Vol. 181, No. 2, 1991 December 16. 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 644-649
Molecular cloning and sequencing of a cDNA clone encoding a new calcium binding protein, named calgizzarin, from rabbit lung+ Masato Watanabe, Yuhko Ando, Hitoshi Todoroki, Hiroyuki Minami, and Hiroyoshi Hidaka* Department of Pharmacology, Nagoya University School of Medicine, Tsurumai 65, Showa-ku, Nagoya 466, Japan Received
October
27,
1991
We purified a new EF-hand type calcium binding protein from chicken gizzard smooth muscle, tentatively named calgizzarin (Todoroki, H., et al. J. Biol. Chem. (1991) in press (1)). Based on the internal pcptide sequence of calgizzarin, we isolated and sequenced a cDNA clone coding for calgizzarin from a rabbit lung cDNA library. This clone (pCALG) has 309 nucleotides of open reading frame including termination codon TGA, 621 nucleotides of the 5’ leader and 186 nucleotides of the 3’ noncoding region. The polypeptides deduced from the open reading frame were consisted of 102 amino acid residues with a molecular weight of 11,429. Computer aided homology analysis revealed that calgizzarin exhibits a 43.2% homology to S-lOOa, 38.6% to S1OOgand 40.0% to annexin II light chain, ~10. By Northern blot analysis, with pC!ALG, a band of 1.1 kbp was detected in rabbit lung, suggesting pCALG contains nearly full length of mBNA. 0 1991
Academic
Press,
Inc.
Intracellular calcium ion is involved in regulating various biochemical events in excitable cells. These events are mediated by a family of calcium binding proteins which exhibit a general structural principle, so called EF-hand structure (2). These EF-hand type calcium binding proteins can be classified at least into two families, calmodulin and S-100 protein families (3). In contrast with calmodulin, proteins of the S-100 protein family are present in limited and specified tissues. This would suggest that there are at least two different intracellular calcium signal transduction systems; one is the general system modulated by calmodulin which is present in all cells and the other is the specific system modulated by the members of the S-100 protein family present in specific cells. We reported the purification of a new calcium binding protein from chicken gizzard (calgizzarin) with an apparent molecular weight of 13,000, using a calcium dependent affinity chromatographic technique (1). The partial peptide sequence of calgizzarin has approximately a
+The nucleotide sequence in this paper has been submitted to the DDBJ, EMBL and Genbank database with the accession number D9053 1. * To whom all correspondence should be addressed. . . Abbrevlanons PCR, polymerase chain reaction, SDS; sodium dodecyl sulfate, PAGE; polyacrylamide gel electrophoresis. 0006-291x/91 Copyright All rights
$1.50
0 I991 by Academic Press, Inc. of reproduction in any form reserved.
644
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
50% homology to S-1OOa and 47% to S-1008. Application of the same affinity chromatographic technique to various tissues revealed that calgizzarin-like protein was abundantly present in rabbit lung. The major objective of the research concerned with proteins of S- 100 family reveal existence of the cell type specific calcium signaling system and its precise molecular mechanism. A first approach to this purpose is to determine the primary structure of a cDNA coding for calgizzarin using molecular biological technique. In this report, we isolated and sequenced a cDNA clone encoding calgizzarin from a rabbit lung cDNA library by screening with the PCR amplified authentic cDNA as a probe. Materials
and
Methods
Materials. [a-32P]dCTP (1 lOTBq/mmol) was purchased from Amersham International. T4 DNA polymerase, Taq polymerase (Ampli Taq), restriction endonucleases and other modifying enzymes were obtained from Takara Shuzo. Oligonucleotides were synthesized on a Model 392A DNA synthesizer (Applied Biosystems Ins.). A rabbit lung @tlO library was purchased from Clonetech. All other chemicals and reagents were of analytical grade obtained from commercial suppliers. Purification of calgizzarin from rabbit lung and protein sequencing. Calgizzarin was partially purified from rabbit lung (about 100 g) on a affinity column using a calmodulin antagonist, W-7 as a affinity ligand (0.8 x 5 cm) according to the method of Todoroki et al (1). Twenty nl of concentrated fraction of calgizzarin was separated by Tricine-SDS PAGE according to Schtigger and Jagow (4). Gel was stained with Coomassie brilliant blue and destained. Calgizzarin band was excised with a laser blade and was washed with distilled water. The excised gel was digested with 1:20 (w/w) of lysylendopeptidase in a 2OOp.lof 5OmM sodium biammonium. After 20hr incubation at 37’C. the resulting peptides were separated on a C-18 reverse phase column. The amino acid sequence was determined using automated protein sequencer (Model 473A, Applied Biosystems Inc.). Isolation of a calgizzarin cDNA clone. A cDNA library synthesized from mRNA of rabbit lung using a cDNA synthesis kit (Pharmacia Corp.) according to the manufacture’s manual was used as a template for synthesizing the authentic cDNA probe of calgizzarin. Two PCR primers, For and Rev, were designed according to the peptide sequence of calgizzarin (Fig. 1A). Twenty five cycles of PCR amplification were performed in a lOOpI reaction volume containing 1Ot.ddouble stranded cDNA library solution, 10 pmol of the primers and the reagents &scribed in the amplification protocol of Perkin-Elmer/Cetus. PCR