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Originally published online as doi:10.1189/jlb.0504287 on November 12, 2004

Published online before print November 12, 2004
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(Journal of Leukocyte Biology. 2005;77:209-218.)
© 2005 by Society for Leukocyte Biology

Properties and expression of human tartrate-resistant acid phosphatase isoform 5a by monocyte-derived cells

Anthony J. Janckila*,{dagger},1, Ranga N. Parthasarathy{ddagger},§,¶, Latha K. Parthasarathy{ddagger}, Ratnam S. Seelan{ddagger}, Yi-Cheung Hsueh*, Jukka Rissanen||, Sari L. Alatalo**, Jussi M. Halleen||,** and Lung T. Yam*,{dagger}{dagger}

* Special Hematology Laboratory and the
{ddagger} Neuroscience and Bioinformatics Laboratory of the U.S. Department of Veterans Affairs Medical Center, Louisville, Kentucky; Departments of
{dagger} Microbiology and Immunology,
§ Biochemistry and Molecular Biology,
Psychiatry, and
{dagger}{dagger} Medicine, University of Louisville School of Medicine, Kentucky;
|| Pharma Test Services, Ltd., Turku, Finland; and
** Department of Anatomy, Institute of Biomedicine, University of Turku, Finland

1 Correspondence: U.S. Department of Veterans Affairs Medical Center, 800 Zorn Avenue, Louisville, KY 40206. E-mail: anthony.janckila{at}med.va.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human serum tartrate-resistant acid phosphatase exists as two enzyme isoforms (TRACP 5a and 5b), derived by differential, post-translational processing of a common gene product. Serum TRACP 5b is from bone-resorbing osteoclasts (OC) and becomes elevated in diseases of increased bone resorption. TRACP 5a is secreted by macrophages (M{Phi}) and dendritic cells (DC) and is increased in many patients with rheumatoid arthritis. Our purpose was to fully characterize the properties of human TRACP isoforms and to produce an antibody specific to TRACP 5a for use as a biomarker in chronic inflammatory diseases. Partially purified, natural serum TRACP isoforms and recombinant TRACP 5a (rTRACP 5a) were compared with respect to specific activity and subunit structure and presence of sialic acid. Mice were immunized with rTRACP 5a, and resulting hybridomas were screened for monoclonal antibody to serum TRACP 5a. One antibody, 220, was tested for its epitope specificity and use in various immunological techniques. rTRACP 5a had properties identical to serum TRACP 5a. Antibody 220 was specific for the trypsin-sensitive epitope in the loop peptide, present only in TRACP 5a. Antibody 220 was effective for specific immunoprecipitation, immunoassay, and immunoblot of TRACP 5a. Intact TRACP was present in M{Phi}, DC, and OC. TRACP 5a was the predominant isoform secreted by M{Phi} and DC, whereas TRACP 5b was the predominant isoform secreted by OC. TRACP isoforms 5a and 5b may have different functions inside and outside of monocyte-derived cells. Antibody 220 is an important resource for studies of the biosynthetic relationship among TRACP isoforms and of the significance of serum TRACP 5a as a marker in diseases of bone metabolism and inflammation.

Key Words: macrophage • dendritic cell • osteoclast • immunoassay • immunocytochemistry


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human acid phosphatases are a diverse group of enzymes that hydrolyze a variety of natural and synthetic phosphoesters at an optimal pH of 5–6. There are seven acid phosphatases in hematopoietic tissues that can be separated electrophoretically and identified by histochemical staining [1 ]. Only the most cationic, band-5 or type-5, is resistant to inhibition by L(+) tartaric acid; hence, it is called tartrate-resistant acid phosphatase (TRACP). Type-5 TRACP is expressed intracellularly to high levels in differentiated cells of monocytic lineage including osteoclasts (OC) [2 ], macrophages (M{Phi}) [3 ], and dendritic cells (DC) [4 ].

Type-5 TRACP is a multifunctional metalloenzyme containing a pair of spin-coupled iron ions in the active site, similar to related purple acid phosphatases (PAP) throughout the plant and animal kingdoms [5 ]. In vitro, TRACP can function as a protein phosphatase, active against phosphotyrosine-containing peptides [6 ] and phosphoserine-containing osteopontin [7 ]. One member of the family, porcine uteroferrin, functions to transport iron transplacentally to provide the developing fetus with iron for hematopoiesis [8 ]. TRACP also can generate reactive oxygen species (ROS) through its redox active iron by way of the Fenton reaction [9 , 10 ]. Evidence indicates that ROS-mediated intracellular collagen fragmentation within transcytotic vesicles of bone-resorbing osteoclasts is facilitated by redox active iron in TRACP [11 ]. An analogous function of macromolecular fragmentation by TRACP may exist in M{Phi} within their antigen-processing compartments [12 , 13 ]. Therefore, TRACP could be an important functional link between the skeletal and immune systems and a useful biomarker for disease.

