From Laboratorium voor Fysiologie, Katholieke
Universiteit Leuven, Campus Gasthuisberg, Herestraat 49, B-3000,
Leuven, Belgium and
Division of Medical and Molecular Genetics,
Guy's Hospital, London, SE1 9RT, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human chromosome 17-specific genomic clones
extending over 90 kilobases (kb) of DNA and coding for
sarco/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3)
were isolated. The presence of the D17S1828 genetic marker in the
cosmid contig enabled us to map the SERCA3 gene (ATP2A3) 11 centimorgans from the top of the short arm p of chromosome
17, in the vicinity of the cystinosis gene locus. The SERCA3 gene
contains 22 exons spread over 50 kb of genomic DNA. The exon/intron
boundaries are well conserved between human SERCA3 and SERCA1 genes,
except for the junction between exons 8 and 9 which is found in the
SERCA1 gene but not in SERCA3 and SERCA2 genes. The transcription start
site (+1) is located 152 nucleotides (nt) upstream of the AUG codon.
The 5'-flanking region, including exon 1, is embedded in a 1.5-kb CpG
island and is characterized by the absence of a TATA box and by the
presence of 14 putative Sp1 sites, 11 CACCC boxes, 5 AP-2-binding
motifs, 3 GGCTGGGG motifs, 3 CANNTG boxes, a GATA motif, as well as
single sites for Ets-1, c-Myc, and TFIIIc. Functional promoter analysis
indicated that the GC-rich region (87% G + C) from 135 to
31 is of
critical importance in initiating SERCA3 gene transcription in Jurkat
cells. Exon 21 (human, 101 base pairs; mouse, 86 base pairs) can be
alternatively excluded, partially included, or totally included, thus
generating, respectively, SERCA3a (human and mouse, 999 amino acids
(aa)), SERCA3b (human, 1043 aa; mouse, 1038 aa), or SERCA3c (human,
1024 aa; mouse, 1021 aa) isoforms with different C termini. Expression of the mouse SERCA3 isoforms in COS-1 cells demonstrated their ability
to function as active pumps, although with different apparent affinities for Ca2+.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs),1 mediating the uptake of Ca2+ into intracellular stores such as sarcoplasmic and endoplasmic reticulum, are encoded by three distinct genes in higher vertebrates (reviewed in Ref. 1). SERCA1 is expressed only in the fast-twitch skeletal muscle as one of its developmentally spliced variants: the adult SERCA1a (994 aa) or the neonatal SERCA1b (1001 aa). Both isoforms present identical amino acid sequences up to amino acid 993. As a result of retention/excision of the penultimate exon (42 bp), respectively, in the SERCA1a/SERCA1b splice variants, the last amino acid (Gly) in SERCA1a is replaced in SERCA1b by a highly charged octapeptide sequence DPEDERRK (2). COS cell expression studies showed no functional differences between SERCA1a and SERCA1b isoforms. The complete structures of the rabbit (2) and human (3) SERCA1 genes have been elucidated. The SERCA1 gene (ATP2A1) has been mapped to human chromosome 16p12.1 (4) and a deficiency in SERCA1 is responsible for at least one autosomal recessive form of Brody disease (5). Tissue-specific processing of the SERCA2 gene primary transcript generates up to four mRNA classes (6), which code for two isoenzymes as follows: a cardiac/slow-twitch skeletal muscle protein (SERCA2a) and a ubiquitously expressed isoform (SERCA2b). As a result of alternative splicing, the SERCA2a-specific C terminus comprising the sequence AILE (aa 994-997) is replaced by a variant tail of 49 or 50 amino acids in SERCA2b (7-9). This extended tail contains a very hydrophobic stretch, which is suggested to represent a possible 11th transmembrane segment (7-9). The divergence in the C-terminal part is responsible for functional differences between SERCA2a and SERCA2b (10, 11); these differences were recently ascribed to the presence of the last 12 amino acids in SERCA2b (12). Thus far, the complete structure of a SERCA2 gene is lacking, but partial characterization of the 5'- and/or 3'-ends of the gene has been reported for human (7, 13), rabbit (14), pig (15), and rat (16). The SERCA2 gene (ATP2A2) has been mapped to human chromosome 12q23-q24.1 (17). Structural and functional analyses of the SERCA2 gene promoter in rabbit (18-20), rat (16), and human (13) identified the promoter regions required for transcriptional activity in NIH3T3 fibroblasts, primary cultured rat cardiomyocytes, C2C12 and Sol8 muscle cells. Several putative cis-acting elements have been described, among which Sp1 sites and thyroid-responsive elements have been proven to exert an important role in transcriptional regulation of the SERCA2 gene (20, 21). Unique SERCA genes have also been described in invertebrates, such as the crustacean Artemia franciscana (22) and the insect Drosophila melanogaster (23). The gene primary transcript is alternatively spliced in Artemia, and the expression of the two isoforms is regulated by tissue-specific alternative promoters (24).
