Complete Hemocyanin Subunit Sequences of the Hunting SpiderCupiennius salei
2002; Elsevier BV; Volume: 277; Issue: 17 Linguagem: Inglês
10.1074/jbc.m111368200
ISSN1083-351X
AutoresPia Ballweber, Jürgen Markl, Thorsten Burmester,
Tópico(s)Physiological and biochemical adaptations
ResumoHemocyanins are large copper-containing respiratory proteins found in many arthropod species. Scorpions and orthognath spiders possess a highly conserved 4 × 6-mer hemocyanin that consists of at least seven distinct subunit types (termed a to g). However, many “modern” entelegyne spiders such as Cupiennius salei differ from the standard arachnid scheme and have 2 × 6-mer hemocyanins. Here we report the complete primary structure of the 2 × 6-mer hemocyanin of C. salei as deduced from cDNA sequencing, gel electrophoresis, and matrix-assisted laser desorption spectroscopy. Six distinct subunit types (1 through 6) and three additional allelic sequences were identified. Each 1 × 6-mer half-molecule most likely is composed of subunits 1–6, with subunit 1 linking the two hexamers via a disulfide bridge located in a C-terminal extension. TheC. salei hemocyanin subunits all belong to the arachnidg-type, whereas the other six types (a–f) have been lost in evolution. The reconstruction of a complex hemocyanin from a single g-type subunit, which commenced about 190 million years ago and was completed about 90 million years ago, might be explained by physiological and behavioral changes that occurred during the evolution of the entelegyne spiders. Hemocyanins are large copper-containing respiratory proteins found in many arthropod species. Scorpions and orthognath spiders possess a highly conserved 4 × 6-mer hemocyanin that consists of at least seven distinct subunit types (termed a to g). However, many “modern” entelegyne spiders such as Cupiennius salei differ from the standard arachnid scheme and have 2 × 6-mer hemocyanins. Here we report the complete primary structure of the 2 × 6-mer hemocyanin of C. salei as deduced from cDNA sequencing, gel electrophoresis, and matrix-assisted laser desorption spectroscopy. Six distinct subunit types (1 through 6) and three additional allelic sequences were identified. Each 1 × 6-mer half-molecule most likely is composed of subunits 1–6, with subunit 1 linking the two hexamers via a disulfide bridge located in a C-terminal extension. TheC. salei hemocyanin subunits all belong to the arachnidg-type, whereas the other six types (a–f) have been lost in evolution. The reconstruction of a complex hemocyanin from a single g-type subunit, which commenced about 190 million years ago and was completed about 90 million years ago, might be explained by physiological and behavioral changes that occurred during the evolution of the entelegyne spiders. Hemocyanins are large allosteric respiratory proteins that occur freely dissolved in the hemolymph of many arthropod and molluscan species (1.Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar, 2.van Holde K.E. Miller K.I. Adv. Protein Chem. 1995; 47: 1-81Crossref PubMed Google Scholar). Oxygen binding of hemocyanins is mediated by a pair of copper atoms that are coordinated by six histidine residues (2.van Holde K.E. Miller K.I. Adv. Protein Chem. 1995; 47: 1-81Crossref PubMed Google Scholar, 3.Linzen B. Soeter N.M. Riggs A.F. Schneider H.J. Schartau W. Moore M.D. Behrens P.Q. Nakashima H. Takagi T. Nemoto T. Vereijken J.M. Bak H.J. Beintema J.J. Volbeda A. Gaykema W.P.J. Hol W.G.J. Science. 1985; 229: 519-524Crossref PubMed Scopus (217) Google Scholar, 4.Cuff M.E. Miller K.I. van Holde K.E. Hendrickson W.A. J. Mol. Biol. 1998; 278: 855-870Crossref PubMed Scopus (341) Google Scholar). Arthropod and molluscan hemocyanins differ essentially in structure and sequence and are most likely of independent evolutionary origin (1.Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar,5.Burmester T. Mol. Biol. Evol. 2001; 18: 184-195Crossref PubMed Scopus (185) Google Scholar, 6.Lieb B. Altenhein B. Markl J. Vincent A. van Olden E. van Holde K.E. Miller K.I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4546-4551Crossref PubMed Scopus (79) Google Scholar, 7.Burmester T. J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 2002; 172: 95-117Crossref PubMed Scopus (225) Google Scholar, 8.van Holde K.E. Miller K.I. Decker H. J. Biol. Chem. 2001; 276: 15563-15566Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). These proteins have been proven to be an excellent topic of functional, structural, and evolutionary studies (1.Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar, 2.van Holde K.E. Miller K.I. Adv. Protein Chem. 1995; 47: 1-81Crossref PubMed Google Scholar, 7.Burmester T. J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 2002; 172: 95-117Crossref PubMed Scopus (225) Google Scholar, 8.van Holde K.E. Miller K.I. Decker H. J. Biol. Chem. 2001; 276: 15563-15566Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 9.Harris J.R. Markl J. Micron. 