Comparative Biochemistry and Physiology Part B 127 (2000) 223 – 233
www.elsevier.com/locate/cbpb
Cloning of a neonatal calcium atpase isoform (SERCA 1B)
from extraocular muscle of adult blue marlin (Makaira
nigricans)
Richard L. Londraville a,*, Tyson D. Cramer a, Jens P.C. Franck b,
Alexa Tullis c, Barbara A. Block d
a
Uni6ersity of Akron, Department of Biology, Akron, OH 44325 -3908, USA
Occidental College, Department of Biology, 600 Campus Rd., Los Angeles, CA 90041, USA
c
The Uni6ersity of Puget Sound, Department of Biology, 1500 N. Warner, Tacoma, WA 98416, USA
d
Stanford Uni6ersity, Hopkins Marine Station, Ocean 6iew Bl6d., Pacific Gro6e, CA 93950, USA
b
Received 21 March 2000; received in revised form 28 May 2000; accepted 8 June 2000
Abstract
Complete cDNAs for the fast-twitch Ca2 + -ATPase isoform (SERCA 1) were cloned and sequenced from blue marlin
(Makaira nigricans) extraocular muscle (EOM). Complete cDNAs for SERCA 1 were also cloned from fast-twitch
skeletal muscle of the same species. The two sequences are identical over the coding region except for the last five codons
on the carboxyl end; EOM SERCA 1 cDNA codes for 996 amino acids and the fast-twitch cDNAs code for 991 aa.
Phylogenetic analysis revealed that EOM SERCA 1 clusters with an isoform of Ca2 + -ATPase normally expressed in
early development of mammals (SERCA 1B). This is the first report of SERCA 1B in an adult vertebrate. RNA
hybridization assays indicate that 1B expression is limited to extraocular muscles. Because EOM gives rise to the
thermogenic heater organ in marlin, we investigated whether SERCA 1B may play a role in heat generation, or if 1B
expression is common in EOM among vertebrates. Chicken also expresses SERCA 1B in EOM, but rat expresses
SERCA 1A; because SERCA 1B is not specific to heater tissue we conclude it is unlikely that it plays a specific role in
intracellular heat production. Comparative sequence analysis does reveal, however, several sites that may be the source
of functional differences between fish and mammalian SERCAs. © 2000 Elsevier Science Inc. All rights reserved.
Keywords: Fish; Thermogenesis; Excitation–contraction coupling; Heater tissue; Endothermy; Calcium cycling; Protein structure;
Phylogenetic analysis
1. Introduction
The sarcoplasmic/endoplasmic reticulum calcium ATPases (SERCAs) play a critical role in
regulation of intracellular calcium, and they are
among the best-studied of all enzymes (MacLen* Corresponding author. Tel.: + 1-330-9727151; fax: + 1330-9728445.
E-mail address: londraville@uakron.edu (R.L. Londraville).
nan, 1990; MacLennan et al., 1992; Tada, 1992;
Wu et al., 1995). Calcium is pumped against its
concentration gradient by SERCA into the lumen
of the sarcoplasmic (muscle) or endoplasmic
(other cells) reticulum; this stored Ca2 + is selectively released to initiate a number of cellular
events, including muscle contraction, developmental events, and second messenger pathways. Extensive data exists on their tissue-specific
expression (Wu et al., 1995), interactions with
0305-0491/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S 0 3 0 5 - 0 4 9 1 ( 0 0 ) 0 0 2 5 6 - X
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other proteins that modulate their function (Tada,
1992), and structure – function relationships (reviewed in MacLennan, 1990; MacLennan et al.,
1992). The SERCA family consists of five isoforms coded for by three genes. The SERCA 1
and SERCA 2 genes each produce two gene products via alternative splicing (SERCA 1A, 1B and
SERCA 2A, 2B). The SERCA 3 gene produces a
single gene product (SERCA 3). Each pump has a
tissue-specific distribution; SERCA 1 is expressed
in fast-twitch muscle, 2A in cardiac and slow
twitch, and 2B and 3 are co-expressed in many
tissues (Wu et al., 1995). SERCA 1 is by far the
best studied Ca2 + -ATPase, with over 20% of its
amino acids individually studied via site-directed
mutagenesis followed by expression and in vitro
assay (MacLennan, 1990; Block, 1994).
