Characterization of two protein disulfide isomerases from the endocytic pathway of bloodstream forms of Trypanosoma brucei
Characterization of two protein disulfide isomerases from the endocytic pathway of bloodstream forms of Trypanosoma brucei
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DOI：
10.1074/jbc.m409375200
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发表时间：
2005-03-18
影响因子：
4.8
通讯作者：
Nolan, DP
中科院分区：
文献类型：
--
作者：
Rubotham, J;Woods, K;Nolan, DP
血流形式的布氏锥虫内吞途径的蛋白质通过添加线性聚-N-乙酰基乳糖胺侧链进行修饰，这使得它们可以通过番茄凝集素亲和层析进行分离。使用针对该番茄凝集素结合部分的抗体从血流形式的布氏锥虫中筛选cDNA表达文库。其中有两个 cDNA 最为突出。这些 cDNA 编码两种假定的蛋白质二硫键异构酶 (PDI)，分别包含一个和两个双半胱氨酸氧化还原活性位点，对应于单结构域 PDI 和 1 类 PDI。对纯化的重组蛋白的分析表明，两种蛋白都具有异构酶活性，但只有单结构域 PDI 具有还原活性。这些 PDI 具有许多不寻常的特征，与之前描述的 PDI 不同。两者的表达都受到发育调节，它们都与内吞途径的标记物共定位，并且都通过 N-糖基化进行修饰。较大的 PDI 具有含有聚 N-乙酰基乳糖胺的 N-聚糖，这种修饰表明在高尔基体中进行加工，并表明锥虫中存在 PDI 的新运输途径。尽管通常认为 PDI 是必需的，但这两种活性似乎都不是锥虫生长所必需的，至少在体外是这样。
Proteins from the endocytic pathway in bloodstream forms of Trypanosome brucei are modified by the addition of linear poly-N-acetyllactosamine side chains, which permits their isolation by tomato lectin affinity chromatography. Antibodies against this tomato lectin binding fraction were employed to screen a cDNA expression library from bloodstream forms of T. brucei. Two cDNAs were prominent among those selected. These cDNAs coded for two putative protein disulfide isomerases (PDIs) that respectively contained one and two double-cysteine redox-active sites and corresponded to a single domain PDI and a class 1 PDI. Assays of the purified recombinant proteins demonstrated that both proteins possess isomerase activity, but only the single domain PDI had a reducing activity. These PDIs possess a number of unusual features that distinguish them from previously characterized PDIs. The expression of both is developmentally regulated, they both co-localize with markers of the endocytic pathway, and both are modified by N-glycosylation. The larger PDI possesses N-glycans containing poly-N-acetyllactosamine, a modification that is indicative of processing in the Golgi and suggests the presence of a novel trafficking pathway for PDIs in trypanosomes. Although generally PDIs are considered essential, neither activity appeared to be essential for the growth of trypanosomes, at least in vitro.