©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purified Horseshoe Crab Factor G
RECONSTITUTION AND CHARACTERIZATION OF THE (13)-beta-D-GLUCAN-SENSITIVE SERINE PROTEASE CASCADE (*)

(Received for publication, July 20, 1994)

Tatsushi Muta (1) (2) Noriaki Seki (2) Yoshie Takaki (1) Ryuji Hashimoto (1) Toshio Oda (1) Atsufumi Iwanaga (1) Fuminori Tokunaga (1)(§) Sadaaki Iwanaga (1) (2)(¶)

From the  (1)Department of Biology, Faculty of Science, and the (2)Department of Molecular Biology, Graduate School of Medical Science, Kyushu University 33, Fukuoka 812, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Horseshoe crab hemocyte lysate responds to (13)-beta-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 (13)-beta-D-glucans and which initiates the hemolymph clotting cascade. Factor G is a heterodimeric protein composed of two noncovalently associated subunits alpha (72 kDa) and beta (37 kDa). In the presence of (13)-beta-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 alpha is cleaved to 55-kDa and 17-kDa fragments, and the 37-kDa subunit beta is shortened to 34 kDa. Longer incubations with (13)-beta-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 (13)-beta-D-glucan-sensitive coagulation pathway in the horseshoe crab hemocyte lysate.


INTRODUCTION

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), (^1)peptideglycan, and (13)-beta-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)-beta-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, (13)-beta-D-glucans have been shown to evoke antitumor activity caused by the stimulation of the reticuloendothelial system(3) . Arthropods also exhibit several well characterized (13)-beta-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, (13)-beta-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)-beta-D-glucans. In 1981, we and others reported the presence of a (13)-beta-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)-beta-D-glucan-mediated cascade.


EXPERIMENTAL PROCEDURES

Materials

Hemocyte lysate from the Japanese horseshoe crab (Tachypleus tridentatus) and dextran sulfate-Sepharose CL-6B were prepared as described(12) . Factor C(8) , proclotting enzyme(12) , coagulogen(14) , and limulus intracellular coagulation inhibitor-1 (LICI-1) (23) and -2 (24) were purified as described. Factor B was partially purified by dextran sulfate-Sepharose CL-6B, Sepharose CL-6B, benzamidine-CH-Sepharose 4B, and Sephacryl S-200 HR column chromatography(10) . alpha(1)-Antitrypsin and alpha(2)-antiplasmin were the same as those used in (25) ). ConA-Sepharose, Sephacryl S-200 HR, Superose 6 HR 10/30, and molecular weight standards (blue dextran 2000, ovalbumin, and chymotrypsinogen A) were purchased from Pharmacia Biotech Inc. Curdlan, carboxymethylcurdlan, and paramylon were obtained from Wako Pure Chemical Industries Ltd. (Osaka); laminaran oligosaccharides (degree of polymerization 2-7) from Seikagaku Corp. (Tokyo); bovine serum albumin (BSA), Laminaria digitata laminarin, bakers' yeast beta-D-glucan, Cetraria islandica and Usnea barbata lichenans, barley beta-D-glucan, Aspergillus awamori nigeran, Saccharomyces cerevisiae alpha-D-mannan, soybean trypsin inhibitor, and hirudine from Sigma; Schizophyllan (Sonifilan) from Kaken Chemical Co. (Tokyo); lentinan from Ajinomoto Co. (Tokyo), oat spelt (14)-beta-D-xylan from Fluka Chemika-Biochemika (Buchs, Switzerland); antithrombin III from Hoechst Japan (Tokyo); protein C inhibitor from Boehringer Mannheim; and heparin from Nakalai Tesque, Inc., (Kyoto). Krestin and (13)-beta-D-galactan from gum arabic were kindly provided by Dr. M. Suzuki and Dr. Y. Hashimoto, respectively. Aprotinin was provided by Mochida Pharmaceutical Co., Ltd. t-Butyloxycarbonyl--benzyl-L-glutamyl-glycyl-L-arginine 4-methyl-coumaryl-7-amide (Boc-E(OBzl)GR-MCA) was obtained from Peptide Institute Inc. (Osaka). t-Butyloxycarbonyl-L-leucyl-glycyl-L-arginine p-nitroanilide (Boc-LGR-pNA) was a gift from Dr. S. Tanaka. Pyrogen-free distilled water was from Otsuka Pharmaceutical Co. Ltd. (Tokyo).