1999; 30: 597-623Crossref PubMed Scopus (299) Google Scholar). Arthropod hemocyanins are hexamers (6-mers) composed of distinct although related subunits in the 75-kDa range that may combine to multimers up to 8 × 6 subunits, depending upon the taxon or the physiological conditions (1.Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar, 10.Markl J. Biol. Bull. (Woods Hole). 1986; 171: 90-115Crossref Google Scholar). The sequences of various hemocyanin subunits have been determined from all euarthropod subphyla, including the Chelicerata, Crustacea, Myriapoda, and Hexapoda (7.Burmester T. J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 2002; 172: 95-117Crossref PubMed Scopus (225) Google Scholar). Phylogenetic analyses demonstrate that subunit evolution took place independently within each subphylum (5.Burmester T. Mol. Biol. Evol. 2001; 18: 184-195Crossref PubMed Scopus (185) Google Scholar, 7.Burmester T. J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 2002; 172: 95-117Crossref PubMed Scopus (225) Google Scholar, 10.Markl J. Biol. Bull. (Woods Hole). 1986; 171: 90-115Crossref Google Scholar, 11.Kusche K. Burmester T. Mol. Biol. Evol. 2001; 18: 1566-1573Crossref PubMed Scopus (65) Google Scholar). The estimated rate of amino acid replacement in the chelicerate hemocyanins is about the half of that found in the crustaceans or myriapods (7.Burmester T. J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 2002; 172: 95-117Crossref PubMed Scopus (225) Google Scholar), which is most likely linked to the conserved structure of these proteins (1.Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar, 12.Voit R. Feldmaier-Fuchs G. Schweikardt T. Decker H. Burmester T. J. Biol. Chem. 2000; 275: 39339-39344Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The hemocyanins of orthognath spiders, scorpions, and related Arachnida are 4 × 6-mer protein complexes consisting of two identical 2 × 6-mers (1.Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar, 10.Markl J. Biol. Bull. (Woods Hole). 1986; 171: 90-115Crossref Google Scholar). The 8 × 6-mer hemocyanin of xiphosurs (Merostomata) such as Limulus polyphemus consists of two identical 4 × 6 halves that correspond structurally to the 4 × 6-mer arachnid hemocyanins. Typically, seven distinct subunit types, termeda–g, are present in a chelicerate hemocyanin, which have been immunologically correlated between L. polyphemus, the scorpion Androctonus australis, and the tarantula Eurypelma californicum (13.Lamy J. Lamy J. Weill J. Markl J. Schneider H.J. Linzen B. Hoppe-Seyler's Z. Physiol. Chem. 1979; 360: 889-895Crossref PubMed Scopus (41) Google Scholar, 14.Markl J. Gebauer W. Runzler R. Avissar I. Hoppe-Seyler's Z. Physiol. Chem. 1984; 365: 619-631Crossref PubMed Scopus (14) Google Scholar, 15.Kempter B. Markl J. Brenowitz M. Bonaventura C. Bonaventura J. Biol. Chem. Hoppe-Seyler. 1985; 366: 77-86Crossref PubMed Scopus (14) Google Scholar). The sequences of these subunits have been determined in E. californicum (12.Voit R. Feldmaier-Fuchs G. Schweikardt T. Decker H. Burmester T. J. Biol. Chem. 2000; 275: 39339-39344Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In this species, formation of the 4 × 6-mer requires a stoichiometric association of four copies of subunits a,d, e, f, and g and two copies of subunits b and c (16.Markl J. Bonaventura C. Bonaventura J. Hoppe-Seyler's Z. Physiol. Chem. 1981; 362: 429-437Crossref PubMed Scopus (12) Google Scholar, 17.Markl J. Savel A. Linzen B. Hoppe-Seyler's Z. Physiol. Chem. 1981; 362: 1255-1262Crossref PubMed Scopus (27) Google Scholar). Each subunit occupies a specific position within the native 4 × 6-mer molecule (17.Markl J. Savel A. Linzen B. Hoppe-Seyler's Z. Physiol. Chem. 1981; 362: 1255-1262Crossref PubMed Scopus (27) Google Scholar, 18.Markl J. Kempter B. Linzen B. Bijlholt M.M.C. van Bruggen E.F. Hoppe-Seyler's Z. Physiol. Chem. 1981; 362: 1631-1641Crossref PubMed Scopus (68) Google Scholar, 19.Decker H. Schmid R. Markl J. Linzen B. Hoppe-Seyler's Z. Physiol. Chem. 1980; 361: 1707-1717Crossref PubMed Scopus (21) Google Scholar). Estimates assuming a molecular clock have led to the conclusion that the diversification of the seven distinct subunits commenced early in evolution, more than 500 million years ago (MYA), 1The abbreviations used are: MYAmillion years agoMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flight mass spectrometryRTAretrolateral tibial apophysisPAMaccepted point mutations per site and was completed about 420 MYA (5.Burmester T. Mol. Biol. Evol. 2001; 18: 184-195Crossref PubMed Scopus (185) Google Scholar, 12.Voit R. Feldmaier-Fuchs G. Schweikardt T. Decker H. Burmester T. J. Biol. Chem. 2000; 275: 39339-39344Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). million years ago matrix-assisted laser desorption/ionization time-of-flight mass spectrometry retrolateral tibial apophysis accepted point mutations per site 4 × 6-mer hemocyanins are also present in a number of labidognath spider families such as Araneidae, Linyphiidae, and Theridionidae (10.Markl J. Biol. Bull. (Woods Hole). 