Tarantula Hemocyanin Shows Phenoloxidase Activity

Abstract

An enzyme generally catalyzes one well defined reaction with high specificity and efficiency. We report here in contrast that the copper protein hemocyanin of the tarantulaEurypelma californicum exhibits two different functions. These occur at the same active site. While hemocyanin usually is an oxygen carrier, its function can be transformed totally to monophenoloxidase and o-diphenoloxidase activity after limited proteolysis with trypsin or chymotrypsin.N-acetyldopamine (NADA) is more effectively oxidized thanl-dopa or dopamine. This irreversible functional switch of tarantula hemocyanin function is limited to the two subunitsb and c of its seven subunit types. A conserved phenylalanine in the hemocyanin molecule acts as a placeholder for other substrates that are phenylalanine derivatives. The proteolytic cleavage removes an N-terminal fragment, including the critical phenylalanine residue, which opens an entrance for substrates. Therefore no new arrangement of the active site, with its two copper atoms and the μ − η2:η2 bound O2 molecule, is necessary to develop the catalytic function. An enzyme generally catalyzes one well defined reaction with high specificity and efficiency. We report here in contrast that the copper protein hemocyanin of the tarantulaEurypelma californicum exhibits two different functions. These occur at the same active site. While hemocyanin usually is an oxygen carrier, its function can be transformed totally to monophenoloxidase and o-diphenoloxidase activity after limited proteolysis with trypsin or chymotrypsin.N-acetyldopamine (NADA) is more effectively oxidized thanl-dopa or dopamine. This irreversible functional switch of tarantula hemocyanin function is limited to the two subunitsb and c of its seven subunit types. A conserved phenylalanine in the hemocyanin molecule acts as a placeholder for other substrates that are phenylalanine derivatives. The proteolytic cleavage removes an N-terminal fragment, including the critical phenylalanine residue, which opens an entrance for substrates. Therefore no new arrangement of the active site, with its two copper atoms and the μ − η2:η2 bound O2 molecule, is necessary to develop the catalytic function. N-acetyldopamine. Although hemocyanins and phenoloxidases, both extracellular proteins in the hemolymph of arthropods, bind oxygen in a μ − η2:η2 coordination, their biological functions are very different (1Kitajima N. Morooka Y. Chem. Rev. 1994; 94: 737-757Crossref Scopus (876) Google Scholar, 2Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1996; 96: 25-26Crossref Scopus (3246) Google Scholar). Hemocyanins serve as oxygen carriers for many chelicerates and crustaceans (3van Holde K.E. Miller K.I. Adv. Protein Chem. 1995; 47: 1-81Crossref PubMed Google Scholar, 4Salvato B. Beltramini M. Life Chem. Rep. 1990; 8: 1-47Google Scholar, 5Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar). This function is well understood on the basis of the known structure of several hemocyanins (6Gaykema W.P.J. Hol W.G. Vereijken J.M. Soeter N.M. Bak H.J. Beintema J.J. Nature. 1984; 309: 23-29Crossref Scopus (308) Google Scholar, 7Volbeda A. Hol W.G.J. J. Mol. Biol. 1989; 209: 249-279Crossref PubMed Scopus (382) Google Scholar, 8Hazes B. Magnus K.A. Bonaventura C. Bonaventura J. Dauter Z. Kalk K.H. Hol W.G. Protein Sci. 1993; 2: 597-619Crossref PubMed Scopus (322) Google Scholar, 9Magnus K.A. Hazes B. Ton-That H. Bonaventura C. Bonventura J. Hol W.G.J. Proteins Struct. Funct. Genet. 1994; 19: 302-309Crossref PubMed Scopus (391) Google Scholar). Phenoloxidases are found in an inactive form in the hemolymph of Crustacea and insects (10Hernandez-Lopez J. Gollas-Galvan T. Vargas-Albores F. Comp. Biochem. Physiol. 