Biochhnica et Biophysica Acta, 1129 (1992) 249-250 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
249
Short Sequence-Paper
BBAEXP 90303
Nucleotide sequence of endothelin-1 c D N A from rabbit endothelial cells Philip A. Marsden i, Parvez Sultan 2, Myron Cybulsky 2, Michael A. Gimbrone, Jr. ~, Barry M. Brenner 3 and Tucker Collins 2 t Renal Dicision, Dt,partment of Medicine, St. Michad's Hospital and Unicersity of Toronto, Toronto (Canada), " Vascular Research Dicision, Departmou of Pathology, Brigham and Womo~ ~ Hospital, Harcard Medical School, Boston, MA (U.S.A.) and "~Renal Dicision, Department of Medicine, Brighton and Women's Hospital and Harcard Medical School and Harcard Center for the Study of Kidney Diseases, Boston, MA (U.S.A.) (Received 4 November 1991 )
Key words: Atherosclerosis: Endothelium: Endothelin: Hypertension; Vasoconstrictor: (Rabbit)
A eDNA encoding rabbit endothelin-I (ET-I) was isolated by plaque hybridization from a rabbit inferior vena caval endothelial cell Agtll cDNA library using human ET-I cDNA as the hybridization probe. DNA sequence analysis indicates that mature 21 amino acid rabbit ET-I is derived from a 202 amino acid precursor, via a 39 amino acid intermediate 'big' ET-I.
1 ¢¢GGGACCGGAGCC¢GGGCCGAGGATCCGAG¢¢A¢GAGAAGCGCTGAGGCTGTTTTT¢~¢GTCTGCTTTTGGGTTCAGT~GCCGGAG~TTTTTTTTA
101
GACCTTTTTCAGMTGGATiACTTCTC~G~GGTCTCiCTGCT~TGGTGGCTTTCCATGGAGcTcCc~AGACAG¢GG~CTCGGG¢ACGuG¢TCAGC fl O y F S fl fl V S L L L V A F H G A P E T A A S G T E L S
201 ACAGGAGCGGAAAACCCAGG6GAGAAGCCCCC6GCCA6C6CACCTTGGCGGCCGCGCC6GTCCMGCGGTGCTCCTGCTCCTC6CT6ATG~CMGGAGT T G A E N P G E K P P A S A P W R P R R $ K R $ C S S L fl 0 K E 301 GTGTCTACTT£TGCCA¢CTGGA¢&~CAT¢TGG~AA~ACCCcCGGG¢ACATCGTcc¢GTATGGACTTGGMG~cC~cCAGGTCCMG~TCCTTGM V y F C tl L 0 1 1 W V N T P G H I V P Y G L G $ P S R $ K R S L K 401 ~ATTTMTT~¢TAc¢AGGGCAG¢~TACCAcMGMcAGGTGcCA~GC~GAGCc~A£AcGACM6MGT6CT6GMT~6~M6~6AAc66M 0 L F p T R A A Y H K N R C Q C T S P H 0 K K C H N F ¢ Q A 6 T E 501 ~TCA~Cc~AAGAGACTATGGAGAMG~CA~GM~M~TTM~/U~AGGCAAA~A~T~T~CMG~TT~GAM6AA6T6TATT~TT~ULA6CT~G~ L R A Q E T fl E K G R N N L K K G K 0 C $ K L 6 K K C Z L Q K L fl Q 501 AA~G~AGAAAA~AAGA~G~TG~AGGC~CAG¢MCAG~CM~TTTc~Gc~GCC~AG¢T~GC~C~CT~C~C~G~A~AAAGT G R K I R R L E A 5 N S I K T S F H A A Q L R A Q L H R E Q K V 701 GA~ACAA¢~A~A~T~M~M~TCCTGG~C~T~GGA~cCGT~T~A~TCT~¢T~ACT~T~T~TC~T~TTT~C~¢~T~T THNRTH t 801 GCCTGGGACCA~A~CTG~AGCT~TCTCCCTGCCAGACT~ATC~¢TTCC~C6G~GGA~CCA~C~TCCTTA~6~C~C¢CA~6ACCA 901 A~6A~GCTCGCACCT~CTTCCTT6GCTTCCTA¢GGCA~GAACTGCTCGGGTCTCCTCT~TGGCTCGG~T~GT~T~A¢CTCTCA6AA~GCA~A~A 1001 CACACAGTG~CTTTGGAGCCTCCAGCAG~AtCAGGGTGG~TT~T~AAG~CCT~GG~TT~CCT~CTC~C~¢CGG~AC~CA 1102 GG~A~GCACTGCAGAGTCCA¢GC~C~TTTGCT~GG~TGCACAAATT~AACACACTCAMGGAC~CA~C~G~AJW~AGAM~CTCTGT 1201 ~T~TT~TTTTTG~TC~AAA~GCAAA~ACT~TACTACT~AAATC~G~CT~rFC~GAAT~TCTACCTCACCTC~GCAcTC~ 1301 GA~TGCCTCCCCAAACTCTCTCCCTCCTGAGCCT~CA~GCATACTT~GC~GTTCCTT~GAA~TGAT6A~A~CTCTC~C~GTGG 1401 GGA¢CAGT~G~G~CGAGGAGCAAACACACC~GTCAACTTATCCAGC~CA~GCTTTGGA~CTGCTACT¢GC~CAGAT~C~C~GGGACCACGAG 1501 CC~GGCGCCTCGCCGGAGGAGTCACCTCGGACCCTGGTCTCTCCCAGGGCAGGTCCCGTCTGTCC~[GC6TC~C~6G6TCCT6CT6AC¢CACAGGCC 1601 TTTG~TCCCTCAGCCCTTGGC~CCAGCTTGCGGCCGC
Fig. 1. Nucleotide and deduced amino acid sequence of rabbit ET-I.
Correspondence: P.A. Marsden, Rm 7360, Medical Sciences Building, University of Toronto, i King's College Circle, Toronto, Ontario. Canada, M5S IA8.
Endothelin-I (ET-I), originally purified from the conditioned medium of cultured porcine aortic endothelial cells, is a 21 amio acid r,eptide with potent
~0 ~elT HImAN PORCINE RAT BOVINE CANINE RABBIT H~ PORCINE RAT BOVINE CANINE
~KOKKC~FCQAGKE~RDQOTI4~KKGTKSKLGEKCIHRQLVNGRKIRR,. . . . . . --'LF,AISNSIKTSFHIAKLKAELYROKKVTHHRTHHNFC~E~RAQS'IIqQKG~FI(KGK~LGI~K¢IYQQLVEG~LRR.. . . . . . . . . LEAISNSIKTSFRV~L~ELYROQKLIHHRAHHNFC~GKE~RAQOSH~KRGKOCSKLGEKCLHQQLVAGRKTRR . . . . . . . . . LEAISNSIKTSFRVAKLKAQLYROKKVIYHRAH-
Fig. 2, Deduced amino acid sequence of preproendothelin-I derived from various species. N-terminus enclosed sequences denote mature ET-i, whereas C-terminus enclosed sequences denote" ET- l-like' peptide.
