Fig 2: Deglycosylation of LRG1 and the LRG1 N325D mutant restores erectile dysfunction under diabetic conditions.a Representative intracavernous pressure (ICP) responses in STZ-induced diabetic mice and age-matched nondiabetic controls 2 weeks after repeated intracavernous injection of PBS (negative control), DG-LRG1 (5 µg/20 µl), or the LRG1 N325D mutant (5 µg/20 µl) on Days 0 and 3. The cavernous nerve was stimulated at 5 V. The stimulus interval is indicated by a solid bar. The ratios of the mean maximal ICP and total ICP (area under the curve) to mean systolic blood pressure (MSBP) were calculated for each group (n = 5). b Representative immunofluorescence (IF) of platelet/endothelial cell adhesion molecule-1 (PECAM-1, green) and neuron-glial antigen 2 (NG2, red) in cavernous tissue from control and STZ-induced diabetic mice 2 weeks after repeated intracavernous injection of PBS (negative control), DG-LRG1 (5 µg/20 µl), or LRG1 N325D mutant (5 µg/20 µl) on Days 0 and 3. Scale bar, 100 µm. c Representative IF of ßIII-tubulin (red) in dorsal nerve bundle (DNB) tissue from control and STZ-induced diabetic mice 2 weeks after repeated intracavernous injections as indicated in b. Scale bars, 25 µm. Nuclei were stained with DAPI (blue). d The PECAM-1 (green), NG (red), or ßIII-tubulin (red)-immunopositive areas in the corpus cavernosum and dorsal nerve bundle of control and diabetic mice were quantified by ImageJ (N = 5 and 6, respectively). Data are plotted as the mean ± SEM. Significance is indicated using one-way ANOVA with Bonferroni’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig 3: Deglycosylated LRG1 potentiates angiogenesis and neurite outgrowth through increased binding to LPHN2.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 ßIII-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 ßIII-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).

Fig 4: LRG1 N325 is the key glycosylation site for attenuating LRG1/LPHN2-mediated angiogenesis and neurite outgrowth.a Tube formation assay using HUVECs after treatment with 1 µg/ml LRG1, DG-LRG1, or LRG1 glycan mutants (N79D, N186D, N269D, N325D). Representative images of tube formation and Transwell cell migration. Scale bars, 200 µm (top). Bottom, quantification of the number of master junctions and migrated cells using ImageJ. b ßIII-tubulin immunostaining in mouse DRG explants after treatment with 1 µg/ml LRG1, DG-LRG1, or LRG1 glycan mutants (N79D, N186D, N269D, N325D). Representative images of DRG explants. Scale bars, 200 µm (top). Bottom, quantification of ßIII-tubulin-immunopositive neurite length from DRG explants (200 cells/field) using ImageJ. In the bar graphs, data are plotted as the mean ± SEM (n = 4). Significance is indicated using Student’s t test (***p < 0.001). The relative ratio of the nontreated group was defined as 1. N.S., not significant. Western blot with HUVECs (c) and mouse primary DRG explants d after treatment with LRG1 (1 µg/ml) or LRG1 N325D mutant (1 µg/ml) in the presence of shCon vs. shLPHN2 lentivirus (n = 3).