ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 345, No. 2, September 15, pp. 230–236, 1997Article No. BB970250

Recombinant Human and Mouse Purple AcidPhosphatases: Expression and Characterization

Karen Marshall,* Kevin Nash,* George Haussman,*,1 Ian Cassady,† David Hume,†John de Jersey,* and Susan Hamilton**Centre for Protein Structure, Function, and Engineering, Department of Biochemistry, and †Centre for Molecular andCellular Biology, University of Queensland, Queensland 4072, Australia

Received February 5, 1997, and in revised form June 16, 1997

Purple acid phosphatases are a family of nonspecificThe mammalian purple acid phosphatases (also enzymes distinguished from other mammalian phos-

called tartrate-resistant acid phosphatases) are ex- phatases by their characteristic color, low pH optimapressed primarily in actively resorbing osteoclasts (Ç5), high isoelectric points (Ç9), and insensitivity toand activated macrophages. The enzymes are charac- inhibition by L(/)-tartrate. Purple acid phosphatasesterized by the presence of a binuclear iron center at catalyze the hydrolysis of a range of activated phos-the active site. Recent studies on transgenic mice lack- phate esters and anhydrides including ATP, pyrophos-ing purple acid phosphatase implicate the osteoclast phate, and phosphotyrosyl proteins (1–3). In addition,enzyme in both bone resorption and bone mineraliza- they are able to catalyze the production of hydroxyltion. To characterize the mammalian enzymes in more radicals via Haber–Weiss–Fenton chemistry (4, 5).detail, particularly with respect to their substrate The relevance of these activities to the biological func-specificity at the low pH of the osteoclastic resorptive tion of the enzyme is not known.space (2.5–3), we have purified the recombinant hu- Expression of the mammalian enzyme is restrictedman and mouse enzymes from baculovirus-infected in- to differentiated cells of nuclear phagocytic lineage,sect cells. The properties of the recombinant mouse

principally osteoclasts and activated macrophages (6–enzyme are compared with those of the nonrecombi-9). In osteoclasts involved in active bone resorption,nant enzyme isolated from mouse spleen. The kineticsthe enzyme is secreted and accumulates in the boneof hydrolysis of the substrates p-nitrophenyl phos-matrix facing the ruffled border (10). Recent studies onphate, phosphotyrosine, and pyrophosphate and atransgenic mice lacking purple acid phosphatase indi-phosphotyrosyl peptide by the recombinant humancate that the osteoclast enzyme is required for normaland mouse enzymes and the nonrecombinant mousemineralization of cartilage in developing bone and boneand pig enzymes were analyzed. For all the enzymesmatrix resorption in adult bone (11). Bone matrix con-the ratio kcat/Km was typically Ç106 M01 s01 and wastains several potential substrates for the enzyme: ithigher at pH 2.5 than at 4.9. The increase was attribut-

able to a large decrease in Km at the lower pH value. is rich in pyrophosphate, a known inhibitor of boneThe results indicate that the enzyme exhibits high cat- resorption, as well as acidic phosphoproteins such asalytic efficiency toward substrates such as pyrophos- osteopontin and sialoprotein (12, 13). Proposed func-phate and acidic phosphotyrosine-containing pep- tions in macrophages include microbial killing by thetides, particularly at low pH values typical of the bone generation of reactive oxygen species (5), the generalresorptive space. The implications of the results for degradation of infectious material (in the case of alveo-the physiological function of the enzyme are dis- lar macrophages) (8, 9), and the hydrolysis of erythro-cussed. q 1997 Academic Press cyte membrane phosphoproteins (in the case of spleen

Key Words: acid phosphatase; tartrate-resistant; re- macrophages) (7, 14).combinant enzyme; bone resorption; metalloenzyme. Early studies on the purple acid phosphatases fo-

cused on the unusual spectral properties of the en-zymes, resulting from the presence of a binuclear ironcenter (FeIII–FeII in the catalytically active form).1 To whom correspondence should be addressed at Department ofThe enzyme was purified from pig allantoic fluid andBiochemistry, University of Queensland, St. Lucia, Queensland

4072, Australia. Fax: 61-7-33654699. bovine spleen in milligram quantities (15, 16), with

230 0003-9861/97 $25.00Copyright q 1997 by Academic Press

All rights of reproduction in any form reserved.

