The complement system in vertebrates plays an important role in host defense against and clearance of invading microbes, in which complement component C3 plays an essential role in the opsonization of pathogens, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. In an effort to understand the molecular activation mechanism of invertebrate C3, we isolated and characterized an ortholog of C3 (designated TtC3) from the horseshoe crab Tachypleus tridentatus. Flow cytometric analysis using an Ab against TtC3 revealed that the horseshoe crab complement system opsonizes both Gram-negative and Gram-positive bacteria. Evaluation of the ability of various pathogen-associated molecular patterns to promote the proteolytic conversion of TtC3 to TtC3b in hemocyanin-depleted plasma indicated that LPS, but not zymosan, peptidoglycan, or laminarin, strongly induces this conversion, highlighting the selective response of the complement system to LPS stimulation. Although originally characterized as an LPS-sensitive initiator of hemolymph coagulation stored within hemocytes, we identified factor C in hemolymph plasma. An anti-factor C Ab inhibited various LPS-induced phenomena, including plasma amidase activity, the proteolytic activation of TtC3, and the deposition of TtC3b on the surface of Gram-negative bacteria. Moreover, activated factor C present on the surface of Gram-negative bacteria directly catalyzed the proteolytic conversion of the purified TtC3, thereby promoting TtC3b deposition. We conclude that factor C acts as an LPS-responsive C3 convertase on the surface of invading Gram-negative bacteria in the initial phase of horseshoe crab complement activation.

Multicellular organisms are endowed with a variety of host defense systems directed against invading microbes. The complement system in vertebrates is a well-characterized innate immune system of over thirty identified components that play roles ranging from the modulation of local inflammatory responses to the promotion of phagocytosis and microbial lysis (1). The vertebrate complement system consists of three activation pathways: the classical, lectin, and alternative pathways (2, 3). The classical and the lectin pathways are serine protease cascades initiated by the recognition of microbes by Abs and lectins, respectively. Although the alternative pathway can be activated spontaneously, it is potently activated upon microbial infection. Activation of each of the three pathways results in generation of a proteolytic complex known as the C3 convertase (C4bC2a in the classical and lectin pathways, or C3bBb in the alternative pathway) that plays a central role in promoting downstream responses. The C3 convertase cleaves C3 to generate fragment C3a, a peptide also known as anaphylatoxin that attracts inflammatory cells to sites of infection. The C3 convertase concomitantly generates C3b, a large polypeptide that covalently attaches to microbial surfaces and promotes C3 receptor-dependent phagocytosis by leukocytes and formation of the membrane attack complex.

Vertebrate complement factors D (Df)5 and B (Bf) are involved in the activation of the alternative pathway. Df is a single-chain serine protease circulating in the blood as an active protease that cleaves Bf, resulting in the formation of C3bBb. The indispensability of Df in initiating the alternative pathway is demonstrated by Df-deficient mice (4). Although several homologues of vertebrate complement factors are known to be present in ascidians, cyclostomes, and echinoderms, the functional analog of Df that triggers the alternative pathway has not been identified in lower deuterostomes (5, 6).

We have previously demonstrated the central role that hemocytes play in the innate immune system of the horseshoe crab Tachypleus tridentatus (7, 8, 9). Granular hemocytes, which constitute 99% of all hemocytes, store a variety of defense molecules, including four serine protease zymogens of coagulation cascade (factor C, factor G, factor B, and proclotting enzyme), the clottable protein coagulogen, several serine protease inhibitors, antimicrobial peptides, and lectins. In response to stimulation by LPS, these defense molecules are rapidly secreted from hemocytes via a heterotrimeric G-protein-dependent exocytic pathway that critically depends on the proteolytic activity of activated factor C (10, 11, 12). In addition to promoting the rapid release of defense molecules from hemocytes, factor C is autocatalytically activated by LPS (or by its essential component, lipid A) in the initiation of hemolymph coagulation. LPS-activated factor C activates coagulation factor B, which in turn converts the proclotting enzyme into the clotting enzyme. The clotting enzyme then promotes the proteolytic conversion of coagulogen to coagulin, which spontaneously forms an insoluble polymer. In this manner, factor C serves to couple the recognition of pathogen-associated molecular patterns (PAMPs), such as LPS, to the formation of a physical barrier at the site of microbial invasion (7, 8).

Several hemolymph plasma proteins from T. tridentatus and the American horseshoe crab Limulus polyphemus have been identified and functionally characterized (13), including hemocyanin (14, 15), tachylectin-5 (16, 17), C-reactive proteins (18), and α2-macroglobulin (19). Recently, homologues of complement components C3 and C2/Bf have been identified in the South Asian horseshoe crab Carcinoscorpius rotundicauda (designated CrC3 and CrC2/Bf, respectively), indicating the presence, in primitive protostomes, of a complement system capable of promoting the phagocytosis of invading microbes (20). CrC3 shows the highest similarity to C3 sequences of the lower deuterostomes and forms a clade with amphioxus C3, cnidaria C3-like protein, and sea urchin C3 in phylogenetic tree of thioester-containing proteins identified in vertebrates and invertebrates (20). However, despite the identification of key complement components in invertebrates, the molecular mechanism underlying C3 activation has remained unknown.

