Molecular isolation and expression analysis of hemocyanin isoform 2 of Macrobrachium rosenbergii.

OBJECTIVE: Hemocyanin is a copper-bearing protein in the hemolymph of many arthropods and mollusks and functions as an oxygen transport and important nonspecific immune protein. METHODS: In this study, complementary DNA of hemocyanin isoform 2 of the prawn Macrobrachium rosenbergii (MrHc2) was isolated by rapid amplification of cDNA ends and mRNA expression was characterized to elucidate molecular basis of its function. RESULT: With a molecular mass of 77.3 kDa, MrHc2 contained three domains: hemocyanin-all-alpha, hemocyanin-copper-containing, and hemocyanin-immunoglobulin-like domains. Molecular phylogenetic analysis revealed that MrHc2 belongs to the γ-type subunit and is closely related to hemocyanin subunit 1 of the palaemonid shrimp Macrobrachium nipponense. In addition, MrHc2 resided in a different clade relative to hemocyanin (MrHc) of M. rosenbergii (α-type subunit) and in a different subclade relative to the hemocyanin proteins of penaeid shrimp. The messenger RNA transcript of MrHc2 was highly expressed in the hepatopancreas and weakly expressed in the gills, intestine, stomach, muscle, and hemocytes. Upon challenge with M. rosenbergii nodavirus (MrNV), the expression of MrHc2 was 1.96-, 2.93-, and 1.96-fold on days 3, 4, and 5, respectively, and then gradually declined to basal levels on day 7. CONCLUSION: This study suggests that MrHc2 plays an important role in the innate immune response of M. rosenbergii to MrNV.

Responsiveness of metallothionein and hemocyanin genes to cadmium and copper exposure in the garden snail Cornu aspersum.

Terrestrial gastropods express metal-selective metallothioneins (MTs) by which they handle metal ions such as Zn2+ , Cd2+ , and Cu+ /Cu2+ through separate metabolic pathways. At the same time, they depend on the availability of sufficient amounts of Cu as an essential constituent of their respiratory protein, hemocyanin (Hc). It was, therefore, suggested that in snails Cu-dependent MT and Hc pathways might be metabolically connected. In fact, the Cu-specific snail MT (CuMT) is exclusively expressed in rhogocytes, a particular molluscan cell type present in the hemocoel and connective tissues. Snail rhogocytes are also the sites of Hc synthesis. In the present study, possible interactions between the metal-regulatory and detoxifying activity of MTs and the Cu demand of Hc isoforms was explored in the edible snail Cornu aspersum, one of the most common European helicid land snails. This species possesses CdMT and CuMT isoforms involved in metal-selective physiological tasks. In addition, C. aspersum expresses three different Hc isoforms (CaH ɑD, CaH ɑN, CaH ß). We have examined the effect of Cd2+ and Cu2+ exposure on metal accumulation in the midgut gland and mantle of C. aspersum, testing the impact of these metals on transcriptional upregulation of CdMT, CuMT, and the three Hc genes in the two organs. We found that the CuMT and CaH ɑD genes exhibit an organ-specific transcriptional upregulation in the midgut gland of Cu-exposed snails. These results are discussed in view of possible interrelationships between the metal-selective activity of snail MT isoforms and the synthesis and metabolism of Hc isoforms.

Design and evaluation of a hypoallergenic peptide-based vaccine for Salsola kali allergy.

