Institute of Biochemistry, University of Giessen, D-35392 Giessen, Germany, and 2School of Biological Sciences, University of Wales, Bangor, Wales LL57 2UW, UK
Received on May 6, 1999; revised on May 19, 1999. accepted on May 23, 1999.
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Abstract |
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Key words: CD15/oligosaccharide structural analysis/on-target enzymatic cleavage/Schistosoma mansoni antigenic glycolipids/stage-specific expression
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Introduction |
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The aim of this study was to structurally analyze the oligosaccharide chains present in the biosynthetic series of neutral glycolipids from cercariae, the stage-specific expression of Lex-containing glycolipids in cercariae and to compare the pattern of cercarial glycolipid structures identified with those present in S.mansoni eggs.
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Results |
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Quantitative defucosylation of PA-hexasaccharide 14-3 required a 6 d-enzymatic treatment with -fucosidase, while a 3 d-treatment of PA-oligosaccharide 12 removed only 20% of the fucose residues. The chemically and enzymatically defucosylated products were pooled and designated 12-Fuc and 14-3-Fuc, respectively. The resultant PA-oligosaccharides were purified by HPLC and analyzed by MALDI-TOF-MS (Figure 5). Aliquots of 12-Fuc and 14-3-Fuc, as well as the PA-tetrasaccharide 10-2, were treated with ß-galactosidase to achieve almost complete cleavage by overnight incubation; these samples were also HPLC-purified and designated 12-Fuc-Gal, 14-3-Fuc-Gal, and 10-2-Gal, respectively. The products following fucose and/or galactose removal were analyzed by MALDI-TOF-MS (Figure 5, Table II), and also for linkage (Table IV).
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On-target enzymatic cleavage
In order to determine the anomeric configuration of the N-acetylhexosamine backbone linkages, PA-oligosaccharides were cleaved on-target with ß-N-acetylhexosaminidases. The PA-disaccharide 6-1 could be cleaved by bovine kidney ß-N-acetylhexosaminidase. The cleavage product was a PA-monosaccharide, which was observed by MALDI-TOF-MS in its protonated form (259.2 Da; Figure 6D). The pseudomolecular ion at m/z 259 did not occur in the PA-disaccharide sample (Figure 6A, insert) or in the enzyme control lacking substrate PA-oligosaccharide (Figure 6G, insert). Though the product PA-monosaccharide signal is located in the matrix ion region of the spectrum, the major ion at m/z 259.2 in Figure 6D, together with the control measurements in Figure 6A and G, clearly documented that the PA-disaccharide was cleaved by the ß-N-acetylhexosaminidase applied. The PA-trisaccharide 8-5 ([M+Li]+ 671.3 Da) was analyzed in the same way and resulted in product ions of the same mass ([M+H]+ 259.3 Da). This indicated that both the terminal GlcNAc and the subterminal, 3-substituted GalNAc (Table IV) were ß-linked, with the resulting structure for 8-5 of GlcNAcß3GalNAcß4Glc-PA (Table V). For 10-2-Gal, which is a PA-trisaccharide with the same mass (Table II) and linkages (Table IV) as 8-5, on-target cleavage with bovine kidney ß-N-acetylhexosaminidase resulted again in a pseudomolecular ion at m/z 259, so that its structure was identical to the PA-trisaccharide 8-5 (data not shown). The PA-trisaccharide 12-Fuc-Gal was converted following on-target cleavage from the PA-trisaccharide lithium-adduct (671.8 Da; Figure 6B) to the protonated PA-monosaccharide (259.2 Da; Figure 6E, insert). Together with the linkage data (Table IV), 12-Fuc-Gal could be assigned the structure GlcNAcß3GalNAcß4Glc-PA, which is identical to 8-5 and 10-2-Gal. Also, in the PA-tetrasaccharide 14-3-Fuc-Gal, all linkages could be cleaved by ß-N-acetylhexosaminidase, as was shown by the observed protonated PA-monosaccharide product (Figure 6F and inset). Together with the linkage data (Table IV), the proposed structure was GlcNAcß3GlcNAcß3GalNAcß4Glc-PA. All these on-target enzymatic cleavage experiments were also performed with the ß-N-acetylhexosaminidases of Diplococcus pneumoniae and jack bean. Like the bovine kidney enzyme, both these enzymes were able to cleave the PA-oligosaccharides 6-1, 8-5, 10-2-Gal, 12-Fuc-Gal, and 14-3-Fuc-Gal in a 150 min on-target cleavage experiment (data not shown). While the jack bean enzyme was approximately as efficient as the bovine kidney ß-N-acetylhexosaminidase, cleavage with the D.pneumoniae enzyme was less complete, due to the reduced amount of enzyme applied (see Materials and methods: On-target enzymatic cleavage). The latter treatment yielded pseudomolecular ions of the parent compound and the final cleavage product Glc-PA (m/z 259). Only 8-5 generated significant pseudomolecular ions of the intermediate cleavage product GalNAcß4Glc-PA, in addition to, Glc-PA as the dominant product.
