Effect of C-terminal sequence on competitive semaphorin binding to neuropilin-1

Abstract

Neuropilins (Nrp) are type I transmembrane proteins that function as receptors for vascular endothelial growth factor (VEGF) and class III Semaphorin (Sema3) ligand families. Sema3s function as potent endogenous angiogenesis inhibitors but require proteolytically processing by furin to compete with VEGF for Nrp binding. This processing liberates a C-terminal arginine (CR) that is necessary for binding to the b1 domain of Nrp, a common feature shared by Nrp ligands. The CR is necessary but not sufficient for potent Nrp inhibition, and the role of upstream residues is unknown. We demonstrate that the second-to-last residue (C-1), immediately upstream of the CR, plays a significant role in controlling competitive ligand binding by orienting the C-terminus for productive Nrp binding. With the use of a peptide library derived from Sema3F, C-1 residues that preferentially adopt an extended bound-like conformation, including proline and β-branched amino acids, were found to produce the most avid competitors. Consistent with this, analysis of the binding thermodynamics revealed that more favorable entropy is responsible for the observed binding enhancement of C-1 proline. We further tested the effect of the C-1 residue on Sema3F processing by furin and found an inverse relationship between processing and inhibitory potency. Analysis of all Sema3 family members reveals two non-equivalent furin processing sites differentiated by the presence of either a C-1 proline or a C-1 arginine and resulting in up to a 40-fold difference in potency. These data reveal a novel regulatory mechanism of Sema3 activity and define a fundamental mechanism for preferential Nrp binding.

Keywords: AP; CHO; Chinese hamster ovary; ITC; Sema3; VEGF; alkaline phosphatase; angiogenesis; class III Semaphorin; furin; isothermal titration calorimetry; peptide library; proteolysis; vascular endothelial growth factor.

Figure 1. Inhibitory potency of C-1 peptide variants

(A) Peptides were titrated to determine their inhibitory potency versus AP-VEGF-A. Shown are representative inhibition curves for wild type peptide (S3F-RR, black line) and the variants with maximum and minimum potency, S3FPR (green line) and S3F-DR (red line), respectively. (B) Table of all C-1 variants with their respective IC₅₀. Values are reported as the mean ± standard deviation.

Figure 2. The C-1 residue of Nrp1 ligands adopts an extended backbone conformation

(A) Superimposition of the product bound structures of VEGF-A (green) (PDB=4DEQ) and Tuftsin (gold) (PDB=2ORZ) in complex with Nrp1 (PDB=2ORZ). There are two distinct interaction interfaces, one of which is mediated by the C_R and the other by the C-2 carbonyl of the ligand. The C-1 residue serves the critical role of tethering the adjacent interacting residues and correctly orienting them with respect to one another within the binding pocket. (B) Ramachandran plot of the φ and ψ angles of the C-1 VEGF-A (C-1 R) and Tuftsin (C-1 P) residue reveals that they adopt an extended conformation. (C) Plotting the IC₅₀ for each C-1 variant against the β-sheet propensity of each amino acid reveals a correlation between potency and the inherent preference of amino acids to adopt an extended conformation. Proline was excluded from the fit due to its inability to conform to the backbone hydrogen bonding pattern present in β-sheets and, therefore, its low β-sheet propensity.

Figure 3. Analysis of peptide binding by ITC

Using ITC the thermodynamic parameters of Nrp1 binding were measured for wild type peptide (S3F-RR, A) and the C-1 proline variant (S3F-PR, B). A representative binding isotherm is shown for each peptide. Values are reported as the mean ± standard deviation of four independent experiments.

Figure 4. Mutation of the Sema3F C-1 residue alters processing efficiency

(A) The Igbasic domains of Sema3F were expressed as a C-terminal Hgh fusion. In addition to wild type protein (S3F-Hgh), a mutant with the C-1 arginine residue of the furin consensus site mutated to proline (R778P-Hgh) was expressed. The arrow indicates the site of furin proteolysis. (B) S3F-Hgh and R778P-Hgh were expressed in CHO, furin deficient (FD11), and furin over expressing (Furin) cells and the efficiency of processing was measured by blotting for Hgh. S3F-Hgh was processed in CHO and Furin cells, as detected by the presence of the processed form, but significantly less processing was seen for R778P-Hgh in these cell types. Neither construct was processed in FD11 cells.

Figure 5. Furin proteolysis of Sema3 generates divergent C-terminal sequences

(A) The Sema3 family of ligands are furin processed at multiple sites within their C-terminus that result in either a C-terminal arginine-arginine motif (site 1 and 2) or a proline-arginine motif (site 3). (B) Peptides corresponding to all furin cleavage sites (black arrow) within the basic domain of Sema3 family members were synthesized with a leading tryptophan (grey). Sema3 variants are labeled according to family member (A–G) and cleavage site number (1–3). (C) An alignment of the four C-terminal residues of all Sema3 peptides illustrating the either-or preference for proline and arginine at the C-1 position. The height of the amino acids at each position represents their relative conservation within the alignment.

Figure 6. Sema3 family variation in potency is explained by the C-1 amino acid

(A) The ability of all Sema3 ligand variants to inhibit VEGF-A binding to Nrp1 was measured. The data are reported as the mean IC₅₀ ± standard deviation. (B) The IC₅₀ for each peptide was plotted against the amino acid present at the C-1 position. Peptides with a C-1 proline (average IC₅₀ = 2.4 ± 1.4 μM) were significantly more potent than those with a C-1 arginine (average IC₅₀ = 19 ± 11 μM). (C) The C-1 residue inversely effects furin consensus strength and Nrp affinity.