Intramolecular localization of epitopes within an oligomeric protein by immunoelectron microscopy and image processing

Three epitopes have been localized by immunoelectron microscopy on subunit Aa6 of the 4 x 6-meric hemocyanin of the scorpion Androctonus australis. Soluble immunocomplexes composed of monoclonal antibodies and of native hemocyanin were purified, negatively stained with uranyle acetate by the single-layer technique, and examined under the electron microscope (EM). The molecule images were digitized, aligned, and submitted to correspondence analysis according to the method of Van Heel and Frank (Ultramicroscopy 6:187-194, 1981). A high-precision localization of the attachment point of the Fab arm to the antigen was achieved through a careful analysis of the average images. This method easily allowed the discrimination of epitopes located in different domains (Mr 20 kDa) of the same subunit. Nonoverlapping epitopes located in the same structural domain of subunit Aa6 could be distinguished by the stain exclusion patterns of their Fab arms. The method is general and may be used for epitope mapping in any antigen producing definite EM views.     

Immunological correlates between multiple isolated subunits of Androctonus australis and Limulus polyphemus hemocyanins: an evolutionary approach

Immunological cross-reactivities between isolated subunits of the scorpion Androctonus australis (Aa) and of the horseshoe crab Limulus polyphemus (Lp) hemocyanins were studied using subunit-specific antibodies prepared through immunoadsorption to pure immobilized subunits. Rocket immunoelectrophoreses of the various subunits of both hemocyanins were carried out at constant antigen concentration against the various subunit-specific antibody preparations. Then the data were analyzed through factorial correspondence analysis and compared to the respective intramolecular locations of the subunits in both hemocyanins. The results show that the dimeric subunits located in the central part of each (4 X 6)meric structure (Aa whole molecule and Lp half molecule) were strongly preserved. In addition, the (8 X 6)mer-forming subunit of Lp hemocyanin (LpIV) and the subunit occupying the same intramolecular position in Aa hemocyanin (Aa5A) were also strongly preserved. Besides the strong antigenic relatedness, less pronounced crossed immunoprecipitations or no precipitation at all were observed between subunits with homologous positions suggesting a minor structural and/or functional roles for these subunits. All the antigen-antibody combinations leading to an absence of immunoprecipitation were screened for the presence of soluble immunocomplexes by radioimmunological tests. In all cases, soluble immunocomplexes were observed. These results suggest the following evolution scenario. First, the central dimeric subunits, responsible of the dodecamer aggregation (Aa3C and 5B and LpV and VI) were already differentiated when Merostomata diverged from Arachnida. Second, the differentiation of the (8 X 6)mer-forming subunit occurred in the Merostomata ramification in a preserved subunit already possessing a functional advantage. Third, the differentiation of subunits Aa3A and Aa3B recently occurred in the scorpion ramification.     

Isolation and partial characterization of the N-terminal functional unit of subunit RtH1 from Rapana thomasiana grosse hemocyanin

Two different structural subunits were identified in Rapana thomasiana hemocyanin: RtH1 and RtH2. RtH1-a is the N-terminal functional unit in the subunit RtH1 and its stability toward temperature and chemical denaturation by guanidinium hydrochloride (Gdn.HCl) are studied and compared with the structural subunit RtH1 and the whole Rapana hemocyanin molecule. The conformational changes, induced by the various treatments, were monitored by CD and fluorescence spectroscopy. The critical temperatures (T(c)) for RtH1-a, the structural subunits and the native Hc, determined by fluorescence spectroscopy, coincide closely with the melting temperatures (T(m)), determined by CD spectroscopy. The free energy of stabilization in water, DeltaG(D)(H(2)O), determined from (Gdn. HCl) denaturation studies, is about two times higher for the structural subunit RtH1 and the whole hemocyanin molecule as compared to the functional unit RtH1-a. The oligomerization between the structural subunits or the eight functional units, assembled in subunit RtH1, has a stabilizing effect on the whole molecule as well as the structural subunits.     

