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INTRODUCTION |
Ionized calcium (Ca2+) serves as a ubiquitous second
messenger in a vast array of signaling cascades occurring in cells
ranging from bacteria to differentiated mammalian tissues.
Ca2+ signaling involves complex spatial and temporal
changes in cytosolic [Ca2+] that depend upon an increase
mediated by release from intracellular storage sites in the endoplasmic
reticulum (ER)1 or
sarcoplasmic reticulum (SR) or by entry across the plasma membrane, and
the subsequent decrease as Ca2+ is removed from the cytosol
(1). The sarco/endoplasmic calcium ATPase pumps (SERCA) play a pivotal
role in the maintenance of intracellular Ca2+ homeostasis
by lowering the cytoplasmic Ca2+ concentration and
replenishing the ER/SR stores following release (2).
Calcium pumps of the SERCA family are encoded by three distinct, but
homologous, genes whose primary transcripts are alternatively spliced,
yielding isoforms that differ at their C termini and are expressed in a
tissue-dependent and developmentally regulated manner. The
SERCA1 gene is expressed exclusively in fast twitch skeletal muscle,
and alternative splicing yields two isoforms, SERCA1a (994 amino acids)
and SERCA1b (1001 amino acids), present in adult and neonatal tissues,
respectively (3). The SERCA2 gene yields primary transcripts that are
processed in a tissue-specific manner, giving rise to two isoenzymes,
SERCA2a (997 amino acids), which is expressed in cardiac/slow twitch
skeletal muscle, and SERCA2b (1046 amino acids), which is expressed
ubiquitously (4, 5).
SERCA3, the isoform of interest for this study, displays a selective
tissue distribution. Alternative splicing of the SERCA3 gene results in
five different isoforms, SERCA3a-SERCA3e, that are expressed
abundantly in only a select number of adult tissue and cell types.
SERCA3 is not expressed alone but is always accompanied by the
expression of the ubiquitous SERCA2b. The highest level of SERCA3
expression is observed in cells of hematopoietic lineage (platelets and
lymphoid cells), embryologically related endothelial cells, secretory
epithelial cells (of the large and small intestine and the thymus), and
Purkinje neurons in the cerebellum (6-10). In addition, the expression
of SERCA3 mRNA during ontogeny in the rat indicates a
tissue-specific and developmentally regulated pattern in the
cardiovascular system (11). Studies on SERCA3-deficient mice have
indicated that lack of SERCA3 alters endothelium and epithelium-dependent relaxation in vascular smooth muscle
(12, 13). Down-regulation of SERCA expression is suggested to be responsible for the impaired glucose responses in the islets of Langerhans in diabetic mouse and rat models of
non-insulin-dependent diabetes mellitus, a metabolic
disease associated with abnormal insulin secretion (14). In humans,
missense mutations in the SERCA3 gene are thought to render patients
more susceptible to type II diabetes mellitus (15).
Antibodies developed against intracellular platelet membranes have been
used widely as a tool to study the multi-SERCA system in platelets. One
such antibody is PL/IM430 (platelet
intracellular membrane 430), which
recognizes all alternatively spliced SERCA3 isoforms (9). This antibody
was reported to inhibit Ca2+ uptake into, but not ATP
hydrolysis by, highly purified platelet intracellular membranes,
suggesting that PL/IM430 was able to uncouple Ca2+
transport from ATP hydrolysis in the SERCA3 protein (16). This observation, if confirmed, would be of fundamental importance for the
further understanding of structure-function relationships involved in
conformational coupling accompanying active ion transport. Identification of the PL/IM430 epitope is an essential key step toward
this goal.
In this paper, we have utilized recombinant human SERCA3 expressed in a
heterologous mammalian system to extend the studies of the effect of
PL/IM430 on human SERCA3 activity. We confirmed the previous
observation that PL/IM430 inhibits human SERCA3 Ca2+ uptake
activity but did not observe an uncoupling effect. Furthermore, we have
identified the epitope for this inhibitory antibody. These data have
important implications in understanding the conformational changes
accompanying active Ca2+ transport mediated by the SERCA pumps.
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EXPERIMENTAL PROCEDURES |
GST Fusion Proteins--
Glutathione S-transferase
(GST) fusion proteins encoding different segments of the human SERCA3
protein were constructed in the vector, pGEX-4T1 (Amersham
Biosciences), using PCR and/or restriction endonuclease digestion. The
following four constructs were made by PCR amplification of human
SERCA3 cDNA from a full-length SERCA3 template (clone hS3II (9),
which corresponds to the alternatively spliced human SERCA3 (gene
ATP2A3) transcript, SERCA3a (17)): amino-terminal domain
(amino acids 11-369 and 1-364); nucleotide/phosphorylation domain
(amino acids 369-626); carboxyl-terminal domain (amino acids
627-998); and actuator domain (amino acids 141-238). In addition, the
rat SERCA3 cDNA (6) was used as the template for GST fusion
constructs encoding amino acids 1-364 of rat SERCA3. PCR was conducted
under the following stringent conditions in order to accommodate the
GC-rich nature of the templates: initial denaturation at 98 °C for 2 min, followed by 25 cycles of denaturation at 98 °C for 15 s
and annealing/extension at 72 °C for 1.5 min, followed by a final
cycle of extension at 72 °C for 5 min. PCR products were digested
with BamHI and XhoI (sites incorporated into
5'-end and 3'-end primers, respectively), ligated between the
BamHI and XhoI sites of pGEX-4T1 vector, and
transformed into Escherichia coli strain DH5
.
