Functional and Phylogenetic Analyses of Phenoloxidases from Brachyuran (Cancer magister) and Branchiopod (Artemia franciscana, Triops longicaudatus) Crustaceans

Reference: Biol. Bull. 210: 38 –50. (February 2006) © 2006 Marine Biological Laboratory

Functional and Phylogenetic Analyses of Phenoloxidases from Brachyuran (Cancer magister) and Branchiopod (Artemia franciscana, Triops longicaudatus) Crustaceans NORA B. TERWILLIGER* AND MARGARET C. RYAN Oregon Institute of Marine Biology, University of Oregon, PO Box 5389, Charleston, Oregon 97420

Introduction

Abstract. 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 oxygenbinding capabilities.

Phenoloxidase is an enzyme that protects an arthropod both externally and internally. The protein catalyzes the hardening, or sclerotization, of an animal’s newly formed or repaired exoskeleton, and it also mounts an immune response internally against invading organisms in the bloodstream (Ashida and Brey, 1995; So¨derha¨ll and Cerenius, 1998; Terwilliger, 1999). Phenoloxidase activity has been described in many arthropods, including crustaceans, insects, and chelicerates (Ashida and Yamazaki, 1990; Aspa´n and So¨derha¨ll, 1991; Nellaiappan and Sugumaran, 1996). It circulates in the hemolymph in an inactive form, prophenoloxidase, in crustacean and insect hemocytes. The conversion of the proenzyme to the active form is an extensively studied system of enzymatic checks and balances known as the phenoloxidase cascade that closely regulates the release and activation of phenoloxidase at the appropriate time and place (Ashida and Yamazaki, 1990; Sugumaran and Kanost, 1993; So¨derha¨ll and Cerenius, 1998). Phenoloxidases catalyze the hydroxylation of monophenols to o-diphenols (tyrosinase or monophenolase activity) and the oxidation of o-diphenols to o-quinones (catecholase or diphenolase activity). Enzymes that can catalyze both reactions are sometimes referred to as tyrosinases (EC 1.14.18.1), while those that carry out only the latter reaction may be defined as catecholoxidases (EC 1.10.3.1). Phenoloxidases are widely distributed among almost all organisms and participate in several critical functions; they are commonly recognized for their role in the browning of fruit or mushrooms (Sa´nchez-Ferrer et al., 1995; Van Gelder et al., 1997). In arthropods, the highly reactive o-quinones participate in cross-linking, or sclerotizing, the proteins in the soft, flexible exoskeleton after the molt (Andersen et al.,

Received 13 October 2005; accepted 13 December 2005. * To whom correspondence should be addressed. E-mail: nterwill@ darkwing.uoregon.edu Abbreviations: HAC, HEPES anticoagulent buffer; HFH, hemocyte-free hemolymph; PAGE, polyacrylamide gel electrophoresis. 38

HEMOCYANIN AND HEMOCYTE PHENOLOXIDASES

1996; Sugamaran, 1998). The o-quinones are also on the pathway of melanin synthesis, a compound with antimicrobial, antifungal, and antiviral properties, and thus phenoloxidases are important components of the innate immune response in invertebrates. The enzymatic activity of all phenoloxidases relies on the dioxygen-copper binding site that is common to both phenoloxidase and the oxygen transport protein, hemocyanin (Solomon et al., 1994; Decker and Terwilliger, 2000; Jaenicke and Decker, 2004). Phenoloxidases can be separated into two types on the basis of their sequence similarities to the hemocyanins, to which they are phylogenetically related. One type is more closely related to molluscan hemocyanins; the other, found in arthropods, shows high similarity to arthropod hemocyanins (Durstewitz and Terwilliger, 1997; Van Gelder et al., 1997; van Holde et al., 2001; Burmester, 2002). Hemocyanin can undergo a functional transition from reversibly binding oxygen to oxidizing diphenols into o-quinones (Bhagvat and Richter, 1938; Zlateva et al., 1996; Salvato et al., 1998; Decker and Rimke, 1998; Decker et al., 2001). Clotting proteins and endogenous antimicrobial peptides have each been shown to induce phenoloxidase activity in hemocyanin of the horseshoe crab Tachypleus tridentatus, results that suggest the phenomenon could occur in vivo (Nagai and Kawabata, 2000; Nagai et al., 2001). Regulation of the conversion of hemocyanin function from oxygen transport to phenoloxidase activity is unknown, however, as are the physiological circumstances under which hemocyanin might be utilized as a phenoloxidase in crustaceans. The source of phenoloxidase activity differs among various groups of arthropods. The primary activity in insects, shrimp, and crayfish is due to a prophenoloxidase protein found in the hemocytes (Aspa´n and So¨derha¨ll, 1991). In some crustaceans that have hemocyanin circulating as extracellular hexamers and multihexamers in the hemolymph, the hemocyanin can be induced to also show phenoloxidase activity (Decker and Jaenicke, 2004). In certain other crustaceans and in chelicerates, phenoloxidase activity in the hemolymph appears to be due only to the hemocyanin, since no o-diphenolase activity has been observed in hemocytes (Decker et al., 2001; Pless et al., 2003). These overlapping differences in source of phenoloxidase are intriguing, in part because they do not fall into a strictly phyletic pattern, and they stimulate questions about the evolution of hemocyanin and phenoloxidase. Here we compare the phenoloxidase activities and substrate specificities of proteins that co-occur in the hemolymph of the brachyuran crab Cancer magister, as a step toward understanding their different roles. We find that the primary phenoloxidase activity is in the hemocytes, but both 2-hexamer and 1-hexamer oligomers of crab hemocyanin are capable of catecholase activity. We describe the complete cDNA sequence of the hemocyte phenoloxidase from C. magister. We have also obtained partial sequences of

