Environmental hypoxia influences hemoglobin subunit composition in the branchiopod crustacean Triops longicaudatus
1 College of Osteopathic Medicine, Touro University, Nevada, Henderson, NV
89014, USA
2 Biology Department, Dominican University, Chicago, IL 60305,
USA
3 Department of Biological Sciences, University of Nevada Las Vegas, 4505
Maryland Parkway, Las Vegas, NV 89154-4004, USA
* Author for correspondence (e-mail: reiber{at}ccmail.nevada.edu)
Accepted 13 July 2005
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Summary |
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Key words: hypoxia, hemoglobin, invertebrate, Triops longicaudatus
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Introduction |
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Hemoglobin structure varies widely among branchiopods. It is a large
extracellular molecule ranging from 220 kDa inArtemia salina
(Moens and Kondo, 1978) to
nearly 800 kDa in Lepidurus apus lubbocki
(Ilan and Daniel, 1979
) and is
composed of various sized subunits. Among the Cladocera and Notostraca, Hb
subunit chains range from 30 to 37 kDa
(Peeters et al., 1990
), with
two heme groups per chain (Ilan and
Daniel, 1979
). In the notostracans Lepidurus apus
lubbocki and Lepidurus bilobatus, the native Hb molecules have
molecular masses of approximately 798 kDa and 680 kDa, respectively, with
subunits in the 33-34 kDa range (Dangott
and Terwilliger, 1979
; Ilan
and Daniel, 1979
). Triops longicaudatus Hb has a
molecular mass of approximately 600 kDa, and Horne and Beyenbach
(1974
) estimated the molecular
mass of the subunits at approximately 20.5 kDa.
The natural history of many crustaceans includes regular bouts of hypoxia,
the response to which includes, but is not limited to, increased ventilation
and perfusion over oxygen exchange tissues, increased cardiac output and/or
reduction in metabolism and the demand for oxygen
(Wheatly and Taylor, 1981;
Hochachka and Lutz, 2001
).
During periods of chronic hypoxia, certain branchiopods such as Daphnia
magna (Fox, 1955
; Zeis,
2003), A. salina (Gilchrist,
1954
; Heip et al.,
1978
) and T. longicaudatus
(Scholnick and Snyder, 1996
;
Harper, 2003
) increase Hb
content; this response has been observed in both experimental and natural
populations (Kobayashi and Hoshi,
1982
; DeWachter et al., 1992). Moreover, branchiopods modify Hb
structure and functional properties in response to hypoxia
(Wolf et al., 1983
; Zeis et
al., 2003). In D. magna and T. longicaudatus, hypoxia
induces an increased Hb oxygen-binding affinity
(Wolf et al., 1983
;
Kobayashi et al., 1994
;
Harper, 2003
; Zeis et al.,
2003). The branchiopod hypoxic response may also include differential Hb
subunit assembly (Kimura et al.,
1999
) and differential subunit expression, as demonstrated in
D. magna (Zeis et al., 2003).
In this study, we assess the hypoxic response and plasticity of Hb expression in a notostracan by examining differences in Hb concentration and subunit expression in T. longicaudatus reared under hypoxic or normoxic conditions. We show that Hb subunit expression changes significantly during developmental hypoxia and when normoxic-reared adults are transferred to a hypoxic environment. Interestingly, the adult response is apparently fixed, as a reversal to normoxic Hb expression patterns does not occur when hypoxic-reared adults are returned to a normoxic environment.
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Materials and methods |
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Hemolymph collection, purification and protein content
Hemolymph samples were collected from sexually mature females (12-13 days
after re-hydration). To induce a hypoxic response in adults, females were
reared to sexual maturity under normoxic conditions and on day 12 were
transferred to hypoxic conditions (5 kPa O2). A normoxic hemolymph
sample was collected on day 12 (prior to transfer), and hypoxic hemolymph
samples were collected once every 24 h post-transfer for 7 days. To assess Hb
changes in hypoxic-reared adults subsequently exposed to normoxia, the reverse
experiments were conducted in which female Triops reared to sexually
maturity under hypoxic conditions were switched to normoxic conditions.