The mammalian SERCA 1 primary transcript is
alternatively spliced into 1A and 1B; SERCA 1B
is expressed in fast-twitch skeletal muscle during
early development and is replaced by SERCA 1A
soon after birth (Brandl et al., 1986, 1987). Mammalian SERCA 1A differs from SERCA 1B by
only five positively charged amino acids on its
C-terminus (1B is longer than 1A; Zhang et al.,
1995). The two isoforms function identically when
expressed in COS cells (Maruyama and MacLennan, 1988). It is reasonable to speculate, however,
that an in vivo functional difference exists between the two isoforms as evidenced by their
developmentally regulated expression (Brandl et
al., 1986, 1987).
We sought to uncover SERCA’s role (if any) in
cellular heat production. Cellular heat is produced
in vertebrates by a variety of mechanisms. These
include proton leak across the inner mitochondrial membrane, either mediated by a specific
uncoupling protein (Klingenberg and Huang,
1999) or passive proton leak (Brand et al., 1991);
calcium leak across the sarcoplasmic reticulum
(Block, 1994); and Na+/K+ leak across the
plasma membrane (Hulbert and Else, 1990). Each
of these leaks creates an ATP demand, which is
met by catabolic pathways that release heat as
they break bonds. Which mechanism(s) is dominant depends upon the phylogeny of the organism
and the specific tissue involved. In skeletal muscles, two types of heat production occur: shivering
and non-shivering thermogenesis (Block, 1994).
Both involve increased metabolic activity of the
tissue, coupled with high turnover of the myosin
and Ca2 + -ATPases.
The blue marlin (Makaira nigricans) offers a
unique model-system for investigations regarding
SERCA’s involvement in heat production. All
billfish (Istiophoridae and Xiphiidae, along with
one scombrid Gasterochisma melampus) have a
thermogenic organ (heater organ) derived from
extraocular muscles (EOM, Block, 1994). The
heater organ warms the brain and eyes significantly above ambient water temperatures (up to
20°C above ambient, Block, 1991), while the body
temperature remains at ambient. Heat production
in the heater organ is associated with expression
of a muscle cell phenotype that is highly aerobic
(Tullis et al., 1991), has a relatively high percentage of cell volume occupied by sarcoplasmic
reticulum and transverse-T system, and is enriched in SERCA and calcium release channel
(RYR, Block et al., 1988, 1994). The heater organ
itself is not contractile, but is derived from the
superior rectus extraocular muscle, which is contractile (Block, 1986). Other contractile muscles in
the body, such as epaxial (swimming) muscle, are
not thermogenic. By comparing superior rectus
and epaxial muscles, we may be able to identify
cell components that are unique to heat production. SERCA may play a role in heat production,
because it can account for over 50% of the total
ATP turnover in muscle (Simonides and van
Hardeveld, 1988) and is abundant in heater cells
(Block et al., 1988, 1994).
In a previous study, we identified the specific
isoform of SERCA expressed in superior rectus
and heater tissue though amplification and sequencing of partial cDNAs (Tullis and Block,
1996). In this study we sought to determine the
complete sequence of SERCA 1 from superior
rectus, and compare it to that of the SERCA 1
from epaxial muscle. Because the structure/function relationship of SERCA 1 is so well characterized (MacLennan, 1990), we could then predict
the functional consequences of any difference in
primary sequence between the two tissues. For
example, a substitution at residue c 275 results in
inefficient calcium pumping in mammals (Klingenberg and Huang, 1999). If that same position
was variant between heater and non-heater, it
may indicate that calcium pumping requires more
ATP in heater, and this greater ATP turnover
could contribute to the mechanism of heat
production.
