Intermolecular autocatalytic activation of serine proteasezymogen factor C through an active transition stateresponding to lipopolysaccharideReceived for publication, February 7, 2018, and in revised form, April 30, 2018 Published, Papers in Press, June 4, 2018, DOI 10.1074/jbc.RA118.002311

X Toshio Shibata‡§1, Yuki Kobayashi‡1,2, Yuto Ikeda§, and Shun-ichiro Kawabata‡§3

From the ‡Department of Biology, Faculty of Science and the §Graduate School of Systems Life Sciences, Kyushu University,Fukuoka 819-0395, Japan

Edited by George N. DeMartino

Horseshoe crab hemolymph coagulation is believed to be trig-gered by the autocatalytic activation of serine protease zymogenfactor C to the active form, �-factor C, belonging to the trypsinfamily, through an active transition state of factor C respondingto bacterial lipopolysaccharide (LPS), designated factor C*.However, the existence of factor C* is only speculative, and itsproteolytic activity has not been validated. In addition, itremains unclear whether the proteolytic cleavage of the Phe737–Ile738 bond (Phe737 site) of factor C required for the conversionto �-factor C occurs intramolecularly or intermolecularlybetween the factor C molecules. Here we show that the Phe737

site of a catalytic Ser-deficient mutant of factor C is LPS-depen-dently hydrolyzed by a Phe737 site– uncleavable mutant, clearlyindicating the existence of the active transition state of factor Cwithout cleavage of the Phe737 site. Moreover, we found the fol-lowing facts using several mutants of factor C: the autocatalyticcleavage of factor C occurs intermolecularly between factor C*molecules on the LPS surface; factor C* does not exhibit intrin-sic chymotryptic activity against the Phe737 site, but it may rec-ognize a three-dimensional structure around the cleavage site;and LPS is required not only to complete the substrate-bindingsite and oxyanion hole of factor C* by interacting with the N-ter-minal region but also to allow the Phe737 site to be cleaved byinducing a conformational change around the Phe737 site or byacting as a scaffold to induce specific protein–protein interac-tions between factor C* molecules.

The molecular mechanism underlying the proteolytic activa-tion of serine protease zymogens has been established in tryp-sinogen, which is activated by the activator enteropeptidasethrough limited proteolysis of the Arg15–Ile16 peptide bond inchymotrypsinogen numbering. The limited proteolysis inducesthe insertion of the newly appearing N-terminal Ile16 into theactivation pocket known as the Ile16 cleft to form a salt bridge

between the �-amino group of Ile16 and the �-carboxyl group ofAsp194. The salt bridge results in conformational changes in thesubstrate-binding site and the oxyanion hole to hydrolyze spe-cific peptide bonds of substrates, whereas the conformationalchanges elsewhere in the molecule, including the catalytic triadof Ser195, His57, and Asp102, are very small (1–3).

Factor C is a serine protease zymogen involved in the hemo-lymph coagulation cascade of horseshoe crabs and is autocata-lytically activated to �-factor C on bacterial LPS4 (4 –6). Theresulting �-factor C activates coagulation factor B to activatedfactor B (7, 8), which activates the proclotting enzyme to theclotting enzyme (9, 10) to convert coagulogen into coagulin gel(11). Alternatively, factor G autocatalytically activated in thepresence of �-1,3-D-glucans directly activates the proclottingenzyme to the clotting enzyme (6). Factor C is also located onhemocytes as a pattern recognition receptor for LPS, and theresulting �-factor C triggers hemocyte exocytosis through aprotease-activated G protein– coupled receptor to induce Ca2�

signaling (12). On the other hand, homologs of mammaliancomplement factors C3 and B/C2 have been identified in hemo-lymph of horseshoe crabs (13), and �-factor C also acts as thecomplement C3 convertase on microbes in close cooperationwith several lectins (14 –16).

Factor C is biosynthesized as a single-chain form of thezymogen containing six N-linked glycosylation sites, whereas atwo-chain form of the zymogen of the H and L chains producedby cleavage of the Arg665–Ser666 bond by an unknown proteaseis principally purified from hemocytes (6, 17–19) (Fig. 1). AnLPS-binding site is located in the N-terminal Cys-rich domain,and a tripeptide sequence of Arg36–Trp37–Arg38 in this domainis essential for LPS binding (20). Based on its amino acidsequence, �-factor C belongs to the trypsin family and exhibitstrypsin-like amidase activity against a peptide substrate for�-thrombin, t-butoxycarbonyl (Boc)–Val–Pro–Arg–p-nitroa-nilide (pNA), or against Boc–Val–Pro–Arg– 4-methylcou-maryl-7-amide but not against a peptide substrate forchymotrypsin, succinyl–Ala–Pro–Phe– 4-methylcoumaryl-7-amide (8). Autocatalytic activation of factor C bound to LPSoccurs through proteolytic cleavage of the Phe737–Ile738 bond

This study was supported by the Joint Research Fund of Seikagaku Corp. Theauthors declare that they have no conflicts of interest with the contents ofthis article.