39

phenoloxidases from the branchiopods Triops longicaudatus and Artemia franciscana, crustaceans that synthesize extracellular hemoglobin for oxygen transport. We present phylogenetic comparisons of the deduced amino acid sequences of these phenoloxidases with those of other arthropod hemolymph proteins, including phenoloxidases, hemocyanins, cryptocyanins, hexamerins, and the putative phenoloxidase from the tunicate Ciona intestinalis to further elucidate the evolution of protein and function in the hemocyanin gene family. Materials and Methods Sample preparation for phenoloxidase assays Cancer magister (Dana) intermolt male adult crabs were caught in Coos Bay, Oregon. Hemolymph from C. magister was collected into a 10-fold volume of ice-cold HEPES anticoagulant buffer (HAC) (Vargas-Albores et al., 1996) using an ice-cold syringe and needle. The hemolymph was centrifuged at 200 ⫻ g for 5 min at 4 °C. The hemocyte pellet was separated from the hemolymph supernatant and washed three times with cold HAC to obtain a preparation of hemocytes free from other hemolymph components. Washed hemocytes were resuspended in a volume of HAC equivalent to their original hemolymph volume; i.e., cells from 1 ml of hemolymph were resuspended in 1 ml of HAC. The hemolymph supernatant was also centrifuged three more times to remove any remaining cells, resulting in a preparation of hemocyte-free hemolymph (HFH). All samples were stored on ice until use that same day. Total protein concentration was determined by the Bradford method, using bovine serum albumin as a standard. Gel filtration chromatography was used to separate twohexamer 25S hemocyanin from one-hexamer 16S hemocyanin. A sample of HFH prepared as described above in a five-fold volume of HAC was concentrated with a Centricon-100 (Amicon) centrifugal filter. The concentrate was applied to a 1.6 ⫻ 88 cm column of Sephadex G-200 equilibrated with 0.1 M Tris-HCl, 0.1 M NaCl, 10 mM CaCl2, 10 mM MgCl2, pH 7.5, at 4 °C. Leading and trailing edges of the peak were concentrated and re-chromatographed to obtain samples that contained primarily twohexamer or one-hexamer hemocyanin. The concentrations of the samples were determined using the extinction coefficient for C. magister hemocyanin, where 1.0 OD280 ⫽ 1 mg/ml. Gel assays for phenoloxidase activity Samples were electrophoresed on pH 7.4, non-denaturing, non-dissociating, 5% polyacrylamide gels (PAGE) (Terwilliger and Terwilliger, 1982) or pH 8.9, non-denaturing, dissociating, 7.5% PAGE (Davis, 1964). A phenoloxidase activity reaction using dopamine as the substrate was

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N. B. TERWILLIGER AND M. C. RYAN

done immediately after electrophoresis (Nellaiappan and Vinayakam, 1993). After the reaction results were photographed, the gel was stained in Coomassie Blue to identify protein bands. Two-hexamer 25S and one-hexamer 16S hemocyanins of C. magister were used as calibrant proteins on both pH 7.4 and 8.9 PAGE (Terwilliger and Terwilliger, 1982). Spectrophotometric assays for phenoloxidase activity An assay for phenoloxidase activity was run according to the dopachrome method (Mason, 1947) using a Beckman DU 640 spectrophotometer. The diphenol substrates tested were dopamine, L-dopa and catechol, and the monophenol substrate tested was tyramine. Final concentrations of assay components were 100 mM sodium phosphate buffer, pH 7.5; 1 mM substrate; and either hemocytes, hemocyanin, or HFH samples. Hemocyanin and HFH samples were at a final concentration of 1 mg/ml protein, and an equal volume of resuspended hemocytes was used. After mixing and determination of baseline, 0.1% sodium dodecyl sulfate (SDS) was added and the reaction was immediately monitored for 10 min for diphenolase and up to 40 min for monophenolase activity. In certain monophenol reactions, 1 ␮M dopamine was added as a cofactor to decrease the lag period (Naraoka et al., 2003). The reactions were monitored at 475 nm for dopamine, L-dopa, and tyramine and at 400 nm for catechol. As negative controls, assays minus SDS or protein sample were monitored over the same time periods. Phenoloxidase-specific activity is defined as the change in optical density over 10 min per milligram of total protein. Vmax and Km were determined using the Beckman DU 640 Enzyme Mechanism Analysis software. The reaction mixture was as described above and consisted of a constant amount of enzyme (either hemocytes or HFH) and substrate concentrations ranging from 0.01 to 1.25 mM for dopamine and from 0.05 to 10 mM for L-dopa and catechol. The reactions were initiated with 0.1% SDS and monitored at 10-s intervals over 120 s at the appropriate wavelength. Spectrophotometric assay reactions were done in triplicate and reported as mean ⫾ standard deviation (SD). cDNA sequences Hemocyte pellets were prepared from C. magister hemolymph in HAC as described above. Pellets were frozen in liquid nitrogen and stored at ⫺80 °C until RNA isolation with TRI Reagent (Chomczynski and Sacchi, 1987). Artemia franciscana and Triops longicaudatus cysts (Carolina Biologicals) were hatched and cultured in the laboratory. Live specimens of Cyamus scammoni were collected from freshly dead gray whales (Eschrictius robustus) that had washed ashore near Coos Bay, Oregon. Whole specimens of A. franciscana, T. longicaudatus, and C. scammoni were frozen and ground in liquid nitrogen, and total RNA was

isolated with TRI Reagent. Total RNA isolation was followed by Dnase I digestion (Ambion, DNA-free). The RNA was reverse-transcribed with PowerScript reverse transcriptase (BD Biosciences). Hemocyanin sequences were initially amplified with degenerate crustacean hemocyanin primers based on conserved regions near the Copper A site: MNE (Sense): GAA YGA RGG NGA RTT YGT NTA YGC and CuA (Antisense): CGV ACN GTN ARY TGR TGR TGV ACC CA. Amplification resulted in 350-bp products that were cloned (Invitrogen TA cloning kit) and sequenced (Oregon State University Center for Gene Research and Biotechnology). The sequences were used to design additional primers to further amplify hemocyanin using the Clontech SMART RACE protocol (BD Biosciences). We also obtained a cDNA sequence for another C. magister cryptocyanin subunit, Cc2. Since arthropod phenoloxidase sequences are more similar to those of the chelicerate hemocyanins than to crustacean hemocyanins (Durstewitz and Terwilliger, 1997; Terwilliger et al., 1999), we used degenerate chelicerate hemocyanin primers (Parkinson et al., 2001; Kusche et al., 2002) to amplify crustacean prophenoloxidases. The resulting 1100-bp products were cloned and sequenced as described above. Using these sequences, 5⬘ and 3⬘ RACE primers were designed for C. magister prophenoloxidase, resulting in the complete cDNA sequence. Partial prophenoloxidase sequences were obtained for A. franciscana and T. longicaudatus, using the same chelicerate hemocyaninbased primers. Nucleotide and deduced amino acid sequences were compared with NCBI BLAST search programs (Altschul et al., 1990). Analysis for the presence of a signal peptide in C. magister prophenoloxidase was performed with SignalP 3.0 (Bendtsen et al., 2004).

