Fig. 3.
Cltx binds to NRP1 only when its C-terminal arginine is de-amidated; Cltx de-amidation occurs in tumor. Cltx-CONH2 (native Cltx with amidated C-terminal arginine) does not bind to NRP1 at concentrations from 0 to 1750 nM (a) while Cltx-COOH (Cltx with a carboxylated C-terminal arginine) demonstrated dose responsive NRP1 binding with an affinity (KD) of ~ 240 nM (c). Graphs are output from BLI assay with KD determined by octet data analysis software version 8.2; graphs are representative of multiple assays. b, peptides identified in PC-3 tumor lysate by MS analysis 1 h post dose of Cltx, in order of abundance. Peptides represented in black or red have amidated versus de-amidated C-terminal arginine residue respectively. Peptides 1 and 3 are full length Cltx with amidated (native) versus de-amidated C-terminal arginine respectively. d, VEGF binding to NRP1 (fixed concentration at 390 nM) was dose dependently inhibited in the presence of increasing concentration of Cltx-COOH (0 to 800 μM), R2 = 0.98; suggests that Cltx binding to NRP1 occurs at VEGF binding site