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• W2807449069 abstract "Horseshoe crab hemolymph coagulation is believed to be triggered by the autocatalytic activation of serine protease zymogen factor C to the active form, α-factor C, belonging to the trypsin family, through an active transition state of factor C responding to bacterial lipopolysaccharide (LPS), designated factor C*. However, the existence of factor C* is only speculative, and its proteolytic activity has not been validated. In addition, it remains unclear whether the proteolytic cleavage of the Phe737–Ile738 bond (Phe737 site) of factor C required for the conversion to α-factor C occurs intramolecularly or intermolecularly between the factor C molecules. Here we show that the Phe737 site of a catalytic Ser-deficient mutant of factor C is LPS-dependently hydrolyzed by a Phe737 site–uncleavable mutant, clearly indicating the existence of the active transition state of factor C without cleavage of the Phe737 site. Moreover, we found the following facts using several mutants of factor C: the autocatalytic cleavage of factor C occurs intermolecularly between factor C* molecules on the LPS surface; factor C* does not exhibit intrinsic chymotryptic activity against the Phe737 site, but it may recognize a three-dimensional structure around the cleavage site; and LPS is required not only to complete the substrate-binding site and oxyanion hole of factor C* by interacting with the N-terminal region but also to allow the Phe737 site to be cleaved by inducing a conformational change around the Phe737 site or by acting as a scaffold to induce specific protein–protein interactions between factor C* molecules. Horseshoe crab hemolymph coagulation is believed to be triggered by the autocatalytic activation of serine protease zymogen factor C to the active form, α-factor C, belonging to the trypsin family, through an active transition state of factor C responding to bacterial lipopolysaccharide (LPS), designated factor C*. However, the existence of factor C* is only speculative, and its proteolytic activity has not been validated. In addition, it remains unclear whether the proteolytic cleavage of the Phe737–Ile738 bond (Phe737 site) of factor C required for the conversion to α-factor C occurs intramolecularly or intermolecularly between the factor C molecules. Here we show that the Phe737 site of a catalytic Ser-deficient mutant of factor C is LPS-dependently hydrolyzed by a Phe737 site–uncleavable mutant, clearly indicating the existence of the active transition state of factor C without cleavage of the Phe737 site. Moreover, we found the following facts using several mutants of factor C: the autocatalytic cleavage of factor C occurs intermolecularly between factor C* molecules on the LPS surface; factor C* does not exhibit intrinsic chymotryptic activity against the Phe737 site, but it may recognize a three-dimensional structure around the cleavage site; and LPS is required not only to complete the substrate-binding site and oxyanion hole of factor C* by interacting with the N-terminal region but also to allow the Phe737 site to be cleaved by inducing a conformational change around the Phe737 site or by acting as a scaffold to induce specific protein–protein interactions between factor C* molecules. The molecular mechanism underlying the proteolytic activation of serine protease zymogens has been established in trypsinogen, which is activated by the activator enteropeptidase through limited proteolysis of the Arg15–Ile16 peptide bond in chymotrypsinogen numbering. The limited proteolysis induces the insertion of the newly appearing N-terminal Ile16 into the activation pocket known as the Ile16 cleft to form a salt bridge between the α-amino group of Ile16 and the β-carboxyl group of Asp194. The salt bridge results in conformational changes in the substrate-binding site and the oxyanion hole to hydrolyze specific peptide bonds of substrates, whereas the conformational changes elsewhere in the molecule, including the catalytic triad of Ser195, His57, and Asp102, are very small (1.Freer S.T. Kraut J. Robertus J.D. Wright H.T. Xuong N.H. Chymotrypsinogen: 2.5-Å crystal structure, comparison with α-chymotrypsin, and implications for zymogen activation.Biochemistry. 