Mutation of human plasminogen kringle 1–5 enhances antiangiogenic action via increased interaction with integrin α_vβ₃

Summary

Angiogenesis plays a primary role in tumor growth and metastasis. Angiostatin, a proteolytic fragment containing the first four kringle domains of human plasminogen, can inhibit angiogenesis. The anti-angiogenic activities of kringle 1–5 (K_1–5) and kringle 5 fragments of plasminogen are greater than angiostatin in inhibiting angiogenesis and angiogenesis-dependent tumor growth. To further optimize kringle fragment anti-angiogenic activities, mutations were created at the potential glycosylation sites Asn-289 and Thr-346 and the Lys binding site, Leu-532, at kringle 5, including K_1–5N289A (replacing Asn by Ala at residue 289), K_1–5T346A, K_1–5L532R, K_1–5N289A/T346A, K_1–5T346A/ L532R, K_1–5N289A/L532R, and K_1–5N289A/T346A/L532R. Wild-type and mutant K_1–5 proteins were expressed successfully by the Pichia pastoris expression system. Native K_1–5 from proteolytic cleavage and wild-type K_1–5 have similar activity in inhibiting basic fibroblast growth factor-induced endothelial cell proliferation. Among these mutated proteins, K_1–5N289A/ T346A/L532R exhibited the greatest effect in inhibiting endothelial cell proliferation and in inducing endothelial cell apoptosis. Integrin α_vβ₃-mediated adhesion of K_1–5N289A/ T346A/L532R to endothelial cells was more greatly enhanced when compared to wild type K_1–5. Furthermore, K_1–5N289A/ T346A/L532R was most potent in inhibiting basic fibroblast growth factor-induced angiogenesis in Matrigel assay in vivo. Angiogenesis-dependent tumor growth was inhibited by systemically injected K_1–5N289A/T346A/L532R into mice. These results demonstrate that alteration of glycosylation and Lys binding properties could increase the anti-angiogenic action of K_1–5, possibly via enhanced interaction with integrin α_vβ₃ in endothelial cells.

Keywords

Angiogenesis and inhibitors - endothelial cells - integrins - plasminogen (-fragments)

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