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Biomedical subjects

M Fountoulakis

Publications and source records attributed to M Fountoulakis.

At least 91 records · Page 5Linked to original sources
PubMed1996

Apparent heterogeneity of recombinant interferon gamma receptors produced in prokaryotic and eukaryotic expression systems.

Recombinant proteins show several types of heterogeneity and post-translational modifications which are usually related to their production system. The apparent heterogeneity of recombinant interferon gamma receptors and interferon gamma receptor-immunoglobulin G fusion proteins expressed in Escherichia coli, baculovirus-infected insect cells and Chinese hamster ovary cells have been studied. In general, all proteins tested showed some type of heterogeneity which was detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The E. coli-derived receptor included non-native conformations involving mis-paired or non-formed disulfides. This type of heterogeneity affected the biological activity of the protein. In addition, the prokaryotic protein had trapped phosphoric acid during downstream processing. The phosphoric acid entrapment did not affect ligand binding capacity. The eukaryotic proteins showed heterogeneity because of the unequal cleavage of the signal peptide and because of differences in glycosylation. The latter types of heterogeneity did not affect activity. Glycosylation-related heterogeneity was partially derived from the unequal utilization of the potential N-glycosylation sites and differently affected the apparent molecular masses and migrations of the proteins on polyacrylamide gels. The results may be useful in characterization studies of recombinant proteins.

Amino Acid Sequence↗
PubMed1995

Quantification of cysteine residues following oxidation to cysteic acid in the presence of sodium azide.

Quantification of cysteines by amino acid composition analysis is inaccurate because of decomposition of these residues during protein hydrolysis. Cysteine (and cystine) residues are oxidized to cysteic acid following hydrochloric acid hydrolysis in the presence of sodium azide. Using selected native and recombinant proteins, containing different numbers of cysteine residues, we investigated the conditions for the quantitative oxidation of cysteines to cysteic acid in the presence of sodium azide. Protein hydrolysis with hydrochloric acid in the presence of 0.20% sodium azide resulted in 87-100% oxidation of the cysteines to cysteic acid which was easily quantified. The results were highly reproducible so that the azide-induced oxidation can be used as a general method to determine cysteine residues in a given protein. The sodium azide-dependent oxidation is superior to oxidation with performic acid because (i) it can be performed in solution not requiring protein lyophilization and in approximately half of the time; (ii) it delivers slightly higher yields of cysteic acid; and (iii) it does not affect tyrosine residues, which can be modified during the performic acid treatment.

Amino Acids↗
PubMed1995

Interferon gamma receptor extracellular domain expressed as IgG fusion protein in Chinese hamster ovary cells. Purification, biochemical characterization, and stoichiometry of binding.

Agents that antagonize the functions of interferon gamma (IFN gamma) are potential pharmaceuticals against several immunological and inflammatory disorders. IFN gamma receptor-immunoglobulin G fusion proteins (IFN gamma R-IgG) function as antagonists of endogenous IFN gamma and have longer half-lives in vivo in comparison with soluble IFN gamma receptors (sIFN gamma R), consisting of the extracellular region of the native sequence. A fusion protein comprising the extracellular domain of the human IFN gamma receptor and the hinge, CH2 and CH3 domains of the human IgG3 constant region, was expressed in Chinese hamster ovary cells. The IFN gamma R-IgG3 fusion protein was secreted into the culture medium as a 175-kDa glycoprotein and was purified over Protein G-Sepharose, DEAE-Sepharose, and size exclusion chromatography. IFN gamma R-IgG3 bound IFN gamma in solid phase assays and ligand blots, competed for the binding of radiolabeled IFN gamma to the cell surface receptor of Raji cells, and inhibited the IFN gamma-mediated antiviral activity with an efficiency at least one order of magnitude higher than that of the soluble receptor produced in the same expression system. Two IFN gamma R-IgG3 fusion proteins bound two IFN gamma dimers forming a complex of approximately 380 kDa. In immunodiffusion assays, the IFN gamma R-IgG3 fusion protein did not precipitate IFN gamma. Dissociation of bound IFN gamma from IFN gamma R-IgG3 was 2-fold slower than from the sIFN gamma R produced in insect cells.

Animals↗
PubMed1995

Oxidation of cysteine and methionine residues during acid hydrolysis of proteins in the presence of sodium azide.

Sodium azide is widely used as bacteriostatic agent during downstream processing of proteins. Amino acid composition analysis of protein samples, subjected to hydrolysis with hydrochloric acid in a buffer containing sodium azide, revealed the presence of cysteic acid, methionine sulfoxide, and methionine sulfone in addition to the expected reaction products. Hydrolysis with methanesulfonic acid in the presence of sodium azide resulted in detection of only methionine sulfoxide in addition to the expected products. When the proteins were hydrolyzed in a buffer containing no sodium azide or after its removal by dialysis, no oxidation products were detected (except for minor amounts of methionine sulfoxide). The generation of the particular oxidation products was affected by the concentration of sodium azide in the protein solution. Therefore, presence of sodium azide in protein samples intended for amino acid composition analysis may lead to wrong conclusions concerning oxidation of cysteine and methionine residues.