1986; 171: 90-115Crossref Google Scholar). However, many other labidognath spider families such as Agelenidae, Salticidae, Thomisidae, Dysderiidae, Clubionidae, Lycosidae, and Ctenidae diverge from this standard scheme of chelicerate hemocyanin structure (10.Markl J. Biol. Bull. (Woods Hole). 1986; 171: 90-115Crossref Google Scholar). They possess a 2 × 6-mer hemocyanin with only two hemocyanin subunit types identified by immunological means, suggesting a severe rearrangement of the subunits. A carefully studied example is the Central American ctenid spider, Cupiennius salei (Fig. 1). In the hemolymph of this species, a mixture of 1 × 6- and 2 × 6-mer hemocyanins occurs in an approximate 1:2 ratio (20.Markl J. Schmid R. Czichos-Tiedt S. Linzen B. Hoppe-Seyler's Z. Physiol. Chem. 1976; 357: 1713-1725Crossref PubMed Scopus (49) Google Scholar,21.Markl J. J. Comp. Physiol. B. 1980; 140: 199-207Crossref Scopus (34) Google Scholar). Although both hemocyanin forms include five electrophoretically distinct but immunologically identical monomer subunits, an additional subunit dimer is present in the 2 × 6 molecules (21.Markl J. J. Comp. Physiol. B. 1980; 140: 199-207Crossref Scopus (34) Google Scholar, 22.Markl J. Markl A. Schartau W. Linzen B. J. Comp. Physiol. B. 1979; 130: 283-292Crossref Scopus (78) Google Scholar). This subunit dimer is immunologically distinct from the monomers (23.Markl J. Kempter B. Lamy J. Lamy J. Invertebrate oxygen-binding Proteins. Dekker, New York1981: 125-137Google Scholar) and is linked by a cysteine-mediated disulfide bridge (21.Markl J. J. Comp. Physiol. B. 1980; 140: 199-207Crossref Scopus (34) Google Scholar, 22.Markl J. Markl A. Schartau W. Linzen B. J. Comp. Physiol. B. 1979; 130: 283-292Crossref Scopus (78) Google Scholar). It is responsible for the formation of the 2 × 6-mer by connecting two hexamers. To understand the architecture of the C. salei hemocyanin and the evolutionary processes that led to the construction of the hemocyanin multimer, we have cloned and sequenced the cDNAs of all subunits of this hemocyanin. This is the second chelicerate hemocyanin for which the full subunit sequence has been elucidated (12.Voit R. Feldmaier-Fuchs G. Schweikardt T. Decker H. Burmester T. J. Biol. Chem. 2000; 275: 39339-39344Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The Central American ctenid spider,Cupiennius salei (Chelicerata, Araneae, Ctenidae; Fig. 1) was obtained from Prof. E.-A. Seyfarth (Institute of Zoology, Frankfurt, Germany). The animals were kept at 28 °C with a 12/12 h light-dark cycle and fed on insects. Specimens used in this study had ∼3–3.5-cm body length and 10–12-cm leg span. Adult spiders were immobilized for 2 h at 4 °C. The hemolymph was withdrawn from the median-dorsal region of the opisthosoma by a syringe and centrifuged for 10 min at 10,000 × g to remove the hemocytes. For some experiments, the hemolymph was dialyzed overnight at 4 °C in a buffer containing 130 mm glycine-NaOH, pH 9.6. SDS-PAGE analyses were carried out according to Laemmli (24.Laemmli U.K. Nature. 1970; 277: 680-685Crossref Scopus (207537) Google Scholar) on a 7.5% gel, either under reducing (with 2.5% β-mercaptoethanol) or nonreducing conditions (without β-mercaptoethanol). Native PAGE was performed on 5% polyacrylamide gels without SDS and β-mercaptoethanol. For Western blotting, the proteins were transferred to nitrocellulose at 0.8 mA/cm2. Nonspecific binding sites were blocked by 5% nonfat dry milk in TBS-T (10 mm Tris-HCl, pH 7.4, 140 mm NaCl, 0.25% Tween 20). Incubation with the anti-C. salei hemocyanin antibodies (21.Markl J. J. Comp. Physiol. B. 1980; 140: 199-207Crossref Scopus (34) Google Scholar) diluted 1:10,000 in 5% nonfat dry milk/TBS-T was carried out overnight at 4 °C. The filters were washed three times for 10 min in TBS-T and subsequently were incubated for 1 h with goat anti-rabbit Fab fragments conjugated with alkaline phosphatase (Dianova) diluted 1:10.000 in 5% nonfat dry milk/TBS-T. The membranes were washed as above and the detection was carried out using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. RNA was prepared from two ∼18-month-old adult C. salei specimens 10 days after induction of hematopoiesis by bleeding. The specimens were shock-frozen in liquid nitrogen and ground to a fine powder under continuous addition of nitrogen. Total RNA was extracted according to the guanidinium thiocyanate method (25.Chirgwin J.M. Przbyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16652) Google Scholar), and the poly(A)+RNA was purified by the aid of the Poly(A)Tract kit (Promega). A directionally cloned cDNA expression library was established applying the Lambda ZAP-cDNA synthesis kit (Stratagene). The library was amplified once and screened with anti- C. salei hemocyanin antibodies. Positive phage clones were converted to pBK-CMV plasmid vectors with the material provided by Stratagene according to the manufacturer's instructions and sequenced by the commercial GEnterprise (Mainz, Germany) sequencing service. Complete hemocyanin cDNA sequences were obtained by primer walking using specific oligonucleotides. MALDI-TOF experiments were performed by Dr. Christian Hunziger (Proteosys, Mainz, Germany) on proteins that had been separated by native PAGE, stained with Coomassie Brilliant Blue, and digested with trypsin. The MALDI-TOF data were evaluated using the program PEPTIDE-MASS. 2Available at the ExPASy web server: www.expasy.ch. Sequence analyses were carried out with the programs provided by software package 9.0 from the Genetics Computer Group (GCG, Wisconsin) and the ExPASy web server.2 Sequences were added by hand to an alignment of the published hemocyanin sequences (12.Voit R. Feldmaier-Fuchs G. Schweikardt T. Decker H. Burmester T. J. Biol. Chem. 2000; 275: 39339-39344Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) using GeneDoc, Version 2.6. 