Chi Sulene L, Wahl Miriam L, Mowery Yvonne M, Shan Siqing, Mukhopadhyay Somnath, Hilderbrand Susana C, Kenan Daniel J, Lipes Barbara D, Johnson Carrie E, Marusich Michael F, Capaldi Roderick A, Dewhirst Mark W, Pizzo Salvatore V
Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA.
Cancer Res. 2007 May 15;67(10):4716-24. doi: 10.1158/0008-5472.CAN-06-1094.
The antiangiogenic protein angiostatin inhibits ATP synthase on the endothelial cell surface, blocking cellular proliferation. To examine the specificity of this interaction, we generated monoclonal antibodies (mAb) directed against ATP synthase. mAb directed against the beta-catalytic subunit of ATP synthase (MAb3D5AB1) inhibits the activity of the F(1) domain of ATP synthase and recognizes the catalytic beta-subunit of ATP synthase. We located the antibody recognition site of MAb3D5AB1 in domains containing the active site of the beta-subunit. MAb3D5AB1 also binds to purified Escherichia coli F(1) with an affinity 25-fold higher than the affinity of angiostatin for this protein. MAb3D5AB1 inhibits the hydrolytic activity of F(1) ATP synthase at lower concentrations than angiostatin. Like angiostatin, MAb3D5AB1 inhibits ATP generation by ATP synthase on the endothelial cell surface in acidic conditions, the typical tumor microenvironment where cell surface ATP synthase exhibits greater activity. MAb3D5AB1 disrupts tube formation and decreases intracellular pH in endothelial cells exposed to low extracellular pH. Neither angiostatin nor MAb3D5AB1 showed an antiangiogenic effect in the corneal neovascularization assay; however, both were effective in the low-pH environment of the chicken chorioallantoic membrane assay. Thus, MAb3D5AB1 shows angiostatin-like properties superior to angiostatin and may be exploited in cancer chemotherapy.
抗血管生成蛋白血管抑素可抑制内皮细胞表面的ATP合酶，从而阻断细胞增殖。为了研究这种相互作用的特异性，我们制备了针对ATP合酶的单克隆抗体（mAb）。针对ATP合酶β催化亚基的单克隆抗体（MAb3D5AB1）可抑制ATP合酶F(1)结构域的活性，并识别ATP合酶的催化β亚基。我们将MAb3D5AB1的抗体识别位点定位在含有β亚基活性位点的结构域中。MAb3D5AB1还能与纯化的大肠杆菌F(1)结合，其亲和力比血管抑素对该蛋白的亲和力高25倍。MAb3D5AB1在比血管抑素更低的浓度下就能抑制F(1) ATP合酶的水解活性。与血管抑素一样，MAb3D5AB1在酸性条件下可抑制内皮细胞表面ATP合酶产生ATP，酸性条件是典型的肿瘤微环境，此时细胞表面ATP合酶活性更高。MAb3D5AB1可破坏内皮细胞的管腔形成，并降低暴露于低细胞外pH值环境中的内皮细胞的细胞内pH值。在角膜新生血管形成试验中，血管抑素和MAb3D5AB1均未显示出抗血管生成作用；然而，在鸡胚绒毛尿囊膜试验的低pH环境中，二者均有效。因此，MAb3D5AB1具有优于血管抑素的血管抑素样特性，可用于癌症化疗。
