Menz Beatrice, Sester Martina, Koebernick Katja, Schmid Ralf, Burgert Hans-Gerhard
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
Mol Immunol. 2008 Nov;46(1):16-26. doi: 10.1016/j.molimm.2008.06.019. Epub 2008 Aug 9.
The E3/19K protein of human adenovirus type 2 (Ad2) was the first viral protein shown to interfere with antigen presentation. This 25 kDa transmembrane glycoprotein binds to major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER), thereby preventing transport of newly synthesized peptide-MHC complexes to the cell surface and consequently T cell recognition. Recent data suggest that E3/19K also sequesters MHC class I like ligands intracellularly to suppress natural killer (NK) cell recognition. While the mechanism of ER retention is well understood, the structure of E3/19K remains elusive. To further dissect the structural and antigenic topography of E3/19K we carried out site-directed mutagenesis and raised monoclonal antibodies (mAbs) against a recombinant version of Ad2 E3/19K comprising the lumenal domain followed by a C-terminal histidine tag. Using peptide scanning, the epitopes of three mAbs were mapped to different regions of the lumenal domain, comprising amino acids 3-13, 15-21 and 41-45, respectively. Interestingly, mAb 3F4 reacted only weakly with wild-type E3/19K, but showed drastically increased binding to mutant E3/19K molecules, e.g. those with disrupted disulfide bonds, suggesting that 3F4 can sense unfolding of the protein. MAb 10A2 binds to an epitope apparently buried within E3/19K while that of 3A9 is exposed. Secondary structure prediction suggests that the lumenal domain contains six beta-strands and an alpha-helix adjacent to the transmembrane domain. Interestingly, all mAbs bind to non-structured loops. Using a large panel of E3/19K mutants the structural alterations of the mutations were determined. With this knowledge the panel of mAbs will be valuable tools to further dissect structure/function relationships of E3/19K regarding down regulation of MHC class I and MHC class I like molecules and its effect on both T cell and NK cell recognition.
人腺病毒2型（Ad2）的E3/19K蛋白是首个被证明能干扰抗原呈递的病毒蛋白。这种25 kDa的跨膜糖蛋白在内质网（ER）中与主要组织相容性复合体（MHC）I类分子结合，从而阻止新合成的肽-MHC复合物转运至细胞表面，进而抑制T细胞识别。近期数据表明，E3/19K还能在细胞内隔离MHC I类样配体，以抑制自然杀伤（NK）细胞识别。虽然内质网滞留机制已得到充分理解，但E3/19K的结构仍不清楚。为了进一步剖析E3/19K的结构和抗原拓扑结构，我们进行了定点诱变，并制备了针对Ad2 E3/19K重组体的单克隆抗体（mAb），该重组体包含腔内结构域及C末端组氨酸标签。通过肽扫描，三种单克隆抗体的表位分别定位到腔内结构域的不同区域，分别包含氨基酸3 - 13、15 - 21和41 - 45。有趣的是，单克隆抗体3F4与野生型E3/19K的反应较弱，但与突变型E3/19K分子（如二硫键断裂的分子）的结合显著增加，这表明3F4能感知蛋白质的去折叠。单克隆抗体10A2结合的表位显然埋藏在E3/19K内部，而3A9的表位是暴露的。二级结构预测表明，腔内结构域包含六条β链和一条与跨膜结构域相邻的α螺旋。有趣的是，所有单克隆抗体都结合在非结构化环上。使用大量E3/19K突变体确定了突变的结构改变。基于这些认识，该单克隆抗体组将成为进一步剖析E3/19K在MHC I类和MHC I类样分子下调方面的结构/功能关系及其对T细胞和NK细胞识别影响的有价值工具。
