Vol. 184, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
May 15, 1992
Pages 1219-1225
eDNA C L O N I N G O F A N E U R A L V I S I N I N - L I K E Ca2÷-BINDING P R O T E I N ~
Takayoshi Kuno, Yasuo Kajimoto, Takeshi Hashimoto, Hideyuki Mukai, Yutaka Shirai, Shuichi Saheki *, and Chikako Tanaka
Department of Pharmacology, Kobe University School of Medicine, Chuo-ku, Kobe 650, Japan ~Department of Clinical Laboratory Medicine, Ehime University School of Medicine, Onsen-gun, Ehime 791-02, Japan
Received March 30, 1992
Summary: A 21,000-dalton Ca2+-binding protein (Walsh, M. P., Valentine, K. A., Ngai, P. K., Carruthers, C. A., and Hollengerg, M. D. (1984) Biochem. J. 224, 117-127) was purified from the rat brain and through the use of oligonucleotide probe ,based on partial amino acid sequence, cDNA clones were obtained from rat brain cDNA library. The complete amino acid sequence deduced from the cDNA contains 191 residues and has a calculated molecular mass of 22,142 daltons. There are three potential Ca2+-binding sites like the EF hands in the sequence. It displays striking sequence homology with visinin and recoverin, retina-specific Ca2÷-binding proteins. Northern blot analysis revealed that the protein is highly and specifically expressed in the brain. © 1992Academic P..... Inc.
Visinin, a cone-specific protein from chicken retina (1), has been identified as a Ca z÷binding protein with three repeated domains of approximately 30 residues called "EF hand" (2). Another retinal protein, recoverin is sharing 59% identity with visinin and has been reported to regulate retinal guanylate cyclase (3). These proteins are thought to be members of a family of Ca>-sensitive regulators that, through cooperative interactions, act as switches at submicromolar Ca 2÷ level (3). Walsh et al. (4) reported the purification of 21,000-dalton Ca2+-binding protein (21kDa CaBP) from bovine brain using Ca2+-dependent hydrophobic-interaction chromatography. The study of the purified 21-kDa CaBP has led to the conclusion that it belongs to the
*Sequence data from this article have been deposited with the EMBL/ GenBank Data Libraries under Accession No. DI0666.
The abbreviationsused are: 21-kDa CaBP, 21,000-d~ton Ca2+-binding protein; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel ele~rophomsis. 0006-291X/92 $4.00 1219
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 184, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
calmodulin "superfamily" (5). However, its primary structure and tissue distribution have not been reported. Here we report cDNA cloning of the 21-kDa CaBP from rat brain cDNA library, and the evidence indicating that it is a neural visinin-like Ca2+-binding protein.
MATERIALS AND METHODS
Materials: Restriction endonucleases, the Klenow fragment of DNA polymerase, T4 DNA ligase, a M13 sequencing kit, and a random primer DNA labeling kit were purchased from Takara Shuzo Co., Ltd., Kyoto, Japan; lysyl endopeptidase (EC 3.4.21.50) from Wako Pure Chemical Industries (Osaka, Japan); and 32P-dCTP from Amersham. All other reagents were of analytical grade. General Methods in Molecular Biology: Preparation of plasmid DNA, restriction enzyme digestion, agarose gel electrophoresis of DNA, labeling of DNA, DNA ligation, and bacterial transformation were carried out using standard methods (6), Purification of the 21-kDa CaBP: The 21-kDa CaBP was purified from rat brain as described by Walsh et al. (4). The purified sample was subjected to SDS-PAGE, and the 21kDa CaBP was directly recovered from gels to eliminate contaminating proteins and was cleaved enzymatically to prepare peptide samples for microsequencing as follows. Extraction of the 21-kDa CaBP from Gels: SDS-PAGE of the purified sample was carried out according to Laemmli (7). After staining with Coomassie brilliant blue R and destaining, the gel was washed with distilled water thoroughly to remove acetic acid. The stained bands of 2 t - k D a CaBP were excised from the gel and diced into pieces of 1 to 2 cubic millimeters with a razor blade. The gel pieces were placed in a capped test tube and resuspended in water. Fine gel fragments were removed by aspiration. Then, gels were resuspended in 5ml of 2% SDS, 0.4M ammonium bicarbonate and the proteins were extracted from the gel pieces by gentle continuous agitation overnight. The extract was lyophilized and was dissolved in 2rnl of water and mixed with 40ml of acetone. After centrifugation at 2,000 rpm for 5min, the pellet containing the protein and ammonium bicarbonate was dissolved in lml of water, and the ammonium bicarbonate/acetone precipitation was repeated two more times to remove SDS and Coomassie blue. A significant amount of the remaining ammonium bicarbonate could be decomposed to gas by heating in steam from a boiling water after moistening the pellet with 0.1ml of water. The pellet from acetone precipitation was dissolved in 4ml of water and mixed with 0.1ml of 3% sodium deoxycholate. The protein was co-precipitated with deoxycholate by acidifying the solution with addition of 0.2ml 100% trichloracetic acid. This step is necessary to remove cations that inhibit lysyl endopeptidase. After washing the pellet twice with 5ml of water by centrifugation, the