Recombinant Expression, Isolation, and Proteolysis of Extracellular Matrix-Secreted Phosphoprotein-24 kDa

Abstract

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.

Full‐length bovine spp24 [spp24 (24‐203)] inhibits BMP‐2 induced bone formation

“…We can speculate, but cannot demonstrate, that the 24 kDa protein that Urist and colleagues observed was fl spp24 and that some of the smaller proteins were spp24 degradation products similar to those identified in E. coli (e.g., spp14.5 and spp16). 5 It should be noted that phosphorylation and glycosylation of eukaryotic proteins in DBM may give rise to spp24 degradation products with higher M r s than those identified in E. coli extracts, where these posttranslational processes do not occur. In any case, the 34 kDa, 22 kDa, 17.5 kDa, 17 kDa, and 14 kDa proteins did not form bone alone or in combination, but the semipurified 18.5 kDa (variously reported as 18 kDa to 19 kDa or 18.5 kDa) protein did form bone.…”

“…5 Effect of spp24 on BMP-2 Induced Ectopic Bone Formation The effect of Met(His) 6 -spp24 (24-203) on BMP-2 induced ectopic bone formation is shown in Table 1. Spp24 alone did not induce bone formation (data not shown).…”

“…An intermediate dose had an intermediate effect. The relative and absolute amounts of spp24 and rhBMP-2 tested were selected based on the amounts previously shown to demonstrate a stimulatory effect of cBBP (cyclic BMP binding peptide) on rhBMP-2-stimulated ectopic bone formation in the mouse, 5 where spp24 contains approximately 10-fold more amino acids than BBP.…”

“…3 To date, the osteogenic properties of the full-length (fl) parental protein (spp24) and its secretory isoform [e.g., bovine spp24 (24-203)] have not been determined. We have generated fulllength recombinant bovine spp24 and its stable Met (His) 6 -tagged secretory isoform 5 and report the inhibitory effects of these recombinant spp24 isoforms on BMP-2 stimulated bone formation in two distinct models; in female transgenic mice overexpressing fl spp24 under the control of the osteocalcin promoter and in the ectopic bone-forming bioassay in male mice treated with BMP-2 and bovine Met(His) 6 -spp24 (24-203).…”

“…3 Like fetuin, spp24 and its degradation products (e.g., spp18.5) 3 may bind members of the TGF-b family of cytokines because they both possess an internal TGF-b receptor II homology-1 (TRH1) domain. 4,5 The cyclic, synthetic 19 amino acid residue peptide derived from the sequence of the TRH1 domain that is shared by spp24 and spp18.5 binds rhBMP-2 and enhances BMP-2 mediated bone formation. 3 This TRH1 domain of spp24 and spp18.5 corresponds to the second internally disulfide-bonded domain of bovine spp24 (residues 110-128).…”