The first report describing the cloning of the SERCA3 cDNA from rat kidney (25) indicated a broad expression pattern for its 4.8-kb transcript. Recent studies demonstrated that SERCA3 is always co-expressed along with the ubiquitous SERCA2b isoform (26), and high levels of SERCA3 mRNA have been documented in the hematopoietic cell lineage, arterial endothelial and secretory epithelial cells, as well as in cerebellar Purkinje neurons (27-30). Upon expression in COS-1 cells, SERCA3 presents a much lower apparent affinity for Ca2+, when compared with the other members of the SERCA family (10). We have previously identified the 97-kDa SERCA3 (999 aa) in both human and rat platelets using a set of SERCA3-specific antisera (27). Additionally, we cloned the human SERCA3 cDNA, isolated and partially characterized a genomic clone encoding all but the 5'-end of the gene, and localized the SERCA3 gene (ATP2A3) on human chromosome 17p13.3 (31). Until very recently, there were no indications that the SERCA3 pre-mRNA was subject to alternative splicing. Two mouse nucleotide sequences coding for SERCA3a and SERCA3b have been deposited in the EMBL/GenBankTM data bank.2 So far, no indications regarding the alternative splicing mechanism were published.
We now document the complete exon/intron organization of the human SERCA3 gene. The transcription initiation site and several upstream putative cis-regulatory elements were identified. The functional promoter analysis delineates the minimal promoter region responsible for efficient transcriptional activity and suggests the involvement of the Sp1 transcription factor. We also provide evidence that the human and mouse SERCA3 gene primary transcripts are alternatively spliced, thereby generating not two but three distinct isoforms with altered C termini as follows: SERCA3a, SERCA3b, and SERCA3c. Furthermore, the three mouse SERCA3 isoforms were overexpressed in COS cells and shown to be functionally active but with different apparent affinities for Ca2+.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation and Characterization of Genomic Clones-- To isolate the entire gene, a human chromosome 17-specific library from Reference Library Data Base, ICRF (32), was screened with the 1482-bp EcoRI insert of the human SERCA3 partial cDNA clone Z8 (31). The EcoRI fragment comprised 6 bp of the 5'-untranslated region and the first 1476 bp of the coding region. Six new positive clones (Fig. 1) were isolated and further characterized according to standard restriction mapping and sequencing protocols. Analysis of repetitive sequences was carried out using the CENSOR server.3 The computer-assisted analysis of the putative transcription factor binding sites was performed using the Wisconsin Package Version 9.0 program from Genetics Computer Group (GCG), Madison, WI.
Primer Extension Analysis-- Poly(A)+ RNA was isolated from human tonsils (31). Primer extension analysis was essentially performed as described (33). The extension primer used (5'-GAGGCCATGTCCGTGCTGGGAC-3') corresponds to the inverse complement of nucleotides 25-46 (numbering relative to the determined transcription site; see Fig. 5b). The 35S-labeled sequencing products (used as size markers) of a 5' genomic fragment primed with the same extension primer and the extension products were separated on a 6% polyacrylamide, 7 M urea sequencing gel.
Analysis of Promoter Activity--
A 6.6-kb
BamHI-KasI genomic fragment containing the
5'-flanking region of the human SERCA3 gene (Fig. 1) was subcloned
between the BglII and HindIII restriction sites
of the luciferase expression vector pGL3 basic (Promega, Madison, WI).
The resulting plasmid, p6.6BK, was used as a template for further
generation of controlled deletions by making use of restriction sites
present within the genomic insert and the luciferase cloning vector
(Fig. 5c). The 5'-end of each of the deletion constructs was
confirmed by sequencing. The human SERCA3 promoter-luciferase
constructs and the unmodified, promoterless pGL3 basic reporter vector
were used for transient transfection of the human cell line Jurkat E6.1
cells by electroporation as described (34). To evaluate transfection
efficiencies, the cells were co-transfected with 150 or 500 ng of a
pEL1-gal vector, containing the
-galactosidase reporter gene
driven by the elongation factor 1 promoter (pEL1-
gal vector is a
gift from Dr. F. Bulens).4
Reporter enzyme activities were assayed 40 h after electroporation according to the manufacturer's instructions. The measurements were
performed with the MicroLumat LB 96P luminometer (EG & G, Berthold, Bad
Wildbad, Germany) and corrected for protein concentration, as
determined by the bicinchoninic acid method (Pierce), using bovine
serum albumin as standard. Luciferase activities are expressed relative
to the
-galactosidase activities and normalized to the value
obtained with the promoterless pGL3 basic vector which is set at
1.
Tissue Distribution of Human SERCA3 mRNA-- The human RNA Master BlotTM (CLONTECH, Palo Alto, CA), to which high quality poly(A)+ RNAs from 50 different adult and fetal tissues have been immobilized along with several controls (Fig. 6), was hybridized following the manufacturer's protocol. The synthesis of a 3'-end probe by PCR was described earlier (31). The probe corresponds to the nucleotides 3033-3405 (accession number Z69881) found in the 3'-untranslated region of human SERCA3 cDNA. The blot was analyzed by means of a PhosphorImager model STORM 840 (Molecular Dynamics, Sunnyvale, CA). A common SERCA3b/SERCA3c probe (90-bp long) was PCR-synthesized using a 5' primer N+ (5'-GCACGGCCTTCTCAGGACAGTCT-3') and the 3' primer P1 (5'-GGCTCATTTCTTCCGGTGTGGTCTGG-3') and the GHS3 clone as template DNA; these primers (Fig. 8a) span the exon/intron junctions involved in the alternative splicing. PCR amplification was carried out for 20 cycles, each cycle consisting of 30 s at 94 °C, 30 s at 65 °C, and 30 s at 72 °C.
