Purification and Characterization of a Catalytic Domain of Rat Intestinal Phospholipase B/Lipase Associated with Brush Border Membranes*

Hiromasa TojoDagger , Tetsuichi Ichida, and Mitsuhiro Okamoto

From the Department of Molecular Physiological Chemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

A brush border membrane-associated phospholipase B/lipase was solubilized from the distal two-thirds of rat small intestine by autolysis during storage at -35 °C over 1 month, and then the enzyme was purified to homogeneity and characterized enzymatically and structurally. The purified enzyme exhibited broad substrate specificity including esterase, phospholipase A2, lysophospholipase, and lipase activities. SDS-gel electrophoretic and reverse-phase high performance liquid chromatographic analyses demonstrated that a single enzyme catalyzes these activities. It preferred hydrolysis at the sn-2 position of diacylphospholipid and diacylglycerol without strict stereoselectivity, whereas it apparently exhibited no positional specificity toward triacylglycerol. Diisopropyl fluorophosphate, an irreversible inhibitor of serine esterases and lipases, inhibited purified enzyme. When the position of enzyme on SDS-gel electrophoresis under the non-reducing conditions was determined by assaying the activity eluted from sliced gels, brush border membrane-associated enzyme corresponded to a ~150-kDa protein; autolysis gave a 35-kDa product, in agreement with the results of immunoblot analysis. The purified 35-kDa enzyme consisted of a 14-kDa peptide and a glycosylated 21-kDa peptide. Their NH2-terminal amino acid sequences were determined and found in the second repeat of 161-kDa phospholipase B/lipase with 4-fold tandem repeats of ~38 kDa each, which we cloned and sequenced in the accompanying paper (Takemori, H., Zolotaryov, F., Ting, L., Urbain, T., Komatsubara, T., Hatano, O., Okamoto, M., and Tojo, H. (1998) J. Biol. Chem. 273, 2222-2231). These results indicate that the purified enzyme is the catalytic domain derived from the second repeat of brush border membrane-associated phospholipase B/lipase.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Phospholipase A2 (PLA2)1 catalyzes the hydrolysis of fatty-acyl ester bond at the sn-2 position of glycerophospholipids and plays major roles in a variety of biological processes such as digestion, membrane phospholipid metabolism, inflammatory reactions, and eicosanoid synthesis. PLA2s represent a diverse family of enzymes and can be classified into broad two classes according to requirement of Ca2+ ions for activity, Ca2+-dependent and Ca2+-independent PLA2s. Ca2+-dependent PLA2s have been extensively studied so far in connection with important roles of Ca2+ ions in regulation of intracellular signaling (1). To date, at least three isoenzymes are known, i.e. group I and II secretory PLA2s and 85-kDa cytosol PLA2 (cPLA2). In the secretory PLA2s Ca2+ ions involve in both catalysis and substrate binding, whereas in cPLA2 calcium does not function in the catalytic machinery, but enhances the enzyme's affinity for membrane phospholipids (2, 3). On the other hand, Ca2+-independent PLA2s, especially those of mammalian origins, have not been so extensively studied. They were recently purified from several mammalian sources, and their involvement in phospholipids metabolism is now becoming evident; for example, a 40-kDa myocardial PLA2 may participate in alkyl and alkenyl phospholipid metabolism (4), a 80-kDa macrophage PLA2 in membrane phospholipid remodeling (5), and a 15-kDa lung acidic PLA2 in surfactant metabolism (6). Very recently, some information on primary structures has become available for these PLA2s (7-9).

During the course of a study on the tissue distribution of rat group I and II PLA2s, we found a high Ca2+-independent PLA2 activity in rat distal intestine (10), and we decided to purify and characterize this enzyme to better understand the detailed structural and enzymatic properties of one of Ca2+-independent PLA2s. This enzyme was similar to those reported in rat and guinea pig brush border membranes, which were partially purified after solubilized from those membranes by detergent or papain treatment and characterized (11, 12). They had the characteristics of a digestive ectoenzyme and exhibited lysophospholipase and lipase activities as well as PLA2 activity (13), therefore being hereafter named phospholipase B/lipase (PLB/LIP). Unfortunately, there was no structural information available on PLB/LIP, because it has not yet been purified to homogeneity in a sufficient quantity.

In this study, we purified to homogeneity a truncated form of PLB/LIP solubilized from rat intestine by autolysis during its storage at -35 °C for over 1 month, and characterized it both enzymatically and structurally. The purified enzyme exhibited PLA2, lysophospholipase, and lipase activities as a single 35-kDa molecule, which consisted of a 14-kDa and a glycosylated 21-kDa peptides. Molecular size analyses by SDS-gel electrophoresis suggested that the purified enzyme was a catalytic domain derived from a ~150-kDa membrane-associated enzyme. The accompanying paper (14) reports cDNA cloning of PLB/LIP and expression of its entire protein, a protein lacking COOH-terminal membrane anchoring domain, and the catalytic domain in COS-7 cells. The results demonstrate that the enzyme purified here is indeed the catalytic domain of membrane-bound PLB/LIP.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- The following glycerolipids were obtained from Avanti Polar Lipids, Inc.: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphocholine (1-O-hexadecyl-OPC), and 1,2-dioleoyl-sn-glycerol. 2,3-Dioleoyl-sn-glycerol was purchased from Serdary Research Laboratories, Inc. Monoolein was obtained from Nu-Check Prep, Inc. 1-Stearoyl-2-linoleoyl-sn-glycerol (SLDG) was purchased from Biomol Research Laboratories, Inc. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 2,3-dipalmitoyl-sn-glycero-1-phosphocholine, 1-palmitoyl-sn-glycero-3-phosphocholine (GPC), 1-myristoyl-GPC, 1,2-dioleoyl-rac-glycerol, 1,3-dipalmitoyl-2-oleoyl-rac-glycerol (POP), 1-palmitoyl-2-oleoyl-3-stearoyl-rac-glycerol (POS), 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC), diisopropyl fluorophosphate (DFP), and Rhizopus arrhizus lipase were obtained from Sigma. Triolein, hydroxyapatite, trifluoroacetic acid, and octaethylene glycol dodecyl ether (C12E8) were purchased from Nacalei Tesque, Ltd. (Kyoto, Japan). Manoalide and p-bromophenacyl bromide (BPB) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). HPLC-grade acetonitrile was purchased from Katayama Chemical Industries Co., Ltd. (Osaka, Japan). Vectastain ABC kit was obtained from Vector Laboratories, Inc. ConA-Sepharose was obtained from Pharmacia Biotech Inc.

