A Link between Blood Coagulation and Prophenol Oxidase Activation in Arthropod Host Defense

Phenol oxidase, a copper-containing enzyme, is widely distributed not only in animals but also in plants and fungi, which is responsible for initiating the biosynthesis of melanin. Activation of prophenol oxidase in arthropods is important in host defense. However, the prophenol oxidase-activating system remains poorly understood at the molecular level. Here we show that the coagulation cascade of the horseshoe crab Tachypleus tridentatus is linked to prophenol oxidase activation, with the oxygen carrier hemocyanin functioning as a substitute for prophenol oxidase. Tachypleus clotting enzyme functionally transforms hemocyanin to phenol oxidase, and the conversion reaches a plateau at 1:1 stoichiometry without proteolytic cleavage. The active site-masked clotting enzyme also has the same effect, suggesting that complex formation of the clotting enzyme with hemocyanin is critical for the conversion. The two systems of blood coagulation and prophenol oxidase activation may have evolved from a common ancestral protease cascade. Phenol oxidase, a copper-containing enzyme, is widely distributed not only in animals but also in plants and fungi, which is responsible for initiating the biosynthesis of melanin. Activation of prophenol oxidase in arthropods is important in host defense. However, the prophenol oxidase-activating system remains poorly understood at the molecular level. Here we show that the coagulation cascade of the horseshoe crab Tachypleus tridentatus is linked to prophenol oxidase activation, with the oxygen carrier hemocyanin functioning as a substitute for prophenol oxidase. Tachypleus clotting enzyme functionally transforms hemocyanin to phenol oxidase, and the conversion reaches a plateau at 1:1 stoichiometry without proteolytic cleavage. The active site-masked clotting enzyme also has the same effect, suggesting that complex formation of the clotting enzyme with hemocyanin is critical for the conversion. The two systems of blood coagulation and prophenol oxidase activation may have evolved from a common ancestral protease cascade. Invertebrates lack adaptive immunity and must rely completely on innate immune systems for host defense (1Hoffmann J.A. Kafatos F.C. Janeway Jr., C.A. Ezekowitz R.A.B. Science. 1999; 284: 1313-1318Crossref PubMed Scopus (2137) Google Scholar). Two such systems, the blood (hemolymph) coagulation system in the horseshoe crab Tachypleus tridentatus and the prophenol oxidase-activating system in insects and crustaceans, are sensitive nonself-recognizing cascades triggered by microbial cell wall constituents (2Iwanaga S. Kawabata S. Muta T. J. Biochem. ( Tokyo ). 1998; 123: 1-15Crossref PubMed Scopus (251) Google Scholar, 3Ratcliffe N.A. Leonard C. Rowley A.F. Science. 1984; 226: 557-559Crossref PubMed Scopus (179) Google Scholar, 4Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1082) Google Scholar). In invertebrates, two kinds of protease cascades have been well characterized at the molecular level, the Tachypleus coagulation cascade (2Iwanaga S. Kawabata S. Muta T. J. Biochem. ( Tokyo ). 1998; 123: 1-15Crossref PubMed Scopus (251) Google Scholar) and the morphogenetic cascade for determining embryonic dorsal-ventral polarity in the fly Drosophila melanogaster (5Belvin M.P. Anderson K.V. Annu. Rev. Cell. Dev. Biol. 1996; 12: 393-416Crossref PubMed Scopus (671) Google Scholar). The structural similarity of their target proteins, a Tachypleus clottable protein coagulogen and a Drosophila Toll ligand Spätzle, as well as the sequence homology between the serine proteases of the two cascades, suggests that these two functionally different cascades may have a common origin (6Bergner A. Oganessyan V. Muta T. Iwanaga S. Typke D. Huber R. Bode W. EMBO J. 1996; 15: 6789-6797Crossref PubMed Scopus (68) Google Scholar, 7Smith C.L. DeLotto R. Protein Sci. 1992; 1: 1225-1226Crossref PubMed Scopus (52) Google Scholar, 8Smith C.L. DeLotto R. Nature. 1994; 368: 548-551Crossref PubMed Scopus (69) Google Scholar). The microbial polysaccharide-mediated coagulation cascade in Tachypleusinvolves four-serine protease zymogens (2Iwanaga S. Kawabata S. Muta T. J. Biochem. ( Tokyo ). 