5 Bijvoet Center, Department of Bio-Organic Chemistry, Section of Glycoscience and Biocatalysis, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands
Received on February 24, 2003; revised on November 19, 2003; accepted on December 30, 2003
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Abstract |
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Key words: HPLC profiling / interaction / lectins / oligosaccharides / surface plasmon resonance
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Introduction |
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In this study the potential of SPR to monitor interactions taking place in solution under dynamic conditions at lectin-coated surfaces was combined with the resolving power of high-performance liquid chromatography (HPLC) for the detection of fluorescently labeled (2-aminobenzamide; 2AB) high-affinity carbohydrate epitopes from complex mixtures. In the development of the method, oligomannose-type N-glycans binding to concanavalin A (Con A) were used. With the authentic mixture of glycans, derived from RNase B, and in a single experiment the preferential binding of Man7GlcNAc2 (Man7), Man8GlcNAc2 (Man8), and Man9GlcNAc2 (Man9) could be demonstrated. These findings were corroborated employing well-defined structures and are in agreement with earlier observations (Mega et al., 1992), thus validating this approach. Subsequently, using the fucose-binding lectin from Lotus tetragonolobus purpureaus (LTA) and a mixture of fucosylated milk oligosaccharides, the selectivity and sensitivity of the combination method was demonstrated. Finally, the experimental setup was exploited to identify fucose-containing oligosaccharides in complex O-glycan mixtures derived from bovine submaxillary gland mucin.
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Results |
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To more accurately evaluate the interaction of oligomannose-type N-glycans with Con A, oligosaccharides Man5, Man6, Man7'/7'', Man8, and Man9 (Figure 1) were isolated, 2AB-labeled, mixed in equimolar amounts, and investigated by SPR combined with HPLC (Man7 was not included). The HPLC profiles (solvent gradient 2) of the collected fractions during the SPR experiment are depicted in Figure 4. The injection (Figure 4A) and three wash fractions (Figure 4BD) showed a gradual decrease of all peaks. Evaluation of the material dissociated from the Con A surface during regeneration (Figure 4EH), via the fluorescence of the signals in the HPLC profiles showed that significantly more Man8 and Man9 bound to the Con A surface (total amount of material in regeneration Man8/Man9:Man5/Man6/Man7'/7'' 2:1). Man5, Man6, and Man7'/7'' glycans were completely removed from the surface following the first regeneration step with 2 mM methyl
-D-mannopyranoside (Figure 4E), whereas Man8 and Man9 were only slowly washed from the surface using 10 mM methyl
-D-mannopyranoside (Figure 4GH). A separate experiment with Man7 and Man7',7'' illustrated that Man7 bound preferentially to Con A. On regeneration of the surface with 2 mM methyl
-D-mannopyranoside, Man7' and Man7'' were efficiently and completely removed from the surface. In the case of Man7, regeneration with 10 mM methyl
-D-mannopyranoside was not sufficient to completely remove Man7 from the Con A surface. It is therefore evident that Man7 does indeed contain the high-affinity epitope present in Man8 and Man9 that is absent in Man7' and Man7''.
Evaluation of the interaction kinetics of Man59GlcNAc2Con A binding
The kinetics of the interaction between the isolated oligomannose-type glycans Man5 to Man9 (using free and 2AB-labeled compounds) and Con A were performed at a surface containing a low amount of immobilized dimeric lectin (150 RU, bound at pH 4.5; less than 1 Con A molecule every 1000 Å). The phenomenon of mass transport was assessed by using a standard protocol (Myszka, 1999
). By saturating the Con A surface with each of the isolated oligosaccharides in separate experiments and recording the level of response in each sensorgram, cooperative binding effects could be completely ruled out. The gradual increase in SPR response from Man5 (10 RU) to Man9 (20 RU) is indicative of comparable surface coverage, the response being linearly dependent on the molecular mass of the glycan, demonstrating a similar concentration of bound material in each case. Divalent binding would reduce the number of bound molecules by two, and for example Man9 would have produced a response of
