Integrative and Comparative Biology Advance Access originally published online on July 12, 2006
Integrative and Comparative Biology 2006 46(6):991-999; doi:10.1093/icb/icl012
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Crustacean hemocyanin gene family and microarray studies of expression change during eco-physiological stress
* Oregon Institute of Marine Biology, University of Oregon Charleston, OR 97420, USA
Department of Biology, University of Oregon Charleston, OR 97420, USA
Correspondence: 1E-mail: nterwill{at}uoregon.edu
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 Synopsis IntroductionHemocyanin gene familycDNA sequences of hemocyanin...Phylogenetic analysis:...Conservation of subunit...Microarray analysis of...SummaryREFERENCES |
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Proteins in the arthropod hemocyanin gene family are involved in major physiological processes, including aerobic respiration, the innate immune response, and molting. Members of this family, hemocyanin, cryptocyanin, and phenoloxidase, are multisubunit molecules that assemble into hexamers and higher aggregates. The hemocyanin hexamers show species-specific subunit heterogeneity. It is hypothesized that this subunit diversity is maintained as a mechanism of selection for functional diversity under changing developmental and environmental conditions. There is good evidence for a strong relationship between subunit composition and functional diversity in the hemocyanins. We have amplified, cloned, and sequenced the complete cDNAs of the 6 hemocyanin genes, 2 cryptocyanins, and 1 phenoloxidase of Cancer magister. Alignment of the amino acid sequences provides the first opportunity to assess in 1 species of brachyuran crustacean the similarities and differences among all the hemocyanin subunits and compare them with cryptocyanin and phenoloxidase. A phylogeny of sequences of crustacean members of the arthropod hemocyanin gene family is described. Construction of a cDNA library for C. magister microarray studies is in progress. The microarrays will be queried using transcriptional profiles from crabs sampled during developmental, molting, and physiological perturbations. The combination of genomics, proteomics, and gene-by-gene analyses will help us dissect how much a gene sequence in this hemocyanin family can vary while conserving function and which aspects of preservation of shape and structural flexibility are essential for functional stability. Integrating focused gene studies with global-expression profiling can eventually lead to the identification of functional networks at the level of the gene, the multisubunit molecule, and the whole organism.
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Introduction |
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SynopsisIntroductionHemocyanin gene familycDNA sequences of hemocyanin...Phylogenetic analysis:...Conservation of subunit...Microarray analysis of...SummaryREFERENCES |
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Organisms in the clade Ecdysozoa share the highly integrated processes of development, growth, and reproduction with other phyla. When the obligate molt cycles of the ecdysozoans are superimposed on these fundamental processes, however, Arthropoda and other members of the clade experience additional complexities of gene regulation (Skinner 1985
; Aguinaldo and others 1997). Genomics and proteomics are powerful new tools to study holistic views of the activity of a cell or the whole organism and to learn more about the evolution of genes and proteins. To identify groups of co-regulated genes in the entire genome of these molting animals, to see where and when these genes are expressed, and to define functional networks in the organism, researchers recognize the value of global gene expression profiling. In conjunction with focused studies on the hemocyanin gene family, we are currently developing a genomic profile of Cancer magister, the Dungeness crab, to monitor the crab's transcriptional response to changes in development, molt cycle, and environmental stresses. |
Hemocyanin gene family |
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SynopsisIntroductionHemocyanin gene familycDNA sequences of hemocyanin...Phylogenetic analysis:...Conservation of subunit...Microarray analysis of...SummaryREFERENCES |
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One family of genes that shows significant changes in expression during development and molting is the hemocyanin gene family (Terwilliger and Brown 1993
; Terwilliger and others 1999, 2005; Brown-Peterson and others 2005). Like other gene families, it is composed of a group of genes whose sequences can be aligned and that share a high degree of amino acid similarity (Thornton and DeSalle 2000). Some of the genes in this family are orthologues, genes in different genomes that have been created by the splitting of taxonomic lineages (Fitch 1970). They are versions of the same gene in different organisms that have slowly differentiated from one another after speciation. Others are paralogues, genes in the same genome that have arisen by gene duplication. While the hemocyanin gene family is strongly conserved among the Arthropoda, it has also undergone multiple gene duplications as evidenced by the number of different gene products that assemble to form the hexameric and multihexameric aggregates circulating in the hemolymph of a crab or tarantula (Markl and Decker 1992). Some gene duplications and subsequent mutations have led to the acquisition of new functions as well as the loss of old functions (Terwilliger and others 2005). Thus the gene family is composed of related protein types with diverse functions.