Purification of Factor G

The lysate (1,250 ml) prepared from 113 g (wet weight) of hemocytes was applied to a dextran sulfate-Sepharose CL-6B column (ø (diameter) 5.0 times 17 cm), which was pre-equilibrated with 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl. The column was washed with the same buffer and then sequentially eluted with 20 mM Tris-HCl (pH 8.0) containing 0.15 M, 0.25 M, 0.5 M, and 2.0 M NaCl. Fractions exhibiting factor G activity were pooled and applied to a ConA-Sepharose column (ø 2.0 times 16 cm) pre-equilibrated with 20 mM Tris-HCl (pH 8.0) containing 0.25 M NaCl. The column was washed extensively with 0.5 M NaCl and then eluted with the same buffer containing 0.5 M methyl-alpha-D-glucoside. The eluted fractions were pooled and concentrated to 18.0 ml by Diaflow ultrafiltration using a PM-10 membrane (Amicon Corp., Ireland). The concentrate was then fractionated on a Sephacryl S-200 HR column (ø 2.7 times 98 cm) equilibrated with 50 mM sodium phosphate (pH 6.5). All procedures were performed under sterile conditions.

Assay of Factor G

The amidase activity of factor G was measured after activation by curdlan. One mg/ml curdlan was dissolved in 0.1 M NaOH and then diluted with pyrogen-free distilled water. Each sample (10 µl in a typical assay) was preincubated with 125 ng/ml curdlan at 37 °C for 20 min in 0.1 M Tris-HCl (pH 8.0) containing 0.5 mg/ml BSA, in a total volume of 200 µl. Ten µl of 2 mM Boc-E(OBzl)GR-MCA was then added, and the reaction mixture was further incubated for 30 min. The reaction was terminated by adding 0.79 ml of 0.6 M acetic acid, and the fluorescence derived from 7-amino-4-methylcoumarin was measured at 440 nm with excitation at 380 nm by a Hitachi 650 10-M fluorescence spectrophotometer. One unit of the enzyme activity was defined as the amount that liberated 1 nmol of 7-amino-4-methylcoumarin/min.

Estimation of Native Molecular Mass of Factor G

Two hundred thirty µg of partially purified factor G was applied to a Superose 6 HR 10/30 column equilibrated with 20 mM Tris-HCl (pH 8.0) containing 0.15 M NaCl. The elution was carried out with the same buffer at a flow rate of 0.4 ml/min using a fast protein liquid chromatography system (Pharmacia). The molecular mass was estimated from the retention time of blue dextran 2000, ovalbumin, and chymotrypsinogen A eluted under the same conditions.

Activation of Factor G by Curdlan and Various Glucans

Various concentrations of curdlan or glucans were incubated with the indicated concentration of factor G at 37 °C for 20 min in 0.1 M Tris-HCl (pH 8.0) containing 0.5 mg/ml BSA. Amidase activity was measured using Boc-E(OBzl)GR-MCA as described above. The percentage of maximal activity at each concentration of factor G was determined.

Time Course for Activation of Zymogen Factor G

A reaction mixture containing 3.1 µg/ml factor G and 125 ng/ml curdlan in 0.1 M Tris-HCl (pH 8.0) was incubated at 37 °C. Amidase activity was then measured in aliquots removed at the indicated times. Simultaneously, an aliquot containing 1.2 µg of the protein was precipitated with 10% trichloroacetic acid and subjected to 12.5% SDS-polyacrylamide gel electrophoresis according to Laemmli's method (26) .

Substrate Specificity of Factor G

Factor G (0.31 µg/ml, 2.8 nM) was activated by 125 ng/ml curdlan at 37 °C for 20 min in 200 µl of 0.1 M Tris-HCl (pH 8.0) containing 0.5 mg/ml BSA. Then, 8 µl of varying concentrations of Boc-E(OBzl)-GR-MCA was added to the mixture. After incubation for 5 min, 792 µl of 0.6 M acetic acid was added, and the fluorescence was measured. k and K(m) for the substrate were calculated by the direct linear plotting(27) . k/K(m) values for other substrates were obtained by measuring the initial velocity of factor G (0.43 µg/ml, 3.77 nM) prepared as above.

Effect of Various Inhibitors on the Activity of Factor G

Factor G was preincubated in the presence and absence of inhibitor in 200 µl of 0.1 M Tris-HCl (pH 8.0) containing 0.5 mg/ml BSA at 37 °C for 5 min. Amidase activity was then measured as described above.

