Glycans have several properties that make them excellent targets for disease biomarkers. Firstly, their location on cell surfaces makes them the first point of contact for cellular interactions, thus they are crucial in the control of normal metabolic processes. Cell surface molecules are also strategically exposed for surveillance by the immune system allowing for recognition of abnormal cells. Also, glycan structures that are not present, or are in low amounts, in normal states proliferate in disease states, such as cancer. Lastly, glycosylation involves many highly abundant proteins. Therefore, a single change in a cell’s glycosylation machinery can affect many different glycoconjugates. Since a cell’s glycosylation state is so significant to its health, highly-specific reagents are urgently needed to effectively employ and discover glycan disease markers. Enzymes evolve substrate affinities sufficient to allow for catalysis then release of product, meaning they have rapid turnover and suggesting they have low binding affinities. However, they can be altered to remove their catalytic function and increase their affinity for their ligands, yielding binding reagents specific to the substrates of the scaffold enzyme. This process begins with the computational methods molecular dynamics simulations and molecular mechanics and generalized Born surface area calculations, which generate a per residue decomposition of energetic contributions to binding. Residues in the binding site may contribute favorably or unfavorably to binding. We hypothesize residues making weakly favorable contributions in the inactive enzyme can be mutated to other residues that may make stronger interactions, thus improving the overall binding capacity of the protein. PNGase F-II catalyzes the cleavage of N-linked glycans, whether or not the glycan is α-1,3 or α-1,6 core fucosylated. Three residues in the binding site of PNGase F-II were considered viable for mutation for developing a reagent that could specifically bind to N-linked glycans. A library of mutants (selected by visual inspection for their potential to improve binding) has been constructed for these residues (selected computationally), and the mutants are being expressed in Escherichia coli. The mutants will be assayed to identify those that result in an enhanced binding capacity. Therefore, if these experiments are successful, they will constitute a novel strategy of developing similar carbohydrate binding reagents.

Development of novel binding reagent for the detection of N-linked glycan disease biomarkers by computational and experimental methods

256th National Meeting (2018)

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