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SIGNIFICANCE OF THE PUTATIVE UPSTREAM POLYBASIC NUCLEAR LOCALISATION SEQUENCE FOR THE BIOLOGICAL ACTIVITY OF HUMAN INTERFERON-GAMMA Stefan Petrov*, Maya Boyanova*, Alfredo Berzal-Herranz+, Andrey Karshikoff#, Genoveva Nacheva* and Ivan Ivanov* * Institute of Molecular Biology, Bulgarian Academy of Sciences, “Acad. G. Bonchev” Str., 21, 1113 Sofia, Bulgaria; + Instituto de Parasitologia y Biomedicina "Lopez-Neyra", CSIC, Avda. del Conocimiento s/n Armilla, 18100 Granada, Spain; # Department of Biosciences at Novum, Karolinska Institutet, Huddinge, S-14157 Huddinge, Sweden Correspondence to: Stefan Petrov, e-mail: [email protected] ABSTRACT Interferon-gamma (IFNγ) accomplishes its multiple biological effects by activating the STAT transcription factors, which are translocated to the nucleus through a specific nuclear localization sequence(s) (NLS) located in the IFNγ molecule. Two putative NLS have been pointed out in the human interferon gamma (hIFN): an upstream located in helix E (residues 83-89) and a downstream located at the C-terminal unstructured region (residues 124-132). To investigate the significance of the putative upstream NLS for the biological activity of hIFN we have introduced a point mutation in the hIFN gene to disturb the polybasic sequence typical for the conventional NLS. In the new gene a gln codon was substituted for the Lys88 codon and the mutated gene was cloned and expressed in E. coli LE392. This mutation led to a 1000-fold decrease in both hIFN antiviral and antiproliferative activities. When co-incubated with the wild-type hIFN (standard), the mutant hIFN competed for the cellular receptors that led to a 30% inhibition of the standard activity. This indicates that the mutation does not interfere with the interaction of the protein to the receptor but probably affects the intracellular signal transduction pathway. To avoid any putative compensatory effect in the function of the upstream NLS caused by the downstream C-terminal NLS, 21 C-terminal codons were deleted from the mutant hIFN gene. The latter resulted in only 10-fold additional decrease in biological activity and 50% inhibition of the standard activity in the competition assay. Our data indicate that the upstream NLS is endowed with a greater functional significance for the hIFNmediated signal transduction rather than the downstream NLS. Keywords: human interferon gamma; nuclear localization sequence; biological activity Introduction Human interferon gamma (hIFNγ) is a cytokine secreted in minute amounts by the T lymphocytes upon induction with viruses, bacteria, fungal antigens, immunotoxins, mitogens, etc. It is endowed with multiple biological activities and plays a key role in the modulation of the immune response (21). hIFN is a 17 kDa single chain protein consisting of 143 amino acids (16). The active form of hIFN is a non-covalent homodimer 1 organised in 6 -helixes linked by short unstructured regions (6). It activates the target cells via interaction with the extracellular domain of the hIFNγ receptor complex (5) followed by activation of the receptor associated JAK kinases (12). The JAK mediated phosphorylation of tyrosine residues at specific receptor sites provides a docking motif for STAT, which is itself also phosphorylated (2, 15). The activated STAT dissociates from the receptor, dimmerizes and is then translocated to the nucleus through a Ran/importin transport system (17). The active nuclear import of the protein complex is directed by a short amino acid sequence called nuclear localization sequence (NLS). The NLS generally consists of one or two clusters of basic amino acids separated by a spacer of variable length (8). Several groups have attempted to locate the elusive NLS in STATs trough mutagenesis of arginine/lysine rich motifs in their molecules (9, 17, 18). The mutational analyses, however, failed to reveal any conventional NLS in STATs, which is in accordance with a concept that the necessary basic NLS is provided by the STAT ligands. A similar examination of hIFNγ revealed two putative