Gene, 109 (1991) 275-279 © 1991 Elsevier Science Publishers B.V. All rights reserved,0378-1119/91/$03.50
275
GENE 06170
Cloning and characterization of the gene encoding rabbit cardiac calsequestrin (Gene expression; myogenesis; myogenin; nucleotide sequencing; recombinant DNA)
Masashi Arai*, Norman R. Alpert* and Muthu Periasamy ",b '° Department of Physiology and Biophysics, and b Department of Microbiology and Molecular Genetics, University of Vermont College of Medicine, Burh'ngton, VT 05403 (U.S.A.)
Received by R. Padmanabhan: 3 June 1991
Revised/Accepted: 12 August/21 August 1991 Received at publishers:17 September1991
SUMMARY A cDNA encoding rabbit cardiac calsequestrin was isolated and characterized. The deduced nascent cardiac calsequestrin contains 409 amino acids of which 26% are acidic residues, and had 93% and 67% aa identity with canine cardiac caisequestrin and rabbit fast-twitch skeletal muscle calsequestrin, respectively. RNA blot analyses indicate that this mRNA is expressed in atrium, ventricle and to a lesser amount in slow-twitch skeletal muscle. This mRNA transcript is not expressed in adult fast-twitch skeletal muscle, smooth muscle, or nonmuscle tissues. Analysis of in vitro skeletal muscle myogenesis using a mouse myoblast cell line C2C 12, demonstrates that both cardiac and skeletal calsequestrin isoforms are coproduced during muscle differentiation.
INTRODUCTION Calsequestrin is a high-capacity, moderate-affinity Ca 2 + binding protein located in the luminal space of junctional sarcoplasmic reticulum (MacLennan et al., 1983). The calsequestrin acts as a Ca 2 + buffer, and the release of Ca 2 + bound to calsequestrin through a Ca2+-release channel triggers muscle contraction (Lytton and MacLennan, 1991). Recently, the major sarcoplasmic reticulum proteins have been isolated and their primary structures determined. Most of these proteins have isoforms and each isoform is expressed in a tissue-specific manner (Lytton and Correspondence to: Dr. M. Periasamy, Department of Physiology and Biophysics, Universityof Vermont Collegeof Medicine, Burlington, VT 05405 (U.S.A.) Tel. (802)656-4433; Fax (802)656-0747.
Abbreviations: aa, aminoacid(s); bp, base pair(s); cDNA, DNA complementary to RNA; DMEM, Dulbecco's modified Eagle's medium; kb, kilobase(s)or 1000bp; nt, nucleotide(s);ORF, open readingframe; SDS, sodium dodecyl sulfate; SSC,0.15 M NaCI/0.015M Na3"citrate pH 7.6.
MacLennan, 1991). So far the primary structure of rabbit fast-twitch skeletal calsequestrin (Fliegel et al., 1987)and of canine cardiac calsequestrin (Scott et al., 1988) have been determined. Earlier studies (Scott et al., 1988; Fliegel et al., 1989) indicated that the fast-twitch muscle calsequestrin is the major isoform expressed in both fast- and slow-twitch skeletal muscle, and cardiac calsequestrin is mainly expressed in heart. However, the precise tissue distribution of both cardiac and fast-twitch skeletal isoforms of caisequestrin mRNAs remains to be determined. Furthermore, it is of interest to determine the expression of both cardiac and fast-twitch caisequestrin isoforms during in vitro myogenesis. This study was designed (i) to characterize the rabbit cardiac calsequestrin by cDNA cloning analysis, (ii) to study the tissue-specific expression of both cardiac and fast-twitch calsequestrin mRNAs using genespecific probes in rabbit muscle and nonmuscle tissues, and (ill) to determine the temporal expression of the two isoforms ofcalsequestrin during in vitro myogenesis of skeletal muscle cell lines L6E9 and C2C12.
276 EXPERIMENTAL AND DISCUSSION
(a) Nucleotide and deduced aa sequence of rabbit cardiac caisequestrin An adult rabbit cardiac eDNA library was screened with the last exon of the rabbit fast-twitch muscle calsequestrin gene (108 bp, SstII-PvuII fragment, nt 7721-7828, ZarainHerzberg et al., 1988). Nine positive clones were obtained
from about 5000 plaques. A partial restriction map of the longest eDNA clone encoding the rabbit cardiac calsequestrin is shown in Fig. 1A. This eDNA clone was 2510 bp long and included a single ORF of 1227 bp long. It also contained l198-bp 3'-untranslated and 85-bp 5'untranslated regions (Fig. 1B). The nt sequence of rabbit cardiac calsequestrin showed 78.4~o identity with canine cardiac calsequestrin (Scott et al., 1988) and 58.6?/0 identity
