The structure of a functional unit from the wall of a gastropod hemocyanin offers a possible mechanism for cooperativity

Structure-function relationships in a molluscan hemocyanin have been investigated by determining the crystal structure of the Rapana thomasiana (gastropod) hemocyanin functional unit RtH2e in deoxygenated form at 3.38 A resolution. This is the first X-ray structure of an unit from the wall of the molluscan hemocyanin cylinder. The crystal structure of RtH2e demonstrates molecular self-assembly of six identical molecules forming a regular hexameric cylinder. This suggests how the functional units are ordered in the wall of the native molluscan hemocyanins. The molecular arrangement is stabilized by specific protomer-to-protomer interactions, which are probably typical for the functional units building the wall of the cylinders. A molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins is proposed on the basis of the molecular interactions between the protomers. In particular, the deoxygenated RtH2e structure reveals a tunnel leading from two opposite sides of the molecule to the active site. The tunnel represents a possible entrance pathway for dioxygen molecules. No such tunnels have been observed in the crystal structure of the oxy-Odg, a functional unit from the Octopus dofleini (cephalopod) hemocyanin in oxygenated form.     

Minireview: Recent progress in hemocyanin research

This review summarizes recent highlights of our joint work on the structure, evolution, and function of a family of highly complex proteins, the hemocyanins. They are blue-pigmented oxygen carriers, occurring freely dissolved in the hemolymph of many arthropods and molluscs. They are copper type-3 proteins and bind one dioxygen molecule between two copper atoms in a side-on coordination. They possess between 6 and 160 oxygen-binding sites, and some of them display the highest molecular cooperativity observed in nature. The functional properties of hemocyanins can be convincingly described by either the Monod-Wyman-Changeux (MWC) model or its hierarchical extension, the Nested MWC model; the latter takes into account the structural hierarchies in the oligomeric architecture. Recently, we applied these models to interpret the influence of allosteric effectors in detailed terms. Effectors shift the allosteric equilibria but have no influence on the oxygen affinities characterizing the various conformational states. We have shown that hemocyanins from species living at different environmental temperatures have a cooperativity optimum at the typical temperature of their natural habitat. Besides being oxygen carriers, some hemocyanins function as a phenoloxidase (tyrosinase/catecholoxidase) which, however, requires activation. Chelicerates such as spiders and scorpions lack a specific phenoloxidase, and in these animals activated hemocyanin might catalyse melanin synthesis in vivo. We propose a similar activation mechanism for arthropod hemocyanins, molluscan hemocyanins and tyrosinases: amino acid(s) that sterically block the access of phenolic compounds to the active site have to be removed. The catalysis mechanism itself can now be explained on the basis of the recently published crystal structure of a tyrosinase. In a series of recent publications, we presented the complete gene and primary structure of various hemocyanins from different molluscan classes. From these data, we deduced that the molluscan hemocyanin molecule evolved ca. 740 million years ago, prior to the separation of the extant molluscan classes. Our recent advances in the 3D cryo-electron microscopy of hemocyanins also allow considerable insight into the oligomeric architecture of these proteins of high molecular mass. In the case of molluscan hemocyanin, the structure of the wall and collar of the basic decamers is now rapidly becoming known in greater detail. In the case of arthropod hemocyanin, a 10-? structure and molecular model of the Limulus 8 × 6mer shows the amino acids at the various interfaces between the eight hexamers, and reveals histidine-rich residue clusters that might be involved in transferring the conformational signals establishing cooperative oxygen binding.     

