Investigating proteolytic activity of engineered trypsin variants using naturally occurring macromolecular substrates

Abstract

Serine proteases constitute numerous physiological roles that include but are not limited to the breakdown of food, the participation in immune responses, and with the triggering of cell apoptosis Engineered serine proteases as therapeutic agents constitute a new class of drugs for potential clinical applications. However, one main complication faced when using serine proteases as therapeutic agents is their relatively short half‐life due to the abundance of tight binding inhibitors in the human body that target serine proteases to regulate proteolytic activity and rapidly remove them from circulation. For a protease to be effective it must display resistance towards inhibitors. Previously engineered trypsin variants K60A, K60V, Y39A, Y39F have shown to diminish prime side interactions, affecting micromolecular inhibition through the disruption of hydrogen bond networks occurring at residue positions 39 and 60. These variants displayed resistance towards certain naturally occurring inhibitors, bovine pancreatic trypsin inhibitor and M84R ecotin. Kinetic data was performed using commercially available tripeptide Z‐GPR‐pNA, also indicating no change of specificity. In order to ascertain the viability of these engineered variants as potential therapeutics we must study the interactions between naturally available macro molecular substrates. Experimental methods required the design and utilization of a novel spectroscopicassay tomonitor the proteolytic activity of trypsin variants using chymotrypsinogen (Cg) as our naturally occurring macromolecular substrate. Within the body, trypsin cleaves triggering the activation of zymogen Cg into its active form. Fluorescence will be measured using chymotrypsin specific 4‐methylumbelliferyl p‐tri‐methylammoniocinnamate chloride (MUTMAC). Preliminary data has shown that a 10:1 concentration ratio (100nM Trypsin to 10μM Cg) proved most efficient for activation. However, only ~22% of chymotrypsinogen has been activated using wild‐type trypsin, suggesting that further optimization of this assay is still required. Future work will aim to elucidate the proteolytic rates of these trypsin variant K60A, K60V, Y39A, Y39F where we hope to obtain a better understanding of the interactions between these variants and naturally occurring substrates.Support or Funding InformationNIH MS/PhD Bridge: R25‐GM048972This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.