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231RECOMBINANT MOUSE AND HUMAN PURPLE ACID PHOSPHATASES

man and mouse purple acid phosphatase cDNAs including the codingmuch smaller amounts obtained from other sourcesregions for the signal sequences (25, 28) were ligated into the EcoRIincluding human hairy cell leukemia spleen,or SmaI sites, respectively, of pBacPAK9 (designated pBacPAK9/Gaucher spleen, human and rat bone, and human PAP) (29). The sequences of both cDNAs were confirmed by dideoxy

osteoclastoma (3, 14, 17–20). terminator cycle sequencing. BacPAK6 DNA (100 ng) linearized byBsu36 1 digestion and 1 mg of pBacPAK9/PAP were cotransfectedHuman purple acid phosphatase is an important tar-into 1.5 1 106 Sf-9 cells in the presence of 8 mg lipofectin reagentget for further study, particularly in view of the evi-(Life Technologies Inc.). Five days post-cotransfection virions in thedence implicating it in bone resorption. Human purpleculture supernatant were analyzed by plaque assay with neutral red

acid phosphatase is also of interest clinically because and X-gal (5-bromo-4-chloro-3-indolyl-b-D galactopyranoside) stain-elevated serum levels are associated with increased ing (30). Plaque lysates of putative recombinant viruses were used

for small-scale cultures, and the viral DNA was extracted from in-bone resorption during normal physiological bonefected cell lysates and analyzed by polymerase chain reaction. Prim-growth as well as many metabolic bone diseases anders both internal and external to the phosphatase cDNA sequencemalignancies (21–24). The mouse enzyme is of interest were used to identify positive recombinant viruses. High titer stocks

because of the development of transgenic mouse model (Ç1 1 108 plaque-forming units per milliliter) were raised (27).systems (11), the use of mouse osteoclasts in in vitro Insect cells from Spodoptera frugiperda pupal ovarian tissue (Sf-

9) were used to support baculoviral growth. Sf-9 cells were culturedbone resorption assays, and the use of mouse macro-at 287C as monolayer cultures in TC-100 medium (Life Technologiesphage cell lines for studying function in macrophages.Inc.) supplemented with 10% (v/v) heat-inactivated fetal calf serum,The mouse enzyme has not previously been isolated or as shaker cultures in TC-100 medium supplemented with 10% (v/v)

characterized. We have therefore expressed recombi- heat-inactivated fetal calf serum and 0.1% (w/v) pluronic F-68, or asshaker cultures in unsupplemented Sf-900 II medium (Life Technolo-nant mouse purple acid phosphatase in baculovirus-gies Inc.). Sf-9 cells for growth in serum-containing media were ob-infected insect cells and compared its properties withtained from Invitrogen Corp. Sf-9 cells adapted for growth in serum-those of the nonrecombinant mouse spleen enzyme.free medium were kindly donated by Dr. Steve Reid, University ofThe recombinant human enzyme has also been ex- Queensland. For enzyme expression Sf-9 cells of greater than 99%

pressed as described previously (5) and its molecular viability were used in shaker cultures at a density of 3 1 106 cells/ml in Sf-900-II medium. Recombinant virus was added to a multiplic-and kinetic properties have been further characterized.ity of infection of three plaque-forming units per cell, and the cellmedium was harvested 3 or 6 days postinfection for the human and

EXPERIMENTAL PROCEDURES mouse enzymes, respectively. In initial experiments, expression andpurification were undertaken essentially as described previously (5).Materials. All reagents were analytical grade unless otherwiseIn subsequent experiments the tissue culture medium was supple-specified. The peptide FmocEEY(P)AA was synthesized as describedmented with 25 and 100 mM FeSO4. Supplementation at these con-previously (2).centrations resulted in a 1.5- to 2-fold increase in enzyme activityBacterial expression. Mouse purple acid phosphatase cDNA (25),in the culture supernatant (measured after reduction) compared tominus the 21-amino-acid signal sequence, was subcloned into theunsupplemented controls. Iron concentrations above 100 mM resultedEcoRI site of the vector pGEX-2T (Amrad-Pharmacia). An inactivein a decrease in enzyme activity and were noted to have detrimentalGST-fusion protein was expressed in Escherichia coli DH5a cellseffects on the insect cells.containing the chaperonins GroEL and GroES (Biomolecular Re-

