Brakenhoff J P, de Hon F D, Fontaine V, ten Boekel E, Schooltink H, Rose-John S, Heinrich P C, Content J, Aarden L A
Department of Autoimmune Diseases, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam.
J Biol Chem. 1994 Jan 7;269(1):86-93.
Neutralizing monoclonal antibodies specific for human interleukin-6 (IL-6) bind two distinct sites on the IL-6 protein (sites I and II). Their interference with IL-6 receptor binding suggested that site I is a receptor-binding site of IL-6, whereas site II is important for signal transduction. Mutagenesis of site II could therefore result in the isolation of IL-6 receptor antagonists. To test this hypothesis, a panel of IL-6 mutant proteins was constructed that did not bind to a site II-specific monoclonal antibody. One such site II mutant protein (with double substitution of Gln-160 with Glu and Thr-163 with Pro) was found to be an antagonist of human IL-6. It was inactive on human CESS cells, weakly active on human HepG2 cells, but active on mouse B9 cells. It could specifically antagonize the activity of wild-type IL-6 on CESS and HepG2 cells. The binding affinity of this variant for the 80-kDa IL-6 receptor was similar to that of wild-type IL-6. High affinity binding to CESS cells, however, was abolished, suggesting that the mutant protein is inactive because the complex of the 80-kDa IL-6 receptor and the mutant protein cannot associate with the signal transducer gp130. The human IL-6 antagonist protein may be potentially useful as a therapeutic agent.
针对人白细胞介素-6（IL-6）的中和性单克隆抗体可结合IL-6蛋白上两个不同的位点（位点I和位点II）。它们对IL-6受体结合的干扰表明，位点I是IL-6的受体结合位点，而位点II对信号转导很重要。因此，对位点II进行诱变可能会分离出IL-6受体拮抗剂。为了验证这一假设，构建了一组不与位点II特异性单克隆抗体结合的IL-6突变蛋白。发现一种这样的位点II突变蛋白（Gln-160被Glu双取代且Thr-163被Pro取代）是人IL-6的拮抗剂。它对人CESS细胞无活性，对人HepG2细胞活性较弱，但对小鼠B9细胞有活性。它可以特异性拮抗野生型IL-6对CESS和HepG2细胞的活性。该变体对80 kDa IL-6受体的结合亲和力与野生型IL-6相似。然而，它与CESS细胞的高亲和力结合被消除，这表明突变蛋白无活性是因为80 kDa IL-6受体与突变蛋白的复合物无法与信号转导子gp130结合。人IL-6拮抗剂蛋白可能有作为治疗剂的潜在用途。
