Thiol-dependent antioxidant activity of interphotoreceptor retinoid-binding protein

Abstract

Interphotoreceptor retinoid-binding protein (IRBP), which is critical to photoreceptor survival and function, is comprised of homologous tandem modules each ~300 amino acids, and contains 10 cysteines, possibly 8 as free thiols. Purification of IRBP has historically been difficult due to aggregation, denaturation and precipitation. Our observation that reducing agent 1,4-dithiothreitol dramatically prevents aggregation prompted investigation of possible functions for IRBP’s free thiols. Bovine IRBP (bIRBP) was purified from retina saline washes by a combination of concanavalin A, ion exchange and size exclusion chromatography. Antioxidant activity of the purified protein was measured by its ability to inhibit oxidation of 2,2'-azinobis [3-ethylbenzothiazoline-6-sulfonate] by metmyoglobin. Homology modeling predicted the relationship of the retinoid binding sites to cysteine residues. As a free radical scanvenger, bIRBP was more active than ovalbumin, thioredoxin, and vitamin E analog Trolox. Alkylation of free cysteines by N-ethylmaleimide inhibited bIRBP’s antioxidant activity, but not its ability to bind all-trans retinol. Structural modeling predicted that Cys 1051 is at the mouth of the module 4 hydrophobic ligand-binding site. Its free radical scavenging activity points to a new function for IRBP in defining the redox environment in the subretinal space.

Structure of zebrafish IRBP reveals fatty acid binding

“…It is, therefore, possible that disruption of the salt bridge destabilizes the ligand-binding site compromising IRBP’s ability to efficiently bind, be effectively secreted (Li et al, 2013), release and/or protect retinoids from degradation. Furthermore, the appreciation of a novel hydrophobic ligand-binding cavity binding fatty acids in Domain B may be important to understanding IRBP’s ability to efficiently clear retinol from bleached outer segments (Tsina et al, 2004b; Wu et al, 2007) while protecting from photodecomposition (Crouch et al, 1992; Gonzalez-Fernandez et al, 2015; Gonzalez-Fernandez et al, 2014; Parker et al, 2011). Efficient clearance of all- trans retinol from the outer segment could minimize bisretinoid toxic intermediates, early events in the pathogenesis of age-related macular degeneration (Sparrow et al, 2003).…”

“…Furthermore, IRBP effectively delivers all- trans retinol and 11- cis retinal to the RPE (Flannery et al, 1990; Jin et al, 2009; Okajima et al, 1989), and rod and cone outer segments (Duffy et al, 1993; Jin et al, 2009; Okajima et al, 1990; Parker et al, 2011). Finally, an important emerging function of IRBP is protect visual-cycle retinoids from photodecomposition within the interphotoreceptor matrix (Crouch et al, 1992; Gonzalez-Fernandez et al, 2015; Gonzalez-Fernandez et al, 2014). …”

Single particle cryo‐EM of the complex between interphotoreceptor retinoid‐binding protein and a monoclonal antibody

“…Our strategy for IRBP purification (Figure S1A,B) is similar to previous methods 50‐53 . However, we isolated retinas in house and prepared the extract without freezing, two attributes necessary for cryo‐EM imaging of bovine IRBP.…”

Interphotoreceptor Retinoid-Binding Protein Mitigates Cellular Oxidative Stress and Mitochondrial Dysfunction Induced by All-trans-Retinal

“…Since oxidative and nitrosative stresses are pathologic mechanisms involved in rod and cone photoreceptor degeneration in retinal degenerative diseases, 35 , 51 – 53 our results suggest that IRBP plays an important role in protection of photoreceptors from oxidative stress through suppression of a t RAL accumulation and its thiol-dependent antioxidant activity. 54 …”