1 Both authors contributed equally to this work.2 Present address: LAL Research and Development Dept., Seikagaku Corp.,

Higashiyamato, Tokyo 207-0021, Japan.3 To whom correspondence should be addressed: Dept. of Biology, Faculty of

Science, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395,Japan. Tel.: 81-92-802-4288; Fax: 81-92-802-4288; E-mail: skawascb@kyudai.jp.

4 The abbreviations used are: LPS, lipopolysaccharide; Boc, t-butoxycarbonyl;pNA, p-nitroanilide; HEK, human embryonic kidney; GnTI, N-acetylgluco-saminyltransferase I; PA, a dodecapeptide derived from human podopla-nin; tPA, tissue-type plasminogen activator.

croARTICLE

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(Phe737 site) (Fig. 1), corresponding to the Arg15–Ile16 bond inchymotrypsinogen numbering (21). It remains unknown howfactor C* displays specific chymotryptic activity against thePhe737 site despite its general trypsin-like primary substratespecificity.

Previously, we prepared a recombinant factor C with anN-glycan of Man5GlcNAc2 at each N-glycosylation site,expressed in an HEK293S mutant cell line lacking N-acetylglu-cosaminyltransferase I (GnTI�) (22). In the absence of LPS,factor C is artificially activated by chymotrypsin through cleav-age of the Phe737 site (23). Using WT factor C, we found thatchymotrypsin-activated factor C contains an additional proteo-lytic cleavage of the Tyr40–Cys41 bond in the N-terminal Cys-rich domain, designated �-factor C (Fig. 1) (22). The resulting�-factor C exhibits amidase activity against the synthetic sub-strates for �-thrombin with �70% specific activity comparedwith that of �-factor C but has neither the LPS-binding activitynor the activating activity for factor B (22). These data suggestthat LPS interaction with �-factor C is required to maintain itsproteolytic activity against factor B. Interestingly, the proteo-lytic conversion of factor B to activated factor B occurs throughcleavage of the Ile126–Ile127 bond (7); in addition, factor B is alsoan LPS-binding protein, and the LPS-bound form of factor B isessentially required for its proteolytic activation by �-factor C(8). Factor C*, however, is only speculative, and its proteolyticactivity has not been validated. Moreover, it remains unclearwhether the autocatalytic cleavage at the Phe737 site occursintramolecularly within factor C bound to LPS or intermolecu-larly between the molecules of factor C. Here we show that theautocatalytic activation of factor C is the unidentified intermo-lecular event between the factor C* molecules on the LPSsurface.

Results

Replacement of the active-site Ser941 to Ala preventsautocatalytic activation of factor C

WT factor C prepared in the HEK293S GnTI� cell line is thetwo-chain form of the zymogen consisting of the H and L chains(22). The autocatalytic activation occurs through cleavage ofthe Phe737 site of the L chain to be converted into the A and Bchains (Fig. 1) (19, 21). Therefore, the proteolytic activation of

factor C was followed by appearance of the B chain, which wasdetected by using a polyclonal antibody against the B chain. TheL chain on Western blotting was observed in a doublet becausethe fourth N-glycosylation site located in the A chain is partiallymodified (Fig. 1, open diamond) (8, 22).

To ensure that the LPS-dependent autocatalytic cleavagereaction of factor C was accurately quantified by Western blot-ting under the conditions used, WT factor C was incubated atvarying concentrations from 12.5 to 100 nM for 30 min at 37 °Cin the presence or absence of LPS and subjected to Westernblotting. WT factor C was autocatalytically cleaved in the pres-ence of LPS but not in the absence of LPS (Fig. 2A). The relativeband density of Western blotting by densitometric analysis wasin direct proportion to the concentration of factor C underthese conditions (Fig. 2B). After 30-min incubation with LPS,factor C at every concentration was cleaved by �80% underthese conditions (Fig. 2C). These results indicate that the LPS-dependent autocatalytic cleavage was quantified by the densi-tometric analysis of Western blotting, at least under the condi-tions used.

To determine whether the catalytic serine residue in the pro-tease domain of factor C is involved in the autocatalytic activa-tion, the Ser941 residue of factor C, corresponding to Ser195 inchymotrypsinogen numbering, was substituted to Ala (S941A).WT factor C or the S941A mutant was incubated at 37 °C in thepresence of LPS and subjected to Western blotting (Fig. 3, Aand B). The autocatalytic cleavage rate was quantitated densi-tometrically (Fig. 3C). As expected, the L chain of WT factor Cwas converted to the B chain by �80% within 30 min under theconditions employed (Fig. 3, A and C), but the appearance of theB chain of the S941A mutant was not detected (Fig. 3, B and C).These results indicate that the Ser941 residue of factor C plays akey role in the LPS-dependent autocatalytic cleavage of thePhe737 site.

