Confirmation Of The Interaction Between Angiogenin And ADAM33

Angiogenin(ANG) can strongly stimulate blood vessel formation and play an important role in tumorigenesis. Data showed that ANG induces a wide range of cellular responses, including cell migration, invasion, proliferation, and formation of tubular structure. However, the underlying mechanism of ANG-induced angiogenesis remains elusive.Since protein-protein interactions are essential in every aspect of cellular activities, we reason that protein-protein interactions should be critical during the biological processes induced by angiogenin. Therefore, our laboratory has interest in exploring its potential interacting molecules. In our previous study, we have found several candidates from human heart cDNA library through yeast two-hybrid screening, and in which one important protein is a disintegrin and metalloproteinase 33 (ADAM33). The purpose of this study was to identify the interaction between angiogenin and ADAM33 through pull down co-immunoprecipitation(Co-IP) and fluorescence resonance energy transfer (FRET) methods. First, we ampilified ADAM33 fragment gene from plasmid pPC86-ADAM33, and subcloned it into theproeukaryotic expression vector pET28a and in vitro expression vector pCMVTnT to give plasmids pET28a-ADAM33 and pCMVTnT-ADAM33. We then employed E. coli BL21(DE3) and the Promega TNT? Quick Coupled Transcription/Translation Systems to produce [His]6 tagged and ~35[S] Met labeled ADAM33 protein, respectively. Data showed that ANG could be pulled down by [His]6-ADAM33 and ADAM33 could be co-precipitated by anti-ANG antibody, revealing that ADAM33 binds to ANG in vitro. We also constructed the plasmids of pECFP-ADAM33 and pEYFP-ANG, co-transfected both plasmids into COS-7 cells, and confirmed the interaction in vivo using FRET approach. Since ADAM33 was involved in the processes of cell adhesion and signal conduction, we proposed that ANG may involve in cell migration, invasion, proliferation, and formation of tubular structure via the interaction with ADAM33 and then induce angiogenesis. The interaction between these two proteins may provide a new interfering target for antiangiogenesis and anti-tumor therapy.