1970; 9 (5442169): 1997-200910.1021/bi00811a022Crossref PubMed Scopus (295) Google Scholar, 2.Bode W. Huber R. Induction of the bovine trypsinogen-trypsin transition by peptides sequentially similar to the N-terminus of trypsin.FEBS Lett. 1976; 68 (10181): 231-23610.1016/0014-5793(76)80443-7Crossref PubMed Scopus (122) Google Scholar, 3.Huber R. Bode W. Structural basis of the activation and action of trypsin.Acc. Chem. Res. 1978; 11: 114-12210.1021/ar50123a006Crossref Scopus (608) Google Scholar). Factor C is a serine protease zymogen involved in the hemolymph coagulation cascade of horseshoe crabs and is autocatalytically activated to α-factor C on bacterial LPS 4The abbreviations used are: LPSlipopolysaccharideBoct-butoxycarbonylpNAp-nitroanilideHEKhuman embryonic kidneyGnTIN-acetylglucosaminyltransferase IPAa dodecapeptide derived from human podoplanintPAtissue-type plasminogen activator. (4.Nakamura T. Morita T. Iwanaga S. Lipopolysaccharide-sensitive serine-protease zymogen (factor C) found in Limulus hemocytes.Eur. J. Biochem. 1986; 154 (3512266): 511-52110.1111/j.1432-1033.1986.tb09427.xCrossref PubMed Scopus (120) Google Scholar, 5.Kawabata S. Muta T. Sadaaki Iwanaga: discovery of the lipopolysaccharide- and β-1,3-d-glucan-mediated proteolytic cascade and unique proteins in invertebrate immunity.J. Biochem. 2010; 147 (20406733): 611-61810.1093/jb/mvq026Crossref PubMed Scopus (28) Google Scholar, 6.Kawabata S. Söderhäll K. Invertebrate Immunity. Springer Science and Business Media, New York2011: 122-136Google Scholar). The resulting α-factor C activates coagulation factor B to activated factor B (7.Muta T. Oda T. Iwanaga S. Horseshoe crab coagulation factor B: a unique serine protease zymogen activated by cleavage of an Ile–Ile bond.J. Biol. Chem. 1993; 268 (8407978): 21384-21388Abstract Full Text PDF PubMed Google Scholar, 8.Kobayashi Y. Takahashi T. Shibata T. Ikeda S. Koshiba T. Mizumura H. Oda T. Kawabata S. Factor B is the second lipopolysaccharide-binding protease zymogen in the horseshoe crab coagulation cascade.J. Biol. Chem. 2015; 290 (26109069): 19379-1938610.1074/jbc.M115.653196Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), which activates the proclotting enzyme to the clotting enzyme (9.Nakamura T. Morita T. Iwanaga S. Intracellular proclotting enzyme in Limulus (Tachypleus tridentatus) hemocytes: its purification and properties.J. Biochem. 1985; 97 (4030738): 1561-157410.1093/oxfordjournals.jbchem.a135213Crossref PubMed Scopus (73) Google Scholar, 10.Muta T. Hashimoto R. Miyata T. Nishimura H. Toh Y. Iwanaga S. Proclotting enzyme from horseshoe crab hemocytes: cDNA cloning, disulfide locations, and subcellular localization.J. Biol. Chem. 1990; 265 (2266134): 22426-22433Abstract Full Text PDF PubMed Google Scholar) to convert coagulogen into coagulin gel (11.Kawasaki H. Nose T. Muta T. Iwanaga S. Shimohigashi Y. Kawabata S. Head-to-tail polymerization of coagulin, a clottable protein of the horseshoe crab.J. Biol. Chem. 2000; 275 (10948203): 35297-3530110.1074/jbc.M006856200Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Alternatively, factor G autocatalytically activated in the presence of β-1,3-d-glucans directly activates the proclotting enzyme to the clotting enzyme (6.Kawabata S. Söderhäll K. Invertebrate Immunity. Springer Science and Business Media, New York2011: 122-136Google Scholar). Factor C is also located on hemocytes as a pattern recognition receptor for LPS, and the resulting α-factor C triggers hemocyte exocytosis through a protease-activated G protein–coupled receptor to induce Ca2+ signaling (12.Ariki S. Koori K. Osaki T. Motoyama K. Inamori K. Kawabata S. A serine protease zymogen functions as a pattern-recognition receptor for lipopolysaccharides.Proc. Natl. Acad. Sci. U.S.A. 2004; 101 (14722355): 953-95810.1073/pnas.0306904101Crossref PubMed Scopus (72) Google Scholar). On the other hand, homologs of mammalian complement factors C3 and B/C2 have been identified in hemolymph of horseshoe crabs (13.Zhu Y. Thangamani S. Ho B. Ding J.L. The ancient origin of the complement system.EMBO J. 2005; 24 (15616573): 382-39410.1038/sj.emboj.7600533Crossref PubMed Scopus (216) Google Scholar), and α-factor C also acts as the complement C3 convertase on microbes in close cooperation with several lectins (14.Ariki S. Takahara S. Shibata T. Fukuoka T. Ozaki A. Endo Y. Fujita T. Koshiba T. Kawabata S. Factor C acts as a lipopolysaccharide-responsive C3 convertase in horseshoe crab complement activation.J. Immunol. 