Interaction and direct palmitoylation of MT1-MMP by ZDHHC13.

<p>HEK293 cells were co-transfected with ZDHHC13-myc (ZDWT-myc) and MT1-MMP-V5. Co-Immunoprecipitation (IP) were performed to determine interaction between ZDHHC13 and MT1-MMP (<b>A</b>) IP using myc antibody (IP: myc) pulled down both ZDWT-myc and MT1-MMP-V5 (left). Arrow indicates the band of ZDWT. Anti-V5 antibody IP (IP: V5) pulled down both MT1-MMP-V5 and ZDWT-myc (right). Vector (PCDNA4-myc) IP group was used as control. Protein lysate before IP was used as input. The cartoon illustrated the constructs and grouping of co-IP experiments. PAT: palmitoyl acyltransferase. (<b>B</b>) Palmitoylation level of MT1-MMP examined by acyl-biotin exchange (ABE). ZDHHC13-flag (ZDWT) or ZDK-flag (mimic mutation in our mice) were co-transfected with MT1-MMP-V5 in HEK293 cells. Streptavisdin-HRP signal represented palmitoylation level. Purified MT1-MMP was analyzed by V5 antibody and used as input normalization control. The quantitative palmitoylation level from three independent repeats was shown as fold change to vector (right). MT1-MMP-V5 has multiple molecular weights of ∼66 KDa and ∼45 KDa (self-processed form). The cartoon illustrated the construct of ZDWT and ZDK. Number represented the amino acid position. DQHC is the domain with PAT activity. (<b>C</b>) WB examination of ZDWT, ZDK (enzyme), and MT1-MMP-V5 (substrate) expression using flag and V5 antibody respectively. (<b>D</b>) Palmitoylation of MT1-MMP in WT and mutant distal femur epiphysis. MT1-MMP was directly purified from P14 WT and mutant epiphysis protein lysate to perform ABE. Streptavidin-HRP signal represented palmitoylation level. Purified MT1-MMP was analyzed by specific antibody. Quantitative result from three independent experiments of three P14 littermate pairs was shown as palmitoylation percentage (right). *<i>P</i><0.05.