Originally published In Press as doi:10.1074/jbc.M111368200 on February 12, 2002
J. Biol. Chem., Vol. 277, Issue 17, 14451-14457, April 26, 2002
Complete Hemocyanin Subunit Sequences of the Hunting Spider
Cupiennius salei
RECENT HEMOCYANIN REMODELING IN ENTELEGYNE SPIDERS*
Pia
Ballweber,
Jürgen
Markl
, and
Thorsten
Burmester
From the Institute of Zoology, Johannes Gutenberg University,
D-55099 Mainz, Germany
Received for publication, November 28, 2001, and in revised form, February 8, 2002
 |
ABSTRACT |
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. The
C. salei hemocyanin subunits all belong to the arachnid
g-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.
| |
INTRODUCTION |
Hemocyanins are large allosteric respiratory proteins that occur
freely dissolved in the hemolymph of many arthropod and molluscan species (1, 2). Oxygen binding of hemocyanins is mediated by a pair of
copper atoms that are coordinated by six histidine residues (2-4).
Arthropod and molluscan hemocyanins differ essentially in structure and
sequence and are most likely of independent evolutionary origin (1,
5-8). These proteins have been proven to be an excellent topic of
functional, structural, and evolutionary studies (1, 2, 7-9).
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, 10). The sequences of various hemocyanin
subunits have been determined from all euarthropod subphyla, including
the Chelicerata, Crustacea, Myriapoda, and Hexapoda (7). Phylogenetic
analyses demonstrate that subunit evolution took place independently
within each subphylum (5, 7, 10, 11). The estimated rate of amino acid
replacement in the chelicerate hemocyanins is about the half of that
found in the crustaceans or myriapods (7), which is most likely linked to the conserved structure of these proteins (1, 12). The hemocyanins
of orthognath spiders, scorpions, and related Arachnida are 4 × 6-mer protein complexes consisting of two identical 2 × 6-mers
(1, 10). 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, termed
a-g, are present in a chelicerate hemocyanin,
which have been immunologically correlated between L. polyphemus, the scorpion Androctonus australis, and the
tarantula Eurypelma californicum (13-15). The sequences of these subunits have been determined in E. californicum (12). 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, 17). Each subunit
occupies a specific position within the native 4 × 6-mer molecule
(17-19). 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),1 and was completed
about 420 MYA (5, 12).
4 × 6-mer hemocyanins are also present in a number of labidognath
spider families such as Araneidae, Linyphiidae, and Theridionidae (10).
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). 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,
21). 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, 22). This
subunit dimer is immunologically distinct from the monomers (23) and is
linked by a cysteine-mediated disulfide bridge (21, 22). 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).
| |
EXPERIMENTAL PROCEDURES |
Animals--
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.
Protein Biochemistry--
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) on a 7.5% gel,
either under reducing (with 2.5% 
-mercaptoethanol) or nonreducing
conditions (without -mercaptoethanol). Native PAGE was performed on
5% polyacrylamide gels without SDS and -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) 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.
Cloning and Sequencing of Hemocyanin cDNAs--
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), 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.
Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass
Spectrometry (MALDI-TOF)--
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.2
Sequence Analysis and Phylogenetic Studies--
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) using GeneDoc,
Version 2.6.3 The alignment
is available from the authors upon request. The PHYLIP 3.6b2 software
package was used for phylogenetic analyses (27). Distances between
pairs of protein sequences were calculated and corrected for multiple
changes according to Dayhoff's empirical PAM 001 matrix (28) 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) with 100 replications (SEQBOOT program). To
estimate the divergence times, the PAM matrix was imported into the
Microsoft Excel 2000 spreadsheet program (30). 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, 31). The confidence limits were estimated using the
observed standard deviation of the protein distances.
| |
RESULTS |
Subunit Composition of C. salei Hemocyanin--
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. 2A). 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). 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. 2B). 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.
2B).
View larger version (41K):
[in this window]
[in a new window]
|
Fig. 2.