cycle consists of a I-min denaturation at 94’C, a l-mm annealing at 37X!, and a 2-min polymerization at 72’C in a thermocycler (PerkinElmerKetus). The PCR product was cloned into pUC8 and used as the authentic cDNA probe. About 2.4 x 101 plaques from a rabbit lung XgtlO cDNA library were screened with the authentic cDNA probe labeled by multi priming method (5) using PCR primer sets as primers. Hybridization was performed overnight at 5o’C, and the final wash was carried out with 0.5 x SSC (20 x SSC; 3M NaCl, 0.3M sodium citrate) containing 0.1% SDS at 5o’C. DNA sequencing. The nucleotide sequences were analyzed by the dideoxy chain termination method (6) as modified by Hattori and Sakaki (7) with [a-32P] dCTP and the Sequenase Ver.2 kit (United States Biochemical Corp.). The double strands plasmid was firstly sequenced from both strands using two vector primers flanking the cDNA insert. The complete sequence was obtained by custom primer-directed DNA sequencing using specific primers complementary to internal cDNA sequences. RNA blotting and hybridization. Total RNA is extracted from rabbit lung by the guanidium isothiocyanate/cesium chloride method (8). Poly (A)+ RNA was preparated on a oligo (dT) cellulose column. Twenty pg of total RNA and five hundreds ng of poly (A)+ RNA were denatured and electrophoresed on a formaldehyde-containing agarose gel. The RNA was transferred onto a Hybond-N (Amersham Corp.) and hybridized in a solution containing !lOmM Ttis-HCl, pH 7.5,0.9M NaCl, 6mM EDTA, 50% formamide, 1 x Denhart (5 x Denhart; 1% bovine serum albumin,1 8 polyvinylpyrollidone,l 96 Ficoll400), 0.1% SDS and 2OOmg/ml heatdenatured salmon testis DNA at 42’C with [a-32P] dCTP labeled cDNA fragments. Finally, the filter was washed in 0.2 x SSC containing 0.1% SDS at 55-C and exposed to imaging plate. The image was analyzed and visualized with Bioimage analyzer (Model Fujix BAS2000, Fuji Film Corp.> 645
Vol.
181,
No.
2, 1991
BIOCHEMICAL
Results
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
and Discussion
We reported the purification of a new calcium binding protein from chicken gizzard, which molecular weight is estimated 13,000 by Tricine-SDS PAGE. The partial amino acid sequence analysis revealed that a new calcium binding protein, named calgizzarin, have two putative Ca2+binding structure (EF-hand) in the molecule. The overall amino acid sequence identity within the region covered by peptides obtained is 50% to S-1OOo and47% S-lOOj3 (1). We decided to isolate a cDNA clone coding for calgizzarin, to determine the characteristics of primary structure of calgizzarin. Firstly, we obtained the authentic cDNA probe of calgizzarin by PCR technique. PCR amplification was performed using two primers (For and Rev) synthesized according to the peptide sequence of calgizzarin (Fig.lA, upper line). PCR product was electrophoresed on a 4% Nu-Sieve (FMC) agarose gel. A 72bp band of expected size was isolated from the agarose gel and cloned into pUC8. The 72bp cDNA fragment identified by sequencing was used as the authentic probe (Fig. 1A, lower line). About 2.4 x 10s plaques from a rabbit lung XgtlO cDNA library were screened with the authentic probe. One positive clone, 457 bp in length, was obtained. This clone appeared to be incomplete, since the open reading frame encoding calgizzarin was not preceded by a 5’ untranslated region or an initiation codon ATG. A 457 bp insert of the clone was used to screen an additional 2 x l@ individual plaques of a rabbit lung library purchased from Clontech. Hybridization and wash were performed according to the procedure described in the Materials and Methods, except the final wash was carried out with 0.1 x SSC at 6X. Finally, one positive clone was isolated and further analyzed. Fig.lB shows the nucleotide and the deduced amino acid sequences of the entire cDNA insert from the recombinant plasmid pCALG. pCALG is 1116bp in length and contains 621bp of 5’ leader region, 309bp coding region including termination codon TGA and 186bp of 3’ nontranslated region. The sequences derived from Edman degradation arc found at amino acid positions #25-49 and #53-62. The polyadenylation signal sequence (AATAAA) is shown at nucleotide position 1056-1061. The calculated molecular weight of pCALG was 11,429 and this value was in good accordance with that of the purified chicken calgizzarin judged by Tricine-SDS PAGE. Northern blot analysis was performed using the entire insert of pCALG as a probe. Probing of total RNA and poly (A)+ RNA derived form rabbit lung revealed that a major band at 1.1-1.0 kbp was detected, suggesting that pCALG contains nearly full length of mRNA (Fig.2). By computer aided homology analysis, there is very limited homology of the pCALG with known sequences in Genbank, EMBL and NBRF databases. Fig.3 shows the results of the homology search. pCALG exhibits a 43.2% homology to S-1OOaof bovine (9,10), 38.6% to S1OOpof human (11,12) and 40.0% to annexin II light chain, ~10, of porcine (13). All these results show that the cDNA of pCALG is derived from mRNA for the rabbit calgizzarin and that calgizzarin is a new member of S- 100 protein family. Two putative EF hand structures appeared at amino acid positions #23-53 and #65-76. The second EF hand is more conserved among three S-100 family proteins than the first structure (Fig.3). It is, at least, thought that the second EF hand has calcium binding ability, since calcium can bind to chicken calgizzarin (1). The deduced polypeptides have 50% of hydrophobic amino 646
Vol.
