The soluble extracellular portion of the human interferon-gamma receptor is a valid substitute for evaluating binding characteristics and for neutralizing the biological activity of this cytokine.
In order to have a reliable and reproducible source of soluble human interferon-gamma (HuIFN-gamma) receptor at our disposal both for studying binding phenomena and for evaluating its neutralizing potential towards the cytokine, we expressed the extracellular part of the receptor in J558L mouse myeloma cells as a fusion protein with the C-terminal c-myc TAG (HuECR-gamma-TAG). It is expected that the receptor will undergo post-translational modifications comparable to that in humans. The affinity purified soluble receptor was subjected to mass spectrometry analysis resulting in a molecular size of 31 to 40 kDa and showed heterogeneous N-glycosylation with an M(r)-contribution of 4 to 13 kDa. Its HuIFN-gamma binding affinity, determined by real time biospecific interaction (BIAcore) analysis, resulted in a value of Kd = 2 x 10(-9) M, which is in agreement with the high affinity described for the cell anchored complete HuIFN-gamma receptor (Kd = 5-35 x 10(-9) M). HuECR-gamma-TAG was able to neutralize the biological activity of HuIFN-gamma in an in vitro antiviral assay. Furthermore, we report for the first time the association and dissociation rate constants, which were, respectively, 2.4 x 10(5) M-1 s-1 and 4.8 x 10(-4) s-1. In conclusion, this mammalian source of the extracellular soluble HuIFN-gamma receptor represents a valuable tool for extensive in vitro studies of the HuIFN-gamma receptor interaction. Furthermore, in view of its expected low or nonimmunogenicity it opens new ways for immunomodulation in vivo.
Michiels
L
L
Laboratory for Biochemistry, University of Leuven, Heverlee, Belgium.
Haelewyn
J
J
Proost
P
P
De Ley
M
M
D000818:Animals; D001665:Binding Sites; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D051379:Mice; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D011869:Radioligand Assay; D017471:Receptors, Interferon / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection; D014407:Tumor Cells, Cultured; D000096743:Interferon gamma Receptor
D007371:Interferon-gamma; D016271:Proto-Oncogene Proteins c-myc; D017471:Receptors, Interferon; D011993:Recombinant Fusion Proteins; D000096743:Interferon gamma Receptor