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.
Stüber
D
D
Department of Biology, Pharmaceutical Research-New Technologies, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Friedlein
A
A
Fountoulakis
M
M
Lahm
H W
HW
Garotta
G
G
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007915:Lepidoptera / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017471:Receptors, Interferon / Q000737:chemistry* / Q000378:metabolism; D000096743:Interferon gamma Receptor
D000911:Antibodies, Monoclonal; D004220:Disulfides; D017471:Receptors, Interferon; D000096743:Interferon gamma Receptor