Figure 5
N-glycosylation of dimeric CMP-Ang1 is critical for protein activity.
(A) Schematic diagram of N-glycosylation mutants of CA1-3 and COMP-Ang1 (CA1-Q and COMP-Q, respectively). For CA1-Q, the N-glycosylation site, asparagine (N) at 106 residue of CA1-3 is substituted to glutamine (Q) to inhibit N-glycosylation. Similarly, for COMP-Q, the N-glycosylation site at 109 residue of COMP-Ang1 is substituted to glutamine (Q). (B) CA1-3 and CA1-Q were separated by SDS-PAGE under non-reduced (NR) and reduced (R) conditions and immunoblotted with anti-Ang1 antibody. Molecular masses (kDa) are indicated on the left. (C) In vitro binding assay of CA1-3, CA1-Q, COMP-Ang1 and COMP-Q to Tie2. (D) HUVECs were starved and then stimulated with 500 ng/ml of indicated proteins for 15 min and the phosphorylation of Tie2 was detected by immunoprecipitation and immunoblotting. (E) Tie2 phosphorylation after treatment with either control buffer, CA1-3, or CA1-3 and PNGase F. (F) SDS-PAGE analysis of COMP-Ang1 and COMP-Q. (G) Tie2 and Akt phosphorylation activity of COMP-Ang1 and COMP-Q.
