Heterogeneity and Differential Expression under Hypoxia of
Two-domain Hemoglobin Chains in the Water Flea, Daphnia
magna*
Shoko
Kimura
§,
Shin-ichi
Tokishita
,
Toshihiro
Ohta
,
Michiyori
Kobayashi§, and
Hideo
Yamagata
¶
From the
Laboratory of Environmental and Molecular
Biology, Environmental Science Division, School of Life Science, Tokyo
University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan and the § Department of Biology, Faculty of Science,
Niigata University, 8050 Ninocho, Ikarashi,
Niigata 950-2181, Japan
 |
ABSTRACT |
Hemoglobin (Hb) purified from the water
flea, Daphnia magna, reared under hypoxia was analyzed by
two-dimensional gel electrophoresis. The Hb was shown to be composed of
six major subunit chain species (designated as DHbA to DHbF). The
NH2-terminal amino acid sequences of DHbA, DHbB, DHbC, and
DHbF are different from one another, indicating that at least four Hb
genes are present in D. magna. The NH2-terminal
amino acid sequences of DHbD and DHbE are the same as those of DHbA and
DHbB, respectively. The six Hb chains were also found in the animal
reared under normoxia in small amounts and with altered composition;
the extent of decrease under normoxia was higher in the amounts of
DHbC, DHbD, and DHbF than those of others. These results indicate that
the Hb genes are differentially regulated by the ambient oxygen
concentration. Four Hb genes constituting a cluster in the order,
dhb4, dhb3, dhb1, and
dhb2, were found on the chromosome of D. magna.
The complete nucleotide sequences of the dhb1,
dhb2, and dhb3 genes and their cDNAs showed
that the genes have a seven-exon, six-intron structure. The structure consists of an intron separating an exon encoding a secretory signal
sequence, two large repeated regions of a three-exon, two-intron structure that encode each a domain containing a heme-binding site, and
an intron bridging the two repeated regions. The deduced amino acid
sequences of the gene products showed higher than 79% identity to one
another and showed unique features conserved in D. magna Hb
chains. The analysis also suggested that DHbB (or DHbE), DHbF, and DHbC
are encoded by the dhb1, dhb2, and
dhb3 genes, respectively.
 |
INTRODUCTION |
Hemoglobins (Hb)1 are
widely distributed among eukaryotes and also in prokaryotes (1). Their
amino acid sequences reveal that the globin fold has been conserved
throughout evolution. Vertebrates have intracellular tetrameric Hb of
very similar structures. Invertebrate Hb are more diverse in quaternary
structure and oxygen binding properties. They are mostly large
extracellular proteins classifiable by the number of chains and that of
globin domains in each chain. They include single-domain chains
organized as single or multiple subunits, two-domain chains arranged as
multiple subunits, and multidomain chains, also organized as multiple
subunits (2-4). Two-domain or multidomain Hb chains are only found in invertebrates, and their physiological roles and structures are interesting from the viewpoint of Hb evolution. cDNA clones
encoding two-domain intracellular Hb chains from the clam
Barbatia reeveana (5) and Barbatia lima (6) and
two-domain extracellular Hb chain from nematode Pseudoterranova
decipiens (7) have been isolated and their nucleotide sequences
determined. The amino acid sequence of the two-domain extracellular Hb
chain of nematode Ascaris suum has been determined by
protein chemistry (8). The nematode Hb are very unusual: they show the
affinity for oxygen 2 orders of magnitude higher than that of other Hb.
Clam two-domain Hb are also unusual and have disadvantageous properties
as oxygen carrier protein: their two oxygen-binding sites can bind
oxygen but the resulting oxyhemoglobins undergo very rapid autoxidation and tend to precipitate (9). Physiological roles of these unusual Hb
are not clear.
Extracellular Hb composed of multiple two-domain chains with relatively
normal oxygen binding activity playing important roles in oxygen
transport are found in hemolymph of Cladocera, such as water fleas
Daphnia magna and Moina macrocopa (10). These animals show a drastic increase in Hb synthesis in response to a
decrease in the ambient oxygen concentration, which results in a change
in the body color from colorless to red (11), providing an excellent
model system for studying the environmental control of gene expression.