Factor C* does not exhibit intrinsic chymotryptic activityagainst the Phe737 site

To examine whether factor C* recognizes the hydrophobicside chain of the Phe737 site, the Phe737 residue was substitutedto Ala (F737A), Glu (F737E), Arg (F737R), or Pro (F737P). Inaddition, to examine whether the side chain of Ile738 affects the

Figure 1. A schematic domain structure of factor C. a, Cys-rich domain. b, epidermal growth factor domain. c, complement control protein domain. d, Limulusfactor C-Coch 5b2-Lg11 domain. e, C-type lectin domain. f, serine protease domain (23). The two interchain disulfide bonds are indicated by bars. PA indicatesthe position of the PA tag inserted between Trp758 and Leu759. Closed diamonds show N-linked glycosylation sites (19), and the fourth N-glycosylation siteindicated by the open diamond is partially modified (8).

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cleavage reaction, the Ile738 residue was substituted to Ala(I738A). All of the mutants, except the F737R mutant, wereexpressed in HEK293S GnTI� cells, but the expression level ofthe F737R mutant was too low for purification.

LPS-dependent autocatalytic cleavage reactions for the P1mutants (F737A, F737E, and F737P) and the P1� mutant(I738A) were performed using a concentration of 50 nM foreach mutant within the range of the linearity of the densitomet-ric analysis of Western blotting, as shown in Fig. 2 for WTfactor C. Each of the mutants was incubated at 37 °C in thepresence of LPS and subjected to Western blotting with anti-Bchain antibody. All of the mutants, except the F737P mutant,were autocatalytically cleaved in the presence of LPS (Fig. 4,A–D), and more than 70% of the L chain of each mutant wasconverted into the B chain by the 30-min incubation (Fig. 4E).Interestingly, the F737A mutant was more efficiently cleavedautocatalytically than WT factor C (Fig. 4E). These results sug-gest that factor C* does not exhibit an intrinsic chymotrypticactivity against the Phe737 site and that the side chain of Ile738

has little effect on the proteolytic cleavage of the Phe737 site.The cleaved form of the F737A mutant exhibited high levels

of specific amidase activities against Boc–Val–Pro–Arg–pNA,

comparable with the levels of WT �-factor C, and the cleavedform of the F737E mutant also had sufficient amidase activity(Fig. 4F). In contrast, the cleaved form of the I738A mutant,corresponding to Ile16 in chymotrypsinogen numbering, exhib-ited no amidase activity (Fig. 4F), indicating an essential inter-action of Ile738 with the Ile16 cleft to induce conversion of thezymogen form to the active form. These data support the pre-vious report of trypsin recombinants with Ile16 mutations,demonstrating that the hydrophobic interaction of the Ile16

side chain is the primary force to stabilize the substrate-bindingsite and the oxyanion hole rather than the electrostatic interac-tion of the Asp194–Ile16 salt bridge (24). As expected, the indi-vidual incubation of the F737P mutant or S941A mutant did notresult in any detectable amidase activity against the syntheticsubstrate in the presence of LPS (Fig. 4F).

Evidence for the existence of factor C*, the active transientstate of zymogen factor C, without cleavage of the Phe737 site

The F737P mutant prevented the cleavage reaction at thePhe737 site in the autocatalytic activation (Fig. 4, C, E, and F),whereas its Ser941 residue remained intact. On the other hand,the S941A mutant lost its authentic serine protease activity(Figs. 3B and 4F), whereas its Phe737 site remained intact. If theautocatalytic cleavage occurs intermolecularly between the fac-tor C* molecules but not intramolecularly within individualmolecules of factor C*, then the Phe737 site of the S941A mutant

Figure 2. LPS-dependent autocatalytic activation is quantified by densi-tometric analysis of Western blotting. A, WT factor C at varying concentra-tions from 12.5 to 100 nM was incubated for 30 min at 37 °C in the presence orabsence of a 20-fold molar excess of LPS and subjected to Western blottingwith anti-B chain antibody. Data are representative of three independentexperiments. B, the relative band intensity of the L and B chains at each factorC concentration to the L and B chains at 100 nM factor C, as analyzed byImageJ software. C, the rate of product formation is shown as the relativeband density of the B chain to the L and B chains at each concentration. Errorbars indicate � S.E. (n � 3).

Figure 3. The catalytic Ser941 residue of factor C is required for LPS-de-pendent autocatalytic activation. A and B, the WT (A) or the S941A mutant(B) (50 nM) was incubated for the indicated duration at 37 °C in the presence of0.68 �M LPS, and aliquots were subjected to Western blotting with anti-Bchain antibody. Data are representative of three independent experiments. C,the autocatalytic cleavage rate is shown as the relative band density of the Bchain to the L and B chain, as analyzed by ImageJ software. Error bars indi-cate � S.E. (n � 3).

An active transition state of zymogen factor C

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could be cleaved by the proteolytic activity of the active transi-tion state of the F737P mutant bound to LPS.

To examine this hypothesis, the F737P and S941A mutantswere mixed and incubated for 30 min at different concentrationratios in the presence of LPS, and the aliquots were subjected toWestern blotting. First, under conditions of a fixed concentra-tion of the S941A mutant at 50 nM and increasing concentra-tions of the F737P mutant from 1 to 5 nM, the autocatalyticcleavage rate increased as the concentrations of the F737Pmutant increased (Fig. 5, A, left, and B, left). Next, under con-ditions of a fixed concentration of the F737P mutant at 50 nM

and increasing concentrations of the S941A mutant from 1 to 5nM, the autocatalytic cleavage reaction was not observed (Fig. 5,A, right, and B, right), indicating that the Phe737 site of theS941A mutant is cleaved by the F737P mutant bound to LPS(F737P*). These results also indicate the presence of the activetransition state of factor C without cleavage of the Phe737 site.