1998; 123: 1-15Crossref PubMed Scopus (251) Google Scholar). Factor C is a biosensor against lipopolysaccharides and is autocatalytically activated to factor C̄, which then activates factor B and, in turn, factorB̄ converts the proclotting enzyme to the clotting enzyme. Another biosensor, factor G, is activated in the presence of β-1,3-glucans to factor Ḡ, which directly activates the proclotting enzyme. In insects and crustaceans, the prophenol oxidase activation system is an important part of the host defense, where it functions to detect and kill invading pathogens, as well as to synthesize melanin for wound healing and encapsulation of pathogens (3Ratcliffe N.A. Leonard C. Rowley A.F. Science. 1984; 226: 557-559Crossref PubMed Scopus (179) Google Scholar, 4Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1082) Google Scholar). However, the molecular mechanism of the prophenol oxidase activation remains poorly understood. Prophenol oxidase-activating enzymes recently cloned from insects have been shown to be homologous to Tachypleusclotting enzyme, factor B̄, or Drosophila Easter (9Muta T. Hashimoto R. Miyata T. Nishimura H. Toh Y. Iwanaga S. J. Biol. Chem. 1990; 265: 22426-22433Abstract Full Text PDF PubMed Google Scholar, 10Muta T. Oda T. Iwanaga S. J. Biol. Chem. 1993; 268: 21384-21388Abstract Full Text PDF PubMed Google Scholar, 11Lee S.-Y. Cho M.Y. Hyun J.H. Lee K.M. Homma K. Natori S. Kawabata S. Iwanaga S. Lee B.L. Eur. J. Biochem. 1998; 257: 615-621Crossref PubMed Scopus (107) Google Scholar, 12Jiang H. Wang Y. Kanost M.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12220-12225Crossref PubMed Scopus (236) Google Scholar, 13Satoh D. Horii A. Ochiai M. Ashida M. J. Biol. Chem. 1999; 274: 7441-7453Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). In the American horseshoe crab Limulus polyphemusthe induction of prophenol oxidase activity in the blood is evident by treatment with either detergents or lipids (14Nellaiappan K. Sugumaran M. Comp. Biochem. Physiol. 1996; 133B: 163-168Crossref Scopus (61) Google Scholar), but prophenol oxidase(s) from horseshoe crabs has not been identified at the molecular level. Here we show that the Tachypleuscoagulation cascade is linked to prophenol oxidase activation, and we propose that the two host defense systems of blood coagulation and prophenol oxidase activation have evolved from a common ancestral protease cascade.RESULTS AND DISCUSSIONTachypleus coagulation factors were tested to determine whether they could produce phenol oxidase activity in plasma free from hemocytes, using 4-methylcatechol as substrate (Fig.1 A). The clotting enzyme and factor B̄ efficiently produced phenol oxidase activity. However, factor C̄ and factor Ḡ, as well as trypsin, could not significantly produce phenol oxidase activity, indicating that the specific proteases are required for the production of phenol oxidase activity in Tachypleus plasma.The phenol oxidase activity in plasma disappeared with the removal of hemocyanin by ultracentrifugation, suggesting thatTachypleus hemocyanin is originally involved in the prophenol oxidase-activating system. Prophenol oxidases and hemocyanins display significant sequence similarity, and both contain two functional copper-binding sites capable of reversibly binding an oxygen molecule (21Burmester T. Scheller K. J. Mol. Evol. 1996; 42: 713-728Crossref PubMed Scopus (115) Google Scholar, 22van Holde K.E. Miller K.I. Adv. Protein Chem. 1995; 47: 1-81Crossref PubMed Google Scholar, 23Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1996; 96: 2563-2605Crossref PubMed Scopus (3135) Google Scholar). Tachypleus hemocyanin is composed of at least six subunits having the same molecular mass of 70 kDa on SDS-polyacrylamide gel electrophoresis but that are separable by an anion-exchanger column chromatography (15Takagi T. Nemoto T. J. Biochem. ( Tokyo ). 