10 RU.
The sensorgrams for the binding of each nonlabeled sample are shown in Figure 5. Differences between the binding characteristics of Man5, Man6, and Man7'/7'' on the one hand, and Man7, Man8, and Man9 on the other, are the slower association and dissociation (shallower slope of the curves) of the latter structures. Similar sensorgrams and kinetics were also obtained for the corresponding 2AB-labeled oligomannose-type structures (Figure 6). A comparison of both labeled and unlabeled Man6 and Man9 at identical concentrations proved that the 2AB-label does not influence the interaction between the oligomannose-type structures and Con A. Calculation of the kinetics by fitting the sensorgram to a 1:1 Langmuir binding profile produced 2 values with a good fit for the binding of Man5, Man6, and Man7'/7'' and KA values of between 13 x 105 M1 (Table II). The other sensorgrams, that is, those of Man7, Man8, and Man9, proved more difficult to fit to the usual binding models as generated by the BIAevaluation software, however it was clear from the sensorgrams that a more stable complex was formed. The typical structural difference between the group of Man7/Man8/Man9 structures as compared to the group of Man5/Man6/Man7'/7'' is the presence of a Man-D3 unit at the B(A)4' fragment. An explanation for the poor fit to usual binding models could include a distortion of the glycosidic bond between Man-D3 and Man-B. An analogous suggestion has been made for Man
6(Man
3)Man extended with a GlcNAc residue (Moothoo and Naismith, 1998
). An approximation of the kinetics indicated Man7, Man8, and Man9 to have a 10-fold higher affinity (KA 13 x 106 M1) than Man5, Man6, and Man7'/7'' (KA 13 x 105 M1), with the increase in affinity most likely originating from the lower dissociation rates recorded (Table II). As expected, the Man7'/7'' mixture has similar binding characteristics to Man5 and Man6, whereas Man7 has a similar profile to Man8 and Man9. In addition to the care taken to avoid possible cooperative binding effects at the Con A surface, the higher-affinity binding of Man7 cannot have been caused by this phenomenon because only the high- [Man
2Man
6(Man
3)Man, D3B(A)4'] but not the low- (Man
2Man
2Man, D1C4) affinity oligomannose binding site epitope (Mandal and Brewer, 1993
; Mandal et al., 1994
) is present in this molecule (Figure 1).
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A comparison of the relative peak areas, representing the absolute amounts of different oligosaccharides, indicated that the structures lacking fucose or carrying fucose -1, 3/4-linked to N-acetylglucosamine were more prominent in the injection. This implied that these oligosaccharides had no or weaker interaction with the lectin than the oligosaccharides containing fucose
-1,2-linked to galactose. During the washing procedure, most bound material was retained on the surface (Figure 7D, E). The HPLC profile of the first regeneration fraction (Figure 7F) showed the increase in relative peak areas (Table III) of the structures containing fucose
-1,2-linked to galactose and the absence of structures lacking fucose. These findings are consistent with the specificity of the LTA lectin, exhibiting an increased affinity for the blood group H determinant Fuc(
1-2)Gal and an especially high affinity for the Fuc(
1-2)Gal(ß1-4)GlcNAc fragment (Pereira and Kabat, 1974
).
In the HPLC profile of regeneration 2, using 10 mM methyl -L-fucopyranoside, only traces of the most abundant peak (2-fucosyl-lacto-n-hexaose) were evident (Figure 7G), indicating almost complete regeneration of the surface with 2 mM methyl
-L-fucopyranoside. Repetitive experiments yielded identical results, validating the sensitivity, accuracy, and potential of the combination of SPR and HPLC profiling. In addition, these experiments show that even if no appropriate regeneration conditions are available, a comparison of the HPLC profiles from the native mixture and the injection would allow a prediction of the interacting structure.
