The N-Terminal Integrity Is Critical for the Stability and Biological Functions of Endostatint
The N-Terminal Integrity Is Critical for the Stability and Biological Functions of Endostatint
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DOI：
10.1021/bi100489x
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发表时间：
2010-08-03
期刊：
影响因子：
2.9
通讯作者：
Luo, Yongzhang
中科院分区：
文献类型：
--
作者：
Fu, Yan;Luo, Yongzhang
内皮抑素是一种内源性血管生成抑制剂，其N末端的氨基酸残基HI、H3、H11和D76与一个锌离子配位。在毕赤酵母表达过程中N末端截短的重组内皮抑素在之前的研究中被广泛使用，并产生了不一致的抗肿瘤结果。然而，很少有人关注N端截短可能对内皮抑素的稳定性和活性造成的改变。在本研究中，巴斯德毕赤酵母表达的内皮抑素的 N 末端截短形式被鉴定为 N-1、N-3 和 N-4，其中四个锌结合残基中的一个或两个丢失。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活性的显着变化，而前三个氨基酸残基 (FISH) 或前四个氨基酸残基 (HSHR) 的截短会显着降低尿素诱导的解折叠和内皮抑素体外和体内生物活性所测得的热力学稳定性。有趣的是，在 N 末端设计了一个短的额外锌结合肽 (ZBP) 的 ZBP-内皮抑素表现出更紧密堆积的三级结构，增加了热力学稳定性和对尿素的协同性，比野生型内皮抑素具有更有效的抗内皮和抗肿瘤活性。这些发现表明，N端完整性对于内皮抑素的稳定性和生物学功能至关重要，这为各种研究中内皮抑素的抗肿瘤活性不一致，包括临床试验中内皮抑素和ZBP-内皮抑素的不同治疗效果提供了根本解释。
Endostatin is an endogenous angiogenesis inhibitor, and amino acid residues HI, 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 (FISH) 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.