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Discussion |
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For enzymatic digestion of PA-oligosaccharides, besides the conventional incubation in solution, on-target cleavage was performed in the presence of a 6-aza-2-thiothymine matrix, which allowed the anomeric configuration determination of the N-acetylhexosamine backbone with minimal sample consumption (Geyer et al., 1999).
While the surface of S.mansoni cercariae reacts with an anti-Lex-mAb only at the acetabular gland opening, the Lex epitope is expressed in patches over the whole surface of the parasite after transformation (Köster and Strand, 1994). We have identified glycolipid-bound Lex in the cercarial stage (Table V). It is not yet clear, however, whether lipid- and/or protein-bound glycoconjugates are responsible for the surface-located expression of this epitope after transformation. Its presence at the surface of the schistosome blood fluke conforms with the concept of molecular mimicry, as Lex is expressed in a wide range of mouse and human tissues (Fox et al., 1983
) and is thus an autoantigen. Human granulocytes have Lex-, often in parallel with sialyl-Lex-integrated glycoproteins (Fukuda et al., 1984
; Spooncer et al., 1984
) and glycolipids (Fukuda et al., 1985
; Symington et al., 1985
). The epitope has been shown to interact homophilically in cellular adhesion (Eggens et al., 1989
) and has been structurally identified on the N-glycans of leukocyte cell adhesion molecules (Asada et al., 1991
). As this epitope is shared by the parasite and its definitive host, an anti-Lex immune response occurred during schistosome infection (Nyame et al., 1995
, 1996, 1997) and putatively caused complement-dependent lysis of host neutrophils; high anti-Lex antibody titers were correlated with the severity of the resultant neutropenia (Borojevic et al., 1983
).
Besides the Lex and pseudo-Ley structures found in S.mansoni cercariae for the first time (Table V), glycolipids similar to those found in eggs (Khoo et al., 1997) were also present in the cercarial life-cycle stage in amounts that did not allow detailed structural analysis. The high amount of 2-substituted Fuc revealed by methylation analysis of the cercarial complex glycolipid fraction could not be substantiated by the PA-oligosaccharides analyzed, except for 13-3 (Table IV). Some 2-substituted fucose could be present in the fractions 15 to 17, but they have not been analyzed further due to their heterogeneity and lack of material. Glycosphingolipid biosynthesis in cercariae appears to differ significantly from that in eggs (Khoo et al., 1997
), as deduced from the structures detected. There are different structures present in the N-acetylhexosamine backbone. In the egg stage, the chain is built up with repeating units of 4GlcNAcß- linked to the schisto-core structure, 3GalNAcß4Glc1Cer, and GalNAcß- serves as a termination signal for the N-acetylhexosamine backbone (Khoo et al., 1997
). The cercarial Lex ceramide hexahexoside (corresponding to the 14-3 PA-hexasaccharide; Table V) was found to have a GlcNAcß3GlcNAc linkage, not previously described in schistosomes, in contrast to egg stage glycosphingolipids with their dominant GlcNAcß4GlcNAc linkages (Khoo et al., 1997
). This was paralleled by the presence of the 3-substituted N-acetylglucosamine in the cercarial complex glycolipid fraction and PA-oligosaccharide fraction 16 (cf. Figure 2 and Table IV) and its absence from egg complex glycolipids (Khoo et al., 1997
). Schistosome ß4- or ß3-N-acetylglucosaminyltransferases have not yet been described, but for the snail host Lymnea stagnalis of the bird schistosome Trichobilharzia ocellata one of these enzymes has been cloned and characterized (Bakker et al., 1994