The following GST fusion constructs were made by deleting segments from
the 3'-end of the 1-364 human SERCA3 GST-construct using convenient
restriction sites located within its sequence combined with
XhoI digestion at the 3'-end and Klenow treatment before
blunt-ended ligation. The restriction enzymes AccI,
Kpn2I, SmaI, and SacI were used to
create constructs encoding amino acids 1-293, 1-236, 1-143, and
1-39, respectively. A chimeric fusion construct, 1-39/144-236, was
made by digesting the 1-236 construct with SacI and
SmaI to remove the segment corresponding to amino acids
40-143, treating with Klenow fragment of DNA polymerase, and blunt
end-ligating the construct to generate the in-frame fusion that has an
extra Gly residue at the junction site.
The PCR overlap extension technique was used to create GST fusion
constructs encoding amino acids 1-364 of human SERCA3 mutated at
selected sites. The hS3II cDNA clone was used as the template, and
the same primers used for the fusion construct encoding amino acids
1-364 were employed as outside primers. Two complementary internal
primers containing mutations to introduce the desired amino acid change
were then used in combination with the outside primers in a two-step
protocol. All of the PCRs were set up as described above. The following
nucleotides were mutated to create human SERCA3 constructs encoding the
amino acid changes P8S (CCG
TCG), E192D (GAG
GAT), and the
double change P8S/E192D. The converse mutations to those described
above were introduced into the rat SERCA3 1-364 construct to create
changes S8P, D192E, and the double change S8P/D192E. PCR products
corresponding to the mutated constructs were cloned into pGEX-4T1. All
of the above constructs were confirmed by DNA sequencing, performed
using fluorescent dye terminators, and analyzed at the University of
Calgary Core DNA Services Facility.
For all GST fusion constructs, protein induction was carried out as
described in the GST handbook from Amersham Biosciences. Briefly,
E. coli were grown overnight in 2× YT medium
containing 100 µg/ml ampicillin in a 37 °C shaker. Cultures were
diluted 1:25 into 2× YT, and protein induction was carried out by the addition of 3 mM
isopropyl-1-thio-
-D-galactopyranoside when the cells
reached an absorbance of 0.6-0.8 measured at 600 nm. Two hours
following induction, whole cell lysates were prepared by sonication in
a phosphate-buffered saline (PBS; 130 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7.2). Aliquots of
these lysates were then subjected to SDS-PAGE and analyzed by
immunoblot using PL/IM430 and anti-GST antibodies.
Full-length Expression Constructs--
Constructs encoding
full-length wild-type and mutated human and rat SERCA3a constructs were
made in the mammalian expression vectors pMT2 (18) and pcDNA3.1(
)
(Invitrogen), respectively. Mutated human SERCA3a constructs were
prepared by isolating the segments from their respective amino acid
1-364 GST fusion constructs using EcoRI and
Kpn2I digestion. These fragments were then ligated into the
full-length pMT2-human SERCA3a construct (9) from which the
corresponding wild-type segment had been removed. The wild-type and
mutated rat SERCA3a constructs were prepared by first linearizing the
corresponding GST fusion construct with EcoRI and then
partially digesting with BamHI to isolate the 5' fragments
that contained the mutations. The 3' part of the rat SERCA3 cDNA
was isolated from the parent construct pBR.RK8-13 (6) using
BamHI and BglII digestion. These two isolated
fragments were then inserted into EcoRI- and
BamHI-digested pcDNA3.1(
) to create the full-length
rat SERCA3a in a three-part ligation process. All of the constructs
were confirmed by DNA sequencing.
Recombinant Expression and Functional Analysis in HEK293
Cells--
Microsomes or postnuclear nonionic detergent lysates were
prepared from HEK293 cells 2 days following transfection with
Qiagen-purified plasmid cDNA encoding the various wild-type and
mutated rat or human SERCA3a proteins, as described previously (9, 19). Briefly, for microsomes, the cells were swollen in hypotonic medium and
lysed by homogenization using a Dounce homogenizer. The resulting cellular homogenate was separated by differential centrifugation to
isolate a postmitochondrial particulate fraction enriched in endoplasmic reticulum that we refer to as "microsomes." For
postnuclear lysates, cells were incubated on ice in buffer containing
1% octaethylene glycol dodecyl ether (C12E8)
and then sedimented in a microcentrifuge for 5 min at 15,000 × g to remove the nuclei. Protein concentration was determined
using a Coomassie dye-binding assay (Bio-Rad) with bovine
-globulin
as the standard. Microsome and postnuclear fractions were stored in
aliquots at
80 °C.