181, No. 2, 1991
A For
primers
sequence
peptide authentic
B
cDNA
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
G A c C C GC TTTATGAATACTGAATT
Rev
cc G GATTATATGATGAAA
FMNTELAAFTKnqkDPGVLDYMMK
probe
TTCATWLACACGGAGCTGGCTGCCTTCACAAACJlACCAGG
gatctcattgatggggtggcctcccgtctctggcccctgccccctccccaccctggggta ttcgacatggagccccctgcccagggatgccgctcttgatgtctgtctagtctgctctag gatggccccaactcctactccttgtccagggtgaggttggaggcagaagacgggagaaac agcacttcttaaatcctatgaagccataatgtaaccccggtggactgatggcagatcagc tggtcctgtgtgctactttgcacggcagaactgcagggaggggctggaggctgtgaaagc ggaccccacccctgcttgcgctggttccagagtccaaggtggtcccctggctggggagaa agagagcagctggaggccggaggttgtggttgtgcagggctcatctcggcctggagggcc aggtcccctcccagctgccctctgttctctgaggctgactttttctgcggtccacccatc catcctctcctttagtgccttgactctcactcagaagcagcccccttcagtccctccgtc ctccctctgactcgctccgctccgctcgccgcgccgcccgcctcgcgcagctccgctcgg CggtcttctccaaaggcagcaATGTCCAGGCCCACAGAGACCGAGCGCTGCATCG~TCC M S R P TETERCIES 1 CTGATCGCCGTGTTCCAGATACGCTGGAAAGGATGGGCACAGCGTCACCCTCTCCAAG L IAVFQKYAGKDGHSVTLSK 14 ACCGAGTTCCTGTCCTTTATGAACACACAGAGCTGCCTGAC TEFLSFMNTELAAFTKNQKP 34 CCCGGCGTCCTCGACCGCATGATGAAGAAATTGGACCTCACAGTGACGGGCAGCTGGAT PGVLDRMMKKLDLNSDGQLD 54 TTCCAAGAATTTCTCAATCTCATTGGTGGCTTGGCCGTGGCTTGCCATGAGTCCTTTGTC FQEFLNLI GGLAVACHESFV 74 AAGGCCGCCCCTCCCCAGACGTTTCTGAggccgccgtggctctggcccgcagacccac K A A P PQKRF* 94 102 ccctactgctgccagcctgccagccctcgccactcctcacggcctgccctgagcccccac tccacacaggccactcctgccggcagtgataaaacaataagcagtatctttttttatga cacacaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
60 120 180 240 300 360 420 480 540
600 660
13 720
33 780
53 840
73 900 93 960 1020 1080 1116
Fig.lA Peptide and oligo nucleotide sequences.Upper line indicatesthe two primers synthesized by Model 392A DNA synthesizer.Forwardmimer(For) is a 17-meroligonucleotide c&responding to FMNTEL and-reverseprimer (Rev) is a 1%mer anti-olig&mcleotide cormsoondintz to DYMMK. Nucleotideseuuence of amulifledPCRnroductis shownin lower line. Middleiine indicatesthe peptidesequ&cederivedt+omEdman*&gradation(capitalletter) andthededucedaminoacidsequence fromPCRproduct(lowercaseletter). Fig.lB The nucleotide sequenceof cDNA coding for rabbit calgizzarin and the deducedamino acid sequence.The nucleotidesequence is numberedstartingat initiationof thiscIone(right side)andthe predictedaminoacidat initiationmentioning(underthesequence). The translatedregionis shownin capitallettersandthe S-, 3’- noncodingregionsareshownin lowercaseletters. Thepredictedaminoacidsequence is shownbelowthenucleotidesequence by one-lettercode. Thin underlinesindicatethepeptidesequence derivedfrom Edmandegradation andthick underlineindicatesthepolyadenylation signal.*, terminationcodon.