pellet was shaken in 5ml of watersaturated diethylether several times to remove deoxycholic acid. Then the water phase and the interphase on the bottom were dried by a gentle nitrogen stream to prepare for protease digestion. Protease Digestion, Peptide Isolation, and Amino Acid Sequencing: The protein sample was digested with lysyl endopeptidase. The protease was dissolved in water and stored at 20°C until use. For digestion, the protein sample on the bottom of the tube described above was soaked with 100~tl of 20mM Tris, pH 10, added with the protease solution in a volume of 10~tl at an enzyme/substrate (w/w) ratio of 1/200, and incubated at room temperature (20°C) overnight under gentle rotary swirling. The reaction was stopped through freezing. The digested sample was mixed with 50[xl of 6M guanidine hydrochloride, transferred to a 1.5ml microeentrifuge tube, and centrifuged at 10,000 rpm for 5rain. The supematant was chromatographed using an high performance liquid chromatography system (Shimadzu, Kyoto, Japan) equipped with a reverse-phased column (Aquapore octyl, RP-300, 2.1x220mm, Brownlee Labs). The peptides were eluted with a linear gradient of acetonitrile from 1 to 1220
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70% (v/v) in 0.1% trifluoroacctic acid, pH 2 with monitoring their absorption at 230nm. Pcptide fractions were desiccated under thc vacuum in a desiccator containing bottles of sulfuric acid and sodium hydroxide on the bottom. Some peptide fractions were rechromatographed on the same column eluted with a different gradient of acetonitrilc from 0 to 70% (v/v) in 10mM ammonium acetate, pH 7.0. Thc peptidc samples were dissolved in 100~tl of 20% (v/v) acetic acid and spotted on a glass fiber disc. Amino acid sequencing from the amino-terminus was carried out at up to 20 cycles by a gas-phase sequencer (Applied Biosystems, Model 477/120A). Screening of the Library and Sequence Analysis: A rat brain cDNA library in lambda gtl0 (8) was screened on nitrocellulose filters with a 32p-labelcd synthetic 33-base oligonucleotide, 5'-CTI'CACCAGGTCCTCCATCACCTCAGGGGCCAG-3', which was designed and synthesized based on the 11 amino acid sequence of peptidc I in Fig. 1, according to the codon usage frequencies (9). Hybridization was carried out at 40°C in a solution containing 5 x SSPE, 5 x Denhardt's solution (6), 100gg/ml sonicated heat-denatured salmon sperm DNA, 0.1% SDS, 20% formamide, and thc probe for one overnight. After hybridization, the filters werc washed with 3 x SSC (6) containing 0.1% SDS at 55°C. The filtcrs were then dried and autoradiographed. Positive clones were purified and cDNA inserts were subcloned into EcoRI site of pUC18. Nuclcotide sequence analysis was performed using the cnzymatic chain termination method in conjugation with M13-dcrived vectors (10). The sequence was determined from both strands by using synthetic oligonucleotide primers sclected at convenicnt intervals (Fig. 2). Northern Blot Analysis: Total RNA from the tissues specified was isolated by the guanidine thiocyanate method of Chirgwin et al. (11), elcctrophoresed in a formaldehyde-l% (w/v) agarose gel, and blotted onto a nitrocellulose paper. After baking at 80°C for 2h and prehybridization, thc blot was hybridized with the labeled cDNA probe in a solution containing 4 x SSC, 40% (v/v) formamide, 10% (w/v) dextran sulfate, 20mM Tris-HC1, pH 7.4, I x Denhardt's solution, 250~g/ml yeast tRNA, and 201.tg/ml salmon sperm DNA at 42°C, and washed in 0.1 x SSC, 0.1% SDS at 60°C. Thc papers were then dried and autoradiographed.
RESULTS AND DISCUSSION
Amino-Acid Sequence of Peptides Derived from the Digested 21-kDa CaBP: Initial attempts to obtain N-terminal sequence data on thc intact 21-kDa CaBP failed, presumably because the N-terminus of this protein was blockcd. Thcreforc, pcptide fragments gcncrated by lysyl endopeptidase treatment of the cxtractcd protein sample from SDS-PAGE gel were isolated by reverse-phase high pcrformancc liquid chromatography. Eight distinct amino-acid sequences were determined as shown in Fig. 1. Later analysis revealed that all of these eight peptide sequences were found in the deduced amino acid sequence from cDNA (Fig. 3).
cDNA Cloning and Sequencing: Approximately 2 x 105 recombinant phages were screcned, and five positive clones wcre isolated. These clones were subcloned into the EcoRI site of the pUC18 vector and analyzcd by restriction mapping (Fig. 2). All of these clones showed similar rcstriction pattcrns indicating that they wcrc dcdvcd from the same mRNA species. The longest clone was sclecte~l for sequence analysis as shown in Fig. 2. The nuclcotide and deduced amino acid sequence is presented in Fig. 3. The eDNA clone consists of 1,051 base
1221
Vol. 184, No. 3, 1992
q)
1 2 3 4 5 6 7 8
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
LAPEVMEDLVK STEFNEHELK QWYK FFPYGDASK LNWAFNIVlYDLDGDGK ITRVEMLEIIEAIYK MNEDGLTPEQRVDK DDQITLDEFK
5'
Q
P S
S
EP
B
II
I
II
I
3'
1,051 base pairs
I
Fig. 1. Amino-acid sequence of peptides derived from the lysyl endopeptidase treated 21-kDa CaBP. Protease-treated sample was applied to high performance liquid chromatography and seParated peptides were sequenced IYomthe amino-terminus by a gasphase sequencer.
