A Designed Angiopoietin-1 Variant, Dimeric CMP-Ang1 Activates Tie2 and Stimulates Angiogenesis and Vascular Stabilization in N-glycan Dependent Manner.

Nuri Oh,Young Suk Seo,Soo Jin Kim,Kangsan Kim,Jung-Eun Lee,Intae Park,Ho Min Kim,Gou Young Koh,Hyun Joo An

doi:10.1038/srep15291

Abstract

Angiopoietin-1 (Ang1), a potential growth factor for therapeutic angiogenesis and vascular stabilization, is known to specifically cluster and activate Tie2 in high oligomeric forms, which is a unique and essential process in this ligand-receptor interaction. However, highly oligomeric native Ang1 and Ang1 variants are difficult to produce, purify, and store in a stable and active form. To overcome these limitations, we developed a simple and active dimeric CMP-Ang1 by replacing the N-terminal of native Ang1 with the coiled-coil domain of cartilage matrix protein (CMP) bearing mutations in its cysteine residues. This dimeric CMP-Ang1 effectively increased the migration, survival, and tube formation of endothelial cells via Tie2 activation. Furthermore, dimeric CMP-Ang1 induced angiogenesis and suppressed vascular leakage in vivo. Despite its dimeric structure, the potencies of such Tie2-activation-induced effects were comparable to those of a previously engineered protein, COMP-Ang1. We also revealed that these effects of dimeric CMP-Ang1 were affected by specified N-glycosylation in its fibrinogen-like domain. Taken together, our results indicate that dimeric CMP-Ang1 is capable of activating Tie2 and stimulating angiogenesis in N-glycan dependent manner.

Highlights

Angiopoietin-1 (Ang1) is a secretory ligand of tyrosine kinase with Immunoglobulin and epidermal growth factor homology domain 2 (Tie2) that is mainly expressed by vascular endothelial cells and hematopoietic cells[1,2,3]
Because cysteine residues are essential for the disulfide bonds, and these disulfide bonds have critical roles in maintaining oligomeric structures, we sought to test how the cysteine residues in the linker of Ang[1] and in the coiled-coil domain of cartilage matrix protein (CMP) affect the oligomeric status of CMP-Ang[1] by replacing them with alanine with different combinations (Fig. 1C), and the newly created CMP-Ang[1] variants were generated and analyzed using the same methods
SDS-PAGE and TEM analysis showed that Fc-Ang[1], GCN-Ang[1], and cartilage oligomeric matrix protein (COMP)-Ang[1] mainly formed dimers, dimers, and pentamers, respectively, which is consistent with previous findings[9,13,15] (Fig. 1E,F)

Summary

Introduction

Angiopoietin-1 (Ang1) is a secretory ligand of tyrosine kinase with Immunoglobulin and epidermal growth factor homology domain 2 (Tie2) that is mainly expressed by vascular endothelial cells and hematopoietic cells[1,2,3]. The oligomeric status of Ang[1] was readily accepted as a critical determinant for the activation of Tie[2] Based on this unique interaction between secretory ligand and membrane receptor, our group previously generated a soluble, stable, and potent pentameric Ang[1] variant by replacing the N-terminal domain of Ang[1] (245 amino acids) with the short, pentameric coiled-coil domain (45 amino acids) of the cartilage oligomeric matrix protein (COMP), and named it as “COMP-Ang1”11. Our findings show that dimeric CMP-Ang[1] is a potent Ang[1] variant that activates Tie[2] receptor and stimulates angiogenesis, and could be developed as a therapeutic protein

Methods

Results

Conclusion