Here we report that the message for SERCA
1A and 1B are both present in the superior rectus
R.L. Londra6ille et al. / Comparati6e Biochemistry and Physiology, Part B 127 (2000) 223–233
eye muscle of adult blue marlin, (M. nigricans),
while a single isoform, SERCA 1A is expressed in
epaxial (body) fast-twitch muscle. Primary sequence of the two isoforms is invariant except for
the last five amino acids at the C-terminal end.
We also demonstrate that SERCA 1B is expressed
in eye muscle of adult birds (chicken), but only
1A is present in mammals (rat). Vertebrate
SERCA 1B expression is not limited to neonates,
and may contribute to the unique functional
properties of tissues that express it.
2. Experimental
2.1. Animals and tissues
Blue marlin (M. nigricans) were caught off
Kona, HI, by fishermen, and tissues were removed within 1–2 h of landing the fish. Dissected
tissues were immediately freeze clamped with copper tongs cooled with liquid nitrogen. Tissues
were stored at − 80°C. Adult Wistar – Kyoto
(WKY), King Holtzman (KH) and Spontaneously
Hypertensive (SHR) strains of rat (Rattus nor6egicus) were obtained from the Biology Animal Resource Center at The University of Akron. Adult
chickens (Gallus domesticus) were obtained from a
local poultry farm. Animals were decapitated and
EOMs (superior and lateral rectus muscles) were
quickly dissected and frozen at liquid nitrogen
temperatures. Similarly, muscle from the leg and
back of a 4-day-old WKY rat, and pectoralis
muscle from adult chicken, was dissected and
frozen for RNA isolation.
2.2. RNA isolation and cDNA library
construction
Total RNA used to construct the blue marlin
superior rectus cDNA library was extracted with
guanidinium isothiocyanate and cesium chloride
(Chirgwin et al., 1979) and used as substrate for
commercial synthesis of a random/oligo dTprimed Lambda ZAP II cDNA library (Stratagene, La Jolla, CA). For all other cDNA synthesis
(fast-twitch muscle cDNA library and RT-PCR),
total RNA was isolated with Tri-Reagent (Molecular Research Center, Cincinnati, OH). Total
RNA was primed with an oligo (dT) primer and
synthesized with either avian myeloblastosis virus
(AMV) or Moloney murine leukemia virus M/
225
MLV reverse transcriptase (Promega, Madison,
WI) as detailed in Franck et al., (1998). RNA
from blue marlin fast-twitch muscle was used to
construct a cDNA library using Stratagene’s
Lambda–Zap kit (Franck et al., 1998).
2.3. RT-PCR
Total RNA was extracted with Tri-Reagent and
primed with an oligo-dT primer for first-strand
cDNA synthesis. Unless otherwise specified, reverse-transcription coupled (RT) PCR was performed under the following conditions: 200 ng
1st-strand cDNA template, 200 mM dNTPs, 1 mM
forward and reverse primers, 3 mM MgCl2, and
0.5 U Taq DNA polymerase (Promega). Samples
were cycled 35 times through denaturing at 95°C
for 1 min, annealing at 50°C for 1 min, and
extension at 72°C for 1.5 min, followed by a
7-min extension at 72°C at the end of the cycling.
PCR products were separated by agarose gel electrophoresis (1–2%) and stained with ethidium
bromide. For determination of rat and chicken
SERCA expression, primers were designed to amplify approximately 150 bp 5% and 3% of the stop
site for the SERCA 1 transcript. For rat SERCA
1 (GenBank accession c M99 223) the forward
primer (Ratfor 5%-GACCCCCTGCCGATGATC3%) corresponds to nucleotides 3016–3033, and the
reverse primer (Ratrev 5%-GAAGGGAACGAGGGTGGGG-3%) corresponds to nucleotides
3335–3317. For chicken SERCA 1 (GenBank accession c M26 064) the forward primer (Chickfor
5%-ACCCCCTGCCCATGATCTTT-3%)
corresponds to nucleotides 2873–2892, and the reverse
primer (Chickrev 5%-TAAGCGGCGCCCATTATGGG-3%) to nucleotides 3165–3146. Amplification of chicken cDNA was done with 1.5 mM
MgCl2 and annealing at 45°C.