Phylogenetic analyses Additional sequences for phylogenetic analyses were retrieved from GenBank and aligned with Clustal W (Thompson et al., 1994). The alignments of the conserved Copper A- and Copper B-binding site histidines were verified. Trees were constructed using parsimony with the PAUP program version 4 Beta 10 (Swofford, 2002). Maximum parsimony was used to estimate the evolutionary relationships of these proteins because of its ability to perform substantially better than current parametric methods over a wide range of conditions tested (Kolaczkowski and Thornton, 2004). Bootstrap percentages were based on 1000 replicates. The crustacean phenoloxidase trees were generated with alignments of the three partial amino acid sequences that we obtained by amplification using the chelicerate primers described above and the corresponding region in six other

HEMOCYANIN AND HEMOCYTE PHENOLOXIDASES

published crustacean phenoloxidases. A total of 426 characters were aligned in this analysis. Another phylogenetic analysis included 73 sequences consisting of 21 crustacean, 3 insect, 13 chelicerate, 4 myriapod, and 1 onychophoran hemocyanins; 4 crustacean cryptocyanins; 7 crustacean and 9 insect phenoloxidases; 9 insect hexamerins; and 2 putative phenoloxidases from the tunicate Ciona intestinalis. Amino acid sequences that corresponded to the 5⬘ sequence of C. intestinalis 1 (missing in C. intestinalis 2) (Immisberger and Burmester, 2004) and the 3⬘ regions of C. intestinalis 1 and 2 that do not align with other hemocyanins and phenoloxidases were removed from the data set, resulting in a total of 712 aligned characters. Results Circulating hemocytes are the predominant source of phenoloxidase activity in fresh hemolymph of the crab Cancer magister. After electrophoresis of samples on pH 7.4 PAGE and activation by SDS, a strong phenoloxidase reaction can be seen in a slowly migrating band in whole hemolymph (Fig. 1b) and in washed hemocytes (Fig. 1h), but not in HFH (Fig. 1e). Hemocyanin electrophoreses as two bands on pH 7.4 PAGE—a moderately slow migrating band corresponding to 25S two-hexamers and a faster migrating band of 16S one-hexamers (Fig. 1) (Terwilliger et al., 1999). Phenoloxidase activity by two-hexamer and onehexamer hemocyanins in either whole hemolymph (Fig. 1b) or HFH (Fig. 1e) is not detectable at the gel loading concentrations used. Lanes 1c, f, and i confirm the relative electrophoretic mobilities of the phenoloxidase and hemocyanin proteins. In whole hemolymph, the protein concentration of hemocyte phenoloxidase is much lower than that of the hemocyanins (Fig. 1a), while the enzyme activity of

Figure 1. Phenoloxidase activity of Cancer magister hemolymph proteins after pH 7.4 PAGE. Lanes a, b, c are whole hemolymph; d, e, f are hemocyte-free hemolymph; g, h, i are washed hemocytes. Lanes a, d, g are stained for protein with Coomassie Blue; b, e, h are phenoloxidase-activity reactions using dopamine as the substrate; c, f, i are stained with Coomassie Blue after the phenoloxidase-activity reaction to confirm the positions of the bands.

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the hemocytes is greater (Fig. 1b). The total protein concentration of hemocytes in this hemolymph sample was 0.5 mg/ml as compared to 29 mg/ml in the HFH. We deliberately avoided using premolt crabs that contained the copperfree protein cryptocyanin in their hemolymph (Terwilliger et al., 2005) or ovigerous females with circulating vitellogenins (Terwilliger, 1991b), because the presence of these proteins would have diluted the proportion of hemocyanin in the total protein measurements of hemolymph. The absence of a band showing phenoloxidase activity in HFH (Fig. 1e) confirms that neither lysed nor intact hemocytes were present in the sample. Although phenoloxidase activity of the oligomeric hemocyanins is not detectable here, individual hemocyanin subunits do show activity after dissociation and electrophoresis on pH 8.9 PAGE (data not shown). In spectrophotometric assays, both hemocytes and hemocyanin demonstrate phenoloxidase activity, but with marked differences in substrate specificity and relative reactivity. Washed hemocytes show a rapid and strong response to the diphenol substrates dopamine and L-dopa, and a slightly lower response to catechol (Fig. 2a). The magnitudes of the diphenol reactions are much lower for HFH than for hemocyte phenoloxidase when assayed at concentrations equivalent to those found in the hemolymph of that crab (Fig. 2b). HFH shows a stronger response to dopamine and catechol than to L-dopa. Comparison between purified hemocyanins shows two-hexamer hemocyanin to have a lower phenoloxidase activity to the three diphenol substrates tested than does one-hexamer hemocyanin when measured at the same protein concentration (Fig. 3). Kinetic analyses indicate that hemocyte phenoloxidase has high specific activities for all diphenol substrates, with L-dopa and dopamine greater than that of catechol (Table 1). HFH shows much lower activities overall, and L-dopa is a poorer substrate than dopamine or catechol. Vmax values of hemocytes are fairly similar for all substrates, although the Km values reflect differences in affinity (Table 2). The Vmax values of the HFH, although lower than those of the hemocytes, are also similar to one another, while the Km values reflect a broad range of affinities for the three substrates. Both hemocytes and HFH show a higher affinity for dopamine, followed by catechol and L-dopa. Hemocyte phenoloxidase is capable of converting the monophenol substrate tyramine to a diphenol (Fig. 4). The lag time for the monophenolase reaction, which can take as long as 40 min, can be decreased by adding 1 ␮M dopamine to the reaction mixture (Naraoka et al., 2003). Hemocytefree hemolymph, however, shows no reactivity to tyramine (data not shown). Thus hemocyte phenoloxidase shows both monophenolase and diphenolase reactivity, while hemocyanin has only the diphenolase reactivity under these experimental conditions. A prophenoloxidase was amplified from hemocyte mRNA

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The cDNA for C. magister prophenoloxidase is 2880 bp and translates into a protein of 700 amino acids with a predicted molecular mass of 80,531 kDa. The N-terminal region of the deduced amino acid sequence lacks a signal peptide. Alignment of the amino acid sequence with other prophenoloxidases (Fig. 5) shows that the two copper-binding sites are conserved, including the six histidines, along with a potential proteolytic cleavage site and a thiol ester site (Hall et al., 1995; Hartzer et al., 2005). We also obtained partial sequences for prophenoloxidases from Artemia franciscana (Genbank DQ230984) and Triops longicaudatus (Genbank DQ230985) that included the copper-binding sites. The Artemia sequence had an apparent 225-bp deletion near the 3⬘ end of the fragment. In addition, we amplified and sequenced a second cryptocyanin from C. magister (GenBank DQ230982) and a hemocyanin from Cyamus scammoni (GenBank DQ230983). These sequences were used in the phylogenetic analysis (see below). All of the sequences we amplified were analyzed by BLAST search programs and ClustalW alignments. In a phylogenetic comparison of an 1100-bp region conserved in seven crustacean prophenoloxidases, the enzymes from the Branchiopoda are distinguished from those of the malacostracan Decapoda (Fig. 6). The putative prophenoloxidase sequences of the ascidian chordate Ciona intestinalis were used as the outgroup because they are outside the arthropod phylogeny. A phylogenetic comparison of the complete prophenoloxidase sequences among multiple members of the arthropod copper-dioxygen gene family reveals distinct patterns of clustering (Fig. 7). One clade of arthropod proteins is composed of the crustacean and insect hemocyanins, crustacean cryptocyanins, and insect hexamerins. Crustacean and insect hemocyte phenoloxidases form another clade. Other clusters include the chelicerate, myriapod, and onychophoran hemocyanins. Discussion