Recent aspects of the subunit organization and dissociation of hemocyanins

1. The hemocyanins of the arthropod phylum are built of multiples of hexamers consisting of 1,2,4,6 and 8 of such basic assemblies. Their molecular weights range from about 0.45 x 10(6) to 3.9 x 10(6) daltons. The basic hexameric unit consists of bean-shaped monomers organized in the form of two layers of trimers placed on top of one another. The subunits are heterogeneous, in most cases consisting of four or more electrophoretically different polypeptide chains. 2. Molluscan hemocyanins have an entirely different structure and pattern of assembly from the arthropodan hemocyanins. The basic assembly of the molluscan hemocyanins are decamers organized in the form of right-handed cylinders approximately 300 A in diameter and 140-190 A in height. Different species have one, two and sometimes more than two such assemblies forming correspondingly longer cylindrical particles with molecular weights ranging from about 3.3 x 10(6) to 13 x 10(6) daltons. Cephalopod and chiton hemocyanins consist of single decameric particles, while gastropods have hemocyanins organized of di-decamers or higher assemblies. The subunits of these hemocyanins are elongated protein chains with seven or eight folded globular domains, each housing a binuclear copper center capable of binding and delivering oxygen. 3. The dissociation behavior of the arthropod hemocyanin hexamers and di-hexamers with the hydrophobic urea series of reagents suggest polar and ionic interactions as the main sources of stabilization of the hexamers and the hexamer to hexamer contacts within the di-hexamers. 4. Dissociation studies with the same urea probes with the molluscan hemocyanins, however, suggest a different pattern of stabilization. The stabilization of the decamer to decamer contacts within the gastropod di-decamers appear to be predominantly polar and ionic with relatively few hydrophobic interaction sites. The dimer contacts within the decamers and the monomer to monomer contacts within the dimers observed in the octopus and chiton hemocyanins appear to be predominantly hydrophobic in nature. 5. The urea and the pH dissociation profiles of the single decameric assemblies of some of the octopus and chiton hemocyanins investigated by light-scattering molecular weight methods, have been fitted using either a two-species, decamer to dimer and decamer to monomer scheme of subunit dissociation or a three-species, decamer to dimer to monomer scheme of dissociation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evolution of molluscan hemocyanin structures

Hemocyanin transports oxygen in the hemolymph of many molluscs and arthropods and is therefore a central physiological factor in these animals. Molluscan hemocyanin molecules are oligomers composed of many protein subunits that in turn encompass subsets of distinct functional units. The structure and evolution of molluscan hemocyanin have been studied for decades, but it required the recent progress in DNA sequencing, X-ray crystallography and 3D electron microscopy to produce a detailed view of their structure and evolution. The basic quaternary structure is a cylindrical decamer 35 nm in diameter, consisting of wall and collar (typically at one end of the cylinder). Depending on the animal species, decamers, didecamers and multidecamers occur in the hemolymph. Whereas the wall architecture of the decamer seems to be invariant, four different types of collar have been identified in different molluscan taxa. Correspondingly, there exist four subunit types that differ in their collar functional units and range from 350 to 550 kDa. Thus, molluscan hemocyanin subunits are among the largest polypeptides in nature. In this report, recent 3D reconstructions are used to explain and visualize the different functional units, subunits and quaternary structures of molluscan hemocyanins. Moreover, on the basis of DNA analyses and structural considerations, their possible evolution is traced. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Higher order assemblies of molluscan hemocyanins

1. The hemocyanins of the Fissurellidae, Naticidae and Melongenidae families of marine gastropods as well as some other molluscs including some members of the Opistobranchia and Bivalvia groups have hemocyanins which exist in solution as tri-decameric and mixed, multi-decameric aggregates characterized by sedimentation coefficients close to 100 S, 130 S, 150 S, 170 S and 200 S to 230 S. 2. The particle masses of the molluscan hemocyanins appear to be integral multiples close to 4.4 x 10(6) daltons. Thus, particle mass values of 4.47 x 10(6), 8.67 x 10(6) and 13.40 x 10(6) daltons were obtained for representative decameric, di-decameric, and tri-decameric components of Stenoplax conspicua, Fasciolaria tulipa and Euspira (Lunatia) heros hemocyanins. For Busycon contrarium, a gastropod with a mixed multidecameric hemocyanin, scanning transmission electron microscopic (STEM) measurements gave particle masses ranging from 8.89 x 10(6) and 13.20 x 10(6) for the di- and tri-decameric components to 38.87 x 10(6) and 43.40 x 10(6) daltons for highest nano- and deca-decameric aggregates. 3. The electron microscopic images of both uranyl acetate-stained and unstained specimens of hemocyanin aggregates indicate a non-random mode of assembly of the multi-decameric particles. This is most apparent from the electron micrographs of the moon snail hemocyanins. The tri-decameric and tetra-decameric particles seem to be assembled from a single di-decameric unit of the Mellema and Klug arrangement, with the collar ends facing outward, to which decameric units have been added from one or both ends, in a unidirectional tail-to-head to tail-to-collar manner. Consequently, all the aggregates including the higher, Melongenidae polymers have the appearance of closed cylinders terminating with the collar ends. 4. The radial distribution of the end-on views of the hemocyanin of the moon-snail Calinatioina oldroydii, show that the radial mass drops to zero at the center of the cylindrical particles consisting of one, two, or three decamers. This suggests that no caps are present at the ends of the hemocyanin particles which would inhibit or terminate their linear assembly. 5. The light-scattering behavior of B. contrarium and Marisa cornarietis hemocyanins examined as a function of increasing reagent concentration using the hydrophobic urea and Hofmeister salt series of reagents, show distinct aggregation and increase in molecular weights at low concentrations of reagent. Together with the stabilizing influence of Mg2+ and Ca2+ ions, this suggests polar and ionic stabilization of the inter-decameric contacts between the central di-decamers and the added decameric units of the higher aggregates of molluscan hemocyanins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Subunit structure and higher order assembly of the hemocyanins of five members of the Naticidae family of marine gastropods: Calinaticina oldroydii (Dall), Euspira heros (Say), Neverita duplicata (Say), Polinices draconis (Dall), and Polinices lewisii (Gould)

