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INTRODUCTION |
The body fluid of many arthropod species contains large copper
proteins that serve to transport oxygen and are referred to as
hemocyanins (1, 2). The principal structure of a hexamer of six similar
or identical subunits in the 75 kDa range is conserved within all
hemocyanins, although in many species, these hexamers associate to
quaternary structures of up to 8 × 6 subunits (3). Each subunit
carries one oxygen molecule by virtue of two copper ions that are
coordinated by six histidine residues (4, 5).
Arthropod hemocyanins are members of a growing protein superfamily that
also comprises the arthropod tyrosinases (prophenoloxidases), the
insect hexamerins, and the dipteran hexamerin receptors (6-10). Hexamerins share about 20-25% sequence identity with the hemocyanins (7, 10) and have similar tertiary and quaternary structures (11, 12)
but do not bind oxygen. The loss of the oxygen transport function is
accompanied by the replacement of the coordinating histidine residues
in the copper-binding center by other amino acids. Hexamerin-type
proteins have been discovered in the hemolymph or storage tissues in
all insects investigated (13). They are generally assumed to act as
storage proteins that provide energy and amino acids for nonfeeding periods.
Occasionally, in the hemolymph of some decapodan Crustacea, similar
proteins were observed that resemble the hemocyanins and hexamerins in
their specific appearance but do not bind oxygen (14, 15). In view of
the recently proposed sister group relationship of the Insecta and
Crustacea (e.g. 16, 17), these proteins were considered as
possible ancestors of the hexamerins (7, 10, 12, 13). However, here I
report two sequences of such a protein and show that, although they
likely represent storage proteins as well, they evolved independently
from crustacean hemocyanins. Because this protein closely resembles the
crustacean hemocyanins but lacks copper and the oxygen-binding
function, here it is termed pseudo-hemocyanin
(PHc).1
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EXPERIMENTAL PROCEDURES |
Animals--
Living adult Homarus americanus
(Crustacea, Malacostraca, Decapoda, and Homaridae) were purchased from
a local seafood dealer, anesthetized, and immediately dissected. The
hemolymph was withdrawn from the abdominal sinus by a syringe and
centrifuged for 10 min at 10,000 × g to remove
hemocytes, tissue contamination, and clotted material. The supernatant
was used as purified hemolymph. Tissues were either immediately used
for RNA preparation or frozen in liquid nitrogen and stored at
80 °C for no longer than 4 weeks.
Purification of PHc and Preparation of Antibodies--
About
0.5-1 ml (approximately 50-200 mg of total protein) of freshly
collected hemolymph was applied to a Biogel A5 m (Bio-Rad) column
(1.5 × 100 cm) as described by Markl et al. (14).
Elution was performed with 100 mM Tris-HCl, pH 7.5, 10 mM MgCl2, and 5 mM
CaCl2. The eluted material was analyzed by SDS-PAGE for the presence of PHc and purity. The oxygen binding capacity of the different fractions was analyzed spectrometrically by reading the
absorption at 280 and 340 nm. The second large peak (16 S) contains the
PHc fraction. Electron microscopy of the different fractions was
performed by negative staining with uranyl-acetate (18). Polyclonal
antibodies against the PHc were raised in guinea pigs and checked for
specificity by Western blotting.
Gel Electrophoresis and Western Blotting--
SDS-PAGE was
performed on a 7.5% gel according to standard procedures (19). For
Western blotting, the proteins were transferred to nitrocellulose at
0.8 mA/cm2. Nonspecific binding sites were blocked by 5%
non-fat dry milk in TBST. Incubation with the anti-PHc-IgGs (diluted
1:10,000 in 5% non-fat dry milk and TBST) was carried out for 2 h
at room temperature. The filters were washed three times for 20 min
each in TBST and subsequently incubated for 1 h with goat
anti-guinea pig Fab fragments conjugated with alkaline phosphatase
(Dianova) diluted in 5% non-fat dry milk and Tris-buffered saline. The
membranes were washed as described above, and the detection was carried out using nitroblue tetrazolium and bromo-chloro-indolyl-phosphate. N-terminal sequencing was performed by a commercial service (H. Heid,
Deutsches Krebsforschungszentrum, Heidelberg, Germany) using protein
samples separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane as described previously. The PHc and hemocyanin bands were excised and submitted to Edman degradation.
