SDS-induced phenoloxidase activity of hemocyanins from Limulus polyphemus, Eurypelma californicum, and Cancer magister

Phenoloxidase, widely distributed among animals, plants, and fungi, is involved in many biologically essential functions including sclerotization and host defense. In chelicerates, the oxygen carrier hemocyanin seems to function as the phenoloxidase. Here, we show that hemocyanins from two ancient chelicerates, the horseshoe crab Limulus polyphemus and the tarantula Eurypelma californicum, exhibit O-diphenoloxidase activity induced by submicellar concentrations of SDS, a reagent frequently used to identify phenoloxidase activity. The enzymatic activity seems to be restricted to only a few of the heterogeneous subunits. These active subunit types share similar topological positions in the quaternary structures as linkers of the two tightly connected 2 x 6-mers. Because no other phenoloxidase activity was found in the hemolymph of these animals, their hemocyanins may act as a phenoloxidase and thus be involved in the primary immune response and sclerotization of the cuticle. In contrast, hemolymph of a more recent arthropod, the crab Cancer magister, contains both hemocyanin with weak phenoloxidase activity and another hemolymph protein with relatively strong phenoloxidase activity. The chelicerate hemocyanin subunits showing phenoloxidase activity may have evolved into a separate phenoloxidase in crustaceans.     

Hemocyanin-derived phenoloxidase activity in the spiny lobster Panulirus argus (Latreille, 1804)

Hemocyanin and phenoloxidase belong to the type-3 copper protein family, sharing a similar active center whereas performing different roles. In this study, we demonstrated that purified hemocyanin (450 kDa) from the spiny lobster Panulirus argus shows phenoloxidase activity in vitro after treatment with trypsin, chymotrypsin and SDS (0.1% optimal concentration), but it is not activated by sodium perchlorate or isopropanol. The optimal pHs of the SDS-activated hemocyanin were 5.5 and 7.0. Hemocyanin from spiny lobster behaves as a catecholoxidase. Kinetic characterization using dopamine, L-DOPA and catechol shows that dopamine is the most specific substrate. Catechol and dopamine produced substrate inhibition above 16 and 2 mM respectively. Mechanism-based inhibition was also evidenced for the three substrates, being less significant for L-DOPA. SDS-activated phenoloxidase activity is produced by the hexameric hemocyanin. Zymographic analysis demonstrated that incubation of native hemocyanin with trypsin and chymotrypsin, produced bands of 170 and 190 kDa respectively, with intense phenoloxidase activity. Three polypeptide chains of 77, 80 and 89 kDa of hemocyanin monomers were identified by SDS-PAGE. Monomers did not show phenoloxidase activity induced by SDS or partial proteolysis.     

Hemocyanin-derived phenoloxidase activity in the spiny lobster Panulirus argus (Latreille, 1804)

Hemocyanin and phenoloxidase belong to the type-3 copper protein family, sharing a similar active center whereas performing different roles. In this study, we demonstrated that purified hemocyanin (450 kDa) from the spiny lobster Panulirus argus shows phenoloxidase activity in vitro after treatment with trypsin, chymotrypsin and SDS (0.1% optimal concentration), but it is not activated by sodium perchlorate or isopropanol. The optimal pHs of the SDS-activated hemocyanin were 5.5 and 7.0. Hemocyanin from spiny lobster behaves as a catecholoxidase. Kinetic characterization using dopamine, L-DOPA and catechol shows that dopamine is the most specific substrate. Catechol and dopamine produced substrate inhibition above 16 and 2 mM respectively. Mechanism-based inhibition was also evidenced for the three substrates, being less significant for L-DOPA. SDS-activated phenoloxidase activity is produced by the hexameric hemocyanin. Zymographic analysis demonstrated that incubation of native hemocyanin with trypsin and chymotrypsin, produced bands of 170 and 190 kDa respectively, with intense phenoloxidase activity. Three polypeptide chains of 77, 80 and 89 kDa of hemocyanin monomers were identified by SDS-PAGE. Monomers did not show phenoloxidase activity induced by SDS or partial proteolysis.     

