Activation of legumain involves proteolytic and conformational events, resulting in a context- and substrate-dependent activity profile

Abstract

Localized mainly to endo/lysosomes, legumain plays an important role in exogenous antigen processing and presentation. The cysteine protease legumain, also known as asparaginyl endopepetidase AEP, is synthesized as a zymogen and is known to undergo pH-dependent autoproteolytic activation whereby N-terminal and C-terminal propeptides are released. However, important mechanistic details of this pH-dependent activation as well as the characteristic pH activity profile remain unclear. Here, it is shown that all but one of the autocatalytic cleavage events occur in trans, with only the release of the C-terminal propeptide being relevant to enzymatic activity. An intriguing super-activation event that appears to be exclusively conformational in nature and enhances the enzymatic activity of proteolytically fully processed legumain by about twofold was also found. Accepting asparagines and, to lesser extent, aspartic acid in P1, super-activated legumain exhibits a marked pH dependence that is governed by the P1 residue of its substrate and conformationally stabilizing factors such as temperature or ligands. The crystallization and preliminary diffraction data analysis of active legumain are presented, which form an important basis for further studies that should clarify fundamental aspects of activation, activity and inactivation of legumain, which is a key target in (auto-)immunity and cancer.

Figure 1

Activation intermediates of legumain. (a) pH-dependent autoproteolytic processing shown by SDS–PAGE. SDS–PAGE of wild-type (wt) prolegumain and D303E/D309E prolegumain after incubation at the indicated pH values (310 K, 20 h). Lane M, molecular-weight marker (labelled in kDa); band 1, prolegumain (no cleavage); band 2, C-­terminal propeptide cleaved (cleavage after Asn323); band 3, N- and C-terminal propeptides cleaved (after Asp25 and Asn323); lane 4, N-terminal propeptide cleaved and additionally processed at the C-terminus (Asp25 and Asp303/309); band 5, C-terminal propeptide. Band 4 was not observed for the D303E/D309E double mutant. (b) Schematic representation of autolytic cleavage intermediates. Activity is expressed as Bz-Asn-pNA turnover normalized to that of super-activated legumain. Autocatalytic cleavage sites are indicated by arrows. Δ-(Val18–Asp25) refers to the N-terminal truncation variant and D303E/D309E to the double mutant with disrupted C-terminal cleavage site. The variant at the bottom (pH > 6) illustrates irreversible inactivation of super-activated legumain after exposure to neutral pH. NPP, N-terminal propeptide (Val18–Asp25); CPP, C-terminal propeptide (Asp324–Tyr433); grey stars, super-activated legumain; filled stars, catalytic His148 and Cys189 residues.

Figure 2

The pH controls the activation and inactivation of legumain. (a) pH-dependent activation of legumain. Wild-type (black) and Δ-(Val18–Asp25) prolegumain (grey) were incubated at different pH values ranging from 7.0 to 4.0 (310 K) for 20 h. Bz-Asn-pNA turnover by the resulting activation intermediates in legumain activity-assay buffer at pH 5.5 was measured after autoprocessing was complete as judged by SDS–PAGE (4 h at pH 4.0; 20 h for all other pH values). White bars: prolegumain activity after incubation with Bz-Asn-pNA at the indicated pH. Prolegumain is inactive from pH 7 to pH 4. Activities are normalized to that of super-activated legumain. (b) Active legumain is irreversibly inactivated when incubated at neutral pH. Super-activated legumain was incubated at pH 4 and pH 7 (30 min, 310 K) and enzymatic activity was measured in legumain reaction buffer at pH 5.5. Neutral pH leads to irreversible inactivation of legumain. Activity values are averaged over three independent measurements and are shown together with the corresponding standard deviations.

Figure 3

Autocatalytic cleavage of prolegumain occurs in trans. SDS–PAGE of C189S prolegumain incubated with super-activated wild-type legumain at pH values as indicated (4 h, 310 K). M, molecular-weight marker (labelled in kDa); band 1, C189S prolegumain; band 2, C-terminal propeptide cleaved (cleavage after Asn323); band 3, N-terminal and C-terminal propeptides cleaved (cleavage after Asp25 and Asn323); band 4, C-terminal propeptide.

Figure 4

pH-dependent asparaginyl-peptidase and aspartyl-peptidase activity of legumain. Bz-Asn-pNA (filled rectangles) and Ac-Tyr-Val-Ala-Asp-pNA (open triangles) turnover of super-activated legumain was measured in legumain reaction buffer at the indicated pH values at 310 K. Activity is represented by k _cat/K _m normalized to maximum activity (100%), which is obtained at pH 5.5 (n = 3, standard deviations indicated). The maximum activity was determined as k _cat/K _m = 7.3 ± 0.05 × 10² s⁻¹  M ⁻¹, with k _cat = 2 ± 0.5 s⁻¹ and K _m = 2.4 ± 0.1 mM.

Figure 5

P1-Asp is preferred at pH 4.0 over pH 5.5. The inhibition profile suggests that P1-­Asp is only tolerated in its protonated form. Mature legumain (15 µM) was incubated for 1 min with YVAD-CMK and AAN-CMK (15 µM each) at pH 4 and pH 5.5 and residual activity was measured. Activity was normalized to the activity of the uninhibited control at the respective pH. Black, uninhibited; grey, inhibited with YVAD-CMK; white, inhibited with AAN-CMK (n = 3, standard deviations indicated).

Figure 6

Active legumain is conformationally destabilized at neutral pH. pH-dependent melting curves of super-activated legumain in the presence and absence of an active site-directed inhibitor are shown. Activated legumain was incubated at the indicated pH values and thermal denaturation was measured by the Thermofluor method. An increase in fluorescence indicates the exposure of hydrophobic protein segments which accompanies protein unfolding. Melting points are indicated.

Figure 7

Mercury-soaked crystals of mature legumain diffracted to beyond 2.5 Å resolution. Super-activated legumain was crystallized in complex with the covalent legumain-specific inhibitor AAN-CMK in a tetragonal lattice. (a) Legumain crystals are about 100 × 100 × 100 µm in size. (b) Diffraction image taken at ID14-4 (ESRF). Resolution at the edge, 2.47 Å; resolution at the corner, 1.86 Å. The diffraction patterns of native and Hg-derivative crystals are isomorphous (<1%) and similar in diffraction quality, e.g. resolution and mosaicity.