HUMAN BONE MORPHOGENETIC PROTEIN-2 CONTAINS A HEPARIN-BINDING SITE WHICH MODIFIES ITS BIOLOGICAL-ACTIVITY

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 M(r) 26 000 was prepared with a purity greater 98%. The rec ombinant BMP-2 was functionally active as demonstrated by the inductio n of alkaline phosphatase activity in the C3H10T1/2 fibroblast cell li ne (EC(50) of 70 nM) and proteoglycan synthesis in embryonic chicken l imb 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 o bservations suggested that the N-termini reduce the specific activity of BMP-2, probably by interacting with heparinic sites in the extracel lular matrix. This conclusion was supported by a variant EHBMP-2, wher e the N-terminal residues 1-12 of BMP-2 had been substituted by a dumm y 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 phys ical interaction between BMP-2 and heparin could be seen in biosensor experiments, where BMP-2 bound to immobilized heparin with a dissociat ion constant, K-d, 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 n ot obligatory for receptor activation but modulate its biological acti vity.