Soluble Interferon Gamma Receptor Research Articles

Urinary evaluation of the balance between soluble interferon-gamma receptor (IFN-γR1) and interleukin-4 receptor (IL-4Rα)

To elucidate the usefulness of the simultaneous analysis of multiple kinds of soluble cytokine receptors in urine specimens, we determined the levels of both the soluble interferon-gamma receptor α chain (sIFN-γR1, Th1-type cytokine receptor) and the soluble interleukin 4-receptor α chain (sIL-4Rα, Th2-type cytokine receptor) in the urine of healthy subjects as reference values and preliminarily applied this method to evaluate patients with diarrhea positive (D+) hemolytic uremic syndrome (HUS) as the diagnostic parameters. The urinary sIFN-γR levels of children were significantly lower than those of adults (p < 0.01, n = 107). On the other hand, there was no significant difference between the urine sIL-4R levels of adults and children. Statistical correlation between sIFN-γR and sIL-4R values was not observed (p = 0.705). On the day of onset of HUS, the urine sIFN-γR levels of the patients (n = 6) with HUS were higher than those of the healthy control group (n = 67) (p < 0.01); however, there was no significant difference in the sIL-4R levels between both groups. The urine evaluation of the balance between the soluble cytokine receptors might be informative for the immune states of HUS patients.

Age-Dependent Decrease in Serum Soluble Interferon-Gamma Receptor (sIFN-γR) in Healthy Japanese Individuals; Population Study of Serum sIFN-γR Level in Japanese

We planned to investigate the clinical significance of serum soluble interferon-gamma receptor (sIFN-γR) level in pediatric patients. The diagnostic application of the measurement of serum sIFN-γR level depends critically on the control value. However, there is no information of the control value of serum sIFN-γR for children. In the present study, we determined the serum sIFN-γR level of healthy Japanese children using an ELISA. The serum sIFN-γR level of children (0–14 years old) was significantly higher than that of adults (over 15 years old) (p < 0.01, n = 104). Thus, it is recommended that, when the serum sIFN-γR level of patients is evaluated, it should be compared against age-matched controls. We also preliminarily applied this assay as a diagnostic parameter for the patients with diarrhea positive (D +) hemolytic uremic syndrome (HUS).

The vaccinia virus soluble interferon-gamma receptor is a homodimer.

The vaccinia virus (VV) interferon (IFN)-gamma receptor (IFN-gammaR) is a 43 kDa soluble glycoprotein that is secreted from infected cells early during infection. Here we demonstrate that the IFN-gammaR from VV, cowpox virus and camelpox virus exists naturally as a homodimer, whereas the cellular IFN-gammaR dimerizes only upon binding the homodimeric IFN-gamma. The existence of the virus protein as a dimer in the absence of ligand may provide an advantage to the virus in efficient binding and inhibition of IFN-gamma in solution.

High levels of soluble IFN gamma receptor alpha chain in the plasma of rheumatoid arthritis patients.

Soluble receptors for hormones and cytokines have been described. They can serve as natural blockers of their respective ligands. The natural soluble interferon gamma receptor (sIFN gamma R) has been isolated and characterized only in urine. Chromatography of human (hu) plasma from rheumatoid arthritis (RA) patients and controls on immobilized hu IFN gamma or antibodies against IFN gamma R alpha chain permitted us to isolate the sIFN gamma R. The receptor isolated from one control is a protein with a molecular weight between 60-67 kDa depending on the presence of reducing agents. We detected a significantly higher level of plasma sIFN gamma R in patients with rheumatoid arthritis than in apparently healthy subjects.

The effects of a nonimmunogenic form of murine soluble interferon-gamma receptor on the development of autoimmune diabetes in the NOD mouse

The effects of a nonimmunogenic form of murine soluble interferon-gamma receptor on the development of autoimmune diabetes in the NOD mouse

Phosphoric acid entrapment leads to apparent protein heterogeneity.

Recombinant proteins produced in prokaryotes or eukaryotes show certain types of heterogeneity due to post-translational modifications. Some preparations of a soluble interferon gamma receptor, produced in Escherichia coli, appeared as a double band with slightly different mobilities in non-reducing sodium dodecylsulfate and native polyacrylamide gels. Ion spray mass spectrometry showed that the two forms had a mass difference of one to three multiples of 97 +/- 2 D. Gas chromatography-mass spectrometry analysis revealed the presence of phosphoric acid in the hydrolysate and in the intact protein. The more slowly migrating protein species had trapped molecules of phosphoric acid during the protein extraction. Most of the trapped phosphoric acid was loosely associated with the protein. One to three molecules were tightly, but non-covalently linked per receptor molecule. Phosphoric acid entrapment did not affect biological activity and most likely did not affect protein conformation. The species carrying phosphoric acid showed higher solubility. Trapping of phosphoric acid by proteins may be a general phenomenon and the results reported here thus useful in the characterization of other recombinant proteins.