Hemocyanin in the exoskeleton of crustaceans: enzymatic properties and immunolocalization

We investigated the enzymatic properties and immunohistochemical localization of cuticular hemocyanin, a known oxygen transporter in the prawn Penaeus japonicus. The molecular weight of hemocyanin purified from the cuticle was estimated to be 67-77 k using SDS-PAGE, and the purified protein was effectively converted into a phenoloxidase-like enzyme by an SDS-treatment. The activated enzyme catalyzed the o-hydroxylation of monophenols and the oxidation of o-diphenols and was inhibited by typical inhibitors of phenoloxidase. These characteristics were nearly identical to the enzymatic properties of hemolymph hemocyanin. Immunological detection showed a diffuse distribution of hemocyanin over the exocuticle and endocuticle, and a higher signal level was observed in the latter. Based on these results, roles of hemocyanin in various physiological processes such as immune response and sclerotization of the cuticle were discussed.     

Functional and phylogenetic analyses of phenoloxidases from brachyuran (Cancer magister) and branchiopod (Artemia franciscana, Triops longicaudatus) crustaceans

Arthropod phenoloxidases catalyze the melanization and sclerotization of the new postmolt exoskeleton, and they function in the immune response. Hemocyanin, phylogenetically related to phenoloxidase, can function as a phenoloxidase under certain conditions. We investigated the relative contributions of hemocyte phenoloxidase and hemocyanin in the brachyuran crab Cancer magister, using the physiological ratio at which they occur in the hemolymph, and found that hemocyte phenoloxidase has higher activity. They both convert diphenols to o-quinones, but only the hemocyte phenoloxidase is able to catalyze the conversion of monophenols to diphenols. The quaternary structure of hemocyanin affects its reactivity as phenoloxidase. We suggest that prophenoloxidase is released from hemocytes and moves across epidermis into new exoskeleton during premolt and is activated in early postmolt. In addition to functional studies, we have determined the complete cDNA sequence of C. magister hemocyte prophenoloxidase and partial sequences from the branchiopods Artemia franciscana and Triops longicaudatus. We also sequenced C. magister cryptocyanin 2 and a hemocyanin from the amphipod Cyamus scammoni and used these and other members of the arthropod hemocyanin superfamily for phylogenetic analyses. The phylogenies presented here are consistent with the possibility that a common ancestral molecule had both phenoloxidase and reversible oxygen-binding capabilities.     

Processing of crayfish hemocyanin subunits into phenoloxidase

Hemocyanin and phenoloxidase are both copper-binding proteins involved in the immune system for a wide range of animal species. In crayfish, these proteins were purified and characterized from plasma and hemocytes, respectively. Recently, we have reported that the processing of one of the hemocyanin subunits occurs by a proteolytic cleavage under acidic conditions which results in the release of an antibacterial peptide designated as astacidin 1 from the C-terminus [J. Biol. Chem. 278 (2003) 7927]. In the present paper, we show that cleavage of crayfish hemocyanin subunit 2 at the N-terminal part results in that the processed hemocyanin exhibits phenoloxidase activity. The calculated mass of the cloned hemocyanin 2 is 78,372Da, which corresponds to the size obtained after SDS-PAGE under reducing conditions of the purified hemocyanin and pI is estimated to be 5.70. The complete hemocyanin 2 sequence shows 74% and 44% similarity with hemocyanin 1 and prophenoloxidase of crayfish, respectively. Crayfish hemocyanin exhibited phenoloxidase activity in presence of trypsin, but no activity could be detected if treated with sodium dodecyl sulfate. These results show that hemocyanin of crayfish is involved in several immune responses such as an oxygen carrier protein, as a precursor for an antibacterial peptide, and a molecule with phenoloxidase function.     