BACKGROUND: The Salsola kali (S. kali) pollen is one of the most important causes of allergic rhinitis in the deserts and semi-desert areas. Immunotherapy with allergen extracts remains the only available treatment addressing the underlying mechanism of allergy. However, given the low efficacy of this method, it is necessary to find more effective and alternative therapeutic interventions using molecular biology and bioinformatics tools. In this study, a hypoallergenic vaccine was designed on the basis of B-cell epitope approach for S. kali immunotherapy. METHODS: Using the Immune Epitope Database (IEDB), a 35-mer peptide was selected and chemically conjugated to a keyhole limpet hemocyanin (KLH) molecule. Specific IgG and IgE from immunized BALB/c mice sera against the vaccine (Sal k 1-KLH), S. kali extract and the recombinant protein, rSal k 1, were measured using ELISA. Also, inhibition of IgE by mouse IgG was evaluated using an inhibitory ELISA. Finally, the IgE reactivity and T-cell reactivity of the designed vaccine were evaluated by dot blot assay and MTT assay. RESULTS: Vaccination with the vaccine produced high levels of protective IgG in mice, which inhibited the binding of patients IgE to recombinant proteins. The result showed that the designed vaccine, unlike the recombinant protein and extract, did not induce T-cell lymphocytes response and also exhibited decreased IgE reactivity. CONCLUSION: The designed vaccine can be considered as a promising candidate for therapeutic allergen-specific immunotherapy.

A peptide immunization approach to counteract a Staphylococcus aureus protease defense against host immunity.

Pathogens that induce acute and chronic infections, as well as certain cancers, employ numerous strategies to thwart host cellular and humoral immune defenses. One proposed evasion mechanism against humoral immunity is a localized expression of extracellular proteases that cleave the IgG hinge and disable host IgG functions. Host immunity appears to be prepared to counter such a proteolytic tactic by providing a group of autoantibodies, denoted anti-hinge antibodies that specifically bind to cleaved IgGs and provide compensating functional restoration in vitro. These respective counter-measures highlight the complex interrelationships among pathogens and host immunity and suggested to us a possible means for therapeutic intervention. In this study, we combined an investigation of pathogen-mediated proteolysis of host IgGs with an immunization strategy to boost host anti-hinge antibodies. In a Staphylococcus aureus infection model using an artificial tissue cage (wiffle ball) implanted into rabbits, cleaved rabbit IgGs were detected in abundance in the abscesses of untreated animals early after infection. However, in animals previously immunized with peptide analogs of the cleaved IgG hinge to generate substantial anti-hinge antibody titers, S. aureus colony formation was markedly reduced compared to control animals or those similarly immunized with a scrambled peptide sequence. The results of this study demonstrate that extensive local proteolysis of IgGs occurs in a test abscess setting and that immunization to increase host anti-hinge antibodies provided substantial acute protection against bacterial growth.

Molecular cloning of hemocyanin cDNA from Fenneropenaeus chinensis and antimicrobial analysis of two C-terminal fragments.

Peptides derived from shrimp hemocyanin have antimicrobial properties. This is the first report of hemocyanin cDNA (FCHc) cloned from Fenneropenaeus chinensis and recombinant expression of two C-terminal fragments. Based on sequence analysis of Fenneropenaeus chinensis hemocyanin FCHc, we subcloned two FCHc fragments by designing special primers. Two antimicrobial peptides (AMPs) were derived from FCHc (FCHc-C1 and FCHc-C2). The recombinant sequence of FCHc-C1 consisted of 207 bp encoding 69 amino acids and the recombinant sequence of FCHc-C2 consisted of 120 bp encoding 40 amino acids. The results of Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting indicated that recombinant FCHc-C1 and FCHc-C2 peptides (rFCHc-C1 and rFCHc-C2) were expressed successfully. An inhibition assay showed that FCHc-C1 and FCHc-C2 were anionic AMPs with antifungal and antibacterial activities.

Identification and characterization of the related immune-enhancing proteins in crab Scylla paramamosain stimulated with rhubarb polysaccharides.

