Shedding of interleukin-6 receptor and tumor necrosis factor α -: Contribution of the stalk sequence to the cleavage pattern of transmembrane proteins
Shedding of interleukin-6 receptor and tumor necrosis factor α -: Contribution of the stalk sequence to the cleavage pattern of transmembrane proteins
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DOI：
10.1046/j.1432-1327.2000.01278.x
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发表时间：
2000-05-01
期刊：
影响因子：
--
通讯作者：
Müllberg, J
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文献类型：
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作者：
Althoff, K;Reddy, P;Müllberg, J
一组功能和结构上不同的跨膜蛋白，包括介体或受体的跨膜形式，可以被蛋白水解切割以形成可溶性生长因子或受体。最近，已经鉴定了负责促肿瘤坏死因子α（proTNF α）加工的蛋白水解活性，并将其命名为TACE（TNF α转化酶）。在用TACE缺陷（TACB（-/-））成纤维细胞的实验中，我们发现4 β-佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）诱导的白细胞介素-6受体（IL-6 R）的脱落强烈减少。然而，仍然可以检测到IL-6 R的基础异羟肟酸敏感性释放。该结果表明TACE在IL-6 R加工中起作用，并且可能涉及另外的金属蛋白酶。PMA诱导的TACE缺陷小鼠成纤维细胞中IL-6 R的脱落可以通过稳定转染TACE cDNA来恢复。为了表征IL-6 R和proTNF α的脱落之间的差异，我们产生了嵌合IL-6 R和proTNF α蛋白，其中内源性切割位点（CS）分别被proTNF α和IL-6 R的相应区域取代。有趣的是，proTNF α嵌合蛋白仅显示最小的脱落。相反，含有proTNF α CS的IL-6 R嵌合体自发脱落，PMA未进一步诱导加工。因此，通过引入proTNF α CS转移的切割模式与proTNF α本身的切割模式相似。我们得出结论，在蛋白水解CS的氨基酸序列有助于蛋白质的切割特性。然而，仅此信息不足以转移可切割性，如用含有IL-6 R CS且对脱落有抗性的proTNF α嵌合体所见。
A functionally and structurally diverse group of transmembrane proteins including transmembrane forms of mediators or receptors can be proteolytically cleaved to form soluble growth factors or receptors. Recently, the proteolytic activity responsible for pro-tumor necrosis factor a (proTNF alpha) processing has been identified and named TACE (TNF alpha converting enzyme). In experiments with TACE deficient (TACB(-/-)) fibroblasts we found that 4 beta-phorbol 12-myristate 13-acetate (PMA)-induced shedding of the interleukin-6 receptor (IL-6R) is strongly reduced. A basal hydroxamate sensitive release of IL-6R, however, could still be detected. This result demonstrates that TACE plays a role in IL-6R processing and that additional metalloproteases might be involved. PMA-induced shedding of IL-6R in TACE deficient mouse fibroblasts could be restored by stable transfection of a TACE cDNA. To characterize differences between shedding of IL-6R and proTNF alpha we generated chimeric IL-6R and proTNF alpha proteins wherein the endogenous cleavage sites (CS) had been replaced by the corresponding region of proTNF alpha and IL-6R, respectively. Interestingly, proTNF alpha chimeric proteins showed only minimal shedding. In contrast, IL-6R chimeras containing the proTNF alpha CS were shed spontaneously, processing was not further induced by PMA. Thus, the cleavage pattern transferred by the introduction of the proTNF alpha CS is similar to that of proTNF alpha itself. We conclude that the amino-acid sequence at the proteolytic CS contributes to the cleavage characteristics of a protein. However, this information alone is not sufficient to transfer cleavability as seen with proTNF alpha chimeras containing the IL-6R CS and which were resistant to shedding.