Fig. 2. The restriction map and DNA sequencing strategy. B, BamHI; E, EcoRI; P, PstI; S, SacI. Arrows show the direction and length of the DNA sequences obtained.
pairs, and has 299 base pairs of 5'-non-coding region, an open reading frame of 573 base pairs, and 239 base pairs of 3'-non-coding region, assuming that the initiating ATG codon is at nucleotide 240.
The ATG codon at 240 is thought to be an initiator because o f
following reasons: (i) the region on the 5' side of the ATG at 240 has no other ATG codon in any flame, and contains two stop codons in-frame with this first ATG; (ii) the nucleotide sequence surrounding this ATG agrees with the consensus sequence for initiation of translation often found in eukaryotes (12); (iii) the peptide #1 in Fig. 1 contains methionine which was coded by the second ATG codon at 276 (Fig. 3). The open reading frame encodes a polypeptide of 191 amino acids whose molecular mass and isoelectric point were calculated to be 22,142 daltons and 5.1, respectively. These data agree with those determined by gel electrophoresis for the purified 21-kDa CaBP (4). The primary structure of the 21-kDa CaBP deduced from the cDNA is strikingly homologous to those of visinin (1) and recoverin (3) as shown in Fig. 4. The amino-acid alignment of the deduced sequence of the 21-kDa CaBP with chicken visinin demonstrates 39% sequence identity and 84% homology, and that with bovine recoverin demonstrates 43% identity and 79% homology (Fig. 4). These three proteins contain three potential Ca2+-binding domains called "EF-hand" (2) (Fig. 4). Northern Blot Analysis:
On Northern blot analysis, we examined total RNA from various
rat tissues, using labeled cDNA probe (Fig. 5). In normal rat tissues, there was a large amount of mRNAs for the 21-kDa CaBP in the brain, and very low or undetectable levels in heart, lung, liver, kidney, spleen, skeletal muscle, small intestine, thymus, and testis. The probe hybridized to major species with an approximate size of 3 kilobase pairs. In this study, we obtained evidence that the 21-kDa CaBP described by Walsh et al. (4) is a neural visinin-like Ca2+-binding protein. The 21-kDa CaBP has been reported to 1222
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10 30 50 GGGGGATTCCCAATCTGCAGCAGAGATTCACCCGAGCGTGTTGCAGCGGCCACTGGGCT 70 90 ii0 TGCAAGGCGCAATCCAAGAGGGATTTAAGCAACCCGGAGCTCCCAG~AAGAAGCGAGAG 130 150 170 AG~CCACTCACAGAGACGGTTTAGCCGTTAGTCTGAATTAAATATATATATTTTTTTGA 190 210 230 AAGAACTGTTTAGTTTTATCATTTTCCTTAAGTGACGGTGCACAGCGCTATAACTGCAAA 250 270 290 ATGGGGAAACAGAATAGC~ACTGGCCCCAGAAGTCATGGAGGACCTGGTGAAGAGCACT M G K Q N S K L A P E V M E D L V K S T 310 330 350 GAGTTCAATGAGCATGAGCTC~GCAGTGGTACAAGGGCTTCCTCAAGGACTGTCCGAGC E F N E H E L K q W Y K G F L K D C P S 370 390 410 GGGAGGCTGAATCTCGAAGAGTTCCAGCAGCTCTATGTG~GTTCTTCCCCTATGGAGAT G R L N L E E F Q Q L Y V K F F P Y G D 430 450 470 GCCTCCAAGTTCGCTCAGCACGCCTTCCGCACCTTCGACAAGAATGGTGATGGCACCATC A S K F A Q H A F R T F D K N G D G T I 490 510 530 GACTTCCGAGAGTTCATCTGTGCTCTGTCCATCACCTCCAGAGGCAGCTTTGAGCAGAAA D F R E F I C A L S I T S R G S F E Q K 550 570 590 CTGAACTGGGCTTTCAACATGTATGATCTGGATGGTGACGGC~GATCACCAGGGTGGAG L N W A F N M Y D L D G D G K I T R 610 630 650 ATGCTGGAGATTATCGAGGCTATCTACAAGATGGTGGGCACTGTGATCATGATGAAAATG M L E I I E A I Y K M V G T V I M M 670 690 710 AATGAGGATGGCCTCACGCCAG~CAGCGAGTGGACAAGATTTTCAGCAAGATGGATAAG N E D G L T P E q R V D K I F S K M 730 750 770 AACAAAGATGACCAGATTACATTGGATGAATTCA~GAAGCTGCAAAAAGCGACCCTTCC N K D D @ I T L D E F K E A A K S D P S 790 810 830 ATTGTATTACTCCTGCAGTGCGACATTCAGAAATGAGCTGATGTCAATGCTATGGACTGC I V L L L Q C D I Q K 850 870 890
V
E
K
M
D
K
ACAGAAGTCTTGATGTTCCATTCAGTCTGCAGCTACACACACACACACACACACACACAC 910 930 950 ACACACACACACACTACACACACACACAATTGCTTGGACTACCTATAAATGGACTTGCTT 970 990 1010
CTTGTGTTTGA~CACTTGTGTGCATGAGAATGTCATTTGCTAATGAATTTTAAAAGCAT 1030 1050 ATATTATA~AACCCATGGTACCCGGATCCTC
Fig. 3, Nucleotide and deduced amino acid sequence of the neural visinin-like Ca 2÷binding protein (21-kDa CaBP). The deduced amino acid sequence of an open reading frame is given in single letter amino acid symbols below the nucleotide sequence. Amino acid sequences derived from purified peptides are underlined. The initiation and termination codons are also underlined.