2.4. Analysis
Sequence identity was confirmed by the BLAST
(Altschul et al., 1990) subroutine in GenBank.
Contigs of overlapping nucleotide sequence were
constructed in MacVector (International Biotechnologies, New Haven, CT), and from consensus
sequence, amino acid sequence was deduced.
Alignment of deduced fish amino-acid sequence
with previously determined SERCA sequences
was performed with ClustalW (Higgins, 1988).
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2.5. Library screening strategy
All probes were labeled with [a32P]-dCTP via
random priming with a Ready-To-Go Priming
Kit (Pharmacia, Piscataway, NJ). The superior
rectus library was initially screened with a PCR
product amplified from superior rectus cDNA
and the N1,N2 primer pair (open reading frame,
ORF 1310–1333) from Tullis and Block (1996).
This probe identified clone LSERC/B-1(ORF
990 – 1349), which was then labeled and used as a
probe, resulting in clone LSERC/B-2. LSERC/B2 was estimated to be over 2 kbp in length,
however sequencing via primer walking revealed
that this clone actually had a section of cytochrome oxidase inserted as part of its sequence
(presumably during the ligation reaction of cDNA
construction); only the 5% SERCA 1 portion of
this clone is represented in Fig. 1 (-379-ORF 580).
Next LSERC/B-1, the N1,N2 PCR product, and
a P1,P2 PCR product (ORF 1026 – 1336, primers
from Tullis and Block, 1996), were each labeled
and used in concert to screen the superior rectus
library; resulting in clones LSERC/B-3 (ORF 8123% UTR) and LSERC/B-4 (ORF 998-3% UTR).
The gap between LSERC/B-1 and LSERC/B-2
was closed by PCR with flanking primers, and
first-strand cDNA from superior rectus muscle.
The PCR product was cloned into T-vector
(Promega), and eight separate clones sequenced to
guard against random sequence error introduced
by Taq infidelity. This completed the contiguous
sequence of SERCA 1B. SERCA 1A was cloned
from the glycolytic muscle library using the PCR
product generated from primers U2549, MR and
clone LSERC/B-3. This probe pulled the fulllength cDNA clone LSERC/A-1.
2.6. RNAse protection assays
Two antisense probes were constructed; one
that hybridized with both SERCA 1A and 1B
transcripts, and a second that protected only
SERCA 1B transcripts. For the SERCA 1A/1B
probe, clone LSerc/B-2 was amplified using M13
(upper, U strand) and U599 (5%-GGCCAGAGAAAGGAGTAG-3%, lower, L strand) primers.
The product was subcloned into T vector
(Promega, Madison, WI) and sequenced to determine orientation of the insertion. Plasmid was
linearized with Apa I, and a 32P dUTP-labeled
probe synthesized with SP6 RNA polymerase according to the Ambion Maxiscript protocol (Albion, Austin, TX). The SERCA 1B probe was
amplified from primers 2991U (5%-GGCTCATGGTCTTTAAGC-3%, U strand), MR, and clone
LSerc/B-3 as a template. Products were cloned
into T-vector and sequenced to determine orientation. Plasmid was linearized with Pst I and a
probe synthesized with T7 RNA polymerase using
Ambion’s kit. Probes were purified on polyacrylamide gels. Hybridizations of labeled probe with
total RNA from each tissue were incubated at
37°C, and were separated on denaturing gels.
Fig. 1. Contig map of clones used to determine full-length cDNA sequence for SERCA 1A and 1B from blue marlin (M. nigricans).
Large, open rectangle indicates the region of cDNA that is translated (small shaded rectangles indicate 5% and 3% untranslated regions)
and numbers under top line indicate number of basepairs. SERCA 1B clones are indicated by solid lines and SERCA 1A (single clone)
by a dashed line. Restriction endonuclease sites are indicated over the coding region.