Hemolymph samples were collected from adult animals (12-20 days old)
via dorsal puncture of the heart. Specifically, animals were netted,
blotted dry and the carapace folded back to reveal the dorsal location of the
heart. The heart was punctured with a 28-gauge needle, and hemolymph samples
were collected into capillary tubes, transferred into ice-cold
micro-centrifuge tubes and frozen at -20°C until use. As previously
demonstrated by Horne and Beyenbach
(1971), the major detectable
protein in Triops hemolymph is Hb. To confirm this, samples were
partially purified on a 10 cm gel exclusion column packed with Sephacryl S-300
(Amersham Biosciences, Piscataway, NJ, USA) in buffer containing 0.05 mol
l-1 Tris, 0.1 mol l-1 NaCl, 0.01 mol l-1
MgCl2 and 1 mmol l-1 PMSF and collected in 0.5 ml
fractions. Absorbance peaks of each fraction were measured at 280 nm for the
determination of protein and 415 nm for determination of Hb. Hb samples from
both normoxic- and hypoxic-reared animals were run through the column, and
these partially purified samples were run on SDS-PAGE gels and compared with
non-purified samples. After confirming that the primary protein in
Triops hemolymph is Hb, protein concentrations of whole hemolymph
samples were used to estimate changes in hemolymph Hb content. Protein content
was determined colormetrically, based on the method of Bradford with a kit
from BioRad (500-0001; Hercules, CA, USA).
One-dimensional gel electrophoresis
SDS-PAGE was performed using the Protean II xi Gel Electrophoresis system
(BioRad) with a 10% separating and 4% stacking gel as described by Laemmli
(1970). After determination of
protein content, Hb was diluted in sample buffer containing 62.5 mmol
l-1 Tris HCl (pH 6.8), 25% glycerol, 2% SDS, 0.01% bromophenol blue
and 5% ß-mercaptoethanol. Samples were heated at 95°C for 5 min and
then loaded onto gels. One lane of each gel was loaded with Precision Plus
Dual Color Protein Standards (Bio-Rad #161-0374) with a molecular mass range
from 250 kDa to 10 kDa. Specifically, molecular mass markers at 25 kDa and 37
kDa were used for determination of the molecular mass of the Hb bands.
Following electrophoresis, proteins were visualized using Coomassie Brilliant
Blue and scanned on a Typhoon 9410 Phosphorimager (Amersham Biosciences).
Two-dimensional gel electrophoresis
Hemolymph samples containing 200 µg protein were added to 300 µl of
rehydration media consisting of 7.9 mol l-1 urea, 4.0% CHAPS (w/v),
84.4 mmol DTT, 35 mmol Tris base, 0.0025% bromophenol blue, 2 mol
l-1 thiourea and 0.8% 3.5-10 ampholytes and shaken for 2 h at
30°C. Isoelectric focusing was performed using pre-cast gel strips with a
3-10 immobilized pH gradient (Amersham Biosciences). The samples were then
applied to the gel strips and the strips were rehydrated overnight. Proteins
were focused (Multiphor IEF; Amersham Biosciences) at 500 V for 30 min, 1500 V
for 1 h, 2500 V for 1 h and 3500 V for 48 h. Gel strips were removed and laid
perpendicularly over a 10% SDS-PAGE to separate the proteins by molecular
mass. Two-dimensional gels were stained with Coomassie Brilliant Blue and
scanned on a Typhoon 9410 Phosphorimager (Amersham Biosciences) for later
analysis of the spots using ImageQuant software (Amersham Biosciences).
Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE)
The experimental design and protocol was followed as per manufacturer's
instructions (Amersham Biosciences). Briefly, CyDyes were reconstituted in 1.5
volumes of high-grade N,N-dimethylformamide-d7 (DMF) to a
concentration of 400 pmol ml-1 CyDye. One hemolymph sample each
from a normoxic-reared and a hypoxic-reared animal was labeled with a
fluorescence dye at a ratio of 50 µg protein labeled with 400 pmol fluor.
Samples were labeled as follows: Cy2, a mixture hemolymph from a
normoxic-reared and a hypoxic-reared adult; Cy3, the normoxic-reared hemolymph
sample; Cy5, the hypoxic-reared hemolymph sample. The Cy2-labeled pooled
sample served as an internal control on the gel for the DeCyder analysis.
