Structure, function, and regulation of tartrate-resistant acid phosphatase

Abstract

The tartrate-resistant acid phosphatases (TRAPs) are a class of metalloenzymes that catalyze the hydrolysis of various phosphate esters and anhydrides under acidic reaction conditions. Because the bound metal ions confer an intense color on these enzymes they are also known as purple acid phosphatases (PAPs). Resistance to inhibition by high concentrations of the competitive inhibitor L 1 tartrate distinguishes TRAP from acid phosphatases of lysosomal or prostatic origin present in many mammalian cells and tissues. TRAP enzymes have been isolated from many mammalian sources, including: bovine and rat spleen; the spleens of patients affected with hairy cell leukemia and Gaucher’s disease; human and rat bone; and human lungs and placenta. The TRAP purified from porcine allantoic fluid, which is also known as uteroferrin, was originally recognized as an abundant basic protein in uterine secretions induced by progesterone. The catalytic mechanism, structure, and properties of the iron center of porcine TRAP have been studied extensively by our group. Mammalian isolated TRAP enzymes all have similar physical properties, including a molecular weight of about 35 kDa, a basic isoelectric point (pI 7.6–9.5), and optimal enzyme activity at an acidic pH. The enzyme can be isolated as a single chain polypeptide, but a dimeric nicked form arises from posttranslational cleavage of the single chain enzyme. Cleavage occurs in an exposed loop that is conserved in all mammalian TRAP enzymes and leads to an increase in Vmax/kcat of the enzyme by an unknown mechanism. Several proteolytic enzymes are able to cleave the exposed loop, but only the cysteine proteinases papain and cathepsin B have been able to cause activation among several tested. Ljusberg et al. put forth the view that TRAP, like several other hydrolases, is synthesized as a relatively inactive proenzyme, and cleavage is the physiological mechanism of proenzyme activation in osteoclasts. Mammalian TRAP enzymes are glycoproteins and, like most lysosomal enzymes, possess the mannose-6-phosphate lysosomal targeting sequence, which must presumably be cleaved or modified to permit secretion. TRAP isolated from allantoic fluid of the pig showed a single, unphosphorylated, high-mannose-type oligosaccharide composed of five or six mannose residues and two N-acetylglucosamine residues. In contrast, recombinant porcine TRAP secreted by Chinese hamster ovary (CHO) cells possessed N-linked, high-mannose oligosaccharide chains that were phosphorylated and could not be dephosphorylated by alkaline phosphatase treatment in vitro. This suggests that the uteroferrin oligosaccharide phosphates were not exposed, perhaps as a result of blocking by an N-acetylglucosamine residue. The glycoprotein structure of human bone TRAP was analyzed by lectin binding and, in agreement with the prior analysis of native uteroferrin, contained only N-linked high-mannose carbohydrates, implying that the native secreted protein is normally dephosphorylated. Analysis of TRAP activity present in electrophoretically separated human serum revealed two isoforms, termed 5a and 5b, with each isoform having a different pH optimum (5a: pH 4.9; 5b: pH 5.5–6.0). The carbohydrate content of the isoforms also differed with only isoform 5a containing sialic acid. TRAP contains two iron atoms at its active site, and the intense purple color of the enzyme results from a tyrosinate Fe(III) charge transfer. Reduction of the active site binuclear center to a mixed valency Fe(III)-Fe(II) form is required for activation and this corresponds to a shift in color from purple to pink. Further reduction or the presence of iron chelators can lead to reversible inactivation and formation of a colorless form of the enzyme. The enzyme is also inhibited noncompetitively by incubation with vanadate or simply following more extended incubation at 37°C. The latter case at least, produced a “yellowish” form of the enzyme. TRAP may become irreversibly inactivated by oxidation in the presence of ascorbate. The recent availability of monoclonal antibodies against TRAP has permitted the identification of an inactivated “yellowish” form of the enzyme as the major form in the circulation.