acid residue in the molecule. Hydropathicity plot suggeststhat hydrophilic portion and hydrophobicportion appearedin turn from aminoterminal to carboxyl terminal of the moleculeasa cluster (data not shown). When calcium binds to calgizzarin, calgizzarin changedrastically in its conformation. This conformational changemakesthe migration of hydrophobic region(s) to the surface of the molecule. These hydrophobic regions may interact with target protein(s) or an affinity ligand. 647
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
28s 18s
F&.& Northern blot analysis of calgizzarin in rabbit lung. Twenty pg of total RNA and 0.5 pg of poly (A)+ RNA were electmphoresed and transferred to Hybond-N. Hybridization with the entire insert of pC!ALG was carried out under the condition described in the Materials and Methods.
The unique tissue distribution of proteins of S-100 family leads one to believe that each protein of S-100 family involves a specific signal transduction system. Calgizzarin reported in this paper also distributes in limited tissues, suggesting the possibility that there is a new and specific calcium signal transduction system. Identification and characterization of target protein(s) of calgizzarin and expression of the recombinant calgizzarin will aid future studies aimed to recognition of a new calcium signal transduction system.
1 CidgiZZViD
60
MSRPTETERCIESLlAVFQKYAGKDGHSVTLSKTEFLSKTEFLSF~TEL~T~QKDPGVLD~
s-looa
GS*L*TAM*T**N**HAHS**E*DKYK***K**~K*LKELLQ***SG*LQA***~AV*KV
s-mop
S*L*KAWA**D**HQ*S*RE*DKHK*K*S*LKLI*N**SH*LEEI*EQE*V*KV
PI0
*SQM*HAM*TMMFT*H*F**DK*Y---*T*EDLRVL*EK*FPG*LE*****LAV*KI 61
102
calgizzalin
MKKLDLNSDGOLDFQEFLNLIGGLAVACHESFVKAAPPQKRF
s-loou
**E**EDG**EV****YW*VAA*T***NNF*WENS
s-loop
*ET**SDG**EC*****MAFVAMITT****F*EHE
PlO
**D**QCR**KVG**S*FS**A**TI**NDY**VHMKQKGK
143.2%] [38.6%] [40.0%]
Fi .3 Comparison of the amino acid sequence of rabbit calgizzarin with S-1OOa bovine, S-loop of human, bovine and snnexin II light chain of porcine. Identical amino acid with rabbit calgizzarin are indicated by asterisks (*). Gaps (-) have inserted to achieve a maximum homology. Underlines indicate the putative calcium binding structures (EF hand structure).
+
648
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Acknowledgments We wish to thank Ryoji Kobayashi for technical advisement of protein sequencing and Etsuko Shimoda, Kiyo Harada for typing this manuscript and Naoko Watanabe for her help in technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
References 1. Todoroki, H., Kobayashi, R., Watanabe, M., Minami, H., and Hidaka, H. (1991) J. Biol. Chem. in press. 2. Kretsinger, R.H. (1980) CRC Crir, Rev, Biochem. 8, 119-174. 3. K&man, D., and Hilt, D.C. (1988) Trends in Biochem. Sci. 13,437-443. 4. Schtigger, H., and Jagow, G. (1987) Anal. Biochem., 166, 368-379 5. Feinberg, A.P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. 6. Sanger, F., Coulson, A.R., and Barre& B.G. (1980) J. Mol. Biol. 143, 161-178. 7. Hattori, M., and Sal&i, Y. (1986) Anal. Biochem. 152,232-238. 8. Sambrook, J., Fritsch, E.F., and Mania& T. (1987) Molecular Cloning, A Laboratory ManuaU2nd ed. Cold Spring Habor Laboratory, Cold Spring Habor, NY. 9. Isobe, T., and Okuyama, T. (1981) Eur. J. Biochem. 116,79-86. 10. Kuwano, R., Usui, H., Mae&, T., Fukui, T., Yamanari, N., Ohtsuka, E., Ikeyama, M., and Takahashi, Y. (1984) Nucleic Acid Res. 12,7455-7456. 11. Jensen, R., Marshak, D.R., Anderson, C., Lukas, T.J., and Watterson, D.M. (1985) 1. Neurochem.
45, 700-705.
12. Kuwana, R., Mac&, T., Usui, H., Araki, K., Yamakuni, T., Oshima, Y., Kurihara, T., and Takahashi, Y. (1986) FEBS Lett. 202, 97-101. 13. Gerke, V., and Weber, K. (1985) EMBO J. 4,2917-2920.