3K. B. Nicholas and H. B. Nicholas, Jr. (1997) www.psc.edu/biomed/genedoc/. The alignment is available from the authors upon request. The PHYLIP 3.6b2 software package was used for phylogenetic analyses (27.Felsenstein J. PHYLIP (Phylogeny Inference Package), Version 3, 6.b2, distributed by the author. Department of Genetics, University of Washington, Seattle2001Google Scholar). Distances between pairs of protein sequences were calculated and corrected for multiple changes according to Dayhoff's empirical PAM 001 matrix (28.Dayhoff M.O. Schwartz R.M. Orcutt B.C. Dayhoff M.O. Atlas of Protein Sequence and Structure. 5, Suppl. 3. National Biomedical Research Foundation, Washington, D. C.1978: 345-352Google Scholar) with the PROTDIST program. Phylogenetic trees were constructed either by the neighbor-joining method or the maximum parsimony method implemented in the PROTPARS program. The reliability of the trees was tested by bootstrap analysis (29.Felsenstein J. Evolution. 1985; 39: 783-791Crossref PubMed Google Scholar) with 100 replications (SEQBOOT program). To estimate the divergence times, the PAM matrix was imported into the Microsoft Excel 2000 spreadsheet program (30.Burmester T. Massey Jr., H.C. Zakharkin S.O. Beneš H. J. Mol. Evol. 1998; 47: 93-108Crossref PubMed Scopus (101) Google Scholar). A linearized tree that corresponds to the phylogeny of the chelicerate hemocyanins was calculated on the basis that Merostomata and Arachnida separated about 450 MYA (5.Burmester T. Mol. Biol. Evol. 2001; 18: 184-195Crossref PubMed Scopus (185) Google Scholar, 31.Dunlop J.A. Selden P.A. Fortey R.A. Thomas R.H. Arthropod Relationships. Systematic Association Special Volume Series 55, Chapman and Hall, London1997: 221-236Google Scholar). The confidence limits were estimated using the observed standard deviation of the protein distances. The hemolymph proteins of six adult individuals of C. salei were extracted and subjected to electrophoretic studies (Fig. 2). The total protein content of the hemolymph of these individuals varied between 28 and 65 mg/ml. In the first set of experiments, the proteins were applied to a native PAGE immediately after the bleeding of the animals (Fig. 2 A). The bands represent the 2 × 6- and 1 × 6-mer hemocyanins, as well as a non-respiratory protein and a significant amount of a hemocyanin heptamer, which is formed by partial dissociation of the 2 × 6-mer molecule (21.Markl J. J. Comp. Physiol. B. 1980; 140: 199-207Crossref Scopus (34) Google Scholar). In a second experiment, the hemolymph proteins were dialyzed in an alkaline glycine buffer at pH 9.6 before the PAGE to ensure the dissociation of 2 × 6- and 1 × 6-mer hemocyanin into subunits (Fig. 2 B). In both types of analysis, no detectable variation in the migration of the hemocyanin multimers and subunits was observed, although in specimens 5 and 6 the relative amount of the subunits varies slightly (Fig. 2 B). SDS-PAGE analyses under both reducing and nonreducing conditions revealed a single prominent hemocyanin band in the 70-kDa range (Fig. 3, lanes 1 and 2). The non-respiratory protein of C. salei forms a single band of about 110 kDa under nonreducing condition (lane 2), and a double band of 100 and 120 kDa, respectively, when β-mercaptoethanol was added (lane 1; cf. Ref. 21.Markl J. J. Comp. Physiol. B. 1980; 140: 199-207Crossref Scopus (34) Google Scholar). An additional band of 140 kDa appears only under nonreducing conditions (lane 2), which was suspected to be the dimeric hemocyanin subunit that is linked by a disulfide bridge (21.Markl J. J. Comp. Physiol. B. 1980; 140: 199-207Crossref Scopus (34) Google Scholar). This was confirmed by Western blotting using anti-C. salei hemocyanin antibodies, which stain the 70-kDa subunits as well as the 140-kDa dimer (lanes 3 and 4). A minor cross-reaction with the 110-kDa non-respiratory protein was observed, probably due to some contamination in the hemocyanin preparation used for immunization. A cDNA library was constructed according to Kempter (32.Kempter B. Naturwissenschaften. 