Hemocyanin Isoforms Research Articles

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

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. 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. 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. This study suggests that MrHc2 plays an important role in the innate immune response of M. rosenbergii to MrNV.

Resolving hemocyanin isoform complexity in haemolymph of black tiger shrimp Penaeus monodon - implications in aquaculture, medicine and food safety

Hemocyanin (Hc) is a multifunctional macromolecule involved in oxygen transport and non-specific immunity in shrimp. Hc is crucial in physiology and nutrition linked with optimal performance in aquaculture production systems. In medicine, Hc has been approved for clinical use in humans as adjuvant and anticancer therapeutic. In contrast, Hc has also been identified as one of the proteins causing anaphylaxis following shrimp consumption. The role of individual Hc isoforms remains unknown due to a lack of resolved Hc isoforms. We successfully identified eleven different Penaeus monodon hemocyanin (PmoHc) γ isoforms including two truncated isoforms (50 and 20 kDa) and one PmoHc β isoform in haemolymph using proteomics informed by transcriptomics. Amino acid sequence homology ranged from 24 to 97% between putative PmoHc gene isoforms. Hc isoforms showed specific patterns of transcript expression in shrimp larval stages and adult hepatopancreas. These findings enable isoform level investigations aiming to define molecular mechanisms underpinning Hc functionality in shrimp physiology and immunity, as well as their individual immunogenic role in human allergy. Our research demonstrates the power of proteomics informed by transcriptomics to resolve isoform complexity in non-model organisms and lay the foundations for improved performance within the aquaculture industry and advance allergenic applications in medicine. SignificanceThe roles of hemocyanin (Hc) in shrimp homeostasis and immunity as well as in human allergy are not well understood because the complexity of Hc isoforms has remained unresolved. Our results have confirmed the existence of at least 12 individual Hc isoforms in shrimp haemolymph and validated putative Hc gene assemblies from transcriptomics. Our findings will enable monitoring the expression of specific Hc isoforms in shrimp haemolymph during different environmental, nutritional and pathogenic conditions, thus providing insights into isoform specific functional roles. In medicine, the potential allergenicity of each Hc isoform could be determined and advance allergenic applications. Lastly, since Hc comprises up to 95% of the total protein in haemolymph, these isoforms become ideal targets for prawn provenance, traceability and food contamination studies.

Protein-Level Evidence of Novel β-Type Hemocyanin and Heterogeneous Subunit Usage in the Pacific Whiteleg Shrimp, Litopenaeus vannamei

The functional diversity of crustacean hemocyanins is broad, encompassing O2 delivery, innate immune response, metabolite storage, and osmolyte balance, all in a heterogeneous protein structure. As such, the sequence diversity of this class of proteins and its subunit composition are the focus of many studies on crustacean adaptation to environmental challenges. Recent transcriptomic and genomic assemblies on the Pacific whiteleg shrimp Litopenaeus vannamei have identified unique isoforms of hemocyanin including an ancestral β-type subunit thought to be lost in penaeid shrimp. However, it is unknown the degree to which these isoforms are translated as proteins, and their abundances within the hemolymph. The present study uses proteomic approaches to characterize the protein-level abundance and organization of these hemocyanin isoforms within their native oligomeric structures. Fractions of each hemocyanin oligomeric form were purified by size-exclusion high performance liquid chromatography for identification of subunit isoforms using tandem mass spectrometry at <1% protein false discovery rate. Data are available via ProteomeXchange with identifier PXD014575. Relative abundances of hemocyanin oligomers and monomeric subunits from hemolymph and fractions were also quantified by polyacrylamide gel electrophoresis with and without denaturation for comparison of subunit heterogeneity. Hemocyanin subunits were organized primarily as hexamers (95-99% relative abundance) as opposed to dodecamers. Ten unique hemocyanins predicted by transcriptome and genome assemblies were identified by tandem mass spectrometry in both oligomer fractions including the first protein-level evidence of a β-type subunit in penaeid shrimp. Identified hemocyanins mapped to four genomic scaffolds and had two or three exons. A single small (75 kDa) subunit constituted half or more of all isoforms identified in each oligomer. The dodecameric fraction exhibited greater subunit heterogeneity utilizing a significantly lower ratio (1.04:1) of small subunit to large subunit compared to hexamers (2.05:1). One isoform, XP_027232115.1, appeared to be more dominant within dodecamers than hexamers. The ability to distinguish and quantify hemocyanin isoforms within oligomeric structures will aid future studies linking hemocyanin genes to transcripts to function and physiology as well as offer insight into the evolutionary history of crustaceans.