649
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 644-649
Molecular cloning and sequencing of a cDNA clone encoding a new calcium binding protein, named calgizzarin, from rabbit lung+ Masato Watanabe, Yuhko Ando, Hitoshi Todoroki, Hiroyuki Minami, and Hiroyoshi Hidaka* Department of Pharmacology, Nagoya University School of Medicine, Tsurumai 65, Showa-ku, Nagoya 466, Japan Received
October
27,
1991
We purified a new EF-hand type calcium binding protein from chicken gizzard smooth muscle, tentatively named calgizzarin (Todoroki, H., et al. J. Biol. Chem. (1991) in press (1)). Based on the internal pcptide sequence of calgizzarin, we isolated and sequenced a cDNA clone coding for calgizzarin from a rabbit lung cDNA library. This clone (pCALG) has 309 nucleotides of open reading frame including termination codon TGA, 621 nucleotides of the 5’ leader and 186 nucleotides of the 3’ noncoding region. The polypeptides deduced from the open reading frame were consisted of 102 amino acid residues with a molecular weight of 11,429. Computer aided homology analysis revealed that calgizzarin exhibits a 43.2% homology to S-lOOa, 38.6% to S1OOgand 40.0% to annexin II light chain, ~10. By Northern blot analysis, with pC!ALG, a band of 1.1 kbp was detected in rabbit lung, suggesting pCALG contains nearly full length of mBNA. 0 1991
Academic
Press,
Inc.
Intracellular calcium ion is involved in regulating various biochemical events in excitable cells. These events are mediated by a family of calcium binding proteins which exhibit a general structural principle, so called EF-hand structure (2). These EF-hand type calcium binding proteins can be classified at least into two families, calmodulin and S-100 protein families (3). In contrast with calmodulin, proteins of the S-100 protein family are present in limited and specified tissues. This would suggest that there are at least two different intracellular calcium signal transduction systems; one is the general system modulated by calmodulin which is present in all cells and the other is the specific system modulated by the members of the S-100 protein family present in specific cells. We reported the purification of a new calcium binding protein from chicken gizzard (calgizzarin) with an apparent molecular weight of 13,000, using a calcium dependent affinity chromatographic technique (1). The partial peptide sequence of calgizzarin has approximately a
+The nucleotide sequence in this paper has been submitted to the DDBJ, EMBL and Genbank database with the accession number D9053 1. * To whom all correspondence should be addressed. . . Abbrevlanons PCR, polymerase chain reaction, SDS; sodium dodecyl sulfate, PAGE; polyacrylamide gel electrophoresis. 0006-291x/91 Copyright All rights
$1.50
0 I991 by Academic Press, Inc. of reproduction in any form reserved.
644
Vol.
181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
50% homology to S-1OOa and 47% to S-1008. Application of the same affinity chromatographic technique to various tissues revealed that calgizzarin-like protein was abundantly present in rabbit lung. The major objective of the research concerned with proteins of S- 100 family reveal existence of the cell type specific calcium signaling system and its precise molecular mechanism. A first approach to this purpose is to determine the primary structure of a cDNA coding for calgizzarin using molecular biological technique. In this report, we isolated and sequenced a cDNA clone encoding calgizzarin from a rabbit lung cDNA library by screening with the PCR amplified authentic cDNA as a probe. Materials
and
Methods
Materials. [a-32P]dCTP (1 lOTBq/mmol) was purchased from Amersham International. T4 DNA polymerase, Taq polymerase (Ampli Taq), restriction endonucleases and other modifying enzymes were obtained from Takara Shuzo. Oligonucleotides were synthesized on a Model 392A DNA synthesizer (Applied Biosystems Ins.). A rabbit lung @tlO library was purchased from Clonetech. All other chemicals and reagents were of analytical grade obtained from commercial suppliers. Purification of calgizzarin from rabbit lung and protein sequencing. Calgizzarin was partially purified from rabbit lung (about 100 g) on a affinity column using a calmodulin antagonist, W-7 as a affinity ligand (0.8 x 5 cm) according to the method of Todoroki et al (1). Twenty nl of concentrated fraction of calgizzarin was separated by Tricine-SDS PAGE according to Schtigger and Jagow (4). Gel was stained with Coomassie brilliant blue and destained. Calgizzarin band was excised with a laser blade and was washed with distilled water. The excised gel was digested with 1:20 (w/w) of lysylendopeptidase in a 2OOp.lof 5OmM sodium biammonium. After 20hr incubation at 37’C. the resulting peptides were separated on a C-18 reverse phase column. The amino acid sequence was determined using automated protein sequencer (Model 473A, Applied Biosystems Inc.). Isolation of a calgizzarin cDNA clone. A cDNA library synthesized from mRNA of rabbit lung using a cDNA synthesis kit (Pharmacia Corp.) according to the manufacture’s manual was used as a template for synthesizing the authentic cDNA probe of calgizzarin. Two PCR primers, For and Rev, were designed according to the peptide sequence of calgizzarin (Fig. 1A). Twenty five cycles of PCR amplification were performed in a lOOpI reaction volume containing 1Ot.ddouble stranded cDNA library solution, 10 pmol of the primers and the reagents &scribed in the amplification protocol of Perkin-Elmer/Cetus. PCR cycle