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Cloning of a cDNA encoding the ovine interleukin-2 receptor 55-kDa protein, CD25
Gene, 113 (1992) 283-284 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00
283
GENE 06414
Cloning of a cDNA encoding the ovine interleukin-2 receptor 55-kDa protein, CD25" (Recombinant DNA; nucleotide sequence; homology to bovine, human and murine genes)
Raymond Bujdoso, David Sargan, Mairi Williamson and lan McConnell Department of Veterinary Pathology, Summerhail, University of Edinburgh, Edinburgh, EH9 I OH (U.K.) Received by K.F. Chater: 27 June 1991 Revised/Accepted: 19 December/20 December 1991 Received at publishers: 4 February 1992
SUMMARY
A 1.3-kb cDNA that encodes the entire 825-bp coding region of ovine CD25, the interleukin-2 receptor 55-kDa protein, has been isolated. Comparison of the deduced amino acid sequence with CD25 proteins from other species shows the ovine sequence to have the greatest homology with that of the bovine species.
Interleukin-2 (IL-2) receptors are transmembrane proteins expressed by activated T cells during an immune response (Waldman, 1989). The fun~:tions of these proteins include the binding of IL-2, a lymphokine secreted by T cells that functions in the growth and differentiation of lymphocytes. In the human system receptor proteins capable of binding IL-2 exist in a low-, intermediate- or high-affinity form on the surface of T cells. The low affinity receptor is a 55-kDa protein, designated CD25, and has a binding constant (Kd) of 10- a M for IL-2, whilst the intermediate affinity receptor is a 75-kDa protein with a Kd of 10- 9 M. The high affinity form is a non-covalent complex of at least the 55-kDa and the 75-kDa protein chains and has a binding constant of 10-~s M for IL-2 (Wang and Smith, 1987). The binding of IL-2 to the high affinity form of the receptor results in signal transduction mediated by the 75-kDa protein, internalisation of the ligand-receptor
Correspondenceto: Dr. R. Bujdoso, Dept. of Veterinary Pathology, University of Edinburgh, Summerhail, Edinburgh, EH19 1QH (U.K.) Tel. (44-31)650-6169; Fax (44-31)650-6511. * On request, the author(s) will supply detailed experimental evidence for the conclusions reached in this Brief Note. Abbreviations: aa, amino acid(s); bp, base pair(s); Con-A, concanavalin A; lI., interleukin; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction.
complex, and the progression of antigen-specific activated T cells through the S phase of the cell cycle and on to cell division. The cDNA prepared from total RNA derived from 16-h Con-A-stimulated ovine lymphocytes was used as a substrate for the PCR using primers of common sequences found at the 5' and 3' ends of cDNAs encoding bovine, human and murine IL-2 receptor 55-kDa protein (Fig. 1). A predicted 1.3-kDa reaction product was generated, cloned into the vectors pTZI8R and pTZI9R then sequenced in both directions by the di-deoxy method. A total of three clones from two independent polymerase chain reactions were sequenced. The nt and predicted aa sequence is shown in Fig. 1. The 1.3-kb fragment has an ORF, with potential initiation sites at nt 77 or 107, which terminates at nt 931. Bestfit alignments of database nt sequences with the ORF from nt 107-931 show this sequence has 93?