Mechanistic basis for the potent anti-angiogenic activity of semaphorin 3F.
Neuropilin-1 (Nrp1), an essential type I transmembrane receptor, binds two secreted ligand families, vascular endothelial growth factor (VEGF) and class III Semaphorin (Sema3). VEGF-A and Sema3F have opposing roles in regulating Nrp1 vascular function in angiogenesis. VEGF-A functions as one of the most potent pro-angiogenic cytokines, while Sema3F is a uniquely potent endogenous angiogenesis inhibitor. Sema3 family members require proteolytic processing by furin to allow competitive binding to Nrp1. We demonstrate that the furin-processed C-terminal domain of Sema3F (C-furSema) potently inhibits VEGF-A-dependent activation of endothelial cells. We find that this potent activity is due to unique heterobivalent engagement of Nrp1 by two distinct sites in the C-terminal domain of Sema3F. One of the sites is the C-terminal arginine, liberated by furin cleavage, and the other is a novel upstream helical motif centered on the intermolecular disulfide. Using a novel chimeric C-furSema, we demonstrate that combining a single C-terminal arginine with the helical motif is necessary and sufficient for potent inhibition of binding of VEGF-A to Nrp1. We further demonstrate that the multiple furin-processed variants of Sema3A, with the altered proximity of the two binding motifs, have dramatically different potencies. This suggests that furin processing not only switches Sema3 to an activated form but also, depending on the site processed, can also tune potency. These data establish the basis for potent competitive binding of Sema3 to Nrp1 and provide a basis for the design of bivalent Nrp inhibitors.
Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky , Lexington, Kentucky 40536, United States.
Hou-Fu
HF
Guo
Xiaobo
X
Li
Matthew W
MW
Parker
Johannes
J
Waltenberger
Patrice M
PM
Becker
Craig W
CW
Vander Kooi
D020533:Angiogenesis Inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D045683:Furin / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D018919:Neovascularization, Physiologic; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D039942:Neuropilin-1 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D034421:Sus scrofa; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D020533:Angiogenesis Inhibitors; D008565:Membrane Proteins; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C491230:SEMA3F protein, human; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D039942:Neuropilin-1; D040301:Vascular Endothelial Growth Factor Receptor-2; C468731:FURIN protein, human; D045683:Furin