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M Fountoulakis

Publications and source records attributed to M Fountoulakis.

At least 109 records · Page 6Linked to original sources
PubMed1992

Unfolding intermediates of the extracellular domain of the interferon gamma receptor.

Reduction of proteins which require disulfide bonds to be stable in the folded state is accompanied by step-wise unfolding. A soluble human interferon gamma receptor produced in Escherichia coli was used to investigate the kinetics of formation of unfolding intermediates. The protein includes 8 cysteine residues forming four disulfide bonds. It was reduced by using either dithiothreitol or the thioredoxin reduction system. Reduction with dithiothreitol resulted in formation of mainly four monomeric unfolding species as visualized by sodium dodecyl sulfate-polyacrylamide gels. The enzymatically catalyzed reaction produced only small amounts of two monomeric products and mostly delivered oligomeric and polymeric forms. In both cases, the ligand binding capacity of the receptor was significantly reduced immediately after appearance of the first intermediate. The intermediates involved interchange of disulfide bonds and did not show ligand binding capacity. Some of them were recognized by specific antibodies which detect conformational epitopes on the native interferon gamma receptor. On the basis of the antibody binding, a preliminary characterization of the formed intermediates was attempted. When the soluble receptor was reduced in the presence of denaturing agents, the reduction products were different from the unfolding intermediates generated in the absence of denaturants.

Blotting, Western↗
PubMed1992

A novel solid-phase test to study the binding of IFN-gamma to its receptor.

A novel solid phase assay for interferon-gamma (IFN-gamma) binding to the human IFN-gamma receptor was developed. The receptor binding assay is carried out using a soluble form of the recombinant IFN-gamma receptor protein corresponding to the extracellular portion of the IFN-gamma receptor. Using different IFN molecules and anti-IFN monoclonal antibodies, we show that the specificity of the soluble IFN-gamma receptor coated to the plastic surface is not altered. In consequence, this new generation binding test can be used to characterize the interactions with the specific ligand under controlled conditions. In comparison with ELISA or RIA tests using antibodies specific for IFN-gamma, the solid-phase binding assay has the advantage of detecting only the active molecules. Finally, since the test has a large capacity, it is being applied for the screening of agents that are able to neutralize the IFN-gamma activity either by blocking the active site of the lymphokine or the binding site of the specific receptor.

Animals↗
PubMed1992

Comparison of the Coomassie brilliant blue, bicinchoninic acid and Lowry quantitation assays, using non-glycosylated and glycosylated proteins.

The concentrations of several non-glycosylated and glycosylated recombinant and native proteins were determined by three widely used colorimetric methods: Coomassie brilliant blue, bicinchoninic acid and Lowry, and, for comparison, by amino acid composition analysis. The colorimetric methods gave results differing from the values derived from the amino acid analysis, in some cases by up to 60%. For the non-glycosylated recombinant proteins, the results were in relatively good agreement with each other and with the values determined on the basis of the amino acid analysis. The Coomassie blue method was strongly dependent on the hydrophobicity of the individual protein. The bicinchoninic acid method gave results closest to those of the amino acid analysis. For the glycosylated proteins, both recombinant and native, the Coomassie blue assay gave values lower, whereas the two other methods gave values higher than those determined on the basis of the amino acid analysis. The concentration of a recombinant interferon gamma receptor produced in two differently glycosylated forms was underestimated by the Coomassie blue assay and overestimated by the bicinchoninic acid and Lowry methods, while for the non-glycosylated form of the same protein, the three colorimetric methods delivered comparable values. The results suggest a potential interference of protein glycosylation with the colorimetric assays.

Carbohydrates↗
PubMed1992

Effect of glycosylation on properties of soluble interferon gamma receptors produced in prokaryotic and eukaryotic expression systems.

We investigated the influence of glycosylation on solubility, chromatographic behavior and resistance to heat- and chaotrope-dependent denaturation and proteolytic digestion of three recombinant human interferon gamma receptors produced in Escherichia coli, Spodoptera frugiperda and Chinese hamster ovary cells. The proteins produced in the eukaryotic expression systems were glycosylated, carrying different, heterogeneous carbohydrate moieties. They were assayed fully glycosylated and after removal of the oligosaccharides. Although glycosylation influenced the chromatographic behavior of the tested proteins, it did not protect against proteolysis and heat- or chaotrope-induced denaturation. The glycosylated receptors were slightly more sensitive to certain proteolytic cleavages and slightly less resistant to chaotropes, than the soluble receptor produced in Escherichia coli.

