Molecular characterisation and evolution of the hemocyanin from the European spiny lobster,<Emphasis Type="Italic"> Palinurus elephas</Emphasis>

The hemocyanin of the European spiny lobster Palinurus elephas (synonym: Palinurus vulgaris) is a hexamer composed by four closely related but distinct subunits. We have obtained the full cDNA sequences of all four subunits, which cover 2275-2298 bp and encode for native polypeptides of 656 and 657 amino acids. The P. elephas hemocyanin subunits belong to the alpha-type of crustacean hemocyanins, whereas beta- and gamma-subunits are absent in this species. An unusual high ratio of non-synonymous versus synonymous nucleotide substitutions was observed, suggesting positive selection among subunits. Assuming a constant evolution rate, the P. elephas hemocyanin subunits emerged from a single hemocyanin gene around 25 million years ago. The alpha-type hemocyanins of P. elephas and the American spiny lobster Panulirus interruptus split around 100 million years ago. This is about five times older than the assumed divergence time of the species and suggests that the genera may have split with the formation of the Atlantic Ocean. The application of the Bayesian method for phylogenetic inference allows for the first time a solid reconstruction of the evolution of the decapod hemocyanins, showing that the beta-subunit types diverged first and that the crustacean pseudo-hemocyanins are associated with the gamma-type subunits.     

Hemocyanin-derived phenoloxidase activity in the spiny lobster Panulirus argus (Latreille, 1804)

Hemocyanin and phenoloxidase belong to the type-3 copper protein family, sharing a similar active center whereas performing different roles. In this study, we demonstrated that purified hemocyanin (450 kDa) from the spiny lobster Panulirus argus shows phenoloxidase activity in vitro after treatment with trypsin, chymotrypsin and SDS (0.1% optimal concentration), but it is not activated by sodium perchlorate or isopropanol. The optimal pHs of the SDS-activated hemocyanin were 5.5 and 7.0. Hemocyanin from spiny lobster behaves as a catecholoxidase. Kinetic characterization using dopamine, L-DOPA and catechol shows that dopamine is the most specific substrate. Catechol and dopamine produced substrate inhibition above 16 and 2 mM respectively. Mechanism-based inhibition was also evidenced for the three substrates, being less significant for L-DOPA. SDS-activated phenoloxidase activity is produced by the hexameric hemocyanin. Zymographic analysis demonstrated that incubation of native hemocyanin with trypsin and chymotrypsin, produced bands of 170 and 190 kDa respectively, with intense phenoloxidase activity. Three polypeptide chains of 77, 80 and 89 kDa of hemocyanin monomers were identified by SDS-PAGE. Monomers did not show phenoloxidase activity induced by SDS or partial proteolysis.     

Subunits of Panulirus japonicus hemocyanin. 1. Isolation and properties

Structural and functional diversities of the subunits of Panulirus japonicus (spiny lobster) hemocyanin were investigated. The hemocyanin mostly exists as a hexamer in the native state. It was found that the hemocyanin is composed of three major subunits (Ib, II and III) and one minor subunit (Ia), which differ in N-terminal sequence. In the dissociated state, the major subunits (Ib, II and III) showed no or very small Bohr effects. The O2 affinity of the subunit III was about three times as high as those of the other two. The subunits could be reassociated into homogeneous and heterogeneous hexamers, which exhibited the cooperativity in O2 binding. The homohexamers were similar to each other in O2 affinity and the Bohr effect, though some differences were observed in the magnitude of the cooperativity. In particular, the subunit II homohexamer exhibited a high cooperativity, which was comparable to that of the native protein. The heterohexamers showed slightly higher O2 affinities and slightly lower cooperativity, as compared with the parent homohexamers. It was concluded that there is no essential difference among the three major subunits of P. japonicus hemocyanin in the O2 binding and assembly properties.     

cDNA and gene sequence of Manduca sexta arylphorin, an aromatic amino acid-rich larval serum protein. Homology to arthropod hemocyanins

