Fig. 2
MMP12 cleaves IFN-γ removing the IFN-γ receptor-binding site. a Silver stained 15% SDS-PAGE analysis of in vitro cleavage of human (h) IFN-γ by 10 or 100 ng hMMP12 catalytic domain at 1:10 and 1:100 enzyme to substrate ratios, incubated over 18 h at 37 °C. Revealing C-terminal cleavage, N-terminal sequencing identified an intact N-terminus commencing at 1MQDPY both in IFN-γ and in the two cleavage products (red arrows). The MMP12-specific inhibitor, Rxp470.1, blocked IFN-γ cleavage and autocatalytic cleavage of MMP12 resulting in stabilized MMP12 protein levels over 18 h. Molecular weight marker positions are shown. b Q-TOF-MS analysis of IFN-γ cleavage reaction products revealed C-terminal cleavage first between 157Met↓Leu158 and then at 135Glu↓Leu136 (see Supplementary Fig. 4). The kcat/KM values calculated for each cleavage event in human and mouse (m) IFN-γ are shown. c Based on the crystal structures of the IFN-γ homodimer (pdb entry: 1HIG)33, 51, a secondary complex consisting of the IFN-γ dimer, two high-affinity IFN-γ receptor 1 molecules (IFNGR1(pdb entry: 1FG9); 29Val-Ser241; dark gray), and two low affinity IFN-γ receptor 2 chains (IFNGR2 (pdb entry: 1FYH); 30Leu-Thr237; light pink) were modeled. The IFN-γ C-terminal peptide (135Glu–158Leu) responsible for IFN-γ receptor interaction and signaling was modeled (green). The transmembrane peptide and JAK1/2 are shown in cartoon form. d Frontal view of the structured region of the IFN-γ homodimer with the C-terminal non-structured flexible region (from 146Ala to Gln166) cartooned in green. The two MMP12 cleavage sites are shown: 157Met↓Leu158 and 135Glu↓Leu136