Members of the arthropod hemocyanin family are significantly involved in at least three major processes of development and adaptation. First, aerobic respiration requires an adequate supply of oxygen, which is provided in many crustaceans by the copper-containing hemocyanin through its role in oxygen transport. Second, the innate immune response uses the enzymatic activity of phenoloxidase, a related copper protein (Söderhäll and Cerenius 1998). Phenoloxidase catalyzes the hydroxylation of monophenols such as tyrosine to diphenols such as dopamine and further oxidizes diphenols to highly reactive o-quinones. These o-quinones are on the pathway of melanin synthesis, a compound with antimicrobial and antifungal properties that is critical in the arthropod immune response. Oxygen used by phenoloxidase may be provided by hemocyanin, and hemocyanin can even function as a phenoloxidase under certain conditions (Zlateva and others 1996; Decker and others 2001). The third process in which this gene family takes part is molting or ecdysis. Phenoloxidase and perhaps hemocyanin play a key role in cross-linking or sclerotizing proteins in the initially flexible new exoskeleton after molting and exoskeleton repair as well as in the encapsulation of foreign material (Sugumaran 1998). Another member of the gene family, cryptocyanin, that has no oxygen transport or oxidase function since it has a reduced number of the histidine residues critical for copper-binding, is produced in high concentrations during premolt and helps form the new exoskeleton (Terwilliger and others 1999; Terwilliger and others 2005). Since these proteins are functionally well characterized and are key players in major physiological processes, we expect that they will be one of the many gene clusters to demonstrate marked transcriptional changes in microarray analyses. We hope to integrate detailed knowledge of protein and gene expression in this gene family with global-expression patterns. It is anticipated that the combined gene-by-gene and global approaches will yield unexpected patterns of protein evolution and conservation of structure and function.
Hemocyanin, like hemoglobin and many other proteins, is a multisubunit molecule. A single hemocyanin gene product or subunit is folded in a characteristic pattern into 3 regions or domains, and the 2 copper atoms are bound in the center of domain II (Hazes and others 1993; Decker and Jaenicke 2004). This region is the functional center of the molecule with respect to oxygen interactions. It contains the 2 copper-binding sites, A and B, each with 3 highly conserved histidines in the hemocyanins and phenoloxidases. Even the cryptocyanins, missing some of these histidines, show a high degree of overall similarity in amino acid sequence in the Copper A and B sites (Burmester 1999). Additional functions that have been described recently for hemocyanin involve the other domains of the protein. The carboxy terminus in domain III can be cleaved in the hemocyanin of penaeid shrimp and crayfish, and the resulting fragments have antifungal or antibacterial properties (Destoumieux-Garzon and others 2001; Lee and others 2003). When the N-terminal portion of domain I of crayfish hemocyanin is removed by selected proteolysis, the remaining molecule becomes an active phenoloxidase (Lee and others 2004). These post-translational modifications of the first and third domains of the hemocyanin protein probably occur in vivo in at least some species.
The hemocyanin subunits self-assemble into hexamers, 2-hexamers, 4-hexamers, 6-hexamers, and 8-hexamers (Markl and Decker 1992; Van Holde and Miller 1995). The level of assembly is specific for different phyletic groups of arthropods. Cryptocyanin subunits also assemble into hexamers that circulate in the hemolymph (Terwilliger 1999). The related copper-free proteins found in hemolymph of numerous insects form hexamers, hence the collective name of "hexamerin" (Telfer and Kunkel 1991; Beintema and others 1994). Crustacean phenoloxidases occur as inactive proenzymes within circulating hemocytes in the hemolymph and are released from the cell upon activation by proteolytic cleavage or conformational change. Despite this different mode of transport through the hemolymph, purified phenoloxidase from the spiny lobster Palinurus elephas and the crayfish Astacus leptodactylus are also hexamers (Jaenicke and Decker 2003). Although the three dimensional structures of cryptocyanin and arthropod phenoloxidase are unknown, their sequence similarities and hexameric quaternary structures suggest that there is a shared motif or "hemocyanin fold" among all these proteins just as there is a "myoglobin fold" for invertebrate and vertebrate hemoglobins and myoglobins. These patterns of subunit folding and hexameric assembly in the hemocyanin gene family demonstrate how conservation of sequence results in a high degree of conservation of folding patterns, binding sites on subunit surfaces for aggregation into multimers, and general molecular shape.