have no effect on the activity of cyclic nucleotide phosphodiesterase, myosin light-chain kinase, and protein kinase C (4). Although the functional role of visinin has not yet been clarified, another visinin-like protein, recoverin, has been reported to be a Ca2+ sensitive activator that must be liberated from Ca2÷ before it activates its target (3). Therefore, it is suggested that the neural visinin-like Ca2+-binding protein also activates its unknown target molecule when Ca2+ is lowered within the submicromolar range. Its restricted distribution to neural tissue suggests that it plays a role in Ca2÷-mediated processes in neural cells. 1223
Vol
1 8 4 , N o . 3, 1 9 9 2
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
Vis
1" MGNSRSSALSREVLQELRASTRYTEEELSRWYEGFQRQCSDGRIRCDEFERIYGNFFPNS
NVP
1'
Re¢
1" MGNSKSGALSKEILEELQLNTKFTEEELSSWYQSFLKECPSGRITRQEFQTIYSKFFPEA
Vis
61" EPQGYARHVFRSFDTNDDGTLDFREYIIALHLTSSGKTHLKLEWAFSLFDVDRNGEVSKS
NVP
60' DASKFAQHAFRTFDKNGDGTIDFREFICALSITSRGSFEQKLNWAFNMYDLDGDGKITRV
Rec
61" DPKAYAQHVFRSFDANSDGTLDFKEYVIALHMTSAGKTNQKLEWAFSLYDVDGNGTISKN
VLS
121" EVLEIITAIFKMIPEEERLQLPEDENSPQKRADKLWAYFNKGENDKIAEGEFIDGVMKND
NVP
120' EMLEIIEAIYKMVGTVIMMKMNEDGLTPEQRVDKIFSKMDKNKDDQITLDEFKEAAKSDP
Rec
121" EVLEIVTAIFKMISPEDTKHLPEDENTPEKRAEKIWGFFGKKDDDKLTEKEFIEGTLANK
Vis
181" AIMRLIQYEPKK
NVP
180' SIVLLLQCDIQK
Rec
181" E ILRLI QFEPQKVKEKLKEKKL
MGKQNSKLAPEVYIEDLVKSTEFNEHELKQWYKGFLKDCPSGRLNLEEFQQLYVKFFPYG
Fig. 4. Amino acid sequence comparison of the neural visinin-like Ca2÷-binding protein (21-kDa CaBP) versus visinin and recoverin. Sequence alignment was done using GENETYX (Software Development Co., Ltd., Tokyo, Japan). The deduced amino acid sequences are shown in one-letter code. The amino acid sequence of visinin and recoverin are from the report by Yamagata et al. (1) and Dizhoor et al. (3), respectively. The matched residues are connected by asterisks. Conservative substitutions are indicated by dots. The three potential Ca2+-binding sites are underlined. NVP, the neural visinin-like Ca2+-binding protein (21-kDa CaBP); Vis, visinin; Rec, recoverin.
28S 18S
1
2
3
4
5
6
7
8
9 10
Fig. 5. Northern blot analysis. Total RNA (20p.g) from various rat tissues was eleetrophoresed and blotted onto a nitrocellulose filter, and the filter was hybridized with 32p_ labeled cDNA. Lane 1, brain; lane 2, heart; lane 3, lung; lane 4, liver; lane 5, kidney; lane 6, spleen; lane 7, skeletal muscle; lane 8, small intestine; lane 9, thymus; lane 10, testis. The 28S and 18S rat ribosomal RNAs are indicated on the left. 1224
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Acknowledgments: These studies were supported by research grants from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare, Japan.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Yamagata, K., Goto, K., Kuo, C. H., Kondo, H., and Miki, N. (1990) Neuron 2, 469476. Kretsinger, R. H. (1980) CRC Crit. Rev. Biochem. 8, 119-174. Dizhoor, A. M., Ray, S.,-Kumar, S., Niemi, G., Spencer, M., Brolley, D., Walsh, K. A., Philipov, P. P., Hurley, J. B., and Stryer, L. (1991) Science 251, 915-918. Walsh, M. P., Valentine, K. A., Ngai, P. K., Carruthers, C. A., and Hollenberg, M. D. (1984) Biochem. J. 224, 117-127. McDonald, J. R., Walsh, M. P., McCubbin, W. D., and Kay, C. M. (1987) Methods Enzymol. 139, 88-105. Sambrook, J., Fritsch, E. Fi, and Maniatis, T. (1989). Molecular cloning: A Laboratory Manual (Cold Spring Harbor, New York; Cold Spring Harbor Laboratory). Laemmli, U. K. (1970) Nature 227, 680-685. Ito, A., Hashimoto, T., Hirai, M., Takeda, T., Shuntoh, H., Kuno, T., and Tanaka, C. (1989) Biochem. Biophys. Res. Commun, 163, 1492-1497. Lathe, R. (1985) J. Mol. Biol. 183, 1-12. Sanger, A. F., Nicklen, S., and Coulson, A. R. (1977). Proc. Natl. Acad. Sci. USA 74, 5463-5467. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299. Kozak, M. (1984) Nucleic Acid Res. 12, 857-872.