Identification and characterization of a hemocyanin-derived phenoloxidase from the crab Charybdis japonica

To determine effective activators of crab hemocyanin (Hc) and the properties of Hc-derived phenoloxidase (HdPO), Hc, for the first time, was purified from hemolymph of Charybdis japonica, and the properties of activated HdPO were studied by using L-DOPA as a substrate. Three distinct subunits were isolated, and each had a molecular mass of about 80, 75 and 70 kDa, respectively. SDS and HLS were much effective in conversion of Hc into HdPO whose PO activity was optimal at pH 7.0 and temperature of 40 °C. The Km value of the HdPO was 2.90 mM for L-DOPA and 7.33 mM for tyrosine. The PO activity of HdPO was most sensitive to 1-phenyl-2-thiourea, cysteine and ascorbic acid, and much sensitive to thio urea and sodium sulfite. Based on its inhibition characteristics and the substrate specificity, this HdPO could be classified as a kind of tyrosinase-type phenoloxidase. The PO activity of HdPO was also strongly inhibited by Cu²⁺, Zn²⁺, ethylenediaminetetraacetic acid (EDTA) and diethyldithiocarbamate (DETC). The results with EDTA, DETC, and some metal ions, combined with the perfect recovery effect of Cu²⁺ on DETC-inhibited PO activity, indicate that the HdPO is a kind of copper-containing metalloenzyme. All these imply that the Hc, as an oxygen carrier, can be activated to have PO activities by SDS or HLS, and the activated HdPO has the properties of a tyrosinase-type copper-containing phenoloxidase. This study makes us to understand more easily the multifunctions of crustacean Hc in oxygen carrier and melaninization at certain stresses in host defence as well.     

Hemocyanins and the immune response: defense against the dark arts

The innate immune response is a conserved trait shared by invertebratesand vertebrates. In crustaceans, circulating hemocytes playsignificant roles in the immune response, including the releaseof prophenoloxidases. Activated phenoloxidase (tyrosinase) participatesin encapsulation and melanization of foreign organisms as wellas sclerotization of the new exoskeleton after wound-repairor molting. Hemocyanin functions as a phenoloxidase under certainconditions and thus also participates in the immune responseand molting. The relative contributions of hemocyte phenoloxidaseand hemocyanin in the physiological ratio at which they occurin hemolymph have been investigated in the crab Cancer magister.Differences in activity, substrate affinity, and catalytic abilitybetween the two enzymes indicate that hemocytes are the predominantsource of phenoloxidase activity in crabs. In contrast, hemocyaninis the primary source of phenoloxidase activity in isopods andchelicerates whose hemocytes show no phenoloxidase activity.Quantitative PCR studies on the distribution of prophenoloxidasemRNA in the tissues of Carcinus maenas showed little effectrelative to salinity stress. Phylogenetic analysis of hemocyanin,phenoloxidase, and other members of this arthropod gene familyare consistent with the possibility that a common ancestralmolecule had both phenoloxidase and oxygen-binding capabilities.     

A potential role for water in the modulation of oxygen-binding by tarantula hemocyanin

Hemocyanin from the tarantula Eurypelma californicum is a large respiratory protein with an exceptional high cooperativity. In contrast to hemocyanins from other species, no physiological allosteric effectors other than protons have been identified so far for this 24-meric oligomer. Here we report for the first time the mediating effects of water activity on the oxygen binding properties of a hemocyanin. Oxygen binding curves were measured in presence of several concentrations of glycine and sucrose since both substances reduce water activity. A pronounced shift of the p(50) was observed in both cases but in different directions: adding sucrose shifts the p(50) towards lower values whereas presence of glycine shows the same tendency as for human hemoglobin. Furthermore, prolonged incubation in sucrose slightly distorts the oxygen binding characteristics of spider hemocyanin. Therefore, only the influence of glycine was further analysed. An analysis based on the nested MWC model indicates, that presence of glycine leads to a preferential population of the two states with lower oxygen affinity (tR and tT) compared to the high affinity states rT and rR. The results corroborate the presence of hierarchically organized interactions in this hemocyanin.     

