Rickers A, Brockstedt E, Mapara M Y, Otto A, Dörken B, Bommert K
Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany.
Eur J Immunol. 1998 Jan;28(1):296-304. doi: 10.1002/(SICI)1521-4141(199801)28:01<296::AID-IMMU296>3.0.CO;2-4.
Apoptosis (programmed cell death) is instrumental in the process of controlling lymphocyte growth and selection. Negative selection, mediated by surface IgM (sIgM) signaling after encountering self antigen, eliminates autoreactive B cells. To identify proteins which are potentially involved in anti-IgM-mediated apoptosis, we used an anti-IgM-sensitive subclone of the human Burkitt lymphoma cell line BL60. After anti-IgM treatment and separation of apoptosis-committed cells, we performed high resolution two-dimensional gel electrophoresis (2-DE). Comparison of the 2-DE protein patterns from apoptotic and non-apoptotic cells showed differences in approximately 80 spots. Subsequent analysis of these proteins was performed by mass spectrometry and Edman microsequencing. We report that one of these spots which disappears after sIgM cross-linking turned out to be D4-GDI. D4-GDI is an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPase. D4-GDI was rapidly truncated to a 23-kDa fragment in BL60 cells. By using a Rho-GDI-specific antiserum, which cross-reacts with D4-GDI, we observed the onset of cleavage after 8 h of stimulation with anti-IgM. Cleavage and apoptosis could be completely inhibited by z-DEVD-fmk, a selective irreversible inhibitor of CPP32 (caspase-3), whereas ac-YVAD-cmk, an inhibitor for interleukin-1beta-converting enzyme-like proteases, did not block cleavage of D4-GDI or apoptosis. Our results revealed the functional importance of caspases and a new target protein in the process of anti-IgM-mediated apoptosis.
细胞凋亡（程序性细胞死亡）在控制淋巴细胞生长和选择过程中起重要作用。负选择由遇到自身抗原后表面IgM（sIgM）信号介导，可消除自身反应性B细胞。为了鉴定可能参与抗IgM介导的细胞凋亡的蛋白质，我们使用了人伯基特淋巴瘤细胞系BL60的抗IgM敏感亚克隆。在抗IgM处理并分离出凋亡相关细胞后，我们进行了高分辨率二维凝胶电泳（2-DE）。比较凋亡细胞和非凋亡细胞的2-DE蛋白质图谱，发现约80个斑点存在差异。随后通过质谱分析和埃德曼微测序对这些蛋白质进行分析。我们报告说，其中一个在sIgM交联后消失的斑点原来是D4-GDI。D4-GDI是一种丰富的造血细胞GDP解离抑制剂，作用于Ras相关的Rho家族GTP酶。在BL60细胞中，D4-GDI迅速截短为一个23 kDa的片段。通过使用与D4-GDI发生交叉反应的Rho-GDI特异性抗血清，我们观察到在用抗IgM刺激8小时后开始出现裂解。z-DEVD-fmk（一种选择性不可逆的CPP32（半胱天冬酶-3）抑制剂）可完全抑制裂解和细胞凋亡，而白细胞介素-1β转化酶样蛋白酶抑制剂ac-YVAD-cmk则不能阻断D4-GDI的裂解或细胞凋亡。我们的结果揭示了半胱天冬酶在抗IgM介导的细胞凋亡过程中的功能重要性以及一个新的靶蛋白。