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CTGGCTCATCCAGGCCTCCAGGAATCTGCCGTGCTCCCTCGGGCACACAGCACGTTTGGG GAAGGAGACGGGAAACTTGTCCCAGATGAAGAGAGCTCACCTGTTCGTCGTGGGGGTCTA M K R A H L F V V G V Y CCTGCTGTCCTCTTGCAGGGCCGAAGAGGGTCTCAACTTCCCCACGTACGACGGGAAGGA L L S S C R A E E G L N F P T ¥ D G K D CCGGGTGGTGAGTCTTTCGGAGAAGAACTTCAAGCAGATTTTGAAGAAATACGACTTGCT R V V S L S E K N F I( Q I L K K ¥ D L L GTGCCTCTACTACCACGCGCCGGTGTCGGCCGACAAGGTGGCCCAGAAGCAGTTCCAGCT C L ¥ Y H A P V 5 A D K V I% Q K Q F Q L GAAGGAGATTGTGCTCGAGCTGGTGGCCCAGGTCCTGGAACACAAAGAGATAGGCTTTGT K E I V L E L V A Q V L E H K E I G F V GATGGTGGATGCCAAGAAAGAAGCCAAACTTGCCAAGAAACTGGGTTTTGATGAAGAAGG M V D A K K E A K L A K K L G F D E E G AAGCCTGTATATTCTTAAGGGTGATCGCACKATTGAGTTTGATGGCGAGTTTGCGGCTGA S L ¥ I L K G D R T I E F D G E F A A D TGTCCTGGTGGAGTTTCTCTTGGACCTGATTGAAGACCCCGTGGAGATCATCAACAGCAA V L V E F L L D L I E D P V E I I N S K GCTGGAAGTGCAGGCCTTTGAGCGCATCGAGGACCACATCAAACTCATCGGCTTTTTCAA L E V Q /% F E R I E D H Z K L I G F P K GAGCGCGGACTCAGAATATTACAAGGCTTTTGAAGAAGCAGCGGAACACTTCCAGCCCTA S A D S E Y ¥ K A F E E A A E H F Q P Y CATCAAATTCTTTG CCACCTTCG ACAAAGGGGTTG CAAAGAAACTGTCTTTGAAGATGAA I K F F A T F D K G V A K K L 5 L K M N TGAAGTCGATTTCTATGAGCCCTTTATGGATGAGCCCACTCCCATCCCCAACAAACCTTA E %2 D F Y E P P M D E P T P I P N K P CACGGAAGAC, GAGCTCGTGGAGTTTGTGAAGGAGCACCAGAGACCCACTCTACGCCGGCT T E E E L V E F V K E H Q R P T L R R L GCGCCCGGAGGATATGTTTGAGACCTGGGAAGATGATTTGAATGGGATCCACATTGTGCC R P E D M F E T W E D D L N G I H I V P CTTTGCAGAGAAGAGTGACCCAGATGGCTATGAGTTCCTGGAGATCCTGAAACAGGTGGC F A E K S D P D G Y E F L E ~ L I( Q V A CCGTGACAATACCGACAACCCGGACCTGAGCATCGTGTGGATCGACCCGGACGACTTTCC R ~D N T D N P D L S I V W I D P D D F P TCTGCTTGTTGCCTATTGGGAGAAGACTTTCAAGATTGACCTGTTCAAGCCCCAGATTGG L L V A Y W E K T F K I D L F K P Q I G GGTGGTGAATGTGACGGACGCTGACAGTGTCTGGATGGAC, ATTCCAGACGACGATGACCT V V .N.~V,,T, D A D S V W M E ~[ P D D D D I'. GCCCACAGCTGAGGAGCTGGAAGACTGGAI'TGAGGACGTGCTCTCCGGAAAGATCAACAC P T A E E T. E D W T E D V I, S G K T N T CGAAGATGATGACAATGAAGATG AAGATGACGACGATGACAATGATGACGACGATGACGA E D D D N E D E D O D D D N D D D D D D CAATGGCAACTCCGATGAGGAGGATAATGATGACAGTGATGAGGATGATGAATAGCCCCA N G N S D E E D N D D S D E D D E ATTTTATAAGACTCCG CTGAAAACAAAGTCACAGCTATCCACTACTGTG C A G A C A G C A C G AGGCAGCAGTGAGTAGCCCTGCCCCACGCCCAGCCAGCTCCTTTCCCTTTCCCGGCCTCT CTTTCCACTGCCAACTCGCACGGAGCGGATCCGGGTACCCAGCTGCCTTGCCGGGTCCAG CCATCCCACTCACCGGGGACAGGGGAGGAATCACTCTCACACGAACTGCTTTGACCGTCA TGGAAAGAAAG CTCCTGTTCTTTGTTAGATTGTTAATAGTCATGACTTTGCTGGGGTCTG GCAGCCTGGGGAGAACAAG CCCCCGG CACCTCGCTTCGGGTTTCCTTGTTGTCTTTGG CA ATGGCAGTAACAGAACTAACAGCTTGCTAACTCACAAGCGTGGAGTGACCTTGTACACAG AGCCGGGGAAGACACACGATCAGGCAGTTTATCTCCCCTGCCTAGCACATCAACTGAGGG TCCTTCCTAAGTGAAGAGGAGCCCCTGGAGGAAGGCTGAGCTTCTAAACAGGTTTATGAA TCAG CTTCTCTGGCTCTATTAAGCATTTCATCCCACATGTGAGTCAGACCATGATGAACT CAGGTCCAAGCCTTTGAGTGACTGCGGAGAGGCCTTGAAACAGGTGTGCAGGTGCCACCC TCATTTCCAGCAGATTCCAATACTGGGGTGGGAGGTTAATTTGCCCTCTGAACTTCCCGG TATGTGACAAGTCTCCAGTATTCAGCAGTGGTGGGGGAGGGATACAAGATTTTCCTTACT TTGATGGGGTTAATGTCTCTGAATGGGTCAGCTGGCTTCCGGGGCTGAG C T G G G A C C T G A ATGCCCG CTCTCTGTCGCTGCTCATGTGCCTTCCCACTTAGACTAAAGATTGCTCTGTCC AGCAGTCAATGAATGCTCCCACGGCTGACCAGGGCAACCATAATTGGAGTTTATAATGAT GCCCTGGGGCACTGAAAAAATTCCCAAGTGCCTTCCAAATGGTCTGAACATTACACTGTG CTTGGTAGTTCTAGTGCTAAGTGTGCAGTATGATG CTCTAATTTATTTTCTCATTCTCTC AACACAAGTGTAAAACACCGTGCAAACTTTTTGAAAGTCAGGAAATCCTTTTGTGGAATT ATAGCTAATTAACTCTGTCATTAAACTCATATTTTGAAAATAAAAAAAAA
Fig. !. Molecular cloning of rabbit cardiac calsequestrin eDNA. An adult rabbit cardiac muscle eDNA library made in ~gtl0 was screened with a 108-bp SstII-Pvull fragment (nt 7721-7828) derived from the last exon of the fast-twitch muscle calsequestrin-encoding gene (Zarain-Herzberg et al., 1988). Hybridization was carried out in 30% formamide/5 x SSC/10 x Denhardt's solution/0.1% SDS/100 ng per ml salmon testes DNA at 42°C overnight. The eDNA containing the longest insert was chosen for further analysis. The insert was subcloned into M13mpl8 and Ml3mpl9 vectors to obtain single-stranded cDNA, and DNA sequencing was performed by the dideoxy chain-termination methods of Sanger et al. (1977). (A)Partial restriction map and sequencing strategy of rabbit cardiac calsequestrin eDNA clone. The closed box indicates the entire protein-coding region. Poly(A)-indicates the poly(A)addition site. Arrows show the extent of sequencing. (B) Nucleotide and deduced aa sequence of rabbit cardiac caisequestrin eDNA. The 2510-bp fragment ofrabbit cardiac calsequestrin encoding DNA is numbered + 1 corresponding to the first nt of the ATG start codon. The aa are numbered (in parentheses) starting at Met + n. The putative polyadenylation signal is underlined. The bracketed aa indicate the putative glycosylation site. These sequence data are available from EMBL/GenBank/DDBJ under accession No. X55040.