Figure 2. Diphenolase activity of (a) washed hemocytes and (b) hemocyte-free hemolymph of individual specimens of Cancer magister. Spectrophotometric assay using 1 mM o-diphenolic substrates. Hemocytefree hemolymph was adjusted to 1 mg/ml final reaction concentration; aliquot of washed, resuspended hemocytes from same hemolymph sample was diluted to equivalent volume for this concentration of hemolymph. Reactions were performed at 475 nm for dopamine and L-dopa and at 400 nm for catechol. Each curve represents a mean of three reactions. Note that the y-axis scale in (a) is 10-fold greater than that in (b).

of C. magister, and the complete cDNA sequence was determined (GenBank DQ230981). No product was obtained using RNA from hepatopancreas or hypodermis, indicating that the primary site of synthesis of prophenoloxidase is the hemocyte.

The brachyuran crab Cancer magister has two distinct sources of phenoloxidase, an enzyme that is crucial for immunodefense and successful molting. Both sources of enzyme activity, hemocytes and hemocyanin, circulate in the hemolymph. When one compares the relative contributions of the two in an aliquot of crab hemolymph, the enzyme in the hemocytes clearly contributes most of the phenoloxidase activity, even though the hemocyanin is present in much higher concentration. Accurate measurement of the phenoloxidase activity of hemocyanin, therefore, necessitates immediate and careful removal of intact hemocytes from the hemolymph to avoid contamination by a highly active hemocyte lysate. The prophenoloxidasespecific activities differ markedly between the hemocyte and HFH samples. The enzymes in the two samples each show a higher affinity for dopamine than for L-dopa, both of which are naturally occurring substrates. The data, although

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HEMOCYANIN AND HEMOCYTE PHENOLOXIDASES

Figure 3. Diphenolase activity of (a) two-hexamer hemocyanin and (b) one-hexamer hemocyanin of Cancer magister. Spectrophotometric assay using 1 mM o-diphenolic substrates; hemocyanins adjusted to 1 mg/ml final concentration. Reactions were performed at 475 nm for dopamine and L-dopa and at 400 nm for catechol.

obtained on lysed hemocytes and cell-free hemolymph, suggest that the functional differences between hemocyte phenoloxidase and hemocyanin may be related to conformational change and the catalytic rate constants and not simply to enzyme-substrate affinity. Carrying these experiments out at the physiological ratios of hemocyte phenoloxidase and hemocyanin that occur in the whole hemolymph of the crab provides information on how the two function in vivo. Further studies to compare the molecular structure and functional properties of purified phenoloxidase and hemocyanin would also be useful. The hemocyte phenoloxidase of C. magister differs from the hemocyanin in its catalytic ability as well as its relative activity. Both monophenolase and diphenolase reactions

can be carried out by hemocyte phenoloxidase of the crab. This discovery allies brachyuran hemocyte phenoloxidase with those of penaeidin and astacean crustaceans and insects (Aspa´n et al., 1995; Ashida and Brey, 1997; Gollas-Galva´n et al., 1999; Jaenicke and Decker, 2003). In contrast, activated hemocyanin from the crab is capable of catalyzing only the diphenolase reaction. This more limited catalytic ability is similar to that in other arthropod hemocyanins that have been tested for both types of activity (Jaenicke and Decker, 2003; Nagai et al., 2001; Pless et al., 2003). A few examples of hemocyanins that sometimes show both monoTable 2 Diphenolase kinetics of Cancer magister hemocytes and hemocyte-free hemolymph

Table 1

Sample

Phenoloxidase-specific activity of Cancer magister hemocytes and hemocyte-free hemolymph

Hemocytes

Substrate

Vmax (mM min⫺1)

Km (mM)

Dopamine

0.158 ⫾ 0.024 0.236 ⫾ 0.006 0.221 ⫾ 0.022 0.015 ⫾ 0.001 0.012 ⫾ 0.002 0.009 ⫾ 0

0.025 ⫾ 0.004 1.705 ⫾ 0.311 0.572 ⫾ 0.117 0.049 ⫾ 0.020 2.875 ⫾ 0.919 0.192 ⫾ 0.049

L-dopa

Substrate

Hemocytes

Hemocyte-free hemolymph HFH

Dopamine L-dopa

Catechol

267.16 ⫾ 32.33 300.31 ⫾ 15.17 176.19 ⫾ 18.16

1.55 ⫾ 0.19 0.51 ⫾ 0.21 0.88 ⫾ 0.27

Specific phenoloxidase activity is defined as the change in optical density over 10 minutes per milligram of total protein. Reactions were performed at 475 nm for dopamine and L-dopa and at 400 nm for catechol. Values given are means ⫾ SD.

Catechol Dopamine L-dopa Catechol

Reactions were performed at 475 nm for dopamine and L-dopa and at 400 nm for catechol. Hemocyte-free hemolymph was adjusted to 1 mg/ml final reaction concentration; the same sample volume was used for resuspended hemocytes. Values are the means of three sets of substrate concentrations ⫾ SD.

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Figure 4. Monophenolase activity of washed hemocytes of Cancer magister. Spectrophotometric assay at 475 nm using 1 mM monophenolic substrate tyramine. Catalytic amount of diphenol dopamine, 1 ␮M. Each curve represents a mean of three reactions.

phenolase and diphenolase activities under in vitro conditions have been reported (Adachi et al., 2005; Decker and Jaenicke, 2004)—results that hint of a potential inhibitor or unknown required co-factor of hemocyanin phenoloxidase activity. A model for conversion of hemocyanin oxygen-binding function to phenoloxidase activity has been proposed on the basis of comparisons of phenoloxidase and hemocyanin sequences and hemocyanin crystal structure (Decker and Rimke, 1998; Decker et al., 2001; Jaenicke and Decker, 2003). The model suggests that proteolytic cleavage, conformational change, or both could open an entrance for larger phenolic substances into the binuclear copper-oxygen binding pocket. Experimental support for the model includes activation of hemocyanins and hemocyte phenoloxidases by means of in vitro effectors such as detergents and salts (Zlateva et al., 1996; Decker et al., 2001), and in vivo factors including antimicrobial peptides (Nagai and Kawabata, 2000; Nagai et al., 2001) and a prophenoloxidase cascade of proteolytic enzymes initiated by bacterial cell wall fragments or lipopolysaccharides (Jiang et al., 1998; Wang et al., 2001). The higher phenoloxidase activity levels of one-hexamer hemocyanin versus two-hexamer hemocyanin of C. magister are probably a function of conformational changes needed for activation, and they may be due to easier access of the diphenol substrate to the copper-binding sites in the less constrained one-hexamers. PAGE analysis of C. ma-

