J Biol Chem, Vol. 274, Issue 41, 29071-29074, October 8, 1999
Identification, Structure, and Properties of Hemocyanins from
Diplopod Myriapoda*
Elmar
Jaenicke
,
Heinz
Decker,
Wolfgang
Gebauer§,
Jürgen
Markl§, and
Thorsten
Burmester§¶
From the Institute of Molecular Biophysics and the
§ Institute of Zoology, University of Mainz,
D-55099 Mainz , Germany
 |
ABSTRACT |
Hemocyanins are copper-containing, respiratory
proteins that occur in the hemolymph of many arthropod species. Here we
report for the first time the presence of hemocyanins in the diplopod Myriapoda, demonstrating that these proteins are more widespread among
the Arthropoda than previously thought. The hemocyanin of Spirostreptus sp. (Diplopoda: Spirostreptidae) is composed
of two immunologically distinct subunits in the 75-kDa range that are
most likely arranged in a 36-mer (6 × 6) native molecule. It has
a high oxygen affinity (P50 = 4.7 torr) but low
cooperativity (h = 1.3 ± 0.2).
Spirostreptus hemocyanin is structurally similar to the
single known hemocyanin from the myriapod taxon, Scutigera coleoptrata (Chilopoda), indicating a rather conservative
architecture of the myriapod hemocyanins. Western blotting demonstrates
shared epitopes of Spirostreptus hemocyanin with both
chelicerate and crustacean hemocyanins, confirming its identity as an
arthropod hemocyanin.
| |
INTRODUCTION |
In the hemolymph of many arthropod species oxygen is transported
by large respiratory copper proteins that are termed hemocyanins (1,
2). The principal structure of a hexamer of six similar or identical
subunits in the 75-kDa range is conserved within all arthropod
hemocyanins, although in many cases these hexamers associate to
quaternary structures containing up to 8 × 6 subunits (3). Each
subunit carries one oxygen molecule by the virtue of two copper ions
that are coordinated by six histidine residues (4). Hemocyanins are
members of a functionally and structurally diverse protein superfamily
that includes arthropod tyrosinases (prophenoloxidases), crustacean
nonrespiratory pseudo-hemocyanins (cryptocyanins), insect hexamerins,
and dipteran hexamerin receptors (5-10).
Hemocyanins had long been unknown among the Myriapoda (classes:
Chilopoda, Diplopoda, Symphyla, and Pauropoda), whereas these proteins
have been studied in detail in many different chelicerate and
crustacean species. Respiratory proteins were considered unnecessary in
the Myriapoda because these animals possess, similar to the insects, a
well developed tracheal system (11). However, several years ago Mangum
et al. (12) demonstrated the existence of a true, 36-mer
hemocyanin in the hemolymph of the common house centipede Scutigera coleoptrata. The exceptional presence of a
hemocyanin in the scutigeramorph Chilopoda has been attributed to the
high activity and peculiar tracheal system of these species rather than
representing a general feature of the Myriapoda (13). However, here we
show that high amounts of hemocyanins are also present in the
Diplopoda, indicating that these respiratory proteins are more
widespread among the Myriapoda than previously thought.
| |
EXPERIMENTAL PROCEDURES |
Animals--
Living male and female specimens of three diplopod
species from the family of Spirostreptidae (two different species of
the genus Spirostreptus, named "A" and "B" here,
Telodeinopus aoutii) were obtained from the Aquazoo in
Düsseldorf (Germany). The hemolymph was withdrawn from the dorsal
intersegmental regions with a syringe, immediately diluted in one
volume of 100 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, 5 mM CaCl2, and centrifuged
for 10 min at 10,000 × g to remove hemocytes and
tissue contamination. Purified hemolymph was stored at 4 °C or
frozen at 
20 °C. Protein concentrations were determined according
to the method of Bradford (14).
Hemocyanin Purification and Analysis--
About 30 µl
(~1.5-2 mg of total protein) of freshly collected hemolymph of
Spirostreptus B were applied to a Superose 6 HR 10/30 column
(Amersham Pharmacia Biotech) and eluted with 100 mM
Tris-HCl, pH 7.5, 20 mM MgCl2, 20 mM CaCl2 at a flow rate of 0.2 ml/min. Proteins
were detected in a flow cell at 280 nm. The molecular masses of the
fractions were estimated after calibration with native hemocyanin
molecules with known sizes: Limulus polyphemus (48-mer, 3600 kDa), Eurypelma californicum (24-mer, 1800 kDa), Astacus leptodactylus (12-mer, 900 kDa), and Panulirus
interruptus (6-mer, 450 kDa). Electron microscopic analysis of the
hemolymph samples was performed by negative staining with
uranyl-acetate (15). UV-visible spectra were recorded on a Hitachi
U-3000 spectrometer. Oxygen-binding curves at pH 7.5 in 100 mM Tris-HCl, 20 mM MgCl2, 20 mM CaCl2 were obtained using the
polarographic-fluorometric method described by Loewe (16).