In serum, TRACP exists as two related glycoprotein isoforms derived post-translationally by differential processing of a single gene product. TRACP 5a is a continuous polypeptide of 33 kD, in which one of its asparaginyl oligosaccharides is processed to complex acidic units containing N-acetyl neuraminic acid (sialic acid) [14 ]. TRACP 5b, conversely, contains no discernable sialic acid and is processed proteolytically to yield disulfide-linked 16 kD and 23 kD subunits [15 ]. Through studies of recombinant TRACP (rTRACP), it is determined that the protease-sensitive site is within a so-called "loop peptide," which interacts with the active site through an aspartate residue at position 146 [16 ]. The loop peptide is believed to regulate the catalytic properties of TRACP. Thus, intact TRACP 5a has a pH optimum of 5.2 and a low specific activity of ~200 U/mg, whereas cleaved TRACP 5b has a pH optimum of 5.8 and high specific activity of ~2000 U/mg [17 ].

During bone resorption, OC release TRACP 5b into the circulation, where it can be quantified by biochemical assay or immunoassay. Serum TRACP 5b activity correlates to other biochemical markers of bone turnover and can be used as a surrogate marker of the number of osteoclasts in diseases of bone metabolism [18 ] and to monitor antiresorptive treatment in osteoporosis [19 ]. TRACP 5a normally accounts for 85–90% of the total TRACP protein in serum. In vitro, M{Phi} and DC secrete only TRACP 5a, not TRACP 5b [20 ]. It is possible that TRACP 5a may be a marker for these cells and that serum TRACP 5a may have clinical relevance in inflammatory diseases. Indeed, serum TRACP 5a protein levels are increased in about one-third of patients with rheumatoid arthritis (RA), but because of its low specific activity, elevated levels are not apparent with current assays for activity. Therefore, we have cloned human TRACP into Chinese hamster ovary cells (CHOTRACP 8F5), which now secrete a functional protein with properties such as serum isoform 5a. Using this rTRACP 5a, we developed a monoclonal antibody (mAb) that reacts with intact TRACP proteins, including natural serum isoform 5a but not natural serum isoform 5b. We intend to use this antibody along with other TRACP-specific antibodies to study TRACP isoform expression in chronic inflammatory, autoimmune, and metabolic bone diseases. However, before we can use this antibody as a cell or disease marker, we must understand more thoroughly the biochemical and cell biological relationships between TRACP isoforms. Our purpose in these studies was to characterize TRACP isoforms, confirm their cell sources, and document the isoform specificity, target epitope, and spectrum of useful applications for our novel anti-TRACP antibody. Our long-term goal is to investigate the mechanisms of regulated expression of TRACP isoforms and their functions within the skeletal and immune systems.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Purification of natural and rTRACP isoforms
De-identified waste specimens of blood from therapeutic phlebotomy of patients with hemochromatosis were obtained from the hematology clinic at the Veterans Affairs Medical Center (Louisville, KY). Hemochromatosis specimens were used because of their abundant supply, which facilitated purification of sufficient, natural TRACP 5a and 5b for biochemical analyses. Blood plasma was converted to serum by adding calcium chloride to 10 mM and thrombin to 0.1 unit/mL. After clotting overnight at 4°C, the samples were centrifuged, and the supernatant serum was stored at –70°C. Serum was subjected to SP-sepharose chromatography as described previously [17 ]. Total TRACP activity and protein were detected by a rapid biochemical assay using 4-nitrophenyl phosphate (4-NPP) as substrate and by enzyme immunoassay (EIA) [21 ], respectively. The enzyme peaks were pooled separately, made to 40% saturation in ammonium sulfate, and further purified by hydrophobic interaction chromatography. Samples were added to a 1.6 x 12-cm column of phenyl-sepharose (Pharmacia-Amersham, Little Chalfont, UK), equilibrated to 10 mM sodium citrate, 40% ammonium sulfate saturation, pH 5.0. After washing the column with equilibration buffer, TRACP was eluted by a 100-mL linear gradient from 40% to 0% ammonium sulfate, collecting 100 equal fractions. TRACP activity peaks were identified by a rapid colorimetric assay at the optimal pH for each isoform. The partially pure TRACP isoforms were dialyzed against 100 mM sodium acetate buffer, pH 5.5, containing 2% glycerol and then concentrated by filter centrifugation (Amicon, Lexington, MA). Partially purified serum TRACP 5a and 5b was determined by immunoassay to have 300 ng/mL and 50 ng/mL, respectively.

rTRACP 5a was purified from the culture supernatant of exponentially growing CHOTRACP 8F5 cells [22 ] in a manner identical to that for serum TRACP isoforms. After hydrophobic interaction chromatography on phenyl sepharose, the rTRACP 5a was further purified on an antibody 14G6-agarose affinity column. Purified rTRACP 5a was dialyzed and concentrated like the serum TRACP enzymes. rTRACP 5a was determined to contain 1.1 mg/mL, according to the method of Lowry et al. [23 ].

Production of anti-TRACP 5a mAb
Female Balb/c mice were immunized with affinity-purified rTRACP 5a protein according to previously published protocol [24 ]. Immune spleen cells were fused with NS-1 plasmacytoma at a ratio of three spleen cells to one NS-1 cell using 50% polyethylene glycol-4000. Hybridomas were grown and selected in hypoxanthine-aminopterin-thymidine medium. On day 12 after seeding, wells showing significant growth were screened for antibody against the immunizing rTRACP 5a protein by EIA. Positive wells were rescreened against partially purified serum 5a and 5b (see below) to detect those with isoform specificity. Wells showing specificity for isoform 5a were expanded and subcloned by three rounds of limiting dilution to confirm monoclonality. Clones having the highest antibody titer were expanded and implanted intraperitoneally into mice for the production of ascites. Immunoglobulin G (IgG) was purified from ascites using protein G affinity columns according to the manufacturer’s protocol (Pierce Chemical Co., Rockford, IL).