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May 15, 1992
Pages 1219-1225
eDNA C L O N I N G O F A N E U R A L V I S I N I N - L I K E Ca2÷-BINDING P R O T E I N ~
Takayoshi Kuno, Yasuo Kajimoto, Takeshi Hashimoto, Hideyuki Mukai, Yutaka Shirai, Shuichi Saheki *, and Chikako Tanaka
Department of Pharmacology, Kobe University School of Medicine, Chuo-ku, Kobe 650, Japan ~Department of Clinical Laboratory Medicine, Ehime University School of Medicine, Onsen-gun, Ehime 791-02, Japan
Received March 30, 1992
Summary: A 21,000-dalton Ca2+-binding protein (Walsh, M. P., Valentine, K. A., Ngai, P. K., Carruthers, C. A., and Hollengerg, M. D. (1984) Biochem. J. 224, 117-127) was purified from the rat brain and through the use of oligonucleotide probe ,based on partial amino acid sequence, cDNA clones were obtained from rat brain cDNA library. The complete amino acid sequence deduced from the cDNA contains 191 residues and has a calculated molecular mass of 22,142 daltons. There are three potential Ca2+-binding sites like the EF hands in the sequence. It displays striking sequence homology with visinin and recoverin, retina-specific Ca2÷-binding proteins. Northern blot analysis revealed that the protein is highly and specifically expressed in the brain. © 1992Academic P..... Inc.
Visinin, a cone-specific protein from chicken retina (1), has been identified as a Ca z÷binding protein with three repeated domains of approximately 30 residues called "EF hand" (2). Another retinal protein, recoverin is sharing 59% identity with visinin and has been reported to regulate retinal guanylate cyclase (3). These proteins are thought to be members of a family of Ca>-sensitive regulators that, through cooperative interactions, act as switches at submicromolar Ca 2÷ level (3). Walsh et al. (4) reported the purification of 21,000-dalton Ca2+-binding protein (21kDa CaBP) from bovine brain using Ca2+-dependent hydrophobic-interaction chromatography. The study of the purified 21-kDa CaBP has led to the conclusion that it belongs to the
*Sequence data from this article have been deposited with the EMBL/ GenBank Data Libraries under Accession No. DI0666.
The abbreviationsused are: 21-kDa CaBP, 21,000-d~ton Ca2+-binding protein; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel ele~rophomsis. 0006-291X/92 $4.00 1219
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 184, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
calmodulin "superfamily" (5). However, its primary structure and tissue distribution have not been reported. Here we report cDNA cloning of the 21-kDa CaBP from rat brain cDNA library, and the evidence indicating that it is a neural visinin-like Ca2+-binding protein.
MATERIALS AND METHODS
Materials: Restriction endonucleases, the Klenow fragment of DNA polymerase, T4 DNA ligase, a M13 sequencing kit, and a random primer DNA labeling kit were purchased from Takara Shuzo Co., Ltd., Kyoto, Japan; lysyl endopeptidase (EC 3.4.21.50) from Wako Pure Chemical Industries (Osaka, Japan); and 32P-dCTP from Amersham. All other reagents were of analytical grade. General Methods in Molecular Biology: Preparation of plasmid DNA, restriction enzyme digestion, agarose gel electrophoresis of DNA, labeling of DNA, DNA ligation, and bacterial transformation were carried out using standard methods (6), Purification of the 21-kDa CaBP: The 21-kDa CaBP was purified from rat brain as described by Walsh et al. (4). The purified sample was subjected to SDS-PAGE, and the 21kDa CaBP was directly recovered from gels to eliminate contaminating proteins and was cleaved enzymatically to prepare peptide samples for microsequencing as follows. Extraction of the 21-kDa CaBP from Gels: SDS-PAGE of the purified sample was carried out according to Laemmli (7). After staining with Coomassie brilliant blue R and destaining, the gel was washed with distilled water thoroughly to remove acetic acid. The stained bands of 2 t - k D a CaBP were excised from the gel and diced into pieces of 1 to 2 cubic millimeters with a razor blade. The gel pieces were placed in a capped test tube and resuspended in water. Fine gel fragments were removed by aspiration. Then, gels were resuspended in 5ml of 2% SDS, 0.4M ammonium bicarbonate and the proteins were extracted from the gel pieces by gentle continuous agitation overnight. The extract was lyophilized and was dissolved in 2rnl of water and mixed with 40ml of acetone. After centrifugation at 2,000 rpm for 5min, the pellet containing the protein and ammonium bicarbonate was dissolved in lml of water, and the ammonium bicarbonate/acetone precipitation was repeated two more times to remove SDS and Coomassie blue. A significant amount of the remaining ammonium bicarbonate could be decomposed to gas by heating in steam from a boiling water after moistening the pellet with 0.1ml of water. The pellet from acetone precipitation was dissolved in 4ml of water and mixed with 0.1ml of 3% sodium deoxycholate. The protein was co-precipitated with deoxycholate by acidifying the solution with addition of 0.2ml 100% trichloracetic acid. This step is necessary to remove cations that inhibit lysyl endopeptidase. After washing the pellet twice with 5ml of water by