A proteomic analysis of the statocyst endolymph in common cuttlefish (Sepia officinalis): an assessment of acoustic trauma after exposure to sound

Recent studies, both in laboratory and sea conditions, have demonstrated damage after sound exposure in the cephalopod statocyst sensory epithelium, which secretes endolymph protein. Here, the proteomic analysis of the endolymph was performed before and after sound exposure to assess the effects of exposure to low intensity, low frequency sounds on the statocyst endolymph of the Mediterranean common cuttlefish (Sepia officinalis), determining changes in the protein composition of the statocyst endolymph immediately and 24 h after sound exposure. Significant differences in protein expression were observed, especially 24 h after exposure. A total of 37 spots were significantly different in exposed specimens, 17 of which were mostly related to stress and cytoskeletal structure. Among the stress proteins eight spots corresponding to eight hemocyanin isoforms were under-expressed possible due to lower oxygen consumption. In addition, cytoskeletal proteins such as tubulin alpha chain and intermediate filament protein were also down-regulated after exposure. Thus, endolymph analysis in the context of acoustic stress allowed us to establish the effects at the proteome level and identify the proteins that are particularly sensitive to this type of trauma.

Gene expression and phenoloxidase activities of hemocyanin isoforms in response to pathogen infections in abalone Haliotis diversicolor.

Hemocyanins (Hc), the main protein components of hemolymph in invertebrates, are not only involved in oxygen transport but also linked to non-specific immune responses. In this study, we used abalone (Haliotis diversicolor) Hc to study the basis of its diversified functions through gene, protein, peptides, and phenoloxidase (PO) activity levels. Three complete hemocyanin gene (HdH) sequences were cloned for the first time. By comparing the copies and location of HdH between abalone and other mollusks, we propose that Hc gene duplication and linkage is likely to be common during the evolution of mollusk respiratory proteins. We further demonstrate that all three genes could be expressed in abalone, with expression varying based on the developmental stages, tissue types, and different pathogen infections. However, HdH1 and HdH2 appear to be synthesized by the same cells by fluorescence in situ hybridization. Furthermore, the PO activity of HdH can be induced by trypsin, urea, and SDS in vitro. Viral infection can stimulate its PO activity in vivo by cleaving the protein into fragments. Consequently, we present a comprehensive study of abalone hemocyanin, providing important evidence for an in-depth understanding of the physiological and immune functions of Hc in mollusks.

Hemocyanin genes as indicators of habitat shifts in Panpulmonata?

Hemocyanin is the primary respiratory protein for the majority of the Mollusca and therefore directly interfaces with the physiological requirements of each species and the environments to which they are adapted. Hemocyanin is therefore likely to have been evolutionarily imprinted by significant habitat shifts. In the gastropod clade Panpulmonata (>30,000 species) major realm transitions have occurred multiple times independently and may have contributed to the diversification of this group. Yet, little is known about the adaptive changes linked to these habitat shifts. In order to gain deeper insight into the evolution of panpulmonate hemocyanins and to infer possible impacts associated with those scenarios, we have assembled and analysed hemocyanin isoforms from 4 panpulmonate species: (i) Helix pomatia, (ii) Cantareus aspersus (both Helicidae, Stylommatophora), (iii) Arion vulgaris (Arionidae, Stylommatophora) and (iv) Lymnaea stagnalis (Lymnaeidae, Hygrophila). Additionally, we describe a new hemocyanin isoform within the genome of the euopisthobranch Aplysia californica. Using these newly acquired hemocyanin data, we performed a phylogenetic analysis that reveals independent duplication events of hemocyanin within lineages that correlate with significant habitat shifts.

