Hemocyanin from the Australian freshwater crayfish Cherax destructor. Electron microscopy of native and reassembled molecules.

Examination and measurement of electron micrographs of negatively stained hemocyanin molecules from Cherax destructor show that the predominant aggregated forms, the 16S and 24S components, are typical structures for arthropod hexamers and dodecamers, respectively. In Cherax hemocyanin the hexamers are formed from the monomeric (Mr congruent to 75,000) subunits, M1 and M2, while the dodecamers contain in addition a dimeric (Mr congruent to 150,000) subunit, M3'. Studies of the composition of solutions of the subunits M1 and m2 to which calcium ions have been added at pH 7.8 show that, under these conditions, reassembly occurs to particles indistinguishable from native hexamers. It is noteworthy that dodecamers are not seen since this confirms the previous suggestion that incorporation of the dimeric subunit in the assembly process is necessary for their formation. The results obtained from Cherax hemocyanin are related to those of previous structural studies of arthropod hemocyanins. In particular, the possible controlling role of certain specific subunits in arthropod hemocyanin oligomers containing more than one kind of subunit is illustrated with a model for the Cherax dodecamer, in which the dimeric subunit is shared between the two halves of the molecule.     

Dissociation and reassembly of Limulus polyphemus hemocyanin.

Limulus polyphemus hemocyanin is a 3.3 x 10(6)-Mr protein containing 48 subunits in an assemblage of eight hexamers. The molecule can be dissociated into monomers and dimers at pH 8.9 in the presence of 0.01 M EDTA. These subunits are heterogeneous and can be separated into five zones (I--V) by DEAE-Sephadex chromatography. Reassembly experiments were carried out with varied subunit mixtures, based on different combinations of the five chromatographic zones, in order to study the structural role of the diverse subunits in the eight-hexamer molecule. The reassembly products were analysed by electron microscopy and ultracentrifugation. No structural role for zone I could be found. Zone V and possibly zone II are needed to form structures larger than hexamers. Absence of zone III causes irregular aggregation of hexamers. Zone IV and perhaps zone II are needed to make eight-hexamer molecules from four-hexamer molecules. From these results we conclude that there is a high degree of subunit specificity in the inter-subunit contacts in the native Limulus hemocyanin molecule.     

Structure and function of the hemocyanin from a semi-terrestrial crab,Ocypode quadrata

Summary Structural and functional studies of the hemocyanin of the semi-terrestrial ghost crab,Ocypode quadrata, demonstrate a variety of differences in comparison to the hemocyanin of aquatic crabs. These differences may be related to the terrestrial habit of this crab. Unlike aquatic crabs, the major (56%) blood component is the hexamer; the remaining 44% is dodecamer. The hexamers and dodecamers are not in rapid equilibrium. Electrophoretic analysis of the subunit composition indicates three major components referred to as 1, 3, and 4, and one minor component referred to as component 2. These components, although electrophoretically distinct, are alike immunologically. Components 1 and 2 are essentially absent from purified hexamers, whereas they compose 1/3 of the subunits in dodecamers. These results suggest that they are involved in linking hexamers to form dodecamers, and that two, rather than one, subunits are involved in the bridge. Oxygen-binding measurements show a higher degree of cooperativity, and a much reduced allosteric effect ofl-lactate on the dialyzed hemocyanin as compared to the hemocyanin of aquatic crabs. Exercise rapidly, induces a large drop in hemolymph pH (0.5 units) and a corresponding increase in lactate concentrations (to 10 mM).     

Hemocyanin from Tachypleus gigas. II. Cooperative interactions of the subunits

Six subunits (I to VI) were isolated from hemocyanin of an Asian horseshoe crab, Tachypleus gigas, by anion exchange chromatography of the dissociated hemocyanin. The subunit preparations were nearly homogeneous as judged by alkaline electrophoresis, but they still showed the presence of isoproteins in isoelectric focusing. The subunits were reassembled (in 10 mM CaCl2 at pH 7.5) and tested for restoration of the cooperativity in O2 binding. The reassembly of the subunits gave equilibrium mixtures of the monomer and hexamer with small amounts of larger molecules. Homogeneous and heterogeneous hexamers were prepared by reassembling a single kind or two kinds of subunits, followed by isolation of the hexamer fraction by gel filtration. Among the homohexamers, only the subunit V hexamer showed cooperativity in O2 binding with the Hill coefficient of 1.6. Among the heterohexamers the subunit I/V hybrid was most noteworthy, showing a Hill coefficient (1.7) higher than that of any other heterohexamer examined. It was concluded that there are specific interactions between the subunits I and V. It is suggested that their interactions are important for the cooperativity in the native hemocyanin.     