ATP Hydrolysis--
This assay was performed as described
previously with some modifications (9). The rate of ATP hydrolysis was
determined at 23 °C using an enzyme-coupled spectrophotometric assay
that utilizes a regenerative system, where hydrolysis of ATP is coupled to the oxidation of NADH, which is measured as a decrease in absorption at 340 nm in a Beckman DU-640 spectrophotometer. The reaction mixture
was set up by adding 2 µl of microsomes isolated from transfected
HEK293 cells to 98 µl of assay solution (120 mM KCl, 25 mM MOPS, 2 mM MgCl2, 1 mM ATP, 1.5 mM phosphoenol pyruvate, 1 mM dithiothreitol, 0.32 mM NADH, 10 units/ml
pyruvate kinase, 10 units/ml lactate dehydrogenase, and 2 µM ionophore A23187). The reaction was started with the
addition of 90 µl of the microsomes/assay solution to a cuvette
containing 10 µl of 4.5 mM CaCl2 and 5.0 mM EGTA, yielding a final free Ca2+
concentration of 3 µM. The absorption at 340 nm was
followed for 9 min. The reaction was then paused for a minute while a
10-µl aliquot of PL/IM430 (obtained from Research Diagnostics, Inc.) diluted in PBS was added and mixed well in the cuvette. Absorption was
followed for another 20 min following antibody addition. Control experiments were conducted using microsomes from cells transfected with
a control vector in which the SERCA3 coding region had been inserted in
the reverse direction and by adding either 10 µl of PBS or 10 µl of
monoclonal antibody R3F1 (which recognizes the Na+/Ca2+ exchanger, NCX1, and not SERCA (20))
in place of PL/IM430. The slopes of the linear portion of these traces
were then determined by least square fit, and the slope after the
antibody addition was reported as a percentage of the slope before
antibody addition.
Ca2+ Uptake--
The Ca2+ uptake
activity of the SERCA3 protein was measured in an ATP-mediated,
oxalate-dependent assay, as described previously, with
slight modifications (9). All reactions were carried out at 23 °C in
a total volume of 110 µl. Microsomes from transfected HEK293 cells (2 µl) were preincubated for 5 min with different amounts of PL/IM430
diluted in 10 µl of PBS. The reaction was started by adding 98 µl
of assay buffer (120 mM KCl, 23 mM MOPS, 3 mM ATP, 3 mM MgCl2, 0.5 mM EGTA, 0.45 mM CaCl2 (free
[Ca2+] = 3 µM), 5 mM potassium
oxalate, pH 7.0, and 1 µCi/ml 45CaCl2).
Following a 10-min incubation period, an aliquot of 90 µl was
withdrawn from the assay mix, and the reaction was terminated by
dilution into a 3-ml quench buffer solution (150 mM KCl, 1 mM LaCl3) on ice. The quenched reactions were
filtered through PHWP-025 filters in a Millipore filtration manifold
and washed with 5 ml of quench buffer. Filters were removed from the
manifold, and radioactivity was measured by liquid scintillation
counting. Control experiments were carried out as described above. The
10-min time point was chosen because it was found to be in the linear portion of the uptake time course, as established in pilot experiments.
Proteolytic Fingerprinting--
Microsomes from
SERCA3-transfected HEK293 cells were partially digested using trypsin.
All reactions were performed on ice. 25 µg of microsomes were treated
with 10 µg/ml trypsin in 100 µl of 160 mM KCl, 17 mM HEPES/KOH, 0.1 mM dithiothreitol, and 0.05 mM CaCl2, pH 7.2. The reaction was terminated
after 1 and 2 min by the addition of 30 µl of 100 mM
phenylmethylsulfonyl fluoride in isopropyl alcohol and 30 µl of 5.9 mg/ml soy bean trypsin inhibitor. Five micrograms of digested
microsomes were then resolved on a 9% SDS-PAGE gel, transferred to a
nitrocellulose membrane, and analyzed by immunoblotting. These
conditions were arrived at empirically to produce an informative
digestion pattern. The extent of proteolysis we observed was greater
than that obtained previously (21), presumably because under these
conditions proteolysis can continue following denaturation of the SERCA
protein prior to and during electrophoresis (22).
Immunoprecipitation--
All steps were conducted at 4 °C.