Oxygen transport proteins: III. Structural studies of the scorpion (Buthus sindicus) hemocyanin, partial primary structure of its subunit Bsin1

The hemocyanin (Hc) from Buthus sindicus, studied in the native state, demonstrated to be an aggregate of eight different types of subunits arranged in four cubic hexamers. Both, the 'top' and the 'side' views of the native molecule have been identified from the negatively stained specimens using transmission electron microscopy. Out of these, eight different polypeptide chains, the partial primary structure (68%) of a subunit Bsin1 (Mr = 72422.7 Da) was established using a combination of automated Edman degradation and mass spectrometry. A multiple sequence alignment with other closely related cheliceratan Hc subunits revealed average identities of ca. 60%. Most of the structurally important residues, i.e. copper and calcium-binding ligands, as well as the residues involved in the presumed oxygen entrance pathway, proved to be strictly conserved in Bsin1. Sequence variations have been observed around the functionally important chloride-binding site, not only for the B. sindicus subunit Bsin1, but also for the subunit Aaus-6 of the scorpion A. australis and the subunit Ecal-a from the spider Eurypelma californicum Hcs. Deviation in the primary structure related to the chloride-binding site suggest that the effect of chloride ions may vary in different hemocyanins. Furthermore, the secondary structural contents of the Hc subunit Bsin1 were determined by circular dichroism revealing ca. 33% alpha-helix, 18%, beta-sheet, 19% beta-turn, and 30% random coil composition. These values are in good agreement with the crystal structure of the closely related Hc subunit Lpol-II from horseshoe crab L. polyphemus. Electron microscopic studies of the purified Hc subunit under native conditions revealed that Bsin1 has self aggregation properties. Results of these studies are discussed.     

The structure of NADH:ubiquinone oxidoreductase from beef-heart mitochondria. Crystals containing an octameric arrangement of iron-sulphur protein fragments

We have investigated the structure of two-dimensional crystals from preparations of NADH:ubiquinone oxidoreductase from beef-heart mitochondria. The crystal structure of these crystals was previously determined to be equivalent with two native enzyme molecules per unit cell, i.e. a p2 symmetry [Boekema, E. J., Van Heel, M. G. & Van Bruggen, E. F. J. (1984) Biochim. Biophys. Acta 787, 19-26]. However, the optical diffraction patterns of the crystals displayed a clear fourfold symmetry. A Fourier analysis carried out on the calculated diffraction pattern proved unambiguously that the crystal symmetry was p42(1)2. Following crystallographic rules the unit cell therefore contained eight identical molecules. As a consequence, only a subcomplex of the enzyme rather than the intact enzyme formed the crystal. Electron microscopy of isolated, single molecules of the iron-sulphur protein, a dissociation product of complex I, revealed the presence of square complexes with sides of approximately 15 nm. Since these complexes were indistinguishable from the building blocks (unit cells) of the two-dimensional crystals, the crystals could be composed of Fe-S protein fragments only. The nature of the fragments in the unit cell was probed by immuno-labelling with monovalent antibodies (Fab's), raised against the 75-kDa subunit from the Fe-S protein, followed by image analysis. We found at least four binding sites for the anti-(75-kDa subunit) Fab per unit cell, indicating the presence of at least four copies of the antigen. In order to account for these observations we postulate the hypothesis that the two-dimensional crystals obtained from complex I are composed of iron-sulphur protein molecules in an octameric arrangement.     

Low-resolution structure of the proteolytic fragments of the Rapana venosa hemocyanin in solution

Rapana venosa hemocyanin (Hc) is a giant oxygen-binding protein consisting of different subunits assembled in a hollow cylinder. The polypeptide chain of each subunit is believed to be folded in several oxygen-binding functional units of molecular mass 50 kDa, each containing a binuclear copper active site. Limited proteolysis with alpha-chymotrypsin of native R. venosa hemocyanin allows the separation of three functional proteolytic fragments with molecular masses of approximately 150, 100, and 50 kDa. The functional fragments, purified by combining gel filtration chromatography and ion-exchange FPLC, were analyzed by means of small-angle X-ray scattering (SAXS). The gyration radius of the 50-kDa Rapana Hc fraction (2.4 nm) agrees well with that calculated on the basis of the dimensions determined by X-ray crystallography for one functional unit of Octopus Hc (2.1 nm). Independent shape determination of the 50- and 100-kDa proteolytic fragments yields consistent low-resolution models. Simultaneous fitting of the SAXS data from these fragments provides a higher-resolution model of the 100-kDa species made of two functional units tilted with respect to each other. The model of the 150-kDa proteolytic fragment consistent with the SAXS data displays a linear chain-like aggregation of the 50-kDa functional units. These observations provide valuable information for the reconstruction of the three-dimensional structure of the minimal functional subunit of gastropod hemocyanin in solution. Furthermore, the spatial relationships among the different functional units within the subunit will help in elucidation of the overall quaternary structure of the oligomeric native protein.     