After labeling, 50 µg of each of the three labeled samples was added to sample buffer (as described above in 2D gel methodology) and loaded onto pre-cast gel strips with a 3-10 immobilized pH gradient. The samples were then subject to 2D gel electrophoresis as described previously and scanned with the phosphorimager in the 2D-DIGE mode at wavelengths appropriate for each of the CyDyes. All spot picking and image analysis of the gel was performed using DeCyder software (Amersham Biosciences) developed specifically for 2D-DIGE gel analysis using an internal standard experimental design. DeCyder software scans the entire gel and outlines all detectable areas. Areas that are upregulated greater than 2.5 times are outlined in blue and those downregulated greater than 2.5 times are outlined in red.
NH2-terminal sequencing
Hemolymph proteins were isolated using 2D gel electrophoresis under
denaturing conditions. Gels were electro-transferred to polyvinylidine
fluoride (PVDF) membranes (TransBlot electrophoretic transfer cell; BioRad).
Transferred spots were visualized with Coomassie Brilliant Blue, cut and sent
to the Nevada Proteomics Facility (Reno, NV, USA). Sequence analysis was
performed using automated Edman degradation on an Applied Biosystems (Foster
City, CA) Precise 492 sequencer.
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Results |
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Hypoxic-reared animals had a significantly greater [Hb protein] (37.7±1.3 mg ml-1; N=23) than normoxic-reared animals (29.3±1.6 mg ml-1; N=20; P<0.001) (Fig. 2). In the transfer experiments, normoxic-reared adults showed no change in [Hb protein] during the first 3 days after transfer to hypoxia but significantly increased [Hb protein] 4-7 days after transfer (F2,25=5.14, P=0.01; Fig. 3A). In the reverse experiments, there were no significant differences in [Hb protein] when hypoxic-reared animals were transferred to normoxic conditions even after 14 days (Fig. 3B; F2,23=0.99; P=0.4).
|
One-dimensional gel electrophoresis
One-dimensional gel electrophoresis revealed that Hb from normoxic-reared
Triops consisted of three primary subunits: two dark staining bands
at 34 kDa (Hb) and 33 kDa (Hbß) and a lighter staining band at 32
kDa (Hb
) (Fig. 4A, lanes
3 and 6). From ImageQuant analysis, the relative proportions of Hb
,
Hbß and Hb
in normoxic animals were 45.5±1.3%,
37.3±2.1% and 14.5±1.1% of total Hb, respectively
(N=7). A faint band at 30 kDa (Hb
), which accounts for less
than 3% of total Hb subunit composition, was present in some normoxic-reared
animals. The same four bands are present in hypoxic-reared animals, but the
relative contribution from each band changes dramatically
(Fig. 4A, lane 12). The higher
molecular mass subunits Hb
and Hbß drop to 30.9±2.1% and
12.6±1.6% of total Hb, respectively, whereas lower-molecular-mass
subunits Hb
and Hb
increase their contributions to
33.4±3.0% and 23.1±1.9%, respectively (N=8).
|
ImageQuant analysis of separated Hb subunits from 4-6 animals per treatment
per day was used to determine the time course of Hb subunit induction in
normoxic-reared animals that were transferred to hypoxia upon sexual maturity.
The intensity of each band (Hb, Hbß, Hb
and Hb
) is
expressed as a percentage of the total intensity of all the bands in each
lane. The relative contribution of Hb
decreases significantly from
normoxic controls by 2 days post-transfer, from 45.5±1.3% to
34.8±1.3% (F8,36=13.17, P<0.001), and
thereafter is not different from hypoxic control values
(Fig. 5A). After 3 days of
hypoxic exposure, the contribution of Hbß decreases significantly from
the normoxic value of 37.3±2.1% to 14.1±1.9%
(F8,36=37.59, P<0.001) and thereafter is not
different from hypoxic-reared animals (Fig.
5B). Hb
increases significantly by 2 days post-transfer,
from 14.5±1.1% to 25.3±2.1% (F8,36=12.27,
P<0.001) (Fig. 5C).