search Institute) by growing cells containing the plasmid for 90 min Purification of insect cell-expressed purple acid phosphatases. Re-at 377C and then inducing with IPTG (0.1 mM) and growing for a combinant enzyme secreted into the tissue culture medium (1 liter)further 4–5 h at the same temperature. Cells were pelleted, resus- was purified by sequential rounds of ion-exchange chromatographypended in 0.01 M Tris, 0.15 M NaCl, 1 mM EDTA (pH 8.0), and lysed as described previously for the human enzyme (5) or by ion-exchangewith lysozyme (1 mg/ml). DTT2 (5 mM) was added, and the sample chromatography followed by gel filtration in the case of the mousesonicated and then centrifuged. Triton X-100 (3% v/v) was added to enzyme. The pH of the culture supernatant was adjusted to 8.0 bythe supernatant which was then applied to GSH–Sepharose (Amrad addition of NaOH and incubated overnight at 47C. Following centrif-Pharmacia). After washing with phosphate-buffered saline the en- ugation, the supernatant was applied to a 5-ml Bio-Scale S FPLCzyme was eluted with 0.05 M Tris buffer containing 0.15 M NaCl, 10 column (Bio-Rad Laboratories) equilibrated with 50 mM HepesmM GSH, and 5 mM DTT (26). A rabbit anti-mouse PAP polyclonal buffer, pH 8.0. The human enzyme was eluted with a 0–1.0 M linearantibody was generated against the purified recombinant fusion pro- NaCl gradient (150 ml). Active fractions were pooled, diluted 10-foldtein for use in Western blotting. in buffer, and rechromatographed on the same column using a linear

0–1.0 M NaCl gradient (200 ml). The mouse enzyme was eluted withExpression in baculovirus-infected insect cells. Recombinant ba-a linear 0–1.0 M NaCl gradient (200 ml), and active fractions wereculovirus containing human or mouse purple acid phosphatase cDNApooled, concentrated, and rechromatographed on a Superose 12under the control of the polyhedron promoter was generated by ho-FPLC column (Pharmacia) equilibrated with 0.1 M acetate buffer,mologous recombination between the transplacement vector pBac-pH 4.9. Purified enzyme was stored at 47C in the dark.PAK9 (Clontech Laboratories, Inc.), which contained purple acid

phosphatase cDNA flanked by viral sequence, and the nonessential Purification of nonrecombinant pig and mouse enzymes. Pig al-polyhedron gene of the Autographa californica nuclear polyhedrosis lantoic fluid purple acid phosphatase was purified according tovirus BacPAK6 (Clontech Laboratories, Inc.). BacPAK6 virions were Campbell et al. (15). Mouse spleen purple acid phosphatase was par-first purified on a 26 to 56% sucrose gradient and the DNA was tially purified as follows. Spleens were excised from 500 mice. To theisolated by a proteinase K digestion procedure (27). Full-length hu- spleens (Ç40 g) was added 1.5 vol of 0.05 M Tris–acetate buffer, pH

7.5, containing 1 mM PMSF, 2% Nonidet P-40, 1 mM EDTA, and0.02% sodium azide. This was homogenized with an Ultraturrax ho-mogenizer (Kika Werk) at half speed. The homogenate was centri-

2 Abbreviations used: DTT, dithiothreitol; p-NPP, p-nitrophenyl fuged at 30,000g for 20 min at 47C. The supernatant (Ç80 ml) wasdialyzed overnight at 47C against 2.0 liters of 0.05 M Hepes buffer,phosphate.

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232 MARSHALL ET AL.

pH 8.0. CM cellulose equilibrated with the same buffer was incubated NPP, the discontinuous assay was used at pH 2.5 and the continuousassay at pH 4.9, as described above. Phosphotyrosine assays werewith the dialyzed supernatant for 1.5 h with intermittent stirring.

The resin was washed extensively and then loaded into a column performed by monitoring the production of tyrosine at 280 nm asdescribed previously (2). The hydrolysis of pyrophosphate was mea-and the protein eluted with a salt gradient (0–1 M NaCl) in 40 ml.

Fractions containing activity were pooled and diluted 1:10 with 0.05 sured as described by Shatton et al. (33) and the hydrolysis ofFmocEEY(P)AA by discontinuous HPLC assay (2). Values of V andM Hepes buffer, pH 8.0, and loaded onto a Bio-Rad Bio-Scale S FPLC

column (5-ml capacity). The column was washed with the same buffer Km were determined from initial rate data using nonlinear regressionanalysis. Values of kcat were calculated from the equation VÅ kcat[E]0 .and the protein eluted with a gradient (0–1 M NaCl) in 200 ml.