gister hemolymph offers further insight into phenoloxidase activity and aggregation state. Detection of enzyme activity by pH 7.4 PAGE, where the hemocyanin is in its native oneand two-hexamer configurations, is difficult, although a faint reaction can be elicited with prolonged reaction times. Analysis at a similar protein concentration by pH 8.9 PAGE, however (a technique that dissociates native hemocyanin into individual subunits without denaturing them), results in all C. magister hemocyanin subunits showing robust phenoloxidase activity (Decker et al., 2001). The 5S subunits of Carcinus maenas hemocyanin also had higher levels of phenoloxidase activity than did the hexameric hemocyanin (Zlateva et al., 1996). Thus dissociation into subunits seems to allow even better access of the phenol substrate to the hemocyanin active site than does the hexamer. The hemocyte phenoloxidase also assembles into a hexamer (Jaenicke and Decker, 2003). When the three-dimensional structure of an arthropod phenoloxidase subunit has been determined and aligned with known structures of chelicerate (Limulus) and crustacean (Panulirus interruptus) hemocyanins (Hazes et al., 1993; Magnus et al., 1994; Volbeda and Hol, 1989), the results should expand our understanding about the marked differences in catalytic ability and activity between arthropod hemocyanin and phenoloxidase. We have observed an accumulation of hemocytes in the connective tissue below the epidermis during premolt. We suggest that these hemocytes are stimulated to release prophenoloxidase immediately before or after ecdysis, and the proenzyme moves across the epidermis into the layers of the newly secreted exoskeleton. Movement of cryptocyanin, another hexameric member of the hemocyanin gene family, from the hemolymph to the new exoskeleton during this time has been shown (Terwilliger et al., 2005). The identification of a cDNA for a prophenoloxidase-activating factor in the epidermis of Callinectes sapidus (Buda and Shafer, 2005) supports the idea that prophenoloxidase is cleaved into the active form in the new exoskeleton space. This study demonstrates that crab hemocyanin does have phenoloxidase activity, albeit low compared to crab hemocyte phenoloxidase. Whether the transition in hemocyanin function occurs under physiological conditions in the animal is as yet unknown. Multiple functions have been reported for arthropod hemocyanins recently, including antibacterial as well as phenoloxidase activity (DestoumieuxGarzon et al., 2001; Lee et al., 2003). The hemocyanin phenoloxidase activity could be a residual function maintained in the molecule’s structural history and demonstrated only in a laboratory setting. A case can be made for a physiological switch between oxygen transport and phenoloxidase activity in chelicerate and isopod crustacean hemocyanins, since the demonstrated phenoloxidase activity of the hemolymph of these organisms is due only to

HEMOCYANIN AND HEMOCYTE PHENOLOXIDASES

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Figure 5. Deduced amino acid sequence of brachyuran crab Cancer magister prophenoloxidase (CmagPPO, DQ230981) aligned with prophenoloxidases of lobster Homarus americanus (HamePPO, AY655139), penaeid shrimp Penaeus monodon (PmonPPO, AF099741), and insect Sarcophaga bullata (SbulPPO, AF161260). Conserved histidines of two copper-binding sites (*), probable site of proteolytic cleavage to the active phenoloxidase (⫹⫹), putative thiol ester region (underlined). Conserved amino acids are black; similar amino acids are gray.

hemocyanin (Nellaiappan and Sugumaran, 1996; Decker et al., 2001; Pless et al., 2003). The hemocytes of horseshoe crabs show no phenoloxidase activity, but they contribute endogenous antimicrobial peptides that induce phenoloxi-

dase activity in hemocyanin of Tachypleus tridentatus (Nagai and Kawabata, 2000; Nagai et al., 2001). Since most crustaceans, unlike the chelicerates, have an ample supply of hemocyte phenoloxidase, in vivo activation of crustacean

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Figure 6. Phylogeny of crustacean phenoloxidases. Single most-parsimonious tree based on partial sequences of crustacean phenoloxidases. Bootstrap support values (⬎50%) out of 1000 replications are represented at each node. GenBank accession numbers in parentheses. Ascidian putative prophenoloxidases are designated as the outgroup.

hemocyanin might be less crucial. Under extreme stress, however—for example, at ecdysis when an entire exoskeleton must be rapidly hardened or during a severe bacterial infection when hemocyte numbers are depleted (Holman et al., 2004; Martin et al., 1993; White and Ratcliffe, 1982)— sufficient upstream activating agents might convert the function of crustacean hemocyanin from the reversible binding of oxygen to the oxidation of diphenols. The low phenoloxidase activity of hemocyanin could be offset by its high concentration. It may be that dopamine release into the hemolymph from neurosecretory sites such as the pericardial organs is accelerated during these times, and the dopamine serves as a diphenol substrate for quinone formation by the hemocyanin phenoloxidase. The complete cDNA sequence that we have obtained for C. magister prophenoloxidase is the first reported for a brachyuran crab. The deduced amino acid sequence does not have a signal peptide, indicating that there is no transmembrane transport of the protein. This is consistent with results found for other arthropod hemocyte prophenoloxidases (Aspa´n et al., 1995; Hall et al., 1995). The thiol ester site in the sequence, conserved in other arthropod hemocyanins and prophenoloxidases, is similar to a region found in members of a gene family within the complement cascade of innate immunity in both invertebrates and vertebrates (Hall et al., 1995). The complement proteins C3 and C4 that participate in both innate and adaptive immunity are thought to have gradually evolved through gene duplication events from alpha-2-macroglobulin, a serine protease inhibitor (Bartl et al., 2003; Dodds and Law, 1998). The thiol ester site has been suggested to result in immobilization of those proteins on cell surfaces as part of an immune function (Hall

et al., 1995). The degenerate primers that we used to amplify the initial cDNA fragment of prophenoloxidase were chosen because they encompass portions of the conserved copper A-binding site and the thiol ester site (Hall et al., 1995; Kusche et al., 2002; Parkinson et al., 2001). The proteolytic cleavage site found in insect prophenoloxidases (Hall et al., 1995; Sritunyalucksana et al., 1999) aligns well with a putative cleavage site in C. magister prophenoloxidase. Phenoloxidase activity has been described in the water flea Daphnia magna (Mucklow and Ebert, 2003) and in hemocytes of the brine shrimp Artemia franciscana (Martin et al., 1999), although there are no complete cDNA sequences published for the branchiopod proteins. Here we present partial sequences of prophenoloxidases from two members of the Branchiopoda, the anostracan A. franciscana and the notostracan Triops longicaudatus. The 1100-bp fragments of the branchiopod prophenoloxidases were amplified using the same primers that amplified the initial C. magister prophenoloxidase fragment. The crustacean prophenoloxidases cluster into two distinct groups when the partial sequences are compared. The proteins from the two Branchiopoda (recently reclassified into Anostraca (A. franciscana) and Phyllopoda (T. longicaudata)) form a single clade, and the malacostracan Decapoda form another. Within the Decapoda, prophenoloxidases group along phyletic lines into the dendrobranchiatan Penaeidea and the pleocyamatan Astacidea and Brachyura. In a more extensive comparison of complete sequences of crustacean phenoloxidases with other members of the arthropod hemocyanin superfamily, the four major clades plus an onychophoran protein all branch from a common