centrifugation, the pellet was shaken in 5ml of watersaturated diethylether several times to remove deoxycholic acid. Then the water phase and the interphase on the bottom were dried by a gentle nitrogen stream to prepare for protease digestion. Protease Digestion, Peptide Isolation, and Amino Acid Sequencing: The protein sample was digested with lysyl endopeptidase. The protease was dissolved in water and stored at 20°C until use. For digestion, the protein sample on the bottom of the tube described above was soaked with 100~tl of 20mM Tris, pH 10, added with the protease solution in a volume of 10~tl at an enzyme/substrate (w/w) ratio of 1/200, and incubated at room temperature (20°C) overnight under gentle rotary swirling. The reaction was stopped through freezing. The digested sample was mixed with 50[xl of 6M guanidine hydrochloride, transferred to a 1.5ml microeentrifuge tube, and centrifuged at 10,000 rpm for 5rain. The supematant was chromatographed using an high performance liquid chromatography system (Shimadzu, Kyoto, Japan) equipped with a reverse-phased column (Aquapore octyl, RP-300, 2.1x220mm, Brownlee Labs). The peptides were eluted with a linear gradient of acetonitrile from 1 to 1220
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70% (v/v) in 0.1% trifluoroacctic acid, pH 2 with monitoring their absorption at 230nm. Pcptide fractions were desiccated under thc vacuum in a desiccator containing bottles of sulfuric acid and sodium hydroxide on the bottom. Some peptide fractions were rechromatographed on the same column eluted with a different gradient of acetonitrilc from 0 to 70% (v/v) in 10mM ammonium acetate, pH 7.0. Thc peptidc samples were dissolved in 100~tl of 20% (v/v) acetic acid and spotted on a glass fiber disc. Amino acid sequencing from the amino-terminus was carried out at up to 20 cycles by a gas-phase sequencer (Applied Biosystems, Model 477/120A). Screening of the Library and Sequence Analysis: A rat brain cDNA library in lambda gtl0 (8) was screened on nitrocellulose filters with a 32p-labelcd synthetic 33-base oligonucleotide, 5'-CTI'CACCAGGTCCTCCATCACCTCAGGGGCCAG-3', which was designed and synthesized based on the 11 amino acid sequence of peptidc I in Fig. 1, according to the codon usage frequencies (9). Hybridization was carried out at 40°C in a solution containing 5 x SSPE, 5 x Denhardt's solution (6), 100gg/ml sonicated heat-denatured salmon sperm DNA, 0.1% SDS, 20% formamide, and thc probe for one overnight. After hybridization, the filters werc washed with 3 x SSC (6) containing 0.1% SDS at 55°C. The filtcrs were then dried and autoradiographed. Positive clones were purified and cDNA inserts were subcloned into EcoRI site of pUC18. Nuclcotide sequence analysis was performed using the cnzymatic chain termination method in conjugation with M13-dcrived vectors (10). The sequence was determined from both strands by using synthetic oligonucleotide primers sclected at convenicnt intervals (Fig. 2). Northern Blot Analysis: Total RNA from the tissues specified was isolated by the guanidine thiocyanate method of Chirgwin et al. (11), elcctrophoresed in a formaldehyde-l% (w/v) agarose gel, and blotted onto a nitrocellulose paper. After baking at 80°C for 2h and prehybridization, thc blot was hybridized with the labeled cDNA probe in a solution containing 4 x SSC, 40% (v/v) formamide, 10% (w/v) dextran sulfate, 20mM Tris-HC1, pH 7.4, I x Denhardt's solution, 250~g/ml yeast tRNA, and 201.tg/ml salmon sperm DNA at 42°C, and washed in 0.1 x SSC, 0.1% SDS at 60°C. Thc papers were then dried and autoradiographed.
RESULTS AND DISCUSSION
Amino-Acid Sequence of Peptides Derived from the Digested 21-kDa CaBP: Initial attempts to obtain N-terminal sequence data on thc intact 21-kDa CaBP failed, presumably because the N-terminus of this protein was blockcd. Thcreforc, pcptide fragments gcncrated by lysyl endopeptidase treatment of the cxtractcd protein sample from SDS-PAGE gel were isolated by reverse-phase high pcrformancc liquid chromatography. Eight distinct amino-acid sequences were determined as shown in Fig. 1. Later analysis revealed that all of these eight peptide sequences were found in the deduced amino acid sequence from cDNA (Fig. 3).
cDNA Cloning and Sequencing: Approximately 2 x 105 recombinant phages were screcned, and five positive clones wcre isolated. These clones were subcloned into the EcoRI site of the pUC18 vector and analyzcd by restriction mapping (Fig. 2). All of these clones showed similar rcstriction pattcrns indicating that they wcrc dcdvcd from the same mRNA species. The longest clone was sclecte~l for sequence analysis as shown in Fig. 2. The nuclcotide and deduced amino acid sequence is presented in Fig. 3. The eDNA clone consists of 1,051 base
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LAPEVMEDLVK STEFNEHELK QWYK FFPYGDASK LNWAFNIVlYDLDGDGK ITRVEMLEIIEAIYK MNEDGLTPEQRVDK DDQITLDEFK
5'
Q
P S
S
EP
B
II
I
II
I
3'
1,051 base pairs
I
Fig. 1. Amino-acid sequence of peptides derived from the lysyl endopeptidase treated 21-kDa CaBP. Protease-treated sample was applied to high performance liquid chromatography and seParated peptides were sequenced IYomthe amino-terminus by a gasphase sequencer.
