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J. Biol. Chem., Vol. 280, Issue 12, 11303-11312, March 25, 2005

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Contributions of Disulfide Bonds in a Nested Pattern to the Structure, Stability, and Biological Functions of Endostatin^*

Hao Zhou, Wei Wang, and Yongzhang Luo

From the Laboratory of Protein Chemistry, Ministry of Education Laboratory of Protein Science, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China

Endostatin can inhibit the proliferation and migration of endothelial cells. It contains two pairs of disulfide bonds in a nested pattern. We constructed three mutants, C33A/C173A, C135A/C165A, and all-Ala, to evaluate the contributions of individual disulfide bonds to the structure, stability, and biological functions of endostatin. Both tryptophan emission fluorescence spectrum and ¹H nuclear magnetic resonance spectrum show that C135A/C165A and all-Ala, the two mutants lacking disulfide bond Cys¹³⁵–Cys¹⁶⁵, lost nearly their entire tertiary structure. Although C33A/C173A appears to retain some native-like structures, it is less stable and has a higher helical content, which confirms our earlier hypothesis that the polypeptide backbone of endostatin has a high helical propensity. C135A/C165A and all-Ala mutants lost most of their inhibitory activities both on the migration and proliferation of human microvascular endothelial cells, whereas C33A/C173A is partially active. The mutants without disulfide bond Cys¹³⁵–Cys¹⁶⁵ can hardly be internalized and localized to cytoskeleton and nucleus in the cell, which probably contributes to their loss of inhibition on the migration and proliferation of endothelial cells. Our studies provide a structural basis for the two disulfide bonds on the biological functions of endostatin.

Received for publication, October 25, 2004 , and in revised form, January 4, 2005.

^* This research was supported by the Major Program of National Science Foundation of China Grant 30291000, National Science Fund for Distinguished Young Scholars in China Grant 30225014, and National 863 Program in China Grant 2002AA2Z345D. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 86-10-6277-2897; Fax: 86-10-6279-4691; E-mail: protein{at}tsinghua.edu.cn.

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