Preparation of a factor C recombinant containing an epitopetag in an internal portion of the B chain

To obtain further evidence for the intermolecular event ofautocatalytic activation, a newly developed epitope tag system,PA tag, which is a dodecapeptide derived from human podopla-nin and recognized by a high-affinity mAb, NZ-1 (anti-PA anti-body) (25), was introduced into the production of factor Cmutants. Addition of a peptide or an epitope tag to the N ter-minus of WT factor C inhibits the LPS-dependent autocatalytic

Figure 4. The side chain of Phe737 at the cleavage site is not recognizedin autocatalytic activation. A–D, the F737A (A), F737E (B), F737P (C), orI738A (D) mutant (50 nM) was incubated for the indicated duration at 37 °Cin the presence of 0.68 �M LPS and subjected to Western blotting withanti-B chain antibody. Data are representative of three independentexperiments. E, the autocatalytic activation rate is shown as the relativeband density of the B chain to the L and B chains in A–D, as analyzed byImageJ software. Error bars indicate � S.E. (n � 3). F, the WT or eachmutant (50 nM) was incubated for 30 min at 37 °C in the presence of 0.68�M LPS, and the specific amidase activity against Boc–Val–Pro–Arg–pNAwas measured. Each specific activity was corrected by the conversion rate

at 30 min in E. Data are the means � S.E. of three independent experi-ments. ND, not detectable.

Figure 5. The Phe737 site of the S941A mutant is proteolytically cleavedby the F737P mutant in the presence of LPS. A, the F737P and S941Amutants were mixed at the indicated concentrations (1, 2, 5, or 50 nM), incu-bated at 37 °C for 30 min in the presence of 0.68 �M LPS, and subjected toWestern blotting with anti-B chain antibody. Data are representative of threeindependent experiments. B, the autocatalytic cleavage rate is shown as therelative band density of the B chain to the L and B chain, as analyzed byImageJ software. Error bars indicate � S.E. (n � 3).

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activation of factor C (22). In contrast, with the PA tag system itis possible to insert the PA tag into internal portions of a recom-binant protein or boundary portions between the domains in amosaic protein to maintain not only the antigenicity of the PAtag but also the physiologic activities of the tag-containing pro-teins (26). Accordingly, the PA tag was inserted into the peptidebond between Trp758–Leu759 in the B chain of WT factor C (PAfactor C), corresponding to the linker position between the two�-barrel structures composing the catalytic domain of serineproteases (1–3).

To examine the time-dependent cleavage reaction, PA factorC or WT factor C was incubated at 37 °C in the presence of LPSand then subjected to Western blotting with an anti-B chainantibody. The LPS-dependent autocatalytic cleavage of PA fac-

tor C occurred in correlation with that of WT factor C (Fig. 6, Aand B). On the other hand, the autocatalytically activated PAfactor C exhibited �30% lower amidase activity against the syn-thetic peptide substrate compared with that of the WT �-factorC (Fig. 6C), indicating that insertion of the PA tag at this posi-tion partially inhibits the amidase activity of �-factor C.

Further evidence of intermolecular cleavage at the Phe737 sitein autocatalytic activation

The LPS-dependent autocatalytic conversion of PA factor Cwas monitored on Western blotting using the anti-PA antibody(Fig. 7A); the results corresponded to those by Western blottingwith the anti-B chain antibody (Fig. 6A). To confirm that thePhe737 site of the S941A mutant is cleaved by the F737P mutantbound to LPS (Fig. 5), the PA tag was inserted into the Trp758–Leu759 bond of the F737P and S941A mutants, yielding thePA-F737P and PA-S941A mutants, respectively. As antici-pated, the resulting PA-F737P and PA-S941A mutants were notautocatalytically converted by individual incubation in thepresence of LPS (Fig. 7, A and B). Then, the PA-F737P andS941A mutants were mixed at a 1:1 ratio, incubated in the pres-ence of LPS for 30 min, and subjected to Western blotting withanti-PA antibody. As expected, the L chain of the PA-F737Pmutant was not converted to the B chain (Fig. 7C, center lane).Next, Western blotting experiments were carried out using a1:1 mixture of the PA-S941A and F737P mutants in the pres-ence of LPS, and the B chain derived from the PA-S941Amutant was detected by anti-PA antibody (Fig. 7C, right lane),clearly indicating that the Phe737 site of the PA-S941A mutantis intermolecularly cleaved by the F737P mutant bound to LPS.