In an effort to elucidate the mechanism of C3 activation in arthropods, we have isolated and characterized an ortholog of C3 (designated TtC3) from T. tridentatus. We observed that LPS is one of the most effective PAMPs to activate the horseshoe crab complement system, leading to the deposition of TtC3b on the surface of Gram-negative bacteria. Further, we provide evidence that factor C acts as an LPS-responsive C3 convertase in the initial phase of complement activation.

The horseshoe crabs T. tridentatus were collected in Hakata Bay and maintained in tanks with aerated with running seawater at 23°C. Factor C was purified as described previously (21). Benzamidine, laminarin, and lipoteichoic acid were from Sigma-Aldrich. LPS (Salmonella minnesota R595) was from List Biological Laboratories. O-saccharides of LPS are not necessary to activate factor C and therefore, the rough LPS from S. minnesota R595 was used in this study (22). Soluble peptidoglycan was the gift of Dr. Bok Luel Lee (Pusan National University, Korea) (23). Micro bicinchoninic acid protein assay reagent kit was from Pierce. Polyclonal Abs against factor C and the proclotting enzyme, mAbs against factor C (2C12 and 2B7), coagulogen (14E7), and factor G (K5574) were prepared previously (24).

The degenerate nucleotide sequences of the primers used for RT-PCR with cDNA of the hepatopancreas or muscle as a template were based on the amino acid sequences of C. rotundicauda CrC3 (20). The conditions used for RT-PCR and 5′- or 3′-RACE analysis were as described previously (25). The obtained ortholog for CrC3 was designated as TtC3.

Hemolymph plasma was collected from a single animal into a sterilized plastic tube and centrifuged at 50 × g for 10 min to remove hemocytes. The resulting plasma was further centrifuged at 100,000 × g for 4 h to remove hemocyanin and dialyzed against 20 mM Tris-HCl (pH 7.0) containing 0.15 M NaCl. For complement activation, HDP was supplemented with 50 mM MgCl2 and 10 mM CaCl2, equivalent to the concentrations in horseshoe crab hemolymph plasma (26). The protein concentration of HDP was ∼2.5 mg/ml and at least 95% of hemocyanin (∼80 mg/ml in hemolymph plasma) was removed from hemolymph plasma by the centrifugation.

HDP was diluted 5-fold with 20 mM Tris-HCl (pH 7.0) containing 1 mM benzamidine. The diluted sample was applied to an S-Sepharose fast flow column (2.6 × 17.5 cm) equilibrated with the same buffer containing 0.05 M NaCl. After washing with 300 ml of the same buffer, elution was conducted with a linear NaCl gradient of 0.05 to 0.5 M. Aliquots of every fifth fraction were subjected to SDS-PAGE, electroblotting onto a polyvinylidene fluoride (PVDF) membrane, and N-terminal amino acid sequence analysis.

Purified TtC3 was subjected to SDS-PAGE under the reducing conditions, and stained using negative staining. The protein band of the α-chain was cut out from the gel and recovered by electroelution for immunization of rabbits (Asahi Techno Glass Company). A polyclonal Ab (anti-TtC3α Ab) was purified sequentially from the anti-serum by using protein A-Sepharose and Ag-conjugated Affigel-10 (Bio-Rad). A fraction of the purified Ab preparation was fluorescently labeled with an AlexaFluor 488 Protein Labeling Kit (Invitrogen).

Escherichia coli K12 and Staphylococcus aureus P209 were incubated with hemolymph plasma at 37°C for 30 min. After being washed with 10 mM sodium phosphate (pH 7.0) containing 0.1% SDS and then with the same buffer without SDS, the microorganisms were incubated with the Alexa 488-conjugated anti-TtC3α Ab at 37°C for 30 min. After washing with the buffer, the labeled microorganisms were analyzed by a flow cytometer (BD Biosciences).

SDS-PAGE was performed in 8 or 10% slab gel, according to Laemmli (27). Western blotting was performed by standard procedures. Precision Plus Protein Standards (Bio-Rad) were used for determination of apparent molecular masses. Samples were subjected to SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% milk, the membrane was incubated with primary Ab (1.0 μg/ml), and then with 5,000-fold diluted HRP-conjugated goat anti-rabbit IgG (Bio-Rad), followed by development with Chemi-lumi One (Nacalai Tesque).

Microtiter plates were coated with the anti-factor C Ab 2C12 (2 μg/ml, 50 μl) by incubating at 37°C for 1 h. After washing with 20 mM Tris-HCl (pH 7.0) containing 0.15 M NaCl, the plates were blocked with 5% milk in the same buffer, and 2-fold serial dilutions (50 μl each) of HDP or purified factor C for making a standard curve were added, incubated at 37°C for 1 h, and then washed. The anti-factor C polyclonal Ab (2 μg/ml, 50 μl) was added, incubated at 37°C for 1 h, and washed. The 200-fold diluted secondary Ab was added and incubated at 37°C for 1 h. The enzyme activity of HRP was detected with O-phenylenediamine at 490 nm, using a microplate reader, model 3550 (Bio-Rad).

