Complete subunit sequences, structure and evolution of the 6 · 6-mer hemocyanin from the common house centipede, Scutigera coleoptrata Kristina Kusche*, Anne Hembach, Silke Hagner-Holler, Wolfgang Gebauer and Thorsten Burmester Institute of Zoology, Molecular Animal Physiology, University of Mainz, Germany Hemocyanins are large oligomeric copper-containing pro- teins that serve for the transport of oxygen in many arth- ropod species. While studied in detail in the Chelicerata and Crustacea, hemocyanins had long been considered unnec- essary in the Myriapoda. Here we report the complete molecular structure of the hemocyanin from the common house centipede Scutigera coleoptrata (Myriapoda: Chilo- poda), as deduced from 2D-gel electrophoresis, MALDI- TOF mass spectrometry, protein and cDNA sequencing, and homology modeling. This is the first myriapod hemo- cyanin to be fully sequenced, and allows the investigation of hemocyanin structure–function relationship and evolution. S. coleoptrata hemocyanin is a 6 · 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 · 6-mer hemocyanin. Phylogenetic analyses show that myriapod hemocyanins are monophyletic, but at least three distinct subunit types evolved before the separation of the Chilo- poda 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. Keywords: hemocyanin; subunit diversity; structure; Arthro- poda; evolution. Oxygen transport in the body fluid of many arthropod and molluscan species is mediated by large copper-containing proteins that are referred to as hemocyanins [1–3]. Mollu- scan hemocyanins, however, are not significantly related to the arthropod proteins and are most likely of divergent evolutionary origin [3–5]. Arthropod hemocyanins typically form hexamers or oligo-hexamers (up to 8 · 6) of subunits with about 620–660 amino acids [1,2,6]. Oxygen binding is mediated by a pair of copper atoms that are coordinated by six histidine residues per monomer. In the past 30 years, hemocyanins have been studied thoroughly in the Chelicerata and Crustacea in terms of function, structure, sequence and evolution [1,2,6]. By contrast, little is known about hemocyanins in Onycho- phora, Myriapoda and Hexapoda. Oxygen supply in these taxa is mediated via trachea [7]. Therefore, the presence of respiratory proteins had long been considered unnecessary, with the exception of some insect species that live under hypoxic conditions and possess extracellular hemoglobins [8, but also see 9]. A putative hemocyanin, however, has been identified in the embryonic hemolymph of the grasshopper Schistocerca americana [10], although this respiratory protein is absent in adult animals and hemocy- anins were lost later in hexapod evolution [6]. Hemocyanins also occur in the Onychophora, suggesting an ancient evolutionary origin of this type of respiratory protein [11]. Mangum et al. [12] were the first who unambiguously demonstrated the occurrence of hemocyanins in the Myr- iapoda. The centipede Scutigera coleoptrata (Chilopoda) possesses a 36-mer (6 · 6) hemocyanin that closely resem- bles other arthropod hemocyanins [12,13]. This protein is composed of four electrophoretically and immunologically distinct subunits (termed a : b : c : d) in the range of 74– 80 kDa, which occur in a ratio of approximately a/b/c/d ¼ 2 : 2 : 1 : 1 [14]. Structurally similar 6 · 6-mer hemocya- nins also occur in at least in one family of the Diplopoda, i.e. the Spirostreptidae, suggesting that despite the well-devel- oped tracheal system hemocyanins are widespread among the Myriapoda [15,16]. Myriapod, crustacean, and chelicerate hemocyanins strikingly differ in subunit composition and quaternary structure [1]. In previous analyses, the complete subunit sequences of one crustacean [17–19] and two chelicerate hemocyanins [20,21] have been determined. Based on sequence comparison and immunological studies, a remark- ably different pattern of hemocyanin subunit evolution has been observed in these two arthropod subphyla [4,6,22], but little was known about the Myriapoda. Here we report the complete cDNA-cloning and biochemical analyses of the Correspondence to T. Burmester, Institute of Zoology, University of Mainz, D-55099 Mainz, Germany. Fax: + 49 6131 3924652, Tel.: + 49 6131 3924477, E-mail: burmeste@uni-mainz.de Abbreviation: Hc, hemocyanin. *Present address: Institute of Animal Physiology, University of Mu ¨ nster, Germany. (Received 21 March 2003, revised 7 May 2003, accepted 12 May 2003) Eur. J. Biochem. 