Involvement of cathepsin E in exogenous antigen processing in primary cultured microglia

组织蛋白酶E(Cthepsin E，CE，EC 3.4.23.34)是胃酶家族的一种胞内天冬氨酸蛋白酶，与溶酶体天冬氨酸蛋白酶D(Cd，EC 3.4.23.5)高度同源。与CD相比，CE在组织和细胞类型中的分布有限，如淋巴组织、胃肠道、泌尿器官和小胶质细胞。CE主要定位于小胶质细胞的内体结构，而CE定位于其他细胞类型和组织的质膜、内质网和高尔基体等多种细胞室。因此，可以想象，成熟形式的CE与该酶的内体内化密切相关，因为CE的ProForm似乎只有在进入酸性隔室后才转化为成熟形式。已知小胶质细胞与中枢神经系统中分离的CD4+T辅助细胞相互作用。通过这些相互作用，小胶质细胞可能有助于自身免疫性疾病期间的组织损伤和修复，…更多的病毒感染和慢性炎症。主要组织相容性复合体(MHC)II类抗原提呈需要内体/溶酶体蛋白参与胞内途径，以降解外源抗原和与MHC II类相关的不变链(Li)。尽管CE具有战略定位，但尚不清楚CE是否参与小胶质细胞MHC II类抗原提呈。在本研究中，我们研究了组织蛋白的高选择性抑制剂，包括天冬氨酸蛋白酶抑制剂胃蛋白酶A，对原代培养的小鼠小胶质细胞呈递卵清蛋白(OVA)或卵清蛋白多肽(OVA 267-282)的影响。经干扰素-g刺激后，CE基因的表达水平显著升高，而对其他内分泌组织蛋白包括组织蛋白酶D(CD)和组织蛋白酶B(CB)的表达水平无明显影响。当胃抑素A处理小胶质细胞时，干扰素-g处理的小胶质细胞经未成熟OVA刺激后，OVA特异性Th细胞杂交瘤细胞产生IL-2的能力明显受到抑制。但胃抑素A不能抑制OVA多肽的提呈。组织蛋白酶S(CS)的特异性抑制剂CLK-060和CB的特异性抑制剂CA-074Me对幼稚卵清蛋白和卵清蛋白多肽的递呈均有抑制作用。天冬氨酸蛋白酶参与了幼虫卵抗原肽的加工过程，进一步支持了CE或CD消化的幼虫卵蛋白片段可被OVA特异性Th细胞杂交瘤识别并产生IL-2的事实。用CD缺乏症小鼠制备的微孔板，对T细胞呈递OVA抗原肽的效果仅略有降低。通过使用抗锂抗体的免疫印迹分析，进一步证实了锂降解的最后阶段对CS和CB的需求。这些结果表明，CE而不是CD是从OVA产生抗原表位所必需的，但对小胶质细胞中LI的加工不是必需的。较少

Cathepsin E (CE, EC 3.4.23.34) is an intracellular aspartic protease of pepsin family, which is highly homologous to the lysosomal aspartic protease cathepsin D (CD, EC 3.4.23.5). In contrast to CD, CE has a limited distribution in tissues and cell types such as lymphoid tissues, gastrointestinal tracts, urinary organs and microglia. CE was mainly localized in the endosomal structures of microglia, whereas CE was localized in various cellular compartments such as the plasma membrane, the endoplasmic reticulum and the Golgi apparatus of the other cell types and tissues. Thus it is conceivable that the mature form of CE is closely associated with endosomal locarization of this enzyme because proform of CE appears to be converted into mature form only after entering in acidic compartments. Microglia are known to interact with ivaded CD4+ T helper cells in the central nervous system. Through these interactions, microglia may contribute to tissue damage and repair during autoimmune disease, … More viral infections and chronic inflammatory disease. Major histocompatibility complex (MHC) class II antigen presentation requires the participation of endosomal/lysosomal proteases in endocytic route to degrade exogenous antigens and invariant chain (li) associated with MHC class II.Despite the strategic localization of CE, no information is available about the involvement of CE in MHC class II antigen presentation of microglia. In the present study, we have investigated the effect of highly selective inhibitors of cathepsins including the aspartic protease inhibitor pepstain A on the presentation of ovalbumin (OVA) or OVA peptide (OVA 267-282) by primary cultured murine microglia to OVA-specific I-Ab restricted helper T (Th) cell hybridomas. Upon stimulation with IFN-g, expression level of CE mRNA was significantly increased without affecting the expression levels of other house keeing cathepsins including cathepisn D (CD) and cathepsin B (CB). When microglia were treated with pepstatin A, IL-2 production from an OVA-specific Th cell hybridomas upon stimulation with naive OVA presented by IFN-g-treated microglia was markedly suppressed. However, pepstatin A failed to inhibit the presentation of OVA peptide. CLIK-060, a specific inhibitor of cathepsin S (CS), and CA-074Me, a specific inhibitor of CB, showed an inhibitory effect on the presentation of both naive OVA and OVA peptide. The involvement of aspartic proteases in the processing of antigenic peptide of naive OVA was further supported by the fact that digested fragments of naive OVA by CE or CD could be recognized by OVA-specific Th cell hybridomas to produce IL-2. When microgla prepared from CD deficinet mice were used, the efficacy for presenting OVA antigenic peptide to T cells was only slightly reduced. The requirements for CS and CB in the final stage of li-degradation were further substantiated by immunoblot analyses by using anti-li antibody. These results indicate that CE rather than CD are necessary for the generation of an antigenic epitope from OVA but not for the processing of li in microglia. Less