2008; 181 (19017991): 7994-800110.4049/jimmunol.181.11.7994Crossref PubMed Scopus (54) Google Scholar, 15.Le Saux A. Ng P.M. Koh J.J. Low D.H. Leong G.E. Ho B. Ding J.L. The macromolecular assembly of pathogen-recognition receptors is impelled by serine proteases, via their complement control protein modules.J. Mol. Biol. 2008; 377 (18279891): 902-91310.1016/j.jmb.2008.01.045Crossref PubMed Scopus (20) Google Scholar, 16.Tagawa K. Yoshihara T. Shibata T. Kitazaki K. Endo Y. Fujita T. Koshiba T. Kawabata S. Microbe-specific C3b deposition in the horseshoe crab complement system in a C2/factor B-dependent or -independent manner.PLoS ONE. 2012; 7 (22611464): e3678310.1371/journal.pone.0036783Crossref PubMed Scopus (21) Google Scholar). lipopolysaccharide t-butoxycarbonyl p-nitroanilide human embryonic kidney N-acetylglucosaminyltransferase I a dodecapeptide derived from human podoplanin tissue-type plasminogen activator. Factor C is biosynthesized as a single-chain form of the zymogen containing six N-linked glycosylation sites, whereas a two-chain form of the zymogen of the H and L chains produced by cleavage of the Arg665–Ser666 bond by an unknown protease is principally purified from hemocytes (6.Kawabata S. Söderhäll K. Invertebrate Immunity. Springer Science and Business Media, New York2011: 122-136Google Scholar, 17.Nakamura T. Tokunaga F. Morita T. Iwanaga S. Kusumoto S. Shiba T. Kobayashi T. Inoue K. Intracellular serine-protease zymogen, factor C, from horseshoe crab hemocytes.Eur. J. Biochem. 1988; 176 (3166424): 89-9410.1111/j.1432-1033.1988.tb14254.xCrossref PubMed Scopus (58) Google Scholar, 18.Nakamura T. Tokunaga F. Morita T. Iwanaga S. Interaction between lipopolysaccharide and intracellular serine protease zymogen, factor C, from horseshoe crab (Tachypleus tridentatus) hemocytes.J. Biochem. 1988; 103 (3163690): 370-37410.1093/oxfordjournals.jbchem.a122276Crossref PubMed Scopus (51) Google Scholar, 19.Muta T. Miyata T. Misumi Y. Tokunaga F. Nakamura T. Toh Y. Ikehara Y. Iwanaga S. Limulus factor C, an endotoxin-sensitive serine protease zymogen with a mosaic structure of complement-like, epidermal growth factor-like, and lectin-like domains.J. Biol. Chem. 1991; 266 (2007602): 6554-6561Abstract Full Text PDF PubMed Google Scholar) (Fig. 1). An LPS-binding site is located in the N-terminal Cys-rich domain, and a tripeptide sequence of Arg36–Trp37–Arg38 in this domain is essential for LPS binding (20.Koshiba T. Hashii T. Kawabata S. A structural perspective on the interaction between lipopolysaccharide and factor C, a receptor involved in recognition of Gram-negative bacteria.J. Biol. Chem. 2007; 282 (17135239): 3962-3967Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Based on its amino acid sequence, α-factor C belongs to the trypsin family and exhibits trypsin-like amidase activity against a peptide substrate for α-thrombin, t-butoxycarbonyl (Boc)–Val–Pro–Arg–p-nitroanilide (pNA), or against Boc–Val–Pro–Arg–4-methylcoumaryl-7-amide but not against a peptide substrate for chymotrypsin, succinyl–Ala–Pro–Phe–4-methylcoumaryl-7-amide (8.Kobayashi Y. Takahashi T. Shibata T. Ikeda S. Koshiba T. Mizumura H. Oda T. Kawabata S. Factor B is the second lipopolysaccharide-binding protease zymogen in the horseshoe crab coagulation cascade.J. Biol. Chem. 2015; 290 (26109069): 19379-1938610.1074/jbc.M115.653196Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Autocatalytic activation of factor C bound to LPS occurs through proteolytic cleavage of the Phe737–Ile738 bond (Phe737 site) (Fig. 1), corresponding to the Arg15–Ile16 bond in chymotrypsinogen numbering (21.Tokunaga F. Miyata T. Nakamura T. Morita T. Kuma K. Miyata T. Iwanaga S. Lipopolysaccharide-sensitive serine-protease zymogen (factor C) of horseshoe crab hemocytes.Eur. J. Biochem. 