Cytoplasmic carbonic anhydrase isozymes in rainbow trout Oncorhynchus mykiss: comparative physiology and molecular evolution
1 Department of Biology, Queen's University, Kingston, ON, Canada K7L
3N6
2 Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N
6N5
3 Department of Biology, Carleton University, Ottawa, ON, Canada K1S
5B6
* Author for correspondence (e-mail: esbaugha{at}biology.queensu.ca)
Accepted 21 February 2005
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Summary |
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Key words: carbonic anhydrase, red blood cell, gill, isozyme, evolution, anaemia
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Introduction |
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CA activity was originally found in the gills of fish, over 60 years ago
(Sobotka and Kann, 1941).
Branchial CA activity is thought to be largely cytoplasmic, occurring
primarily in pavement cells and chloride cells
(Lacy, 1983
;
Conley and Malalatt, 1988
;
Rahim et al., 1988
;
Flügel et al., 1991
;
Sender et al., 1999
;
Wilson et al., 2000
), with the
exception of membrane bound isozymes found in the gills of elasmobranchs
(Swenson and Maren, 1987
;
Gilmour et al., 1997
,
2001
,
2002
;
Henry et al., 1997
;
Wilson et al., 2000
), and
Antarctic fishes, Chaenocephalus aceratus and Notothenia
coriiceps (Tufts et al.,
2002
). Evidence is mixed on whether the same CA isozyme is present
in both rbcs and gill tissue. Rahim et al.
(1988
) provided immunological
evidence of a gill CA isozyme in rainbow trout Oncorhynchus mykiss
and carp Cyprinus carpio that was distinct from the rbc CA isozyme.
By contrast, Sender et al.
(1999
) found that the gill and
rbc CA enzymes in the flounder Platichthys flesus were not
immunologically distinct. Recently, however, Esbaugh et al.
(2004
) provided further
evidence of CA activity in the gill cytoplasm of rainbow trout that was not
that of the rbc CA isozyme. It is therefore unclear what isozyme is
responsible for the gill cytoplasmic CA activity in teleosts.
Blood and branchial CA activities serve different functions. The primary
physiological role of rbc CA is to catalyse the hydration of CO2 to
HCO3- at the tissue site of production, and dehydration
of HCO3- to CO2 at the respiratory surface,
to facilitate the transport and excretion of CO2 from the body
(Perry, 1986;
Perry and Laurent, 1990
;
Henry and Heming, 1998
;
Tufts and Perry, 1998
;
Henry and Swenson, 2000
).
Selective pressures preventing the rate of these reactions from limiting
CO2 transport and excretion are believed to be the primary forces
driving the increase in rbc CA catalytic rate that is apparent through the
fish lineage (Henry et al.,
1993
; Tufts et al.,
2003
). In contrast, the primary purpose of cytoplasmic gill CA is
to catalyse the hydration/dehydration reactions of CO2 within the
branchial epithelium to provide counter ions for ion exchange processes that
regulate acid-base balance and ionic homeostasis
(Henry and Heming, 1998
;
Henry and Swenson, 2000
;
Marshall, 2002
). A notable
exception, however, is the membrane-associated CA in the gills of dogfish,
which contributes to CO2 excretion
(Gilmour et al., 2001
).
The main objective of this study was to determine if branchial CA activity
in rainbow trout was the result of a general cytoplasmic CA isozyme, with
kinetic properties, tissue distribution and physiological functions distinct
from those of the rbc-specific CA isozyme. In particular, the trend from
agnathans to teleosts towards a faster rbc CA isozyme
(Henry et al., 1993;
Tufts et al., 2003
), leads to
the prediction that a trout general cytoplasmic CA isozyme with a wide tissue
distribution will exhibit a lower turnover number (slower catalytic rate) than
the rbc-specific isozyme. In addition, a second series of experiments tested
the differential regulation of the two cytoplasmic CA isozymes in response to
a physiological disturbance, anaemia.
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Materials and methods |
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To induce anaemia, rainbow trout (61.5±1.5 g, N=23; mean ± S.E.M.) were lightly anaesthetised in a solution of ethyl-p-aminobenzoate (0.1 g l-1) and 1 ml of blood was removed by caudal puncture. Fish were then placed in a single 0.5 m diameter holding tank. At each of 12 h, 72 h, and 15 days, six fish were removed from the holding tank, and blood and gill tissues were sampled as described below. Haematocrit was measured in duplicate at the time of sampling, using microcapillary tubes centrifuged at 6000 g for 6 min.