1996; 113C: 61-66Google Scholar, 11Kopacek P. Weise C. Gotz P. Insect Biochem. Mol. Biol. 1995; 25: 1081-1091Crossref PubMed Scopus (96) Google Scholar, 12Nellaiappan K. Sugumaran M. Comp. Biochem. Physiol. 1996; 113B: 163-168Crossref Scopus (62) Google Scholar, 13Sanchez-Ferrer A. Rodriguez-Lopez J.N. Garcia-Canovas F. Garcia-Carmona F. Biochim. Biophys. Acta. 1995; 1247: 1-11Crossref PubMed Scopus (1134) Google Scholar, 14Fujimoto K. Okino N. Kawabata S. Iwanaga S. Ohnishi E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7769-7773Crossref PubMed Scopus (139) Google Scholar, 15Aspan A. Huang T.S. Cerenius L. Soderhall K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 939-943Crossref PubMed Scopus (232) Google Scholar, 16Brivio M.F. Mazzei C. Scari G. Comp. Biochem. Physiol. 1996; 113B: 281-287Crossref Scopus (17) Google Scholar, 17van Gelder C.W.G. Flurkey W.H. Wicherts H.J. Phytochemistry. 1997; 45: 1309-1323Crossref PubMed Scopus (412) Google Scholar, 18Söderhäll K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1127) Google Scholar). After limited proteolysis, the phenoloxidase so produced shows both a monophenoloxidase and ano-diphenoloxidase activity. Phenoloxidase is widespread in animals and plants as well as in fungi; it starts the synthesis of melanin, is involved in defense reactions (19Prota G. Melanins and Melanogenesis. Academic Press, San Diego1992Google Scholar, 20Sugumaran M. Kanost M. Beckage N.E. Thompson S.N. Federici A.B. Parasites and Pathogens of Insects. Academic Press, San Diego1993: 317-342Crossref Scopus (81) Google Scholar), and is also crucial for arthropod sclerotization, using N-acetyldopamine (NADA)1 as substrate (21Anderson S.O. Binnington K. Retnakan A. Physiology of the Insect Epidermis Sclerotization. 1991: 123-140Google Scholar,22Sugumaran M. Binnington K. Retnakan A. Physiology of the Insect Epidermis Sclerotization. 1991: 141-168Google Scholar). Although much is known about the biological functions of phenoloxidase, its molecular mechanism and regulation are not well understood because of the lack of any known structure (2Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1996; 96: 25-26Crossref Scopus (3246) Google Scholar, 13Sanchez-Ferrer A. Rodriguez-Lopez J.N. Garcia-Canovas F. Garcia-Carmona F. Biochim. Biophys. Acta. 1995; 1247: 1-11Crossref PubMed Scopus (1134) Google Scholar). o-Diphenoloxidase and monophenoloxidase activity were measured spectrophotometrically by recording the formation of dopachrome, with an absorption maximum at 475 nm. Iterative spectra in the region of 300–600 nm were recorded to exclude other by-products. The assay medium contained 4 mml-dopa in the case of o-diphenol oxidation and 2 mm tyrosine in the case of tyrosinase activity, which includes the monophenoloxidase and o-diphenoloxidase activities, in 0.1 m potassium phosphate buffer at pH 6.8 at 20 °C. Native polyacrylamide gel electrophoresis (5%) and crossed immuno gel electrophoresis were performed according to Lamyet al. (32Lamy J. Lamy J. Weill J. Markl J. Schneider H.-J. Linzen B. Hoppe-Seyler's Z. Physiol. Chem. 1979; 360: 889-895Crossref PubMed Scopus (42) Google Scholar) and stained by incubation in 4 mml-dopa, pH 6.8, and 0.1 m potassium phosphate at room temperature. When the protein was to be activated by the staining solution itself, then 1 mg/ml trypsin was also added. After staining, the gels were dried or stored in methanol/acetic acid/water. Limited proteolysis was performed with bovine trypsin and bovine chymotrypsin (Sigma) in 0.1 mTris/HCl buffer, pH 7.8 (in the case of the native 24-mer) and pH 9.6 (in the case of subunits) at room temperature. The ratio between hemocyanin and protease varied between 1:1 and 10:1 (w/w). Proteolysis was terminated by addition of soybean trypsin inhibitor (Sigma). The x-ray structures of subunit II of Limulus polyphemus hemocyanin(Protein Data