1987; 167 (3308457): 405-41610.1111/j.1432-1033.1987.tb13352.xCrossref PubMed Scopus (51) Google Scholar). It remains unknown how factor C* displays specific chymotryptic activity against the Phe737 site despite its general trypsin-like primary substrate specificity. Previously, we prepared a recombinant factor C with an N-glycan of Man5GlcNAc2 at each N-glycosylation site, expressed in an HEK293S mutant cell line lacking N-acetylglucosaminyltransferase I (GnTI−) (22.Kobayashi Y. Shiga T. Shibata T. Sako M. Maenaka K. Koshiba T. Mizumura H. Oda T. Kawabata S. The N-terminal Arg residue is essential for autocatalytic activation of a lipopolysaccharide-responsive protease zymogen.J. Biol. Chem. 2014; 289 (25077965): 25987-2599510.1074/jbc.M114.586933Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). In the absence of LPS, factor C is artificially activated by chymotrypsin through cleavage of the Phe737 site (23.Tokunaga F. Nakajima H. Iwanaga S. Further studies on lipopolysaccharide-sensitive serine-protease zymogen (factor C): its isolation from Limulus polyphemus hemocytes and identification as an intracellular zymogen activated by α-chymotrypsin, not by trypsin.J. Biochem. 1991; 109 (2016264): 150-15710.1093/oxfordjournals.jbchem.a123337Crossref PubMed Scopus (26) Google Scholar). Using WT factor C, we found that chymotrypsin-activated factor C contains an additional proteolytic cleavage of the Tyr40–Cys41 bond in the N-terminal Cys-rich domain, designated β-factor C (Fig. 1) (22.Kobayashi Y. Shiga T. Shibata T. Sako M. Maenaka K. Koshiba T. Mizumura H. Oda T. Kawabata S. The N-terminal Arg residue is essential for autocatalytic activation of a lipopolysaccharide-responsive protease zymogen.J. Biol. Chem. 2014; 289 (25077965): 25987-2599510.1074/jbc.M114.586933Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The resulting β-factor C exhibits amidase activity against the synthetic substrates for α-thrombin with ∼70% specific activity compared with that of α-factor C but has neither the LPS-binding activity nor the activating activity for factor B (22.Kobayashi Y. Shiga T. Shibata T. Sako M. Maenaka K. Koshiba T. Mizumura H. Oda T. Kawabata S. The N-terminal Arg residue is essential for autocatalytic activation of a lipopolysaccharide-responsive protease zymogen.J. Biol. Chem. 2014; 289 (25077965): 25987-2599510.1074/jbc.M114.586933Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). These data suggest that LPS interaction with α-factor C is required to maintain its proteolytic activity against factor B. Interestingly, the proteolytic conversion of factor B to activated factor B occurs through cleavage of the Ile126–Ile127 bond (7.Muta T. Oda T. Iwanaga S. Horseshoe crab coagulation factor B: a unique serine protease zymogen activated by cleavage of an Ile–Ile bond.J. Biol. Chem. 1993; 268 (8407978): 21384-21388Abstract Full Text PDF PubMed Google Scholar); in addition, factor B is also an LPS-binding protein, and the LPS-bound form of factor B is essentially required for its proteolytic activation by α-factor C (8.Kobayashi Y. Takahashi T. Shibata T. Ikeda S. Koshiba T. Mizumura H. Oda T. Kawabata S. Factor B is the second lipopolysaccharide-binding protease zymogen in the horseshoe crab coagulation cascade.J. Biol. Chem. 2015; 290 (26109069): 19379-1938610.1074/jbc.M115.653196Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Factor C*, however, is only speculative, and its proteolytic activity has not been validated. Moreover, it remains unclear whether the autocatalytic cleavage at the Phe737 site occurs intramolecularly within factor C bound to LPS or intermolecularly between the molecules of factor C. Here we show that the autocatalytic activation of factor C is the unidentified intermolecular event between the factor C* molecules on the LPS surface. WT factor C prepared in the HEK293S GnTI− cell line is the two-chain form of the zymogen consisting of the H and L chains (22.Kobayashi Y. Shiga T. Shibata T. Sako M. Maenaka K. Koshiba T. Mizumura H. Oda T. Kawabata S. The N-terminal Arg residue is essential for autocatalytic activation of a