Tissues were collected from individual rainbow trout that were
anaesthetized in either CO2-saturated water (kinetic analyses), or
0.1 g l-1 of ethyl-p-aminobenzoate (molecular analyses).
Blood was collected into a heparinized syringe by caudal puncture and
transferred to a microfuge tube. The rbcs and plasma were then separated by
centrifugation and immediately frozen in liquid nitrogen. The rbc pellets used
in kinetic analyses were washed three times with saline prior to being frozen,
to ensure that no other blood components were present. The gills, along with
other tissues (heart, brain, gut, liver, spleen, anterior and posterior
kidney), were removed after perfusing the body with saline to clear it of
blood. Perfusions were performed by exposing and cannulating the bulbus
arteriosus with polyethylene tubing (PE 160; Clay-Adams, Missaussauga, ON,
Canada), and using a peristaltic pump to pump 100 ml of heparinized (50 i.u.
ml-1 sodium heparin) Cortland's saline
(Wolf, 1963) into the body,
followed by 1 l of non-heparinized Cortland's saline. Immediately after
cannulating the bulbus arteriosus, the ventricle was severed to allow fluid in
the circulatory system to drain from the body. Upon sampling, all tissues were
carefully examined for blood clots; any observed were removed. Tissue samples
were then frozen in liquid nitrogen and stored at -80°C.
Series I: kinetic analysis of rainbow trout gill and rbc cytoplasmic CA
Tissue homogenization and fractionation
To facilitate homogenization, adult trout tissues (1-2 g;N=4) were
cut into fine pieces using scissors and a scalpel. The tissue was then added
to 8 volumes of refrigerated Tris buffer (in mmol l-1: 225
mannitol, 75 sucrose, 10 Tris base, adjusted to pH 7.4 using 10% phosphoric
acid) per gram tissue and homogenized using a motor-driven Teflon-glass
homogenizer until no pieces of tissue remained (approximately 5 passes). Next,
the crude homogenate was centrifuged (100 000 g for 90 min;
Beckman L8-55M ultracentrifuge; Henry et
al., 1993) at 4°C to remove cellular debris, mitochondria and
membrane fractions from the tissue cytoplasmic fraction. The cytoplasmic
fractions were then examined to determine the relative levels of CA and
haemoglobin.
Measurement of carbonic anhydrase activity and haemoglobin concentration
Carbonic anhydrase activity was measured using the electrometric pH
method (Henry, 1991
;
Henry et al., 1993
). The
reaction medium consisted of 10 ml of Tris buffer kept at 4°C. After the
enzyme source was added, the reaction was started by the addition of 400 µl
of CO2-saturated distilled water from a 1000 µl gas-tight
Hamilton syringe. The reaction velocity was measured over a pH change of 0.15
units. To obtain the true catalysed reaction rate, the uncatalyzed rate was
subtracted from the observed rate, and the buffer capacity was taken into
account to convert the rate from pH units time-1 to mol
H+ time-1. The pH was measured using a Radiometer GK2401
C combined pH electrode connected to a Radiometer PHM64 research pH meter.
Haemoglobin concentration was measured using Drabkin's method (Sigma,
Oakville, CA, USA) with cyanomethaemoglobin (Sigma) as a standard.
Kinetic analysis
To determine the kinetic properties of the rbc and gill cytoplasmic CAs,
experiments were conducted to examine the velocity of CO2 hydration
at increasing concentrations of CO2. The reciprocals of these
values were plotted on a Lineweaver-Burke plot
(Maren et al., 1980;
Henry et al., 1993
), from
which the Vmax and Km values were
obtained. The enzyme units (eu) were kept between 1 and 2
(Maren et al., 1960
), and
these values were recorded for each trial.
The enzyme concentration was obtained by measuring CA activity in the
presence of different concentrations of acetazolamide (Az), a potent CA
inhibitor. These data were then plotted on an Easson-Stedman plot
(Easson-Stedman, 1937), using
the equation:
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where Io is the inhibitor concentration, i is
the fractional inhibition at a given inhibitor concentration,
Ki is the inhibition constant, and Eo
is the concentration of enzyme (Maren et al.,
1960,
1980
;
Henry et al., 1993
). For each
inhibitor concentration, assays were performed in duplicate and the mean
activity was plotted. Eo and Ki Az
values were calculated for each sample. For each trial, the eu value was
determined and a ratio of Eo/eu was then calculated; the
Eo of further samples could then easily be determined
based on the calculated eu (Maren et al.,
1980
,
1993
).