Assembly and calcium-induced cooperativity of Limulus IV hemocyanin: a model system for analysis of structure-function relationships in the absence of subunit heterogeneity

Hemocyanins, the high molecular weight copper proteins which serve as oxygen carriers in many arthropods and molluscs, are representative of multisubunit complexes which are capable of reversible dissociation and assembly. Although reversible, in many hemocyanins these processes are not in true thermodynamic equilibria, and it has been suggested that there is "microheterogeneity" among the molecules in solution. An alternative explanation is that their complex behavior is due to the existence of quaternary interactions between structurally distinct types of subunits within the native molecule which have varying pH and ionic strength sensitivity. Limulus IV hemocyanin was used as a model system to examine structure-function relationships in the absence of subunit heterogeneity. Purified subunit IV of Limulus polyphemus hemocyanin is homogeneous by a number of electrophoretic and immunological criteria and is capable of undergoing pH-dependent self-assembly into hexamers. The monomer-hexamer transition was found to be an equilibrium whose rate is dependent on the presence or absence of calcium ions. The observation that the assembly of this homopolymer behaves as a true equilibrium suggests that the nonequilibrium dissociation profiles observed for native Limulus hemocyanin are related to the extensive subunit heterogeneity of the native protein. In calcium-containing buffers, the monomer-hexamer transitions of Limulus IV hemocyanin can be described by a cooperative mechanism with approximately six protons per hexamer lost on assembly from acid pH and three protons gained on assembly from alkaline pH. Increased ionic strength or increased temperature favors dissociation. Like the native molecule, Limulus IV hemocyanin behaves as an allosteric protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural study of Carcinus maenas hemocyanin by native ESI‐MS: Interaction with L‐lactate and divalent cations

The interaction of L ‐lactate and divalent cations with Carcinus maenas hemocyanin has been probed by electrospray ionization mass spectrometry under conditions preserving noncovalent interactions (native ESI‐MS). C. maenas native hemocyanin in the hemolymph occurs mainly as dodecamers and to a lesser extent as hexamers. A progressive acidification with formic acid after alkaline dissociation resulted in the preferential recruitment of the two lightest subunits into light dodecamers, a molecular complex absent from native hemolymph, in addition to regular dodecamers and hexamers. Addition of L ‐lactic acid also induced the recruitment of these subunits, even at alkaline pH. A dodecamer‐specific subunit is needed to enable aggregation over the hexameric state. Experiments with EDTA suggested the existence of different binding sites and association constants for divalent cations within hexameric structures and at the interface between two hexamers. L ‐lactic acid specific interaction with the lightest subunits was not inhibited by removal of the divalent cations. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Molecular heterogeneity of the hemocyanin isolated from the king crab Paralithodes camtschaticae.

Native Paralithodes camtschaticae hemocyanin is found as a mixture of dodecamers (24S; 80%) and hexamers (16S; 20%). Removal of Ca2+ ions by dialysis against EDTA-containing buffer solution at neutral pH induces complete dissociation of the 24S form into the 16S form. Under these conditions, a further increase in pH to 9.2 produces complete dissociation of the hexamers into monomers (5S). In both cases, the dissociation process is reversible. The dodecamer (24S) is composed of two different hexamers which can be discriminated only by ion-exchange chromatography in the presence of Ca2+ ions. At alkaline pH and in the presence of EDTA, two major monomeric fractions can be separated by ion-exchange chromatography: ParcI (60%) and ParcII (40%). The reassociation properties of the two fractions were studied separately to define their ability to form hexamers and dodecamers. The oxygen-binding properties of the different aggregation states were investigated. Native hemocyanin binds O2 co-operatively (nH = 3) and with low affinity (p50 approximately 103 Torr). The two monomeric fractions, ParcI and ParcII, are not co-operative and the affinity is twice that of the native protein (p50 approximately 65 and 52 Torr). Oxygen-binding measurements of native hemocyanin carried out at different pH values indicate a strong positive Bohr effect within the pH range 6.5-8.0 and an increase in oxygen affinity at pH below 6.5.     

Low-resolution molecular structures of isolated functional units from arthropodan and molluscan hemocyanin

Synchrotron x-ray scattering measurements were performed on dilute solutions of the purified hemocyanin subunit (Bsin1) from scorpion (Buthus sindicus) and the N-terminal functional unit (Rta) from a marine snail (Rapana thomasiana). The model-independent approach based on spherical harmonics was applied to calculate the molecular envelopes directly from the scattering profiles. Their molecular shapes in solution could be restored at 2-nm resolution. We show that these units represent stable, globular building blocks of the two hemocyanin families and emphasize their conformational differences on a subunit level. Because no crystallographic or electron microscopy data are available for isolated functional units, this study provides for the first time structural information for isolated, monomeric functional subunits from both hemocyanin families. This has been made possible through the use of low protein concentrations (< or = 1 mg/ml). The observed structural differences may offer advantages in building very different overall molecular architectures of hemocyanin by the two phyla.     