Gel Electrophoresis and Western Blotting--
About 400 µg of
purified Spirostreptus B hemocyanin were used to raise
polyclonal antibodies in a guinea pig. Crossed immunoelectrophoresis was carried out according to Weeke (17).
SDS-PAGE1 was performed on a
7.5% gel (18). 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 TBST (10 mM
Tris-HCl, pH 7.4, 140 mM NaCl, 0.25% Tween 20). Incubation
with different anti-hemocyanin antisera in 5% nonfat dry milk/TBST was
carried out for 2 h at room temperature. The filters were washed
three times for 20 min in TBST, incubated for 1 h with the
appropriate secondary antibody conjugated with alkaline phosphatase,
and diluted in 5% nonfat dry milk/TBST. The membranes were washed as
above and the detection was carried out using nitro blue tetrazolium
and 5-bromo-4-chloro-3-indolyl phosphate.
| |
RESULTS |
Identification of Putative Hemocyanins in Diplopoda--
The
hemolymph of male and female adult specimens of three juliform
Diplopoda from the family of Spirostreptidae (Spirostreptus A and B, T. aoutii) was investigated for the presence of
hemocyanin-like proteins. In all species, prominent polypeptides with
apparent molecular masses between 70 and 85 kDa were observed in
SDS-PAGE (Fig. 1A), which is
in the expected range of arthropod hemocyanin subunits. In all three
species, electron microscopic investigations of the hemolymph revealed
the presence of protein particles that resemble the unique appearance
of 6 × 6 S. coleoptrata hemocyanin (12, 19). This was
the first indication that hemocyanin-like proteins may be present in
the Diplopoda. In Fig. 1B, an electron micrograph of the
hemolymph of Spirostreptus species B is displayed. This
species was chosen for further investigations because of the large size
of the adult animals (about a 10-15-cm body length, 1-cm diameter) and
the high content of putative hemocyanin molecules in the hemolymph
(about 40-50 mg/ml).
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Fig. 1.
Putative hemocyanins of Diplopoda.
A, 5-10 µg of hemolymph proteins of male and female
individuals of three different diplopod species (A,
Spirostreptus A; B, Spirostreptus B;
C, T. aoutii) were separated by SDS-PAGE and
stained with Coomassie Brilliant Blue. The molecular mass standard is
given on the left side. B, the hemolymph of
Spirostreptus B was diluted and investigated by electron
microscopy. The length of the bar corresponds to 50 nm, and
the arrows indicate putative hemocyanin particles. On the
right side, the images are enlarged by a factor of 2 and
show the hemocyanins in different orientations.
|
|
Purification and Properties of Spirostreptus
Hemocyanin--
Size-exclusion gel chromatography on a Superose 6 HR
10/30 column yields three peaks of about 2400, 1250, and 390 kDa (Fig. 2A). The first two peaks
(P1 and P2) both contain two polypeptides of
about 75 kDa, whereas the third peak consists of four other proteins
between 85 and 200 kDa (Fig. 2B). Electron-microscopic investigations show no differences between the proteins of peak one and
two with particles resembling the putative hemocyanin observed in the
raw hemolymph samples, but also particles that probably correspond to
monohexamers (not shown). The third peak does not contain any
hemocyanin-like structures. Assuming a mean molecular mass of about 75 kDa/subunit (as revealed by SDS-PAGE), the first peak indicates a
protein consisting of more than 30 subunits. Because arthropod
hemocyanins only elute as multimers of six subunits and considering the
similarities to Scutigera hemocyanin, a 36-mer (6 × 6)
molecule is the most probable structure of Spirostreptus
hemocyanin. This assumption is also consistent with the appearance of
this molecule in the electron microscopic images. The estimated
molecular mass of the second peak (P2) is about half of the
first, suggesting a stable 18-mer dissociation product (3 × 6).
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Fig. 2.