vasoconstrictor properties [1], ET-1 represents one member of a family of structurally related peptides. The three endothelins: ET-I, ET-2 and ET-3, each contain free amino- and carboxy-termini and four cys. teine residues forming two sets of intrachain disulfide bonds [2], it appears that vascular endothelial cells do not produce ET-2 or ET-3. This report describes the nuclcotid¢ and deduced amino acid sequences of rabbit preproendothelin-l, A eDNA clone was isolated from a random-primed Agtll eDNA library derived from lipopolysaccharide-treated (5 h) rabbit vena caval endothelial cells. The eDNA denoted in Fig. 1 has an open reading frame of 606 base pairs which predicts a protein of 202 amino acids. Analysis of amino acid homology indicates that rabbit preproendothelin-I shares 66.5, 70.8, 73.9 and 74.8% identity with human [3], rat [4], porcine [1] and bovine [5] sequences, respectively, Shown in Fig. 2, mature rabbit ET-1 sequence is identical to human [3], porcine [!], rat [4], bovine [5] and canine ET, I [6] (enclosed sequence), Also shown in Fig, 2, a dibasic cleavage site at Lys-90-Arg-91 suggests rabbit 'big' ET-I contains 39 amino acids, Moreover, as noted with other mammalian sequences, amino acid residues 110-131 of rabbit preproendothe-
lin-I encode an 'ET-like' peptide (enclosed sequence). This seque,ce contains four Cys residues at relative positions 1, 3, 11 and 15. These residues are identical to the Cys residues in mature ET-I. Given that the rabbit is an important species model for the study of cardiovascular disease processes, the isolation and characterization of a full-length rabbit preproendothelin-I eDNA will serve as a useful reagent in the study of ET-I pathophysiology. Supported by N.I.H. grants DK 35930 (B.M.B.), HL 36028 (M.C., M.A.G., T.C.) and HL 35716 (T.C.). P.A.M. is a Medical Research Council of Canada Fellowship Recipient. T.C. is a fellow of the Pew Scholars Program and an Established Investigator of the American Heart Association. References I 2 3 4
Yanagisawa, M., et al. (1988) Nature 332, 41 !-415. lnoue, A,, et al. (1989) Proc. Natl. Acad. Sci. USA 86, 2863-2867. ltoh, Y., et al. (1988) FEBS Left. 231,440-444. Sakurai, T., et al. (1991) Biochem. Biophys. Res. Commun. 175, 44-47, 5 EIshourbagy, N,A., et al. (1990) Nucleic Acids Res. 18, 4273. 6 Kimura, C., et al. (1989) Nucleic Acids Res. 17, 3290.
249
Short Sequence-Paper
BBAEXP 90303
Nucleotide sequence of endothelin-1 c D N A from rabbit endothelial cells Philip A. Marsden i, Parvez Sultan 2, Myron Cybulsky 2, Michael A. Gimbrone, Jr. ~, Barry M. Brenner 3 and Tucker Collins 2 t Renal Dicision, Dt,partment of Medicine, St. Michad's Hospital and Unicersity of Toronto, Toronto (Canada), " Vascular Research Dicision, Departmou of Pathology, Brigham and Womo~ ~ Hospital, Harcard Medical School, Boston, MA (U.S.A.) and "~Renal Dicision, Department of Medicine, Brighton and Women's Hospital and Harcard Medical School and Harcard Center for the Study of Kidney Diseases, Boston, MA (U.S.A.) (Received 4 November 1991 )
Key words: Atherosclerosis: Endothelium: Endothelin: Hypertension; Vasoconstrictor: (Rabbit)
A eDNA encoding rabbit endothelin-I (ET-I) was isolated by plaque hybridization from a rabbit inferior vena caval endothelial cell Agtll cDNA library using human ET-I cDNA as the hybridization probe. DNA sequence analysis indicates that mature 21 amino acid rabbit ET-I is derived from a 202 amino acid precursor, via a 39 amino acid intermediate 'big' ET-I.