Calorimetric Study of Helix aspersa Maxima Hemocyanin Isoforms.

The thermal unfolding of hemocyanin isoforms, β-HaH and αD+N-HaH, isolated from the hemolymph of garden snails Helix aspersa maxima, was studied by means of differential scanning calorimetry (DSC). One transition, with an apparent transition temperature (Tm) at 79.88°C, was detected in the thermogram of β-HaH in 20 mM HEPES buffer, containing 0.1 M NaCl, 5 mM CaCl2, and 5 mM MgCl2, pH 7.0, at scan rate of 1.0°C min−1. By means of successive annealing procedure, two individual transitions were identified in the thermogram of αD+N-HaH. Denaturation of both hemocyanins was found to be an irreversible process. The scan-rate dependence of the calorimetric profiles indicated that the thermal unfolding of investigated hemocyanins was kinetically controlled. The thermal denaturation of the isoforms β-HaH and αD+N-HaH was described by the two-state irreversible model, and parameters of the Arrhenius equation were calculated.

Uncovering Hemocyanin Subunit Heterogeneity in Penaeid Shrimp using RNA-Seq.

Aquatic crustaceans can experience low levels of O2 alone but more often in combination with high levels of CO2 both in natural estuaries and in aquaculture ponds. Hemocyanin, the respiratory pigment in many crustacean species, facilitates O2 transport and is documented to change in abundance, structure, and function in response to low O2 The impacts of high CO2 on the respiratory pigment are less clear. In this synthesis we bring together data from recently published and new RNA-Seq studies toward the aims of defining the full repertoire of hemocyanin subunits, as well as their differential expression and regulation in the Penaeoidea family in response to low O2 with or without high CO2 RNA-Seq data were collected from the hepatopancreas tissues of aquacultured Pacific whiteleg shrimp Litopenaeus vannamei, wild-caught L. vannamei, and wild-caught Atlantic brown shrimp Farfantepenaeus aztecus. De novo assembly yielded high-quality stranded transcriptomes. Manual curation of the hemocyanin subunits from all three groups of penaeid shrimp confirmed the existence of a small γ-type hemocyanin subunit (HcS), greater sequence diversity in the large γ-type hemocyanin subunit than previously identified (HcL1-3 isoforms) and expression of a β-type hemocyanin subunit (HcB) previously unidentified in Penaeid shrimp. Relative abundance of transcripts encoding these hemocyanin isoforms differed within and among the three species/strains. Exposure to low O2 induced expression of all of the subunits in aquacultured L. vannamei With concurrent exposure to high CO2, the number of γ-type hemocyanin transcripts decreased while the expression of β-type transcripts remained unchanged. Together with functional data for hemocyanins in the same shrimp species/strains, the RNA-Seq approach shows great promise to provide new insights into the connection between sequence, protein structure, and physiological function of respiratory pigments in this decapod crustacean family.

Comparison of Haliotis rubra hemocyanin isoforms 1 and 2

We have obtained for the first time the complete cDNA sequences encoding the two known hemocyanin isoforms from the mollusc Haliotis rubra. Based on the predicted amino acid sequence each isoform has an almost identical estimated molecular mass of ~390kDa. The isoforms share an amino acid sequence similarity of 66% and consist of eight functional units (FUs a–h) separated by variable linker regions. As observed in other molluscan hemocyanins, FU-h has a C-terminal extension of ~100 amino acids. The presence of a conserved di-copper binding domain in each FU, which contributes to hemocyanin's role as an oxygen transporter, was also confirmed. The most closely related sequences to H. rubra were those of the hemocyanin isoforms from Haliotis tuberculata with a homology of >90%. Like the hemocyanin isoforms from H. tuberculata, the H. rubra versions contain several predicted N-linked glycosylation sites. The predicted N-linked glycosylation pattern of H. rubra hemocyanin isoforms is highly similar but not identical to H. tuberculata hemocyanin isoforms. Such differences may translate to variations in structural organisation of these oligomeric proteins or in other known functions of hemocyanin such as innate immunity or antiviral activity. Knowledge of the complete cDNA sequences of H. rubra hemocyanin isoforms will allow mapping of minimal domain(s) required for these functions.

