Tartrate-resistant acid phosphatase (TRAP) is a metallophosphoesterase participating in osteoclast-mediated bone turnover. Activation of TRAP is associated with the redox state of the di-iron metal center as well as with limited proteolytic cleavage in an exposed loop domain. The cysteine proteinases cathepsin B, L, K, and S as well as the matrix metalloproteinase-2, -9, -13, and -14 are expressed by osteoclasts and/or other bone cells and have been implicated in the turnover of bone and cartilage. To identify proteases that could act as activators of TRAP in bone, we report here that cathepsins K and L, in contrast to the matrix metalloproteinases, efficiently cleaved and activated recombinant TRAP in vitro. Activation of TRAP by cathepsin K/L was because of increases in catalytic activity, substrate affinity, and sensitivity to reductants. Processing by cathepsin K occurred sequentially by an initial excision of the loop peptide Gly Tartrate-resistant acid phosphatase (TRAP), 1 also known as type 5 acid phosphatase (EC 3.1.3.2) or uteroferrin, belongs to the purple acid phosphatase (PAP) subfamily of the non-heme dinuclear metallophosphatases (1-3). The metals of the catalytic center of all PAPs consist of a common ferric ion and a divalent metal cation in an active enzyme, where mammalian PAPs characteristically contain a redox-active iron in the M(II) site (4 -6).The TRAP enzyme is abundantly expressed by bone-resorbing cells, osteoclasts, and certain subpopulations of monocytes/ macrophages and dendritic cells (7-10). The precise role of osteoclastic TRAP is not fully understood, but studies on TRAP knock-out mice showed disturbed endochondral ossification with decreased resorptive activity of osteoclasts (11, 12), whereas overexpression of TRAP was associated with increased bone turnover (10). Different functions have been suggested for TRAP, e.g. as an osteopontin phosphatase (13-15), generation of reactive oxygen species (16 -19), iron transport (20 -24), and as a growth/differentiation factor for hematopoietic (25) and osteoblastic (26) cells.Mammalian PAPs are synthesized as 35-37-kDa monomers but are commonly isolated from tissues as proteolytically cleaved two-subunit forms consisting of a 23-kDa N-terminal domain disulfide-linked to a 16-kDa C-terminal domain. The monomeric form exhibits properties of a proenzyme with low phosphatase activity that is converted to a high activity, twosubunit form upon proteolytic cleavage in the intervening loop domain with either serine proteases, e.g. trypsin or chymotrypsin (27), or members of the cathepsin family (28,29). Mutagenesis studies suggested that proteolysis removes or alters repressive interactions between loop amino acids and active site residues because replacement of Asp 146 of the exposed loop domain with Ala resulted in activation of unproteolyzed TRAP (30).Several lines of evidence indicate a role for cathepsin K in bone resorption. Cathepsin K is highly expressed in osteoclasts near the ruffled border membrane and has been shown to participate i...

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Phosphorylated osteopontin (OPN) inhibits hydroxyapatite crystal formation and growth, and bone alkaline phosphatase (BALP) promotes extracellular mineralization via the release of inorganic phosphate from the mineralization inhibitor inorganic pyrophosphate (PPi). Tartrate-resistant acid phosphatase (TRAP), produced by osteoclasts, osteoblasts, and osteocytes, exhibits potent phosphatase activity towards OPN; however, its potential capacity as a regulator of mineralization has not previously been addressed. We compared the efficiency of BALP and TRAP towards the endogenous substrates for BALP, i.e., PPi and pyridoxal 5′-phosphate (PLP), and their impact on mineralization in vitro via dephosphorylation of bovine milk OPN. TRAP showed higher phosphatase activity towards phosphorylated OPN and PPi compared to BALP, whereas the activity of TRAP and BALP towards PLP was comparable. Bovine milk OPN could be completely dephosphorylated by TRAP, liberating all its 28 phosphates, whereas BALP dephosphorylated at most 10 phosphates. OPN, dephosphorylated by either BALP or TRAP, showed a partially or completely attenuated phosphorylation-dependent inhibitory capacity, respectively, compared to native OPN on the formation of mineralized nodules. Thus, there are phosphorylations in OPN important for inhibition of mineralization that are removed by TRAP but not by BALP. In conclusion, our data indicate that both BALP and TRAP can alleviate the inhibitory effect of OPN on mineralization, suggesting a potential role for TRAP in skeletal mineralization. Further studies are warranted to explore the possible physiological relevance of TRAP in bone mineralization.

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TRACP is synthesized as a latent proenzyme requiring proteolytic processing to attain maximal phosphatase activity. Excision of an exposed loop domain abolishes the interaction between the loop residue Asp146 and a ligand to the redox-sensitive iron of the active site, most likely Asn91, providing a mechanism for the enzyme repression. Both cathepsin K and L efficiently cleave in the loop domain and activate the latent enzyme, and we propose that cathepsin K acts as a physiological activator of TRACP in osteoclasts, whereas cathepsin L might fulfill a similar role in different types of macrophages. Considering the rather broad substrate specificity of TRACP, a tight regulation of its activity in the cell appears warranted. Besides proteolytic cleavage, the enzyme should need a specific local environment with a slightly acidic pH and reducing equivalents to keep the enzyme fully active. Cellular subcompartments where these required conditions prevail are potential subcellular site ( M AMMALIAN TARTRATE-RESISTANT purple acid phosphatase (TRACP/PAP) is an iron-containing, cationic glycoprotein with a molecular weight of around 35 kDa. Although the enzyme is translated as a single polypeptide, the protein isolated from different tissues commonly exist as a disulfide-linked two-subunit structure with an N-terminal fragment of 20 -23 kDa joined to the 16-to 17-kDa C-terminal part.(1) It now appears clear that the two-subunit form is generated by proteolytic excision of a loop domain protruding between helix 5 and 6 at the surface of the molecule, and that its removal leads to a significant increase in the enzymatic activity of the molecule at pH Ͼ5. Besides serine proteases (e.g., trypsin and chymotrypsin), several cysteine proteinases of the cathepsin family have been shown to cleave in the loop region, with the latter proteases leading to markedly higher enzyme activation. (3,4) We have focused our interest on the cysteine proteinases as potential physiological regulators of TRACP enzyme activation in osteoclasts and macrophages, primarily because members of this family have been implicated in resorption of bone as well as in lysosomal protein degradation. Interestingly, both cathepsin K and L are highly efficient activators of the latent monomeric TRACP in vitro.(5,6) Cathepsins B, H, and S were much less effective, and MMP 2 and 9 were completely ineffective.(5) Of the two cathepsins, only cathepsin K is expressed to a significant extent in osteoclasts, while cathepsin L predominate in other types of macrophages.(7) That cathepsin K is involved in processing of TRACP in bone was shown by use of cathepsin K knockout mice, where an increased content of monomeric TRACP was recovered from their bones.(5) Thus, members of the cathepsin family can process TRACP to a more active enzyme, and we propose that cathepsin K fulfills this role in osteoclasts while in other TRACP expressing cells of the macrophage lineage cleavage can be accomplished by cathepsin L.

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