Structure and expression of a cloned cDNA for human interleukin-2

Parts used

Key findings

• The human interleukin-2 (IL-2) precursor is a 153-residue polypeptide of calculated molecular weight 17,631.7, encoded by a single open reading frame whose first ATG sits at nucleotides 48-50 of the pIL2-50A cDNA and is followed by 152 codons before the TGA stop at nucleotides 507-509. An AATAAA hexanucleotide at positions 771-776 precedes the poly(A) tract.
• The N-terminal 20 residues of the human IL-2 precursor, Met-Tyr-Arg-Met-Gln-Leu-Leu-Ser-Cys-Ile-Ala-Leu-Ser-Leu-Ala-Leu-Val-Thr-Asn-Ser, are strongly hydrophobic and act as a secretory signal peptide. Signal peptidase cleaves between Ser at position 20 and Ala at position 21, leaving a mature IL-2 of 133 amino acids with a calculated molecular weight of 15,420.5, close to the 15,000 reported for IL-2 purified from Jurkat cells. Cleavage at an alternative nearby position cannot be excluded.
• Human IL-2 carries no potential N-glycosylation site: neither AsN-X-Ser nor AsN-X-Thr appears in the deduced sequence, consistent with IL-2 from Jurkat cells being non-glycosylated in vivo. O-glycosylation and sialylation are not excluded, and the previously described molecular heterogeneity of human IL-2 is attributed at least partly to differing extents of sialylation.
• Human IL-2 has a neutral isoelectric point. The deduced sequence contains equal numbers of basic (Arg plus Lys) and acidic (Asp plus Glu) residues, matching the pI of 6.8 to 7.1 measured for partially purified IL-2.
• The IL-2 polypeptide shares little homology with interferons or growth hormones. Against human IFN-gamma the only match is three consecutive residues, Lys-Asn-Phe at IFN-gamma positions 61-63 and IL-2 positions 96-98. Against human IFN-beta1, IL-2 residues 83-90 (Leu-Lys-Pro-Leu-Glu-Glu-Val-Leu) resemble IFN-beta1 residues 98-106 (Leu-Lys-Thr-Val-Leu-Glu-Glu-Lys-Leu).
• Placing the IL-2 cDNA downstream of the SV40 early promoter produces biologically active, correctly processed IL-2 from mammalian cells. The pCEIL-2 plasmid carries the cDNA at a single PstI site between the SV40 early promoter and a rabbit beta-globin genomic fragment containing one intron, together with the SV40 replication origin and the SV40 early polyadenylation site. Transfected into COS-7 cells it yields 1.5 units per ml of IL-2 at 48 h and up to 14 units per ml at 72 h, against below 0.1 units per ml for the empty pCE-1 vector, with a maximum of 70 units per ml obtained.
• Digesting an SV40-origin expression plasmid with HhaI before transfection raises cDNA expression in COS cells, because sequences that hamper replication of the transfected DNA are removed from the part of the plasmid needed for expression. Omitting this step lowered expression roughly 5- to 10-fold for human IFN-beta1 and IFN-gamma cDNAs expressed the same way. A G-C tract between the SV40 promoter and the cDNA, left by G-C tailing, further limits expression.
• IL-2 secreted by COS-7 cells from the cloned cDNA is indistinguishable from authentic human IL-2: it stimulates 3H-thymidine incorporation by the IL-2-dependent CTLL-2 line and in vitro T-cell growth, is neutralized by a monoclonal antibody raised against purified human IL-2, loses activity on trypsin treatment, and chromatographs on Sephadex G-100 at a molecular weight of about 15,000, matching IL-2 from Con A-stimulated Jurkat cells.
• IL-2 mRNA is rare and inducible. Jurkat-111 cells make no detectable IL-2 mRNA without a stimulus such as concanavalin A, and the cloned cDNA hybridizes only to mRNA from induced cells. IL-2-specific colonies occur at 1:2,000 in a cDNA library built from sucrose-gradient-fractionated poly(A) RNA, implying a frequency of about 1:20,000 in the unfractionated poly(A) mRNA given the roughly 10-fold enrichment the gradient provides.
• Human IL-2 mRNA sediments at 11.5S. A 13S peak of IL-2 mRNA activity appears occasionally, but on RNA blot analysis it yields a single band co-migrating with the 11.5S species, identifying the 13S form as an aggregate rather than a distinct transcript. The human genome carries the IL-2 gene in a single copy.

Abstract

A cDNA coding for human interleukin-2 (IL-2) has been cloned from a cDNA library prepared from partially purified IL-2 mRNA. The DNA sequence codes for a polypeptide which consists of 153 amino acids including a putative signal sequence. A biologically active polypeptide, characteristic of human IL-2, was produced when the cDNA was fused to a simian virus 40 promoter sequence and used to transfect cultured monkey COS cells.