Hemolymph plasma was incubated with 1.0 μg/ml of selected PAMPs at 37°C for 20 min in a total volume of 55 μl. The sample was then mixed with 5 μl of various 4-methylcoumaryl-7-amide (MCA) substrates (the final concentration of 0.83 mM), and further incubated at 37°C for 5 min, after which reactions were terminated by addition of 940 μl of 5% acetic acid. The fluorescence of 7-amino-4-methylcoumarin (AMC) was measured at an excitation of 380 nm and emission of 440 nm. The following substrates were used: N-tert-butoxycarbonyl (Boc)-V-P-R-MCA and Boc-E(OBzl)-G-R-MCA for the activated factor C, Boc-M-T-R-MCA for the activated coagulation factor B, and Z-L-G-R-MCA for the clotting enzyme (21).

Proteins were subjected to SDS-PAGE and transferred to PVDF membrane. After staining with Coomassie Brilliant Blue R-250, protein bands of interest were excised and subjected to amino acid sequence analysis using an Applied Biosystems Procise 491-HT gas-phase protein sequencer.

The interaction of factor C with TtC3 was examined using the 27-MHz quartz-crystal microbalance, Affinix Q (Initium). TtC3 (0.1 mg/ml) was immobilized onto 27-MHz electrodes in 20 mM Tris-HCl (pH 7.5) containing 0.15 M NaCl. The electrodes were dried at 25°C, washed with water several times for removal of the excess TtC3, and then soaked in the same buffer, and monitored continuously for frequency changes at 25°C. The frequency changes (ΔF) in response to the various concentrations of factor C in the same buffer were assessed. The dissociation constant of factor C against TtC3 was determined by the published method (28).

α-Chymotrypsin directly converts factor C to the active protease through the cleavage of a specific Phe-Ile bond in the absence of LPS (29). To avoid contamination with LPS, factor C was activated by α-chymotrypsin, and then the remaining α-chymotrypsin activity was inactivated by 1.0 μM Ala-Ala-Phe-chloromethylketone at 37°C for 10 min. To prepare an inactivated factor C without the proteolytic activity, the chymotrypsin-activated factor C was treated with 10 μM D-Phe-Pro-Arg-chloromethylketone (PPACK) at 37°C for 10 min.

We identified an ortholog of C3 in T. tridentatus, which we named TtC3 (Fig. 1 and Supplemental Data Fig. S1,6 and GenBank database accession no. AB353279). TtC3 consisted of 1,716 amino acid residues with the identical domain architecture to vertebrate C3, including α2-macroglobulin domains, complement-urchin-bone domains, a thioester-containing domain, an anaphylatoxin domain, and a C345C domain. A thioester motif (-C1004-G1005-E1006-Q1007-) and a catalytic His residue (H1116) were conserved in the deduced amino acid sequence. TtC3 had a high sequence identity with CrC3 (97.7%).

FIGURE 1.

Schematic representation of the structural organizaion of TtC3. The amino acid sequences of the α-, β-, and γ-chains and the sequence encompassing the site of cleavage by horseshoe crab convertase within the α-chain of TtC3 are indicated.

FIGURE 1.

Schematic representation of the structural organizaion of TtC3. The amino acid sequences of the α-, β-, and γ-chains and the sequence encompassing the site of cleavage by horseshoe crab convertase within the α-chain of TtC3 are indicated.

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TtC3 was purified from HDP by ion-exchange column chromatography on an S-Sepharose column (Fig. 2,A). The second peak contained a protein with an apparent molecular mass of 185 kDa on nonreducing SDS-PAGE that gave rise to three bands under reducing conditions (Fig. 2 B). Polypeptides corresponding to the 80, 70, and 35 kDa bands were respectively identified as α-, β-, and γ-chains of TtC3 by the N-terminal sequence analysis: E649-I650-M651- for the α-chain, A1-N2-I3-F4-V5- for the β-chain, and D1410-N1411-R1412- for the γ-chain. The αβ and αγ processing sites of TtC3 contained the cleavage motif of -R-X-K/R-R-, which are recognized by Kex2-like family of serine proteases, a member of which has been identified previously in T. tridentatus (30). Therefore, cleavage at these processing sites may occur during the protein maturation rather than as the result of nonphysiological cleavages during the purification, suggesting that TtC3 forms a three-chain structure, unlike vertebrate C3 which is present in a two-chain form (31). Approximately 70 mg of TtC3 was purified from 200 ml of HDP, indicating that TtC3 was present in hemolymph plasma at a concentration of > ∼0.3 mg/ml.

FIGURE 2.

Purification of TtC3. A, Diluted HDP was applied to an S-Sepharose column, and after washing, proteins were eluted by a linear NaCl gradient and collected (10 ml/tube). The fractions indicated with the solid bar were pooled. B, Purified TtC3 was subjected to SDS-PAGE under reducing (lane 1) and nonreducing (lane 2) conditions.

FIGURE 2.

Purification of TtC3. A, Diluted HDP was applied to an S-Sepharose column, and after washing, proteins were eluted by a linear NaCl gradient and collected (10 ml/tube). The fractions indicated with the solid bar were pooled. B, Purified TtC3 was subjected to SDS-PAGE under reducing (lane 1) and nonreducing (lane 2) conditions.

