Screening And Confirmation Of Angiogenin-Interacting Proteins

Angiogenin is one of the angiogenic proteins to be identified solely based on its ability to induce blood vessel formation. It is the only angiogenic protein that is a ribonuclease and is also the only member of the ribonuclease superfamily that is angiogenic. Angiogenin is a very basic, single-chain protein with molecular weight about 14 kD. It shares 33% sequence identity with bovine pancreatic RNaseA and the crystal structures of both proteins are very similar. The ribonucleolytic activity of angiogenin is about ~106-fold less than that of pancreatic ribonuclease. However, this seemingly weak but characteristic ribonucleolytic activity is necessary for angiogenin to induce angiogenesis.Besides an absolute requirement of enzymatic activity, angiogenin must also interact with endothelial and smooth muscle cells to induce a wide range of cellular responses. It is known that angiogenin activates both cells through binding with membrance proteins and nuclear translocation, thus induces cell migration, invasion, proliferation, and formation of tubular structures. Data show that aniogenin binds to actin or a putative 170 kDa protein under different cell density, activates ERK1/2 inhuman umbilical vein endothelial cells or SAPK/JNK in human umbilical artery smooth muscle cells. Meanwhile, exogenous angiogenin is translocated to the nucleus through an unknown mechanism and binds to nucleolar DNA, hence enhances rRNA transcription. However, these lines of evidence are fragmentary and their inter-connections remain to be established.We reason that protein interactions should be critical during these biological processes. Therefore, yeast two-hybrid screening was employed to identify its potential interacting molecules. We found several partners which relate with ECM digestion, cell motility and cell adhesion, such as laminin-2 chain, fibulinl, 2 and EGF-containing fibulin-like extracellular matrix protein 1 and 2(EFEMP1 and EFEMP 2), fibronectin, latent transforming growth factor beta binding protein 3 and 4(LTBP-3,-4), a disintegrin and metalloproteinase domain 33, a-actinin-2, interferon alpha-inducible protein 27(IFI27/P27), granulin, and so on. The interaction between angiogenin and a-actinin-2 was confirmed through pull down, co-immunoprecipitation and FRET analysis.The protein interactions we found here imply the involvement of angiogenin in cell migration and adhesion during the process of angiogenesis. We think each interaction might be a novel interference target for anti-angiogenesis and anti-tumor therapy.