Aliquots of postnuclear lysates from transfected HEK293 cells
containing 500 µg of protein were diluted to 1 ml with 1%
C12E8, 0.1 mg/ml ovalbumin, 140 mM NaCl, 25 mM Tris-Cl, 10 mM EDTA, pH 7.5, and
precleared by incubating for 30 min with 20 µl of Protein A-Sepharose
beads (Sigma) followed by sedimentation at 3000 × g
for 2 min. The supernatant was then incubated with 5 µg of PL/IM430
antibody for 1 h followed by the addition of 20 µl of Protein
A-Sepharose beads and a further incubation of 30 min. The beads were
then washed three times with buffer containing 0.3%
C12E8 and finally suspended in Laemmli gel
sample buffer before loading on SDS-polyacrylamide gels.
Immunoblotting--
Four different SERCA3 antibodies were used
for immunoblotting experiments: the PL/IM430 monoclonal antibody (16)
(obtained from Research Diagnostics Inc.) and three other
SERCA-specific polyclonal antibodies, C4 (4, 9), PA-910 (9) (obtained from Affinity BioReagents Inc.), and N1. The last was prepared by
immunizing rabbits with a purified amino-terminal hexa-His-tagged recombinant fragment of rat SERCA2 encompassing amino acids 362-704. This antibody recognizes both SERCA2 and SERCA3 products (data not
shown). The anti-GST antibody employed was raised in a rabbit injected
with a GST-Na+/Ca2+ + K+ exchanger
fusion protein. The serum from this rabbit recognizes selectively the
GST portion of the fusion protein and does not react with SERCA
proteins. All immunoblotting procedures were carried out at room
temperature. Nitrocellulose membranes were blocked for 30 min with 5%
dried skim milk dissolved in PBS containing 0.1% Tween 20 (PBS-T),
followed by incubation with the primary antibody in PBS-T for 1 h.
The membranes were washed three times in PBS-T for 5 min each and
incubated with the horseradish peroxidase-conjugated secondary antibody
for 30 min followed by washing as above. The immunoblots were then
developed using ECL Super Signal Plus reagent from Pierce.
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RESULTS |
SERCA Activity in Transfected HEK293 Cells--
To confirm the
previously reported effect of PL/IM430 on human SERCA3 function (16),
recombinant human SERCA3 was expressed transiently in HEK293 cells
using the construct huS3-II, which encodes the SERCA3a protein (9). As
illustrated in Fig. 1, microsomes from
HEK cells expressing human SERCA3a exhibited an ~80-fold higher rate
of ATP-mediated, oxalate-dependent Ca2+ uptake
than microsomes from the control transfected cells. There was also an
~20-fold increase in activity for
Ca2+-dependent ATP hydrolysis in
SERCA3a-transfected cells when compared with the control transfected
cells. That these measurements represented SERCA activity was confirmed
by the effect of the specific SERCA inhibitor, thapsigargin (23). In
all subsequent experiments, the activity of the control microsomes,
corresponding to less than 10% of the activity of the microsomes
expressing SERCA3a, has been subtracted. Thus, the HEK293 microsome
system allows the measurement of SERCA activity corresponding
unambiguously to the protein encoded by the transfected cDNA.

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Fig. 1.
SERCA activity in microsomes from transfected
HEK293 cells. Microsomes prepared from HEK293 cells transfected
with either a cDNA construct encoding human SERCA3 (HS3)
or a construct in which the SERCA3 sequence had been inserted in the
reverse orientation (RC) were analyzed for either
ATP-mediated, oxalate-dependent
45Ca2+ uptake (A) or
Ca2+-dependent ATP hydrolysis (B).
In each case, the right-hand column (Tg)
indicates the activity of microsomes containing human SERCA3 following
treatment with 0.5 µM thapsigargin. The values shown are
the mean ± S.E. for either two (RC) or three
(HS3 and Tg) different microsome preparations.
The relatively large errors associated with human SERCA3 activity
reflect the variation in the level of expression from experiment to
experiment typically observed in transient transfection systems.
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The Effect of PL/IM430--
SERCA3a-expressing
microsomes were then treated with increasing amounts of purified
PL/IM430 monoclonal antibody and the effect on Ca2+ uptake
activity was determined. Fig.
2A shows that PL/IM430
inhibited Ca2+ uptake with a maximal inhibition of 81% at
a half-maximal concentration of 8.3 µg/ml antibody. The
isotype-matched anti-Na+/Ca2+-exchange
antibody, R3F1 (20), was used as a control to demonstrate that the
addition of IgG did not have an inhibitory effect on the assay itself.
The effect of PL/IM430 on SERCA3a ATP hydrolysis activity was then
tested using an enzyme-coupled spectrophotometric assay. As seen in
Fig. 2B, the effect of PL/IM430 on ATP hydrolysis activity
was essentially indistinguishable from that on Ca2+ uptake
activity with a maximal inhibition of 73% at a half-maximal concentration of 5.9 µg/ml antibody. Thus, in the recombinant system,
where activity of SERCA3a can be isolated from other components, PL/IM430 had a comparable inhibitory effect on both Ca2+
uptake and ATP hydrolysis and did not appear to uncouple the two. Since
the inhibitory effect of PL/IM430 on SERCA activity had been confirmed,
the next objective was to identify the epitope for this antibody.