270, 2860–2868 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03664.x hemocyanin subunits from S. coleoptrata, being the first myriapod hemocyanin of which the complete molecular structure is known. This allows us to compare structure, and intra- and intermolecular evolution of hemocyanins from Myriapoda, Crustacea and Chelicerata. Materials and methods Protein biochemistry Scutigera coleoptrata (Myriapoda, Chilopoda, Scutigero- morpha) were captured on Lesbos, Greece. The hemolymph was withdrawn from the dorsal intersegmental regions withasyringeandstoredfrozenat)20 °C until use. The hemocyanin was purified by ultracentrifugation in a Beck- man airfuge for 16 h at 120 000 g.SDS/PAGEand Western blotting were carried out using standard methods as previously described [14,15]. Two-dimensional gel elec- trophoresis with pH 3.5–10 ampholines was performed according to O’Farrell et al. [23,24]. For antibody prepar- ation, the hemocyanin subunits were separated by SDS/ PAGE and stained by Coomassie brilliant blue. The two hemocyanin bands were cut out and dispersed. About 100 lg of hemocyanin was used for the immunization of rabbits. For determination of the N-terminal ends, samples were separated by SDS/PAGE and transferred to a poly(vinylidene difluoride) membrane by electroblotting. The hemocyanin bands were excised and submitted to Edman degradation. MALDI-TOF mass spectrometry experiments were performed by Proteosys (Mainz, Germany) on hemocyanin subunits that had been separated by 2D PAGE, stained with Coomassie brilliant blue and digested with trypsin. The MALDI-TOF data were evalu- ated using the program PEPTIDE - MASS at the ExPASy web server (http://www.expasy.org). Cloning of S. coleoptrata hemocyanin cDNA The total RNA was extracted from a single specimen and poly(A) + RNA was purified from total RNA by the aid of the PolyATract kit (Promega). About 5 lg poly(A) + RNA were used to construct a directionally cloned cDNA expression library applying the Lambda ZAP-cDNA syn- thesis kit (Stratagene). The library was amplified using the material provided by Stratagene and screened with the anti- (S. coleoptrata hemocyanin) Igs. Positive phage clones were converted into pBK-CMV plasmid vectors and sequenced by a commercial sequencing service (Genterprise, Mainz, Germany). The missing 5¢ ends of two clones [hemocyanin (Hc)B and HcD; see below] were obtained from the library by a PCR approach using two nested clone-specific oligonucleotides primers and the T3 vector primer. The sequences were obtained after the cloning of the PCR products into the pCR4-TOPO TA vector (Invitrogen). Protein structure modeling Homology models of the S. coleoptrata hemocyanin sub- units were built applying the online facility SWISSMODEL (GlaxoWellcome) [25] at the following address: http:// www.expasy.ch/spdbv/using the known arthropod hemo- cyanin crystal structures [26,27] as templates. Sequence data analyses and phylogenetic studies The tools provided with Genetics Computer Group (GCG) Software Package 10 and by the ExPASy Molecular Biology Server of the Swiss Institute of Bioinformatics (http://www.expasy.ch) were used for the analyses of DNA and amino acid sequences. Signal peptides were predicted using the online version of SIGNALP V1.1 [28]. The amino acid sequences of the S. coleoptrata hemocyanins were added to a previously published alignment of the arthropod hemocyanin superfamily [4,11] by the aid of GENEDOC 2.6 [29]. Only selected hemocyanin and phenoloxidase sequences were used for phylogenetic inference. The signal peptides were eliminated from the final data set. Distances between pairs of sequences were calculated