Active fractions were pooled, diluted 1:5, and then loaded onto a Enzyme concentrations were calculated from the A280 , an A1%1 cm of

14.20, and molecular weights of 36,000 and 35,000 for the recombi-Pharmacia Mono S Smart system column (0.1-ml capacity) equili-brated with 0.05 M Hepes buffer, pH 8.0. The enzyme was eluted nant human and mouse enzymes, respectively (based on the results

of SDS–PAGE). In cases where the enzyme lost activity in storagewith 1 M NaCl. The purification yielded Ç8 U of enzyme of specificactivity 36 U/mg after reduction with iron/b-mercaptoethanol. SDS– (in particular the human enzyme), the enzyme concentration was

adjusted according to the decrease in specific activity.PAGE showed that the enzyme was not pure. Further purificationwas not practicable in view of the small amount of enzyme available.The purity was estimated to be Ç18% by comparing the A280/A530 RESULTSratio (91.5) with that of the pure recombinant mouse enzyme (16.3).The specific activity for the pure spleen enzyme, based on this figure, The elution profile for the final chromatography ofis Ç200 U/mg. the insect cell-expressed human enzyme from iron-sup-

SDS–gel electrophoresis and Western blotting. SDS–PAGE was plemented medium is shown in Fig. 1A. The proteinperformed with a 10 or 15% acrylamide separating gel (31) and the

peak was biphasic, with the activity measured prior togel silver stained (32). For nonreducing gels b-mercaptoethanol wasreduction of the enzyme displaced to the right of theomitted from the loading buffer. For Western blots, proteins were

electroblotted onto a nitrocellulose membrane (Hybond ECL, Amer- main A280 peak. The results suggested that there maysham Life Science), and the membrane was blocked with 3% (w/v) have been a proportion of oxidized, inactive enzymebovine serum albumin, incubated with an appropriate dilution of the present, which eluted prior to the active enzyme. Fol-antisera in phosphate-buffered saline–0.05% (v/v) Tween 20 for 1 h

lowing reduction, the activity in all fractions was in-at room temperature, and detected using anti-mouse immunoglobu-creased, with the greatest increase in early fractions.lin conjugated to horseradish peroxidase and reagents of the en-

hanced chemical luminescence kit of Amersham Life Science. All The highest specific activity obtained for the humanwashings were performed in phosphate-buffered saline with 0.5% enzyme was 350 U/mg, equivalent to a specific activity(v/v) Tween 20. of 562 U/mg under the assay conditions employed by

Enzyme activity assays. Acid phosphatase activity was rou- Hayman and Cox (5). SDS–PAGE showed the presencetinely determined using a discontinuous p-nitrophenyl phosphateof two closely related species of Ç35.0 and 36 kDa and(p-NPP) assay (15). An aliquot of enzyme was added to 5 mM p-

NPP in 0.1 M acetate buffer, pH 4.9, equilibrated at 257C. Aliquots essentially no contaminating proteins (Fig. 2). From awere withdrawn at 2, 4, and 6 min and added to 2 vol of 0.1 M 1-liter culture Ç8 mg of purified enzyme was obtained.NaOH. p-Nitrophenol was determined spectrophotometrically at Sequence analysis showed a single amino-terminal se-400 nm (e Å 18,320 M01 cm01). Alternatively, continuous p-NPP

quence ATPALR starting at /22 from the initiatingassays were performed in 0.1 M acetate buffer, pH 4.9, at 257C asmethionine (28). The sequence is identical to that ofdescribed previously (2). One unit of activity (U) is defined as the

amount of enzyme which catalyzed the hydrolysis of 1 mmol of p- the human osteoclastoma enzyme (20), indicating thatNPP per minute. Specific activity is defined as units of activity the signal sequence is being cleaved correctly in theper milligram of protein. insect cells. Upon storage at 47C two major bands of

Protein determination. Protein concentrations were determined Ç18 and Ç15.5 kDa formed over a period of months.by measuring the absorbance at 280 nm. For pure enzyme an A1%1 cm

The same pattern was observed in both reducing andequal to that of pure pig allantoic fluid purple acid phosphatase(14.20) was used to calculate the concentration of protein from the nonreducing gels, indicating that the fragments wereA280 (15). not disulfide linked (Fig. 2). Sequence analysis of this

Oxidation and reduction of enzyme. Reduction in the presence of enzyme indicated a second N-terminal sequence pres-ferrous ion was performed by incubating the enzyme overnight in ent at low level: XT/PRLSW.0.1 M acetate buffer, pH 4.9, with 58 mM ferrous ammonium sulfate

The elution profile for the final gel filtration stepand 135 mM b-mercaptoethanol at 47C. Where specified, excess re-in the purification of the insect cell-expressed mouseducing reagents were removed by gel filtration on a Superose 12

