Identification of an ADAM17 cleavage region in human CD16 (FcγRIII) and the engineering of a non-cleavable version of the receptor in NK cells.
Identification of an ADAM17 cleavage region in human CD16 (FcγRIII) and the engineering of a non-cleavable version of the receptor in NK cells.
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DOI：
10.1371/journal.pone.0121788
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Walcheck B
中科院分区：
文献类型：
--
作者：
Jing Y;Ni Z;Wu J;Higgins L;Markowski TW;Kaufman DS;Walcheck B
CD 16 a和CD 16 b分别是由人自然杀伤（NK）细胞和嗜中性粒细胞表达的IgG Fc受体。两种CD 16亚型在各种刺激激活细胞后通过ADAM 17介导的蛋白水解裂解进行表达的快速下调。我们通过质谱分析检测了活化NK细胞和中性粒细胞释放的可溶性CD 16，并在P1/P1′位置（丙氨酸195/缬氨酸196、缬氨酸196/丝氨酸197和苏氨酸198/异亮氨酸199）附近确定了三个独立的切割位点，揭示了CD 16的近膜切割区域。在基于细胞的测定中，将切割区域中间的位置197处的丝氨酸替换为脯氨酸（S197 P）有效地阻断了CD 16 a和CD 16 b切割。我们还表明，当在人NK细胞系NK 92和来源于基因工程人诱导多能干细胞的原代NK细胞中表达时，CD 16 a/S197 P对切割具有抗性。CD 16 a是一种有效的活化受体，尽管阻断了CD 16 a脱落，但S197 P突变并没有破坏受体与IgG的结合或抗体处理的肿瘤细胞对NK 92细胞的活化。我们的研究结果提供了由ADAM 17切割的CD 16的进一步表征，并且它们证明了CD 16 a的不可切割形式可以在工程化NK细胞中表达。
CD16a and CD16b are IgG Fc receptors expressed by human natural killer (NK) cells and neutrophils, respectively. Both CD16 isoforms undergo a rapid down-regulation in expression by ADAM17-mediated proteolytic cleavage upon cell activation by various stimuli. We examined soluble CD16 released from activated NK cells and neutrophils by mass spectrometric analysis, and identified three separate cleavage sites in close proximity at P1/P1′ positions alanine195/valine196, valine196/serine197, and threonine198/isoleucine199, revealing a membrane proximal cleavage region in CD16. Substitution of the serine at position 197 in the middle of the cleavage region for a proline (S197P) effectively blocked CD16a and CD16b cleavage in cell-based assays. We also show that CD16a/S197P was resistant to cleavage when expressed in the human NK cell line NK92 and primary NK cells derived from genetically-engineered human induced pluripotent stem cells. CD16a is a potent activating receptor and despite blocking CD16a shedding, the S197P mutation did not disrupt IgG binding by the receptor or its activation of NK92 cells by antibody-treated tumor cells. Our findings provide further characterization of CD16 cleavage by ADAM17 and they demonstrate that a non-cleavable version of CD16a can be expressed in engineered NK cells.
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