using the PAM matrix [30] implemented in the PHYLIP 3.6a2 package [31]. Tree constructions were performed by the neighbor-joining method. The reliability of the trees was tested by the bootstrap procedure with 100 replications [32]. Replacement rates were estimated from the PAM distances assuming that Chilopoda and Diplopoda diverged 450 million years ago [33,34]. Alternative models of sequence evolution were tested using T REE -P UZZLE [35], and the WAG model [36] was chosen on the basis of the highest likelihood values. Bayesian phylogenetic analyses were performed with MRBAYES 3.0 [37]. The WAG model with gamma distribu- tion of rates was applied. Metropolis-coupled Markov chain Monte Carlo sampling was performed with four chains that were run for 200 000 generations. Prior probabilities for all trees were equal, starting trees were random, tree sampling was performed every 10 generations. Posterior probability densities were estimated on 5000 trees (burnin ¼ 15 000). Results Purification and analyses of S. coleoptrata hemocyanin The 6 · 6-mer hemocyanin of S. coleoptrata (55S) [12] was purified from total hemolymph by ultracentrifugation. After separation by SDS/PAGE, the hemocyanin fraction shows two distinct polypeptide bands with apparent molecular masses in the range of 75 kDa, but no other contaminating protein detectable by Coomassie staining, suggesting that the preparation results in >95% pure protein (Fig. 1). Antibodies raised against the hemocyanin fraction show staining of the two 75 kDa bands in Western blot, but do not recognize any other proteins of the hemolymph. Although we expected that each of these bands contains two distinct polypeptides [14], we determined their N-terminal sequences. In fact both are heterogeneous, but in each case a predominant sequence was obtained: The first 18 amino acids of the major sequence in the upper band are DQEPAVPADTKDKLEKIL, the lower band gave rise to the sequence DKEPXATD(QKI)EAKQKXMLE. By 2D gel electrophoresis a total of 10 distinct protein spots were identified in the hemocyanin sample. The appar- ent molecular masses are about 75–80 kDa, the isoelectric points (pI) range from 5 to 6.5 (Fig. 2). MALDI-TOF analyses of tryptic peptides show that the spots represent four distinct subunit types, as deduced from the pattern of identical peptide masses. The subunits were named HcA to Ó FEBS 2003 Centipede hemocyanin structure (Eur. J. Biochem. 270) 2861 HcD, in agreement with the previous data on Scutigera hemocyanin subunit masses and charges [14]. HcA and HcD were each found to form two spots that slightly differ in their sizes, whereas each HcB and HcC form three spots that probably represent charge variants. cDNA-sequences of S. coleoptrata hemocyanin subunits A cDNA expression library was constructed from about 5 lg poly(A) + RNA extracted from an adult S. coleoptrata specimen. The library was screened with the antibodies raised against the S. coleoptrata hemocyanin. In a total of about 20 clones, five distinct hemocyanin subunit cDNAs were identified and fully sequenced (Fig. 3). The cDNA sequences were assigned to distinct subunits (HcA to HcD; Fig. 2) by the aid of the MALDI-TOF data. About 30 peptides that cover a total of at least 40% of each subunit could be unambiguously identified for each subunit. How- ever, the fifth cDNA sequence could not be allocated to any of the spots found in the native 6 · 6-mer hemocyanin, and thus has been termed HcX. As deduced from the N-terminal sequences obtained by conventional protein sequencing and from comparison with other arthropod hemocyanins, the cDNAs cover the complete coding regions for the four hemocyanin subunits and HcX, plus 6–45 bp of the respective 5¢ untranslated regions and the entire 3¢ untranslated regions. The standard polyadenylation signals (AATAAA) and the poly(A)-tails of different lengths are present in each clone. The open reading frames of the five sequences translate into distinct polypeptides of 656–685 amino acids (Table 1; Fig. 3). Signal peptides required for the transmembrane