Structural Mapping of Post-translational Modifications in Human Interleukin-24
2009; Elsevier BV; Volume: 284; Issue: 44 Linguagem: Inglês
10.1074/jbc.m109.036061
ISSN1083-351X
AutoresKerry L. Fuson, Mingzhong Zheng, Molly Craxton, Abujiang Pataer, Rajagopal Ramesh, Sunil Chada, R. Bryan Sutton,
Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoHuman interleukin-24 (IL-24) is unique among the IL-10 superfamily as there is considerable evidence that it possesses multiple anti-cancer properties, including direct tumor cell cytotoxicity, helper T cell (TH1) immune stimulation, and anti-angiogenic activities. The primary sequence of human IL-24 differs from homologous cytokines, because it possesses three consensus N-linked glycosylation sites and the potential for a single disulfide bond. To address the significance of these modifications in human IL-24, we analyzed the relationship between post-translational modifications and the cytokine activity of the human IL-24 protein. In contrast to related interleukins, we identified a relationship between net glycosylation, protein solubility, and cytokine activity. In addition, abrogation of the two cysteine residues by mutagenesis dramatically altered the ability of IL-24 to secrete from host cells and resulted in the concomitant loss of IL-24 activity. We conclude that, unlike other IL-10 family members, human IL-24 must be glycosylated to maintain solubility and bioavailability. Further, a single, unique disulfide bond is required for secretion and activity. These structure-function relationships show that, although IL-24 is a member of the IL-19 subfamily of IL-10-like cytokines by sequence similarity, its surface properties and its distinctive disulfide arrangement make it unique. These observations could explain the novel biological activities measured of this cytokine. Understanding the structural basis of IL-24 activity will be important in the interpretation of the function of this cytokine and in the development of scale-up strategies for biophysical and clinical applications. Human interleukin-24 (IL-24) is unique among the IL-10 superfamily as there is considerable evidence that it possesses multiple anti-cancer properties, including direct tumor cell cytotoxicity, helper T cell (TH1) immune stimulation, and anti-angiogenic activities. The primary sequence of human IL-24 differs from homologous cytokines, because it possesses three consensus N-linked glycosylation sites and the potential for a single disulfide bond. To address the significance of these modifications in human IL-24, we analyzed the relationship between post-translational modifications and the cytokine activity of the human IL-24 protein. In contrast to related interleukins, we identified a relationship between net glycosylation, protein solubility, and cytokine activity. In addition, abrogation of the two cysteine residues by mutagenesis dramatically altered the ability of IL-24 to secrete from host cells and resulted in the concomitant loss of IL-24 activity. We conclude that, unlike other IL-10 family members, human IL-24 must be glycosylated to maintain solubility and bioavailability. Further, a single, unique disulfide bond is required for secretion and activity. These structure-function relationships show that, although IL-24 is a member of the IL-19 subfamily of IL-10-like cytokines by sequence similarity, its surface properties and its distinctive disulfide arrangement make it unique. These observations could explain the novel biological activities measured of this cytokine. Understanding the structural basis of IL-24 activity will be important in the interpretation of the function of this cytokine and in the development of scale-up strategies for biophysical and clinical applications. Melanoma differentiation-associated gene 7 was identified as a novel tumor suppressor gene in human melanoma cells (1Jiang H. Lin J.J. Su Z.Z. Goldstein N.I. Fisher P.B. Oncogene. 1995; 11: 2477-2486PubMed Google Scholar). Because of its physical location within the IL-10 4The abbreviations used are: IL-10interleukin-10STAT3signal transducers and activators of transcription 3ERendoplasmic reticulumIFNinterferon. gene cluster, its homology with the IL-10 protein and its cytokine activity, melanoma differentiation-associated gene 7 was reclassified as interleukin-24 (IL-24) (2Caudell E.G. Mumm J.B. Poindexter N. Ekmekcioglu S. Mhashilkar A.M. Yang X.H. Retter M.W. Hill P. Chada S. Grimm E.A. J. Immunol. 