ABSTRACT

Carbonic anhydrase (CA)-IX is an extracellular enzyme that is essential in the adaptation of tumor cells to their increasingly more hypoxic and acidic microenvironment. Within the family of carbonic anhydrases, CA-IX is unique in that it is the only CA with an N-terminal intrinsically disordered region (IDR) containing a proteoglycan (PG)-like domain. This PG-like IDR has been described to be instrumental in CA-IX’s enzyme activity, as well as tumor cell motility and invasion. We have characterized the antibody–epitope interactions of two novel and unique antibodies (11H9 and 12H8) that are specific for the human CA-IX’s IDR. Binding interactions of these antibodies to the intact IDR were studied by surface plasmon resonance and high-resolution nuclear magnetic resonance (NMR) spectroscopy, while the specific epitopes were determined by both NMR and yeast surface display (YSD). Our data show that 12H8 binds to the N-terminus of CA-IX, while 11H9 has a high affinity for an epitope located in the central region of the IDR containing three GEEDLP repeats in a manner that is different from the previously described M75 antibody. Titration NMR spectroscopy using CA-IX’s entire IDR in addition identified a secondary epitope of 11H9 at the beginning of the PG-like domain that remains exposed and available for further binding events after the engagement at its primary epitope at the center of the PG-like domain. Transverse relaxation optimized NMR spectroscopy of 11H9-F(Ab) in complex with the CA-IX IDR outlines structural rigidification of a linear epitope, while the rest of the IDR remains largely unstructured upon complex formation. This study illustrates how high-resolution NMR and YSD are used as complementary tools for a comprehensive characterization of antibody–epitope interactions involving intrinsically unstructured antigen domains with highly repetitive sequences.

摘要

碳酸酐酶 (CA)-IX 是一种细胞外酶，对于肿瘤细胞适应日益缺氧和酸性的微环境至关重要。在碳酸酐酶家族中，CA-IX 的独特之处在于它是唯一具有 N 端固有无序区域 (IDR) 且含有蛋白聚糖 (PG) 样结构域的 CA。这种 PG 样 IDR 已被描述为有助于 CA-IX 的酶活性以及肿瘤细胞的运动和侵袭。我们已经表征了两种新颖且独特的抗体（11H9 和 12H8）的抗体-表位相互作用，这两种抗体对人类 CA-IX 的 IDR 具有特异性。通过表面等离子共振和高分辨率核磁共振 (NMR) 光谱研究了这些抗体与完整 IDR 的结合相互作用，同时通过 NMR 和酵母表面展示 (YSD) 确定了特定表位。我们的数据显示，12H8 与 CA-IX 的 N 末端结合，而 11H9 对位于包含三个 GEEDLP 重复的 IDR 中心区域的表位具有高亲和力，其方式与之前描述的 M75 抗体不同。使用 CA-IX 的整个 IDR 的滴定 NMR 光谱还鉴定了 PG 样结构域起始处的 11H9 二级表位，该二级表位在与 PG 样结构域中心的初级表位接合后仍然暴露并可用于进一步的结合事件领域。与 CA-IX IDR 复合的 11H9-F(Ab) 的横向弛豫优化 NMR 光谱概述了线性表位的结构刚性，而 IDR 的其余部分在复合物形成时仍然基本上是非结构化的。这项研究说明了如何使用高分辨率 NMR 和 YSD 作为补充工具来全面表征抗体-表位相互作用，涉及具有高度重复序列的本质上非结构化抗原结构域。