Biochemical assays for TRACP
Biochemical activity of TRACP was determined by a rapid colorimetric microplate method [25 ] modified by using 10 mM 4-NPP as substrate in the presence of 50 mM sodium tartrate in a total reaction volume of 200 µL. After incubation of sample with substrate for 1 h, the reaction was stopped by adding 50 µL 3M NaOH. Routinely, 100 mM sodium acetate with 50 mM tartrate was used as substrate buffer. TRACP 5a activity was measured at pH 5.2 and TRACP 5b activity at pH 5.8. For some experiments, activity was determined over a pH range of 4.0–8.0 for which 100 mM sodium citrate was used as substrate buffer from pH 4.0 to pH 6.5, and 100 mM Tris was used from pH 7.0 to pH 8.0.

Immunoassays for TRACP activity and protein
Immunoassays for TRACP activity and protein were done according to previously published methods with some minor modifications [21 ]. For isoform 5a assays, NeutrAvidin microwells (Pierce Chemical Co.) were coated for 2 h at room temperature with 1 µg antibody 220 in 100 µL phosphate-buffered saline (PBS). After washing three times with PBS, samples were added in a final volume of 100 µL immunoassay buffer (20 mM Tris, 150 mM NaCl, 10 mM EDTA, 2% glycerol, pH 7.5) and were incubated overnight at 4°C. For isoform 5a activity, wells were washed four times with Tris-buffered saline containing 0.05% Tween-20 and then filled with 200 µL substrate solution (10 mM 4-NPP in 100 mM sodium acetate/50 mM sodium tartrate, pH 5.8). The reaction was carried out for 1 h and then stopped with 50 µL 3M NaOH. Absorbance was read at 405 nm. For isoform 5a protein, wells were washed four times with PBS containing 0.05% Tween-20 (PBST) and then filled with 100 µL peroxidase-conjugated anti-TRACP antibody J1B [J1B-horseradish peroxidase (HRP)], optimally diluted in PBST. Second antibody was incubated for 1 h at room temperature. Wells were washed four times with PBST and then filled with 200 µL peroxidase substrate solution (4 mg o-phenylene diamine dihydrochloride and 40 µL 3% H2O2 in 10 mL 50 mM sodium citrate/25 mM sodium phosphate buffer, pH 5.0). Wells were incubated 15 min at room temperature, and the reaction was stopped by adding 50 µL 2 M H2SO4. Absorbance was read at 490 nm. Immunoassays for isoform 5b activity and total protein were done similarly, except that streptavidin wells were coated with 0.5 µg antibody 14G6 to capture all TRACP. Isoform 5b activity was detected with 10 mM 4-NPP substrate at pH 6.1. Total TRACP protein was detected with J1B-HRP. TRACP activity was read from a standard curve constructed with serial dilutions of 4-nirtrophenolate made from 10 to 0.16 U/L. TRACP protein was read from a standard curve constructed from serial dilutions of rTRACP made from 5 to 0.08 ng/mL. The calibration curves for total TRACP protein and isoform 5a protein made with antibody 14G6 and antibody 220 were practically identical; therefore, TRACP 5b protein was calculated as total TRACP protein minus TRACP 5a protein.

Polyacrylamide gel electrophoresis (PAGE) and Western blotting
Nondenaturing PAGE in 7.5% acrylamide, pH 4.0, was conducted according to a published method [1 ]. Activity bands were stained with naphthol ASBI phosphate as substrate and fast garnet GBC as coupler at pH 5.5. Sodium dodecyl sulfate-PAGE, under nonreducing and reducing conditions, was conducted according to a published method [26 ] in 12% polyacrylamide slab gels. Proteins were stained directly with Coomassie blue or transferred to polyvinyl difluoride membranes and detected by an alkaline phosphatase-antialkaline phosphatase (APAAP) method using anti-TRACP antibody 9C5 to detect both isoforms [24 ] or the newly developed anti-TRACP antibody 220 to detect only isoform 5a (this paper).

Digestion of rTRACP with sialidase and trypsin
Purified rTRACP 5a protein (1 µg) was digested with 180–720 mU N-acetyl neuraminidase (sialidase; Sigma Chemical Co., St. Louis, MO) for 60 min at 37°C in 200 µL digest buffer (100 mM Na acetate, pH 5.0, containing 0.1% Triton X-100). Enzyme digests were analyzed directly by electrophoresis and immunoassay or stored at –70°C for later analyses. Purified rTRACP 5a protein (1 µg) was also digested with 100–400 mU trypsin-agarose for 60 min at room temperature in 200 µL digest buffer (10 mM Tris, pH 7.0, containing 0.1% Triton X-100). After digestion, the solid-phase trypsin was removed by centrifugation, and the supernatant digest was analyzed directly by gel electrophoresis and immunoassay or stored at –70°C for later analyses.

