Structural, Thermal, and Storage Stability of Rapana Thomasiana Hemocyanin in the Presence of Cholinium-Amino Acid-Based Ionic Liquids.

Novel biocompatible compounds that stabilize proteins in solution are in demand for biomedical and/or biotechnological applications. Here, we evaluated the effect of six ionic liquids, containing mono- or dicholinium [Chol]1or2 cation and anions of charged amino acids such as lysine [Lys], arginine [Arg], aspartic acid [Asp], or glutamic acid [Glu], on the structure, thermal, and storage stability of the Rapana thomasiana hemocyanin (RtH). RtH is a protein with huge biomedicinal potential due to its therapeutic, drug carrier, and adjuvant properties. Overall, the ionic liquids (ILs) induce changes in the secondary structure of RtH. However, the structure near the Cu-active site seems unaltered and the oxygen-binding capacity of the protein is preserved. The ILs showed weak antibacterial activity when tested against three Gram-negative and three Gram-positive bacterial strains. On the contrary, [Chol][Arg] and [Chol][Lys] exhibited high anti-biofilm activity against E. coli 25213 and S. aureus 29213 strains. In addition, the two ILs were able to protect RtH from chemical and microbiological degradation. Maintained or enhanced thermal stability of RtH was observed in the presence of all ILs tested, except for RtH-[Chol]2[Glu].

Purification, characterization and antibacterial activities of red color-related protein found in the shell of kuruma shrimp, Marsupenaeus japonicus.

The well-known red color change plays a significant role in consumer acceptability of crustacean species. In this study, we described the purification of the red color-related protein named MjRCP75 from the shell of Marsupenaeus japonicus. It was a homogeneous monomer with molecular mass of 75 kDa and rich in α-helix conformation. The α-helix content decreased within the increasing of heating temperature and was transformed dominantly to ß types. Identification and structural analysis revealed that MjRCP75 belonged to hemocyanin family. The released pigment from heated MjRCP75 showed a λmax at 483 nm in acetone. MjRCP75 showed clearly antibacterial activity against Escherichia coli, Staphylococcus aureus, and Vibrio parahaemolyticus. These findings identify MjRCP75 as the red color-related protein in M. japonicus shell and reveal its involvement in antibacterial activities.

Intranasal Peptide-Based FpvA-KLH Conjugate Vaccine Protects Mice From Pseudomonas aeruginosa Acute Murine Pneumonia.

Pseudomonas aeruginosa is an opportunistic pathogen causing acute and chronic respiratory infections associated with morbidity and mortality, especially in patients with cystic fibrosis. Vaccination against P. aeruginosa before colonization may be a solution against these infections and improve the quality of life of at-risk patients. To develop a vaccine against P. aeruginosa, we formulated a novel peptide-based P. aeruginosa subunit vaccine based on the extracellular regions of one of its major siderophore receptors, FpvA. We evaluated the effectiveness and immunogenicity of the FpvA peptides conjugated to keyhole limpet hemocyanin (KLH) with the adjuvant curdlan in a murine vaccination and challenge model. Immunization with the FpvA-KLH vaccine decreased the bacterial burden and lung edema after P. aeruginosa challenge. Vaccination with FpvA-KLH lead to antigen-specific IgG and IgM antibodies in sera, and IgA antibodies in lung supernatant. FpvA-KLH immunized mice had an increase in recruitment of CD11b+ dendritic cells as well as resident memory CD4+ T cells in the lungs compared to non-vaccinated challenged mice. Splenocytes isolated from vaccinated animals showed that the FpvA-KLH vaccine with the adjuvant curdlan induces antigen-specific IL-17 production and leads to a Th17 type of immune response. These results indicate that the intranasal FpvA-KLH conjugate vaccine can elicit both mucosal and systemic immune responses. These observations suggest that the intranasal peptide-based FpvA-KLH conjugate vaccine with curdlan is a potential vaccine candidate against P. aeruginosa pneumonia.

Facile synthesis of haemocyanin-capped zinc oxide nanoparticles: Effect on growth performance, digestive-enzyme activity, and immune responses of Penaeus semisulcatus.