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cDNA sequences of hemocyanin gene family in C. magister |
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SynopsisIntroductionHemocyanin gene familycDNA sequences of hemocyanin...Phylogenetic analysis:...Conservation of subunit...Microarray analysis of...SummaryREFERENCES |
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In addition to their multisubunit quaternary structure, crustacean hemocyanins show extensive subunit heterogeneity. The number of types of subunits is species-specific. For example, based on polyacrylamide gel electrophoresis, hemocyanin of Petrolisthes cinctipes is composed of 4 subunits, while that of Petrolisthes eriomerus contains 5 (Schmitt 2002
). Whether each subunit is the product of a distinct gene or the result of gene splicing or post-translational modification is not known for most crustacean hemocyanins. A combination of modified 2-D gel electrophoresis and N-terminal sequencing of the 6 electrophoretically distinct subunits of C. magister hemocyanin had indicated that the subunits were separate gene products (Larson and others 1981; Durstewitz and Terwilliger 1997b). We have recently amplified, cloned, and sequenced the complete cDNAs of these 6 hemocyanin subunits, as well as 2 cryptocyanins, and 1 prophenoloxidase. We are also sequencing the genomic DNA. The data confirm their identities as unique gene products.
Each of the 9 cDNA sequences encompassed the complete coding region plus 5' and 3' untranslated regions. The open reading frames translate into putative 662–677 amino acid proteins with predicted molecular masses of 72 637–77 325 kDa (Table 1). All 6 hemocyanin sequences and 2 cryptocyanin sequences include signal peptides of 15–20 amino acids, as expected for proteins synthesized in hepatopancreas tubule cells and secreted into the hemolymph (Bendtsen and others 2004; Terwilliger and others 2005). Prophenoloxidase does not have a signal peptide, consistent with hemocyte prophenoloxidase sequences of other arthropods (Aspán and others 1995; Hartzer and others 2005; Terwilliger and Ryan 2006) and reflecting its location in the hemolymph in circulating hemocytes rather than as an extracellular protein.
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Alignment of the deduced amino acid sequences of hemocyanin subunits 1–6 of C. magister provides the first opportunity to assess the similarities and differences among all the subunits that make up the multihexameric hemocyanin of one species of brachyuran crustacean (Fig. 1). It also affords a unique comparison of hemocyanin with cryptocyanin and prophenoloxidase in the same species. Those amino acids that are functionally similar across all 9 sequences and 3 protein types are highlighted in blue to illustrate the high degree of conservation in this family.
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The 6 CuA and CuB binding site histidines in the second domain are completely conserved in the hemocyanins and prophenoloxidase. In contrast, the cryptocyanins lack the full complement of histidines, as mentioned above. A phenylalanine in domain I is conserved across all the C. magister sequences except the cryptocyanins; this residue corresponds to Phe49 in hemocyanin of the chelicerate Limulus polyphemus that has been thought to play an important role in regulation of oxygen affinity, along with 2 conserved phenylalanines in domain II (Hazes and others 1993
). The phenylalanine in domain I may also participate in allowing access of larger phenolic substrates into the oxygen-binding pocket and thus help control the phenoloxidase activity of hemocyanin and of hemocyte phenoloxidase (Decker and Tuczek 2000). Therefore, the residues necessary for oxygen binding, either transport or oxidase activity, are present in the hemocyanins and prophenoloxidase of C. magister but are absent or partially conserved in the cryptocyanins.
A putative proteolytic cleavage site found in insect and crustacean prophenoloxidases that results in activation of the enzyme (Aspán and others 1995; Hall and others 1995; Sritunyalucksana and others 1999) is present in prophenoloxidase of C. magister (Terwilliger and Ryan 2006) but not in the hemocyanin or cryptocyanin subunits of this species. Similarly, a conserved thiol-ester motif (GCGWPQHM/L) found in chelicerate hemocyanins and prophenoloxidases of insects and other crustaceans (Hall and others 1995; Parkinson and others 2001; Kusche and others 2002) is seen in prophenoloxidase of C. magister and not in its hemocyanins or cryptocyanins. Another interesting feature of the prophenoloxidase is that it is lacking an alpha helical region in domain 1 that is present in the hemocyanin and cryptocyanin sequences of C. magister and in other crustacean hemocyanins and insect hexamerins. This region is also missing in chelicerate, myriapod, and onychophoran members of the hemocyanin gene family. Thus, crustacean and insect phenoloxidases share a number of features with chelicerate hemocyanins. The evolutionary implications are enhanced when one considers that hemocyanin, and not a separate hemocyte phenoloxidase, appears to be the source of phenoloxidase activity in chelicerates (Decker and others 2001).