The serum (storage) proteins produced by insect larvae at the end of the feeding cycle are hexameric blood proteins with one or more type of subunits. The cDNA and gene structure of the aromatic amino acid-rich larval serum protein arylphorin from the tobacco hornworm, Manduca sexta, has been determined. In M. sexta arylphorin there are two subunits alpha and beta, which have 686 and 687 amino acids, respectively, and whose amino acid sequences are 68% identical. The two genes, separated by 7.1 kilobases of chromosomal DNA, are transcribed in the same direction. Based on the alignment of the amino acid sequence, the rate of nucleotide substitution between the two coding regions predicts that the two genes diverged about 100 million years ago. Both genes contain 5 exons and the upstream region contains a sequence, TGATAAA, which is similar to a sequence found in all other storage protein genes for which information is available. When the National Biomedical Research Foundation protein sequence data base was searched, it was found that the arylphorin subunits showed significant similarity to the arthropod hemocyanins, which are hexameric oxygen-carrying proteins. Based on the alignment of the sequence of M. sexta arylphorin and the hemocyanin from the spiny lobster (Panulirus interruptus), for which a 3.2 A structure has been determined, it was observed that the highest concentration of conserved residues were found in those regions of the sequence which are involved in subunit interactions in the hexameric protein. It is suggested that the insect storage proteins and the arthropod hemocyanins have evolved from a common ancestor.     

Quaternary structure of the European spiny lobster (Palinurus elephas) 1x6-mer hemocyanin from cryoEM and amino acid sequence data

Arthropod hemocyanins are large respiratory proteins that are composed of up to 48 subunits (8 x 6-mer) in the 75kDa range. A 3D reconstruction of the 1 x 6-mer hemocyanin from the European spiny lobster Palinurus elephas has been performed from 9970 single particles using cryoelectron microscopy. An 8A resolution of the hemocyanin 3D reconstruction has been obtained from about 600 final class averages. Visualisation of structural elements such as alpha-helices has been achieved. An amino acid sequence alignment shows the high sequence identity (>80%) of the hemocyanin subunits from the European spiny lobster P.elephas and the American spiny lobster Panulirus interruptus. Comparison of the P.elephas hemocyanin electron microscopy (EM) density map with the known P.interruptus X-ray structure shows a close structural correlation, demonstrating the reliability of both methods for reconstructing proteins. By molecular modelling, we have found the putative locations for the amino acid sequence (597-605) and the C-terminal end (654-657), which are absent in the available P.interruptus X-ray data.     

Common origin of arthropod tyrosinase,arthropod hemocyanin,insect hexamerin,and dipteran arylphorin receptor

Dipteran arylphorin receptors, insect hexamerins, cheliceratan and crustacean hemocyanins, and crustacean and insect tyrosinases display significant sequence similarities. We have undertaken a systematic comparison of primary and secondary structures of these proteins. On the basis of multiple sequence alignments the phylogeny of these proteins was investigated. Hexamerin subunits, hemocyanin subunits, and tyrosinases share extensive similarities throughout the entire amino acid sequence. Our studies suggest the origin of arthropod hemocyanins from ancient tyrosinase-like proteins. Insect hexamerins likely evolved from hemocyanins of ancient crustaceans, supporting the proposed sister-group position of these subphyla. Arylphorin receptors, responsible for incorporation of hexamerins into the larval fat body of diptera, are related to hexamerins, hemocyanins, and tyrosinase. The receptor sequences display extensive similarities to the first and third domains of hemocyanins and hexamerins. In the middle region only limited amino acid conservation was observed. Elements important for hexamer formation are deleted in the receptors. Phylogenetic analysis indicated that dipteran arylphorin receptors diverged from ancient hexamerins, probably early in insect evolution. Correspondence to: T. Burmester     

Biochemical and molecular characterisation of hemocyanin from the amphipod <Emphasis Type="Italic">Gammarus roeseli</Emphasis>: 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 α-type of crustacean hemocyanin subunits. Rather, amphipod hemocyanins split from the clade leading to α and γ-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 β-type subunits and other crustacean hemocyanins occurred around 315 million years ago (MYA) in the malacostracan stemline, while α- and γ-type subunits separated 258 MYA, and pseudohemocyanins and γ-subunits 210 million years ago.     

