328 Biochimica et Biophysica Acta, 1171 (1993) 328-330 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00

BBAEXP 90441 Short Sequence-Paper

Nucleotide and deduced amino acid sequence of porcine interleukin 4 cDNA derived from lamina propria lymphocytes

Michael Bailey a, Anthony C.F. Perry b, Paul W. Bland a, Christopher R. Stokes " and Len Hall b

Departments of a Veterinary Medicine and b Biochemistry, Unit,ersity of Bristol, Bristol (UK)

(Received 18 September 1992)

Key words: Interleukin 4; Cytokine; Lymphocyte; Intestine; (Pig)

Total RNA was isolated from in vitro activated lamina propria lymphocytes and used to direct the synthesis of cDNA. Interleukin 4 transcripts were then specifically amplified by PCR. Comparison of the nucleotide sequence with its human homologue demonstrates deletion within the coding region of pig interleukin 4 centred around amino acid residue 70 in the mature human protein.

Interleukin 4 (IL-4) is a product of activated T lymphocytes originally identified as a costimulator of B lymphocyte proliferation [1]. It has subsequently been shown to induce expression of MHC class II antigens on small, resting B cells [2], to enhance their expression of the low affinity Fc epsilon receptor and to promote isotype switching to IgG1 and IgE [3-6]. In addition, IL-4 can maintain proliferation of at least some T cells [7,8] and has variable effects on the expression of receptors for the major T cell growth factor, inter- leukin 2 (IL-2) [9,10]. The ability to secrete interleukins has been used to group cloned mouse T cells into subsets, Th l cells producing predominantly IL-2 and y-interferon and Th2 cells producing interleukins 4, 5 and 6 [11]. While this classification may be an oversim- plification, there is evidence from studies in mice that the outcome of some infectious diseases does depend upon the spectrum of cytokines produced by activated T cells in vivo [12,13]. We have recently reported the sequence of porcine IL-2 [14] and in order to facilitate studies of immune responses in pigs, we have now cloned and sequenced cDNA coding for porcine IL-4 by polymerase chain reaction (PCR).

Activation of splenic lymphocytes results in only limited production of IL-4 m R N A [15]. However, sev- eral groups have observed a bias towards the produc-

Correspondence to: M. Bailey, Department of Veterinary Medicine, Langford House, Langford, Bristol, BS18 7DU, UK. The sequence data in this paper have been submitted to the EMBL Data Bank under the accession number X68330.

tion of IL-4 protein or m R N A by intestinally-derived lymphocytes activated in vitro [16,17]. cDNA was therefore prepared from porcine lamina propria lym- phocytes activated in vitro with concanavalin A.

Two areas towards the ends of the IL-4 coding region, largely conserved between the human and bovine sequences [8,18], were used to design oligo- nucleotide primers (primers B and E; see Fig. 1) which successfully allowed the amplification of a product from this cDNA. To obtain the 5' sequence encoding the N-terminus, one primer was synthesised from within the initial PCR product (primer D) and a second primer within the 5' noncoding region (primer A) based on the human/bov ine consensus sequence. The design of a suitable 3' primer to amplify the 3' end of IL-4 cDNA, encoding the C-terminus of the peptide, proved more difficult due to the high A + T composi- tion of the 3' noncoding region. Eventually a longer primer based on the human/bov ine sequences and containing the polyadenylat ion/cleavage signal and part of the poly(A) tail (primer F), together with a primer (primer C) from within the initial PCR product, proved successful. Two independent PCR products were fully sequenced on both DNA strands, using a Du Pont Genesis 2000 automated sequencer, and were found to be identical.

The collated cDNA sequence obtained and the de- duced amino acid sequence is shown in Fig. 1. Compar- ison with the human protein suggests a 24 amino acid signal sequence and a mature peptide of 109 amino acid residues. Alignment of the porcine, bovine [18], human [8] and murine [7] amino acid sequences (Fig. 2)

329

PRIMER A PRIMER B 1

cctggtaaactaattgtctcacattgTCAGTGCAAATAGAGCTCTATTC ATG GGT CTC ACC TCC CAA CTG ATC CCA ACC CTG GTC TGC TTA

1oo CTG GCA TGT ACC AGC AAC TTC GTC CAC GGA CAC AAG TGC Leu Ala Cys Thr Ser Asn Phe Val His Gly His Lys Cys - i 0 - I +I

ACA GCG AGA AAG AAC TCG TGC ATG GAG CTG CCC GTG ACG Thr Ala Arg Lys Asn Ser Cys Met Glu Leu Pro Val Thr

20 30

TTC TGC CGG GCC TCG ACT GTG CTT CGG CAC ATC TAC AGA Phe Cys Arg Ala Set Thr Val Leu Arg His l l e Tyr Arg