), which was shown to transfer GlcNAc to GlcNAcß- to yield the chitobiose structure GlcNAcß4GlcNAcß-. Besides the different N-acetylhexosamine backbones, egg and cercarial glycolipids demonstrate differences in the degree of galactosylation: while egg glycosphingolipids lack galactose, our structural data would indicate a ß4-galactosyltransferase to act on the terminal N-acetylglucosamine of the glycosphingolipid N-acetylhexosamine backbone. A ß4-galactosyltransferase activity has been measured in extracts from adult worms and found to be able to synthesize N-acetyllactosamine structures (Rivera-Marrero and Cummings, 1990
). This enzyme activity could be involved in the galactosylation of circulating cathodic antigen in the adult worm and, also, hypothetically in the biosynthetic pathway of the cercarial glycolipids. Galactosylation may be accompanied by fucosylation to yield the Lex structures. An
3-fucosyltransferase activity has been detected in extracts of adult S.mansoni worms (DeBose-Boyd et al., 1996
) and shown to act on N-acetyllactosamine to yield Lex. In addition, a S.mansoni fucosyltransferase highly homologous to mouse and human fucosyltransferase VII has been cloned and characterized, but a physiological substrate of this enzyme has not yet been identified (Marques et al., 1998
). In cercarial homogenates of the bird schistosome T. ocellata, an
3-fucosyltransferase was detected (Hokke et al., 1998
) and was found to be active on the GlcNAc of N-acetyllactosamine and GalNAcß4GlcNAcß- (LacdiNAc). An
2-fucosyltransferase activity has also been described (Hokke et al., 1998
), which is thought to be responsible for the synthesis of oligofucosyl side-chains (Khoo et al., 1995
, 1997; Hokke et al., 1998
). A schistosomal fucosyltransferase acting on the GalNAc of LacdiNAc structures has not yet been identified. This yet to be identified enzyme is a candidate for the synthesis of the pseudo-Ley structure found in our study. We would assume this enzyme to have a side-activity which would allow it to transfer fucose to the 3-position of both GalNAc and Gal.
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Materials and methods |
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Isolation and purification of complex, neutral glycolipids
Glycolipids were isolated by consecutive extractions using approximately 100 ml of organic solvent per gram dry weight of parasite material: extraction was performed twice with chloroform:methanol:water (10:10:1, by volume) with sonication of the suspension (Branson sonifier B15; Branson, Danbury, CT) for 30 min and incubation at 50°C for 30 min; once with chloroform:methanol:0.8 M aqueous sodium acetate (30:60:8, by volume) with a 30 min sonication step and overnight incubation at 4°C; and twice with 2-propanol:n-hexane:water (55:20:25, by volume), followed by a 30 min sonication step and 30 min incubation at 50°C. After each extraction step, the sample was centrifuged at 10,000 x g for 10 min and the resultant supernatant rotary evaporated to dryness. Raw extracts were mildly saponified using methanolic 0.1 M sodium hydroxide for 2 h at 37°C. Salt and hydrophilic contaminants were removed by reverse-phase chromatography (Chromabond C18ec, Macherey and Nagel, Düren, Germany) as described elsewhere (Dennis et al., 1996). The preparation was then dissolved in chloroform:methanol:water (30:60:8, by volume) and applied to a QAE-Sephadex column (10 x 80 mm, acetate form; Pharmacia, Freiburg, Germany), as described elsewhere (Itonori et al., 1991
). Briefly, the neutral glycolipid fraction was collected as the flow-through with 50 ml of chloroform:methanol:water (30:60:8, by volume), and the acidic lipid fraction was eluted with 50 ml 0.45 M ammonium acetate in methanol. The neutral fraction glycolipids were purified by Florisil chromatography (Dennis et al., 1998
), resolved on a silica-gel cartridge (Waters, Eschborn, Germany; Dennis et al., 1995
) and analyzed by HPTLC and orcinol/H2SO4-staining, as well as HPTLC-immunostaining. CMH/CDH and complex, neutral glycolipids were separated into two pools.