Reverse Transcriptase-PCR Analyses--
Total RNA (0.5 µg)
from mouse pancreatic islets (gift from D. L. Eizirik and D. Pipeleers, Department of Metabolism and Endocrinology, Vrije
Universiteit, Brussels, Belgium) and 0.5 µg of poly(A)+
RNA from human kidney (CLONTECH) were
reverse-transcribed in an oligo(dT)-primed reaction as described (27).
The mouse SERCA3 primers used are as follows: a 5' primer M + 1 (5'-GGGGTGGTGCTTCAGATGTCTCTGC-3') corresponding to nucleotides
2948-2972 in mouse SERCA3a and SERCA3b nucleotide sequences (accession
numbers U49394 and U49393, respectively) and a 3' primer M 1 (5'-GGACAAATGCCTGGATGCTCTCAGT-3') corresponding to the inverse
complement of nucleotide stretches 3086-3110 and 3159-3183 in mouse
SERCA3a and SERCA3b cDNA nucleotide sequences, respectively. A
specific 3' primer for the mouse SERCA3c isoform P3
(5'-CTTCAGGTCCTTTTTTTCCAAGAAGCCAAC-3') spans the splice boundary
between the last exon and an optional exon. PCR amplifications were
carried out for 35 cycles, each cycle consisting of 30 s at
94 °C, 30 s at 68 °C, and 30 s at 72 °C for both M + 1/M
1, and M + 1/P3 pairs. The human SERCA3 primers used are as
follows: a common 5' primer 22+ (5'-CTGCACTTCCTCATCCTGCTCG-3')
corresponding to nucleotides 2833-2854 and a 3' primer 1
(5'-ATGGGCACCATCAGTCTGAGG-3') corresponding to the inverse complement
of the nucleotide stretch 3040-3060; numbering according to the
nucleotide sequence deposited under accession number Z69881. Two
additional 3' primers specific for the human SERCA3b and SERCA3c
isoforms were designed as follows: the above mentioned primer P1 and
the primer P2 (5'-GGCTCATTTCTTCAAAGAGGCCAAC-3'), respectively. The PCR
conditions were the same for the 3 pairs of primers (22+/1
, 22+/P1,
and 22+/P2): 35 cycles, each cycle consisting of 30 s at 94 °C,
30 s at 65 °C, and 30 s at 72 °C. All PCR
amplifications were performed using a mixture of Pwo
(proofreading activity) and Taq polymerases from Boehringer
Mannheim, Brussels, Belgium. M + 1, M
1, P2, and P3 primers are
also represented in Fig. 8a. PCR fragments were gel-purified
and subcloned, and for each fragment several individual clones were
sequenced.
Various Analyses Using PCR Techniques--
Amplification of the
genetic marker D17S1828 (accession number, 602622) from human genomic
DNA, ICRFc105-G1035 and -F10124 cosmid clones was performed using the
5' primer L+ (5'-TGCACTCACAGATTTGCC-3') and the 3' primer L
(5'-TTAAGCCAAGTTCGGATTTG-3') for 35 cycles, each cycle consisting of
30 s at 94 °C, 30 s at 55 °C, and 30 s at
72 °C.
Construction and Expression of SERCA3 cDNAs in COS-1
Cells--
The entire coding regions of the mouse SERCA3a, SERCA3b,
and SERCA3c cDNAs were amplified by PCR from mouse pancreatic
islets first-strand cDNA using a common 5' primer MMLD
(5'-AGAAGCGACCTGGACGTCGCGGAC-3') corresponding to nucleotides 8-31 in
mouse SERCA3a and SERCA3b cDNA sequences (numbering according to
accession numbers U49394 and U49393, respectively) in combination with
either the primer M 1 (for SERCA3a and SERCA3b amplifications)
or the SERCA3c-specific primer P3. PCR reactions were carried out for
35 cycles, each cycle consisting of 1 min at 94 °C and 4 min at
72 °C for both MMLD/M
1 and MMLD/P3 primer pairs. PCR
products were separated by 1% agarose gel electrophoresis,
gel-purified, blunt-ended, phosphorylated, and transferred into the
EcoRI-cut, dephosphorylated, and blunt-ended mammalian
expression vector pMT2 (from R. J. Kaufman, Genetics Institute,
Boston, MA). The cloning of the pig SERCA2b cDNA in pSV57
expression vector was described earlier (11). COS-1 cell culture and
DEAE-dextran-mediated DNA transfections were performed as described
(11).