1980; 87: PubMed Scopus Google Scholar). the different phenol oxidase activity the for the sequence is A.F. W. H. T. Nemoto T. A. Science. PubMed Scopus Google the prophenol oxidases a proteolytic for activation by a specific the prophenol oxidase with a molecular mass of kDa is an active with kDa by the prophenol oxidase-activating enzyme A. L. K. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). The hemocyanin recently to phenol oxidase activity with or H. T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the with the clotting enzyme the molecular mass of 70 kDa on SDS-polyacrylamide gel electrophoresis for the that the significant phenol oxidase activity sequence of the the that the were with of the protein A.F. W. H. T. Nemoto T. A. Science. PubMed Scopus Google Scholar), indicating that proteolytic not required for the functional conversion of hemocyanin to phenol conversion of hemocyanin to phenol oxidase. the with the clotting enzyme at and the at and were for SDS-polyacrylamide gel the phenol oxidase activity of the as and the stoichiometry between the and the clotting enzyme or the proclotting enzyme. The with different of the clotting enzyme or the proclotting enzyme and the phenol oxidase activity clotting proclotting enzyme. oxidase activity of the by the active site-masked The with the clotting enzyme the active site-masked clotting enzyme factor B̄ or the active site-masked factor B̄ at 1:1 The activity of the is shown by stoichiometry of the conversion by the of the clotting enzyme a of the The clotting enzyme the in a and the phenol oxidase activity a plateau at 1:1 The specific activity to that of the oxidase, from the Lee Lee J.H. Kawabata S. Iwanaga S. Lee B.L. Mol. Cell. Scholar). for phenol such as and completely the activity of the by the clotting active site-masked clotting enzyme and factor B̄, with the for activation that the a complex with the clotting enzyme or factor B̄ to a of the and phenol oxidase activity. The stoichiometry also suggests that the Tachypleus prophenol oxidase-activating system is with a mechanism to phenol oxidase activity at the of and to of activity a proteolytic the of the clotting enzyme also the phenol oxidase activity, and the of the at an of common structural of the clotting enzyme and factor the presence of an the of (9Muta T. Hashimoto R. Miyata T. Nishimura H. Toh Y. Iwanaga S. J. Biol. Chem. 1990; 265: 22426-22433Abstract Full Text PDF PubMed Google Scholar, 10Muta T. Oda T. Iwanaga S. J. Biol. Chem. 1993; 268: 21384-21388Abstract Full Text PDF PubMed Google Scholar). are also in the of prophenol oxidase-activating enzymes S.-Y. Cho M.Y. Hyun J.H. Lee K.M. Homma K. Natori S. Kawabata S. Iwanaga S. Lee B.L. Eur. J. Biochem. 1998; 257: 615-621Crossref PubMed Scopus (107) Google Scholar, 12Jiang H. Wang Y. Kanost M.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12220-12225Crossref PubMed Scopus (236) Google Scholar, 13Satoh D. Horii A. Ochiai M. Ashida M. J. Biol. Chem. 1999; 274: 7441-7453Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) but not in of Tachypleus factor C̄ T. Miyata T. Y. T. Toh Y. Y. Iwanaga S. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and factor Muta T. Oda T. D. K. Miyata T. Iwanaga S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). the of the clotting enzyme and factorB̄ may the of these proteases with hemocyanin to on the for functional phenol the functionally an oxidase activity. and were but and were not significantly A). prophenol oxidases are to be activated by treatment with or M. H. H. and Scholar). from the and the significant phenol oxidase activity in the presence of T. M. 1996; PubMed Scopus Google Scholar). and also the phenol oxidase activity of the and the specific activity of the for to that activated by the clotting enzyme The substrate of the also to that with the activated by the clotting enzyme of the hemocyanin the with the clotting enzyme at 1:1 and the activity using and as 4-methylcatechol and phenol oxidase activity of the by a and a The with or for at and the activity using The activity of the is shown by a using the as for the substrate of the that the Tachypleus coagulation cascade is linked to prophenol oxidase activation the of the specific coagulation factors with which to the functional