Sorting Motifs in the Cytoplasmic Tail of the Immunomodulatory E3/49K Protein of Species D Adenoviruses Modulate Cell Surface Expression and Ectodomain Shedding.
Sorting Motifs in the Cytoplasmic Tail of the Immunomodulatory E3/49K Protein of Species D Adenoviruses Modulate Cell Surface Expression and Ectodomain Shedding.
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D 种腺病毒免疫调节 E3/49K 蛋白细胞质尾部的分选基序调节细胞表面表达和胞外域脱落。
DOI：
10.1074/jbc.m115.684787
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发表时间：
2016
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影响因子：
--
通讯作者：
Windheim M
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文献类型：
--
作者：
Windheim M
人类C种腺病毒（Ads）的E3转录单元编码介导感染细胞直接保护的免疫调节蛋白。最近，我们描述了一种新的E3蛋白E3/ 49k的免疫调节功能，这是一种由D Ads物种唯一表达的E3蛋白。Ad19a/Ad64（一种引起流行性角膜结膜炎的血清型）的E3/ 49k被合成为高度糖基化的I型跨膜蛋白，随后被切割，导致其大的外膜结构域（sec49K）的分泌。sec49K与白细胞上的CD45结合，损害自然杀伤细胞和T细胞的活化和功能。E3/49K定位于高尔基/反式高尔基网络（TGN）、早期核内体和质膜上，但E3/49K被切割的细胞室和参与的蛋白酶仍不清楚。我们发现tgn定位的E3/49K既有新合成的分子，也有回收的分子。晚期核内体/溶酶体中未检测到全长E3/49K，但c端片段在感染后期在该室中积累。抑制剂研究表明，裂解发生在tgn后腔室，溶酶体增厚剂促进分泌。有趣的是，E3/49K的细胞质尾部包含两个潜在的分选基序YXXΦ（其中Φ代表一个大体积的疏水氨基酸）和LL，它们对于体外结合网格蛋白接头蛋白AP-1和ap -2很重要。令人惊讶的是，突变LL基序，无论是单独还是与YXXΦ一起，都不会阻止蛋白水解过程，但会增加细胞表面的表达和分泌。在brefeldin A处理后，细胞表面表达迅速丧失，甚至对于缺乏所有已知内吞作用基序的突变体也是如此。结合免疫荧光数据，我们提出了细胞内E3/49K转运模型，其中基质金属蛋白酶在细胞表面进行切割。
The E3 transcription unit of human species C adenoviruses (Ads) encodes immunomodulatory proteins that mediate direct protection of infected cells. Recently, we described a novel immunomodulatory function for E3/49K, an E3 protein uniquely expressed by species D Ads. E3/49K of Ad19a/Ad64, a serotype that causes epidemic keratokonjunctivitis, is synthesized as a highly glycosylated type I transmembrane protein that is subsequently cleaved, resulting in secretion of its large ectodomain (sec49K). sec49K binds to CD45 on leukocytes, impairing activation and functions of natural killer cells and T cells. E3/49K is localized in the Golgi/trans-Golgi network (TGN), in the early endosomes, and on the plasma membrane, yet the cellular compartment where E3/49K is cleaved and the protease involved remained elusive. Here we show that TGN-localized E3/49K comprises both newly synthesized and recycled molecules. Full-length E3/49K was not detected in late endosomes/lysosomes, but the C-terminal fragment accumulated in this compartment at late times of infection. Inhibitor studies showed that cleavage occurs in a post-TGN compartment and that lysosomotropic agents enhance secretion. Interestingly, the cytoplasmic tail of E3/49K contains two potential sorting motifs, YXXΦ (where Φ represents a bulky hydrophobic amino acid) and LL, that are important for binding the clathrin adaptor proteins AP-1 and AP-2in vitro. Surprisingly, mutating the LL motif, either alone or together with YXXΦ, did not prevent proteolytic processing but increased cell surface expression and secretion. Upon brefeldin A treatment, cell surface expression was rapidly lost, even for mutants lacking all known endocytosis motifs. Together with immunofluorescence data, we propose a model for intracellular E3/49K transport whereby cleavage takes place on the cell surface by matrix metalloproteases.