Recently, considerable interest has been focused on immunostimulants to reduce diseases in crab aquaculture. However, information regarding to the related immune-enhancing proteins in crabs is not available yet. In this study, rhubarb polysaccharides were tested for enhancement of the immune activity in crab Scylla paramamosain. Compared with those in the control group, values of, phenoloxidase (PO), alkaline phosphatase (AKP) and alkaline phosphatasein (ACP) activity in the, experimental group were improved significantly 4 d after the treatment. Furthermore, 15 and 17 altered proteins from haemocytes and hepatopancreas, respectively, were found in rhubarb polysaccharide-treated crabs using 2-DE approach. Of these, hemocyanin, chymotrypsin, cryptocyanin, C-type lectin receptor, and ferritin protein were identified by mass spectrometry. In addition, RT-PCR, analysis showed that the mRNA levels of hemocyanin and chymotrypsin increased about 2.4- and 1.4-fold in the experiment group. Moreover, the hemocyanin gene in S. paramamosain (SpHMC) was, cloned and characterized. SpHMC contains one open reading frame of 2022 bp and encodes a polypeptide of 673 amino acids. It is clustered into one branch along with crab hemocyanin in a phylogenetic tree. The mRNA transcripts of SpHMC were detected mainly in the tissues of, hepatopancreas, hemocyte and intestines, and its levels were up-regulated significantly in hemocytes, of S. paramamosain treated with Vibrio parahemolyticus, Beta streptococcus or poly I:C for 6-48 h. Taken together, these studies found 5 related immune-enhancing proteins and a novel heomcyanin homologue with potential pathogen-resistant activities in crab.

Molecular characterization and evolution of haemocyanin from the two freshwater shrimps Caridina multidentata (Stimpson, 1860) and Atyopsis moluccensis (De Haan, 1849).

Haemocyanin (Hc) is a copper-containing respiratory protein, floating freely dissolved in the hemolymph of many arthropod species. A typical haemocyanin is a hexamer or oligohexamer of six identical or similar subunits, with a molecular mass around 75 kDa each. In the crustaceans, the haemocyanins appear to be restricted to the remipedes and the malacostracans. We have investigated the haemocyanins of two freshwater shrimps, the Amano shrimp Caridina multidentata and the bamboo shrimp Atyopsis moluccensis. We obtained three full-length and one partial cDNA sequences of haemocyanin subunits from the Amano shrimp, which were assigned to the α- and γ-types of decapod haemocyanin subunits. Three complete and two partial haemocyanin cDNA sequences were obtained from the bamboo shrimp, which represent subunit types α, ß and γ. This is the first time that sequences of all three subunit types of the decapod haemocyanins were obtained from a single species. However, mass spectrometry analyses identified only α- and γ-type subunits, suggesting that a ß-subunit is not a major component of the native haemocyanin of the bamboo shrimp. Phylogenetic and molecular clock analyses showed that malacostracan haemocyanins commenced to diversify into distinct subunit types already ~515 million years ago. ß-subunits diverged first, followed by α- and γ-type subunits ~396 million years ago. The haemocyanins of phyllocarids and peracarids form distinct clades within the α/γ-cluster. Within the Caridea, an early divergence of distinct α-type subunits occurred ~200 MYA. The tree of the γ-subunits suggests a common clade of the Caridea (shrimps) and Penaeidae (prawns).

Molecular and functional characterization of hemocyanin of the giant African millipede, Archispirostreptus gigas.

In contrast to other terrestrial arthropods, where gaseous O2 that fuels aerobic metabolism diffuses to the tissues in tracheal tubes, and most other metazoans, where O2 is transported to tissues by circulating respiratory proteins, the myriapods (millipedes and centipedes) strikingly have tracheal systems as well as circulating hemocyanin (Hc). In order to elucidate the evolutionary origin and biological significance of millipede Hc, we report the molecular structure (subunit composition and amino acid sequence) of multimeric (36-mer) Hc from the forest floor-dwelling giant African millipede Archispirostreptus gigas and its allosteric oxygen-binding properties under various physico-chemical conditions. Archispirostreptus gigas Hc consists of only a single subunit type with differential glycosylation. Phylogenic analysis revealed that millipede Hc is a sister group to centipede HcA, which supports an early divergence of distinct Hc subunits in myriapods and an ancient origin of multimeric Hcs. Archispirostreptus gigas Hc binds O2 with a high affinity and shows a strong Bohr effect. O2 binding is, moreover, modulated by Ca(2+) ions, which increase the O2 affinity of the Hc in the tense (T; deoxygenated) as well as the relaxed (R; oxygenated) states, and by (l)-lactate, which modulates Hc-O2 affinity by changing the allosteric equilibrium constant, L. Cooperativity in O2 binding at half O2 saturation (n50) is pH dependent and maximal at ~pH 7.4, and the number of interacting O2-binding sites (q) is markedly increased by binding Ca(2+). The data are discussed in the light of the mutually supplementary roles of Hc and the tracheal system for tissue O2 supply.

