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

Demineralized bone matrix (DBM) is a complex mixture of osteoinductive bone morphogenetic proteins (BMPs), as well as BMP-binding proteins that regulate BMP bioactivity and localization. Our aim was to use modern proteomic methods to identify additional BMP-binding proteins in DBM, with initial emphasis on the most abundant. Relatively large, water-soluble noncollagenous proteins (NCPs) were preferentially extracted from DBM with alkalinized urea. The insoluble residue, which contained the BMP activity, was extracted with GuHCl/CaCl2, dialyzed versus citrate, defatted, resuspended in GuHCl, dialyzed sequentially against Triton X-100 and water, pelleted, and lyophilized. The proteins in this pellet were fractionated by hydroxyapatite affinity chromatography. Proteins that copurified with BMP bioactivity were separated by SDS-PAGE. Distinct bands were excised, and the proteins in them were reduced and alkylated, digested with trypsin, eluted, and subjected to MALDI/ToF MS (matrix-assisted laser-desorption ionization time-of-flight mass spectrometry). Computer-assisted peptide fingerprint analysis of the MS profiles was used to identify C-terminal lysine-6-oxidase; dermatopontin (DPT); histones H2A2, H2A3, and H2B; and trace amounts of gamma-actin. DPT is a 22-kDa, tyrosine-rich acidic matrix protein not previously recognized to be among the most abundant small proteins to copurify with BMP bioactivity in DBM. We tested the effects of DPT on BMP-2 stimulation of alkaline phosphatase (ALP) activity in C2C12 cells. BMP-2 stimulated ALP activity in C2C12 cells by 6.2-fold above basal levels. DPT alone had no effect on ALP activity in C2C12 cells. When added with BMP-2, DPT blocked 40% of the stimulatory effect of BMP-2 on ALP activity in C2C12 cells. DPT is an abundant protein in DBM, and it can inhibit the stimulatory effects of BMP-2 on ALP activity in C2C12 cells.

Bone is subjected to a variety of physiological, as well as cell‐deforming biomechanical stresses, including hydrostatic compression and fluid flow. However, little is known about the molecular mechanisms that protect bone cells from mechanical, ischemic, or oxidative damage. Crystallins are 20 kD heat shock proteins that function as molecular chaperones. We tested the hypothesis that alpha B‐crystallin (αB‐crystallin), the most widely expressed vertebrate crystallin, is present in bone and osteoblast‐like cells. Noncollagenous proteins (NCPs) were extracted from human demineralized bone matrix with 4 M guanidine HCl containing 0.5 M CaCl2 and protease inhibitors, defatted, dialyzed against 0.2% (v/v) Triton X‐100 in 100 mM Tris‐HCI (pH 7.2) and water, centrifuged, and lyophilized. The NCPs were separated by 2D IEF/SDS‐PAGE. The two most abundant 20 kD spots, with apparent pIs of 7.85 and 7.42 in urea gels, were excised, subjected to matrix‐assisted laser desorption ionization/time‐of‐flight mass spectrometry, and identified as αB‐crystallins. Indirect immunofluorescence localized αB‐crystallin to the interphase nucleus, cytoskeleton and cytoplasm of proliferating MC3T3‐El mouse osteoblast‐like cells, as well as the cytoskeleton and cytoplasm of confluent cells. In conclusion, αB‐crystallin is present in bone and osteoblast‐like cells. We hypothesize that αB‐crystallin may play a role in protecting the osteoblast cytoskeleton from mechanical stress and may be important in modulating nuclear or cellular functions, such as transcription or apoptosis, as observed in other tissues. Published by Elsevier Science Ltd. On behalf of Orthopaedic Research Society.

Secreted phosphoprotein-24 kDa (spp24) is an extracellular matrix protein first cloned from bone. Bovine spp24 is transcribed as a 203 amino acid residue protein that undergoes cleavage of a secretory peptide to form the mature protein (spp24, residues 24 to 203). While not osteogenic itself, spp24 is degraded to a pro-osteogenic protein, spp18.5, in bone. Both spp18.5 and spp24 contain a cyclic TRH1 (TGF-beta receptor II homology-1) domain similar to that found in the receptor itself and in fetuin. A synthetic peptide corresponding to the TRH1 domain of spp18.5 and spp24 specifically binds BMP-2 and enhances the rate and magnitude of BMP-2-induced ectopic bone formation in vivo. The parental protein, spp24, exhibits a high affinity for bone and mineral complexes, but its abundance there is low, suggesting that it is rapidly degraded. The availability of recombinant spp24 and its degradation products would facilitate the elucidation of their structure:function relationships. We describe here the expression of His(6)-tagged bovine spp24 (residues 24 to 203) in E. coli, its purification by high-resolution IMAC (immobilized metal affinity chromatography), and the characterization of the full-length recombinant 21.5 kDa protein and its two major 16 kDa and 14.5 kDa degradation products (spp24, residues 24 to 157, and spp24, residues 24 to 143) by mass spectroscopy. The recombinant spp24 protein was resistant to proteolysis by MC3T3-E1 osteoblastic cell extracts in the absence of calcium; however, in the presence of 4 mM Ca, it can undergo essentially complete proteolysis to small peptides, bypassing the 16 kDa and 14.5 kDa intermediates. This confirms the proteolytic susceptibility of spp24. It also suggests that the levels of spp24 in bone may be regulated, in part, by calcium-dependent proteolysis mediated by osteoblastic cells.