The N-Terminal Integrity Is Critical for the Stability and Biological Functions of Endostatint

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.