The Alzheimer’s disease–linked protease BACE2 cleaves VEGFR3 and modulates its signaling
Andree Schmidt, … , Bettina Schmid, Stefan F. Lichtenthaler
Andree Schmidt, … , Bettina Schmid, Stefan F. Lichtenthaler
Published June 18, 2024
Citation Information: J Clin Invest. 2024;134(16):e170550. https://doi.org/10.1172/JCI170550.
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The Alzheimer’s disease–linked protease BACE2 cleaves VEGFR3 and modulates its signaling

Abstract

The β-secretase β-site APP cleaving enzyme (BACE1) is a central drug target for Alzheimer’s disease. Clinically tested, BACE1-directed inhibitors also block the homologous protease BACE2. Yet little is known about physiological BACE2 substrates and functions in vivo. Here, we identify BACE2 as the protease shedding the lymphangiogenic vascular endothelial growth factor receptor 3 (VEGFR3). Inactivation of BACE2, but not BACE1, inhibited shedding of VEGFR3 from primary human lymphatic endothelial cells (LECs) and reduced release of the shed, soluble VEGFR3 (sVEGFR3) ectodomain into the blood of mice, nonhuman primates, and humans. Functionally, BACE2 inactivation increased full-length VEGFR3 and enhanced VEGFR3 signaling in LECs and also in vivo in zebrafish, where enhanced migration of LECs was observed. Thus, this study identifies BACE2 as a modulator of lymphangiogenic VEGFR3 signaling and demonstrates the utility of sVEGFR3 as a pharmacodynamic plasma marker for BACE2 activity in vivo, a prerequisite for developing BACE1-selective inhibitors for safer prevention of Alzheimer’s disease.

Authors

Andree Schmidt, Brian Hrupka, Frauke van Bebber, Sanjay Sunil Kumar, Xiao Feng, Sarah K. Tschirner, Marlene Aßfalg, Stephan A. Müller, Laura Sophie Hilger, Laura I. Hofmann, Martina Pigoni, Georg Jocher, Iryna Voytyuk, Emily L. Self, Mana Ito, Kana Hyakkoku, Akimasa Yoshimura, Naotaka Horiguchi, Regina Feederle, Bart De Strooper, Stefan Schulte-Merker, Eckhard Lammert, Dieder Moechars, Bettina Schmid, Stefan F. Lichtenthaler

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Figure 2

Cleavage of VEGFR3 by BACE2.

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Cleavage of VEGFR3 by BACE2. (A) Schematic of VEGFR3 fragments. From lef...
(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.