Preparation of M{Phi}, DC, and OC
M{Phi} and DC were prepared by culture of blood monocytes obtained from waste specimens from therapeutic phlebotomy of patients with hemochromatosis. Monocytes were isolated from the mononuclear cell fraction by overnight adherence on plastic tissue-culture dishes. M{Phi} or DC were developed by culture for 6 days in the absence (M{Phi}) or presence (DC) of 20 ng/mL interleukin-4 and 25 ng/mL granulocyte M{Phi}-colony stimulating factor (GM-CSF), according to previously described methods [27 ]. After the culture period, the supernatant medium was reserved, and the cells were washed three times in Hanks’ balance salt solution. Washed cells were pelleted by centrifugation and resuspended at 107/mL in lysis buffer of 10 mM Tris, pH 7.4, containing 300 mM sodium chloride, 0.5% Nonidet P-40, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1x protease inhibitor cocktail (BioVision, Inc., Mountain View, CA). Lysates were incubated 30 min on ice and then centrifuged to remove insoluble debris. Lysates and supernatant medium were stored at –70°C until analyzed by electrophoresis and immunoassay.

Human osteoclast precursor cells (PoieticsTM, Cambrex Bio Science Walkersville, Inc., Walkersville, MD) were suspended into {alpha}-minimal essential medium (MEM) containing 10% fetal bovine serum, penicillin/streptomycin, and HEPES. Cells were allowed to attach to bovine bone slices (20,000 cells/slice) for 1 h. Bone slices were then transferred to 24-well tissue-culture plates containing {alpha}-MEM medium with receptor activator of nuclear factor-{kappa}B ligand (RANKL; PeproTech EC Ltd., London, UK) and M-CSF (R&D Systems, Minneapolis, MN). Cells were incubated in a CO2 incubator in a humidified atmosphere of 95% air and 5% CO2 at 37°C. A single addition of transforming growth factor-ß1(TGF-ß1; R&D Systems) was made on day 3. After complete osteoclast differentiation at day 7, the medium was completely replaced. Cells were cultured for an additional 2 days, allowing the formed OC to resorb bone. One set of triplicate wells was used to harvest culture supernatant for TRACP measurements and to prepare fixed cells on the bone slices for cytochemistry and immunocytochemistry (see below). A second set of triplicate wells was used to prepare cell lysates from the cells attached to the bone slices, which were washed with three changes of cold PBS and then lysed by incubating in 200 µL lysis buffer [10 mM Tris-HCl, pH 7.4, containing 300 mM NaCl, 0.5% Triton X-100, 1 mM EGTA, 1 mM PMSF, and 1x protease inhibitor cocktail (CompleteTM, Mini, EDTA-free, Boehringer Mannheim, Mannheim, Germany)] for 10 min on ice. Cells in lysis buffer were repeatedly frozen in dry ice and thawed three times. Cell lysates were centrifuged to remove insoluble debris. Culture supernatants and cell lysates were stored at –70°C until analyzed. In one experiment, we compared TRACP isoform secretion by RANKL-differentiated OC to undifferentiated OC precursors. An additional, complete medium change was done on day 9 of culture, and medium was harvested from quadruplicate wells on day 12 for TRACP isoform immunoassay.

Cytochemistry and immunocytochemistry
Cytocentrifuge smears were prepared from cultured M{Phi} and DC and fixed with buffered formal acetone. Bone slices containing attached OC were washed with PBS and fixed in a solution of 3% paraformaldehyde. TRACP activity was stained using naphthol ASBI phosphate as substrate and hexazotized pararosaniline in a buffer of 100 mM sodium acetate/50 mM sodium tartrate, pH 5.5 [28 ]. Immunocytochemistry for intact TRACP protein was performed with antibody 220, according to published methods [3 ], using the APAAP technique after heat-induced epitope retrieval. Calcitonin receptors (CTR) on OC were stained with a commercial antibody (Serotec, Oxford, UK). Commercial antibodies to CD64 (Ancell Corp., Bayport, MN) and CD86 (Leinco Technologies, St. Louis, MO) were used to stain these lineage markers on M{Phi} and DC, respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characteristics of TRACP isoforms
To document that our rTRACP 5a is a faithful representative of the natural TRACP 5a, we conducted a series of experiments to compare the structure and function of rTRACP 5a to serum TRACP isoforms. Differential migration of serum TRACP isoforms in acidic, nondenaturing PAGE gels is the original criterion used to define serum TRACP 5a and 5b. The slower migration of isoform 5a is a result of the presence of sialic acid [14 ]. Figure 1A shows after digestion with increasing amounts of sialidase, a dose-dependent conversion of rTRACP 5a from a slow migrating form to a fast migrating form such as serum TRACP 5b. We have previously documented that serum TRACP 5a is an intact protein of 33 kD, and serum TRACP 5b is cleaved into two disulfide-linked subunits of 16 and 23 kD [15 , 22 ]. Limited proteolysis with increasing amounts of trypsin converted rTRACP 5a from an intact protein to a cleaved 5b-like protein (Fig. 1B) . Only the 16-kD fragment is seen as a result of the specificity of antibody 9C5 antibody used for Western blot. Figure 2A shows that intact rTRACP 5a has a pH optimum of 5.2, like that of serum 5a, whereas serum TRACP 5b has a pH optimum of 5.8. Sialidase digestion had no significant effect on biochemical activity of rTRACP 5a, whereas trypsin proteolysis caused an increase in activity and pH optimum, consistent with conversion from isoform 5a to 5b (Fig. 2B and 2C) .



