HEPARIN BINDING DOMAINS IN BMP6: STUDY ON THE SYNTHETIC PEPTIDES AND THE RECOMBINANT PROTEIN

Hepcidin is the master regulator of iron homeostasis and its expression is mainly attributed to BMP6 protein which triggers the phosphorylation of SMAD1/5/8. Our previous results have shown that heparins strongly repress the BMP6/SMAD signaling resulting in a decrease of hepcidin expression. This finding suggested that not only heparin, but also endogenous heparan sulfates (HSs), ubiquitous heparin-like polysaccharides exposed on cells surface, may participate in the BMP6/SMAD pathway by facilitating the binding of BMP6 to its receptors. How BMP6 binds heparin is not clear, but different Heparin Binding Domains (HBDs) have been already characterized in BMP2 and BMP4 isoforms as lysine and arginine-rich regions.The aim of this work is to define the heparin/HS binding domains of BMP6 to clarify the mechanism of action of heparin in hepcidin inhibition and to verify if HSs are involved in hepcidin expression.BMP6 presents three basic regions rich in Arg and Lys, that are putative HBDs. The first is located at the unstructured N-terminus (HBD1), a second in central portion (HBD2) and the third at C-Terminus (HBD3). Synthetic 15-residue peptides representing the three domains were produced in fusion with biotin. They were used to track the energetics of binding of each HBD peptide to heparin adsorbed onto microcantilever biosensors. We found that N-terminus domain (HBD1) showed higher heparin affinity than the others and that it was dose dependent. Then we assayed their binding to heparan sulfates (HS) using monolayers of cells that express or not membrane HS. The results showed that also with this approach the N-terminus HBD peptide bound with higher affinity the surface of cells expressing HS, and that this binding was reduced in the presence of heparin. These findings prompted us to produce the recombinant mature form of human BMP6 in a prokaryotic system in order to study its heparin and HS binding activity in a completely folded protein. Human mature BMP6 with his-tag was cloned into the pASK-IBA43plus vector and expressed in E. Coli RosettaTM (DE3) host strain, as insoluble inclusion bodies. BMP6 was solubilized and then purified obtaining a highly pure denatured BMP6 on its monomeric form. Finally, we used an in vitro oxidative renaturation protocol to obtain the dimeric functional form of BMP6.We found that the produced dimeric BMP6 was able to bind heparin and that it binds more effectively to cells expressing HS. The biological activity of BMP6 seems to require the glycosylation, thus we cloned the mature human and mouse BMP6 in eucariotic vectors. We transfected the Hek293 with these plasmids and treated HepG2 cells with their supernatant. The transcription of hepcidin was induced by the BMP6 transfected Hek supernatant supporting the expression and secretion of BMP6. Mutagenesis of arginine in these prokaryotic and eukaryotic clones are in progress to identify the major HBD in the BMP6; the results will be present.