Improved biological activity of a mutant endostatin containing a single amino-acid substitution.
Improved biological activity of a mutant endostatin containing a single amino-acid substitution.
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DOI：
10.1038/sj.bjc.6601745
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发表时间：
2004-04-19
影响因子：
8.8
通讯作者：
中科院分区：
文献类型：
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作者：
人内皮抑素在位置 126-128 处有一个内部 Asn-Gly-Arg (NGR) 基序，后面是位置 125 处的脯氨酸。含有 Asn-Gly-Arg 的肽已被证明可以靶向肿瘤血管系统并抑制氨肽酶 N 活性。我们之前比较了天然内皮抑素和脯氨酸丙氨酸突变型内皮抑素（P125A-内皮抑素）的体外和体内生物活性。与天然内皮抑素相比，P125A-内皮抑素对内皮细胞增殖和人卵巢癌生长表现出更大的抑制作用。在这里，我们进一步探讨了 P125A 突变对生物活性的影响，并表明氨肽酶 N 不参与其中。为了确定突变体生物活性的增加是否是由于下游 NGR 序列的暴露所致，研究了内皮抑素对氨肽酶 N 活性的影响。天然内皮抑素和P125A-内皮抑素均不抑制氨肽酶N。然而，由内皮抑素的S118-T131区域组成的合成肽抑制氨肽酶N。这些结果表明天然或突变内皮抑素中的内部NGR位点不能被氨肽酶N接近，并且这种活性不涉及P125A形式的增强的生物活性。 P125A-内皮抑素比天然内皮抑素更有效地与内皮细胞结合，并且不仅对内皮细胞的增殖而且对内皮细胞的迁移表现出更大的抑制作用。 P125A-内皮抑素也比天然蛋白更高程度地定位到肿瘤组织中，并且对无胸腺小鼠中的结肠癌生长表现出更大的抑制作用。两种蛋白均能有效抑制肿瘤细胞诱导的血管生成。实时 PCR 分析表明，天然内皮抑素和 P125A-内皮抑素均可降低关键促血管生成生长因子的表达。血管内皮生长因子和血管生成素1被突变体更多地下调。这些研究表明，可以对 P125 周围的区域进行修饰，以提高内皮抑素的生物活性。
Human endostatin has an internal Asn-Gly-Arg (NGR) motif at position 126–128 following a proline at position 125. Asn-Gly-Arg-containing peptides have been shown to target tumour vasculature and inhibit aminopeptidase N activity. We previously compared the in vitro and in vivo biological activities of native endostatin and endostatin with a proline to alanine mutation (P125A-endostatin). P125A-endostatin exhibited greater inhibition of both endothelial cell proliferation and human ovarian cancer growth compared to native endostatin. Here we explore further the effects on biological activity of the P125A mutation, and show that aminopeptidase N is not involved. To determine whether the increased biological activity of the mutant was due to unmasking of downstream NGR-sequence, effect of endostatin on aminopeptidase N activity was investigated. Neither the native nor the P125A-endostatin inhibited aminopeptidase N. However, synthetic peptides consisting of the S118-T131 region of endostatin inhibited aminopeptidase N. These results suggest that the internal NGR site in native or mutant endostatin is not accessible to aminopeptidase N, and that this activity is not involved in the enhanced biological activity of the P125A form. P125A-endostatin bound to endothelial cells more efficiently than native endostatin and exhibited greater inhibition of not only proliferation but also migration of endothelial cells. P125A-endostatin also localised into tumour tissue to a higher degree than the native protein, and displayed greater inhibition of growth of colon cancer in athymic mice. Both proteins inhibited tumour cell-induced angiogenesis effectively. Real-time PCR analysis showed that both native and P125A-endostatin decreased expression of key proangiogenic growth factors. Vascular endothelial growth factor and angiopoietin 1 were downregulated more by the mutant. These studies suggest that the region around P125 can be modified to improve the biological activity of endostatin.
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DOI：
10.1083/jcb.200203064
发表时间：
2002-08-05
期刊：
The Journal of cell biology
影响因子：
--
作者：
Hanai J;Gloy J;Karumanchi SA;Kale S;Tang J;Hu G;Chan B;Ramchandran R;Jha V;Sukhatme VP;Sokol S
通讯作者：
Sokol S
影响因子：
3.1
作者：
Wild, R;Ramakrishnan, S;Griffioen, AW
通讯作者：
Griffioen, AW
影响因子：
11.4
作者：
Yamaguchi, N;Anand-Apte, B;Olsen, BR
通讯作者：
Olsen, BR
影响因子：
11.4
作者：
Sasaki, T;Larsson, H;Timpl, R
通讯作者：
Timpl, R
影响因子：
4.8
作者：
Shichiri, M;Hirata, Y
通讯作者：
Hirata, Y