FPLC column (Pharmacia) equilibrated with 0.1 M acetate buffer, enzyme is shown in Fig. 1B. The highest specific activ-pH 4.9, or by repeated concentration and redilution using a micro- ity following reduction with iron/b-mercaptoethanolconcentrator (Amicon Microcon 10). Oxidation of the enzyme was was 383 U/mg. Approximately 4 mg of enzyme of thisperformed by addition of H2O2 (10 mM) and incubation for 10 min

specific activity was obtained from a 1-liter cultureat room temperature.with overall recovery of activity of 50%. SDS–PAGE ofMetal ion analysis and sequence analysis. Metal ion analyses

were performed on a Spectra Analytical Instruments Model M / the purified enzyme showed the presence of two closelyP inductively coupled plasma atomic emission spectrophotometer. related species of Ç36 and 35.5 kDa plus additionalSamples and standards were prepared in 0.1 M acetate buffer, pH bands at Ç22.5, 20.5, and 15.5 kDa. All bands were4.9. Sequence analysis was performed by Edman degradation using

recognized by the antiserum generated against thean API 470A protein sequencer.GST–mouse purple acid phosphatase fusion proteinKinetics. Kinetic constants were determined in either 0.062 or

0.1 M glycine buffer, pH 2.5, or 0.1 M acetate buffer, pH 4.9. For p- expressed in E. coli (Fig. 2). The smaller fragments

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233RECOMBINANT MOUSE AND HUMAN PURPLE ACID PHOSPHATASES

preparation was analyzed by SDS–PAGE, only intactenzyme was seen. The specific activity of the pure en-zyme thus depended on the extent to which the enzymewas nicked. Based on the measured specific activitiesfor the intact and Ç50% nicked enzymes, the specificactivity for the fully nicked enzyme was estimated tobe Ç570 U/mg. A similar increase in specific activityupon nicking was observed for the nonrecombinantmouse spleen enzyme. Immediately after purification,three immunoreactive peptides were present in thepartially purified preparation, corresponding to speciesof Ç35, Ç20, and Ç14 kDa on SDS–PAGE. Upon stor-age for 1 month at 47C, the Ç35-kDa species was lostcompletely and the specific activity increased 2.5-fold.

Both recombinant enzymes exhibited visible absorp-tion spectra typical of the mammalian purple acid phos-phatases. For the pure enzymes as isolated, the visiblelmax was Ç540 nm. Upon reduction with b-mercapto-ethanol and ferrous ion and removal of excess reagents,the visible spectrum exhibited a lmax at Ç515 nm. TheA280/A530 ratios for the reduced human and mouse en-zymes were 16.5 and 16.3, respectively. These valuesare very similar to those determined for the fully re-duced pig allantoic fluid enzyme (lmax Å 515 nm, A280/A530 Å 16.0) (15). Metal ion analysis of nonreduced hu-man enzyme expressed in the absence of added ironshowed the presence of Ç1.3 Fe per enzyme and forthe enzyme expressed in the presence of 100 mM iron,1.5 Fe per enzyme. The mouse enzyme expressed inthe presence of 100 mM iron contained 1.93 Fe per en-zyme. In all cases the level of zinc was at or belowthe detection limit of the instrument (corresponding to£0.3 Zn atoms per molecule) and there was no detect-FIG. 1. Elution profiles from final chromatographies of recombi-able copper. Fully reduced pig allantoic fluid purplenant proteins. (A) Chromatography of human purple acid phospha-acid phosphatase was analyzed under the same condi-tase, expressed in the presence of 100 mM iron, on final Bio-Scale S

FPLC column (5 ml) equilibrated with 0.05 M Hepes buffer, pH 8.0, tions and found to contain 2.0 Fe atoms per enzyme.and eluted with a 0–1.0 M NaCl gradient in the same buffer. (h)[protein] (mg/ml): (s) activity before reduction, (l) activity after re-duction with Fe/b-mercaptoethanol. (B) Chromatography of mousepurple acid phosphatase, expressed in the presence of 100 mM iron,on Superose 12 column (10 ml) equilibrated with 0.1 M acetate buffer,pH 4.9. (h) [protein] (mg/ml): (s) activity before reduction, (l) activ-ity after reduction with Fe/b-mercaptoethanol.

were apparently derived from the intact enzyme byproteolytic nicking. The extent of nicking was esti-mated by densitometry to be Ç50%. Under nonreduc-ing conditions the enzyme remained intact, suggestingthat the fragments were linked by a disulfide bond (Fig. FIG. 2. SDS–PAGE of purified enzymes. (A, B) Reducing gels of2). Sequencing of the enzyme demonstrated the pres- unnicked human and mouse enzymes, respectively. The human en-ence of two amino termini with the sequences TAPTPT- zyme was analyzed immediately after purification. The mouse en-

zyme was from the small-scale preparation. (C, D) Reducing gels ofLRFV and VARTQLSWLK corresponding to regionspartially nicked human and mouse enzymes, respectively. The hu-starting at /22 and /182 from the N-terminal methio-man enzyme was analyzed after storage for several months at 47C.nine (25). In a small-scale expression and purification The mouse enzyme was from the large-scale purification. (E, F) Non-

of the enzyme a lower specific activity for the pure en- reducing gels of partially nicked human and mouse enzymes, respec-tively. A–F are silver stained. (G) Western blot corresponding to D.zyme was obtained (184 U/mg). When enzyme from this

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234 MARSHALL ET AL.