High CO2 alters the hypoxia response of the Pacific whiteleg shrimp (Litopenaeus vannamei) transcriptome including known and novel hemocyanin isoforms

Acclimation to low O2 in many organisms involves changes at the level of the transcriptome. Here we used high-throughput RNA sequencing (RNA-Seq) to explore the global transcriptomic response and specific involvement of a suite of hemocyanin (Hc) subunits to low O2 alone and in combination with high CO2, which naturally co-occurs with low O2. Hepatopancreas mRNA of juvenile L. vannamei exposed to air-saturated water, low O2, or low O2/high CO2 for 4 or 24 h was pooled, sequenced (HiSeq 2500) and assembled (Trinity: 52,190 contigs) to create a deep strand-specific reference transcriptome. Annotation of the assembly revealed sequences encoding the previously described small Hc subunit (HcS), and three full-length isoforms of the large subunit (HcL1-3). In addition to this, a previously unidentified full-length Hc subunit was discovered. Phylogenetic analysis demonstrated the subunit to be a β-type Hc subunit (denoted HcB), making this the first report of a β-type hemocyanin subunit in the Penaeoidea. RNAs of individual shrimp were sequenced; regulated genes identified from pairwise comparisons demonstrated a distinct pattern of regulation between prolonged low O2 and low O2/high CO2 treatments by GO term enrichment analysis (Roff-Bentzen, P < 0.0001), showcasing the stabilization of energetically costly translational machinery, mobilization of energy stores, and downregulation of the ubiquitin/proteasomal degradation machinery. Exposure to hypoxia for 24 h resulted in an increase in all of the full-length hemocyanin subunits (HcS, HcL1, HcL2, HcL3, and HcB). The addition of CO2 to hypoxia muted the transcriptomic response of all the Hc subunits to low O2, except for the β-type subunit.

Proteomic analysis of differentially expressed protein in hemocytes of wild giant freshwater prawn Macrobrachium rosenbergii infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV)

Epizootic diseases cause huge mortality and economical loses at post larvae stages in freshwater prawn aquaculture industry. These prawns seem less susceptible to viral diseases except for infectious hypodermal and hematopoietic necrosis virus (IHHNV). During viral infection in prawns, hemocytes are the primary organ that shows immunological response within the early stages of infection. We applied proteomic approaches to understand differential expression of the proteins in hemocytes during the viral disease outbreak. To aid the goal, we collected Macrobrachium rosenbergii broodstocks from the local grow out hatchery which reported the first incidence of IHHNV viral outbreak during larvae stage. Primarily, application of the OIE primer targeting 389bp fragments of IHHNV virus was used in identification of the infected and non-infected samples of the prawn breeding line. Analysis of two-dimensional gel electrophoresis showed specific down-regulation of Arginine kinase and Sarcoplasmic calcium-binding protein and up/down-regulation of Prophenoloxidase1 and hemocyanin isoforms. These proteins were validated using semi quantitative RT-PCR and gene transcripts at mRNA level. These identified proteins can be used as biomarkers, providing a powerful approach to better understanding of the immunity pathway of viral disease with applications in analytic and observational epidemiology diagnosis. Proteomic profiling allows deep insight into the pathogenesis of IHHNV molecular regulation and mechanism of hemocyte in freshwater prawns.