Fig. 2. The restriction map and DNA sequencing strategy. B, BamHI; E, EcoRI; P, PstI; S, SacI. Arrows show the direction and length of the DNA sequences obtained.
pairs, and has 299 base pairs of 5'-non-coding region, an open reading frame of 573 base pairs, and 239 base pairs of 3'-non-coding region, assuming that the initiating ATG codon is at nucleotide 240.
The ATG codon at 240 is thought to be an initiator because o f
following reasons: (i) the region on the 5' side of the ATG at 240 has no other ATG codon in any flame, and contains two stop codons in-frame with this first ATG; (ii) the nucleotide sequence surrounding this ATG agrees with the consensus sequence for initiation of translation often found in eukaryotes (12); (iii) the peptide #1 in Fig. 1 contains methionine which was coded by the second ATG codon at 276 (Fig. 3). The open reading frame encodes a polypeptide of 191 amino acids whose molecular mass and isoelectric point were calculated to be 22,142 daltons and 5.1, respectively. These data agree with those determined by gel electrophoresis for the purified 21-kDa CaBP (4). The primary structure of the 21-kDa CaBP deduced from the cDNA is strikingly homologous to those of visinin (1) and recoverin (3) as shown in Fig. 4. The amino-acid alignment of the deduced sequence of the 21-kDa CaBP with chicken visinin demonstrates 39% sequence identity and 84% homology, and that with bovine recoverin demonstrates 43% identity and 79% homology (Fig. 4). These three proteins contain three potential Ca2+-binding domains called "EF-hand" (2) (Fig. 4). Northern Blot Analysis:
On Northern blot analysis, we examined total RNA from various
rat tissues, using labeled cDNA probe (Fig. 5). In normal rat tissues, there was a large amount of mRNAs for the 21-kDa CaBP in the brain, and very low or undetectable levels in heart, lung, liver, kidney, spleen, skeletal muscle, small intestine, thymus, and testis. The probe hybridized to major species with an approximate size of 3 kilobase pairs. In this study, we obtained evidence that the 21-kDa CaBP described by Walsh et al. (4) is a neural visinin-like Ca2+-binding protein. The 21-kDa CaBP has been reported to 1222
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10 30 50 GGGGGATTCCCAATCTGCAGCAGAGATTCACCCGAGCGTGTTGCAGCGGCCACTGGGCT 70 90 ii0 TGCAAGGCGCAATCCAAGAGGGATTTAAGCAACCCGGAGCTCCCAG~AAGAAGCGAGAG 130 150 170 AG~CCACTCACAGAGACGGTTTAGCCGTTAGTCTGAATTAAATATATATATTTTTTTGA 190 210 230 AAGAACTGTTTAGTTTTATCATTTTCCTTAAGTGACGGTGCACAGCGCTATAACTGCAAA 250 270 290 ATGGGGAAACAGAATAGC~ACTGGCCCCAGAAGTCATGGAGGACCTGGTGAAGAGCACT M G K Q N S K L A P E V M E D L V K S T 310 330 350 GAGTTCAATGAGCATGAGCTC~GCAGTGGTACAAGGGCTTCCTCAAGGACTGTCCGAGC E F N E H E L K q W Y K G F L K D C P S 370 390 410 GGGAGGCTGAATCTCGAAGAGTTCCAGCAGCTCTATGTG~GTTCTTCCCCTATGGAGAT G R L N L E E F Q Q L Y V K F F P Y G D 430 450 470 GCCTCCAAGTTCGCTCAGCACGCCTTCCGCACCTTCGACAAGAATGGTGATGGCACCATC A S K F A Q H A F R T F D K N G D G T I 490 510 530 GACTTCCGAGAGTTCATCTGTGCTCTGTCCATCACCTCCAGAGGCAGCTTTGAGCAGAAA D F R E F I C A L S I T S R G S F E Q K 550 570 590 CTGAACTGGGCTTTCAACATGTATGATCTGGATGGTGACGGC~GATCACCAGGGTGGAG L N W A F N M Y D L D G D G K I T R 610 630 650 ATGCTGGAGATTATCGAGGCTATCTACAAGATGGTGGGCACTGTGATCATGATGAAAATG M L E I I E A I Y K M V G T V I M M 670 690 710 AATGAGGATGGCCTCACGCCAG~CAGCGAGTGGACAAGATTTTCAGCAAGATGGATAAG N E D G L T P E q R V D K I F S K M 730 750 770 AACAAAGATGACCAGATTACATTGGATGAATTCA~GAAGCTGCAAAAAGCGACCCTTCC N K D D @ I T L D E F K E A A K S D P S 790 810 830 ATTGTATTACTCCTGCAGTGCGACATTCAGAAATGAGCTGATGTCAATGCTATGGACTGC I V L L L Q C D I Q K 850 870 890
V
E
K
M
D
K
ACAGAAGTCTTGATGTTCCATTCAGTCTGCAGCTACACACACACACACACACACACACAC 910 930 950 ACACACACACACACTACACACACACACAATTGCTTGGACTACCTATAAATGGACTTGCTT 970 990 1010
CTTGTGTTTGA~CACTTGTGTGCATGAGAATGTCATTTGCTAATGAATTTTAAAAGCAT 1030 1050 ATATTATA~AACCCATGGTACCCGGATCCTC
Fig. 3, Nucleotide and deduced amino acid sequence of the neural visinin-like Ca 2÷binding protein (21-kDa CaBP). The deduced amino acid sequence of an open reading frame is given in single letter amino acid symbols below the nucleotide sequence. Amino acid sequences derived from purified peptides are underlined. The initiation and termination codons are also underlined.