TABLE I

Effect of Oxidation and Reduction on Specific Activities of Pure Recombinant Enzymesa

Human Mouse

Iron supplementation of medium (mM) 0 100 100

Specific activity untreated 100 (161)b 67 54Specific activity after oxidationc 100 20 38Specific activity after reductiond 350 (562)b 291 383Specific activity after reduction/oxidatione 45 ND f 38

a Specific activity in mmol/min/mg.b Specific activity measured under the conditions of Hayman and Cox (5).c Oxidation in the presence of 10 mM hydrogen peroxide.d Reduction in the presence of 58 mM ferrous ammonium sulfate/135 mM b-mercaptoethanol.e Enzyme treated as in d followed by removal of excess reagents and treatment with 10 mM hydrogen peroxide.f ND, not determined.

Table I summarizes the effect of oxidation and reduc- trophoretic behavior with those described earlier. How-tion on the specific activities of the enzymes. Oxidation ever, we observed that the activity of the enzyme ob-with hydrogen peroxide had no effect on the specific tained from cultures which were not supplementedactivity of the human enzyme expressed without iron with iron was completely insensitive to inhibition bysupplementation. In contrast, peroxide treatment re- peroxide, and metal ion analysis showed the presencesulted in a decrease in activity of the human and mouse of only 1.3 g atom of iron per mole of enzyme [cf. 2enzymes expressed with iron supplementation. Upon reported by Hayman and Cox (5)]. The results suggestreduction with b-mercaptoethanol and ferrous ion, the that the activity of this enzyme is attributable to aspecific activity of the human enzyme expressed with nonredox active form, probably the FeIII–ZnII enzyme.and without iron supplementation increased 4.3- and Metal ion analysis was not sufficiently sensitive to3.5-fold, respectively. The specific activity of the mouse measure less than Ç0.2–0.3 g atom of Zn/mol of en-enzyme after reduction with b-mercaptoethanol and zyme at the enzyme concentrations available. Assum-ferrous ion was Ç7-fold higher than that of the un- ing that the FeIII–ZnII form of the human enzyme,treated enzyme. When the enzymes were reconstituted like the pig and red kidney bean enzymes, has a specificwith b-mercaptoethanol and ferrous ion and excess re- activity approximately the same as that of the FeIII–agents removed by gel filtration, subsequent treatment FeII enzyme (34), a level of zinc of 0.28 g atom/molwith hydrogen peroxide resulted in a much larger loss would account for the specific activity of the enzyme asin activity: 87 and 90% for human and mouse enzymes, isolated and also for the insensitivity to oxidation.respectively. A sample of the Fe3//Fe2/ pig allantoic The specific activity of the human enzyme isolatedfluid enzyme treated similarly exhibited Ç93% loss of from iron-supplemented medium was lower than thatactivity upon oxidation. Visible spectra for the oxidized obtained from unsupplemented medium, but was morehuman, mouse, and pig enzymes exhibited lmax values sensitive to inhibition by peroxide. The metal ion con-at Ç560 nm. tent increased from 1.3 to 1.5 g atom/mol as a result

The fully reduced human and mouse enzymes were of supplementation. Metal ion analysis of the mousecharacterized kinetically using the substrates p-ni- enzyme isolated from supplemented medium showedtrophenyl phosphate, phosphotyrosine, pyrophosphate, the presence of a full complement of iron. Both enzymesand FmocEEY(P)AA. These results are summarized in were activated at least 10-fold on treatment with iron/Table II, together with data for the nonrecombinant

b-mercaptoethanol. The results are consistent with thepig and mouse enzymes. proposition that when the medium is supplementedwith iron, there is an increase in the amount of enzyme

DISCUSSION synthesized in the FeIII–FeII form, and this becomessubstantially oxidized to the inactive FeIII–FeIII formIn the first report of the expression and purificationafter secretion into the culture medium and/or duringof active recombinant purple acid phosphatase (5), thethe purification.human enzyme was expressed in baculovirus-infected

Both the human and mouse enzymes underwent pro-insect cells unsupplemented with metal ions. The prop-teolytic cleavage either during the isolation procedureerties of the enzyme isolated in the present study are

comparable in terms of yield, specific activity, and elec- or upon storage of the purified enzyme. It was esti-

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235RECOMBINANT MOUSE AND HUMAN PURPLE ACID PHOSPHATASES

TABLE II

Kinetic Constants for Recombinant and Nonrecombinant Purple Acid Phosphatases

kcat (s01), Km (mM)