Latent phenoloxidase activity and N-terminal amino acid sequence of hemocyanin from Bathynomus giganteus,a primitive crustacean

N-terminal amino acid sequences for the two hemocyanin subunits from the deep-sea crustacean Bathynomus giganteus have been determined by Edman degradation, providing the first sequence information for a hemocyanin from an isopod. In addition, purified hemocyanin from B. giganteus exhibited phenoloxidase activity in the presence of sodium dodecyl sulfate. Although a natural activator has not yet been identified, a preliminary study of the enzyme indicated a K(m) of 5mM for dopamine and an initial rate of 0.1 micromol per min per mg protein, values consistent with a significant role for this enzyme in the innate immune system of B. giganteus. Moreover, after separation of hemolymph by alkaline polyacrylamide gel electrophoresis, the only detectable phenoloxidase activity coincided with the two hemocyanin subunits. The hemocyanin of this primitive crustacean may fulfill dual functions, both as oxygen carrier and as the phenoloxidase crucial for host defense.     

Functional conversion of hemocyanin to phenoloxidase by horseshoe crab antimicrobial peptides

Arthropod hemocyanins and phenoloxidases serve different physiological functions as oxygen transporters and enzymes involved in defense reactions, respectively. However, they are equipped with a structurally similar oxygen-binding center. We have shown that the clotting enzyme of the horseshoe crab, Tachypleus tridentatus, functionally converts hemocyanin to phenoloxidase by forming a complex without proteolytic cleavage (Nagai, T., and Kawabata, S. (2000) J. Biol. Chem. 275, 35297-35301). Here we show that chitin-binding antimicrobial peptides of the horseshoe crab induce the intrinsic phenoloxidase activity of hemocyanin. Tachyplesin, a major Tachypleus antimicrobial peptide with an amphiphilic structure, converted the hemocyanin to phenoloxidase. Surface plasmon resonance analysis revealed the specific interaction of tachyplesin with hemocyanin at K(d) = 3.4 x 10(-)6 m. The chemical modification of Trp or Tyr in tachyplesin, but not Lys or Arg, dramatically reduced the affinity to hemocyanin, suggesting that the binding site is located in the hydrophobic face of tachyplesin. Hemocyanin has no affinity with chitin, but it significantly binds to tachyplesin-coated chitin, leading to the expression of phenoloxidase activity. The chitin coated with antimicrobial peptides may serve as a scaffold for the binding of hemocyanin, and the resulting phenoloxidase activity appears to function as a trigger of exoskeleton wound healing.     

Immunological correspondence between arthropod hemocyanin subunits. I. Scorpion (Leiurus, Androctonus) and spider (Eurypelma, Cupiennius) hemocyanin

The hemocyanins of the scorpions Leiurus quinquestriatus and Androctonus australis, the tarantula Eurypelma californicum (all 24-mers), and the lycosid spider Cupiennius salei (dodecamer) were dissociated into subunits, the subunits isolated and studied by two-dimensional immunoelectrophoresis for interspecific cross-reactivities. Androctonus hemocyanin yielded a pattern of 8 subunit types in agreement with data from Lamy et al. (1979, Arch. Biochem. Biophys. 193, 140-149). Leiurus hemocyanin is also composed of 8 immunologically distinct subunits which could be assigned to the pattern of Androctonus in a subunit-to-subunit correlation. The subunit designations 1 to 6 of Lamy et al. could be adopted for both scorpion hemocyanins; however, in the present communication, Lamy's subunits 3A/3B are designated as 3'/3", because we could not unequivocally decide if 3' = 3A and 3" = 3B or vice versa. The 7 subunit types a to g of Eurypelma hemocyanin could be correlated with the scorpion hemocyanin subunits as follows: a = 3', b = 5B, c = 3C, d = 5A, e = 6, f = 2, g = 4. Additional cross-reactivities were detected between e/4, and f/5A, respectively. No subunit of Eurypelma hemocyanin is homologous to scorpion 3", which could not be precipitated by anti-Eurypelma antiserum. Antiserum against Cupiennius hemocyanin precipitated subunit f of Eurypelma and subunits 2 and 5A of scorpion hemocyanin. The published models of quaternary structure and a possible subunit phylogeny of arachnidan hemocyanins are discussed in view of the present results.     

