An enzyme-based assay for the detection of carboxyethyl lysine-protein adducts

PROJECT SUMMARY Advanced glycation endproducts (AGEs) are hallmarks of mammalian aging and disease. Nε-carboxyethyl-lysine (CEL) in particular, is among the most abundant AGEs accumulating in tissue proteins with aging, and is an important marker of glycation, oxidative stress, and disease. Current techniques for reliable measurement of CEL are slow, expensive (>$120/sample), and not readily available to many academic research labs nor easily applied for routine analysis of clinical samples. Development of a convenient method for measurement of CEL would democratize the research tools used to study AGEs and advance the tools available to study and treat diseases at the interface of aging, glycation and oxidative stres. Revel has discovered a CEL oxidase enzyme which oxidizes CEL to lysine. The newly discovered CEL oxidase provides an opportunity to develop enzymes for diagnostic use in monitoring CEL-modifications. The goal of this Phase I SBIR is to develop a rapid, one-pot, inexpensive enzymatic test (<$10/sample) to measure CEL. The proposed assay will be developed analogous to the enzymatic HbA1c test which is used to assess patient blood sugar levels and diagnose diabetes. In Phase II, we will develop a workflow for CEL detection in patient samples. Eventually, further engineering of the enzyme for removal of CEL from endogenous proteins could yield a ground-breaking tool for reversing glycoxidatively damaged proteins in clinical medicine.