Although the physiological roles, oxygen binding properties, and
quaternary structures of cladoceran Hb have been extensively
investigated (11-13), information on their amino acid sequences has
been scarce. Only the NH2-terminal amino acid sequence of
the second domain of the M. macrocopa Hb chain has been
reported (9). No information about the cladoceran Hb gene was available until recently, when we cloned and analyzed a cDNA encoding a two-domain Hb chain of D. magna (14). D. magna Hb
was reported to be composed of 16 polypeptide chains each carrying two
heme-binding domains (12), whereas the purified Hb was separated into
at least six multimeric species on nondenatured isoelectric focusing (15). These results suggest that D. magna contains several
Hb subunit chains giving rise to several multimeric Hb species. No chemical analysis has been done on the subunit chains.
In this work, we analyzed the heterogeneity and properties of D. magna Hb chains by separating the chains with the aid of two-dimensional gel electrophoresis and by determining the
nucleotide sequences of their genes and cDNAs.
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EXPERIMENTAL PROCEDURES |
Preparation of Hb-containing Crude Extracts and Purification of
Hb from D. magna--
The D. magna strain isolated in
Matsuyama, Japan (16) was used. Hb-rich (red) and Hb-poor (pale)
D. magna were obtained by rearing animals under hypoxia (1.2 ml of O2/liter of water) and normoxia (4.8 ml of
O2/liter of water), respectively, as described previously
(17). Frozen animals were homogenized on ice in 50 mM
Tris-HCl (pH 7.4) containing 1 mM CaCl2 and 1 mM PMSF. After filtration through MIRACLOTH (Calbiochem),
the homogenate was centrifuged at 10,000 × g for 30 min at 4 °C. The supernatant was bubbled with carbon monoxide gas to
convert Hb into the carboxyform and then applied on a DE52 column
(Whatman) equilibrated with 25 mM Tris-HCl (pH 7.4). The
adsorbed Hb was washed with 25 mM Tris-HCl (pH 7.4)
supplemented with 1 mM PMSF and bubbled with carbon
monoxide gas and then eluted with 50 mM Tris-HCl (pH 7.4) containing 100 mM NaCl. The eluted fractions with a red
color containing Hb were collected and combined (Hb-containing crude extract). For the purification of Hb, the crude extract prepared from
10.7 g wet weight of red animals was subjected to gel filtration with 50 mM Tris-HCl (pH 7.4) containing 1 mM
CaCl2 on a column (2.5 cm diameter × 80 cm length) of
Sepharose 6B (Amersham Pharmacia Biotech). Fractions with
A420/A280 values higher
than 3 were collected. The collected fractions were diluted with an
equal volume of water and then applied to a column (1.4 cm
diameter × 21 cm length) of DE52. The adsorbed Hb was washed with 25 mM Tris-HCl (pH 7.4) and then eluted from the column with a
linear gradient of 0-100 mM NaCl in 25 mM
Tris-HCl (pH 7.4) containing 1 mM CaCl2. The fractions eluted at about 40 mM NaCl with
A420/A280 values higher than 4 were collected and combined. The combined fractions (30 ml)
contained 0.087 mg/ml Hb with a purity higher than 95%, as judged from
the profile on sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) followed by staining with Coomassie Brilliant Blue. It was concentrated with the aid of a spin column, UFC3TGC00 (Nihon Millipore, Japan) and used as a sample for electrophoresis.
Gel Electrophoresis--
SDS-PAGE was performed using 8%
separating and 4.5% stacking gels as described by Laemmli (18).
Western blot analysis was performed as described by Burnette (19). For
this analysis, rabbit antiserum produced against M. macrocopa Hb and goat anti-rabbit IgG conjugated with peroxidase
were used for detection. Two-dimensional gel electrophoresis was
performed by the method of O'Farrell (20) with minor modifications.
12.5 µl of the sample solution containing D. magna Hb was
mixed with 15 mg of urea, 3 µl of 10% Nonidet P-40 (Nacalai Tesque,
Kyoto, Japan), and 1.5 µl each of 40% Byolyte (pH 3-10, Bio-Rad)
and 2-mercaptoethanol and then loaded onto a 11-cm isoelectric focusing
tube gel consisting of 8 M urea, 4% polyacrylamide, 2%
Nonidet P-40, 0.4% Biolyte (pH 3-10), and 1.6% Biolyte (pH 5-8).