The catalytic rate constant (kcat) was then calculated
using the formula:
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as described by Maren et al.
(1980). The inhibition
constant for chloride was also calculated, using the method of Dixon
(1953
). Mean values of
kcat, Km, Ki Az
and Ki Cl- were obtained for the four samples
utilised. To examine the sensitivity of each CA isozyme to the rainbow trout
plasma inhibitor of CA (pICA), CA activity in the cytoplasm of the rbcs and
gills was assayed in the presence of increasing volumes of separated trout
plasma.
Series II: molecular analysis of rainbow trout gill cytoplasmic CA
Determination of cDNA sequence
The following procedures were performed independently by groups at Queen's
University (using gill tissue), and the University of Ottawa (using whole
blood). Total RNA was extracted from rainbow trout gills and whole blood by
the acid/phenol method of Chromczynski and Sacchi
(1987), as modified for fish
blood by Currie et al. (1999
),
or using Trizol (Invitrogen, Burlington, ON, Canada).
First strand cDNA was synthesized from purified rainbow trout RNA from gill and whole blood using AMV reverse transcriptase (RT) and random primers. A 333 bp internal segment of rainbow trout CA coding region was amplified by PCR at an annealing temperature of 50°C, using a forward primer (CA-F; 5'-CAG TTC CAT TTC CAT TGG GG-3') and reverse primer (CA-R; 5'-CAG AGG AGG GGT GGT CAG-3'). All PCR reactions involved an initial denaturation at 94°C for 30 s followed by 30 cycles of: 94°C for 30 s; annealing temperature for 60 s; 72°C for 90 s, and ending with a final extension for 10 min at 72°C. Both the forward and reverse primers were designed on the basis of high sequence identity among zebrafish CA (GenBank, U55177), gar Lepisosteus osseus rbc CA (GenBank, AY125007), human CA I (GenBank, X05014), and human CA II (GenBank, J03037). The resulting PCR product was ligated into a pDrive vector (Qiagen, Missaussauga, ON, Canada) and sequenced. This sequence information was used to perform 3' rapid amplification of cDNA ends (RACE). The cDNA for 3' RACE was amplified using the 3' RACE adapter primer (Invitrogen) and Superscript II (Invitrogen). The 3' sequence was amplified with nested PCR using the Abridged Universal Amplification primer (Invitrogen), and CA forward primers (1st round) (5'-CCT TGC TGT TGT AGG AGT CTT C-3') and (2nd round) (5'-GGT CCT TGA TGC TTT TGA TG-3'). The 3' RACE product was ligated into a PCR 2.1 vector (Invitrogen) and sequenced.
The 3' cDNA sequence and a GenBank 5' cDNA sequence for a rainbow trout CA homologue (CB94032) were combined to yield a 780 bp coding region (TCAc). The complete coding region sequence was entered in GenBank (AY514870).
Northern blot analysis
For northern blots, 10 µg of total RNA was fractionated by
glyoxal/dimethyl sulphoxide (DMSO) denaturing electrophoresis on a 1% agarose
gel and transferred to a Duralon nylon membrane (Stratagene, Missaussauga, ON,
Canada) using 20x standard saline citrate (SSC). Membranes were
ultraviolet-crosslinked (Fisher UV crosslinker) twice at optimal setting prior
to hybridization.
Probes for rbc CA (TCAb) and ß-globin (a haemoglobin subunit) were
generated from first strand cDNA from rainbow trout rbc mRNA. A 446 base pair
probe for ß-globin was amplified as described by Lund et al.
(2000). Both the TCAb and TCAc
probes were 333 base pair fragments that were amplified using the CA-F and
CA-R primers, as previously described. Probes were labelled using
[
-32P]dCTP (specific activity 109 cts
min-1 µg-1 DNA) and the Ready-To-Go labelling system
(Pharmacia, Piscataway, NJ, USA). Membranes were prehybridized at 60°C for
3 h in Church's buffer. Blots were then hybridized overnight in the same
solution at 60°C, with approximately 109 cts min-1
of denatured probe. The blots were then washed twice using 1xSSC/0.1%
SDS solution (20 min, 60°C) and once using 0.25xSSC/0.1% SDS (20
min, 60°C). Finally, blots were exposed to a phosphor screen (Kodak,
Rochester, NY, USA) and visualized and quantified using a phosphoimager
(Molecular Devices, Sunnyvale, CA, USA) driven by ImageQuant software. All
membranes were also probed with a human 18S rRNA probe
(Battersby and Moyes, 1998
) to
correct blots for loading differences, and were expressed relative to the band
with the greatest density.