ZnO nanoparticles (NPs) synthesized using haemocyanin (Hc-ZnONPs) purified from haemolymph of Penaeus semisulcatus were characterized using various techniques. HR-TEM and SEM microscopy indicated Hc-ZnONPs had a typical size of 20-50 nm and were spherical. The objective of current investigation was to assess the effects of dietary supplementation of Hc-ZnONPs on the development and activity of digestive and metabolic enzymes, as well as the antioxidant levels in P. semisulcatus. Trial basal diets were supplemented with Hc-ZnONPs at rates of 0, 10, 20, 40, 60, and 80 mg kg-1 (dry feed weight) and were fed to P. semisulcatus for 30 d. For 60 mg kg-1 Hc-ZnONPs-supplemented feed, significantly (P < 0.05) enhanced endurance, development, and activity of the digestive enzyme were observed. The enzymatic antioxidants and metabolic enzymes activities in the muscle exhibited no significant changes when 10-60 mg kg-1 Hc-ZnONPs-supplemented feed was fed to P. semisulcatus. Conversely, feeding the P. semisulcatus with 80 mg kg-1 Hc-ZnONPs produced a harmful outcome, with significant increase in the enzymatic antioxidants and metabolic enzymes. Consequently, 80 mg kg-1 Hc-ZnONPs was identified as lethal to P. semisulcatus. Hence, it is proposed that the diet of P. semisulcatus can be supplemented with up to 60 mg kg-1 Hc-ZnONPs for improving the endurance, development and immunity.

Synthesis and characterization of a novel chemically designed (Globo)3-DTPA-KLH antigen.

In recent years, many experiments have been conducted for the production and evaluation of anticancer glycoconjugated vaccines in developed countries and many achievements have been accomplished with Globo H derivatives. In the current experiment, a new chemically designed triplicate version of (Globo H)3-diethylenetriamine pentaacetic acid (DTPA)-KLH antigen was synthesized and characterized. Immunization with (Globo H)3-DTPA-KLH, a hexasaccharide that is a member of a family of antigenic carbohydrates that are highly expressed in various types of cancers conjugated with DTPA and KLH protein, induced a high level of antibody titer along with an elevated level of IL-4 in mice. Treatment of tumors with the collected sera from immunized mice decreased the tumor size in nude mice as well. None of the immunized mice illustrated any sign of tumor growth after injection of MCF-7 cells compared to the control animals. These findings, based on the newly presented structure of the Globo H antigen, lend exciting and promising evidence for clinical advancement in the development of a therapeutic vaccine in the future.

Hemocyanin-derived phenoloxidase activity: a contributing factor to hyperpigmentation in Nephrops norvegicus.

The phenomenon of hyperpigmentation (melanosis) in shellfish has long been attributed to phenoloxidase enzymes. Over the last number of years, the oxygen carrier hemocyanin, has demonstrated several immune- and physiological functionalities, most notably, inducible phenoloxidase activity. In this study, hemocyanin purified from the hemolymph of Nephrops norvegicus displays diphenoloxidase activity in the presence of a number of elicitors and retains structural and functional integrity throughout the process of freeze-thawing (at -25 °C). Conversely, cellular phenoloxidase activity (present in cell-lysates), demonstrates >98% reduction in activity after freeze-thawing. We present evidence that hemocyanin may act as a causative agent of hyperpigmentation in N. norvegicus. The inhibition of hemocyanin-derived phenoloxidase activity is discussed, and for the first time, the biophysical interactions of shellfish hemocyanin with known phenoloxidase inhibitors are presented.

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.

Preparation and characterization of artificial antigens for cadmium and lead.

Cadmium and lead were conjugated to two carrier proteins using a bifunctional chelator [2-(4-aminobenzyl)-diethylenetriaminepentaacetic acid] to synthesize artificial antigens for cadmium and lead. The techniques, including ultraviolet spectrometry, circular dichroism, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and inductively coupled plasma optical emission spectroscopy, were utilized for characterizing the artificial antigens. The results of ultraviolet spectrometry showed characteristic absorption peak shifts between conjugates and carrier proteins. Circular dichroism resulted that the second structure of the conjugates was α-helix. The sodium dodecyl sulfate polyacrylamide gel electrophoresis results revealed the differences of band migration and molecular weight among antigens, chelator protein conjugate, and carrier proteins. The result of coupling ratios revealed that the metal content of the antigens was much higher than that of carrier proteins. These results indicated that the artificial antigens of cadmium and lead were synthesized successfully and had potential application in immunoassays of cadmium and lead ions.

