the activity of factor G,
LICI-2 strongly inhibited its factor G amidase activity (Table 5). Other known serine protease inhibitors exhibited
moderate inhibitory activities toward factor G (Table 5). Among
them,
-antiplasmin showed strong inhibition. We also
examined the effects of monosaccharides (D-glucose, D-galactose, D-mannose, D-xylose, D-glucosamine, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and N-acetylneuraminic acid), oligosaccharides (
-lactose and N-acetylallolactosamine), and mucopolysaccharides (chondroitin
sulfates A, B, and C) on factor G, none of which inhibited the amidase
activity. p-Nitrophenol derivatives of oligosaccharides used
for substrates of glucanases, such as p-nitrophenyl-
-D-galactopyranoside, p-nitrophenyl-N-acetyl-
-D-glucosaminide, p-nitrophenyl-
-cellobioside, p-nitrophenyl-
-D-glucoside, and p-nitrophenyl-tri-N-acetyl-
-chitotrioside were
also without effect.
Recently, the presence of (13)-
-D-glucan
binding proteins, which are responsible for the activation of
prophenoloxidase, have been reported in crayfish(29) ,
cockroaches(30) , and silkworms (31) . The molecular
identity of these proteins, however, remains to be determined.
Horseshoe crab factor G as described here is the first
(1
3)-
-D-glucan-responsive protein whose
characteristics have been biochemically analyzed on the molecular
level.
We conclude that both the 72- and the 37-kDa polypeptides are
subunits of zymogen factor G based on the following observations: 1)
the two subunits are co-purified during the procedure ( Fig. 1and 2A); 2) the native molecular mass as
estimated by gel filtration is very close to the sum of the two
subunits (Fig. 2B); and 3) only the two subunits among
many proteins in the crude ConA eluate undergo structural changes after
the addition of curdlan (data not shown and Fig. 4B).
Furthermore, the deduced amino acid sequence analysis of factor G cDNA (22) suggests that the known functions of factor G, glucan
binding and amidase activity, would require the presence of both
subunits for its full activity; subunit contains a
-1,3-glucanase A1-like domain, three xylanase A-like domains, and
two xylanase Z-like domains, and subunit
is a serine protease
zymogen.
Although subunit contains glucanase-like sequences,
any glucanase activity was undetectable when curdlan and laminarin were
used as substrates under the previously described
conditions(33) , in which the activity of the same amount of
laminarinase was easily detected (data not shown). It is noteworthy
that the
-1,3-glucanase domain is rapidly digested in the middle
after incubating factor G with curdlan, as described below, which may
result in the loss of any glucanase activity.
Purified factor G was
easily activated by various glucans but not by LPS. The most effective
activators examined were linear (13)-
-D-glucans,
such as curdlan and paramylon (Table 3). Branching of the linear
chain with (1
4)-
or (1
6)-
linkages appears to
reduce the factor G-activating activity. These results agree well with
a previous report in which crude factor G was used(34) .
Mannan, galactan, and xylan preparations used in this study also
induced the activation of factor G. However, we cannot exclude the
possibility of a trace amount of contamination of
(1
3)-
-D-glucans in the test samples, since their
ED
values were very high (>10
g/ml).
In 1985, it was pointed out by another group that carboxymethylated
(1
3)-
-D-glucan but not native
(1
3)-
-D-glucans activate Limulus polyphemus amebocyte lysate coagulation(35) . However,
carboxymethylation of curdlan significantly reduces the factor
G-activating activity (Table 3).
The shift in optimal factor G
activity relative to its substrate concentration suggests that the
molar ratio of factor G to (13)-
-D-glucan is
important in the activation (Fig. 3). The kinetics also indicate
that the activation of factor G occurs through an intermolecular
interaction between factor G molecules bound to
-glucan. Similar
phenomena have been observed in the activation of horseshoe crab factor
C by LPS (36) and mammalian coagulation factor XII by a
negatively charged surface(37) .
Activation of factor G
causes limited proteolysis of both subunits and
. The
optimal pH for the activation (approximately pH 7.5) is almost the same
as that of the amidase activity of factor G (data not shown). Based on
the amino-terminal sequence of the 55-kDa fragment, the first cleavage
of the 72-kDa subunit
was found to occur between Arg
and Glu
, which are located in the middle of the
glucanase A1-like domain(22) . Simultaneously, an
Arg
-Ile
bond in subunit
is
cleaved to form an active serine protease linked with the
amino-terminal short light chain(22) . In both cases, the
carboxyl-terminal sides of arginine residues are cleaved, consistent
with the substrate specificity of factor G (Table 4). In fact,
factor G subunit
has an aspartic acid at the primary substrate
binding site(22) . On the other hand, neither trypsin nor any
other proteases examined activated factor G in the absence of
(1
3)-
-D-glucan.
These characteristics of factor
G allow us to speculate as to the activation mechanism of factor G. In
the zymogen form, subunit sterically hinders the activation site
of subunit
;
-glucan binding to subunit
then exposes
the active site of subunit
, allowing autocatalytic activation
through intermolecular interaction between subunit
s. The active
subunit
then quickly hydrolyzes the Arg
-Glu
bond in subunit
. Then, another site in the same subunit is
cleaved, which is followed by the inactivation of the protease
activity. Since this cleavage in subunit
appears to reduce the
amidolytic activity, subunit
is likely to regulate the catalytic
activity of the subunit
.
We frequently encountered spontaneous
activation followed by the inactivation of the amidase activity during
the attempts for purification. When the ConA-Sepharose eluate was
applied to a Sephacryl S-200 HR column at pH 8.0, factor G was often
activated, and the active form was eluted in fractions between the two
major protein peaks with several contaminating proteins (data not
shown). Since factor G should exist as a stable zymogen in the granules
of hemocytes, and these secretory granules are known to maintain a low
pH(32) , we therefore tested the effect of lowering the pH
during Sephacryl S-200 HR chromatography. At pH 6.5, factor G was
stable and, unexpectedly, it was eluted in very late fractions well
separated from other proteins (Fig. 1C), probably
because of an interaction with the resin. The activation at pH 8.0 may
reflect the dissociation of the two subunits caused by a low protein
concentration, resulting in autocatalytic activation by subunit .
The requirement for lower pH may result from its stabilization of
subunit interaction or from reduction of the endogenous amidase
activity in subunit
. Using the cDNAs for both subunits
and
, these hypotheses could be examined in expression experiments of
the native and mutated forms of factor G.
In this study, we have
established the role of factor G in the coagulation cascade; factor G
induces clot formation through the activation of proclotting enzyme
followed by the activation of coagulogen. In our reconstitution
experiments, factor G, proclotting enzyme, and coagulogen are the
minimum requirements for clot formation. Similar to the factor C/LPS
system, this enzymatic cascade, stimulated by
(13)-
-D-glucans located on the cell surface of
invading fungi, allows the horseshoe crab to defend itself by
immobilizing microorganisms in an insoluble protein matrix. Because
these organisms are also pathogenic for humans, an assay system for
quantifying (1
3)-
-D-glucans based on this novel
heterodimeric serine protease zymogen may be relevant for the early and
sensitive diagnosis of fungal sepsis. Furthermore, this
(1
3)-
-D-glucan-sensitive factor G may be useful as
a unique tool to analyze other biological reactions stimulated by the
glucans.