Real-time PCR
Total RNA was extracted from 30 mg aliquots of powdered tissue samples
using the Absolutely RNA RT-PCR Miniprep Kit (Stratagene). To remove any
remaining genomic DNA, the RNA was treated on-column using RNase-free DNase (5
µl) for 15 min at 37°C. The RNA was eluted in 70 µl of nuclease-free
H2O and its quality was assessed by gel electrophoresis and
spectrophotometry (Eppendorf, Missaussauga, ON, Canada). cDNA was synthesized
from 2 µg of RNA using random hexamer primers and Stratascript reverse
transcriptase (Stratagene).
TCAb, TCAc and haemoglobin mRNA levels were assessed by real time PCR on samples of cDNA (0.5 µl) using a Brilliant SYBR Green QPCR Master Mix Kit (Stratagene) and a Stratagene MX-4000 multiplex quantitative PCR system. ROX (Stratagene) was used as a reference dye. The PCR conditions (final reaction volume, 25 µl) were as follows: 0.5 µl cDNA template, 300 nmol l-1 forward and reverse primer, 12 µl 2xMaster Mix, 1:30000 ROX final dilution. The annealing and extension temperatures over 40 cycles were 58°C (30 s) and 72°C (30 s), respectively. The following primer pairs were designed using Primer3 software: ß-actin forward (5'-CCA ACA GAT GTG GAT CAG CAA-3'), ß-actin reverse (5'-GGT GGC ACA GAG CTG AAG TGG TA-3'), TCAc forward (5'-CAG TCT CCC ATT GAC ATC GTA-3'), TCAc reverse (5'-CGT TGT CGT CGG TGT AGG T-3'), TCAb forward (5'-TTG GCT TTG TGG ATG ATG TT-3'), TCAb reverse (5'-AGG GGA ACT TGA TTC CAT TG-3'), haemoglobin forward (5'-ATG GTC GAC TGG ACA GAT CC-3'), haemoglobin reverse (5'-CTG AGT CCA TGG AGA CAC GA-3').
The specificity of the primers was verified by the cloning (TOPO TA cloning
kit; Invitrogen) and sequencing of amplified products. To ensure that SYBR
green was not being incorporated into primer dimers or non-specific amplicons
during the real-time PCR runs, the PCR products were analysed by gel
electrophoresis in initial experiments. Single bands of the expected size were
obtained in all instances. Furthermore, the construction of SYBR green
dissociation curves after completion of 40 PCR cycles revealed the presence of
single amplicons for each primer pair. To ensure that residual genomic DNA was
not being amplified, control experiments were performed in which reverse
transcriptase was omitted during cDNA synthesis. Relative expression of mRNAs
was determined (using actin as an endogenous standard) by a modification of
the delta-delta Ct method (Pfaffl,
2001). Amplification efficiencies were determined from standard
curves generated by serial dilution of plasmid DNA.
Sequence analysis
The TCAc sequence was compared with TCAb (GenBank, AY307082), gar rbc CA,
zebrafish retina CA, and dace (Tribolodon hakonensis) gill CA
(GenBank, AB055617) sequences, as well as human CA I, CA II and CA VII
(GenBank, AY075019). Alignment of the amino acid sequences was performed using
ClustalW (version 1.8) multiple sequence alignment. In addition, a comparative
analysis of the active sites was performed between TCAc, TCAb, gar rbc CA and
dace gill CA, as well as human CA VII and consensus CA I and CA II sequences,
as reported by Tashian et al.
(2000).