Cloning and Sequencing of the PHc cDNAs--
Total RNA was
either isolated from the complete thorax of a single lobster without
the legs and the cuticle or prepared from defined tissues by the method
of Scheller and Karlson (20). Poly(A)+ RNA was purified
from total RNA using the Poly(A)Tract kit (Promega). About 5 µg of
poly(A)+ RNA were used for the construction of a
directionally cloned cDNA expression library applying the Lambda
ZAP cDNA synthesis kit (Stratagene). The library was amplified once
and screened with
-PHc antibodies. Positive phage clones were
converted to plasmid vectors using the material provided by Stratagene
in the cDNA synthesis kit. The PHc cDNAs inserted in the
pBK-CMV vector were sequenced on both strands by the commercial SeqLab
(Göttingen) sequencing service.
Northern Blotting--
Equal amounts of RNA from different
tissues were denatured and subjected to electrophoresis in a 1%
agarose gel containing 1 M formaldehyde. After
electrophoresis, gels were rinsed in 20× SSC and transferred to a
nitrocellulose membrane in 20× SSC. Digoxigenin-UTP-labeled antisense
RNA probes were transcribed from the complete cDNA clones using the
Roche Molecular Biochemicals RNA in vitro transcription kit.
The filters were pre-hybridized in 50% formamide, 5× SSC, 0.1%
N-laurylsarcosine, 0.02% SDS, and 1% blocking agent (Roche Molecular
Biochemicals) for 1 h and then hybridized in the same solution
containing the labeled probe overnight at 60 °C. Immunodetection was
carried out using the Roche Molecular Biochemicals digoxigenin detection kit as described by the manufacturer.
Sequence Analysis and Phylogenetic Studies--
The programs
provided with Sequence Analysis Software Package 8.0 from the Genetics
Computer Group, Wisconsin were used for sequence analysis and
manipulation. For phylogenetic inference, the deduced PHc protein
sequence was aligned by hand to the previously published alignment of
hemocyanins and hexamerins (12), including other sequences that became
available only recently. The complete alignment is available from the
author upon request. Distances between the pairs of protein sequences
were calculated and corrected for multiple changes according to
Dayhoff's empirical PAM 001 matrix by using the PROTDIST program of
the PHYLIP 3.5c package (21). Phylogenetic inference was carried out
using either the neighbor joining method (22) or the maximum parsimony
method implemented in the PROTPARS program of the PHYLIP 3.5c software package (23). The robustness of the trees was tested by bootstrap analysis (24) with 100 replications (SEQBOOT program). Majority role
consensus trees were obtained using the CONSENSE program.
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RESULTS |
Purification and Analysis of PHc--
The separation of the
hemolymph proteins of adult H. americanus by gel filtration
on a Biogel A5 m column yields two major peaks corresponding to the
sedimentation coefficients in the analytical ultracentrifugation of
about 16 S and 24 S (Fig. 1A;
Ref. 14). The dodecameric (2 × 6) hemocyanin elutes at 24 S (14,
25). This peak displays an UV spectrum with a 280 nm:340 nm absorption ratio of about 5, which is typical for a native hemocyanin and indicates the formation of the copper-oxygen complex that causes increased absorption at a wavelength of 340 nm. The second major peak
that elutes at 16 S has a 280 nm:340 nm ratio of >20 (Fig. 1B), indicating a protein that does not bind oxygen by a
copper complex and that has therefore been referred to as
non-respiratory protein (NRP) (14) However, to avoid possible confusion
with chelicerate NRPs that are not related to hemocyanin (26), the H. americanus NRP has been renamed PHc here.

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Fig. 1.
Isolation and spectroscopic properties of
Homarus hemocyanin and PHc. Total hemolymph proteins
of a female H. americanus were separated on a Biogel A5 m
column as described previously (A). Two major peaks of 24 S
(dodecameric hemocyanin) and 16 S (hexameric PHc) were observed. The
absorption spectrums of these fractions were analyzed over a range of
250-360 nm (B). Only hemocyanin (24 S) displays an
additional absorption maximum at 340 nm.
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Electron microscopic examination of the 24 S fraction shows the
presence of the dodecameric hemocyanin, consisting of two hexamers of
10 nm in diameter each (Fig.
2A) (25). The 16 S fraction
(Fig. 2B) contains a protein with a diameter of about 12 nm
that resembles a typical hexameric hemocyanin, with six subunits
arranged as a trigonal antiprism (4). After separation on SDS-PAGE
(Fig. 3A), the 24 S fraction
shows three bands with apparent molecular masses of 76-80 kDa, and the
16 S fraction separates into two PHc subunits of about 85 and 86 kDa
(14). Antibodies were raised against the 16 S fraction in guinea pigs. In Western blotting, these antibodies recognize only the PHcs and not
the hemocyanin subunits, whereas the anti-hemocyanin antibodies (25) do
not stain PHc (Fig. 3B). After transfer to a polyvinylidene difluoride membrane, 20-22 N-terminal amino acids of the PHc and hemocyanin subunits were determined by microsequencing (Fig.