Bank code 1oxy) and the hexameric hemocyanin of Panulirus interruptus (Protein Data Bank code 1hcy) were obtained from the Protein Data Bank, Brookhaven National Laboratory. Sequence comparison was performed using DNAstar, based on the clustal method. The various peptide sequences were obtained from the Protein Data Bank as indicated previously (28Beintema J.J. Stam W.T. Hazes B. Smidt M.P. Mol. Biol. Evol. 1994; 11: 493-503PubMed Google Scholar, 29Burmester T. Scheller K. J. Mol. Evol. 1996; 42: 713-728Crossref PubMed Scopus (115) Google Scholar, 30Basyoni M. On the Sequence of Subunit b from Tarantula HemocyaninPh.D. thesis. University of Munich, 1990Google Scholar). The hemocyanin of Eurypelma californicum hemolymph was obtained by dorsal puncture of the heart. All samples were immediately diluted 1:2 (v/v) with 0.2 m Tris/HCl, pH 8.0, 10 mmCaCl2, 10 mm MgCl2 to stabilize the protein. The samples were then centrifuged at low speed for 10 min at room temperature using a table centrifuge to remove blood cells. The hemocyanin was purified by gel filtration (TSK-HW 55 F; 0.1m Tris/HCl, pH 8.0, 5 mm CaCl2, 5 mm MgCl2 at 20 °C). The large leading peak contained purified 24-meric hemocyanin, as verified by UV spectroscopy and two-dimensional immuno gel electrophoresis. 24-meric hemocyanin was dissociated by dialysis against 0.02 m glycine/NaOH buffer, pH 9.6, at 4 °C for 4–5 days. The protein concentration was below 1 mg/ml to avoid reassociation. The native 24-meric hemocyanin of the tarantula E. californicum (23Markl J. Kempter B. Linzen B. Bijlholt M.M.C. van Bruggen E.F.J. Hoppe-Seyler's Z. Physiol. Chem. 1981; 362: 1631-1641Crossref PubMed Scopus (69) Google Scholar, 24Decker H. Sterner R. J. Mol. Biol. 1990; 211: 281-293Crossref PubMed Scopus (64) Google Scholar) seems incapable of oxidizingo-diphenols such as l-dopa and monophenols such as l-tyrosine, even after addition of urea or perchlorate at any concentration. An activity similar to phenoloxidase has recently been observed for the hemocyanins from the crustaceans Homarus americanus and Carcinus maenas and from the molluscOctopus vulgaris using catechol (25Zlateva T. di Muro P. Salvato B. Beltramini M. FEBS Lett. 1996; 384: 251-254Crossref PubMed Scopus (84) Google Scholar, 26Salvato B., Santamaria, M., Beltramini, M., Alzuet, G., and Casella, L. (1998) Biochemistry, in pressGoogle Scholar). We have now observed monophenoloxidase activity as well aso-diphenoloxidase activity for a hemocyanin from a chelicerate, the tarantula E. californicum, after limited proteolysis with serine proteases such as trypsin and chymotrypsin, by following the enzyme-specific formation of dopachrome at 475 nm (Fig. 1, A and B). Monophenoloxidase activity of activated tarantula hemocyanin starts with a long lag phase that is also characteristic for tyrosinases but not well understood (2Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1996; 96: 25-26Crossref Scopus (3246) Google Scholar, 13Sanchez-Ferrer A. Rodriguez-Lopez J.N. Garcia-Canovas F. Garcia-Carmona F. Biochim. Biophys. Acta. 1995; 1247: 1-11Crossref PubMed Scopus (1134) Google Scholar). The o-diphenoloxidase activity has no lag phase although the active sites are identical for both activities, assessed by means of absorption spectroscopy (2Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1996; 96: 25-26Crossref Scopus (3246) Google Scholar). Compared with the activity of tyrosinase from mushrooms, the activity of hemocyanin is lower by almost two orders of magnitude (Fig. 1 C). To prove that an oxygen consuming process occurs during phenoloxidase activity, oxygen consumption was followed using protease-treated 24-meric hemocyanin with various o-diphenol derivatives such as l-dopa, dopamine, and NADA (Fig. 1 D). These data represent an absolute measure for comparing the phenoloxidase