LPS is also required to allow the Phe737 site to be cleaved byinducing a conformational change or by acting as a scaffold toinduce interactions between factor C* molecules

The N-terminal tripeptide of Arg36–Trp37–Arg38 of factor Cis essential for LPS recognition, and the replacement of bothArg36 and Arg38 to Glu (the RE factor C mutant (factor Cmutants with the replacement of both Arg36 and Arg38 to Glu))causes the loss of LPS-binding activity (20). Previously, wefound that incubation of factor C with LPS in the presence ofthe chemical cross-linker dimethyl adipimidate induces auto-catalytic activation of factor C through the formation of a dimeror its multiple species of factor C, resulting in chemical cross-linking of factor C molecules (22). However, it remains unclearwhether LPS-binding to the N-terminal region of factor C isalso required to allow other factor C* molecules to undergoprotein–protein interactions and thereby cleave the Phe737 site.To answer this question, three types of LPS-binding activity–deficient mutants, RE-factor C, RE-F737P, and RE-S941Amutants, were prepared. These mutants were incubated for 30min individually or in a 1:1 combined mixture in the presence ofLPS and subjected to Western blotting with anti-B chain anti-body. The autocatalytic conversion of WT factor C was �80%under the conditions employed (Fig. 8A, lane 1, and B, column1), whereas that of the 1:1 mixture of WT factor C and theRE-factor C mutant was reduced to �40%, suggesting that theRE-factor C mutant is not involved in LPS-dependent autocat-alytic activation (Fig. 8A, lane 3, and B, column 3). The RE-fac-

Figure 6. LPS-dependent autocatalytic activation of the PA tag–containing recombinants. A, WT factor C (top panel) or PA-factor C (bottompanel) (50 nM) was incubated for the indicated duration at 37 °C in the pres-ence of 0.68 �M LPS, and aliquots were subjected to Western blotting withanti-B chain antibody. B, the autocatalytic cleavage rate is shown as the rela-tive band density of the B chain to the L and B chain, as analyzed by ImageJsoftware. Error bars indicate � S.E. (n � 3). C, WT factor C or PA-factor C (50 nM)was incubated for 30 min at 37 °C in the presence of 0.68 �M LPS, and thespecific amidase activity against Boc–Val–Pro–Arg–pNA was measured. Eachspecific activity was corrected by the conversion rate at 30 min in B. Data arethe means � S.E. of three independent experiments.

An active transition state of zymogen factor C

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tor C mutant was not autocatalytically cleaved when incubatedindividually (Fig. 8A, lane 2, and B, column 2).

The mixture of the S941A and Phe737P mutants in the pres-ence of LPS yielded an autocatalytic conversion of �35% (Fig.8A, lane 4, and B, column 4) because the Phe737 site of theS941A mutant was cleaved by the F737P mutant bound to LPS.In contrast, the 1:1 mixture of the S941A and RE-F737P

Figure 7. Further evidence for intermolecular cleavage at the Phe737 sitein autocatalytic activation. A, PA-factor C (top panel) and the PA-F737P (cen-ter panel) or PA-S941A mutant (bottom panel) (50 nM) was incubated at 37 °Cfor the indicated duration in the presence of 0.68 �M LPS and subjected toWestern blotting with anti-PA antibody. B, the autocatalytic cleavage rate isshown as the relative band density of the B chain to the L chain and B chain, asanalyzed by ImageJ software. Error bars indicate � S.E. (n � 3). C, PA-factor Cand each recombinant was incubated at 37 °C for 30 min individually or in amixture at the indicated concentration in the presence of 0.68 �M LPS andsubjected to Western blotting with anti-PA antibody. Figure 8. The active transition state induced by LPS binding is required in

autocatalytic activation. A, WT factor C or each mutant was incubated at37 °C for 30 min individually or in a mixture at the indicated concentrationand subjected to Western blotting with anti-B chain antibody. Data arerepresentative of three independent experiments. B, the autocatalyticcleavage rate is shown as the relative band density of the B chain to the Lchain and B chain, as analyzed by ImageJ software. Error bars indicate �S.E. (n � 3). C, WT factor C or the RE-factor C mutant (50 nM) was incubatedwith 40 nM chymotrypsin for 30 min at 37 °C, and the specific amidaseactivities of the resulting �-factor C and �-RE-factor C against Boc–Val–Pro–Arg–pNA were measured.

An active transition state of zymogen factor C

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mutants in the presence of LPS did not yield the B chain (Fig.8A, lane 5, and B, column 5). Moreover, the 1:1 mixture of theRE-S941A and F737P mutants in the presence of LPS also didnot yield the B chain (Fig. 8A, lane 6, and Fig. 8B, column 5).These results suggest that LPS is required not only to completethe substrate-binding site and oxyanion hole of factor C* byinteracting with the N-terminal region but also to allow thePhe737 site to be cleaved by inducing a conformational changearound the Phe737 site or by acting as a scaffold to induce spe-cific protein–protein interactions between factor C* molecules.

There is a possibility that the replacement of both Arg36 andArg38 to Glu impedes the expression of the enzymatic activity ofthe catalytic domain of the RE-factor C mutant. To confirmthat the catalytic domain of the RE-factor C mutant is func-tional, the RE-factor C mutant was incubated with chymotryp-sin. The resulting chymotrypsin-treated RE-factor C mutant(�-RE-factor C) exhibited a level of specific amidase activityagainst Boc–Val–Pro–Arg–pNA equivalent to that of �-factorC, a chymotrypsin-treated WT factor C, indicating that thecatalytic domain of �-RE-factor C remains intact (Fig. 8C).