Membrane Preparations, Immunoblotting Analysis, and Ca2+ Transport Assays-- Microsomes were isolated from COS-1 cells expressing mouse SERCA3a, SERCA3b, SERCA3c, and pig SERCA2b according to Verboomen et al. (11). Preparation of the N89 anti-SERCA3 antibody, denaturing gel electrophoresis on 0.75-mm-thick 7.5% polyacrylamide slab gels, semi-dry blotting onto Immobilon-P membranes (Millipore, Brussels, Belgium), and immunostaining of the blots were done as reported earlier (27). Oxalate-stimulated Ca2+ uptake was measured by a rapid filtration method in the absence or presence of 5 mM ATP at 27 °C as described (12).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation and Characterization of Human SERCA3 Genomic Clones-- We have previously described the isolation and partial characterization of the first genomic clone (GHS3, approximately 40 kb in length) specifying the 3' region of the human SERCA3 gene and localized the gene by fluorescence in situ hybridization to human chromosome 17 (31). Subsequent screening of a human chromosome 17-specific cosmid library from the Reference Library Data Base, ICRF (32) with a probe corresponding to the 5'-coding region of human SERCA3 cDNA resulted in the isolation of six new overlapping cosmid clones, whose restriction maps are illustrated in Fig. 1. The cosmid contig covers a genomic region of about 90 kb. The exon/intron organization of the human SERCA3 gene and the sizes of all exons are shown in Fig. 1. The gene is divided in 22 exons distributed across 50 kb of genomic DNA. Exon 21 is optional and, when retained, consists of 88 or 101 bp due to the use of an internal donor splice site (see below). The positions and sizes of introns were determined by PCR analyses using SERCA3-specific primers derived from exonic sequences homologous to those flanking the exon/intron junctions in human (3) and rabbit SERCA1 (2), for which the complete gene structures are known. The exon sequences obtained by genomic sequencing perfectly match those from the cDNA (accession number Z69881), except for a single polymorphism in which a C replaces a T at position 1361 of the cDNA sequence. This point mutation does not change the amino acid sequence at position 453 (Asn). The nucleotide sequences of the exon/intron junctions as well as the position and size of each intron in relation to the amino acid stretch are shown in Fig. 2. However, the last intron (intron 20, 3.218 kb long) contains an optional exon (exon 21). The inset in Fig. 1 documents the presence of the D17S1828 marker in the cosmid clones ICRFc105-G1035 and -F10124 via PCR analysis. D17S1828, containing the dinucleotide repeat (CA)22, has been mapped by Genethon 11 cM from the top of the short arm p of chromosome 17. The amplified product (215 bp) was used as a probe in Southern blot hybridization analysis and assigned to a position approximately 20 kb downstream of the SERCA3 gene (Fig. 1).
|
|
Comparison of Exon/Intron Boundaries of SERCA Genes-- The exon/intron structure of the human SERCA3 gene is compared in Fig. 3a with that of the indicated SERCA genes. The positions of 13 out of 20 introns present in the human SERCA3 gene occur in equivalent positions in the Artemia gene, whereas in the Drosophila gene only six introns occur in the same position as in human SERCA3. Two introns in Drosophila and four in Artemia (arrowheads in Fig. 3a) are not present in human SERCA3. Prior to this elucidation of the human SERCA3 gene structure, all known exons of mammalian SERCA genes were found in conserved positions, with one minor exception at the junction between exons 12 and 13 in the human and rabbit SERCA1 genes, where splicing in the human transcript occurs one nucleotide downstream of the rabbit splicing site (2). We found that this junction is conserved in both human SERCA1 and SERCA3 (Fig. 3a). The complete analysis of the exon/intron layout of the human SERCA1 and SERCA3 genes indicated that the positions of all junctions are conserved except for one boundary, which is found in the SERCA1 gene between exons 8 (298 bp) and 9 (167 bp), but not in the SERCA3 gene. This boundary is also absent from the Artemia and Drosophila genes. PCR amplifications from both human genomic DNA and human kidney first-strand cDNA with human SERCA2-specific primers (Fig. 3b) documented the absence of an intervening sequence in human SERCA2 at this position, too.
|
Structural and Functional Analyses of the 5'-End of the
Gene--
The cosmid contig described in Fig. 1 contains approximately
22 kb of genomic DNA upstream of the translation initiation site, of
which the proximal 4447 nt were sequenced. In order to determine the
transcription initiation site for the SERCA3 mRNA, primer extension
analysis was performed with an antisense 22-nt long extension primer,
stretching from 107 to
128 nt upstream of the ATG site, and using
poly(A)+ RNA from human tonsils. The result in Fig.
4 shows a single extension product of 46 nucleotides, thus locating the transcription initiation site (referred
to as nt +1) at position 152 upstream of the translation initiation
site (Fig. 5b). The
transcription initiation site determined in the present study is found
in the vicinity of the sequence 5'-CCACTGC-3' (represented
as a box in Fig. 5b) matching the consensus
initiator (Inr) sequence YYAN(T/A)YY (35), where the
A indicates the transcription start site frequently used in
other genes.
|
|
Tissue Distribution of Human SERCA3 mRNA-- To determine the relative levels of human SERCA3 mRNA in different tissues, we used a human mRNA Master Blot (dot blot), on which the applied mRNA amounts are normalized for eight housekeeping genes, thus minimizing the tissue-specific variations often related to the expression of any single housekeeping gene. Our dot blot hybridization analysis, using a 3'-end probe, first demonstrated that SERCA3 mRNA is expressed in the human adult and fetal non-muscle tissues shown in Fig. 6, and second, revealed that the expression levels dramatically vary from tissue to tissue as follows: with high levels in thymus, trachea, salivary gland, spleen, bone marrow, lymph node, peripheral leukocytes, pancreas, and colon and intermediate to low levels in the rest of the tissues.