Molecular cloning, characterization and mRNA expression of copper-binding protein hemocyanin subunit in Chinese mitten crab, Eriocheir sinensis.

Hemocyanin is a copper-binding protein and plays a crucial role in the physiological processes in crustacean. In this study, the cDNA encoding hemocyanin subunit from Chinese mitten crab Eriocheir sinensis (EsHc) was cloned by using EST analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of EsHc was 2573 bp, consisting of a 5' untranslated region of 51 bp, a 3' untranslated region of 458 bp, and an open reading frame of 2064 bp. The deduced protein had 688 amino acid residues with molecular mass of 77,997.31 Da. Quantitative real-time RT-PCR analysis showed that the EsHc gene was expressed in haemocytes, hepatopancreas, muscles, gills, and intestines with the highest level of expression in the hepatopancreas and the lowest in the muscles. After Aeromonas hydrophila challenge, the relative expression level of EsHc in hemolymph was up-regulated at 3 h post-injection of bacteria followed by a gradual recovery from 12 to 24 h. In the second set of transcriptional studies, the mRNA expression patterns of EsHc in haemocytes and hepatopancreas were measured by quantitative real-time RT-PCR after the Chinese mitten crab were fed six diets containing different levels of copper (0, 10, 20, 40, 80 and 400 mg kg(-1)) for 8 weeks, respectively. The feeding trial showed that the expression levels of EsHc mRNA significantly increased at the copper levels of 20-40 mg kg(-1). This study implies that the expression levels of EsHc could be affected by dietary copper in the hepatopancreas and haemocytes, and hemocyanin may be potentially involved in the immune responses of the Chinese mitten crab.

Initial analysis of the hemocyanin subunit type 1 (Hc1 gene) from Locusta migratoria manilensis.

Hemocyanins are copper-containing (Cu(+)) proteins that transport oxygen in many arthropods hemolymph. We characterized Hc1 gene from the grasshopper species Locusta migratoria manilensis. In particular, we cloned and sequenced the corresponding cDNAs and studied their expression at different developmental stages. The cDNA of Hc1 gene (GenBank accession no.:HQ213937) is 2271 bp in length and the open reading frame is 2016 bp, which encodes a 672 amino acids protein with a calculated molecular mass of 77.9 kD and the isoelectric point of 6.06. Sequence alignment analysis result showed that this gene shares 94.7% identity with Schistocerca americana EHP. In addition, analysis of quantitative RT-PCR indicated that, LmiHc1 was expressed in the embyro (24, 39, 62, 86, 144, and 193 h after hatch), nymphs (1st instar, 2nd instar, 3rd instar, 4th instar and 5th instar) and in adult. These results showed that Hc1 plays an important role in grasshopper, which may be related to an enhanced oxygen supply. Phylogenetic analysis of insecta based on Hc1 are basically consistent with the morphology.

The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum.

Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α(D)-HlH, α(N)-HlH and ß-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α(D)-HlH and ß-HlH was obtained, including the 5' and 3' UTR, 180bp of the 5' end and around 900bp at the 3' end are missing for the third subunit. The subunits α(D)-HlH and ß-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α(N)-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α(D)-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry.

A putative hexamerin from a Campodea sp. suggests an independent origin of haemocyanin-related storage proteins in Hexapoda.