Native PAGE of the hemolymph proteins from
C. salei. A, 15 µg of hemolymph proteins of six
different individuals (lanes 1-6) were applied per lane.
I, 2 × 6-hemocyanin; II, 1 × 7-hemocyanin; III, non-respiratory protein; IV,
1 × 6-mer hemocyanin. B, proteins were dialyzed
against alkaline glycine-NaOH buffer before PAGE; 15 µg of total
protein was applied. V, hemocyanin subunit dimer
(CsaHc-1); VI, dissociated non-respiratory protein;
VII, hemocyanin subunits (see panel C).
HL, hemolymph. C, Coomassie-stained gel with
subunit bands after dissociation in alkaline buffer as used for
MALDI-TOF. The numbers 2-6 refer to the different
hemocyanin subunits, CsaHc-2-6.
|
|
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 -mercaptoethanol
was added (lane 1; cf. Ref. 21). 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). 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.
View larger version (41K):
[in this window]
[in a new window]
|
Fig. 3.
SDS-PAGE and Western blot analyses of the
hemolymph proteins with and without
-mercaptoethanol. 10 µg of total protein was
applied. Lanes 1 and 2, Coomassie-stained gels;
lanes 3 and 4, Western blot employing
anti-C. salei hemocyanin antibodies; lanes 1 and
3, SDS-PAGE with -mercaptoethanol; lanes 2 and
4, SDS-PAGE without -mercaptoethanol. On the
left side, the molecular size standard is given.
|
|
Cloning and Sequencing of the C. salei Hemocyanin cDNAs--
A
cDNA library was constructed according to Kempter (32) from
C. salei 10 days after induction of hematopoiesis by
bleeding. The library was screened with specific anti-C.
salei hemocyanin antibodies (21). 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). 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 of
C. salei.
View this table:
[in this window]
[in a new window]
|
Table II
Pairwise sequence identities of the C. salei hemocyanin subunits
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). To assign these cDNA sequences to distinct subunits that have
been identified by gel electrophoresis, the Coomassie-stained bands
from a native gel (Fig. 2C) 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.
Hemocyanin Sequence Comparison and Evolution--
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, 12). 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. californicum
hemocyanin subunit g (EcaHc-g) (71.2-72.8% identity at the
amino acid level), whereas other sequences were lower than 66% (data
not shown).
View larger version (131K):
[in this window]
[in a new window]
|
Fig. 4.
Amino acid alignment of the amino acid
sequences of the C. salei hemocyanin subunits.
Strictly conserved residues are shaded. Above
the sequences, the secondary structure elements as
deduced from L. polyphemus subunit II (36) are indicated:
A,  -helices; B, -sheets. Other features are
given below the sequences: *, copper ligand;
c, disulfide bridges. CsaHc-1-6, hemocyanin
subunits 1-6 of C. salei (see Table I for accession
numbers). Polymorphic sites in alleles of subunits 5 and 6 are
underlined.
|
|
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,
10). 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, 12, 15) and that the Merostomata and Arachnida
diverged about 450 MYA in the Ordovician period (31). 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, 12). 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.
View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
Phylogenetic relationship among the
chelicerate hemocyanins. The simplified trees were deduced by a
neighbor-joining analysis based on the alignment of the amino acid
sequences. The crustacean hemocyanins are considered as the out-group
(26). The numbers at the nodes represent the
statistical confidence estimates computed by the bootstrap procedure
(29). HamHc-a, Homarus americanus hemocyanin
subunit A (GenBankTM accession no. AJ272095),
PinHc-a-c, Panulirus interruptus hemocyanin
subunits a through c (P04254, P10787, and P80096, respectively);
AauHc-6, Androctonus australis hemocyanin
6 (P80476); LpoHc-2, subunit II of L. polyphemus
(P04253); TtrHc-a, Tachypleus tridentatus
hemocyanin (3); EcaHc-a-g, E. californicum
hemocyanin subunits a through g (X16893,
AJ290429, AJ277489, AJ290430, X16894, AJ277491, and AJ277492,
respectively). For other abbreviations, see Table I.
|
|
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 6.