Bovine submaxillary gland mucin type I O-glycans interacting with LTA lectin
Using the same fucose-binding lectin, the SPR behavior of a mixture of 2AB-labeled O-glycans from bovine submaxillary gland mucin type I (BSM-I) was studied. The O-glycans have been previously isolated and characterized by NMR spectroscopy as their corresponding alditols (Chai et al., 1992). Using the NMR data and published glucose unit (GU) values for 2AB-labeled O-linked glycans (Mattu et al., 1998
; Royle et al., 2002
; Rudd et al., 1999
), several peaks in the HPLC profile could be assigned. The HPLC profile (solvent gradient 3) of the original mixture of O-glycans and the injection fraction (
2 pmol of carbohydrate were injected) were identical (Figure 8B, C). In the two subsequent washing steps, residual material was washed away (Figure 8D, E). The HPLC profile of the first regeneration step, employing 2 mM methyl
-L-fucopyranoside (40 µl), showed four major peaks (ad in Figure 8F) with an intensity order of d > b > a > c. The second regeneration step, using 10 mM methyl
-L-fucopyranoside (40 µl), contained the same four peaks but with an intensity order of a > b > d > c (Figure 8G). The sum of the peaks from both regeneration steps represented
1% (50100 fmol) of the total amount of material used for the SPR experiment, and the elution positions of ad corresponded to elution positions of minor components in the original profile (Figure 8B). Based on the elution positions (GU values) of peaks ad, they could not belong to difucosylated core 2 type {GlcNAc(ß1-6)[Gal(ß1-3)]GalNAc} or core 3 type [GlcNAc(ß1-3)GalNAc] structures.
Using this restriction, the NMR data (Chai et al., 1992) and the observed differences in affinity for peaks ad, it is evident that peaks ad correspond to relatively short glycans containing fucose residues and that the linkage type is most likely different in peaks a and b (
-1,2-) when compared to d (
-1,3-). This approach not only allowed the identification of fucose-containing structures that might have been ignored if conventional techniques had been used but also permitted direct quantification of glycans in a mixture. The combination of the SPR and HPLC approach may prove particularly valuable for identifying trace amounts of epitopes responsible for specific interactions as, for example, the inhibition of Escherichia coli adhesion by a fuco-oligosaccharide present in milk at a concentration of 20 pmol/L (Cravioto et al., 1991
).
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Discussion |
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In the oligosaccharideCon A study, oligomannose-type glycans were allowed to compete for the binding sites on Con A in a dynamic situation, and the course of interaction was quantitatively monitored. We observed that the monovalent interaction of oligomannose-type structures containing the Man2 unit D3 connected to Man-B of the B(A)4' fragment has a higher affinity for the binding site of dimeric Con A than that of the B(A)4' structure alone, and therefore the optimum binding epitope is actually this tetrasaccharide unit (Figure 1); a preliminary communication about the ranking of Man5GlcNAc2 to Man9GlcNAc2 has appeared (Haseley et al., 2001
). Over the past 20 years, the specificity and the thermodynamics of interaction of Con A have been studied extensively (e.g., Clegg et al., 1981
; Dam et al., 2000
; Derewenda et al., 1989
; Goldstein et al., 1974
; Goldstein and Poretz, 1986
; Gupta et al., 1997
; Mandal and Brewer, 1993
; Mandal et al., 1994
; Mega et al., 1992
). These studies have disclosed the different affinities that mono- (KA 8.2 x 103 M1), di- (KA
4 x 104 M1) and trisaccharides (KA 1.53.4 x 105 M1) built up from mannose display toward Con A. Crystallographic studies of the binding of well-defined oligosaccharides to Con A (Bouckaert et al., 1999
; Moothoo and Naismith, 1998
; Moothoo et al., 1999
; Naismith and Field, 1996
) revealed that the binding site is an elongated cleft on the surface of the protein. This site is sufficiently large to accommodate a pentasaccharide [GlcNAcß2Man
6(GlcNAcß2Man
3)Man: KA 1.4 x 106 M1], flexible enough to allow the binding of a Man
2Man
OMe unit in two different ways, and requires at least one monosaccharide in the appropriate orientation to allow interactions with Asn-14, Leu-99, Tyr-100, Asp-208, and Arg-228 of Con A. It is generally assumed that the high-affinity binding epitope for Con A consists of a trisaccharide [B(A)4' or 4(4')3].