Reconstitution of the Glucan-mediated Pathway

A series of reaction mixtures containing factor G (0.31 µg/ml), proclotting enzyme (16.2 µg/ml), or both was incubated with or without 125 ng/ml curdlan at 37 °C for 15 min in 200 µl of 0.1 M Tris-HCl (pH 8.0). Then, 50 µl of 2 mM Boc-LGR-pNA was added and incubated further at 37 °C for 5 min. The reaction was terminated by the addition of 790 µl of 0.6 M acetic acid, and the absorbance at 405 nm was measured.

Reconstitution of the Gel Formation

A series of reaction mixtures containing a combination of curdlan (100 ng/ml), factor G (0.16 µg/ml), factor C (0.075 µg/ml), proclotting enzyme (1.62 µg/ml), and/or partially purified factor B (1.6 µg/ml) was incubated with coagulogen (0.78 mg/ml) at 37 °C for 60 min in 400 µl of 0.1 M Tris-HCl (pH 8.0). Then, 1 ml of saline was added, and turbidity was measured spectrophotometrically at 360 nm as described(20) .

Amino Acid Analysis and Sequencing

Protein concentration of the purified factor G was obtained from amino acid analysis on 24-h hydrolysates with 6 M HCl at 110 °C. Amino acid analysis was performed on a Hitachi L-8500 automatic analyzer. The amino-terminal sequence of the 55-kDa fragment derived from subunit alpha was analyzed by the method of Matsudaira (28) using an Applied Biosystems 477A protein sequencer(11) .


RESULTS

Purification of the Zymogen Factor G

Because the amidase activity of activated factor G is unstable, it was purified in its stable zymogen form; potential activating factors such as Sephadex-based matrix or dialysis tubing were avoided during the purification process. During the first purification step on dextran sulfate-Sepharose CL-6B, as shown in Fig. 1A, factor G activity was eluted with 0.25 M NaCl, thereby separating it from the horseshoe crab coagulation factors proclotting enzyme(12) , factor B(10) , and factor C(8) . Factor G was then separated from the major clottable protein coagulogen by applying this fraction to ConA-Sepharose (Fig. 1B) and eluting bound factor G with methyl-alpha-D-glucoside. Size fractionation using Sephacryl S-200 HR at pH 6.5 (Fig. 1C) separated factor G from the majority of the remaining contaminating proteins, which were eluted in earlier fractions. Interestingly, the factor G activity was detected near the column volume. This unusual elution behavior of factor G was not observed at pH 8.0 (data not shown, see discussion).


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-alpha-D-glucoside (alpha-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 alpha (the 72 kDa-subunit) and beta (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).

Activation of the Zymogen Factor G by Various Glucans

Factor G was activated by a wide variety of glucans containing (13)-beta linkages (Table 3) but was insensitive to smaller oligosaccharides as well as LPS, steroids, and a variety of steroid sulfates (not shown). Curdlan, a linear (13)-beta-D-glucan from the soil bacterium Alcaligenes faecalis var. myxogenes, was the most effective activator among the glucans examined, with an ED of 4 times 10 g/ml; mannan, galactan, and xylan were at least 10,000 times less active. Carboxymethylation of curdlan essentially eliminated its ability to activate factor G.



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 alpha and the 37-kDa subunit beta 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 alpha(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 beta was converted to a 34-kDa fragment during activation. Under nonreducing conditions, the activated subunit beta 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 alpha and beta, 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 alpha-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).

Specificity of Active Factor G toward Synthetic Substrates

The activated factor G, factor G, showed amidase activity against many small synthetic substrates with different kinetics, as shown in Table 4. Boc-E(OBzl)GR-MCA, which was usually used in the purification procedure, was the best among 50 other peptide substrates tested (data not shown). Its K(m) and k were 470 µM and 67.3 s, respectively. This amidase activity was pH- and temperature-dependent, with an optimum at around pH 7.5 and 40 °C, respectively (data not shown). Factor G was unstable and quickly inactivated at temperatures greater than 40 °C.



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. (^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, alpha(2)-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 (alpha-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-beta-D-galactopyranoside, p-nitrophenyl-N-acetyl-beta-D-glucosaminide, p-nitrophenyl-beta-cellobioside, p-nitrophenyl-beta-D-glucoside, and p-nitrophenyl-tri-N-acetyl-beta-chitotrioside were also without effect.