excretion into the hemolymph were found in all sequences and cover 18–20 amino acids, as predicted by the SIGNALP computer program [28]. The estimated molecular masses of the native subunits (without signal peptides; 74.4–77.7 kDa) and the theoretical isoelectric points (pI 5.44–6.18) agree well with those observed in SDS/PAGE and in 2D PAGE (Figs 1 and 2; Table 1). The N-terminal amino acid sequences (see above) allow the assignment of the HcA clone to the major sequence of the upper hemocyanin band in the SDS/PAGE gel (Fig. 1). Two nonmatching amino acids at positions 3 (Glu instead of Cys) and 5 (Ala instead of Pro) may be explained by the presence of an additional subunit in the sample that most likely corresponds to HcC, as also inferred from the minor sequence in the sample (not shown). The lower protein band was assigned to HcB, whereas minor amino acid peaks most likely derive from HcD. Again, no indication for the presence of a HcX-like subunit was found. Sequence comparison The four S. coleoptrata hemocyanin subunits (HcA-D) share 49.5–55.5% of the amino acids (Table 2) with 240 amino acids ( 35%) being strictly conserved (Figs 3 and 4). The HcX sequence is highly diverged, as deduced from the lower identity scores (42.1–48.1% identity with HcA to HcD) and several short sequence insertions (Table 2; Fig. 3). It is not included in the calculations below. Arthropod hemocyanins are divided into three structural domains [26,27]. While 119 strictly conserved residues ( 52%) have been found in domain 2 of subunits HcA to D, there is less sequence conservation in domains 1 (51 conserved amino acids;  30%) and 3 (70 conserved residues;  27%). As expected, the Scutigera hemocyanin subunits (HcA-D) show the highest degree of sequence similarity to the previously determined hemocyanin from the diplopod Spirostreptus (44.9–51.0% identity). Lower scores were observed with the chelicerate hemocyanins (38–46%), the onychophoran hemocyanin ( 34–37%), the phenoloxidases of Crustacea and insects ( 31–38%), the crustacean hemocyanins ( 27–35%) and the insect hemo- cyanin ( 34–38%). Secondary and tertiary structure of Scutigera hemocyanin subunits The S. coleoptrata hemocyanin subunits contain the six copper-coordinating histidines necessary for copper binding (Fig. 3), which are strictly conserved in all arthropod hemocyanins [4,6,38]. No long insertions or deletions were observed upon comparison with the other hemocyanins. Fig. 2. Identification of S. coleoptrata hemocyanin subunits. About 20 lg of purified hemocyanin was separated by two-dimensional PAGE. The anode (+) and cathode (–) are indicated, the molecular mass marker is on the right side. The spots were submitted to MALDI- TOF analysis and assigned to distinct subunits. Fig. 1. SDS/PAGE and immunoblotting of S. coleoptrata hemocyanin. About 10 lg of total hemolymph protein (HL) and 3 lgofpurified hemocyanin (Hc) were separated on SDS/PAGE and stained with Coomassie brilliant blue R-250. A Western blot analysis of total hemolymph using anti-(S. coleoptrata hemocyanin) Ig is shown on the rightside(Western).Themolecularmassmarkerproteinsareonthe left side (kDa). 2862 K. Kusche et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Fig. 3. Alignment of the myriapod hemocyanins. The conserved amino acids are shaded, the signal peptides are underlined and the potential N-glycosylation sites are in italics/bold face. Above the alignment: Copper-binding histidines of the CuA and CuB sites (*); putative disulfide bridges (c). Below the alignment: Secondary structure elements derived from a modeling approach of ScoHcA. The nomenclature follows the standard convention for hemocyanin structure [26,27]; a, a helix; b, b sheet. Note that a helix 1.2 is missing. The abbreviations used are: ScoHcA to D, S. coleoptrata hemocyanin subunits A to D; ScoHcX, S. coleoptrata hemocyanin X; SpiHc1, Spirostreptus spec. hemocyanin subunit 1. Table 1. Properties of myriapod hemocyanin subunits. Accession numbers are the EMBL/GenBank DNA data accession number. CDNA lengths refer to the sequence without the poly(A) tail; protein length is including the signal peptide but molecular mass and pI are calculated without the signal peptide. Subunit Accession number cDNA (bp) Protein (amino acids) Molecular mass (kDa) pI Replacement rates (per site per year) ScoHcA AJ344359 2258 656 74.44 5.44 0.66 · 10 )9 ScoHcB AJ512793 2283 659 73.93 5.57 0.92 · 10 )9 ScoHcC AJ431379 2236 673 75.91 5.68 1.07 · 10 )9 ScoHcD AJ344360 2271 668 74.75 6.18 0.89 · 10 )9 ScoHcX AJ431378 2209 685 77.74 5.79 1.89 · 10 )9 SpiHc1 AJ297738 2082 653 73.74 6.16 1.13 · 10 )9 Ó FEBS 2003 Centipede hemocyanin structure (Eur. J. Biochem. 