An 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.
|
|
| |
DISCUSSION |
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 (20-21). 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), 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.
Cupiennius Hemocyanin Structure--
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). 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 the C. 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 the
E. californicum hemocyanin subunits (12), 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, 34). 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).
As already observed with various hemocyanin sequences (1, 5, 12), 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. salei
hemocyanin 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). 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). 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) 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).
View larger version (96K):
[in this window]
[in a new window]
|
Fig. 7.
A model of the C. salei Hc-1
subunit. The arrow indicates the position of the
C-terminal extension implied to be responsible for the formation of the
2 × 6-mer.
|
|
The Evolution of the Cupiennius Hemocyanin--
Seven different
subunit types (a-g) and a typical 4 × 6-mer hemocyanin are present in most chelicerate orders (10). The subunit types diverged from an ancestral hemocyanin gene as early as
550 MYA (5, 12). 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 single
g-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 the
Cupiennius type 2 × 6-mer hemocyanin appears to be
restricted to the "higher" entelegyne Araneae of the retrolateral
tibial apophysis (RTA) clade (37), 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.
Implications for the Evolution of the Araneae--
Hemocyanin
sequences have been used successfully to infer a time scale of the
evolution of arthropod taxa (5, 7, 8, 11). Given the sparse fossil
record (38), 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). 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). 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). So far, only species that belong to the RTA
clade possess a Cupiennius-type hemocyanin (10). 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 an
Eurypelma-type hemocyanin (10). 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). 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). We have calculated that the formation of the
Cupiennius-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. salei
hemocyanin 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.
| |
ACKNOWLEDGEMENTS |
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.
| |
FOOTNOTES |
*
This work was supported by grants from the Deutsche
Forschungsgemeinschaft (Bu956/3-2 and 5-1, Ma843/4-4) and by the
Feldbauschstiftung Mainz.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ307903, AJ307904, AJ307905, AJ307906, AJ307907, AJ307908,
AJ307909, AJ307910, and AJ307911 (C. salei hemocyanin subunits
1-5, 5', 6, 6', and 6", respectively).
To whom correspondence should be addressed: Institute of
Zoology, University of Mainz, Müllerweg 6, D-55099 Mainz,
Germany. Tel.: 49-6131-392-2314; Fax: 49-6131-392-4652; E-mail:
markl@mail.uni- mainz.de.
Published, JBC Papers in Press, February 12, 2002, DOI 10.1074/jbc.M111368200
2
Available at the ExPASy web server:
www.expasy.ch.
3
K. B. Nicholas and H. B. Nicholas, Jr.
(1997) www.psc.edu/biomed/genedoc/.
| |
ABBREVIATIONS |
The abbreviations used are:
MYA, million years
ago;
MALDI-TOF, matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry;
RTA, retrolateral tibial apophysis;
PAM, accepted point mutations per site.
| |
REFERENCES |
| 1.
|
Markl, J.,
and Decker, H.
(1992)
Adv. Comp. Environ. Physiol.
13,
325-376
|
| 2.
|
van Holde, K. E.,
and Miller, K. I.
(1995)
Adv. Protein Chem.
47,
1-81[Medline]
[Order article via Infotrieve]
|
| 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.,
and Hol, W. G. J.
(1985)
Science
229,
519-524[Abstract/Free Full Text]
|
| 4.
|
Cuff, M. E.,
Miller, K. I.,
van Holde, K. E.,
and Hendrickson, W. A.
(1998)
J. Mol. Biol.
278,
855-870[CrossRef][Medline]
[Order article via Infotrieve]
|
| 5.
|
Burmester, T.
(2001)
Mol. Biol. Evol.
