Legumain Functions as a Transient TrkB Sheddase

Abstract

While primarily found in endo-lysosomal compartments, the cysteine protease legumain can also translocate to the cell surface if stabilized by the interaction with the RGD-dependent integrin receptor αVβ3. Previously, it has been shown that legumain expression is inversely related to BDNF-TrkB activity. Here we show that legumain can conversely act on TrkB-BDNF by processing the C-terminal linker region of the TrkB ectodomain in vitro. Importantly, when in complex with BDNF, TrkB was not cleaved by legumain. Legumain-processed TrkB was still able to bind BDNF, suggesting a potential scavenger function of soluble TrkB towards BDNF. The work thus presents another mechanistic link explaining the reciprocal TrkB signaling and δ-secretase activity of legumain, with relevance for neurodegeneration.

Keywords: dimerization; functional processing; lysomale proteases; neurotrophins; tyrosine receptor kinase.

Figure 1

Preparation of rat TrkB and proBDNF. (A) Domain organization of TrkB. CC1/2: cysteine cluster 1/2; LRM: leucine-rich-motif; Ig1/2: Ig-like domain 1/2; ML: membrane linker; KD: kinase domain. (B) SDS-PAGE of TrkB variants. Lane 1: purified TrkB ECD; Lane 2: purified TrkB Ig2 domain with different glycosylation isoforms. (C) HPLC-MS analysis of the relative fractional abundance of the correct disulfide isoform Cys302-Cys345 in TrkB Ig2. Fractional abundances of the disulfide bonds, as detected by mass spectrometry, are normalized frequencies, i.e., they add up to 100%. (D) SDS-PAGE of (pro-)BDNF. Lane 1: purified proBDNF; Lane 2: furin-activated BDNF; the 70 kDa band corresponds to inactivated furin.

Figure 2

Legumain processes TrkB. A+B: Coomassie-stained SDS PAGE gel (A) and corresponding Immunoblot (B) of TrkB ECD and Ig2 domain in the presence (+) and absence (−) of legumain, denoted as LEG. An anti-His6 antibody is used in the Immunoblot to detect the C-terminal His-tag of the uncleaved TrkB variants. M represents the molecular marker lane. (C) Relative fractional abundance of three detected cleavage sites CS1 (Asp385), CS2 (Asn389), and CS3 (Asn391) at pH 5.5 and 7.0. Fractional abundances of the individual cleavage events, as detected by mass spectrometry, are normalized frequencies, i.e., they add up to 100%. (D) Cartoon representation of the TrkB ECD with the three detected cleavage sites CS1, CS2, and CS3 (Asp385, Asn389, and Asn391) in the membrane linker segment with their cleavage predominance indicated by their color intensity.

Figure 3

TrkB in complex with BDNF is protected from processing by legumain. M represents the protein marker. In the next three lanes, the presence or absence (+/−) of BDNF and legumain (“LEG”) on the processing of TrkB ECD is tested, followed by three lanes on the processing of TrkB Ig2 domain. The outer right lanes represent controls of BDNF and legumain (LEG) only, migrating at approximately 14 kDa and 36 kDa, respectively. Note the presence of cell medium-derived BSA migrating at 60 kDa. Additionally, in the BDNF sample, inactivated furin is visible at 70 kDa, which was used to activate proBDNF.

Figure 4

Legumain-processed TrkB variants bind BDNF. Non-processed TrkB variants bind proBDNF. (A) The legumain-processed TrkB ECD binds BDNF in solution with a K_d of 188 nM. (B) The legumain-processed TrkB Ig2 binds BDNF in solution with a K_d of 300 nM. Of note, in both cases, secondary binding events at micromolar concentrations were observed. (C) TrkB ECD binds proBDNF in solution with a K_d of 306 nM. (D) TrkB Ig2 binds proBDNF in solution with a K_d of 441 nM. Contrasting the situation with mature BDNF, no secondary binding events were observed.

Figure 5

Possible impact of TrkB shedding by Legumain. On the left side, BDNF mediates a heterotetrameric complex with membrane-bound TrkB (TrkB-BDNF)₂, which prevents shedding by legumain (“LEG”). In the absence of BDNF, TrkB can be shed from the membrane. Soluble TrkB can still bind BDNF, resulting in a soluble (TrkB-BDNF)₂ complex.