A phylogenetic analysis of amino acid sequences was also carried out, which
included rainbow trout TCAb and TCAc, gar rbc CA, dace gill CA and zebrafish
retina CA. This analysis also included: mouse CA I (GenBank, NM_009799), CA II
(GenBank, BC055291), CA III (GenBank, NM_007606), CA Vb (GenBank, NM_019513)
and CA VII (GenBank, NM_053070); human CA I, CA II, CA III (GenBank,
NM_005181), CA Va (GenBank, NM_001739), CA Vb (GenBank, NM_007220) and CA VII;
rat CA I (GenBank, XM_226922), CA II (GenBank, NM_019291), CA III (GenBank,
NM_019292), CA V (GenBank, NM_019293) and CA VII (GenBank, XM_226204), and
chicken CA II (GenBank, X12639), Xenopus CA II (GenBank, BC041213)
and zebrafish CA VII (BC049309). Alignment used for the phylogenetic analysis
was performed by ClustalX (version 1.81). Phylogenetic hypotheses were
constructed using both neighbour joining (NJ;
Saitou and Nei, 1987) and
maximum parsimony (MP) as performed by PAUP* (beta test version
4.0b10; Swofford, 2000
). MP
analysis consisted of a heuristic search with TBR branch swapping and ACCTRAN
character state optimization enforced, and with random stepwise addition and
1000 random addition replicates. NJ was performed on a matrix of mean
character distances. Support for nodes for both analytical procedures was
performed using the bootstrap analysis with 1000 pseudoreplicates. All
analyses were performed using mouse and human CA VII as outgroups, as
previously described by Hewitt-Emmett and Tashian (1996).
Gaps in sequence alignment were accounted for in three distinct series of analyses. In the first analysis, all possibly informative gaps were included and treated as missing data. In the second analysis, all gaps were removed, and in the third analysis, all gaps were treated as a distinct character state. The final analysis could only be performed using MP analysis. All subsequent trees were compared qualitatively for differences, with no major differences arising.
Statistical analysis
Values are expressed as means ±
S.E.M. Statistical differences in the kinetic
properties and inhibitor sensitivities of TCAb and TCAc were analysed using
unpaired Student's t-tests. One-way analysis of variance (ANOVA)
followed by post hoc multiple comparisons using the Bonferroni test,
as appropriate, were used to statistically analyse the effect of anaemia or
acid infusion on relative rbc or gill mRNA expression of TCAb and TCAc. In all
analyses, the fiducial level of significance was 5%.
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Results |
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NJ and MP analyses of vertebrate cytoplasmic CAs produced generally well supported phylogenetic trees of similar topology (Fig. 2). These analyses suggested that CAs I, II and III constitute a single monophyletic clade, while the fish cytoplasmic CAs (with the exception of zebrafish CA VII) constitute a separate clade. The fish cytoplasmic CA clade is basal to that of other vertebrate CAs (CA I, II, and III), but appears after the divergence of CA V and VII. Within the fish CA group, slight differences in topology were obtained with the two different approaches. NJ analysis revealed that TCAb and zebrafish retina CA were closely grouped, and TCAc and dace gill CA grouped together. The gar rbc CA was the most ancestral sequence. The tree formed by MP analysis showed a similar topology (tree not shown), with the exception that dace gill CA and TCAc did not group together, but diverged after gar rbc CA and prior to the TCAb/zebrafish retina CA group. It should also be noted that zebrafish CA VII grouped most closely with mammalian CA VII rather than with the fish cytoplasmic CA clade.
|
The last aspect of the sequence analyses involved a comparison of the
active site of TCAc with those of other fish CAs, as well as those from
consensus CA I and II (Tashian et al.,
2000) and human CA VII sequences. The active site of the TCAc
sequence was most similar to the TCAb and dace gill CA sequences, differing at
only one amino acid residue, while differing at two amino acid residues from
the gar rbc CA sequence (Fig.
3). When compared to the mammalian CA active sites, the TCAc
sequence was most similar to CA VII, differing by three amino acid
residues.
|
Tissue distribution
The tissue distributions of both TCAb and TCAc were examined using northern
blot analysis (Fig. 4) and
real-time RT-PCR (Fig. 5) of
perfused trout tissues. These analyses indicated that the TCAb isozyme was
expressed almost exclusively in the rbc. Low levels of expression in the
spleen and anterior kidney (Fig.
4), or heart and brain (Fig.
