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. 2020 Feb 7;10:2099. doi: 10.1038/s41598-020-58964-x

Figure 2.

Figure 2

The C-terminus of TIMP-1 interacts with the extracellular large loop (LEL) of CD63. (A) 2-dimensional (left) and 3-dimensional (right) structure of TIMP-1, showing T1ΔC (17aa deletion, light orange) and T1ΔC2 (9aa deletion, light blue). 2D structural diagram of TIMP-1 adapted from Bodden et al.21. The N-terminal and C-terminal domains of TIMP-1 consist of loops 1–3 and 4–6, respectively. 3D structural diagram of TIMP-1 was made using PyMOL Molecular Graphics System based on the PDB entry 1UEA entered by Gomis-Ruth et al.18. The site occupied by MMP-3 catalytic domain in the crystal structure of MMP-3/TIMP-1 complex is shown in magenta in the N-terminal MMP inhibitory domain; helix 2 and helix 3 are shown in green and yellow respectively, and the connector between helices 2 and 3 is shown in orange; disulfide bridges are depicted in pale yellow. (B) PCA using cell lysates prepared from HEK293FT cells transfected with GLucN-TIMP-1 (T1), GLucN-T1ΔC (T1ΔC), or GLucN-T1ΔC2 (T1ΔC2) together with CD63-GLucC (CD63); GLucN-HNF4 and HNF4-GLucC (HNF4 dimer), GLucN-HNF4 and CD63-GLucC (HNF4/CD63), GLucN-TIMP-1 and HNF4-GLucC (T1/HNF4), GLucN-T1ΔC and HNF4-GLucC (T1ΔC/HNF4), GLucN-T1ΔC2 and HNF4-GLucC (T1ΔC2/HNF4), or without transfection (293FT NT). Immunoblot analysis of TIMP-1 using cell lysates or conditioned media from HEK293FT cells transfected with GLucN-TIMP-1 (T1), GLucN-T1ΔC (T1ΔC), GLucN-T1ΔC2 (T1ΔC2) or without transfection (293FT NT). (C) A diagram of CD63 depicting mutagenesis in the small extracellular loop (SEL) and the large extracellular loop (LEL) domains. Amino acid residues highlighted in color in the SEL were mutated to alanine residues and designated as 7AA, IIQ and TPGS accordingly. Separately, the entire LEL domain was removed for PCA domain analysis (ΔLEL). (D) PCA using cell lysates of HEK293FT cells transfected with luciferase fusion vectors as indicated (see Supplemental Fig. 1). Immunoblot analysis using anti-Gaussia Luciferase pAb in cell lysates of HEK293FT cells transfected with indicated wild type or mutant CD63-GLucC vectors (Right panel). Lanes are splitted because two separate immunoblots were used (see full immunoblot in Supplemental Fig. 2). Transferrin receptor (TfR) was used as loading control. For panels B and D, all values shown are the average of at least triplicate measurements. Error bars represent standard deviation. Significance was assessed as described in Materials and Methods with P < 0.05 being considered as significant.