1983; 70: 255-256Crossref Scopus (12) Google Scholar) fromC. salei 10 days after induction of hematopoiesis by bleeding. The library was screened with specific anti-C. salei hemocyanin antibodies (21.Markl J. J. Comp. Physiol. B. 1980; 140: 199-207Crossref Scopus (34) Google Scholar). A total of 27 positive clones was identified and partially sequenced; 17 of the clones encoded hemocyanin. Comparison of the 5′ and 3′ sequences revealed that they represent a total of nine distinct hemocyanin cDNAs. The complete sequences of these clones were obtained on both strands by primer walking. The full-length cDNAs cover the complete coding regions for the different subunits together with 44 to 67 bp of the respective 5′ untranslated regions and the complete 3′ untranslated regions comprising the standard polyadenylation signals (AATAAA) and the poly(A)-tails of different lengths (Table I). In each case, the presence of 3 purines upstream of the putative initiator codons (ATG) fulfills the minimum criteria for an eukaryotic translation start site (33.Kozak M. Nucleic Acids Res. 1984; 12: 857-872Crossref PubMed Scopus (2383) Google Scholar). Because three of the sequences are almost identical (>99.2%) with other cDNAs at the nucleotide level, and the distances among the others are in about the same range (Table II), we assume that these sequences represent alleles in the C. salei gene pool. Thus we identified a total of six sequences that are expected to encode the individual hemocyanin subunits ofC. salei.Table IMolecular properties of the C. salei hemocyanin subunitsSubunitAccession number1-aGenBank™/EBI DNA data accession number.cDNA1-bWithout poly(A)-tail.Protein1-cIncluding the initiator methionine. (No. of amino acids)Molecular mass1-dWithout initiator methionine.pI1-dWithout initiator methionine.bpkDaCsaHc-1AJ307903208463472.696.02CsaHc-2AJ307904204862671.025.58CsaHc-3AJ307905203962671.345.59CsaHc-4AJ307906203062671.065.43CsaHc-5AJ307907210162671.405.65CsaHc-5′AJ307908210162671.505.63CsaHc-6AJ307909226662671.615.55CsaHc-6′AJ307910226462671.545.59CsaHc-6“AJ307911226562671.585.591-a GenBank™/EBI DNA data accession number.1-b Without poly(A)-tail.1-c Including the initiator methionine.1-d Without initiator methionine. Open table in a new tab Table IIPairwise sequence identities of the C. salei hemocyanin subunitsCsaHc-1CsaHc-2CsaHc-3CsaHc-4CsaHc-5CsaHc-5′CsaHc-6CsaHc-6′CsaHc-6“CsaHc-177.277.076.476.776.778.077.777.8CsaHc-281.679.678.483.983.978.879.179.0CsaHc-380.586.479.479.479.286.186.086.0CsaHc-479.785.683.777.877.882.181.681.8CsaHc-580.287.985.585.599.280.180.280.1CsaHc-5′80.288.285.585.699.280.080.180.0CsaHc-679.984.789.085.985.885.899.399.4CsaHc-6′79.784.588.885.885.685.699.799.6CsaHc-6“79.684.388.785.685.585.599.599.8Nucleotide identities (within the coding region) are above and amino acid identities below the diagonal. Open table in a new tab Nucleotide identities (within the coding region) are above and amino acid identities below the diagonal. The open reading frames translate into five distinct polypeptides of 626 amino acids and a single one of 634 amino acids with calculated molecular masses in the range of 72 kDa (Table I), which agrees well with observations made by SDS-PAGE (Fig. 3; cf. Ref.21.Markl J. J. Comp. Physiol. B. 1980; 140: 199-207Crossref Scopus (34) Google Scholar). To assign these cDNA sequences to distinct subunits that have been identified by gel electrophoresis, the Coomassie-stained bands from a native gel (Fig. 2 C) were excised and submitted to MALDI-TOF analyses. Using a theoretical digest of the hemocyanin polypeptides deduced from the cDNAs, between 4 and 14 unique peptides from each band were unambiguously allocated. The hemocyanin subunits were named according to their apparent migration in the native gel, with CsaHc-1 being the subunit dimer, CsaHc-2 the slowest, and CsaHc-6 the fastest migrating subunit. Pairwise sequence comparison (excluding the allelic sequences) of the C. salei hemocyanin subunits revealed that 76.4–86.1% of the nucleotides and 79.2–88.8% of the amino acids are identical (Fig. 4; Table II). The nucleotide and amino acid sequences of the C. salei hemocyanin subunits were added to the previously published alignments of the chelicerate hemocyanins (5.Burmester T. Mol. Biol. Evol. 2001; 18: 184-195Crossref PubMed Scopus (185) Google Scholar, 12.Voit R. Feldmaier-Fuchs G. Schweikardt T. Decker H. Burmester T. J. Biol. Chem. 2000; 275: 39339-39344Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Although the amino acid identity score among the distinct C. salei hemocyanin subunits is higher than 79.2%, the lower scores were obtained with other known hemocyanin sequences. The highest score was found with the E. californicumhemocyanin subunit g (EcaHc-g) (71.2–72.8% identity at the amino acid level), whereas other sequences were lower than 66% (data not shown). For phylogenetic inference, four selected crustacean hemocyanin sequences were included in the alignment. Crustacean and chelicerate hemocyanins form two distinct branches, which separated at the time of the divergence of the subphyla in the Cambrian or an earlier period (5.Burmester T. Mol. Biol. Evol. 2001; 18: 184-195Crossref PubMed Scopus (185) Google Scholar,10.Markl J. Biol. Bull. (Woods Hole). 