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

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:
Abbreviation: Hc, hemocyanin.
*Present address: Institute of Animal Physiology, University of
Mu
¨

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 ().
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

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

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

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

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

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

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

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-

, 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

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