1. The hemocyanins of the Naticidae family, E. heros, N. duplicata, P. draconis, P. lewisii and C. oldroydii were investigated by sedimentation velocity and scanning transmission electron microscopy. 2. At pH 8.0, 0.05 M Mg2+ E. heros hemocyanin is found to be predominantly in the tri-decameric state with a sedimentation coefficient (So20,w) of 131.3 (+/- 0.6) S. While the hemocyanin of N. duplicata is also mainly in the 130 S form, the hemocyanin of C. oldroydii is largely in the di-decameric form with a sedimentation coefficient close to 100 S. Other Naticidae hemocyanins, those of P. lewisii and P. draconis, have mixtures of the 100 S and 130 S di- and tri-decamers, and minor amounts of 150 S and faster sedimenting components. 3. The average particle masses based on STEM measurements are 8.85 x 10(6), 1303 x 10(6), and 17.1 x 10(6) da for the di-, tri-, and tetra-decameric assemblies of hemocyanin. 4. The subunit mol. wts of C. oldroydii hemocyanin and the published values for E. heros hemocyanin at alkaline pHs and in the presence of 8.0 M urea range from 4.2 x 10(5) to 4.8 x 10(5), suggesting the same decameric organization of the sub-assemblies of the Naticidae hemocyanins as for other molluscan hemocyanins. 5. The appearance of the larger hemocyanin particles in the electron micrographs support the hypothesis for their assembly that was based on similar studies of the hemocyanins of the Melongenidae family. According to this scheme the formation of higher aggregates is accomplished by the tail-to-head addition of each decameric unit to a central di-decamer which itself has the tail-to-tail Mellema and Klug arrangement of decamers. In this model all the higher aggregates terminate from either end with the same "collar" ends.     

Nautilus pompilius hemocyanin: 9 A cryo-EM structure and molecular model reveal the subunit pathway and the interfaces between the 70 functional units

Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs. Nautilus pompilius (Cephalopoda) hemocyanin is a cylindrical decamer of a 350 kDa polypeptide subunit that in turn is a “pearl-chain” of seven different functional units (FU-a to FU-g). Each globular FU has a binuclear copper centre that reversibly binds one O₂ molecule, and the 70-FU decamer is a highly allosteric protein. Its primary structure and an 11 Å cryo-electron microscopy (cryo-EM) structure have recently been determined, and the crystal structures of two related FU types are available in the databanks. However, in molluscan hemocyanin, the precise subunit pathway within the decamer, the inter-FU interfaces, and the allosteric unit are still obscure, but this knowledge is crucial to understand assembly and allosterism of these proteins. Here we present the cryo-EM structure of Nautilus hemocyanin at 9.1 Å resolution (FSC_1/2-bit criterion), and its molecular model obtained by rigid-body fitting of the individual FUs. In this model we identified the subunit dimer, the subunit pathway, and 15 types of inter-FU interface. Four interface types correspond to the association mode of the two protomers in the published Octopus FU-g crystal. Other interfaces explain previously described morphological structures such as the fenestrated wall (which shows D5 symmetry), the three horizontal wall tiers, the major and minor grooves, the anchor structure and the internal collar (which unexpectedly has C5 symmetry). Moreover, the potential calcium/magnesium and N-glycan binding sites have emerged. Many interfaces have amino acid constellations that might transfer allosteric interaction between FUs. From their topologies we propose that the prime allosteric unit is the oblique segment between major and minor groove, consisting of seven FUs from two different subunits. Thus, the 9 Å structure of Nautilus hemocyanin provides fundamentally new insight into the architecture and function of molluscan hemocyanins.     