1 ¢¢GGGACCGGAGCC¢GGGCCGAGGATCCGAG¢¢A¢GAGAAGCGCTGAGGCTGTTTTT¢~¢GTCTGCTTTTGGGTTCAGT~GCCGGAG~TTTTTTTTA
101
GACCTTTTTCAGMTGGATiACTTCTC~G~GGTCTCiCTGCT~TGGTGGCTTTCCATGGAGcTcCc~AGACAG¢GG~CTCGGG¢ACGuG¢TCAGC fl O y F S fl fl V S L L L V A F H G A P E T A A S G T E L S
201 ACAGGAGCGGAAAACCCAGG6GAGAAGCCCCC6GCCA6C6CACCTTGGCGGCCGCGCC6GTCCMGCGGTGCTCCTGCTCCTC6CT6ATG~CMGGAGT T G A E N P G E K P P A S A P W R P R R $ K R $ C S S L fl 0 K E 301 GTGTCTACTT£TGCCA¢CTGGA¢&~CAT¢TGG~AA~ACCCcCGGG¢ACATCGTcc¢GTATGGACTTGGMG~cC~cCAGGTCCMG~TCCTTGM V y F C tl L 0 1 1 W V N T P G H I V P Y G L G $ P S R $ K R S L K 401 ~ATTTMTT~¢TAc¢AGGGCAG¢~TACCAcMGMcAGGTGcCA~GC~GAGCc~A£AcGACM6MGT6CT6GMT~6~M6~6AAc66M 0 L F p T R A A Y H K N R C Q C T S P H 0 K K C H N F ¢ Q A 6 T E 501 ~TCA~Cc~AAGAGACTATGGAGAMG~CA~GM~M~TTM~/U~AGGCAAA~A~T~T~CMG~TT~GAM6AA6T6TATT~TT~ULA6CT~G~ L R A Q E T fl E K G R N N L K K G K 0 C $ K L 6 K K C Z L Q K L fl Q 501 AA~G~AGAAAA~AAGA~G~TG~AGGC~CAG¢MCAG~CM~TTTc~Gc~GCC~AG¢T~GC~C~CT~C~C~G~A~AAAGT G R K I R R L E A 5 N S I K T S F H A A Q L R A Q L H R E Q K V 701 GA~ACAA¢~A~A~T~M~M~TCCTGG~C~T~GGA~cCGT~T~A~TCT~¢T~ACT~T~T~TC~T~TTT~C~¢~T~T THNRTH t 801 GCCTGGGACCA~A~CTG~AGCT~TCTCCCTGCCAGACT~ATC~¢TTCC~C6G~GGA~CCA~C~TCCTTA~6~C~C¢CA~6ACCA 901 A~6A~GCTCGCACCT~CTTCCTT6GCTTCCTA¢GGCA~GAACTGCTCGGGTCTCCTCT~TGGCTCGG~T~GT~T~A¢CTCTCA6AA~GCA~A~A 1001 CACACAGTG~CTTTGGAGCCTCCAGCAG~AtCAGGGTGG~TT~T~AAG~CCT~GG~TT~CCT~CTC~C~¢CGG~AC~CA 1102 GG~A~GCACTGCAGAGTCCA¢GC~C~TTTGCT~GG~TGCACAAATT~AACACACTCAMGGAC~CA~C~G~AJW~AGAM~CTCTGT 1201 ~T~TT~TTTTTG~TC~AAA~GCAAA~ACT~TACTACT~AAATC~G~CT~rFC~GAAT~TCTACCTCACCTC~GCAcTC~ 1301 GA~TGCCTCCCCAAACTCTCTCCCTCCTGAGCCT~CA~GCATACTT~GC~GTTCCTT~GAA~TGAT6A~A~CTCTC~C~GTGG 1401 GGA¢CAGT~G~G~CGAGGAGCAAACACACC~GTCAACTTATCCAGC~CA~GCTTTGGA~CTGCTACT¢GC~CAGAT~C~C~GGGACCACGAG 1501 CC~GGCGCCTCGCCGGAGGAGTCACCTCGGACCCTGGTCTCTCCCAGGGCAGGTCCCGTCTGTCC~[GC6TC~C~6G6TCCT6CT6AC¢CACAGGCC 1601 TTTG~TCCCTCAGCCCTTGGC~CCAGCTTGCGGCCGC
Fig. 1. Nucleotide and deduced amino acid sequence of rabbit ET-I.
