Proteolytic processing of ovalbumin and beta-galactosidase by the proteasome to a yield antigenic peptides.
Proteolytic processing of ovalbumin and beta-galactosidase by the proteasome to a yield antigenic peptides.
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DOI：
10.4049/jimmunol.152.8.3884
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发表时间：
1994-04
影响因子：
4.4
通讯作者：
Lawrence;-R.;Dick;S. C. Jameson;li Carolyn;Moomaw;Bikash C. Pramanik;C. Kuyler;Doyle;George;DeMartino;Michael;-J.;Bevan;Clive A. Slaughter
中科院分区：
文献类型：
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作者：
Lawrence;-R.;Dick;S. C. Jameson;li Carolyn;Moomaw;Bikash C. Pramanik;C. Kuyler;Doyle;George;DeMartino;Michael;-J.;Bevan;Clive A. Slaughter
在MHC的II类区域中与编码蛋白酶体的亚基的基因同源的基因的鉴定已经导致了对这种酶在多肽Ag的蛋白水解加工中的可能作用的强烈兴趣。我们已经测试了20 S蛋白酶体产生肽的能力，所述肽可以由I类分子呈递作为用于通过OVA特异性和β-半乳糖苷酶特异性CTL克隆杀伤的靶。完整的卵清蛋白和β-半乳糖苷酶的样品进行消化，在体外由20 S蛋白酶体纯化牛红细胞和所得的肽混合物进行分级分离，通过反相HPLC。测试所述级分通过特异性CTL克隆使适当的小鼠靶细胞对裂解敏感的能力。在这两种情况下，发现在所有色谱条件下以与代表天然加工蛋白质的已知表位的合成肽不可区分的保留时间洗脱的组分能够致敏靶细胞。此外，在OVA的情况下，通过氨基酸序列和质谱分析直接证明了预期的靶肽的存在。结果表明，纯的20 S蛋白酶体能够从两种蛋白质产生抗原肽，用于由I类分子呈递，而没有蛋白质降解系统的其他组分的参与。这一发现与蛋白酶体参与体内Ag加工的假设一致。
The identification of genes in the class II region of the MHC that are homologous to genes encoding subunits of the proteasome has led to intense interest in the possible role of this enzyme in the proteolytic processing of polypeptide Ags. We have tested the ability of the 20S proteasome to produce peptides that can be presented by class I molecules as targets for killing by OVA-specific and beta-galactosidase-specific CTL clones. Samples of intact OVA and beta-galactosidase were subjected to digestion in vitro by 20S proteasome purified from bovine red cells and the resulting peptide mixtures were fractionated by reverse-phase HPLC. The fractions were tested for their ability to sensitize appropriate mouse target cells for lysis by specific CTL clones. In both cases, components that under all chromatographic conditions eluted with retention times indistinguishable from synthetic peptides representing known epitopes of the naturally processed proteins were found to be able to sensitize the target cells. Moreover, in the case of OVA, the presence of the expected target peptides was demonstrated directly by amino acid sequence and mass spectrometric analysis. The results demonstrate that the pure 20S proteasome is capable of generating antigenic peptides from two proteins for presentation by class I molecules without the participation of additional components of the protein degradation system. This finding is consistent with the hypothesis of proteasome involvement in Ag processing in vivo.