Subunits of Panulirus japonicus hemocyanin. 2. Cooperativity of the homogeneous hexamers

Hexameric hemocyanin from a spiny lobster, Panulirus japonicus, comprises three major subunits (Ib, II and III) and one minor subunit (Ia), as reported in the preceding paper in this journal. It has previously been shown that the O2 equilibria of Panulirus hemocyanin can be described by a concerted model extended to three affinity states [Makino, N. (1986) Eur. J. Biochem. 154, 49-55]. In this study the equilibrium binding of O2 to the reassociated subunits (Ib, II and III) was examined at various pH in the presence or absence of Ca2+ in order to test the applicability of the three-state model to the homogeneous hexamers. The hexameric structure of the reassembled subunits was less stable than that of the native protein under the conditions examined. The model could be fitted to the O2-binding isotherms of the homohexamers composed of the subunits II or III, if the molecular dissociation of the protein was taken into account. It was postulated that the monomeric hemocyanin has the same ligand affinity as that of the hexamer in the intermediate-affinity state (S). The fitting of the model to the O2 binding of the subunit I was unsuccessful mainly because of the low cooperativity of the assembled subunits.     

Hemocyanin-derived phenoloxidase activity in the spiny lobster Panulirus argus (Latreille, 1804)

Hemocyanin and phenoloxidase belong to the type-3 copper protein family, sharing a similar active center whereas performing different roles. In this study, we demonstrated that purified hemocyanin (450 kDa) from the spiny lobster Panulirus argus shows phenoloxidase activity in vitro after treatment with trypsin, chymotrypsin and SDS (0.1% optimal concentration), but it is not activated by sodium perchlorate or isopropanol. The optimal pHs of the SDS-activated hemocyanin were 5.5 and 7.0. Hemocyanin from spiny lobster behaves as a catecholoxidase. Kinetic characterization using dopamine, L-DOPA and catechol shows that dopamine is the most specific substrate. Catechol and dopamine produced substrate inhibition above 16 and 2 mM respectively. Mechanism-based inhibition was also evidenced for the three substrates, being less significant for L-DOPA. SDS-activated phenoloxidase activity is produced by the hexameric hemocyanin. Zymographic analysis demonstrated that incubation of native hemocyanin with trypsin and chymotrypsin, produced bands of 170 and 190 kDa respectively, with intense phenoloxidase activity. Three polypeptide chains of 77, 80 and 89 kDa of hemocyanin monomers were identified by SDS-PAGE. Monomers did not show phenoloxidase activity induced by SDS or partial proteolysis.     

Oxygenation-linked changes in the ultraviolet absorption and circular dichroism spectra of spiny lobster hemocyanin

As an approach to elucidate the mechanism of the protein structure change in the cooperative ligand binding, the UV difference and CD spectra of aromatic residues in Panulirus japonicus (spiny lobster) hemocyanin were examined. The native hemocyanin showed an O2-induced narrow-banded change in the absorption spectrum around 290 nm, which was not affected by pH in the range of 7.5 to 9.5. When the native hexameric protein was stripped of divalent cations with EDTA (at pH 7.5), the magnitude of the narrow-banded difference was reduced to about half, whereas it was almost completely abolished on dissociation into subunits (stripped at pH 9.5). The magnitude of the absorption change was found to be proportional to the degree of O2 saturation in the native and stripped hemocyanins. It was inferred that the spectral difference reflects a tertiary structure change directly linked to the oxygenation, though it depends greatly on the subunit association. Panulirus hemocyanin showed negative CD bands in the region of 260 to 300 nm, the intensities of which were considerably reduced by oxygenation and also by dissociation into subunits.     

