Most proteins expressed in mammalian cells are post-translationally modified in manners that affect their function, localization, and role within the cell. Of these post-translational modifications, attachment of glycans is often required for the protein to function as intended. However, the sites of attachment of these glycans, and the chemical nature of the glycans attached are not templated and are difficult to predict. Protein glycans are attached either through N- or O-linked means, and are elaborated in many ways. N-linked glycans include many notable complex glycan structures which serve as immune regulators and cancer markers, and are regulated through reversible attachment at an asparagine's carboxamide. The enzymes that catalyze the reaction to remove the entirety of these glycan structures from proteins are called N-glycanases or N-glycosidases, and the enzyme leaves a scar" by hydrolyzing the amide bond liberating an aspartic acid and an amino-glycan. Typically, the reactions of these enzymes are monitored with gel-shift assays for potential substrates, a fairly-low throughput assay format. Diazo compounds uniquely prefer to react with protonated carboxylic acids, even in aqueous buffers. The reaction occurs at rapid rates, forming an ester bond. As such, we have envisioned utilizing a suite of diazo compounds to assay the specificity, kinetics, and identity of N-glycanases and their substrates. Our current platform has been optimized utilizing the known enzyme Peptide:N-Glycosidase F (PNGaseF) and glycoprotein standard Ribonuclease B (RNaseB). Our current assay platforms utilize a non-fluorescent, rapidly-reacting diazo compound to first mask carboxylic acids present in protein sidechains, termini, and glycans. PNGase F is added for 15 minutes, followed by another diazo compound attached to a fluorophore. Fluorescence readout by gel or by plate reader is facilitated by bright fluorophores attached in a modular fashion to a common diazo core. The same diazo core can also be appended to isotopically-labeled amines that enable rapid mass-spectrometry identification of formerly N-glycanated sites, or be used to increase positive charges on protein and glycan fragments during intact glycoproteomic studies, mitigating much of the signal loses in complex glycan structures such as poly-sialic acid modifications. We have been applying these chemical tools to advance our understanding of human N-glycanase 1, and its role in physiology and pathology.<br/><br/><br/>

Diazo compounds as tools to assay the specificity, kinetics, and identity of N-glycanases and their substrates

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.