Assay for Lipolytic Activities-- PLA2, lysophospholipase, and lipase activities were determined by a non-radiometric HPLC method based on precolumn derivatization with 9-anthryldiazomethane (ADAM) as described previously (10). Individual fatty acids released from mixed-acyl glycerophospholipids and tri- and diacylglycerols were simultaneously determined by this method. Substrate stock solutions used were as follows: mixed micelles of 5 mM diradyl-phospholipid and various concentrations of a bile salt, cholate, or deoxycholate (DOC); 1-acyl-GPC (5 mM) micelles; and emulsions of 5 mM triacylglycerol and 5% gum arabic. In a typical experiment, the assay mixtures contained 10 mM EDTA, substrate micelles or emulsion (10-µl stock solution), 0.1 M NaCl, 0.1 M Tris-HCl (pH 8.5), and the enzyme sample in a final volume of 50 µl.

Cholesterol esterase activity was also determined by the ADAM method. Cholesterol oleate (5 mM) was emulsified with 3.6 mg/ml mineral oil (15), 7.5 mM DHPC, or 5% arabic gum. DHPC was used in place of phosphatidylcholine (16) because PLB/LIP has a high PLA2 activity. The assay mixtures contained cholesterol ester emulsion (10 µl), either 10 mM DOC or 10 mM sodium taurocholate, 0.1 M NaCl, either 0.1 M MES (pH 6.5) or 0.1 M Tris-HCl (pH 8.5), and the enzyme sample.

Solubilization of PLB/LIP from Brush Border Membranes-- Rats anesthetized with pentobarbital were sacrificed by drawing blood from the abdominal aorta, then the proximal half of the ileum was removed and its mucosa was scraped off with a glass plate. Brush border membrane fractions were prepared according to Hauser et al. (17). The brush border membrane fraction was incubated with an equal volume of either 2 M KCl, 2 M KBr, 1% DOC, or 2% Triton X-100 on ice for 1 h, and then 2 ml of the mixture was centrifuged at 100,000 × g for 60 min. The pellets were resuspended in 2 ml of 20 mM Tris-HCl containing 1 mM EDTA and 0.15 M NaCl (pH 7.4), and PLA2 activity of the supernatant and pellet fractions was determined with POPE/DOC mixed micelles as substrate. For autolysis experiments, the brush border membrane fraction was kept at -35 °C, and at the indicated times aliquots were taken, and handled in the same manner as described above.

Purification of PLB/LIP-- Rat small intestines (lower 2/3 portions) were kept at -35 °C for at least 1 months, and then used for the purification. The frozen tissues (80 g) were incubated in 800 ml of 10 mM Tris-HCl (pH 7.4), containing 2 mM EDTA with stirring for 1 h at room temperature to remove the mucosal cells and then the extract was sonicated for 5 min on ice. After removing the residual intestine tissues, the extract was centrifuged at 23,000 × g for 1 h. The pH of the supernatant was adjusted to 8.5, and then the solution was applied to a QAE-Toyopearl column (3 × 20 cm) pre-equilibrated with 10 mM Tris-HCl (pH 8.5), containing 0.1% Triton X-100. The PLA2 activity was eluted with the same buffer containing 0.5 M NaCl. Since the affinity of PLB/LIP for the column was rather weak, the pass-through fraction was, if necessary, applied again to the same QAE-Toyopearl column pre-equilibrated in the same manner as described above. To the pooled PLA2 fraction was added 1 M lithium sulfate, and then the solution was applied to a phenyl-Sepharose column preequilibrated with 20 mM Tris-HCl (pH 7.4), containing 1 M lithium sulfate. The PLA2 activity bound to this column under the conditions, and was eluted with 10 mM Tris-HCl containing 10% ethylene glycol and 0.1% Triton X-100. The pooled PLA2 fraction was dialyzed against 10 mM Tris-HCl containing 50 mM NaCl, and then applied to a QAE-Toyopearl column preequilibrated with 10 mM Tris-HCl. The PLA2 activity was recovered in the flow-through fractions, whereas a large amount of contaminants bound to the column. The resultant PLA2-active fractions were dialyzed against 10 mM MES (pH 6.8), and then subjected to hydroxyapatite column chromatography. The column was developed with a linear gradient of potassium phosphate from 10 to 500 mM (pH 7.4), and the PLA2 activity was eluted as a broad peak in the phosphate concentration range of 30-150 mM. The pooled PLA2 fraction was applied to a ConA-Sepharose affinity column (1.5 × 7 cm) pre-equilibrated with 10 mM Tris-HCl (pH 7.4). A major part (51%) of the PLA2 activity bound to the column under these conditions, while a small part (34%) did not. The column was washed with 10 mM Tris-HCl (pH 7.4), containing 0.1% C12E8 to remove strongly uv-absorbing Triton X-100. The major bound enzyme activity was eluted in a stepwise manner with the same buffer containing 0.5 M methylmannoside, and the resultant solution was dialyzed against 20 mM Tris-HCl (pH 8.5), and further purified by HPLC on a Cosmogel QA column (0.7 × 10 cm, Nacalei Tesque) preequilibrated with 20 mM Tris-HCl (pH 8.5), containing 0.1% C12E8. The column was developed with a linear concentration gradient of NaCl from 0 to 0.5 M in 60 min. The PLA2 activity was eluted as a single peak at the retention time of 18 min. The pooled PLA2 fraction was rechromatographed on the same column; the pH of the eluent was decreased to 8.0, and a shallower gradient of NaCl from 0 to 0.1 M in 60 min was used. The enzyme activity was eluted at the retention time of 31 min, coinciding well with a protein peak detected at 280 nm.

