Retinal glycoprotein enrichment by concanavalin a enabled identification of novel membrane autoantigen synaptotagmin-1 in equine recurrent uveitis.

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.