The bottom buffer (anode) consisted of 10 mM phosphoric
acid and the top buffer (cathode) of 20 mM sodium hydroxide. Proteins were focused at 400 V for 12 h, and then at 800 V for 1 h, after which the gel was layered perpendicularly over an SDS-polyacrylamide slab gel consisting of 4.5% stacking and
8% separating gels.
Quantitation of Hb and Hb Chains--
A crude extract prepared
from red D. magna was subjected to SDS-PAGE and Western
blotting as described above. Total Hb content was determined by
densitometric analysis of the intensity of the detected bands, using
Dual Wavelength Flying Spot Scanning Densitometer CS-9300-PC (Shimadzu
Corp., Japan). Hb purified from red D. magna was used as a
control. The intensity was proportional to the amount of Hb within the
range of amounts used. Hb chain composition was determined also by
densitometric analysis of Hb chains after separation of them by
two-dimensional electrophoresis of the purified Hb and staining with
Coomassie Brilliant Blue. Content of each Hb chain in red animals was
calculated from the total Hb content and the Hb chain composition thus
determined. Content of each Hb chain in pale animals was calculated
from the determined content in red animals and the ratio of intensity
of each chain detected after Western blotting of the crude extract
prepared from pale animals to that detected after Western blotting of
the crude extract prepared from red animals.
Determination of NH2-terminal Amino Acid
Sequences--
Hb chains separated by two-dimensional gel
electrophoresis were transferred to a PVDF membrane and then their
NH2-terminal amino acid sequences were determined as
described by Matsudaira (21) using a protein sequencer, Applied
Biosystems model 477A-120A. For the detection of Cys residues, the
pyridylethylation method described by Cavins and Friedman (22) was
used. Insulin B chain (Wako Pure Chemical Industries, Osaka, Japan)
containing Cys at the 7th position from its NH2 terminus
was used in a control experiment.
Isolation of cDNA Clones and Determination of Their
Nucleotide Sequences--
The construction of a cDNA
expression library and the isolation of clones carrying cDNAs for
Hb chains were described previously (14). Because the 5' region of the
DHb2 cDNA was truncated, the sequence was extended by 5' rapid
amplification of cDNA ends using a Marathon cDNA Amplification
Kit (CLONTECH). Total cDNA prepared from red
D. magna and oligonucleotides, 5'-GGTTCTTTAAGGTCTTTGAT-3' and 5'-GAGAATGCCGGTGCTTTCCT-3', were used as a template and specific primers, respectively. Various restriction fragments obtained from the
cDNA clones were subcloned into the M13 mp10, mp11, and pUC119
vectors (23) and then their nucleotide sequences were determined by the
dideoxy chain termination method using an ABI-377 DNA sequencer (PE
Applied Biosystems). The sequencing was performed for the entire
lengths of both strands, and all the ends of the restriction fragments
used overlapped with one another.
Preparation of Total DNA from D. magna--
Manipulation of DNA
was carried out as described by Sambrook et al. (24) except
where otherwise noted. 4.2 g wet weight of animals frozen in
liquid nitrogen was ground in a prechilled mortar with a pestle and
then suspended in 15 ml of extraction buffer (10 mM
Tris-HCl (pH 9.4), 300 mM EDTA, 1% SDS). The suspension was supplemented with proteinase K (Wako Pure Chemical Industries) to
50 µg/ml and then incubated for 9 h at 50 °C (proteinase K was added at 4 h to a final concentration of 100 µg/ml). After the incubation, the suspension was centrifuged at 3000 × g for 10 min at 25 °C. The supernatant was extracted
three times with an equal volume of phenol at room temperature (30 min,
overnight, and 30 min, respectively), and then extracted once with a
mixture of chloroform, phenol, and isoamyl alcohol (25:24:1). DNA was precipitated with ethanol and then dissolved in 5 ml of 10 mM Tris-HCl (pH 8.0) containing 10 mM EDTA and
1% SDS. The solution was supplemented with proteinase K to 100 µg/ml, incubated for 6 h at 50 °C, and then extracted with
phenol and a mixture of chloroform, phenol, and isoamyl alcohol as
described above. DNA was precipitated with ethanol, dissolved in 2.5 ml
of 10 mM Tris-HCl (pH 8.0) containing 1 mM
EDTA, and then purified by equilibrium centrifugation in a CsCl
gradient. The yield was 12 µg/g wet weight of D. magna.
