Schneider P, Bodmer J L, Holler N, Mattmann C, Scuderi P, Terskikh A, Peitsch M C, Tschopp J
Institute of Biochemistry, University of Lausanne, BIL Biomedical Research Center, CH-1066 Epalinges, Switzerland.
J Biol Chem. 1997 Jul 25;272(30):18827-33. doi: 10.1074/jbc.272.30.18827.
The death-inducing receptor Fas is activated when cross-linked by the type II membrane protein Fas ligand (FasL). When human soluble FasL (sFasL, containing the extracellular portion) was expressed in human embryo kidney 293 cells, the three N-linked glycans of each FasL monomer were found to be essential for efficient secretion. Based on the structure of the closely related lymphotoxin alpha-tumor necrosis factor receptor I complex, a molecular model of the FasL homotrimer bound to three Fas molecules was generated using knowledge-based protein modeling methods. Point mutations of amino acid residues predicted to affect the receptor-ligand interaction were introduced at three sites. The F275L mutant, mimicking the loss of function murine gld mutation, exhibited a high propensity for aggregation and was unable to bind to Fas. Mutants P206R, P206D, and P206F displayed reduced cytotoxicity toward Fas-positive cells with a concomitant decrease in the binding affinity for the recombinant Fas-immunoglobulin Fc fusion proteins. Although the cytotoxic activity of mutant Y218D was unaltered, mutant Y218R was inactive, correlating with the prediction that Tyr-218 of FasL interacts with a cluster of three basic amino acid side chains of Fas. Interestingly, mutant Y218F could induce apoptosis in murine, but not human cells.
死亡诱导受体Fas在被II型膜蛋白Fas配体（FasL）交联时被激活。当人可溶性FasL（sFasL，含细胞外部分）在人胚肾293细胞中表达时，发现每个FasL单体的三个N-连接聚糖对于有效分泌至关重要。基于密切相关的淋巴毒素α-肿瘤坏死因子受体I复合物的结构，使用基于知识的蛋白质建模方法生成了与三个Fas分子结合的FasL同三聚体的分子模型。在三个位点引入了预测会影响受体-配体相互作用的氨基酸残基的点突变。模拟功能丧失型小鼠gld突变的F275L突变体表现出高度聚集倾向，并且无法与Fas结合。P206R、P206D和P206F突变体对Fas阳性细胞的细胞毒性降低，同时对重组Fas-免疫球蛋白Fc融合蛋白的结合亲和力也降低。尽管Y218D突变体的细胞毒性活性未改变，但Y218R突变体无活性，这与FasL的Tyr-218与Fas的三个碱性氨基酸侧链簇相互作用的预测相关。有趣的是，Y218F突变体可在小鼠细胞而非人细胞中诱导凋亡。
