Mutation of human plasminogen kringle 1-5 enhances anti-angiogenic action via increased interaction with integrin alpha(v)beta(3).
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.
Chang
Po-Chiao
PC
Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, No. 1, University Rd, Tainan, Taiwan, 701.
Chang
Yu-Jia
YJ
Wu
Hua-Lin
HL
Chang
Chin-Wei
CW
Lin
Chung-I
CI
Wang
Wei-Chih
WC
Shi
Guey-Yueh
GY
D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D018827:Carcinoma, Lewis Lung / Q000098:blood supply / Q000188:drug therapy; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D006031:Glycosylation; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000502:physiology*; D018082:Kringles / Q000235:genetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009389:Neovascularization, Pathologic / Q000517:prevention & control; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology / Q000502:physiology*; D010958:Plasminogen / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
D017931:DNA Primers; D039302:Integrin alphaVbeta3; D010446:Peptide Fragments; D011994:Recombinant Proteins; C491499:plasminogen kringle 5; D010958:Plasminogen