HPLC-- The HPLC system consisted of two Gilson model 302 liquid delivery modules, and a Gilson model 811 dynamic mixer. For a Cosmogel-QA column of 7 × 100 mm (Nacalai Tesque), the system equipped with a 1.8-ml mixing chamber was operated at 0.5 ml/min. When a Cosmosil 5C8-300 column of 2.1 × 100 mm or 2.1 × 30 mm was used, the system equipped with a 65-µl mixing chamber was operated in the microflow mode at 0.1 ml/min. Cosmosil 5C8-300 (Nacalai Tesque) was slurry-packed into a column (2.1 × 100 mm or 2.1 × 30 mm) in our laboratory.

Extraction of Enzyme Activities from SDS-Polyacrylamide Gel Electrophoresis-- SDS-PAGE was performed according to Laemmli et al. (18) under the nonreducing conditions The gels were cut into slices of 3-mm length, which were immersed in 200 µl of buffer containing 0.25 M Tris-HCl and 0.25 M NaCl (pH 8.5), overnight at 4 °C to extract PLB/LIP. PLA2 and lysophospholipase activities in the extracts were determined as described in the standard assay. The recovery of enzyme activity was 52%.

Determination of the Positional Specificity and Stereospecificity of PLB/LIP-- The positional specificity of PLB/LIP toward glycerophospholipids was determined using the mixed micelles of 6 mM DOC and 1 mM DOPC, a symmetric acyl phospholipid, as substrate. The reaction was followed under the standard conditions, and the released lysophospholipids, 1-oleoyl- and 2-oleoyl-GPC, were separated by reverse-phase HPLC on a LiChrosorb RP-18 column (4 × 150 mm, Merck) at the flow rate of 1 ml/min and room temperature as reported (19). The eluent used was methanol/acetonitrile/water (57:20:23) containing 20 mM choline chloride, and the effluent was monitored at 205 nm. The regiospecificity toward di- and triacylglycerols was determined with mixed acyl glycerides, SLDG, POP, and POS, as substrates. The stereospecificity was also assessed with enantiomeric pairs of DPPC and dioleoylglycerol. The released fatty acids were derivatized, separated, and quantitated by the same method as in the ADAM assay.

Protein Sequencing-- The purified PLB/LIP was reduced, S-carboxymethylated, and purified as described in the legend of Fig. 4. The amino acid sequences were analyzed with an Applied Biosystems 477A sequencer and a 120A PTH analyzer. To identify a phenylthiohydantoin S-carboxymethylated Cys precisely, both S-carboxymethylated and unmodified proteins were analyzed and the resultant data were compared with each other.

Other Analytical Methods-- Phospholipid phosphorus was determined according to the method of Vaskovsky et al. (20). ConA-peroxidase staining was performed as recommended by the manufacturer's instructions (Honen, Tokyo, Japan). Immunoblotting was performed as reported (21) and immunoreacted bands were visualized with a Konica immunostain kit (Konica). Protein concentrations of crude samples during purification were measured with a bicinchoninic acid (BCA) protein assay kit (Pierce), and those of purified enzyme were estimated by microbore reverse phase HPLC based on protein concentrations determined by amino acid analysis with beta -alanine as an internal standard (22).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Solubilization of PLB/LIP from Brush Border Membranes-- We determined the distribution of Ca2+-independent PLA2 activity, which was immunochemically distinct from Ca2+-dependent group I and group II PLA2s, along rat intestine's length, and along the villus-crypt units according to the method of Pinkus (23), and its subcellular distribution. The results confirmed that Ca2+-independent PLA2 is located in brush border membranes of the absorptive cells of the lower intestine, especially the proximal ileum (24). Hereafter, we will use the words "PLA2 activity" and "Ca2+-independent PLA2 activity" as the same meaning unless it is essential to distinguish between Ca2+-dependent and Ca2+-independent PLA2 activities.

A high concentration of KCl or KBr and phosphatidylinositol-specific phospholipase C treatment (0.57-570 milliunits/ml) did not solubilize PLA2 activity at all, but treatment with 1% Triton X-100 solubilized 88% of PLA2 activity in the membrane fractions. Limited proteolysis by endogenous and exogenous proteases has been used to solubilize proteins associated with brush border membranes successfully (25). We found that autolysis gave an active fragment that accounted for 50% of the total PLA2 activity of brush border membrane fractions during storage at -35 °C for 1 month.

The molecular size of the solubilized enzymes was determined by SDS-PAGE under the non-reducing conditions, where PLA2 and lysophospholipase activities being recoverable from gel slices allowed us to estimate the RF values of enzyme (Fig. 1). The size of Triton X-100-solubilized enzyme (corresponding to 150 kDa) was identical to that of the enzyme in brush border membrane fractions. Autolysis produced an active 35-kDa enzyme and active PLB/LIP species of another size were not detectable under the conditions used. PLA2 and lysophospholipase activities in all solubilized enzyme solutions co-migrated on SDS-PAGE as those in untreated membrane fractions, suggesting that a single enzyme catalyzes those activities and that the 35-kDa autolytic product is derived from the enzyme associated with brush border membrane.


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Fig. 1.   Solubilization of Ca2+-independent PLA2 and lysophospholipase activities from rat intestinal brush border membrane fractions. The membrane fractions were prepared, extracted, and subjected to SDS-PAGE on a 7.5% gel as described under "Experimental Procedures." Enzyme activities were eluted from sliced gels, and then assayed as described under "Experimental Procedures." The substrates used were 1 mM POPE plus 6 mM DOC mixed micelles and 1 mM 1-myristoyl-GPC micelles. A, brush border membrane fraction; B, 1% Triton X-100 extract; C, autolytic extract.