NLS: a downstream, located in the unstructured C-terminus of the protein between amino acid residues 124-132 (20) and an upstream (resembling the NLSs of the SV40 large tumour antigen, polyoma virus and the steroid hormone receptors) identified between the amino acid residues 83-89 (23). Larkin and co-workers (11) found that the upstream NLS is much less efficient than the Cterminal one, which was consistent with the observed loss of more than 90% of biological activity in the protein lacking the downstream NLS. The finding that the deletion of the last 23 C-terminal amino acids resulted in inactivation of the hIFNγ was explained by the absence of C-terminal NLS (19). Nacheva et al., (14) demonstrated that the removal of the entire unstructured (21 amino acids) C-terminal part from the molecule of hIFNγ led to a 10-fold decrease in its biological activity only. It was postulated that the absence of the downstream NLS in hIFNγ can be compensated (at least partly) by its upstream analogue. It was assumed also that the unstructured C-terminal domain in hIFNγ plays modulating but not crucial role for the biological activity of this cytokine. The present study aims to investigate the significance of the putative upstream NLS in hIFNγ for its biological activity. To this end two new variants of the hIFNγ gene are constructed. The first one (Lys/Gln88) contains a gln substitution for the Lys88 in order to disturb the corresponding polybasic NLS and the second one (Lys88/Gln/ΔC) carries both the same substitution plus a deletion of the last 21 3’-terminal codons (these amino acid residues carry the entire downstream NLS). The two hIFN derivative proteins are studied for the basic hIFNγ biological activities (antiviral and antiproliferative) as well as for their affinity to the hIFNγ receptor. Materials and methods Chemicals and Bacterial Strains Restriction endonucleases and other DNA modifying enzymes were purchased from Gibco-BRL (Life Technologies, Inc., Gaithersburg, MD, USA). All other reagents for electrophoresis, DNA manipulations, antiviral and kynurenine assays were products of Merck (Darmstadt, Germany) and Sigma (USA). E.coli LE 392 cells were used for expression of the hIFNγ genes. 2 DNA Manipulations The oligonucleotides necessary for the modification of the hIFNγ gene were synthesized on a Millipore Cyclon Plus DNA synthesizer (MilliGene, Division of Millipore, Burlington, MA, USA) by the phosphoramidite method and purified by electrophoresis on a polyacrylamide (PAA) urea gel. Their nucleotide sequences are as follows: Forward primer: 5’-CCC AAG CTT ATG CAG GAC C - 3’ Reverse primer: 5’-GCTT TTC GAA GTC ATC ACG TTG CT T TTT GTT G -3’ The forward primer carries a HindIII cloning site (shown in italic) and an ATG codon followed by a nucleotide sequence identical to that of the IFN gene. The reverse primer carries an AsuII cloning site (shown in italic) and introduces a point mutation (in bold) designed to substitute a gln codon (CAA) for the Lys88 codon AAA in the hIFNγ gene. To obtain the mutant hIFNγ genes, the genes encoding for the full size (143 amino acids) and the 3’-end truncated (122 amino acids) hIFNγ (14) were amplified by a two step PCR including 5 cycles of annealing at 500C for 1 min and 35 cycles at 600C for 1 min. The PCR products were digested with HindIII and AsuII and cloned in a pBR322-based expression vector (Fig.1). The primary structure of the derivative hIFNγ genes was verified by DNA sequencing on a DNA Cycle Sequencing System (Gibco–BRL). Bacterial Transformation E.coli LE 392 cells were transformed with the expression plasmids by the CaCl2 method. PAA-SDS gel electrophoresis PAA-SDS gels (15%) were prepared according to Laemmly (10) and run at 20 mA for 1.5 h. After electrophoresis, the gels were stained by Coomassie blue R250. Protein yield determination Bacterial cells (6 OD595) from overnight cultures of transformed E.coli LE 392 cells were harvested by centrifugation, resuspended in 1 ml 0.14 M NaCl, 10 mM Tris-HCl, 0.1 mM PMSF and disrupted by sonication. After centrifugation (15 min, 14,000g) the supernatants (clear