Asparaginyl Endopeptidase (Legumain) Supports Human Th1 Induction via Cathepsin L-Mediated Intracellular C3 Activation

Abstract

Autocrine activation of the complement receptors C3aR and CD46 by complement activation components C3a and C3b produced through C3 cleavage by the protease cathepsin L (CTSL) during T cell stimulation is a requirement for IFN-γ production and Th1 induction in human CD4+ T cells. Thus, lack of autocrine CD46 activation, such as in CD46-deficient patients, is associated with defective Th1 responses and recurrent infections. We have identified LGMN [the gene coding for legumain, also known as asparaginyl endopeptidase (AEP)] as one of the key genes induced by CD46 co-stimulation during human CD4+ T cell activation. AEP processes and activates a range of proteins, among those α1-thymosin and CTSL, which both drive intrinsically Th1 activity—but has so far not been described to be functionally active in human T cells. Here we found that pharmacological inhibition of AEP during activation of human CD4+ T cells reduced CTSL activation and the CTSL-mediated generation of intracellular C3a. This translated into a specific reduction of IFN-γ production without affecting cell proliferation or survival. In line with these findings, CD4+ T cells isolated from Lgmn−/− mice also displayed a specific defect in IFN-γ secretion and Th1 induction. Furthermore, we did not observe a role for AEP-driven autocrine α1-thymosin activation in T cell-derived IFN-γ production. These data suggest that AEP is an “upstream” activator of the CTSL-C3-IFN-γ axis in human CD4+ T cells and hence an important supporter of human Th1 induction.

Complement C3 overexpression activates JAK2/STAT3 pathway and correlates with gastric cancer progression

“…Several studies have shown that it could facilitate cellular proliferation and regeneration by dysregulating mitogenic signaling pathways, sustaining angiogenesis and oncogenesis [30,31]. The internal synthesis and secretion of C3 were observed in various immune cells and GC cells [32][33][34]. Our study confirmed that both SGC-7901 and MGC-803 cell lines produced intracellular C3.…”

Proteomics Reveals Extracellular Matrix Injury in the Glomeruli and Tubulointerstitium of Kidney Allografts with Early Antibody-Mediated Rejection

“…LGMN inhibition reduced IFNγ production, CTSL activation and C3a generation in activated human CD4 + T-cells; moreover, CD4+ T-cells from Lgmn-deficient mice showed defective IFNγ production and Th1 induction. 84 CTSS and LGMN also mediate HLA class-II antigen presentation, promoting the generation of competent HLA class-II molecules for peptide binding, 85,86 and processing of exogenous peptides for HLA class-II-presentation, [87][88][89] respectively. CTSS inhibition prevented HLA class-II-mediated antigen presentation in autoimmunity, 90 and reduced the generation of competent HLA class-II and the infiltration of IFNɣ-expressing CD4+ T-cells in a neuropathy model.…”

Glycosylation is important for legumain localization and processing to active forms but not for cystatin E/M inhibitory functions

“…In induced regulatory T cells, blocking legumain imparts regulatory function of these cells [112]. Legumain is required for normal Th1 induction in human and mouse CD4 + T cells, through processing of cathepsin L and subsequently cathepsin L-mediated complement C3 activation [113]. Also, legumain can process prothymosin α ( Table 1) into active α1thymosin [78], which is implicated in human CD4 + T cell activation and modulation of cytokine production [114].…”

“…To proteolytic cleave substrates, prolegumain must be activated. Maturation and stability of legumain, the presence of endogenous inhibitors and/or GAGs, environmental conditions (especially pH and redox potential) and subcellular localization are factors that regulate the legumain activity [27,29,34,113] (Figure 9). In paper I, very low levels of legumain were observed in the cytosolic fraction of both untreated and tunicamycin-treated cells after cell fractionation, showing both glycosylated and unglycosylated legumain in the cytosol.…”