National Engineering Laboratory for Anti-tumor Protein Therapeutics, Beijing Key Laboratory of Protein Therapeutics, and Cancer Biology Laboratory, School of Life Sciences, Tsinghua University, Beijing, P. R. China.
Biochemistry. 2010 Aug 3;49(30):6420-9. doi: 10.1021/bi100489x.
Endostatin is an endogenous angiogenesis inhibitor, and amino acid residues H1, H3, H11, and D76 at its N-terminus coordinate with one zinc ion. Recombinant endostatin suffering from N-terminal truncations during Pichia pastoris expression was widely used in previous studies and generated inconsistent antitumor results. However, little attention was paid to the possible alteration on the stability and activity of endostatin caused by N-terminal truncations. In this study, N-terminally truncated forms of endostatin expressed by P. pastoris are identified as N-1, N-3, and N-4, in which one or two of the four zinc-binding residues are lost. The N-terminal truncation of the first amino acid residue (H) does not result in a significant change in the conformation, zinc-binding capacity, thermodynamic stability, or biological activity, while truncations of the first three amino acid residues (HSH) or the first four amino acid residues (HSHR) dramatically decrease the thermodynamic stability measured by urea-induced unfolding and biological activities of endostatin both in vitro and in vivo. Intriguingly, ZBP-endostatin with a short extra zinc-binding peptide (ZBP) engineered at the N-terminus exhibits a more tightly packed tertiary structure and increased thermodynamic stability and cooperativity against urea, with more potent antiendothelial and antitumor activities than the wild-type endostatin. These findings demonstrate that the N-terminal integrity is essential for the stability and biological functions of endostatin, which provides fundamental explanations for the inconsistent antitumor activities of endostatin in a variety of studies, including the different therapeutic efficacies of endostatin and ZBP-endostatin in clinical trials.
内皮抑素是一种内源性血管生成抑制剂，其 N 端的 H1、H3、H11 和 D76 氨基酸残基与一个锌离子配位。在毕赤酵母表达过程中，内皮抑素会遭受 N 端截短，这种截短形式在以前的研究中被广泛应用，但产生的抗肿瘤结果并不一致。然而，人们很少关注 N 端截短可能对内皮抑素稳定性和活性的影响。在本研究中，通过毕赤酵母表达鉴定出内皮抑素的 N 端截短形式为 N-1、N-3 和 N-4，其中一个或两个锌结合残基丢失。第一个氨基酸残基（H）的 N 端截短不会导致构象、锌结合能力、热力学稳定性或生物学活性发生显著变化，而截短第一个三个氨基酸残基（HSH）或前四个氨基酸残基（HSHR）则会显著降低通过脲诱导展开测量的热力学稳定性以及内皮抑素的体外和体内生物学活性。有趣的是，在 N 端工程化带有短额外锌结合肽（ZBP）的 ZBP-内皮抑素表现出更紧密的三级结构和增加的热力学稳定性和对脲的协同性，与野生型内皮抑素相比，具有更强的抗血管生成和抗肿瘤活性。这些发现表明，N 端的完整性对于内皮抑素的稳定性和生物学功能至关重要，这为不同研究中内皮抑素抗肿瘤活性不一致提供了基本解释，包括临床试验中内皮抑素和 ZBP-内皮抑素的不同治疗效果。
