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• W2890524303 abstract "IFNϵ and IFNκ are interferons that induce microbial immunity at mucosal surfaces and in the skin. They are members of the type-I interferon (IFN) family, which consists of 16 different IFNs, that all signal through the common IFNAR1/IFNAR2 receptor complex. Although IFNϵ and IFNκ have unique expression and functional properties, their biophysical properties have not been extensively studied. In this report, we describe the expression, purification, and characterization of recombinant human IFNϵ and IFNκ. In cellular assays, IFNϵ and IFNκ exhibit ∼1000-fold lower potency than IFNα2 and IFNω. The reduced potency of IFNϵ and IFNκ are consistent with their weak affinity for the IFNAR2 receptor chain. Despite reduced IFNAR2-binding affinities, IFNϵ and IFNκ exhibit affinities for the IFNAR1 chain that are similar to other IFN subtypes. As observed for cellular IFNAR2 receptor, the poxvirus antagonist, B18R, also exhibits reduced affinity for IFNϵ and IFNκ, relative to the other IFNs. Taken together, our data suggest IFNϵ and IFNκ are specialized IFNs that have evolved to weakly bind to the IFNAR2 chain, which allows innate protection of the mucosa and skin and limits neutralization of IFNϵ and IFNκ biological activities by viral IFN antagonists. IFNϵ and IFNκ are interferons that induce microbial immunity at mucosal surfaces and in the skin. They are members of the type-I interferon (IFN) family, which consists of 16 different IFNs, that all signal through the common IFNAR1/IFNAR2 receptor complex. Although IFNϵ and IFNκ have unique expression and functional properties, their biophysical properties have not been extensively studied. In this report, we describe the expression, purification, and characterization of recombinant human IFNϵ and IFNκ. In cellular assays, IFNϵ and IFNκ exhibit ∼1000-fold lower potency than IFNα2 and IFNω. The reduced potency of IFNϵ and IFNκ are consistent with their weak affinity for the IFNAR2 receptor chain. Despite reduced IFNAR2-binding affinities, IFNϵ and IFNκ exhibit affinities for the IFNAR1 chain that are similar to other IFN subtypes. As observed for cellular IFNAR2 receptor, the poxvirus antagonist, B18R, also exhibits reduced affinity for IFNϵ and IFNκ, relative to the other IFNs. Taken together, our data suggest IFNϵ and IFNκ are specialized IFNs that have evolved to weakly bind to the IFNAR2 chain, which allows innate protection of the mucosa and skin and limits neutralization of IFNϵ and IFNκ biological activities by viral IFN antagonists. IFNϵ and IFNκ are part of the human type-I interferon (IFN) 2The abbreviations used are: IFNinterferonISGIFN-stimulated geneFRTfemale reproductive trackIAiodoacetamideROreceptor occupancyNAbneutralizing antibodySPRsurface plasmon resonanceSEAPsecretion of embryonic alkaline phosphataseRUresponse unitsPDBProtein Data Bank. family that consists of 16 different cytokines whose signaling properties are critical for the control and elimination of microbial pathogens (1.Samuel C.E. Antiviral actions of interferons.Clin. Microbiol. Rev. 2001; 14 (11585785, table of contents): 778-80910.1128/CMR.14.4.778-809.2001Crossref PubMed Scopus (2139) Google Scholar, 2.Ivashkiv L.B. Donlin L.T. Regulation of type I interferon responses.Nat. Rev. Immunol. 2014; 14 (24362405): 36-4910.1038/nri3581Crossref PubMed Scopus (1761) Google Scholar, 3.Pestka S. Langer J.A. Zoon K.C. Samuel C.E. Interferons and their actions.Annu. Rev. Biochem. 