2002; 168: 6041-6046Crossref PubMed Scopus (267) Google Scholar). Gene delivery of IL-24 using plasmid or adenoviral vectors has demonstrated that the product of the human IL-24 gene exhibits powerful tumor specific pro-apoptotic, growth inhibitory, and anti-angiogenic activities (3Ramesh R. Mhashilkar A.M. Tanaka F. Saito Y. Branch C.D. Sieger K. Mumm J.B. Stewart A.L. Boquoi A. Dumoutier L. Grimm E.A. Renauld J.C. Kotenko S. Chada S. Boquio A. Cancer Res. 2003; 63: 5105-5113PubMed Google Scholar). In primary human endothelial cells, IL-24 interacts with a unique set of cell surface receptors, IL-22R1/IL-20R2 (4Wang M. Tan Z. Zhang R. Kotenko S.V. Liang P. J. Biol. Chem. 2002; 277: 7341-7347Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 5Dumoutier L. Leemans C. Lejeune D. Kotenko S.V. Renauld J.C. J. Immunol. 2001; 167: 3545-3549Crossref PubMed Scopus (357) Google Scholar), resulting in potent anti-angiogenic activity (3Ramesh R. Mhashilkar A.M. Tanaka F. Saito Y. Branch C.D. Sieger K. Mumm J.B. Stewart A.L. Boquoi A. Dumoutier L. Grimm E.A. Renauld J.C. Kotenko S. Chada S. Boquio A. Cancer Res. 2003; 63: 5105-5113PubMed Google Scholar). In tumor cells that express these receptors, IL-24 induces apoptosis; however, no cytotoxicity is observed in normal cells that also express the IL-24 receptors (6Zheng M. Bocangel D. Doneske B. Mhashilkar A. Ramesh R. Hunt K.K. Ekmekcioglu S. Sutton R.B. Poindexter N. Grimm E.A. Chada S. Cancer Immunol. Immunother. 2007; 56: 205-215Crossref PubMed Scopus (52) Google Scholar). Given these properties, IL-24 is now being considered as a promising new bio-therapeutic agent in the treatment of various cancers (7Tong A.W. Nemunaitis J. Su D. Zhang Y. Cunningham C. Senzer N. Netto G. Rich D. Mhashilkar A. Parker K. Coffee K. Ramesh R. Ekmekcioglu S. Grimm E.A. van Wart Hood J. Merritt J. Chada S. Mol. Ther. 2005; 11: 160-172Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 8Inoue S. Shanker M. Miyahara R. Gopalan B. Patel S. Oida Y. Branch C.D. Munshi A. Meyn R.E. Andreeff M. Tanaka F. Mhashilkar A.M. Chada S. Ramesh R. Curr. Gene Ther. 2006; 6: 73-91Crossref PubMed Scopus (36) Google Scholar). interleukin-10 signal transducers and activators of transcription 3 endoplasmic reticulum interferon. IL-24 has been categorized as a member of the IL-19 subfamily of IL-10-like cytokines (9Zdanov A. Vitam. Horm. 2006; 74: 61-76Crossref PubMed Scopus (11) Google Scholar). This subfamily includes IL-19, IL-20, IL-22, and IL-24. The three-dimensional structures of two of the four IL-19 subfamily members, IL-19 and IL-22, have been determined to high resolution (10Chang C. Magracheva E. Kozlov S. Fong S. Tobin G. Kotenko S. Wlodawer A. Zdanov A. J. Biol. Chem. 2003; 278: 3308-3313Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 11Nagem R.A. Colau D. Dumoutier L. Renauld J.C. Ogata C. Polikarpov I. Structure. 2002; 10: 1051-1062Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 12Xu T. Logsdon N.J. Walter M.R. Acta Crystallogr. D. Biol. Crystallogr. 2005; 61: 942-950Crossref PubMed Scopus (41) Google Scholar). Like IL-10, both IL-19 and IL-22 are composed of a bundle of 6 α-helices; however, unlike IL-10, these cytokines are active as monomers. Each of these cytokines possesses a set of highly conserved disulfide bonds, which are characteristic of each cytokine. Presumably, these disulfide bridges stabilize the α-helical structure for efficient receptor engagement in the extracellular environment (13Thornton J.M. J. Mol. Biol. 1981; 151: 261-287Crossref PubMed Scopus (677) Google Scholar). IL-24 is the lone exception, because it is missing two complementary cysteine residues that form consensus disulfide bonds in the other members of this family. In many of the known IL-10-like cytokines (IL-10, IL-20, IL-22, and others), glycosylation is not required for activity. Either the proteins are not glycosylated, as is the case for IL-10 and IL-20, or the cytokine is glycosylated, but the glycan can be removed either enzymatically or by mutagenesis without compromising activity, as is the case for IL-22. Refolding IL-22 from bacterially expressed protein has no effect on the cytokine activity or the overall three-dimensional structure (11Nagem R.A. Colau D. Dumoutier L. Renauld J.C. Ogata C. Polikarpov I. Structure. 