Substrate pH Human Mouse (recomb) Mouse (spleen) Pig

p-NPP 2.5 24 { 1, 23 { 4 25 { 1, 42 { 5a ND 52 { 1, 35 { 34.9 296 { 6, 2443 { 150 207 { 5, 710 { 70a 370 { 7,c 240 { 20 380 { 10, 750 { 70

Phosphotyrosine 2.5 54 { 2, 94 { 11 41 { 3, 115 { 25 ND 42 { 0.5, 64 { 44.9 Ç205, Ç14,000 220 { 40, 7000 { 2500 ND 203 { 8, 4250 { 150

Pyrophosphate 2.5 25 { 2, 4.3 { 1.2 18 { 1, 8 { 2.b ND 38 { 1, 13 { 14.9 120 { 3, 800 { 100 92 { 2, 370 { 30b 130 { 20,c 300 { 100 160 { 5, 890 { 80

FmocEEY(P)AA 2.5 ND ND ND 35 { 2, 5.6 { 0.94.9 ND 5 { 0.2, 12 { 2b 37 { 4,c 35 { 7 100 { 17, 120 { 5

a Determined using fully reduced enzyme from the large-scale preparation of enzyme from iron-supplemented medium. The enzyme wasÇ50% nicked.

b Determined using fully reduced enzyme from the small-scale preparation. The enzyme was intact.c kcat values for the pure mouse spleen enzyme were calculated from the values obtained for the partially purified enzyme using the

estimated purity of the enzyme (18%).

mated that the specific activity of the mouse enzyme mouse Cys 163), thus eliminating the possibility of di-sulfide bond linkage of the two large fragments.increased threefold upon nicking. Similar nicking of

the nonrecombinant bovine, rat, and human purple The available data show that the recombinant andnonrecombinant mouse enzymes are similar in termsacid phosphatases has been reported previously (3, 35,

36). The specific activity of the nicked form of the bo- of their molecular and enzymatic properties. They havesimilar sizes, undergo a similar pattern of proteolyticvine enzyme is also threefold higher than that of the

intact enzyme. Sequence analysis of the mouse enzyme nicking with accompanying increase in specific activity,and have similar Km values for the substrates p-NPP,indicated that the nicking occurs after Gly 181, which

corresponds closely to the cleavage site identified in the pyrophosphate, and FmocEEY(P)AA. Comparison ofkcat values is less precise because of the effect of nickingbovine enzyme (35). Although no crystal structure has

been determined for the mammalian purple acid phos- and the low level of purity of the spleen enzyme. Giventhe difficulties associated with the isolation of thephatase, a structure has been modeled based on the

structure of the red kidney bean enzyme (37). In this mouse enzyme, further studies will be facilitated bythe availability of the recombinant protein.model, the region where the mouse and bovine enzymes

are nicked forms an exposed loop near the active site. Kinetic studies on the human, mouse, and pig en-zymes were undertaken at pH 4.9 and also at pH 2.5The two larger, diffuse fragments seen on SDS–PAGE

probably correspond to differently glycolysated forms (Table II). The latter pH is close to the pH of the boneresorptive space. The enzymes lost negligible activityof the N-terminal fragment. Two forms of the intact

enzyme are also present, consistent with the presence over several hours at 257C at pH 2.5. For all substratesexamined, and for all of the enzymes, both the kcat andof differently glycosylated species. The N- and C-termi-

nal fragments are apparently disulfide-linked, as has Km decreased as the pH decreased. The ratio kcat/Km,which provides a measure of catalytic efficiency, wasalso been observed for the nonrecombinant bovine, rat,

and human enzymes. The disulfide must involve Cys significantly higher at pH 2.5 than 4.9. For example,for the human enzyme catalyzing the hydrolysis of py-163 (mouse enzyme numbering), the only Cys residue

in the larger N-terminal fragment, and one of either rophosphate, kcat/Km was 5.8 1 106 at pH 2.5 comparedwith 0.151 106 at pH 4.9. Of note also are the micromo-Cys 211 or 230 in the smaller C-terminal fragment. In

the case of the human enzyme, nicking occurred to a lar Km values for the phosphotyrosyl peptide. Previousstudies on the pig enzyme (2) showed that the enzymesmall extent during storage, to form fragments which

were not disulfide crosslinked. Limited N-terminal se- prefers phosphotyrosyl peptide substrates containingacidic residues and that the presence of the Fmoc groupquencing showed that cleavage occurred after Ala 181.

