Watarai H, Nozawa R, Tokunaga A, Yuyama N, Tomas M, Hinohara A, Ishizaka K, Ishii Y
Pharmaceutical Research Laboratory, Kirin Brewery Company, Ltd., 3. Miyahara-cho, Takasaki 370-1295, Gunma, Japan.
Proc Natl Acad Sci U S A. 2000 Nov 21;97(24):13251-6. doi: 10.1073/pnas.230445397.
Glycosylation inhibiting factor (GIF) and macrophage migration inhibitory factor (MIF) share an identical structure gene. Here we unravel two steps of posttranslational modifications in GIF/MIF molecules in human suppressor T (Ts) cell hybridomas. Peptide mapping and MS analysis of the affinity-purified GIF from the Ts cells revealed that one modification is cysteinylation at Cys-60, and the other is phosphorylation at Ser-91. Cysteinylated GIF, but not the wild-type GIF/MIF, possessed immunosuppressive effects on the in vitro IgE antibody response and had high affinity for GIF receptors on the T helper hybridoma cells. In vitro treatment of wild-type recombinant human GIF/MIF with cystine resulted in preferential cysteinylation of Cys-60 in the molecules. The cysteinylated recombinant human GIF and the Ts hybridoma-derived cysteinylated GIF were comparable both in the affinity for the receptors and in the immunosuppressive activity. Polyclonal antibodies specific for a stretch of the amino acid sequence in alpha2-helix of GIF bound bioactive cysteinylated GIF but failed to bind wild-type GIF/MIF. These results strongly suggest that cysteinylation of Cys-60 and consequent conformational changes in the GIF/MIF molecules are responsible for the generation of GIF bioactivity.
糖基化抑制因子（GIF）和巨噬细胞移动抑制因子（MIF）共享一个相同的结构基因。在此，我们揭示了人类抑制性T（Ts）细胞杂交瘤中GIF/MIF分子的两步翻译后修饰。对Ts细胞中亲和纯化的GIF进行肽图谱分析和质谱分析显示，一种修饰是第60位半胱氨酸的半胱氨酸化，另一种是第91位丝氨酸的磷酸化。半胱氨酸化的GIF，而非野生型GIF/MIF，对体外IgE抗体反应具有免疫抑制作用，并且对T辅助杂交瘤细胞上的GIF受体具有高亲和力。用胱氨酸对野生型重组人GIF/MIF进行体外处理导致分子中第60位半胱氨酸优先发生半胱氨酸化。半胱氨酸化的重组人GIF与Ts杂交瘤来源的半胱氨酸化GIF在受体亲和力和免疫抑制活性方面相当。针对GIFα2螺旋中一段氨基酸序列的多克隆抗体可结合具有生物活性的半胱氨酸化GIF，但不能结合野生型GIF/MIF。这些结果强烈表明，第60位半胱氨酸的半胱氨酸化以及GIF/MIF分子随之发生的构象变化是GIF生物活性产生的原因。
