Regulation of tartrate-resistant purple acid phosphatase by proteolytic processing in rat

Sammanfattning: Purple acid phosphatase (PAP) is an iron-containing binuclear acid metallohydrolase with a characteristic purple colour, also referred to as tartrate-resistant acid phosphatase (TRAP), uteroferrin or type 5 acid phosphatase. PAP is highly expressed in osteoclasts and has a role in bone resorption. The aims of the present investigation were to further characterize the PAP enzyme regarding repressive interactions responsible for enzyme latency and to elucidate the role of proteolytic processing as a biologically relevant regulatory mechanism for enzymatic activation. Recombinant rat TRAP was expressed using Baculovirus-infected insect cells and a monomeric protein with characteristic PAP features regarding colour, specific activity, protein phosphatase activity, molecular mass, iron content and light absorption spectrum was purified and characterized. EPR spectroscopy established that the recombinant enzyme belongs to the purple acid phosphatase family. The data validated that recombinant rat PAP is a useful tool in future studies substituting for the native rat bone TRAP. There is an exposed loop sequence in the monomeric recombinant PAP involved in inhibitory interactions with catalytic residues and the divalent Fe 2+ active site metal leading to a catalytically latent enzyme. To investigate the role of proteolytic processing of recombinant PAP as an activation mechanism, various proteases were tested in proteolysis studies. The results demonstrated that cleavage with cathepsin K or L increase the enzymatic activity, shift the pH optimum and increase sensitivity to reducing agents to the same extent as for native bone PAP. PAP activation was due to an extensive excision of the loop region, and the putative repressive amino acids of the loop region could be mapped to short peptide segments by N- and C-terminal protein sequencing. Site-directed mutagenesis of monomeric recombinant PAP was performed in order to identify the amino acid(s) in the loop region involved in catalytic inhibitory interactions to active site residues affecting the Fe2+. Conversion of Ser1 45 or Asp 146 into Ala, which should eliminate the putative interaction of the side chains with the active site residues Asn91 or His92, resulted in increased enzyme activity but neither of the two mutations alone reached the activity level of the native bone enzyme. Together these results indicate that Ser145 and Asp 146 are important amino acids in the repressive function of the loop. A role for cathepsin K as an activator of PAP in vivo was strongly suggested by immunohistochemical studies indicating that cathepsin K cleavage of PAP is taking place in close proximity to the osteoclastic ruffled border in the extracellular bone resorption area. In summary, proteolytic activation of PAP requires excision of loop region amino acids Ser145 and Asp146 in order to obtain a fully activated PAP enzyme. Cathepsin K is responsible for proteolytic processing of secreted PAP in vivo during bone resorption.