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Fig. 2.
Effect of PL/IM430 on human SERCA3
activity. Microsomes isolated from HEK293 cells transfected with
the cDNA construct encoding human SERCA3 were treated with the
indicated concentration of monoclonal antibodies PL/IM430 ( ) or R3F1
( ) and assayed for ATP-mediated, oxalate-dependent
45Ca2+ uptake (A) or
Ca2+-dependent ATP hydrolysis (B).
R3F1 is a monoclonal antibody directed against the
Na+/Ca2+ exchanger, NCX1, that does not
recognize SERCA3; it was used as an IgG isotype control. Background
activity due to endogenous SERCA2b present in the microsomes (obtained
from microsomes isolated from HEK293 cells transfected with the reverse
control construct) has been subtracted, and the values have been
normalized to the activity in the absence of antibody. The means ± S.E. are plotted against the final antibody concentration present
during the uptake assay for seven determinations on five different
preparations of microsomes for Ca2+ uptake and for three
determinations on three different microsome preparations in the case of
ATP hydrolysis. Note that the ATP hydrolysis data were obtained from
different preparations of microsomes than those used for the
Ca2+ uptake assays. The curves fitted to the PL/IM430 data
were obtained from least squares fit of a cooperative inhibition model.
For Ca2+ uptake, maximal inhibition was 81 ± 2%, the
half-maximal dose of antibody was 8.3 ± 0.4 µg/ml, and the Hill
coefficient was 1.5 ± 0.1; for ATP hydrolysis, maximal inhibition
was 73 ± 1%, the half-maximal dose of antibody was 5.9 ± 0.3 µg/ml, and the Hill coefficient was 1.4 ± 0.1.
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Proteolytic Fingerprinting--
The general location of the
PL/IM430 epitope was determined by proteolytic fingerprinting using
trypsin digestion of microsomes isolated from HEK293 cells transfected
with human SERCA3a cDNA. The digests were resolved on SDS-PAGE gels
and immunoblotted with PL/IM430 and other SERCA-specific antibodies
directed against different parts of the SERCA3 protein: antibody C4
(directed against the fragment encompassing amino acids 17-215) and
antibody N1 (directed against the fragment encompassing amino acids
362-704). The specific patterns of fragments recognized by PL/IM430
and C4 were very similar, whereas that of N1 was quite different (Fig. 3). These results suggested that the
PL/IM430 epitope was located within the same region as that of the C4
epitope (i.e. the amino-terminal 215 amino acids).

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Fig. 3.
Mapping the PL/IM430 epitope by partial
proteolysis. Microsomes isolated from HEK293 cells transfected
with the cDNA construct encoding human SERCA3 were incubated on ice
for 2 min with or without 10 µg/ml trypsin. The reaction was stopped
with phenylmethylsulfonyl fluoride and soy bean trypsin inhibitor, and
5 µg of microsomes were resolved on a 9% SDS-PAGE gel.
Immunoblotting was carried out on three parallel blots using the SERCA
antibodies PL/IM430, C4, and N1, as indicated.
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GST Fusion Proteins--
The PL/IM430 epitope was further
localized using GST fusion proteins encoding different regions of the
SERCA3a protein. Consistent with the proteolysis data, the construct
encoding the amino-terminal region (amino acids 1-364) of SERCA3 was
PL/IM430-reactive, whereas those constructs encoding the
nucleotide/phosphorylation domain loop (amino acids 369-626) or the
carboxyl-terminal hydrophobic region (amino acids 627-998) were not
reactive, as seen in Fig. 4. Whereas the
1-364 construct was positive for PL/IM430 reactivity, the construct
encoding amino acids 11-369 was not, suggesting that the
amino-terminal 10 amino acids were necessary for epitope recognition.
When this region was expressed by itself in the 1-39 construct,
however, reactivity was not observed, although the fusion protein was
clearly expressed, as seen in the anti-GST panel
(
-GST) of Fig. 4.

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Fig. 4.
Mapping the PL/IM430 epitope using GST fusion
protein constructs. Full-length human SERCA3 (expressed in HEK293
cells; top row) and extracts from E. coli expressing various GST-human SERCA3 fusion constructs were
analyzed for PL/IM430 immunoreactivity by immunoblot. The constructs
are illustrated schematically on the left, with the
black bar showing SERCA3 and the
numbers corresponding to the amino acids present. The
gray bar corresponds to GST, whereas the
thin line indicates regions deleted from SERCA3.
On the right side are two parallel immunoblots
(rotated 90 ° counterclockwise for presentation purposes) probed
with PL/IM430 and an anti-GST antibody ( -GST), as
indicated. The anti-GST blot confirms that the fusion proteins were
expressed in each lane.