/0, 72% and 67?/o similarity with nt sequences encoding the precursor protein of bovine (Weinberg et al., 1988), human (Nikaido et al., 1984) and murine (Miller et al., 1985) CD25, respectively. The ORF between nt 77-931 would encode a protein with an extra 10 aa which are not seen in the CD25 molecules from any of the other species. In the proposed 5' non-coding region of the ovine sequence there is an area of high homology with bovine and human IL-2 receptor cDNA where 24 out of 26 nt are identical between all three species. As discussed by Weinberg et al. (1988) this region comprises
284 1 TC~cGGTG~GATC~cAAcAAGAGG~TG~GG~GCG~T~TGcAGAG~GAcA~T~T~T~TTc~GG~c~GATGT~GGcAGT~AT~AGGGG~A -21 , -i0 +I M E P S L L M W R F F V F I V V P G C V T E A C H D D P P S L R 101GGAGGATGGAGCCAAGcTTGCTGATGTGGAGGTTCTTcGTATTcATcGTGGTACcTGGcTGcGTGACAGAGGCTTGTcATGATGACCcTCcGAGTcTCAG 20 40 N A M F K V L R Y E V G T M I N C D C K A G F R R V S A V M R C V 201AAACGCcATGTTCAAGGTCcTcAGGTACGAGGTGGGCACcATGATAAAcTGCGAcTGcAAGGCAGGcTTccGcAGGGTGTCGGCCGTCATGcGcTGCGTG 60 G D S S H S A W N N R C F C N S T S P A K N P V K P V T P G S E E Q 301 GGGGAcTCCAGCcAcTcTGCcTGGAACAACAGATGCTTCTGcAAcAGcACCTCCcCTGCTAAGAACCcAGTAAAACcAGTTAcTCCTGGATCcGAAGAAC 80 100 R E R K P T D A Q S Q T Q P P E Q A D L P G H C E E P P P W E H E 401 AGAGGGAGAGAAAAcccAcAGATGCACAGAGCcAAAcGcAGcCTccGGAGCAAGCTGACCTTcCAGGTCAcTGCGAGGAAcCACCGcCATGGGAACATGA 120 140 R E P L K R V Y H F T L G Q T V H Y Q C A Q G F R A L H T G P A E 501 ACGTGAAcCTTTGAAGAGAGTcTATCATTTcAcGCTGGGGcAGACGGTTcACTAccAGTGTGcccAGGGATTcAGGGCCCTACACACCGGTCCTGCTGAA 160 T T C T M I H G E M R W T R P R L K C I S E G A N S Q A P D E A E P 601 A•GA••TGCA•GATGATC•A•GGGGAGATGAGGTGGACCAGGcccAGGcT•AAGTGCATAAGTGAAGGGGcGAACAGTCAGGCTCCAGATGAAGCAGAGC 180 200 P E S T E A P P G S G T F L T T R M A G T T D F Q K P T D V V A T 701 cTccGGAGAGcAcAGAAGCTcCcccTGGGAGTGGAAcTTTcTTAAcAACcAGGATGGcAGGGAccACAGATTTCcAGAAGccCAcAGACGTGGTTGCAAC 220 240 L D T F I F T T E Y Q I A V A G C I L L L S S I L L L S C L T W Q 801 G~TGGATACGTTcATATTTAcCAccGAGTAccAGATcGcAGTGGccGGcTGCATccTcCYGcTcTCcAGCATccTccTCCTGAGCTGccTCACGTGGCAG 250 R R W K K N R R T I * 901CGGAGATGGAAGAAGAACAGAAGGAcAATcTAGAAAACcAAGGGCCAGAAGAAGTCAAGAAcAGCccACAGGTGTCACGGACcACAATCAGAATCAAAGA i001AGCTAAACACTCATccAAGAGGcATcTcCTGATccGGTGGGcTcTGGAAAG•TCTGAAGTcACGTAACAGAACACTGGGCAACTGCAGcC•CATcGTGAA II01 GCcAGcTcTGTAGTATcAAcccTTGAGGcTGAcccGTcTAGGcAGcAAGTcCAAGGTCGCTGGAGGAACGGGGAGAGGCAAcCAGAACTCTTTCTC•TGT 1201TTTcATGTATATGTGATCAcTAGATcAcAAGTAGccTGGAGcTcTcTCCAcAcCATGTAGTGTAAAGAAGAGTAGTTTCATGCTAAGAGCATCccGAcTT 1301 ~CCAGGTTAGAAATCCCA
Fig. 1. The nt sequence of ovine CD25 and deduced aa sequence. The nt are numbe~d ~om the first nt of the sequence and an asterisk marks the stop codon. The Glu residue of the predicted mature polypeptidc sequence is numbered I and aa on the N-terminal side ofthis are indicated by negative numbers. The nt sequences of primers used in the PCR are underlined. The EMBL accession No. of this sequence is X60149.