R.L. Londra6ille et al. / Comparati6e Biochemistry and Physiology, Part B 127 (2000) 223–233
Fig. 2. Nucleotide and deduced amino acid sequence for SERCA 1A and SERCA 1B from blue marlin, M. nigricans. Nucleotide
sequence is numbered on the left relative to the start codon; amino acid sequence is numbered on the right. Sequences for SERCA
1A and SERCA 1B are identical except where indicated: denotes the beginning of the SERCA 1B sequence ( −68) and indicates where SERCA 1A and 1B diverge. Sequence above solid black line is that of SERCA 1A cloned from a fast-twitch muscle
library; sequence below line is that of SERCA 1B cloned from the eye muscle library.
227
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Fig. 3. Phylogenetic relationships among SERCA sequences. A
multiple alignment of amino acid sequences was performed
using CLUSTAL W (Higgins, 1988) and used as input for a
parsimony-based tree-construction program (ProtPars within
PHYLIP, Felsenstein, 1993). Numbers at nodes indicated percent of 500 bootstrapped replicates that identify grouping.
Drosophila SERCA sequence is the designated outgroup and
serves to root the tree.
2.7. Sequencing
Plasmid preps from excised clones were sequenced with an Applied Biosystems (ABI/Perkin
Elmer; Foster City, CA) 373 automated DNA
sequencer. Templates were cycle sequenced using
a Prism Ready Reaction Dye Deoxy kit (ABI)
and electrophoresed through 6% denaturing gels.
Clones were initially sequenced with the M13
forward and reverse primers. Primers were subsequently synthesized from the deduced sequence
until the entire clone was sequenced (primer walking). All clones were sequenced at least three
times each on both strands.
2.8. Results
The complete cDNA for SERCA 1 from fish
was determined from sequencing overlapping
clones isolated from the superior rectus cDNA
library (Figs. 1 and 2). The compiled contiguous
sequence includes 3307 nucleotides (nt), with an
open reading frame (ORF) of 2988 nt, coding for
996 amino acids (Fig. 2). This sequence is five
amino acids longer than the ORF coded for in the
complete cDNA for SERCA 1 isolated from the
glycolytic muscle library (4167 total nt, ORF of
2973 nt, 991 amino acids). The two sequences
(superior rectus and glycolytic) are identical except for the untranslated regions (UTRs) and the
last six codons of the ORF.
Amino acid alignment with published SERCA
sequences (ClustalW, Higgins, 1988) followed by
phylogenetic analysis (PHYLIP, Felsenstein,
1993) clusters both fish sequences with the
SERCA 1 isoforms (Fig. 3). SERCA 1 primary
sequence is highly conserved, with 81% sequence
identity among fish, frog, chicken, rabbit, and
human sequences (alignment not shown). Assignment of the fish sequences to SERCA 1A and
SERCA 1B was made according to the human
sequence (Brand et al., 1991). The human SERCA
1 gene has an intron/exon border at nt c 2980,
where alternative splicing includes one of two
exons to result in 1A or 1B. If exon c 22 is
spliced in the resulting transcript translates into
SERCA 1A, if exon c 22 is spliced out then the
transcript results in SERCA 1B. The two fish
SERCA 1 sequences diverge at a similar position
(nucleotide c 2971, Fig. 2), therefore this was
defined as the alternative splicing site in fish. In
fact, considering that fish SERCA 1A sequence is
three residues shorter than human, the nucleotide
position of the SERCA 1A/1B divergence is identically positioned in both fish and mammals. According to the mammalian convention, the shorter
isoform was labeled SERCA 1A and the longer
isoform SERCA 1B. The same criteria were used
to assign isoform identity for sequences amplified
from rat and chicken. Primers flanking the alternative splice site were used to amplify short ( :
300 bp) products from extraocular and axial
muscles; these products were cloned, sequenced,
and translated to determine the position of the
stop codon. Longer ORFs that ended in 5–9
charged residues were deemed SERCA 1B;
shorter ORFs were deemed SERCA 1A.