lipopolysaccharide-responsive protease zymogen.J. Biol. Chem. 2014; 289 (25077965): 25987-2599510.1074/jbc.M114.586933Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The autocatalytic activation occurs through cleavage of the Phe737 site of the L chain to be converted into the A and B chains (Fig. 1) (19.Muta T. Miyata T. Misumi Y. Tokunaga F. Nakamura T. Toh Y. Ikehara Y. Iwanaga S. Limulus factor C, an endotoxin-sensitive serine protease zymogen with a mosaic structure of complement-like, epidermal growth factor-like, and lectin-like domains.J. Biol. Chem. 1991; 266 (2007602): 6554-6561Abstract Full Text PDF PubMed Google Scholar, 21.Tokunaga F. Miyata T. Nakamura T. Morita T. Kuma K. Miyata T. Iwanaga S. Lipopolysaccharide-sensitive serine-protease zymogen (factor C) of horseshoe crab hemocytes.Eur. J. Biochem. 1987; 167 (3308457): 405-41610.1111/j.1432-1033.1987.tb13352.xCrossref PubMed Scopus (51) Google Scholar). Therefore, the proteolytic activation of factor C was followed by appearance of the B chain, which was detected by using a polyclonal antibody against the B chain. The L chain on Western blotting was observed in a doublet because the fourth N-glycosylation site located in the A chain is partially modified (Fig. 1, open diamond) (8.Kobayashi Y. Takahashi T. Shibata T. Ikeda S. Koshiba T. Mizumura H. Oda T. Kawabata S. Factor B is the second lipopolysaccharide-binding protease zymogen in the horseshoe crab coagulation cascade.J. Biol. Chem. 2015; 290 (26109069): 19379-1938610.1074/jbc.M115.653196Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 22.Kobayashi Y. Shiga T. Shibata T. Sako M. Maenaka K. Koshiba T. Mizumura H. Oda T. Kawabata S. The N-terminal Arg residue is essential for autocatalytic activation of a lipopolysaccharide-responsive protease zymogen.J. Biol. Chem. 2014; 289 (25077965): 25987-2599510.1074/jbc.M114.586933Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). To ensure that the LPS-dependent autocatalytic cleavage reaction of factor C was accurately quantified by Western blotting under the conditions used, WT factor C was incubated at varying concentrations from 12.5 to 100 nm for 30 min at 37 °C in the presence or absence of LPS and subjected to Western blotting. WT factor C was autocatalytically cleaved in the presence of LPS but not in the absence of LPS (Fig. 2A). The relative band density of Western blotting by densitometric analysis was in direct proportion to the concentration of factor C under these conditions (Fig. 2B). After 30-min incubation with LPS, factor C at every concentration was cleaved by ∼80% under these conditions (Fig. 2C). These results indicate that the LPS-dependent autocatalytic cleavage was quantified by the densitometric analysis of Western blotting, at least under the conditions used. To determine whether the catalytic serine residue in the protease domain of factor C is involved in the autocatalytic activation, the Ser941 residue of factor C, corresponding to Ser195 in chymotrypsinogen numbering, was substituted to Ala (S941A). WT factor C or the S941A mutant was incubated at 37 °C in the presence of LPS and subjected to Western blotting (Fig. 3, A and B). The autocatalytic cleavage rate was quantitated densitometrically (Fig. 3C). As expected, the L chain of WT factor C was converted to the B chain by ∼80% within 30 min under the conditions employed (Fig. 3, A and C), but the appearance of the B chain of the S941A mutant was not detected (Fig. 3, B and C). These results indicate that the Ser941 residue of factor C plays a key role in the LPS-dependent autocatalytic cleavage of the Phe737 site. To examine whether factor C* recognizes the hydrophobic side chain of the Phe737 site, the Phe737 residue was substituted to Ala (F737A), Glu (F737E), Arg (F737R), or Pro (F737P). In addition, to examine whether the side chain of Ile738 affects the cleavage reaction, the Ile738 residue was substituted to Ala (I738A). All of the mutants, except the F737R mutant, were expressed in HEK293S GnTI− cells, but the expression level of the F737R mutant was too low for purification. LPS-dependent autocatalytic cleavage reactions for the P1 mutants (F737A, F737E, and F737P) and