277 with rabbit fast-twitch calsequestrin (Fliegel et al., 1987). The' rabbit cardiac calsequestrin sequence did not contain the second ORF reported in the 3'-untranslated region of canine cardiac calsequestrin by Scott et al. (1988). This eDNA sequence revealed a single poly(A)-addition site, whereas in the case of canine cardiac calsequestrin, two distinct poly(A)-addition sites that generate two distinct mRNA sizes were identified (Scott et al., 1988). The deduced aa sequence of rabbit cardiac calsequestrin revealed a protein of 409 aa residues with an Mr of 47 356 (Fig. 2). A striking feature of the rabbit cardiac calsequestrin is the high content of acidic residues. This protein contained 58 Asp and 49 Glu residues (26% acidic residues), and the calculated isoelectric point was 4.01. The C-end is rich in acidic residues, suggesting the involvement of this region in large Ca 2 + binding capacity. A cluster of three closely spaced Ser at the C-end is also known to be the site of rapid phosphorylation by casein kinase II in canine cardiac calsequestrin (Cala and Jones, 1991). Interestingly, the second Ser residue (Ser4°°) is replaced by Asp in rabbit cardiac calsequestrin, suggesting slower phosphorylation than canine cardiac calsequestrin. Cardiac calsequestrin is a glycoprotein (Cala and Jones, 1983; Campbell et al., 1983) and a potential glycosylation site was identified at aa residues 335-337, Asn-Val-Thr (Fig. IB). Rabbit cardiac calsequestrin showed 93% aa sequence identity with the canine cardiac calsequestrin (Scott et al., 1988) and 67% aa identity with rabbit fast-twitch calseRC CC RF
MKRAHLFVVGVYLL ......... SSCRAEEGLNFPTYDGKDRVVS LSEKN ---T---IA-L---. ........ A-W .................... T---NA- DRMGAR-A--LLLVLGS PQ-GVHG .... D--E---V---INVNA--
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FKQ I LKKY DLLCLYYHAPVSADKVAQKQFQLKE I V L E L V A Q V L E H K E IG F ---V ..... V ...... ES--S ......................... D--Y-NVF---EV-A-L--E-PED--AS-R--EME-LI---A ..... D-GV--
91 91 100
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V M V D A K K E A K T A K K L G F D E E G S LY I L K G D R T I E F D G E F A A D V L V E F L L D L ........................ V ......................... GL--SE-D-AV ..... LT--D-I-VF-E-EV--Y .... S--T ....... V
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I EDPVE I INSKLEVQAFERI EDHIKLIGFFKSADSEYYKAFEEAAEHFQP ...................... Q ......... EE ................ L ..... L-EGER-L .... N---E ..... Y--NK---H .... K .... E-H-
19] 191 200
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y I KFFATFDKGVAKKLS LKMNEVDFYEPFMDEPTPI PNKPYTEEELVEFV ................................. IA--D ............ --P ...... SK ..... T--L--I .... '--'--''-'''-''-'''-'--
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VWME I PDDDDLPTAEELEDWI EDVLSGKI NTEDDDNEDEDDDDDNDDDDD ..................................... EG--G--DE .... .... MD-EE---S ....... L .... E-E ....... D--D ..... D
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I H IV P F A E R S D P D G ¥ E F L E I L K Q V A---R ............... ...... MD ..... A---EA ............ S-
Fig. 2. Amino acid sequence alignment of calsequestrin cDNAs. Sequence comparison was done using a Digital VAX computer and the University of Wisconsin Genetics Computer Group (UWGCG) software. Identical aa are indicated by a dashed line. Gaps (dots) have been introduced to achieve maximum homology. RC, rabbit cardiac calsequestrin (see Fig. IB); CC, canine cardiac calsequestrin (Scott et al., 1988); RF, rabbit fast-twitch skeletal muscle calsequestrin (Fliegel et al., 1987).
questrin (Fliegel et al., 1987) (Fig. 2). Thus the rabbit cardiac calsequestrin was more similar to canine cardiac calsequestrin than ~o rabbit fast-twitch calsequestrin, suggesting that there are "i~nctional similarities between the cardiac isoforms.