270) 2863 Similar to the Onychophora and Chelicerata, a-helix 1.2 is missing in the myriapod hemocyanins. Tentative 3D structures of the subunits were constructed by a homology modeling approach using the hemocyanins of Limulus polyphemus and Panulirus interruptus as templates [26,27]. The modeling process was straightforward, with the excep- tion of the first  20–30 amino acids of subunits HcC and D, which could not be recovered. The positions of the amino acids conserved among all subunits were super- imposed on the model of subunit HcA (Fig. 4). Strong conservation was found around the copper-binding sites, most notably in the a-helices 2.1 and 2.2 (CuA site) and 2.5 and 2.6 (CuB site). The subunits all contain the cysteines forming a disulfide bridge that stabilize domain 3 [27] (Figs3and4).One(HcA,HcB,HcC)ortwo(HcDand HcX) potential N-glycosylation sites (NXT/S) are present, which are, however, not conserved in any other arthropod hemocyanin subunit. Nevertheless, the glycosylation site at a-sheet 2E (Fig. 3) is located at the surface of the putative hemocyanin hexamer, as deduced from the comparison with the L. polyphemus and P. interruptus hemocyanin structures. Hemocyanin molecular phylogeny Phylogenetic trees were calculated using an amino acid alignment of the six myriapod hemocyanins, 24 selected hemocyanins from other arthropod species and eight prophenoloxidase sequences [4]; the prophenoloxidases are considered as an outgroup [4,39]. Various tree-building methods were applied; the results of a neighbor-joining and a Bayesian approach are presented in Fig. 5. The mono- phyly of the myriapod hemocyanins was recovered by all types of analyses with 100% support. There is solid bootstrap support (82%) and Bayesian posterior probability (0.87) for an association of the myriapod hemocyanins with the crustacean and insect hemocyanins. All analyses support a pancrustacean taxon of the insect and crustacean proteins that excludes the myriapod hemocyanins. Within the clade of myriapod hemocyanins, there is strong support that Scutigera HcA is associated with the Spirostreptus hemocy- anin 1. This branch joins a clade formed by HcB and HcX, whereas the branch leading to HcC and HcD splits earlier. Replacement rates of myriapod hemocyanins were diffi- cult to calculate because the time of divergence of the Chilopoda and Diplopoda is essentially unknown. Both chilopod and diplopod fossils that already belong to distinct extant orders have been identified in lower Devonian and upper Silurian strata [33,34]. Thus these taxa must have diverged more than 410–420 million years ago, although this date may be underestimated [40]. For further calcula- tions we assumed a time of divergence of 450 million years ago. Then the estimated replacement rates of the different myriapod hemocyanin subunits show a large variance, Table 2. Comparison of myriapod hemocyanin subunits. Amino acid identities (%) are given. ScoHcB ScoHcC ScoHcD ScoHcX SpiHc1 ScoHcA 55.5 53.7 53.1 44.6 51.0 ScoHcB 53.0 49.5 48.1 46.0 ScoHcC 54.4 42.1 44.9 ScoHcD 43.3 45.3 ScoHcX 38.9 Fig. 4. Stereo view of a model of S. coleoptrata hemocyanin subunit A. The structure was deduced by comparative modeling. Positions strictly conserved in all S. coleoptrata subunits are displayed in red, the copper atoms are blue, the coordinating histidines green and the disulfide bridge- forming cysteines yellow. Fig. 5. Phylogenetic analysis of the arthropod hemocyanins. A simpli- fied phylogenetic tree was calculated by the neighbor-joining method of the amino acids based on PAM distances [30]. The numbers at the branches represent the confidence limits computed by the bootstrap procedure (left number) [32] or the Bayesian posterior probabilities (right number) [37] based on the WAG model [36]. 