Expression and characterization of a des-methionine mutant interleukin-2 receptor (Tac protein) with interleukin-2 binding affinity.

A gene coding for the Tac protein (interleukin-2 receptor alpha-subunit, IL-2R alpha) of the interleukin-2 receptor was constructed by chemoenzymatic gene synthesis. The gene designed for mutagenesis codes for a receptor protein where all 10 methionines are substituted by alanine, valine, leucine, and isoleucine. In addition, aspartate at position 6 is substituted by glutamate. This desmethionine IL-2R alpha and the wild-type IL-2R alpha genes were integrated into a eukaryotic expression vector and transferred into different cell lines. The recipient cell lines express both wild-type and mutant receptor proteins on their cell surfaces which are recognized equally by different monoclonal antibodies. It was possible to establish cell lines with high level IL-2R alpha chain expression by fluorescence-activated cell sorting. The wild-type IL-2R alpha expressed in LTK- cells is a glycoprotein with an apparent molecular size of about 60 kDa and a typical low interleukin-2 binding affinity of KD = 12 nM. Despite the fact that 11 amino acids are altered, no significant difference in the mutant IL-2R alpha is observed, exhibiting the same molecular size and a low interleukin-2 binding affinity of KD = 26 nM.

only transiently expressed on antigen-or mitogen-stimulated T-cells (5). Since neither IL-2 nor IL-2R is expressed constitutively, the induction of these two genes is of eminent importance for the immune response. The constitutive expression of IL-2R is induced by infection of T-cells with HTLV-I and is correlated with their tumorous growth by autocrine regulation (6-8).
With the aid of the monoclonal antibody anti-Tac (3, 9), which recognizes human IL-2R, the cDNA for an IL-2R subunit (Tac protein, IL-2Ra, or p55) has been cloned and verified by heterologous expression (10-12). IL-2Ra is a glycoprotein which migrates on SDS-polyacrylamide gel electrophoresis with a molecular size of =60 kDa. The precursor protein of 28.5 kDa is co-and post-translationally modified by glycosylation, phosphorylation, and sulfation (4, 13, 14).
Radiolabel binding studies indicate a high (KD = 10 pM) and low (KO = 10 nM) affinity for IL-2 binding to IL-2R (15, 16). Transfer and expression of IL-2Ra cDNA in nonlymphoid cells generates only low affinity binding sites (17)(18)(19). In contrast, expression of human IL-2Ra cDNA upon transfer into mouse T-cell lines CTLL-2 or EL-4 results in the exhibition of high affinity receptors as well as the reconstitution of typical functionality (proliferation response to IL-2) in CTLL-2 cells (20,21). This discrepancy seems to be explained by the demonstration of a second IL-2-binding protein, the IL-2R /+subunit (IL-2R@, p70, or converter) which is only found in T-lymphocytes and related cell lines (22-24). Different studies suggest that high affinity IL-2R is a membrane complex composed of at least the converter protein p70 and the Tac protein p55 (25-27). Although IL-2Ra has been studied extensively, little is known about the IL-2-binding site(s) and IL-2Ra function. Site-directed mutagenesis of both IL-2Ra and ligand IL-2 has not yet helped to answer these questions (28-30). Nothing is known about the three-dimensional structure of the Tac protein which is essential for understanding IL-2 binding and interaction with the converter protein.
As a prerequisite for solving these problems, we have devised a synthetic gene allowing simple modifications for mutagenesis, which codes for an IL-2Ra protein without methionines, coined des-Met IL-2Ra (31). The replacements of methionines were done to maintain the predicted secondary structure (32-34). The acid-labile sequence Asp-Asp-Asp-Pro at positions 4-7 was stabilized by substitution of aspartate at position 6 by glutamate. In this report, we show that this des-Met IL-2Ra is expressed as an IL-2-binding protein in eukaryotic cells. In parallel, we have expressed des-Met IL-2Ra in Escherichia coli as a fusion with P-galactosidase in order to isolate the receptor protein after chemical cleavage with cyanogen bromide (35). (Tad   IL-2 REC L   MET ASP SER TYR LEU LEU MET TRP GLY LEU LEU THR PHE ILE MET VAL PRO GLY CYS ASN  ALA   -20  -10  -1   5'"  TTC ATG GAT TCA TAC CTG CTG ATG TGG GGA CTG CTC ACG TTC ATC ATG GTG CCT GGC TGC CAG GCA GAG  C  T  '   a   3'   G TAC CTA ACT ATG GAC GAC TAC ACC CCT GAC GAG TGC AAG TAG TAC CAC GGA CCG ACG GTC

FIG. 2. Plasmid constructions for expression of interleukin-2 receptors in eukaryotic cells. The synthetic gene segments IL-2RecI-111 (finely stippled boxes)
were inserted into pUC8/9 for subcloning and verification of DNA sequence (step 1). Gene segments with the correct DNA sequence were ligated to the total receptor gene and cloned with the leader segment (IL-2RecL or PstI-Sac1 leader sequence from pGL2) into the expression vector pBEH (step 2). The cDNA for human IL-2Ra in pGL2 (stippled bores) was also inserted into the exmession vector pBEH. Details of the plasmid constructions are described under "Experimental Procedures." Functional parts of the vectors as indicated.