activity of different substrates. Although dopamine is oxidized at the same rate as l-dopa, NADA is oxidized significantly faster. The phenoloxidase activity of tarantula hemocyanin is not dependent on the quaternary structure of the 24-mer. When investigated on polyacrylamide gel electrophoresis under conditions where destabilized 24-mers tend to dissociate, pH 8.8, 24-mers are detected as well as various intermediates and monomers (27Savel-Niemann A. Markl J. Linzen B. J. Mol. Biol. 1988; 204: 385-395Crossref PubMed Scopus (31) Google Scholar). The 24-, the 12-, and the 7-mer dissociation intermediates show phenoloxidase activity after incubation with a phenoloxidase-specific activity staining assay (Fig. 2 panel A). Among the subunits, only the broad band containing the subunit d and heterodimerbc shows phenoloxidase activity. Using crossed immuno gel electrophoresis (Fig. 2, panel B) only the heterodimer bc shows phenoloxidase activity. This observation was also confirmed by separating the heterodimerbc from the monomeric subunits a, d, e, f, and g by size-exclusion gel chromatography (TSK HW 55), where again only the heterodimerbc showed phenoloxidase activity. We believe that both theb and c subunits possess enzymic activity. Incubation in 3 and 4 m urea results in a partial dissociation of b and c from the heterodimer (Fig. 2, panel B). At 3 m urea, subunitsb and c are both visible and both show phenoloxidase activity. At higher concentrations of urea, however, all subunits become unstable, in particular subunit c becomes undetectable. This exclusive phenoloxidase activity of the heterodimerbc of tarantula hemocyanin is observed after limited proteoloysis with trypsin as well as with chymotrypsin. Based on sequence alignments (8Hazes B. Magnus K.A. Bonaventura C. Bonaventura J. Dauter Z. Kalk K.H. Hol W.G. Protein Sci. 1993; 2: 597-619Crossref PubMed Scopus (322) Google Scholar, 28Beintema J.J. Stam W.T. Hazes B. Smidt M.P. Mol. Biol. Evol. 1994; 11: 493-503PubMed Google Scholar, 29Burmester T. Scheller K. J. Mol. Evol. 1996; 42: 713-728Crossref PubMed Scopus (115) Google Scholar), including the sequence of subunitb (30Basyoni M. On the Sequence of Subunit b from Tarantula HemocyaninPh.D. thesis. University of Munich, 1990Google Scholar) (subunit c is not sequenced yet), and the x-ray structures of L. polyphemus hemocyanin subunit II (chelicerate) and of P. interruptus hemocyanin (crustacean) (6Gaykema W.P.J. Hol W.G. Vereijken J.M. Soeter N.M. Bak H.J. Beintema J.J. Nature. 1984; 309: 23-29Crossref Scopus (308) Google Scholar, 7Volbeda A. Hol W.G.J. J. Mol. Biol. 1989; 209: 249-279Crossref PubMed Scopus (382) Google Scholar, 8Hazes B. Magnus K.A. Bonaventura C. Bonaventura J. Dauter Z. Kalk K.H. Hol W.G. Protein Sci. 1993; 2: 597-619Crossref PubMed Scopus (322) Google Scholar, 9Magnus K.A. Hazes B. Ton-That H. Bonaventura C. Bonventura J. Hol W.G.J. Proteins Struct. Funct. Genet. 1994; 19: 302-309Crossref PubMed Scopus (391) Google Scholar), several cleavage positions for chymotrypsin and trypsin have been detected that are located on the surface of a chelicerate hemocyanin subunit. According to the sequence numbering of Limulus subunit II, trypsin may cleave at Arg-81 (subunitb: Arg-68), Lys-130 (subunit b: Lys-0117) and Arg-133 (subunit b: Arg-120). Chymotrypsin may cleave at Phe-83 (subunit b: Tyr-70). The most promising candidate seems to be Arg-120 on subunit b, which corresponds to Arg-176 of the closely related prophenoloxidases of the crustaceanPacifastacus leniusculus (15Aspan A. Huang T.S. Cerenius L. Soderhall K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 939-943Crossref PubMed Scopus (232) Google Scholar). This enzyme, withM r = 81,000, is activated by trypsin at Arg-176, yielding an active fragment of about 60 kDa. The other tarantula hemocyanin