Fish SERCA 1A has an expression pattern that
is distinct from SERCA 1B (Fig. 4). Singlestranded RNA probes, one designed to recognize
both SERCA 1A and 1B and another to recognize
only 1B (by utilizing its unique UTR) hybridized
to transcripts in fish muscle. Heater organ, superior rectus, and glycolytic muscles, but not oxidative or cardiac muscles, hybridized to the SERCA
1 probe. Only heater organ and superior rectus
hybridized to the SERCA 1B probe. Therefore, it
R.L. Londra6ille et al. / Comparati6e Biochemistry and Physiology, Part B 127 (2000) 223–233
229
3. Discussion
3.1. 1B is expressed in adult muscle
Fig. 4. Tissue distribution of SERCA 1 mRNA. A ribonuclease protection assay was performed on isolated RNA from
marlin tissues (HO, heater organ; SR, superior rectus; FT,
fast-twitch muscle; SO, slow-oxidative muscle; CM, cardiac
muscle; yeast, control RNA). 1B (left) or 1A/1B (right)-specific
RNA probes were reverse transcribed from 1B and 1A clones
in the presence of 32P-dUTP and hybridized to 5 mg total RNA
from each tissue. Each sample was then digested with RNAse
and separated on 5% polyacrylamide gels.
appears that SERCA 1A is expressed in all fasttwitch fibers, whereas SERCA 1B expression is
restricted to eye muscles (including heater organ)
of adult marlin.
In adult chickens, we identified only SERCA
1B transcripts in EOMs, and only 1A in pectoral
muscle. The SERCA 1A ORF from pectoral muscle ends in YLEA*, whereas the 1B ORF in
extraocular muscle ends in YLEADAEDLRKKRK*. In rat, however, SERCA 1B
(YLEGDPEDERRK*) could only be amplified in
neonates (trunk), and 1A (YLEG*) was the only
isoform detected in EOMs (Fig. 5).
Fig. 5. Amplification of SERCA 1 from rat muscles. RT-PCR
was performed on mRNA isolated from extraocular muscle of
adult King – Holtzman rat (lane 2), Wistar–Kyoto rat (lane 3),
spontaneously hypertensive rat (lane 4), and skeletal muscle
from neonatal King – Holtzman rat (lane 5), using SERCA 1
primers that flanked the coding region where 1A and 1B differ
(lane 1, 100 bp ladder). PCR products were separated on a 2%
agarose gel and stained with ethidium bromide. All products
were cloned and sequenced to confirm their identities. Products in lanes 2 – 4 were SERCA 1A fragments (all identical
sequence), the product in lane 5 is a SERCA 1B fragment.
Here we report the first instance of significant
and dominant SERCA 1B expression in tissue of
an adult vertebrate (some very minor expression of
Serca 1B in adults was recently reported by Peters
et al., 1999). Mature blue marlin express both
SERCA 1B and 1A in eye muscle, but only 1A in
epaxial muscle (ectothermic). It is possible, therefore, that because SERCA 1B expression is specific
to heater tissue and the muscle from which it is
derived (superior rectus), this specific isoform may
contribute to heat generation more than another
SERCA isoform. An alternate hypothesis is that
SERCA 1B expression is not specific to tissues that
generate heat, but rather to any extraocular muscle. Because EOMs are relatively uncommon subjects of study, this expression pattern may have
been overlooked in general surveys of SERCA
isoform expression (Wu et al., 1995). To resolve
these hypotheses, we investigated SERCA expression in some representative vertebrates.
We can address the hypothesis that SERCA 1B
is exclusively expressed in eye muscles of vertebrates by comparing the expression pattern of
SERCA in a mammal, a bird and a fish. These
species are certainly not all vertebrates, but they do
give insight as to SERCA’s expression pattern
across diverse classes of vertebrates. SERCA 1B is
expressed in EOM of chicken and fish, but not rat,
where we could only amplify SERCA 1A from
EOM. This result is not biased by the primers’
inability to amplify SERCA 1B in rat, because 1B
is easily amplified from neonatal rat muscle (Fig.
5). Adult chickens, however, exclusively produce
SERCA 1B in extraocular, and 1A in pectoral
muscles. Therefore we can make two assertions
about the expression pattern of SERCA 1 isoforms
and its relationship to endothermy. SERCA 1B’s
expression is not unique to heater tissue in blue
marlin; it is also present in bird extraocular muscle
(an endotherm). Secondly, SERCA 1B expression
is not characteristic of all (adult) EOMs, because
mammals express SERCA 1A. Because all the
EOMs in this study are either thermogenic tissues
or tissues from endotherms, and because these
EOMs express either SERCA 1A or 1B, our data
do not support the hypothesis that SERCA 1
isoform expression is diagnostic of heat production
in a tissue.
