Figure 2. Mutation of the SERPINB4 RCL at the P2-P1-P1′ positions completely abolishes complex formation with human GrM.

(A) A RCL-mutant of SERPINB4 was employed in which the amino acids at the putative P2(Glu³⁵³)-P1(Leu³⁵⁴)-P1′(Ser³⁵⁵) positions were mutated into P2(Gln³⁵³)-P1(Gly³⁵⁴)-P1′(Ala³⁵⁵). Purified recombinant GrM (0.9 µM), GrM-SA (0.9 µM), SERPINB4 (0.9 µM), and SERPINB4 RCL-mutant (0.9 µM) were incubated for 1 h at 37°C. All samples were treated with PNGase F, separated by SDS-PAGE, and immunoblotted for GrM. Bound antibodies were visualized using DAB. (B) Cell lysates of 293T cells transfected with C-terminal GFP-conjugated SERPINB4 or an empty vector (mock) were incubated with recombinant GrM (0.5 µM) for 1 h at 37°C. Subsequently, samples were immunoblotted for GFP. SERPINB4-GFP represents the full length protein, whereas SERPINB4*-GFP depicts the C-terminal cleavage product. (C) Schematic representation of C-terminal GFP-conjugated SERPINB4, including the GrM cleavage site after Leu³⁵⁴ at the P1-position in the RCL.