The mean value of Hb
from 3 days post-transfer to 7 days post-transfer
is 30.8±1.4%, a value not significantly different from hypoxic
controls. The induction of Hb
occurs by 3 days post-transfer, when it
increases significantly from normoxic values of 2.78% to 25.1±1.3%
(F8,36=23.01, P<0.001), which is not
significantly different from hypoxic-reared animals
(Fig. 5D). These data
collectively show that the Hb subunit composition of normoxic-reared, sexually
mature animals becomes indistinguishable from hypoxic-reared animals by 3 days
after transfer to chronic hypoxia (Fig.
4A).
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Two-dimensional gel electrophoresis
Hemolymph samples collected from normoxic-reared, hypoxic-reared and
hypoxic-transferred Triops were run on 2D gels to further elucidate
changes in Hb subunit expression. Representative gels of individual animals
are shown in Fig. 6. Based on
an average of four 2D gels from each group, Hb and Hb
increase
from 12% and 0% in normoxic-reared animals to 28% and 22% in hypoxic-reared
animals, respectively. Each 2D gel revealed several isoelectric forms of Hb
subunits, suggesting a typical phosphorylation train
(Halligan et al., 2004
).
|
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NH2-terminal sequencing
Spots were removed from 2D gels of normoxic- and hypoxic-reared animals for
NH2-terminal sequencing, as shown in
Fig. 6. Examination of
NH2-terminal amino acid sequences
(Table 1) revealed differences
in amino acid sequences between Hb subunits, with the sequences for Hb,
Hbß and Hb
being distinctly different. The `trains' of spots that
are of similar molecular mass have similar NH2-terminal sequences,
supporting the hypothesis that these trains of spots are derived from
post-translational events, perhaps phosphorylation. Both spots of Hb
have identical sequences, while there is minor Hb
spot sequence
variation.
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Discussion |
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Increasing Hb concentration affords the animal an increase in
oxygen-carrying capacity (Gilchrist, 1955;
Pirow et al., 2001) and, in
both A. salina and D. magna, the increase in Hb
concentration during hypoxic stress is accompanied by an increased oxygen
affinity (Heip, 1978
;
Kobayashi et al., 1988
).
Similarly, using whole hemolymph samples, under physiological conditions,
T. longicaudatus Hb demonstrates an increase in O2 binding
affinity from P50=1.14 kPa O2 in
normoxic-reared animals to P50=0.5 kPa O2 in
hypoxic-reared animals (1-3 kPa; Harper,
2003
). Horne and Beyenbach
(1971
) reported a
P50 value of
0.91 kPa O2 for hemolymph
samples collected from field populations of T. longicaudatus.
The differential Hb subunit expression between normoxic- and hypoxic-reared
individuals may account for the differences in Hb oxygen-binding affinity in
hypoxic-reared individuals. When exposed to chronic hypoxia, normoxic-reared
adult Triops alter their Hb subunit structure to match that of
hypoxic-reared animals. During this transition, hypoxia induces a nearly
10-fold increase in Hb by 3 days post-transfer and a mean 3-fold
increase in Hb
. Variation in the concentration of these two subunits
largely explains the increases in Hb concentration during hypoxia (Figs
5C,D,
7). DeCyder analysis indicates
no downregulation of the heavier subunits, Hb
and Hbß, and
supports the findings of the ImageQuant analysis that the relative drop in
Hb
and Hbß is due to the upregulation of the lower molecular mass
subunits, Hb
and Hb
.
The relative contribution of Hb drops 2 days post-transfer but then
stabilizes and continues to be an important component of Hb, contributing over
30% to total Hb. Alternatively, the contribution of Hbß drops sharply
from 35% of total Hb to a mean of only 15%. These changes, combined with those
of Hb
and Hb
, result in Hb profiles that do not differ between
normoxic animals 3 days post-transfer to a hypoxic environment and
hypoxic-reared animals. Hypoxic induction of differential Hb subunit
expression has been demonstrated in the cladoceran D. magna, with
both up- and downregulation of different Hb subunits
(Kobayashi et al., 1988
; Zeis
et al., 2003). In A. salina, all three Hb subunits are upregulated
when adult animals are exposed to hypoxia, with the greatest increase in the
subunit with the greatest O2 affinity, Hb III
(van den Branden et al.,
1978
), which is not normally present in adult animals
(Heip et al., 1978
;
Vandenberg et al., 2002
).