the P1′ mutant (I738A) were performed using a concentration of 50 nm for each mutant within the range of the linearity of the densitometric analysis of Western blotting, as shown in Fig. 2 for WT factor C. Each of the mutants was incubated at 37 °C in the presence of LPS and subjected to Western blotting with anti-B chain 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 was converted into the B chain by the 30-min incubation (Fig. 4E). Interestingly, the F737A mutant was more efficiently cleaved autocatalytically than WT factor C (Fig. 4E). These results suggest that factor C* does not exhibit an intrinsic chymotryptic activity 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 α-factor C, and the cleaved form 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, exhibited no amidase activity (Fig. 4F), indicating an essential interaction of Ile738 with the Ile16 cleft to induce conversion of the zymogen form to the active form. These data support the previous 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-binding site and the oxyanion hole rather than the electrostatic interaction of the Asp194–Ile16 salt bridge (24.Hedstrom L. Lin T.Y. Fast W. Hydrophobic interactions control zymogen activation in the trypsin family of serine proteases.Biochemistry. 1996; 35 (8605201): 4515-452310.1021/bi951928kCrossref PubMed Scopus (93) Google Scholar). As expected, the individual incubation of the F737P mutant or S941A mutant did not result in any detectable amidase activity against the synthetic substrate in the presence of LPS (Fig. 4F). The F737P mutant prevented the cleavage reaction at the Phe737 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 the autocatalytic cleavage occurs intermolecularly between the factor C* molecules but not intramolecularly within individual molecules of factor C*, then the Phe737 site of the S941A mutant could be cleaved by the proteolytic activity of the active transition state of the F737P mutant bound to LPS. To examine this hypothesis, the F737P and S941A mutants were mixed and incubated for 30 min at different concentration ratios in the presence of LPS, and the aliquots were subjected to Western blotting. First, under conditions of a fixed concentration of the S941A mutant at 50 nm and increasing concentrations of the F737P mutant from 1 to 5 nm, the autocatalytic cleavage rate increased as the concentrations of the F737P mutant increased (Fig. 5, A, left, and B, left). Next, under conditions of a fixed concentration of the F737P mutant at 50 nm and increasing concentrations of the S941A mutant from 1 to 5 nm, the autocatalytic cleavage reaction was not observed (Fig. 5, A, right, and B, right), indicating that the Phe737 site of the S941A mutant is cleaved by the F737P mutant bound to LPS (F737P*). These results also indicate the presence of the active transition state of factor C without cleavage of the Phe737 site. To obtain further evidence for the intermolecular event of autocatalytic activation, a newly developed epitope tag system, PA tag, which is a dodecapeptide derived from human podoplanin and recognized by a high-affinity mAb, NZ-1 (anti-PA antibody) (25.Kato Y. Kaneko M.K. Kuno A. Uchiyama N. Amano K. Chiba Y. Hasegawa Y. Hirabayashi J. Narimatsu H. Mishima K. Osawa M. Inhibition of tumor cell-induced platelet aggregation using a novel anti-podoplanin antibody reacting with its platelet-aggregation-stimulating domain.Biochem. Biophys. Res. Commun. 2006; 349 (16979138): 1301-130710.1016/j.bbrc.2006.08.171Crossref PubMed Scopus (169) Google Scholar), was introduced into the production of factor C mutants. Addition of a peptide or an epitope tag to the N terminus of WT factor C inhibits the LPS-dependent autocatalytic activation of factor C (22.Kobayashi Y. Shiga T. Shibata T. Sako M. Maenaka K. Koshiba T. Mizumura H. Oda T. Kawabata S. The N-terminal Arg residue is essential for autocatalytic activation of a lipopolysaccharide-responsive protease zymogen.J. Biol. Chem. 2014; 289 (25077965): 25987-2599510.1074/jbc.M114.586933Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). In contrast, with the PA tag system it is possible to insert the PA tag into internal