TRACP as an Osteopontin Phosphatase

Abstract

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.

Secretion of tartrate‐resistant acid phosphatase by osteoclasts correlates with resorptive behavior

“…One proposal is that the enzyme generates free radicals that help to dissolve collagen fragments released from bone by cathepsin K [Halleen et al, 1999]. Alternative roles include actions as an osteopontin phosphatase [Andersson et al, 2003], in iron transport [Saunders et al, 1985], and as a growth factor [Bazer et al, 1991;Sheu et al, 2003]. Whatever its function, it has long been known that TRAP levels are increased in the serum of individuals who have a high rate of bone resorption [Stepan et al, 1989;Halleen et al, 2002].…”

Proteolytic Excision of a Repressive Loop Domain in Tartrate-resistant Acid Phosphatase by Cathepsin K in Osteoclasts

“…After extraction, the RNA was treated with 5 units of DNase I (Invitrogen) for 30 min at room temperature. The reverse transcriptase reaction was performed with 5 g of total RNA and 200 units of Superscript TM II reverse transcriptase (Invitrogen) at 42°C, using (dT) [12][13][14][15][16][17][18] as primer (Invitrogen), according to the protocol of the manufacturer. In negative controls, reverse transcriptase was excluded from the reaction mixture.…”

“…Different functions have been suggested for TRAP, e.g. as an osteopontin phosphatase (13)(14)(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.…”

Expression and secretion of the novel adipokine tartrate-resistant acid phosphatase from adipose tissues of obese and lean women

“…5,6 In vivo, TRAP 5b has previously been proposed to function as a generator of reactive oxygen species and as a protein phosphatase regulating the bioactivity of osteopontin. [7][8][9] Recently, we showed that TRAP 5a, but not TRAP 5b, induces obesity in mice and is increased in the adipose tissue of obese humans. 10 Furthermore, TRAP 5a, but not TRAP 5b, induces both proliferation and differentiation of mouse and human adipocyte precursors in vitro.…”