Fattorusso R, Pellecchia M, Viti F, Neri P, Neri D, Wüthrich K
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland.
Structure. 1999 Apr 15;7(4):381-90. doi: 10.1016/s0969-2126(99)80051-3.
The process of angiogenesis (i.e. the formation of new blood vessels from pre-existing ones) is fundamental to physiological processes such as reproduction, development and repair, as well as to pathological conditions such as tumor progression, rheumathoid arthritis and ocular disorders. The oncofoetal ED-B domain, a specific marker of angiogenesis, consists of 91 amino acid residues that are inserted by alternative splicing into the fibronectin (FN) molecule.
The NMR structure of the ED-B domain is reported and reveals important differences from other FN type III domains. A comparison of the ED-B domain with the crystal structure of a four-domain FN fragment shows the novel features of ED-B to be located in loop regions that are buried at interdomain interfaces, and which therefore largely determine the global shape of the FN molecule. The negatively charged amino acids in this highly acidic protein are uniformly distributed over the molecular surface, with the sole exception of a solvent-exposed hydrophobic patch that represents a potential specific recognition site. Epitope mapping with 82 decapeptides that span the ED-B sequence revealed that three ED-B-specific monoclonal antibodies, which selectively target newly forming blood vessels in tumor-bearing mice, bind to adjacent regions on the ED-B surface.
The NMR structure enables the identification of a large surface area of the ED-B domain that appears to be accessible in vivo, opening up new diagnostic and therapeutic opportunities. Furthermore, the mapping of specific monoclonal antibodies to the three-dimensional structure of the ED-B domain, and their use in angiogenesis inhibition experiments, provides a basis for further investigation of the role of the ED-B domain in the formation of new blood vessels.
血管生成过程（即从已有的血管形成新的血管）对于诸如生殖、发育和修复等生理过程，以及诸如肿瘤进展、类风湿性关节炎和眼部疾病等病理状况而言至关重要。癌胚ED-B结构域是血管生成的一种特异性标志物，由91个氨基酸残基组成，这些残基通过选择性剪接插入到纤连蛋白（FN）分子中。
报道了ED-B结构域的核磁共振结构，揭示了其与其他FN III型结构域的重要差异。将ED-B结构域与一个四结构域FN片段的晶体结构进行比较，结果表明ED-B的新特征位于埋于结构域间界面的环区，因此在很大程度上决定了FN分子的整体形状。在这种高度酸性的蛋白质中，带负电荷的氨基酸均匀分布在分子表面，唯一的例外是一个暴露于溶剂中的疏水斑块，它代表一个潜在的特异性识别位点。用跨越ED-B序列的82个十肽进行表位作图显示，三种选择性靶向荷瘤小鼠新生血管的ED-B特异性单克隆抗体与ED-B表面的相邻区域结合。
核磁共振结构能够识别ED-B结构域在体内似乎可及的大面积区域，从而带来新的诊断和治疗机会。此外，将特异性单克隆抗体定位到ED-B结构域的三维结构，并将其用于血管生成抑制实验，为进一步研究ED-B结构域在新血管形成中的作用提供了基础。