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  		Figure 1. Structural comparisons among serum TRACP 5a, serum TRACP 5b, and rTRACP. (A) rTRACP (1 µg) was digested with increasing amounts of sialadase in 200 µL. Each digest (10 µL, 50 ng) was then subjected to nondenaturing PAGE. For comparison, 5 µL partially pure serum isoform 5a and isoform 5b were run simultaneously. Gels were stained for TRACP activity using naphthol ASBI-P as substrate and fast garnet GBC as coupler at pH 5.5. (B) rTRACP (1 µg) was digested with increasing amounts of trypsin in 200 µL. Each digest (10 µL, 50 ng) was then subjected to Western blot under nonreducing or reducing conditions. Blots were labeled by the APAAP method using anti-TRACP antibody 9C5 as probe and stained with nitroblue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP), as described in Materials and Methods.

 


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  		Figure 2. pH optima of rTRACP, serum TRACP 5a, and serum TRACP 5b. (A) Purified rTRACP and partially purified serum TRACP isoforms were assayed for biochemical activity over a pH range of 4.0–8.0. The effects of sialidase (B) and trypsin (C) on the activity and pH optimum of rTRACP were also determined.

 
Production of mAb to rTRACP 5a
Having demonstrated that rTRACP 5a from CHOTRACP 8F5 cells bears the structural and functional properties of serum TRACP 5a, we used it to immunize mice to generate mAb to epitopes specific to isoform 5a. Of 1000 wells plated with cell fusion products, 440 wells showed clonal growth after 2 weeks. Of these, 25 produced specific antibody to immunizing rTRACP. Four of these clones produced antibody that reacted with serum isoform 5a but not serum isoform 5b. Two of these survived expansion and three rounds of subcloning by limiting dilution. Antibody from clone 220 captured equivalent TRACP activity as antibody 14G6 in immunoassay, and antibody from clone 89 captured about half as much activity. Clone 220 was used to prepare purified antibody from ascites for further experiments. The isotype of antibody 220 is IgG1-{kappa}.

Specificity of anti-TRACP antibody 220 for isoform 5a
Antibody14G6 IgG and antibody 220 IgG were conjugated to Amino-Link Plus agarose (Pierce Chemical Co.) at 2.5 mg IgG/mL gel to make solid-phase antibodies for immunoprecipitation of TRACP from serum. IgG from nonimmune control mouse ascites (NMA) was also conjugated to agarose. Antibody-agarose (10 µL) was added to 200 µL serum sample and incubated with mixing at 4°C for 16 h. The immune complexes were removed by centrifugation, and the supernatant sera were subjected to nondenaturing PAGE (Fig. 3 ). Untreated serum (Nil) and serum from control immunoprecipitation (NMA) contain activity bands in 5a and 5b positions. The bands stained for TRACP activity after specific immunoprecipitations show that antibody 14G6 reacted with and removed isoforms 5a and 5b (14G6), and antibody 220 reacted with and removed only isoform 5a (220). These data substantiate that antibody 220 is specific for serum TRACP 5a, as originally defined by a nondenaturing PAGE technique.



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  		Figure 3. Specific immunoprecipitation of serum TRACP 5a by antibody 220. Human serum (50 µL) was subjected to nondenaturing PAGE after removal of TRACP by immunoprecipitation with nothing (Nil), NMA IgG, control antibody 14G6 IgG (14G6), or antibody 220 IgG (220). Gels were stained for TRACP activity using naphthol ASBI-P as substrate and fast garnet GBC as coupler at pH 5.5.

 
Pooled serum (400 mL) from hemochromatosis patients was subjected to chromatographic separation on SP-sepharose. TRACP activity and protein peaks were identified by immunoassay of chromatographic fractions using antibody 14G6 or antibody 220 to capture TRACP enzymes. Figure 4 shows that TRACP 5a and TRACP 5b were easily separated and detected by immunoassay for activity (Fig. 4A) or protein (Fig. 4B) with antibody 14G6. Antibody 220 reacted only with the isoform 5a peak.



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  		Figure 4. Specificity of antibody 220 for serum TRACP 5a. Human serum (400 mL) was chromatographed on a 1.6 x 25-cm SP-sepharose column. Fractions were assayed for total TRACP activity at pH 5.5 (A) and total TRACP protein (B) by enzyme-capture immunoassay and two-site immunoassay, respectively, using control antibody (Ab) 14G6 or antibody 220 to capture enzyme antigen.

 
Identification of the epitope recognized by antibody 220
To determine which isoform 5a epitope—sialic acid or loop peptide—was the target for antibody 220, purified rTRACP 5a was subjected to Western blot and immunoassay before and after sialidase or trypsin digestion. Western blots of undigested and digested rTRACP 5a show that control antibody 9C5 reacted with a unique epitope on the 16-kD subunit, which is unaffected by digestions (Fig. 5A ). Antibody 220 reacted with rTRACP 5a after digestion with sialidase but not after digestion with trypsin, whether under nonreducing or reducing conditions (Fig. 5B) . Therefore, the specificity of antibody 220 for isoform 5a is based on its binding to the trypsin-sensitive site within the loop peptide of intact TRACP and not to sialic acid on the oligosaccharides. Removal of sialic acid had little effect on the specific activity of rTRACP 5a (Fig. 6A ) or the ability of either antibody to capture TRACP protein (Fig. 6B) in immunoassays. rTRACP 5a activity was approximately sixfold higher when bound by antibody 220 compared with antibody 14G6, whether sialic acid had been removed or not. Trypsin cleavage increased over threefold the specific activity of rTRACP captured by antibody 14G6 but destroyed the 220 binding site, causing a loss of antibody 220 reactivity in enzyme activity and protein immunoassays.