5) could be accounted for by blood that was not removed during
saline perfusion. Blood contamination is, in fact, indicated by corresponding
expression of haemoglobin in the spleen, anterior kidney
(Fig. 4), brain and heart
(Fig. 5). By contrast, the TCAc
isozyme exhibited a wider tissue distribution. Although gill was the
predominant site of expression of TCAc, brain tissue also displayed
substantial expression, with low levels found in the kidney, gut, liver and
muscle (Figs 4 and
5). Unlike northern blot
analysis, real-time RT-PCR also revealed low TCAc expression in the rbcs. A
comparison of the abundance of each isozyme in the rbcs, using real-time
RT-PCR, indicated that TCAb was 666±183 times (N=6) more
abundant than TCAc.
|
|
Kinetic analysis
The kinetic properties and inhibitor sensitivities of cytoplasmic CA
isozymes from gill and rbc lysates were examined
(Table 1). The
Ki values for Az and chloride were similar for both the
rbc and gill CA isozymes. However, when the cytoplasmic fractions of gill and
rbc lysates were assayed in the presence of increasing volumes of trout
plasma, which contains an endogenous CA inhibitor
(Dimberg, 1994;
Haswell and Randall, 1976
;
Henry et al., 1997
), the rbc
CA isozyme was found to be significantly more sensitive than the gill CA
isozyme (Fig. 6). Moreover,
both the turnover value (kcat) and substrate affinity
value (Km) of the rbc CA isozyme were significantly higher
than corresponding values for the gill CA isozyme
(Table. 1); thus, the
rbc-specific isozyme appears to be a faster enzyme with a lower substrate
affinity than the cytoplasmic isozyme.
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|
Functional analysis
Changes in mRNA expression in whole blood and gill tissue for both TCAb and
TCAc in response to the induction of anaemia were examined using real-time
RT-PCR. Withdrawal of 1 ml of blood was sufficient to decrease haematocrit
significantly (one-way ANOVA, P<0.05) by approximately 45-55%,
from the control value of 47.6±3.7% (N=6) to 28.0±2.8%
(N=6) at 12 h, 23.5±1.8% (N=6) at 72 h and
21.5±4.1% (N=5) at 15 days. The response to this induction of
anaemia was a significant increase in rbc TCAb mRNA expression at all sample
times (Fig. 7A), in the absence
of any significant change in rbc or gill TCAc mRNA expression
(Fig. 7A and B).
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Discussion |
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The general cytoplasmic CA isozyme has a coding region of 780 base pairs
(259 amino acids; Fig. 1), and
high sequence identity to other known fish CA sequences (74-76%). The high
sequence identity between the TCAb and TCAc isozymes (76%) may explain the
results of Sender et al.
(1999), who found no evidence
for the presence of two distinct CA isozymes in the gill cytoplasm and rbc of
flounder (Platichthys flesus). The gill CA activity in flounder was
believed to be caused by the same isozyme found in the rbc, because antibodies
produced to CA from both sources were cross-reactive. Assuming that two
isozymes do, in fact, occur in flounder, cross-reactivity between antibodies
could be the result of high protein similarities.
To date, molecular analyses of the phylogeny of the -CA gene family
have been limited by the small amount of CA sequence information from
non-mammalian vertebrate species (Lund et
al., 2002
; Tufts et al.,
2003
). When TCAc was included in a phylogenetic analysis of
cytoplasmic
-CA isozymes, it joined a previously described fish CA
monophyletic clade (Peterson et al.,
1997
; Lund et al.,
2002
; Tufts et al.,
2003
; Esbaugh et al.,
2004
). These findings suggest that the current trend of using
mammalian nomenclature (slow type CA I and fast type CA II) to identify fish
CA isozymes may be inappropriate (Tufts et
al., 2003
). In agreement with previous studies, the fish
cytoplasmic CA group emerged prior to the gene duplication event that gave
rise to the mammalian CA I, II and III genes, as well as the divergence of
other tetrapod vertebrate isozymes, such as Xenopus and chicken CA II
(Fig. 2). The analyses also
agree with previous studies that show that the fish cytoplasmic CA group
diverged after the emergence of CA V and VII in vertebrates. Unlike previous
studies, however, this analysis contains two cytoplasmic CA isozymes from a
single species. Interestingly, the two cytoplasmic CA isozymes from rainbow
trout do not group most closely together; rather, TCAc groups closely with a
gill isozyme from dace, whereas TCAb is most closely associated with a
zebrafish retina sequence. There is evidence that the common ancestor of
salmonid fish underwent a genome duplication event
(Hinegardner and Rosen, 1972
;
Allendorf and Thorgaard, 1984
).