1987; 56 (2441659): 727-77710.1146/annurev.bi.56.070187.003455Crossref PubMed Scopus (1599) Google Scholar, 4.Pfeffer L.M. Dinarello C.A. Herberman R.B. Williams B.R. Borden E.C. Bordens R. Walter M.R. Nagabhushan T.L. Trotta P.P. Pestka S. Biological properties of recombinant α-interferons: 40th anniversary of the discovery of interferons.Cancer Res. 1998; 58 (9635566): 2489-2499PubMed Google Scholar, 5.Borden E.C. Sen G.C. Uze G. Silverman R.H. Ransohoff R.M. Foster G.R. Stark G.R. Interferons at age 50: past, current and future impact on biomedicine.Nat. Rev. Drug Discov. 2007; 6 (18049472): 975-99010.1038/nrd2422Crossref PubMed Scopus (872) Google Scholar). IFNs activate innate immunity through the induction of IFN-stimulated genes (ISGs) that exhibit antiviral activity (2.Ivashkiv L.B. Donlin L.T. Regulation of type I interferon responses.Nat. Rev. Immunol. 2014; 14 (24362405): 36-4910.1038/nri3581Crossref PubMed Scopus (1761) Google Scholar, 6.Der S.D. Zhou A. Williams B.R. Silverman R.H. Identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays.Proc. Natl. Acad. Sci. U.S.A. 1998; 95 (9861020): 15623-1562810.1073/pnas.95.26.15623Crossref PubMed Scopus (1532) Google Scholar, 7.Schoggins J.W. Wilson S.J. Panis M. Murphy M.Y. Jones C.T. Bieniasz P. Rice C.M. A diverse range of gene products are effectors of the type I interferon antiviral response.Nature. 2011; 472 (21478870): 481-48510.1038/nature09907Crossref PubMed Scopus (1641) Google Scholar). IFNs also promote adaptive immunity through up-regulation of major histocompatibility complex and the induction of chemokines (8.Yang C.H. Wei L. Pfeffer S.R. Du Z. Murti A. Valentine W.J. Zheng Y. Pfeffer L.M. Identification of CXCL11 as a STAT3-dependent gene induced by IFN.J. Immunol. 2007; 178 (17202361): 986-99210.4049/jimmunol.178.2.986Crossref PubMed Scopus (42) Google Scholar, 9.Gray R.C. Kuchtey J. Harding C.V. CpG-B ODNs potently induce low levels of IFN-αβ and induce IFN-αβ-dependent MHC-I cross-presentation in DCs as effectively as CpG-A and CpG-C ODNs.J. Leukoc. Biol. 2007; 81 (17227820): 1075-1085Crossref PubMed Scopus (29) Google Scholar, 10.Loh J.E. Chang C.H. Fodor W.L. Flavell R.A. Dissection of the interferon γ-MHC class II signal transduction pathway reveals that type I and type II interferon systems share common signalling component(s).EMBO J. 1992; 11 (1314162): 1351-136310.1002/j.1460-2075.1992.tb05180.xCrossref PubMed Scopus (49) Google Scholar, 11.Zhao W. Cha E.N. Lee C. Park C.Y. Schindler C. Stat2-dependent regulation of MHC class II expression.J. Immunol. 2007; 179 (17579067): 463-47110.4049/jimmunol.179.1.463Crossref PubMed Scopus (37) Google Scholar). They also control cell growth and apoptosis (12.Chawla-Sarkar M. Lindner D.J. Liu Y.F. Williams B.R. Sen G.C. Silverman R.H. Borden E.C. Apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis.Apoptosis. 2003; 8: 237-24910.1023/A:1023668705040Crossref PubMed Scopus (667) Google Scholar, 13.Herzer K. Hofmann T.G. Teufel A. Schimanski C.C. Moehler M. Kanzler S. Schulze-Bergkamen H. Galle P.R. IFN-α-induced apoptosis in hepatocellular carcinoma involves promyelocytic leukemia protein and TRAIL independently of p53.Cancer Res. 2009; 69 (19141642): 855-86210.1158/0008-5472.CAN-08-2831Crossref PubMed Scopus (70) Google Scholar), B-cell lineage commitment (14.de Goër de Herve M.G. Durali D. Dembele B. Giuliani M. Tran T.A. Azzarone B. Eid P. Tardieu M. Delfraissy J.F. Taoufik Y. Interferon-α triggers B cell effector 1 (Be1) commitment.PLoS ONE. 2011; 6 (21559410): e1936610.1371/journal.pone.0019366Crossref PubMed Scopus (24) Google Scholar), and induction of T regulatory cells (15.Liu Y. Carlsson R. Comabella M. Wang J. Kosicki M. Carrion B. Hasan M. Wu X. Montalban X. Dziegiel M.H. Sellebjerg F. Sørensen P.S. Helin K. Issazadeh-Navikas S. FoxA1 directs the lineage and immunosuppressive properties of a novel regulatory T cell population in EAE and MS.Nat. Med. 2014; 20 (24531377): 272-28210.1038/nm.3485Crossref PubMed Scopus (113) Google Scholar, 16.Delgoffe G.M. Vignali D.A. A Fox of a different color: FoxA1 programs a new regulatory T cell subset.Nat. Med. 2014; 20 (24603791): 236-23710.1038/nm.3493Crossref PubMed Scopus (4) Google Scholar). Due to the importance of IFNs, medical applications of IFNs have been developed including the treatment of viral infections (17.Foster G.R. Pegylated interferons for the treatment of chronic hepatitis C: pharmacological and clinical differences between peginterferon-α-2a and peginterferon-α-2b.Drugs. 