3C). Because the first 12 amino acids of the two PHc
subunits are identical, only the 86-kDa subunit was sequenced up to the
20th residue. Database research shows that this sequence clearly
belongs to the hemocyanin superfamily.

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Fig. 2.
Electron microscopic images. Negative
stains of Homarus hemocyanin (24 S peak; A) and
PHc (16 S peak; B). Bar, 10 nm.
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Fig. 3.
Electrophoretic properties and N-terminal
sequences of Homarus hemocyanin and PHc subunits.
A, proteins of total hemolymph (HL), the 24 S
peak (25), and the 16 S peak (16) were separated on SDS-PAGE and
stained with Coomassie Blue. B, Western blotting analysis of
total hemolymph using anti-hemocyanin antibodies (25)
( Hc) or anti-PHc antibodies ( PHc).
C, total hemolymph was separated by SDS-PAGE, and the
N-terminal sequences of the two PHcs (PHc1 and
PHc2) and the three hemocyanin subunits (Hc1,
Hc2, and Hc3) were determined.
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Cloning and Sequencing of Homarus PHc cDNA--
A cDNA
library was constructed from poly(A)+ RNA isolated from
thorax tissue of Homarus. The library was amplified once,
and about 3 × 105 clones were screened with the
anti-PHc antibodies. 29 positive clones were identified. Three clones
(clones 11, 31, and 42) with cDNA inserts between 2.3 and 2.5 kb
were subjected to further analysis and sequenced (Fig.
4). Whereas clones 31 and 42 are identical (except in the length of the 5' region), clone 11 represents another PHc clone. The identity score on the nucleotide level within
the coding region of the native polypeptides is 96%. However, there
are fundamental differences in the 3' noncoding region, and only
limited conservation is seen in the putative signal peptide. Restriction enzyme analysis (EcoRI/XhoI) of other
positive clones allowed the assignment to either one of the clone
types. Conceptual translation of the PHc clone shows that both
sequences cover the actual N terminus of the native polypeptide,
whereas they are not complete in the 5' sequences that very likely
correspond to the signal peptide of the nascent protein. Clone 11 (referred to as PHc-1 in the remainder of the text) has 71 nucleotides
of the putative signal peptide, clone 31 (PHc-2) has 65 nucleotides of
the putative signal peptide, and clone 42 has 53 nucleotides of the
putative signal peptide.

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Fig. 4.
cDNA and deduced protein sequences of the
pseudo-hemocyanins of H. americanus. DNA is numbered
according to the nucleotide sequence of PHc-1; nucleotide substitutions
in PHc-2 within the coding region are depicted by the
letters in the top row, and bases missing in that
clone are indicated by a dot (.). Amino acid numbering
starts with the N terminus of the native polypeptide, and the sequences
of the putative signal peptide are underlined. Amino acid
changes in PHc-2 resulting from base exchanges are not shown. The stop
codon (TAA) is indicated by three asterisks, and the
polyadenylation site is double-underlined. Potential
N-glycosylation sites are italic.
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The deduced N termini of both PHcs are identical, match exactly to
those that have been determined by microsequencing of the proteins, and
confirm the identity of the clones. The inferred primary structures of
the native PHc proteins yield polypeptides of 661 (PHc-1) and 660 (PHc-2) amino acids with deduced molecular masses of 77,313 and 76,970 Da, respectively. The PHc amino acid sequences share 623 common
residues (94.4% identity; 97.6% similarity, considering conservative
replacements). Computer analysis reveals the presence of four conserved
potential N-glycosylation sites (NXT) in both PHc (Fig. 7).
PHc Expression--
The expression of PHc mRNA in different
tissues was examined by Northern blotting (Fig.
5). A single strong PHc signal of about
2.5 kb was observed in the ovaries, whereas less PHc mRNA is
present in the heart tissues. Except for the ovaries, no difference was
observed between the sexes. No PHc expression was detected in the
hepatopancreas, the site of hemocyanin synthesis in H. americanus (27), and in three other tissues analyzed (gills, connective tissue, and muscle). When comparing the level of PHc in the
hemolymph of different individuals, significant differences in the
amount of this protein were observed (Fig.