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Further experiments revealed that successive C-terminal deletions of
the immunopositive 1-364 construct led to the loss of PL/IM430
reactivity between constructs 1-236 and 1-143, suggesting that the
region between amino acids 144 and 236 was critical for the PL/IM430
epitope (Fig. 4). However, when this region was expressed alone in the
141-238 construct, reactivity was not observed, although the protein
was expressed.
Since two critical regions had been identified as necessary but not
sufficient for creation of the PL/IM430 epitope, a construct combining
these two regions was prepared. As seen in Fig. 4, the 1-39/144-236
construct encoded a protein that could be recognized by PL/IM430,
suggesting that the complete epitope for PL/IM430 is a noncontiguous
combination of two regions that come together in three-dimensional
space. The plausibility of this result is confirmed by examining the
three-dimensional crystal structure of rabbit SERCA1 (24), which
illustrates that the actuator domain comprises the amino-terminal 40 amino acids and amino acids 126-230, folded together into a stable
structure (Fig. 5).

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Fig. 5.
The PL/IM430 binding site. The human
SERCA3 protein sequence was threaded onto the three-dimensional
structure of SERCA1 in the E1-Ca2+ conformation (24),
refined by energy minimization using Swiss-Model (31), and displayed
graphically in stereo using RasMol (33). The path of the polypeptide
chain is shown in ribbon form to illustrate secondary structural
features. The residues within the actuator domain of human SERCA3 that
are different from the rat enzyme are shown in space
fill and labeled. The region of the actuator domain corresponding
to amino acids 1-40 is red, whereas that corresponding to
amino acids 126-230 is green.
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Site-directed Mutagenesis--
To localize the epitope further, we
utilized an important characteristic feature of the monoclonal antibody
PL/IM430, that it is highly specific to human SERCA3 and does not
recognize the rat SERCA3 isoform, although these proteins are about
97% identical. Sequence comparison between the two SERCA3 orthologs in
the region encompassing the PL/IM430 epitope reveals that there are
seven differences in the first 40 amino acids and three more between amino acids 144 and 236. The only pair of amino acids in these two
regions that lie in three-dimensional proximity, based on the SERCA1
crystal structure, are Pro8 and Glu192 of human
SERCA3, as seen in Fig. 5. Thus, these residues were mutated to the
corresponding amino acids of rat SERCA3 to determine whether this would
destroy PL/IM430 binding. The converse experiment, the conversion of
the rat residues, Ser8 and Asp192, to their
human counterparts, was done with the expectation that these changes
would render rat SERCA3 PL/IM430- reactive.
GST fusion constructs corresponding to these mutations were prepared,
expressed, and analyzed by immunoblotting with PL/IM430, as illustrated
in Fig. 6. The double mutation,
P8S/E192D, introduced into human SERCA3 resulted in the complete loss
of PL/IM430 recognition. Proteins containing the individual mutations
(P8S or E192D) still retained very weak binding, evident upon long
exposure of the immunoblots. Moreover, the rat SERCA3 protein
containing the double mutation, S8P/D192E, was recognized by PL/IM430,
although the individual mutations, S8P or D192E, were not. Thus, it is
clear that Pro8 and Glu192 play a crucial role
in forming the PL/IM430 epitope, as predicted.

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Fig. 6.
Mapping the PL/IM430 epitope using mutations
to GST-fusion constructs. Extracts from E. coli
expressing GST fusion constructs encoding amino acids 1-364 from
either human or rat SERCA3 harboring the indicated mutations were
separated by SDS-PAGE and analyzed by immunoblot with PL/IM430
(top panel) and anti-GST antibody
(lower panel). The right
two lanes contained lysates from the wild-type
human SERCA3 GST fusion segment (hS3:1-364) and the empty
GST construct (Control), respectively. The PL/IM430 panel
has purposely been overexposed to illustrate the weak signal in the
leftmost two lanes. The anti-GST blot
confirms that comparable amounts of fusion protein have been loaded in
each lane.
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Full-length Mutant SERCA3--
To confirm that Pro8
and Glu192 are necessary and sufficient for conferring
PL/IM430 reactivity in the context of the whole SERCA3 protein, the
corresponding mutations were created in full-length rat and human
SERCA3a cDNA constructs. Following expression of these constructs
in HEK293 cells, all of the constructs demonstrated Ca2+-dependent ATP hydrolysis activity (data
not shown), indicating that mutation at positions 8 and 192 can be
tolerated in the SERCA structure, as anticipated. Postnuclear extracts
from transfected cells were then tested for PL/IM430 immunoreactivity,
by both immunoblot and immunoprecipitation. As shown in Fig.