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.     

Contribution of disulfide bonds and calcium to Molluscan hemocyanin stability

Disulfide bonds and calcium ions contribute significantly to the stability of the hemocyanin from the mollusc Rapana thomasiana grosse (gastropod). An extremely powerful protective effect of Ca2+ at a concentration of 100 mM (100% protection) against the destructive effect of reductants like dithiothreitol was observed. This is important for the practical application of molluscan hemocyanins in experimental biochemistry, immunology and medicine. The reduction of the disulfide bonds in the Rapana hemocyanin leads to a 20% decrease of the a-helical structure. The S-S bonds contribute significantly to the free energy of stabilization in water increasing delta G(D)H2O by 6.9 kJ mol (-1) The data are related to the X-ray model of the Rapana hemocyanin functional unit RtH2e. The results of this study can be of common validity for related respiratory proteins because the cysteine residues are conserved in all sequences of molluscan hemocyanins published so far.     

Complete amino-acid sequence of a functional unit from a molluscan hemocyanin (Helix pomatia)

From the beta c-hemocyanin (beta c-Hc) of the vineyard snail, Helix pomatia, the functional unit d (Mr approximately equal to 50,000-55,000) was isolated by limited proteolysis and gel chromatography. A small quantity of functional unit d was obtained intact, but the major part in the form of two peptides (Mr approximately equal to 43,000 and 10,000, respectively) connected by a disulfide bridge. After reduction and carboxymethylation, these were separated from each other and cleaved by conventional methods. The peptides were isolated by gel chromatography and HPLC, and sequenced manually or automatically. The complete sequence of Helix beta c-Hc d comprises 410 residues plus 3 residues at the N-terminus seemingly resulting from incomplete cleavage. There is apparently only one carbohydrate side-chain. Comparison of this gastropodan hemocyanin sequence to the partial sequence of a cephalopodan Hc C-terminal unit revealed sufficient identities to state that the functional units of molluscan hemocyanins have arisen by a series of gene duplications. On the other hand, there is practically no homology with arthropodan hemocyanins except for one section of 42 residues which is clearly homologous. This section corresponds to the "Copper B" site of Panulirus interruptus hemocyanin. It is also found in tyrosinases from Neurospora crassa, Streptomyces glaucescens, and mouse. In the N-terminal half of Helix beta c-Hc d there are other sections clearly homologous to the tyrosinases, but overall homology is limited. The second copper-binding site was not identified but must be completely distinct from the "Copper A" binding site of arthropodan hemocyanins. It is suggested that molluscan and arthropodan hemocyanins have evolved independently from a common ancestral mononuclear copper protein.     

Conformational states of the Rapana thomasiana hemocyanin and its substructures studied by dynamic light scattering and time-resolved fluorescence spectroscopy

Hemocyanins are dioxygen-transporting proteins freely dissolved in the hemolymph of mollusks and arthropods. Dynamic light scattering and time-resolved fluorescence measurements show that the oxygenated and apo-forms of the Rapana thomasiana hemocyanin, its structural subunits RtH1 and RtH2, and those of the functional unit RtH2e, exist in different conformations. The oxygenated respiratory proteins are less compact and more asymmetric than the respective apo-forms. Different conformational states were also observed for the R. thomasiana hemocyanin in the absence and presence of an allosteric regulator. The results are in agreement with a molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins including transfer of conformational changes from one functional unit to another.     