HEMOCYANIN AND HEMOCYTE PHENOLOXIDASES

Figure 7. Evolutionary relationships among the arthropod hemocyanin gene superfamily. Single mostparsimonious tree based on complete coding sequences of 73 members of the family. Hc, hemocyanins; Cc and Ph, cryptocyanins; SL, SP, and Hex, hexamerins; PPO, prophenoloxidases. ␣, ␤, and ␦ refer to immunologically defined crustacean hemocyanin subunit types (Markl, 1986). Bootstrap support values (⬎50%) out of 1000 replications are represented at each node. GenBank accession numbers in parentheses. Ascidian putative prophenoloxidases are designated as the outgroup.

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ancestor, and all show high bootstrap values. The phenoloxidases form a separate, strongly supported branch, and within this, the crustacean phenoloxidases are clearly distinct from the insect phenoloxidases. Chelicerate hemocyanins and myriapod hemocyanins each compose a unique clade. The crustacean and insect hemocyanins, cryptocyanins, and hexamerins form another clade, with the hexamerins differentiated from the hemocyanins and cryptocyanins. Relationships within the clade of crustacean hemocyanins are interesting, as they tend to group according to the immunologically defined subunit types described by Markl (1986). The entire sequences of all six of the hemocyanin subunits of C. magister are represented here— the first complete example in a brachyuran crab. The ensemble of C. magister hemocyanins includes two beta and four gamma subunit types but no alpha, in contrast to the prediction that alpha subunits would be common to all crustacean hemocyanins (Burmester, 2002). Based on our tree, the beta-type C. magister Hc 1 and 2 diverged early from the other types. The cryptocyanins of C. magister and Homarus americanus may also be gamma types. The hemocyanin of Cyamus scammoni, the cyamid amphipod whose hemolymph contains high concentrations of a highmolecular-weight extracellular hemoglobin (Terwilliger, 1991a), aligns with another amphipod hemocyanin. Most amphipods express hemocyanin as their oxygen transport protein, and this is the first example of a higher crustacean that expresses both hemoglobin and hemocyanin. These results provide evidence for the evolution of new proteins with new functions—and the retention of old functions. One of the intriguing questions about the hemocyanin gene family concerns the ancestral molecule. Did it have both phenoloxidase activity and reversible oxygen-binding capabilities that have been retained in contemporary crustacean and chelicerate hemocyanins? A subsequent gene duplication and mutation could have resulted in a new gene product, the specialized phenoloxidase expressed in hemocytes of some crustaceans and insects. The phylogeny we present here supports this possibility. The absence of a hemocyte phenoloxidase and the presence of a hemocyanin with reversible oxygen-binding and phenoloxidase activity in chelicerates are consistent with this hypothesis. At least one peracaridan crustacean, Bathynomus giganteus, resembles the chelicerates in having its phenoloxidase activity restricted to the hemocyanin (Pless et al., 2003), and preliminary experiments suggest this may be true for other isopods (Arellano and Terwilliger, 2004). In addition, when we examined the peracaridan amphipod Cyamus scammoni, we could amplify a hemocyanin from cDNA by using crustacean degenerate hemocyanin primers but were unable to amplify a phenoloxidase, even though we used the same chelicerate hemocyanin-based primers that gave positive results for other crustacean phenoloxidases. Apparently isopods and amphipods lack a hemocyte phenoloxidase.

An alternate hypothesis supported by other phylogenetic analyses is that the phenoloxidases are the most basal group in the arthropod hemocyanin superfamily and that variable patterns of gain and loss of functions (reversible oxygenbinding, phenoloxidase activity) have occurred across the phyla with time and gene duplications (Decker and Terwilliger, 2000; Burmester, 2001; Decker and Jaenicke, 2004; Immisberger and Burmester, 2004). Many have postulated that defense against oxygen toxicity preceded the need for oxygen binding. The two functions may have arisen together, however; and as we probe further back into evolutionary history, we should continue to consider both hypotheses. It will be interesting to determine the pattern of phenoloxidase expression in the hemocyanin-expressing myriapods and other members of the Ecdysozoa, as well as in members of the pre-Bilateria. Acknowledgments This work was supported by NSF grant IAB-9984202. Literature Cited Adachi, K., H. Endo, T. Watanabe, T. Nishioka, and T. Hirata. 2005. Hemocyanin in the exoskeleton of crustaceans: enzymatic properties and immunolocalization. Pigm. Cell Res. 18: 36 –143. Altschul, S., W. Gish, W. Miller, E. Myers, and D. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403– 410. Andersen, S.O., M.G. Peter, and P. Roepstorff. 1996. Cuticular sclerotization in insects. Comp. Biochem. Physiol. B 113: 698 –705. Arellano, S., and N. Terwilliger. 2004. Hemocyanin, cryptocyanin and phenoloxidase in deep sea (Bathynomus giganteus) and intertidal (Cirolana harfordi) isopods. Integr. Comp. Biol. 43: 961A. Ashida, M., and P. T. Brey. 1995. Role of the integument in insect defense: pro-phenol oxidase cascade in the cuticular matrix. Proc. Natl. Acad. Sci. USA 92: 10698 –10702. Ashida, M., and P. T. Brey. 1997. Recent advances in research on the insect prophenoloxidase cascade. Pp. 135–172 in Molecular Mechanisms of Immune Responses in Insects, P. T. Brey and D. Hultmark, eds. Chapman and Hall, London. Ashida, M., and H. I. Yamazaki. 1990. Biochemistry of the phenoloxidase system in insects: with special reference to its activation. Pp. 239 –265 in Molting and Metamorphosis, E. Ohnishi and H. Ishizaki, eds. Springer-Verlag, Berlin. Aspa´n, A., and K. So¨derha¨ll. 1991. Purification of prophenoloxidase from crayfish blood cells, and its activation by an endogenous serine proteinase. Insect Biochem. 21: 363–373. Aspa´n, A., T.-S. Huang, L. Cerenius, and K. So¨derha¨ll. 1991. cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation. Proc. Natl. Acad. Sci. USA 92:939 –943. Bartl, S., M. Baish, I. Weissman, and M. Diaz. 2003. Did the molecules of adaptive immunity evolve from the innate immune system? Integr. Comp. Biol. 43: 338 –346. Bendtsen, J. D., H. Nielsen, G. von Heijne. and S. Brunak. 2004. Improved prediction of signal peptides. J. Mol. Biol. 340: 783–795. Bhagvat, H., and D. Richter. 1938. Animal phenolases and adrenaline. Biochem. J. 32: 1397. Buda, E. S., and T. H. Shafer. 2005. Expression of a serine proteinase homolog prophenoloxidase-activating factor from the blue crab, Callinectes sapidus. Comp. Biochem. Physiol. B 140: 521– 673.

