Design of cell-permeable, fluorescent activity-based probes for the lysosomal cysteine protease asparaginyl endopeptidase (AEP)/legumain

Abstract

Asparaginyl endopeptidase (AEP), also known as legumain, is a cysteine protease that has been ascribed roles in antigen presentation yet its exact role in human biology remains poorly understood. We report here the use of a positional scanning combinatorial library of peptide AOMKs containing a P1 aspartic acid to probe the P2, P3 and P4 subsite specificity of endogenous legumain. Using inhibitor specificity profiles of cathepsin B and legumain, we designed fluorescent ABPs that are highly selective, cell permeable reagents for monitoring legumain activity in complex proteomes.Asparaginyl Endopeptidase (AEP), or legumain, was originally identified in leguminous seeds as a cysteine protease with specificity for asparagine residues in the P1 position [1]. The mammalian legumain homologue is a lysosomal cysteine protease that is a member of the clan CD protease family which includes the caspases, separase and the gingipains [2]. Mammalian legumain has been ascribed a role in the initiation of invariant chain processing during MHC class II mediated antigen presentation [3,4]. Although the nature of this activity remains controversial, legumain is undoubtedly a key player in lysosomal proteolysis, contributing to the processing of antigenic peptides as well as the processing of the papain family cathepsins [5].Like all endocytic proteases, legumain is synthesized as an inactive zymogen, and its activity is regulated by post-translational activation events. Therefore, tools that can be used to monitor legumain's activity are necessary in order to understand its functional role. Activity based probes (ABPs) are reagents that can specifically label active proteases, thus allowing their activity, and more importantly their regulation, to be directly monitored [6,7]. Our laboratory recently reported a synthesis strategy based on the general solid phase methods developed by Ellman and co-workers [8] for the production of peptidyl acyloxymethyl ketone ABPs for diverse cysteine protease activities [9].We have previously demonstrated that the biotinylated ABP b-hex-D-AOMK efficiently labels endogenous legumain in 816 B cell lysates [9]. However this reagent lacks cell permeability and its overall selectivity towards legumain had not been extensively examined. We therefore Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Fig. 1a, b). Both ABPs potently labeled a ∼38 kDa protein in acidic proteomes from 816 B cells or RAW264.7 monocytes. This activity was confirmed to be legumain by immunoprecipitation using antisera specific for legumain. In addition to the ∼38 kDa predominant ...

Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination

“…The additional P2 = Pro prevents the labeling of PLCPs, which prefer hydrophobic residues at this position. Similar probes carrying a Pro-Asp dipeptide and an AOMK warhead were previously used to label mammalian legumains, which are orthologous to VPEs (Sexton et al, 2007).…”

“…Chemical synthesis of JOPD1 is much less complicated. JOPD1 is a bodipy version of the Pro-Asp-AOMK probe described by Sexton et al (2007) and carries an Asp at the P1 position, while the Pro at the P2 position prevents the labeling of PLCPs. JOGDA1 and JOPD1 have not been described before, and the details of their synthesis are provided (Supplemental Text S1).…”

Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling

“…Proteases define one of the largest groups of enzymes, and it has proven difficult to selectively measure their activity in biological samples because of the presence of multiple family members with overlapping specificity (1,19,28). To overcome this problem, substantial effort has been exerted by many groups over the years to understand the principles of recognition of natural and artificial substrates and to leverage this information to produce definitive diagnostic tools for in vivo and in vitro work.…”

Protease signaling in animal and plant‐regulated cell death

“…Although VPEs preferentially cleave after an asparagine residue, they have been shown to process substrates after an aspartic acid and have been linked to caspase-like activities in plants [161–163]. In particular, VPEs were active against caspase-1 substrates and were blocked by a caspase-1 inhibitor, much as seen in their mammalian counterpart [164]. In addition, VPEs were shown to act as positive regulators of virus- or bacteria-induced HR cell death [162, 163] and to participate in seed coat formation, a developmental process featuring cell death [165].…”