1986; 171: 90-115Crossref Google Scholar). Therefore, the crustacean hemocyanins may be used as the out-group to infer the phylogeny of chelicerate proteins. Tree construction was performed assuming maximum parsimony or by the neighbor-joining method based on a PAM matrix (Fig. 5). In both analyses, the six C. salei hemocyanin subunits form a single, well supported clade (100% bootstrap support) nested within the other chelicerate hemocyanins. The C. salei branch is associated with the g-subunit of E. californicum(100 and 99% bootstrap support, respectively). A time scale of chelicerate hemocyanin evolution was inferred under the assumption that the L. polyphemus hemocyanin subunit II and E. californicum hemocyanin subunit a are orthologous proteins (5.Burmester T. Mol. Biol. Evol. 2001; 18: 184-195Crossref PubMed Scopus (185) Google Scholar, 12.Voit R. Feldmaier-Fuchs G. Schweikardt T. Decker H. Burmester T. J. Biol. Chem. 2000; 275: 39339-39344Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 15.Kempter B. Markl J. Brenowitz M. Bonaventura C. Bonaventura J. Biol. Chem. Hoppe-Seyler. 1985; 366: 77-86Crossref PubMed Scopus (14) Google Scholar) and that the Merostomata and Arachnida diverged about 450 MYA in the Ordovician period (31.Dunlop J.A. Selden P.A. Fortey R.A. Thomas R.H. Arthropod Relationships. Systematic Association Special Volume Series 55, Chapman and Hall, London1997: 221-236Google Scholar). Assuming a PAM substitution matrix, we calculated a mean amino acid replacement rate of 0.65 ± 0.03 × 10−9 substitutions per site per year, which is good agreement with the previous estimates using fewer sequences (5.Burmester T. Mol. Biol. Evol. 2001; 18: 184-195Crossref PubMed Scopus (185) Google Scholar, 12.Voit R. Feldmaier-Fuchs G. Schweikardt T. Decker H. Burmester T. J. Biol. Chem. 2000; 275: 39339-39344Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Thus the time of divergence of the branches leading to the C. salei hemocyanin subunits and E. californicum subunit g (EcaHc-g) occurred around 279 ± 5 MYA (Fig. 6). CsaHc-1 and the precursor of the other Cupiennius subunits split 186 ± 10 MYA. Subunit CsaHc-4 diverged 127 ± 5 MYA, CsaHc-2 and CsaHc-5 split 97 ± 1.5 MYA, and CsaHc-3 and CsaHc-6 split 93 ± 1.4 MYA.Figure 6An approximate time scale of the evolution in the chelicerate hemocyanins. The linearized tree was obtained on the basis of corrected protein distance data (PAM 001 matrix; 28). The divergence times were estimated as described.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The hemocyanin of the ctenid spider C. salei is represented by a 1 × 6 oligomer formed by five distinct subunits and a 2 × 6 multimer with six subunits, one of which forms a dimer that links the two hexamers (20.Markl J. Schmid R. Czichos-Tiedt S. Linzen B. Hoppe-Seyler's Z. Physiol. Chem. 1976; 357: 1713-1725Crossref PubMed Scopus (49) Google Scholar, 21.Markl J. J. Comp. Physiol. B. 1980; 140: 199-207Crossref Scopus (34) Google Scholar). We have cloned and sequenced six distinct hemocyanin cDNAs (plus three allelic sequences). The MALDI-TOF data allow the unambiguous assignment of each of these polypeptides to one of the protein bands observed in gel electrophoresis and also demonstrate that all subunits were covered by the cDNA data. Besides the 4 × 6-mer hemocyanin of the North American tarantula E. californicum, which consists of seven distinct subunits (12.Voit R. Feldmaier-Fuchs G. Schweikardt T. Decker H. Burmester T. J. Biol. Chem. 2000; 275: 39339-39344Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), C. salei hemocyanin is the second chelicerate hemocyanin for which subunit sequences have been completely determined. However, the structures of these two respiratory proteins essentially differ. Although the 4 × 6-mer hemocyanin of E. californicum represents the standard type of an arachnid hemocyanin, C. salei is the representative of another 2 × 6-mer hemocyanin form only found in a distinct group of the Araneae. Based on the cross-reactions of the C. salei subunits with antibodies raised against the various hemocyanin subunits of E. californicum, the monomers were previously assigned to the chelicerate subunit type f, whereas the dimer appeared to belong to the d-type subunits (10.Markl J. Biol. Bull. (Woods Hole). 1986; 171: 90-115Crossref Google Scholar). However, both sequence comparison and phylogenetic analyses clearly show that the dimer as well as the monomers do in fact belong to the chelicerate subunit type g (Fig. 5). The C. salei hemocyanin sequences closely resemble those of the other chelicerates. It is, however, noteworthy that in theC. salei hemocyanins as well as in EcaHc-g the copper-binding site A carries a conserved HHWYWH motif, whereas in the other chelicerate hemocyanin subunits this sequence is HHWHWH. The functional consequences of this mutation are unknown. As in theE. californicum hemocyanin subunits (12.Voit R. Feldmaier-Fuchs G. Schweikardt T. Decker H. Burmester T. J. Biol. Chem. 2000; 275: 39339-39344Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), no signal peptides except putative initiator methionines were found in the C. salei hemocyanin subunits, although these polypeptides are extracellular proteins. This is explained by the fact that spider hemocyanins are synthesized by free ribosomes and most likely released by holocrine secretion (32.Kempter B. Naturwissenschaften. 