Chromosome Walking Analysis--
The genomic DNA fragments,
a to e shown in Fig. 5, were amplified by PCR
using a TaKaRa LA PCR Kit Version 2 (Takara Shuzo, Tokyo, Japan). 250 ng of D. magna DNA was used as a template. The nucleotide
sequences of the oligonucleotide primers used were 5'-GGAAGCTTCGCCAAATTCGCT-3' and 5'-CAAAATTTGTTTGCTACGATC-3' (for amplification of fragment a), 5'-GGCCGG-TTTGAACGTCGTCAT-3' and 5'-TTCGATCGCAATTCGTACG-3' (for amplification of fragment b),
5'-GGAAGCTTCGCCAAATTCGCTAATG-3' and 5'-CGACGCGTTCAGTTGGTTGGCCATC-3'
(for amplification of fragment c), 5'-CGCAATTCTACGGAAGAGCATC-3' and
5'-TTTGAGTGCCACGTTTGAAT-3' (for amplification of fragment d), and
5'-AAATTCAAACGTGGCACTCA-3' and 5'-CCATTAGCCGAGGTTGAAAT-3' (for
amplification of fragment e). The nucleotide sequences of the amplified
fragments were directly determined with a Big Dye Terminator Cycle
Sequencing Ready Reaction Kit (PE Applied Biosystems).
 |
RESULTS |
Separation by Two-dimensional Gel Electrophoresis and Determination
of the NH2-terminal Amino Acid Sequences of Hb
Chains--
Hb was purified from D. magna reared under
hypoxia (red). Total amount of Hb contained in red animals was
determined to be 3 mg/g wet weight of animals by Western blotting
analysis of the crude extract using the purified Hb as a standard. To
determine whether D. magna contains heterogeneous Hb chains
or not, the purified Hb was subjected to two-dimensional gel
electrophoresis followed by staining with Coomassie Brilliant Blue. Six
predominant spots were found on the gel (Fig.
1A, denoted as
A-F). Hb chains constituting the six spots were designated
as DHbA to DHbF, respectively, and further analyzed. Although minor
spots were found at the basic side of spot A, and the acidic side of
spots D, E, and F, they have not been characterized. Composition of the
six major Hb chains was determined by a densitometric analysis of Fig.
1A and then the content of each chain in red animals was
calculated. The amounts of DHbA to DHbD were about two times higher
than those of DHbE and DHbF (Table I).
The NH2-terminal amino acid sequences of the six Hb chains
were determined after blotting them onto a PVDF membrane (Fig.
1B). All of the determined sequences contained unusual
repeats of Thr clusters with a Val in between. The sequences of DHbA
and DHbB were the same as those of DHbD and DHbE, respectively. The
sequences of DHbA (or DHbD), DHbB (or DHbE), DHbC, and DHbF were
different from one another, indicating that at least four Hb chain
species produced from different genes are present in D. magna. The first residues of DHbB, DHbE, and DHbF could not be
determined by the phenyl-isothiocyanate method of Edman (25) employed
here, suggesting that they are Cys. These NH2-terminal Cys
were confirmed by comparison of the amino acid sequences with the
nucleotide sequences of cDNAs, as described below.

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Fig. 1.
D. magna Hb chains separated by
two-dimensional gel electrophoresis and their NH2-terminal
amino acid sequences. A, 32 µg of Hb purified from
D. magna reared under hypoxia was subjected to
two-dimensional gel electrophoresis and then stained with Coomassie
Brilliant Blue. The six major Hb chains were designated as DHbA to DHbF
and denoted as A-F, respectively. B, the
determined NH2-terminal amino acid sequences of the Hb
chains, DHbA to DHbF.