2002; 10: 1051-1062Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 14de Oliveira Neto M. Ferreira Jr., J.R. Colau D. Fischer H. Nascimento A.S. Craievich A.F. Dumoutier L. Renauld J.C. Polikarpov I. Biophys. J. 2008; 94: 1754-1765Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Although similar claims have been reported for IL-24 (15Sauane M. Gupta P. Lebedeva I.V. Su Z.Z. Sarkar D. Randolph A. Valerie K. Gopalkrishnan R.V. Fisher P.B. Cancer Res. 2006; 66: 11869-11877Crossref PubMed Scopus (51) Google Scholar, 16Mumm J.B. Ekmekcioglu S. Poindexter N.J. Chada S. Grimm E.A. J. Interferon Cytokine Res. 2006; 26: 877-886Crossref PubMed Scopus (21) Google Scholar, 17Sauane M. Gopalkrishnan R.V. Choo H.T. Gupta P. Lebedeva I.V. Yacoub A. Dent P. Fisher P.B. Oncogene. 2004; 23: 7679-7690Crossref PubMed Scopus (61) Google Scholar), these analyses relied on very small amounts of protein, so it was difficult to accurately associate protein structure with function. It is also clear that IL-24 is not as robust as other related cytokines, because several groups have shown varied responses of this cytokine in different biological assays (18Kreis S. Philippidou D. Margue C. Rolvering C. Haan C. Dumoutier L. Renauld J.C. Behrmann I. PLoS ONE. 2007; 2: e1300Crossref PubMed Scopus (29) Google Scholar, 19Kreis S. Philippidou D. Margue C. Behrmann I. J. Cell. Mol. Med. 2008; 12: 2505-2510Crossref PubMed Scopus (29) Google Scholar). One possibility to explain the discrepancy between IL-24 and the other IL-19-like cytokines is that the IL-24 protein possesses unique structural features that contribute both to its distinctive biology and to the characteristic properties of the protein. In this study, we utilized cytokine activity assays in tandem with genomic and biophysical analyses to assess the role of post-translational modifications in IL-24. We found that, unlike the other members of the IL-19 subfamily, human IL-24 requires at least two contiguous glycosylated sites for efficient secretion and activity. These neighboring glycan groups likely mask a non-polar region on the surface of IL-24 located near the putative receptor binding site on helix B. Further, the IL-24 protein possesses a novel pairing of disulfide bonds that has not been identified in related interleukins. Understanding the structural characteristics of human IL-24 will be essential for its development as a therapeutic cytokine. IL-24 sequences were identified by tblastn searches of the primary nucleotide sequences at NCBI using the Homo sapiens amino acid sequence as the probe. Scientific names, common names, and data base accessions for translated gene sequences that correlate with the putative secreted IL-24 proteins are as follows: H. sapiens (human) IL-24, AC098935.2; Pan troglodytes (chimp), AACZ02012631.1; Pongo abelii (orangutan) ABGA01259979.1; Macaca mulatta (rhesus monkey), AC194488.2; Microcebus murinus (gray mouse lemur), ABDC01084535.1; Tupaia belangeri (northern tree shrew), AAPY01334263.1; Cavia porcellus (Guinea pig), AAKN02017883.1; Dipodomys ordii (kangaroo rat), ABRO01173591.1; Mus musculus (mouse), AC165322.12; Rattus norvegicus (Norway rat), AAHX01075042.1; Spermophilus tridecemlineatus (ground squirrel), AAQQ01504599.1, AAQQ01504600.1, and AAQQ01786339.1; Canis familiaris (dog), AAEX02025893.1; Felis catus (domestic cat), ACBE01499449.1; Bos taurus (cow), AAFC03056217.1; Tursiops truncatus (dolphin), ABRN01323706.1; Myotis lucifugus (brown bat), AAPE01426984.1 and AAPE01426909.1; Equus caballus (horse), AAWR02036827.1; Loxodonta africana (African elephant), AAGU02213483.1; Monodelphis domestica (short-tailed opossum (20Wong E.S. Young L.J. Papenfuss A.T. Belov K. Immunome Res. 2006; 2: 4Crossref PubMed Google Scholar)), AAFR03023107.1; H. sapiens IL-20, AAH74948; H. sapiens IL-19, Protein Data Bank (pdb) code 1N1F; H. sapiens IL-22, pdb code 1M4R; H. sapiens IL-10, pdb code 1ILK; and cytomegalovirus IL-10, pdb code 1LQS_A. Sequences were aligned using salign in Modeler (21Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10636) Google