1983; 70: 255-256Crossref Scopus (12) Google Scholar, 34.Markl J. Stumpp S. Bosch F.X. Voit R. Preaux G. Lontie R. Invertebrate Dioxygen Carriers. University Press, Louvain, Belgium1990: 497-502Google Scholar). Thus, the proteins do not pass the Golgi apparatus and the putative N-glycosylation sites (NX(T/S)), which, although present in the primary structure of all subunits, are probably not used. There are four strictly conserved cysteine residues in all C. salei hemocyanins in positions 531, 533, 574, and 581 in domain 3. They most likely form two disulfide bridges that make up a flexible hinge stabilizing the three-dimensional structure of the subunit, as deduced from other hemocyanins (35.Topham R. Tesh S. Westcott A. Cole G. Mercatante D. Kaufman G. Bonaventura C. Arch. Biochem. Biophys. 1999; 369: 261-266Crossref PubMed Scopus (8) Google Scholar). As already observed with various hemocyanin sequences (1.Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar, 5.Burmester T. Mol. Biol. Evol. 2001; 18: 184-195Crossref PubMed Scopus (185) Google Scholar, 12.Voit R. Feldmaier-Fuchs G. Schweikardt T. Decker H. Burmester T. J. Biol. Chem. 2000; 275: 39339-39344Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), most variations are present in the first and third structural domains. The second domain, which forms the core of the hemocyanin subunit and includes the copper-binding sites, is strikingly conserved within the different C. salei hemocyanin subunits (79.7% identical amino acids) and between those and the other hemocyanins. Although in general the first domain is the least conserved region among different hemocyanins, we found the third domain of the C. saleihemocyanin subunits to be the most variable, with only 63.7% strictly conserved residues (71.4% in the first domain). Based on the MALDI-TOF data, CsaHc-1 was found to form the homodimeric hemocyanin subunit. In this protein, the two polypeptide chains are linked by a disulfide bridge (20.Markl J. Schmid R. Czichos-Tiedt S. Linzen B. Hoppe-Seyler's Z. Physiol. Chem. 1976; 357: 1713-1725Crossref PubMed Scopus (49) Google Scholar). The hemocyanin dimer is responsible for the formation of the 2 × 6-mer hemocyanin and mediates a flexible but stable interhexamer contact that is readily visible in electron micrographs (21.Markl J. J. Comp. Physiol. B. 1980; 140: 199-207Crossref Scopus (34) Google Scholar). The dimer-forming subunit, CsaHc-1, is longer than the other subunits and contains eight additional amino acids at its C-terminal end, which includes a cysteine at position 631 (Fig. 4). Comparison with the known three-dimensional structure of subunit II from L. polyphemus (36.Hazes B. Magnus K.A. Bonaventura C. Bonaventura J. Dauter Z. Kalk K.H. Hol W.G. Protein Sci. 1993; 2: 597-619Crossref PubMed Scopus (319) Google Scholar) reveals that this is the only cysteine available at the subunit surface (Fig. 7). Thus we assume that Cys-631 of CsaHc-1 is in fact responsible for the formation of the disulfide link between two CsaHc-1 subunits and the creation of the 2 × 6-mer hemocyanin. We cannot conclude from our data the position of the other subunits in the native 2 × 6-mer hemocyanin. It should be noted, however, that in reassembly experiments the monomeric subunits (CsaHc-2 to-6) were individually able to form hexamers and to combine with the dimeric subunit (CsaHc-1) to form 2 × 6-mers (23.Markl J. Kempter B. Lamy J. Lamy J. Invertebrate oxygen-binding Proteins. Dekker, New York1981: 125-137Google Scholar). Seven different subunit types (a–g) and a typical 4 × 6-mer hemocyanin are present in most chelicerate orders (10.Markl J. Biol. Bull. (Woods Hole). 1986; 171: 90-115Crossref Google Scholar). The subunit types diverged from an ancestral hemocyanin gene as early as 550 MYA (5.Burmester T. Mol. Biol. Evol. 2001; 18: 184-195Crossref PubMed Scopus (185) Google Scholar, 12.Voit R. Feldmaier-Fuchs G. Schweikardt T. Decker H. Burmester T. J. Biol. Chem. 2000; 275: 39339-39344Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). By contrast, the hemocyanin of C. salei is a rather recent derivative of this ancient chelicerate hemocyanin structure, which emerged only about 90–190 MYA from a singleg-type subunit (Fig. 6). It can be assumed that the ancestor of the Cupiennius-type hemocyanin was a simple hexamer, which contained a single g-like subunit type. The reasons why the other subunits have been lost is essentially unknown, but it might be speculated that morphological changes made the highly complex 4 × 6-mer hemocyanin unnecessary. It is noteworthy that theCupiennius type 2 × 6-mer hemocyanin appears to be restricted to the “higher” entelegyne Araneae of the retrolateral tibial apophysis (RTA) clade (37.Coddington J.A. Levi H.W. Annu. Rev. Ecol. Syst. 1991; 22: 565-592Crossref Scopus (599) Google