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The complement system in vertebrates is activated by PAMPs, such as LPS of Gram-negative bacteria, and zymosan of fungi. To evaluate whether PAMPs trigger activation of the horseshoe crab complement system, HDP was incubated with LPS, peptidoglycan, laminarin, and zymosan. To chase the conversion of TtC3 to TtC3b, a polyclonal Ab against the α-chain was prepared. The anti-TtC3α Ab specifically recognized the α-chain of TtC3 in HDP (data not shown). The proteolytic conversion of TtC3 to TtC3b was evaluated by Western blotting. In this experiment, hemolymph plasma was not used, because a large amount of hemocyanin subunits with apparent molecular mass of 70 kDa interrupted the detection of the α-chain and its fragment of TtC3b by Western blotting. Among the PAMPs examined, LPS effectively triggered the proteolytic conversion of TtC3 (Fig. 3). The N-terminal sequence of the fragment of 72 kDa (α′) produced in the LPS-induced complement activation was determined to be G722-R723-F724-G725-, indicating that horseshoe crab C3 convertase cleaves at the R721-G722 bond (Fig. 1).

FIGURE 3.

PAMP-induced complement activation in HDP. HDP was incubated with various PAMPs (1.0 μg/ml), including LPS, peptidoglycan (PGN), laminarin (LAM), and zymosan (ZYM) at 37°C for 30 min. Samples were analyzed by Western blotting with the anti-TtC3α Ab, and complement activation was evaluated by the production of the α′-chain.

FIGURE 3.

PAMP-induced complement activation in HDP. HDP was incubated with various PAMPs (1.0 μg/ml), including LPS, peptidoglycan (PGN), laminarin (LAM), and zymosan (ZYM) at 37°C for 30 min. Samples were analyzed by Western blotting with the anti-TtC3α Ab, and complement activation was evaluated by the production of the α′-chain.

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Opsonization by C3b is one of the most important responses of the vertebrate complement system to invading microbes. To evaluate the opsonization ability of the horseshoe crab complement system, E. coli and S. aureus were mixed with hemolymph plasma, and the deposition of TtC3b was quantified by flow cytometric analysis, using the Alexa 488-conjugated anti-TtC3α Ab (Fig. 4, C and D). The fluorescence-conjugated Ab did not show the cross-reactivity against the surface substances on E. coli (Fig. 4,A) and S. aureus (Fig. 4,B). The deposition of TtC3b on the surface of E. coli was relatively homogeneous, but showed a bimodal distribution on the surface of S. aureus. It appears that TtC3b binds uniformly to substances on the surface of E. coli. At present, the reason for this discrepancy is unclear. Pretreatment of hemolymph plasma with hydroxylamine to inactivate the thioester bond on TtC3 dramatically decreased the deposition of TtC3b on the microbes, indicating the importance of covalent cross-linking in the opsonization of microbes (dotted lines in Fig. 4, C and D).

FIGURE 4.

Deposition of TtC3b on microbial surfaces. To check the cross-reactivity of the anti-TtC3α Ab against the surface substances on bacteria, E. coli (A) and S. aureus (B) were incubated with the fluorescence-conjugated anti-TtC3α Ab in the absence of hemolymph plasma (dotted line). The solid line (A and B) indicates a negative control in which bacteria were incubated with buffer (autonomous fluorescence of bacteria). Then, E. coli (C) and S. aureus (D) were incubated with hemolymph plasma, and the deposition of TtC3b on microbial surfaces was analyzed by flow cytometry using the Alexa-conjugated anti-TtC3α Ab (solid line). Alternatively, hemolymph plasma was incubated with hydroxylamine (0.2M) at 4°C for 1 h to inactivate the reactive thioester bond of TtC3 before incubation with bacteria (dotted line). The gray line (C and D) indicates a negative control in which bacteria were incubated with buffer instead of plasma.

FIGURE 4.

Deposition of TtC3b on microbial surfaces. To check the cross-reactivity of the anti-TtC3α Ab against the surface substances on bacteria, E. coli (A) and S. aureus (B) were incubated with the fluorescence-conjugated anti-TtC3α Ab in the absence of hemolymph plasma (dotted line). The solid line (A and B) indicates a negative control in which bacteria were incubated with buffer (autonomous fluorescence of bacteria). Then, E. coli (C) and S. aureus (D) were incubated with hemolymph plasma, and the deposition of TtC3b on microbial surfaces was analyzed by flow cytometry using the Alexa-conjugated anti-TtC3α Ab (solid line). Alternatively, hemolymph plasma was incubated with hydroxylamine (0.2M) at 4°C for 1 h to inactivate the reactive thioester bond of TtC3 before incubation with bacteria (dotted line). The gray line (C and D) indicates a negative control in which bacteria were incubated with buffer instead of plasma.