Separation of Hb on 2D gels revealed a number of isoelectric forms for
three of the four different molecular mass subunits. The observed pattern was
characteristic of post-translational phosphorylation
(Halligan et al., 2004) and was
similar to a train of spots that has been reported for Moina
macrocopa and D. magna
(Kimura et al., 1999
;
Kato et al., 2001
; Zeis et
al., 2003). While post-translational modification may play a role in
regulating Hb oxygen affinity, these spots may just as likely be due to
differential gene expression, as already demonstrated in D. magna
exposed to hypoxia (Kimura et al.,
1999
).
Sequencing of the NH2-terminal was performed in an effort to
assess whether the isoelectric forms were due to post-translational
modification or differential gene expression. The differences in
NH2-terminal sequences between Hb, Hbß and HB
suggest that these Hb subunits are produced from different genes that may be
differentially expressed upon hypoxic exposure. The similarity in sequence
between Hb
spots 1 and 3 suggests that these two spots have different
isoelectric points due to post-translational modifications. The slight amino
acid differences between Hb
2, 4 and 5 are inconclusive and may be due
to either post-translational modification and/or additional gene regulation.
NH2-terminal sequences of Hb in the daphnid species M.
macrocopa indicate at least three different subunits represented by three
genes (Kato et al., 2001
).
This was confirmed by comparing the amino acid sequences with the amino acid
sequences derived from the translation of nucleotide sequences of M.
macrocopa Hb-encoding genes (Kato et
al., 2001
). There are currently no genetic data available for such
a comparison in Triops.
While differential Hb subunit expression is plastic during development and
inducible in adulthood, the response was not reversed upon transfer of
hypoxic-reared Triops to normoxia. Hypoxic-reared Triops,
when returned as adults to a normoxic environment, showed no changes in the
pattern of Hb subunit expression up to 14 days after return to normoxia, nor
did Hb concentration decrease significantly (Figs
3B,
4B). In D. magna and
A. salina, differences in Hb concentration are often associated with
changes in coloration (Gilchrist,
1954; Kobayashi and Gonoi,
1985
). Hypoxic-reared adult Triops have a visibly deeper
red coloration of their ventral appendages compared with their normoxic-reared
counterparts. When normoxic-reared animals are transferred to hypoxia, there
is a visible increase in the redness of their ventral appendages; however,
there is no obvious decrease in redness when hypoxic-reared animals are
transferred to normoxia. In our experiments, coincident with the hatching of
Triops we observed hatching of the anastrocan Thamnocephalus
platyurus, and we therefore transferred hypoxic-reared T.
platyurus along with Triops. The T. platyurus response
is bidirectional in that hypoxic-reared individuals show a decrease in color
and reduction in hemolymph protein concentration from 28.3±3.6% to
8.4±1.1% after 10 days in normoxia; hypoxic-reared T.
platyurus not transferred to normoxic conditions remain dark red (J.A.G.,
unpublished). Similarly, A. salina and D. magna lower Hb
concentration after a return to high oxygen concentration
(Kobayashi and Hoshi, 1982
).
The mechanism of Hb turnover and/or degradation in branchiopods is not well
understood, although hypoxic-reared Triops are clearly less
responsive to a return to normoxia than T. platyurus, A. salina or
D. magna.
Even so, Triops longicaudatus demonstrates remarkable
developmental plasticity when reared in different oxygen environments, and a
hallmark of this response is differential Hb subunit expression. Adults
transferred to hypoxia are sensitive to changes in oxygen tension, which
induce a change in Hb subunit composition that is similar to the Hb subunits
expressed by hypoxic-reared animals. These subunits may be crucial to
increasing Hb oxygen-binding affinity of hypoxic animals. The recent discovery
that hypoxia-induced Hb synthesis in D. magna is HIF (hypoxia
inducible factor) dependent (Gorr et al.,
2004) could explain the mechanism by which hypoxia induces
differential subunit expression in Triops as well. The finding that
Hb concentration and subunit expression are not reversed upon a return to
normoxia in Triops merits further investigation into the possible
mechanisms and regulation of Hb turnover in branchiopods.
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Acknowledgments |
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