7, the results obtained from both assays
were identical. Mutation of either P8S or E192D alone in human SERCA3a
reduced PL/IM430 binding substantially, whereas both mutations combined
abolished reactivity completely. Conversely, the acquisition of
PL/IM430 immunoreactivity in rat SERCA3a required the conversion of
both S8P and D192E. These data confirm the earlier results using the
GST fusion proteins and establish that Pro8 and
Glu192 are critical components of the PL/IM430 binding site
in the intact human SERCA3 protein.

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Fig. 7.
Mapping the PL/IM430 epitope using mutations
to full-length SERCA3. Postnuclear lysates prepared from HEK293
cells transfected with constructs encoding the full-length wild
type and mutated human and rat SERCA3 proteins, as indicated, were
analyzed by immunoprecipitation and by immunoblot using the
antibodies PL/IM430, N1, or PA-910. Control indicates the
lysate from cells transfected with the reverse construct. The
top row illustrates an N1 (pan-SERCA-specific)
immunoblot of a PL/IM430 immunoprecipitation (IP) from 500 µg of cell lysate. Note that the Control lane does not
contain sample in this case. In the lower two
rows, 15 µg of lysate was loaded in each lane and probed
directly with either PL/IM430 or with PA-910, a SERCA3-specific
antibody that recognizes rat SERCA3 with higher affinity than human
SERCA3 due to a single amino acid difference within the epitope. The
PA-910 blot confirms that comparable amounts of SERCA3 protein have
been loaded in each lane.
|
|
 |
DISCUSSION |
In this paper, we utilized a mammalian expression system to
examine the effects of PL/IM430 on human SERCA3 activity. We confirmed the previously reported observation that PL/IM430 inhibited human SERCA3 Ca2+ uptake activity (16). Unlike that report,
however, we observed that ATP hydrolysis activity was also inhibited by
PL/IM430. Furthermore, both the extent of inhibition and the
half-maximal doses of antibody required were almost identical for the
two measurements. We obtained a maximal level of 81% inhibition for
Ca2+ uptake activity and 73% inhibition for ATP hydrolysis
activity, with a half-maximal dose of 5.9-8.3 µg/ml PL/IM430. We
conclude from our data that PL/IM430 does not uncouple ATP hydrolysis
from Ca2+ uptake but that it inhibits both activities equally.
Although our results contradict the observations made previously, we
believe that differences in the experimental systems can account for
these observations. In the present study, we used recombinantly
expressed SERCA3a to obtain a high level of activity specific to SERCA3
and to minimize background activity. As seen in Fig. 1, expression in
HEK293 microsomes allows unambiguous determination of SERCA3 activity.
Hack et al. (16) used platelet intracellular membranes to
study the effect of PL/IM430, since SERCA3 had not been identified or
cloned at that time. In the platelet system, both SERCA3 and SERCA2b
are present (25), and it is likely that SERCA3 is predominantly
responsible for the measured Ca2+ transport activity, since
Hack et al. found that PL/IM430 inhibited Ca2+
uptake to a maximal extent of 80% with a half-maximal dose of about 10 µg/ml, almost identical values to those we observe.
On the other hand, whereas we observed very similar inhibition by
PL/IM430 of both SERCA3-dependent Ca2+ uptake
and ATPase activities in HEK293 microsomes, Hack et al. observed no significant inhibition of ATPase activity in platelet membranes. We believe this difference can be explained by a high level
in platelet membranes of Ca2+-dependent ATPase
activity that is not SERCA-related. It is well known that platelet
membranes express ecto-ATP-diphosphohydrolase (apyrase) activity (26),
a powerful ATP hydrolysis pathway. Furthermore, the relative ratio
of ATP hydrolysis to Ca2+ uptake catalyzed by SERCA
measured under conditions similar to those used by Hack et
al. is no greater than 5 (27). Thus, assuming that all of the
Ca2+ uptake (~1.5 nmol/min/mg of protein) they observed
was due to SERCA activity, one can calculate a maximum corresponding
ATP hydrolysis activity of 7.5 nmol/min/mg of protein. Consequently, even 100% inhibition of SERCA ATP hydrolysis by PL/IM430 would only
have reduced the total ATP hydrolysis (~100 nmol/min/mg of protein)
measured by Hack et al. by less than 10%, well within the
error of their measurements (16).
