Generation and characterization of anti-glucosepane antibodies enabling direct detection of glucosepane in retinal tissue

Authors: STREETER, MATTHEW - Yale University; ROWAN, SHELDON - Jean Mayer Human Nutrition Research Center On Aging At Tufts University; RAY, JASON - Yale University; MCDONALD, DAVID - Yale University; VOLKIN, JONATHAN - Tufts University; CLARK, JONATHAN - Babraham Institute; TAYLOR, ALLEN - Jean Mayer Human Nutrition Research Center On Aging At Tufts University; SPIEGEL, DAVID - Yale University

Submitted to: ACS Chemical Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/25/2020
Publication Date: 9/25/2020
Citation: Streeter, M.D., Rowan, S., Ray, J., McDonald, D.M., Volkin, J., Clark, J., Taylor, A., Spiegel, D.A. 2020. Generation and characterization of anti-glucosepane antibodies enabling direct detection of glucosepane in retinal tissue. ACS Chemical Biology. 15(10):2655-2661. https://doi.org/10.1021/acschembio.0c00093.
DOI: https://doi.org/10.1021/acschembio.0c00093

Interpretive Summary: With an aging population, there is an increasing need to identify individuals at increased risk of developing advanced disease or complications, including diabetes. One approach to do so is to identify biomarkers, molecular markers that can be analyzed in a laboratory. Prior research suggests that toxic, sugar-derived products called advanced glycation end-products increase with aging and diabetes risk. One of these advanced glycation end-products is called glucosepane, and is of great interest, but lacked analytical tools for easy detection. In this study, we identified antibodies that are capable of recognizing glucosepane - a first step to developing biomarker assays. The new antibody could also detect where glucosepane is found in tissues of the body. We found that glucosepane was present at high levels in a part of the eye that is susceptible to age-related disease. Glucosepane was also present in a part of the cell that has been connected to disease state. The amount of glucosepane in the eye appeared to increase with aging. Our new antibody reagent should be a valuable tool to explore whether glucosepane might be involved with disease formation and whether glucosepane might be a useful biomarker of various diseases.

Technical Abstract: Although there is ample evidence that the advanced glycation end-product (AGE) glucosepane contributes to agerelated morbidities and diabetic complications, the impact of glucosepane modifications on proteins has not been extensively explored due to the lack of sufficient analytical tools. Here, we report the development of the first polyclonal anti-glucosepane antibodies using a synthetic immunogen that contains the core bicyclic ring structure of glucosepane. We investigate the recognition properties of these antibodies through ELISAs involving an array of synthetic AGE derivatives and determine them to be both high-affinity and selective in binding glucosepane. We then employ these antibodies to image glucosepane in aging mouse retinae via immunohistochemistry. Our studies demonstrate for the first time accumulation of glucosepane within the retinal pigment epithelium, Bruch's membrane, and choroid: all regions of the eye impacted by age-related macular degeneration. Co-localization studies further suggest that glucosepane colocalizes with lipofuscin, which has previously been associated with lysosomal dysfunction and has been implicated in the development of age-related macular degeneration, among other diseases. We believe that the anti-glucosepane antibodies described in this study will prove highly useful for examining the role of glycation in human health and disease.