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Horseshoe crab hemocytes secrete factor C, an LPS-sensing serine protease zymogen, by LPS-induced exocytosis, leading to the initiation of the hemolymph coagulation system (7, 8). Factor C is also localized on the surface of hemocytes and acts as the LPS sensor (12, 32). Interestingly, factor C contains tandem-repeated complement control protein domains similar to those found in vertebrate complement factors, as shown in Fig. 5,A (33), a structural feature that prompted us to hypothesize that factor C could mediate LPS-induced complement activation. To determine whether factor C is present in hemolymph plasma, we performed Western blotting on a variety of tissue samples using a mAb (2C12) directed against the H chain of factor C (Fig. 5,B). Protein bands corresponding to the single-chain form of factor C (123 kDa) and the H chain of the two-chain form of factor C (80 kDa) were detected for the sample from hemolymph plasma (Fig. 5,B, lane 2). The two-chain form of factor C was detected in all tissues examined, including muscle, heart, stomach, hepatopancreas, and intestine (Fig. 5 B, lanes 3–7). The functional difference between the two types of zymogen forms of factor C remains unknown (29). In this experiment, the relative abundance of factor C in these tissues could not be quantitatively compared because there is currently no control Ag known that could serve as an appropriate internal control. The amount of factor C in HDP was determined to be 10.7 ± 1.2 μg/ml by sandwich ELISA (data not shown). In contrast, Ags of coagulation factor G and the proclotting enzyme were not detectable in HDP by Western blotting (data not shown).

FIGURE 5.

Tissue specific localization of factor C. A, Schematic domain structures of the single-chain and the two-chain forms of factor C. Cys-rich, Cys-rich domain; CCP, complement control protein domain; EGF, epidermal growth factor domain; LCCL, Limulus factor C, Coch-5b2, and Lg11 domain; Lectin, C-type lectin domain; serine protease, serine protease domain. B, The total proteins (10 μg of each as determined by micro bicinchoninic acid assay) was prepared from the indicated tissues and subjected to Western blotting using the anti-factor C Ab 2C12. Lane 1, hemocytes; lane 2, plasma; lane 3, muscle; lane 4, heart; lane 5, stomach; lane 6, hepatopancreas; lane 7, intestine.

FIGURE 5.

Tissue specific localization of factor C. A, Schematic domain structures of the single-chain and the two-chain forms of factor C. Cys-rich, Cys-rich domain; CCP, complement control protein domain; EGF, epidermal growth factor domain; LCCL, Limulus factor C, Coch-5b2, and Lg11 domain; Lectin, C-type lectin domain; serine protease, serine protease domain. B, The total proteins (10 μg of each as determined by micro bicinchoninic acid assay) was prepared from the indicated tissues and subjected to Western blotting using the anti-factor C Ab 2C12. Lane 1, hemocytes; lane 2, plasma; lane 3, muscle; lane 4, heart; lane 5, stomach; lane 6, hepatopancreas; lane 7, intestine.

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Factor C is activated autocatalytically upon formation of a complex with LPS through its lipid A portion (22). The L chain-specific mAb (2B7) inhibited the amidase activity of factor C against Boc-V-P-R-MCA (data not shown). Preincubation of HDP with the anti-factor C Ab (2B7) dose-dependently inhibited the proteolytic conversion of TtC3 to TtC3b in the presence of LPS (Fig. 6,A, lanes 2–4). As a negative control, the anti-coagulogen Ab (14E7) had no effect on the conversion of TtC3 (Fig. 6,A, lane 5). Moreover, the Ab (2B7) inhibited the deposition of TtC3b on E. coli in hemolymph plasma (Fig. 6 B). These data clearly indicate that the proteolytic activity of factor C mediates LPS-induced complement activation. In addition, Abs against coagulation factor G (monoclonal) and the proclotting enzyme (polyclonal) did not inhibit the deposition of TtC3b on E. coli, suggesting that these coagulation factors are not involved in LPS-induced complement activation (data not shown). In contrast, the Ab (2B7) exhibited no inhibitory effect on the deposition of TtC3b on S. aureus under the same conditions, raising the possibility that a factor C-independent pathway is responsible for opsonization of Gram-positive bacteria (data not shown).

FIGURE 6.

Factor C is involved in LPS-induced complement activation. A, HDP was preincubated with the anti-factor C Ab (2B7) (0, 10, or 50 μg/ml) or the anti-coagulogen Ab (14E7) (50 μg/ml) at 4°C for 1 h. Samples were then incubated with 1 μg/ml LPS at 37°C for 30 min (lanes 2–5), and subjected to Western blotting using the anti-TtC3α Ab. Complement activation was evaluated by production of the α′-chain. Anti-FC, anti-factor C Ab (2B7); Anti-Coagulogen, anti-coagulogen Ab (14E7). B, E. coli was incubated with hemolymph plasma, and the deposition of TtC3b on the microbial surface was analyzed by flow cytometry with the anti-TtC3α Ab (solid line). The dotted line indicates flow cytometric data obtained following pretreatment of hemolymph plasma with the anti-factor C Ab (2B7) at 10 μg/ml for 30 min at 4°C. The gray line indicates the negative control in which bacteria were incubated with buffer instead of hemolymph plasma.

FIGURE 6.

