Mechanism of selective VEGF-A binding by neuropilin-1 reveals a basis for specific ligand inhibition

Abstract

Neuropilin (Nrp) receptors function as essential cell surface receptors for the Vascular Endothelial Growth Factor (VEGF) family of proangiogenic cytokines and the semaphorin 3 (Sema3) family of axon guidance molecules. There are two Nrp homologues, Nrp1 and Nrp2, which bind to both overlapping and distinct members of the VEGF and Sema3 family of molecules. Nrp1 specifically binds the VEGF-A(164/5) isoform, which is essential for developmental angiogenesis. We demonstrate that VEGF-A specific binding is governed by Nrp1 residues in the b1 coagulation factor domain surrounding the invariant Nrp C-terminal arginine binding pocket. Further, we show that Sema3F does not display the Nrp-specific binding to the b1 domain seen with VEGF-A. Engineered soluble Nrp receptor fragments that selectively sequester ligands from the active signaling complex are an attractive modality for selectively blocking the angiogenic and chemorepulsive functions of Nrp ligands. Utilizing the information on Nrp ligand binding specificity, we demonstrate Nrp constructs that specifically sequester Sema3 in the presence of VEGF-A. This establishes that unique mechanisms are used by Nrp receptors to mediate specific ligand binding and that these differences can be exploited to engineer soluble Nrp receptors with specificity for Sema3.

Figure 1. Nrp1 selectively inhibits VEGF-A binding.

The ability of Nrp1 and Nrp2 to selectively sequester AP-VEGF-A from Nrp1 adsorbed on affinity plates was assessed. The amount of retained AP-VEGF-A was measured and the Nrp concentration recorded where half-inhibition was achieved. Nrp1 inhibited the binding of VEGF-A with an IC₅₀ = 1.8 µM (black line). Inhibition by Nrp2 was seen only at the highest concentration of protein attainable with an estimated IC₅₀≈310 µM (grey line). Experiments were performed in triplicate and reported as the mean ±1 S.D.

Figure 2. Nrp1 residues mediate specific VEGF-A binding.

(A) Alignment of orthologous Nrp1 and Nrp2 b1 domains shows conservation of residues critical for C-terminal arginine binding (marked with a *) but variability within regions surrounding the interloop cleft (orange: Nrp1:E285/Nrp2:R287 ; green: Nrp1∶299-TN-300/Nrp2∶301-DGR-303; blue: Nrp1∶304-ER-305/Nrp2∶307-QQ-308; purple: Nrp1∶350-KKK-352/Nrp2∶353-QNG-355). Below the alignment is a conservation histogram illustrating identity across the displayed sequences. (B) Surface representation of the Nrp b1 domain reveals that the direct VEGF-A binding region (gold) is closely associated with the selected regions (colored according to 2A) in three-dimensional space. (C) VEGF-A binding of Nrp1 mutants reveals loss of binding for each mutant protein compared to wild-type. Retained AP-Nrp1 binding is reported as the percent of retained wild-type AP-Nrp1. (D) Determination of the VEGF-A binding capacity of Nrp2 mutants reveals that three of the four Nrp2 chimeras show enhanced VEGF-A binding compared to wild-type. Retained AP-Nrp2 binding is reported as the percent of retained wild-type AP-Nrp2. Experiments were performed in triplicate and reported as the mean ±1 S.D.

Figure 3. Nrp^Chimera molecules exhibit reversed VEGF-A specificity.

(A) The secondary structure of WT Nrp and Nrp^Chimera was assessed by CD. The overlapping spectra of Nrp^Chimera with wild-type Nrp demonstrate that the incorporated mutations are not structurally deleterious. (B) Nrp1^Chimera (blue line) and Nrp2^Chimera (green line) were tested for their ability to selectively sequester AP-VEGF-A from Nrp1 adsorbed on affinity plates. The Nrp^Chimera molecules show reversed VEGF-A specificity with Nrp1^Chimera having a marked reduction in inhibitory potency (IC₅₀ = 62 µM) and Nrp2^Chimera exhibiting a significant gain in potency (IC₅₀ = 3.9 µM). Wild-type Nrp1 (black dotted line, IC₅₀ = 1.8 µM) and Nrp2 (grey dotted line, IC₅₀≈310 µM) are shown for comparison (data from Figure 1). Experiments were performed in triplicate and reported as the mean ±1 S.D.

Figure 4. Nrp inhibits Sema3F binding through interaction with the C-terminal basic domain.

(A) Nrp1 and Nrp2 dependent inhibition of AP-Sema3F binding to Nrp1 affinity plates was measured. Both Nrp homologues showed similar ability to compete for AP-Sema3F, with Nrp1 inhibiting with an IC₅₀ = 2.0 µM and Nrp2 with an IC₅₀ = 2.7 µM. (B) The ability of Nrp1 and Nrp2 to selectively sequester AP-Sema3F-Ig-basic from Nrp1 adsorbed on affinity plates was assessed. The amount of retained AP-Sema3F-Ig-basic was measured and the Nrp concentration recorded where half-inhibition was achieved. Nrp1 (black line) and Nrp2 (grey line) had similar ability for inhibiting Sema3F binding with IC₅₀ = 1.2 µM and IC₅₀ = 6.2 µM, respectively. (C) Two peptides corresponding to the C-terminal basic domain of Sema3A (C-furSema3A) and Sema3F (C-furSema3F) were analyzed for their ability to inhibit AP-VEGF-A binding to Nrp1 affinity plates. Both peptides potently inhibited binding with an IC₅₀ = 22 nM and 67 nM for C-furSema3F and C-furSema3A, respectively. Experiments were performed in triplicate and reported as the mean ±1 S.D.

Figure 5. Nrp2 and Nrp1^Chimera preferentially sequester Sema3F.

The ability of Nrp1, Nrp2, and Nrp1^Chimera to selectively sequester C-furSema was determined through combined incubation of Nrp, C-furSema3F, and AP-VEGF-A in Nrp1 adsorbed affinity plates. Nrp1 was unable to relieve C-furSema-dependent inhibition and completely abolished VEGF-A binding (black line). Conversely, titration with Nrp2 promoted 63% recovery of VEGF-A binding demonstrating selective sequestration of Sema3F (grey line). Nrp1^Chimera also relieved C-furSema-dependent inhibition, promoting recovery of VEGF-A binding to 37% the level of uninhibited VEGF-A (blue line). Experiments were performed in triplicate and reported as the mean ±1 S.D.