a mini-cistron that potentially codes for a peptide of five aa. We believe the true start codon is at nt 107 and, as proposed by Kozak (1986) that the upstream ATG codon may play a role in regulation of mRNA translation. The predicted aa sequence of the ovine ORF from nt 107-931 gives a protein of 275 aa which appears to be the precursor protein of ovine CD25, as this polypeptide has 94%, 72~o and 637~, similarity with the predicted aa sequences of bovine, human and murine CD25, respectively. The N-terminal 244 aa make up an extracellular domain with a signal sequence of 21 aa. A potential cleavage site for the signal sequence (Von Heijne, 1983) is between Thr- t and Glu + t as is the case in the bovine, human and murine species. The ovine protein sequence maintains 12 out of a possible 13 Cys residues found in all four species and, like bovine CD25, has a potential N-linked glycosylation site at aa residues 59-61. A presumptive transmembrane region extends from aa 221-241 in the ovine sequence and is similar to that found in all four species. The ovine CD25 has a cytoplasmic tail of 13 aa (residues 242-254) of which six are basic. The cytoplasmic tail of the CD25 molecule is relatively small and unlikely to participate in enzymatic
function although it may itself be modified as potential phosphorylation sites (Ser, Thr, but not Tyr) exist in this region. In particular, Thr + 253 is conserved in all four species. REFERENCES Kozak, M.: Bifunctional RNAs in eukaryotes. Cell 47 (1986) 481-483. Miller, J., Malek, T.R., Leonard, W.J., Greene, W.C., Shevach, E.M. and Germain, R.N.: Nucleotide sequence and expression of a mouse interleukin 2 receptor eDNA. J. lmmunol. 134 (1985) 4212-4217. Nikaido, T., Shimizu, A., Ishida, N., Sabe, H., Teshigawara, K., Maeda, M., Uchiyama, T., Yodoi, J. and Honjo, T.: Molecular cloning of eDNA encoding human interleukin-2 receptor. Nature 311 (1984) 631-635. yon Heijne, G.: Patterns of amino acids near signal sequence cleavage sites. Eur. J. Biochem. 133 (1983) 17-21. Waldman, T.A.: The multi-subunit interleukin-2receptor. Ann. Rev. Biochem. 58 (1989) 875-911. Wang, H.-M. and Smith, K.A.: The interleukin 2 receptor. Functional consequences of its bimolecular structure. J. Exp. Med. 166 (1987) 1055-1069. Weinberg, A.D., Shaw, J., Paetkau, V., Bleackley,R.C., Magnuson, N.S., Reeves, R. and Magnuson, J.A.: Cloning ofcDNA for the bovine IL-2 receptor (bovine Tac antigen). Immunology 63 (1988) 603-610.
283
GENE 06414
Cloning of a cDNA encoding the ovine interleukin-2 receptor 55-kDa protein, CD25" (Recombinant DNA; nucleotide sequence; homology to bovine, human and murine genes)
Raymond Bujdoso, David Sargan, Mairi Williamson and lan McConnell Department of Veterinary Pathology, Summerhail, University of Edinburgh, Edinburgh, EH9 I OH (U.K.) Received by K.F. Chater: 27 June 1991 Revised/Accepted: 19 December/20 December 1991 Received at publishers: 4 February 1992
SUMMARY
A 1.3-kb cDNA that encodes the entire 825-bp coding region of ovine CD25, the interleukin-2 receptor 55-kDa protein, has been isolated. Comparison of the deduced amino acid sequence with CD25 proteins from other species shows the ovine sequence to have the greatest homology with that of the bovine species.
Interleukin-2 (IL-2) receptors are transmembrane proteins expressed by activated T cells during an immune response (Waldman, 1989). The fun~:tions of these proteins include the binding of IL-2, a lymphokine secreted by T cells that functions in the growth and differentiation of lymphocytes. In the human system receptor proteins capable of binding IL-2 exist in a low-, intermediate- or high-affinity form on the surface of T cells. The low affinity receptor is a 55-kDa protein, designated CD25, and has a binding constant (Kd) of 10- a M for IL-2, whilst the intermediate affinity receptor is a 75-kDa protein with a Kd of 10- 9 M. The high affinity form is a non-covalent complex of at least the 55-kDa and the 75-kDa protein chains and has a binding constant of 10-~s M for IL-2 (Wang and Smith, 1987). The binding of IL-2 to the high affinity form of the receptor results in signal transduction mediated by the 75-kDa protein, internalisation of the ligand-receptor
Correspondenceto: Dr. R. Bujdoso, Dept. of Veterinary Pathology, University of Edinburgh, Summerhail, Edinburgh, EH19 1QH (U.K.) Tel. (44-31)650-6169; Fax (44-31)650-6511. * On request, the author(s) will supply detailed experimental evidence for the conclusions reached in this Brief Note. Abbreviations: aa, amino acid(s); bp, base pair(s); Con-A, concanavalin A; lI., interleukin; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction.