have no effect on the activity of cyclic nucleotide phosphodiesterase, myosin light-chain kinase, and protein kinase C (4). Although the functional role of visinin has not yet been clarified, another visinin-like protein, recoverin, has been reported to be a Ca2+ sensitive activator that must be liberated from Ca2÷ before it activates its target (3). Therefore, it is suggested that the neural visinin-like Ca2+-binding protein also activates its unknown target molecule when Ca2+ is lowered within the submicromolar range. Its restricted distribution to neural tissue suggests that it plays a role in Ca2÷-mediated processes in neural cells. 1223
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Vis
1" MGNSRSSALSREVLQELRASTRYTEEELSRWYEGFQRQCSDGRIRCDEFERIYGNFFPNS
NVP
1'
Re¢
1" MGNSKSGALSKEILEELQLNTKFTEEELSSWYQSFLKECPSGRITRQEFQTIYSKFFPEA
Vis
61" EPQGYARHVFRSFDTNDDGTLDFREYIIALHLTSSGKTHLKLEWAFSLFDVDRNGEVSKS
NVP
60' DASKFAQHAFRTFDKNGDGTIDFREFICALSITSRGSFEQKLNWAFNMYDLDGDGKITRV
Rec
61" DPKAYAQHVFRSFDANSDGTLDFKEYVIALHMTSAGKTNQKLEWAFSLYDVDGNGTISKN
VLS
121" EVLEIITAIFKMIPEEERLQLPEDENSPQKRADKLWAYFNKGENDKIAEGEFIDGVMKND
NVP
120' EMLEIIEAIYKMVGTVIMMKMNEDGLTPEQRVDKIFSKMDKNKDDQITLDEFKEAAKSDP
Rec
121" EVLEIVTAIFKMISPEDTKHLPEDENTPEKRAEKIWGFFGKKDDDKLTEKEFIEGTLANK
Vis
181" AIMRLIQYEPKK
NVP
180' SIVLLLQCDIQK
Rec
181" E ILRLI QFEPQKVKEKLKEKKL
MGKQNSKLAPEVYIEDLVKSTEFNEHELKQWYKGFLKDCPSGRLNLEEFQQLYVKFFPYG
Fig. 4. Amino acid sequence comparison of the neural visinin-like Ca2÷-binding protein (21-kDa CaBP) versus visinin and recoverin. Sequence alignment was done using GENETYX (Software Development Co., Ltd., Tokyo, Japan). The deduced amino acid sequences are shown in one-letter code. The amino acid sequence of visinin and recoverin are from the report by Yamagata et al. (1) and Dizhoor et al. (3), respectively. The matched residues are connected by asterisks. Conservative substitutions are indicated by dots. The three potential Ca2+-binding sites are underlined. NVP, the neural visinin-like Ca2+-binding protein (21-kDa CaBP); Vis, visinin; Rec, recoverin.
28S 18S
1
2
3
4
5
6
7
8
9 10
Fig. 5. Northern blot analysis. Total RNA (20p.g) from various rat tissues was eleetrophoresed and blotted onto a nitrocellulose filter, and the filter was hybridized with 32p_ labeled cDNA. Lane 1, brain; lane 2, heart; lane 3, lung; lane 4, liver; lane 5, kidney; lane 6, spleen; lane 7, skeletal muscle; lane 8, small intestine; lane 9, thymus; lane 10, testis. The 28S and 18S rat ribosomal RNAs are indicated on the left. 1224
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Acknowledgments: These studies were supported by research grants from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare, Japan.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Yamagata, K., Goto, K., Kuo, C. H., Kondo, H., and Miki, N. (1990) Neuron 2, 469476. Kretsinger, R. H. (1980) CRC Crit. Rev. Biochem. 8, 119-174. Dizhoor, A. M., Ray, S.,-Kumar, S., Niemi, G., Spencer, M., Brolley, D., Walsh, K. A., Philipov, P. P., Hurley, J. B., and Stryer, L. (1991) Science 251, 915-918. Walsh, M. P., Valentine, K. A., Ngai, P. K., Carruthers, C. A., and Hollenberg, M. D. (1984) Biochem. J. 224, 117-127. McDonald, J. R., Walsh, M. P., McCubbin, W. D., and Kay, C. M. (1987) Methods Enzymol. 139, 88-105. Sambrook, J., Fritsch, E. Fi, and Maniatis, T. (1989). Molecular cloning: A Laboratory Manual (Cold Spring Harbor, New York; Cold Spring Harbor Laboratory). Laemmli, U. K. (1970) Nature 227, 680-685. Ito, A., Hashimoto, T., Hirai, M., Takeda, T., Shuntoh, H., Kuno, T., and Tanaka, C. (1989) Biochem. Biophys. Res. Commun, 163, 1492-1497. Lathe, R. (1985) J. Mol. Biol. 183, 1-12. Sanger, A. F., Nicklen, S., and Coulson, A. R. (1977). Proc. Natl. Acad. Sci. USA 74, 5463-5467. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299. Kozak, M. (1984) Nucleic Acid Res. 12, 857-872.
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