Immunization of beef heifers against gonadotropin-releasing hormone prevents luteal activity and pregnancy: Effect of conjugation to different proteins and effectiveness of adjuvants.

Three experiments were conducted to evaluate methods of immunization against GnRH on antibody titer, luteal activity, and pregnancy in beef heifers. Experiment 1 evaluated the efficacy of adjuvants with 30 heifers. Control heifers were immunized against human serum albumin (HSA) emulsified in Freund's complete adjuvant (FCA). The other 4 treatments contained GnRH conjugated to HSA (HSA-GnRH) emulsified in FCA, Freund's incomplete adjuvant (FIA), DEAE dextran (DD) + mineral oil (MO), or DD+FIA. Treatment was in the mammary gland for all experiments. Titers against GnRH for heifers immunized against HSA-GnRH with FCA, DD+MO, or DD+FIA were greater than titers for HSA-GnRH with FIA or control heifers (P < 0.01). Body weight was reduced (P < 0.05) in control and FCA heifers compared with FIA, DD+MO, and DD+FIA heifers. Heifers immunized with DD+MO and DD+FIA had fewer granulomas in mammary glands than heifers treated with FCA (P < 0.01). In Exp. 2, 36 heifers were used to determine the effect of the protein conjugated to GnRH on titers against GnRH. Heifers (6/treatment) received a primary immunization against GnRH conjugated to HSA (HSA-GnRH), ovalbumin (OA-GnRH), or keyhole limpet hemocyanin (KL-GnRH), or heifers were immunized against each carrier protein. Antigens were emulsified in DD+FIA. Immunization of heifers against OA-GnRH, KL-GnRH, or HSA-GnRH suppressed luteal activity (P < 0.01) for 23, 16, and 12 wk, respectively, and antibody titers against GnRH were greater (P < 0.01) for 19, 5, and 7 wk, respectively, compared with heifers immunized against the carrier proteins. In Exp. 3, 90 heifers were used to determine the effect of immunization against GnRH on ovarian activity and pregnancy rate. Heifers (30/treatment) received a primary and 2 or 3 booster immunizations against GnRH conjugated to OA, and controls received a primary and 2 booster immunizations against OA. All antigens were emulsified in DD+FIA. At 8 wk after primary immunization, heifers were exposed to fertile bulls for 24 wk. Pregnancy rate was less (P < 0.01) for 3-booster heifers (13%) compared with control (83%) and 2-booster (62%) heifers. We conclude that immunization against GnRH, conjugated to OA and emulsified in DD+FIA, does not influence ADG and produces sufficient titers against GnRH to prevent estrous cycles with few mammary granulomas. Immunization against GnRH with 3 booster immunizations prevented luteal activity and pregnancy in most beef heifers for more than 4 mo.

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.

Marine gastropod hemocyanins as adjuvants of non-conjugated bacterial and viral proteins.

Killed viral vaccines and bacterial toxoids are weakly immunogenic. Numerous compounds are under evaluation as immunological adjuvants and peptide-carriers to improve the immune response. The hemocyanins, giant extracellular copper proteins in the blood of many mollusks, are widely used as immune stimulants. In the present study we investigated the adjuvant properties of hemocyanins isolated from marine gastropods Rapana thomasiana and Megathura crenulata. An immunization with Influenza vaccine or tetanus toxoid combined with Rapana thomasiana hemocyanin (RtH) and Keyhole limpet hemocyanin (KLH) in mice induced an anti-influenza cytotoxic response lasting at least 5 months and an antibody response to viral proteins. The IgG antibody response to the tetanus toxoid (TT) combined with RtH or KLH was comparable to the response of the toxoid in complete Freund's adjuvant. The results obtained demonstrate that the both hemocyanins are acceptable as potential bio-adjuvants for subunit vaccines.

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.

Folate-targeted hapten immunotherapy of adjuvant-induced arthritis: comparison of hapten potencies.

