Biosynthetic Processing of Cathepsins and Lysosomal Degradation Are Abolished in Asparaginyl Endopeptidase-deficient Mice

Abstract

Asparaginyl endopeptidase (AEP)/legumain, an asparagine-specific cysteine proteinase in animals, is an ortholog of plant vacuolar processing enzyme (VPE), which processes the exposed asparagine residues of various vacuolar proteins. In search for its physiological role in mammals, here we generated and characterized AEP-deficient mice. Although their body weights were significantly reduced, they were normally born and fertile. In the wild-type kidney where the expression of AEP was exceedingly high among various organs, the localization of AEP was mainly found in the lamp-2-positive late endosomes in the apical region of the proximal tubule cells. In these cells of AEP-deficient mice, the lamp-2-positive membrane structures were found to be greatly enlarged. These aberrant lysosomes, merged with the late endosomes, accumulated electron-dense and membranous materials. Furthermore, the processing of the lysosomal proteases, cathepsins B, H, and L, from the single-chain forms into the two-chain forms was completely defected in the deficient mice. Thus, the AEP deficiency caused the accumulation of macromolecules in the lysosomes, highlighting a pivotal role of AEP in the endosomal/lysosomal degradation system.

Diverse regulatory roles for lysosomal proteases in the immune response

“…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]. Thus, targeting a single enzyme may have an impact on the activity or at least the form and stability of other enzymes.…”

“…Also as noted above, loss of one enzyme can affect others. For example, a substantial accumulation of cathepsin L is observed in lysosomes of AEP-deficient mice and this may partially compensate for the predicted shortfall in tetanus toxin processing [7], Matthews et al (submitted for publication). In addition, there is strong evidence that the protease content of primary cells and immortalised cell lines may be very different [35,36], Matthews et al (submitted for publication).…”

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

“…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.…”

“…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].…”

The expression of legumain, an asparaginyl endopeptidase that controls antigen processing, is reduced in endotoxin-tolerant monocytes

“…As deduced from real-time PCR on reverse transcribed mRNA (RT-PCR), monocytes clearly expressed legumain, although this expression was much lower than that of the other tested proteases that play a role in antigen processing, cathepsin B, cathepsin H, cathepsin L, cathepsin F and cathepsin S (Figure 1a). This low expression and the fact that legumain activates lysosomal cathepsins and unlocks the antigens for splitting 18,25 suggests that the level and activity of legumain regulates the antigen cleavage. Figure 1 The expression of legumain is reduced in endotoxintolerant monocytes.…”