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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.

Hemocyanins are large oligomeric copper-containing proteins that serve for the transport of oxygen in many arthropod species. While studied in detail in the Chelicerata and Crustacea, hemocyanins had long been considered unnecessary in the Myriapoda. Here we report the complete molecular structure of the hemocyanin from the common house centipede Scutigera coleoptrata (Myriapoda: Chilopoda), as deduced from 2D-gel electrophoresis, MALDI-TOF mass spectrometry, protein and cDNA sequencing, and homology modeling. This is the first myriapod hemocyanin to be fully sequenced, and allows the investigation of hemocyanin structure-function relationship and evolution. S. coleoptrata hemocyanin is a 6 x 6-mer composed of four distinct subunit types that occur in an approximate 2 : 2 : 1 : 1 ratio and are 49.5-55.5% identical. The cDNA of a fifth, highly diverged, putative hemocyanin was identified that is not included in the native 6 x 6-mer hemocyanin. Phylogenetic analyses show that myriapod hemocyanins are monophyletic, but at least three distinct subunit types evolved before the separation of the Chilopoda and Diplopoda more than 420 million years ago. In contrast to the situation in the Crustacea and Chelicerata, the substitution rates among the myriapod hemocyanin subunits are highly variable. Phylogenetic analyses do not support a common clade of Myriapoda and Hexapoda, whereas there is evidence in favor of monophyletic Mandibulata.

Keyhole limpet (Megathura crenulata) hemocyanin (KLH), an extracellular respiratory protein, is widely used as hapten carrier and immune stimulant. Although it is generally accepted that the sugar constituents of this glycoprotein are likely to be implicated in the antigenicity and biomedical properties of KLH, knowledge of its carbohydrate structure is still limited. Therefore, we have investigated the N-linked oligosaccharides of KLH. Glycan chains were enzymatically liberated from tryptic glycopeptides, pyridylaminated and separated by two-dimensional HPLC. Only neutral oligosaccharides were obtained and characterized by carbohydrate constituent and methylation analyses, MALDI-TOF-MS, ESI-ion trap-MS and sequential exoglycosidase digestion. The results revealed that KLH is carrying high mannose-type glycans and truncated sugar chains derived thereof. As a characteristic feature, a number of the studied N-glycans contained a Gal(beta1-6)Man-unit which has not been found in glycoprotein-N-glycans so far. Hence, our studies demonstrate that this marine mollusk glycoprotein is characterized by a unique oligosaccharide pattern comprising, in part, novel structural elements.

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.

111Ag(I) perturbed angular correlations of gamma-rays (PAC) has been used to investigate the binuclear metal site of 111Ag(I)-substituted Carcinus aestuarii deoxyhemocyanin. The studies have shown that apo-hemocyanin is able to bind 2 mol of Ag(I) per mol of protein and that the binding is specific for the metal ion sites. The PAC spectra show pronounced changes when the stoichiometry of Ag(I) to protein is increased from 0.1 to 2.0. These changes have been interpreted as evidence of interactions between the two sites in terms of a structural destabilization of the first occupied site caused by the occupation of the second site. The experimental data for the Ag(I)-substituted metal sites do not agree well with the three-coordinated structure found in the Cu(I) holo-protein. However, if a water molecule is included as a coordinating ligand in the Ag(I) metal site a successful interpretation of the experimental data can be obtained.