However, the topology of the trees formed by both NJ and MP analysis suggest
that the duplication event that gave rise to the two cytoplasmic CA isozymes
in trout occurred in the ancestor common to both zebrafish and trout, and
possibly also dace. Either a gene duplication event that occurred at some
point in the evolution of teleost fish, or a genome duplication event that
occurred at the origin of modern fishes, could account for the presence of two
cytoplasmic CA isozymes in trout (Amores et
al., 1998
; Meyer and Schartl,
1999
; Robinson-Rechavi et al.,
2001
; Taylor et al.,
2001
). It is therefore necessary to examine whether multiple
cytoplasmic CAs are present in more teleost families, as well as in other fish
lineages, such as agnathans, dipnoans and non-teleost actinopterygians.
Examination of the amino acid residues that are specific to the active site
pocket, as described by Tashian et al.
(2000), can also yield insight
into the functional evolution of CA isozymes. A comparison of the active site
amino acid residues (Fig. 3)
indicates not only that the fish cytoplasmic CA isozymes are highly similar at
the amino acid level, but that the critical elements of enzyme function are
almost entirely conserved, with only one amino acid difference between the
teleost CA isozymes. In addition, the active site of TCAc and gar rbc CA
differ by only two amino acids. It is also noteworthy that none of these amino
acid differences fall within sections of the sequence that have been directly
implicated in the catalytic mechanism (reviewed by
Stams and Christianson,
2000
).
Despite the high sequence similarity between the two trout cytoplasmic CA
isozymes, significant differences were detected in their kinetic properties
and inhibitor sensitivities, supporting the contention that the sequences
represent discrete isozymes, with potentially physiologically distinct roles.
It should be noted that because the mRNA, and presumably protein, level of
TCAb in the rbc greatly exceeded that of TCAc, the kinetic properties of rbc
lysates were taken to be representative of TCAb. TCAc was significantly more
sensitive to the endogenous inhibitor in trout plasma than was TCAb
(Fig. 6), while both isozymes
exhibited similar inhibition constants when exposed to Az and chloride ions
(Table 1). The inhibition
constant for Az for both isozymes was within the range previously described
for rainbow trout rbc CA (Henry et al.,
1993; Esbaugh et al.,
2004
), while the value for chloride was slightly lower than that
reported by Henry et al.
(1993
). Calculation of the
turnover (kcat) and substrate affinity constant
(Km) revealed that, as predicted, both were lower for the
general cytoplasmic CA than for TCAb (Table
1). Indeed, the rbc-specific TCAb reaction rate was over three
times faster than that of the more widely distributed TCAc. This difference in
catalytic efficiency is most probably related to the different roles of the
two isozymes. The primary role of the faster TCAb isozyme is to catalyse the
dehydration/hydration reactions of CO2 for the purpose of
CO2 transport and excretion
(Perry, 1986
;
Tufts and Perry, 1998
;
Henry and Swenson, 2000
). A
single pass of blood through the gill vasculature (approximate transit time of
0.5-2.5 s; Cameron and Polhemus,
1974
) can remove up to 35% of the total blood CO2 load
(Perry, 1986
). Thus, an
emphasis on the rapid translation of HCO3- from the
plasma to the rbc and its conversion to molecular CO2 is critical.
Current theory contends that CO2 excretion is rate-limited by the
speed of the Cl-/HCO3- exchanger
(Perry, 1986
;
Perry and Gilmour, 1993
;
Desforges et al., 2001
), and
Henry et al. (1993
) proposed
that the evolution of faster rbc CA isozymes was related to the incorporation
of a rapid Cl-/HCO3- exchanger into the rbc
membrane (see also Tufts et al.,
2003
). The data from the present study are certainly consistent
with this hypothesis - a faster rbc isozyme related to the presence of a rapid
rbc membrane anion exchanger (Cameron,
1978
; Romano and Passow,
1984
) and involved in CO2 transport and excretion,
versus a slower cytoplasmic isozyme that may play a role in numerous
different functions (Henry,
1996
). For example, within the gill, the general cytoplasmic
isozyme probably plays a major role in acid-base balance and ionic regulation,
where high activity may not be as crucial as it is to CO2
excretion. It should also be noted that TCAb and TCAc do not display the
dramatic differences in catalytic and kinetic properties associated with
mammalian CA I and II, suggesting that TCAc may play a more catalytically
demanding role in fish than the low activity CA I isozyme plays in
mammals.