Animals↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

Purification and biochemical characterization of a soluble mouse interferon-gamma receptor produced in insect cells.

The extracellular domain of the mouse interferon gamma receptor comprising amino acids 17-243 of the protein was produced in Spodoptera frugiperda cells infected with a recombinant baculovirus. The receptor was mainly secreted into the culture medium and was purified to homogeneity in several hundred milligram amounts. The purification procedure involved four chromatography steps and delivered a soluble and active receptor with an overall recovery of 30%. From each purification run, two pools of soluble receptor with the same interferon gamma binding capacity were isolated. Under reducing electrophoretic conditions the protein of pool I migrates as two bands of molecular masses 32 and 34 kDa and of pool II as two bands of 30 and 32 kDa. The soluble receptor of both pools carries a heterogeneous glycosylation. After deglycosylation it appears as one protein band of 27 kDa. N-linked carbohydrates contribute about 6 kDa and O-linked carbohydrates 1 kDa to its molecular mass. The nonreduced protein specifically binds interferon gamma on ligand blots and in a solid-phase binding system and competes for the binding of radiolabeled interferon gamma to the cell surface receptor. The soluble mouse interferon gamma receptor exists as a monomer in physiological buffer and binds interferon gamma in its dimeric form. It is stable at room temperature and against tryptic digestion, but is very sensitive to proteinase K digestion. The soluble mouse interferon gamma receptor produced in the insect/baculovirus expression system may prove useful to study the function of interferon gamma receptor as an antagonist of endogenous interferon gamma in the treatment of immunological and inflammatory disorders.

Amino Acids↗
PubMed1991

High-affinity receptor for interferon-gamma (IFN-gamma), a ubiquitous protein occurring in different molecular forms on human cells: blood monocytes and eleven different cell lines have the same IFN-gamma receptor protein.

High-affinity receptors for human IFN-gamma were analyzed using 13 different cells, including blood monocytes. Scatchard analysis showed one high-affinity binding site for each cell. One cross-linked complex between IFN-gamma and the receptor was detected, although their apparent molecular masses were variable in different cells, as also confirmed in immunoblots of membrane extracts. Variations in molecular masses were abolished if N-linked glycosylation was absent. Stable tryptic fragments contained the intact binding site for IFN-gamma and antibody epitopes characteristic of the extracellular domain of the IFN-gamma receptor of Raji cells and were of different sizes only if glycosylated. In addition, Northern analysis showed the same mRNA encoding the high-affinity IFN-gamma receptor in each cell analyzed. Thus, all cells including blood monocytes express the same high-affinity IFN-gamma receptor protein. N-linked sugars may give structural stability to the IFN-gamma receptor and are unlikely to be directly involved in IFN-gamma binding.

Antibodies, Monoclonal↗
PubMed1990

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.

Cell Line↗
PubMed1990

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.

Chromatography, Affinity↗
PubMed1990

Human interferon-gamma receptor. Mapping of epitopes recognized by neutralizing antibodies using native and recombinant receptor proteins.

Monoclonal antibodies produced against native interferon-gamma receptor (IFN gamma-R) have been characterized for their capacity to react with purified receptor and receptor-positive cells, to inhibit the binding of IFN gamma to cellular receptor, to precipitate the receptor protein when cross-linked to IFN-gamma, and to recognize the recombinant interferon-gamma receptor and 19 overlapping fragments of this protein expressed in Escherichia coli. The results of this analysis showed that: (i) the extracellular portion of human IFN gamma-R is located between the N terminus and the transmembrane region (amino acids 18-246). (ii) The intracellular domain is between the transmembrane region and the C terminus (amino acids 269-489). (iii) The monoclonal antibodies that react with the IFN gamma-R intracellular domain recognize small linear epitopes. (iv) The human IFN gamma-R binding site is located between the N terminus and the transmembrane region. (v) The monoclonal antibodies that react with IFN gamma-R extracellular domain and inhibit the binding of IFN gamma recognize two different epitopes. One of these epitopes (included between amino acids 26 and 133) is very close to the binding site for IFN gamma. The second (included between amino acids 70 and 210) is related to the binding site for IFN gamma without including it. (vi) These two functional epitopes are conformational and need S-S bridges to maintain their architecture. (vii) These conformational epitopes are formed in receptor fragments expressed in E. coli.