Scholar). We used Modeler (21Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10636) Google Scholar) to construct and refine a homology model of human IL-24 based on the known structures of IL-19 and IL-22. As the pairing of disulfide bonds is unique in IL-24, the software was explicitly instructed to construct the model including a single disulfide bond between Cys-59 and Cys-106 (Fig. 1). Structural representations were created using PyMOL (22DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific LLC, San Carlos, CA2002Google Scholar). A cDNA encoding residues 52–206 of human IL-24 was subcloned into pET28A using the NcoI and BamHI restriction sites and overexpressed as a non-His tagged, insoluble protein. The bacterial cells were lysed and centrifuged at 5,000 rpm in an SS-34 rotor at 4 °C. The resulting pellet was dissolved in 2 m Urea and re-centrifuged at 5,000 rpm at 4 °C. This step was repeated three times. The final pellet was dissolved in 6 m Urea, 10 mm Tris, pH 8.0, and refolded overnight at 4 °C by dilution to 50 μg/ml into 2 mm reduced glutathione:0.2 mm oxidized glutathione with 0.5% decyl-maltoside. Misfolded, aggregated protein was removed by centrifugation at 10,000 rpm for 20 min at 4 °C. Protein purity was assayed by SDS-PAGE gel electrophoresis and stained with Coomassie Blue (data not shown) and/or Western blot analysis using both polyclonal and monoclonal IL-24 specific antibodies. Protein was concentrated to 3 mm IL-24 using an Amicon pressure cell. All CD measurements were carried out at room temperature on an Aviv 215 spectropolarimeter using a quartz cell with a path length of 1 mm. Samples were scanned from 195 nm to 260 nm in 1 nm steps in 5 mm Tris, pH 8.0. For CD analysis, the IL-24 was diluted from 3 mm to 50 μm in 5 mm Tris, pH 8.0. The spectrum is an average of 10 scans. Background corrections for variance within the buffer were made in all spectra. The H1299 lung cancer cell line was obtained from the America Type Culture Collection (ATCC, Manassas, VA) and was maintained in Dulbecco's modified Eagle's medium (HyClone, Inc., Logan, UT) supplemented with 10% fetal bovine serum (Invitrogen), 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine, and HEPES buffer (Invitrogen). The cells were routinely screened to verify lack of mycoplasma contamination and were used in the log phase of growth. Monoclonal anti-IL-24 antibody was prepared as described previously (23Poindexter N.J. Walch E.T. Chada S. Grimm E.A. J. Leukocyte Biol. 2005; 78: 745-752Crossref PubMed Scopus (61) Google Scholar). Rabbit phospho-STAT3 (Tyr-705) was purchased from Cell Signaling Technology Inc. (Beverly, MA), β-actin monoclonal antibody was purchased from Oncogene Research Products (San Diego, CA). H1299 cells were transfected with 4 μg of expression plasmid DNAs using Lipofectamine 2000 (Invitrogen) for 18–48 h. Stable cell lines were obtained by cotransfection with psv2neo and selection in G418 (800 ng/ml) for 10 days. Supernatants were harvested from the culture of transfected cells and centrifuged at 14,000 rpm for 10 min to measure only soluble, non-aggregated IL-24 protein. Supernatant was saved for Western blot analysis or used to treat tumor cells. Immunoblotting using various antibodies and standard procedures were performed as described previously (24Chada S. Mhashilkar A.M. Ramesh R. Mumm J.B. Sutton R.B. Bocangel D. Zheng M. Grimm E.A. Ekmekcioglu S. Mol. Ther. 2004; 10: 1085-1095Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Proteins were visualized using enhanced chemiluminescence (Amersham Biosciences) after incubation with primary or secondary antibodies. Activation of STAT3 was determined by immunofluorescence assay using a phospho-STAT3-specific antibody (24Chada S. Mhashilkar A.M. Ramesh R. Mumm J.B. Sutton R.B. Bocangel D. Zheng M. Grimm E.A. Ekmekcioglu S. Mol. Ther. 2004; 10: 1085-1095Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Pictures were taken using a fluorescence microscope 1–2 h after staining. Cell killing was determined by the trypan blue exclusion assay (25Beintema J.J. J. Mol. Evol. 1986; 24: 118-120Crossref PubMed Scopus (7) Google Scholar). At designated times after treatment with IL-24 (wild type and mutants), cells were harvested