Characterization of hemocyanin from the peacock mantis shrimp Odontodactylus scyllarus (Malacostraca:Hoplocarida)

Hemocyanin is the blue respiratory protein of many arthropod species. While its structure, evolution, and physiological function have been studied in detail in Decapoda, there is little information on hemocyanins from other crustacean taxa. Here, we have investigated the hemocyanin of the peacock mantis shrimp Odontodactylus scyllarus, which belongs to the Stomatopoda (Hoplocarida). O. scyllarus hemocyanin forms a dodecamer (2 × 6-mer), which is composed of at least four distinct subunit types. We obtained the full-length cDNA sequences of three hemocyanin subunits, while a fourth cDNA was incomplete at its 5′ end. The complete full-length cDNAs of O. scyllarus hemocyanin translate into polypeptides of 650–662 amino acids, which include signal peptides of 16 or 17 amino acids. The predicted molecular masses of 73.1–75.1 kDa correspond well with the main hemolymph proteins detected by SDS-PAGE and Western blotting using various anti-hemocyanin antibodies. Phylogenetic analyses show that O. scyllarus hemocyanins belong to the β-type of malacostracan hemocyanin subunits, which diverged from the other subunits before the radiation of the malacostracan subclasses around 520 million years ago. Molecular clock analysis revealed an ancient and complex pattern of hemocyanin subunit evolution in Malacostraca and also allowed dating divergence times of malacostracan taxa.     

Structure of arthropod hemocyanin

Hemocyanins are large multi-subunit copper proteins that transport oxygen in many arthropods and molluscs. The amino acid sequence of subunit a of Panulirus interruptus hemocyanin (657 residues) has been completed and fitted to the electron-density map (3.2 Å resolution). Comparison of amino acid sequence data for several different hemocyanin subunits of arthropod species indicated that the general features of the polypeptide architecture as found in spiny lobster hemocyanin occur in all arthropods. This structure must therefore be at least as old as the estimated time of divergence of crustaceans and chelicerates, 540–600 million years ago.     

Structure of Panulirus interruptus hemocyanin at 5 Å resolution

The hemocyanin from the spiny lobster Panulirus interruptus, a hexamer with a molecular weight of approximately 540,000, was crystallized in space group P2₁ with two molecules in the unit cell and cell dimensions $\text{a = 119.8}\text{A}\text{?}\text{, b = 193.1}\text{A}\text{?}\text{, c = 122.2}\text{A}\text{?}\text{and}\text{β = 118.1 °}$. With screened precession photographs a three-dimensional set of reflections was collected up to 10 Å resolution. Both the conventional and the fast rotation function programs were applied and gave results that were in excellent agreement with each other. The hemocyanin hexamer has 32 point group symmetry. Its 3-fold molecular axis runs approximately parallel to the crystallographic 2-fold screw axis.X-ray diffraction data to 5 Å resolution were collected by the oscillation method. Rotation function studies with data between 7 and 5 Å resolution confirmed the 10 Å studies and, furthermore, showed that the rotation axes relating subunits within one hexameric molecule can be distinguished from the rotation axes relating subunits belonging to different hexamers in the unit cell. The local 3-fold axis in the hexamer makes an angle of about 6 ° with the crystallographic 2-fold screw axis.For a mercury and a platinum derivative three-dimensional data sets were collected to 5 Å by the oscillation method. The difference Patterson of the platinum derivative could be solved. The eventual number of heavy-atom sites was 36 for the platinum derivative and 70 for the mercury derivative. From the well-occupied sites the point-group symmetry of the molecule could be established accurately. In addition, the centre of the hexamer could be located within 0.2 Å.Protein phases were obtained from isomorphous as well as anomalous differences. A “best” electron density map calculated with these phases showed the shape of the hexameric molecule as well as the boundaries of the six subunits. Correlation coefficients between the densities of the subunits showed little variation, suggesting a random distribution of the different subunit types (Van Eerd & Folkerts, 1981) over the six positions in the hexamer.The subunits are positioned at the corner of an antiprism. When viewed along the 3-fold axis the hexamer is roughly hexagonal in shape, with a diameter of approximately 120 Å. Viewed along one of the 2-fold axes the molecule is of rectangular shape with dimensions 95 Å × 120 Å. The subunit can be described as an ellipsoid of irregular shape with axes of 80 Å, 55 Å and 48 Å. Each subunit makes extensive contacts with three other subunits in the hexamer and, possibly, a much weaker contact with a fourth subunit.     

