表位
单克隆抗体
突变
跨膜结构域
跨膜蛋白
化学
主要组织相容性复合体
内质网
分子生物学
突变体
抗原
细胞生物学
生物
氨基酸
生物化学
抗体
基因
遗传学
受体
作者
Beatrice Menz,Martina Sester,Katja Koebernick,Ralf Schmid,Hans-Gerhard Burgert
标识
DOI:10.1016/j.molimm.2008.06.019
摘要
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 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 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 -strands and an -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.
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