There is significant interest in protein glycation, a non-enzymatic post-translational modification in which side chain amino groups (e.g., lysine and arginine) react with sugar aldehyde groups in a Maillard reaction to produce Schiff’s bases, Amadori products and eventually, advanced glycation endproducts (AGEs). Glycation impacts protein function, and several diseases including Alzheimer’s, atherosclerosis, and diabetes can be linked to the binding of AGEs by the transmembrane protein RAGE (receptor for advanced glycation endproducts). Many factors that mediate protein glycation still remain elusive. Recently, Scheck and coworkers performed a methodical <i>in vitro</i> study of protein glycation to elucidate the relative glycation levels and AGEs for sites on various proteins, as well as the effect of local sequence, structure, and other chemical features. We seek to explore the recognition of glycated peptide sequences by the RAGE receptor using phage display. Phage-displayed peptide libraries are composed of bacteriophage virions with randomized peptides displayed via their fusion to a coat protein. We plan to glycate three commercially available phage display libraries (Ph.D.-7, -12, -C7C) such that the chemically modified displayed peptides mimic typical ligands of RAGE. The glycated libraries can be panned against the target RAGE to identify glycated peptides that are preferentially bound by the receptor. As the coat proteins are also subject to glycation, which may compromise the infectivity of the phage, we have performed preliminary glycation experiments on control phages, one displaying an arginine-containing peptide (HGHHRHH) and another with no displayed peptide. Various concentrations of methylglyoxal were used, and the virulent phage were quantified following infection of <i>E. coli</i> cells. The maximum concentration of methylglyoxal, above which phage infectivity drops significantly, was determined. Following the optimization of all reaction conditions, the phage libraries will be glycated and panned against RAGE to probe the binding interactions between the receptor and its natural ligands.

Exploration of advanced glycation end products through the glycation of phage-displayed peptide libraries

ACS Spring 2022

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