Characterization of a novel allergenic protein from the octocoral Scleronephthya gracillima (Kuekenthal) that corresponds to a new GFP‐like family named Akane

Certain marine organisms have been known to cause allergic reactions among occupational fishermen. We have previously reported that bronchial asthma among the workers engaged in spiny lobster fishing in Japan was caused by octocorals such as Dendronephthya sp. and Scleronephthya gracillima (previously named Alcyonium gracillimum). Now we have found another octocoral, Scleronephthya gracillima (Kuekenthal), which causes the allergic disease in fishermen. The octocoral was characterized as a new green fluorescent protein (GFP)‐like family. The new allergen has a molecular mass of 27 kDa in 1D and 2D SDS‐PAGE under reduced conditions. The 27 kDa component was determined to be an allergen by western blotting, ECL immune staining method and absorption of patient sera with the antigen. Furthermore, the combination of analysis with LC‐ESI‐MS/MS and MASCOT search in the NCBInr database concluded the 27 kDa component had the sequence YPADI/LPDYFK, and that the 22 kDa component had the sequence QSFPEGFSWER, which both matched a GFP‐like protein in Acropora aculeus and in Montastraea annularis. Further analysis by MALDI‐TOF/MS/MS and MASCOT search in the NCBInr database of all 27 kDa eight spot components from 2D SDS‐PAGE indicated that the sequence QSFPEGFSWER also matched as GFP‐like protein in Lobophyllia hemprichii and Scleractinia sp. To our knowledge, this is the first report of the new allergenic protein that corresponds to a new GFP‐like protein named Akane, and which has fluorescent emissions in the red and green part of the spectra at 628 nm and 508 nm, respectively.     

Isolation, Characterisation and Molecular Imaging of a High-Molecular-Weight Insect Biliprotein, a Member of the Hexameric Arylphorin Protein Family

The abundant blue hemolymph protein of the last instar larvae of the moth Cerura vinula was purified and characterized by protein-analytical, spectroscopic and electron microscopic methods. Amino acid sequences obtained from a large number of cleavage peptides revealed a high level of similarity of the blue protein with arylphorins from a number of other moth species. In particular, there is a high abundance of the aromatic amino acids tyrosine and phenylalanine amounting to about 19% of total amino acids and a low content of methionine (0.8%) in the Cerura protein. The mass of the native protein complex was studied by size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and scanning transmission electron microscopy and found to be around 500 kDa. Denaturating gel electrophoresis and mass spectrometry suggested the presence of two proteins with masses of about 85 kDa. The native Cerura protein is, therefore, a hexameric complex of two different subunits of similar size, as is known for arylphorins. The protein was further characterized as a weakly acidic (pI ∼ 5.5) glycoprotein containing mannose, glucose and N-acetylglucosamine in an approximate ratio of 10:1:1. The structure proposed for the most abundant oligosaccharide of the Cerura arylphorin was the same as already identified in arylphorins from other moths. The intense blue colour of the Cerura protein is due to non-covalent association with a bilin of novel structure at an estimated protein subunit-to-ligand ratio of 3:1. Transmission electron microscopy of the biliprotein showed single particles of cylindrical shape measuring about 13 nm in diameter and 9 nm in height. A small fraction of particles of the same diameter but half the height was likely a trimeric arylphorin dissociation intermediate. Preliminary three-dimensional reconstruction based on averaged transmission electron microscopy projections of the individual particles revealed a double-trimeric structure for the hexameric Cerura biliprotein complex, suggesting it to be a dimer of trimers.     