HPTLC
For HPTLC, the complex, neutral glycolipids were separated on silica-gel 60 plates (Merck, Darmstadt, Germany) with chloroform:methanol: 0.25% KCl (50:40:10, by volume) as the developing solvent in an automatic HPTLC-developing tank (DC-MAT; Baron, Reichenau, Germany). Glycosphingolipids were visualized chemically by orcinol/H2SO4-staining (Dennis et al., 1998) or, alternatively, by immunostaining (Baumeister et al., 1994
). The globoside standard was purchased from ICT (Bad Homburg, Germany). A Lex-neoglycolipid (LexD) was used as a positive control in immunostaining and was prepared according to the literature (Feizi et al., 1994
) by reductive amination of lacto-N-fucopentaose III (Dextra Laboratories, Reading, England) with dihexadecanoyl-L-
-phosphatidylethanolamine (Sigma, Deisenhofen, Germany). For immunostaining, the developed HPTLC plates were coated with polyisobutylmethacrylate (Plexigum P28; Aldrich, Steinheim, Germany), blocked with bovine serum albumincontaining phosphate-buffered saline and incubated with the primary antibody for at least 2 h at room temperature. The primary antibodies used were: sera from 8 mice with chronic S.mansoni infection (CIS1-8); mouse-mAb anti-CD15 BRA4F1 (Biogenex, San Ramon, CA; IgM, recognizing the Lex-epitope); mouse-mAb anti-CD15 4D1 (IgM; kindly provided by Dr. B.Kniep); mouse-mAb G8G12 (kindly provided by Dr. Q.Bickle; generated in a CBA mouse against irradiated cercariae followed by a booster against non-irradiated cercariae prior to fusion of the spleen cells; Bickle et al., 1986
). Horseradish peroxidase-coupled, rabbit anti-mouse Ig (Dako Diagnostics, Hamburg, Germany) was used as secondary antibody. As a modification to the described method (Baumeister et al., 1994
), following secondary antibody incubation the plate was washed twice with phosphate-buffered saline and equilibrated once (5 min) with sodium citrate buffer (100 mM, pH 6.0). For staining, 240 µl of a substrate stock solution (97.5 mg of chloronaphthol (Sigma) and 60 mg diethylphenylenediamine (Sigma) in a mixture of 9 ml acetonitrile and 1 ml methanol, and stored at 20°C) and 8 µl of 30% H2O2 (Merck) were added to 10 ml of sodium citrate buffer. The plate was overlaid with this substrate solution and bound secondary antibody was visualized by a blue precipitate (Conyers and Kidwell, 1991
). Alternatively, alkaline phosphatasecoupled, goat anti-mouse Ig (Sigma) was applied as secondary antibody (Bethke et al., 1986
; Müthing, 1998
) and binding visualized by use of 10 mg of 5-bromo-4-chloro-3-indolyl phosphate (Biomol, Hamburg, Germany) and 5 mg nitro-blue tetrazolium chloride (Sigma) as substrates in 10 ml glycine buffer, 100 mM, pH 10.4, containing 1 mM ZnCl2 and 1 mM MgCl2.
Preparation of CDH and CPH
For the isolation of individual glycosphingolipids, HPLC fractionation on a porous silica gel column (Iatrobeads 6RS-8010, 10 µm, 4.6 x 500 mm; Macherey and Nagel) at a flow rate of 1 ml/min was performed. The column was equilibrated with 2% methanol in chloroform (by volume) and the sample was dissolved in chloroform:methanol (9:1, v/v). After injection, the column was run isocratically for 15 min, then within a further 60 min period the methanol content of the eluting solvent was increased to 38% (by volume). The column was washed with methanol.