(b) Tissue-specific expression of cardiac calsequestrin To determine whether the cardiac caisequestrin mRNA was expressed in other than cardiac muscle, RNA blot analysis was performed using total RNA from various tissues. As shown in Fig. 3, this eDNA hybridized to a single 2.9-kb mRNA species in rabbit tissues, contrary to two (2.9 and 2.2 kb) mRNAs reported in canine cardiac muscle (Scott et al., 1988). Cardiac calsequestrin mRNA was detectable in atrium, ventricle and slow-twitch (soleus) skeletal muscle. This mRNA was undetectable in fasttwitch (plantaris) skeletal muscle and smooth/nonmuscle tissues. However, we have found that cardiac calsequestrin is transiently expressed in the fetal stages of fast-twitch skeletal muscle development (data not shown). The expression of fast-twitch calsequestrin mRNA was also examined on the same blot using rabbit fast-twitch skeletal calsequestrin-specific probe (Fig. 3). The expression of fast-
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Fig. 3. Northern blots showing tissue distribution of cardiac and fasttwitch calsequestrin mRNAs. Total cellular RNA from various tissues was isolated using the guanidine-thiocyanate method (Chomczyuski and Sacchi, 1987). Total RNA (15 #g) from various tissues was size-fractionated on ! % agarose gel containing 2.2 M formaldehyde, blotted onto nitrocellulose membrane, and sequentially hybridized with randomprimed DNA probe for rabbit cardiac calsequestrin [the longest clone of 2.5 kb cDNA (EcoRI linker-Y end)] and for rabbit fast-twitch calsequestrin [108-bp genomic DNA containing last exon, nt "/721-7828 (Sstll-Pvull), Zarain-Herzberg et al., 1988]. The hybridizations were done in 50% formamide and the membranes were was,ed with I x S SC/0. 1% SDS at 55 °C for 30 min and exposed overnight to Kodak X-Omat-AR film using an intensifying screen at -70°C. Sm Intestine, small intestine; U Bladder, urinary bladder.
278
-. sequestrin (MacLennan and Wong, 1971; lkemoto et al., 197!) and 35-40 too! of Ca 2 +/mol of cardiac calsequestrin (Mitchell et al., t987)).
twitch isoform was restricted to fast-twitch and slow-twitch skeletal muscle and to a lesser extent in esophagus. Esophagus muscle has been shown to contain a mixture of skeletal and smooth muscle. Tile expression pattern of eardiac calsequestrin is similar to the cardiac/slow-twitch Ca2+-ATPase, which is also expressed in slow-twitch soleus muscle in addition to cardiac muscle (Brandl et al., 1986). These results show that the cardiac muscle expresses exclusively the cardiac isoform, while the fast-twitch skeletal muscle expresses exclusively the fast-twitch isoforal in adult stages. In contrast, the slow-twitch skeleta~ muscle expresses both slow-twitch and fast-twitch isoforms of calsequestrin. However, the functional significance of these tissue specific expression of both isoforms is not fully understood, since the Ca 2 + storage capacity of both isoforms is quite similar (40-50 tool of Ca 2 '~/tool of fast skeletal cal-
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(c) Cardiac and fast-twitch calsequestrin mRNA expression during in vitro skeletal muscle myogenesis To determine the temporal expression of both cardiac ~nd fast-twitch calsequestrin m R N A in skeletal muscle development, we analyzed in vitro myogenesis of L6E9 ¢rat) and C2C 12 (mouse) skeletal muscle cell lines. During L6E9 myogenesis, the cardiac calsequestrin mRNA was detectable from day-1 myotube stage, but fast-twitch calsequestrin m R N A was not detectable (days 1-7) (Fig. 4). The data also revealed that L6E9 cells expressed low levels of calsequestrin m R N A as compared to C2C12 cells, indicating that L6E9 cells do not form well developed sarco-
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Fig. 4. Expression of cardiac and fast-twitch skeletal calsequestrin mRNAs in L6E9 and C2C12 muscle cells. C2C12 mouse myoblasts and L6E9 rat myoblasts were maintained in DMEM supplemented with 20% fetal bovine serum. To induce differentiation,myoblastsat subconfluencywere transferred to DMEM supplemented with 5~ adult horse serum. Subconfluent myoblasts and differentiating myotubes were harvested for RNA isolation. Total RNA (30 pg) from LrE9 cells (A) and C2C!2 cells (B) from myoblasts (Mb) and differentiating myotubes (Mt at days 1, 2, 3, 4, 5, and 7) were size-fractionated and hybridized with rabbit calsequestrin-encoding probes as described in Fig. 3 legend. The cDNA and genomic probes used for this analysisincluded most ofthe C terminus, a highlyconserved regionin the calsequestrin protein. We have found that the rabbit probes hybridizespecifically to rat L6E9 and C2C12 mRNA with little or no background. In addition the mouse myogenin [l.l-kb cDNA, pEMSVmyoS, nt 1-1107 (5'-EcoRl linker-EcoRI), Edmondson and Olson, 1989], and rat ~-skeletal actin [0.53kb cDNA, pAC-16, nt 705-1235 (PstI.Pstl), Garfinkel et al., 1982] were also used (see corresponding panels as marked on the right margin).