Structure of a molluscan hemocyanin didecamer (HtH1 from Haliotis tuberculata) at 12 A resolution by cryoelectron microscopy

A 12 A resolution three-dimensional density map of the Haliotis tuberculata hemocyanin type 1 (HtH1) didecamer has been obtained by cryoelectron microscopy of unstained molecules and angular reconstitution. The dyad symmetry of the 8 MDa D5 HtH1 didecamer, formed by the pairing of two asymmetric 4 MDa ring-like C5 decamers, is emphasised. The major and minor surface helical grooves of the didecamer are well defined, in agreement with earlier data on molluscan hemocyanins. The location of the obliquely orientated repeating unit, a subunit dimer, within the decamer has been defined. Following interactive extraction of this dimer, several new structural features of the dimer and of the subunit have now emerged with improved detail. The subunit dimer possesses pseudo 2-fold symmetry, resulting from the steric arrangement of the wall domains/functional units (FUs-abcdef) of the two subunits. The arc and collar FUs (g and h) depart from this inherent 2-fold symmetry and are thereby responsible for the asymmetry of the C5 decamer, with the internalised collar/arc complex at one edge of the decamer. The FU heterodimers forming the wall morphological units have a hollow centre, and thus create a series of repeating channels that extend within the wall through all three tiers of the decamer. The connections between the wall and the arc are defined with improved clarity, and evidence is provided to indicate that the arc and collar FU pairs have a homodimeric composition (gg and hh, respectively). Two possibilities for the subunit path within the subunit dimer are presented, which correlate with the available structural, immunolabelling and protease cleavage data from HtH1 and other molluscan hemocyanins.     

Molecular evolution of the arthropod hemocyanin superfamily

Arthropod hemocyanins are members of a protein superfamily that also comprises the arthropod phenoloxidases (tyrosinases), crustacean pseudohemocyanins (cryptocyanins), and insect storage hexamerins. The evolution of these proteins was inferred by neighbor-joining, maximum-parsimony, and maximum-likelihood methods. Monte Carlo shuffling approaches provided evidence against a discernible relationship of the arthropod hemocyanin superfamily and molluscan hemocyanins or nonarthropodan tyrosinases. Within the arthropod hemocyanin superfamily, the phenoloxidase probably emerged early in the (eu-)arthropod stemline and thus form the most likely outgroup. The respiratory hemocyanins evolved from these enzymes before the radiation of the extant euarthropodan subphyla. Due to different functional constraints, replacement rates greatly vary between the clades. Divergence times were thus estimated assuming local molecular clocks using several substitution models. The results were consistent and indicated the separation of the cheliceratan and crustacean hemocyanins close to 600 MYA. The different subunit types of the multihexameric cheliceratan hemocyanin have a rather conservative structure and diversified in the arachnidan stemline between 550 and 450 MYA. By contrast, the separation of the crustacean (malacostracan) hemocyanin subunits probably occurred only about 200 MYA. The nonrespiratory pseudohemocyanins evolved within the Decapoda about 215 MYA. The insect hemocyanins and storage hexamerins emerged independently from the crustacean hemocyanins. The time of divergence of the insect proteins from the malacostracan hemocyanins was estimated to be about 430-440 MYA, providing support for the notion that the Hexapoda evolved from the same crustacean lineage as the Malacostraca.     

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 β-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 β-HlH was obtained, including the 5′ and 3′ UTR, 180 bp of the 5′ end and around 900 bp at the 3′ end are missing for the third subunit. The subunits α_D-HlH and β-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.     

Light-scattering and scanning transmission electron microscopic investigation of the hemocyanin of the bivalve, Yoldia limatula (Say)

1. The hemocyanin of the bivalve, Yoldia limatula (Say) was found by light-scattering to have a mol. wt of 8.0 +/- 0.6 x 10(6). Mass measurements by scanning transmission electron microscopy (STEM) gave a particle mass of 8.25 +/- 0.42 x 10(6) for the native particle and 4.09 +/- 0.20 x 10(6) for the half-molecule. 2. The hemocyanin subunits fully dissociated in 8.0 M urea and 6.0 M GdmCl at pH 8.0, and at pH 11.0, 0.01 M EDTA have mol. wts of 4.38 x 10(5), 4.22 x 10(5) and 4.71 x 10(5), close to one-twentieth of the parent molecular weight of Y. limatula hemocyanin and most gastropod hemocyanins. 3. Analyses of the urea dissociation transitions studied at pH 8.0, 1 x 10(-2) M Mg2+, 1 x 10(-2) M Ca2+ and pH 8.0, 3 x 10(-3) M Ca2+ suggest few hydrophobic amino acid groups, of the order of 10 to 15 at the contact areas of each half-molecule or decamer. 4. The further dissociation of the decamers to dimers and the dimers to monomers indicates the presence of a larger number of amino acid groups of ca 35-40/dimer and 100-120/monomer. 5. This suggests hydrophobic stabilization of the dimer to dimer and monomer to monomer contacts within the decamers, as observed with other molluscan hemocyanins.     