consists of a I-min denaturation at 94’C, a l-mm annealing at 37X!, and a 2-min polymerization at 72’C in a thermocycler (PerkinElmerKetus). The PCR product was cloned into pUC8 and used as the authentic cDNA probe. About 2.4 x 101 plaques from a rabbit lung XgtlO cDNA library were screened with the authentic cDNA probe labeled by multi priming method (5) using PCR primer sets as primers. Hybridization was performed overnight at 5o’C, and the final wash was carried out with 0.5 x SSC (20 x SSC; 3M NaCl, 0.3M sodium citrate) containing 0.1% SDS at 5o’C. DNA sequencing. The nucleotide sequences were analyzed by the dideoxy chain termination method (6) as modified by Hattori and Sakaki (7) with [a-32P] dCTP and the Sequenase Ver.2 kit (United States Biochemical Corp.). The double strands plasmid was firstly sequenced from both strands using two vector primers flanking the cDNA insert. The complete sequence was obtained by custom primer-directed DNA sequencing using specific primers complementary to internal cDNA sequences. RNA blotting and hybridization. Total RNA is extracted from rabbit lung by the guanidium isothiocyanate/cesium chloride method (8). Poly (A)+ RNA was preparated on a oligo (dT) cellulose column. Twenty pg of total RNA and five hundreds ng of poly (A)+ RNA were denatured and electrophoresed on a formaldehyde-containing agarose gel. The RNA was transferred onto a Hybond-N (Amersham Corp.) and hybridized in a solution containing !lOmM Ttis-HCl, pH 7.5,0.9M NaCl, 6mM EDTA, 50% formamide, 1 x Denhart (5 x Denhart; 1% bovine serum albumin,1 8 polyvinylpyrollidone,l 96 Ficoll400), 0.1% SDS and 2OOmg/ml heatdenatured salmon testis DNA at 42’C with [a-32P] dCTP labeled cDNA fragments. Finally, the filter was washed in 0.2 x SSC containing 0.1% SDS at 55-C and exposed to imaging plate. The image was analyzed and visualized with Bioimage analyzer (Model Fujix BAS2000, Fuji Film Corp.> 645
Vol.
181,
No.
2, 1991
BIOCHEMICAL
Results
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
and Discussion
We reported the purification of a new calcium binding protein from chicken gizzard, which molecular weight is estimated 13,000 by Tricine-SDS PAGE. The partial amino acid sequence analysis revealed that a new calcium binding protein, named calgizzarin, have two putative Ca2+binding structure (EF-hand) in the molecule. The overall amino acid sequence identity within the region covered by peptides obtained is 50% to S-1OOo and47% S-lOOj3 (1). We decided to isolate a cDNA clone coding for calgizzarin, to determine the characteristics of primary structure of calgizzarin. Firstly, we obtained the authentic cDNA probe of calgizzarin by PCR technique. PCR amplification was performed using two primers (For and Rev) synthesized according to the peptide sequence of calgizzarin (Fig.lA, upper line). PCR product was electrophoresed on a 4% Nu-Sieve (FMC) agarose gel. A 72bp band of expected size was isolated from the agarose gel and cloned into pUC8. The 72bp cDNA fragment identified by sequencing was used as the authentic probe (Fig. 1A, lower line). About 2.4 x 10s plaques from a rabbit lung XgtlO cDNA library were screened with the authentic probe. One positive clone, 457 bp in length, was obtained. This clone appeared to be incomplete, since the open reading frame encoding calgizzarin was not preceded by a 5’ untranslated region or an initiation codon ATG. A 457 bp insert of the clone was used to screen an additional 2 x l@ individual plaques of a rabbit lung library purchased from Clontech. Hybridization and wash were performed according to the procedure described in the Materials and Methods, except the final wash was carried out with 0.1 x SSC at 6X. Finally, one positive clone was isolated and further analyzed. Fig.lB shows the nucleotide and the deduced amino acid sequences of the entire cDNA insert from the recombinant plasmid pCALG. pCALG is 1116bp in length and contains 621bp of 5’ leader region, 309bp coding region including termination codon TGA and 186bp of 3’ nontranslated region. The sequences derived from Edman degradation arc found at amino acid positions #25-49 and #53-62. The polyadenylation signal sequence (AATAAA) is shown at nucleotide position 1056-1061. The calculated molecular weight of pCALG was 11,429 and this value was in good accordance with that of the purified chicken calgizzarin judged by Tricine-SDS PAGE. Northern blot analysis was performed using the entire insert of pCALG as a probe. Probing of total RNA and poly (A)+ RNA derived form rabbit lung revealed that a major band at 1.1-1.0 kbp was detected, suggesting that pCALG contains nearly full length of mRNA (Fig.2). By computer aided homology analysis, there is very limited homology of the pCALG with known sequences in Genbank, EMBL and NBRF databases. Fig.3 shows the results of the homology search. pCALG exhibits a 43.2% homology to S-1OOaof bovine (9,10), 38.6% to S1OOpof human (11,12) and 40.0% to annexin II light chain, ~10, of porcine (13). All these results show that the cDNA of pCALG is derived from mRNA for the rabbit calgizzarin and that calgizzarin is a new member of S- 100 protein family. Two putative EF hand structures appeared at amino acid positions #23-53 and #65-76. The second EF hand is more conserved among three S-100 family proteins than the first structure (Fig.3). It is, at least, thought that the second EF hand has calcium binding ability, since calcium can bind to chicken calgizzarin (1). The deduced polypeptides have 50% of hydrophobic amino 646
Vol.