279 plasmic reticulum. Similarly low levels of expression were also noted for sarcoplasmic reticulum Ca 2+ -ATPase in these cells (M.A. and M.P., unpublished). In contrast, during C2C 12 myotube formation, both cardiac and fast-twitch ealsequestrin mRNAs were co-expressed from day 1. With the progression ofmyogenesis the cardiac isoform gradually decreased, while the skeletal isoform increased, indicating a switch in isoforms (Fig. 4). These data suggest that L6E9 and C2C 12 cells show intrinsic differences in their ability to express sarcoplasmic reticulum proteins. To check the activation pattern of other muscle-specific genes in both cell lines, we further examined the expression of mRNA for myogenin, a master regulatory gene involved in muscle differentiation (Weintraub et al., 1991), and for actins (Fig. 4). In both muscle cell lines, myogenin mRNA was expressed at high levels from day-1 myotube stage, and actin showed switching from nonmuscle p/~ type to sarcometic a type, a phenotypic change associated with myotube formation. The expression pattern of these genes was very similar in both cell lines, suggesting that both cell lines undergo a similar process of myogenesis. However, it is unclear at this time why the L6E9 cells alone fail to express the fast-twitch calsequestrin mRNA.
(d) Conclusions (1) We have cloned the eDNA sequence (approx. 2.4 kb long) encoding the rabbit cardiac calsequestrin. The protein is 409 aa long of which 26?/0 are acidic residues and shows high homology (93 ?/o) with canine cardiac calsequestrin. (2) Cardiac calsequestrin mRNA is expressed in the heart (atrium and ventricle) and in slow-twitch skeletal muscle. But it is also transiently expressed in fast-twitch skeletal muscle during fetal stages. (3) The mRNAs encoding cardiac and fast-twitch skeletal calsequestrin are coexpressed during in vitro skeletal muscle myogenesis, which might suggest that the two genes share some common regulatory mechanisms.
ACKNOWLEDGEMENTS
We are grateful to Dr. Eric N. Oison for myogenin eDNA, and Drs. Jonathan Lytton, Angel Zarain-Herzberg and David H. MaeLennan for rabbit fast-twitch calsequestrin eDNA and rabbit cardiac eDNA library. We are also grateful to Dr. Larry R. Jones for critical reading of this manuscript. This work was supported by National Institutes of Health Grants R.O.I HL-39303 and P.O.I HL-28001 to M.P. and N.R.A.M.A. is supported by a postdoctoral fellowship from the American Heart Association (Vermont). M.P. is an established investigator of the American Heart Association.
REFERENCES Brandl, C.J., Green, N.M., Korczak, B. and MacLennan, D.H.: Two Ca2÷-ATPase genes: homologies and mechanistic implications of deduced amino acid sequences. Cell 44 (1986) 597-607. Cala, S.E. and Jones, L.R.: Rapid purification of calsequestrin from cardiac and skeletal muscle sarcoplasmic reticulum vesicles by Ca 2 +-dependent elution from phenyl-sepharose. J. Biol. Chem. 258 (1983) 11932-11936. Cala, S.E. and Jones, L.R.: Phosphorylation of cardiac and skeletal muscle calsequestrin isoforms by casein kinase, II. Demonstration of a cluster of unique rapidly phosphorylated sites in cardiac calsequestrin. J. Biol. Chem. 266 (1991) 391-398. Campbell, K.P., MacLennan, D.H., Jorgensen, A.O. and Mintzer, M.C.: Purification and characterization of calsequestrin from canine cardiac sarcoplasmic reticulum and identification of the 53000 dalton glycoprotein. J. Biol. Chem. 258 (1983) 1197-1204. Chomczynski, P. and Sacchi, N.: Single step method of RNA isolation by acid guanidine thiocyanate.phenol-ehloroform extraction. Analyt. Biochem. 162 (1987) 156-169. Edmondson, D.G. and Olson, E.N.: A gene with homology to the myc similarity region of MyoD! is expressed during myogenesis and is sufficient to activate the muscle differentiation program. Genes Develop. 3 (1989) 628-640. Fliegel, L., Ohnishi, M., Carpenter, M.R., Kbanna, V.J., Reithmeier, R.A.F. and MacLennan, D.H.: Amino acid sequence of rabbit fasttwitch skeletal muscle calsequestrin deduced from eDNA and peptide sequencing. Proc. Natl. Acad. Sci. USA 84 (1987) 1167-1171. Fliegel, L., Leberer, E., Green, N.M. and MacLennan, D.H.: The fasttwitch muscle calsequestrin isoform predominates in rabbit slowtwitch soleus muscle. FEBS Lett. 242 (1989) 297-300. Garfinkel, L.I., Periasamy, M. and Nadal-Ginard, B.: Cloning and characterization of eDNA sequences corresponding to myosin light chain 1, 2, and 3, troponin-C, troponin-T, a-tropomyosin, and aactin. J. Biol. Chem. 257 (1982) 11078-11086. Ikemoto, N., Bhatnager, G.M. and Gergely, J.: Fractionation of solubilized sarcoplasmic reticulum. Biochem. Biophys. Res. Commun. 44 (1971) 1510-1516. Lytton, J. and MacLennan, D.H.: Sarcoplasmic reticulum. In: Fozzard, H.A., Haber, E., Jenning, R.B., Katz, A.M. and Morgan, H.E. (Eds.), The Heart and Cardiovascular System, Vol. 1, 2rid ed. Raven Press, New York, 1991. MacLennan, D.H. and Wong, P.T.S.: Isolation of a calcium-sequestering protein from sarcoplasmic reticulum. Proc. Natl. Acad. Sci. USA 68 (1971) 1231-1235. MacLennan, D.H., Campbell, K.P. and Reithmeier, R.A.F.: Calsequestrin. In: Cheung, W.Y. (Ed.), Calcium and Cell Function, Vol. IV. Academic Press, New York, 1983, pp. 151-173. Mitchell, R.D., Simmerman, H.K.B. and Jones, L.R.: Ca :+ binding effects on protein conformation and protein interaction of canine cardiac calsequestrin. J. Biol. Chem. 263 (1987) 1376-138 !. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Scott, B.T., Simmerman, H.K.B., Collins, J.H., Nadal-Ginard, B. and Jones, L.B.: Complete amino acid sequence of canine cardiac caisequestrin deduced by eDNA cloning. J. Biol. Chem. 263 (1988) 8958-8964. Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M" Bonezra, R., Biackwell, T.K., Turner, D., Rupp, R., Hollenberg, S., Zhuang, Y. and Lassar, A.: The MyoD gene family: nodal point during specification of the muscle cell lineage. Science 251 (1991) 761-766. Zarain-Herzberg, A., Fliegel, L. and MacLennan, D.H.: Structure of the rabbit fast-twitch skeletal calsequestrin gene. J. Biol. Chem. 263 (1988) 4807-4812.