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  		Figure 5. Identification of trypsin cleavage site as target epitope for antibody 220. Undigested (Cont), sialidase-digested (Sial), or trypsin-digested (Tryp) rTRACP 5a (50 ng) was subjected to Western blot. Samples were run under nonreducing and reducing conditions and probed with control antibody 9C5 (A) or antibody 220 (B). Blots were disclosed by APAAP method and stained with NBT-BCIP as described in Materials and Methods.

 


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  		Figure 6. Antibody 220 specificity for intact TRACP isoforms. After digestion of rTRACP with 640 mU sialidase or 400 mU trypsin, equal aliquots were subjected to immunoassays for TRACP activity (A) and protein (B) with control antibody 14G6 or antibody 220 as capture antibody. Bound TRACP activity was measured with 4-NPP at pH 5.8. Bound TRACP protein was detected with antibody J1B-HRP.

 
Figure 7 illustrates that in addition to increasing its specific activity, antibody 220 binding caused an increase in the pH optima of rTRACP 5a and serum TRACP 5a to that of serum TRACP 5b. Therefore, antibody 220, through its binding to loop peptide at the trypsin-sensitive site of intact TRACPs, has the same effects as trypsin cleavage. The nature of the target epitope for antibody 220 indicates that it will react with intact TRACP polypeptide precursors as well as the processed, mature isoform 5a.



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  		Figure 7. Effect of antibody 14G6 and antibody 220 binding on the pH optima of TRACP isoforms in EIA. Purified rTRACP ({circ}) or partially purified serum isoforms 5a (•) and 5b ({blacksquare}) were assayed for bound activity from pH 4.0 to pH 7.5 using antibody (Ab) 14G6 (A) or antibody 220 (B) to capture enzyme antigen.

 
Localization of intact TRACP
With an antibody specific to the loop peptide of TRACP 5a, we sought to strengthen and expand our previous hypothesis that TRACP isoforms are compartmentalized differently in M{Phi}, DC, and OC [20 ]. Smears of day 6 M{Phi} and DC, as well as RANKL-differentiated OC were stained cytochemically for TRACP activity and immunocytochemically for TRACP protein. Figure 8 shows that M{Phi}, DC, and OC express TRACP activity (A–C). Intact TRACP protein disclosed with antibody 220 (D–F) was also expressed by all cells. The lineage markers CD64 (G), CD86 (H), and CTR (I) were expressed on M{Phi}, DC, and OC, respectively. These data show that not only can antibody 220 be used for immunoassay and Western blotting, it is also useful for immunocytochemical localization of intact TRACP.



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  		Figure 8. Cytochemical and immunocytochemical localization of TRACP and differentiation markers in human M{Phi}, DC, and OC. (A–C) Cytochemical stain for TRACP activity. (D–F) Immunocytochemical stain for intact TRACP using antibody (Ab) 220. (G–I) Immunocytochemcial stain for differentiation markers CD64 on M{Phi}, CD86 on DC, and CTR on OC. For M{Phi} and DC, bar = 10 µm; for OC, bar = 100 µm.

 
M{Phi}, DC, and OC cultures were also examined by immunoassay for expression of intact TRACP isoforms, including TRACP 5a intracellularly and extracellularly. With the understanding that "TRACP 5a" in these experiments also includes all intact TRACP molecules, TRACP 5a activity was estimated as the enzymatic activity bound by antibody 220 measured at pH 5.8. TRACP 5b activity was estimated as the enzymatic activity bound by antibody 14G6 measured at pH 6.1. TRACP 5a protein was estimated as the amount of TRACP bound by antibody 220 and detected with anti-TRACP J1B-HRP. TRACP 5b protein was estimated as total TRACP protein bound by antibody 14G6 and detected with antibody J1B-HRP minus TRACP 5a protein. Figure 9A and 9B , shows that the proportions of TRACP isoforms expressed by M{Phi} and DC are equivalent in supernatant medium and cell lysates. TRACP 5a activity and protein predominate in culture supernatants, and TRACP 5b activity and protein predominate intracellularly. Although M{Phi} and DC secreted similar amounts of both isoforms into the supernatant, DC synthesized and kept much more of both isoforms intracellularly. In contrast to M{Phi} and DC, isoform 5b was the predominant TRACP protein in OC supernatants, although significant isoform 5a was also present (Fig. 9C) . The isoform 5a could have come from osteoclasts or contaminating mononuclear precursors. Note as well that the amount of isoform 5a activity in OC supernatants is higher than isoform 5b; this is because isoform 5a activity is significantly activated when bound by antibody 220. To determine whether the measured TRACP 5a in OC supernatants was derived from OC or contaminating pre-OC, a follow-up experiment was done, whereby pre-OC were cultured in the absence of RANKL, and OC were differentiated from the same number of precursors in the presence of RANKL. Media were changed on days 7 and 9 and harvested on day 12. Figure 10 shows that the amounts of TRACP 5a activity secreted by OC and their precursors were about the same, and TRACP 5b activity secreted by differentiated OC was clearly greater. However, when TRACP protein was measured, OC appeared to secrete more intact protein than did their precursors. This suggests that intact TRACP 5a-like protein released by OC was a totally inactive form, perhaps a proenzyme.