Scholar), which are active hunters with a complex tracheal system. It is conceivable that the loss of the 4 × 6-mer hemocyanin is linked to the evolution of such respiratory organs. It is also possible that the ancestors of the RTA clade passed a period of dwarfism in which a simple 1 × 6-mer hemocyanin acted as a high-affinity oxygen storage protein rather than a sophisticated oxygen carrier. Later, an increase in body size rendered simple tracheal respiration inefficient to sustain the metabolic need of an active hunter. By a gene duplication event about 186 MYA they regained a more complex hemocyanin with an additional dimeric subunit, enabling the formation of 2 × 6-mer hemocyanin. This in turn would allow more sophisticated allosteric regulations as well as a higher oxygen transport capacity while maintaining blood osmolarity and viscosity. The later diversification of the different subunits by gene duplication 90–120 MYA might either be related simply to the need of more hemocyanin polypeptides or may again enhance the regulation capacity of the protein. Hemocyanin sequences have been used successfully to infer a time scale of the evolution of arthropod taxa (5.Burmester T. Mol. Biol. Evol. 2001; 18: 184-195Crossref PubMed Scopus (185) Google Scholar, 7.Burmester T. J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 2002; 172: 95-117Crossref PubMed Scopus (225) Google Scholar, 8.van Holde K.E. Miller K.I. Decker H. J. Biol. Chem. 2001; 276: 15563-15566Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 11.Kusche K. Burmester T. Mol. Biol. Evol. 2001; 18: 1566-1573Crossref PubMed Scopus (65) Google Scholar). Given the sparse fossil record (38.Selden P.A. Benton M.J. The Fossil Record 2. Chapman and Hall, London1993: 297-320Google Scholar), the present knowledge of the evolution of the spiders (Araneae) is poor. The Araneae probably emerged in the Devonian period some 400 MYA. Recent cladistic analyses treat the suborders Mygalomorpha (represented here by E. californicum) and Araneomorpha (C. salei) as sister taxa, which are joined as Opisthothelae (37.Coddington J.A. Levi H.W. Annu. Rev. Ecol. Syst. 1991; 22: 565-592Crossref Scopus (599) Google Scholar). The first fossils of the Mygalomorpha derive from the early Triassic period (some 240 MYA), whereas the lower bound of the fossil record of the Araneomorpha is about 160 MYA (38.Selden P.A. Benton M.J. The Fossil Record 2. Chapman and Hall, London1993: 297-320Google Scholar). The time of divergence of the C. salei hemocyanins and EcaHc-g most likely coincides with the split of the Mygalomorpha and Araneomorpha. Assuming a molecular clock, we calculated this date to be about 280 MYA, which agrees with the fossil data. The most successful subgroup within the Araneomorpha are the Entelegynae, which are subdivided in the Orbicularidae and the spiders of the RTA clade (37.Coddington J.A. Levi H.W. Annu. Rev. Ecol. Syst. 1991; 22: 565-592Crossref Scopus (599) Google Scholar). So far, only species that belong to the RTA clade possess a Cupiennius-type hemocyanin (10.Markl J. Biol. Bull. (Woods Hole). 1986; 171: 90-115Crossref Google Scholar). However, it is uncertain whether it can be considered as a molecular synapomorphy of this taxon, because its occurrence outside of the RTA clade is still possible. If this is the case it would provide an excellent trait for tracing the closest relatives of the RTA clade. On the other hand, various families of the Orbicularidae possess anEurypelma-type hemocyanin (10.Markl J. Biol. Bull. (Woods Hole). 1986; 171: 90-115Crossref Google Scholar). Thus the formation of the 2 × 6-mer hemocyanin must have occurred after the separation of the Orbicularidae and the progenitors of the RTA clade. The fossil records of both taxa are poor and are mainly restricted to specimens from the Tertiary period preserved in Baltic amber (38.Selden P.A. Benton M.J. The Fossil Record 2. Chapman and Hall, London1993: 297-320Google Scholar). The lower bound of divergence of the Orbicularidae and the RTA clade is set by an orbicularian spider from the early Cretaceous period, some 140 MYA (38.Selden P.A. Benton M.J. The Fossil Record 2. Chapman and Hall, London1993: 297-320Google Scholar). We have calculated that the formation of theCupiennius-type hemocyanin commenced about 190 MYA, which should be considered as the lower boundary for the time of emergence of the RTA clade. The different subunits of the C. saleihemocyanin have existed as distinct genes for at least 90 MYA. Future studies will elucidate the distribution and relationship of hemocyanin subunits in the Entelegynae and other Araneae and will help to infer the evolution of spiders. We thank Prof. E.-A. Seyfarth for the spiders, Kristina Kusche for assistance with the cDNA library construction, Wolfgang Gebauer for help with the native gels, Michael Schaffeld and Christian Hunziger for help with the analyses of the MALDI data, and Robin Harris for critical reading of the manuscript and correcting the language. AJ307904AJ307905AJ307906AJ307907AJ307908AJ307909AJ307910AJ307911
Referência(s)