A model for the architecture of the hemocyanin from the arthropod Squilla mantis (Crustacea, Stomatopoda)

Squilla mantis hemocyanin is composed of two hexameric subunits but has electron microscopic profiles different from other bis-hexameric hemocyanins, e.g. Astacus and Homarus. We distinguished three different electron microscopic profiles of S. mantis hemocyanin: two sideviews and a topview. These profiles were studied using computer image alignment and correspondence analysis [Van Heel, M. and Frank, J. (1981) Ultramicroscopy 6, 187 - 194]. With the results of this analysis we were able to build a three-dimensional model for the quaternary structure of this hemocyanin. In this model the two hexamers are stacked in such a way that their hexagonal surfaces overlap to about 60% of their width. In the overlap area four subunits are arranged in two different interhexameric pairs, each forming a bridging area between the two hexamers.     

Three-Dimensional Immunoelectron Microscopy of Scorpion Hemocyanin Labeled with a Monoclonal Fab Fragment

An immunocomplex of the 4 × 6-meric hemocyanin of the scorpion Androctonus australis with the monoclonal Fab fragment L104 was reconstructed from electron micrographs of a negatively stained specimen, using the double-carbon-layer technique. The resulting structure enables a clear visualization of the Fab fragments bound to the four copies of the Aa6 subunit and directly confirms a previous localization of the L104 epitope deduced from two-dimensional image processing. Despite a strong flattening effect produced by the negative-staining technique the orientations of the Fab fragments are well characterized. Moreover, the observation of a central hole within the elbow bends of the Fab fragments provides information about the disposition of the Fabs around their main axis.     

Co(II)-substituted Octopus vulgaris hemocyanin

Co(II)-substituted hemocyanin (Co(II)Hc) of the octopus, Octopus vulgaris, has been prepared by dialysis of apohemocyanin against Co(II·) ion and subsequent Chelex-treatment. The blue 50%-Co(II)Hc (half-apo Co(II)Hc), in which binuclear coppers are replaced in the hemocyanin by a single Co(II), exhibits two absorption maxima at 560 (?_Co=250) and 594 nm (?_Co=320 M^?1 cm^?1) and a shoulder near 610 nm, all of which are attributed to a dd transition of high spin Co(II) (S=3/2) with a tetrahedral geometry. The magnetic circular dichroism (MCD) spectrum in this region also suggests the existence of a tetrahedral Co(II) species in the protein. The visible absorption and MCD spectra of octopus 50%-Co(II)Hc are quite similar to those of squid 50%-Co(II)Hc described in the previous paper (S. Suzuki, J. Kino, M. Kimura, W. Mori and A. Nakahara, Inorg. Chim. Acta, 66, 41 (1982)). The formation of half-apo Co(II)Hc demonstrates that the binuclear copper sites in native octopus hemocyanin may differ from each other in coordination geometry, as in other molluscan hemocyanins, squid and snail hemocyanins. The coordination environment of the active-site Co(II) substituted for Cu in the octopus hemocyanin is the same as that of the corresponding active site of the squid hemocyanin.     

Topological mapping of 13 epitopes on a subunit of Androctonus australis hemocyanin.

A topological localization of epitopes on the surface of the Aa6 subunit of Androctonus australis hemocyanin has been carried out. First, immunocomplex strings composed of native hemocyanin and monoclonal antibodies were examined in the electron microscope and submitted to an image processing by correspondence analysis. The average images were then compared to a three-dimensional model of the 24-mer suggesting that 11 of the 13 epitopes are located in three zones of the subunit surface. Second, the overlaps between the epitopes were then studied by polyacrylamide gel electrophoresis, competitive binding inhibition, and immunoelectron microscopy. Four groups of epitopes were identified. One group was capable of binding exclusively to the free subunit. The other three groups were identical to those found in immunoelectron microscopy. The data are consistent with the existence of a small number of immunodominant regions on the surface of the Aa6 subunit.     

Characterization and epitope mapping of monoclonal antibodies directed against the beta' subunit of the Escherichia coli RNA polymerase.

