Fig. 2: Deglycosylated LRG1 potentiates angiogenesis and neurite outgrowth through increased binding to LPHN2.
From: Crystal structure of LRG1 and the functional significance of LRG1 glycan for LPHN2 activation
a SDS‒PAGE of recombinant LRG1 and DG-LRG1 (incubation of LRG1 with 40 µg/ml PNGase F at 4 °C overnight). b Left, schematic of the ___domain architecture of LPHN2. Lec, lectin; Olf, olfactomedin-like; HomoR, hormone receptor motif; GAIN/GPS, GPCR autoproteolysis-inducing/GPCR proteolysis site. Right, the binding of DG-LRG1 to the LPHN2 ectodomain (Lec, Olf, Lec+Olf, or Ecto-full ___domain) was determined by a solid-phase binding assay. c Binding of Alexa 647-conjugated LRG1 or DG-LRG1 (1 μM) to parental and LPHN2-knockdown HUVECs was analyzed by FACS. d, e Tube formation assay (e) and Transwell cell migration assay (f) after treatment of HUVECs with LRG1 (1 μg/ml) or DG-LRG1 (1 μg/ml) in the presence of shControl or shLPHN2 lentivirus (5 × 104 TU/ml culture medium). Top, representative images of tube formation and Transwell cell migration. Scale bars, 200 µm. Bottom, quantification of the number of master junctions and migrated cells using ImageJ. f βIII-tubulin immunostaining in mouse DRG explants after treatment with LRG1 (1 μg/ml) or DG-LRG1 (1 μg/ml) in the presence of shCon or shLPHN2 lentivirus (5 × 104 TU/ml culture medium). Top, representative images of DRG explants. Scale bars, 200 µm. Bottom, quantification of βIII-tubulin-immunopositive neurite length from DRG explants (200 cells/field) using ImageJ. d, f Data in bar graphs are plotted as the mean ± SEM (n = 4). Significance is indicated using Student’s t test (**p < 0.01; ***p < 0.001). The relative ratio of the nontreated group was defined as 1. Western blot with HUVECs (g) and mouse primary DRG explants (h) after treatment with LRG1 (1 μg/ml) or DG-LRG1 (1 μg/ml) in the presence of shCon vs. shLPHN2 lentivirus (n = 3).