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Differential Expression of Hb Chains under Hypoxia and
Normoxia--
Hb-containing crude extracts prepared from D. magna reared under hypoxia (red) and normoxia (pale) were
subjected to two-dimensional gel electrophoresis and then the Hb chains
separated on the gel were detected by Western blot analysis. The
pattern of spots obtained with the extract prepared from red animals
(Fig. 2A) was essentially the
same as that shown in Fig. 1A. This indicates that the
antiserum used in this analysis recognized all of the heterogeneous
D. magna Hb chains. The efficiencies of recognition seemed
to differ depending on Hb chain species. For example, the intensity of
spot E seems to be nearly equal to those of spots A-D in
Fig. 2A, while its intensity was about half of the spots
A-D when the spots were stained with Coomassie Brilliant
Blue (Fig. 1A). The six Hb chains were detected with altered
composition when the extract prepared from pale animals was analyzed
(Fig. 2B), clearly showing that the Hb chain composition was
affected by the ambient oxygen concentration. It must be noted that the
extract used in the analysis shown in Fig. 2B corresponds to
seven times larger amount of animals compared with that used in the
analysis shown in Fig. 2A. Although the efficiency of
detection in Western blotting analysis differs depending on Hb chain
species, the amounts in pale animals of each Hb chain could be roughly
calculated from the ratio of intensity of each spot compared between
Fig. 2A and B, on the basis of the amount of each
chain in red animals determined by staining with Coomassie Brilliant
Blue (see "Experimental Procedures" and Table I). As shown in Table
I, all Hb chains were up-regulated by hypoxia. The amounts of the three
Hb chains, DHbC, DHbD, and DHbF, were relatively smaller than those of
other chains under normoxia and highly increase (14-21-fold) under
hypoxia, while other chains show 5-10-fold increase under hypoxia.
Total amount of Hb chains in pale animals was
of that in
red animals (0.3 mg/g wet weight of animals).

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Fig. 2.
Effect of the ambient oxygen concentration on
the composition of Hb chains. Hb-containing crude extracts were
subjected to two-dimensional gel electrophoresis followed by Western
blot analysis. A, the extract corresponding to 0.33 mg wet
weight of red D. magna (reared under hypoxia) was loaded on
the gel. B, the extract corresponding to 2.3 mg wet weight
of pale D. magna (reared under normoxia) was loaded on the
gel.
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cDNA Analysis--
We previously isolated 11 clones that carry
cDNAs of about 1.2 kb length encoding D. magna Hb
chains, one of which was analyzed (
-DHb1) (14). On analyzing the
other 10 clones, we found two clones containing new species of Hb
cDNAs. The entire nucleotide sequences of the cDNAs contained
by the two clones, designated as
-DHb2 and
-DHb3, have been
determined. The nucleotide sequences of the
-DHb2 and
-DHb3
cDNAs showed 79.4 and 86.6% identity, respectively, with that of
-DHb1 cDNA. Fig. 3 compares the
deduced amino acid sequences of the Hb chains, DHb1, DHb2, and DHb3,
encoded by the three cDNAs. Both DHb2 and DHb3 had a signal
peptide-like sequence at their NH2 terminus and a
two-domain structure similar to DHb1. The NH2-terminal
amino acid sequences of the predicted mature forms of DHb1, DHb2, and
DHb3 exactly matched the above described NH2-terminal
sequences of DHbB (or DHbE), DHbF, and DHbC, respectively. The
calculated molecular weights of the mature forms of DHb2 and DHb3 were
36,177 and 36,217, respectively, which was very close to that of DHb1
(36,228). Several key amino acids that are invariant in all or most Hb
from other organisms and required for functional heme binding are
conserved in each of the two domains. The identities of the amino acid
sequences between DHb1 and DHb2 and DHb1 and DHb3 are 83.1 and 92.4%,
respectively. Fig. 4 shows the identities
of the amino acid sequences among the Hb domains of invertebrates
containing two-domain Hb chains. The reported identity between the
amino acid sequences of the Hb domains of invertebrates belonging to
different genera is generally low (less than 21% identity), while that
between the amino acid sequences of two domains of the same Hb chain is
much higher (60 to 80%; Fig. 4, underlined). A unique
feature conserved in D. magna Hb chains is that the identity
between the amino acid sequences of the first and second domains,
either in the same chain or in different chains, is exceptionally low
(21-24%; Fig. 4, shadowed), while the identity between the
amino acid sequences of the corresponding domains in different chains
(D1/D1 or D2/D2) is high (78-92%).

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Fig. 3.
The amino acid sequences of D. magna Hb chains deduced from the nucleotide sequences of
cDNAs. The amino acid position numbers are shown at the
right. The gaps denoted by hyphens were
introduced into the amino acid sequences to obtain maximum matching.