Factor C is involved in LPS-induced complement activation. A, HDP was preincubated with the anti-factor C Ab (2B7) (0, 10, or 50 μg/ml) or the anti-coagulogen Ab (14E7) (50 μg/ml) at 4°C for 1 h. Samples were then incubated with 1 μg/ml LPS at 37°C for 30 min (lanes 2–5), and subjected to Western blotting using the anti-TtC3α Ab. Complement activation was evaluated by production of the α′-chain. Anti-FC, anti-factor C Ab (2B7); Anti-Coagulogen, anti-coagulogen Ab (14E7). B, E. coli was incubated with hemolymph plasma, and the deposition of TtC3b on the microbial surface was analyzed by flow cytometry with the anti-TtC3α Ab (solid line). The dotted line indicates flow cytometric data obtained following pretreatment of hemolymph plasma with the anti-factor C Ab (2B7) at 10 μg/ml for 30 min at 4°C. The gray line indicates the negative control in which bacteria were incubated with buffer instead of hemolymph plasma.

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Amidase activities in the PAMPs-treated hemolymph plasma were measured using synthetic peptide substrates for activated factor C, Boc-V-P-R-MCA and Boc-E(OBzl)-G-R-MCA. Only LPS induced these amidase activities in hemolymph plasma, whereas other PAMPs, such as lipoteichoic acid, peptidoglycan, and laminarin had no effect on hydrolysis of these peptide substrates (Fig. 7,A). The LPS-induced amidase activities toward these substrates were diminished by the preincubation of hemolymph plasma with the anti-factor C Ab (2B7) in a dose-dependent manner (Fig. 7 B).

FIGURE 7.

Amidase activities in PAMP-treated hemolymph plasma. A, Hemolymph plasma was incubated with various PAMPs (1.0 μg/ml), including LPS, lipoteichoic acid (LTA), peptidoglycan (PGN), and laminarin (LAM) at 37°C for 20 min. B, Hemolymph plasma was preincubated with the anti-factor C Ab (2B7) (0, 10, or 50 μg/ml) at 4°C for 1 h, followed by incubation with 1 μg/ml LPS at 37°C for 20 min. The resulting amidase activities in A and B were analyzed, using Boc-V-P-R-MCA (▪) and Boc-E(OBzl)-G-R-MCA (□) as described in Materials and Methods.

FIGURE 7.

Amidase activities in PAMP-treated hemolymph plasma. A, Hemolymph plasma was incubated with various PAMPs (1.0 μg/ml), including LPS, lipoteichoic acid (LTA), peptidoglycan (PGN), and laminarin (LAM) at 37°C for 20 min. B, Hemolymph plasma was preincubated with the anti-factor C Ab (2B7) (0, 10, or 50 μg/ml) at 4°C for 1 h, followed by incubation with 1 μg/ml LPS at 37°C for 20 min. The resulting amidase activities in A and B were analyzed, using Boc-V-P-R-MCA (▪) and Boc-E(OBzl)-G-R-MCA (□) as described in Materials and Methods.

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The sequence around the cleavage site of TtC3 by C3 convertase (-E719-G720-R721↓-G722-) was consistent with the substrate specificity of activated factor C (21). To test whether factor C directly cleaves TtC3, both purified components were incubated together in the presence of LPS. As expected, activated factor C directly cleaved TtC3 (Fig. 8,A). To determine whether factor C on the surface of Gram-negative bacteria could induce the deposition of TtC3b, E. coli was coated with purified factor C and the binding of factor C on the surface of E. coli was confirmed by flow cytometry (data not shown). Then, factor C-coated E. coli was incubated with purified TtC3. Flow cytometric analysis showed that the factor C-coated E. coli enhance the deposition of TtC3b on the bacteria (solid line in Fig. 8,B). Interestingly, the incubation of TtC3 with noncoated E. coli (dotted line in Fig. 8,B) did not reduce the signal to that to the buffer-only baseline in noncoated E. coli (gray line in Fig. 8,B), suggesting autoactivation activity of TtC3. An inactivated factor C (PPACK-factor C) without the proteolytic activity was prepared as described under Materials and Methods. PPACK-factor C was incubated with E. coli. The resulting PPACK-factor C-coated E. coli did not enhance the deposition of TtC3b (dotted line in Fig. 8,C), which corresponded to the opsonization level with noncoated E. coli (dotted line in Fig. 8 B), indicating that the proteolytic activity of factor C on the surface of E. coli is essential for the deposition of TtC3b.

FIGURE 8.

Direct conversion of TtC3 to TtC3b by activated factor C. A, Purified TtC3 (50 μg/ml) was incubated with factor C (10 μg/ml) at 37°C for the indicated times in the presence of LPS (1.0 μg/ml). Following incubation, samples were subjected to Western blotting using the anti-TtC3α Ab. B, The factor C-coated (solid line) or noncoated (dotted line) E. coli was incubated with purified TtC3 (300 μg/ml) at 37°C for 30 min and then washed with the buffer containing 0.1% SDS. The deposition was analyzed by flow cytometry with an anti-TtC3α Ab. A gray line indicates the negative control in which bacteria were incubated with buffer instead of purified TtC3. C, E. coli was coated with PPACK-factor C (20 μg/ml), and the resulting PPACK-factor C-coated E. coli was incubated with the purified TtC3 (dotted line). The solid line indicates a positive control in which the factor C-coated bacteria were incubated with TtC3, as shown in Fig. 8 B.