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Table 1
Subset of non-conserved blue marlin SERCA 1 residues studied by site-directed mutagenesis in rabbita
Residuec
Rabbit
Result of mutagenesis in rabbit
Marlin
Reference
114
192
245
696 (R), 693 (M)
895 (R), 892 (M)
696 (R), 693 (M)
N
E
D
E
E
D
Reduced function
No effect
No effect
No effect
No effect
No effect
D
D
E
D
P
T
Clarke et al., 1990
Clarke et al., 1990
Clarke et al., 1990
Vilsen et al., 1991
Clarke et al., 1989a,b
MacLennan, 1990
a
Residue numbering scheme is that of rabbit SERCA 1 until residue c503; fish sequence is missing mammalian residues 503–505
(see Fig. 2, and Tullis and Block, 1996). After residue c 503, sequence is listed as rabbit (R) and marlin (M).
3.2. Fish-specific aspects of primary structure and
possible functional correlates
Blue marlin SERCA 1A and 1B sequences (nucleotide and amino acid) are identical over the
coding sequence, with the exception of the last
five residues of 1B (Fig. 2). Therefore, any aspects
of fish-specific SERCA 1 function is likely shared
by SERCA 1A and 1B, and if SERCA 1B has a
distinct function, it must be linked to the charged
carboxyl tail. One distinct difference between fish
and mammal sequence is a three-amino acid deletion near the fluoroscein-isothiocyanate (FITC)
binding site (c 503– 505), first reported by Tullis
and Block (1996) from partial cDNAs. FITC is a
competitor for ATP, and fish SERCA does have
10-fold higher sensitivity to FITC than that of
rabbit, although ATP turnover is similar between
the two enzymes (Hieu et al., 1992).
Other functional clues from the primary structure come from the site-directed mutagenesis studies of SERCA 1 (MacLennan, 1990). Rabbit and
fish sequences vary at 155 sites; of those six sites
have been studied by site-directed mutagenesis
(Table 1). At one site (c114) an N for A mutation in rabbit SERCA results in a pump with
: 50% the pumping efficiency (calcium pumped
per ATP hydrolyzed) as the wild-type pump. Aspartate (D) occupies the homologous position in
marlin. If substituting a polar residue (N) for a
non-polar residue (A) results in reduced function
in rabbit SERCA (Clarke et al., 1989a,b), then it
is likely that a charged residue (D) at this site in
marlin may also result in a reduced (relative to
rabbit) function SERCA. This is precisely the type
of mutation that could contribute to heat generation at a cellular level. However, both thermogenic
(heater)
and
non-thermogenic
(fast-twitch) muscles have the same residue at this
position. Therefore, if aspartate at this position in
marlin results in an inefficient pump, it is equally
inefficient between heater and non-heater, and
thus is not a ‘smoking gun’ for the cellular source
of heat.
In studies on rabbit SERCA, five of these six
sites were found to have no effect when mutated
(Table 1), and we speculate that these sites generally have little influence on SERCA function. The
difference in properties of the amino acid between
fish and rabbit, however, is more severe than
imposed on the mammal sequence via site-directed mutagenesis. In general rabbit SERCA experiments substitute the residue being studied
with alanine, a small non-polar residue. In the fish
sequence, a charged residue substituted in or out
(D at 192 and 696, and T for D at 963) may
contribute to any taxon-specific differences in
SERCA function (if indeed they exist). Interestingly, four of the six sites (including proline,
which typically induces great influence on structure) map close to the sarcoplasmic membrane on
proposed two-dimensional maps of SERCA
(MacLennan et al., 1992). Because fish have a
membrane phospholipid composition that is distinct from that of mammals (Hazel et al., 1991),
these sites may reflect fish-specific membrane constraints on fish SERCA structure. Indeed, phospholipid composition has been shown to
dramatically affect SERCA function (Lee, 1998).