Although PL/IM430 does not uncouple ATP hydrolysis from
Ca2+ transport, the location of the antibody binding site
is still of interest for understanding the structure-function relation,
since antibody binding does inhibit SERCA3 activity. Our data
demonstrate that PL/IM430 binds to the so-called actuator domain, which
comprises a tightly folded independent structure containing amino acids 1-40 and 126-230 (24). We took advantage of the specificity of
PL/IM430 for human, and not rat, SERCA3 to define two residues within
this domain that are critical for antibody binding to human SERCA3:
Pro8 and Glu192. Thus, PL/IM430 recognizes a
linearly noncontiguous set of amino acids that are clustered together
in three-dimensional space (Fig. 5). This site is located between the
conserved Thr-Gly-Glu-Ser184 motif (TGES) and the T2
tryptic cleavage site at Arg198. Comparison of the SERCA1
crystal structure determined in the E1-Ca conformation (24) with that
in the E2(Tg) conformation (28) reveals a large rotational motion of
the actuator domain. In the E2(Tg) conformation, both the TGES motif
and Arg198 are buried at the interface between the actuator
and nucleotide binding domains. Such an arrangement is also confirmed
by the fact that the T2 tryptic site is inaccessible in the E2(Tg)
conformation (21). Furthermore, mutations to Thr, Gly, or Glu of the
TGES motif prevent E1-P to E2-P interconversion and thus SERCA activity (29). This conformationally sensitive three-dimensional rearrangement of the actuator domain provides a clear explanation for the PL/IM430 effect on activity. Binding of PL/IM430 to its site in proximity to the
TGES motif and Arg198 will interfere sterically with the
position of the actuator domain in the E2(Tg) conformation, where these
sites are buried. Although movement of the actuator domain between
different conformations during the catalytic cycle has already been
clearly established, the effect of PL/IM430 on activity provides strong
evidence that this motion is essential for, rather than merely
coincident with, catalytic activity. A recent proteolytic study in
which SERCA1 was cleaved by proteinase K to selectively remove five
amino acids at the C-terminal side of the actuator domain (30) is
consistent with such a proposal.
We were surprised that PL/IM430 recognizes human SERCA3 by immunoblot,
although the epitope is linearly noncontiguous. This implies either
that the actuator domain fold is sufficiently stable to resist
SDS-denaturation, electrophoresis, and blotting or that there is
sufficient mobility in these regions even on the blotted membrane so
that the antibody can induce the conformation needed for binding. It is
interesting to note that PL/IM430 appears to react more weakly with the
chimeric GST fusion (1-39/144-236) than it does with the intact
(1-236) construct. The chimeric construct is lacking the first 18 amino acids that form two lateral
-strands of the M2-M3 portion of
the actuator domain and are in contact with amino acids 21-25 from the
N-terminal region. Consequently, one would expect the domain to be less
stable without these 18 amino acids, which supports the notion that
recognition by PL/IM430 on blots involves the domain remaining folded.
We have identified two amino acids, Pro8 and
Glu192, that, when altered individually, do not abolish but
greatly reduce the sensitivity (and thus presumably the affinity for
binding) of PL/IM430 detection. Proline residues reduce the rotational
flexibility of the polypeptide chain and can thus promote different
conformations of neighboring amino acids. Thus, proline may contribute
only indirectly to the PL/IM430 epitope. We think that is not likely to
be the case here, since position 8 is at the amino-terminal end of a
short helix. Insertion of a proline in place of the serine found at
this site in the SERCA1 structure does not dramatically influence the
dihedral angles or change the position of the neighboring residues, as assessed by threading the human SERCA3 sequence onto the rabbit SERCA1
crystal coordinates followed by energy minimization using the
Swiss-Model automated modeling service (31) available from the Expasy
server (available on the World Wide Web at www.expasy.org). Also,
the P8S mutation does not influence the stability of the protein when
expressed in HEK293 cells, which might be anticipated if the structure
of the actuator domain were disturbed. When Pro8 and
Glu192 were introduced into the rat SERCA3 protein, both
changes were required to gain PL/IM430 recognition, whereas removal of
these residues individually in human SERCA3 only reduced, and did not abolish, PL/IM430 reactivity. Moreover, PL/IM430 appears to bind more
tightly to human SERCA3 than to the doubly mutated rat protein (Fig.
7). These data suggest that, whereas Pro8 and
Glu192 are clearly essential for the PL/IM430 epitope,
other amino acids (especially those different between rat and human
SERCA3 proteins, such as Val220 (Fig. 5)) are also part of
the epitope.
Even at saturating concentrations of the PL/IM430 antibody, activity of
human SERCA3 could only be inhibited to a maximum of 80%. Although it
is not evident precisely why this is so, several alternative
possibilities exist. First, since the E2(Tg) position of the actuator
domain would not be compatible with PL/IM430 binding, there might be a
competition between these two states, such that at any given time, 20%
of the SERCA enzyme is in the E2 conformation and has no antibody
bound. This fraction of the enzyme may then turn over briefly before
interacting with PL/IM430 in the E1 conformation. Second, as it has
been suggested that rotation of the actuator domain accompanies the
rate-limiting E1-P to E2-P transition (32), PL/IM430 binding may simply
slow down rotation of the actuator domain without interfering with
catalysis per se. Third, the position that the actuator
domain normally adopts in the E2 conformation may not be reached with
PL/IM430 bound. However, the enzyme may still be capable of turning
over in this strained state, but at only about 20% of the normal rate.
Further examination to distinguish among these possibilities will help
clarify the conformational movements associated with
Ca2+ transport in the SERCA family of pumps.