Comparison of the relative contributions of �-factor C andfactor C* to autocatalytic activation

To compare the relative contributions of �-factor C and fac-tor C* to the LPS-dependent autocatalytic activation, �-factorC and factor C* were prepared by individually incubating WTfactor C and the F737P mutant for 30 min at 37 °C in the pres-ence of LPS. An aliquot of the resulting �-factor C or F737P*(factor C*) was incubated with the PA-S941A mutant for theindicated duration at 37 °C and subjected to Western blottingwith anti-PA antibody (Fig. 9A). Interestingly, the densitomet-ric analyses of Western blotting suggest that there was no sig-nificant difference in the degree of contribution between �-fac-tor C and factor C* to the autocatalytic cleavage rate of thePA-S941A mutant (Fig. 9B).

Discussion

Proteolytic cascades in blood/hemolymph coagulation in-volve serine protease zymogens and are triggered through theinteraction of initiator zymogens with biologic substancesderived from host tissues or microbes to amplify and propagatebiologic reactions (27–30). In the horseshoe crab coagulationcascade, the initiator zymogen factor C is thought to be trig-gered by autocatalytic activation through the active transitionstate of factor C (factor C*) bound to LPS. However, factor C* isonly speculative, and it remains unclear whether the proteolyticcleavage of the Phe737 site of factor C required for the conver-sion to �-factor C occurs intramolecularly or intermolecularlybetween the factor C molecules. As for the mammalian extrin-sic coagulation pathway, the catalytic activity of factor VIIa pro-teolytically converted from zymogen factor VII by factor Xa isaccelerated by the interaction with the cofactor protein tissuefactor to form a 1:1 complex on phospholipids in the presenceof Ca2� (31). Higashi et al. (32) proposed a molecular model inwhich a tissue factor traps one of the conformational states offactor VIIa, converting the zymogen state into an active transi-tion state.

Here we used several mutants of factor C to show that thePhe737 site of the S941A mutant was cleaved LPS-dependentlyby the F737P mutant (Fig. 5), clearly indicating the presence ofthe active transition state of factor C with an active conforma-tion induced by the interaction with LPS without cleavage ofthe Phe737 site. Interestingly, the autocatalytic cleavage rateincreased in proportion to the concentration of the F737Pmutant when the concentration of the S941A mutant was keptconstant (Fig. 5), indicating multiple sequential catalytic turn-overs of the active transition state of the F737P mutant boundto LPS, F737P*. Moreover, the LPS-dependent proteolytic con-version of the PA-S941A mutant was clearly detected byanti-PA antibody in the mixture of the F737P and PA-S941Amutants, indicating that the Phe737 site of the PA-S941Amutant is cleaved by F737P* (Fig. 7). Therefore, the autocata-lytic activation of factor C occurs intermolecularly between thefactor C* molecules but not intramolecularly.

The results of our previous studies suggested that factor C*may exhibit intrinsic chymotryptic activity to cleave the Phe737

site despite being a member of the trypsin family (21, 22). Unex-pectedly, as shown in Fig. 4, the two factor C mutants with thereplacement of the Phe737 site, the F737A and F737E mutants,were autocatalytically activated with an efficient conversionrate comparable with that of WT factor C but the F737Pmutant was not, indicating that factor C* does not have

Figure 9. The degree of contribution of �-factor C and factor C* toautocatalytic activation. A, WT factor C or the F737P mutant (500 nM) wasincubated for 30 min at 37 °C in the presence of 6.8 �M LPS to prepare�-factor C and factor C*. An aliquot of the resulting �-factor C or factor C*(50 nM) was incubated with the PA-S941A mutant (50 nM) for the indicatedduration at 37 °C and subjected to Western blotting with anti-PA anti-body. Data are representative of three independent experiments. B, theautocatalytic cleavage rate is shown as the relative band density of the Bchain to the L chain and B chain, as analyzed by ImageJ software. Error barsindicate � S.E. (n � 3).

An active transition state of zymogen factor C

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intrinsic chymotryptic activity against the Phe737 site. To thebest of our knowledge, there are no other examples of serineproteases targeting a specific substrate with a P1 site distinctfrom their characterized primary substrate specificities. Fac-tor C* may recognize a three-dimensional structure aroundthe Phe737 site in the case of the LPS-dependent autocata-lytic activation.