275
GENE 06170
Cloning and characterization of the gene encoding rabbit cardiac calsequestrin (Gene expression; myogenesis; myogenin; nucleotide sequencing; recombinant DNA)
Masashi Arai*, Norman R. Alpert* and Muthu Periasamy ",b '° Department of Physiology and Biophysics, and b Department of Microbiology and Molecular Genetics, University of Vermont College of Medicine, Burh'ngton, VT 05403 (U.S.A.)
Received by R. Padmanabhan: 3 June 1991
Revised/Accepted: 12 August/21 August 1991 Received at publishers:17 September1991
SUMMARY A cDNA encoding rabbit cardiac calsequestrin was isolated and characterized. The deduced nascent cardiac calsequestrin contains 409 amino acids of which 26% are acidic residues, and had 93% and 67% aa identity with canine cardiac caisequestrin and rabbit fast-twitch skeletal muscle calsequestrin, respectively. RNA blot analyses indicate that this mRNA is expressed in atrium, ventricle and to a lesser amount in slow-twitch skeletal muscle. This mRNA transcript is not expressed in adult fast-twitch skeletal muscle, smooth muscle, or nonmuscle tissues. Analysis of in vitro skeletal muscle myogenesis using a mouse myoblast cell line C2C 12, demonstrates that both cardiac and skeletal calsequestrin isoforms are coproduced during muscle differentiation.
INTRODUCTION Calsequestrin is a high-capacity, moderate-affinity Ca 2 + binding protein located in the luminal space of junctional sarcoplasmic reticulum (MacLennan et al., 1983). The calsequestrin acts as a Ca 2 + buffer, and the release of Ca 2 + bound to calsequestrin through a Ca2+-release channel triggers muscle contraction (Lytton and MacLennan, 1991). Recently, the major sarcoplasmic reticulum proteins have been isolated and their primary structures determined. Most of these proteins have isoforms and each isoform is expressed in a tissue-specific manner (Lytton and Correspondence to: Dr. M. Periasamy, Department of Physiology and Biophysics, Universityof Vermont Collegeof Medicine, Burlington, VT 05405 (U.S.A.) Tel. (802)656-4433; Fax (802)656-0747.
Abbreviations: aa, aminoacid(s); bp, base pair(s); cDNA, DNA complementary to RNA; DMEM, Dulbecco's modified Eagle's medium; kb, kilobase(s)or 1000bp; nt, nucleotide(s);ORF, open readingframe; SDS, sodium dodecyl sulfate; SSC,0.15 M NaCI/0.015M Na3"citrate pH 7.6.
MacLennan, 1991). So far the primary structure of rabbit fast-twitch skeletal calsequestrin (Fliegel et al., 1987)and of canine cardiac calsequestrin (Scott et al., 1988) have been determined. Earlier studies (Scott et al., 1988; Fliegel et al., 1989) indicated that the fast-twitch muscle calsequestrin is the major isoform expressed in both fast- and slow-twitch skeletal muscle, and cardiac calsequestrin is mainly expressed in heart. However, the precise tissue distribution of both cardiac and fast-twitch skeletal isoforms of caisequestrin mRNAs remains to be determined. Furthermore, it is of interest to determine the expression of both cardiac and fast-twitch caisequestrin isoforms during in vitro myogenesis. This study was designed (i) to characterize the rabbit cardiac calsequestrin by cDNA cloning analysis, (ii) to study the tissue-specific expression of both cardiac and fast-twitch calsequestrin mRNAs using genespecific probes in rabbit muscle and nonmuscle tissues, and (ill) to determine the temporal expression of the two isoforms ofcalsequestrin during in vitro myogenesis of skeletal muscle cell lines L6E9 and C2C12.
276 EXPERIMENTAL AND DISCUSSION
(a) Nucleotide and deduced aa sequence of rabbit cardiac caisequestrin An adult rabbit cardiac eDNA library was screened with the last exon of the rabbit fast-twitch muscle calsequestrin gene (108 bp, SstII-PvuII fragment, nt 7721-7828, ZarainHerzberg et al., 1988). Nine positive clones were obtained
from about 5000 plaques. A partial restriction map of the longest eDNA clone encoding the rabbit cardiac calsequestrin is shown in Fig. 1A. This eDNA clone was 2510 bp long and included a single ORF of 1227 bp long. It also contained l198-bp 3'-untranslated and 85-bp 5'untranslated regions (Fig. 1B). The nt sequence of rabbit cardiac calsequestrin showed 78.4~o identity with canine cardiac calsequestrin (Scott et al., 1988) and 58.6?/0 identity
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Fig. !. Molecular cloning of rabbit cardiac calsequestrin eDNA. An adult rabbit cardiac muscle eDNA library made in ~gtl0 was screened with a 108-bp SstII-Pvull fragment (nt 7721-7828) derived from the last exon of the fast-twitch muscle calsequestrin-encoding gene (Zarain-Herzberg et al., 1988). Hybridization was carried out in 30% formamide/5 x SSC/10 x Denhardt's solution/0.1% SDS/100 ng per ml salmon testes DNA at 42°C overnight. The eDNA containing the longest insert was chosen for further analysis. The insert was subcloned into M13mpl8 and Ml3mpl9 vectors to obtain single-stranded cDNA, and DNA sequencing was performed by the dideoxy chain-termination methods of Sanger et al. (1977). (A)Partial restriction map and sequencing strategy of rabbit cardiac calsequestrin eDNA clone. The closed box indicates the entire protein-coding region. Poly(A)-indicates the poly(A)addition site. Arrows show the extent of sequencing. (B) Nucleotide and deduced aa sequence of rabbit cardiac caisequestrin eDNA. The 2510-bp fragment ofrabbit cardiac calsequestrin encoding DNA is numbered + 1 corresponding to the first nt of the ATG start codon. The aa are numbered (in parentheses) starting at Met + n. The putative polyadenylation signal is underlined. The bracketed aa indicate the putative glycosylation site. These sequence data are available from EMBL/GenBank/DDBJ under accession No. X55040.