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  		Figure 9. TRACP isoform expression by cultured M{Phi} (A), DC (B), and bone-resorbing OC (C) in tissue-culture supernatants (TCSN) and cell lysates (lysate). TRACP isoforms were estimated by immunoassays as described in Materials and Methods. Isoform 5a activity is defined as TRACP activity bound by antibody 220 and measured at pH 5.8. Isoform 5b activity is defined as TRACP activity bound by antibody 14G6 and measured at pH 6.1. Isoform 5a protein is defined as TRACP protein bound by antibody 220 and detected with antibody J1B-HRP. Isoform 5b protein is defined as total TRACP protein bound by antibody 14G6 and detected with J1B-HRP minus TRACP 5a protein. Bars represent the mean ± SD of three separate experiments with different donor cells for M{Phi} and DC and for three separate culture wells for OC.

 


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  		Figure 10. Secretion of TRACP isoforms by pre-OC and OC differentiated on bovine bone slices. Pre-OC were cultured in the presence of M-CSF and TGF-ß. OC were differentiated from pre-OC by culture in the presence of M-CSF, TGF-ß, and RANKL as described in Materials and Methods. Fully supplemented media were changed on days 7 and 9. Culture media were harvested on day 12 for immunoassay of TRACP isoform activity (A) and protein (B). Quadruplicate wells from pre-OC were pooled; therefore, bars represent the average TRACP secreted by four cultures. Quadruplicate wells from differentiated OC were harvested separately; therefore, bars represent the mean ± SD of four cultures.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
If TRACP isoforms are to be used as separate cell or disease-specific markers, it is important to know their biochemical and cell biological relationships. Generally, TRACP, which is not cleaved into subunits, has been considered to be a latent proenzyme precursor to cleaved TRACP [29 ]. This concept is based on results from biochemical studies of natural and recombinant enzymes in which the cleaved forms have significantly higher catalytic efficiency. Most natural TRACPs purified from mammalian tissues are processed proteins containing disulfide-linked subunits of ~16 kD and ~23 kD with relatively high specific activity [30 31 32 33 ]. Orlando et al. [33 ] carefully purified beef spleen PAP and showed that in addition to a major cleaved form with high specific activity, an intact form existed as a minor component with little or no catalytic activity. This "native" form was activated significantly in vitro by limited trypsin digestion of an exposed antigenic loop peptide to yield the two-subunit form. Recombinant rat TRACP, made in insect cells infected with TRACP-baculovirus constructs (BacTRACP), is secreted as an intact enzyme with relatively low specific activity. Ljusberg et al. [29 ] have shown that proteolytic processing of BacTRACP with a variety of proteases increases the specific activity and pH optimum to that of purified, natural OC TRACP 5b. X-ray crystallographic studies [34 ] and mutational analyses [16 ] have confirmed that the aspartate residue at position 146 in the loop peptide interacts with the active site, thus having a repressive function. Proteolytic processing of loop peptide relieves this interaction, thereby explaining the increased, specific activity of the cleaved TRACP.

Although intact TRACP protein derived from tissues may indeed be a latent precursor to intracellular, cleaved isoform 5b, there are at least two examples in which an intact TRACP enzyme naturally exists in extracellular fluids. Porcine allantoic fluid and culture supernatant from porcine endometrial explants contain large amounts of uteroferrin, a member of the family of tartrate-resistant PAP. Uteroferrin is secreted as a mature, uncleaved glycoprotein with low specific phosphatase activity and low pH optimum [35 ]. Analogous to uteroferrin, the vast majority of circulating TRACP in human serum is uncleaved isoform 5a with low phosphatase activity and low pH optimum [17 ]. Therefore, in addition to an inactive proenzyme, uncleaved TRACP, after becoming fully glycosylated and modified, such as serum isoform 5a, may be a mature protein with extracellular functions distinct from cleaved TRACP 5b. Human serum has been used rarely as a source of TRACP for detailed study, as it contains only micrograms per liter. To obtain sufficient, natural serum enzymes for comparative analyses, we used an abundant source from therapeutic phlebotomy specimens from hemochromatosis patients. Although the relative levels of TACP 5a and 5b may deviate from normal in these samples, the characteristics of the enzymes are normal and should not have affected our results or interpretations.

After demonstrating the similarities between natural TRACP 5a and recombinant human TRACP 5a, including the presence of sialic acid on asparaginyl oligosaccharides and an intact loop peptide, we prepared sufficient rTRACP 5a in CHO cells to generate a specific mAb to TRACP 5a. One resulting antibody, 220, was shown to be specific for natural TRACP 5a and has potential for specific immunoassay of isoform 5a activity and protein in clinical settings of chronic inflammation. Antibody 220 had the same effects on activity and pH optimum of rTRACP 5a and natural TRACP 5a as trypsin cleavage, suggesting that the target epitope was the trypsin-sensitive site in the loop peptide and not the sialic acid residues. Immunoassay and Western blot of rTRACP after digestion with sialidase or trypsin proved that the target epitope of antibody 220 was indeed the trypsin cleavage site. To our knowledge, only one other antibody (4E6) has been described to be isoform 5a-specific [36 ]. The specificity was reported to be dependent on the sialic acid of TRACP 5a, as antigen-antibody complexes were sensitive to sialidase digestion in a dose-dependent manner [37 ]. It is curious that this antibody was raised against a BacTRACP preparation, which would be exceedingly rare to contain sialic acid. Lepidopterin cells, including Sf9 cells, lack the biosynthetic capacity to synthesize and/or transfer sialic acid to glycoproteins [38 ]. One possible explanation for the apparent sialic acid specificity of antibody 4E6 is that the sialidase enzyme used contained some protease activity.

