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Jianzhong Chen, Dongfeng Cao, Seth Fortmann, Christine A Curcio, Richard Feist, Jason Crosson; Intact transthyretin proteoforms of intraocular origin in human subretinal fluid from rhegmatogenous retinal detachment. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1183 – A0037.
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© ARVO (1962-2015); The Authors (2016-present)
Rhegmatogenous retinal detachment leads to fluid between the photoreceptor outer segments and retinal pigment epithelium (RPE). Knowledge of the protein composition in this fluid may provide clues to retinal diseases such as age-related macular degeneration, which is believed to involve dysregulated lipid transfer between photoreceptors, RPE, and choroid.
Intact proteins were extracted from the subretinal fluids of five patients with rhegmatogenous retinal detachment and analyzed by tandem mass spectrometry. A publicly available retinal gene expression database was searched. Two human donor eyes with normal maculas were immunoprobed for transthyretin (TTR) with appropriate controls.
Albumin, TTR, and apolipoprotein A-I were the three most abundant proteins in subretinal fluid. Remarkably, TTR relative to the other proteins was more abundant than its serum counterpart, suggestive of TTR being predominantly synthesized locally. Six post-translationally modified protein forms (proteoforms) of TTR were detected, with the relative amount of glutathionylated TTR being much higher in the subretinal fluid (12 - 43%) than values reported for counterparts in serum (< 5%) and cerebrospinal fluid (0.4 - 13%). Moreover, a putative TTR dimer of 32,428, most likely with glycosylation modifications, was detected as the fourth most abundant protein. The high abundance of TTR and putative TTR dimer in subretinal fluid was supported by single-cell RNA sequencing analysis, which showed strong signal for TTR in RPE. Immunohistochemistry further showed strong diffuse TTR immunoreactivity in choroidal stroma that contrasted with vertically aligned signal in the outer segment zone of the subretinal space and negligible signal in RPE cell bodies. Lower relative amounts of glutathionylated TTR and a putative TTR dimer in one AMD eye merits further investigation in more samples.
The high percentage of glutathionylated TTR suggests that glutathionylation is crucial for normal TTR function in the retina. RPE expression of TTR gene and compartmentalized immunoreactivity suggests that TTR is made intraocularly as well as in the liver for plasma. Lipid carriers like TTR may participate in pathways that when dysregulated lead to subretinal drusenoid deposit. Subretinal fluid from patients undergoing retinal detachment repair is a rich biofluid for discovery.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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