FIGURE 8.

Direct conversion of TtC3 to TtC3b by activated factor C. A, Purified TtC3 (50 μg/ml) was incubated with factor C (10 μg/ml) at 37°C for the indicated times in the presence of LPS (1.0 μg/ml). Following incubation, samples were subjected to Western blotting using the anti-TtC3α Ab. B, The factor C-coated (solid line) or noncoated (dotted line) E. coli was incubated with purified TtC3 (300 μg/ml) at 37°C for 30 min and then washed with the buffer containing 0.1% SDS. The deposition was analyzed by flow cytometry with an anti-TtC3α Ab. A gray line indicates the negative control in which bacteria were incubated with buffer instead of purified TtC3. C, E. coli was coated with PPACK-factor C (20 μg/ml), and the resulting PPACK-factor C-coated E. coli was incubated with the purified TtC3 (dotted line). The solid line indicates a positive control in which the factor C-coated bacteria were incubated with TtC3, as shown in Fig. 8 B.

Close modal

Quartz-crystal microbalance analysis was conducted to examine the interaction of factor C with TtC3. The dissociation constant of factor C for TtC3 immobilized on the sensor tip was Kd = 4.9 × 10−8 M (Fig. 9). It is therefore possible that in hemolymph plasma, factor C may exist in a complex with TtC3, and that formation of this complex is a prerequisite for the immediate activation of TtC3 by factor C on the surface of Gram-negative bacteria.

FIGURE 9.

Interaction of factor C with TtC3. The dissociation constant of factor C with TtC3 was determined by quartz-crystal microbalance analysis. The double-reciprocal plot of 1/(-ΔF) against 1/[factor C] gave a straight line with an intercept on the abscissa equal to −1/Kd.

FIGURE 9.

Interaction of factor C with TtC3. The dissociation constant of factor C with TtC3 was determined by quartz-crystal microbalance analysis. The double-reciprocal plot of 1/(-ΔF) against 1/[factor C] gave a straight line with an intercept on the abscissa equal to −1/Kd.

Close modal

The flow cytometric analysis presented in this study indicates that complement components required for the deposition of TtC3b on invading microbes including Gram-negative and positive bacteria exist in hemolymph plasma (Fig. 4). In the horseshoe crab C. rotundicauda, phagocytosis of bacteria by hemocytes both in vivo and in vitro is inhibited in the presence of a mixture of protease inhibitors (20), suggesting that bacteria opsonized with TtC3b via the proteolytic activity of factor C increase hemocyte phagocytosis.

We found that factor C, an LPS-sensing serine protease zymogen originally characterized as an initiator of hemolymph coagulation, acts as an LPS-responsive C3 convertase. The anti-factor C Ab (2B7) inhibited the deposition of TtC3b on E. coli (Fig. 6 B), whereas the same Ab exhibited no effect on the deposition of TtC3b on S. aureus, suggesting the presence of a factor C-independent pathway to initiate the opsonization of Gram-positive bacteria.

LPS strongly induced the proteolytic conversion of TtC3 in HDP, but other PAMPs, such as zymosan, peptidoglycan, and laminarin had little effect on the conversion of TtC3 (Fig. 3). The innate immune responses elicited by LPS are very important for the innate immune system in horseshoe crabs. For instance, hemocytes respond only to LPS through surface-bound factor C that triggers G protein-coupled exocytosis (12, 32). The majority of microbes in the world’s oceans are Gram-negative bacteria (34). Given that the native marine environment of the horseshoe crab is abundant in Gram-negative bacteria, it is reasonable to expect that horseshoe crabs would have a developed a highly sensitive LPS-induced innate immune response. In contrast, PAMPs or complement components in hemolymph plasma required for the deposition of TtC3b on Gram-positive bacteria remain to be examined. Recently, Saux et al. showed that factor C and CrC2/Bf from C. rotundicauda interact with pathogen-recognition lectins including galactose-binding protein, Carcinolectin-5, and C-reactive protein by yeast two-hybrid and pull-down methods (35). In T. tridentatus, several pathogen-recognition lectins such as tachylectin-5 and three types of C-reactive proteins have been also identified (9, 16, 17, 18). Therefore, the protease-lectin complexes on the surface of S. aureus may enhance the deposition of TtC3b.

We have demonstrated in this study that factor C also plays a key role in LPS-induced complement activation in hemolymph plasma. Previously, we have proposed that factor C on hemocytes acts as an LPS sensor that activates a protease-activated G protein-coupled receptor leading to hemocyte exocytosis, analogous to the thrombin-thrombin receptor signaling axis in mammalian platelets (12). Horseshoe crab transglutaminase is also secreted from hemocytes in response to stimulation by LPS (36, 37). The relatively high concentration of TtC3 in hemolymph plasma (∼0.3 mg/ml) and its binding affinity to factor C (Fig. 9) suggest that TtC3 is incorporated into a coagulation plug to form transglutaminase-dependent coagulation mesh at injured sites.