Characterization of four hemocyanin isoforms in Litopenaeus vannamei

In this study, the gene encoding hemocyanin subunit L, LvHcL, was cloned from Litopenaeus vannamei and the genomic organization was characterized. This gene was diverse with many SNPs and also had at least four isoforms, while one of them (LvHcL4) only had two exons and the exon2 was missed. Transcription analysis showed that these isoforms of LvHcL were up-regulated after WSSV challenge in WSSV-resistant shrimp, while the transcriptions were decreased constantly in WSSV-susceptible shrimp. It is suggested that the hemocyanin had rich polymorphism and was involved in the antiviral response. These results could extend our previous findings and provide insights into the immune feature of hemocyanin, which would be helpful for further studies aimed at antiviral mechanism in invertebrate.

Cryo-EM Structure of a Molluscan Hemocyanin Suggests Its Allosteric Mechanism

Hemocyanins are responsible for transporting O2 in the arthropod and molluscan hemolymph. Haliotis diversicolor molluscan hemocyanin isoform 1 (HdH1) is an 8 MDa oligomer. Each subunit is made up of eight functional units (FUs). Each FU contains two Cu ions, which can reversibly bind an oxygen molecule. Here, we report a 4.5 A° cryo-EM structure of HdH1. The structure clearly shows ten asymmetric units arranged with D5 symmetry. Each asymmetric unit contains two structurally distinct but chemically identical subunits. The map is sufficiently resolved to trace the entire subunit Ca backbone and to visualize densities corresponding to some large side chains, Cu ion pairs, and interaction networks of adjacent subunits. A FU topology path intertwining between the two subunits of the asymmetric unit is unambiguously determined. Our observations suggest a structural mechanism for the stability of the entire hemocyanin didecamer and 20 ‘‘communication clusters’’ across asymmetric units responsible for its allosteric property upon oxygen binding.

Influence of Temperature, Hypercapnia, and Development on the Relative Expression of Different Hemocyanin Isoforms in the Common Cuttlefish Sepia officinalis

The cuttlefish Sepia officinalis expresses several hemocyanin isoforms with potentially different pH optima, indicating their reliance on efficient pH regulation in the blood. Ongoing ocean warming and acidification could influence the oxygen-binding properties of respiratory pigments in ectothermic marine invertebrates. This study examined whether S. officinalis differentially expresses individual hemocyanin isoforms to maintain optimal oxygen transport during development and acclimation to elevated seawater pCO(2) and temperature. Using quantitative PCR, we measured relative mRNA expression levels of three different hemocyanin isoforms in several ontogenetic stages (embryos, hatchlings, juveniles, and adults), under different temperatures and elevated seawater pCO(2). Our results indicate moderately altered hemocyanin expression in all embryonic stages acclimated to higher pCO(2), while hemocyanin expression in hatchlings and juveniles remained unaffected. During the course of development, total hemocyanin expression increased independently of pCO(2) or thermal acclimation status. Expression of isoform 3 is reported for the first time in a cephalopod in this study and was found to be generally low but highest in the embryonic stages (0.2% of total expression). Despite variable hemocyanin expression, hemolymph total protein concentrations remained constant in the experimental groups. Our data provide first evidence that ontogeny has a stronger influence on hemocyanin isoform expression than the environmental conditions chosen, and they suggest that hemocyanin protein abundance in response to thermal acclimation is regulated by post-transcriptional/translational rather than by transcriptional modifications.

Role of blood-oxygen transport in thermal tolerance of the cuttlefish, Sepia officinalis

Mechanisms that affect thermal tolerance of ectothermic organisms have recently received much interest, mainly due to global warming and climate-change debates in both the public and in the scientific community. In physiological terms, thermal tolerance of several marine ectothermic taxa can be linked to oxygen availability, with capacity limitations in ventilatory and circulatory systems contributing to oxygen limitation at extreme temperatures. The present review briefly summarizes the processes that define thermal tolerance in a model cephalopod organism, the cuttlefish Sepia officinalis, with a focus on the contribution of the cephalopod oxygen-carrying blood pigment, hemocyanin. When acutely exposed to either extremely high or low temperatures, cuttlefish display a gradual transition to an anaerobic mode of energy production in key muscle tissues once critical temperatures (T(crit)) are reached. At high temperatures, stagnating metabolic rates and a developing hypoxemia can be correlated with a progressive failure of the circulatory system, well before T(crit) is reached. However, at low temperatures, declining metabolic rates cannot be related to ventilatory or circulatory failure. Rather, we propose a role for hemocyanin functional characteristics as a major limiting factor preventing proper tissue oxygenation. Using information on the oxygen binding characteristics of cephalopod hemocyanins, we argue that high oxygen affinities (= low P(50) values), as found at low temperatures, allow efficient oxygen shuttling only at very low venous oxygen partial pressures. Low venous PO(2)s limit rates of oxygen diffusion into cells, thus eventually causing the observed transition to anaerobic metabolism. On the basis of existing blood physiological, molecular, and crystallographical data, the potential to resolve the role of hemocyanin isoforms in thermal adaptation by an integrated molecular physiological approach is discussed.