Although we have identified several sites of
structural disparity between the fish and mammal
sequence, functional consequences need to be
confirmed with in vitro assays of the enzyme.
Unlike the site-directed mutagenesis experiments,
these amino-acid ‘substitutions’ do not occur in
isolation; because many residues differ between
the fish and mammal sequences, a functional
change introduced at one site may be compen-
R.L. Londra6ille et al. / Comparati6e Biochemistry and Physiology, Part B 127 (2000) 223–233
sated for by other sites. Comparative sequence
analysis is useful, however, because it highlights
which regions are most likely the sources of any
functional difference.
3.3. Is SERCA 1B typical of a single muscle
fiber-type?
It is reasonable to assume that SERCA 1A and
1B are not functionally equivalent, because 1B
expression persists through vertebrate evolution
(this report). Their function is equivalent, however, when expressed in COS cells (rabbit SERCA
1, Clarke et al., 1989a,b). Therefore, some aspect
of the in vivo environment must distinguish
SERCA 1A from 1B functionally. For example,
SERCA 1B expression may be restricted to one
muscle fiber type. EOM is typified by many diverse fiber types, including those that are fast-contracting, non-fatiguing, and highly aerobic (Fast
Oxidative Glycolytic, FOG fibers, Porter and
Karathanasis, 1999) and slow oxidative (SO)
fibers. Other muscle fibers that fit the phenotype
of FOG also express neonatal isoforms. In adult
chicken pectoralis, fast-twitch ‘red’ fibers express
an embryonic fast isoform of myosin (Shear et al.,
1988). Perhaps the pattern of SERCA 1B expression in skeletal muscle of developing mammals
(Brandl et al., 1986, 1987) indicates the presence
of FOG fibers, that later become fast-glycolytic
(FG) fibers (and switch to SERCA 1A) after
birth. The exact origin of the heater tissue phenotype remains unknown but previous studies indicate that both types of aerobic fibers (FOG and
SO) in the extraocular muscle may be contributing to this thermogenic muscle cell type (Tullis
and Block, 1997). While the SERCA expression
pattern (Fig. 4) would suggest a fast-twitch fiber
derivation, results from expression studies with
the sarcoplasmic reticulum calcium release channel (RYR) indicate the involvement of slowtwitch fibers (Tada, 1992; Block, 1994; Franck et
al., 1998).
SERCA 1B may also be characteristic of another, as yet uncharacterized muscle fiber type.
EOM contains muscle fibers that do not fit into
classical muscle fiber types (Jacoby et al., 1990;
Jacoby and Ko, 1993). EOM is also spared in
neuromuscular diseases (such as Duchenne Muscular Dystrophy) that target all other skeletal
muscles (Porter et al., 1998), indicating that the
muscle fibers themselves are fundamentally differ-
231
ent. Interestingly, EOM that is spared in muscular
dystrophy maintains intracellular calcium levels,
whereas pectoral muscle does not (Khurana et al.,
1995). Although ability to maintain calcium
homeostasis is not the mechanism of EOM sparing in muscular dystrophy (Porter and
Karathanasis, 1998), it does suggest that calcium
cycling, and thus SERCA function, may be fundamentally different in EOM. Now that isoformspecific probes are available, we can determine the
specific fiber-type distribution of SERCA 1B with
in situ hybridization. Then we can critically evaluate if SERCA 1B is expressed in a specific fibertype, and if it confers unique functions upon that
fiber type.
Acknowledgements
The authors acknowledge the support of the
National Science Foundation (IBN-9 507 499 to
BAB) and the Research (Faculty Projects) Committee of the University of Akron (RLL). The
authors also gratefully acknowledge the help of
Howard Reisman. Complete cDNA sequences for
blue marlin SERCA 1A and 1B are listed in
GenBank under accession numbers U65 228 and
U65 229, respectively. Address reprint requests to
Richard Londraville, University of Akron, Department of Biology, Akron, OH 44325-3908.
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