Purification of PLB/LIP-- We purified to homogeneity PLB/LIP solubilized by autolysis during storage of the intestines at -35 °C. The results of purification are summarized in Table I. The enzyme activity was separated into the flow-through and bound fractions by ConA affinity chromatography. We purified PLB/LIP from the latter fraction because of its higher specific activity. Among several chromatographic steps involved in this purification strategy, Cosmogel QA HPLC is the most effective (Fig. 2), leading to the overall purification and yield of 22,000-fold and 4.2%, respectively. Inclusion of a non-ionic detergent such as C12E8 in the eluent of the HPLC was essential for improving the recovery, otherwise the yield of enzyme activity was less than 10% on a single chromatographic run. The ratios of lysophospholipase to PLA2 activities at each purification step were rather constant (0.36-0.53), except for that of the supernatant of intestine extracts (0.13), suggesting a single enzyme catalyzes those reactions.

                              
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Table I
Purification of PLB/LIP from rat small intestine
The PLA2 and lysophospholipase activities were determined as described under "Experimental Procedures" using 1 mM POPE plus 6 mM DOC mixed micelles and 1 mM 1-palmitoyl-GPC as substrates. Enzyme concentrations of crude preparations were estimated by the BCA method, and that of the purified PLB/LIP was determined as described under "Experimental Procedures."


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Fig. 2.   HPLC profile of protein and lipolytic activities on a Cosmogel QA column at pH 8.0. PLA2 and lysophospholipase activities were measured as described under "Experimental Procedures" using 1 mM POPE plus 6 mM DOC mixed micelles (bullet ) and 1 mM 1-palmitoyl-GPC (triangle ) as substrates. The sample volume applied was 6 ml, and 0.5-ml fractions were collected. The column was developed at the flow rate of 0.5 ml/min as described under "Experimental Procedures," and the eluent contained 0.1% C12E8.

On SDS-PAGE analysis, purified PLB/LIP gave a single band corresponding to 35 kDa, which is the same as that in the autolytic extracts on a 10% gel under the non-reducing conditions, whereas reduction with 2-mercaptoethanol caused its separation into 14-kDa and 21-kDa proteins. To examine whether the 35-kDa protein indeed represented PLB/LIP, the same enzyme samples were loaded to adjacent two lanes on a 10% gel. A gel strip of one lane was stained with Coomassie Brilliant Blue, and the other strip was sliced into 3-mm pieces from which PLA2 and lysophospholipase activities were eluted and assayed as described under "Experimental Procedures." The enzyme activities were observed only in fraction 12 corresponding to a 35-kDa protein band under the non-reducing conditions (Fig. 3A), whereas no activity was detectable in the reduced protein lane. Furthermore, reverse-phase HPLC analysis of purified enzyme on a Cosmosil 5C8-300 column (2.1 × 30 mm) showed a single protein peak coeluted with PLA2, lysophospholipase, and lipase activities (Fig. 4A). These results demonstrate that a single enzyme catalyzes the three lipolytic activities.


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Fig. 3.   SDS-PAGE analyses of purified PLB/LIP. A, co-migration of a single Coomassie staining band (0.2 µg) with both PLA2 (open circle ) and lysophospholipase (triangle ) activities under the non-reducing conditions (lane 1). Enzyme activities were eluted from sliced gels and then assayed as in Fig. 1. Lane 2, an aliquot of the same sample was reduced with 5% 2-mercaptoethanol at 80 °C for 1 min. B, ConA-peroxidase staining of purified PLB/LIP (50 ng each). Lane 1, non-reduced; lane 2, reduced. Large and small arrowheads and an arrow indicate the 35-kDa PLB/LIP, 14-kDa fragment, and 21-kDa fragment, respectively. C, comparison of purified and intestine extract PLB/LIPs by immunoblot analyses using a specific IgG raised against amino acids 450-1450 of PLB/LIP (14). Tissue extracts were prepared as described under "Experimental Procedures." Lane 1, dye-labeled markers, of which molecular masses are indicated by small arrowheads, from top to bottom, 175, 83, 62, 47, 32, 25, and 16.5 kDa as reference of lanes 2-5; lane 3, purified PLB/LIP (2.4 ng); lanes 2, 4, and 5, homogenate, supernatant, and pellet fractions of stored whole intestinal mucosa, respectively (8 µl each). A small arrow indicates an inactive digested product of PLB/LIP, as confirmed by assaying of PLA2 activity of materials eluted from sliced gels in a separate SDS-PAGE experiment, and a large arrowhead the 35-kDa active enzyme. Freshly prepared ileum tissues (8 µl each) were applied to lanes 6-9. Lane 6, brush border membrane fractions, reduced; lane 7, ileum homogenate, reduced; lane 8, brush border membrane fractions, non-reduced; lane 9, ileum homogenate, non-reduced. The small arrowheads indicate the positions of the same molecular mass markers as in lane 1.