As established in trypsinogen, the proteolytic activity in ser-ine protease zymogens can be switched on by highly localizedconformational changes to complete the substrate-binding siteand oxyanion hole. These changes are induced by the cleavageof the Arg15–Ile16 bond, leading to salt bridge formationbetween the Ile16 and Asp194 residues (1–3). It has been pro-posed that salt bridge formation at the Ile16 cleft is importantfor the nonproteolytic activation of prothrombin by complexformation with staphylocoagulase (33–36). Staphylocoagulase,a staphylococcal nonenzymatic protein, interacts with pro-thrombin to form a 1:1 complex to convert the zymogen state ofprothrombin into a thrombin-like active state; the N-terminalportion (Ile1–Val2–) of staphylocoagulase is inserted into theIle16 cleft of bound prothrombin to form a salt bridge betweenthe �-amino group of Ile1 of staphylocoagulase and the �-car-boxyl group of Asp194 of prothrombin, resulting in the activetransition state without proteolytic cleavage of the Arg15–Ile16

bond of prothrombin. We clearly identified the existence offactor C*, the active transition state of factor C without cleavageof the Phe737 site, because the Phe737 site of the S941A orPA-S941A mutant was LPS-dependently cleaved by a Phe737

site–replacing mutant, the F737P mutant bound to LPS (Figs. 3and 5).

The N-terminal Arg of factor C plays an important role inprotein–protein interactions between the factor C mole-cules to form the dimer or its multimers on LPS for autocat-alytic activation; a factor C mutant with a replacement ofArg1 with Lys, but not replacement of Arg1 with other aminoacids residues, including Ala, Leu, or His, maintains LPS-de-pendent autocatalytic activation, suggesting that the positiveside chain of Arg1 or Lys1 is essential for inducing LPS-de-pendent autocatalytic activation (22). Interestingly, unlikeother serine protease zymogens, tissue-type plasminogenactivator (tPA) is proteolytically active in a single-chain formwithout cleavage of the Arg15–Ile16 bond in chymotrypsino-gen numbering because the �-amino group of Lys156 locatedin the catalytic domain of tPA forms a salt bridge with the�-carboxyl group of Asp194, promoting an active conforma-tion in the single-chain form of tPA to maintain its lowzymogenicity (37). Also, in the oligomeric factor C on theLPS surface, a salt bridge may form between the basic sidechain of Arg1 or Lys1 and the side chain of Asp194 to promotethe active transition state of factor C without proteolyticcleavage of the Phe737 site.

Based on the above findings and our present results, we pro-pose the following model for autocatalytic activation and initi-ation of hemolymph coagulation by factor C* on the LPS sur-face derived from Gram-negative bacteria (Fig. 10). Factor C,which is secreted by LPS-induced hemocyte exocytosis, bindsto LPS through the tripeptide motif of the Arg36–Trp37–Arg38

sequence in the Cys-rich domain to cause the conformational

changes in factor C, resulting in the active transition state, fac-tor C*. Factor C* forms the dimer or its multimers on LPS, andautocatalytic activation occurs intermolecularly between thefactor C* molecules in the complex. The resulting �-factor C inthe complex may be exchangeable with noncomplexed factor Con LPS.

Homologs of mammalian complement factors C3 and B/C2were identified in horseshoe crabs (13–16), and an arachnidhomolog of complement factor B/C2 was also identified in thespider Loxosceles laeta (38). Moreover, a homolog of horseshoecrab factor C was reported from the tick Ixodes ricinus and mayplay a role in the tick primordial complement system (39).Therefore, the nomenclatures for the zymogens involved in thehorseshoe crab coagulation cascade, including factor C, factorB, and factor G, are becoming confused because the identical orsimilar terms are used for the zymogens in the complementsystem in Chelicerata. Therefore, we propose new terms forthree protease zymogens in the horseshoe crab coagulation cas-cade: prochelicerase C, prochelicerase B, and prochelicerase G,each of which is activated into the corresponding chelicerase inthe proteolytic cascade (Fig. 11).

A Limulus reagent prepared from hemocyte lysates of horse-shoe crabs is used in a sensitive assay to detect LPS. The use ofthis Limulus test has increased dramatically along withadvancements to monitor LPS contamination in parenteraldrugs, medical devices, and biologics (40, 41). However, theLimulus test is totally dependent on the limited naturalresource of horseshoe crab hemocytes. As an alternative ap-proach, we recently developed a next-generation Limulus testusing recombinant coagulation factors (42). We are convincedthat further detailed functional studies of recombinant coagu-lation factors will contribute to the development of more sen-sitive and convenient assays for LPS.

Figure 10. A proposed model for the autocatalytic activation of factor Cand initiation of the coagulation cascade on LPS. Coagulation factors,including factor C and factor B, are secreted from hemocytes at injured sites inresponse to LPS stimulation. Autocatalytic activation and initiation of thecoagulation cascade occur through the active transition state of factor C,factor C*, as described under “Discussion.”

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Experimental procedures

Materials

The HEK293S GnTI� cell line was obtained from the ATCC.LPS derived from Salmonella minnesota R595 (Re) (Mr ��2,500) was purchased from List Biological Laboratories(Campbell, CA) and used for the factor C activation assays.Boc–Val–Pro–Arg–pNA was provided by Seikagaku (Tokyo).The polyclonal antibody against the B chain of factor C wasprepared previously (8).

Mutagenesis

The pCA7 plasmid encoding the full-length of factor C,which contains a six-histidine tag and a cleavage site of factorXa at the N-terminal end, was used for expression andmutagenesis (8). Mutations were introduced using site-di-rected mutagenesis by inverse PCR. The PA tag (25) wasinserted into the plasmid at the site between Trp758 and Leu759

of factor C with the In-Fusion� HD Cloning Kit (Takara Bio,Shiga, Japan).