portions of a recombinant protein or boundary portions between the domains in a mosaic protein to maintain not only the antigenicity of the PA tag but also the physiologic activities of the tag-containing proteins (26.Fujii Y. Matsunaga Y. Arimori T. Kitago Y. Ogasawara S. Kaneko M.K. Kato Y. Takagi J. Tailored placement of a turn-forming PA tag into the structured domain of a protein to probe its conformational state.J. Cell Sci. 2016; 129 (26872787): 1512-152210.1242/jcs.176685Crossref PubMed Scopus (35) Google Scholar). Accordingly, the PA tag was inserted into the peptide bond between Trp758–Leu759 in the B chain of WT factor C (PA factor C), corresponding to the linker position between the two β-barrel structures composing the catalytic domain of serine proteases (1.Freer S.T. Kraut J. Robertus J.D. Wright H.T. Xuong N.H. Chymotrypsinogen: 2.5-Å crystal structure, comparison with α-chymotrypsin, and implications for zymogen activation.Biochemistry. 1970; 9 (5442169): 1997-200910.1021/bi00811a022Crossref PubMed Scopus (295) Google Scholar, 2.Bode W. Huber R. Induction of the bovine trypsinogen-trypsin transition by peptides sequentially similar to the N-terminus of trypsin.FEBS Lett. 1976; 68 (10181): 231-23610.1016/0014-5793(76)80443-7Crossref PubMed Scopus (122) Google Scholar, 3.Huber R. Bode W. Structural basis of the activation and action of trypsin.Acc. Chem. Res. 1978; 11: 114-12210.1021/ar50123a006Crossref Scopus (608) Google Scholar). To examine the time-dependent cleavage reaction, PA factor C or WT factor C was incubated at 37 °C in the presence of LPS and then subjected to Western blotting with an anti-B chain antibody. The LPS-dependent autocatalytic cleavage of PA factor C occurred in correlation with that of WT factor C (Fig. 6, A and B). On the other hand, the autocatalytically activated PA factor C exhibited ∼30% lower amidase activity against the synthetic peptide substrate compared with that of the WT α-factor C (Fig. 6C), indicating that insertion of the PA tag at this position partially inhibits the amidase activity of α-factor C. The LPS-dependent autocatalytic conversion of PA factor C was monitored on Western blotting using the anti-PA antibody (Fig. 7A); the results corresponded to those by Western blotting with the anti-B chain antibody (Fig. 6A). To confirm that the Phe737 site of the S941A mutant is cleaved by the F737P mutant bound to LPS (Fig. 5), the PA tag was inserted into the Trp758–Leu759 bond of the F737P and S941A mutants, yielding the PA-F737P and PA-S941A mutants, respectively. As anticipated, the resulting PA-F737P and PA-S941A mutants were not autocatalytically converted by individual incubation in the presence of LPS (Fig. 7, A and B). Then, the PA-F737P and S941A mutants were mixed at a 1:1 ratio, incubated in the presence of LPS for 30 min, and subjected to Western blotting with anti-PA antibody. As expected, the L chain of the PA-F737P mutant was not converted to the B chain (Fig. 7C, center lane). Next, Western blotting experiments were carried out using a 1:1 mixture of the PA-S941A and F737P mutants in the presence of LPS, and the B chain derived from the PA-S941A mutant was detected by anti-PA antibody (Fig. 7C, right lane), clearly indicating that the Phe737 site of the PA-S941A mutant is intermolecularly cleaved by the F737P mutant bound to LPS. The N-terminal tripeptide of Arg36–Trp37–Arg38 of factor C is essential for LPS recognition, and the replacement of both Arg36 and Arg38 to Glu (the RE factor C mutant (factor C mutants with the replacement of both Arg36 and Arg38 to Glu)) causes the loss of LPS-binding activity (20.Koshiba T. Hashii T. Kawabata S. A structural perspective on the interaction between lipopolysaccharide and factor C, a receptor involved in recognition of Gram-negative bacteria.J. Biol. Chem. 2007; 282 (17135239): 3962-3967Abstract Full Text Full Text PDF P" @default.
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• W2807449069 title "Intermolecular autocatalytic activation of serine protease zymogen factor C through an active transition state responding to lipopolysaccharide" @default.
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