Key residues revealed in a major conformational epitope of the U1-70K protein.
Epitopes depending on three-dimensional folding of proteins have during recent years been acknowledged to be main targets for many autoantibodies. However, a detailed resolution of conformation-dependent epitopes has to date not been achieved in spite of its importance for understanding the complex interaction between an autoantigen and the immune system. In analysis of immunodominant epitopes of the U1-70K protein, the major autoantigen recognized by human ribonucleoprotein (RNP)-positive sera, we have used diversely mutated recombinant Drosophila melanogaster 70K proteins as antigens in assays for human anti-RNP antibodies. Thus, the contribution of individual amino acids to antigenicity could be assayed with the overall structure of the major antigenic domain preserved, and analysis of how antigenicity can be reconstituted rather than obliterated was enabled. Our results reveal that amino acid residue 125 is situated at a crucial position for recognition by human anti-RNP autoantibodies and that flanking residues at positions 119-126 also appear to be of utmost importance for recognition. These results are discussed in relation to structural models of RNA-binding domains, and tertiary structure modeling indicates that the residues 119-126 are situated at easily accessible positions in the end of an alpha-helix in the RNA binding region. This study identifies a major conformation-dependent epitope of the U1-70K protein and demonstrates the significance of individual amino acids in conformational epitopes. Using this model, we believe it will be possible to analyze other immunodominant regions in which protein conformation has a strong impact.
Welin Henriksson
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E
Department of Medicine, Rheumatology Research Unit, Karolinska Hospital, Karolinska Institutet, S-171 76 Stockholm, Sweden. Elisabet.Welin@cmm.ki.se
Wahren-Herlenius
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M
Lundberg
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I
Mellquist
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E
Pettersson
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I
D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000097:blood; D000939:Epitopes; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D017433:Protein Structure, Secondary; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry* / Q000276:immunology*
D001323:Autoantibodies; D000939:Epitopes; D017412:Ribonucleoprotein, U1 Small Nuclear