Purification and subunit composition of the
hemocyanin of Spirostreptus. A, total
hemolymph proteins were applied to a Superose 6 HR 10/30 column as
described and eluted at 0.2 ml/min. B, the three peaks
(named P1, P2, and P3; H,
hemolymph control) were analyzed by SDS-PAGE. C, crossed
immunoelectrophoresis of sample P1 with
anti-Spirostreptus hemocyanin antibodies. The
arrows indicate the two precipitation lines interpreted as
subunits. The arrowhead marks the material considered as
undissociated hemocyanin.
|
|
Column-purified hemocyanin was used to raise specific antibodies in
guinea pigs (Charles River, Germany). Then the hemocyanin was
dissociated into subunits by dialysis against 0.13 M
glycine/NaOH buffer for 2 days at pH 9.6 and applied to crossed
immunoelectrophoresis (17). Although the dissociation of the hemocyanin
was incomplete even after prolonged incubation at a high pH, we
observed several precipitation lines, indicating the presence of at
least two immunologically distinct polypeptides in the hemocyanin
sample (Fig. 2C).
Oxygen-binding Properties of Spirostreptus Hemocyanin--
In
spectroscopic analysis, Spirostreptus hemocyanin shows a
peak at 336 nm, which is typical for oxygenated hemocyanins and reflects the formation of the copper-oxygen complex (Fig.
3A). However, as already
observed in the centipede S. coleoptrata, the
A340/A280 ratio is much
lower than in the other arthropod hemocyanins. This may be explained in
part by its high extinction coefficient at 280 nm (1 mg/ml corresponds
to 1.8-2 OD), which indicates a highly aromatic protein. The oxygen
affinity of Spirostreptus hemocyanin was measured in Tris
buffer at pH 7.4 according to Loewe (16) and yields a
P50 of 4.7 torr (Fig. 3B). A low
cooperativity (Hill coefficient, h = 1.3 ± 0.2)
was detected. The hemolymph of Spirostreptus has an
extraordinarily high protein content up to 100 mg/ml. We estimated that
hemocyanin represents about 40-50% of the total hemolymph proteins
(Figs. 1 and 2), indicating a hemocyanin content up to 50 mg/ml.
Because every subunit of about 75 kDa can carry one oxygen molecule, an
oxygen transport capacity of about 20 ml/liter hemolymph was
calculated. This value is comparable with those of other
Arthropoda.
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Fig. 3.
Oxygen-binding properties of the
Spirostreptus hemocyanin. A, the
absorption spectrum of purified hemocyanin was measured in a range from
250 to 450 nm. Note that the right part of the spectrum is
enlarged by a factor of 10. B, oxygen binding was determined
by a polarographic-fluorometric method. Hemocyanin concentration was
0.1 mg/ml, the temperature was 20 °C, and the pH was 7.5. The
inset shows the Hill plot.
|
|
Spirostreptus Hemocyanin Cross-reacts with Antibodies against Other
Arthropod Hemocyanins--
Several specific antibodies directed
against hemocyanins of Crustacea and Chelicerata have been described in
the literature; in this study, five different rabbit antibodies have
been tested for cross-reaction with the Spirostreptus
hemocyanin by Western blotting (Fig. 4).
Two of the antisera were raised against chelicerate hemocyanins
(E. californicum and Androctonus australis), and
three were raised against hemocyanins of malacostracan Crustacea
(A. leptodactylus, Carcinus maenas, and
Homarus americanus). About 2.5 µg of total hemolymph
proteins from Spirostreptus species B were applied/lane; the
different anti-hemocyanin antibodies were used in 1:5000 to 1:10,000
dilutions. Signals in the 75-kDa range were observed in all cases,
although most of the antibodies cross-react differentially with the two
hemocyanin polypeptides. This is consistent with the assumption that
they correspond to immunologically distinct hemocyanin subunits (see
also Fig. 2C). None of the other proteins in the
Spirostreptus hemolymph were detected by the anti-hemocyanin
antibodies, demonstrating the specificity of the immunological
relationship.
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Fig. 4.
Spirostreptus hemocyanin cross-reacts with
antibodies raised against crustacean and chelicerate hemocyanins.
About 2.5 µg of total hemolymph proteins were applied/lane and tested
for cross-reaction with the specific  -Spirostreptus
hemocyanin antibody and antibodies against other arthropod hemocyanins
as indicated. A Coomassie Brilliant Blue-stained gel of total hemolymph
is given on the left.
|
|
| |
DISCUSSION |
The hemocyanins of Arthropoda have been the subject of detailed
functional, structural, and evolutionary studies mainly in the
Chelicerata and Crustacea (1-5). This paper is the first report of the
presence of hemocyanins in the diplopod Myriapoda. All three
investigated species belong to the Spirostreptidae, a large family of
mainly Afrotropical and Neotropical juliform Diplopoda. The hemocyanin
of Spirostreptus sp. displays the typical arthropod
hemocyanin features in terms of subunit size, structural appearance,
and spectroscopic properties. Moreover, it shares similar antigenic
determinants with crustacean and chelicerate hemocyanins. Therefore,
the identity of the O2-binding protein of
Spirostreptus as a genuine hemocyanin should be considered as conclusive.
