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 Esclzerichia coli, and after renaturation a dimeric BMP-2 protein of M , 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 C3HlOT1/2 fibroblast cell line (EC,, of 70 nM) and proteoglycan synthesis in embryonic chicken limb bud cells (EC,,(15)(16)(17)(18)(19)(20). 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-termini 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,, 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.Keywords: human bone morphogenetic protein ; bacterial expression ; heparin binding; limb bud; proteoglycan synthesis ; variant bone morphogenetic protein 2.Bone morphogenetic protein 2 (BMP-2) is one of the proteins originally identified as factors extractable from bone and triggering ectopic bone formation at non-skeletal sites in vivo (Urist, 1965;Sampath and Reddi, 1981;Urist et al., 1982; for review see Wozney, 1992;Rosen and Thies, 1992;Reddi, 1994). After cloning and expression in Chinese hamster ovary (CHO) cells Wang et al., 1990) purified BMP-2 has been established to induce de novo bone formation and bone repair in adult animals (see, for example, Toriumi et al., 1991), and chondrogenic and osteogenic differentiation in various cellular systems in vitro. In addition, BMP-2 regulates, similarly to its nearest homologue BMP-4, diverse fundamental processes during embryonic development (see, for example, Kingsley, 1994). The mature BMP-2 protein is closely related to the Drosophila dpp protein (Padgett et al., 1993) and the Xenopus BMP-2 (Plessow et al., 1991). The human BMP-2 can substitute the Drosophila homologue dpp during dorsalventral patterning, and, vice versa, a recombinant dpp protein induces ectopic bone formation in rats (Padgett et al., 1993;Sampath et al., 1993). BMP-2 and the other BMP have great potential for medical therapeutic applications, in particular beCorrespondence to W. Sebald, Physiologisc...

Efficacy of interspinous process lumbar fusion with recombinant human bone morphogenetic protein-2 delivered with a synthetic polymer and β-tricalcium phosphate in a rabbit model

“…One possible solution to this problem is high-efficiency, lowcost production of monomer rhBMPs in BMP-gene-transfected E. coli and conversion of the monomer rhBMPs to dimeric rhBMP using ex vivo biochemical processing. The ectopic induction of new bone formation by dimeric E-BMP-2 has already been reported elsewhere [22,32]. Recent studies in our laboratory found that characteristics of E-BMP-2 are satisfactory for clinical use to promote bone healing in place of the currently available BMP-2 produced by BMP-2-expressing CHO cells [12,43].…”

“…Dimeric E-BMP-2 was purified using several steps of chromatography. Details of the procedures for dimerization of cytokines have already been reported [12,32,42,43].…”

Sustained release and osteogenic potential of heparan sulfate-doped fibrin glue scaffolds within a rat cranial model

“…HS has been implicated in facilitating and stabilising the interaction of FGFs with their receptors. The more complicated interaction with BMPs have been demonstrated to include a stabilizing and storage role in the extracellular space (Ruppert et al 1996), maintaining the growth factor spatially away from it's receptor, thus limiting signaling potential (Takeda et al 2003) and preventing diffusion from a site of injury (Depprich et al 2005), however these interactions act in unison to modulate the activity and it has not yet been shown that HS interacts directly with the BMP2/receptor complex. Nontheless, a synergistic interaction seems apparent between HS and BMP2, although the exact mechanism driving this is still unknown.…”

Proteoglycans: Master regulators of molecular encounter?

“…Perhaps, but a more likely explanation is that the biological significance of the interactions of these growth factors with HS is simply not captured by the kinds of in vitro assays that are typically used to study growth factor signaling. Support for this idea has come from genetic analysis of PG core protein mutants in vivo, in which HSPGs appear to regulate signaling by growth factors, such as members of the BMP family, and components of the insulin-like growth factor pathway (Pilia et al, 1996;Jackson et al, 1997), which in vitro assays have suggested are not HS-dependent (e.g., Ruppert et al, 1996).…”