Subunit sequences of the 4 x 6-mer hemocyanin from the golden orb-web spider, Nephila inaurata.

The transport of oxygen in the hemolymph of many arthropod and mollusc species is mediated by large copper-proteins that are referred to as hemocyanins. Arthropod hemocyanins are composed of hexamers and oligomers of hexamers. Arachnid hemocyanins usually form 4 x 6-mers consisting of seven distinct subunit types (termed a-g), although in some spider taxa deviations from this standard scheme have been observed. Applying immunological and electrophoretic methods, six distinct hemocyanin subunits were identified in the red-legged golden orb-web spider Nephila inaurata madagascariensis (Araneae: Tetragnathidae). The complete cDNA sequences of six subunits were obtained that corresponded to a-, b-, d-, e-, f- and g-type subunits. No evidence for a c-type subunit was found in this species. The inclusion of the N. inaurata hemocyanins in a multiple alignment of the arthropod hemocyanins and the application of the Bayesian method of phylogenetic inference allow, for the first time, a solid reconstruction of the intramolecular evolution of the chelicerate hemocyanin subunits. The branch leading to subunit a diverged first, followed by the common branch of the dimer-forming b and c subunits, while subunits d and f, as well as subunits e and g form common branches. Assuming a clock-like evolution of the chelicerate hemocyanins, a timescale for the evolution of the Chelicerata was obtained that agrees with the fossil record.     

A new theoretical approach to the investigation of the symmetry of protein oligomers with bifunctional reagents

The use of bifunctional reagents to form cross-links between subunits in protein oligomers and subsequent disruption of noncovalent interactions with SDS allows comment upon the number of subunits and the symmetry in the original assembly. In existing treatments the number of equations needed to describe theoretically the proportions of all the cross-linked species that can be formed as a function of time in this way makes the analysis of the system unmanageable for proteins with more than four subunits. A method is presented that allows the required equations for any oligomer to be formulated as an algorithm suitable for solution by computer. Its application is illustrated with reference to experimental results obtained with two protein hexamers, Jasus hemocyanin and alpha-urease from jack bean.     

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.     

All hierarchical levels are involved in conformational transitions of the 4 x 6-meric tarantula hemocyanin upon oxygenation

The respiratory protein of the tarantula Eurypelma californicum is a 4 x 6-meric hemocyanin that binds oxygen with high cooperativity. This requires the existence of different conformations which have been confirmed by small angle X-ray scattering (SAXS). Here we present reconstructed 3D-models of the oxy- and deoxy-forms of tarantula hemocyanins, as obtained by fitting small angle X-rays scattering curves on the basis of known X-ray structures and electron microscopy of related hemocyanins. For the first time, the involvement of movements at all levels of the quaternary structure was confirmed for an arthropod hemocyanin upon oxygenation. The two identical 2 x 6-meric half-molecules of the native 4 x 6-mer were shifted in the oxy-state along each other compared with the deoxy-state by about 14 A. In addition, the angle between the two 2 x 6-meric half-molecules increased by 13 degrees. Within these 2 x 6-mers the two hexamers were rotated against each other by about 26 degrees with respect to the deoxy-state. In addition, the distance between the two trimers of each hexamer increased upon oxygenation by about 2.5 A. These strongly coupled movements are based on the particular hierarchical structure of the 4 x 6-mer. It also shows a concept of allosteric interaction in hierarchically assembled proteins to guarantee the involvement of all subunits of a native oligomer to establish very high Hill coefficients.     

Structural and functional properties of hemocyanin from Cyanagraea praedator, a deep-sea hydrothermal vent crab.