Three predicted N-glycosylation sites (NXT/S) are located in the 2 cryptocyanin sequences, consistent with the strong PAS (Periodic Acid-Schiff) staining reactivity of the protein (Terwilliger and others 1999). A fourth potential site is present in Cryptocyanin 2. Hemocyanin subunits 3–6 each have one potential n-glycosylation site in domain III.
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Phylogenetic analysis: hemocyanin gene family |
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SynopsisIntroductionHemocyanin gene familycDNA sequences of hemocyanin...Phylogenetic analysis:...Conservation of subunit...Microarray analysis of...SummaryREFERENCES |
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The relatedness among the members of the hemocyanin gene family in C. magister, as determined by phylogenetic analysis using parsimony (Swofford 2002
), outlines the distinctions among the proteins (Fig. 2). In this tree, C. magister prophenoloxidase was used as the outgroup. The separation of hemocyanin subunits into 2 groups, 1 and 2 versus 3, 4, 5, and 6, is clear. The high degree of amino acid identity between hemocyanin subunits 1 and 2 seen in Figure 1 is also evident in the phylogeny and suggests a recent gene duplication. In these 2 subunits, domains I and II are nearly identical; sequence differences are much more marked in domain III. The cryptocyanins, also very similar to one another, form a separate group from the hemocyanins.
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In a phylogenetic comparison of the sequences of crustacean members of the arthropod hemocyanin gene family, the prophenoloxidases, including that of C. magister, form a strongly supported clade separate from the hemocyanins and cryptocyanins (Fig. 3). The putative prophenoloxidases of the ascidian Ciona intestinalis, a deuterostome, were used as the outgroup (Immesberger and Burmester 2004
). Crustacean hemocyanin sequences group into a pattern of alpha, beta, and gamma subunits first described on the basis of immunological cross-reactivities (Markl 1986). Hemocyanin subunits 1 and 2 of C. magister cluster in the beta group, while the other 4 C. magister hemocyanin subunits are clearly allied with gamma hemocyanin subunits of shrimp, spiny lobster, and another crab. Hemocyanin of C. magister apparently lacks alpha subunits in contrast to hemocyanins of other decapods, including lobsters and spiny lobsters (Kusche and others 2003). Crab and lobster cryptocyanins (Burmester 1999; Terwilliger and others 1999) are included in the hemocyanin clade. A comparison of hemocyanin gene family members from all classes of arthropods has been presented (Terwilliger and Ryan 2006); the phylogeny in Figure 3 specifically includes only hemocyanins, cryptocyanins, and phenoloxidases of crustaceans.
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, ß, and 
refer to immunologically defined crustacean hemocyanin subunit types (Markl 1986 |
Conservation of subunit diversity |
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SynopsisIntroductionHemocyanin gene familycDNA sequences of hemocyanin...Phylogenetic analysis:...Conservation of subunit...Microarray analysis of...SummaryREFERENCES |
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Hemocyanins from all arthropod groups studied to date are composed of multiple subunit types (Van Holde and Miller 1995
). The reasons for conservation of such subunit diversity are not known. It is possible that sloppy gene replication, a tendency toward duplicity, and a high degree of tolerance toward multiple copies are common among arthropods. An alternate hypothesis is that maintenance of gene duplications is a mechanism for selection for environmental diversity of function and for evolution toward multisubunit molecules with a broad spectrum of functional properties and responses. This has been a central theme of research on hemocyanins and hemoglobins (Mangum 1992). Experiments in which hemocyanin hexamers and multihexamers have been dissociated and reassembled in different combinations of monomers and tested functionally have shown that subunit heterogeneity is important for both structural aggregation state and cooperative oxygen binding (Sullivan and others 1974; van Bruggen and others 1980; Decker and others 1989). Changes in subunit stoichiometry in the whole animal in response to external or ontogenic stimuli have also reflected the relationship between heterogeneity and function. In experiments illustrating the effect of environmental perturbations on subunit composition and function, hemolymph of Callinectes sapidus contained more hemocyanin 1-hexamers than 2-hexamers after exposure of the crabs to hypoxia. The oligomers differed in subunit composition and oxygen-binding properties (Mangum and others 1991). Studies where developmental changes in subunit expression of hemocyanin resulted in marked differences in subunit composition and oxygen affinity have also demonstrated that subunit diversity affects function (Terwilliger and Brown 1993; Brown and Terwilliger 1998).