|
Alternative Splicing of the SERCA3 Primary Transcript Generates
Three Variants in Human and Mouse--
Recently, two nucleotide
sequences encoding mouse SERCA3a and SERCA3b isoforms were deposited in
the EMBL/GenBankTM data base under accession numbers U49394
and U49393, respectively, and RT-PCR analysis indicated that SERCA3b is
co-expressed with SERCA3a in mouse pancreatic islets of
Langerhans.5 Insertion of a
73-bp optional exon in SERCA3b occurs immediately after nt 2980 (relative to the ATG codon) which, interestingly, also represents the
point of divergence between the different splice variants in the
related SERCA1 and SERCA2 genes. Retention of this additional
nucleotide stretch results in a shift in the open reading frame, so
that the last 6 amino acids of SERCA3a are replaced by a 45-aa tail in
the SERCA3b isoform. We confirmed the existence of the two SERCA3
transcripts in mouse islets by means of RT-PCR using M + 1 and M 1 as primers (Fig. 7a, lane 1). Equally intense bands of 163 and 236 bp were detected.
Subcloning and subsequent sequencing confirmed that the 163-bp
fragment, indeed, corresponded to SERCA3a. Remarkably, the 236-bp band
proved to represent a heterogeneous population, consisting of a
fragment of 236 bp (SERCA3b-specific) contaminated with a 249-bp long
fragment. The latter represented a novel variant, SERCA3c, in which the 3'-end of the SERCA3b optional exon is extended with an additional 13-bp stretch (see also Fig.
8a). The SERCA3c-specific
amplification from mouse islets became possible by using a mouse
SERCA3c-specific primer, P3 (Fig. 7a, lane 2). Analysis of
the genomic sequence of the 3'-end of the human SERCA3 gene indicated
that the generation of the three SERCA3 splice variants is
theoretically possible. The hybridization of the human mRNA Master
Blot with a common human SERCA3b/SERCA3c probe indicated that SERCA3b
and/or SERCA3c are mainly expressed in human kidney, thymus, salivary
gland, trachea, and colon but at much lower levels than the predominant SERCA3a mRNA (Fig. 7b). RT-PCR from human kidney
performed with the human-specific primers, 22+ and 1
(Fig. 7c,
lane 1), encompassing the optional exon(s), could in principle
amplify all three SERCA3-specific variants (expected lengths: 228, 316, and 329 bp for SERCA3a, SERCA3b, and SERCA3c, respectively). However,
only a 228-bp SERCA3a-specific product was detected; amplification of
SERCA3b- and SERCA3c-specific products (Fig. 7c, lanes 2 and
3, respectively) became possible by using primer 22+ in
combination with splice variant-specific primers P1 and P2,
respectively. Fig. 8a compares a 3218-bp long human genomic
fragment spanning the intervening region between exons 20 and 22 (31),
with a partial mouse genomic sequence derived from a 3-kb PCR product
amplified from mouse genomic DNA with the M + 1 and M
1 primers
(data not shown). An optional exon (exon 21) is found 334 bp (in human)
and 387 bp (in mouse) downstream of the conserved point of divergence
(nt 2980). The human exon 21 is 15 nt longer than the mouse one,
because an additional 3' acceptor splice site is found in the human
sequence 15 nt upstream of the one used in mouse. Exon 21 contains an
internal 5' donor splice site (designated D1 in Fig. 8, a
and b). D1 and D2 are conserved in human and mouse. If D1 is
used, the size of exon 21 is 88 or 73 bp in human or mouse,
respectively, giving rise to the SERCA3b isoforms. If D2 is used, exon
21 reaches its maximum size in both human (101 bp) and mouse (86 bp),
thereby giving rise to SERCA3c. An illustration of how SERCA3 splice
variants are generated is shown in the upper part of Fig.
8b.