Purification and characterization of three major hemolymph proteins of an insect,Calpodes ethlius (lepidoptera,hesperiidae)

Like many other Lepidoptera, fifth-stage Calpodes larvae have three major hemolymph proteins. Their molecular weights were estimated by 3-15% nondenaturing polyacrylamide gel electrophoresis (N-PAGE) as 470,000 (arylphorin; Ar), 580,000 (storage protein 2; SP2) and 720,000 (storage protein 1; SP1). Carbohydrate is associated with all three, but only Ar has lipid. The three proteins have been purified by preparative N-PAGE and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. On 3-15% SDS gels, Ar dissociated into 82,000 M_r subunits, SP2 into 86,000 M_r subunits, and SP1 into both 86,000 and 90,000 M_r subunits. The 470,000 M_r protein is identified as Ar because it is rich in aromatic amino acids. The 580,000 and 720,000 M_r proteins are rich in glycine and are called storage proteins. Electron microscopy of negatively stained preparations shows that each polymer has a different geometrical arrangement of subunits. SP1 is a cube made from eight subunits. SP2 is a hexamer in the form of a pentahedral prism. Ar is probably an octahedron made from six subunits. All three geometrical arrangements could permit the presence of a central carrying space.     

Hexamers of subunit II from Limulus hemocyanin (a 48-mer) have the same quaternary structure as whole Panulirus hemocyanin molecules

Hemocyanins are copper-containing proteins that transport oxygen in a variety of invertebrates. Considerable evidence has accumulated that arthropodan hemocyanins are multimers of a fundamental hexameric unit. X-Ray crystallographic structure determination has revealed that the hemocyanin molecule from the spiny lobster Panulirus interruptus is a single hexamer having 32 point group symmetry. Using crystals of subunit II, one of 8 polypeptide types comprising the octahexameric hemocyanin of the horseshoe crab Limulus polyphemus, and the molecular replacement method for crystallographic phase determination we show that subunit II forms assemblies with the same hexameric quaternary structure as the whole Panulirus hemocyanin molecule. Observation of the same hexameric motif in two widely separated species provides strong additional evidence that this quaternary structural unit is a universal building block of arthropodan hemocyanins.     

Purification and characterization of the clotting protein from the white shrimp Penaeus vannamei

The protein responsible for clot formation was isolated from plasma of the white shrimp Penaeus vannamei by affinity chromatography in a heparin–agarose column. The protein, named clotting protein (CP), was found to be a lipoglycoprotein, composed of two 210-kDa subunits covalently bound by disulfide bridges. CP formed large polymers when incubated with hemocyte lysate. Dansylcadaverine can be incorporated into CP by a hemocyte lysate or guinea pig transglutaminase mediated reaction. The amino acid composition and the amino terminal sequence were determined and compared with the clotting protein of the crayfish and the spiny lobster.     

Quantum mechanical analysis of oxygenated and deoxygenated states of hemocyanin: theoretical clues for a plausible allosteric model of oxygen binding.

Abstract: In this work with ab initio computations, we describe relevant interactions between protein active sites and ligands, using as a test case arthropod hemocyanins. A computational analysis of models corresponding to the oxygenated and deoxygenated forms of the hemocyanin active site is performed using the Density Functional Theory approach. We characterize the electron density distribution of the binding site with and without bound oxygen in relation to the geometry, which stems out of the crystals of three hemocyanin proteins, namely the oxygenated form from the horseshoe crab Limulus polyphemus, and the deoxygenated forms, respectively, from the same source and from another arthropod, the spiny lobster Panulirus interruptus. Comparison of the three available crystals indicate structural differences at the oxygen binding site, which cannot be explained only by the presence and absence of the oxygen ligand, since the geometry of the ligand site of the deoxygenated Panulirus hemocyanin is rather similar to that of the oxygenated Limulus protein. This finding was interpreted in the frame of a mechanism of allosteric regulation for oxygen binding. However, the cooperative mechanism, which is experimentally well documented, is only partially supported by crystallographic data, since no oxygenated crystal of Panulirus hemocyanin is presently available. We address the following question: is the local ligand geometry responsible for the difference of the dicopper distance observed in the two deoxygenated forms of hemocyanin or is it necessary to advocate the allosteric regulation of the active site conformations in order to reconcile the different crystal forms? We find that the difference of the dicopper distance between the two deoxygenated hemocyanins is not due to the small differences of ligand geometry found in the crystals and conclude that it must be therefore stabilized by the whole protein tertiary structure.