Monoclonal antibodies (mAbs) raised against the beta' subunit of the Escherichia coli RNA polymerase were used to probe the structure and function of this subunit. Of the five anti-beta' monoclonal antibodies studied, only mAb 311G2 is a strong inhibitor of RNA polymerase activity. This antibody binds to an epitope which is exposed in both the assembled holoenzyme and isolated beta' subunit. In contrast, the null antibodies bind to the free beta' subunit but very weakly to native RNA polymerase. It would appear that the beta' domain in which their epitopes reside is either conformationally altered or blocked due to interaction with other subunits in native RNA polymerase. In order to locate the positions of the epitopes for these five monoclonal antibodies, a series of overlapping deletion mutants have been constructed by partial restriction and religation of the beta' gene present in pT7 beta' (Zalenskaya, K., Lee, J., Gujuluva, C. N., Shin, Y. K., Slutsky, M., nd Goldfarb, A. (1990) Gene 89, 7-12). The presence of the epitopes for each of the anti-beta' monoclonal antibodies was assessed by Western blotting. The results indicate that the epitopes for mAb 340F11, mAb 370F3, mAb 371D6, and mAb 372B2 are located between amino acids 817-876. This region may be important in enzyme assembly or subunit-subunit interaction. The epitope for the inhibitory antibody, mAb 311G2, is located between amino acids 1047-1093. This region may be involved in the catalytic function of RNA polymerase.     

A monoclonal antiidiotypic antibody with rheumatoid factor activity defines a cross-reactive idiotope on murine anticollagen antibodies.

A syngeneic antiidiotypic mAb, C1C3, was characterized as to its binding to monoclonal anti-collagen II (-CII) auto-antibodies reactive with different epitopes of the native CII molecule. Both by direct binding and by inhibition ELISA studies, the anti-idiotypic antibody was shown to react with a cross-reactive idiotope present on Fab fragments of most, but not all, tested anti-CII mAb, whereas the binding to Fab fragments from normal mouse IgG was low. As previously described, C1C3 bound to isolated Fc fragments from normal mouse IgG. The binding to intact normal mouse IgG was, however, weak, and only isolated Fc-gamma fragments, not intact IgG, competed efficiently with Fab fragments of anti-CII antibodies for binding to the antiidiotypic antibody. The antibody was shown to self-associate, i.e., to behave similarly to certain IgG rheumatoid factors obtained from patients with rheumatoid arthritis. The presented data indicate that the described anti-anti-CII mAb may be representative of antibodies involved in the physiologic regulation of autoimmunity to CII and, consequently, may be used as a tool for further studies on idiotypic regulation in CII-induced arthritis.     

Cultured human fibroblasts contain a large pool of precursor beta 1-integrin but lack an intracellular pool of mature subunit

Previous work has shown the presence of an important intracellular pool of beta 1-integrin subunit in human skin fibroblasts as detected with monoclonal antibody DH12 [De Strooper, B., Van der Schueren, B., Jaspers, M., Saison, M., Spaepen, M., Van Leuven, F., Van den Berghe, H. & Cassiman, J. J. (1989) J. Histochem. Cytochem. 37,299-307]. To analyze this more quantitatively, a radioimmunoassay with radioiodinated monoclonal antibody was developed. The total amount of specific binding sites for monoclonal antibody DH12 on skin fibroblasts was between 0.8-1.5 x 10(6)/cell. After permeabilizing the cells with digitonin, a threefold increase in specific binding was observed, which suggested that about 60% of the total amount of beta 1-subunit was localized intracellularly. From pulse/chase experiments, it was deduced that an important pool of precursor subunit, as defined by its sensitivity to endoglycosidase treatment, existed in fibroblasts. Since in steady-state-labeling conditions, at least three to four times more precursor than mature subunit was immunoprecipitated with monoclonal antibody DH12, we suggested that the intracellular pool of beta 1-integrin subunit is mainly precursor pool. This precursor pool contains a degradation compartment and a maturation compartment. Other investigators have found evidence for a recirculating pool of mature integrin in Chinese hamster ovary cells. Therefore, the presence of a recirculating pool of integrin in human fibroblasts was also considered. The data obtained with mAb DH12 showed that less than 10% of the surface pool of integrin was internalized by endocytosis. Since, however, cross linking of beta 1-integrins with polyclonal antibodies leads to rapid endocytosis of most of the integrin, it remains possible that the quantitatively small effect was actually an artefact induced by the divalent mAb. We conclude that the intracellular pool of beta 1-integrins observed in our previous studies consists of precursor and that in skin fibroblasts no mature beta 1-integrin is available intracellularly for rapid quantitative modulations at the cell surface.     