Using pyridylamino-derivatized oligosaccharides Mega et al. (1992) conducted a study employing microequilibrium dialysis at pH 7.0 followed by HPLC. Our observations concord with their findings in the identification of the contribution of Man-D3 to the increased affinity and of the order of magnitude for the difference in affinity between oligomannose-type structures containing the D3B(A)4' epitope and those without. However, it is very likely that the reported KA values reported in that study are overestimated by a factor of 35, judging from other literature data and our own results; also, they could not completely rule out cooperative binding as the experiments were performed with tetrameric Con A.
In view of differences between our observations, in agreement with one other report (Mega et al., 1992) and other literature, it was decided to examine the interaction between Con A and different oligomannose-type structures in more detail. The first and most important item to rule out was that of cooperative binding events at the surface of the SPR biosensor. To avoid the appearance of cooperative binding, Con A was immobilized to the SPR sensor chip at pH 4.5, at which the lectin associates as a dimer. In this way divalent binding, as has been reported for the interaction to the tetramer, could be avoided. However, to exclude any effects of a lower pH on the interaction, the glycanlectin binding experiments were performed at a physiological pH. The lectin was immobilized with a low level (150 RU) of Con A, representing less than one Con A molecule every 1000 Å, so that the possibility of a glycan bridging two Con A dimers could be completely ruled out. Moreover, it was shown that saturation of the surface with Man5 to Man9 brought about a gradual increase of the maximum response level, a result in agreement with the absence of cooperative binding events. Finally, the higher-affinity binding of Man7 but not of Man7' and Man7'' confirmed that the difference in affinity was a result of binding at one binding site.
The conformation of the lectin has not been changed by immobilization to the surface because the calculated affinities are in agreement with those reported by using other methods (Dam et al., 1998, 2000
; Gupta et al., 1997
; Mandal and Brewer, 1993
; Mandal et al., 1994
). Finally, the possible effect of the 2AB label in the initial experiments was completely ruled out by observing identical kinetics of labeled and nonlabeled glycans.
In conclusion, we suggest here that the Man2Man element in D3B(A)4', present in Man7 to Man9, binds in a fashion similar to the GlcNAcß2Man
6 epitope in the pentasaccharide GlcNAcß2Man
6(GlcNAcß2Man
3)Man (Moothoo and Naismith, 1998
), thereby contributing significantly to the higher affinity. We propose the optimum binding carbohydrate epitope of Con A to be Man
2 Man
6(Man
3)Man [D3B(A)4', Figure 1], rather than the Man
6(Man
3)Man structure [B(A)4']. The tetrasaccharide does not appear to bind via a simple 1:1 interaction model but is more likely to undergo a conformational change to incorporate the mannose D3 residue. Interestingly, Bachhawat et al. (2001)
, using SPR, noted a comparable binding phenomenon between oligomannose-type structures (free oligosaccharides and glycopeptides) and the garlic lectin from Allium sativum, although a discrimination between D3B(A)4' and B(D2A)4' was not made.
Improvements of the profiling system could involve the development of an SPR surface capable of binding more molecules, which at present is in the picomole to femtomole range. In addition, the implementation of already commercially available micro flow cells in the fluorescence detector could enhance the sensitivity even further. The combination of SPR and HPLC could be interfaced in the future without too many difficulties. The collected SPR fractions never exceeded the maximum injectable volume for the analytical HPLC column; the buffer systems of both techniques are compatible, and no major purification is needed after SPR. Finally, the volatile buffer system applied for the HPLC profiling permits amplification of the system with an in-line coupled mass spectrometer. This would facilitate detailed structural information and further broaden the scope of the technique.
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Materials and methods |
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Preparation of oligosaccharides
N-glycans of RNase B (2 mg) were released by PNGase F digestion as described elsewhere (Van Rooijen et al., 1998). The mixture of liberated oligosaccharides was separated from detergent, protein, and salts in a single step, on graphitized carbon columns (Packer et al., 1998
). The O-glycans of BSM-I (25 mg) were released by manual hydrazinolysis (4 h, 65°C) and further purified as described (Patel et al., 1993
). Human milk oligosaccharides were a gift from Prof. H. H. Baer (University of Ottawa, Canada).