subunits do not have an Arg but a Lys at this position, but surprisingly they are not activated. The prophenoloxidase subunits from the insect Galleria mellonella are of similar sizes toPacifastacus prophenoloxidase, with masses of 80 and 83 kDa. After proteolysis with chymotrypsin, two active fragments with 67 and 50 kDa were found. Phenoloxidase-active Eurypelma hemocyanin fragments also have comparable masses. SDS-gel electrophoretic analysis reveals that the heterodimer bc splits into peptides with two major components of M r ≈ 50,000 and M r ≈ 55,000 after cleavage with chymotrypsin and one major component with M r ≈ 55,000 after cleavage with trypsin (Fig. 2, panel C). However, any procedure to obtain a purified pseudo-native 55-kDa fragment with full enzymatic properties has not been successful so far. In the case of tarantula hemocyanin from E. californicum, the cleavage of an N-terminal peptide including Phe-49 from the two subunits b and c seems to open the entrance to the active site, with its copper atoms, for phenolic substrates. This is independent of the association status of the subunits (Fig. 3). This highly conserved phenylalanine in hemocyanins is at a distance of 3.5 Å from the active site and serves as an allosteric trigger during the oxygenation process (8Hazes B. Magnus K.A. Bonaventura C. Bonaventura J. Dauter Z. Kalk K.H. Hol W.G. Protein Sci. 1993; 2: 597-619Crossref PubMed Scopus (322) Google Scholar, 9Magnus K.A. Hazes B. Ton-That H. Bonaventura C. Bonventura J. Hol W.G.J. Proteins Struct. Funct. Genet. 1994; 19: 302-309Crossref PubMed Scopus (391) Google Scholar). We think that Phe-49 may also act as a placeholder for phenolic substrates during the time when hemocyanin is acting only as an oxygen carrier, to conserve the hidden function as a phenoloxidase in the native but uncleaved state. Assuming the same of substrates for the phenoloxidase activity as the almost to the with of the to one of the two oxygen atoms (Fig. In this the may bind to the copper atoms to the cleavage of the oxygen molecule (2Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1996; 96: 25-26Crossref Scopus (3246) Google Scholar). The arrangement of the substrate may be by to the numbering of L. polyphemus This the low activity of hemocyanin with to other phenoloxidase and of a substrate Phe-49 at the oxygen site. The hemocyanin subunit II is K.A. Hazes B. Ton-That H. Bonaventura C. Bonventura J. Hol W.G.J. Proteins Struct. Funct. Genet. 1994; 19: 302-309Crossref PubMed Scopus (391) Google Scholar). The two copper atoms are by The is bound in a μ − η2:η2 A is to Phe-49 to a that Phe-49 as well as the are to the and to the for the of phenoloxidase activity of tarantula hemocyanin by limited proteolysis seems to be based not on a of the active site but by a for various substrates to the active site, following of the substrate The of the active site of tyrosinase with hemocyanin can also be from of the copper atoms by in both proteins M. Salvato B. M. K. Biochim. Biophys. Acta. 1990; PubMed Scopus Google Scholar). This about a entrance is by a (25Zlateva T. di Muro P. Salvato B. Beltramini M. FEBS Lett. 1996; 384: 251-254Crossref PubMed Scopus (84) Google Scholar) which shows that for catechol to the active site is by the of of the such as high concentrations of The the low phenoloxidase activity of hemocyanins americanus and C. by a of two (25Zlateva T. di Muro P. Salvato B. Beltramini M. FEBS Lett. 1996; 384: 251-254Crossref PubMed Scopus (84) Google Scholar). However, have not been to any phenoloxidase activity in the hemocyanins from P. H. americanus by proteolysis, using tyrosine as substrates. This in the hemocyanins from Crustacea and may be on the basis of their x-ray structures (8Hazes B. Magnus K.A. Bonaventura C. Bonaventura J. Dauter Z. Kalk K.H. Hol W.G. Protein Sci. 1993; 2: 597-619Crossref PubMed Scopus (322) Google Scholar, 9Magnus K.A. Hazes B. Ton-That H. Bonaventura C. Bonventura J. Hol W.G.J. Proteins Struct. Funct. Genet. 