Chen Daming, Nicholas John
Johns Hopkins Oncology Center, 1650 Orleans Street, Room 309, Baltimore, MD 21231,USA.
J Virol. 2006 Oct;80(19):9811-21. doi: 10.1128/JVI.00872-06.
Human herpesvirus 8 interleukin-6 (vIL-6) displays 25% amino acid identity with human IL-6 (hIL-6) and shares an overall four-helix-bundle structure and gp130-mediated STAT/mitogen-activated protein kinase signaling with its cellular counterpart. However, vIL-6 is distinct in that it can signal through gp130 alone, in the absence of the nonsignaling gp80 alpha-subunit of the IL-6 receptor. To investigate the structural requirements for gp80 independence of vIL-6, a series of expression vectors encoding vIL-6/hIL-6 chimeric and site-mutated IL-6 proteins was generated. The replacement of hIL-6 residues with three vIL-6-specific tryptophans implicated in gp80 independence from crystallographic studies or the A and C helices containing these residues did not confer gp80 independence to hIL-6. The N- and C-terminal regions of vIL-6 could be substituted with hIL-6 sequences with the retention of gp80-independent signaling, but substitutions of other regions of vIL-6 (helix A, A/B loop, helix B, helix C, and proximal half of helix D) with equivalent sequences of hIL-6 abolished gp80 independence. Interestingly, the B helix of vIL-6 was absolutely required for gp80 independence, despite the fact that this region contains no receptor-binding residues. Point mutational analysis of helix C, which contains residues involved in physical and functional interactions with gp130 domains 2 and 3 (cytokine-binding homology region), identified a variant, VI120EE, that was able to signal and dimerize gp130 only in the presence of gp80. gp80 was also found to stabilize gp130:g130 dimers induced by a distal D helix variant of vIL-6 that was nonetheless able to signal independently of gp80. Together, our data reveal the crucial importance of overall vIL-6 structure and conformation for gp80-independent signaling and provide functional and physical evidence of the stabilization of vIL-6-induced gp130 signaling complexes by gp80.
人类疱疹病毒8型白细胞介素-6（vIL-6）与人类白细胞介素-6（hIL-6）有25%的氨基酸同源性，并且与其细胞对应物共享整体的四螺旋束结构以及gp130介导的信号转导和转录激活因子/丝裂原活化蛋白激酶信号通路。然而，vIL-6的独特之处在于，在缺乏白细胞介素-6受体的无信号传导的gp80α亚基的情况下，它可以仅通过gp130进行信号传导。为了研究vIL-6不依赖gp80的结构要求，构建了一系列编码vIL-6/hIL-6嵌合和位点突变白细胞介素-6蛋白的表达载体。用三个vIL-6特异性色氨酸取代hIL-6残基（这些色氨酸在晶体学研究中与不依赖gp80有关），或者取代含有这些残基的A螺旋和C螺旋，并没有赋予hIL-6不依赖gp80的特性。vIL-6的N端和C端区域可以被hIL-6序列取代，同时保留不依赖gp80的信号传导能力，但是用hIL-6的等效序列取代vIL-6的其他区域（A螺旋、A/B环、B螺旋、C螺旋和D螺旋近端一半）会消除不依赖gp80的特性。有趣的是，尽管vIL-6的B螺旋区域不包含受体结合残基，但它对于不依赖gp80却是绝对必需的。对C螺旋进行点突变分析，该螺旋包含与gp130结构域2和3（细胞因子结合同源区域）发生物理和功能相互作用的残基，鉴定出一个变体VI120EE，它仅在存在gp80的情况下才能进行信号传导并使gp130二聚化。还发现gp80能够稳定由vIL-6的远端D螺旋变体诱导的gp130:g130二聚体，而该变体仍然能够独立于gp80进行信号传导。总之，我们的数据揭示了vIL-6整体结构和构象对于不依赖gp80的信号传导至关重要，并为gp80稳定vIL-6诱导的gp130信号复合物提供了功能和物理证据。