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Fig. 4.   Reverse phase HPLC profiles of purified PLB/LIP on a Cosmosil 5C8-300 column (2.1 × 30 mm). The eluents used were as follows: eluent A, 0.1% trifluoroacetic acid in water; eluent B, 95% acetonitrile containing 0.1% trifluoroacetic acid. A, purified PLB/LIP (~2 µg) was applied to the column, and the enzyme was eluted with a linear concentration gradient of eluent B: from 0 to 25% in 5 min; from 25 to 60% in 45 min; from 60 to 100% in 10 min, at a flow rate of 0.1 ml/min. PLA2 (bullet ), lipase (square ), and lysophospholipase (triangle ) activities in the fractions collected with a Gilson 201 fraction collector in a manual mode were measured as described under "Experimental Procedures." B, reduced and S-carboxymethylated enzyme. L and S indicate the large and small fragments of PLB/LIP, respectively. The enzyme (~5 µg) purified as in A was reduced with tributylphosphine in 0.5 M ammonium bicarbonate (pH 8.5)/1-propanol (1:1, v/v) under nitrogen for 2 h, and then reacted with 3-fold molar excess of sodium iodoacetate over the reductant for 30 min at 37 °C. The reaction was stopped by adding 5 µl of 2-mercaptoethanol. To the reaction mixture was added 0.5 volume of formic acid and 2 volumes of 0.1% trifluoroacetic acid to reduce the concentration of organic solvent and improve the solubility of denatured PLB/LIP. The resultant solution (0.5 ml) was directly injected onto the column preequilibrated with eluent A at a flow rate of 0.4 ml/min, and the column was washed with the same solvent until the absorbance at 210 nm returned to the base-line level. Then it was developed with a linear concentration gradient of eluent B: from 0 to 30% in 5 min; from 30 to 60% in 50 min; from 60 to 100% in 10 min, at a flow rate of 0.1 ml/min.

The majority of PLA2 and lysophospholipase activities bound to a ConA-Sepharose column, suggesting the purified enzyme is a glycoprotein that contains ConA-recognizable sugar chain(s). Moreover, a ConA-peroxidase stain of the purified enzyme transferred on a polyvinylidene difluoride membrane after SDS-PAGE showed that its large fragment was indeed glycosylated (Fig. 3B). The presence of PLB/LIP that did not bind to the column indicated a minor fraction of PLB/LIP is either unglycosylated or modified with ConA-unrecognizable sugar chain(s).

Fig. 3C shows the results of immunoblot analysis using specific antibody raised against amino acids 450-1450 of PLB/LIP expressed in Escherichia coli (14). Under non-reducing conditions a 35-kDa band corresponding to purified enzyme was clearly observed together with other degraded products in the supernatant and membrane fractions of intestine homogenate prepared from stored tissues. On the other hand, brush border membrane fractions and intestine homogenate prepared freshly contained a single ~200-kDa2 form of enzyme under the same conditions. SDS-PAGE in combination with detection by enzyme assay gave a molecular mass estimation of ~150 kDa (Fig. 1). This difference was due to a lower resolution of the latter method. Denaturation treatment led to its splitting into 90- and 130-kDa proteins.

Enzymatic Properties of PLB/LIP-- Purified PLB/LIP did not require calcium ions for activity; the addition of calcium up to 10 mM did not affect PLA2, lysophospholipase, and lipase activities significantly. We examined the substrate specificity of PLB/LIP in the presence and absence of DOC using diradylphospholipids (POPC, POPE, POPG, or 1-O-hexadecyl-OPC), 1-palmitoyl-GPC, glycerides (triolein, diolein, or monoolein) and cholesterol oleate as substrates (Table II). The enzyme exhibited a broad substrate specificity, and the apparent order of activity under the standard conditions in the presence of 6 mM DOC was as follows: diolein > POPC ~ POPE > 1-O-hexadecyl-OPC ~ POPG > L-palmitoyl-GPC ~ triolein > monoolein. The enzyme also had esterase activity toward p-nitrophenyl fatty acid esters. Cholesterol oleate, which was emulsified by three different methods as described under "Experimental Procedures," was not a substrate for PLB/LIP. PLB/LIP required a bile salt for the activities toward diradylphospholipids and triacylglycerols, but not for those toward lysophospholipids and mono- and diacylglycerols (Table II). Fig. 5 shows typical dependence of the PLA2, lysophospholipase, and lipase activities on the DOC/glycerolipid molar ratio. PLB/LIP exhibited virtually no activity toward POPC and TOG in the absence of the bile salt, but its presence dramatically enhanced the activities with increasing the bile salt/glycerolipid molar ratio. The activity toward POPC reached a maximum at the molar ratio of 6 and then gradually decreased, whereas that toward TOG exhibited saturation and did not decrease at higher ratios (up to 100). In contrast, the enzyme was active to 1-palmitoyl-GPC micelles and the bile salt maximally increased the activity 1.8 times that in its absence at the molar ratio of 2, and then the activity gradually decreased (Fig. 5). The optimal activity toward POPC, 1-palmitoyl-GPC, or triolein was observed over the pH range of 8-9, and activity was hardly detected below pH 4. 

                              
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Table II
Substrate specificity of PLB/LIP
Lipolytic activities were determined by the ADAM method in the presence or absence of 6 mM DOC as described under "Experimental Procedures." Activity toward p-nitrophenyl pentanoate was determined by continuously monitoring absorbance changes at 400 nm in the presence or absence of 10 mM DOC at pH 8.0. The total p-nitrophenol released was calculated with its pK value of 6.98.---, not detectable.


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Fig. 5.   Effects of DOC on lipolytic activities of PLB/LIP. Substrates (1 mM each) used were POPC, 1-palmitoyl-GPC (square ), and TOG (×). POPC/DOC mixed micellar solutions were prepared as described previously (52). DOC dissolved in water was added to the reaction mixtures for assaying lysophospholipase and lipase activities. Oleic acid-releasing (bullet ) and palmitic acid-releasing (open circle ) activities from POPC were simultaneously monitored as described under "Experimental Procedures."

To determine the positional specificity of PLB/LIP toward diacylphospholipids, diacylglycerols and triacylglycerols, the time courses for release of the individual fatty acids from mixed acyl phospholipid and glycerides, i.e. POPC, SLDG, POP, and POS, by the enzyme's action were followed by the ADAM method as described under "Experimental Procedures" (Fig. 6). The results were compared with those with an sn-2-specific rat pancreatic PLA2 and an sn-1(3)-specific R. arrhizus lipase. PLB/LIP produced oleic acid and linoleic acid at much higher rate than palmitic acid and stearic acid from POPC and SLDG as substrate, respectively, suggesting its preference to sn-2 hydrolysis of diacylphospholipid and diacylglycerol. In contrast, the rates of fatty acid release from each ester bonds of POP and POS were practically identical, suggesting low regiospecificity as to triacylglycerol hydrolysis of PLB/LIP. The stereospecificity of this enzyme was also examined by the ADAM method with enantiomeric pairs of DPPC and dioleoylglycerol as substrates. (Table III). PLB/LIP hardly discriminated between these enantiomeric pairs.