Correspondence: P.A. Marsden, Rm 7360, Medical Sciences Building, University of Toronto, i King's College Circle, Toronto, Ontario. Canada, M5S IA8.
Endothelin-I (ET-I), originally purified from the conditioned medium of cultured porcine aortic endothelial cells, is a 21 amio acid r,eptide with potent
~0 ~elT HImAN PORCINE RAT BOVINE CANINE RABBIT H~ PORCINE RAT BOVINE CANINE
~KOKKC~FCQAGKE~RDQOTI4~KKGTKSKLGEKCIHRQLVNGRKIRR,. . . . . . --'LF,AISNSIKTSFHIAKLKAELYROKKVTHHRTHHNFC~E~RAQS'IIqQKG~FI(KGK~LGI~K¢IYQQLVEG~LRR.. . . . . . . . . LEAISNSIKTSFRV~L~ELYROQKLIHHRAHHNFC~GKE~RAQOSH~KRGKOCSKLGEKCLHQQLVAGRKTRR . . . . . . . . . LEAISNSIKTSFRVAKLKAQLYROKKVIYHRAH-
Fig. 2, Deduced amino acid sequence of preproendothelin-I derived from various species. N-terminus enclosed sequences denote mature ET-i, whereas C-terminus enclosed sequences denote" ET- l-like' peptide.
vasoconstrictor properties [1], ET-1 represents one member of a family of structurally related peptides. The three endothelins: ET-I, ET-2 and ET-3, each contain free amino- and carboxy-termini and four cys. teine residues forming two sets of intrachain disulfide bonds [2], it appears that vascular endothelial cells do not produce ET-2 or ET-3. This report describes the nuclcotid¢ and deduced amino acid sequences of rabbit preproendothelin-l, A eDNA clone was isolated from a random-primed Agtll eDNA library derived from lipopolysaccharide-treated (5 h) rabbit vena caval endothelial cells. The eDNA denoted in Fig. 1 has an open reading frame of 606 base pairs which predicts a protein of 202 amino acids. Analysis of amino acid homology indicates that rabbit preproendothelin-I shares 66.5, 70.8, 73.9 and 74.8% identity with human [3], rat [4], porcine [1] and bovine [5] sequences, respectively, Shown in Fig. 2, mature rabbit ET-1 sequence is identical to human [3], porcine [!], rat [4], bovine [5] and canine ET, I [6] (enclosed sequence), Also shown in Fig, 2, a dibasic cleavage site at Lys-90-Arg-91 suggests rabbit 'big' ET-I contains 39 amino acids, Moreover, as noted with other mammalian sequences, amino acid residues 110-131 of rabbit preproendothe-
lin-I encode an 'ET-like' peptide (enclosed sequence). This seque,ce contains four Cys residues at relative positions 1, 3, 11 and 15. These residues are identical to the Cys residues in mature ET-I. Given that the rabbit is an important species model for the study of cardiovascular disease processes, the isolation and characterization of a full-length rabbit preproendothelin-I eDNA will serve as a useful reagent in the study of ET-I pathophysiology. Supported by N.I.H. grants DK 35930 (B.M.B.), HL 36028 (M.C., M.A.G., T.C.) and HL 35716 (T.C.). P.A.M. is a Medical Research Council of Canada Fellowship Recipient. T.C. is a fellow of the Pew Scholars Program and an Established Investigator of the American Heart Association. References I 2 3 4
Yanagisawa, M., et al. (1988) Nature 332, 41 !-415. lnoue, A,, et al. (1989) Proc. Natl. Acad. Sci. USA 86, 2863-2867. ltoh, Y., et al. (1988) FEBS Left. 231,440-444. Sakurai, T., et al. (1991) Biochem. Biophys. Res. Commun. 175, 44-47, 5 EIshourbagy, N,A., et al. (1990) Nucleic Acids Res. 18, 4273. 6 Kimura, C., et al. (1989) Nucleic Acids Res. 17, 3290.
