Figure 1
Design and characterization of CMP-Ang1 and its variants.
(A) Axial and side views of CMP structure (PDB: 1AQ5). Coiled-coil domain of CMP are trimerized and stabilized by disulfide bonds. Orange, the three disulfide bonds. (B) Schematic diagram of native Ang1 and CMP-Ang1. Ang1 consists of the secretory signal sequences (S), super-clustering domain (SCD), coiled-coil domain (CCD), linker (L) and fibrinogen-like domain (FLD). Crystal structure of Ang1-Tie2 complex (PDB: 4K0V) indicates that FLD of Ang1 is essential to bind to Tie2 receptor. Bluish green, FLD of Ang1; purple, extracellular domain (ECD) of Tie2. The SCD and CCD of Ang1 are replaced with the short CCD of CMP to generate CMP-Ang1 (denoted CA1-1). (C) Schematic diagrams of CMP-Ang1 variants. In CA1-2, cysteine 76 is replaced with alanine; in CA1-3, cysteines 31 and 76 are replaced with alanine. (D) For hFc-Ang1, GCN-Ang1 and COMP-Ang1, the SCD and CCD of Ang1 are replaced with the Fc portion of human IgG1 and with the short coiled-coil domains of GCN and COMP, respectively. (E) Ang1 variants were separated by SDS-PAGE under non-reduced (NR) and reduced (R) conditions and were immunoblotted with anti-Ang1 antibody. In NR condition, CA1-1 and CA1-2 mainly form trimers (orange thin arrows) but slightly exist as higher order oligomers (orange arrowheads), while CA1-3, GCN-Ang1, Fc-Ang1 and COMP-Ang1 mostly form dimers, dimers, dimers and pentamers, respectively (thick arrow of each different color). Molecular masses (kDa) are indicated on the left. (F) The oligomeric states of recombinant proteins were imaged by electron microscopy; respective schematic depictions are shown below. CA1-1 and CA1-2 mainly form trimers, whereas CA1-3, GCN-Ang1 and Fc-Ang1 form dimeric structures. COMP-Ang1 shows pentameric structures. (G) Oligomeric statuses of COMP-Ang1, CA1-1 and CA1-3 were analyzed using a size-exclusion chromatography. The peaks for COMP-Ang1, CA1-1 and CA1-3 correspond to pentamer (~175 kDa), trimer (~105 kDa) and dimer (~70 kDa), respectively. Arrowheads indicate the molecular masses of standards.