277 with rabbit fast-twitch calsequestrin (Fliegel et al., 1987). The' rabbit cardiac calsequestrin sequence did not contain the second ORF reported in the 3'-untranslated region of canine cardiac calsequestrin by Scott et al. (1988). This eDNA sequence revealed a single poly(A)-addition site, whereas in the case of canine cardiac calsequestrin, two distinct poly(A)-addition sites that generate two distinct mRNA sizes were identified (Scott et al., 1988). The deduced aa sequence of rabbit cardiac calsequestrin revealed a protein of 409 aa residues with an Mr of 47 356 (Fig. 2). A striking feature of the rabbit cardiac calsequestrin is the high content of acidic residues. This protein contained 58 Asp and 49 Glu residues (26% acidic residues), and the calculated isoelectric point was 4.01. The C-end is rich in acidic residues, suggesting the involvement of this region in large Ca 2 + binding capacity. A cluster of three closely spaced Ser at the C-end is also known to be the site of rapid phosphorylation by casein kinase II in canine cardiac calsequestrin (Cala and Jones, 1991). Interestingly, the second Ser residue (Ser4°°) is replaced by Asp in rabbit cardiac calsequestrin, suggesting slower phosphorylation than canine cardiac calsequestrin. Cardiac calsequestrin is a glycoprotein (Cala and Jones, 1983; Campbell et al., 1983) and a potential glycosylation site was identified at aa residues 335-337, Asn-Val-Thr (Fig. IB). Rabbit cardiac calsequestrin showed 93% aa sequence identity with the canine cardiac calsequestrin (Scott et al., 1988) and 67% aa identity with rabbit fast-twitch calseRC CC RF
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Fig. 2. Amino acid sequence alignment of calsequestrin cDNAs. Sequence comparison was done using a Digital VAX computer and the University of Wisconsin Genetics Computer Group (UWGCG) software. Identical aa are indicated by a dashed line. Gaps (dots) have been introduced to achieve maximum homology. RC, rabbit cardiac calsequestrin (see Fig. IB); CC, canine cardiac calsequestrin (Scott et al., 1988); RF, rabbit fast-twitch skeletal muscle calsequestrin (Fliegel et al., 1987).
questrin (Fliegel et al., 1987) (Fig. 2). Thus the rabbit cardiac calsequestrin was more similar to canine cardiac calsequestrin than ~o rabbit fast-twitch calsequestrin, suggesting that there are "i~nctional similarities between the cardiac isoforms.
(b) Tissue-specific expression of cardiac calsequestrin To determine whether the cardiac caisequestrin mRNA was expressed in other than cardiac muscle, RNA blot analysis was performed using total RNA from various tissues. As shown in Fig. 3, this eDNA hybridized to a single 2.9-kb mRNA species in rabbit tissues, contrary to two (2.9 and 2.2 kb) mRNAs reported in canine cardiac muscle (Scott et al., 1988). Cardiac calsequestrin mRNA was detectable in atrium, ventricle and slow-twitch (soleus) skeletal muscle. This mRNA was undetectable in fasttwitch (plantaris) skeletal muscle and smooth/nonmuscle tissues. However, we have found that cardiac calsequestrin is transiently expressed in the fetal stages of fast-twitch skeletal muscle development (data not shown). The expression of fast-twitch calsequestrin mRNA was also examined on the same blot using rabbit fast-twitch skeletal calsequestrin-specific probe (Fig. 3). The expression of fast-
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Fig. 3. Northern blots showing tissue distribution of cardiac and fasttwitch calsequestrin mRNAs. Total cellular RNA from various tissues was isolated using the guanidine-thiocyanate method (Chomczyuski and Sacchi, 1987). Total RNA (15 #g) from various tissues was size-fractionated on ! % agarose gel containing 2.2 M formaldehyde, blotted onto nitrocellulose membrane, and sequentially hybridized with randomprimed DNA probe for rabbit cardiac calsequestrin [the longest clone of 2.5 kb cDNA (EcoRI linker-Y end)] and for rabbit fast-twitch calsequestrin [108-bp genomic DNA containing last exon, nt "/721-7828 (Sstll-Pvull), Zarain-Herzberg et al., 1988]. The hybridizations were done in 50% formamide and the membranes were was,ed with I x S SC/0. 1% SDS at 55 °C for 30 min and exposed overnight to Kodak X-Omat-AR film using an intensifying screen at -70°C. Sm Intestine, small intestine; U Bladder, urinary bladder.