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Fig. 6.   Positional specificity of PLB/LIP toward diacylphospholipid, diacylglycerol, and triacylglycerol. The substrates used were as follows: A and D, 1 mM POPC, 6 mM DOC mixed micelles; B and E, 1 mM SLDG emulsified with 1% arabic gum and 6 mM DOC; C and F, 1 mM POP or POS emulsified with 1% arabic gum and 6 mM DOC. Enzymes used were as follows: A-C, purified PLB/LIP (A and B, 7.1 ng/ml; C, 23.5 ng/ml); D, purified rat pancreatic PLA2 (0.22 µg/ml); E and F, R. arrhizus lipase (0.4 µg/ml). Time courses were followed as described under "Experimental Procedures." Fatty acids released were as follows: bullet , oleic acid from POPC (A and D) and from POP (C and F); open circle , oleic acid from POS (C and F); black-triangle, palmitic acid from POP (C and F); triangle , palmitic acid from POPC (A and D) and from POS (C and F); square , stearic acid from SLDG (B and E) and from POS (C and F); black-down-triangle , linoleic acid from SLDG (B and E).

                              
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Table III
Hydrolysis of diolein and DPPC enantiomers by PLB/LIP
Lipolytic activities were determined in the presence of 6 mM DOC for PLB/LIP and 6 mM cholate for rat pancreatic PLA2 as described under "Experimental Procedures." ---, not detectable; ND, not determined.

We confirmed the sn-2 specificity for diacylphospholipid hydrolysis by PLB/LIP by identifying which lysophospholipid regioisomers, 1-oleoyl- or 2-oleoyl-GPC, the enzyme produces from DOPC. These isomers were well separated by reverse-phase HPLC (Fig. 7A) as reported (19). PLB/LIP time-dependently produced oleic acid and 1-oleoyl-GPC, but not appreciably 2-oleoyl-GPC (Fig. 7, B and C); a small amount of 2-oleoyl-GPC may be formed through base- and silica gel-catalyzed 1,2-acyl migration (26, 27), because the slow production of the isomer was also observed in the reaction with pancreatic PLA2. The rate of oleic acid release was greater than that of 1-oleoyl-GPC release; this is consistent with the fact that 1-oleoyl-GPC is an intermediate which the enzyme further converts to oleic acid and GPC. On the other hand, the time courses for the formation of two products were superimposable on hydrolysis by sn-2-specific rat pancreatic PLA2 (Fig. 7D). These results unequivocally demonstrated that PLB/LIP is specific for the hydrolysis at the sn-2 position of diacylglycerophospholipids.


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Fig. 7.   Confirmation of the positional specificity of PLB/LIP toward DOPC by reverse phase HPLC. A, separation of authentic 1-oleoyl-GPC (peak 3) and 2-oleoyl-GPC (peak 2). 1-Oleoyl- and 2-oleoyl-GPC were prepared from DOPC by the action of rat pancreatic PLA2 and R. arrhizus lipase (53), respectively. B, HPLC profiles of the products formed by the reaction of purified PLB/LIP (23.5 ng/ml) with 2 mM DOPC/12 mM cholate mixed micelles at 37 °C. Peak 1, oleic acid. C, time courses of the formation of oleic acid (square ), 1-oleoyl-GPC (bullet ), and 2-oleoyl-GPC (open circle ). The assay conditions were the same as in B. D, time courses of the formation of oleic acid (square ), 1-oleoyl-GPC (bullet ), and 2-oleoyl-GPC (open circle ) for the reaction of rat pancreatic PLA2 and 2 mM DOPC/12 mM cholate mixed micelles.

Inhibition of PLB/LIP by DFP, Manoalide, and BPB-- Since PLB/LIP exhibited PLA2, lysophospholipase, and lipase activities as a single enzyme, we tested the ability of known potent inhibitors for secretory 14-kDa PLA2 (manoalide and BPB) and lipases (DFP) to inhibit the lipolytic activities of PLB/LIP. When the enzyme was preincubated with various concentrations of inhibitors for 1 h at 37 °C, they dose-dependently inhibited the lipolytic activities. The extent of inhibition was expressed as an inhibitor concentration required for half-maximal inhibition (Table IV). Notably, the dependence of activity on inhibitor concentration was similar irrespective of substrates tested, suggesting a single active site is involved in catalysis. DFP was apparently most effective, and treatment with 0.5 mM DFP completely inhibited activity in all cases.

                              
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Table IV
Inhibition of PLB/LIP by DFP, BPB, and manoalide
PLB/LIP (23.5 ng/ml) was incubated with various concentrations of the inhibitors (DFP, 1 µM to 2 mM; manoalide, 0.01-0.5 mM; BPB, 0.05-2 mM) for 1 h at 37° C and pH 8.5, and then enzyme activity was determined as described under "Experimental Procedures" using the following substrate/DOC combinations: 1 mM POPC plus 6 mM DOC, 1 mM 1-palmitoyl-GPC plus 6 mM DOC, and 1 mM TOG and 1% arabic gum plus 10 mM DOC. Oleic acid-releasing and palmitic acid-releasing activities from POPC were simultaneously monitored.