complex, and the progression of antigen-specific activated T cells through the S phase of the cell cycle and on to cell division. The cDNA prepared from total RNA derived from 16-h Con-A-stimulated ovine lymphocytes was used as a substrate for the PCR using primers of common sequences found at the 5' and 3' ends of cDNAs encoding bovine, human and murine IL-2 receptor 55-kDa protein (Fig. 1). A predicted 1.3-kDa reaction product was generated, cloned into the vectors pTZI8R and pTZI9R then sequenced in both directions by the di-deoxy method. A total of three clones from two independent polymerase chain reactions were sequenced. The nt and predicted aa sequence is shown in Fig. 1. The 1.3-kb fragment has an ORF, with potential initiation sites at nt 77 or 107, which terminates at nt 931. Bestfit alignments of database nt sequences with the ORF from nt 107-931 show this sequence has 93?/0, 72% and 67?/o similarity with nt sequences encoding the precursor protein of bovine (Weinberg et al., 1988), human (Nikaido et al., 1984) and murine (Miller et al., 1985) CD25, respectively. The ORF between nt 77-931 would encode a protein with an extra 10 aa which are not seen in the CD25 molecules from any of the other species. In the proposed 5' non-coding region of the ovine sequence there is an area of high homology with bovine and human IL-2 receptor cDNA where 24 out of 26 nt are identical between all three species. As discussed by Weinberg et al. (1988) this region comprises
284 1 TC~cGGTG~GATC~cAAcAAGAGG~TG~GG~GCG~T~TGcAGAG~GAcA~T~T~T~TTc~GG~c~GATGT~GGcAGT~AT~AGGGG~A -21 , -i0 +I M E P S L L M W R F F V F I V V P G C V T E A C H D D P P S L R 101GGAGGATGGAGCCAAGcTTGCTGATGTGGAGGTTCTTcGTATTcATcGTGGTACcTGGcTGcGTGACAGAGGCTTGTcATGATGACCcTCcGAGTcTCAG 20 40 N A M F K V L R Y E V G T M I N C D C K A G F R R V S A V M R C V 201AAACGCcATGTTCAAGGTCcTcAGGTACGAGGTGGGCACcATGATAAAcTGCGAcTGcAAGGCAGGcTTccGcAGGGTGTCGGCCGTCATGcGcTGCGTG 60 G D S S H S A W N N R C F C N S T S P A K N P V K P V T P G S E E Q 301 GGGGAcTCCAGCcAcTcTGCcTGGAACAACAGATGCTTCTGcAAcAGcACCTCCcCTGCTAAGAACCcAGTAAAACcAGTTAcTCCTGGATCcGAAGAAC 80 100 R E R K P T D A Q S Q T Q P P E Q A D L P G H C E E P P P W E H E 401 AGAGGGAGAGAAAAcccAcAGATGCACAGAGCcAAAcGcAGcCTccGGAGCAAGCTGACCTTcCAGGTCAcTGCGAGGAAcCACCGcCATGGGAACATGA 120 140 R E P L K R V Y H F T L G Q T V H Y Q C A Q G F R A L H T G P A E 501 ACGTGAAcCTTTGAAGAGAGTcTATCATTTcAcGCTGGGGcAGACGGTTcACTAccAGTGTGcccAGGGATTcAGGGCCCTACACACCGGTCCTGCTGAA 160 T T C T M I H G E M R W T R P R L K C I S E G A N S Q A P D E A E P 601 A•GA••TGCA•GATGATC•A•GGGGAGATGAGGTGGACCAGGcccAGGcT•AAGTGCATAAGTGAAGGGGcGAACAGTCAGGCTCCAGATGAAGCAGAGC 180 200 P E S T E A P P G S G T F L T T R M A G T T D F Q K P T D V V A T 701 cTccGGAGAGcAcAGAAGCTcCcccTGGGAGTGGAAcTTTcTTAAcAACcAGGATGGcAGGGAccACAGATTTCcAGAAGccCAcAGACGTGGTTGCAAC 220 240 L D T F I F T T E Y Q I A V A G C I L L L S S I L L L S C L T W Q 801 G~TGGATACGTTcATATTTAcCAccGAGTAccAGATcGcAGTGGccGGcTGCATccTcCYGcTcTCcAGCATccTccTCCTGAGCTGccTCACGTGGCAG 250 R R W K K N R R T I * 901CGGAGATGGAAGAAGAACAGAAGGAcAATcTAGAAAACcAAGGGCCAGAAGAAGTCAAGAAcAGCccACAGGTGTCACGGACcACAATCAGAATCAAAGA i001AGCTAAACACTCATccAAGAGGcATcTcCTGATccGGTGGGcTcTGGAAAG•TCTGAAGTcACGTAACAGAACACTGGGCAACTGCAGcC•CATcGTGAA II01 GCcAGcTcTGTAGTATcAAcccTTGAGGcTGAcccGTcTAGGcAGcAAGTcCAAGGTCGCTGGAGGAACGGGGAGAGGCAAcCAGAACTCTTTCTC•TGT 1201TTTcATGTATATGTGATCAcTAGATcAcAAGTAGccTGGAGcTcTcTCCAcAcCATGTAGTGTAAAGAAGAGTAGTTTCATGCTAAGAGCATCccGAcTT 1301 ~CCAGGTTAGAAATCCCA
