Autoantigenic properties of the U1-70K protein

Abstract

Patients with the autoimmune diseases systemic lupus erythematosus and mixed connective tissue disease produce autoantibodies directed towards specific groups of cellular constituents 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 epitopes of this protein are located in the second quarter of the protein, approximately amino acid residues 99-167, this region being situated within the RNA binding domain required for the 70K-U1 snRNA interaction. The amino-acid sequence of the U1-70K protein is highly conserved and RNP positive patient sera have been shown to react with the 70K protein originating from several other species.

In this thesis, recombinant proteins containing the major antigenic region from the human and the Drosophilia melanogaster 70K (Dm 70K) proteins have been used to explore the specificity of human anti-RNP sera. We found that human anti-RNP sera contain both human specific antibodies, and antibodies cross-reacting with Dm 70K. The cross-reactive anti-Dm 70K antibodies seem to appear secondary to the species-specific antibodies. This supports the hypothesis that the endogenous human 70K protein is the immunogen driving the production of anti-70K autoantibodies, as opposed to polyclonal activation or antibody production stimulated by exogenous proteins. When canine autoimmune sera were examined, the anti-70K response was found to be 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 we thus conclude that the targeting of a specific part of the U1-70K protein is not restricted to the human autoimmune process. It has been proposed that the major antigenic region of the U1-70K protein is constituted by conformational epitopes but the crucial amino acid residues have not been identified.

In this study we have used the highly homologous, but less antigenic Drosophila melanogaster U1 70K protein as a starting point for attempts to reconstitute antigenicity. First, Dm 70K was grafted with human sequence segments and assayed for antigenicity. It was concluded that the major sites for autoantibody binding were situated within amino acid residues 99-128. In addition, proper folding seems to be a prerequisite for antigenicity. Second, in vitro mutagenesis, using both random mutation by DNA shuffling and directed point mutagenesis, was used to pinpoint the contribution of specific residues. Almost complete antigenicity could be reconstituted by replacing residue 125 with the corresponding human residue. Other key residues are located in the 119-126 region. Tertiary structure modelling shows that this part of the protein is situated in the end of an alpha-helix and the beginning of a loop, separated from the residues forming the RNA binding site.

Conclusion: This thesis is focused on the autoantigenic properties of the U1-70K protein, and reveals new insights into the complex interaction between the autoantigen and the immune system. We have established the presence of species-specific autoantibodies in human sera, and show that a specific part of the protein is targeted by the autoimmune process in both man and dog. Residues crucial for antigenicity are clustered in the 119-126 region, and replacement of residue 125 with the human equivalent reconstitutes almost full antigenicity to the homologous Drosophila melanogaster U1-70K protein.