5O PRIMER

Met Gly Leu Thr Ser Gln Leu l l e Pro Thr Leu Val Cys Leu -24 -20

PRIMER C

GAC ATC ACC TTA CAA GAG ATC ATC AAA ACC TTG AAC ATT CTC Asp l l e Thr Leu Gln Glu l l e l l e Lys Thr Leu Ash l l e Leu

I0 2oo

GAC GTC TTT GCT GCC CCA GAG AAC ACG ACG GAG AAG GAA ACC Asp Val Phe Ala Ala Pro Glu Asn Thr Thr Glu Lys Glu Thr

4O 30o

CAC CAC ACG TGC ATG AAG AGC CTC CTG AGC GGA CTT GAC AGG His His Thr Cys Met Lys Ser Leu Leu Ser Gly Leu Asp Arg

60 7O D PRIMER E

AAC CTG AGC AGC ATG GCA AAC ATG ACC TGT TCT GTG CAT GAA GCC AAG AAG AGC ACT TTG AAA GAC TTC TTG GAA AGG CTA Asn Leu Ser Ser Met Ala Asn Met Thr Cys Ser Val His Glu Ala Lys Lys Ser Thr Leu Lys Asp Phe Leu Glu Arg Leu

8O 9O 40o

AAG ACG ATT ATG AAG GAG AAA TAC TCA AAG TGT TGA AGCTGACTATTTTTAATTTATGATTTTTATACACTTATTTTAAAAATATTTATATATTT Lys Thr l l e Met Lys Glu Lys Tyr Ser Lys Cys

100 109 PRIMER F 490

ATAACTcataaaataaagtatatgtagaatctaaaaaa

Fig. l. Nucleotide and predicted amino acid sequence of porcine interleukin 4 cDNA established by PCR. Regions shown in lower case represent the extreme 5' and 3' PCR primers and are based on the h u m a n / b o v i n e IL-4 sequences.

demonstrates major species differences in the number of amino acids present between residues 60 to 81 of the human sequence. NMR studies of the structure of human IL-4 have indicated a bundle of four alpha helices with an up-up-down-down connectivity (shown underlined on the human sequence) [19] and this re- gion corresponds to a major part of the loop between the second and third helices plus approximately half of the third helix. However, deletions of this size could be

accommodated by several of the spatial models pro- posed without major effects on overall structure. All six cysteine residues involved in disulphide bonds in the human sequence are conserved in pig IL-4. Interest- ingly, a tyrosine at position 124 (in the human se- quence), implicated in signal transduction but not in receptor binding [20], is conserved and lies close to a span of eight amino acid residues in the fourth helix of which six are also fully conserved between species.

Porcine IL-4 Bovine IL-4 Murine IL-4 Human IL-4

MGLTSQLIPTLVCLLACTSNFVHGHKCDIT-LQEI IKTLNILTARKNSCMELPVTDVFAAPENTTEKETF

MGLTSQLIPVLVCLLVCTSHFVHGHKCDIT-LAEI IKTLNILTTRKNSCMELPVADVFAAPKNTTEKETF

MGLNPQLVVILLFFLECTRSHIHG--CDKNHLREI IGILNEVTGEGTPCTEMDVPNVLTATKNTTESELV

MGLTSQLLPPLFFLLACAGNFVHGHKCDIT-LOEI IKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETF

- 2 0 I 20 40

CRASTVLRHIYRHH . . . . TCMKS . . . . . . . . . . . . . LLSGLDRNLSSMANM-TCSVHEAKKST--LKDFLERLKTIMKEKYSKC

CRVGIELRRIYRSH . . . . TCLNK . . . . . . . . . . . . . FLGGLDRNLNSLASK-TCSVNEAKTSTSTLKDLLERLKTIMKEKYSKC CRASKVLRIFYLKHGK-TPCLKK . . . . . . . NSSVLMELQRLFRAFRCLDSSlSCTMNESK-STS-LKDFLESLKSIMQMDYS cRAATvLROFYsHHEKDTRCLGATAQOFHRHKQL~RFLKRLDRNLWGLAGLNScPVKEANQST~LENFLERLKT~MREKY~KcSS

60 80 100 120 Fig. 2. Alignment of the amino acid sequences of porcine, bovine, murine and human interleukin 4. Dashes are inserted to optimise the alignment and numbering is based on the human sequence. Asterisks indicate residues conse~ed in all ~ u r sequences, a Helices demonstrated

in human IL-4 [19] have been underlined (see text).

330

Biological activity attributable to interleukin 4 has not yet been described from the pig or the ox and there- fore the degree to which the homologues can act across species is unknown. However, the importance of these residues and of the region containing the deletions in receptor interaction may be worth examining.

This work was supported by the Agricultural and Food Research Council. The authors would like to thank Drs. V. Heussler and D. Dobbelaere of the University of Berne for supplying the sequence of bovine IL-4 prior to publication and Dr. D. Emery and H. Downer for assistance with automated DNA se- quencing.

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