Retinal Glycoprotein Enrichment by Concanavalin A Enabled Identification of Novel Membrane Autoantigen Synaptotagmin-1 in Equine Recurrent Uveitis

Abstract

Complete knowledge of autoantigen spectra is crucial for understanding pathomechanisms of autoimmune diseases like equine recurrent uveitis (ERU), a spontaneous model for human autoimmune uveitis. While several ERU autoantigens were identified previously, no membrane protein was found so far. As there is a great overlap between glycoproteins and membrane proteins, the aim of this study was to test whether pre-enrichment of retinal glycoproteins by ConA affinity is an effective tool to detect autoantigen candidates among membrane proteins. In 1D Western blots, the glycoprotein preparation allowed detection of IgG reactions to low abundant proteins in sera of ERU patients. Synaptotagmin-1, a Ca2+-sensing protein in synaptic vesicles, was identified as autoantigen candidate from the pre-enriched glycoprotein fraction by mass spectrometry and was validated as a highly prevalent autoantigen by enzyme-linked immunosorbent assay. Analysis of Syt1 expression in retinas of ERU cases showed a downregulation in the majority of ERU affected retinas to 24%. Results pointed to a dysregulation of retinal neurotransmitter release in ERU. Identification of synaptotagmin-1, the first cell membrane associated autoantigen in this spontaneous autoimmune disease, demonstrated that examination of tissue fractions can lead to the discovery of previously undetected novel autoantigens. Further experiments will address its role in ERU pathology.

Early development of the nervous system of the eutherian <i>Tupaia belangeri</i>

“…The functional meaning of interspecific differences regarding the physiological expression of α-internexin needs to be clarified in follow-up studies. Knowledge of these expression patterns is also relevant under pathological conditions, e.g., in a spontaneous equine model of autoimmune uveitis where vitreal IgM autoantibodies target NF-M in neuronal processes of the retina (Swadzba et al, 2012).…”

Equine recurrent uveitis—A review

“…The S‐antigen, a major autoantigen in human medicine, resulted in a significantly lower number of uveitis cases when used to experimentally induce uveitis in horses (Deeg et al, 2004b). Two additional retinal autoantigens have been detected more recently: cellular retinaldehyde‐binding protein (cRALBP), part of the rod and cone visual cycle, and synaptotagmin‐1, a protein integral to synaptic vesicle membranes (Deeg et al, 2006a; Swadzba et al, 2012). Deeg et al (2002b) revealed that disease severity, as well as the T cell response, varied amongst the uveitis cases following experimental induction with IRBP.…”

Immunological Insights in Equine Recurrent Uveitis

“…The combination of active downscaling in damaged RMG and release of NF-M fragments into the adjacent vitreous might cause this combination of high vitreal occurrence and low retinal levels of NF-M ( 130 ). A further novel membrane-bound autoantigen, synaptotagmin-1, was identified with decreased expression in ERU ( 131 ), which might be a result of active downscaling of this protein in the retina through impaired neurotransmitter release in ERU. Since Synaptotagmin-1 is also expressed in the pineal gland, and pinealitis is known to concurrently develop in ERU horses ( 132 ), synaptotagmin-1 might play a role in pineal immunopathology in ERU ( 131 ).…”

“…A further novel membrane-bound autoantigen, synaptotagmin-1, was identified with decreased expression in ERU ( 131 ), which might be a result of active downscaling of this protein in the retina through impaired neurotransmitter release in ERU. Since Synaptotagmin-1 is also expressed in the pineal gland, and pinealitis is known to concurrently develop in ERU horses ( 132 ), synaptotagmin-1 might play a role in pineal immunopathology in ERU ( 131 ). Whether these IgM-targeted retinal antigens qualify as autoantigen targets for ERU, however, remains to be proven in further studies.…”