Reconstitution of the beta-D-Glucan-mediated Coagulation Pathway

We reported in 1981 that (13)-beta-D-glucan-sensitive protease zymogen(s) caused hemolymph coagulation through the activation of proclotting enzyme, which is a component of the LPS-mediated pathway(20) . However, it remained to be determined whether factor G directly activated proclotting enzyme or other factor(s) acted as intermediates. We used purified factor G and proclotting enzyme to examine this issue, incubating them in the presence or absence of curdlan. Proclotting enzyme had no amidase activity in the presence or absence of curdlan, and factor G had negligible activity toward this substrate (Table 6). When proclotting enzyme was incubated with factor G in the presence of curdlan, substantial amidase activity was observed, but in the absence of curdlan, the activity was minimal. These results indicate that factor G is intrinsically capable of activating proclotting enzyme. In another set of experiments, (13)-beta-D-glucan-mediated gelation was reconstituted with factor G, proclotting enzyme, and coagulogen (Table 7, experiments 3 and 4). When proclotting enzyme or factor G was omitted from the incubation mixture, no gelation was observed, indicating that both of the components are essential for (13)-beta-D-glucan-mediated gelation (experiments 5 and 6).






DISCUSSION

Recently, the presence of (13)-beta-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 (13)-beta-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 alpha contains a beta-1,3-glucanase A1-like domain, three xylanase A-like domains, and two xylanase Z-like domains, and subunit beta is a serine protease zymogen.

Although subunit alpha 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 beta-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)-beta-D-glucans, such as curdlan and paramylon (Table 3). Branching of the linear chain with (14)-beta or (16)-beta 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 (13)-beta-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 (13)-beta-D-glucan but not native (13)-beta-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)-beta-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 beta-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 alpha and beta. 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 alpha 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 beta 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 beta 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 (13)-beta-D-glucan.

These characteristics of factor G allow us to speculate as to the activation mechanism of factor G. In the zymogen form, subunit alpha sterically hinders the activation site of subunit beta; beta-glucan binding to subunit alpha then exposes the active site of subunit beta, allowing autocatalytic activation through intermolecular interaction between subunit betas. The active subunit beta then quickly hydrolyzes the Arg-Glu bond in subunit alpha. Then, another site in the same subunit is cleaved, which is followed by the inactivation of the protease activity. Since this cleavage in subunit alpha appears to reduce the amidolytic activity, subunit alpha is likely to regulate the catalytic activity of the subunit beta.

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 beta. The requirement for lower pH may result from its stabilization of subunit interaction or from reduction of the endogenous amidase activity in subunit beta. Using the cDNAs for both subunits alpha and beta, 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)-beta-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 (13)-beta-D-glucans based on this novel heterodimeric serine protease zymogen may be relevant for the early and sensitive diagnosis of fungal sepsis. Furthermore, this (13)-beta-D-glucan-sensitive factor G may be useful as a unique tool to analyze other biological reactions stimulated by the glucans.


FOOTNOTES

*
This work was supported by a grant-in- aid for scientific research from the Ministry of Education, Science, and Culture of Japan. 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.

§
Present address: Dept. of Life Science, Faculty of Science, Himeji Inst. of Technology, Harima Science Park City, Kamigori, Hyogo 678-12, Japan.

To whom correspondence should be addressed: Dept. of Biology, Faculty of Science, Kyushu University 33, Fukuoka 812, Japan.

(^1)
The abbreviations used are: LPS, lipopolysaccharide; LICI, limulus intracellular coagulation inhibitor; BSA, bovine serum albumin; Boc-E(OBzl)GR-MCA, t-butyloxycarbonyl--benzyl-L-glutamyl-glycyl-L-arginine 4-methyl-coumaryl-7-amide; Boc-LGR-pNA; t-butyloxycarbonyl-L-leucyl-glycyl-L-arginine p-nitroanilide; ConA, concanavalin A.

(^2)
Y. Miura, S. Kawabata, Y. Wakamiya, T. Nakamura, and S. Iwanaga, manuscript in press.


ACKNOWLEDGEMENTS

We thank Dr. T. Nakamura (Tokushima University) for his contribution to the initial stage of the present study and helpful discussion, Dr. J. Aketagawa (Seikagaku Corp.) for providing glucans, and Dr. D. Sylvestre (Sloan-Kettering Institute) for help in preparing the manuscript. We are also grateful to C. Yano for technical assistance on amino acid analyses.


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