Recombinant Anti-CD4 antibody 13B8.2 blocks membrane-proximal events by excluding the Zap70 molecule and downstream targets SLP-76, PLCγ1, and Vav-1 from the CD4-Segregated Brij 98 detergent-resistant raft domains
Recombinant Anti-CD4 antibody 13B8.2 blocks membrane-proximal events by excluding the Zap70 molecule and downstream targets SLP-76, PLCγ1, and Vav-1 from the CD4-Segregated Brij 98 detergent-resistant raft domains
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DOI：
10.4049/jimmunol.179.1.409
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发表时间：
2007-07-01
影响因子：
4.4
通讯作者：
Chardes, Thierry
中科院分区：
文献类型：
--
作者：
Chentouf, Myriam;Ghannam, Soufiane;Chardes, Thierry
R1gG(1)13B8.2针对CDR3样环的生物学效应部分是由于阻止核转录因子-B-k核转位的信号，但其确切的作用机制，特别是在膜近端信号水平上，仍不清楚。我们支持Rigg(1)13B8.2通过干扰膜筏内信号或受体分子的时空分布而起作用的假设。在Jurkat T淋巴细胞r1gG，13B8.2被发现在37℃下诱导CD4分子在聚氧乙烯-20醚Brij 98耐洗涤剂抗性膜内聚集/保留，以及TCR，CD3 Zeta，p56Lck，Lyn，和Syk p70激酶，T细胞激活的连接物，与神经鞘糖脂适配器蛋白相关的CSK结合蛋白/磷蛋白，以及蛋白激酶C theta，但排除了ZAP70及其下游靶标-76 kDa的含有Src同源2结构域的白细胞蛋白，磷脂酶C-1，和P95(Nu A Nu)。对ZAP70磷酸化等关键上游事件的分析表明，在13B8.2诱导ZAP70从膜筏中排除的同时，Tyr(292)和Tyr(319)磷酸化的调节也发生了。13B8.2诱导的差异RAFT分配依赖于表位、胆固醇和肌动蛋白，但不需要抗体的超交联性。荧光共聚焦成像证实了CD4复合体在R1gG(1)13B8.2结扎后10-30 S内的时空分离和伴随的ZAP70排斥，在1min达到平台期，并持续到1h实验结束。膜筏内CD4受体和ZAP70信号激酶之间的不同时空分割中断了近端的信号串扰，导致随后的NF-B-k核转位，并解释了杆状病毒表达的CD4-CDR3样特异性r1gG(1)13B8.2是如何作用于诱导其生物学效应的。
The biological effects of r1gG(1) 13B8.2, directed against the CDR3-like loop on the D1 domain of CD4, are partly due to signals that prevent NF-B-k nuclear translocation, but the precise mechanisms of action, particularly at the level of membrane proximal signaling, remain obscure. We support the hypothesis that rIgG(1) 13B8.2 acts by interfering with the spatiotemporal distribution of signaling or receptor molecules inside membrane rafts. Upon cross-linking of Jurkat T lymphocytes, r1gG, 13B8.2 was found to induce an accumulation/retention of the CD4 molecule inside polyoxyethylene-20 ether Brij 98 detergent-resistant membranes at 37 degrees C, together with recruitment of TCR, CD3 zeta, p56 Lck, Lyn, and Syk p70 kinases, linker for activation of T cells, and Csk-binding protein/phosphoprotein associated with glycosphingolipid adaptor proteins, and protein kinase C theta, but excluded Zap70 and its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa, phospholipase C gamma 1, and p95(nu a nu). Analysis of key upstream events such as Zap70 phosphorylation showed that modulation of Tyr(292) and Tyr(319) phosphorylation occurred concomitantly with 13B8.2-induced Zap70 exclusion from the membrane rafts. 13B8.2-induced differential raft partitioning was epitope, cholesterol, and actin dependent but did not require Ab hyper-cross-linking. Fluorescence confocal imaging confirmed the spatiotemporal segregation of the CD4 complex inside rafts and concomitant Zap70 exclusion, which occurred within 10-30 s following r1gG(1) 13B8.2 ligation, reached a plateau at I min, and persisted until the end of the 1-h experiment. The differential spatiotemporal partitioning between the CD4 receptor and the Zap70-signaling kinase inside membrane rafts interrupts the proximal signal cross-talk leading to subsequent NF-B-k nuclear translocation and explains how baculovirus-expressed CD4-CDR3-like-specific r1gG(1) 13B8.2 acts to induce its biological effects.