Expression of recombinant proteins

Recombinant proteins were expressed as described previ-ously (8, 22). In brief, the recombinant proteins were expressedin HEK293S GnTI� cells and secreted into culture medium.HEK293S GnTI� cells were maintained in Dulbecco’s modifiedEagle’s medium supplemented with glutamine, penicillin–streptomycin, and 10% fetal bovine serum at 37 °C under 5%CO2. Dulbecco’s modified Eagle’s medium supplemented withplasmids containing inserts of the recombinant proteins (1.8�g/ml), polyethyleneimine (2.7 �g/ml), glutamine, penicillin–streptomycin, and 2% fetal bovine serum was used to transfectHEK293S GnTI� cells at 80 –90% confluence. Culture mediawere collected 5 days after transfection and centrifuged at 6,000rpm for 30 min.

Purification of recombinant proteins

Recombinants were purified as described previously (8, 22).In brief, collected culture media containing factor C recombi-

nants were applied to a nickel-nitrilotriacetic acid–agarose col-umn. The buffer of eluates was exchanged, treated with factorXa, and further applied to a nickel-nitrilotriacetic acid–agarosecolumn to remove the free histidine tag. The concentrations ofrecombinants were determined using the extinction coefficientof 1% solution at A280 nm of 21.3 (4) or a Micro BCA ProteinAssay Kit (Thermo Fisher Scientific, Waltham, MA).

Autocatalytic activation and assay for amidase activity

For autocatalytic activation, factor C recombinants wereincubated with 0.68 �M (1.7 �g/ml) LPS at 37 °C in 20 mM

Tris-HCl (pH 8.0) containing 300 mM NaCl and subjected toWestern blotting with anti-B chain antibody or anti-PA anti-body (Wako, Osaka, Japan). In the case of amidase activityagainst Boc–Val–Pro–Arg–pNA, factor C recombinants wereincubated with 0.68 �M LPS for 30 min at 37 °C in 20 mM Tris-HCl (pH 8.0) containing 300 mM NaCl. One microliter of eachsolution was diluted by 19 �l of 20 mM Tris-HCl (pH 8.0] con-taining 150 mM NaCl and 1.5 �M BSA, and then 5 �l of 2 mM

Boc–Val–Pro–Arg–pNA was added. After incubation for 5min at 37 °C, the reaction was stopped by addition of 75 �l of 0.6M acetic acid, and the liberated pNA was measured by absor-bance at 405 nm. One unit of amidase activity was defined as 1�mol of digested substrate per minute, and the specific activitywas expressed as units per nanomole of factor C.

Western blotting

Samples were subjected to SDS-PAGE in 10% or 12% Laem-mli’s gel and transferred to a polyvinylidene difluoride mem-brane. After blocking with 5% skim milk, the membrane wasincubated with anti-B chain antibody or anti-PA antibody con-jugated with peroxidase (Wako). In the case of anti-B chainantibody, horseradish peroxidase– conjugated secondary anti-body (Bio-Rad) was applied, or horseradish peroxidase wasconjugated with the Lightning-Link HRP Conjugation Kit(Innova Biosciences, Cambridge, UK). This was followed bydevelopment with WesternBright Quantum or Sirius (Advan-sta, Menlo Park, CA). Images were obtained with the Omega

Figure 11. The proteolytic coagulation cascade in horseshoe crabs using newly designated terms. Newly designated terms of the zymogens and theiractive forms are shown in red.

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Lum G imaging system (Aplegen Life Sciences, San Francisco,CA). Precision Plus protein standards (Bio-Rad) were used todetermine the apparent molecular masses.

Author contributions—T. S., Y. K., and S. K. conceptualization; T. S.,Y. K., and S. K. data curation; T. S., Y. K., and S. K. formal analysis;T. S., Y. K., Y. I., and S. K. investigation; T. S., Y. K., Y. I., and S. K.methodology; T. S., Y. K., and S. K. writing-original draft; T. S., Y. K.,and S. K. project administration; T. S., Y. K., and S. K. writing-reviewand editing; Y. K. and S. K. supervision; S. K. funding acquisition;S. K. validation; S. K. visualization.

Acknowledgments—We thank Naoki Hirakawa (Kyushu University,Fukuoka, Japan) for preparation of factor C recombinants containing thePA tag and for preliminary experiments regarding the autocatalytic acti-vation of these recombinants. We also thank Prof. Shouichi Higashi(Yokohama City University, Yokohama, Japan) for helpful discussionsregarding autocatalytic activation of serine protease zymogens.

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Toshio Shibata, Yuki Kobayashi, Yuto Ikeda and Shun-ichiro Kawabatathrough an active transition state responding to lipopolysaccharide

Intermolecular autocatalytic activation of serine protease zymogen factor C

doi: 10.1074/jbc.RA118.002311 originally published online June 4, 20182018, 293:11589-11599.J. Biol. Chem. 

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