1994; 19: 302-309Crossref PubMed Scopus (391) Google Scholar). Although the active sites are highly to the active site of is by the in (Fig. which any substrate oxygen from to the active site, even after of the N-terminal is conserved in In the case of Eurypelma hemocyanin, however, a for the exclusive phenoloxidase activity of subunits b and c We may from may the high of quaternary structure of the 24-meric tarantula hemocyanin in this b and c the two identical 2 (23Markl J. Kempter B. Linzen B. Bijlholt M.M.C. van Bruggen E.F.J. Hoppe-Seyler's Z. Physiol. Chem. 1981; 362: 1631-1641Crossref PubMed Scopus (69) Google Scholar). In the case of subunit are not only and 2 are found in the hemolymph of (3van Holde K.E. Miller K.I. Adv. Protein Chem. 1995; 47: 1-81Crossref PubMed Google Scholar, 4Salvato B. Beltramini M. Life Chem. Rep. 1990; 8: 1-47Google Scholar, 5Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar). In of two large arthropods, Crustacea and have most by of which is indicated by the between phenoloxidase and hemocyanin K. Okino N. Kawabata S. Iwanaga S. Ohnishi E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7769-7773Crossref PubMed Scopus (139) Google Scholar, 15Aspan A. Huang T.S. Cerenius L. Soderhall K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 939-943Crossref PubMed Scopus (232) Google Scholar), and of a phenoloxidase activity. seems to have a function in the hemocyanin from the tarantula Eurypelma This is a in contrast to the one function with to the active site. We that the Eurypelma hemocyanin can develop two different functions using the same active site. the hemocyanins serve as oxygen At a crucial of the of arthropods, the hemocyanin function may switch to phenoloxidase activity by phenoloxidase activity is known to be necessary to the after to for against The of hemocyanin may the in the using NADA as the as is the case for many insect (21Anderson S.O. Binnington K. Retnakan A. Physiology of the Insect Epidermis Sclerotization. 1991: 123-140Google Scholar, M. Binnington K. Retnakan A. Physiology of the Insect Epidermis Sclerotization. 1991: 141-168Google Scholar). This may be by the more oxidation rate of NADA with l-dopa or dopamine (Fig. The enzymatic activity is but it may be for by the high concentration of hemocyanin in the hemolymph of the between 5 and be that were not to other phenoloxidase activity in the hemolymph of tarantula under We hemolymph with and but hemolymph by but not because of the high of the However, not for prophenoloxidase activity any cells. exclude the of any prophenoloxidase in the blood or in other cells. to the of in are in prophenoloxidases have been in the of insects and a but have not been for in chelicerates K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1127) Google Scholar). as hemocyanin are also in the of in contrast to which are in of the J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar), the following very In arthropods, hemocyanin was in and both the oxygen and the oxidation of after proteolytic This is found in the after of chelicerates from other crustaceans and a has leading to hemocyanins and (28Beintema J.J. Stam W.T. Hazes B. Smidt M.P. Mol. Biol. Evol. 1994; 11: 493-503PubMed Google Scholar, 29Burmester T. Scheller K. J. Mol. Evol. 1996; 42: 713-728Crossref PubMed Scopus (115) Google Scholar). These hemocyanins no possess the to become by proteolytic cleavage and become proteins in the The other has the to form and to oxygen and has become the prophenoloxidases in insects and a one may the basis of phenoloxidase activity because no structure of any phenoloxidase is We F. J. and N. for and K. Miller for the and with the