Inhibition of Human SERCA3 by PL/IM430

MOLECULAR ANALYSIS OF THE INTERACTION*

Charukeshi P. Chandrasekera and Jonathan LyttonDagger

From the Cardiovascular Research Group, Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Received for publication, December 13, 2002, and in revised form, January 7, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The monoclonal antibody PL/IM430 has previously been reported to uncouple Ca2+ transport from ATP hydrolysis in platelet membranes (Hack, N., Wilkinson, J. M., and Crawford, N. (1988) Biochem. J. 250, 355-361). More recently, we have demonstrated that this antibody is specific for human SERCA3 (Poch, E., Leach, S., Snape, S., Cacic, T., MacLennan, D. H., and Lytton, J. (1998) Am. J. Physiol. 275, C1449-C1458). In this paper, we have extended the analysis of the PL/IM430-SERCA3 interaction. Using HEK293 cells to express human SERCA3a, we were able to measure both ATP-mediated, oxalate-dependent 45Ca2+ uptake and Ca2+-dependent ATP hydrolysis activities due exclusively to SERCA3. Treatment with PL/IM430 inhibited both activities almost identically, with a maximal inhibition of 81 and 73% and a half-maximal concentration of 8.3 and 5.9 µg/ml, for Ca2+ uptake and ATP hydrolysis, respectively. We conclude that PL/IM430 does inhibit SERCA3 activity but does not uncouple Ca2+ transport from ATP hydrolysis. Using a combination of partial proteolysis, GST fusion protein expression, and mutation of residues that differ between rat and human SERCA3, we have identified human SERCA3 amino acids Pro8 and Glu192 as essential to forming the PL/IM430 epitope. PL/IM430 thus recognizes a linearly noncontiguous set of amino acids within the actuator domain of human SERCA3. We propose that PL/IM430 inhibits SERCA3 activity by sterically preventing movement of the actuator domain into a catalytically critical position in the E2 conformation of the enzyme.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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 DH5alpha . 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 right-arrow TCG), E192D (GAG right-arrow 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-beta -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 gamma -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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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.

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 (open circle ) 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.

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.

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 (alpha -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 (alpha -GST), as indicated. The anti-GST blot confirms that the fusion proteins were expressed in each lane.

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.

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.

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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.

    ACKNOWLEDGEMENTS

We thank Stephen Leach and Ah-Ling Cheng for assistance with the design and preparation of some of the GST fusion constructs.

    FOOTNOTES

* This work was supported by a grant-in-aid from the Heart and Stroke Foundation of Alberta, NWT and Nunavut (to J. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Senior Scholar of the Alberta Heritage Foundation for Medical Research and an Investigator of the Canadian Institutes of Health Research. To whom correspondence should be addressed: University of Calgary Health Science Centre, Rm. 2518, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-2893; Fax: 403-283-4841; E-mail: jlytton@ucalgary.ca.

Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M212745200

    ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; C12E8, octaethylene glycol dodecyl ether; E1-Ca, one of the major conformational states of the SERCA enzyme, with Ca2+ bound, as determined by Toyoshima et al. (24); Tg, thapsigargin; E2(Tg), one of the major conformational states of the SERCA enzyme, which is stabilized by the binding of Tg, as determined by Toyoshima et al. (28); GST, glutathione S-transferase; PBS, phosphate-buffered saline; PBS-T, PBS containing 0.1% Tween 20; SERCA, sarcoplasmic or endoplasmic reticulum Ca2+-ATPase; SR, sarcoplasmic reticulum; MOPS, 4-morpholinepropanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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