Yokoyama Y, Ramakrishnan S
Department of Pharmacology, University of Minnesota, 6-120 Jackson Hall, 321 Church Street, S E, Minneapolis, MN 55454, USA.
Br J Cancer. 2004 Apr 19;90(8):1627-35. doi: 10.1038/sj.bjc.6601745.
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.
人内皮抑素在第125位的脯氨酸之后，第126 - 128位有一个内部天冬酰胺-甘氨酸-精氨酸（NGR）基序。含天冬酰胺-甘氨酸-精氨酸的肽已被证明可靶向肿瘤血管并抑制氨肽酶N的活性。我们之前比较了天然内皮抑素和脯氨酸突变为丙氨酸的内皮抑素（P125A-内皮抑素）的体外和体内生物学活性。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的抑制作用更强。在此，我们进一步探究P125A突变对生物学活性的影响，并表明氨肽酶N不参与其中。为了确定突变体生物学活性的增加是否是由于下游NGR序列的暴露，我们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-内皮抑素均未抑制氨肽酶N。然而，由内皮抑素的S118 - T131区域组成的合成肽可抑制氨肽酶N。这些结果表明，天然或突变型内皮抑素中的内部NGR位点对氨肽酶N不可及，且该活性不参与P125A形式增强的生物学活性。P125A-内皮抑素比天然内皮抑素更有效地结合内皮细胞，不仅对内皮细胞增殖而且对其迁移均表现出更强的抑制作用。P125A-内皮抑素在肿瘤组织中的定位程度也高于天然蛋白，并且在无胸腺小鼠中对结肠癌生长的抑制作用更强。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，天然和P125A-内皮抑素均降低了关键促血管生成生长因子的表达。突变体对血管内皮生长因子和血管生成素1的下调作用更强。这些研究表明，可以修饰第125位周围的区域以改善内皮抑素的生物学活性。