lysates) were collected. The protein concentration was determined by the Bradford method (4). Cell lysates were diluted with phosphate-buffered saline (pH 7.2-7.4) to a final protein concentration of 27 μg/ml. Samples of 50 μl were applied on polyvinylchloride 96-well microplates (Costar Ltd., USA) by overnight incubation at 40C and the content of hIFNγ was determined by ELISA with sequence specific monoclonal antibodies. Bioassays Clear cell lysates were sterilized by filtration and subjected to serial dilutions appropriate for testing of both hIFNγ antiviral and antiproliferative activities. Antiviral activity was determined by measuring the protective effect of the protein on WISH cells against the cytopathic action of the vesicular stomatitis virus (VSV) (7). Antiproliferative activity was determined by a modified kynurenine bioassay on WISH cells as described earlier (3). Competition assay 3 The affinity of the hIFNγ derivative proteins to the cell receptor was analyzed by measuring their capacity to compete with the wild-type protein. Appropriate serial dilutions of clear cell lysates and purified (99.5% purity) recombinant hIFNγ (specific antiviral activity 5x107) were prepared. The mutant proteins (in cell lysates) were mixed in equimollar amounts with pure wild-type protein and the antiproliferative activity of the obtained mixtures was determined in a standard kynurenine bioassay. The results were compared with that of the pure wild-type hIFNγ. Results and Discussion 1. Design of hIFNγ analogs It is a routine approach to study the structure-function relationship by mutating the protein molecule by routine recombinant DNA techniques. As mentioned above, to investigate the functional significance of the putative upstream NLS in hIFNγ, two derivative proteins (Lys/Gln88 and Lys88/Gln/ΔC) were created (Fig. 2). The introduced mutations had to carry minimum or no effect on the secondary structure of hIFNγ and on its ability to bind the cell receptor. Thus hIFNγ derivative proteins were designed on the basis of a model describing the electrostatic interactions in the homodimer (the active form) of hIFNγ as free molecule and hIFNγ bound to the extracellular part of the receptor (1). One of the most general characteristics of the behavior of proteins in solution is the set of pK values of their titratable (ionizing) groups. Because the electrostatic interactions cannot be experimentally measured in the protein molecule, their theoretical prediction is the main tool for their analysis. In the above paper (1) the electrostatic interactions in hIFN are calculated for two different states of the molecule - alone (in the form of homodimer) and hIFNγ bound to the receptor. The study was based on the tree-dimensional structures of hIFNγ (6, 22) and clearly demonstrated that a number of titratable groups (mainly basic) had a remarkable shifts in their pK values after binding to the receptor. It is worth mentioning that the groups having high desolvation energy in both states (free and in a complex with the receptor) usually corresponded to highly conserved amino acids. Four of them, Tyr53, Lys61, Lys88, and Glu112 were conserved in all IFNγ sequences (6, 13), which is an indication for their functional significance. Calculating the ionisation equilibrium of titratable groups in the area of the putative upstream NLS for the two states of hIFNγ we found that most of the basic groups were drastically desolvated. Analysis of the free state of the molecule showed that the protonation states and the solvent accessibility of Lys86 and Lys87 in the putative upstream NLS essentially differ in the two subunits (monomers) of the homodimer and also that the Lys88 was inaccessible to the solvent in both subunits. In the hIFNγ/receptor complex Lys86 and Lys87 had high solvent accessibility in both subunits, while Lys88 was still inaccessible (Table 1). These data indicated that the Lys88 was located at a distance from the receptor binding site and therefore its mutation would not affect the affinity of the protein to the receptor. In order to minimize possible changes in the solubility of the protein due to change of the local polarity it was chosen to substitute 4 Lys88 with glutamine (Fig. 2). Furthermore, to evaluate the potential compensatory effect of the downstream NLS on the function of the upstream one, we constructed a second mutant hIFNγ in witch the last 21 C-terminal amino acids (together with the entire downstream NLS) were deleted (Fig.2). The mutated hIFNγ genes were prepared and expressed in E. coli LE392 as described in Materials and Methods. The expression of the resulted proteins (Fig. 3) is presented in Fig. 4. 