We have developed a HPLC procedure to isolate the two different hemocyanin types (HtH1 and HtH2) of the European abalone Haliotis tuberculata. On the basis of limited proteolytic cleavage, two-dimensional immunoelectrophoresis, PAGE, N-terminal protein sequencing and cDNA sequencing, we have identified eight different 40-60-kDa functional units (FUs) in HtH2, termed HtH2-a to HtH2-h, and determined their linear arrangement within the elongated 400-kDa subunit. From a Haliotis cDNA library, we have isolated and sequenced a cDNA clone which encodes the five C-terminal FUs d, e, f, g and h of HtH2. As shown by multiple sequence alignments, defg of HtH2 correspond structurally to defg from Octopus dofleini hemocyanin. HtH2-e is the first FU of a gastropod hemocyanin to be sequenced. The new Haliotis hemocyanin sequences are compared to their counterparts in Octopus, Helix pomatia and HtH1 (from the latter, the sequences of FU-f, FU-g and FU-h have recently been determined) and discussed in relation to the recent 2.3 A X-ray structure of FU-g from Octopus hemocyanin and the 15 A three-dimensional reconstruction of the Megathura crenulata hemocyanin didecamer from electron micrographs. This data allows, for the first time, an insight into the evolution of the two functionally different hemocyanin isoforms found in marine gastropods. It appears that they evolved several hundred million years ago within the Prosobranchia, after separation of the latter from the branch leading to the Pulmonata. Moreover, as a structural explanation for the inefficiency of the type 1 hemocyanin to form multidecamers in vivo, the additional N-glycosylation sites in HtH1 compared to HtH2 are discussed.

We have identified two separate hemocyanin types (HtH1 and HtH2) in the European abalone Haliotis tuberculata. HtH1/HtH2 hybrid molecules were not found. By selective dissociation of HtH2 we isolated HtH1 which, as revealed by electron microscopy and SDS/PAGE, is present as didecamers of a approximately 400 kDa subunit. Immunologically, HtH1 and HtH2 correspond to keyhole limpet hemocyanin (KLH)1 and KLH2, respectively, the two well-studied hemocyanin types of the closely related marine gastropod Megathura crenulata. On the basis of limited proteolytic cleavage, two-dimensional immunoelectrophoresis, SDS/PAGE and N-terminal sequencing, we identified eight different 40-60 kDa functional units in HtH1, termed HtH1-a to HtH1-h, and determined their linear arrangement within the elongated subunit. From Haliotis mantle tissue, rich in hemocyanin-producing pore cells, we isolated mRNA and constructed a cDNA library. By expression screening with HtH-specific rabbit antibodies, a cDNA clone was isolated and sequenced which codes for the three C-terminal functional units f, g and h of HtH1. Their sequences were aligned to those available from other molluscs, notably to functional unit f and functional unit g from the cephalopod Octopus dofleini. HtH1-f, which is the first sequenced functional unit of type f from a gastropod hemocyanin, corresponds to functional unit f from Octopus. Also functional unit g from Haliotis and Octopus correspond to each other. HtH1-h is a gastropod hemocyanin functional unit type which is absent in cephalopods and has not been sequenced previously. It exhibits a unique tail extension of approximately 95 amino acids, which is lacking in functional units a to g and aligns with a published peptide sequence of 48 amino acids from functional unit h of Helix pomatia hemocyanin. The new Haliotis sequences are discussed with respect to their counterparts in Octopus, the 15 A three-dimensional reconstruction of the KLH1 didecamer from electron micrographs, and the recent 2.3 A X-ray structure of functional unit g from Octopus hemocyanin.

Carcinus aestuarii hemocyanin (Hc) exists in two aggregation forms at pH 7.5 and 20 mM Ca2+: 24S accounting for 90% of total hemocyanin and 16S accounting for 10%. Removal of metal cations by EDTA at neutral pH causes the complete dissociation of 24S hemocyanin into two different 16S. At pH 9.2, 24S hemocyanin dissociates into a pH stable 16S and a 5S component. The 5S component consists of three monomeric fractions named CaeSS1 (10%), CaeSS2 (50%) and CaeSS3 (40%); the latter fraction consisting of two isoforms. The fractions CaeSS1, CaeSS2 and CaeSS3 have been studied as far as their reassociation properties to form hexamers are concerned. We investigated the oxygen-binding properties of the native form (24S), the mixture of the two 16S forms, the pH-stable 16S alone and of purified subunit fractions to define the role of each species on the expression of the allosteric behaviour of the 24S aggregate. The analysis of O2-binding data reveals that 24S-Hc can be well described by the modified Monod Wyman and Changeaux-model (nested MWC-model), while the half-molecules (16S) bind oxygen according to the simple MWC-model. The two hexameric 16S within the dodecameric 24S hemocyanin can be regarded as nested allosteric units. They behave as being functionally coupled in the T-states (tT and rT). In the R-states (tR and rR) the two half-molecules seem to be functionally uncoupled since they have the same values of oxygen binding constants as deduced for isolated 16S hexamers.