2010; 70 (20108989): 147-165Crossref PubMed Scopus (92) Google Scholar), cancer (18.Kirkwood J. Cancer immunotherapy: the interferon-alpha experience.Semin. Oncol. 2002; 29 (12068384): 18-2610.1053/sonc.2002.33078Crossref PubMed Scopus (184) Google Scholar), and multiple sclerosis (19.Jacobs L.D. Cookfair D.L. Rudick R.A. Herndon R.M. Richert J.R. Salazar A.M. Fischer J.S. Goodkin D.E. Granger C.V. Simon J.H. Alam J.J. Bartoszak D.M. Bourdette D.N. Braiman J. Brownscheidle C.M. et al.Intramuscular interferon β-1a for disease progression in relapsing multiple sclerosis: the multiple sclerosis collaborative research group (MSCRG).Ann. Neurol. 1996; 39 (8602746): 285-29410.1002/ana.410390304Crossref PubMed Scopus (2367) Google Scholar). interferon IFN-stimulated gene female reproductive track iodoacetamide receptor occupancy neutralizing antibody surface plasmon resonance secretion of embryonic alkaline phosphatase response units Protein Data Bank. The biological activities of all 16 IFNs are initiated upon binding to the cell-surface receptors IFNAR1 and IFNAR2. IFN-IFNAR interactions activate JAK1 and TYK2 kinases and the transcription factors STAT1 and STAT2 (20.Richter M.F. Duménil G. Uze G. Fellous M. Pellegrini S. Specific contribution of Tyk2 JH regions to the binding and the expression of the interferon α/β receptor component IFNAR1.J. Biol. Chem. 1998; 273 (9733772): 24723-2472910.1074/jbc.273.38.24723Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 21.Colamonici O.R. Uyttendaele H. Domanski P. Yan H. Krolewski J.J. p135tyk2, an interferon-α-activated tyrosine kinase, is physically associated with an interferon-alpha receptor.J. Biol. Chem. 1994; 269 (8106393): 3518-3522Abstract Full Text PDF PubMed Google Scholar, 22.Domanski P. Fish E. Nadeau O.W. Witte M. Platanias L.C. Yan H. Krolewski J. Pitha P. Colamonici O.R. A region of the β subunit of the interferon α receptor different from box 1 interacts with Jak1 and is sufficient to activate the Jak-Stat pathway and induce an antiviral state.J. Biol. Chem. 1997; 272 (9334213): 26388-2639310.1074/jbc.272.42.26388Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 23.Stark G.R. Darnell Jr., J.E. The JAK-STAT pathway at twenty.Immunity. 2012; 36 (22520844): 503-51410.1016/j.immuni.2012.03.013Abstract Full Text Full Text PDF PubMed Scopus (951) Google Scholar). JAK/STAT signaling, and additional kinases and transcription factors (24.Platanias L.C. Mechanisms of type-I- and type-II-interferon-mediated signalling.Nat. Rev. Immunol. 2005; 5 (15864272): 375-38610.1038/nri1604Crossref PubMed Scopus (2316) Google Scholar), ultimately induce IFN gene expression programs that protect the host from virus, bacteria, and even fungi (5.Borden E.C. Sen G.C. Uze G. Silverman R.H. Ransohoff R.M. Foster G.R. Stark G.R. Interferons at age 50: past, current and future impact on biomedicine.Nat. Rev. Drug Discov. 2007; 6 (18049472): 975-99010.1038/nrd2422Crossref PubMed Scopus (872) Google Scholar, 7.Schoggins J.W. Wilson S.J. Panis M. Murphy M.Y. Jones C.T. Bieniasz P. Rice C.M. A diverse range of gene products are effectors of the type I interferon antiviral response.Nature. 2011; 472 (21478870): 481-48510.1038/nature09907Crossref PubMed Scopus (1641) Google Scholar, 25.Li T. Niu X. Zhang X. Wang S. Liu Z. Recombinant human IFNα-2b response promotes vaginal epithelial cells defense against Candida albicans.Front. Microbiol. 