HEMOCYANIN AND HEMOCYTE PHENOLOXIDASES Burmester, T. 2001. Molecular evolution of the arthropod hemocyanin superfamily. Mol. Biol. Evol. 18: 184 –195. Burmester, T. 2002. Origin and evolution of arthropod hemocyanins and related proteins. J. Comp. Physiol. 172B: 95–107. Chomczynski, P., and N. Sacchi. 1987. Single step method of RNA isolation by acid guanidinium thiocyanate phenol-chloroform extraction. Anal. Biochem. 162: 156 –159. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121: 404 – 427. Decker, H., and E. Jaenicke. 2004. Recent findings on phenoloxidase activity and antimicrobial activity of hemocyanins. Dev. Comp. Immunol. 28: 673– 687. Decker, H., and T. Rimke. 1998. Tarantula hemocyanin shows phenoloxidase activity. J. Biol. Chem. 273: 25889 –25892. Decker, H., and N. Terwilliger. 2000. COPs and robbers: putative evolution of copper oxygen binding proteins. J. Exp. Biol. 203: 1777– 1782. Decker, H., M. Ryan, E. Jaenicke, and N. Terwilliger. 2001. SDS induced phenoloxidase activity of hemocyanins from Limulus polyphemus, Eurypelma californicum and Cancer magister. J. Biol. Chem. 276: 17796 –17799. Destoumieux-Garzon, D., D. Saulnier, J. Garniert, C. Jouffrey, P. Bulet, and E. Bachere. 2001. Crustacean immunity. Antifungal peptides are generated from the C terminus of shrimp hemocyanin in response to microbial challenge. J. Biol. Chem. 276: 47070 – 47077. Dodds, A., and S. Law. 1998. The phylogeny and evolution of the thioester bond-containing proteins C3, C4 and alpha 2-macroglobulin. Immunol. Rev. 166: 15–26. Durstewitz, G., and N. B. Terwilliger. 1997. cDNA cloning of a developmentally regulated hemocyanin subunit in the crustacean Cancer magister and phylogenetic analysis of the hemocyanin gene family. Mol. Biol. Evol. 14: 266 –276. Gollas-Galva´n, T., J. Herna´ndez-Lo´pez, and F. Vargas-Albores. 1999. Prophenoloxidase from brown shrimp (Penaeus californiensis) hemocytes. Comp. Biochem. Physiol. 122: 77– 82. Hall, M., T. Scott, M. Sugumaran, K. So¨derha¨ll, and J. Law. 1995. Prophenol oxidase of the hawkmoth Manduca sexta: purification, activation, substrate specificity of the active enzyme, and molecular cloning. Proc. Natl. Acad. Sci. USA 92: 7764 –7768. Hartzer, K. L., K. Y. Zhu, and J. E. Baker. 2005. Phenoloxidase in larvae of Plodia interpunctella (Lepidoptera: Pyralidea): molecular cloning of the proenzyme cDNA and enzyme activity in larvae paralyzed and parasitized by Habrobracon hebetor (Hymenoptera: Braconidae). Arch. Insect Biochem. Physiol. 59: 67–79. Hazes, B., K. A. Magnus, C. Bonaventura, J. Bonaventura, Z. Dauter, K. H. Kalk, and W. G. J. Hol. 1993. Crystal structure of deoxygenated Limulus polyphemus subunit II hemocyanin at 2.18Å resolution: clues for a mechanism for allosteric regulation. Protein Sci. 2: 597– 619. Holman, J. D., K. G. Burnett, and L. E. Burnett. 2004. Effects of hypercapnic hypoxia on the clearance of Vibrio campbellii in the Atlantic blue crab, Callinectes sapidus Rathbun. Biol. Bull. 206: 188 – 196. Immisberger, A., and T. Burmester. 2004. Putative phenoloxidases in the tunicate Ciona intestinalis and the origin of the arthropod hemocyanin superfamily. J. Comp. Physiol. B 174: 169 –180. Jaenicke, E., and H. Decker. 2003. Tyrosinases from crustaceans form hexamers. Biochem. J. 371: 515–523. Jaenicke, E., and H. Decker. 2004. Functional changes in the family of type 3 copper proteins during evolution. ChemBioChem 5: 163–176. Jiang, H., Y. Wang, and M. Kanost. 1998. Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: a bacterial-inducible protein similar to Drosophila easter. Proc. Natl. Acad. Sci. USA 95: 12220 –12225.