18,
184-195[Abstract/Free Full Text]
|
| 6.
|
Lieb, B.,
Altenhein, B.,
Markl, J.,
Vincent, A.,
van Olden, E.,
van Holde, K. E.,
and Miller, K. I.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
4546-4551[Abstract/Free Full Text]
|
| 7.
|
Burmester, T.
(2002)
J. Comp. Physiol. B Biochem. Syst. Environ. Physiol.
172,
95-117[CrossRef][Medline]
[Order article via Infotrieve]
|
| 8.
|
van Holde, K. E.,
Miller, K. I.,
and Decker, H.
(2001)
J. Biol. Chem.
276,
15563-15566[Free Full Text]
|
| 9.
|
Harris, J. R.,
and Markl, J.
(1999)
Micron
30,
597-623[CrossRef][Medline]
[Order article via Infotrieve]
|
| 10.
|
Markl, J.
(1986)
Biol. Bull. (Woods Hole)
171,
90-115[Abstract/Free Full Text]
|
| 11.
|
Kusche, K.,
and Burmester, T.
(2001)
Mol. Biol. Evol.
18,
1566-1573[Abstract/Free Full Text]
|
| 12.
|
Voit, R.,
Feldmaier-Fuchs, G.,
Schweikardt, T.,
Decker, H.,
and Burmester, T.
(2000)
J. Biol. Chem.
275,
39339-39344[Abstract/Free Full Text]
|
| 13.
|
Lamy, J.,
Lamy, J.,
Weill, J.,
Markl, J.,
Schneider, H. J.,
and Linzen, B.
(1979)
Hoppe-Seyler's Z. Physiol. Chem.
360,
889-895[Medline]
[Order article via Infotrieve]
|
| 14.
|
Markl, J.,
Gebauer, W.,
Runzler, R.,
and Avissar, I.
(1984)
Hoppe-Seyler's Z. Physiol. Chem.
365,
619-631[Medline]
[Order article via Infotrieve]
|
| 15.
|
Kempter, B.,
Markl, J.,
Brenowitz, M.,
Bonaventura, C.,
and Bonaventura, J.
(1985)
Biol. Chem. Hoppe-Seyler
366,
77-86[Medline]
[Order article via Infotrieve]
|
| 16.
|
Markl, J.,
Bonaventura, C.,
and Bonaventura, J.
(1981)
Hoppe-Seyler's Z. Physiol. Chem.
362,
429-437[Medline]
[Order article via Infotrieve]
|
| 17.
|
Markl, J.,
Savel, A.,
and Linzen, B.
(1981)
Hoppe-Seyler's Z. Physiol. Chem.
362,
1255-1262[Medline]
[Order article via Infotrieve]
|
| 18.
|
Markl, J.,
Kempter, B.,
Linzen, B.,
Bijlholt, M. M. C.,
and van Bruggen, E. F.
(1981)
Hoppe-Seyler's Z. Physiol. Chem.
362,
1631-1641[Medline]
[Order article via Infotrieve]
|
| 19.
|
Decker, H.,
Schmid, R.,
Markl, J.,
and Linzen, B.
(1980)
Hoppe-Seyler's Z. Physiol. Chem.
361,
1707-1717[Medline]
[Order article via Infotrieve]
|
| 20.
|
Markl, J.,
Schmid, R.,
Czichos-Tiedt, S.,
and Linzen, B.
(1976)
Hoppe-Seyler's Z. Physiol. Chem.
357,
1713-1725[Medline]
[Order article via Infotrieve]
|
| 21.
|
Markl, J.
(1980)
J. Comp. Physiol. B
140,
199-207
|
| 22.
|
Markl, J.,
Markl, A.,
Schartau, W.,
and Linzen, B.
(1979)
J. Comp. Physiol. B
130,
283-292
|
| 23.
|
Markl, J.,
and Kempter, B.
(1981)
in
Invertebrate oxygen-binding Proteins
(Lamy, J.
, and Lamy, J., eds)
, pp. 125-137, Dekker, New York
|
| 24.
|
Laemmli, U. K.
