Structural analysis of the adenovirus type 2 E3/19K protein using mutagenesis and a panel of conformation-sensitive monoclonal antibodies.
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.
Menz
Beatrice
B
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
Sester
Martina
M
Koebernick
Katja
K
Schmid
Ralf
R
Burgert
Hans-Gerhard
HG
D017503:Adenovirus E3 Proteins / Q000737:chemistry* / Q000276:immunology*; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D001667:Binding, Competitive; D002241:Carbohydrates / Q000737:chemistry; D002460:Cell Line; D017124:Conserved Sequence; D003545:Cysteine / Q000235:genetics; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry / Q000276:immunology; D009154:Mutation / Q000235:genetics; D010449:Peptide Mapping; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000276:immunology
D017503:Adenovirus E3 Proteins; D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D000939:Epitopes; D050505:Mutant Proteins; D011994:Recombinant Proteins; D003545:Cysteine; D000409:Alanine