2017; 8 (28473823): 69710.3389/fmicb.2017.00697Crossref PubMed Scopus (7) Google Scholar, 26.Fung K.Y. Mangan N.E. Cumming H. Horvat J.C. Mayall J.R. Stifter S.A. De Weerd N. Roisman L.C. Rossjohn J. Robertson S.A. Schjenken J.E. Parker B. Gargett C.E. Nguyen H.P. Carr D.J. Hansbro P.M. Hertzog P.J. Interferon-ϵ protects the female reproductive tract from viral and bacterial infection.Science. 2013; 339 (23449591): 1088-109210.1126/science.1233321Crossref PubMed Scopus (156) Google Scholar). Due to the critical role IFNs play in protecting the host from infection, many pathogens produce proteins that block IFN activity at multiple steps in the IFN signaling pathway. For example, Dengue, West Nile, and Zika viruses disrupt IFN-mediated STAT2 signaling (27.Jones M. Davidson A. Hibbert L. Gruenwald P. Schlaak J. Ball S. Foster G.R. Jacobs M. Dengue virus inhibits α interferon signaling by reducing STAT2 expression.J. Virol. 2005; 79 (15827155): 5414-542010.1128/JVI.79.9.5414-5420.2005Crossref PubMed Scopus (209) Google Scholar, 28.Grant A. Ponia S.S. Tripathi S. Balasubramaniam V. Miorin L. Sourisseau M. Schwarz M.C. Sanchez-Seco M.P. Evans M.J. Best S.M. Garcia-Sastre A. Zika virus targets human STAT2 to inhibit type I interferon signaling.Cell Host Microbe. 2016; 19 (27212660): 882-89010.1016/j.chom.2016.05.009Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar). In contrast, poxviruses encode IFN-binding proteins that neutralize IFN activity by binding to secreted IFNs, which prevents them from engaging cell-surface IFNAR1 and IFNAR2 (29.Seet B.T. Johnston J.B. Brunetti C.R. Barrett J.W. Everett H. Cameron C. Sypula J. Nazarian S.H. Lucas A. McFadden G. Poxviruses and immune evasion.Annu. Rev. Immunol. 2003; 21 (12543935): 377-42310.1146/annurev.immunol.21.120601.141049Crossref PubMed Scopus (490) Google Scholar). The IFN-binding proteins, B18R and B19R, were identified in vaccinia virus strains Western Reserve and Copenhagen, respectively (30.Symons J.A. Alcamí A. Smith G.L. Vaccinia virus encodes a soluble type I interferon receptor of novel structure and broad species specificity.Cell. 1995; 81 (7758109): 551-56010.1016/0092-8674(95)90076-4Abstract Full Text PDF PubMed Scopus (422) Google Scholar, 31.Colamonici O.R. Domanski P. Sweitzer S.M. Larner A. Buller R.M. Vaccinia virus B18R gene encodes a type I interferon-binding protein that blocks interferon α transmembrane signaling.J. Biol. Chem. 1995; 270 (7608155): 15974-1597810.1074/jbc.270.27.15974Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). B18R/B19R encode the same secreted ∼65-kDa glycoprotein that promiscuously binds to all of the type-I IFNs (32.Huang J. Smirnov S.V. Lewis-Antes A. Balan M. Li W. Tang S. Silke G.V. Pütz M.M. Smith G.L. Kotenko S.V. Inhibition of type I and type III interferons by a secreted glycoprotein from Yaba-like disease virus.Proc. Natl. Acad. Sci. U.S.A. 2007; 104 (17517620): 9822-982710.1073/pnas.0610352104Crossref PubMed Scopus (56) Google Scholar). Deletion of B18R from vaccinia virus resulted in an attenuated virus, emphasizing the importance of the type-I IFNs in controlling vaccinia virus infection (30.Symons J.A. Alcamí A. Smith G.L. Vaccinia virus encodes a soluble type I interferon receptor of novel structure and broad species specificity.Cell. 1995; 81 (7758109): 551-56010.1016/0092-8674(95)90076-4Abstract Full Text PDF PubMed Scopus (422) Google Scholar, 31.Colamonici O.R. Domanski P. Sweitzer S.M. Larner A. Buller R.M. Vaccinia virus B18R gene encodes a type I interferon-binding protein that blocks interferon α transmembrane signaling.J. Biol. Chem. 1995; 270 (7608155): 