Neuropilin-1/Heparan Sulfate Interactions and Angiogenesis-Dependent Tumor Growth
Neuropilin-1 (Nrp1) acts as a coreceptor with Vascular Endothelial Growth Factor Receptor (VEGFR) to facilitate binding of its ligand, Vascular Endothelial Growth Factor (VEGF) and downstream signaling that leads to angiogenesis. Heparan sulfate has been shown to be a cofactor facilitating the interaction of Nrp1 with VEGF/VEGFRs, but the functional significance of this interaction has not been established. Nrp1 mediates angiogenesis including branching and organization of vessels through its role in tip cell function and interactions with VEGFR/VEGFs. This dissertation focuses on how Nrp1 interaction with heparin/heparan sulfate influences its function as a part of the VEGF/HSPG/Nrp1/VEGFR2 complex and the resulting effects on tumor angiogenesis. Chapter 1 is a mini-review on the VEGF/HSPG/Nrp1/VEGFR2 complex, which serves as a model for understanding how binding of glycan ligands to proteins modulate their biological effects. Chapter 2 focuses on the molecular modeling and biochemical mapping of the heparin/heparan sulfate binding site in Nrp1 along with investigations into the biological significance of the interaction in vivo. In order to map the heparin/heparan sulfate binding site in Nrp1, we completed a combinatorial library screening using heparin oligosaccharides followed by molecular dynamic simulations of a best-fit tetradecasaccharide. We then created and expressed a series of recombinant Nrp1 mutants to identify the specific amino acids that interact with heparin and heparan sulfate. Specific mutations in the b1 and b2 domains of Nrp1 decreased binding to heparin and cell surface heparan sulfate. These mutants also prevent heparin-induced stabilization of the protein during thermal denaturation. Heparin-induced dimerization of Nrp1 was found to be length-dependent and dependent on key amino acid residues in the b1 and b2 domains, specifically arginine 513 and lysine 514. Heparin icosasaccharides induced a 1:2 heparin/Nrp1 complex as measured by isothermal calorimetry. Knock-in mice expressing Nrp1 deficient in heparan sulfate binding (Nrp1D) were created using CRISPR/Cas9 gene targeting. Analysis of subcutaneous tumor formation revealed that Nrp1D mice exhibit a reduction in angiogenesis-dependent tumor growth compared to wildtype controls. This finding suggests that the interaction of Nrp1 with heparan sulfate modulates its function in pathological angiogenesis. Chapter 3 is an extended discussion on these findings, their significance and future directions. The work completed in this dissertation confirms and further elucidates the heparin/heparan sulfate binding groove in mouse Nrp1, establishes a 1:2 heparin/protein complex, and demonstrates the biological significance for the heparan sulfate/Nrp1 interaction in vivo.