|
|
Functional Analyses of the Three SERCA3 Isoforms Transiently Expressed in COS-1 Cells-- To characterize functionally the three SERCA3 isoforms, we have employed the COS-1 cell expression system and measured the oxalate-stimulated Ca2+ uptake into the microsomal fraction. For this purpose, we PCR-amplified the coding regions of SERCA3a, SERCA3b, and SERCA3c cDNAs from mouse pancreatic islets first-strand cDNA (data not shown) and subcloned them into the expression vector pMT2. Since the higher GC content of the 5'-untranslated region of the human SERCA3 gene relative to the mouse one causes premature termination of the reverse transcription reaction (data not shown), the amplification of the complete human SERCA3 cDNAs coding for their corresponding isoforms was not possible so far. Therefore, COS-1 cells were transfected with each of the mouse SERCA3 constructs and, for comparison, also with the pig SERCA2b construct. Fig. 9a shows a typical immunoblot analysis of the mouse SERCA3 isoforms expressed in microsomes isolated from COS-1 cells transfected with the corresponding SERCA3 cDNAs. The immunoblot was stained with the polyclonal antibody N89, which was raised against an epitope close to the N terminus of rat SERCA3 (27). The epitope amino acid sequence is also conserved in both human and mouse SERCA3 isoforms. The time course of oxalate-stimulated Ca2+ uptake into microsomal vesicles isolated from COS-1 cells transfected with SERCA2b and each of the SERCA3 cDNAs (Fig. 9b) demonstrates the ability of each of the SERCA3 isoforms to function as a Ca2+ pump. The apparent affinities for Ca2+ of the SERCA3 and SERCA2b isoforms were also deduced (Fig. 9c). We confirm that SERCA3a presents, in this COS-1 cell system, a lower Ca2+ affinity with respect to SERCA2b (K1/2 = 2.2 versus K1/2 = 0.19 µM), but the obtained values differ slightly from those reported earlier: K1/2 = 1.1 µM for rat SERCA3 (10) and K1/2 = 0.27 or 0.24 for SERCA2b (10, 12). Interestingly, SERCA3b and SERCA3c show much lower apparent affinities for Ca2+ than SERCA3a. The K1/2 values for SERCA3b and SERCA3c cannot be determined, since the saturation plateau for either SERCA3b or SERCA3c was not reached under our experimental conditions and the use of still higher free Ca2+ concentrations was incompatible with the calcium oxalate precipitation technique.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have isolated a total of seven genomic clones spanning a DNA region of 90 kb, of which 50 kb encode the human SERCA3 gene. So far, four other SERCA genes have been completely characterized as follows: the rabbit (23 kb; Ref. 2) and human (26 kb; Ref. 3) SERCA1 genes, and the unique SERCA genes in the crustacean Artemia franciscana (65 kb; Ref. 22) and in the fruit fly Drosophila melanogaster (7.1 kb; Ref. 23). For SERCA2 only a partial exon/intron characterization of the human (7), rabbit (14), rat (16), and pig (15) genes has been reported. The estimated size of the mammalian SERCA2 gene is between 45 and 50 kb, which is comparable to that of human SERCA3. The human SERCA3 gene consists of 22 exons with an average exon size of 219 bp. In comparison, the rabbit and human SERCA1 genes count 23 exons. In both genes, the penultimate exon (exon 21 in SERCA3 and exon 22 in SERCA1) is alternatively spliced. Analysis of the exon/intron boundaries showed (Fig. 3a) that all the intron positions are conserved between the SERCA3 and SERCA1 genes, with the exception of one boundary that is present only in SERCA1 between exons 8 and 9. In the SERCA3 gene, the corresponding exonic sequences are joined in one exon, i.e. exon 8. We now provide evidence (Fig. 3b) that this junction is also absent from the human SERCA2 gene. The comparative junction analysis suggests that SERCA2 and SERCA3 would have diverged through gene duplication mechanisms from a common ancestor gene prior to the SERCA1 separation. Recent phylogenetic tree analyses based on the amino acid sequence comparison of the invertebrate and vertebrate SERCA pumps are in line with this conclusion (48). The localization of SERCA3 gene on human chromosome 17 (31) was further confirmed by the isolation of six overlapping genomic clones from a chromosome 17-specific library. In this study (inset in Fig. 1), we showed that the genetic marker D17S1828 was found approximately 20 kb downstream of the SERCA3 gene, which means that the gene encoding the SERCA3 pump can now be mapped 11 cM from the top of the short arm p of chromosome 17. D17S1828 and D17S1798 microsatellite markers have been recently demonstrated to flank a genetic region of 1 cM, which represents the interval where the cystinosis gene locus has been mapped (49). It remains an open question whether ATP2A3 is included in the genomic region associated with cystinosis, an autosomal recessive disorder caused by a defect in the transport of cystine from lysosomes to the cytosol.
