Abstract
Purpose :
Bestrophin is an anion channel and a regulator of intracellular calcium signaling from the basolateral plasma membrane of the retinal pigment epithelium (RPE). Anti-bestrophin autoantibodies (AAbs) were found in patients with vitelliform macular dystrophy (VMD) without known gene mutations. The goal of this study was to identify immunodominant epitopes targeted by anti-bestrophin AAbs and determine their association with autoimmune retinopathy.
Methods :
Sera from patients with VMD were used in the study. Autoantibody titers against bestrophin were determined by immunoblotting and ELISA. Epitope mapping was performed using 49 biotinylated synthetic peptides that overlapped the entire sequence of human bestrophin. The peptides 15-amino acids in length, offset by 12 amino acids were coated onto a streptavidin-treated microplate and bestrophin-specific sera were tested for their ability to bind to immobilized peptides by ELISA.
Results :
Initially, we studied 2 VMD patients with similar clinical phenotype however with different HLA associations. Using overlapping synthetic bestrophin peptides we found that serum 1 strongly bound to the sequence 61-73 of the first external loop. In contrast, serum 2 reacted with two epitopes 326-331 and 517-539 located within the N-terminal cytoplasmic RPE domain. Analyses of additional 14 bestrophin-positive sera of patients with vitelliform macular lesions showed that 100% serum AAbs bound to the epitope 61-73, proposing it as the major determinate for anti-bestrophin AAbs. In addition, 71% AAbs bound to the epitope 325-351 within the cytoplasmic domain and 57% AAbs recognized the epitope 529-543 of the C-terminus. The difference in epitope recognition could be a result of a different mechanism of antibody generation and their HLA involvement.
Conclusions :
: Presumably, the 3 bestrophin regions represent the major immunopathogenic epitopes, involved in pathogenesis of VMD. Over 97% of the disease-causing mutations are also located in the bestrophin N-terminal domain, implying its important functional properties. Since AAbs can block the same important sequences their binding could mimic symptoms related to the bestrophin genetic defects. These findings provided valuable information for understanding bestrophin antigenicity in VMD, which might be useful in the development of epitope-based treatments, and provide insights for understanding the pathogenesis of retinal degeneration.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.