2. Biological activity of the hIFNγ analogs In order to avoid artifacts related to the incorrect folding of the recombinant proteins during their purification, the biological activity of the hIFNγ derivatives was measured directly in clear cell lysates. Thus the insoluble fraction (inclusion bodies) was removed and the activity of the proteins was determined using the soluble (cytosolic fraction) only. Two biological activities, antiviral and antiproliferative, which are typical for the hIFNγ, were measured. The first activity was determined on the basis of the protective effect of hIFNγ on WISH cells against the cytopathic action of the VSV and the second one was measured by the so called “kynurenine bioassay” using the same cell line. To determine the specific activity of the tested proteins, their content in the clear lysates was measured by ELISA using anti-hIFNγ monoclonal antibodies and pure recombinant hIFN as a standard. As seen in Table 2, the single amino acid substitution in the construct Lys/Gln88 had a 1000-fold lower hIFN activity and the activity of the truncated construct (Lys88/Gln/ΔC) was 10-fold lower than that of the Lys/Gln88 mutant. To shed light on the reason for the lower biological activity of the new hIFNγ constructs we have analyzed their affinity to the hIFNγ receptor by measuring their capacity to compete with the wild-type protein. The experiments were performed as described in Material and Methods on WISH cells (known to be enriched in hIFN receptors. The results presented in Table 2 indicate that the coincubation of the mutant Lys/Gln88 with the wild-type hIFN led to a 30% decrease in its antiproliferative activity. We assume that inhibition higher than 30% could not be achieved because of the residual biological activity of the hIFN mutant (see Table 2). The mutant hIFNγ with fully truncated C-terminus (Lys88/Gln/ΔC) showed approximately 50 % inhibition of the standard activity in the competition assay, confirming our previous data that the lack of the C-terminus stabilizes the hIFN/receptor complex (1). These data indicate that the mutant proteins are capable of interacting with the hIFN receptor but are inefficient in triggering the intracellular signal transduction pathway. Conclusions The role of the putative upstream NLS of hIFN was investigated by site directed mutagenesis at the area of both upstream and downstream NLSs. Two derivative proteins were thus obtained containing either a single gln substitution for the Lys88 (Lys/Gln88) or both the single substitution and a 21 aminoacid C-terminal deletion (Lys88/Gln/ΔC). The first one demonstrated a 1000-fold lower biological activity in comparison with the wild-type hIFNγ and the second one was only 10- times less active than the Lys/Gln88 mutant. In a competition bioassay both constructs behaved as efficient competitors of the wild-type hIFN for the cellular receptor. The latter means that 5 the putative upstream NLS is crucial for the biological activity of hIFN but it is dispensable for its interaction of the protein with the cellular receptor. Based on these data we are tempted to speculate that the upstream NLS is of a greater functional importance for the signal transduction as compared to the downstream NLS. REFERENCES 1. Altobelli G., Nacheva G., Todorova K., Ivanov I. and Karshikoff A. (2001) PROTEINS: Structure, Function and Genetics, 43, 125-133 Acknowledgments This work is supported by National Science Fund, grant K-1405 11. Larkin J. P., Subramaniam B. A., Torres Johnson, H. M. (2001) J. Interferon Cytokine Res. 21, 341-348 12. Leaman D. W., Leung S., Li,X., and Stark G.R. (1996) FASEB J. 10, 1578-1588 2. Bach E. A., Aguet M., and Schreiber R. D. (1997) Annu. Rev. Immunol. 