Functional requirements for the lysosomal thiol reductase GILT in MHC class II-restricted antigen processing
Functional requirements for the lysosomal thiol reductase GILT in MHC class II-restricted antigen processing
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DOI：
10.4049/jimmunol.177.12.8569
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发表时间：
2006-12-15
影响因子：
4.4
通讯作者：
Cresswell, Peter
中科院分区：
文献类型：
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作者：
Hastings, K. Taraszka;Lackman, Rebecca L.;Cresswell, Peter
Ag通过MHC II类加工和呈递是激活CD4+ T淋巴细胞所必需的。γ - ifn诱导的溶酶体硫醇还原酶（GILT）存在于MHC II类装载室中，并已被证明可促进II类Ag的加工和对含有二硫键的Ag的召回反应，如鸡蛋溶菌酶（HEL）。假设MHC II类装载舱内蛋白质的减少可以暴露II类结合和蛋白酶修剪的残基。体外分析表明，GILT的活性位点涉及Cys（46）和Cys(49)，它们存在于与硫氧还蛋白家族相似的CXXC基序中。为了确定MHC II类Ag加工中GILT的功能需求，我们产生了一个GILT缺陷的小鼠B细胞淋巴瘤系，并稳定地用GILT野生型和半胱氨酸突变体进行了转导。细胞内流式细胞术、免疫印迹和免疫荧光分析表明，野生型和突变型GILT均能表达并维持溶酶体定位。野生型GILT的转导重建了依赖GILT的HEL表位的MHC II类加工。Cys（46）或Cys（49）的突变都取消了gilt依赖性HEL表位的MHC II类加工。此外，对这些突变体的生化分析表明，活性位点促进了前体GILT向成熟形式的加工。Cys（200）或Cys（211）中携带gilt突变的前体形式，先前发现在体外显示巯基还原酶活性，不能介导银加工。这些研究表明，GILT的硫醇还原酶活性是其在MHC ii类限制性Ag加工中的重要功能。
Ag processing and presentation via MHC class II is essential for activation of CD4+ T lymphocytes. gamma-IFN-inducible lysosomal thiol reductase (GILT) is present in the MHC class II loading compartment and has been shown to facilitate class II Ag processing and recall responses to Ags containing disulfide bonds such as hen egg lysozyme (HEL). Reduction of proteins within the MHC class II loading compartment is hypothesized to expose residues for class II binding and protease trimming. In vitro analysis has shown that the active site of GILT involves Cys(46) and Cys(49), present in a CXXC motif that shares similarity with the thioredoxin family. To define the functional requirements for GILT in MHC class II Ag processing, a GILT-deficient murine B cell lymphoma line was generated and stably transduced with wild-type and cysteine mutants of GILT. Intracellular flow cytometric, immunoblotting, and immunofluorescence analyses demonstrated that wild-type and mutant GILT were expressed and maintained lysosomal localization. Transduction with wild-type GILT reconstituted MHC class II processing of a GILT-dependent HEL epitope. Mutation of either Cys(46) or Cys(49) abrogated MHC class II processing of a GILT-dependent HEL epitope. In addition, biochemical analysis of these mutants suggested that the active site facilitates processing of precursor GILT to the mature form. Precursor forms of GILT-bearing mutations in Cys(200) or Cys(211), previously found to display thiol reductase activity in vitro, could not mediate Ag processing. These studies demonstrate that the thiol reductase activity of GILT is its essential function in MHC class II-restricted Ag processing.