2864 K. Kusche et al. (Eur. J. Biochem. 270) Ó FEBS 2003 ranging from 0.66 · 10 )9 (HcA) to 1.89 · 10 )9 (HcX) substitutions per site per year (Table 1). Discussion In contrast to previous assumptions, hemocyanins are present in the hemolymph of various myriapod species. Hemocyanins have been identified in the Scutigeramorpha (Chilopoda) Scutigera longicornis [41] and S. coleoptrata [12], as well as in various Spirostreptidae (Diplopoda) [15,16], suggesting a universal occurrence of these respirat- ory proteins among the Myriapoda. While hemocyanins have been studied in great detail in the Chelicerata and Crustacea [1,2,6,22], our knowledge on function, structure and evolution of myriapod hemocyanins is sparse. S. coleoptrata hemocyanin is a 36-mer with four subunit types The hemocyanin of S. coleoptrata has a unique structure of 6 · 6 subunits that is unknown in other arthropod subphyla [1,12,13]. Gebauer and Markl [14] identified four distinct subunit types by means of immunological studies, native and denaturating PAGE. Our MALDI-TOF experi- ments and cDNA sequencing confirm their results. By comparison of the patterns of the SDS, native and 2D electrophoresis, we assign HcA and HcB to the a and b polypeptides (that could not be separated in [14]), respect- ively, HcC to the c subunit, and HcD to the d subunit. We observed charge variants for HcA, HcB and HcC, as well as slight variations in the masses of HcA and HcD (Fig. 2). These differences are most likely to be explained by post- translational modifications such as differential phosphory- lation or glycosylation. Gebauer and Markl [14] calculated that the four subunit types occur in a stoichiometric ratio of 2 : 2 : 1 : 1. This is also in basic agreement with the intensities of the spots in the 2D gel, indicating that the native 36-mer is composed of 12 HcA, 12 HcB, six HcC and six HcD polypeptides. Thus the basic building block of the hemocyanin, the hexamer, most likely contains two copies of each HcA and HcB, and single copies of HcC and HcD. Three structural domains can be recognized in an arthropod hemocyanin subunit [26,27,38]. As already observed by the comparison of other hemocyanin sequences [4], most conservation among the four myriapod subunits occurs in the second domain (52% strictly conserved residues), which includes the two copper-sites required for oxygen-binding [6,20,21,38]. The lower degree of conserva- tion in domains 1 and 3 (30% and 27% conserved amino acids) may be explained by less selectional pressure imposed on these sequences, but also by positive selection that have formed distinct protein surfaces required for subunit assembly and interaction [42]. An additional subunit not included in the native hemocyanin The presence of the putative hemocyanin subunit (HcX) in the cDNA library that could not be discovered in the native 6 · 6-mer hemocyanin is surprising. This additional hemo- cyanin is highly diverged, with a substitution rate about two to three times higher than in the other S. coleoptrata hemocyanin subunits (Table 1; Fig. 5). However, all key determinants of arthropod hemocyanins, such as the six copper-coordinating histidines, are strictly conserved. Thus it is unlikely that this protein acts as hemocyanin derived, copper-less storage protein, similar to the crustacean pseudo-hemocyanins or cryptocyanins [43,44], or the insect hexamerins [45]. Further studies are required to elucidate the significance of this sequence. Hemocyanin phylogeny and its implication for arthropod evolution Arthropod hemocyanins belong to a protein superfamily that also includes phenoloxidases, crustacean pseudo- hemocyanins, insect hexamerins and hexamerin receptors [4,6,38,46]. Sequences from the arthropod hemocyanin superfamily