Fig 2: Cleavage of VEGFR3 by BACE2.(A) Schematic of VEGFR3 fragments. From left to right: The immature proVEGFR3 (200 kDa) can be cleaved by BACE2, releasing the immature, soluble ectodomain sol proVEGFR3 (130 kDa). The mature protein consists of 2 subunits linked through a disulfide bridge: VEGFR3α (75 kDa) and VEGFR3β (125 kDa). Upon BACE2 cleavage, the VEGFR3β-CTF (70 kDa) and sVEGFR3 (130 kDa) are generated, the latter of which consists of the VEGFR3α (75 kDa) and VEGFR3β-NTF (55 kDa) fragments. (B) Immunoblot detection of VEGFR3 in lysates and media of HEK293 cells transfected with empty control plasmids (Ctrl), Vegfr3, Vegfr3 + Bace2, and Bace2. B2, BACE2. Data show 3 independent experiments. sVEGFR3 is not detectable under reducing conditions. sol proVEGFR3 in the lysates appears at around 100 kDa and derives from BACE2 cleavage of immaturely glycosylated proVEGFR3 early in the secretory pathway upon BACE2 overexpression. (C) Localization and length of identified individual peptides (black dots) on the canonical VEGFR3 sequence. The ectodomain is indicated in blue, the intracellular domain in green, the signal peptide in orange, and the transmembrane domain in yellow. (D) N-terminal juxtamembrane region of VEGFR3 sequence. The identified semispecific peptide after LysN digestion is marked in yellow, the proposed cleavage site after amino acid alanine with 2 vertical lines, and the transmembrane region in gray. (E) Comparison of the fragment ion spectra of the identified C-terminal peptide of the LysN digestion KGC(cam)VN(+1)SSASVA (lower spectrum) to a synthetic peptide with the same sequence (upper spectrum). Identified y-ions are indicated in red, b-ions in blue, and fragment ions with neutral losses in green. Both spectra match with a dot product (73) of 0.93 for the fragment ion intensities.

Fig 3: Endogenous cleavage of VEGFR3 in LECs.(A) Immunoblot detection after control treatment (–) or upon BACE1 and BACE2 knockdown (+). Lysates were blotted for VEGFR3, BACE1/2, and actin. Conditioned media were blotted for sVEGFR3. (B) Corresponding densitometric quantifications, deriving from VEGFR3β (lysate) and sVEGFR3 (medium). (C) Immunoblots of cells treated with DMSO (–) or 100 nM verubecestat (+). (D) Corresponding densitometric quantifications as in B. Dot plots were normalized on the control mean and depict mean and SD, alongside the calculated P values, calculated by unpaired t test. **P < 0.01; ****P < 0.0001. P values are only indicated where significance was observed. Data are derived from n = 6 biological replicates. Shown are representative data from 3 independent experiments.

Fig 4: BACE2-dependent changes in VEGFR3 signaling in LECs.(A) Schematic for VEGFR3 signaling. Upon ligand binding, VEGFR3 dimerizes, resulting in intracellular autophosphorylation and activation of the downstream genes FOXC2 and DLL4. V, verubecestat, inhibitor of BACE2. Gene expression levels of (B) DLL4 and FOXC2 and (C) VEGFR3 after the application of DMSO, 100 nM verubecestat (V), and VEGF-C. (D and E) Gene expression levels of DLL4, FOXC2, VEGFR3, BACE1, and BACE2 after BACE knockdown (siB1/siB2) without or with (+V) subsequent verubecestat application. All dot plots were normalized on the control mean and depict mean and SD alongside the P values calculated by unpaired t tests against the DMSO control (B and C) or by 1-way ANOVA (D and E), in both cases followed by Bonferroni’s multiple-comparison test. *P < 0.05; **P < 0.01; ****P < 0.0001. P values are only indicated where significance was observed. In B, 1 data point was excluded from the DLL4 expression/VEGF-C156S data set, since it was identified as an outlier via the ROUT method. Data are derived from n = 6 (B and C) or n = 4 (D and E) biological replicates.

Fig 5: Identification of VEGFR3 as a BACE2 substrate candidate.(A) Volcano plot of proteomic analysis of murine plasma from WT and B2KO mice (n = 6). VEGFR3 (FLT4) is highlighted in red. (B) Normalized VEGFR3 LFQ intensities extracted from A. (C) MSD-assay quantifications of sVEGFR3 in the same plasma samples. (D) Immunoblot detection of sVEGFR3 ectodomain in mouse plasma from A, using nonreducing and reducing conditions. (E) Volcano plot of proteomic analysis of murine plasma from an independent B2KO line (n = 9) compared with WT (n =9) and (F) the extracted normalized LFQ values. Volcano plots of the proteomic analyses of Bace1/Bace2 double-knockout (BDKO) mice (n =9) compared with the WT line (n = 9) (G) (corresponding extracted LFQ intensities of sVEGFR3 in F) and B1KO (n = 9) compared with an individual control WT line (n = 9) (H). (I) Normalized LFQ values extracted from H. (J) Localization of identified individual peptides (black dots) on the canonical VEGFR3 sequence. The signal peptide is shown in rose, the ectodomain is indicated in blue, the intracellular domain in green, and the transmembrane domain in yellow. Two sided Student’s t tests with a permutation-based FDR correction (FDR < 0.05; indicated by hyperbolic curves) were used for volcano plots (A, E, G, and H). Proteins with P < 0.05 are shown as red circles. Extracted LFQ quantifications (B, F, and I) of VEGFR3 with significance after FDR correction are labeled with plus signs. All dot plots were normalized on the WT mean and depict mean and SD. MSD-assay data (C) additionally depicts the P value calculated by unpaired t test. ****P < 0.0001.