by trypsinization, and a small aliquot was suspended in a 1:10 volume with 0.1% trypan blue (Invitrogen). Total cell numbers, and the number of dead cells was counted using a hemocytometer under light microscopy. Assays were performed three times. H1299 lung cancer cells (5 × 104 cells/well) were grown on chamber slides to 70% confluence and then transfected with the wild-type or mutant IL-24 cDNA or treated with phosphate-buffered saline as a negative control. Seventy-two hours later, the cells were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde/phosphate-buffered saline for confocal microscopic analysis. Cells were blocked with 1% normal goat serum for 1 h and then incubated overnight at a dilution of 1:100 with the primary mouse monoclonal IL-24 antibody. The slides were then washed to remove primary antibody, rinsed with phosphate-buffered saline, and placed in a prewarmed staining solution containing ER-TrackerTM red dyes or MitoTracker Deep Red 633 (Molecular Probes) for ∼15–20 min at 37 °C. The slides were then washed and incubated with a fluorescein isothiocyanate- or rhodamine-conjugated secondary antibody (Invitrogen) for 1 h. Next, the slides were mounted with ProLong Gold Antifade reagent containing 4′,6-diamidino-2-phenylindole (Invitrogen) and analyzed under an Olympus FluoView FV500 laser confocal microscope (Olympus America, Melville, NY) after adjustment for background staining. We compared the IL-24 protein sequences from 19 mammalian genomes to reveal characteristic glycosylation trends that have been maintained in this cytokine throughout evolution (25Beintema J.J. J. Mol. Evol. 1986; 24: 118-120Crossref PubMed Scopus (7) Google Scholar). Human IL-24 possesses three consensus sites for N-linked glycosylation (Fig. 1). Here, each site is labeled (using the human IL-24 amino acid numbering) as site I (Asn-85), site II (Asn-99), and site III (Asn-126) (Fig. 1). Interestingly, the relatively heavy glycosylation pattern characteristic of the IL-24 in higher primates is not conserved across all species. Site I is represented in 12 of 19 of the available sequences, while site II is represented in 16 of 19 of the sequences; site III is found in only 5 of 19 of the sequences. Furthermore, 8 of 19 of the IL-24 proteins examined in this analysis possess only one consensus glycosylation site. In most cases, the protein sequences with a single glycosylation sequon are coincident with site II, suggesting that this is a unique characteristic of IL-24. Of the other members of the IL-19-like subfamily, IL-20 lacks any post-translational N-linked glycosylation signals, IL-19 possesses two consensus sites, but only one is utilized (10Chang C. Magracheva E. Kozlov S. Fong S. Tobin G. Kotenko S. Wlodawer A. Zdanov A. J. Biol. Chem. 2003; 278: 3308-3313Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), and IL-22 has three consensus N-linked glycosylation sites (Fig. 1). Only IL-24 site I is common to all the glycosylated family members. The crystal structures of the related cytokines, IL-19 and IL-22, have been solved to high resolution (10Chang C. Magracheva E. Kozlov S. Fong S. Tobin G. Kotenko S. Wlodawer A. Zdanov A. J. Biol. Chem. 2003; 278: 3308-3313Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 11Nagem R.A. Colau D. Dumoutier L. Renauld J.C. Ogata C. Polikarpov I. Structure. 2002; 10: 1051-1062Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 12Xu T. Logsdon N.J. Walter M.R. Acta Crystallogr. D. Biol. Crystallogr. 2005; 61: 942-950Crossref PubMed Scopus (41) Google Scholar). Considering only the range of amino acids from the secreted form of these cytokines, IL-19 is ∼37% similar to IL-24, whereas IL-22 is ∼40% similar. Using these two three-dimensional structures and the primary sequence similarity to IL-24, we computed a model of human IL-24 (Fig. 1) using Modeler (21Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10636) Google Scholar, 26Martí-Renom M.A. Stuart A.C. Fiser A. Sánchez R. Melo F. Sali A. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 291-325Crossref PubMed Scopus (2597) Google Scholar). The resulting model (human IL-24 residues 51–206) consists of 6 α-helices (indicated as A–F in Fig. 1), with a single disulfide bond joining helices A and C (Fig. 1). Although the quality of this homology model requires further experimental consideration, it does predict that Lys-77 and Asp-194 can form a highly conserved salt bridge joining helices A and F. The function of this salt bridge in the IL-19 subfamily is not well understood; however, it is highly characteristic of this class of α-helical interleukins (e.g. IL-10, IL-19, IL-20, and IL-22) (27Zdanov A. Curr. Pharm. Des. 2004; 10: 3873-3884Crossref PubMed Scopus (62) Google Scholar). Our homology model of IL-24 also predicts that two of the N-linked glycosylation sites (sites I and III) localize to loops at the "top" of the molecule, whereas an additional site (site II) localizes to a loop at the base of helix B. The cDNA corresponding to the secreted form of human IL-24 (residues 52–206) was cloned into multiple bacterial expression systems. We attempted to produce soluble protein as glutathione S-transferase, maltose-binding protein, and His-tagged fusion proteins; however, only very small amounts of soluble protein could be obtained. In each case, once the fusion partner was separated from IL-24 by protease cleavage, the IL-24 became insoluble. A screen for refolding conditions using the untagged IL-24 identified a detergent-based protocol that yielded human IL-24 with very high solubility (>50 mg/ml). However, upon lowering the detergent concentration by dialysis or dilution, the IL-24 protein quantitatively precipitated. IL-24 produced in Escherichia coli migrated to ∼20 kDa on PAGE gels, which is similar in size to the unglycosylated mammalian IL-24 (∼20 kDa) (Fig. 3). The protein was also immunoreactive with human IL-24 monoclonal antibody. When diluted into culture media, the biological activity of the bacterially expressed, detergent-refolded IL-24 demonstrated no significant cell killing; however, it was difficult to control for the presence bacterial endotoxins and excess detergent in cell assays (data not shown). Other groups have confirmed that bacterially expressed IL-24 does not show significant cytokine activity (18Kreis S. Philippidou D. Margue C. Rolvering C. Haan C. Dumoutier L. Renauld J.C. Behrmann I. PLoS ONE. 2007; 2: e1300Crossref PubMed Scopus (29) Google Scholar). On the other hand, the glycosylated human IL-24 produced in mammalian cells showed specific tumor cell killing (6Zheng M. Bocangel D. Doneske B. Mhashilkar A. Ramesh R. Hunt K.K. Ekmekcioglu S. Sutton R.B. Poindexter N. Grimm E.A. Chada S. Cancer Immunol. Immunother. 2007; 56: 205-215Crossref PubMed Scopus (52) Google Scholar, 24Chada S. Mhashilkar A.M. Ramesh R. Mumm J.B. Sutton R.B. Bocangel D. Zheng M. Grimm E.A. Ekmekcioglu S. Mol. Ther. 2004; 10: 1085-1095Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The cell killing activity of the fully glycosylated protein was inhibited using specific anti-IL-24 antibodies (6Zheng M. Bocangel D. Doneske B. Mhashilkar A. Ramesh R. Hunt K.K. Ekmekcioglu S. Sutton R.B. Poindexter N. Grimm E.A. Chada S. Cancer Immunol. Immunother. 2007; 56: 205-215Crossref PubMed Scopus (52) Google Scholar, 24Chada S. Mhashilkar A.M. Ramesh R. Mumm J.B. Sutton R.B. Bocangel D. Zheng M. Grimm E.A. Ekmekcioglu S. Mol. Ther. 2004; 10: 1085-1095Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). To test whether the detergent-soluble IL-24 possessed measurable secondary structure, we analyzed our re-folded protein by CD. This analysis of refolded human IL-24 confirmed that the cytokine is predominantly α-helical (Fig. 2). Ellman's reagent (28Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21624) Google Scholar) confirmed that two Cys residues were reactive in our detergent-refolded samples (data not shown), indicating that the disulfide bridge was not formed in the bacterial protein. This is consistent with the known secondary structure of related cytokines. In the case of recombinant human IL-10, reduction of the two disulfide bridges by the addition of 1 mm dithiothreitol resulted in a loss of biological activity, but had only a relatively minor effect on the overall secondary structure of the protein (29Windsor W.T. Syto R. Tsarbopoulos A. Zhang R. Durkin J. Baldwin S. Paliwal S. Mui P.W. Pramanik B. Trotta P.P. et al.Biochemistry. 