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

Fountoulakis, Michael; Lahm, Hans‐Werner; Maris, Antony; Friedlein, Arno; Manneberg, Michael; Stueber, Dietrich; Garotta, Gianni

doi:10.1016/s0021-9258(18)98573-x

Cited by 22 publications

(6 citation statements)

References 24 publications

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“…When each domain was tested for its ability to inhibit the binding of IFNy to the native receptor on Raji cells, however, no significant inhibition could be detected up to a concentration of »15 /iM (results not presented). This confirms earlier work (Fountoulakis et al, 1991a) which showed that a 25 kDa stretch of the extracellular portion of the receptor is required for full ligand binding capacity.…”

Section: Discussionsupporting

confidence: 91%

Dissection of the extracellular human interferon .gamma. receptor .alpha.-chain into two immunoglobulin-like domains. Production in an Escherichia coli thioredoxin gene fusion expression system and recognition by neutralizing antibodies

Williams

Ruegg²,

Birch³

et al. 1995

Biochemistry

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The extracellular interferon gamma receptor alpha-chain (IFN gamma R) is believed to comprise two discrete approximately 110 amino acid immunoglobulin-like domains, perhaps similar to those seen in the crystal structure of the extracellular human growth hormone receptor [De Vos, A. M., Ultsch, M., & Kossiakoff, A. (1992) Science 255, 306-312], a distant relative in the cytokine receptor superfamily. In accord with this idea, we show that these IFN gamma R immunoglobulin-like domains can be produced separately in a soluble form with a native-like fold. The N-terminal domain (residues 1-108), with a Cys105 to Ser105 mutation, was produced at a high level, in a soluble form, as a thioredoxin-interferon gamma receptor fragment fusion protein in the cytoplasm of Escherichia coli. Upon extraction, the receptor Cys60-Cys68 disulfide bond formed spontaneously, to generate a native-like structure directly without the need for refolding. Cleavage of the fusion protein by enterokinase released the receptor fragment (approximately 12 kDa), which was recognized by several neutralizing antibodies with affinities, measured using surface plasmon resonance technology, that were essentially indistinguishable from those seen with the full length extracellular IFN gamma R produced in eukaryotic cells. Circular dichroism and 1D 1H nuclear magnetic resonance spectra indicated that the receptor fragment adopts a folded state, with mainly beta-sheet and reverse turn secondary structure. The second membrane-proximal Ig-like domain of the IFN gamma R (residues 90-229) was produced, albeit less efficiently, and characterized in a similar way. The production of these two independently folded proteins provides experimental support for the two domain organization of the IFN gamma R and opens new avenues for structural studies on these Ig-like molecules by NMR and crystallographic methods.

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Section: Discussionsupporting

confidence: 91%

Dissection of the extracellular human interferon .gamma. receptor .alpha.-chain into two immunoglobulin-like domains. Production in an Escherichia coli thioredoxin gene fusion expression system and recognition by neutralizing antibodies

Williams

Ruegg²,

Birch³

et al. 1995

Biochemistry

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“…Digestion with endoproteinase Glu-C yielded peptide a (Figure 2) which includes Cys60 and Cys68. The peptide had the expected mass of4346.9 (within experimental error) which means that Cys60 and Cys68 are connected with a disulfide bond, as we have previously described (Fountoulakis et al, 1991a). The same digestion delivered peptide b comprising the sequences Ile166-Glu201 and Val217-Ser229 (lacking the stretch Gly202-Glu216) and including cysteine residues 178, 183,197, and 218.…”

Section: Resultssupporting

confidence: 68%

“…A mass for this peptide could not be determined. However, since amino acid sequence analysis and mass spectrometry data unambiguously showed the existence of three disulfide bonds between Cys60-Cys68, Cys178-Cys183, and Cys197-Cys218, and the receptor had no free thiol groups (Fountoulakis et al, 1991a), we concluded that Cys105 and Cys150 must be connected with a disulfide bond.…”

Section: Resultsmentioning

confidence: 78%

“…They were generated due to internal starts of translation within the mRNA. Since they also contain the 6XHis sequence, they were copurified with the full-length soluble receptors (Fountoulakis et al, 1991a).…”

Section: Resultsmentioning

confidence: 99%

“…It did not bind the mutant where the four cysteine residues 178, 183, 197, and 218 were substituted (lane 14). The antibody y R99 recognized several bands migrating between 22 and 25 kDa (Figure 4C, lanes 1-7, 13, and 15), representing products of internal translation starts, that lack the IFNy binding region near the amino terminus (Fountoulakis et al, 1991a). These polypeptides were not detected by either radiolabeled IFNy (Figure 4A) or antibody yR38 (Figure 4B).…”

Section: Resultsmentioning

confidence: 99%

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

Stüber¹,

Friedlein²,

Fountoulakis³

et al. 1993

Biochemistry

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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.

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T-Cell Interferon Gamma Binding in Patients With Dementia of the Alzheimer Type

Bongioanni¹,

Boccardi²,

Borgna³

et al. 1997

Archives of Neurology

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

Cited by 22 publications

References 24 publications

Dissection of the extracellular human interferon .gamma. receptor .alpha.-chain into two immunoglobulin-like domains. Production in an Escherichia coli thioredoxin gene fusion expression system and recognition by neutralizing antibodies

Dissection of the extracellular human interferon .gamma. receptor .alpha.-chain into two immunoglobulin-like domains. Production in an Escherichia coli thioredoxin gene fusion expression system and recognition by neutralizing antibodies

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

T-Cell Interferon Gamma Binding in Patients With Dementia of the Alzheimer Type

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