Direct binding of recombinant plasminogen kringle 1–3 to angiogenin inhibits angiogenin-induced angiogenesis in the chick embryo CAM

Abstract

Angiogenin is one of the most potent angiogenesis-inducing proteins. Angiostatin is one of the most potent angiogenesis inhibitors, and it contains the first four kringle domains of plasminogen (K1–4). Recombinant human plasminogen kringle 1–3 (rK1–3) was expressed in Escherichia coli and purified to homogeneity. The binding of t-4-aminomethylcyclohexanecarboxylic acid with the purified kringle 1–3 was determined by changes in intrinsic fluorescence. rK1–3 exhibits comparable ligand-binding properties as native human plasminogen kringle 1–3. The purified rK1–3 inhibits neovascularization in the chick embryo chorioallantoic membrane (CAM) assay. Interaction of angiogenin with rK1–3 was examined by immunological binding assay and surface plasmon resonance kinetic analysis, and the equilibrium dissociation constants for the complex, K d, are 0.89 and 0.18 μM, respectively. rK1–3 inhibits angiogenin-induced angiogenesis in the chick embryo CAM in a concentration-dependent manner. These results indicate that rK1–3 directly binds to angiogenin and thus rK1–3 inhibits the angiogenic activity of angiogenin.