NH2-terminal Amino Acid Sequences of PLB/LIP Fragments-- The purified PLB/LIP was reduced and S-carboxymethylated, and then subjected to reverse-phase HPLC, by which the S-carboxymethylated enzyme was separated into two peaks (Fig. 4B). SDS-PAGE confirmed that the earlier and later peaks corresponded to the small and large fragments of PLB/LIP, respectively. Automated Edman analysis of these fragments revealed the NH2-terminal sequences of 13 residues of the small fragment and of 31 residues of the large fragment. A data base search showed that both sequences were similar to parts of the sequence of AdRab-B protein that has been cloned from an adult rabbit intestine-specific cDNA library (28). AdRab-B contained four internal repeats (designated repeats 1 through 4) of about 350 amino acids each, and the identity between the sequences of either small or large fragment and those of each repeat of AdRab-B was as follows: 22, 67, 40, and 27% for the small fragment versus repeats 1, 2, 3, and 4, respectively; 39, 81, 52, and 45%, for the large fragment versus repeats 1 through 4, respectively (Fig. 8). The sequences of both fragments showed the highest similarity to repeat 2 of AdRab-B.


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Fig. 8.   Comparisons of NH2-terminal amino acid sequences of the small and large fragments of PLB/LIP with the sequences of each repeats of AdRab-B. The numbers on both sides of sequences indicate those of amino acids of rat PLB/LIP (14) and rabbit AdRab-B (28) deduced from their cDNA sequences. The residues conserved among all five sequences are boxed. The asterisks indicate the conserved amino acid residues of the fragments and repeat 2 of Ad-RabB.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Purified Enzyme Is a Catalytic Domain of PLB/LIP-- We purified to homogeneity a phospholipase B/lipase solubilized from rat intestinal brush border membranes by autolytic limited proteolysis. This truncated form of PLB/LIP (35 kDa) has a considerably smaller size than the brush border membrane form, but actively catalyzes PLA2, lysophospholipase, and lipase reactions (Table II). Co-migration of these three activities on reverse phase HPLC and SDS-PAGE of the purified enzyme confirmed that a single enzyme is responsible for such broad specificity (Figs. 3A and 4A). The NH2-terminal amino acid sequences of two peptides derived from the purified enzyme were most similar to the sequence of the second repeat of AdRab-B that contains 4-fold internal repeats with about 38 kDa molecular mass each. In the following paper (14), we deduced the amino acid sequence of PLB/LIP by its cDNA sequencing, and found that it contains the same structural organization as AdRab-B. The sequence of the small fragment was found in the NH2-terminal part of repeat 2 domain and that of the large fragment in its mid-portion, confirming that the purified enzyme is indeed derived from the second repeat; hence, repeat 2 is a catalytic domain of PLB/LIP.

We used mild autolysis conditions, that is, the storage of intact intestine tissues at a low temperature of -35 °C over 1 month, and tissue extracts were prepared without homogenization. This gave an enzymatically active autolytic product, which consisted of the 14-kDa and 21-kDa peptides, as revealed by reverse phase HPLC and SDS-PAGE (Figs. 3 and 4). This raises a question whether these peptides are autolytic products or genetically distinct subunits. The amino acid sequence deduced from cDNAs of rat PLB/LIP showed that the nascent PLB/LIP is a single polypeptide with a molecular weight of 161,070, and that it contains Lys367 and Arg528 just before the NH2 termini of the small and large fragments, respectively (14). This confirmed the two peptides are indeed proteolytic products, but not distinct gene products, and suggested that trypsin or trypsin-like protease rather specifically cleaved PLB/LIP to give the solubilized homogeneous enzyme as judged by NH2-terminal amino acid, SDS-PAGE, and reverse-phase HPLC analyses. Guinea pig phospholipase B similar to rat PLB/LIP was effectively solubilized from brush border membrane fractions with papain treatment, and the solubilized 97-kDa enzyme was partially purified (12).

Immunoblot analysis under non-reducing conditions showed that the autolytically solubilized form was hardly detected in fresh intestine homogenate, which instead contained a single ~200 kDa form; denaturation gave two smaller species (90 and 130 kDa). This suggested that limited proteolysis split the 200-kDa form into a few fragments, which remain associated, like the purified enzyme composed of two peptides. Smaller-sized fragments dissociating on denaturation could not be detected by the antibody used. The formation of nick by proteolysis might modulate enzyme's function; for example, the substrate specificity of rat hepatic lipase was modified by limited proteolysis by collagenase (29). Although the full-length PLB/LIP expressed in COS-7 cells exhibited the substrate specificity similar to the enzyme purified here (14), it is necessary to examine in more detail whether the structural changes caused by proteolytic cleavage modulate PLB/LIP's function.

Catalytic Properties of PLB/LIP-- Purified PLB/LIP exhibited high PLA2 and triacylglycerol lipase activities. Its specific activity toward the mixed micelles of 1 mM POPC and 6 mM cholate (713 µmol/min/mg) was 6.7 times that of rat pancreatic PLA2 (107 µmol/min/mg) under the same conditions (22). The specific activity toward triolein (Fig. 5) was lower than that of rat pancreatic lipase (5330 µmol/min/mg; Ref. 30), but was comparable with those of other rat lipases, such as lingual lipase (230 µmol/min/mg; Ref. 31), hepatic lipase (750 µmol/min/mg; Ref. 32), and adipose tissue lipoprotein lipase (258 µmol/min/mg; Ref. 33). This unique substrate specificity was also found in partially purified guinea pig intestinal phospholipase B (13); the reported specific activities for PLA2 (22.8 µmol/min/mg) and triacylglycerol lipase (19.9 µmol/min/mg) were significantly lower than those obtained in this study under similar conditions (Table II). Since the structural information on the guinea pig enzyme has not been available, it is unknown at present whether it is a homolog of rat PLB/LIP. On the other hand, rabbit AdRab-B homologous to PLB/LIP exhibited virtually no triacylglycerol lipase activity and much lower diacylglycerol lipase activity than PLA2 activity (28), when expressed in COS-7 cells. An AdRab-B preparation purified recently from rabbit intestine by immunoaffinity chromatography was a mixture of several forms of its proteolytic digests, and the specific activity values of purified enzyme have not yet been reported (34). Since the physical states of substrate significantly influence lipolytic activities, the specificities of different enzymes should be compared side by side under the same assay conditions. The positional specificity of AdRab-B toward diacylphospholipids has not yet been firmly established. In this study, we demonstrated the A2 specificity by separating two positional isomers by reverse-phase HPLC with DOPC, a symmetrical acyl phospholipid, as substrate. This avoids the problem on positional impurity of substrate that may be encountered in experiments with a mixed acyl phospholipid or doubly-radiolabeled substrates. Following the time course of the formation of each isomers unequivocally determined the positional specificity of PLB/LIP.