The Myxoma Virus-soluble Interferon-γ Receptor Homolog, M-T7, Inhibits Interferon-γ in a Species-specific Manner

The myxoma virus M-T7 protein contains significant sequence similarity to the ligand binding domain of the mammalian interferon-gamma receptors, and functions as a soluble homolog which can bind and inhibit the biological activities of rabbit interferon-gamma (Upton, C., Mossman, K., and McFadden, G. (1992) Science 258:1369-1372). M-T7, the most abundantly secreted protein from myxoma virus-infected cells, was shown to be expressed in significant biological amounts as a typical poxvirus early gene product, efficiently secreted at early times of infection to levels that exceed 5 x 10(7) molecules/cell, and function as a stable inhibitory protein in infected cell supernatants until late times of infection. M-T7 was specific in binding and inhibiting rabbit interferon-gamma, and did not bind either human or murine interferon-gamma. Scatchard analysis of rabbit interferon-gamma binding curves yielded a single high affinity binding site on M-T7, with a Kd of 1.2 x 10(-9) M, which is comparable to the affinity between soluble forms of cellular interferon-gamma receptors and their cognate ligands. In comparison, rabbit interferon-gamma was shown to bind its cellular receptor with a Kd of 5.9 x 10(-10) M, again comparable to the affinity of membrane bound forms of other mammalian interferon-gamma receptors for interferon-gamma. Thus, the myxoma virus M-T7 protein is a functional soluble interferon-gamma receptor homolog which binds and inhibits interferon-gamma with high affinity in a species-specific manner.

Experimental therapy of systemic lupus erythematosus: the treatment of NZB/W mice with mouse soluble interferon-gamma receptor inhibits the onset of glomerulonephritis.

Female NZB/W F1 mice develop an autoimmune disease similar to human systemic lupus erythematosus (SLE), and ultimately die of glomerulonephritis. Starting at the age of 16 weeks NZB/W F1 mice were treated for a period of 19 weeks with soluble interferon-gamma receptor (sIFN-gamma R), anti-IFN-gamma monoclonal antibody (mAb) or IFN-gamma. All mice treated with sIFN-gamma R or anti-IFN-gamma mAb were alive 4 weeks after the treatment was discontinued, whereas 50% of mice died in the placebo groups and 78% of the mice died in the IFN-gamma-treated group. Histologically, there was severe membrano-proliferative glomerulonephritis in IFN-gamma- and placebo-treated mice, and minimal or no mesangioproliferative disease in mice receiving sIFN-gamma R or anti-IFN-gamma mAb. The renal mononuclear infiltrate (T lymphocytes and monocytes), expression of major histocompatibility complex class II antigen and glomerular immunoglobulin and complement deposition were reduced in those mice. These data suggest that an IFN-gamma inhibitor, such as the soluble IFN-gamma R, can be used for SLE therapy in the early stages of the disease.

Alignment of disulfide bonds of the extracellular domain of the interferon gamma receptor and investigation of their role in biological activity.

The extracellular ligand binding domain of the human interferon gamma receptor includes eight cysteine residues forming four disulfide bonds. Only the nonreduced protein binds interferon gamma. We investigated the alignment of the disulfide bonds, using an enzymatically deglycosylated form of a soluble interferon gamma receptor, produced in baculovirus-infected insect cells. The soluble receptor was digested with endoproteinase Glu-C and proteinase K, and the proteolytic fragments were characterized by amino acid sequence analysis and mass spectrometry. It was found that four consecutive disulfide bonds are formed between residues Cys60-Cys68, Cys105-Cys150, Cys178-Cys183, and Cys197-Cys218. We also investigated the role of the disulfide bonds in biological activity of the receptor, using site-directed mutagenesis and by exchanging the cysteine residues for serines. The mutated proteins were expressed in Escherichia coli and analyzed for ligand binding capacity on protein blots. The assays showed that all disulfide bonds are essential for full ligand binding capacity. Double or quadruple mutations at cysteine residues 60 and 68, and residues 178, 183, 197, and 218, respectively, resulted in complete loss of the activity, whereas double mutations at residues 105 and 150, 178 and 183, and 197 and 218, respectively, resulted in a residual activity about 1 order of magnitude lower than that of the wild type. The specific antibodies gamma R38 and gamma R99 detected conformational epitopes stabilized by disulfide bonds involving cysteine residues 60 and 68, and 178 and 183, respectively.