We have previously reported that disease symptoms can be greatly ameliorated in rodents with adjuvant-induced arthritis (AIA) by first immunizing the rodents against fluorescein and then treating the animals with folate-fluorescein. In this targeted hapten therapy, folate-fluorescein was shown to decorate folate receptor (FR)-expressing activated macrophages with fluorescein (an immunogenic hapten), leading to binding of antifluorescein antibodies and the consequent elimination of the activated macrophages by Fc receptor-expressing immune cells. In the current study, we compare the therapeutic potencies of a variety of FR-targeted haptens in treating the symptoms of AIA in rats. Rats were immunized with either dinitrophenyl (DNP) or trinitrophenyl (TNP) conjugated to keyhole limpet hemocyanin followed by induction of AIA with heat-inactivated Mycobacterium butyricum. Following development of arthritis, rats were treated with one of five folate-hapten conjugates (folate-DNP1, folate-DNP2, folate-DNP3, folate-FITC, or folate-TNP) at two different doses (30 nmol/kg or 200 nmol/kg) 5x/week for 25 days. Symptoms of AIA in treated rats, including paw swelling, arthritis score, splenomegaly, bone erosion, and FR(+) activated macrophage density in inflamed tissues, were quantitated over the course of therapy. Although all folate-hapten conjugates promoted a reduction in disease symptoms, folate-TNP and folate-FITC proved to be more potent than any of the 3 folate-DNP conjugates. We conclude that both folate-TNP and folate-FITC constitute promising haptens for use in FR-targeted immunotherapy of arthritis.

Thermodynamics of effector binding to hemocyanin: influence of temperature.

Hemocyanins are allosterically regulated oxygen carriers freely dissolved in the blood of many crustaceans. The natural modulator urate accumulates under hypoxic conditions in the hemolymph of Homarus vulgaris, and increases the oxygen affinity of the respiratory pigment. Other non-physiological effectors such as caffeine, dimethylxanthines and methylxanthines are also known to modulate the oxygen-binding properties of hemocyanin. In order to gain insight into the thermodynamic driving forces of these interactions we analyzed the binding of several urate analogs to dodecameric hemocyanin at different temperatures by employing isothermal titration calorimetry (ITC). All investigated non-physiological effectors including caffeine, dimethylxanthines and methylxanthines bind exothermically to hemocyanin and the binding processes are enthalpy driven. Furthermore, we demonstrated a strong temperature dependent binding of caffeine and dimethylxanthines to the hemocyanin of the European lobster and could show that the binding properties of the effectors to the urate-binding site depend on the hydrophobicity of a given molecule.

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.

A novel putative tyrosinase involved in periostracum formation from the pearl oyster (Pinctada fucata).

Tyrosinase (monophenol, L-DOPA: oxygen oxidoreductase, EC 1.14.18.1), a kind of copper-containing phenoloxidase, arouses great interests of scientists for its important role in periostracum formation. A cDNA clone encoding a putative tyrosinase, termed OT47 because of its estimated molecular mass of 47kDa, was isolated from the pearl oyster, Pinctada fucata. This novel tyrosinase shares similarity with the cephalopod tyrosinases and other type 3 copper proteins within two conserved copper-binding sites. RT-PCR analysis showed that OT47 mRNA was expressed only in the mantle edge. Further in situ hybridization analysis and tyrosinase activity staining revealed that OT47 was expressed at the outer epithelial cells of the middle fold, different from early histological results in Mercenaria mercenaria, suggesting a different model of periostracum secretion in P. fucata. Taken together, these results suggest that OT47 is most likely involved in periostracum formation. The identification and characterization of oyster tyrosinase also help to further understand the structural and functional properties of molluscan tyrosinase.

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.

Optimization of the modification of carrier proteins with aminated haptens.

In this report we show that succinic groups are far more reactive to amino compounds than the carboxylic groups derived from Asp and Glu on the protein when using coupling via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) (even by an 8 fold factor). Accordingly, a new carrier-protein was designed where both natural amino and carboxylic moieties were transformed into succinic residues. To prepare this hypersuccinylated carrier, all exposed carboxylic acids were first transformed into amino groups by reaction with ethylendiamine after activation with EDCI. Secondly, all these residues together with the ones from Lys were succinylated to prepare a fully succinylated protein. This was even more relevant considering that the amount of Lysine was 2-4 fold lower than Asp and Glu in most of the proteins. These "hyper-succinylated" proteins (KLH or BSA) offer significant improvements in protein reactivity compared to the native proteins (by a factor of 8-10). The optimization of the reaction, in which the presence of dioxane was found to be influential, permitted further improvements in the modification of the protein. Finally, this new strategy was successfully used to develop antibodies against the commercial anti-tumor molecule, ET-637-NH2. Using native KLH no response was found, whereas 1/64,000 serum dilutions gave very high values in ELISA procedures when immunization was performed using the hyper-succinylated KLH.

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.