Keyhole Limpet Hemocyanin: 9-Å CryoEM Structure and Molecular Model of the KLH1 Didecamer Reveal the Interfaces and Intricate Topology of the 160 Functional Units

Hemocyanins are blue copper-containing respiratory proteins in the hemolymph of many arthropods and molluscs. Molluscan hemocyanins are decamers, didecamers, or multidecamers of a 340- to 400-kDa polypeptide subunit containing seven or eight globular functional units (FUs; FU-a to FU-h), each with an oxygen-binding site. The decamers are short 35-nm hollow cylinders, with their lumen narrowed by a collar complex. Our recently published 9-Å cryo-electron microscopy/crystal structure hybrid model of a 3.4-MDa cephalopod hemocyanin decamer [Nautilus pompilius hemocyanin (NpH)] revealed the pathway of the seven-FU subunit (340 kDa), 15 types of inter-FU interface, and an asymmetric collar consisting of five “arcs” (FU-g pairs). We now present a comparable hybrid model of an 8-MDa gastropod hemocyanin didecamer assembled from two asymmetric decamers [isoform keyhole limpet hemocyanin (KLH) 1 of the established immunogen KLH]. Compared to NpH, the KLH1 subunit (400 kDa) is C-terminally elongated by FU-h, which is further extended by a unique tail domain. We have found that the wall-and-arc structure of the KLH1 decamer is very similar to that of NpH. We have traced the subunit pathway and how it continues from KLH1-g to KLH1-h to form an annulus of five “slabs” (FU-h pairs) at one cylinder edge. The 15 types of inter-FU interface detected in NpH are also present in KLH1. Moreover, we have identified one arc/slab interface, two slab/slab interfaces, five slab/wall interfaces, and four decamer/decamer interfaces. The 27 interfaces are described on the basis of two subunit conformers, yielding an asymmetric homodimer. Six protrusions from the cryo-electron microscopy structure per subunit are associated with putative attachment sites for N-linked glycans, indicating a total of 120 sugar trees in KLH1. Also, putative binding sites for divalent cations have been detected. In conclusion, the present 9-Å data on KLH1 confirm and substantially broaden our recent analysis of the smaller cephalopod hemocyanin and essentially solve the gastropod hemocyanin structure.     

Hemocyanin of the horseshoe crab, Limulus polyphemus. Structural differentiation of the isolated components.

The high molecular weight hemocyanin found in the hemolymph of the horseshoe crab, Limulus polyphemus, is composed of at least eight different kinds of subunits. Ion exchange chromatography at high pH in the presence of EDTA yields five major zones, hemocyanins I to V, three of which are electrophoretically heterogeneous. The subunits have similar molecular weights, 65,000 to 70,000, and their amino acid compositions are remarkably similar to each other and to other arthropod and molluscan hemocyanins. Digestion of the native subunits of Limulus hemocyanin by formic acid or trypsin shows considerable structural diversity which is supported by cyanogen bromide cleavage patterns and by peptide mapping of the tryptic peptides prepared from denatured hemocyanin subunits. The structural differentiation of the subunits is accompanied by functional differentiation, as shown in previous investigations of their O2 and CO affinities (Sullivan, B., Bonaventura, J., and Bonaventura, C. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2558-2562; Bonaventura, C., Bonaventura, J., Sullivan, B., and Bourne, S. (1975) Biochemistry 13, 4784-4789). The subunit diversity of Limulus hemocyanin suggests that other electrophoretically heterogeneous hemocyanins may be composed of structurally distinct subunits.     

cDNA cloning of a developmentally regulated hemocyanin subunit in the crustacean Cancer magister and phylogenetic analysis of the hemocyanin gene family

