Characterization of Critical Residues of the Granzyme B Inhibitor, Serpina3n (50.43)

Abstract Granzyme B (GrB) is crucial for the immune system in eliminating tumor and virus-infected cells by caspase-mediated apoptosis. GrB also has detrimental effects in allograft tissue rejection and autoimmune diseases. Our aim was to design effective GrB inhibitors as unique immunosuppressive drugs in treating these disorders. Our lab has defined a novel murine inhibitor of GrB named serpina3n (ser3n). Ser3n is a superior candidate for drug design since it is the only known GrB inhibitor that is secreted extracellularly, making it more plasma stable. We performed scanning mutagenesis of the reactive centre loop (RCL) domain, which confers serpins with their protease specificity, to test which residues have inhibitory properties. Using an in vitro transcribed/translated (IVTT) ser3n protein, we found that the putative P1 residue (Met) and the P3' residue (Lys) are important for ser3n binding to GrB. Human antichymotrypsin (ACT) is highly homologous to ser3n, but is not a GrB inhibitor. Our goal was to generate a human secretory protein that inhibits GrB. We created an ACT-ser3n chimera by replacing the RCL of ACT with that of ser3n. We then created a mutant ACT-ser3n chimera with a Met-to-Asp mutation that resulted in greater GrB binding than the original chimera, to a degree equal to ser3n binding. This inhibitor will work on pathogenic states when secreted GrB is important. This reveals a potential drug for blocking GrB in autoimmune diseases.