5, 563-591. 13. Lundell D.J., Narula Pharmacol. Ther. 64, 1-21 3. Boyanova M., Tsanev R., Ivanov I. (2002) Annal. Biochem. 308, 178-181 14. Nacheva G., Boyanova, M., Todorova, K., Karshikoff, A., Ivanov, I. (2003) ABB 4. Bradford M. M. (1976) Anal. Biochem. 72, 248-254 15. Pestka S., Kotenko S. V., Muthukumaran G., Izotova L. S., Cook J. R., and Garotta G. (1997) Cytokine Growth Factor Rev. 8, 189206. 5. Darnell J.E. Jr., Kerr I. M., Stark G. M. (1994) Science 264, 1415-1421 6. Ealick S.E., Cook W.J., Vijay-Kumar S., Carson M., Nagabhushan T.L., Trotta P.P., Bugg C.E. (1991) Science 252, 698-702 7. Forti R. L., Schuffman S.S., Davies H.A., Mitchell W.M. (1986) Methods Enzymol. 119, 533-540 8. Gorlich D., and Mattaj I. W. (1996) Science 271, 1513-1518 9. Herrington J., Rui L., Luo G., Yu-Lee L., and Carter-Su C. (1999) J. Biol. Chem. 274, 5138–5145 S.K. (1994) 16. Rinderknecht E., O”Connor B.H., Rodriguez H. (1984) J.Biol.Chem. 259, 67906797 17. Sekimoto T., Imamoto N., Nakajima K., Hirano T., and Yoneda Y. (1997) EMBO J. 16, 7067-7077 18. Strehlow I. and Schindler C. (1998) J. Biol. Chem. 273, 28049–28056 19. Subramaniam P. S., Larkin, J., III, Mujtaba, M. G., Walter, M.R., and Johnson, H., M. (2000) J.Cell. Sci. 113, 2771-2781 10. Laemmly U. (1970) Nature 227, 680-685 6 20. Subramaniam P. S., Mujtaba M. G., Paddy M.R., and Johnson H.M. (1999) J. Biol. Chem. 274, 403-407 21. Tsanev R. and Ivanov I. (2002) CRC Press 22. Walter M. R., Windsor W.T., Nagabhushan T.L., Lundell D.J., Lunn C.A., Zauondy P. J., and Narula S.K. (1995) Nature 376, 230-235 23. Zu. X. W., Jay F. (1991) J. Biol. Chem. 266, 6023-6026 7 Figure legends Fig. 1 Schematic structure of the vector pP1SD-IFNγ for expression of hIFNγ gene in E.coli. P1 – strong synthetic constitutive promoter, R9 – ribosome binding site SD. Fig. 2 Mutations introduced in the hIFN-gene. The putative NLSs are marked in orange and yellow and the polybasic sequences are underlined. The amino acid substitution is indicated by arrow. The scissors indicate the truncation of the unstructured C-terminus. Fig. 3 Amino acid sequence of hIFNγ and its mutant derivatives. The C-terminal amino acids are shown in green. Fig. 4 SDS-PAAGE of crude lysates of E. coli LE392 cells expressing hIFNg derivative genes. 1: E.coli LE 392 host (nontransformed) cells; 2-4: bacterial cells expressing the wild type hIFNγ, Lys/Gln88 and Lys88/Gln/ΔC respectively. The arrow indicates the position of the hIFNγ. Table 1 Charge values and solvent accessibility of selected amino acids in the putative upstream NLS in a free state and in a complex with the cellular receptor. Table 2 Specific biological activities of the mutant derivatives and competitive inhibition of hIFNγ by its analogs. 8 Fig.1 1 84 143 94 125 N 134 C Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Gln AAA (Lys88) Lys Thr Gly Lys Arg Lys Arg Ser Gln Met CAA (gln) Lys88/Gln/ΔC Lys88/Gln Fig.2 hIFN Gln1…..Lys86 Lys87 Lys88…..Leu 120 Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Arg140 Ala Ser Gln 143 Lys88/Gln Gln1…..Lys86 Lys87 Gln88…..Leu 120 Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Arg140 Ala Ser Gln 143 Lys88/Gln/C Gln1…..Lys86 Lys87 Gln88…..Leu 120 Ser Pro122 Fig.3 9 1 2 3 4 Fig. 4 10 Table 1 Amino acids Lys 80 Lys 86 Lys 87 Lys 88 Arg 89 Asp 90 Asp 91 Charge A 0.40 0.85 0.99 0.38 1 -1 -1 Charge B 0.99 0 0.09 0.02 1 -1 0 Free state Accessibility A (Å2) 62.91 134.18 122.13 9.02 77.72 56.06 7.52 Accessibility B (Å2) 40.97 69.30 87.71 47.98 83.01 61.65 6.49 Charge A 0.92 1 1 1 1 -1 -0.99 In a complex with the receptor Charge Accessibility Accessibility B A (Å2) B (Å2) 0.28 23.92 37.08 1 128.66 127.53 1 143.96 140.35 1 17.81 23.15 1 63.41 63.34 -1 60.45 63.34 0.91 22.35 19.29 Table 2 Protein Antiviral activity (IU/mg) Antiproliferative Inhibition activity (IU/mg) (%) hIFNγ 6.1 х 107 1.1 х 108 0 Lys88/Gln 5.7 x 104 1.7 x 105 32(±4%) Lys88/Gln/ΔC 1.2 x 103 6.7 x 103 50(±5%) 11