278
-. sequestrin (MacLennan and Wong, 1971; lkemoto et al., 197!) and 35-40 too! of Ca 2 +/mol of cardiac calsequestrin (Mitchell et al., t987)).
twitch isoform was restricted to fast-twitch and slow-twitch skeletal muscle and to a lesser extent in esophagus. Esophagus muscle has been shown to contain a mixture of skeletal and smooth muscle. Tile expression pattern of eardiac calsequestrin is similar to the cardiac/slow-twitch Ca2+-ATPase, which is also expressed in slow-twitch soleus muscle in addition to cardiac muscle (Brandl et al., 1986). These results show that the cardiac muscle expresses exclusively the cardiac isoform, while the fast-twitch skeletal muscle expresses exclusively the fast-twitch isoforal in adult stages. In contrast, the slow-twitch skeleta~ muscle expresses both slow-twitch and fast-twitch isoforms of calsequestrin. However, the functional significance of these tissue specific expression of both isoforms is not fully understood, since the Ca 2 + storage capacity of both isoforms is quite similar (40-50 tool of Ca 2 '~/tool of fast skeletal cal-
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(c) Cardiac and fast-twitch calsequestrin mRNA expression during in vitro skeletal muscle myogenesis To determine the temporal expression of both cardiac ~nd fast-twitch calsequestrin m R N A in skeletal muscle development, we analyzed in vitro myogenesis of L6E9 ¢rat) and C2C 12 (mouse) skeletal muscle cell lines. During L6E9 myogenesis, the cardiac calsequestrin mRNA was detectable from day-1 myotube stage, but fast-twitch calsequestrin m R N A was not detectable (days 1-7) (Fig. 4). The data also revealed that L6E9 cells expressed low levels of calsequestrin m R N A as compared to C2C12 cells, indicating that L6E9 cells do not form well developed sarco-
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Fig. 4. Expression of cardiac and fast-twitch skeletal calsequestrin mRNAs in L6E9 and C2C12 muscle cells. C2C12 mouse myoblasts and L6E9 rat myoblasts were maintained in DMEM supplemented with 20% fetal bovine serum. To induce differentiation,myoblastsat subconfluencywere transferred to DMEM supplemented with 5~ adult horse serum. Subconfluent myoblasts and differentiating myotubes were harvested for RNA isolation. Total RNA (30 pg) from LrE9 cells (A) and C2C!2 cells (B) from myoblasts (Mb) and differentiating myotubes (Mt at days 1, 2, 3, 4, 5, and 7) were size-fractionated and hybridized with rabbit calsequestrin-encoding probes as described in Fig. 3 legend. The cDNA and genomic probes used for this analysisincluded most ofthe C terminus, a highlyconserved regionin the calsequestrin protein. We have found that the rabbit probes hybridizespecifically to rat L6E9 and C2C12 mRNA with little or no background. In addition the mouse myogenin [l.l-kb cDNA, pEMSVmyoS, nt 1-1107 (5'-EcoRl linker-EcoRI), Edmondson and Olson, 1989], and rat ~-skeletal actin [0.53kb cDNA, pAC-16, nt 705-1235 (PstI.Pstl), Garfinkel et al., 1982] were also used (see corresponding panels as marked on the right margin).
279 plasmic reticulum. Similarly low levels of expression were also noted for sarcoplasmic reticulum Ca 2+ -ATPase in these cells (M.A. and M.P., unpublished). In contrast, during C2C 12 myotube formation, both cardiac and fast-twitch ealsequestrin mRNAs were co-expressed from day 1. With the progression ofmyogenesis the cardiac isoform gradually decreased, while the skeletal isoform increased, indicating a switch in isoforms (Fig. 4). These data suggest that L6E9 and C2C 12 cells show intrinsic differences in their ability to express sarcoplasmic reticulum proteins. To check the activation pattern of other muscle-specific genes in both cell lines, we further examined the expression of mRNA for myogenin, a master regulatory gene involved in muscle differentiation (Weintraub et al., 1991), and for actins (Fig. 4). In both muscle cell lines, myogenin mRNA was expressed at high levels from day-1 myotube stage, and actin showed switching from nonmuscle p/~ type to sarcometic a type, a phenotypic change associated with myotube formation. The expression pattern of these genes was very similar in both cell lines, suggesting that both cell lines undergo a similar process of myogenesis. However, it is unclear at this time why the L6E9 cells alone fail to express the fast-twitch calsequestrin mRNA.
(d) Conclusions (1) We have cloned the eDNA sequence (approx. 2.4 kb long) encoding the rabbit cardiac calsequestrin. The protein is 409 aa long of which 26?/0 are acidic residues and shows high homology (93 ?/o) with canine cardiac calsequestrin. (2) Cardiac calsequestrin mRNA is expressed in the heart (atrium and ventricle) and in slow-twitch skeletal muscle. But it is also transiently expressed in fast-twitch skeletal muscle during fetal stages. (3) The mRNAs encoding cardiac and fast-twitch skeletal calsequestrin are coexpressed during in vitro skeletal muscle myogenesis, which might suggest that the two genes share some common regulatory mechanisms.
ACKNOWLEDGEMENTS
We are grateful to Dr. Eric N. Oison for myogenin eDNA, and Drs. Jonathan Lytton, Angel Zarain-Herzberg and David H. MaeLennan for rabbit fast-twitch calsequestrin eDNA and rabbit cardiac eDNA library. We are also grateful to Dr. Larry R. Jones for critical reading of this manuscript. This work was supported by National Institutes of Health Grants R.O.I HL-39303 and P.O.I HL-28001 to M.P. and N.R.A.M.A. is supported by a postdoctoral fellowship from the American Heart Association (Vermont). M.P. is an established investigator of the American Heart Association.
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