Additional cross functionalities exist between the coagulation and complement systems in mammals. Notably, thrombin itself is involved in complement activation (38, 39, 40), and MASP-2, one of the complement proteases in the lectin pathway, is capable of converting prothrombin to thrombin (41). The functional interrelatedness of the clotting and complement cascades may be rooted in their common evolutionary origin. Sequence analysis of serine proteases based on codon bias and lineage-specific markers has revealed extensive overlap between phylogenetic and functional groupings (42). On this basis, factor C occupies a clade on a phylogenetic tree occupied by both vertebrate complement factors (MASP-1, MASP-2, C1r, and C1s) and certain vitamin K-dependent coagulation factors (factors VII, IX and X, and protein C), implying both functional and evolutionary linkages the complement and coagulation systems, particularly with respect to factors involved in their respective initiation phases.

In the alternative pathway of the vertebrate complement system, the interaction between Bb and C3b is essential to form C3 convertase (43, 44, 45). Although physiological roles of Bf in the horseshoe crab complement system remain unknown, horseshoe crab Bf may be responsible for the formation of the second C3 convertase. A schematic representation of the proposed by which factor C potentiates complement activation is depicted in Fig. 10. In this study, factor C is involved in the initial step of complement activation, as it recognizes LPS on Gram-negative bacteria and recruits TtC3. The resulting activated factor C localized on Gram-negative bacteria converts TtC3 to TtC3b, which is rapidly deposited on the surface of the bacterium. Whether the activated factor C can convert horseshoe crab Bf leading to the formation of a vertebrate-like C3 convertase on the surface of Gram-negative bacteria remains to be examined. In both protostomes and lower deuterostomes, the triggering protease corresponding to Df in the vertebrate alternative pathway has not yet been identified (5, 6). Saux et al. (35) postulated that CrC2/Bf could be activated by horseshoe crab factor D. However, despite the fact horseshoe crab factor D is a serine protease homologue, its essential catalytic Ser residue is substituted with a Gly residue (46), indicating that horseshoe crab factor D is catalytically impaired and thus incapable of proteolytically activating C2/Bf.

FIGURE 10.

Proposed mechanism for the activation of TtC3 by factor C localized on the surface of Gram-negative bacteria in the initial phase of the complement system. Factor C is activated on the surface of Gram-negative bacteria through the interaction with LPS via the N-terminal Cys-rich region of factor C (33 ). Activated factor C then acts as a C3 convertase, and the resulting TtC3b is rapidly deposited on the surface of Gram-negative bacteria through covalent cross-linking. Although physiological roles of horseshoe crab Bf remain unknown, horseshoe crab Bf may be responsible for the formation of the second C3 convertase on the microbes.

FIGURE 10.

Proposed mechanism for the activation of TtC3 by factor C localized on the surface of Gram-negative bacteria in the initial phase of the complement system. Factor C is activated on the surface of Gram-negative bacteria through the interaction with LPS via the N-terminal Cys-rich region of factor C (33 ). Activated factor C then acts as a C3 convertase, and the resulting TtC3b is rapidly deposited on the surface of Gram-negative bacteria through covalent cross-linking. Although physiological roles of horseshoe crab Bf remain unknown, horseshoe crab Bf may be responsible for the formation of the second C3 convertase on the microbes.

Close modal

A wide variety of factors are involved in the regulation of the complement system in vertebrates, and defects of the regulatory molecules result in immunological disorders (47, 48, 49). Several serine protease inhibitors (7), α2-macroglobulin (19), and lectins (7, 9) have been identified in T. tridentatus, and these molecules may participate in the regulation of the horseshoe crab complement system. We propose in this study that the LPS-sensing serine protease factor C acts as a C3 convertase on the surface of invading Gram-negative bacteria in the initial phase of the complement system in horseshoe crab hemolymph. Although thematically consistent with features of the coagulation and complement systems of vertebrates, these findings nonetheless elaborate a novel mode of complement activation in primitive protostomes.

We thank Noriko Ichinomiya-Sato for expert technical assistance with peptide sequencing. We also thank Drs. John Kulman (Puget Sound Blood Center, Seattle), Miki Nakao, Nozomu Okino, and Yoko Unoki (Department of Bioscience and Biotechnology, Kyushu University, Japan) for helpful discussions and suggestion on this manuscript, and Dr. Bok Luel Lee (Pusan National University, Korea) for providing soluble peptidoglycan.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Priority Area 839 to S.K. and T.F., No. 13143203 to S.K., No. 13143204 to T.F., and No. 18370045 to S.K.) and by the Naito Foundation (to S.K.).

2

The sequences reported in this article have been deposited in GenBank database (accession no. AB353279 for TtC3).

5

Abbreviations used in this paper: Df, complement factor D; Bf, complement factor B; PAMP, pathogen-associated molecular pattern; HDP, hemocyanin-depleted plasma; PVDF, polyvinylidene fluoride; MCA, 4-methylcoumaryl-7-amide; AMC, 7-amino-4-methylcoumarin; PPACK, D-Phe-Pro-Arg-chloromethylketone; Boc, N-tert-butoxycarbonyl; Bzl, benzyl.

6

The online version of this article contains supplementary material.

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