Legumain/asparaginyl endopeptidase controls extracellular matrix remodeling through the degradation of fibronectin in mouse renal proximal tubular cells

Abstract

Legumain/asparaginyl endopeptidase (EC 3.4.22.34) is a novel cysteine protease that is abundantly expressed in the late endosomes and lysosomes of renal proximal tubular cells. Recently, emerging evidence has indicated that legumain might play an important role in control of extracellular matrix turnover in various pathological conditions such as tumor growth/metastasis and progression of atherosclerosis. We initially found that purified legumain can directly degrade fibronectin, one of the main components of the extracellular matrix, in vitro. Therefore, we examined the effect of legumain on fibronectin degradation in cultured mouse renal proximal tubular cells. Fibronectin processing can be inhibited by chloroquine, an inhibitor of lysosomal degradation, and can be enhanced by the overexpression of legumain, indicating that fibronectin degradation occurs in the presence of legumain in lysosomes from renal proximal tubular cells. Furthermore, in legumain-deficient mice, unilateral ureteral obstruction (UUO)-induced renal interstitial protein accumulation of fibronectin and renal interstitial fibrosis were markedly enhanced. These findings indicate that legumain might have an important role in extracellular matrix remodeling via the degradation of fibronectin in renal proximal tubular cells.

Structural analysis of asparaginyl endopeptidase reveals the activation mechanism and a reversible intermediate maturation stage

“…Additionally, both fully activated AEP at pH 3.5 (AEP pH3. 5 ) and partially activated AEP at pH 4.5 (AEP pH4.5 ) were able to cleave recombinant protein GST-TEV-ubiquitin (Ub) at the asparagine (N) site, as identified by N-terminal sequencing (Supplementary information, Figure S2B and S2C).…”

“…(D) The overall structure of activated AEP pH4.5 (crystallized at pH 8.5), which is similar to the structure of AEP pH7. 5 . The key cleavage sites were indicated as cyan sticks.…”

“…5 and AEP pH4. 5 The structures of AEP pH7.5 and AEP pH4.5 were determined at 2.49 and 2.0 Å resolution, respectively (Table 1). AEP pH7.5 was crystallized at pH 7.0 and adopts a fold composed of 14 α-helices and 11 β-sheets ( Figure 2B).…”

“…5 To study the molecular basis of the AEP activation process, we determined and compared the crystal structures of AEPs that were processed under different pH conditions. The pro-AEP purified at pH 7.5 (AEP pH7.5 ) was incubated at pH 3.5 (AEP pH3.5 ) for complete activation.…”

“…AEP is also involved in the proteolytic maturation of pro-cathepsins B, H and L found in the endo/lysosomes [4]. In addition to the cathepsins, AEP also processes fibronectin [5], pro-gelatinase A [6] and α-thymosin [7].…”