A Canstatin-Derived Peptide Provides Insight into the Role of Capillary Morphogenesis Gene 2 in Angiogenic Regulation and Matrix Uptake

Abstract

Capillary Morphogenesis Gene 2 protein (CMG2) is a transmembrane, integrin-like receptor and the primary receptor for the anthrax toxin. In addition to its role as an anthrax toxin receptor, CMG2 has been repeatedly shown to play a role in angiogenic processes. However, the molecular mechanism mediating observed CMG2-related angiogenic effects has not been fully elucidated. Previous studies have found that CMG2 binds type IV collagen (Col-IV), a key component of the vascular basement membrane, as well as other ECM proteins. Currently, no link has been made between these CMG2-ECM interactions and angiogenesis; however, ECM fragments are known to play a role in regulating angiogenesis. Here, we further characterize the CMG2-Col-IV interaction and explore the effect of this interaction on angiogenesis. Using a peptide array, we observed that CMG2 preferentially binds peptide fragments of the NC1 (non-collagenous domain 1) domains of Col-IV. These domains are also known as the fragments arresten (from the α1 chain) and canstatin (from the α2 chain) and have documented antiangiogenic properties. A second peptide array was probed to map a putative binding epitope. A top hit from the initial array, a canstatin-derived peptide, binds to the CMG2 ligand-binding von Willebrand factor A (vWA) domain with sub-micromolar affinity (peptide S16, Kd = 400 ± 200 nM). This peptide competes with anthrax protective antigen (PA) for CMG2 binding, and does not bind CMG2 in the presence of EDTA. Together these data suggest that, like PA, S16 interacts with CMG2 at the metal-ion 17 dependent adhesion site (MIDAS) of its vWA domain. We demonstrate that CMG2 specifically 18 mediates endocytic uptake of S16, since CMG2-/-endothelial cells show markedly reduced S16 uptake, 19 without reducing total endocytosis. Furthermore, we show that S16 reduces endothelial migration but 20 not cell proliferation. Taken together, our data demonstrate that a Col IV-derived anti-angiogenic 21 peptide acts via CMG2, suggesting a possible link between CMG2-Col IV interactions and 22 angiogenesis. 23

Collagen IV-Related Diseases and Therapies

“…This includes the anti-angiogenic peptide arresten and canstatin, generated by proteolytic cleavage of NC1 domains of α1(IV) and α2(IV), respectively [77]. These molecules can bind integrin α 1 β 1 , α v β 3 and α v β 5 , and transmembrane integrin like receptor CMG2 (capillary morphogenesis gene 2) and may alter angiogenesis by affecting MAPK signalling [77,280]. Therefore, the reduced levels of these peptides due to lower levels of collagen IV in the ECM of Col4a1 mutant mice [180,184,206,221,270], represents one possible mechanism by which these mutations affect angiogenesis.…”