(1970)
Nature
277,
680-685
|
| 25.
|
Chirgwin, J. M.,
Przbyla, A. E.,
MacDonald, R. J.,
and Rutter, W. J.
(1979)
Biochemistry
18,
5294-5299[CrossRef][Medline]
[Order article via Infotrieve]
|
| 26.
|
Burmester, T.,
and Scheller, K.
(1996)
J. Mol. Evol.
42,
713-728[CrossRef][Medline]
[Order article via Infotrieve]
|
| 27.
|
Felsenstein, J.
(2001)
PHYLIP (Phylogeny Inference Package), Version 3, 6.b2, distributed by the author
, Department of Genetics, University of Washington, Seattle
|
| 28.
|
Dayhoff, M. O.,
Schwartz, R. M.,
and Orcutt, B. C.
(1978)
in
Atlas of Protein Sequence and Structure
(Dayhoff, M. O., ed), Vol. 5, Suppl. 3
, pp. 345-352, National Biomedical Research Foundation, Washington, D. C.
|
| 29.
|
Felsenstein, J.
(1985)
Evolution
39,
783-791[CrossRef]
|
| 30.
|
Burmester, T.,
Massey, H. C., Jr.,
Zakharkin, S. O.,
and Bene , H.
(1998)
J. Mol. Evol.
47,
93-108[CrossRef][Medline]
[Order article via Infotrieve]
|
| 31.
|
Dunlop, J. A.,
and Selden, P. A.
(1997)
in
Arthropod Relationships
(Fortey, R. A.
, and Thomas, R. H., eds)
, pp. 221-236, Systematic Association Special Volume Series 55, Chapman and Hall, London
|
| 32.
|
Kempter, B.
(1983)
Naturwissenschaften
70,
255-256
|
| 33.
|
Kozak, M.
(1984)
Nucleic Acids Res.
12,
857-872[Abstract/Free Full Text]
|
| 34.
|
Markl, J.,
Stumpp, S.,
Bosch, F. X.,
and Voit, R.
(1990)
in
Invertebrate Dioxygen Carriers
(Preaux, G.
, and Lontie, R., eds)
, pp. 497-502, University Press, Louvain, Belgium
|
| 35.
|
Topham, R.,
Tesh, S.,
Westcott, A.,
Cole, G.,
Mercatante, D.,
Kaufman, G.,
and Bonaventura, C.
(1999)
Arch. Biochem. Biophys.
369,
261-266[CrossRef][Medline]
[Order article via Infotrieve]
|
| 36.
|
Hazes, B.,
Magnus, K. A.,
Bonaventura, C.,
Bonaventura, J.,
Dauter, Z.,
Kalk, K. H.,
and Hol, W. G.
(1993)
Protein Sci.
2,
597-619[Abstract]
|
| 37.
|
Coddington, J. A.,
and Levi, H. W.
(1991)
Annu. Rev. Ecol. Syst.
22,
565-592[CrossRef]
|
| 38.
|
Selden, P. A.
(1993)
in
The Fossil Record 2
(Benton, M. J., ed)
, pp. 297-320, Chapman and Hall, London
|
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
H. Decker, N. Hellmann, E. Jaenicke, B. Lieb, U. Meissner, and J. Markl
Minireview: Recent progress in hemocyanin research
Integr. Comp. Biol.,
October 1, 2007;
47(4):
631 - 644.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Dolashka-Angelova, A. Dolashki, S. N. Savvides, R. Hristova, J. Van Beeumen, W. Voelter, B. Devreese, U. Weser, P. Di Muro, B. Salvato, et al.
Structure of Hemocyanin Subunit CaeSS2 of the Crustacean Mediterranean Crab Carcinus aestuarii
J. Biochem.,
September 1, 2005;
138(3):
303 - 312.
[Abstract]
[Full Text]
[PDF]
|
|
|
Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
TOP
ABSTRACT
INTRODUCTION
