Abstract
Purpose::
Autoantigens not only hold a critical key in our understanding of the etiology of autoimmune uveitis, but also can serve as disease biomarkers for diagnosis. The purpose of this study is to develop a sensitive and efficient phage display system that will allow us to identify unknown autoantigens directly from uveitis patients using autoreactive antibodies in their blood.
Methods::
Patient or control IgG was immobilized onto 96-well plates through coated protein G. A unique T7 bacteriophage display cDNA library generated from mouse eye was pre-adsorbed with control IgG, followed by selection with patient IgG. Four rounds of subtractive phage panning were performed, and phage enrichment was monitored by PCR. The binding affinity of isolated phage clones, defined by the relative binding activities of clonal phages vs. wild-type phage to patient IgG, and their patient specificity, defined by clonal phage binding to patient IgG vs. control IgG, were analyzed by a phage-based binding assay.
Results::
Among several identified candidate autoantigens, a natural fusion protein of paralemmin and A kinase (PRKA) anchor protein 2 (PARAL-PRKA-AP2) demonstrated significantly higher binding affinity to IgG from all birdshot patients tested so far, but not from healthy subjects or patients with other types of uveitis. Paralemmin and PRKA-AP2 are proteins translated independently from two separate mRNAs. However, sequence analysis indicates that genes encoding these two proteins are adjacently located on mouse chromosome 4, suggesting that this natural fusion protein may be a product of unconventional mRNA splicing. The immunoreactivity of the fusion protein in birdshot patients and its functional role in the retina are currently under investigation.
Conclusions::
Our optimized system of subtractive phage display offers an efficient and sensitive approach to identify previously unknown autoantigens in an unbiased fashion for uveitis patients with minimal invasion. PARAL-PRKA-AP2 fusion protein may serve as a birdshot-specific disease biomarker for disease diagnosis.
Keywords: autoimmune disease • uveitis-clinical/animal model • inflammation