have been successfully used to infer both protein and species evolution [4,11,16,47]. Phylogenetic analyses show that the myriapod hemocyanin sequences form a robust common clade. There is no evidence for a paraphyly of the Myriapoda in respect of the insects, as suggested by some morphological studies [48]. Moreover, the myriapod and insect hemocyanins do not form a common clade, as it may be assumed by the ÔTracheataÕ hypothesis [7,48,49]. As proposed by the ÔPancrustaceaÕ concept [50], there is a well-supported common branch of the crustacean and insect sequences. Such topology is also supported by a number of molecular studies using other markers [40,51–54]. However, conflicting molecular evi- dence on the exact position of the Myriapoda exists, either supporting a common branch with the Pancrustacea [52,54] or with the Chelicerata [40,53]. In contrast to previous calculations using only the single Spirostreptus hemocyanin [16], the inclusion of five additional myriapod sequences resulted in a solid support of a Myriapoda + Pancrustacea clade (Fig. 5). This renders the Mandibulata monophyletic, with the chelicerates being an early offshoot of the euarthropod clade. Thus our results are in agreement with studies using nuclear markers [41,44], although the support values in the present are significantly higher. This observa- tion supports the notion of the usefulness of hemocyanin sequences for the resolution of the arthropod phylogenetic tree [4,6]. Myriapod hemocyanin subunit evolution and diversity One of the most striking features of crustacean and chelicerate hemocyanins is their enormous diversity of subunit sequences [4] and subunit assembly [1,22]. Hemo- cyanin subunit composition and evolution have been studied in detail in the Chelicerata and decapod Crustacea by immunological means and by sequence comparison [6,22]. Most chelicerates have highly conserved 4 · 6-mer or 8 · 6-mer hemocyanins with seven distinct subunit types that separated 550–420 million years ago. Crustacean hemocyanins typically are 6-mers or 2 · 6-mer proteins, whereas higher aggregation states have rarely been observed [1,22]. Three distinct subunit types have been identified in the decapod Crustacea that diverged only some 220 million years ago [6,55], and assemble to quaternary structures that may even differ within species [22]. Ó FEBS 2003 Centipede hemocyanin structure (Eur. J. Biochem. 270) 2865 Although the clades leading to the Diplopoda and Chilopoda diverged at least 420 million years ago [28,29], similar 6 · 6 hemocyanins are present in both the Scutiger- amorpha (Chilopoda) and the Spirostreptidae (Diplopoda). This observation suggests that such quaternary structure is an ancient feature of the myriapod hemocyanins. This also applies, at least in part, to myriapod hemocyanin subunit sequence diversity. There is consistent support that the hemocyanin subunit 1 of Spirostreptus and S. coleoptrata HcA are orthologous and have a more recent common ancestor than the other four S. coleoptrata hemocyanin (Fig. 5). The topology demonstrates that the diversification of the hemocyanin subunits commenced before the Chilo- poda and Diplopoda split more than 420 million years ago and supports the notion of a universal occurrence of hemocyanins in the Myriapoda [15,16]. At least three hemocyanin subunits were present at the time of divergence of the Chilopoda and Diplopoda (HcA, HcB, and HcC/D). According to the N-terminal sequences [16], the second Spirostreptus hemocyanin subunit may be orthologous to Scutigera HcB, while there is no indication for the presence of HcC or HcD-like subunits in the Diplopoda [15,16]. Given the structural similarities of Scutigera and Spirostrep- tus hemocyanins, it may be assumed that HcA and HcB form the core-subunits that are necessary to build a 6 · 6- mer hemocyanin structure. The variability of estimated amino acid replacement rates among the myriapod hemocyanin subunits is unusual among the arthropod hemocyanins. Chelicerate hemo- cyanins evolved with rather constant rates of about 0.5–0.6 · 