The larger fragment was smaller than the equivalent also contributes to the low Km value seen for these sub-strates. Substrates such as FmocEEY(P)AA thus pro-fragment formed from the mouse enzyme, indicating

that a second cleavage at the carboxyl-terminal end of vide the basis for the design of effective competitiveinhibitors of the purple acid phosphatases. We are cur-the larger fragment of the human enzyme may have

occurred. It seems likely that this cleavage excised a rently investigating the ability of a series of stable ana-logs of FmocEEY(P)AA to inhibit the enzymes. Furtherpeptide encompassing Cys 161 (equivalent to the

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236 MARSHALL ET AL.

W. H., Carlton, M. B., Evans, M. J., and Cox, T. M. (1996) Devel-studies of the function of the enzyme in osteoclasts willopment 122, 3151–3162.be facilitated by the availability of specific inhibitors

12. Rifkin, B. R., and Gay, C. V. (1992) Biology and Physiology ofof the type described. Given the apparent importancethe Osteoclast, CRC Press, Boca Raton, FL.

of the enzyme in bone resorption, selective inhibitors13. Ek-Rylander, B., Flores, M., Wendel, M., Heinegard, D., and

may also find therapeutic use in the treatment of bone- Andersson, G. (1994) J. Biol. Chem. 269, 14853–14856.resorptive diseases. 14. Robinson, D. B., and Glew, R. H. (1980) J. Biol. Chem. 255,

The observation that pyrophosphate, phosphotyro- 5964–5870.sine, and the phosphotyrosyl peptide FmocEEY(P)AA 15. Campbell, H. D., Dionysius, D. A., Keough, D. T., Wilson, B. E.,

de Jersey, J., and Zerner, B. (1978) Biochem. Biophys. Res. Com-are efficiently hydrolyzed at low pH is consistent withmun. 82, 615–620.the hypothesis that the osteoclast enzyme functions to

16. Campbell, H. D., and Zerner, B. (1973) Biochem. Biophys. Res.hydrolyze either pyrophosphate or an acidic phospho-Commun. 54, 1498–1503.protein (e.g., osteopontin) in the osteoclastic resorptive

17. Ketcham, C. M., Baumbach, G. A., Bazer, F. W., and Roberts,space during bone resorption. It has been reported that R. M. (1985) J. Biol. Chem. 260, 5768–5776.osteoclasts bind to osteopontin coated onto glass (38) 18. Janckila, A. J., Latham, M. D., Lam, K. W., Chow, K. C., Li,and that when the protein is partially dephosphory- C. Y., and Yam, L. T. (1992) Clin. Biochem. 25, 437–443.lated by purple acid phosphatase it no longer supports 19. Allen, S. H., Nuttleman, P. R., Ketcham, C. M., and Roberts,

R. M. (1989) J. Bone Miner. Res. 4, 47–55.binding (13). The specific residues dephosphorylated20. Stepan, J. J., Lau, K. H., Mohan, S., Singer, F. R., and Baylink,have not been identified. Whether acidic peptides con-

D. J. (1990) Biochem. Biophys. Res. Commun. 168, 792–800.taining phosphoserine/phosphothreonine are also good21. Chen, J., Yam, L. T., Janckila, A. J., Li, C. Y., and Lam, W. K. W.substrates for the enzyme at low pH is not known.

(1979) Clin. Chem. 25, 719–722.However, we have previously noted a preference by the

22. Tavassoli, M., Rizo, M., and Yam, L. T. (1980) Cancer 45, 2400–pig enzyme for phosphate ester substrates possessing 2403.good leaving groups (1) and therefore expect that the 23. Drexler, H. G., and Gignac, S. M. (1994) Leukemia 8, 359–368.kcat values for phosphoseryl/phosphothreonyl peptides 24. Cheung, C. K., Panesar, N. S., Haines, C., Masarei, J., and Swa-will prove to be lower than for the corresponding phos- minathan, R. (1995) Clin. Chem. 41, 679–686.

25. Cassady, A. I., King, A. J., Cross, N. C. P., and Hume, D. A.photyrosyl peptides.(1993) Gene 130, 201–207.

26. Frangioni, J. V., and Neel, B. G. (1993) Anal. Biochem. 210, 179–ACKNOWLEDGMENTS 187.

27. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seid-This work was supported in part by grants from the NH & MRCman, J. D., Smith, J. A., and Struhl, K. (1990) Current Protocolsand the University of Queensland. The CMCB is a Special Researchin Molecular Biology, Wiley, New York.Centre of the Australian Research Council.

28. Lord, D. K., Cross, N. C. P., Bevilacqua, M. A., Rider, S. H., Gor-man, P. A., Groves, A. V., Moss, D. W., Sheer, D., and Cox, T. M.

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