49

Kolaczkowski, B., and J. W. Thornton. 2004. Performance of maximum parsimony and likelihood phylogenetics when evolution is heterogeneous. Nature 431: 980 –984. Kusche, K., H. Rubberg, and T. Burmester. 2002. A hemocyanin from the Onychophora and the emergence of respiratory proteins. Proc. Natl. Acad. Sci. USA 99: 10545–10548. Lee, S. Y., B. L. Lee, and K. So¨derha¨ll. 2003. Processing of an antibacterial peptide from hemocyanin of the freshwater crayfish Pacifasticus leniusculus. J. Biol. Chem. 278: 7927–7933. Magnus, K., B. Hazes, H. Ton-That, C. Bonaventura, J. Bonaventura, and W. Hol. 1994. Crystallographic analysis of oxygenated and deoxygenated states of arthropod hemocyanin shows unusual differences. Proteins Struct. Funct. Genet. 19: 302–309. Markl, J. 1986. Evolution and function of structurally diverse subunits in the respiratory protein hemocyanin from arthropods. Biol. Bull. 171: 90 –115. Martin, G., D. Poole, C. Poole, J. Hose, M. Arias, L. Reynolds, N. McKrell, and A. Whang. 1993. Clearance of bacteria injected into the hemolymph of the penaeid shrimp, Sicyonia ingentis. J. Invertebr. Pathol. 62: 308 –315. Martin, G., H. Lin, and C. Luc. 1999. Reexamination of hemocytes in brine shrimp (Crustacea, Branchiopoda). J. Morphol. 242: 283–294. Mason, H. S. 1947. The chemistry of melanin. II. The oxidation of dihydroxyphenyalanine by mammalian dopa oxidase. J. Biol. Chem. 168: 433– 438. Mucklow, P., and D. Ebert. 2003. Physiology of immunity in the water flea Daphnia magna: environmental and genetic aspects of phenoloxidase activity. Physiol. Biochem. Zool. 76: 836 – 842. Nagai, T., and S. Kawabata. 2000. A link between blood coagulation and prophenol oxidase activation in arthropod host defense. J. Biol. Chem. 275: 29264 –29267. Nagai, T., T. Osaki, and S.-i. Kawabata. 2001. Functional conversion of hemocyanin to phenoloxidase by horseshoe crab antimicrobial peptides. J. Biol. Chem. 276: 27166 –27170. Naraoka, T., H. Uchisawa, H. Mori, H. Matsue, S. Chiba, and A. Kimura. 2003. Purification, characterization and molecular cloning of tyrosinase from the cephalopod mollusk, Illex argentinus. Eur. J. Biochem. 270: 4026 – 4038. Nellaiappan, K., and M. Sugumaran. 1996. On the presence of prophenoloxidase in the hemolymph of the horseshoe crab, Limulus. Comp. Biochem. Physiol. B 113: 163–168. Nellaiappan, K., and A. Vinayakam. 1993. A method for demonstrating prophenoloxidase after electrophoresis. Biotech. Histochem. 68: 193–195. Parkinson, N., I. Smith, R. Weaver, and J. P. Edwards. 2001. A new form of arthropod phenoloxidase is abundant in venom of the parasitoid wasp Pimpla hypochondriaca. Insect Biochem. Mol. Biol. 31: 57– 63. Pless, D., M. Aguilar, A. Falcon, E. Lozano-Alvarez, and E. de la Cotera. 2003. Latent phenoloxidase activity and N-terminal amino acid sequence of hemocyanin from Bathynomus giganteus, a primitive crustacean. Arch. Biochem. Biophys. 409: 402– 410. Salvato, B., M. Santamaria, M. Beltramini, G. Alzuet, and L. Casella. 1998. The enzymatic properties of Octopus vulgaris hemocyanin: o-diphenol oxidase activity. Biochemistry 37: 14065–14077. ´ ., J. N. Rodrı´guez-Lo´pez, F. Garcı´a-Ca´novas, and F. Sa´nchez-Ferrer, A Garcı´a-Carmona. 1995. Tyrosinase: a comprehensive review of its mechanism. Biochim. Biophys. Acta 1247: 1–11. So¨derha¨ll, K., and L. Cerenius. 1998. Role of prophenoloxidase-activating system in invertebrate immunity. Curr. Opin. Immunol. 10: 23–28. Solomon, E., F. Tuczek, D. Root, and C. Brown. 1994. Spectroscopy of binuclear dioxygen complexes. Chem. Rev. 94: 827– 856. Sritunyalucksana, K., L. Cerenius, and K. So¨derha¨ll. 1999. Molecular

50

N. B. TERWILLIGER AND M. C. RYAN

cloning and characterization of prophenoloxidase in the black tiger shrimp, Penaeus monodon. Dev. Comp. Immunol. 23: 179 –186. Sugumaran, M. 1998. Unified mechanism for sclerotization of insect cuticle. Adv. Insect Physiol. 27: 227–334. Sugumaran, M., and M. R. Kanost. 1993. Regulation of insect hemolymph phenoloxidases. Pp. 317–342 in Parasites and Pathogens of Insects, vol. 1, N. E. Beckage, S. N. Thompson, and B. A. Federici, eds. Academic Press, New York. Swofford, D. L. 2002. PAUP*, Phylogenetic Analysis Using Parsimony (*and Other Methods), Ver. 4. Sinauer Associates, Sunderland, MA. Terwilliger, N. B. 1991a. Arthropod (Cyamus scammoni, Amphipoda) hemoglobin structure and function. Pp. 59 – 63 in Structure and Function of Invertebrate Oxygen Carriers, S. Vinogradov and O. Kapp, eds. Springer, Berlin. Terwilliger, N. B. 1991b. Hemocyanins in crustacean oo¨cytes and embryos. Pp. 31–36 in Crustacean Egg Production, A. Wenner and A. Kuris, eds. A. A. Balkema, Rotterdam. Terwilliger, N. B. 1999. Hemolymph proteins and molting in crustaceans and insects. Am. Zool. 39: 589 –599. Terwilliger, N. B., and R. C. Terwilliger. 1982. Changes in the subunit structure of Cancer magister hemocyanin during larval development. J. Exp. Biol. 221: 181–191. Terwilliger, N. B., L. D. Dangott, and M. C. Ryan. 1999. Cryptocyanin, a crustacean molting protein: evolutionary link with arthropod hemocyanins and insect hexamerins. Proc. Natl. Acad. Sci. USA 96: 2013–2018. Terwilliger, N., M. Ryan, and D. Towle. 2005. Evolution of novel

functions: cryptocyanin helps build new exoskeleton in Cancer magister. J. Exp. Biol. 208: 2467–2474. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673– 4680. Van Gelder, C. W. G., W. H. Flurkey, and H. J. Wichers. 1997. Sequence and structural features of plant and fungal tyrosinases. Phytochemistry 45: 1309 –1323. van Holde, K., K. Miller, and H. Decker. 2001. Hemocyanins and invertebrate evolution. J. Biol. Chem. 276: 15563–15566. Vargas-Albores, F., F. Jimenez-Vega, and K. So¨derha¨ll. 1996. A plasma protein isolated from brown shrimp (Penaeus californiensis) which enhances the activation of prophenoloxidase system by beta-1,3Glucan. Dev. Comp. Immunol. 20: 299 –306. Volbeda, A., and W. G. J. Hol. 1989. Crystal structure of hexameric hemocyanin from Panulirus interruptus refined at 3.2Å resolution. J. Mol. Biol. 209: 249 –279. Wang, R., S. Lee, L. Cerenius, and K. So¨derha¨ll. 2001. Properties of the prophenoloxidase activating enzyme of the freshwater crayfish, Pacifastacus leniusculus. Eur. J. Biochem. 268: 895–902. White, K., and N. Ratcliffe. 1982. The segregation and elimination of radio- and fluorescent-labelled marine bacteria from the haemolymph of the shore crab, Carcinus maenas. J. Mar. Biol. Assoc. UK 62: 819 – 833. Zlateva, T., P. Di Muro, B. Salvato, and M. Beltramini. 1996. The o-diphenol oxidase activity of arthropod hemocyanin. FEBS Lett. 384: 251–254.