Forty years ago, Marshall Urist described a partially purified extract of demineralized bone matrix which induced the formation of ectopic bone. This substance, bone morphogenetic proteinhon-collagenous protein (BMPINCP), was never purified to honiogeneity but other investigators used similar starting materials to clone a number of recombinant BMPs. Urist recognized that his material probably contained the BMPs which had been cloned by others but always contended that it contained another, more potent, bone inducing material which differed significantly in its physical and chemical properties from the known BMPs. We have used Urist's protocol to isolate a protein that has the chemical and physical properties of Urist's "BMP". It is an 18.5 k D fragment of the bone matrix protein, SPP-24. This fragment contains the cystatin-like domain of SPP-24. We have located a 19 amino acid region which is similar to the TGF-P/BMP-binding region of fetuin, a member of the cystatin family of protease inhibitors. A cyclic peptide, which we call BMP binding peptide (BBP) was generated using this sequence. The peptide avidly bound rhBMP-2 with a K D of 3 x M. When implanted alone in mouse muscle, the peptide frequently induced dystrophic calcification. When implanted with rhBMP-2, the peptide enhanced the osteogenic activity of the recombinant molecule. We hypothesize that Urist's "BMP" was a fragment of SPP-24 which influenced bone induction by binding to bone morphogenetic proteins. BBP may be clinically useful because of its effects on other bone-inducing substances.

The mechanism of the BBP enhanced osteoinductive properties of BMPs involves the binding and retention of the growth factor, resulting in a prolonged exposure of BMP to the desired fusion site. The use of BBP in conjunction with BMPs may prove to provide satisfactory fusion outcomes, while reducing the costs and side effects associated with BMP use.

Secreted phosphoprotein 24 kDa (spp24) is a bone matrix protein. It contains a TGF-b receptor II homology 1 (TRH1) domain. A cyclic, synthetic 19 amino acid peptide (bone morphogenetic protein binding peptide or BBP) based on the sequence of the TRH1 domain enhances BMP-2 induced osteogenesis. Many observations suggest that different size forms of this protein have very different effects (inhibiting or enhancing) on BMP-2 induced osteogenesis. Using the stable recombinant Met(His) 6 -tagged secretory form of full-length (fl) bovine spp24 [Met(His) 6 -spp24 (residues 24-203)] and transgenic (TG) mice expressing fl bovine spp24 (residues 1-203), we have demonstrated that spp24 inhibits BMP-2 induced bone formation. The effects of Met(His) 6 -spp24 (24-203) were determined in the ectopic bone-forming bioassay in male mice. Implantation of 5 mg of BMP-2 stimulated bone formation, assessed densitometrically as bone area and mineral content. When Met(His) 6 -spp24 (24-203) was implanted with BMP-2, it elicited a dose-dependent decrease in BMP-2-medicated ectopic bone formation. When added at a 50-fold excess (w/w), Met(His) 6 -spp24 (24-203) completely ablated the effects of BMP-2, while addition of a 10-fold excess had no effect. Constitutive expression of fl bovine spp24 (1-203) under the control of the osteocalcin promoter in TG female mice reduced femoral and vertebral bone mineral density at 3 months of age and reduced femoral BMD at 8 months of age, but had no effects in male mice, which can exhibit less osteocalcin-promoter driven gene transcription than females. Histomorphometric analysis demonstrated that bone volume and trabecular thickness were lower in TG female mice at 3 months of age than in sex-and age-matched wild type (WT) controls. Thus, fl spp24 and its secretory isoform (Met(His) 6 -spp24 ), which contain a BMP-binding or TRH1 motif, inhibit ectopic bone formation in male mice and adversely affects BMD and histological parameters related to bone mass and formation in female mice expressing the human transgene. Under these conditions, fl spp24 acts as a BMP antagonist in vivo. Keywords: bone formation; spp24; bone morphogenetic protein Secreted phosphoprotein-24 (spp24) is a 24 kDa protein of unknown function first cloned from bovine bone matrix.1 The calculated molecular weight of the fulllength unprocessed precursor [bovine spp24 (residues 1-203)] is 23.1 kDa (Swiss-Prot database; AC #Q27967). The first 23 amino acid residues of bovine spp24 (1-203) constitute a signal peptide that is cleaved when the protein is secreted in its mature form [spp24 (24-203)]. The protein contains two internal disulfide bonds linking residues 86 to 97 and 110 to 128, respectively, and contains internal domains with sequence homology to cystatins (cysteine protease inhibitors) and cathelicidins (protease-sensitive precursors of endogenous antibiotic peptides).1 Recent work demonstrated that an 18.5 kDa protein in the osteogenic fraction that Urist et al.2 isolated from demineralized bone matrix (DBM) by hy...

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Secreted phosphoprotein-24 kDa (spp24) is a bone morphogenetic protein (BMP)-binding protein isolated from bone. It exists in a number of size forms and is hypothesized to function as a BMP latency protein and/or a ''slow release'' mechanism for BMPs involved in bone turnover and repair. We have examined the hypothesis that proteolytic modification of the C-terminus of spp24 affects its BMP-2-binding properties and bioactivity in the BMP-2-stimulated ectopic bone forming bioassay. Three different size forms of recombinant spp24 that correspond to predicted 18.1 kDa, 16.0 kDa, and 14.5 kDa proteolytic products were compared to full-length (fl) spp24. One of these forms (spp18.1) we hypothesize to be the protein which Urist initially, but apparently inaccurately, called ''BMP.'' Only full-length spp24 completely inhibited BMP-2-induced bone formation. The 18.1 kDa truncated isoform of spp24 which we hypothesize to be Urist's protein did not. The inhibitory capacity of the proteins was correlated with their kinetic constants, assessed by surface plasmon resonance. At the highest, inhibitory, dose of spp24 and its derivatives, k d (''stability'') best predicted the extent of ectopic bone formation whereas at the lowest dose, which was not inhibitory, k a (''recognition'') best predicted the extent of ectopic bone formation. We conclude that proteolytic processing of spp24 affects the interaction of this protein with BMP-2 and this affects the function of the protein. ß