Rapana thomasiana hemocyanin (RtH): Comparison of the two isoforms, RtH1 and RtH2, at 19 Å and 16 Å resolution

Three-dimensional (3D) reconstructions of the two 8.4 MDa Rapana thomasiana hemocyanin isoforms, RtH1 and RtH2, have been obtained by cryoelectron microscopy of molecules embedded in vitreous ice and single particle image processing. The final 3D structures of the RtH1 and RtH2 didecamers at 19 Å and 16 Å resolution, respectively, are very similar to earlier reconstructions of gastropodan hemocyanins, revealing structural features such as the obliquely oriented subunits, the five- and two-fold symmetrical axes. Three new interactions are defined; two of them connecting the arch and the wall while the third is formed between the collar and the wall. The collar–wall connection and one of the arch–wall connections are positioned between two individual subunit dimers, while the second arch–wall connection is located between two subunits within the subunit dimer. All three interactions establish connections to the first tier of the wall. Furthermore, for each interaction we have allocated two first tier functional units most likely involved in forming the connections.

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.

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.

Automated three-dimensional reconstruction of keyhole limpet hemocyanin type 1

We have reconstructed a three-dimensional map of keyhole limpet hemocyanin isoform 1 (KLH1), using our automated data collection software, Leginon, integrated with particle selection algorithms, and the SPIDER reconstruction package. KLH1, a 7.9 MDa macromolecule, is an extracellular respiratory pigment composed of two asymmetric decamers, and presents an overall D 5 point-group symmetry. The reconstruction is in agreement with previous data published on molluscan hemocyanins. The reconstructed map (11.3 Å resolution, 3 σ criterion) was used to fit an available X-ray crystallography structure of Octopus dofleini Odg, solved at 2.3 Å [J. Mol. Biol. 278 (4) (1998) 855], with satisfactory results. The results validate the approach of automating the cryoEM process and demonstrate that the quality of the images acquired and the particles selected is comparable to those obtained using manual methods. Several problems remain to be solved however before these results can be generalized.

C-terminal functional unit of Rapana thomasiana (marine snail, gastropod) hemocyanin isoform RtH1: isolation and characterization

Rapana thomasiana hemocyanin (RtH) is a mixture of two hemocyanin (Hc) isoforms termed RtH1 and RtH2. Both subunit types are built up of eight functional units (FUs). The C-terminal functional unit (RtH1- h) of the Rapana Hc subunit 1 has been isolated by limited trypsinolysis of the subunit polypeptide chain. The oxy- and apo-forms of the unit are characterized by fluorescence spectroscopy. Upon excitation of RtH1- h at 295 or 280 nm, tryptophyl residues buried in the hydrophobic interior of the protein globule determine the fluorescence emission. This is confirmed by quenching experiments with acrylamide, cesium chloride and potassium iodide. The copper–dioxygen system at the binuclear active site quenches the indole emission of the oxy-RtH1- h. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophyl residues in the apo-RtH1- h. The thermal stability of the apo-RtH1- h is characterized fluorimetrically by the “melting” temperature T m (65 °C) and by the transition temperature T m (83 °C) obtained by differential scanning calorimetry for oxy-RtH1- h. The results confirm the role of the copper–dioxygen complex for the stabilization of the Hc structure in solution.