Abstract
We have attempted to elucidate an involvement of cathepsin E (CE) in major histocompatibility complex class II-mediated antigen presentation by microglia. In primary cultured murine microglia, CE was localized mainly in early endosomes and its expression level was markedly increased upon stimulation with interferon-gamma. Pepstatin A, a specific inhibitor of aspartic proteases, significantly inhibited interleukin-2 production from an OVA-(266-281)-specific T helper cell hybridomas upon stimulation with native OVA presented by interferon-gamma-treated microglia. However, pepstatin A failed to inhibit the presentation of OVA-(266-281) peptide. The possible involvement of CE in the processing of native OVA into antigenic peptide was further substantiated by that digested fragments of native OVA by CE could be recognized by OVA-specific Th cells. Cathepsin D also degraded native OVA into antigenic peptide, whereas microglia prepared from cathepsin D-deficient mice retained an ability for antigen presentation. On the other hand, the requirement for cysteine proteases such as cathepsins S and B in the processing of invariant chain (Ii) was confirmed by immunoblot analyses in the presence of their specific inhibitors. In conclusion, CE is required for the generation of an antigenic epitope from OVA but not for the processing of Ii in microglia.
Highlights
Cathepsin E (CE,1 EC 3.4.23.34) is an intracellular aspartic protease of the pepsin family, which is highly homologous to the lysosomal aspartic protease cathepsin D (CD, EC 3.4.23.5)
Our observations here show that cathepsin E (CE) exists in murine microglia/macrophages as the mature enzyme, whereas this enzyme exists in other peripheral antigen presenting cells (APCs) such as splenocytes and Bone marrow cells (BMCs) as enzymatically inactive proform
The difference in molecular form of CE among APCs is considered to reflect the difference in its intracellular localization, because CE was found to be converted into the mature form in an autocatalytic manner only after entering acidic compartments such as endosomes [4]
Summary
Cathepsin E (CE,1 EC 3.4.23.34) is an intracellular aspartic protease of the pepsin family, which is highly homologous to the lysosomal aspartic protease cathepsin D (CD, EC 3.4.23.5). Pepstatin A, a specific inhibitor of aspartic proteases, significantly inhibited interleukin-2 production from an OVA-(266 –281)-specific T helper cell hybridomas upon stimulation with native OVA presented by interferon-␥treated microglia. To elucidate a specific involvement of CE and CD in the MHC class II-mediated antigen presentation by microglia, we conducted experiments utilizing specific inhibitors for cathepsins, purified enzymes and microglia prepared from CDϪ/Ϫ mice.
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