Photo-products of retinal pigment epithelial bisretinoids react with cellular thiols

[摘要] Purpose: Bisretinoids such as A2E thataccumulate as components of the lipofuscin of retinal pigmentepithelial cells are implicated in some retinal disease processes.These compounds undergo light-induced oxidation and cleavage with thelatter releasing of a mixture of aldehyde-bearing fragments, includingdicarbonyl methylglyoxal. We tested for the reactivity ofphotooxidation and photodegradation products of A2E withthiol-containing glutathione (GSH). Methods: In cell-free assays, wemeasured the ability of photooxo-A2E to competitively inhibit theGSH-mediated reduction of the thiol reagent5,5′-dithiobis-(2-nitrobenzoic acid). Cellular GSH was assayedcolorimetrically. Products of GSH reduction and GSH-adducts weredetected by electrospray ionization mass spectrometry (ESI-MS) and GSHand oxidized GSH (glutathione disulfide [GSSG]) were quantified fromchromatographic peak areas. Results: We found that GSH can donatehydrogen atoms to, and form conjugates with, photooxidized forms of thebisretinoid A2E and with its photocleavage products. Reaction withnon-photooxidized A2E was not observed. Chemical reduction by GSHinvolved the donation of a hydrogen atom from each of two GSHs. Theratio of GSH consumed to GSSG formed was consistent with GSH being usedfor both reduction and adduct formation. With the aid of synthesizedstandards, methylglyoxal-GSH adducts were identified within mixtures ofGSH and photooxidized A2E; the adducts formed noncatalytically and byglutathione-S-transferase mediation. Conclusions: Reduction and adductformation by GSH likely limits the reactivity of bisretinoidphotoproducts and may aid their elimination from the cells. Thesefindings are significant to forms of macular degeneration associatedwith bisretinoid formation and maculopathy stemming from GSH synthasedeficiency.

[学科分类] 生物化学/生物物理