Contributions Of Arg Clusters And Zinc Ions To The Structure And Biological Functions Of Endostatin

Endostatin, a C-terminal proteolytic fragment of collagen XVIII, exerts its antiangiogenic and antitumor functions via specifically targeting the proliferating endothelial cells in angiogenic blood vessels.Endostatin can be internalized by endothelial cells, which is essential for its biological functions. Thus the intracellularly stability of endostatin under acidic conditions is of significant physilogical revelence. Here we find that sulfate anions induce endostatin to form a folding intermediate at pH 3.0 and stabilize the folding intermediate more than the native structure of endostatin. In addition, heparin-binding loosens the structure of endostatin especially at low pH values. This study not only demonstrates the sulfate anion effect on the folding of endostatin, but also provides clues to stabilize endostatin under acidic conditions.Endostatin has an Arg-rich surface serving as a heparin-binding site, and the mechanism of the heparin-dependent antiendothelial activity of endostatin remains unclear. The current study shows that certain Arg clusters mediate the interaction between endostatin and its receptor nuceloin without influenceing the conformation of endostatin. Additionally, the interaction of nucleolin and endostatin is positively correlated with the biological activities of endostatin. These observastions not only elucidate the contributions of Arg clusters to the biological functions of endostatin, but also provide strong evidence for the dominant role of nucleoin as the functional receptor for endostatin.Endostatin has a zinc-binding site at its N-terminus. The contribution of zinc-binding to its biological functions is under debate. By parrelel comparison of the intimately related endostatin molecules tested in clinical trials, we report here that zinc-binding is essential for both the structures and functions of endostatin. An intact N-terminus of endostatin is critical for efficient zinc-binding, which results in a conformation with high binding affinity with its receptor nucleolin. This study provides fundamental explanations for the discrpent efficacies of endostatin shown in clinical trials.In sum, the current studies ragrding the folding mechanism and functional motifs of endostatin elucidate the molecular mechanism of the biological functions of endostatin.