The presence of a typical hemocyanin in the Diplopoda is rather
surprising. This protein appears to be structurally very similar to the
hemocyanin from the centipede, S. coleoptrata (12, 13, 20).
In this species, the presence of an oxygen-carrying hemocyanin was
considered as an exception related to the peculiar blind-ending tracheal system in the Scutigeramorpha (13). In contrast, although the
tracheal system of the Spirostreptidae is normal and similar to those
of the other Diplopoda, a high hemocyanin concentration was observed in
most specimens. Therefore, the evolution of trachea is not necessarily
accompanied by a loss of specific respiratory proteins. This suggests
that hemocyanins may be found in other Myriapoda as well and that these
proteins are more widespread among the Arthropoda than previously thought.
Chilopod and diplopod Myriapoda probably diverged in the Devonian or an
earlier period (21). However, whereas the subunit arrangement within
the chelicerate and crustacean hemocyanins is variable, ranging from
1 × 6 to 8 × 6 subunits in the native molecule, the unique
6 × 6 organization of subunits appears to be a conservative
feature of the Myriapoda that has been conserved for at least 400 million years. Nevertheless, the oxygen-binding properties of the
hemocyanins from Scutigera and from Spirostreptus are strikingly different. Whereas Spirostreptus hemocyanin
has a high oxygen affinity (P50 = 4.7 torr at pH
7.5) and a low cooperativity (h = 1.3 ± 0.2), the
situation is reversed in Scutigera hemocyanin, which has a
low oxygen affinity (P50 = 55 torr at pH 7.5)
and high cooperativity (h = 8.9 at pH 7.5) (12). The
high oxygen affinity of Spirostreptus hemocyanin may be
related to the low oxygen conditions of the subterrestical environment,
where these species remain most of the time during daylight hours.
Recent molecular phylogenetic studies (22, 23) shed doubt on the
monophyly of the taxon "Tracheata" or "Antennata," which combines the Myriapoda with the Hexapoda (11). Although there is
increasing evidence for a sister group relationship of the Hexapoda and
Crustacea (23, 24), the actual systematic position of the Myriapoda
became uncertain. Although we observed cross-reactivity of the
Spirostreptus hemocyanin with antibodies that have been raised against either chelicerate or crustacean hemocyanins (Fig. 4),
such data are not sufficient to determine the exact evolutionary relationships among the arthropods. However, at least some antigenic structures have been conserved among myriapod, crustacean, and chelicerate hemocyanins because of their divergence in the Cambrian period more than 520 million years ago. As already mentioned by Mangum
et al. (12), the presence of hemocyanins (and related proteins, e.g. hexamerins) is an autapomorphic character of
the Arthropoda, providing strong evidence in favor for a monophyletic origin of this phylum. These proteins have also been successfully used
to investigate arthropod relationships (5-7). Hence, the determination
of the cDNA sequence of Spirostreptus hemocyanin will
not only provide insights in a complete new class of hemocyanins but
will probably also allow the elucidation of the phylogenetic affinities
of the Myriapoda.
| |
ACKNOWLEDGEMENTS |
We thank S. Löser (Aquazoo,
Düsseldorf) for supplying the animals, H. Enghoff for
determination of the species, W. Conze for the oxygen-binding
curves, J. R. Harris and D. Scheffler for the EM pictures, U. Mei
ner and U. Kolb for their help with the image analysis, and
J. R. Harris for correcting the language.
| |
FOOTNOTES |
*
This work was supported by Grant Bu956/3 from the Deutsche
Forschungsgemeinschaft.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.
¶
To whom correspondence should be addressed. Tel.: (49) 6131-39 4477; Fax: (49) 6131-39 4652; E-mail:
thorsten@uzomai.biologie.uni-mainz.de.
| |
ABBREVIATIONS |
The abbreviations used are:
PAGE, polyacrylamide
gel electrophoresis;
TBST, Tris-buffered saline with Tween 20.
| |
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Copyright © 1999 by the American Society for Biochemistry and Molecular Biology.
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ABSTRACT
INTRODUCTION
