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