1993; 32: 8807-8815Crossref PubMed Scopus (83) Google Scholar). To test the overall role of N-linked glycosylation in human IL-24, we eliminated all of the glycosylation sites in human IL-24 by mutagenesis (N86Q, N99Q, and N126Q; labeled as Δ1,2,3 in Fig. 3). This construct was transfected into H1299 cells, and the IL-24 was screened for solubility and tumor killing activity. We found that little or no mutated IL-24 was secreted from the H1299 cells (Fig. 3, lane Δ1,2,3, supernatant). When we analyzed the intracellular levels of IL-24, we found that it was also markedly diminished compared with the wild-type controls (Fig. 3, lane Δ1,2,3, lysate). This is likely due to rapid ubiquitination and subsequent intracellular degradation of IL-24 (30Gopalan B. Shanker M. Scott A. Branch C.D. Chada S. Ramesh R. Cancer Gene Ther. 2008; 15: 1-8Crossref PubMed Scopus (19) Google Scholar). Although there are three consensus sites for N-linked glycosylation in the human IL-24 sequence, the actual extent of post-translational modification has not been described in this cytokine. To test whether each individual sequon is available for post-translational modification, we mutated all combinations of pairs of sequons, thereby restricting glycosylation to single sites, and tested for secretion and activity. These constructs were labeled according to the sites deleted by mutagenesis (Δ1,2 = ΔN86/99Q; Δ1,3 = ΔN86/126Q; and Δ2,3 = ΔN99/126Q). The resulting secreted protein products ran on SDS-gels at sizes corresponding to singly glycosylated IL-24 (Fig. 3, lanes Δ1,2, Δ1,3, and Δ2,3). Each of the three double mutants was secreted from H1299 cells at similar levels but significantly less than wild-type levels. Because of the marked reduction in secreted IL-24 levels, we can conclude that a single glycosylation event is not sufficient to mediate wild-type secretion levels in human IL-24. This result also demonstrates that each of the three sequons is amenable to post-translational modification, and the mature secreted cytokine utilizes all three sites. To test the contribution of pairs of glycosylation sites, we mutated individual sequons (Fig. 3, lanes Δ1, Δ2, and Δ3). The predominant band in the Δ1 construct corresponds in mass to IL-24 glycosylated at only sites II and III (Fig. 3, Δ1). Levels of expression for this mutant are comparable to the wild-type secreted protein. The secreted amounts of the Δ3 construct in the media were reproducibly higher relative to the wild-type IL-24; however, immunoreactive lower molecular weight bands corresponding to the single glycosylated species and unglycosylated IL-24 were also present. Surprisingly, the Δ2 mutation displayed a marked reduction in secreted levels in the media. Either the Δ2 site is structurally unique compared to the other two sites or two contiguous glycosylation events must cooperate to overcome unfavorable aggregation properties of the protein surface coincident with helix B. Each of the IL-10-like cytokines whose x-ray structure has been solved possesses multiple sets of disulfide bonds (Fig. 1). The cysteine residues that contribute to these disulfide bonds are highly conserved; therefore, one can usually predict the presence of a homologous disulfide bridge in other family members. IL-24 lacks two of these cysteine residues, making alignment-based prediction of disulfide pairs problematic. Although these two cysteine residues are present in the homologous cytokines, they have never been observed forming a bond with each other. This leaves two possible scenarios for the disulfide connectivity of IL-24. In the first case, the cytokine is monomeric like IL-19 and IL-22. This would force a novel arrangement of disulfide bonds between helices A and C and would likely result in a novel structure for the cytokine (27Zdanov A. Curr. Pharm. Des. 2004; 10: 3873-3884Crossref PubMed Scopus (62) Google Scholar). In the second case, IL-24 could be a dimer like IL-10 and use this disulfide to inter-connect the two molecules. However, the disulfide-linked IL-10 dimer has only been observed in the cytomegalovirus IL-10 (31Jones B.C. Logsdon N.J. Josephson K. Cook J. Barry P.A. Walter M.R. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 9404-9409Crossref PubMed Scopus (116) Google Scholar), so this possibility is
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