Integrative and Comparative Biology Advance Access originally published online on June 6, 2007
Integrative and Comparative Biology 2007 47(4):662-665; doi:10.1093/icb/icm039
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Hemocyanins and the immune response: defense against the dark arts
Oregon Institute of Marine Biology and Department of Biology, University of Oregon, Charleston, OR 97420 USA
Correspondence: 1E-mail: nterwill{at}uoregon.edu
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The innate immune response is a conserved trait shared by invertebrates and vertebrates. In crustaceans, circulating hemocytes play significant roles in the immune response, including the release of prophenoloxidases. Activated phenoloxidase (tyrosinase) participates in encapsulation and melanization of foreign organisms as well as sclerotization of the new exoskeleton after wound-repair or molting. Hemocyanin functions as a phenoloxidase under certain conditions and thus also participates in the immune response and molting. The relative contributions of hemocyte phenoloxidase and hemocyanin in the physiological ratio at which they occur in hemolymph have been investigated in the crab Cancer magister. Differences in activity, substrate affinity, and catalytic ability between the two enzymes indicate that hemocytes are the predominant source of phenoloxidase activity in crabs. In contrast, hemocyanin is the primary source of phenoloxidase activity in isopods and chelicerates whose hemocytes show no phenoloxidase activity. Quantitative PCR studies on the distribution of prophenoloxidase mRNA in the tissues of Carcinus maenas showed little effect relative to salinity stress. Phylogenetic analysis of hemocyanin, phenoloxidase, and other members of this arthropod gene family are consistent with the possibility that a common ancestral molecule had both phenoloxidase and oxygen-binding capabilities.
Recent outbreaks of black-shell disease in the lobster Homarus americanus have caused major concern for fisheries in the northeastern United States and Canada (Schwartz 2006
). Shells of lobsters infected with this malady, also known as black spot or chitinolytic bacterial disease, are eroded by gram-negative bacteria that form characteristic lesions as they digest the exoskeletal chiton. The lesions become blackened due to the formation of melanin, resulting in unsightly and unappetizing animals. Many crustacean species are subject to black-shell disease (Vogan et al. 2002). Other crustacean diseases with descriptive names such as pepper spot, bitter crab, cotton crab, black mat, and lethargic crab, result from infection by various organisms (Shields 1994) The increased occurrence of infectious diseases in crustaceans emphasizes the need to better understand how arthropods cope with immunochallenges in natural habitats and aquaculture.
The innate immune response, a conserved trait shared by invertebrates and vertebrates, contributes to defense against pathogens and parasites through detection, signaling pathways and initiation of defense mechanisms (Hoffmann 2003; Leulier et al. 2003). In crustaceans, circulating hemocytes play significant roles in the innate immune response, including release of nonself-recognition proteins, clotting proteins, antimicrobial peptides, and prophenoloxidase. Activated phenoloxidase (tyrosinase) catalyzes the hydroxylation of monophenols to diphenols such as dopamine and dopa. In a second step (catecholoxidase reaction), the enzyme oxidizes the diphenol to an ortho-quinone, a highly reactive molecule that is involved in encapsulation and melanization of foreign organisms. Phenoloxidase is also important in sclerotization (hardening) of the new exoskeleton after wound-repair and molting (Söderhäll and Cerenius 1998; Terwilliger 1999).
The oxygen-transport protein hemocyanin functions as a phenoloxidase under certain conditions (Decker et al. 2001; Jaenicke and Decker 2004). Hemocyanin belongs to the same family of copper proteins as phenoloxidase. Therefore, hemocyanin, hemocytes and phenoloxidase are related players in the crustacean immune response or "defense against the dark arts." The basic mechanisms for activation of arthropod hemocyanins and phenoloxidases and nonarthropod tyrosinases have been discussed recently (Decker et al. 2006; Matoba et al. 2006). We compared the phenoloxidase activities and substrate specificities of hemocyte phenoloxidase and hemocyanin that co-occur in the hemolymph of the Dungeness crab, Cancer magister, to better understand their different roles (Terwilliger and Ryan 2006). Whole hemolymph, washed hemocytes and hemocyte-free hemolymph were assayed using the physiological ratio at which they occur in the crab (Fig. 1). In whole hemolymph, the protein concentration of hemocyte phenoloxidase is much lower than that of hemocyanin, while the enzymic activity of the hemocytes is greater. Hemocytes and hemocyanin show marked differences in substrate specificity and in relative reactivity to diphenol substrates. The two naturally occurring oligomers of hemocyanin differ in activity; one-hexamer aggregates have higher phenoloxidase activity than two-hexamer oligomers. This is consistent with the model of hemocyanin activation via conformational change (Decker and Tuczek 2000; Jaenicke and Decker 2004) and easier access of the diphenol substrate to active sites in the less constrained one-hexamer hemocyanin.