The primary structures of 21 novel monoantennary and diantennary N-glycans of the glycoprotein alphaD-hemocyanin (alphaD-Hc) of Helix pomatia have been determined. Outer oligosaccharide fragments (antennae) were released from the glycoprotein by Smith degradation of an alphaD-Hc pronase digest. The major antenna, obtained following HPLC fractionation on Lichrosorb-NH2, was characterized using 1H-NMR spectroscopy, fast-atom-bombardment mass spectrometry, and linkage analysis, and corresponds to a pentasaccharide fragment. The intact carbohydrate chains of alphaD-Hc were released with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase-F digestion, separated from the protein on Bio-Gel P-100, and subfractionated on Bio-Gel P-4. A portion of subfractions was reduced with sodium borodeuteride, and the non-reduced and reduced samples were further fractionated on CarboPac PA-1, Lichrosorb-NH2/Lichrosphere-NH2, and/or Lichrosphere-C18. Purified oligosaccharides and oligosaccharide-alditols were analyzed using 500/600-MHz 1H-NMR spectroscopy. In total, four novel types of antenna were identified, namely, [structures: see text] which are all attached to O-2 of alphaMan residues of the trimannosyl-N,N'-diacetylchitobiose core element, which is generally beta-1,2-xylosylated and alpha-1,6-fucosylated, Man(alpha1-6)[Man(alpha1-3)][+/-Xyl(beta1-2)]Man(beta1-4)GlcNAc(beta1-4) [+/-Fuc(alpha1-6)]GlcNAc.

In functional units d and g from the betaC-haemocyanin of the gastropod Helix pomatia, aminoacid analysis in the presence of dimethyl sulfoxide showed the occurrence of seven cysteine residues. Titration with 5,5'-dithiobis(2-nitrobenzoic acid), however, did not reveal any free thiol group. Pepsinolysis at pH 2.0 followed by amino acid analysis and partial sequencing of the cysteine-containing peptides showed that six cysteine residues are involved in the formation of three disulfide bridges at corresponding positions. The results indicated that the remaining cysteine residue is linked by a thioether bridge to a histidine residue located two positions further in the sequence (H. pomatia d Cys60 His62; H. pomatia g Cys66 His68). This residue corresponds to one of the three histidine residues considered to be involved in the coordination of the copper A atom of the dinuclear copper group of the functional units. The presence of the thioether bond was further evidenced by an absorption band at 255 nm and by molecular mass determinations with electrospray mass spectrometry on a peptic peptide from H. pomatia d and on peptides obtained by proteolysis of carboxymethylated H. pomatia d with trypsin and pronase. Upon sequence analysis the cysteine-histidine thioether bridge was cleaved into didehydroalanine (polymers) and 2-thiolhistidine. A peptide with a cysteine-histidine thioether bridge was isolated from pepsinolysates of functional units c, e, f, g and h of Sepia officinalis and unit g of Octopus vulgaris, obtained from haemocyanin of the cephalopods S. officinalis and O. vulgaris. A cysteine-histidine thioether bridge, which was reported previously for tyrosinase of Neurospora crassa, thus seems to be a general feature for functional units of molluscan haemocyanins.

