Identification of Glucosepane Cross-Link Breaking Enzymes

Abstract

Glucosepane is a member of the class of advanced glycation end-products (AGEs), which form non-enzymatically in the human body. Glucosepane, is the most abundant AGE found in human collagen and has been implicated in the pathophysiology of various conditions ranging from diabetes to normal human aging. Glucosepane crosslinks impact the structural and mechanical properties of collagen and contribute to stiffening of collagenous tissues and vascular dysfunction. We have previously demonstrated the total synthesis of glucosepane, enabling methods for detecting and targeting glucosepane. The present study seeks to identify enzymes that are capable of catalyzing the decomposition of synthetic glucosepane crosslinks. To identify glucosepane crosslink breaking enzymes, we developed a novel screening technology based on metagenomics. Using this strategy, four enzymes were identified for in vitro validation of glucosepane degradation activity. Thus far, our efforts have focused on identifying metabolites of one class I-like enzyme in particular, since it is heterologously expressed at high levels and does not require any unusual cofactors. In vitro enzymatic assays showed that incubation of glucosepane with the class I-like enzyme led to a product consistent with citrulline. We are currently in the process of evaluating the enzyme’s mechanisms of action and identifying other metabolites generated. This is the first demonstration that glucosepane can be broken down enzymatically. Our findings may provide new insight into the role of glucosepane in aging and disease and aid in the development of novel therapeutic strategies for targeting glucosepane. Disclosure M. Streeter: None. T.N. Goddard: None. J.M. Crawford: None. D.A. Spiegel: None.