Fig. 1. The nt sequence of ovine CD25 and deduced aa sequence. The nt are numbe~d ~om the first nt of the sequence and an asterisk marks the stop codon. The Glu residue of the predicted mature polypeptidc sequence is numbered I and aa on the N-terminal side ofthis are indicated by negative numbers. The nt sequences of primers used in the PCR are underlined. The EMBL accession No. of this sequence is X60149.
a mini-cistron that potentially codes for a peptide of five aa. We believe the true start codon is at nt 107 and, as proposed by Kozak (1986) that the upstream ATG codon may play a role in regulation of mRNA translation. The predicted aa sequence of the ovine ORF from nt 107-931 gives a protein of 275 aa which appears to be the precursor protein of ovine CD25, as this polypeptide has 94%, 72~o and 637~, similarity with the predicted aa sequences of bovine, human and murine CD25, respectively. The N-terminal 244 aa make up an extracellular domain with a signal sequence of 21 aa. A potential cleavage site for the signal sequence (Von Heijne, 1983) is between Thr- t and Glu + t as is the case in the bovine, human and murine species. The ovine protein sequence maintains 12 out of a possible 13 Cys residues found in all four species and, like bovine CD25, has a potential N-linked glycosylation site at aa residues 59-61. A presumptive transmembrane region extends from aa 221-241 in the ovine sequence and is similar to that found in all four species. The ovine CD25 has a cytoplasmic tail of 13 aa (residues 242-254) of which six are basic. The cytoplasmic tail of the CD25 molecule is relatively small and unlikely to participate in enzymatic
function although it may itself be modified as potential phosphorylation sites (Ser, Thr, but not Tyr) exist in this region. In particular, Thr + 253 is conserved in all four species. REFERENCES Kozak, M.: Bifunctional RNAs in eukaryotes. Cell 47 (1986) 481-483. Miller, J., Malek, T.R., Leonard, W.J., Greene, W.C., Shevach, E.M. and Germain, R.N.: Nucleotide sequence and expression of a mouse interleukin 2 receptor eDNA. J. lmmunol. 134 (1985) 4212-4217. Nikaido, T., Shimizu, A., Ishida, N., Sabe, H., Teshigawara, K., Maeda, M., Uchiyama, T., Yodoi, J. and Honjo, T.: Molecular cloning of eDNA encoding human interleukin-2 receptor. Nature 311 (1984) 631-635. yon Heijne, G.: Patterns of amino acids near signal sequence cleavage sites. Eur. J. Biochem. 133 (1983) 17-21. Waldman, T.A.: The multi-subunit interleukin-2receptor. Ann. Rev. Biochem. 58 (1989) 875-911. Wang, H.-M. and Smith, K.A.: The interleukin 2 receptor. Functional consequences of its bimolecular structure. J. Exp. Med. 166 (1987) 1055-1069. Weinberg, A.D., Shaw, J., Paetkau, V., Bleackley,R.C., Magnuson, N.S., Reeves, R. and Magnuson, J.A.: Cloning ofcDNA for the bovine IL-2 receptor (bovine Tac antigen). Immunology 63 (1988) 603-610.