Confirmation And Functional Exploration Of The Interaction Between Angiogenin And Fibulin Protein Family

Angiogenin is one of the angiogenic proteins identified solely based on its strong ability to induce blood vessel formation, and was originally isolated from the conditioned medium of cultured HT-29 human colon adenocarcinoma cells. Angiogenin is a single-chain alkaline protein involved in tumorigenesis with a molecular weight of about 14kDa and was reported to be a key player in tumor angiogenisis and tumorigenesis. Data reveal that Angiogenin can promote cell proliferation, invasiveness, and adhesion. However, the molecular mechanism of Angiogenin-induced angiogenisis remains elusive.Protein-protein interaction is crucial in directing and regulating biological processes. Therefore, we reason that determining angiogenin-interacting proteins is of priority in the study of mechanism of action of angiogenin. For this purpose, yeast two-hybrid technique was employed to screen angiogenin's interacting molecules, and several candidates was found from human heart cDNA library, including Fibulin-1, Fibulin-2, EGF-containing Fibulin-like Extracellular Matrix Protein 1 (EFEMP1, also known as Fibulin-3), EGF-containing Fibulin-like Extracellular Matrix Protein 2 (EFEMP2, also known as Fibulin-4). The interaction between Fibulin-1,-2,-3,-4 and angiogenin was late confirmed by GST-pull down.Fibulin-1,-2,-3,-4 is highly identical to Fibulin-5 in amino acid sequence. On the other hand, all members of Fibulin protein family have the simility in majority of their domains. Accordingly, it is likely that Fibulin-5 may interact with angiogenin as well. Later the interaction was confirmed through yeast two-hybrid, GST-pull down and co-immunoprecipitation. This new finding could help us to further understand the molecular mechanisms of ANG.Fibulin-5 is an extracellular matrix glycoprotein expressed in elastin-rich tissues, mediates these matrix structures by its ability to interact with many extracellular proteins. Recent studies have revealed that Fibulin-5 is an endogenous angiogenesis inhibitor, as well as it support adhesion of cells. Fibulin-5 was also shown to reduce migration of endothelial cells toward fibronectin. Consequently, functional exploration of the interaction between Fibulin-5 and ANG was done based on the model of Fibulin-5 inhibite the migration and adhesion of endothelial cells to fibronectin. Nevertheless, no significant change was observed to effect of ANG addition in the model.In a word, the results from yeast two-hybrid and GST pull down revealed that Fibulin-1,-2,-3,-4,-5 and angiogenin did interact with each other in vitro. A quite conserved sequence was further identified among the Fibulin protein family. However, it was not included in other matrix proteins. Further functional exploration of the interaction was also made with models of cell migration and adhesion. However, no significant change was observed after Fibulin-5 interact with ANG The next step should focus on two sides:firstly, to find the interaction sites between angiogenin and Fibulin protein family; Secondly, to explore the effect of the interaction between angiogenin and Fibulin protein family.