A challenge of both in vitro and in vivo experiments testing the adaptive significance of gene duplications is the ability to distinguish between specific versus system-wide responses to a stimulus or condition. The data we present here illustrate that with the striking similarities among the 9 C. magister sequences, certain regions cannot be differentiated among a hemocyanin, phenoloxidase, or cryptocyanin. However, there are also unique regions in each sequence that can be exploited to design subunit-specific probes. We have already used cDNA sequence differences between C. magister cryptocyanin 1 and hemocyanins 1–2 to develop in situ probes that established the site of synthesis of each protein (Terwilliger and others 2005). The sequence information on this gene family will provide expanded opportunities to analyze subunit-specific changes in mRNA expression and translation on a gene-by-gene level. Combining these focused gene studies with microarray analyses designed to differentiate between specific and general responses will present new opportunities to experimentally assess the adaptive effect of maintaining paralogous genes.
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Microarray analysis of expression change during eco-physiological stress in C. MAGISTER |
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SynopsisIntroductionHemocyanin gene familycDNA sequences of hemocyanin...Phylogenetic analysis:...Conservation of subunit...Microarray analysis of...SummaryREFERENCES |
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Construction of a cDNA library for microarray analysis of C. magister is in progress. A major design criterion was the inclusion of a broad spectrum of expressed genes in the microarray. Samples representing different developmental stages included embryos, megalopas, first through fifth instar juvenile crabs, and adult crabs. Daily samples across a complete molt cycle of first instar crabs were collected under laboratory conditions. Other crabs were exposed to experimental conditions of osmotic, hypoxic, or thermal stress and sampled before, during, and after the stresses. In addition, tissue-specific samples (heart, gill, hypodermis, leg muscle, hemocytes, and hepatopancreas) were collected from adult crabs. The cDNA library is being amplified and will be used to print microarrays. The arrays will then be queried using transcriptional profiles generated from crabs sampled during and after the developmental and physiological perturbations.
These experiments are expected to yield new information comparing those gene networks involved primarily in crustacean development, those that fluctuate through repeated molt cycles, and those that participate in both. The microarray studies may also demonstrate that transcriptome changes seen in response to unexpected challenges of hypoxia, hyposalinity, or hyperthermia do not resemble gene expression patterns of crustaceans during "normal" anticipated challenges such as molting. The analysis of correlated changes between transcriptome profiles during these major physiological events in crustaceans is expected to increase our understanding of the expression of genes in response to life cycle changes and environmental stresses.
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Summary |
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SynopsisIntroductionHemocyanin gene familycDNA sequences of hemocyanin...Phylogenetic analysis:...Conservation of subunit...Microarray analysis of...SummaryREFERENCES |
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Microarray studies that include cDNAs of individual subunits of multisubunit proteins along with expressed sequence tags (ESTs) or a cDNA library will allow us to monitor changes in expression of single subunits of each protein in the gene family in tandem with changes in the rest of the genome. The resulting data analyses in conjunction with directed functional studies will lead to the identification of functional networks at the level of the gene, the whole multisubunit molecule, and the whole organism simultaneously.
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Acknowledgements |
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The authors thank Don Mykles and David Towle, organizers of this SICB symposium. The authors also thank Eric Johnson, University of Oregon. This research was supported by NSF 9984202 to NBT.
Conflict of interest: None declared.
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Footnotes |
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From the symposium "Genomic and Proteomic Approaches in Crustacean Biology" presented at the annual meeting of the Society for Integrative and Comparative Biology, January 4–8, 2006, at Orlando, Florida.
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SynopsisIntroductionHemocyanin gene familycDNA sequences of hemocyanin...Phylogenetic analysis:...Conservation of subunit...Microarray analysis of...SummaryREFERENCES |
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