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Tartrate-resistant purple acid phosphatase is synthesized as a latent proenzyme and activated by cysteine proteinases.

ABSTRACT: Purple acid phosphatases (PAPs) are binuclear acid metallohydrolases also referred to as tartrate-resistant acid phosphatases (TRAPs) or type 5 acid phosphatases. The cDNA sequences of TRAP/PAP enzymes from different species and organs indicate that these enzymes are translated as monomeric polypeptides of approx. 35 kDa, contrasting with the predominantly two-subunit structure observed in purified enzyme preparations. In the present study we have compared certain structural and enzyme-kinetic properties of recombinant rat PAP (monomeric) with those of the native rat bone TRAP/PAP enzyme (two-subunit), and examined effects on these parameters by cleaving the monomeric recombinant PAP with the serine proteinase trypsin or the cysteine proteinases papain or cathepsin B. Cleavage with trypsin resulted in a moderate activation of the recombinant enzyme and shifted the pH optimum to a slightly more basic value (5.0-5.5). Cleavage with papain resulted in complete activation and conferred similar properties to those of the bone PAP variant with regard to pH optimum (5.5-6.0) and sensitivity to reducing agents, as well as in the sizes of the subunits. Substrate specificity studies showed that the two-subunit bone PAP was considerably more active than the monomeric recombinant rat PAP towards a variety of serine-, threonine- and tyrosine-phosphorylated substrates. Of these substrates, bovine milk osteopontin seemed to be the most readily dephosphorylated substrate. In conclusion, the results suggest that the monomeric form of PAP represent a latent proenzyme with low enzymic activity towards both tyrosine- and serine/threonine-containing phosphorylated substrates. Besides being implicated in the catabolism of the extracellular matrix, members of the cysteine proteinase family might also exert a regulatory role in degradative processes involving the PAP enzymes by converting the newly synthesized PAPs to enzymically active and microenvironmentally regulated species.

SUBMITTER: Ljusberg J

PROVIDER: S-EPMC1220524 | biostudies-other | 1999 Oct

REPOSITORIES: biostudies-other

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Project description:The tartrate-resistant acid phosphatase (TRAP) of rat osteoclasts has been shown to exhibit high (85-94%) identity at the amino acid sequence level with the purple acid phosphatase (PAP) from bovine spleen and with pig uteroferrin. These iron-containing purple enzymes contain a binuclear iron centre, with a tyrosinate-to-Fe(III) charge-transfer transition responsible for the purple colour. In the present study, production of rat osteoclast TRAP could be achieved at a level of 4.3 mg/litre of medium using a baculovirus expression system. The enzyme was purified to apparent homogeneity using a combination of cation-exchange, hydrophobic-interaction, lectin-affinity and gel-permeation chromatography steps. The protein as isolated had a purple colour, a specific activity of 428 units/mg of protein and consisted of the single-chain form of molecular mass 34 kDa, with only trace amounts of proteolytically derived subunits. The recombinant enzyme had the ability to dephosphorylate bone matrix phosphoproteins, as previously shown for bone TRAP. Light absorption spectroscopy of the isolated purple enzyme showed a lambda max at 544 nm, which upon reduction with ascorbic acid changed to 515 nm, concomitant with the transition to a pink colour. EPR spectroscopic analysis of the reduced enzyme at 3.6 K revealed a typical mu-hydr(oxo)-bridged mixed-valent Fe(II)Fe(III) signal with g-values at 1.96, 1.74 and 1.60, proving that recombinant rat TRAP belongs to the family of PAPs. To validate the use of recombinant PAP in substituting for the rat bone counterpart in functional studies, various comparative studies were carried out. The enzyme isolated from bone exhibited a lower K(m) for p-nitrophenyl phosphate and was slightly more sensitive to PAP inhibitors such as molybdate, tungstate, arsenate and phosphate. In contrast with the recombinant enzyme, TRAP from bone was isolated predominantly as the proteolytically cleaved, two-subunit, form. Both the recombinant enzyme and rat bone TRAP were shown to be substituted with N-linked oligosaccharides. A slightly higher apparent molecular mass of the monomeric form and N-terminal chain of bone TRAP compared with the recombinant enzyme could not be accounted for by differential N-glycosylation. Despite differences in specific post-translational modifications, the recombinant PAP should be useful in future studies on the properties and regulation of the mammalian PAP enzyme.