Analytical dark-field scanning transmission electron microscopy (STEM) of freeze-dried unstained specimens of keyhole limpet hemocyanin (KLH; from Megathura crenulata, a prosobranch gastropod) gave a molecular mass of 400 kDa for the subunit of KLH1 and of 345 kDa for the subunit of KLH2, which confirms our published values from SDS/PAGE. Within the 400-kDa KLH1 subunit we identified, by limited proteolysis, isolation of fragments and N-terminal sequencing, eight distinct 45-60 kDa functional domains (termed 1a through 1h) and determined their sequential arrangement. The KLH1 domains differ biochemically and immunologically from each other and from the previously characterized seven domains of KLH2 (termed 2a through 2g). Our partial amino acid sequences suggest that a domain, equivalent to the C-terminal domain 1h, is missing in KLH2. This deficiency is believed to be genuine and not an artifact of the subunit preparation procedure, since STEM measurements of the native didecamers yielded a mass difference of about 800 kDa between KLH1 and KLH2 (8.3 MDa versus 7.5 MDa), correlating with 20 copies of a functional 1h domain. It was also shown that the KLH1 didecamer can be rapidly split (minutes) into an almost homogeneous population of stable decamers by increasing the pH of the Tris/saline stabilizing buffer (routinely pH 7.4), which contains 5 mM CaCl2 and 5 mM MgCl2, to pH 8.5. Reformation of the didecamers occurred more slowly (days) upon dialysis against the pH 7.4 stabilizing buffer. Addition of 100 mM calcium and 100 mM magnesium ions to the pH 7.4 stabilizing buffer leads to the more rapid (overnight) formation of didecamers together with a significant number of previously unobserved KLH1 multidecamers, which could be structurally distinguished from the established multidecamers of KLH2.

To establish the competence of the active site of hemocyanin to acquire diverse coordination geometries, the binding of azide to three forms of a crab hemocyanin, the dinuclear cupric or met-hemocyanin, the mononuclear cupric or met-apo-hemocyanin, and the mononuclear Co(II)-substituted derivative has been studied by near-ultraviolet circular dichroism and EPR spectroscopies. The near-ultraviolet circular dichroism spectra of the various derivatives present qualitatively similar features, namely a negative peak around 335 nm in the case of the two copper-containing derivatives and a three-component pattern with the Co(II) derivative. Upon decreasing the pH from 7.0 to 5.5 a decrease of optical activity is observed with all protein samples. The characteristic CD features, attributable to N(imidazole)-to-metal and to OH -to-metal charge-transfer transitions, are strongly affected by azide binding. In particular, the intensity of the negative band exhibited by the two copper-containing protein forms decreases with the onset of a new negative feature with maximum around 400 nm diagnostic for azide-to-Cu(II) charge-transfer transitions. The visible region is affected as well, indicating that changes in the coordination sphere of copper take place. The affinity for azide of the different protein forms is higher at low pH. EPR measurements on the paramagnetic met-apo-hemocyanin derivative as a function of pH demonstrate heterogeneity in the coordination environment at low pH. In the presence of azide an increase of rhombic distortion of the EPR spectra is observed and on the basis of the identified sets of copper hyperfine features in the course of azide titration experiments two different azide bound forms of met-apo-hemocyanin can be detected. The CD and EPR data at the different pH values are consistent with a reaction scheme in which azide replaces a fourth ligand in the metal-coordination sphere, identified as a water or hydroxide molecule.

The primary structure of the hemocyanin Aa6 subunit from the scorpion Androctonus australis was resolved by using protein sequencing and mass spectrometry for analysis of the polypeptide chain and of fragments obtained by CNBr, trypsin, and chymotrypsin cleavage. Due to the high sensitivity of the methodologies used, only a small amount of material, less than 1 mg, was consumed. The complete sequence is composed of 626 amino acid residues and the protein is not glycosylated but probably phosphorylated at Ser374. Its molecular mass measured by mass spectrometry (71,890 +/- 7 Da) is about 30 Da higher than the mass calculated from the sequence data (71,860.1 Da). The origin of this difference is not clear but could result from minor molecular heterogeneities. Within the chelicerates, the Aa6 subunit of the arachnid A. australis shares 405 identical residues with chain e of another arachnid, Eurypelma californicum, and 399 with chain alpha of the merostom Tachypleus tridentatus. The degrees of identity between these three subunits, which are known to occupy the same location in the native hemocyanin oligomers, are significantly higher than those existing between the subunits a, d, and e of E. californicum. This favors the hypothesis that gene duplications, leading to separate chains in one species, have occurred before the divergence between arachnids and merostoms.

