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Radiation Inactivation of Human Y-Interferon: Cellular Activation Requires Two Dimers (Target Size/Receptor) JEROME A

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Radiation Inactivation of Human Y-Interferon: Cellular Activation Requires Two Dimers (Target Size/Receptor) JEROME A Proc. Nati. Acad. Sci. USA Vol. 91, pp. 5818-5822, June 1994 Biophysics Radiation inactivation of human y-interferon: Cellular activation requires two dimers (target size/receptor) JEROME A. LANGER*t, ABBAS RASHIDBAIGI*t, GIANNI GAROTTA§, AND ELLIS KEMPNER¶ *Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854; §Department of Immunology, Hoffmann-La Roche, Basel, Switzerland; and ILaboratory of Physical Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892 Communicated by Martin Rodbell, March 22, 1994 (receivedfor review November 17, 1993) ABSTRACT -Interferon (IFN-y) is a 17-kDa broad- receptor (7, 15-17) and the biochemical pathways leading to spectrum cytokine which exerts its effects on a variety oftarget cellular activation (e.g., refs. 18-22). cells through its interaction with the IFN-y receptor. Although Irradiation of macromolecules leads to their destruction physicochemical studies of Escherichia coUl-derived IFN-y, as and the consequent reduction of measured parameters, such well as its crystal structure, demonstrate that it is a homodimer as biological activity (23). Quantitative measurements of the in solution (Mr 34,000), previous radiation inactivation studies dose-response curve allow the calculation ofa "target size," yielded a functional size for IFN-yof 63-73 kDa in an antiviral understood as the mass of the unit responsible for the assay. To understand the relationship between the solution measured parameter. form ofIFN-yand the moiety that actually binds to the cellular Radiation inactivation techniques have been applied to receptor and activates cells, we examined irradiated nonradio- several IFNs to determine their target size for stimulating active and 32P-labeled IFN-y for its migration in SDS/poly- cellular responses (24, 25). In a standard cytopathic-effect acrylamide gels (to determine its physical integrity), its binding (antiviral) assay, the functional size for purified recombinant to cells, its reactivity in an ELISA, and its antiviral activity. IFN-y from E. coli was 73 ± 6 kDa, about 4 times the The functional size of IFN-y differed in the assays, being 22 ± monomer mass. It was concluded that a molecular tetramer 2 kDa for the physical destruction ofIFN-y, 56 ± 2 kDa for the of IFN-y is required for activation of cells in the antiviral cellular binding assay, 45-50 kDa for reactivity in the ELISA, assay; however, it was noted that " . it is not clear how and 72 ± 6 kDa for antiviral activity. The results from the these functional sizes related to . their receptor interac- binding assays constitute direct evidence that binds to its tions that lead to generation of the antiviral state" (25). IFN-y However, one IFN-y dimer binds to two molecules of a cellular receptor as a dimer. However, for antiviral activity, the soluble form of the receptor, although this seems to be in functional mass is equivalent to a tetramer. This is consistent equilibrium with a complex of one IFN-y dimer with one with models involving ligand-induced receptor dimerization, molecule of soluble receptor (15). These studies suggesting whereby two dimers acting in concert (equivalent to the target the binding of an IFN-ydimer appear to be inconsistent with size of a tetramer) are required to activate cells in the antiviral the previous radiation inactivation studies implicating a func- assay. tional IFN-y tetramer for cellular activation. We have now irradiated highly active 32P-labeled IFN-y ,-Interferon (IFN-y) is a cytokine identified as an IFN by (26, 27) to compare directly the functional size of IFN-y for virtue of its broad-spectrum antiviral activity. It also has a binding to its cell surface receptor and for triggering biolog- range of immunomodulatory activities, being a primary ac- ical (e.g., antiviral) activity. The finding that IFN-y binds to tivator ofmacrophages and a physiological inducer ofclass II cells as a dimer but appears to trigger biological responses as major histocompatibility complex (MHC)-encoded mole- a tetramer leads to the conclusion that biological activity cules (1, 2). requires more than the simple binding of IFN-y to its recep- Recombinant human IFN-y expressed in Escherichia coli tor, leading us to suggest models consistent with these is a physiologically active, unglycosylated molecule with a observations. mass of 17 kDa (3, 4). Purified natural (glycosylated) IFN-y or recombinant human IFN-yfrom E. coli behaves as a dimer EXPERIMENTAL in solution, at or near neutral pH, with no evidence for higher PROCEDURES molecular weight species or for dissociation