15974-1597810.1074/jbc.270.27.15974Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). IFNϵ and IFNκ are unique from the other IFNs based on their amino acid sequences and limited expression in mucosa and skin. IFNϵ and IFNκ share 35% sequence identity with one another and the 14 other IFNs. The additional 14 IFNs consist of 12 IFNα subtypes, IFNω, and IFNβ. The IFNαs share 77–95% sequence identity with one another. IFNβ exhibits 35% sequence identify with all other IFNs, whereas IFNω shares the highest sequence identity (60%) with both IFNϵ/κ/β and the IFNαs. Based on sequence and structural comparisons, all IFNs exhibit an α-helical fold consisting of five helices, which are labeled from the N terminus as helix A, B, C, D, E, and F (33.Pestka S. Krause C.D. Sarkar D. Walter M.R. Shi Y. Fisher P.B. Interleukin-10 and related cytokines and receptors.Annu. Rev. Immunol. 2004; 22 (15032600): 929-97910.1146/annurev.immunol.22.012703.104622Crossref PubMed Scopus (939) Google Scholar, 34.Pestka S. Krause C.D. Walter M.R. Interferons, interferon-like cytokines, and their receptors.Immunol. Rev. 2004; 202 (15546383): 8-3210.1111/j.0105-2896.2004.00204.xCrossref PubMed Scopus (1276) Google Scholar, 35.Walter M.R. Structural analysis of IL-10 and type I interferon family members and their complexes with receptor.Adv. Protein Chem. 2004; 68 (15500862): 171-22310.1016/S0065-3233(04)68006-5Crossref PubMed Scopus (41) Google Scholar). A unique feature of IFNϵ is that it encodes an 18-amino acid C-terminal tail following helix F, whereas IFNκ has a 13-amino acid peptide insertion between helices D and E. In addition to these novel sequence features, IFNϵ and IFNκ are expressed predominantly in the female reproductive track (FRT) and keratinocytes, respectively (26.Fung K.Y. Mangan N.E. Cumming H. Horvat J.C. Mayall J.R. Stifter S.A. De Weerd N. Roisman L.C. Rossjohn J. Robertson S.A. Schjenken J.E. Parker B. Gargett C.E. Nguyen H.P. Carr D.J. Hansbro P.M. Hertzog P.J. Interferon-ϵ protects the female reproductive tract from viral and bacterial infection.Science. 2013; 339 (23449591): 1088-109210.1126/science.1233321Crossref PubMed Scopus (156) Google Scholar, 36.LaFleur D.W. Nardelli B. Tsareva T. Mather D. Feng P. Semenuk M. Taylor K. Buergin M. Chinchilla D. Roshke V. Chen G. Ruben S.M. Pitha P.M. Coleman T.A. Moore P.A. Interferon-κ, a novel type I interferon expressed in human keratinocytes.J. Biol. Chem. 2001; 276 (11514542): 39765-3977110.1074/jbc.M102502200Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). In fact, analysis of the IFNϵ gene identified putative progesterone-binding sites in the promoter, suggesting hormones regulate IFNϵ expression (37.Hardy M.P. Owczarek C.M. Jermiin L.S. Ejdebäck M. Hertzog P.J. Characterization of the type I interferon locus and identification of novel genes.Genomics. 2004; 84 (15233997): 331-34510.1016/j.ygeno.2004.03.003Crossref PubMed Scopus (172) Google Scholar). Subsequent studies confirmed IFNϵ expression is induced by hormones but not by viral infection (e.g. TLRs) like other IFNs (26.Fung K.Y. Mangan N.E. Cumming H. Horvat J.C. Mayall J.R. Stifter S.A. De Weerd N. Roisman L.C. Rossjohn J. Robertson S.A. Schjenken J.E. Parker B. Gargett C.E. Nguyen H.P. Carr D.J. Hansbro P.M. Hertzog P.J. Interferon-ϵ protects the female reproductive tract from viral and bacterial infection.Science. 2013; 339 (23449591): 1088-109210.1126/science.1233321Crossref PubMed Scopus (156) Google Scholar). Consistent with IFNϵ's expression in the reproductive tract, IFNϵ is able to induce important restriction factors that prevent HIV-1 infection (38.Garcia-Minambres A. Eid S.G. Mangan N.E. Pade C. Lim S.S. Matthews A.Y. de Weerd N.A. Hertzog P.J. Mak J. Interferon ϵ promotes HIV restriction at multiple steps of viral replication.Immunol. Cell Biol. 2017; 95 (28045025): 478-48310.1038/icb.2016.123Crossref PubMed Scopus (24) Google Scholar, 39.Tasker C. Subbian S. Gao P. Couret J. Levine C. Ghanny S. Soteropoulos P. Zhao X. Landau N. Lu W. Chang T.L. IFN-ϵ protects primary macrophages against HIV infection.JCI insight. 