Fig. 5.

LOXL2/L2Δ13 catalyzes deacetylation of aldolase A-K13.

(A) Left: acetylation level of total proteins from whole cell lysates of KYSE510 cells depleted for LOXL2; right: schematic diagram of the analytical strategy for SILAC labeling and global profiling of lysine acetylation. Control and LOXL2-depleted KYSE510 cells were separately labeled with “heavy” and “light” arginine and lysine using SILAC, and then proteins were digested for LC-MS/MS analysis. (B) Significant patterns show KEGG enrichment pathways of acetylated proteins in MS analysis. (C) Total proteins from untreated cells (mock), cells with scrambled siRNA or shRNA (siNC or shNC) and LOXL2-silenced cells were immunoprecipitated using acetyl-Lys and probed with indicated antibodies. Stain-Free gels was used as the control for equal protein concentration for the IP. (D) Total proteins from KYSE510 stably-infected cells were immunoprecipitated using acetyl-Lys and probed with indicated antibodies. Beta-Actin served as the control for equal protein concentration for the IP. (E) Endogenous aldolase A was purified from LOXL2-depleted and control KYSE510 cells with SILAC labeling, and acetyl peptides regulated by LOXL2 were identified. Shown are representative MS/MS spectra of acetylated aldolase A-K13. (F) Crystal structure model of human aldolase A (PDB database accession: 1ALD). Lys 12 in the model indicates ALDOA-K13 in our study. (G) Sequence alignment of acetylation sites of aldolase A-K13 from different species. (H, I) Aldolase A acetylated at K13 (aldolase A-K13ac) and total aldolase A expression level in indicated cells, esophagi and livers of mice. (J) Purified GST-aldolase A from bacteria were incubated with increasing amount of purified LOXL2-Flag/L2Δ13-Flag from HEK293T transfectants or recombinant LOXL2 protein for in vitro LOXL2/L2Δ13 deacetylase activity assays. (K) Indicated acetyl peptides of the aldolase A-K13 were incubated with the purified LOXL2-Flag, L2Δ13-Flag and Flag-vector proteins from HEK293T transfected cells. The reaction products were blotted with anti-aldolase A-K13ac or anti-Flag. (L) The acetyl peptides of the aldolase A-K13 were incubated with the purified recombinant LOXL2 protein. (M) HEK293T cells were transfected with the indicated plasmids and treated with or without traditional histone deacetylase inhibitors. Aldolase A acetylation was analyzed with an antibody against acetyl-Aldolase A (K13).