to monomers IFNs and Antiviral Assays. Recombinant human IFN-ywas (5-7). IFN-y in crystals is a dimer (8, 9), and a genetically isolated from E. coli and was radiophosphorylated with engineered covalent IFN-ydimer, with monomers connected [y-32P]ATP by using cAMP-dependent protein kinase (26, tail-to-head by an IgA hinge peptide, has high biological 27). The ability of irradiated or unirradiated IFN- y to protect activity (10). cells from virus killing was assayed on human WISH cells IFN-y stimulates a variety of responses in target cells with vesicular stomatitis virus (VSV) or with encephalomy- through interaction with its cellular receptor (1, 2). The ocarditis virus (EMCV) as described (28). In brief, serially IFN-y receptor (IFN-yR) cDNA has been cloned and ex- diluted irradiated or unirradiated IFN-y was incubated with pressed (11). A receptor-associated accessory factor required cells at 370C for 6-16 hr. Virus was then added and the IFN-y for IFN-a activation has been identified and cloned (12, 13), titer was determined 24-48 hr later as the concentration of and the existence of additional accessory factors has been IFN-y that provided 50% protection of cells, as judged by deduced (14). Biochemical and molecular biological studies have begun to elucidate the interaction of IFN-y with its Abbreviations: IFN, interferon; IFN-yR, IFN-y receptor; VSV, vesicular stomatitis virus; EMCV, encephalomyocarditis virus; BSA, bovine serum albumin. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tPresent address: Interferon Sciences, 783 Jersey Avenue, New in accordance with 18 U.S.C. §1734 solely to indicate this fact. Brunswick, NJ 08901. 5818 Downloaded by guest on September 24, 2021 Biophysics: Langer et al. Proc. Natl. Acad. Sci. USA 91 (1994) 5819 staining live cells with crystal violet. Laboratory standards of total binding for unirradiated samples (2000-7000 cpm in calibrated against international reference standards were various experiments). Cells and IFN-y were incubated at included. either 40C for 2.5 hr or 230C for 1-1.5 hr with gentle rocking. Irradiation and Processing of Samples. Samples of nonra- The slopes ofthe radiation decay curves were the same at 40C dioactive or 32P-labeled IFN-y were prepared for radiation and 230C. In some experiments with unirradiated IFN-'y, inactivation as follows. For IFN-y, samples were at 10o preliminary saturation binding curves were measured. A units/ml (10 ug/ml) in either Dulbecco's modified Eagle's concentration of 32P-labeled IFN-y corresponding to s60% medium (DMEM) or Eagle's minimal essential medium, each of saturation was then used for the subsequent binding containing 10%o fetal bovine serum. For 32P-labeled IFN--y, experiments with irradiated ligand, to ensure a linear dose- -3 pg ofIFN-y was phosphorylated in the presence of 1 mCi response curve. (37 MBq) of [y.32P]ATP to about 0.6 mol Of 32p per mol of ELISA. The ELISA for IFN-y (33) is a sandwich assay, IFN- y (170 t&Ci/,pg) (26, 27). This was diluted to 10-11 ml in employing the same monoclonal antibody (y69; ref. 34) for phosphate-buffered saline (PBS) containing 0.5% bovine binding to the solid support and for detection. The ELISA serum albumin (BSA). Samples of0.5 ml were placed in 2-ml detects the biologically active dimer, but not denatured siliconized glass ampules and frozen on dry ice. Vials were IFN-'y (33, 34). rapidly sealed with an oxygen/gas flame, with no thawing Data Analysis. In the simplest cases, the survival ofactivity observed. Storage of samples was at -70'C or lower prior to as a function of radiation dose is expected to be a simple and following irradiation. Shipment of samples was on dry exponential decay (23, 35). From both fundamental consid- ice. Samples were not thawed until ready for assay. erations and the details of the experimental protocol, it has In several experiments, samples seemed to be at least twice been demonstrated that the target size (mass, M, in kilodal- as sensitive to radiation in all assays (i.e., to have higher tons) can be calculated from the exponential activity decay calculated functional sizes). The addition of ascorbate to the curve from the equation: PBS/BSA formulation or the use of an entirely different medium reduced or eliminated spurious and variable radia- M = (1.79 x 106)/D37 tion sensitization. Thus, improved buffer conditions are (i) PBS with BSA, as above, but supplemented with 10 mM where D37 is the radiation dose (in megarads; 1 rad = 0.01 Gy) ascorbate; or (ii) 10 mM Tris-HCl, pH 7.5, with apotransfer- at which the activity is reduced to 37% of that in the rin at 4 mg/ml (Sigma catalogue no. T2252). It is likely that unirradiated control. the divergent results arose from a radiosensitizing effect of Values reported here were derived from a linear least- phosphate, as observed in several other systems (29).
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