2016; 1 (27942584): e88255Crossref PubMed Scopus (23) Google Scholar). In fact, HIV-1 negative female sex workers express high levels of IFNϵ in their cervical tissues (40.Abdulhaqq S.A. Zorrilla C. Kang G. Yin X. Tamayo V. Seaton K.E. Joseph J. Garced S. Tomaras G.D. Linn K.A. Foulkes A.S. Azzoni L. VerMilyea M. Coutifaris C. Kossenkov A.V. et al.HIV-1-negative female sex workers sustain high cervical IFN-ϵ, low immune activation, and low expression of HIV-1-required host genes.Mucosal Immunol. 2016; 9 (26555708): 1027-103810.1038/mi.2015.116Crossref PubMed Scopus (21) Google Scholar). IFNκ is constitutively expressed in keratinocytes, which are found in skin and in the mucosa of the FRT. Thus, IFNκ and IFNϵ are both found in the FRT, but IFNκ is inducible by virus and dsRNA, whereas IFNϵ is not (36.LaFleur D.W. Nardelli B. Tsareva T. Mather D. Feng P. Semenuk M. Taylor K. Buergin M. Chinchilla D. Roshke V. Chen G. Ruben S.M. Pitha P.M. Coleman T.A. Moore P.A. Interferon-κ, a novel type I interferon expressed in human keratinocytes.J. Biol. Chem. 2001; 276 (11514542): 39765-3977110.1074/jbc.M102502200Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Although it appears that IFNϵ appears to play an important role in immunity against HIV-1, IFNκ expression is rapidly reduced in keratinocytes that are infected with human papilloma virus strains that induce cervical cancer (41.Reiser J. Hurst J. Voges M. Krauss P. Münch P. Iftner T. Stubenrauch F. High-risk human papillomaviruses repress constitutive κ interferon transcription via E6 to prevent pathogen recognition receptor and antiviral-gene expression.J. Virol. 2011; 85 (21849431): 11372-1138010.1128/JVI.05279-11Crossref PubMed Scopus (111) Google Scholar). These data strongly argue that IFNϵ and IFNκ are essential components to the host response against pathogens in the FRT, whereas the specific role of IFNκ, produced by keratinocytes in the skin, remains to be determined. Despite a critical role of IFNϵ and IFNκ signaling in mucosa and skin, their interactions with the IFNARs and their functional activities have not been extensively characterized. To address this issue, we have expressed human IFNϵ and IFNκ for comparative biophysical and functional studies with other IFN family members (often called IFN subtypes). Studies with purified IFNϵ and IFNκ reveal they induce ISFG3-mediated gene expression that is ∼1000-fold weaker than IFNα2 or IFNω. The weaker potency of IFNϵ and IFNκ is consistent with their reduced affinities for the IFNAR2 receptor chain. However, IFNϵ and IFNκ exhibit IFNAR1 binding affinity that is similar to IFNα2 and IFNω. Because poxviruses cause disseminated infections of the skin and mucosa and can block type-I IFN signaling, we evaluated the ability of B18R to neutralize IFNϵ and IFNκ cellular activity. Subsequent kinetic binding studies determined IFNϵ, IFNκ, as well as IFNα1, exhibit reduced binding to B18R, relative to the other IFNs. Sequence and structural models of IFNϵ/κ-IFNAR2 and IFNϵ/κ-B18R complexes identified residues responsible for the disrupted IFNAR2 and B18R binding phenotypes. Our data suggests IFNϵ and IFNκ have evolved to exhibit reduced IFNAR2 binding affinity and biological potency optimized for tissue-specific expression and escape from viral type-I IFN antagonists. Expression plasmids encoding human IFNϵ and IFNκ protein sequences, which also encode C-terminal histidine tags, were synthesized using optimized codons. Expression studies were performed