Thursday, June 1, 2023

Crystal structure of LRG1 and the functional significance of LRG1 glycan for LPHN2 activation – Experimental & Molecular Medicine

Recombinant protein expression and purification

Recombinant human LRG1 protein and human LPHN2 variant proteins (Lec, Olf, Lec/Olf domain, or ecto-full domain) were expressed and purified as described previously20. The constructs used for this study are listed in Supplementary Table 4. LRG1 N-glycan mutants (N79D, N186D, N269D, and N325D) were constructed using the QuikChange Lightning Site-Directed Mutagenesis kit (#210518, Agilent Technologies) with the LRG1-Fc construct as a template. DG-LRG1 was prepared by incubating LRG1 with GST-fused PNGase F at 4 °C overnight and then passing the solution over GST-Sepharose beads to remove GST-fused PNGase F.

Crystallization and structure determination

Crystallization and diffraction data collection were performed as described previously21. The LRG1 crystal belonged to space group P6322 and had the following unit cell dimensions: a = 143.0 Å; b = 143.0 Å; c = 113.7 Å; α = 90°; β = 90°; and γ = 120°. The initial phases were calculated by molecular replacement using PHASER22, and the structure of NGL3 (Protein Data Bank code: 3ZYN) was used as a search probe for structure determination. The atomic model was built after iterative rounds of model building using the program COOT23 and refinement using the program PHENIX24. The final model was validated using MolProbity in PHENIX (Rwork = 18.58%/Rfree = 21.94%). A Ramachandran plot analysis of the LGR1 structure showed that 92.51% and 0.00% of residues were in favored and outlier regions, respectively. The data collection and refinement statistics are shown in Supplementary Table 1. The structure factor and coordinate files have been deposited in the Protein Data Bank under accession code 8H24. All figures for structural models were generated using PyMOL (v2.3.1)25.

Solid-phase binding assay

LPHN2 extracellular domain variants (Lec, Olf, Lec/Olf domain, or ecto-full domain; 100 nM) were added to MaxiSorp 96-well plates (Nunc) and incubated for 1 hour at room temperature. Wells were washed twice with PBS and incubated with 1% bovine serum albumin (BSA) for 2 hours. After blocking, varying amounts (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 μM) of DG-LRG1 or LRG1 N-glycan mutants (N79D, N186D, N269D, and N325D) were added to 96-well plates coated with the indicated LPHN2 extracellular domain variants. We detected DG-LRG1 or LRG1 N-glycan mutants (N79D, N186D, N269D, and N325D) bound to coated LPHN2 proteins by ELISA using an anti-LRG1 antibody (Cat# sc517443, Santa Cruz) and peroxidase-conjugated anti-mouse secondary antibody (Cat# 62-6520, Thermo Fisher Scientific).

Cell culture and lentivirus treatments

Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Cat# CC-2519). HUVECs were cultured in EBM-2 (Cat# CC-3156, Lonza) supplemented with EGM-2 (Cat# CC-3162, Lonza), 2 mM L-glutamine (Cat# 25030081, GibcoTM), 100 U/ml penicillin, and 100 μg/ml streptomycin (Cat# 15140122, GibcoTM) on gelatin (Cat# G1890, Sigma‒Aldrich; 0.1% in PBS)-precoated plates in 5% CO2 in a humidified incubator. The dorsal root ganglia (DRG) were isolated from C57BL/6 mice and digested as described previously26. DRGs were plated onto poly-D-lysine (Cat# A3890401, Thermo Fisher Scientific)-coated dishes in Minimum Essential Medium (Cat# 11095080, Thermo Fisher Scientific) containing N-2 Supplement (Cat# 17502048, Thermo Fisher Scientific), 2 mM L-glutamine (Cat# 25030081, Thermo Fisher Scientific), 100 U/ml penicillin, and 100 μg/ml streptomycin (Cat# 15140122, GibcoTM). Cells were cultured in a humidified 37 °C incubator with 5% CO2. For LPHN2 knockdown in HUVECs and DRG neurons, shLPHN2 lentivirus particles were added to the culture medium at 5 × 104 TU/ml as described previously20. The scrambled shRNA was used as a control.

Apparent binding affinity of LRG1 to HUVECs and HEK293T cells

The apparent binding affinity of LRG1 for parental and LPHN2-knockdown HUVECs was measured by treating 1.0 × 106 cells/200 µl with 1 μM Alexa 647-conjugated LRG1 or Alexa 647-conjugated DG-LRG1. After washing with PBS, cell surface-bound LRG1 or DG-LRG1 was detected by monitoring fluorescence signals by fluorescence-activated cell sorting (FACS) and analyzed using FlowJo 10 software (FlowJo, LLC).