A 25-kDa stretch of the extracellular domain of the human interferon gamma receptor is required for full ligand binding capacity

We investigated which is the shortest fragment of the interferon gamma receptor with ligand binding capacity. A recombinant soluble interferon gamma receptor produced in Escherichia coli was subjected to controlled digestion with several proteolytic enzymes. The fragments generated were assayed by four approaches for interferon gamma binding. A 25-kDa polypeptide comprising residues 6-227 of the mature protein was produced by sequential digestion with trypsin and proteinase K. It was identified as the shortest receptor domain with full interferon gamma binding capacity as judged by ligand blots. The proteolytic fragments were further tested for ligand binding by interferon gamma affinity chromatography. A 15-kDa polypeptide comprising amino acids 94-227 produced by digestion with endoproteinase Glu-C was found to bind with low affinity to immobilized interferon gamma. This fragment, which does not show ligand binding capacity on protein blots, was immunoprecipitated as a complex with interferon gamma by anti-interferon gamma antibodies. It also competed for the binding of radiolabeled interferon gamma to the cell surface receptor when it was assayed as a mixture of the proteolytic products, but not after separation from the cleaved rest of the molecule. The 15-kDa polypeptide probably carries the ligand-binding domain or part of it, but it lacks sequences essential for full interferon gamma binding capacity. The stretch between amino acids 6 and 21 which does not include any disulfide bonds seems to be essential for the receptor to show full activity. The digestion with endoproteinase Glu-C revealed that cysteine residues 60 and 68 of the interferon gamma receptor form a disulfide bond.

One interferon gamma receptor binds one interferon gamma dimer.

We investigated the stoichiometry of the interferon gamma and interferon gamma receptor interaction, using recombinant interferon gamma and recombinant soluble interferon gamma receptor, applying chemical cross-linking and chromatographic techniques, and analyzing the resulting products in denaturing polyacrylamide gels. Interferon gamma cross-linked to itself produced a major band of an apparent molecular mass of 34 kDa, which suggests that it exists as a dimer in physiological buffer and which agrees with published data. Soluble interferon gamma receptor cross-linked to itself produced mainly a 28-kDa band, suggesting that the interferon gamma receptor exists as a monomer. Interferon gamma cross-linked to the soluble interferon gamma receptor resulted in the formation of two main products of apparent molecular masses of 60 and 44 kDa. The predominant 60-kDa band resulted from the cross-linking of one interferon gamma dimer (34 kDa) to one interferon gamma receptor molecule (27 kDa). The 44-kDa band was formed by the cross-linking of one interferon gamma molecule to one interferon gamma receptor. Kinetic studies showed that the cross-linking of interferon gamma dimer to the soluble receptor proceeds through the intermediate formed by cross-linking one molecule of the interferon gamma dimer to the receptor. Reducing and dissociating agents inhibited complex formation. When chromatographed on Sephadex G-100, interferon gamma was eluted as a protein of 34-kDa molecular mass, the soluble interferon gamma receptor as a protein of 40 kDa, and their mixture was eluted in one peak corresponding to an apparent molecular mass of 73 kDa. Sodium dodecyl sulfate-polyacrylamide gel analysis of the eluted mixture showed the presence of both interferon gamma and interferon gamma receptor at a ratio of 2:1. The found results suggest that the interferon gamma receptor binds interferon gamma as a dimer.

Purification and biochemical characterization of a soluble human interferon gamma receptor expressed in Escherichia coli.

We purified and characterized a soluble human interferon gamma receptor expressed in Escherichia coli. The soluble receptor comprises the amino acids 15-246 of the encoded protein (Aguet, M., Dembic, Z., and Merlin, G. (1988) Cell 55, 273-280) and was purified from large scale fermentations through four chromatographic steps with an overall recovery of 28%. The refolded soluble receptor shows some heterogeneity on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, where it appears as the major band of 27 kDa molecular mass, accompanied by a few minor bands with molecular masses between 26 and 30 kDa. On reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis it appears as a homogeneous protein of 32 kDa molecular mass. The soluble interferon gamma receptor is an active and stable protein and is recognized by specific antibodies raised against the native receptor. When nonreduced it has the capacity to specifically bind interferon gamma and to compete for the binding of interferon gamma to the cell surface receptor. The observed heterogeneity of the soluble interferon gamma receptor under nonreducing electrophoretic conditions is probably due to different conformational forms resulting from the formation of non-native intramolecular disulfide bonds among the 8 cysteine residues present in the soluble interferon gamma receptor molecule.