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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（Cathepsin E，CE，EC 3.4.23.34）是胃蛋白酶家族的细胞内天冬氨酸蛋白酶，其与溶酶体天冬氨酸蛋白酶组织蛋白酶D（Cathepsin D，CD，EC 3.4.23.5）高度同源。与CD相比，CE在组织和细胞类型（如淋巴组织、胃肠道、泌尿器官和小胶质细胞）中的分布有限。CE主要定位于小胶质细胞的内体结构中，而CE定位于其他细胞类型和组织的各种细胞隔室，如质膜、内质网和高尔基体中。因此，可以想象，CE的成熟形式与这种酶的内体定位密切相关，因为CE的前体形式似乎只有在进入酸性区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的ivaded CD 4 + 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， ...更多信息病毒感染和慢性炎症性疾病。主要组织相容性复合体（MHC）II类抗原呈递需要内吞途径中的内体/溶酶体蛋白酶的参与，以降解与MHC II类相关的外源性抗原和不变链（li）。尽管CE的战略定位，但没有关于CE参与小胶质细胞MHC II类抗原呈递的信息。在本研究中，我们研究了高选择性的组织蛋白酶抑制剂，包括天冬氨酸蛋白酶抑制剂pepstain A对卵清蛋白（OVA）或OVA肽（OVA 267-282）的介绍原代培养的小鼠小胶质细胞的OVA特异性I-Ab限制性辅助T（Th）细胞杂交瘤的效果。在IFN-g刺激下，CE mRNA的表达水平显著增加，而不影响其他管家组织蛋白酶包括组织蛋白酶D（CD）和组织蛋白酶B（CB）的表达水平。当用胃酶抑素A处理小胶质细胞时，在用IFN-γ处理的小胶质细胞呈递的幼稚OVA刺激后，来自OVA特异性Th细胞杂交瘤的IL-2产生被显著抑制。然而，胃酶抑素A不能抑制OVA肽的呈递。组织蛋白酶S（CS）的特异性抑制剂CLIK-060和CB的特异性抑制剂CA-074 Me对天然卵清蛋白和卵清蛋白肽的呈递均显示出抑制作用。通过CE或CD消化的天然OVA片段可以被OVA特异性Th细胞杂交瘤识别以产生IL-2的事实进一步支持了天冬氨酸蛋白酶参与天然OVA的抗原肽的加工。当使用从CD缺陷小鼠制备的microgla时，将OVA抗原肽呈递给T细胞的功效仅略微降低。通过使用抗li抗体进行免疫印迹分析，进一步证实了在li降解的最后阶段对CS和CB的要求。这些结果表明，CE，而不是CD是必需的抗原表位从OVA的产生，但不是在小胶质细胞中的处理li。少