FIBCD1 is an endocytic GAG receptor associated with a novel neurodevelopmental disorder (2022)
Figure 4

Figure 4: FIBCD1 is an endocytic receptor for hippocampal glycosaminoglycans.

(A) HPLC traces representative of 3 independent experiments of variously sulphated GAGs (as labelled) in adult Fibcd1 WT (top, blue) and KO (bottom, red) CA1 pyramidal cell layer hippocampi. Unsulphated CS = CS-0S; hyaluronic acid = HA; 4-O-sulphated CS = CS-4S; 6-O-sulphated CS = CS-6S.

(B) Immunoblot analysis (left) and quantification of signal intensity (right) of Fibcd1 WT (blue) versus Fibcd1 KO littermates (red) adult hippocampi with antibodies against CS-0S, CS-4S, CS-6S and actin as a loading control. Each lane represents an independent animal (n = 3). Protein marker sizes are indicated.

(C) Top binding pose for in silico docking of CS-4S to FIBCD1 FReD (PDB 4M7F). Inset (left) is the orientation of CS-4S within the FReD binding pocket and (right) binding free energy of CS-4S vs CS-6S. Van der Waals (vdW), electrostatic (Elec) and desolvation (Desolv) components of binding free energy change.

(D) Competitive ELISA with increasing concentrations of CS-4S (blue circles) or -6S (red circles) incubated with recombinant FIBCD1 FReD and acetylated BSA. Inset is IC50 concentrations for CS-4S and CS-6S (n = 4).

(E) Flow cytometric analysis of N2a cells expressing full-length mFIBCD1, mFIBCD1ΔFReD or empty vector control incubated with FITC-tagged chondroitin-4-sulfate (CS-4S), poly-sulphated chondroitin sulphate (CS-PS) or dermatan sulphate (DS) (n = 3).

(F) Confocal images depicting internalisation of FITC-tagged CS-4S by FIBCD1-overexpressing HEK293T lines compared to untransduced cells and unstained cells. Left, representative images; right, quantification. Data is plotted as total puncta per condition (n = 5). Cells are further stained with CellMask Orange (cellular membrane) and Hoechst (nuclei). Scale bar = 50µm.

(G) Internalisation of FITC-tagged CS-4S by HEK293T-FIBCD1 cells treated with inhibitors of endocytosis, Dynole 34-2 and PitStop 2 (+) vs. their respective negative control compounds with no inhibitory properties, Dynole 31-2 and PitStop 2 (-) (n = 6).

(H) Representative (of 2 independent experiments) images of HEK293T cells overexpressing mCherry-FIBCD1 fusion protein (yellow) stained with Hoechst (nuclei, blue), lysosomal vesicles (LysoTracker, red) and FITC-CS-4S (green). White arrows indicate co-localisation of CS-4S, lysosomal vesicles and FIBCD1; blue arrow indicates co-localisation of FIBCD1 and CS-4S but not lysosomal vesicles. Scale bar = 15µm. Inset, digital zoom of HEK293T images showing co-localisation. Scale bar = 7.5µm.

(I) Top, superposition ribbon diagrams of the WT FReD domain (dark blue) with R406C (left) and P456L (right) mutants (in grey). The loops surrounding the ligand binding site (389-399 and 423-448) exhibit the largest structural rearrangement in both mutants. Bottom, comparison of the electrostatic potential mapped onto the solvent-accessible surface between WT and the two variant FReDs.

(J) Top, schematic depiction of FIBCD1 protein and location of patient variants (red) and W6* control (blue). Bottom, flow cytometric analysis of untransduced HEK293T cells (n = 3), or expressing constructs with full-length wild-type human FIBCD1 (n = 3), FIBCD1 with the W6* early stop variant as control (FIBCD1_W6*) (n = 4), or the three patient variants (as labelled, n = 4) incubated with FITC-tagged CS-4S represented as percentage of CS-4S-FITC relative to unstained control.

Data information: For panel B, each data point represent hippocampal protein isolates from an individual mouse, for panel D inset, each data point represents a technical replicate, for panels E, F, G and J each data point represents an individual cell preparation. Data are shown as mean values +/- SEM. p values were calculated using 1-way ANOVA (panels E, F, G, J) or paired Student's t-test (panels B, D). * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.