conversion of hemocyanin to phenol oxidase Phenol oxidases in insects and in wound healing and of the as well as of the 1999; Scopus Google Scholar). Tachypleus hemocyanin functions as an oxygen carrier it may be to phenol oxidase at the of to microbial and to wound The Tachypleus coagulation cascade to be a and system by which the host defense both blood coagulation and prophenol oxidase In insects and crustaceans, an ancestral protease cascade to the cascade in horseshoe crabs may have evolved an system of prophenol oxidase between the horseshoe crab clotting and prophenol oxidase-activating cascades, and a between and of the and prophenol oxidase-activating prophenol phenol Invertebrates lack adaptive immunity and must rely completely on innate immune systems for host defense (1Hoffmann J.A. Kafatos F.C. Janeway Jr., C.A. Ezekowitz R.A.B. Science. 1999; 284: 1313-1318Crossref PubMed Scopus (2137) Google Scholar). Two such systems, the blood (hemolymph) coagulation system in the horseshoe crab Tachypleus tridentatus and the prophenol oxidase-activating system in insects and crustaceans, are sensitive nonself-recognizing cascades triggered by microbial cell wall constituents (2Iwanaga S. Kawabata S. Muta T. J. Biochem. ( Tokyo ). 1998; 123: 1-15Crossref PubMed Scopus (251) Google Scholar, 3Ratcliffe N.A. Leonard C. Rowley A.F. Science. 1984; 226: 557-559Crossref PubMed Scopus (179) Google Scholar, 4Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1082) Google Scholar). In invertebrates, two kinds of protease cascades have been well characterized at the molecular level, the Tachypleus coagulation cascade (2Iwanaga S. Kawabata S. Muta T. J. Biochem. ( Tokyo ). 1998; 123: 1-15Crossref PubMed Scopus (251) Google Scholar) and the morphogenetic cascade for determining embryonic dorsal-ventral polarity in the fly Drosophila melanogaster (5Belvin M.P. Anderson K.V. Annu. Rev. Cell. Dev. Biol. 1996; 12: 393-416Crossref PubMed Scopus (671) Google Scholar). The structural similarity of their target proteins, a Tachypleus clottable protein coagulogen and a Drosophila Toll ligand Spätzle, as well as the sequence homology between the serine proteases of the two cascades, suggests that these two functionally different cascades may have a common origin (6Bergner A. Oganessyan V. Muta T. Iwanaga S. Typke D. Huber R. Bode W. EMBO J. 1996; 15: 6789-6797Crossref PubMed Scopus (68) Google Scholar, 7Smith C.L. DeLotto R. Protein Sci. 1992; 1: 1225-1226Crossref PubMed Scopus (52) Google Scholar, 8Smith C.L. DeLotto R. Nature. 1994; 368: 548-551Crossref PubMed Scopus (69) Google Scholar). The microbial polysaccharide-mediated coagulation cascade in Tachypleusinvolves four-serine protease zymogens (2Iwanaga S. Kawabata S. Muta T. J. Biochem. ( Tokyo ). 1998; 123: 1-15Crossref PubMed Scopus (251) Google Scholar). Factor C is a biosensor against lipopolysaccharides and is autocatalytically activated to factor C̄, which then activates factor B and, in turn, factorB̄ converts the proclotting enzyme to the clotting enzyme. Another biosensor, factor G, is activated in the presence of β-1,3-glucans to factor Ḡ, which directly activates the proclotting enzyme. In insects and crustaceans, the prophenol oxidase activation system is an important part of the host defense, where it functions to detect and kill invading pathogens, as well as to synthesize melanin for wound healing and encapsulation of pathogens (3Ratcliffe N.A. Leonard C. Rowley A.F. Science. 1984; 226: 557-559Crossref PubMed Scopus (179) Google Scholar, 4Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1082) Google Scholar). However, the molecular mechanism of the prophenol oxidase activation remains poorly understood. Prophenol oxidase-activating enzymes recently cloned from insects have been shown to be homologous to Tachypleusclotting enzyme, factor B̄, or Drosophila Easter (9Muta T. Hashimoto R. Miyata T. Nishimura H. Toh Y. Iwanaga S. J. Biol. Chem. 1990; 265: 22426-22433Abstract Full Text PDF PubMed Google Scholar, 10Muta T. Oda T. Iwanaga S. J. Biol. Chem. 1993; 268: 21384-21388Abstract Full Text PDF PubMed Google Scholar, 11Lee S.