Project description:Biallelic mutations in ACP5, encoding tartrate-resistant acid phosphatase (TRACP), have recently been identified to cause the inherited immuno-osseous disorder, spondyloenchondrodysplasia (SPENCD). This study was undertaken to characterize the eight reported missense mutations in ACP5 associated with SPENCD on TRACP expression. ACP5 mutant genes were synthesized, transfected into human embryonic kidney (HEK-293) cells and stably expressing cell lines were established. TRACP expression was assessed by cytochemical and immuno-cytochemical staining with a panel of monoclonal antibodies. Analysis of wild (WT) type and eight mutant stable cell lines indicated that all mutants lacked stainable enzyme activity. All ACP5 mutant constructs were translated into intact proteins by HEK-293 cells. The mutant TRACP proteins displayed variable immune reactivity patterns, and all drastically reduced enzymatic activity, revealing that there is no gross inhibition of TRACP biosynthesis by the mutations. But they likely interfere with folding thereby impairing enzyme function. TRACP exists as two isoforms. TRACP 5a is a less active monomeric enzyme (35kD), with the intact loop peptide and TRACP 5b is proteolytically cleaved highly active enzyme encompassing two subunits (23 kD and 16 kD) held together by disulfide bonds. None of the mutant proteins were proteolytically processed into isoform 5b intracellularly, and only three mutants were secreted in significant amounts into the culture medium as intact isoform 5a-like proteins. Analysis of antibody reactivity patterns revealed that T89I and M264K mutant proteins retained some native conformation, whereas all others were in "denatured" or "unfolded" forms. Western blot analysis with intracellular and secreted TRACP proteins also revealed similar observations indicating that mutant T89I is amply secreted as inactive protein. All mutant proteins were attacked by Endo-H sensitive glycans and none could be activated by proteolytic cleavage in vitro. In conclusion, determining the structure-function relationship of the SPENCD mutations in TRACP will expand our understanding of basic mechanisms underlying immune responsiveness and its involvement in dysregulated bone metabolism.

Project description:Tartrate-resistant acid phosphatase (TRAP) was first identified in cells from patients with hairy cell leukaemia. Subsequently, it has been found in other leukaemias, B-lymphoblastoid cell lines, osteoclasts and subsets of normal lymphocytes, macrophages, and granulocytes. Recent data indicate that TRAP and porcine uteroferrin, a placental iron-transport protein, represent a single gene product. However, the intracellular role of TRAP is unknown. We used a full-length human placental TRAP cDNA probe to examine TRAP expression in human peripheral mononuclear cells (PMCs). TRAP mRNA increased 50-75-fold after 24 h in unstimulated PMC cultures. Cell-fractionation experiments indicated that monocytes were the main cell population accounting for increased TRAP mRNA transcripts, and this was confirmed by histochemical staining for TRAP enzyme activity. Because expression of other iron-binding and -transport proteins is controlled by iron availability, we examined the role of iron in regulating TRAP expression. Increase of TRAP mRNA transcripts in PMCs was inhibited by 50 microM desferrioxamine, a potent iron chelator. The 5' flanking region of the TRAP gene was cloned from a mouse genomic library. In preliminary transient transfection experiments, it was determined that the 5'-flanking region of the TRAP gene contained iron-responsive elements. Therefore, a series of stably transfected HRE H9 cell lines was developed bearing genetic constructs containing various segments of the murine TRAP 5' promoter region driving a luciferase reporter gene. Treatment of transfectants with 100 micrograms/ml iron-saturated human transferrin (FeTF) was performed to assess iron responsiveness of the constructs. Constructs containing a full-length TRAP promoter (comprising base pairs -1846 to +2) responded to FeTF with a 4-5-fold increase of luciferase activity whereas constructs containing only base pairs -363 to +2 of the TRAP promoter did not respond. Constructs containing 1240 or 881 bp of the TRAP promoter gave only a 1.5- to 2-fold increase of luciferase activity with FeTF. In all cases, increase of luciferase activity was blocked by desferrioxamine. Cells transfected with another luciferase construct driven by a simian virus 40 promoter did not show any increase of luciferase activity with FeTF. These data indicate that expression of TRAP is regulated by iron and that this regulation is exerted at the level of gene transcription. The transfection experiments also suggest that the region of the TRAP 5'-flanking sequence between base pairs -1846 and -1240 contains an iron regulatory element.

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Tartrate-resistant purple acid phosphatase is synthesized as a latent proenzyme and activated by cysteine proteinases.

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