Amino Acid Sequence↗
PubMed1989

Structure and membrane topology of the high-affinity receptor for human IFN-gamma: requirements for binding IFN-gamma. One single 90-kilodalton IFN-gamma receptor can lead to multiple cross-linked products and isolated proteins.

We analyzed the high affinity receptor for IFN-gamma of Raji cells and human placenta by combining Scatchard analysis, cross-linking experiments, and receptor purification. Only one high affinity binding site was found, Kd 2.1 X 10(-10). The receptor is a 90-kDa glycoprotein. However, multiple cross-linked products of 110 kDa to about 250 kDa could be generated and proteins of 90, 70, and 50 kDa could be obtained upon purification. These proteins all contained the same 90-kDa receptor, or part of it. We suggest that extensive cross-linking and/or proteolysis may explain many of the conflicting results published thus far. The extracellular domain of the 90-kDa receptor protein was highly resistant to digestion with trypsin or proteinase K. Trypsin digestion neither affected the number of binding sites per cell, nor the Kd for IFN-gamma. A cluster of sites for different proteases was found in the intracellular domain. The 50-kDa fragment created by trypsin digestion had the same characteristics as the isolated 50-kDa receptor fragment. It contained the IFN-gamma binding site and the receptor's extracellular and amino-terminal domain. N-linked glycosylation contributed about 15 kDa to its molecular mass, of which 4 kDa were attributable to sialic acid residues. O-Linked glycosylation was not detected. The number of binding sites per cell and the Kd for IFN-gamma were not affected by the presence or absence of N-linked glycosylation. The receptor contained at least one critical disulfide bridge and the reduced receptor could be reactivated in vitro.

Animals↗
PubMed1985

Analysis of antibody response in patients receiving recombinant Escherichia coli-derived human interferon-gamma.

Recombinant Escherichia coli-derived human interferon-gamma (rIFN-gamma) was given to a total of 20 patients by iv bolus injection at various doses once a week for 4 weeks. The sera obtained 7-10 days after the final injection were analyzed for antibodies against both rIFN-gamma and natural human IFN-gamma. Biological assays demonstrated that the postinoculation sera of the patients did not neutralize the antiviral activities of either rIFN-gamma or natural human IFN-gamma. Enzyme-linked immunosorbent assay showed that no detectable antibodies against rIFN-gamma were elicited. These results indicate that this rIFN-gamma preparation is not a potent antigen and may be suitable for longer-term clinical trials and applications in the future.

Animals↗
PubMed1982

Structure and expression of human IFN-alpha genes.

Copy DNA (cDNA) was prepared from induced leucocyte poly(A) RNA and cloned in Escherichia coli. IFN-alpha cDNA clones were isolated by subculture cloning with the use of a translation hybridization assay. Definitive identification of the clones was based on the production of an interferon-like protein by the transformed bacteria. Different IFN-alpha cDNAs, with characteristic target cell specificities, were identified. The cloned cDNAs typically encode a mature polypeptide of 166 (or, for IFN-alpha 2, 165) amino acids and a signal sequence of 23 amino acids. A human chromosomal library was screened with IFN cDNA and 17 distinct IFN-alpha-related sequences were isolated and identified, of which 7 proved to be nonallelic authentic genes and 4 pseudogenes; 6 sequences remain to be elucidated. Taking into account the work of Goeddel and his colleagues, 13 non-allelic authentic genes and 6 pseudogenes can be distinguished. In addition, 9 genes believed to be allelic to the 13 authentic genes have been sequenced. The IFN-alpha genes may be classified into two major subfamilies, which diverged at least 33 Ma ago, but perhaps much earlier, if sequence rectification occurred. At least one IFN-alpha gene appears to have resulted by a recombinational event between members of the subfamily I and II. IFN-beta is distantly related to IFN-alpha's and may have diverged from a common ancestor at least 500 Ma ago. Both IFN-alpha and IFN-beta genes differ from most other genes of higher organisms by being devoid of introns. The mouse was found to possess an IFN-alpha gene family of a size similar to that of man; the murine genes also do not have introns. IFN-alpha genes devoid of their signal sequence were joined to prokaryotic promoters to produce the mature interferons in E. coli in high yield. IFN-alpha 2, purified to homogeneity, has been crystallized by T. Unge and B. Strandberg (Uppsala). Hybrid genes consisting of IFN-alpha 1 and IFN-alpha 2 segments were constructed and expressed in E. coli; the target cell specificities of such hybrids were dependent on the arrangement of the segments and were different from those of either parent. The chromosomal gene for HuIFN-alpha 1 was introduced into mouse L cells to study the mechanism of its expression. Correct transcription was only detected after induction (with Newcastle disease virus); expression was transient, with the same kinetics as those of the endogenous mouse IFN mRNA. Natural murine IFNs and human IFN-beta and IFN-gamma are glycosylated. Because E. coli cells transformed with the genes of eukaryotic glycoproteins are not expected to yield correctly glycosylated polypeptides, we prepared lines of hamster cells permanently transformed with hybrid plasmids, which contained an IFN gene linked to the SV40 early promoter, as well as dihydrofolate reductase as a selective marker. After intracellular amplification of the introduced genes, cell lines were obtained which constitutively produced IFN at about 40 000 units ml-1 and could be propagated for at least several months.