The complete cDNA sequence and protein reading frame of a developmentally regulated hemocyanin subunit in the Dungeness crab (Cancer magister) is presented. The protein sequence is aligned with 18 potentially homologous hemocyanin-type proteins displaying apparent sequence similarities. Functional domains are identified, and a comparison of predicted hydrophilicities, surface probabilities, and regional backbone flexibilities provides evidence for a remarkable degree of structural conservation among the proteins surveyed. Parsimony analysis of the protein sequence alignment identifies four monophyletic groups on the arthropodan branch of the hemocyanin gene tree: crustacean hemocyanins, insect hexamerins, chelicerate hemocyanins, and arthropodan prophenoloxidases. They form a monophyletic group relative to molluscan hemocyanins and nonarthropodan tyrosinases. Arthropodan prophenoloxidases, although functionally similar to tyrosinases, appear to belong to the arthropodan hexamer- type hemolymph proteins as opposed to molluscan hemocyanins and tyrosinases.      

Hemocyanins Relationships in Their Structure, Function and Assembly

Hemocyanins are high molecular weight oxygen-carrying proteinsthat occur in the molluscs and arthropods. The oxygen-bindingsite in these proteins is a pair of copper atoms bound directlyto ammo acid side chains. The biscopper sites of these proteinsbind single molecules of dioxygen or carbon monoxide. In arthropodsthere are two copper atoms per approximately 70 000 daltonsof protein. This corresponds to the molecular weight of theminimum polypeptide chain. In molluscs however there are twocopper atoms per 50,000 daltons of protein. This does not correspondto the minimum polypeptide chain although it does correspondto the minimal functional unit. The minimal polypeptide chainin molluscan hemocyanins is approximately 400,000 daltons andappears to be composed of eight or more 50 000 dalton unitslinked together like a string of pearls. In the molluscs, thenative hemocyanins found in the hemolymph generally occur asgiant cylindrical molecules 350 Å in diameter, 380 Ålong, with molecular weights of 9 000,000. These large moleculesare composed of approximately 20 polypeptide chains. In thehemolymph of the arthropods depending upon the species, hemocyaminsoccur as hexamers dodecamers 24-mers and 48-mers. The molecularweight of the 48-mers is about 3 600,000. The respiratory functionsof hemocyamns show a wide range of allosteric properties. Thelarge molecules commonly show cooperativity in oxygen binding.The number of interacting subunits is in some cases dependentupon external conditions of pH and ionic composition. The oxygenaffinity is usually sensitive to pH and to low molecular weightcofactors like chloride, calcium and magnesium ions. The intrinsicoxygen binding properties of an organism s hemocyanin and itsallosteric control by modulators allow organisms possessingthese giant molecules to adapt to their environmental conditions.     

Primary structure and unusual carbohydrate moiety of functional unit 2-c of keyhole limpet hemocyanin (KLH)

The complete amino acid sequence of the Megathura crenulata hemocyanin functional unit KLH2-c was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of the protein, and of peptides obtained by cleavage with EndoLysC proteinase, chymotrypsin and cyanogen bromide. This is the first complete primary structure of a functional unit c from a gastropod hemocyanin. KLH2-c consists of 420 amino acid residues. Circular dichroism spectra indicated approx. 31% beta-sheet and 29% alpha-helix contents. A multiple sequence alignment with other molluscan hemocyanin functional units revealed average identities between 41 and 49%, but 55% in case of Octopus hemocyanin functional unit c which is the structural equivalent to KLH2-c. KLH2-c has a molecular mass of approx. 48 kDa as calculated from its sequence and a measured mass of approx. 56 kDa; the mass difference is attributed to the sugar side chains usually decorating molluscan hemocyanin. However, inspection of the sequence of KLH2-c revealed no potential N-linked carbohydrate attachment sites, and this was supported by its inability to bind concanavalin A. Also KLH1-c was unreactive, whereas most, if not all, other functional units of KLH1 and KLH2 reacted positively to this lectin. On the other hand, peanut agglutinin specifically binds KLH2-c, indicating the presence of O-glycosidically linked carbohydrates in this functional unit. This contrasts to all other KLH functional units (including KLH1-c), which lack O-linked glycosides. The present results are discussed in view of the recent X-ray structure of the functional unit g from Octopus hemocyanin, and a published record of the Thomsen Friedenreich tumor antigenic epitope in KLH.