Haemocyanins are copper-containing respiratory proteins in the arthropod haemolymph. In hexapods, haemocyanins gave rise to hexamerins, which have lost the ability to bind copper and thus oxygen. Hexamerins are thought to act mainly as storage proteins in nonfeeding periods. So far, hexamerins have only been identified in ectognathan hexapods, but not in Entognatha. Here we report the identification of a putative hexamerin from Campodea sp. (Diplura). The full-length cDNA of Campodea sp. hexamerin 1 (CspHex1) measures 2188 bp and translates into a native polypeptide of 667 amino acids. As in other hexamerins, the six copper-coordinating histidines are not conserved. However, sequence comparison and phylogenetic analyses demonstrated that CspHex1 is not closely related to other hexapod hexamerins, which derive from hexapod type 1 haemocyanin subunits in the ectognathan lineage, but rather resembles a derivative of hexapod type 2 haemocyanin subunits. Hence, haemocyanin-related storage proteins emerged at least two times independently in Hexapoda.

The first complete cDNA sequence of the hemocyanin from a bivalve, the protobranch Nucula nucleus.

By cDNA sequencing we have achieved the first, and complete, hemocyanin sequence of a bivalve (Nucula nucleus). This extracellular oxygen-binding protein consists of two immunologically distinguishable isoforms, here termed NnH1 and NnH2. They share a mean sequence identity of 61%, both contain a linear arrangement of eight paralogous, ca.50-kDa functional units (FUs a-h), and in both isoforms the C-terminal FU-h possesses an extension of ca. 100 amino acids. The cDNA of NnH1 comprises 11,090 bp, subdivided into a 5'utr of 75 bp, a 3'utr of 791 bp, and an open reading frame for a signal peptide of 19 amino acids plus a polypeptide of 3389 amino acids (Mr = 385 kDa). The cDNA of NnH2 comprises 10,849 bp, subdivided into a 5'utr of 47 bp, a 3'utr of 647 bp, and an open reading frame for a signal peptide of 16 amino acids plus a polypeptide of 3369 amino acids (Mr = 387 kDa). In contrast to other molluscan hemocyanins, which are highly glycosylated, the bivalve hemocyanin sequence exhibits only four potential N-glycosylation sites, and within both isoforms a peculiar indel is present, surrounding the highly conserved copper-binding site CuA. Phylogenetic analyses of NnH1 and NnH2, compared to the known hemocyanin sequences of gastropods and cephalopods, reveal a statistically sound closer relationship between gastropod and protobranch hemocyanin than to cephalopod hemocyanin. Assuming a molecular clock, the last common ancestor of protobranch and gastropods lived 494 million +/- 50 million years ago, in conformity with fossil records from the late Cambrian.

Molecular cloning and characterisation of prophenoloxidase cDNA from haemocytes of the giant freshwater prawn, Macrobrachium rosenbergii, and its transcription in relation with the moult stage.

Expression of prophenoloxidase (proPO) cDNA was determined from haemocytes of the giant freshwater prawn Macrobrachium rosenbergii by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA using oligonucleotide primers based on the proPO sequence of tiger shrimp Penaeus monodon, freshwater crayfish Pacifastacus leniusculus, green tiger shrimp Penaeus semisulcatus, kuruma shrimp Marsupenaeus japonicus, and white shrimp Litopenaeus vannamei. The proPO of M. rosenbergii was constitutively expressed. The 2,547-bp cDNA contained an open reading frame (ORF) of 2,013 bp, a 96-bp 5'-untranslated region, and a 438-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid (aa) sequence (671 aa) was 76.7 kDa with an estimated pI of 7.05. It contained putative copper-binding sites, a complement-like motif (GCGWPRHM), a proteolytic activation site, and a conserved C-terminal region common to all known proPOs. However, no signal peptide sequence was detected in giant freshwater prawn proPO. Comparison of amino acid sequences showed that prawn proPO is similar to the proPO of penaeid, crayfish and lobster. Prawn proPO was only synthesised in haemocytes. The proPO transcript was significantly increased in the A stage and achieved the highest level in the B stage, and then declined sharply in the C stage and reached the lowest level in the D(2)/D(3) stage.

Developmental expression of two Haliotis asinina hemocyanin isoforms.