To establish the full extent and sources of TRACP 5a expression, human M{Phi}, DC, and bone-resorbing OC were stained cytochemically for TRACP activity and immunocytochemically with antibody 220. All cell types stained for TRACP activity and intact TRACP protein. Effective immunostaining with antibody 220 required heat-induced epitope retrieval to re-expose the cross-linked, antigenic determinant after aldehyde fixation [39 ].

We had previously shown by nondenaturing PAGE that M{Phi} and DC preferentially secrete TRACP 5a, and cell lysates contained mostly isoform 5b. [20 ]. Immunoassays of TRACP in culture supernatants and cell lysates provided supporting evidence. In those experiments, the activity in culture supernatants was higher at pH 5.2 than pH 6.1, whereas the reverse was true of TRACP activity in cell lysates. Here, we confirm and extend this finding using the isoform 5a-specific antibody 220 for immunoassay of culture supernatants and cell lysates of M{Phi} and DC. We attempted to use the same immunoassay strategy to determine if OC, unlike M{Phi} and DC, secreted only isoform 5b as we had hypothesized. However, results showed that although isoform 5b was indeed the predominant TRACP secreted by OC cultures, significant isoform 5a was also present. By comparing TRACP isoforms secreted by pre-OC and OC, we were able to determine that some secreted TRACP 5a was derived from the OC themselves but was released as an inactive form, perhaps a proenzyme. Additional, carefully controlled experiments are needed to confirm this possibility. We also showed that an intact TRACP protein was expressed inside OC, M{Phi}, and DC. However, as antibody 220 recognizes the loop peptide and as a noncleaved polypeptide must be a precursor to either isoform, we cannot say whether the intact TRACP within cells was a sialylated, mature isoform 5a or an asialo precursor to isoform 5b. Isoform 5a is the predominant TRACP secreted from M{Phi} and DC in vitro and is the major TRACP isoform in the circulation. This suggests that extracellular TRACP 5a, as an enzymatically active polypeptide, may be a unique, biosynthetic end-product with its own functional and perhaps clinical significance.

We propose a unified, general hypothesis to explain our data regarding differential expression of TRACP isoforms in monocyte-derived cells in context with previously published results. TRACP isoforms 5a and 5b could be important to bone metabolism. Isoform 5b is likely to contribute to intracellular collagen degradation by OC by virtue of its ability to generate ROS [10 , 11 ]. In the process of transcytosis of resorption products, it is released to the circulation. As such, it is an effective biochemical marker of osteoclast number in diseases in which resorbing OC are increased or decreased as postulated by Halleen et al. [40 ]. If OC secrete isoform 5a, it may also have relevance to bone metabolism. It could be secreted into the resorption lacuna through the ruffled border and function there to specifically dephosphorylate osteopontin and other phosphorylated bone matrix proteins in accordance with the postulate of Andersson et al. [41 ]. It has yet to be determined whether the TRACP enzyme that has been identified in the resorption lacuna is isoform 5a or 5b. TRACP isoforms 5a and 5b could also have relevance to the immune response. Similar to OC, DC and M{Phi} may use intracellular TRACP 5b as a mechanism to generate ROS for nonspecific degradation of macromolecules during antigen processing and presentation [12 ]. TRACP 5a is selectively secreted by M{Phi} and DC in vitro and is probably the isoform responsible for increased serum TRACP protein in patients with RA [20 ]. Therefore, secreted TRACP 5a may be a product of activated M{Phi} and an effective biomarker of chronic inflammation. Potential, extracellular phosphoprotein substrates for TRACP 5a, released during inflammation, have yet to be identified.

In conclusion, we have shown by five criteria that our recombinant human TRACP 5a made in CHO cells is a faithful representation of natural serum TRACP 5a. Using this rTRACP 5a preparation, we developed a novel mAb 220, which reacts with intact TRACP and mature serum TRACP 5a. Immunocytochemistry with antibody 220 revealed that an intact TRACP is expressed intracellularly in monocyte-derived cells, which may be the precursor form to mature isoforms 5a and 5b. Immunoassay with antibody 220 has further strengthened our hypothesis that TRACP 5a is a mature glycoprotein, selectively secreted by M{Phi} and DC. In contrast, TRACP 5b is the predominant isoform secreted by OC, whereas M{Phi} and DC selectively retain it intracellularly. Investigations of the mechanism for TRACP isoform expression could provide another interesting link between the skeletal and immune systems. The work that we have presented here to fully characterize the differences between human, natural TRACP isoforms 5a and 5b and to develop an antibody specific for TRACP 5a lays a strong foundation for these investigations and now allows us to investigate the clinical significance of TRACP isoforms in physiologic and inflammatory bone diseases.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Research Service of the U.S. Department of Veterans Affairs (A. J. J., R. N. P.) and The Clinical Research Foundation of Louisville (R. N. P., L. T. Y.). We gratefully acknowledge Ms. Luann Jaggers for her expert technical assistance.

Received May 12, 2004; revised October 5, 2004; accepted October 7, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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