in Escherichia coli where IFNϵ and IFNκ formed insoluble inclusion bodies. The guanidine-solubilized IFNs were refolded by rapid dilution into a refolding buffer. The refolded IFNs were purified using a 2-step strategy consisting of nickel affinity and cation exchange chromatography. Endotoxin levels for IFNϵ and IFNκ were less than 1 EU/μg, which is within the range of values observed in IFNα preparations obtained from commercial sources (42.Kuruganti S. Accavitti-Loper M.A. Walter M.R. Production and characterization of thirteen human type-I interferon-α subtypes.Protein Expr. Purif. 2014; 103 (25149396): 75-8310.1016/j.pep.2014.08.010Crossref PubMed Scopus (11) Google Scholar). SDS-PAGE gel analysis of the final purified IFNϵ and IFNκ protein preparations is shown in Fig. 1. The molecular weight of IFNϵ on SDS-PAGE gels matched its theoretical molecular weight of 23,249. However, IFNκ ran larger than expected (∼25,000 kDa) in SDS-PAGE gels, suggesting the peptide could be a frameshift product (43.Yoon S.I. Walter M.R. Identification and characterization of a +1 frameshift observed during the expression of Epstein-Barr virus IL-10 in Escherichia coli.Protein Expr. Purif. 2007; 53 (17224278): 132-13710.1016/j.pep.2006.12.001Crossref PubMed Scopus (6) Google Scholar) or exhibit aberrant gel migration. To resolve these possibilities, MS was performed on the samples, which demonstrated the molecular masses were consistent with full-length IFNϵ and IFNκ proteins without the N-terminal initiating methionine residues (Table 1).Table 1Mass spectrometry of IFNϵ and IFNκTreatmentObserved massExpected massaCalculated mass without N-terminal methionine.DeltabMass difference between expected and observed masses.InterpretationIFNϵNone23,08223,11836IA23,14523,1396+ 1 IAIA + DTT23,25623,2533+ 3 IAIFNκNone23,20123,22019IA23,25923,2581+ 1 IAIA + DTT23,49623,48610+ 5 IAa Calculated mass without N-terminal methionine.b Mass difference between expected and observed masses. Open table in a new tab The IFNϵ and IFNκ amino acid sequences include three and five cysteine residues, respectively. Based on prior structural and biophysical analysis of other IFNs (42.Kuruganti S. Accavitti-Loper M.A. Walter M.R. Production and characterization of thirteen human type-I interferon-α subtypes.Protein Expr. Purif. 2014; 103 (25149396): 75-8310.1016/j.pep.2014.08.010Crossref PubMed Scopus (11) Google Scholar, 44.Radhakrishnan R. Walter L.J. Hruza A. Reichert P. Trotta P.P. Nagabhushan T.L. Walter M.R. Zinc mediated dimer of human interferon-α 2b revealed by X-ray crystallography.Structure. 1996; 4 (8994971): 1453-146310.1016/S0969-2126(96)00152-9Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar), IFNϵ and IFNκ are predicted to contain one free cysteine and form one and two disulfide bonds, respectively, in their folded forms. To confirm disulfide bond formation had occurred during the refolding process, IFNϵ and IFNκ were treated with iodoacetamide (IA) in the presence, or absence, of the reducing agent dithiothreitol (DTT, Table 1). IA selectively binds to free cysteines, resulting in an increase in mass of 57 Da. Upon IA treatment in the absence DTT, the mass of IFNϵ and IFNκ increased by 57 Da, which is consistent with one free cysteine in the folded proteins (Table 1). However, in the presence of DTT, the mass of IFNϵ and IFNκ increased by three IA and five IA mass units, respectively. This is consistent with the protection of two (IFNϵ) and four (IFNκ) cysteines, due to disulfide bond formation, in the folded proteins (Table 1). Thus, the MS data are consistent with the predicted IFNϵ and IFNκ disulfide bonding patterns. Three preparations of IFNϵ and IFNκ were characterized for their ability to bind to soluble IFNARs (Fig. 2). The IFNs were