Hemocyanins are large copper-containing respiratory proteins that play a role in oxygen transport in many molluscs. In some species only one hemocyanin isoform is present while in others two are expressed. The physiological relevance of these isoforms is unclear and the developmental and tissue-specific expression of hemocyanin genes is largely unknown. Here we show that two hemocyanin genes in the gastropod Haliotis asinina, which encode H. asinina hemocyanin (HaH1) and HaH2 isoforms, are developmentally expressed. These genes initially are expressed in a small number of mesenchyme cells at trochophore and pre-torsional veliger stages, with HaH1 expression slightly preceding HaH2. These cells largely are localized to the visceral mass, although a small number of cells are present in head and foot regions. Following metamorphosis the isoforms show overlapping as well as isoform-specific expression profiles, suggesting some degree of isoform-specific function.

Biochemical and molecular characterisation of hemocyanin from the amphipod Gammarus roeseli: complex pattern of hemocyanin subunit evolution in Crustacea.

Hemocyanin is a copper-containing respiratory protein that is widespread within the arthropod phylum. Among the Crustacea, hemocyanins are apparently restricted to the Malacostraca. While well-studied in Decapoda, no hemocyanin sequence has been known from the 'lower' Malacostraca. The hemocyanin of the amphipod Gammarus roeseli is a hexamer that consists of at least five distinct subunits. The complete cDNA sequence of one subunit and a tentative partial sequence of another subunit have been determined. The complete G. roeseli hemocyanin subunit comprises 2,150 bp, which translates in a protein of 672 amino acids with a molecular mass of 76.3 kDa. Phylogenetic analyses show that, in contrast to previous assumptions, the amphipod hemocyanins do not belong to the alpha-type of crustacean hemocyanin subunits. Rather, amphipod hemocyanins split from the clade leading to alpha and gamma-subunits most likely at the time of separation of peracarid and eucarid Crustacea about 300 million years ago. Molecular clock analyses further suggest that the divergence of beta-type subunits and other crustacean hemocyanins occurred around 315 million years ago (MYA) in the malacostracan stemline, while alpha- and gamma-type subunits separated 258 MYA, and pseudohemocyanins and gamma-subunits 210 million years ago.

Effects of hypoxia on gene and protein expression in the blue crab, Callinectes sapidus.

Increases in hypoxic conditions are one of the major factors responsible for declines in estuarine habitat quality, yet to date there are no indicators for recognizing populations of estuarine organisms that are suffering from chronic hypoxic stress. Here we test the hypothesis that alterations in gene and protein expression of antioxidant enzymes and other stress-specific proteins can be used as molecular indicators of hypoxic stress. Blue crabs, Callinectes sapidus, were exposed to 2-3 ppm DO for 5 days. Gene expression was measured using macroarrays constructed from cDNA of 10 partial gene transcripts cloned from blue crab hepatopancreas. Significant (p< or =0.05) down-regulation of gene expression was found for MnSOD, hemocyanin, ribosomal S15 and L23. Subtractive hybridization using RNA from control and hypoxic hepatopancreas tissues also indicated down-regulation of hemocyanin transcription. In contrast, Western blotting showed a significant (p< or =0.05) increase of hemocyanin protein in the hepatopancreas and cross-linking of MnSOD proteins in hypoxia-exposed crabs. Thus, hypoxia-responsive cDNA arrays and Westerns may be useful diagnostic tools for monitoring effects of hypoxia in estuarine crustacea.

Putative phenoloxidases in the tunicate Ciona intestinalis and the origin of the arthropod hemocyanin superfamily.

