Molecular characterization of the Ro52 autoantigen and its disease related epitopes

The presence of high titers of autoantibodies in patient sera is characteristic for the autoimmune disorder Sjögren s syndrome. The major targets for these autoantibodies are three intracellular proteins Ro52, Ro60 and La. It is still unclear why these proteins become targets for the immune system, and how this autoreactivity is triggered. Antibodies to the Ro52 protein have also been shown to associate with the development of congenital heart block in fetuses of anti-Ro52 positive mothers. The focus of this thesis has been to characterize the Ro52 protein at the molecular level, and to analyze the role of anti-Ro52 antibodies in congenital heart block.

By combining immunologic, biophysical and biochemical methods we have determined the protein domain composition of Ro52. The stable domains found in recombinant Ro52 correspond well with bioinformatic predictions. We have confirmed that the predicted RING-finger, B-box and coiled-coil domains are all functional in Ro52, based on secondary structure analysis and functional studies. The RING and B-box together form a single folding unit. Two Zn2+-binding sites with nanomolar affinities were found within the RING-finger, while the B-box contains one independent Zn2+-binding site with micromolar affinity. The region between RING and B-box appears crucial for Zn2+-dependent subdomain interactions within Ro52.

Secondary structure analysis of the coiled-coil domain by circular dichroism confirmed that the domain has a predominant alpha-helical fold. This domain of Ro52 was determined to consist of two structurally stable coiled-coil formations, separated by a short stretch of exposed amino acid residues. The coiled-coil domain of Ro52 forms weak homodimers, which does not exclude possible heterodimerization with an unknown interaction partner.

As a tool for studying congenital heart block, we identified and cloned two human monoclonal antibodies directed against the stretch of Ro52 recognized by antibodies associated with congenital heart block. The monoclonal antibodies were isolated from an antibody library from autoimmune patients by phage display technology. The specificities of these antibodies were fine mapped and one antibody clone was found to recognize a conformational epitope within the Ro52 coiled-coil domain. This antibody specificity was found in high frequency of sera from children affected by congenital heart block.

In vitro studies with rat cardiomyocytes and in vivo studies in a rat model confirmed the importance of the certain antibody specificity in development of congenital heart block. The antibodies were found to interact with an antigen on the surface of cardiomyocytes, leading to disrupted Ca2+-homeostasis in response to antibody binding. After initial increase of calcium oscillation frequency, the cells were overloaded with Ca2+ and died via apoptosis. This mechanism is proposed as the initiating event in congenital heart block.

In conclusion, this thesis work has revealed the presence and functionality of stable domains found in Ro52. We also suggest a mechanism for the induction of congenital heart block, and further characterized the role of specific anti-Ro52 autoantibodies in this process.

List of scientific papers

I. Ottosson L, Hennig J, Espinosa A, Brauner S, Wahren-Herlenius M, Sunnerhagen M (2005). Structural functional and immunologic characterization of folded subdomains in the Ro52 protein targeted in Sjögrens syndrome. [Submitted]

II. Hennig J, Ottosson L, Andresen C, Wahre-Herlenius M, Sunnerhagen M (2005). Zn2+-binding effects on structure and folding of the Ring-Bbox-Coiled Coil (RBCC) region in Ro52, a protein targeted in Sjögrens syndrome. [Manuscript]

III. Salomonsson S, Ottosson L, Safsten P, Hof D, Brauner H, Sunnerhagen M, Raats J, Wahren-Herlenius M (2004). Cloning and characterization of two human Ro52-specific monoclonal autoantibodies directed towards a domain associated with congenital heart block. J Autoimmun. 22(2): 167-77.
https://doi.org/10.1016/j.jaut.2003.11.004

IV. Ottosson L, Salomonsson S, Hennig J, Sonesson SE, Dorner T, Raats J, Kuchroo VK, Sunnerhagen M, Wahren-Herlenius M (2005). Structurally derived mutations define congenital heart block-realted epitopes within the 200-239 amino stretch of the Ro52 protein. [Accepted]
https://doi.org/10.1111/j.0300-9475.2005.01542.x

V. Salomonsson S, Sonesson SE, Ottosson L, Muhallab S, Olsson T, Sunnerhagen M, Kuchroo VK, Thoren P, Herlenius E, Wahren-Herlenius M (2005). Ro/SSA autoantibodies directly bind cardiomyocytes, disturb calcium homeostasis, and mediate congenital heart block. J Exp Med. 201(1): 11-7.
https://doi.org/10.1084/jem.20041859