Abstract
Purpose :
IRBP may be protective against DR and early DR shows reduced vitreal [IRBP]. Adequate functional vitreal and retinal [IRBP] may therefore have a critical role in the pathogenesis of DR. As an extracellular glycoprotein, it is susceptible to nonenzymatic glycation during hyperglycemia. However, no study has shown whether IRBP can be glycated or if glycation affects its stability and/or function.
Methods :
bIRBP was purified from bovine retina by Concanavalin A, ion-exchange, and size-exclusion chromatography. Purified bIRBP was incubated for 24 days at 37C with 0.2 mg/mL sodium azide (NaN3) in the presence of 0.5M glucose, 5mM methylglyoxal (MGO), or buffer alone. Independently, another set incubated for 5 days with 0.4mg/mL NaN3 and 1mM diethylenetriamine pentaacetate for bacteriostatic control. bIRBP was then analyzed by LC-MS/MS. The modified bIRBP was studied by SDS-PAGE, free radical scavenging of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS assay), and rapid pulse-probe assay with an integrated sphere as a phototoxicity chamber.
Results :
SDS-Page from longer incubation times showed partial protein breakdown compared to control. LC-MS/MS identified multiple AGE’s in protein exposed to glucose and MGO: carboxymethyl lysine (CML) and carboxyethyl lysine (CEL). In the MGO exposed bIRBP sample, >30% of available lysines contained CEL. In the glucose exposed sample, <10% of available lysines contained either CML or CEL. 8/10 of these post-translational modifications involved lysines that are conserved among bovine, human, Xenopus, and zebrafish IRBP. In comparison, similar AGE’s were not found in controls. Lastly, ABTS and rapid pulse probe assay showed a modest decrease in bIRBP’s free-radical scavenging activity, protection of retinol from photodecomposition, and stability.
Conclusions :
Exposure to glucose and MGO resulted in posttranslational modifications of lysine residues. These modifications may be functionally significant as most modified lysines were highly conserved. Glycation resulted in modest inhibition of free radical scavenging, retinol protection, and protein stability. The effect of hyperglycemia on IRBP should not only prompt study of IRBP’s production and turnover, but also the effect of non-enzymatic glycation on stability and function.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.