injected over Biacore chips coupled with IFNAR1-FC, IFNAR2-FC, or an IFNAR1/IFNAR2-FC heterodimer, as previously described (45.Deshpande A. Putcha B.D. Kuruganti S. Walter M.R. Kinetic analysis of cytokine-mediated receptor assembly using engineered FC heterodimers.Protein Sci. 2013; 22 (23703950): 1100-110810.1002/pro.2285Crossref PubMed Scopus (11) Google Scholar). Binding is reported as receptor occupancy (RO) for each IFN. For comparison with other IFNs, binding studies were also performed with IFN subtypes, IFNα2 and IFNω. IFNα2 and IFNω bound to IFNAR2 with RO values of 53 and 69%, respectively (Fig. 2). In contrast, IFNϵ and IFNκ bound very poorly to IFNAR2, exhibiting IFNAR2 occupancies of 3 and 5%, respectively. Despite poor IFNAR2-binding properties, IFNϵ bound to IFNAR1 (RO = 26%) better than IFNα2 (RO = 19%), whereas IFNκ-IFNAR1 RO values were lower than IFNα2 (RO = 11%). In addition to binding to the single IFNARs, we evaluated IFN binding to an IFNAR1/IFNAR2-FC heterodimer, which positions IFNAR1 and IFNAR2 close to one another in space through the FC heterodimer (45.Deshpande A. Putcha B.D. Kuruganti S. Walter M.R. Kinetic analysis of cytokine-mediated receptor assembly using engineered FC heterodimers.Protein Sci. 2013; 22 (23703950): 1100-110810.1002/pro.2285Crossref PubMed Scopus (11) Google Scholar). IFNϵ and IFNκ binding to IFNAR1/IFNAR2-FC remained low, relative to IFNα2 and IFNω. However, IFNκ binding, in particular, was significantly enhanced (RO = 36%) relative to the IFNAR2 and IFNAR1 single receptor experiments. The biological activity of IFNϵ and IFNκ were compared against IFNω and IFNα2 using a reporter cell line (HL116), which contains a firefly luciferase gene downstream of the IFI6 promoter. Dose-response curves were generated from at least six independent measurements, to derive half-maximal effective concentrations (EC50, Fig. 2B, Table 2). Consistent with the receptor binding data, IFNϵ (60 nm ± 26 nm) and IFNκ (22 ± 16 nm) exhibited ∼1000-fold lower EC50 values, compared with IFNα2 (11 ± 3 pm) or IFNω (6 ± 1 pm). The dose-response curves were repeated using a different reporter cell line that contains the ISG54 promoter, with similar results.Table 2Surface plasmon resonance-derived binding constants and ISFG3 assay EC50 valueskakdKDm−1 s−1s−1mIFNα2IFNAR1——2.3 (± 0.5) × 10−6EC50 = 11 pm (±3 pm)IFNAR25.9 (± 0.4) × 1060.031 (± 0.002)5.3 (± 0.2) × 10−9IFNAR1/28 (± 1) × 1064.2 (± 0.4) × 10−454 (± 3) × 10−12IFNϵIFNAR1——2.2 (± 0.3) × 10−6EC50 = 60 nm (±26 nm)IFNAR24 (± 3) × 1050.01 (± 0.01)70 (± 16) × 10−9IFNAR1/23.3 (± 0.8) × 1059 (± 1) × 10−43.5 (± 0.8) × 10−9IFNωIFNAR1——0.5 (± 0.1) × 10−6EC50 = 6 pm (±1 pm)IFNAR27 (± 2) × 1070.01 (± 0.07)237 (± 37) × 10−12IFNAR1/23 (± 2) × 1084 (± 2) × 10−325 (± 8) × 10−12IFNκIFNAR1——0.34 (± 0.13) × 10−6EC50 = 22 nm (±16 nm)IFNAR21.2 (± 0.2) × 1052.1 (± 0.3) × 10−321 (± 4) × 10−9IFNAR1/24 (± 2) × 1058 (± 1) × 10−42 (± 1) × 10−9IFNκ-M2IFNAR1/27 (± 2) × 1053 (± 1) × 10−40.4 (± 0.3) × 10−9EC50 = 4 nm (±2 nm)IFNα1IFNAR1——0.4 × 10−6EC50 = 258 pm (±73 pm)IFNAR25 (± 0.1) × 1050.18 (± 0.02)353 (± 83) × 10−9IFNAR1/21.8 (± 0.2) × 1064 (± 2) × 10−4222 (± 92) × 10−12IFNα1-M1IFNAR1——0.2 × 10−6EC50 = 26 pm (±10 pm)IFNAR22.4 (± 0.1) × 1060.06 (± 0.01)26" @default.
• W2890524303 created "2018-09-27" @default.
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• W2890524303 date "2018-10-01" @default.
• W2890524303 modified "2023-10-17" @default.
• W2890524303 title "Human interferon-ϵ and interferon-κ exhibit low potency and low affinity for cell-surface IFNAR and the poxvirus antagonist B18R" @default.
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• W2890524303 doi "https://doi.org/10.1074/jbc.ra118.003617" @default.
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