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The differences in activity, substrate affinity and catalytic ability between hemocyanin and hemocyte phenoloxidase that we observed indicate that in crabs, hemocytes are the predominant source of phenoloxidase activity and that they can provide a rapid immune response to microbial challenge. We suggest that during ecdysis, prophenoloxidase released from hemocytes moves across the epidermis into the new layers of exoskeleton to participate in sclerotization. Does crab hemocyanin function in vivo as a phenoloxidase? It may be that crab hemocyanin function is converted from oxygen transport to phenoloxidase only during a major bacterial invasion and/or during molting when there is a need for concerted and rapid sclerotization.
Crustaceans infected with bacteria often show a marked increase of hemocytes in the gills (Martin et al. 2000; Vogan et al. 2002). We amplified cDNA coding for prophenoloxidase in the green crab Carcinus maenas and examined the distribution in its tissues of prophenoloxidase gene expression by quantitative PCR relative to salinity stress (Terwilliger and Towle 2007). Hemocytes showed high levels of prophenoloxidase-encoding mRNA, as expected. Exposure to dilute salinity had little effect on prophenoloxidase distribution in any tissue. Thus the accumulation of hemocytes in gills seems to be a specific feature of the immune response to particulates and pathogens and not a generalized stress response.
The oxygen-requiring catalytic activity of phenoloxidase is reflected in the impairment of a crustacean's ability to resist bacterial infection when subjected to environmental hypoxia (Cheng et al. 2002; Holman et al. 2004; Le Moullac et al. 1999). Hypoxia and low pH have been shown to directly suppress phenoloxidase activity in hemocytes from the blue crab Callinectes sapidus (Tanner et al. 2006). Thus, crustaceans living in oxygen-poor coastal waters are apt to experience hypercapnic hypoxia, decreased phenoloxidase activity, and increased susceptibility to infectious pathogens. Determination of differential O2 and pH sensitivities between hemocyte phenoloxidase and hemocyanin could help explain why crabs have both sources of phenoloxidase.
Phenoloxidase activity in isopods, in contrast to crabs, seems to be restricted to hemocyanin and not hemocytes. This was first demonstrated in the large deep-sea isopod Bathynomus giganteus found in the Gulf of Mexico (Pless et al. 2003). We compared the hemolymph of B. giganteus and that of a much smaller isopod Cirolana harfordi that is in the same family as B. giganteus but lives in the low intertidal zone of the Oregon coast (Arellano and Terwilliger 2004). While the predominant hemocyanin oligomer in B. gigantea was the one-hexamer form and in C. harfordi, the two-hexamer form, hemolymph from each species contained both oligomers (Fig. 2). Phenoloxidase activity was strong but observed only in hemocyanin. A similar pattern of phenoloxidase activity in hemocyanin but not in hemocytes has been noted in chelicerates (Decker et al. 2001; Nagai and Kawabata 2000). Therefore, the source of phenoloxidase activity differs among arthropods.
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A phylogenetic comparison of the hemocyte prophenoloxidase sequence of C. magister with other members of the arthropod hemocyanin gene family shows four major clades branching from a common ancestral protein (Terwilliger and Ryan 2006
). Crustacean and insect hemocyte phenoloxidases constitute one clade, crustacean hemocyanins and cryptocyanins, insect hemocyanins, and insect hexamerins form a second, and chelicerate hemocyanins and myriapod hemocyanins each form a clade. The phylogeny is consistent with the possibility of a common ancestral molecule with both phenoloxidase and reversible oxygen-binding capabilities, as is present today in crab, isopod, and chelicerate hemocyanins. A gene duplication and mutation could have resulted in a new gene product, the specialized phenoloxidase in hemocytes of certain crustaceans and insects.
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I thank Margaret Ryan, Shawn Arellano, and David Towle for their contributions, and I thank Heinz Decker, organizer of this ICRB symposium. Supported by NSF 9984202 and Mt. Desert Island Biological Laboratory Salisbury Cove Research Fund.
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From the symposium "Integrative and Evolutionary Physiology of Copper Proteins: From Molecules to Organisms in their Environment" presented at the First International Congress of Respiratory Biology, August 14–16, 2006, at Bad Honnef/Bonn, Germany.
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