Amino acid residues conserved in any two of the three sequences are
shadowed. Those invariant in all or most Hb from other
organisms are indicated by asterisks. The partial amino acid
sequence of DHb4 was predicted from the genomic sequence by assuming
its homology with those of other Hb chains.
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Fig. 4.
Percentage identity between the amino acid
sequences of various invertebrate Hb domains. The amino acid
sequences corresponding to that from Ser20 to
Lys175 of DHb1 shown in Fig. 3 are compared.
Abbreviations: D1, first domain; D2, second
domain; B.l., clam B. lima two-domain
chain (6); A.s., nematode A. suum two-domain
chain (8).
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Genomic Sequences of Hb Chains--
Chromosomal walking analysis
was carried out by means of PCR, for which total DNA prepared from
D. magna was used as a template and oligonucleotides
synthesized according to the nucleotide sequences of the above
described cDNAs were used as primers. Five genomic fragments, a to
e, were amplified. The nucleotide sequences of the amplified fragments
showed that there are four Hb genes (designated as dhb1 to
dhb4) on the D. magna chromosome in the same
direction and in the order of dhb4, dhb3,
dhb1, and dhb2, with very short intergenic
regions of a few kb (Fig. 5A).
dhb1, dhb2, and dhb3 are the genes for
the cDNAs carried by
-DHb1,
-DHb2,
-DHb3, respectively. A
fragment containing the 3' region of the dhb4 gene was
fortuitously amplified by using a 5' primer whose sequence was
conserved in the three cDNAs. Amplification of DNA fragments containing the 5' region of the dhb4 gene was unsuccessful.
The sequences of the dhb1, dhb2, and
dhb3 genes, compared with their cDNAs, showed that they
have a seven-exon, six-intron structure (Fig. 5B). A similar
structure was reported for the gene of a two-domain Hb chain in the
clam, B. reeveana (5). An intron bridging the two large
repeated regions is conserved in both organisms. The organizations of
the two large repeated regions encoding the two heme-binding domains
are similar in both organisms, having the three-exon, two-intron
structure characteristic of animal Hb genes (26). However, in contrast
to the first intron located in the 5'-untranslated region of the
B. reeveana gene, the first intron separates an exon
encoding a secretory signal sequence in D. magna Hb genes.
The lengths of introns were much shorter in D. magna genes
(less than 100 bp on average), which, together with the short lengths
of the intergenic regions, makes the gene cluster very compact. The
positions of introns were perfectly conserved in the three Hb genes.
Several sequences homologous to the octameric binding motif
(TACGTGCT, underlined nucleotides were forced to be
conserved) for mammalian hypoxia inducible factor-1, HIF-1 (27), were
found in each of the intergenic regions (Fig. 5A).

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Fig. 5.
Organization of cluster and structures of
D. magna Hb genes. A, organization of
the D. magna Hb gene cluster. Open arrows
indicate the positions and directions of the Hb genes. Bars
at the top, a-e, denote the positions and
lengths of the genomic DNA fragments amplified by PCR. At the
bottom in parentheses are the lengths of the
genes (transcribed regions) and intergenic regions shown.
Vertical bars in the intergenic regions represent HIF-1
binding motifs (marked + or , depending on the strand they are
located on) and the putative TATA box (denoted by
asterisks). B, the exon, intron structure common
to D. magna Hb genes. The shadowed bars,
E1-E7, and open bars, I1-I6,
indicate exons and introns, respectively. The numbers at the
top in parentheses show the lengths of the exons
and introns in the dhb2 gene (base pairs).
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 |
DISCUSSION |
Two-dimensional gel electrophoresis of Hb purified from D. magna clearly demonstrated that the animal contains six chemically different Hb chains, while analyses of the NH2-terminal
amino acid sequences of Hb chains and the genomic DNA sequences only confirmed the presence of four Hb genes. Analysis is in progress to
determine whether each Hb chain constitutes a homomultimeric Hb or Hb
chains are mixed and constitute heteromultimeric Hb. It is not clear at
present whether DHbA and DHbB are the products of the same genes as
those for DHbD and DHbE, respectively, showing different migration on
two-dimensional gel electrophoresis due to post-translational
modification, or they are the products of different genes having the
same NH2-terminal amino acid sequences. D. magna
may contain more than four Hb genes, or it may contain only four Hb
genes giving rise to six Hb chain species through some
post-translational modification. Assuming that the latter is the case,
one of the possible sites of modification is the NH2-terminal Cys of DHbB, DHbE, and DHbF. Detection of
these NH2-terminal Cys after pyridylethylation of their
sulfhydryl groups (22) was unsuccessful, while Cys in the insulin B
chain was clearly detected in a control experiment. The sulfhydryl
groups of these Cys might have undergone some modification that blocks pyridylethylation.