Hemocyanin conformational changes associated with SDS-induced phenol oxidase activation

The enzymatic activity of phenoloxidase is assayed routinely in the presence of SDS. Similar assay conditions elicit phenoloxidase activity in another type 3 copper protein, namely hemocyanin, which normally functions as an oxygen carrier. The nature of the conformational changes induced in type 3 copper proteins by the denaturant SDS is unknown. This comparative study demonstrates that arthropod hemocyanins can be converted from being an oxygen carrier to a form which exhibits phenoloxidase activity by incubation with SDS, with accompanying changes in secondary and tertiary structure. Structural characterisation, using various biophysical methods, suggests that the micellar form of SDS is required to induce optimal conformational transitions in the protein which may result in opening a channel to the di-copper centre allowing bulky phenolic substrates access to the catalytic site.     

Characterization of microtubule-associated proteins isolated from bovine adrenal gland

Following induction of hemopoiesis, poly(A)-rich RNA was prepared from the heart of the tarantula, Eurypelma californicum, and translated in rabbit reticulocyte lysates. In vitro translation products were immunoprecipitated with antiserum against whole dissociated Eurypelma hemocyanin. Analysis of the immunoprecipitate by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed a set of polypeptides comigrating with authentic Eurypelma hemocyanin. The mRNA was transcribed into cDNA, clones were constructed using the pUC9 vector and probed with a synthetic 17-mer oligonucleotide probe complementary to the amino acid sequence of the 'copper A' binding site of chelicerate hemocyanins. One clone, pHC4, contained a 1.62-kb cDNA insert, which was subcloned into phage M13. Sequence analysis by the dideoxynucleotide chain-termination method yielded a nucleotide sequence coding for 526 amino acids of Eurypelma hemocyanin subunit e.     

Switch between tyrosinase and catecholoxidase activity of scorpion hemocyanin by allosteric effectors

Phenoloxidases and hemocyanins have similar type 3 copper centers although they perform different functions. Hemocyanins are oxygen carriers, while phenoloxidases (tyrosinase/catecholoxidase) catalyze the initial step in melanin synthesis. Tyrosinases catalyze two subsequent reactions, whereas catecholoxidases catalyze only the second one. Recent results indicate that hemocyanins can also function as phenoloxidases and here we show for the first time that hemocyanin can be converted to phenoloxidase. Furthermore, its substrate specificity can be switched between catecholoxidase and tyrosinase activity depending on effectors such as hydroxymethyl-aminomethan (Tris) and Mg(2+)-ions. This demonstrates that substrate specificity is not caused by a chemical modification of the active site.     

Comparative biochemistry of eumelanogenesis and the protective roles of phenoloxidase and melanin in insects

The phenolic biopolymer eumelanin is an important skin pigment found throughout the animal kingdom. The enzyme, tyrosinase, initiates melanogenesis in mammals. The biogenesis is assisted by a number of mammalian protein factors including dopachrome tautomerase and 5,6-dihydroxyindole-2-carboxylate oxidase. Invertebrates, such as insects, employ phenoloxidase and dopachrome (decarboxylating) isomerase for melanin biosynthesis. Recently generated molecular biological and biochemical data indicate that tyrosinase and phenoloxidase are distinctly different enzymes in spite of possessing both monophenol monooxygenase activity as well as o-diphenoloxidase activity. Similarly, insect dopachrome isomerase also differs significantly from its mammalian counterpart in several of its properties including the nature of the enzymatic reaction. In addition, there are considerable differences in the eumelanogenic pathways of these two animal groups that include the utility of substrates, use of dihydroxyindoles and the nature of eumelanin pigment. Thus, the biochemistry and molecular biology of melanogenesis in mammals and insects are significantly different. The advantages of generating different eumelanin pigments and intermediates by the insects are discussed.     