181, No. 2, 1991
A For
primers
sequence
peptide authentic
B
cDNA
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
G A c C C GC TTTATGAATACTGAATT
Rev
cc G GATTATATGATGAAA
FMNTELAAFTKnqkDPGVLDYMMK
probe
TTCATWLACACGGAGCTGGCTGCCTTCACAAACJlACCAGG
gatctcattgatggggtggcctcccgtctctggcccctgccccctccccaccctggggta ttcgacatggagccccctgcccagggatgccgctcttgatgtctgtctagtctgctctag gatggccccaactcctactccttgtccagggtgaggttggaggcagaagacgggagaaac agcacttcttaaatcctatgaagccataatgtaaccccggtggactgatggcagatcagc tggtcctgtgtgctactttgcacggcagaactgcagggaggggctggaggctgtgaaagc ggaccccacccctgcttgcgctggttccagagtccaaggtggtcccctggctggggagaa agagagcagctggaggccggaggttgtggttgtgcagggctcatctcggcctggagggcc aggtcccctcccagctgccctctgttctctgaggctgactttttctgcggtccacccatc catcctctcctttagtgccttgactctcactcagaagcagcccccttcagtccctccgtc ctccctctgactcgctccgctccgctcgccgcgccgcccgcctcgcgcagctccgctcgg CggtcttctccaaaggcagcaATGTCCAGGCCCACAGAGACCGAGCGCTGCATCG~TCC M S R P TETERCIES 1 CTGATCGCCGTGTTCCAGATACGCTGGAAAGGATGGGCACAGCGTCACCCTCTCCAAG L IAVFQKYAGKDGHSVTLSK 14 ACCGAGTTCCTGTCCTTTATGAACACACAGAGCTGCCTGAC TEFLSFMNTELAAFTKNQKP 34 CCCGGCGTCCTCGACCGCATGATGAAGAAATTGGACCTCACAGTGACGGGCAGCTGGAT PGVLDRMMKKLDLNSDGQLD 54 TTCCAAGAATTTCTCAATCTCATTGGTGGCTTGGCCGTGGCTTGCCATGAGTCCTTTGTC FQEFLNLI GGLAVACHESFV 74 AAGGCCGCCCCTCCCCAGACGTTTCTGAggccgccgtggctctggcccgcagacccac K A A P PQKRF* 94 102 ccctactgctgccagcctgccagccctcgccactcctcacggcctgccctgagcccccac tccacacaggccactcctgccggcagtgataaaacaataagcagtatctttttttatga cacacaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
60 120 180 240 300 360 420 480 540
600 660
13 720
33 780
53 840
73 900 93 960 1020 1080 1116
Fig.lA Peptide and oligo nucleotide sequences.Upper line indicatesthe two primers synthesized by Model 392A DNA synthesizer.Forwardmimer(For) is a 17-meroligonucleotide c&responding to FMNTEL and-reverseprimer (Rev) is a 1%mer anti-olig&mcleotide cormsoondintz to DYMMK. Nucleotideseuuence of amulifledPCRnroductis shownin lower line. Middleiine indicatesthe peptidesequ&cederivedt+omEdman*&gradation(capitalletter) andthededucedaminoacidsequence fromPCRproduct(lowercaseletter). Fig.lB The nucleotide sequenceof cDNA coding for rabbit calgizzarin and the deducedamino acid sequence.The nucleotidesequence is numberedstartingat initiationof thiscIone(right side)andthe predictedaminoacidat initiationmentioning(underthesequence). The translatedregionis shownin capitallettersandthe S-, 3’- noncodingregionsareshownin lowercaseletters. Thepredictedaminoacidsequence is shownbelowthenucleotidesequence by one-lettercode. Thin underlinesindicatethepeptidesequence derivedfrom Edmandegradation andthick underlineindicatesthepolyadenylation signal.*, terminationcodon.