Base Sequence↗
PubMed1982

Structure and expression of human alpha-interferon genes.

cDNA was prepared from induced leukocyte poly(A) RNA and cloned in Escherichia coli. Interferon (IFN)-alpha cDNA clones were isolated by subculture cloning using a translation hybridization assay. Definitive identification of the clones was based on the production of an interferon-like protein by the transformed bacteria. Different IFN-alpha cDNAs, with characteristic target cell specificities, were identified. The cloned cDNAs typically encode a mature polypeptide of 166 (or, in the case of IFN-alpha 2, 165) amino acids and a signal sequence of 23 amino acids.

Amino Acid Sequence↗
PubMed1981

Reversible cleavage of the cobalt-carbon bond to coenzyme B12 catalysed by methylmalonyl-CoA mutase from Propionibacterium shermanii. The use of coenzyme B12 stereospecifically deuterated in position 5'.

1. (5'R)-(5'-2H1)Adenosine [(5'R):(5'S) = 85:15] was prepared by a procedure which involved inter alia the reduction of 6-N-benzoyl-2',3'-O-isopropylidene-5'-oxoadenosine with a reagent obtained from LiAl2H4 and (-)-isoborneol. 2. (5'S)-(5'-2H1)AdoCbl [(5'S):(5'R) = 74:26] (AdoCbl = 5'-deoxyadenosylcobalamin) was synthesized by reacting cobal(I)amin with (5'R)-2'-3'-O-isopropylidene-5'-tosyl-(5'-2H1) adenosine followed by acid hydrolysis to remove the isopropylidene protective group. 3. (5'R)-(5'-2H1)AdoCbl [(5'R):(5'S) = 77:23] was prepared by reacting cobalt(I)amin with (5'S)-5'-chloro-5'-(5'-2H1)deoxyadenosine [(5'S):(5'R) = 80:20] obtained in turn from (5'R)-(5'-2H1)adenosine. The reaction sequence involved two consecutive inversions at the C-5' atom of adenosine 4. Comparison of the 500-MHz 1H-NMR spectra of unlabelled, (5'S)- and (5'R)-(5'-2H1)AdoCbl allowed assignment of the triplet at 0.58 ppm and the doublet at 1.525 ppm to the diastereotopic 5'-HRe and 5'-HSi atoms, respectively. On acidification, these two protons gave rise to two triplets at 0.11 ppm and 1.78 ppm indicating that torsion had occurred around the C-4'--C-5' bond. 5. Samples of (5'R)- and (5'S)-(5'-2H1)AdoCbl were incubated with methylmalonyl-CoA mutase from Propionibacterium shermanii. Examination by 1H-NMR spectroscopy at 500 MHz revealed partial loss and stereochemical scrambling of the deuterium at the 5' position. This indicates transient conversion of the C-5' atom into a torsiosymmetric group and hence cleavage of the cobalt-carbon bond during interaction with the enzyme. The mechanism by which deuterium is lost remains to be elucidated.

Deuterium↗
PubMedDate not supplied

Two-dimensional map of human brain proteins.

Samples of human brain from the parietal cortex lobe were analyzed by two-dimensional gel electrophoresis, using immobilized pH gradient strips covering the various pH regions. The protein spots were visualized with colloidal Coomassie blue stain and identified by matrix-assisted laser desorption/ionization mass spectrometry. Approximately 400 spots were identified, corresponding to 180 different brain proteins. The list of identified proteins includes a large number of structural proteins and of enzymes or enzyme subunits with various catalytic activities. The majority of proteins are localized in the cytoplasma and in mitochondria. The two-dimensional map may be useful as a reference database to study changes in the protein level caused by various disorders, such as Alzheimer's disease, major depression and schizophrenia.

Animals↗