Human Bone Morphogenetic Protein 2 Contains a Heparin‐Binding Site which Modifies Its Biological Activity

Abstract

Bone morphogenetic protein 2 (BMP‐2) plays a decisive role during bone regeneration and repair as well as during various stages of embryonal development. A cDNA encoding mature human BMP‐2 could be efficiently expressed in Escherichia coli, and after renaturation a dimeric BMP‐2 protein of Mr 26000 was prepared with a purity greater 98%. The recombinant BMP‐2 was functionally active as demonstrated by the induction of alkaline phosphatase activity in the C3H10T1/2 fibroblast cell line (EC 50 of 70 nM) and proteoglycan synthesis in embryonic chicken limb bud cells (EC 50 of 15–20 nM). A peptide 1–17 representing the N‐terminal basic part of BMP‐2 as well as heparin increased the specific activity of the protein about fivefold in the limb bud assay. These observations suggested that the N‐terminal reduce the specific activity of BMP‐2, probably by interacting with heparinic sites in the extracellular matrix. This conclusion was supported by a variant EHBMP‐2, where the N‐terminal residues 1–12 of BMP‐2 had been substituted by a dummy sequence of equal length and which showed an EC 50 value of around 1 nM which was affected neither by heparin nor by peptide 1–17. A physical interaction between BMP‐2 and heparin could be seen in biosensor experiments, where BMP‐2 bound to immobilized heparin with a dissociation constant, Kd, of approximately 20 nM, whereas the heparin‐binding of variant EHBMP‐2 was negligible. These results identify the basic N‐terminal domains of dimeric BMP‐2 as heparin‐binding sites that are not obligatory for receptor activation but modulate its biological activity.