The reaction that gives met-hemocyanin from Octopus vulgaris oxy-hemocyanin has been reinvestigated under several experimental conditions. Various anions including azide, fluoride and acetate have been found to promote this reaction. Kinetic data indicate that the reaction mechanism is different from that currently accepted involving a peroxide displacement of bound dioxygen through an associative chemistry on an open axial position of the copper ions [Hepp, A. F., Himmelwright, R. S., Eickman, N. C. & Solomon, E. I. (1979) Biochem. Biophys. Res. Commun. 89, 1050-1057; Solomon, E. I. in Copper proteins (Spiro, T. G., ed.) pp. 43-108, J. Wiley, New York]. Our study suggests that the protonated form of the anion is likely to be the species reacting with the oxygenated form of the protein. Furthermore, it is also proposed that protonation of bound dioxygen generates an intermediate hydroperoxo-dicopper(II) complex to which the exogenous anion is also bound. This intermediate in not accumulated and preceds the release of hydrogen peroxide by reaction with water. Upon dialysis it leads to the met-hemocyanin form. The structure of this dinuclear copper(II) derivative contains a di-mu-hydroxo bridge but there is evidence from optical and circular dichroism spectra for partial protonation of these bridges at low pH. As a consequence, while one azide molecule binds in the bridging mode to met-hemocyanin with low affinity (K = 30 M-1) at pH 7.0, it binds with much higher affinity at pH 5.5 (K = 1500 M-1), where a second azide ligand also binds in the terminal mode (K = 20 M-1). The coordination mode of the azide ligands is deduced from the optical and circular dichroism spectra of the protein complexes.

Negatively stained complexes of Panulirus interruptus (spiny lobster) hemocyanin with two different monoclonal antibodies, named E and J, were studied by electron microscopy and image processing. The attachment site of the antibodies to the hexameric hemocyanin molecule was deduced from two perpendicular views of hemocyanin/antibody complexes, in which either the threefold axis or one of the twofold axes was oriented perpendicular to the supporting film. Images of complexes in these orientations were searched with reference images simulated from the known X-ray structure of P. interruptus hemocyanin. The two sites were further characterized by combining our results from electron microscopy with structural data obtained by X-ray diffraction and other methods. These two antibodies recognize different non-overlapping epitopes. The epitope for clone E is located on domain 3 at the surface of the beta barrel and consists of certain loops, which form connections between beta-strand structures. The epitope for clone J is situated on domain 1 at the surface of an alpha-helical region and consists mainly of certain alpha-helices connecting loops. The orientation of the hemocyanin hexamers in the two complexes is very different, as is demonstrated most clearly when they form chains. Clone E forms complexes with the threefold axes perpendicular to the chain direction, while for clone J the threefold axes seem to be parallel to the main direction. The angle between the Fab part of an IgG molecule and the threefold axis of the hexamer is 60 +/- 5 degrees for clone E and 35 +/- 7 degrees for clone J. This observation is clearly related to the difference in orientation of the hexamers for the two complexes.

The amino acid sequence of the hemocyanin subunit c from the spiny lobster, Panulirus interruptus, has been determined. The elucidation was mainly based on three digests, with CNBr, trypsin and endoproteinase Glu-C, respectively. Additional evidence was obtained by sequencing of peptides from an endoproteinase Lys-C digest. Subunit c is a polypeptide with 661 amino acid residues and with a carbohydrate group attached to residue 476 in the third domain. No heterogeneity was observed. The degree of identity with subunit a is 59%. Some differences with subunit a are an N-terminal extension of six residues, a one-residue C-terminal extension, and a three-residue deletion. Furthermore, carbohydrate attachment is in a different position, as are most half-cystine residues. Limited trypsinolysis resulted in cleavage at the same site as in subunits a and b.