To investigate the regulation of the human SERCA3 gene expression,
primer extension, nucleotide sequence, functional promoter, and
mRNA dot blot hybridization analyses were performed. The primer extension analysis indicated that the transcription site (nt +1) is
located 152 nt upstream of the AUG codon. No TATA element was found
25-30 bp upstream of the cap site. We have, however, identified a
sequence 5'-CCACTGC-3' extending from +7 to +13 nt
(represented as a box in Fig. 5b) that matches
the consensus initiator (Inr) sequence YYAN(T/A)YY (35),
where the A represents the transcription start site
frequently used in other genes. For SERCA3, the A is found at position
+9. It has been previously demonstrated that an Inr element can enhance
the promoter strength even if it is shifted a few bases upstream or
downstream with respect to the transcription initiation site (50). The
SERCA3 promoter falls in the TATA Inr+
category of promoters (51), whereas the SERCA1 and SERCA2 genes in
human and rabbit (2, 3, 13, 14) have TATA+
Inr
-type promoters. It should be noted that almost every
Inr element described so far functions in connection with upstream
Sp1-binding sites (52). It has also been shown that a distance of
40-50 bp between the Sp1 element(s) and the cap site is optimal for accurate transcription initiation (53). Analysis of the CpG dinucleotide distribution within the first 11 kb of the SERCA3 gene
(Fig. 5a) showed that the 5'-end of the gene is embedded in
an 1.5-kb well defined CpG island (36). Within the CpG island, a total
of 14 putative DNA-binding sites for Sp1 were identified. Eight of them
were found immediately upstream of the cap site, in the region between
267 and
39. Moreover, three adjacent Sp1 elements were clustered in
the region
57 to
39, i.e. within the optimal distance
range with respect to the Inr element. Transient transfections in
Jurkat cells via electroporation were performed using seven chimeric
promoter constructs. The results obtained with the PstI- and
SmaI-del constructs were the most informative ones. The
PstI-del construct (from +55 to
135) gave the maximum transcriptional activity, whereas a total loss of activity was obtained
for the SmaI-del construct (from +55 to
31). One pertinent conclusion deduced from the functional promoter analysis is that the
GC-rich region (87% G + C) from
135 to
31 is of critical importance in initiating SERCA3 gene transcription. This region contains six putative Sp1 motifs, an inverse complement for the CACCC
box, and single potential binding sites for AP-2 and TFIIIc. The
results of our functional analysis are in line with a transcription model in which Sp1 protein mediates the transcription initiation through complex interactions involving the Inr element and the cellular
transcription machinery. Besides Sp1, several additional transcription
factors are likely to be involved in the modulation of the core
promoter activity. In contrast to the SERCA2 genes, no
thyroid-responsive elements were identified in the 5'-flanking region
of the SERCA3 gene. This suggests that SERCA3 expression is not under
the control of thyroid hormone as is the case for SERCA2. We conclude
that the existence of a TATA
Inr+ promoter,
which seems to be prevalent among the hematopoietic lineage-specific
genes in mammals (54), together with the several putative
cis-regulatory elements identified in the 5'-flanking region
of the SERCA3 gene might account for the observed tissue-restricted expression pattern of SERCA3 (Fig. 6). On the contrary, a
TATA+ Inr
promoter (like the one
characterizing the SERCA2 gene) might be responsible for a
lineage-independent expression (54). Interestingly, the SERCA gene from
A. franciscana comprises two promoters (24) as follows: a
TATA+ Inr+ promoter, controlling the expression
of a housekeeping isoform, and a TATA+ Inr
promoter involved in the expression of the muscle-specific isoform. We
might speculate that the evolution from a unique SERCA gene in
invertebrates to the multigene SERCA family in vertebrates was also
accompanied by rearrangements of the TATA and Inr promoter elements.
The alternative processing of the SERCA3 pre-mRNA can give rise to three SERCA3 isoforms, which, like the other SERCA family members, differ solely in their C-terminal amino acid sequences, found downstream of amino acid 993. Like in SERCA1, an optional SERCA3 exon (exon 21) can be skipped, thereby generating the SERCA3a splice variant, but it can be retained partially (in SERCA3b) or entirely (in SERCA3c) due to the alternative use of an internal 5' donor splice site (D1) as is the case for SERCA2. Moreover, the alternative splicing mode for SERCA3 resembles that of the plasma membrane Ca2+-ATPase 1 gene. In the latter case, four isoforms with different C-terminal parts can be generated by alternative exclusion, inclusion, or partial inclusion of a single exon in the 3'-end of the gene.
Both human and mouse SERCA3 genes are transcribed and processed in the
same way, according to the splicing scheme illustrated in Fig.
8b. We have identified in both human and mouse introns preceding the corresponding exon 21 a sequence, 5'-CTCTGAC-3', that matches the consensus branch point sequence 5'-YNYURAC-3' (complementary to the U2 snRNA sequence) in which the A is involved in
the first transesterification reaction of the pre-mRNA splicing and
lariat RNA formation. However, human SERCA3b and SERCA3c splice variants structurally differ from their mouse counterparts; this difference is caused by the occurrence of an additional 3' acceptor splice site in the human genomic nucleotide sequence, located 15 nt
upstream of the site used in mouse. This explains why the optional exon
(exon 21; 101 bp) in the human gene is 15 bp longer than in mouse (86 bp). Moreover, the 15-nt sequence, representing the 5'-end of exon 21, encodes a new stretch of five amino acids, ACLYP998. This
stretch, present in both human SERCA3b and SERCA3c isoforms, is
inserted immediately downstream of amino acid 993, which is encoded by
the last constitutively spliced exon (exon 20). Both in human and
mouse, when present, SERCA3b and SERCA3c are always co-expressed in the
same tissue along with the SERCA3a isoform. However, in mouse
pancreatic islets of Langerhans, SERCA3a and SERCA3b are expressed at
nearly equal levels, whereas in human kidney SERCA3b is expressed at
lower levels than SERCA3a. SERCA3c was also found to be expressed at
much lower levels than SERCA3a in all human and mouse tissues examined
so far. The tissue expression pattern of SERCA3b and/or SERCA3c
mRNAs seems to be much more restricted than that of SERCA3a. As a
result of alternative splicing, the SERCA3a-specific C terminus
comprising the last six amino acids (from 994 to 999 aa) is replaced
either by a tail of 45 or 50 aa in mouse or human SERCA3b,
respectively, or by a stretch of 33 or 36 aa in mouse or human SERCA3c,
respectively (Fig. 8b). Hydropathy analysis of the
C-terminal primary sequence of SERCA3b did not show any propensity for
a hydrophobic stretch, which might function as an additional
transmembrane domain; this contrasts with the situation in SERCA2b.