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.     

Hemocyte components in crustaceans convert hemocyanin into a phenoloxidase-like enzyme

The functional conversion of hemocyanin (Hc), an oxygen transporter, into an enzyme was investigated in crustaceans. Hc is converted into a phenoloxidase-like enzyme by hemocyte components, which is triggered by beta-1,3-glucan. This activation is severely hampered with leupeptin and E-64 treatment, indicating that the serine/cysteine proteases in the hemocytes are involved in the activation. In a SDS-PAGE-analysis, no change was observed between normal and activated Hc under reduced conditions. However, under non-reduced condition of normal Hc, several minor bands were observed at oligomeric position of Hc subunit, which disappeared upon activation. These results indicate that a split of the reductive bond, such as the disulfide bond between subunits, is essential for Hc activation. This is the first report to show the enzymatic conversion of Hc and the presence of the covalent bond in the Hc subunit of crustaceans.     

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)     

Cell-free synthesis of hemocyanin from the scorpion Androctonus australis. Characterization of the translation products by monospecific antisera

Translation of Androctonus australis poly(A)-RNA in vitro led to a number of polypeptides products (8-10) of 70-73 kDa analyzed by two-dimensional gel electrophoresis and identified by immunoprecipitation with an anti-(dissociated hemocyanin) antiserum. The translated hemocyanin polypeptides have the same physico-chemical characteristics as authentic hemocyanin subunits. Subunits Aa 2 and Aa 4 have been identified with monospecific antisera characterized (a) by their capability of reacting with their homologous subunit and (b) by their inability of binding to cross-reacting subunits. Each polypeptide chain is coded by a different messenger without significant post-translational events. Hemocyanin could be detected among the translation products of the poly(A)-RNA isolated from the cuticle under the carapace.     

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

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

Immunoglobulin binding by the regular surface array of Aeromonas salmonicida

The cell surface of Aeromonas salmonicida is covered by a regular surface array composed of a single species of protein, the A-protein (Phipps, B. M., Trust, T. J., Ishiguro, E. E., and Kay, W. W. (1983) Biochemistry 22, 2934-2939). The array, known as the A-layer, is the key virulence factor for this organism. Cells containing the A-layer specifically bound rabbit IgG and human IgM with high affinity (KD = 1.0 X 10(-6) M and 3.3 X 10(-6) M, respectively), but neither isogenic A-protein-deficient strains nor an Aeromonas hydrophila strain also possessing a regular surface array had binding activity. Selective removal of A-protein at pH 2.2 inactivated IgG binding. Structurally intact IgG was requisite for binding since both Fab and Fc fragments were inactive. Aeromonas A-protein did not share the same IgG binding sites as Staphylococcus aureus protein A. Purified A-protein bound IgG only weakly, but reassembled A-layer regained binding activity. Protein modification and perturbation of the A-layer indicated that no single amino acid residue was critical for binding, and that the binding site consisted of a native arrangement of at least four A-protein monomers in the layer.     

Diplopod hemocyanin sequence and the phylogenetic position of the Myriapoda

Hemocyanins are copper-containing respiratory proteins of the Arthropoda that have so far been thoroughly investigated only in the Chelicerata and the Crustacea but have remained unstudied until now in the Myriapoda. Here we report the first sequence of a myriapod hemocyanin. The hemocyanin of Spirostreptus sp. (Diplopoda: Spirostreptidae) is composed of two distinct subunits that are arranged in a 6 x 6 native molecule. The cloned hemocyanin subunit cDNA codes of for a polypeptide of 653 amino acids (75.5 kDa) that includes a signal peptide of 18 amino acids. The sequence closely resembles that of the chelicerate hemocyanins. Molecular phylogenetic analyses reject with high statistical confidence the integrity of the Tracheata (i.e., Myriapoda + Insecta) but give conflicting results on the position of the myriapod hemocyanin. While distance matrix and maximum-likelihood methods support a basal position of the Spirostreptus hemocyanin with respect to the other hemocyanins, parsimony analysis suggests a sister group relationship with the chelicerate hemocyanins. The latter topology is also supported by a unique shared deletion of an alpha-helix. A common ancestry of Myriapoda and Chelicerata should be seriously considered.