Chemoenzymutic Synthesis of a Gene Coding for Des-Met
IL-2Ra"In order to obtain insight into the structure-function relationship of IL-2 binding to its membrane receptor, we have devised a synthetic gene coding for a des-Met IL-2Ra mutant protein. On the basis of the published cDNA and protein sequence (10-12), this gene was constructed ( Fig. 1) with some particularly designed features. Following the secondary structure prediction of Chou and Fasman (32), we exchanged all 10 methionines of IL-2Ra with the amino acids alanine, valine, leucine, or isoleucine. The replacements were done to maintain the predicted secondary structure, e.g. Met-Ala (@sheet) or Met-Leu (a-helix). Then, the gene was modified on the DNA level due to the degeneracy of the genetic code by introducing new restriction endonuclease recognition sites, e.g. KpnI, SalI, and CZuI, and deleting existing double or triple sites, e.g. NcoI and BglI. These modifications result in a module system facilitating future modifications as sitespecific mutagenesis by cutting out a gene fragment and synthesis of a modified new double strand followed by reinsertion. Besides these features, the sequence was modified in order to guarantee the exact finding of the individual complementary oligonucleotides and their joining with high effi-* "Experimental Procedures" are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. ciency in a one-step test tube reaction as introduced by Khorana (48). This was done with a computer program (51) checking whether the opposite strands alb, c/d, and elf do pair preferentially over, e.g. a/d or a/f (see Fig. 1). In particular, the free overlapping ends of 6-9 bases within the whole segments 1-111 were checked for their unambiguous matchings.
The total gene consists of 834 base pairs and is divided into four DNA segments, 1-111 and L (for leader), defined by individual restriction endonuclease cutting sites (Fig. 2). Each segment is constructed from 10 to 16 individual oligonucleotides, two for the leader fragment, ranging in length from 27 to 73 bases. After a one-step hybridization and ligation of oligonucleotides La-Lb, Ia-Ip, IIa-111, and IIIa-IIIj with good success, we verified the DNA sequence according to Maxam and Gilbert (40). We found four out of five clones of segment IL-2RecIII with the correct sequence. Similar results were obtained with segments IL-2RecI and IL-2RecII and IL-2RecL.
Establishment of Cell Lines with Stable Expression of Wildtype and Des-Met IL-2Ra"The different gene fragments were ligated to the total gene and cloned in the vector pBEH as outlined under "Experimental Procedures." (see Fig. 2). This eukaryotic expression vector contains pBR322/328 and SV40 sequences for replication in E. coli and certain primate cells. A gene inserted into the polylinker from pUC9 is set under the control of the SV40 early promoter. Splicing and polyadenylation signals are from SV40. As a positive control in all experiments, the cDNA gene coding for wild-type IL-2Ra was cloned into the same vector (Fig. 2).

TABLE I Eukmyotic cells with stable expression of wild-type and des-Met
Cells were analyzed with a fluorescence-activated cell sorter after incubation with monoclonal antibody against IL-2Ra. The percentage of Tac positive cells was estimated as described under "Experimental Procedures." Cells were transfected with the indicated IL-2Ra. LTK-, and CHO-dhfr cells with pBEH des-Met IL-2Ra or pBEH IL-2Ra cDNA. After selection for (3418 resistance, mixtures of about 500-1000 clones were tested for IL-2Ra expression by directly or indirectly staining with monoclonal antibodies. We used different antibodies: anti-Tac (3), IL-2R1-Fitc (Coulter clone), and 2A3 (46). They are all similar with regard to competing with IL-2 for binding to the receptor. The flow cytometric analysis of these cells clearly shows the expression of IL-2Ra and the des-Met mutant in all three examined cell lines ( Fig. 3 and Table I). Stained cells had an intensive membrane fluorescence as seen in Fig. 4B. Monoclonal antibodies used for detection of IL-2Ra cannot distinguish between wild-type and mutant receptors showing that des-Met IL-2Ra is stably expressed as an integral membrane protein exposing an epitope detected by these different antibodies.

