1. Mutation of human plasminogen kringle 1-5 enhances anti-angiogenic action via increased interaction with integrin alpha(v)beta(3).
- Author
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Chang PC, Chang YJ, Wu HL, Chang CW, Lin CI, Wang WC, and Shi GY
- Subjects
- Amino Acid Substitution, Animals, Apoptosis drug effects, Base Sequence, Carcinoma, Lewis Lung blood supply, Carcinoma, Lewis Lung drug therapy, Cattle, Cell Proliferation drug effects, Cells, Cultured, DNA Primers genetics, Endothelial Cells cytology, Endothelial Cells drug effects, Endothelial Cells physiology, Glycosylation, Humans, Kringles genetics, Male, Mice, Mice, Inbred C57BL, Mutagenesis, Site-Directed, Neovascularization, Pathologic prevention & control, Peptide Fragments chemistry, Peptide Fragments pharmacology, Plasminogen chemistry, Plasminogen pharmacology, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins pharmacology, Integrin alphaVbeta3 physiology, Mutation, Neovascularization, Physiologic drug effects, Neovascularization, Physiologic genetics, Peptide Fragments genetics, Peptide Fragments physiology, Plasminogen genetics, Plasminogen physiology
- Abstract
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(1-5)N289A (replacing Asn by Ala at residue 289), K(1-5)T346A, K(1-5)L532R, K(1-5)N289A/T346A, K(1-5)T346A/L532R, K(1-5)N289A/L532R, and K(1-5)N289A/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(1-5)N289A/T346A/L532R exhibited the greatest effect in inhibiting endothelial cell proliferation and in inducing endothelial cell apoptosis. Integrin alpha(v)beta(3)-mediated adhesion of K(1-5)N289A/T346A/L532R to endothelial cells was more greatly enhanced when compared to wild type K(1-5). Furthermore, K(1-5)N289A/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(1-5)N289A/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 alpha(v)beta(3) in endothelial cells.
- Published
- 2008
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