Tube formation and cell migration assay

The tube formation assay with HUVECs was performed as described previously27. After treating HUVECs with 1 μg/ml LRG1, DG-LRG1, or LRG1 N-glycan mutants (N79D, N186D, N269D, and N325D), tube formation was monitored for 12–16 hours under a phase-contrast microscope and quantified by counting the number of master junctions from four separate experiments in a blinded manner using ImageJ 1.34. Migration assays were performed using a modified Boyden chamber with polycarbonate filters (8 μm pores, Corning) coated with 0.1% gelatin. A total of 1 × 105 HUVECs in 200 μl of serum-free medium were seeded in the upper chamber of 12-well plates, and 600 μl of culture medium was added to the lower chamber28. HUVECs were treated with 1 μg/ml LRG1, DG-LRG1, or LRG1 N-glycan mutants (N79D, N186D, N269D, and N325D). After culturing for 18 hours at 37 °C, nonmigrating cells on the upper surface of the insert were removed with a cotton swab. Migrated cells on the bottom surface of the filter were fixed with 4% formaldehyde, stained with crystal violet (Cat# V5265, Sigma), and counted at ×400 magnification under a microscope.

DRG explant culture and sprouting assay

Mouse DRGs were dissected and maintained as described previously20. After DRG culture was treated separately with LRG1 (1 µg/ml) or DG-LRG1 (1 µg/ml) or one of the LRG1 mutants (N79D, N186D, N269D, N325D) for 7 days, neurite outgrowth segments were fixed in 4% paraformaldehyde for at least 30 minutes and immunostained with anti-βIII-tubulin (Cat# ab107216, Abcam; 1:100).

Western blot analysis

Cells were lysed in RIPA lysis buffer containing 1X protease inhibitor cocktail (Cat# P3100, GenDEPOT) and phosphatase inhibitors (Cat# P3200, GenDEPOT). Proteins were resolved via SDS‒PAGE and transferred onto PVDF membranes (Cat# 1620177, Bio-Rad). The membranes were incubated in the presence of the primary antibody overnight at 4 °C and then with appropriate HRP-conjugated secondary antibodies (Thermo Fisher Scientific). The antibodies chosen for this western blot analysis are listed in Supplementary Table 4: anti-LPHN2 (Cat# MBS244156, MyBioSource), anti-LRG1 (Cat# HPA001888, Sigma‒Aldrich), anti-phospho-Akt (Cat#9271, Cell Signaling Technology), anti-Akt (Cat# 9272, Cell Signaling Technology), anti-phospho-PI3 kinase p85 (Cat#4282, Cell Signaling Technology), anti-PI3 kinase p85 (Cat# 4292, Cell Signaling Technology), anti-NF-kB (Cat #8242, Cell Signaling Technology), phospho-NF-kB (Cat# MA5-15181, Thermo Fisher Scientific), and an internal control (β-actin; Cat# sc-69879, Santa Cruz Biotechnology).

Animals and measurement of erectile function

Eight-week-old male C57BL/6 (Orient Bio, Korea) mice were used in this study. The mice were age-matched in all experiments. Experiments were conducted with approval from the Inha University Animal Care and Use Committee (Assurance Number: INHA 180523-570). Diabetes was induced by intraperitoneal injection of streptozotocin (STZ; 50 mg/kg body weight) for 5 consecutive days as described previously29. Eight weeks after diabetes induction, the mice were anesthetized with intramuscular injections of ketamine (100 mg/kg) and xylazine (5 mg/kg). Erectile function was measured as described previously29. Briefly, the maximal intracavernous pressure (ICP) was recorded during stimulation (5 V at a frequency of 12 Hz, pulse width of 1 ms, and duration of 1 minute) using bipolar platinum wire electrodes placed around the cavernous nerve. The total ICP was determined as the area under the curve from the beginning of cavernous nerve stimulation to a point 20 seconds after stimulus termination. Systemic blood pressure was measured using a noninvasive tail-cuff system (Visitech Systems) prior to measuring the ICP. The ratios of the maximal ICP and total ICP to the mean systolic blood pressure (MSBP) were calculated to account for variations in the systemic blood pressure.

Histological examination of immunofluorescence

Mouse penile tissues were fixed in 4% paraformaldehyde for 10 minutes at room temperature and then blocked in 1% BSA (Cat# 9048-46-8, Sigma‒Aldrich) for 2 hours at room temperature. Immunohistochemistry was performed as described previously20 using primary antibodies [anti-PECAM1 antibody (Cat# MAB1398Z, Millipore), anti-NG2 antibody (Cat# ab5320, Millipore), anti-βIII-tubulin antibody (Cat# ab107216, Abcam)] and secondary antibodies [goat anti-Armenian hamster fluorescein (FITC) (Cat# 127-095-160, Jackson ImmunoResearch Laboratories), donkey anti-rabbit DyLight® 550 (Cat# ab98489, Abcam), and donkey anti-chicken rhodamine (TRITC) (Cat# 703-025-155, Jackson ImmunoResearch Laboratories)]. Immunofluorescence staining intensity was quantified using ImageJ 1.34.

Statistical analysis

All data are presented as the mean ± SEM. For cell culture data, three independent experiments were performed in triplicate. For normally distributed data, a two-tailed Student’s t test was used to compare the two groups. For comparisons among groups, one-way ANOVA and post hoc Bonferroni testing were performed using Prism 9 software (Graph Pad Software). A p value < 0.05 was considered significant.

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