6). A total of eight adult lobsters (two
female, four male, and two of undetermined sex) weighing around
500 g were investigated. Complete absence of PHc was detected in
one male and in one individual of undetermined sex, whereas hemocyanin
subunits are present in all animals. Therefore, PHc is not a
female-specific protein.

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Fig. 5.
Tissue-specific expression of PHc mRNA
analyzed by Northern blotting. About 10 µg of total RNA from
different tissues were separated on an agarose gel and probed with
digoxigenin-labeled PHc antisense RNA as described. RNA from the
complete thorax (lane 1), hepatopancreas (lane
2), ovary (lane 3), heart (lane 4), gills
(lane 5), connective tissue (lane 6), and muscle
(lane 7) was analyzed. With the exception of muscle tissue,
the tissues were derived from a female animal.
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Fig. 6.
Analysis of the hemolymph proteins of
different individuals by SDS-PAGE. The numbers identify
individual specimens; the sex is indicated at the top of the
lanes. About 20 µl of a 1:100 dilution were applied per lane.
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Sequence Comparison and Phylogenetic Analysis--
Database
searches show that PHc displays the highest similarity with crustacean
hemocyanins (47-56% identity, 74-80% similarity) and the
cryptocyanin of the Dungeness crab Cancer magister (15) (50% identity, 74% similarity), whereas lower similarity scores were
observed with chelicerate hemocyanins, arthropod prophenoloxidases, and
insect hexamerins. Hemocyanins transport oxygen by two copper ions
coordinated by six histidine residues, which are arranged in two
copper-binding sites (1-5). These histidines are also present in the
arthropod prophenoloxidases (8-10). However, whereas five of these
residues are conserved within PHc as well, the first histidine in
copper-binding site A is replaced in both PHc-1 and PHc-2 by a tyrosine
(Fig. 7).

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Fig. 7.
Comparison of the amino acid sequences of the
PHcs and crustacean hemocyanins. The asterisks (*)
indicate the positions that are conserved, the copper-binding residues
are shaded, and the putative N-glycosylation sites are
depicted in bold. Note that the first histidine of
copper-binding site A (position 201) is replaced by a tyrosine in the
PHcs. See the Fig. 8 legend for abbreviations.
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To infer the position of the PHc proteins within the superfamily of
arthropod hemocyanins and hexamerins, the amino acid sequences were
included in a multiple sequence alignment that has been described in an
earlier study (12). Two recently published sequences (15, 28) were
added as well. The hemocyanins of the Chelicerata were considered to be
the most ancient branch and were used here as the outgroup (12),
although no differences in the arrangement of the clades were observed
when using the arthropod prophenoloxidases as the outgroup (10). Both
distance matrix methods and maximum parsimony show that the PHcs are
associated with the cryptocyanin of Cancer magister (Fig.
8). There is strong statistical support that these three non-respiratory proteins are included within the clade
of the crustacean hemocyanins (100% bootstrap value). However, whereas
parsimony analysis indicates a moderately supported association of the
PHcs and cryptocyanin with a clade comprising hemocyanin C of the spiny
lobster Panulirus interruptus, hemocyanin 6 of C. magister, and a hemocyanin of the shrimp Penaeus
vannamei (63% bootstrap support; Fig. 8A), distance
matrix methods show PHc in the sister group position to all sequenced
crustacean hemocyanins (75% bootstrap support; Fig.
8B).

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Fig. 8.
Phylogenetic position of the PHcs within the
hemocyanin superfamily. The simplified trees were deduced by
(A) the parsimony method or (B) neighbor joining
based on the alignment described previously (12). The
numbers at the nodes represent the statistical confidence
estimates computed by the bootstrap procedure (24). HamPHc1,
H. americanus PHc-1; HamPHc2, H. americanus PHc-1; CmaCC1, C. magister
cryptocyanin (15); SamEHP, Schistocerca americana
embryonic hemolymph protein (28); CmaHc6; C. magister hemocyanin 6 (29); PinHcA, P. interruptus hemocyanin a (30); PinHcB, P. interruptus hemocyanin b (31); PinHcC, P. interruptus hemocyanin c (32); PvuHc, Palinurus
vulgaris hemocyanin (33); PvaHc, P. vannamei
hemocyanin (34).
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DISCUSSION |
The hemocyanins of arthropods have been subjected to detailed
functional, structural, and evolutionary studies, mainly in the
Chelicerata and Crustacea (see Refs 1-3 and 7). Among the Crustacea,
only the Malacostraca possess hemocyanins, whereas these proteins are
apparently absent in other crustacean taxa (35). In the American
lobster, H. americanus, dodecameric hemocyanin represents
about 60-90% of the total hemolymph proteins (27). An additional
hexameric protein with similar biophysical properties was identified in
Homarus as well as in other decapodan species and termed NRP
(14). However, the nature of these proteins has long remained obscure.