Preparation of PA-oligosaccharides
Aliquots of the cercarial, complex, neutral glycolipid fraction were dissolved in 200 µl 50 mM sodium acetate buffer (pH 5.0; 0.1% sodium taurodeoxycholate), sonicated for 5 min at 50°C. Recombinant endoglycoceramidase II (20 µl (40 mU); from Escherichia coli encoding the gene of Rhodococcus sp. endoglycoceramidase II; Takara Shuzu Co., Ltd., Otsu, Shiga, Japan) was added, the sample incubated at 37°C for 72 h and 20 mU of fresh enzyme added each day. Samples were applied to a RP-cartridge (500 mg; Chromabond C18ec, Macherey and Nagel), washed with 10 ml of water to obtain the released oligosaccharides, and uncleaved glycolipids and free ceramides were eluted with 10 ml methanol and 20 ml chloroform:methanol (2:1, v/v). Washes and eluates were lyophilized or rotary evaporated to dryness, and released oligosaccharides as well as uncleaved glycolipids were quantitated by carbohydrate composition analysis. Coupling reagent (20 µl; 200 mg sublimation-purified 2-aminopyridine in 53 µl glacial acetic acid) was added to the dry, released oligosaccharides (Natsuka and Hase, 1998). After incubation at 90°C for 60 min, 70 µl of the reduction reagent were added (200 mg dimethylamine-borane complex in 50 µl water and 80 µl acetic acid) and incubated at 80°C for 35 min. Samples were adjusted to pH 10 by addition of NH3 (25% in water) and the volume was adjusted to 400 µl by addition of water. The reaction mixture was extracted six times with 600 µl chloroform to reduce the 2-aminopyridine excess and the 2-aminopyridine-labeled oligosaccharides (PA-oligosaccharides) were lyophilized.
Amino-phase-HPLC
PA-oligosaccharides were fractionated on an amino-phase HPLC column (4.6 x 250 mm, Nucleosil-Carbohydrate; Macherey and Nagel) at a flow rate of 1 ml/min at room temperature (RT) and detected by fluorescence (310/380 nm). The column was equilibrated with 200 mM aqueous triethylamine-acetic acid, pH 7.3: acetonitrile (25%:75%). A gradient of 2560% aqueous triethylamine-acetic acid buffer was applied within a 60 min period and the column was run isocratically for a further 10 min. Fractions of 2 ml were collected and lyophilized.
RP-HPLC
PA-oligosaccharides were fractionated on a RP-HPLC column (C18, 4.6 x 250 mm; Hypersil, Astmoor, Runcorn, Cheshire, UK) at a flow rate of 0.8 ml/min at RT and detected by fluorescence (320/400 nm). The column was equilibrated with aqueous 0.01% trifluoroacetic acid. A gradient from 0 to 3% acetonitrile in a period of 150 min was applied. Fractions of 1.6 ml were collected and lyophilized.
MALDI-TOF-MS
For MALDI-TOF-MS-experiments, the 6-aza-2-thiothymine matrix (Sigma) was spotted at 0.5 µl (5 mg/ml in water) onto the stainless-steel target. Approximately 1 µl of the PA-oligosaccharides dissolved in water was added to the matrix droplet and dried in a gentle stream of cold air. Glycolipids were dissolved in chloroform:methanol:water (10:10:1, by volume), and 1 µl was added to a dry matrix spot under a stream of warm air. MALDI-TOF-MS was performed on a Vision 2000 time-of-flight mass spectrometer (Finningan/MAT, Bremen, Germany) equipped with a UV-nitrogen laser (337 nm). The instrument was operated in the positive-ion reflectron mode throughout. All spectra represent accumulated spectra obtained by 320 laser shots and given molecular masses represent the monoisotopic masses rounded up to the first decimal place. The instrument was calibrated with the monoisotopic peak of angiotensin I (Sigma) and a matrix peak (285.0 Da).