-Y. Cho M.Y. Hyun J.H. Lee K.M. Homma K. Natori S. Kawabata S. Iwanaga S. Lee B.L. Eur. J. Biochem. 1998; 257: 615-621Crossref PubMed Scopus (107) Google Scholar, 12Jiang H. Wang Y. Kanost M.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12220-12225Crossref PubMed Scopus (236) Google Scholar, 13Satoh D. Horii A. Ochiai M. Ashida M. J. Biol. Chem. 1999; 274: 7441-7453Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). In the American horseshoe crab Limulus polyphemusthe induction of prophenol oxidase activity in the blood is evident by treatment with either detergents or lipids (14Nellaiappan K. Sugumaran M. Comp. Biochem. Physiol. 1996; 133B: 163-168Crossref Scopus (61) Google Scholar), but prophenol oxidase(s) from horseshoe crabs has not been identified at the molecular level. Here we show that the Tachypleuscoagulation cascade is linked to prophenol oxidase activation, and we propose that the two host defense systems of blood coagulation and prophenol oxidase activation have evolved from a common ancestral protease cascade. AND DISCUSSIONTachypleus coagulation factors were tested to determine whether they could produce phenol oxidase activity in plasma free from hemocytes, using 4-methylcatechol as substrate (Fig.1 A). The clotting enzyme and factor B̄ efficiently produced phenol oxidase activity. However, factor C̄ and factor Ḡ, as well as trypsin, could not significantly produce phenol oxidase activity, indicating that the specific proteases are required for the production of phenol oxidase activity in Tachypleus plasma.The phenol oxidase activity in plasma disappeared with the removal of hemocyanin by ultracentrifugation, suggesting thatTachypleus hemocyanin is originally involved in the prophenol oxidase-activating system. Prophenol oxidases and hemocyanins display significant sequence similarity, and both contain two functional copper-binding sites capable of reversibly binding an oxygen molecule (21Burmester T. Scheller K. J. Mol. Evol. 1996; 42: 713-728Crossref PubMed Scopus (115) Google Scholar, 22van Holde K.E. Miller K.I. Adv. Protein Chem. 1995; 47: 1-81Crossref PubMed Google Scholar, 23Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1996; 96: 2563-2605Crossref PubMed Scopus (3135) Google Scholar). Tachypleus hemocyanin is composed of at least six subunits having the same molecular mass of 70 kDa on SDS-polyacrylamide gel electrophoresis but that are separable by an anion-exchanger column chromatography (15Takagi T. Nemoto T. J. Biochem. ( Tokyo ). 1980; 87: PubMed Scopus Google Scholar). the different phenol oxidase activity the for the sequence is A.F. W. H. T. Nemoto T. A. Science. PubMed Scopus Google the prophenol oxidases a proteolytic for activation by a specific the prophenol oxidase with a molecular mass of kDa is an active with kDa by the prophenol oxidase-activating enzyme A. L. K. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). The hemocyanin recently to phenol oxidase activity with or H. T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the with the clotting enzyme the molecular mass of 70 kDa on SDS-polyacrylamide gel electrophoresis for the that the significant phenol oxidase activity sequence of the the that the were with of the protein A.F. W. H. T. Nemoto T. A. Science. PubMed Scopus Google Scholar), indicating that proteolytic not required for the functional conversion of hemocyanin to phenol stoichiometry of the conversion by the of the clotting enzyme a of the The clotting enzyme the in a and the phenol oxidase activity a plateau at 1:1 The specific activity to that of the oxidase, from the Lee Lee J.H. Kawabata S. Iwanaga S. Lee B.L. Mol. Cell. Scholar). for phenol such as and completely the activity of the by the clotting active site-masked clotting enzyme and factor B̄, with the for activation that the a complex with the clotting enzyme or factor B̄ to a of the and phenol oxidase activity. The stoichiometry also suggests that the Tachypleus prophenol oxidase-activating system is with a mechanism to phenol oxidase activity at the of and to of activity a proteolytic the of the clotting enzyme also the phenol oxidase activity, and the of the at an of common structural of the clotting enzyme and factor the presence of an the of (9Muta T. Hashimoto R. Miyata T. Nishimura H. Toh Y. Iwanaga S. J. Biol. Chem. 1990; 265: 22426-22433Abstract Full Text PDF PubMed Google Scholar, 10Muta T. Oda T. Iwanaga S. J. Biol. Chem. 1993; 268: 21384-21388Abstract Full Text PDF PubMed Google Scholar). are also in the of prophenol oxidase-activating enzymes S.