Cyanagraea praedator (Crustacea: Decapoda: Brachyura) is an endemic species of the East Pacific Rise hydrothermal vents, living in the upper part of black smoker chimneys. Because we were seeking species that have made respiratory adaptations to the hydrothermal environment, we looked at Cyanograea hemocyanin (Hc) and determined its quaternary structure and the oxygen-binding properties in relation to temperature, pH, and lactate. C. praedator Hc is composed of dodecamers and hexamers, with dodecamers formed by the perpendicular association of two hexamers. The composition of these polymers was determined by electrophoresis and, for the first time, by electrospray mass spectrometry. Dodecamers and hexamers are composed of six subunits common to the two forms, with molecular mass ranging from 75,008 Da to 75,534 Da. In addition, we found two dodecamer-specific subunits, at 75,419 Da and 75,629 Da. The native hemocyanin possesses a high oxygen affinity (P(50) varies between 4 and 10 Torr at pH 7.5, 15 degrees C) and a large Bohr coefficient (Delta log P(50)/DeltapH approximately -1.8). Oxygen affinity is not affected by lactate or, surprisingly, temperature between 5 degrees C and 35 degrees C (DeltaH = 1.16 kJ/mol(1) 5-35 degrees C). Dialysis of native hemolymph elicited a significant increase in Hc-O(2) affinity (DeltaP(50) = 2.5 Torr at pH 7.5), an effect opposite the usual trend observed for crustacean hemocyanins. In this article these functional properties are interpreted in relation to characteristics of the environment.     

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.     

The subunit composition of Portunus trituberculatus hemocyanin polymers

The subunit composition of isolated polymeric forms of Portunus trituberculatus hemocyanin were analysed by immunological techniques. The dodecamers contain four monomeric subunits corresponding to subunits I, II, III and IV, whereas the hexamers are devoid of subunit IV. These results suggest that subunit IV is required as a joining piece for the assembly of dodecamers.     

The allosteric effector l-lactate induces a conformational change of 2x6-meric lobster hemocyanin in the oxy state as revealed by small angle x-ray scattering

Hemocyanins are multisubunit respiratory proteins found in many invertebrates. They bind oxygen highly cooperatively. However, not much is known about the structural basis of this behavior. We studied the influence of the physiological allosteric effector l-lactate on the oxygenated quaternary structure of the 2x6-meric hemocyanin from the lobster Homarus americanus employing small angle x-ray scattering (SAXS). The presence of 20 mm l-lactate resulted in different scattering curves compared with those obtained in the absence of l-lactate. The distance distribution functions p(r) indicated a more compact molecule in presence of l-lactate, which is also reflected in a reduction of the radius of gyration by about 0.2 nm (3%). Thus, we show for the first time on a structural basis that a hemocyanin in the oxy state can adopt two different conformations. This is as predicted from the analysis of oxygen binding curves according to the "nesting" model. A comparison of the distance distribution functions p(r) obtained from SAXS with those deduced from electron microscopy revealed large differences. The distance between the two hexamers as deduced from electron microscopy has to be shortened by up to 1.1 nm to agree well with the small angle x-ray curves.     

Complete hemocyanin subunit sequences of the hunting spider Cupiennius salei: recent hemocyanin remodeling in entelegyne spiders

Hemocyanins are large copper-containing respiratory proteins found in many arthropod species. Scorpions and orthognath spiders possess a highly conserved 4 x 6-mer hemocyanin that consists of at least seven distinct subunit types (termed a to g). However, many "modern" entelegyne spiders such as Cupiennius salei differ from the standard arachnid scheme and have 2 x 6-mer hemocyanins. Here we report the complete primary structure of the 2 x 6-mer hemocyanin of C. salei as deduced from cDNA sequencing, gel electrophoresis, and matrix-assisted laser desorption spectroscopy. Six distinct subunit types (1 through 6) and three additional allelic sequences were identified. Each 1 x 6-mer half-molecule most likely is composed of subunits 1-6, with subunit 1 linking the two hexamers via a disulfide bridge located in a C-terminal extension. The C. salei hemocyanin subunits all belong to the arachnid g-type, whereas the other six types (a-f) have been lost in evolution. The reconstruction of a complex hemocyanin from a single g-type subunit, which commenced about 190 million years ago and was completed about 90 million years ago, might be explained by physiological and behavioral changes that occurred during the evolution of the entelegyne spiders.     

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.     

节肢动物血蓝蛋白家族的组成与演化

血蓝蛋白是动物界的三类呼吸功能蛋白之一,目前仅发现于节肢动物和软体动物等少数动物类群中。不同亚型的血蓝蛋白有不同的理化性质和序列,但均结合氧分子,并以六聚体,甚至更复杂的聚合体结构存在。血蓝蛋白与酚氧化酶、拟血蓝蛋白、昆虫储存蛋白以及昆虫储存蛋白受体等结构类似、进化上近缘的分子共同组成了血蓝蛋白超家族。该文主要介绍了血蓝蛋白家族成员在节肢动物四大类群（螯肢动物、多足动物、甲壳动物和六足动物）中已知的分布、结构和功能,并重点综述了血蓝蛋白家族成员在节肢动物系统演化研究中发挥的独特而有效的作用,进一步强调了在更多节肢动物类群中研究血蓝蛋白家族的功能和演化的重要性。