Autoantigenic properties of the U1-70K protein

Sammanfattning: Autoantigenic properties of the U1-70K protein Elisabet Welin Henriksson Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, S-171 77 Stockholm, Sweden Patients with the autoimmune diseases systemic lupus erythematosus and mixed connectivetissue disease produce autoantibodies directed towards specific groups of cellularconstituents in their own body. Two of the targeted antigens are denoted Sm and RNP,and consist of distinct collections of splicing associated RNA-protein complexes.The main antigen recognised by anti-RNP sera is the U1-70K protein. The major epitopesof this protein are located in the second quarter of the protein, approximately aminoacid residues 99-167, this region being situated within the RNA binding domain requiredfor the 70K-U1 snRNA interaction. The amino-acid sequence of the U1-70K protein ishighly conserved and RNP positive patient sera have been shown to react with the70K protein originating from several other species. In this thesis, recombinant proteins containing the major antigenic region fromthe human and the Drosophilia melanogaster 70K (Dm 70K) proteins have beenused to explore the specificity of human anti-RNP sera. We found that human anti-RNPsera contain both human specific antibodies, and antibodies cross-reacting with Dm70K. The cross-reactive anti-Dm 70K antibodies seem to appear secondary to the species-specificantibodies. This supports the hypothesis that the endogenous human 70K protein isthe immunogen driving the production of anti-70K autoantibodies, as opposed to polyclonanactivation or antibody production stimulated by exogenous proteins. When canine autoimmune sera were examined, the anti-70K response was found tobe directed against the same region of the U1-70K protein as the human response.This pattern is not typical for other shared canine and human autoantigens, and wethus conclude that the targeting of a specific part of the U1-70K protein is notrestricted to the human autoimmune process. It has been proposed that the major antigenic region of the U1-70K protein isconstituted by conformational epitopes but the crucial amino acid residues have notbeen identified. In this study we have used the highly homologous, but less antigenicDrosophila melanogaster U1 70K protein as a starting point for attempts toreconstitute antigenicity. First, Dm 70K was grafted with human sequence segmentsand assayed for antigenicity. It was concluded that the major sites for autoantibodybinding were situated within amino acid residues 99-128. In addition, proper foldingseems to be a prerequisite for antigenicity. Second, in vitro mutagenesis, usingboth random mutation by DNA shuffling and directed point mutagenesis, was used topinpoint the contribution of specific residues. Almost complete antigenicity couldbe reconstituted by replacing residue 125 with the corresponding human residue. Otherkey residues are located in the 119-126 region. Tertiary structure modelling showsthat this part of the protein is situated in the end of an alpha-helix and the beginningof a loop, separated from the residues forming the RNA binding site. Conclusion: This thesis is focused on the autoantigenic properties of the U1-70Kprotein, and reveals new insights into the complex interaction between the autoantigenand the immune system. We have established the presence of species-specific autoantibodiesin human sera, and show that a specific part of the protein is targeted by the autoimmuneprocess in both man and dog. Residues crucial for antigenicity are clustered in the119-126 region, and replacement of residue 125 with the human equivalent reconstitutesalmost full antigenicity to the homologous Drosophila melanogaster U1-70K protein. Keywords: U1-70K protein, ribonucleoproteins, autoantigens, autoantibodies, epitopemapping, connective tissue disease, species specificity, cross reactions, amino acidsequence, recombinant proteins, mutagenesis ISBN 91-628-2818-5