10 )9 substitutions per site per year [4,6,20], whereas crustacean rates are a little more variable and range from 1.2 to 1.5 · 10 )9 [4,6,45,55]. By contrast, the evolution rates among the myriapod hemocyanins differ by a factor of more than two, ranging from about 0.7–1.9 · 10 )9 (Table 1). It must be considered, however, that these values may be in fact overestimated, because we assumed – based on fossil data [33,34] – that Diplopoda and Chilopoda diverged 450 million years ago, whereas mole- cular estimates hint to a more ancient date [40]. Moreover, the extraordinarily high evolution rate of HcX (1.9 · 10 )9 substitutions per site per year) may be in fact explained by its, as yet unknown, divergent function. Ignoring HcX, the replacement rates of the other myriapod hemocyanins are in the range of 0.66–1.13 · 10 )9 , with HcA being the most conservative sequence. The reasons for this still unusual large variability in amino acid replacement rates are essentially unknown, but it may be speculated that different structural or functional constraints have been imposed on the subunits during evolution. Distinct hemocyanin structure and function in Chilopoda and Diplopoda Both the chilopod and diplopod hemocyanins display virtually identical quaternary structures, i.e. 6 · 6 subunits in similar arrangements [13–15], however, the oxygen- binding characteristics differ strikingly. S. coleoptrata hemo- cyanin displays a low oxygen affinity of 55 Torr and allosteric behavior with very high cooperativity (Hill coefficient: h ¼ 8.9) [12]. Such features are typical for an efficient oxygen carrier with large functional plasticity [56,57]. By contrast, the Spirostreptus hemocyanin shows a higher oxygen affinity (4.7 Torr) but low cooperativity (h ¼ 1.3) [15], as typical for an efficient oxygen storage protein. It is known from various Crustacea that an increase in the number of subunits decreases oxygen affinity but increase cooperativity of hemocyanins [1]. Consequently the higher functional plasticity of S. coleoptrata hemocyanin may be related to the presence of two additional subunits (HcC and HcD) in the Scutigera hemocyanin, which cannot be found in Spirostreptus [15,16] and were lost during the evolution of the Spirostreptidae (Fig. 5). The reason for different hemo- cyanin function and thus structure of Scutigera and Spirostreptus may be found in their distinct life styles. In the Spirostreptidae, an oxygen storage protein that may be used for continuous supply of oxygen can be easily related to the moderate hypoxic environment of the subterrestrial habitats in which the animals burrow during daytime. By contrast, the Scutigeramorpha are very fast animals that may require bursts of oxygen in active phases. Both high cooperativity and low affinity guarantee that oxygen is easily released upon demand. Given their occurrence in the Scutigeramorpha and Spirostreptida, it may be assumed that hemocyanins are present in a wide range of myriapod taxa. Future studies may shed light on the occurrence, functional differences, subunit diversity and evolution of hemocyanins in the Myriapoda, which may turn out to be as complex as in the Chelicerata and Crustacea [1,4,6,22]. Acknowledgements We wish thank J. Markl for his generous support, G. Pass for animals, H. Heid for the determination of the N-terminal sequences, C. Hunzinger for the MALDI-TOF analyses, C. Bache and J. Hermanns for their help with the cloning experiments, and J. R. Harris for correcting the language. The nucleotide sequences reported in this paper have been deposited at the GenBank TM /EMBL databases with the accession numbers AJ344359 (HcA), AJ512793 (HcB), AJ431379 (HcC), AJ344360 (HcD), and AJ431378 (HcX). 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Complete subunit sequences, structure and evolution of the 6 · 6- mer hemocyanin from the common house centipede, Scutigera coleoptrata Kristina. sequenced, and allows the investigation of hemocyanin structure function relationship and evolution. S. coleoptrata hemocyanin is a 6 · 6- mer composed of four