On-target enzymatic cleavage
PA-oligosaccharide samples applied to the MALDI-TOF-MS-target were first used to determine the molecular mass of the intact molecule. Then the same sample spot was analyzed by on-target exoglycosidase treatment and subsequent MALDI-TOF-MS measurement of the cleavage product (Geyer et al., 1999). For this, the sample was redissolved in 2 µl of dialyzed enzyme solution. The target was placed in a screw-capped jar containing ammonium acetate buffer at the bottom and incubated at 37°C for 150 min. Subsequently, spots were dried in a cold stream of air and the mass profile of the digestion products was recorded. The enzymes used were ß-N-acetylhexosaminidase from jack bean (133 mU/µl; Sigma), from bovine kidney (50 mU/µl; Boehringer Mannheim, Mannheim, Germany), and from D.pneumoniae (1 mU/µl; Boehringer Mannheim). All enzymes were dialyzed for 4 h against 25 mM ammonium acetate buffer adjusted to the optimal pH for each enzyme (pH 4.5 for the bovine kidney enzyme and pH 5.0 for the D.pneumoniae and jack bean enzymes). The dialyzed enzymes were used undiluted for on-target cleavage.
Exoglycosidase treatment
PA-oligosaccharides were treated with either -fucosidase from bovine kidney (4 mU/µl; Boehringer Mannheim) or with ß-galactosidase from E.coli (500 mU/µl; Sigma). Enzymes were dialyzed for 4 h against 25 mM ammonium acetate solution adjusted to the optimal pH for each enzyme (pH 5.0 for
-fucosidase and pH 7.3 for ß-galactosidase). The dialyzed enzymes (50 µl) were added undiluted to the dried PA-oligosaccharides and the sample was incubated at 37°C. When incubation was continued for more than 24 h, 20 µl of dialyzed enzyme was added to the sample each day. The PA-oligosaccharide 13-3 was incubated with dialyzed
-fucosidase for 20 days in the presence of 0.02% sodium azide in order to inhibit microbial growth, and fresh enzyme was added each week. Following enzymatic cleavage, the PA-oligosaccharides were analyzed for their characteristic pseudomolecular ions by MALDI-TOF-MS.
Partial acid hydrolysis
For partial hydrolysis (Khoo et al., 1995), complex, neutral glycolipids were incubated in 100 µl of TFA-solution (0.10.2 M) at 80°C for 4080 min, and the resultant samples were dried down in a Speed-Vac.
Monosaccharide composition analysis
For composition analysis, samples were hydrolyzed in 100 µl 4 N aqueous TFA (Merck) at 100°C for 4 h, and dried down in a Speed-Vac. For derivatization with anthranilic acid (Anumula, 1994), the samples were dissolved in 10 µl 0.6% sodium acetate solution with sonication. The reagent solution was obtained by dissolving 6 mg anthranilic acid (Sigma) and 20 mg sodium cyanoborohydride (Sigma) in 1 ml methanol containing 2.4% sodium acetate and 2% boric acid, 50 µl of which was added to the sample. After a 45 min incubation at 80°C, the derivatized monosaccharides were resolved by HPLC and detected by fluorescence (360/425 nm) after separation on a Superspher RP 18ec column (4 µm, 4 x 250 mm; Merck) at a flow rate of 1 ml/min. The column was equilibrated in aqueous 0.2% 1-butylamine, 0.5% phosphoric acid and 1% tetrahydrofuran (by volume) containing 2.5% acetonitrile. After injection, the column was run isocratically for 5 min, then within 17 min the acetonitrile content was raised to 9% and followed by a final 15 min wash step at 50% acetonitrile.
Methylation-linkage analysis
PA-oligosaccharides and glycolipids were permethylated (Paz-Parente et al., 1985) and hydrolyzed (4 N aqueous TFA, 100°C, 4 h). Partially methylated alditol acetates obtained after sodium borohydride reduction and peracetylation were analyzed by capillary GC/MS, using the instrumentation and microtechniques described elsewhere (Geyer and Geyer, 1994
). Lacto-N-tetraose was used as a standard for the identification of 4,6-GlcN(Me)AcOH.
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Abbreviations |
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Footnotes |
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References |
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