FIG. 4.

GzmA cleaves recombinant HMG2 to produce a 20-kDa product in vitro. (a) rHMG2 is cleaved by GzmA but not GzmB. rHMG2 (1 μM) was incubated for 2 h at 37°C with the indicated amounts of GzmA or 1 μM S-AGzmA or GzmB, and the reaction products were analyzed by HMG2 immunoblotting. (b) Schematic of HMG2 showing the GzmA cleavage site in HMG box A, one of the DNA binding domains of HMG2. HMG2 contains HMG boxes A and B, a linker region (l), a joining region (j), and an acidic tail. The cleavage site was identified by N-terminal sequencing of the 20-kDa cleavage fragment shown in panel a. (c) Although HMG2 is degraded after adding GzmA to cytoplasmic lysates, HMG1 in cell lysates is not cleaved by GzmA. This is despite the fact that the region around the HMG2 cleavage site differs only by a single amino acid change in the P₁′ site.