Differences between trout and mammals are also apparent in the tissue
distribution of the CA isozymes. In mammals, CA I and CA II are found in the
cytoplasm of the rbcs as well as many other tissues throughout the body
(Chegwidden and Carter, 2000;
Parkkila, 2000
;
Swenson, 2000
). In contrast,
the major trout rbc CA isozyme, TCAb, appears to be found only in the rbc
(Figs 4 and
5). Unlike TCAb, however, TCAc
exhibits a widespread distribution among different tissues, with greatest
expression occurring in the brain and gills, and lower expression in the
liver, gut, white muscle and the anterior and posterior kidney. Presumably,
TCAc has numerous physiological roles
(Henry, 1996
), similar to
mammalian CA I and II (Chegwidden and
Carter, 2000
; Parkkila,
2000
; Swenson,
2000
). The finding that TCAc is present with TCAb in the rbcs of
trout is in contrast to the prevailing belief that teleost rbcs express only
one CA isozyme (Maren et al.,
1980
; Sanyal et al.,
1982
; Kim et al.,
1983
; reviewed by Henry and
Heming, 1998
), but is in accordance with a few studies that
presented evidence for two isozymes
(Girard and Istin, 1975
;
Carter et al., 1976
). Whereas
mammalian rbcs express two cytoplasmic CA isozymes that differ by an order of
magnitude in catalytic activity, both trout rbc cytoplasmic isozymes appear to
be higher activity enzymes. It is unclear why two isozymes with the potential
for overlapping function would be expressed in the same tissue, although it is
possible that their roles are differentiated by protein interactions. Such
protein interactions - for example, with the rbc membrane
Cl-/HCO3- exchanger - might also explain why
the slower TCAc isozyme is the more widespread isozyme in trout, while the
higher activity TCAb is specific to the rbc. In mammalian rbcs, cytoplasmic CA
II interacts with numerous transport proteins, while CA I is unable to do so
(Vince et al., 2000; Reithmeier,
2001
; Sterling et al.,
2001
,
2002a
,b
;
Li et al., 2002
). It remains
to be determined whether an analogous system also evolved in modern fish.
Previous studies indicated that CA expression in fish is regulated in
response to a variety of internal and external conditions, such as salinity
(Girard and Istin, 1975;
Dimberg et al., 1981
;
Kultz et al., 1992
),
temperature (Houston and McCarty,
1978
; Houston and Mearow,
1979
), and acid-base status
(Dimberg and Hoglund, 1987
).
With this in mind, a preliminary study was performed to examine whether the
two cytoplasmic CA isozymes in the rbc were differentially regulated in
response to anaemia.
In the rbc, a dramatic increase in TCAb mRNA expression occurred in
response to the induction of anaemia (Fig.
7A), whereas no significant change in TCAc mRNA expression was
observed in the rbc (Fig. 7A)
or gill (Fig. 7B). It should,
however, be noted that young rbcs were probably released into the bloodstream
over the duration of the experiment, and these young rbcs are known to have
10-fold higher levels of mRNA transcripts
(Lund et al., 2000). The
extent to which young rbcs may affect the results of these experiments is
unclear as the haematocrit of all animals remained depressed throughout the
15-day experimental period, suggesting that few young rbcs were released into
the bloodstream. In addition, if the increased expression levels of TCAb mRNA
observed throughout anaemia were, in fact, due to the increased mRNA
transcript levels inherent in young rbcs, then the levels of TCAc mRNA in the
rbcs would also be expected to increase. The expression levels of TCAc mRNA,
however, did not significantly change throughout the course of the experiment.
Although these data do not unequivocally demonstrate differential regulation
of the two CA isozymes, it remains plausible. The differential regulation of
the two rbc CA isozymes would support the idea that they serve different
functions within the rbc, which is similar to mammalian rbcs where CA II is
responsible for the majority of CO2 hydration/dehydration
(Maren et al., 1976
). Further
experiments should be performed to more carefully quantify the effect of young
rbcs, as well as the roles the two cytoplasmic isozymes may play.
In conclusion, the results of the present study demonstrate that, in rainbow trout, the CA isozyme in the rbcs is distinct in structure, tissue distribution, kinetic properties, and probably physiological role, from a second, more widely distributed or general cytoplasmic isozyme. Although both isozymes exhibited similar inhibitor sensitivities, the catalytic activity of the rbc CA isozyme was threefold higher than that of the general cytoplasmic form, likely owing to selective pressures specific to the demands of CO2 excretion. Selective up-regulation of the rbc-specific CA isozyme within rbcs in response to anaemia supports the hypothesis of two discrete isozymes with different functions.
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Acknowledgments |
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References |
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