Amino Acids↗
PubMed1995

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.

Animals↗
PubMed1995

Crystallization of the complex of human IFN-gamma and the extracellular domain of the IFN-gamma receptor.

A complex of human interferon-gamma (IFN-gamma) with the soluble extracellular domain of the IFN-gamma receptor alpha-chain (IFN-gamma-R) has been crystallised. Crystals of the complex were grown using PEG 4000 as the precipitating agent in the presence of beta-octyl glucoside. The receptor-ligand complex crystallizes in a monoclinic space group and diffracts to about 3.0 A resolution. Isomorphous crystals have been obtained with complex containing selenomethionine and cysteine mutants of IFN-gamma, which may facilitate the ongoing X-ray structure determination.

Amino Acid Sequence↗
PubMed1995

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.

Chemical Phenomena↗
PubMed1995

Interferon-gamma receptor extracellular domain-IgG fusion protein produced in Chinese hamster ovary cells as mixture of glycoforms.

Glycosylation of proteins fulfills important functions and because of its diversity contributes to apparent protein heterogeneity. We investigated the heterogeneity of a fusion protein comprising the extracellular domain of the human interferon-gamma (IFN-gamma) receptor and parts of the human IgG3 constant region, a potential IFN-gamma antagonist. The protein was produced in Chinese hamster ovary (CHO) cells and was secreted into the culture medium as an 175 kD glycoprotein. Glycosylation represented approximately one-third of the apparent molecular mass of the fusion protein, consisted of N- and O-linked carbohydrate moieties, and included sialic acid residues as part of both N- and O-linked oligosaccharides. Fusion protein forms with different apparent molecular masses and charges were separated by ion-exchange chromatography. Preparative electrofocusing revealed a wide spectrum of glycoforms. Glycosylation of the fusion protein and of soluble IFN-gamma receptors, comprising the extracellular domain of the native sequence, expressed in insect and CHO cells did not interfere with affinity of ligand binding.

Amino Acid Sequence↗
PubMed1995

Role of interferon-gamma in mediating the antitumor efficacy of interleukin-12.

Although interleukin-12 (IL-12) has marked antitumor activity against the murine Renca renal cell carcinoma in vivo, no antiproliferative activity with IL-12 was observed against these tumor cells in vitro; in contrast, interferon-gamma (IFN-gamma) had growth inhibitory activity. Since one of the properties of IL-12 is its ability to stimulate production of IFN-gamma, the role of IFN-gamma in mediating the antitumor activity of IL-12 was evaluated. Substantially diminished antitumor activity was observed in mice injected with IL-12 and neutralizing antibody to murine IFN-gamma compared with mice receiving IL-12 alone, indicating that IFN-gamma was required for the optimal antitumor efficacy of IL-12. However, several lines of investigation suggest that the antitumor effect of IL-12 is not mediated solely through the induction of IFN-gamma. Exogenous administration of IFN-gamma to Renca tumor-bearing euthymic mice resulted in less antitumor efficacy than that which could be obtained with IL-12. In addition, the antitumor effect of IL-12 was reduced in nude mice compared with euthymic mice, but an approximately 10-fold higher level of serum IFN-gamma was induced in nude than in euthymic mice. Thus, these results indicate that induction of high serum levels of IFN-gamma is not sufficient to mediate the antitumor efficacy of IL-12.

Animals↗
PubMed1994

Structural analysis and localization of the carbohydrate moieties of a soluble human interferon gamma receptor produced in baculovirus-infected insect cells.

A soluble form of the human interferon gamma receptor that is required for the identification of interferon gamma antagonists was expressed in baculovirus-infected insect cells. The protein carried N-linked carbohydrate and showed a heterogeneity on denaturing polyacrylamide gels. We investigated the utilization of the potential sites for N-linked glycosylation and the structure of the carbohydrate moieties of this soluble receptor. Amino acid sequence analysis and ion spray mass spectrometry revealed that of the five potential sites for N-linked glycosylation, Asn17 and Asn69 were always utilized, whereas Asn62 and Asn162 were utilized in approximately one-third of the protein population. Asn223 was never found to be glycosylated. The soluble receptor was treated with N-glycosidase F and the oligosaccharides released were analyzed by matrix-assisted laser desorption mass spectrometry, which showed that the protein carried six types of short carbohydrate chains. The predominant species was a hexasaccharide of molecular mass 1,039, containing a fucose subunit linked to the proximal N-acetylglucosamine residue: [formula: see text]

Amino Acid Sequence↗
PubMed1993

Mouse soluble IFN gamma receptor as IFN gamma inhibitor. Distribution, antigenicity, and activity after injection in mice.

