Identification of Cellular Compartments Involved in Processing of Cathepsin E in Primary Cultures of Rat Microglia

Abstract

Cathepsin E is a major nonlysosomal, intracellular aspartic proteinase that localizes in various cellular compartments such as the plasma membrane, endosome‐like organelles, and the endoplasmic reticulum (ER). To learn the segregation mechanisms of cathepsin E into its appropriate cellular destinations, the present studies were initiated to define the biosynthesis, processing, and intracellular localization as well as the site of proteolytic maturation of the enzyme in primary cultures of rat brain microglia. Immunohistochemical and immunoblot analyses revealed that cathepsin E was the most abundant in microglia among various brain cell types, where the enzyme existed predominantly as the mature enzyme. Immunoelectron microscopy studies showed the presence of the enzyme predominantly in the endosome‐like vacuoles and partly in the vesicles located in the trans‐Golgi area and the lumen of ER. In the primary cultured microglial cells labeled with [35S]methionine, >95% of labeled cathepsin E were represented by a 46‐kDa polypeptide (reduced form) after a 30‐min pulse. Most of it was proteolytically processed via a 44‐kDa intermediate to a 42‐kDa mature form within 4 h of chase. This processing was completely inhibited by bafilomycin A1, a specific inhibitor of vacuolar‐type H+‐ATPase. Brefeldin A, a blocker for the traffic of secretory proteins from the ER to the Golgi complex, also inhibited the processing of procathepsin E and enhanced its degradation. Procathepsin E, after pulse‐labeling, showed complete susceptibility to endoglycosidase H, whereas the mature enzyme almost acquired resistance to endoglycosidases H as well as F. The present studies provide the first evidence that cathepsin E in microglia is first synthesized as the inactive precursor bearing high‐mannose oligosaccharides and processed to the active mature enzyme with complex‐type oligosaccharides via the intermediate form and that the final proteolytic maturation step occurs in endosome‐like acidic compartments.

Involvement of proteases in porcine reproductive and respiratory syndrome virus uncoating upon internalization in primary macrophages

“…Cathepsin E is an intracellular aspartic protease homologous to cathepsin D [22,35]. Cathepsin D is localized within the lysosomes of most cells [9] while cathepsin E is localized in various non-lysosomal compartments such as the endosome, the endoplasmic reticulum and the Golgi complex and has a limited cell and tissue distribution [26,35,37,41]. Cathepsin E has been detected in mouse, rabbit and rat macrophages [2,9,30,36].…”

“…It is very well possible that this pH dependency arises from the low pH-autocatalytic conversion of procathepsin E to the mature active form [37], which cleaves one of the PRRSV structural proteins. Cathepsin E has a cleavage specificity and maximal activity at around pH 6.0 [3,42].…”

Differential Regulation of the Nature and Functions of Dendritic Cells and Macrophages by Cathepsin E

“…However, it remains unclear whether cathepsin E is essential for MHC class II Ag presentation. Because cathepsin E exists in APC endosomes as the mature enzyme (23,27), and because cathepsin E expression is increased upon stimulation with IFN-␥ and LPS, known to induce MHC class II expression and costimulatory molecules (55), it is relevant to determine to what extent cathepsin E activity influences MHC class II-restricted Ag presentation. The present results showed for the first time that cathepsin E deficiency strongly affected the presentation of OVA by macrophages and DCs to its specific T cell hybridomas, implying that cathepsin E is involved in antigenic presentation.…”

“…In contrast, in vitro studies using purified proteases and their inhibitors have demonstrated that cathepsin D (18 -21) and/or cathepsin E (21-24) might play a role in antigenic peptide generation. Further studies using APC derived from cathepsin D-deficient mice have shown that cathepsin D is not essential for MHC class II Ag presentation (23,(25)(26)(27). However, to date it remains speculative whether cathepsin E is directly involved in the MHC class II Ag presentation in APC.…”

Cathepsin E (EC 3.4.23.34) — a review

“…In a human organism, cathepsin E occurs in: erythrocytes, thymus, dendritic cells, epithelial M cells, microglia cells, Langerhans cells, lymphocytes, epithelium of gastrointestinal tract, urinary bladder, lungs, osteoclasts, spleen and lymphatic nodes [9][10][11][12][13][14]. However, it does not occur in human neutrophiles or bovine erythrocytes [15] (Table 2).…”