Analysis of the binding specificity of dopamine-2 receptor antibodies in paediatric autoimmune movement and psychiatric disorders

Abstract

Over the last decade, multiple autoantibodies targeting brain proteins and receptors have been identified in adults and children. Autoantibody exposure in the brain can lead to transient or permanent behavioural or cognitive abnormalities. Current treatment options for autoimmune brain-reactive autoantibody-associated diseases include global immunosuppressive therapies, but these can have severe side effects, and are not always efficacious. Understanding the nature and specificity of brain autoantibodies may reveal potential therapeutic strategies alleviating or preventing the neurological pathologies and behavioural abnormalities associated with antibody-mediated brain disorders. Recently, anti-dopamine-2 receptor (D2R) antibodies have been identified in a subgroup of children with autoimmune movement and psychiatric disorders. D2R is an important brain receptor involved a variety of functions including voluntary movement, learning, memory, attention, and hormonal regulation. Currently, knowledge of the function of D2R tertiary structures, including the extracellular N-terminus, is limited, and its role in autoantibody binding is unknown. Here we report a major biological role for D2R extracellular N-terminus as a regulator of receptor surface availability, and as a major epitope targeted in brain autoimmunity. Human embryonic kidney cells were transfected with D2R mutants modified in their extracellular domains, and the level of cell surface expression and epitope specificity of 35 anti-D2R antibody-positive patient sera were analysed using a quantitative flow cytometry assay. We found that N-glycosylation at amino acids N5 and/or N17 was critical for high surface expression via interaction with the last 15 residues of extracellular D2R N-terminus. No anti-D2R antibody-positive patient sera bound to the three extracellular loops, but all patient sera (35/35) targeted the extracellular N-terminus. Overall, patient antibody binding was dependent on two main regions encompassing amino acids 20 to 29, and 23 to 37. Residues 20 to 29 contributed to the majority of binding (77 %, 27/35), among which sera from 26 % (7/27) of patients bound to amino acids R20, P21, and F22, 37 % (10/27) patient sera binding was dependent on residues at positions 26 and 29, and 30% (8/27) sera required R20, P21, F22, N23, D26, and A29. Seven patient sera bound to the region 23 to 37 independently of D26 and A29, but most sera exhibited N-glycosylation-independent epitope recognition at N23. Interestingly, no evident segregation of binding pattern according to patient clinical phenotypes was observed. Finally, we describe the optimisation of a method for single-cell isolation and RT-PCR for the generation of monoclonal antibodies. D2R N-terminus is a central epitope in autoimmune movement and psychiatric disorders, and this knowledge could help the design of novel specific immune therapies tailored to improve patient outcome.