With the purpose to use a soluble receptor as an IFN gamma antagonist in vivo, we assessed the immunogenicity, the half-life, the bioavailability, and the activity of the soluble receptor after injection into mice. No significant immunogenicity was detected after multiple chronic injections of mouse (Mo)IFN gamma-R or injections of MoIFN gamma-R in emulsion with CFA. Pharmacokinetic experiments using radiolabeled MoIFN gamma-R revealed that the half-life of the soluble MoIFN gamma-R is 3 h in the blood and 6 h in the lymphoid organs. When the MoIFN gamma-R protein was traced by the capacity of the animal sera to displace the binding of radiolabeled MoIFN gamma-R from affinity purified rabbit anti-MoIFN gamma-R antibodies, a blood half-life of 1 h was determined. Finally, the capacity of the injected receptor to bind and neutralize the IFN gamma-mediated antiviral activity was assessed in vivo. Treatment with IFN-gamma or IFN-alpha A/D protected the mice from the lethal infection with encephalomyocarditis virus. Mice treated with MoIFN-gamma-R neutralized the protective effect of IFN-gamma.

Animals↗
PubMed1993

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.

Amino Acid Sequence↗
PubMed1993

Purification of interferon gamma-interferon gamma receptor complexes by preparative electrophoresis on native gels.

Cytokine-receptor complexes are required for certain studies including crystallization, NMR spectra, and investigation of the biological response mechanism to the cytokine. The purity of the ligand-receptor complex is critical for most of these applications. We investigated the possibility of purifying protein-protein complexes by electrophoresis on native gels. Starting with partially purified mouse and highly purified human proteins, we prepared milligram amounts of interferon gamma-interferon gamma receptor complexes by preparative electrophoresis on nondenaturing polyacrylamide gels. In both cases, pure ligand-receptor complexes with the correct stoichiometry of binding were recovered. Electrophoresis on preparative native gels may prove to be of general interest for the preparation of protein-protein complexes to be used in diverse studies.

Amino Acids↗
PubMed1993

Immunomodulation with soluble IFN-gamma receptor: preliminary study.

Several in vivo experiments support the hypothesis that an IFN-gamma antagonist may have therapeutic applications in autoimmune diseases, hypersensitivities, and alloreactions. IFN-gamma exerts its biological activity through the binding to a single-chain cell surface receptor. The protein that corresponds to the external domain of mouse IFN-gamma receptor was expressed in insect cells infected with recombinant baculovirus; this protein was characterized and used in vivo as a prototype of the IFN-gamma antagonist. This protein does not show any strong antigenicity after in vivo injection in mice. Despite a blood half-life of only 1-3 hr as demonstrated in pharmacokinetic experiments, the mouse soluble IFN-gamma R was able to modify the onset of acute GVHD (alloreaction) and chronic GVHD (lupuslike disease).

Acute Disease↗
PubMed1992

Analysis of soluble human and mouse interferon-gamma receptors expressed in eukaryotic cells.

The extracellular domains of the human and mouse interferon-gamma receptors were produced in insect Spodoptera frugiperda cells infected with recombinant baculoviruses and in mammalian Chinese-hamster-ovary cells. The receptors expressed in both systems are secreted into the culture medium. Their signal peptides are cleaved off and the proteins show heterogeneity in glycosylation which, however, does not affect the capacity to bind interferon gamma or specific antibodies. The soluble mouse receptors exhibit binding capacities similar to those of cell-surface-anchored receptors, whereas the human receptors exhibit a lower binding capacity. All soluble receptors inhibit the binding of interferon gamma to cellular receptors and neutralize the antiviral activity exerted by interferon gamma. These receptors could therefore be useful for structure/function analyses and in vivo studies.

Animals↗
PubMed1992

Stoichiometry of interaction between interferon gamma and its receptor.

The biological response of interferon gamma is mediated by binding to a specific cell-surface receptor. We investigated the stoichiometry of this binding using soluble receptors produced in prokaryotic and eukaryotic expression systems comprising the extracellular ligand-binding domain of the native protein. The ligand-receptor complexes were analyzed by cross-linking, chromatography, analytical ultracentrifugation and laser-light scattering. Cross-linking and chromatography showed that the stoichiometry of the interaction between ligand and receptor depends on the molar ratios of the two components mixed. All approaches confirmed that mixtures of ligand-receptor complexes are formed with one interferon-gamma dimer bound by one or two receptors. The soluble receptor produced in Escherichia coli mainly showed a ligand/receptor stoichiometry of 1:1, while the receptors produced in eukaryotic cells showed a stoichiometry of binding of 1:2. This apparent discrepancy is most likely due to the conformational heterogeneity of the Escherichia-coli-derived protein.

Amino Acid Sequence↗