In addition to the respiratory copper-containing proteins for which it is named, the arthropod hemocyanin superfamily also includes phenoloxidases and various copperless storage proteins (pseudo-hemocyanins, hexamerins and hexamerin receptors). It had long been assumed that these proteins are restricted to the arthropod phylum. However, in their analysis of the predicted genes in the Ciona intestinalis (Urochordata:Tunicata) genome, Dehal et al. (Science 298:2157-2167) proposed that the sea squirt lacks hemoglobin but uses hemocyanin for oxygen transport. While there are, nevertheless, four hemoglobin genes present in Ciona, we have identified and cloned two cDNA sequences from Ciona that in fact belong to the arthropod hemocyanin superfamily. They encode for proteins of 794 and 775 amino acids, respectively. The amino acids required for oxygen binding and other structural important residues are conserved in these hemocyanin-like proteins. However, phylogenetic analyses and mRNA expression data suggest that the Ciona hemocyanin-like proteins rather act as phenoloxidases, possibly involved in humoral immune response. Nevertheless, the putative Ciona phenoloxidases demonstrate that the hemocyanin superfamily emerged before the Protostomia and Deuterostomia diverged and allow for the first time the unequivocal rooting of the arthropod hemocyanins and related proteins. Phylogenetic analyses using neighbor-joining and Bayesian methods show that the phenoloxidases form the most ancient branch of the arthropod proteins, supporting the idea that respiratory hemocyanins evolved from ancestors with an enzymatic function. The hemocyanins evolved in agreement with the expected phylogeny of the Arthropoda, with the Onychophora diverged first, followed by the Chelicerata and Pancrustacea. The position of the myriapod hemocyanins is not resolved.

Expressed sequence tag analysis of vanadocytes in a vanadium-rich ascidian, Ascidia sydneiensis samea.

Some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, which corresponds to about 10(7) times higher than that in seawater. In these species signet ring cells, with a single huge vacuole in which vanadium ion is contained, function as vanadium-accumulating cells, vanadocytes. To investigate the mechanism underlying this phenomenon, we performed an expressed sequence tag (EST) analysis of a complementary DNA library from vanadocytes of a vanadium-rich ascidian, Ascidia sydneiensis samea. We determined the nucleotide sequences of 1000 ESTs and performed a BLAST analysis against the SwissProt database. We found 93 clones of metal-related gene homologues, including the ferritin heavy subunit, hemocyanin, and metallothionein. Two ESTs, in particular, exhibited significant similarity to vanabins that have been extracted from A. sydneiensis samea blood cells as low molecular weight vanadium-binding proteins. We have named the genes encoding these ESTs vanabin3 and vanabin4. Immobilized metal ion affinity chromatography revealed that these novel vanabin homologues bind vanadium(IV) ions.

cDNA sequence, protein structure, and evolution of the single hemocyanin from Aplysia californica, an opisthobranch gastropod.

By protein immunobiochemistry and cDNA sequencing, we have found only a single hemocyanin polypeptide in an opisthobranch gastropod, the sea hare Aplysia californica, which contrasts with previously studied prosobranch gastropods, which express two distinct isoforms of this extracellular respiratory protein. We have cloned and sequenced the cDNA encoding the complete polypeptide of Aplysia californica hemocyanin (AcH). The cDNA comprises 11,433 bp, encompassing a 5'UTR of 77 bp, a 3'UTR of 1057 bp, and an open reading frame for a signal peptide of 20 amino acids plus a polypeptide of 3412 amino acids (Mr ca. 387 kDa). This polypeptide is the subunit of the cylindrical native hemocyanin (Mr ca. 8 MDa). It comprises eight different functional units (FUs: a, b, c, d, e, f, g, h) that have been identified immunobiochemically after limited proteolysis of AcH purified from the hemolymph. Each FU shows a highly conserved copper-A and copper-B site for reversible oxygen binding. FU AcH-h carries a specific C-terminal extension of ca. 100 amino acids that include two cysteines that may be utilized for disulfide bridge formation. Potential N-glycosylation sites are present in six FUs but lacking in AcH-b and AcH-c. On the basis of multiple sequence alignments, phylogenetic trees and a statistically firm molecular clock were calculated. The latter suggests that the last common ancestor of Haliotis and Aplysia lived 373+/-47 million years ago, in convincing agreement with fossil records from the early Devonian. However, the gene duplication yielding the two distinct hemocyanin isoforms found today in Haliotis tuberculata occurred 343+/-43 million years ago.