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

组织蛋白酶 E 参与原代培养小胶质细胞外源抗原加工

基本信息

批准号：
11671845
负责人：
NAKANISHI Hiroshi
金额：
$ 2.5万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671845/
关键词：
microglia cathepsin E cathepsin D antigen presentation ovalbumin antigenic peptide invaliant chain cathepsin D-deficient mice カテプシンD カテプシン阻害剤 pepstatin A インターロイキン-2 インターフェロン-γ

项目摘要

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

组织蛋白酶E（CE，EC 3.4.23.34）是胃蛋白酶家族的细胞内天冬氨酸蛋白，它与溶酶体天冬氨酸蛋白组织蛋白酶D高度同源（CD，EC 3.4.23.5）。与CD相反，CE在组织和细胞类型中的分布有限，例如淋巴组织，胃肠道，尿道器官和小胶质细胞。 CE主要定位于小胶质细胞的内体结构中，而CE则位于各种细胞室中，例如质膜，内质网和其他细胞类型和组织的高尔基体。可以想象，CE的成熟形式与该酶的内体定位密切相关，因为CE的成型仅在进入酸性隔室后才将其转化为成熟形式。已知小胶质细胞与中枢神经系统中的ivad的CD4+ T辅助细胞相互作用。通过这些相互作用，小胶质细胞可能导致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性疾病。主要的组织相容性复合物（MHC）II类抗原呈递要求内体/溶酶体蛋白参与内吞途径，以降解与MHC II相关的MHC II相关的外源性抗原和不变链（LI）的策略本地化，但CE II II级参与MHC Antiglia在CE中无需提供信息。在介绍研究中，我们研究了组织蛋白酶高度选择性抑制剂的影响，包括天冬氨酸蛋白抑制剂pepstain A对卵巢蛋白（OVA）或OVA胡椒（OVA 267-282）的呈现，对原代鼠类小胶质细胞对OVA特异性I-Ab-ab Crestifuct thrber thhs thh hy tunber t（uva）的影响（OVA 267-282）的影响（OVA 267-282）。在用IFN-G刺激后，CE mRNA的表达水平显着升高，而不会影响其他房屋的表达水平，包括饲养组织蛋白酶D（CD）和组织蛋白酶B（CB）（CB）。当小胶质细胞用pepstatin A处理时，用IFN-G处理的小胶质细胞呈现的幼稚OVA刺激后，由OVA特异性TH细胞杂交瘤产生IL-2。然而，pepstatin A未能抑制OVA肽的表现。 Clik-060是CLIK-060（CS）的特异性抑制剂，CA-074ME（一种CB的特异性抑制剂）对呈抑制作用的影响对天真OVA和OVA肽的表现产生抑制作用。天冬氨酸蛋白参与天真OVA的抗原肽加工的参与得到了这样的事实，即CE或CD消化的幼体OVA的消化片段可以被OVA特异性的TH细胞杂交瘤识别以产生IL-2。当使用从CD不足小鼠制备的小og时，仅略微降低了将OVA抗原性胡椒呈现给T细胞的OVA抗原性胡椒的有效性。通过使用抗LI抗体进行免疫印迹分析，进一步证实了在LI降解最后阶段CS和CB的要求。这些结果表明，CE而不是CD对于从OVA产生抗原表位是必需的，而不是在小胶质细胞中加工Li。较少的