-Y. Cho M.Y. Hyun J.H. Lee K.M. Homma K. Natori S. Kawabata S. Iwanaga S. Lee B.L. Eur. J. Biochem. 1998; 257: 615-621Crossref PubMed Scopus (107) Google Scholar, 12Jiang H. Wang Y. Kanost M.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12220-12225Crossref PubMed Scopus (236) Google Scholar, 13Satoh D. Horii A. Ochiai M. Ashida M. J. Biol. Chem. 1999; 274: 7441-7453Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) but not in of Tachypleus factor C̄ T. Miyata T. Y. T. Toh Y. Y. Iwanaga S. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and factor Muta T. Oda T. D. K. Miyata T. Iwanaga S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). the of the clotting enzyme and factorB̄ may the of these proteases with hemocyanin to on the for functional phenol the functionally an oxidase activity. and were but and were not significantly A). prophenol oxidases are to be activated by treatment with or M. H. H. and Scholar). from the and the significant phenol oxidase activity in the presence of T. M. 1996; PubMed Scopus Google Scholar). and also the phenol oxidase activity of the and the specific activity of the for to that activated by the clotting enzyme The substrate of the also to that with the activated by the clotting enzyme of the hemocyanin the with the clotting enzyme at 1:1 and the activity using and as 4-methylcatechol and phenol oxidase activity of the by a and a The with or for at and the activity using The activity of the is shown by a using the as for the substrate of the that the Tachypleus coagulation cascade is linked to prophenol oxidase activation the of the specific coagulation factors with which to the functional conversion of hemocyanin to phenol oxidase Phenol oxidases in insects and in wound healing and of the as well as of the 1999; Scopus Google Scholar). Tachypleus hemocyanin functions as an oxygen carrier it may be to phenol oxidase at the of to microbial and to wound The Tachypleus coagulation cascade to be a and system by which the host defense both blood coagulation and prophenol oxidase In insects and crustaceans, an ancestral protease cascade to the cascade in horseshoe crabs may have evolved an system of prophenol oxidase between the horseshoe crab clotting and prophenol oxidase-activating cascades, and a between and of the and prophenol oxidase-activating prophenol phenol Tachypleus coagulation factors were tested to determine whether they could produce phenol oxidase activity in plasma free from hemocytes, using 4-methylcatechol as substrate (Fig.1 A). The clotting enzyme and factor B̄ efficiently produced phenol oxidase activity. However, factor C̄ and factor Ḡ, as well as trypsin, could not significantly produce phenol oxidase activity, indicating that the specific proteases are required for the production of phenol oxidase activity in Tachypleus The phenol oxidase activity in plasma disappeared with the removal of hemocyanin by ultracentrifugation, suggesting thatTachypleus hemocyanin is originally involved in the prophenol oxidase-activating system. Prophenol oxidases and hemocyanins display significant sequence similarity, and both contain two functional copper-binding sites capable of reversibly binding an oxygen molecule (21Burmester T. Scheller K. J. Mol. Evol. 1996; 42: 713-728Crossref PubMed Scopus (115) Google Scholar, 22van Holde K.E. Miller K.I. Adv. Protein Chem. 1995; 47: 1-81Crossref PubMed Google Scholar, 23Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1996; 96: 2563-2605Crossref PubMed Scopus (3135) Google Scholar). Tachypleus hemocyanin is composed of at least six subunits having the same molecular mass of 70 kDa on SDS-polyacrylamide gel electrophoresis but that are separable by an anion-exchanger column chromatography (15Takagi T. Nemoto T. J. Biochem. ( Tokyo ). 