It was also unique that PLB/LIP preferred the sn-2 hydrolysis of diacylphospholipids and diacylglycerols without clear chiral selectivity, whereas it hydrolyzed triacylglycerols without regiospecificity (Fig. 6). Comparable inhibition of these lipolytic activities by DFP, BPB, and manoalide suggested that a single active site is involved in catalysis and that the binding mode of triacylglycerol to the enzyme differs from that of the former substrates in the same active site. Further extensive structural and functional studies on its active site should be required to clarify the mechanism underlying the complex, at a glance, positional specificity of PLB/LIP.

Recent studies revealed that members of the lipase superfamily (35) exhibit a wide range of ratios of phospholipase A1 to lipase activities; classical pancreatic lipases have no significant phospholipase activity, the guinea pig and coypu lipases belonging to the type 2 pancreatic lipase-related proteins, a pancreatic lipase subfamily, exhibit high phospholipase A1 and lipase activities (36), and new members of the lipase superfamily, hornet venom phospholipase A1 (37) and platelet serine phospholipid-specific phospholipase A1 (38), are devoid of lipase activity. Recent development of x-ray crystallographic studies on natural and mutated lipases and PLA2s provided important clues to unravel how lipolytic enzymes regulate their substrate selectivities (39, 40). However, the studies along this line are still on the starting line. The catalytic domain of PLB/LIP with the unique substrate and positional specificity and the primary structure entirely unrelated to those of the enzymes belonging to the known lipase superfamily provides a good model for studying the mechanism of controlling substrate specificity. Its comparison with recently cloned and sequenced PLA2s with an active site serine, including cPLA2 (41, 42) and PAF acetylhydrolases (43, 44) will provide further insights into the structure-function relationship of lipases and phospholipases A.

DFP, an irreversible inhibitor for serine esterases and lipases (45), completely inhibited the three lipolytic activities of PLB/LIP, suggesting that a serine nucleophile is involved in its catalytic mechanism. In the best-established lipase family (35), the serine is present within the conserved pentapeptide sequence G-X-S-X-G. Very recently, Upton and Buckley proposed another class of lipase family, of which enzymes, such as Aeromonas hydrophila lipase/acyltransferase (46) and E. coli thioesterase I (47), contain the active serine in another conserved G-D-S-L sequence (48). The repeat 2 domain of PLB/LIP (14) contains two serines, Ser414 and Ser459, in the former conserved sequence, and a Ser404 in the latter conserved sequence. We recently studied by chemical modification and site-directed mutagenesis which serine residues were responsible for catalysis, and obtained evidence that Ser404, but not Ser414 and Ser459, was essential for catalysis.3

In contrast to protein and polysaccharide digestion involving coordinated luminal and membrane digestion (49), glycerolipid digestion was believed to proceed exclusively in the lumen by the action of the major secretory enzymes, preduodenal acid lipase, pancreatic lipase, and pancreatic phospholipase A2 until recently (50). The presence of PLB/LIP in ileal brush border membranes suggests that it is the first lipolytic enzyme participating in membrane digestion and indicates a need to reassess the individual roles of these enzymes in lipid digestion and absorption. Recently, a quantitative study on the relative contributions of gastric and pancreatic lipases to lipolysis in the upper gastrointestinal tracts was reported in normal subjects (51). Furthermore, it is interesting to determine its pathophysiological significance in the intestine under diseased conditions, such as pancreatic insufficiency and inflammatory bowel disease. In addition to its digestive roles on the luminal side, the physiological roles of PLB/LIP should be further examined in spermatids, and Paneth cells as described in the following paper (14).

    Addendum

During preparation of these manuscripts, we noted a report that described evidence that DFP reacted with a Ser400 in the G-D-S-L sequence of AdRab-B purified from rabbit intestine by immunoaffinity chromatography (34).

    FOOTNOTES

* This work was supported in part by Research Grant 08670173 from the Ministry of Education, Science, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Molecular Physiological Chemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan. Tel.: 81-6-879-3283; Fax: 81-6-879-3288; E-mail: htojo{at}mr-mbio.med.osaka-u.ac.jp.

1 The abbreviations used are: PLA2, phospholipase A2; ADAM, 9-anthryldiazomethane; BPB, p-bromophenacylbromide; C12E8, octaethylene glycol dodecyl ether; DFP, diisopropyl fluorophosphate; DHPC, 1,2-dihexadecyl-sn-glycero-3-phosphocholine; DOC, deoxycholate; DOPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; cPLA2, cytosolic Ca2+-dependent PLA2; GPC, sn-glycero-3-phosphocholine; 1-O-hexadecyl-OPC, 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphocholine; PLB/LIP, phospholipase B/lipase; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; POP, 1-palmitoyl-2-oleoyl-3-stearoyl-rac-glycerol; POS, 1-palmitoyl-2-oleoyl-3-stearoyl-rac-glycerol; PAGE, polyacrylamide gel electrophoresis; SLDG, 1-stearoyl-2-linoleoyl-sn-glycerol; HPLC, high performance liquid chromatography; ConA, concanavalin A; MES, 4-morpholineethanesulfonic acid.

2 These values are only nominal, because the presence of intact disulfide bridges and hydrated carbohydrate moieties deviates the estimation.

3 T. Lu, H. Takemori, T. Urbain, M. Ito, M. Okamoto, and H. Tojo, manuscript in preparation.

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Results
Discussion
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