Earlier studies concerning the structure-function relationship revealed
that the divergence in the extreme C terminus is responsible for
functional differences between SERCA2a and SERCA2b (10-12). By
expressing the corresponding mouse SERCA3 cDNAs in COS-1 cells, we
now demonstrate that each of the SERCA3 isoforms is able to function as
a Ca2+ pump. We also confirm that SERCA3 (designated now
SERCA3a) displays a reduced apparent affinity for Ca2+ than
SERCA2b. It has been proposed earlier that the reduced Ca2+
affinity of SERCA3 is consistent with an enzyme (E) in which the equilibrium between the E1 (which binds
Ca2+ with high affinity) and E2 (low
affinity for Ca2+) conformations is shifted toward the
E2 conformational state (10). Structural
interactions between the SERCA3-specific nucleotide/hinge and
C-terminal transmembrane domains appear to mediate the shift in the
E1 E2 equilibrium and
thereby the Ca2+ dependence (55). Finally, we report that
SERCA3b and SERCA3c present different apparent affinities for
Ca2+, which are even lower than the one observed for
SERCA3a (SERCA3a > SERCA3b and SERCA3c). The extended tails of
SERCA3b and SERCA3c must, somehow, lower the affinity for
Ca2+, possibly by their direct interactions with other
cytoplasmic domains of the pump. In turn, these interactions, via long
range coupling, may modulate further interactions between the
transmembrane domains of the enzyme. As a result, the equilibrium
E1
E2 would be
shifted even more toward the E2 state. An
example in which the Ca2+ transport activity can be
modulated through intramolecular interactions is that of the plasma
membrane Ca2+-ATPase, in which the C-terminal
calmodulin-binding domain can exert an autoinhibitory effect on the
enzyme activity. These experimental observations strengthen the idea
that similar effects on the Ca2+ dependence may be mediated
in vivo by interactions between the extended tails of
SERCA3b and SERCA3c with other regions of the pump. The reduced
Ca2+ affinity of SERCA3a and, especially, of the SERCA3b
and SERCA3c isoforms raises questions with regard to their
physiological significance. In the normal cellular context, they would
be most likely inactive, unless they are expressed in a cellular
environment characterized by increased Ca2+ concentrations
or their Ca2+ transport activities are regulated by as yet
unidentified modulators, which are normally not present in COS-1 cells.
Recently, it has been documented that SERCA3a is more resistant to
peroxide than SERCA2b (56). Further investigations are needed to
determine to what extent SERCA3b and SERCA3c share this resistance to
oxidative agents. Another issue to be addressed in the future concerns
the functional significance, if any, of the amino acid stretch,
ACLYP998, which is found only in the human SERCA3b and
SERCA3c isoforms.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge the Reference
Library, ICRF, London, UK, for providing both the gridded filters of
the human chromosome 17-specific cosmid library and the positive clones
and Dr. G. I. Bell (Dept. of Biochemistry and Molecular Biology,
Howard Hughes Medical Institute, Chicago, IL) for communicating data to
us prior to their publication. We thank Dr. D. L. Eizirik and Dr.
D. Pipeleers (Dept. of Metabolism and Endocrinology, Vrije Universiteit
Brussel, Brussels, Belgium) for the gift of mouse pancreatic islets
mRNA; Dr. R. J. Kaufman (Genetics Institute, Boston, MA) for
providing the expression vector pMT2; and Dr. Frank Bulens (Center of
Molecular and Vascular Biology, Katholieke Universiteit Leuven, Leuven, Belgium) for the gift of the pEL1-gal vector. We also appreciate the
skillful technical assistance of Yves Parijs, Anja Florizoone, Marina
Crabbé, and Raphael Verbist.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the University Poles of Attraction Programme P4/23, Belgian State, Prime Minister's Office, Federal Office for Scientific, Technical and Cultural Affairs.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y15724-15730 and Y15734-15738.
§ Recipients of fellowships from the Onderzoeksfonds K. U. Leuven.
¶ To whom correspondence should be addressed: Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg, Herestraat 49, B-3000, Leuven, Belgium. Tel.: (32) 16 345834; Fax: (32) 16 345991; E-mail: Leo.Dode{at}med.kuleuven.ac.be.
1 The abbreviations used are: SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; MOPS, 3-(N-morpholino)propanesulfonic acid; RT-PCR, reverse-transcribed-polymerase chain reaction; TFIIIc, transcription factor IIIc; aa, amino acids; bp, base pairs; cM, centimorgans; nt, nucleotides; kb, kilobase pairs; Inr, initiator.
2 Deposited in the EMBL/GenBankTM data bank under the accession numbers U49394 and U49393, respectively, by Y. Tokuyama, X. Chen, M. W. Roe, and G. I. Bell.
3 Available at the following on-line address: censor{at}charon.girinst.org.
4 F. Bulens, I. Van Nerum, P. Merchiers, A. Belayew, and D. Collen, unpublished data.
5 G. I. Bell, unpublished observations.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|