1980; 87: PubMed Scopus Google Scholar). the different phenol oxidase activity the for the sequence is A.F. W. H. T. Nemoto T. A. Science. PubMed Scopus Google Scholar). the prophenol oxidases a proteolytic for activation by a specific the prophenol oxidase with a molecular mass of kDa is an active with kDa by the prophenol oxidase-activating enzyme A. L. K. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). The hemocyanin recently to phenol oxidase activity with or H. T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the with the clotting enzyme the molecular mass of 70 kDa on SDS-polyacrylamide gel electrophoresis for the that the significant phenol oxidase activity sequence of the the that the were with of the protein A.F. W. H. T. Nemoto T. A. Science. PubMed Scopus Google Scholar), indicating that proteolytic not required for the functional conversion of hemocyanin to phenol oxidase. The stoichiometry of the conversion by the of the clotting enzyme a of the The clotting enzyme the in a and the phenol oxidase activity a plateau at 1:1 The specific activity to that of the oxidase, from the Lee Lee J.H. Kawabata S. Iwanaga S. Lee B.L. Mol. Cell. Scholar). for phenol such as and completely the activity of the by the clotting enzyme. The active site-masked clotting enzyme and factor B̄, with the for activation that the a complex with the clotting enzyme or factor B̄ to a of the and phenol oxidase activity. The stoichiometry also suggests that the Tachypleus prophenol oxidase-activating system is with a mechanism to phenol oxidase activity at the of and to of activity a proteolytic the of the clotting enzyme also the phenol oxidase activity, and the of the at an of common structural of the clotting enzyme and factor the presence of an the of (9Muta T. Hashimoto R. Miyata T. Nishimura H. Toh Y. Iwanaga S. J. Biol. Chem. 1990; 265: 22426-22433Abstract Full Text PDF PubMed Google Scholar, 10Muta T. Oda T. Iwanaga S. J. Biol. Chem. 1993; 268: 21384-21388Abstract Full Text PDF PubMed Google Scholar). are also in the of prophenol oxidase-activating enzymes S.-Y. Cho M.Y. Hyun J.H. Lee K.M. Homma K. Natori S. Kawabata S. Iwanaga S. Lee B.L. Eur. J. Biochem. 1998; 257: 615-621Crossref PubMed Scopus (107) Google Scholar, 12Jiang H. Wang Y. Kanost M.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12220-12225Crossref PubMed Scopus (236) Google Scholar, 13Satoh D. Horii A. Ochiai M. Ashida M. J. Biol. Chem. 1999; 274: 7441-7453Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) but not in of Tachypleus factor C̄ T. Miyata T. Y. T. Toh Y. Y. Iwanaga S. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and factor Muta T. Oda T. D. K. Miyata T. Iwanaga S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). the of the clotting enzyme and factorB̄ may the of these proteases with hemocyanin to on the for functional conversion. phenol the functionally an oxidase activity. and were but and were not significantly A). prophenol oxidases are to be activated by treatment with or M. H. H. and Scholar). from the and the significant phenol oxidase activity in the presence of T. M. 1996; PubMed Scopus Google Scholar). and also the phenol oxidase activity of the and the specific activity of the for to that activated by the clotting enzyme The substrate of the also to that with the activated by the clotting enzyme that the Tachypleus coagulation cascade is linked to prophenol oxidase activation the of the specific coagulation factors with which to the functional conversion of hemocyanin to phenol oxidase Phenol oxidases in insects and in wound healing and of the as well as of the 1999; Scopus Google Scholar). Tachypleus hemocyanin functions as an oxygen carrier it may be to phenol oxidase at the of to microbial and to wound The Tachypleus coagulation cascade to be a and system by which the host defense both blood coagulation and prophenol oxidase In insects and crustaceans, an ancestral protease cascade to the cascade in horseshoe crabs may have evolved an system of prophenol oxidase L. Lee for the also S. Iwanaga for and W. for