Mutation of single lysine residues increases half-life of TIMP-3 in the medium of HTB94 chondrosarcoma cells.
A, HTB94 chondrosarcoma cells were incubated (0–24 h) with 1 nm FLAG-tagged wild-type TIMP-3 (black circle), TIMP-3 preincubated with 1 μM RAP (white circle), or TIMP-3 mutant K26A (red circle), K42A (blue square), or K45A (green triangle) in DMEM with 0.1% FCS. Media were concentrated by TCA precipitation and analyzed by immunoblotting with anti-FLAG M2 antibody and densitometry using Phoretix 1D software. The loss of each mutant from the medium was calculated relative to its pixel volume at t = 0 h (defined as 100%). Immunoblots show a representative experiment, and graphs show analysis of three technical replicates (mean ± S.D. (error bars)). B, HTB94 chondrosarcoma cells were incubated (0–24 h) with FLAG-tagged wild-type TIMP-3 (black circle), TIMP-3 preincubated with 1 μM RAP (white circle), or TIMP-3 mutant K71A (red circle), TIMP-3 K110A (blue square), or K157A (green triangle) in DMEM with 0.1% FCS. Media were concentrated and analyzed as in A. Immunoblots show a representative experiment, and graphs show analysis of three technical replicates (mean ± S.D.). C, half-lives of wild-type TIMP-3 and its mutants were calculated from A and B. Wild-type TIMP-3 had a half-life of 3.6 ± 0.3 h. TIMP-3 mutants K42A, K71A, and K157A exhibited significantly increased half-lives of 8.8 ± 1.6, 12.9 ± 0.4, and 7.8 ± 2.5 h, respectively (**, p ≤ 0.01; ***, p ≤ 0.001).