Fluorescence-activated Cell Sorting Generates LTK-Cells with High Level IL-2Ra and Des-Met IL-2Ra Expression-
Both genes were stably expressed in BHK-21, CHO-dhfr, and LTK-cells. In mixtures of cell clones, the expression of receptor varies over a wide range. We enriched a population by sorting out highly fluorescent cells to get a large number of receptor molecules per cell. For this purpose, we used LTKcells since they were more convenient for sorting. We found that more than 90% of the cells were still alive after sorting. The top 5% of stained viable cells at each sorting were collected. After 1 week of cultivation, enough cells were grown to repeat the sorting procedure. Five cycles of sorting were necessary to establish cells with high level expression of antigen as shown in Fig. 4 (C and D). The cells expressing wild-type IL-2Ra were 81% positive; des-Met mutant cells were 97% positive (Table I). Most cells had a high relative fluorescence intensity, and the level of expression was stable during 2 months of cultivation. The population of LTK-cells expressing wild-type IL-2Ra still contained about 20% negative cells.
Immunoprecipitation of Des-Met IL-2Ra with an Apparent Molecular Size of 60 kDa-IL-2Ra on T-lymphocytes is a glycoprotein with a 28.5-kDa peptide backbone which is sequentially processed through at least two intermediate forms to a mature form of 50-60 kDa containing both Nand 0linked carbohydrate moieties (4,14). For this reason, we were interested whether the post-translational modifications of wild-type and mutant receptor protein in transfected LTKcells were distinguishable. It is known that the recognition of potential glycosylation sites is due to several intrinsic parameters of the protein including protein conformation. We examined the molecular size of des-Met and wild-type IL-2Ra by immunoprecipitation of cells ["sSS]cysteine-labeled in vivo with a rabbit polyclonal antibody directed against human IL-2Ra. The result is shown in Fig. 5. The wild-type and des-Met mutant IL-2Ra have identical molecular sizes of about 60 kDa on a discontinuous 12% SDS-polyacrylamide gel (Fig.  5, lanes C-E), suggesting that both molecules are equally glycosylated in mouse LTK-cells. The bands are indistinct, which is typical for a heterogeneity in the carbohydrate moieties. This heterogeneity is found for IL-2Ra in lymphoid cells, too (4,9).
Scatchard Plot Analysis of Interleukin-2 Binding to Recombinant LTK-Cells-Since we did not observe any differences by comparing mutant with wild-type IL-2Ra, we attempted to test whether or not mutant IL-2Ra is capable of binding IL-2.
Competition experiments and estimations of the dissociation constant ( K D ) with '2sI-labeled rIL-2 as tracer were undertaken according to Robb et al. (15,16) with five timessorted LTK-cells. Staining profiles of these cells are shown in Fig. 4 (C and D). Nontransfected LTK-cells served as a negative control. In each experiment, unspecific, nonsaturable binding was determined by addition of 0.1 g/liter anti-Tac mAb (9,16,49) or 10 p~ rIL-2 into the assay medium and is substracted from total binding in Fig. 6 or Table 11. This unspecific binding was usually low and did not vary significantly. The result of different radiolabel binding experiments is that des-Met IL-2Ra can bind IL-2. It was possible to displace 76-94% of lZ5I-labeled rIL-2 binding to LTK-cells expressing the des-Met mutant by 10 ~L M rIL-2 or 0.1 g/liter anti-Tac mAb. As a control, we employed mouse ascites fluid with antibody directed against &galactosidase at a concentration of 0.1 g/liter, which had no effect on IL-2 binding (data not shown). This demonstrated the specific and saturable binding of IL-2 to the mutant receptor. The results of binding experiments to determine the dissociation constant (KD) with different IL-2 concentrations (typically from 0.235 to 60.0 nM) are shown in Fig. 6. As calculated from the slope of the straight line in the Scatchard plot, des-Met 11-2Ra binds IL-2 with KD = 25.8 k 7.0 nM. Authentic IL-2Ra binds IL-2 with an affinity of KD = 11.9 & 2.1 nM (Table 11). If present at all, this difference in affinity is small and correlates well with published KD values for low affinity IL-2 binding (16-18) to the Tac protein. As expected, we could not detect any high affinity binding sites at low IL-2 concentrations on transfected LTK-cells. High affinity binding sites are found on the HTLV-I-infected human T-cell line HUT 102 (Table 11)

Interleukin-2 receptor numbers and affinities on sorted LTKand HUT 102 cells
The results represent the mean & S.D. of multiple binding experimenta (numbers in parentheses).