In our previous work (14), red D. magna was shown to contain
a more than 12 times higher amount of Hb mRNA as compared with pale
animals. The differential up-regulation by hypoxia of Hb chains
demonstrated here is possibly a result of the transcriptional control
of Hb genes. Post-translational modification of Hb chains, if any, may
also be affected by hypoxia. Oxygen-responsive genes are found in a
broad range of organisms, from bacteria to humans, and investigation of
the molecular mechanisms underlying their regulation is one of the
important and rapid-growing fields in current molecular physiology
(27). The analysis of the cis-elements and trans-acting factors
involved in the transcriptional regulation of Hb genes in D. magna is of particular interest. HIF-1 is a trans-acting factor
playing a critical role in the hypoxic induction of several
physiologically important genes in mammalian cells (27). Functional
analysis of HIF-1 binding motifs found in the intergenic regions of the
D. magna Hb gene cluster is in progress.
The amino acid sequences deduced from the nucleotide sequences of the
cloned cDNAs show unique features of D. magna Hb chains. The exceptionally low identity between the amino acid sequences of the
first and second domains suggests that the duplication of a
single-domain Hb gene in D. magna occurred a very long time ago and then the resulting two single-domain Hb genes fused to form a
two-domain Hb gene. The multiplication of the two-domain Hb gene seems
to have occurred relatively recently, because the identity of the amino
acid sequences as a whole between different chains is high. Supporting
this notion, phylogenetic analysis showed that the first domains and
second domains of D. magna Hb chains constitute different
clusters (Fig. 6). The three-exon, two-intron structure encoding the two domains is conserved in all
D. magna Hb genes, further indicating the dominance of this structure among animal globin genes (26). Another unique feature conserved in D. magna Hb chains is the presence of an
unusual NH2-terminal extension. This extension is
reminiscent of the COOH-terminal extension found in the two-domain Hb
chain of a parasitic nematode, A. suum, containing a repeat
of the sequence, Glu-Glu-His-Lys (8, 28). This extension, named the
polar zipper sequence, was proposed to be joined in an eight-stranded
barrel at the center of the molecule. The repeated Thr clusters and
a Val residue in between them in the NH2-terminal extension
of D. magna Hb chains may also play a role in the
multimerization of 16 subunit chains to yield a functional Hb molecule
through hydrophilic and hydrophobic interactions of the side chains of
Thr and Val, respectively.

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Fig. 6.
A phylogenetic tree of the amino acid
sequences of various invertebrate Hb domains. The amino acid
sequences of the Hb domains of clam B. reeveana (5) and
nematode P. decipiens (7), and domain E1 of
Artemia Hb (29), in addition to those compared in Fig. 3,
were analyzed. The tree was constructed by using the PAUP (Phylogenetic
Analysis Using Parsimony) program developed by Swofford (30). The amino
acid sequence of Paramecium Hb (31) was used as an outer
group sequence. The numbers on the bars indicate
the levels of confidence (%).
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FOOTNOTES |
*
This work was supported by Grants-in-aid for Scientific
Research from the Ministry of Education, Science, Sports and Culture of
Japan (09839034 and 09760101). Support was also provided by The
Sumitomo Foundation and Higeta Shoyu Co., Ltd.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/EMBL Data Bank with accession number(s) U67067, AB021134, AB021136, and AB021137.
¶
To whom all correspondence should be addressed. Tel.:
81-426-76-7053; Fax: 81-426-76-7081; E-mail:
yamagata{at}ls.toyaku.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
Hb, hemoglobin(s);
PMSF, phenylmethanesulfonyl fluoride;
PAGE, polyacrylamide gel
electrophoresis;
PVDF, polyvinylidene difluoride;
PCR, polymerase chain
reaction;
kb, kilobase pair(s);
HIF-1, hypoxia-inducible
factor-1.
 |
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