Multistep Autoactivation of Asparaginyl Endopeptidase in Vitro and in Vivo

Li, Dongtao Ni; Matthews, S.; Antoniou, Antony N.; Mazzeo, Daniela; Watts, Colin

doi:10.1074/jbc.m305930200

Cited by 156 publications

(162 citation statements)

References 58 publications

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“…In addition to the ∼38 kDa predominant active form of legumain, a faint ∼40 kDa protein was labeled by both probes and was also immunodepleted by legumain specific antisera. This protein likely corresponds to the p46 intermediate form of legumain that has been reported to retain enzymatic activity [10]. A ∼50 kDa polypeptide was labeled in the RAW264.7 extracts, presumably corresponding to the ∼56 kDa proenzyme of legumain [10].…”

Section: Introductionmentioning

confidence: 89%

“…This protein likely corresponds to the p46 intermediate form of legumain that has been reported to retain enzymatic activity [10]. A ∼50 kDa polypeptide was labeled in the RAW264.7 extracts, presumably corresponding to the ∼56 kDa proenzyme of legumain [10]. Previous studies using saturating concentration of ABPs have demonstrated labeling of inactive protease zymogens due to flexibility of pro-peptide binding in the active site [11].…”

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confidence: 85%

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

Sexton

Witte

Blum

et al. 2007

Bioorganic & Medicinal Chemistry Letters

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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 ...

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Section: Introductionmentioning

confidence: 89%

mentioning

confidence: 85%

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

Sexton

Witte

Blum

et al. 2007

Bioorganic & Medicinal Chemistry Letters

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“…Legumain is a lysosomal protease, appears to be expressed in response to stress (66), and may be secreted from cells under some conditions (67), and like cathepsin L it may be active in the pericellular environment (68). Legumain is expressed in various organs (69) and was found in macrophages (70), dendritic cells (71), and in vivo in tumors with high invasive and metastatic potential (66). Under these conditions, legumain may be responsible for processing of CCL15 to the molecule CCL15 and may than support the immune response or the malignant character of a tumor.…”

Section: Discussionmentioning

confidence: 99%

Quantum Proteolytic Activation of Chemokine CCL15 by Neutrophil Granulocytes Modulates Mononuclear Cell Adhesiveness

Richter¹,

Bistrian

Escher³

et al. 2005

The Journal of Immunology

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Monocyte infiltration into inflammatory sites is generally preceded by neutrophils. We show here that neutrophils may support this process by activation of CCL15, a human chemokine circulating in blood plasma. Neutrophils were found to release CCL15 proteolytic activity in the course of hemofiltration of blood from renal insufficiency patients. Processing of CCL15 immunoreactivity (IR) in the pericellular space is suggested by a lack of proteolytic activity in blood and blood filtrate, but a shift of the retention time (tR) of CCL15-IR, detected by chromatographic separation of CCL15-IR in blood and hemofiltrate. CCL15 molecules with N-terminal deletions of 23 (Δ23) and 26 (Δ26) aa were identified as main proteolytic products in hemofiltrate. Neutrophil cathepsin G was identified as the principal protease to produce Δ23 and Δ26 CCL15. Also, elastase displays CCL15 proteolytic activity and produces a Δ21 isoform. Compared with full-length CCL15, Δ23 and Δ26 isoforms displayed a significantly increased potency to induce calcium fluxes and chemotactic activity on monocytes and to induce adhesiveness of mononuclear cells to fibronectin. Thus, our findings indicate that activation of monocytes by neutrophils is at least in part induced by quantum proteolytic processing of circulating or endothelium-bound CCL15 by neutrophil cathepsin G.

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“…Consequently, the ablation of one activity may impact on others. For example, conversion of pro-AEP involves first autoactivation but then further processing by other enzymes [6]. In turn, AEP is absolutely required for conversion of cathepsins B, L and H from a single chain to the normal two-chain form [7].…”

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confidence: 99%

Diverse regulatory roles for lysosomal proteases in the immune response

et al. 2009

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The innate and adaptive immune system utilise endocytic protease activity to promote functional immune responses. Cysteine and aspartic proteases (cathepsins) constitute a subset of endocytic proteases, the immune function of which has been described extensively. Although historically these studies have focused on their role in processes such as antigen presentation and zymogen processing within the endocytic compartment, recent discoveries have demonstrated a critical role for these proteases in other intracellular compartments, and within the extracellular milieu. It has also become clear that their pattern of expression and substrate specificities are more diverse than was first envisaged. Here, we discuss recent advances addressing the role of lysosomal proteases in various aspects of the immune response. We pay attention to reports demonstrating cathepsin activity outside of its canonical endosome/lysosome microenvironment.Key words: Cathepsins . Endosomes . Immune regulation . Lysosomes IntroductionThe endosome/lysosome compartments, together with the cytosolic proteasomes, are the two major protein degradation systems in cells. Both have emerged as having many important regulatory functions in addition to their role in protein breakdown for amino acid recycling. For example, limited proteolysis within the endocytic pathway can induce activating conformational changes [1,2], release functional proteins from chaperones [3], and cleave soluble bioactive molecules from membrane-anchored precursors [4]. The concept of ''regulation through limited destruction'' is evident in many processes in innate and adaptive immunity. Endosome/lysosome-located proteases play key roles in antigen processing and presentation, cytokine regulation, NKT cell development, activation of serine protease zymogens in regulated secretory granules, integrin activation, induction of apoptosis and TLR signalling. Given that these processes may also be associated with undesirable immune responses and inflammation, the proteases involved may be considered as attractive drug targets. EnzymesEndosomes and lysosomes harbour mainly cysteine and aspartic acid proteases so-named because their active site utilises either a cysteine thiol or an aspartic acid as a key part of the catalytic site. Some endosome/lysosome located serine proteases such as cathepsin G, granzymes and thymus specific serine protease (TSSP) have important roles in the immune system; however, we focus primarily here on the cysteine and aspartic proteases. Most of the lysosomal cysteine proteases are related to papain and belong to the so-called C1 family. These include cathepsins L, S, C, F, H, B, X, K, V and W. Cathepsins D and E are also found in lysosomes but are aspartic acid proteases related to pepsin. An additional cysteine protease is found in lysosomes, which is more closely related to the caspases. This is asparaginyl endopeptidase (AEP) or legumain, a member of the C13 family. Some of these enzymes are endopeptidases (cathepsins S, L, K, F, V, D, E and AEP), where...

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Multistep Autoactivation of Asparaginyl Endopeptidase in Vitro and in Vivo

Cited by 156 publications

References 58 publications

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

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

Quantum Proteolytic Activation of Chemokine CCL15 by Neutrophil Granulocytes Modulates Mononuclear Cell Adhesiveness

Diverse regulatory roles for lysosomal proteases in the immune response

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