Here I report two sequences of the NRP of H. americanus that
clearly show that this protein is related to the hemocyanins as well as
to the other members of the hemocyanin superfamily (Fig. 7). To
emphasize its particular similarity to hemocyanin on one hand and to
avoid confusion with other non-respiratory hemolymph proteins on the
other hand, it has been renamed PHc. This protein is very likely
homologous to the recently described cryptocyanin of C. magister, a non-respiratory, hemocyanin-related protein associated
with the molting cycle (15). The native PHc protein consists of two
different subunits of 660 and 661 amino acids, respectively, that are
termed PHc-1 and PHc-2. In SDS-PAGE, two polypeptides are consistently
present in approximately equimolar amounts that probably correspond to
each of the PHc cDNAs (Fig. 2). Whereas the nucleotide sequences
are very similar within the coding region for the native protein, they
vary greatly in the signal peptide coding region and in the 3'
untranslated part. This observation is consistent with the assumption
that the two cDNAs are encoded by two genes rather than
representing different alleles in the population.
The conservation of two different, expressed genes provides further
evidence that these hemocyanin-like proteins are functional, although
they do not act as respiratory proteins. The inability of PHc to bind
copper ions and, therefore, form the copper-oxygen complex (Fig.
1B) can now be explained by the replacement of a single
histidine with a tyrosine in copper-binding site A in both types of
subunits, whereas the other copper-coordinating residues are conserved
(Fig. 7). Otherwise, the PHcs closely resemble the crustacean
hemocyanins in sequence and structure. The molecular masses deduced
from conceptual translation (77.0 and 77.3 kDa) of the cDNA clones
are significantly lower than those determined experimentally by
SDS-PAGE (85 and 86 kDa; Fig. 2). However, four potential
N-glycosylation sites are present in each of the PHc subunits (Fig. 4),
indicating a heavily glycosylated protein. By contrast, the crustacean
hemocyanins possess either none or only one glycosylation site per
subunit (Fig. 7).
Although the mRNA for the PHcs accumulates in the ovaries of the
adult female (Fig. 5), there is not sufficient evidence to deduce
sex-specific differences of PHc levels in the hemolymph (Fig. 6). One
can speculate that ovary PHc mRNA may code for some storage
proteins that are transported into the developing eggs. Additional
studies are required to elucidate the potential role of the PHcs in
reproduction. Heart tissue appears to be the principle site of PHc
synthesis in both sexes and is sufficient to produce high amounts of
PHc. However, the PHcs are not constitutively expressed because a
complete absence of this protein was observed in two of eight
individuals, demonstrating that this protein is dispensable (Fig. 6).
One may speculate that the appearance of the PHcs is either associated
with nutritional conditions or, similar to the Cancer
cryptocyanin (15), depends on the molting cycle. Unfortunately, due to
the long molting cycle of H. americanus, a possible
stage-specific analysis of the expression of the PHcs is currently
beyond experimental control.
It has been hypothesized the PHcs might represent the ancestors of the
insect hexamerins (7, 10, 12, 13), copper-free hemolymph proteins that
resemble the hemocyanins in structure and sequence (6, 13) but had lost
the ability to bind oxygen. However, the present phylogenetic analyses
clearly show that the PHcs, as well as the cryptocyanin of C. magister (15), are not particularly related to the hexamerins but
are authentic descendants of the crustacean hemocyanins (Fig. 8). The
trees suggest that PHc and cryptocyanin emerged from the hemocyanins
early in decapodan evolution, before the Astacura (Homarus)
and Brachyura (Cancer) diverged. Although other specific
roles of PHc are possible, it likely acts as some kind of storage
protein. It has been repeatedly observed that hemocyanin concentration
decreases drastically during starvation or molting (36). This does not
necessarily mean that hemocyanins actually act as storage proteins
sensu stricto, but they are dispensable, at least in some
species and under particular developmental or environmental conditions.
The evolutionary advantage of more specialized storage proteins like
the PHcs may be to uncouple the synthesis of oxygen transport proteins
and of proteins that specifically accumulate amino acids and energy.
The fact that hemocyanins were used twice in evolution for similar
purposes may hint at some particular structural advantages, which may
be either their stability in the hemolymph or their high molecular mass
that allows the accumulation of many amino acids with low osmotic impact.