acid residue in the molecule. Hydropathicity plot suggeststhat hydrophilic portion and hydrophobicportion appearedin turn from aminoterminal to carboxyl terminal of the moleculeasa cluster (data not shown). When calcium binds to calgizzarin, calgizzarin changedrastically in its conformation. This conformational changemakesthe migration of hydrophobic region(s) to the surface of the molecule. These hydrophobic regions may interact with target protein(s) or an affinity ligand. 647
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28s 18s
F&.& Northern blot analysis of calgizzarin in rabbit lung. Twenty pg of total RNA and 0.5 pg of poly (A)+ RNA were electmphoresed and transferred to Hybond-N. Hybridization with the entire insert of pC!ALG was carried out under the condition described in the Materials and Methods.
The unique tissue distribution of proteins of S-100 family leads one to believe that each protein of S-100 family involves a specific signal transduction system. Calgizzarin reported in this paper also distributes in limited tissues, suggesting the possibility that there is a new and specific calcium signal transduction system. Identification and characterization of target protein(s) of calgizzarin and expression of the recombinant calgizzarin will aid future studies aimed to recognition of a new calcium signal transduction system.
1 CidgiZZViD
60
MSRPTETERCIESLlAVFQKYAGKDGHSVTLSKTEFLSKTEFLSF~TEL~T~QKDPGVLD~
s-looa
GS*L*TAM*T**N**HAHS**E*DKYK***K**~K*LKELLQ***SG*LQA***~AV*KV
s-mop
S*L*KAWA**D**HQ*S*RE*DKHK*K*S*LKLI*N**SH*LEEI*EQE*V*KV
PI0
*SQM*HAM*TMMFT*H*F**DK*Y---*T*EDLRVL*EK*FPG*LE*****LAV*KI 61
102
calgizzalin
MKKLDLNSDGOLDFQEFLNLIGGLAVACHESFVKAAPPQKRF
s-loou
**E**EDG**EV****YW*VAA*T***NNF*WENS
s-loop
*ET**SDG**EC*****MAFVAMITT****F*EHE
PlO
**D**QCR**KVG**S*FS**A**TI**NDY**VHMKQKGK
143.2%] [38.6%] [40.0%]
Fi .3 Comparison of the amino acid sequence of rabbit calgizzarin with S-1OOa bovine, S-loop of human, bovine and snnexin II light chain of porcine. Identical amino acid with rabbit calgizzarin are indicated by asterisks (*). Gaps (-) have inserted to achieve a maximum homology. Underlines indicate the putative calcium binding structures (EF hand structure).
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Acknowledgments We wish to thank Ryoji Kobayashi for technical advisement of protein sequencing and Etsuko Shimoda, Kiyo Harada for typing this manuscript and Naoko Watanabe for her help in technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
References 1. Todoroki, H., Kobayashi, R., Watanabe, M., Minami, H., and Hidaka, H. (1991) J. Biol. Chem. in press. 2. Kretsinger, R.H. (1980) CRC Crir, Rev, Biochem. 8, 119-174. 3. K&man, D., and Hilt, D.C. (1988) Trends in Biochem. Sci. 13,437-443. 4. Schtigger, H., and Jagow, G. (1987) Anal. Biochem., 166, 368-379 5. Feinberg, A.P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. 6. Sanger, F., Coulson, A.R., and Barre& B.G. (1980) J. Mol. Biol. 143, 161-178. 7. Hattori, M., and Sal&i, Y. (1986) Anal. Biochem. 152,232-238. 8. Sambrook, J., Fritsch, E.F., and Mania& T. (1987) Molecular Cloning, A Laboratory ManuaU2nd ed. Cold Spring Habor Laboratory, Cold Spring Habor, NY. 9. Isobe, T., and Okuyama, T. (1981) Eur. J. Biochem. 116,79-86. 10. Kuwano, R., Usui, H., Mae&, T., Fukui, T., Yamanari, N., Ohtsuka, E., Ikeyama, M., and Takahashi, Y. (1984) Nucleic Acid Res. 12,7455-7456. 11. Jensen, R., Marshak, D.R., Anderson, C., Lukas, T.J., and Watterson, D.M. (1985) 1. Neurochem.
45, 700-705.
12. Kuwana, R., Mac&, T., Usui, H., Araki, K., Yamakuni, T., Oshima, Y., Kurihara, T., and Takahashi, Y. (1986) FEBS Lett. 202, 97-101. 13. Gerke, V., and Weber, K. (1985) EMBO J. 4,2917-2920.
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