Purification and characterization of phenoloxidase from the hemocytes of Eurygaster integriceps (Hemiptera: Scutelleridae)

Insects protect themselves from microbial invaders by two main immune activities, namely the cellular and humoral reactions. Phenoloxidases are oxidative enzymes that have an important role in both cell-mediated and humoral immunity. In this study, the purification and biochemical characterization of a phenoloxidase from the hemocytes of the Sunn pest Eurygaster integriceps Puton (Hemiptera: Scutelleridae) were carried out. After the final purification step, the enzyme was purified 7.31-fold with a recovery of 3.94% and a specific activity of 4.95U/mg protein. Results of the biochemical characterization showed that the purified phenoloxidase has a maximum activity at pH 6 and at 30-35°C and is stable for 24-36h. Divalent cations such as Ca(2+) and Cu(2+) significantly increased the enzymatic activity and synthetic inhibitors such as phenylthiourea significantly decreased it. The purified phenoloxidase has a molecular mass of 22kDa. The current paper represents a further step towards the characterization of humoral immunity of E. integriceps in order to develop new strategies for the biological control of the Sunn pest.     

The toxic effects of phenoloxidase from the haemolymph of tenebrionid beetles

The injection of haemolymph originating from several species of tenebrionid beetles into blowfly larvae caused a gradual paralysis accompanied by colour changes in the haemolymph of the injected test insects. It was found that the lethal effect of the haemolymph of the beetle Blaps sulcata was due to phenoloxidase. The enzyme was activated by the exposure and incubation of the haemolymph at room temperature.The identity between the toxic factor and phenoloxidase in the beetle's haemolymph was demonstrated by the following data: (1) A correlation between the rate of lethal and phenoloxidase activities during the activation process of the toxic haemolymph. (2) Phenylthiourea, a well-known inhibitor of phenoloxidase, inhibited both the enzymatic and the toxic action of the beetle's haemolymph. (3) A commercial preparation of phenoloxidase (originating from mushrooms) imitated the lethal effects and the accompanying symptoms of the toxic haemolymph. (4) Sephadex G-100 column separation of the Blaps haemolymph revealed a complete overlap between the enzymatic and lethal regions of the elution pattern.The possible effects of phenoloxidase on the haemolymph of the injected insects are discussed.     

Nested allostery in scorpion hemocyanin (Pandinus imperator)

The oxygen-binding behavior of the 24-meric hemocyanin of the scorpion Pandinus imperator and its dependence on allosteric effectors such as protons can be successfully described by the nesting model; the MWC model is not acceptable. The affinities of the four assumed conformations of the allosteric unit, the 12-meric half-molecule, are not dependent on pH whereas the three allosteric equilibrium constants decrease with decreasing proton concentration. Comparison with the oxygen-binding behavior of the 24-meric tarantula hemocyanin (Eurypelma californicum) reveals that the affinity values for the various conformations seem to be conserved for chelicerata hemocyanin.     

Effect of glutaraldehyde and lead on the activity of hepatic glucose-6-phosphatase. A biochemical and cytochemical study.

The sensitivity of mouse liver glucose-6-phosphatase activity towards glutaraldehyde fixation has been analysed by biochemical and cytochemical means. The degree of enzymatic inhibition and various enzymatic properties have been studied. Several differences have been observed in the Km determination, the sensitivity to pH 5 and the activity related to pH between fixed and unfixed enzymes. The role of Pb++ ions in the cytochemical media has also been estimated. It is concluded that several enzymatic differences appear between fixed and unfixed enzymes and that the inhibition by Pb ions is dependent on the buffer and on the amount of substrate used.