Labeling of the glycans
Oligosaccharides were fluorescently labeled with 2AB essentially as described. Briefly, to a solution of 23.6 mg 2AB in 500 µl dimethylsulfoxide/acetic acid (70:30, v/v) was added 35.4 mg NaCNBH3, and the mixture was heated for 2 min at 65°C to yield a clear solution. An aliquot (5 µl) of the solution was added to dried oligosaccharide (P2O5), and the mixture was incubated twice for 1 h at 65°C with intermediate mixing. After cooling to room temperature, the mixture was transferred onto an acid-preconditioned QMA strip (3 x 10 cm), and the residual reagents were eluted from the labeled glycan mixture by ascending chromatography using acetonitrile. The labeled glycan mixture (remaining at the baseline) was excised from the strip, placed in an ultrafree MC centrifugal Eppendorf filter (5000 nmwl), and recovered by centrifugation with water (3 x 200 µl, 8000 x g, 15 min). The resulting solution was lyophilized and redissolved in 100 µl water; an aliquot was used for SPR and/or HPLC analysis. Quantifications of the 2AB-labeled oligosaccharides are based on 2AB calibration curves.
Isolation, purification, and characterization of oligomannose-type glycans
Oligomannose-type N-glycans were enzymatically released from RNase B (20 mg) using PNGase F and fractionated by high-performance anion exchange chromatography on a Dionex LC system, using a CarboPac PA-1 pellicular anion-exchange column (0.9 x 25 cm, Dionex, Sunnyvale, CA) and a gradient buffer consisting of 0.1 M NaOH/0.5 M sodium acetate (Van Rooijen et al., 1998). Collected fractions were neutralized with diluted acetic acid, desalted by gelfiltration on HiTrap columns (5x5 ml bed volume), then lyophilized. Man5, Man6, Man8, and Man9 were obtained as pure compounds. The Man7 fraction contained 510 mol% Man5 and Man6. Man7' and Man7'' (molar ratio, 2:3) coeluted, and the Man7'/7'' fraction contained
5 mol% Man8. The structures of the oligosaccharides in each fraction were identified by 1H-NMR spectroscopy (Table IV) (Hård et al., 1991
; Priem et al., 1993
; Tseneklidou-Stoeter et al., 1995
). Aliquots of the oligosaccharides were fluorescently labeled with 2AB as reported previously (Stroop et al., 2000
).
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SPR
All SPR experiments were performed on a BIAcore 2000 system, using a running buffer (pH 7.4) consisting of 10 mM Tris, 150 mM NaCl, 1 mM CaCl2, and 1 mM MgCl2. For the experiments, carboxymethylated dextrancoated sensorchips (CM5, Pharmacia, Uppsala, Sweden) were activated (Haseley et al., 1999), and different lectins immobilized. For the preparation of a Con A lectin surface, dimeric Con A (in 10 mM NaOAc, pH 4.5) was attached to all four channels (
8000 RU each). Experiments were performed at a flow rate of 5 µl/min. A solution of the 2AB-labeled Man5 to Man9 (Figure 1) mixture (50 µl) was flowed across the sensor chip (flow cells in series), and the injected sample was recovered manually, while material eluting from the surface by using buffer was collected automatically by the instrument in three fractions of 30 µl each. The surface was regenerated by using 2 mM (15 µl), 5 mM (15 µl), and 2 x 10 mM (15 µl) methyl
-D-mannopyranoside, and the corresponding fractions were recovered for injection on HPLC. Finally, a regeneration step was carried out using 100 mM methyl
-D-mannopyranoside (40 µl), but the eluent was not collected.
For calculation of the kinetics of interaction of Man5 to Man9 glycans, separate experiments with a set of 2AB-labeled and a set of nonlabeled compounds were performed on a flow cell containing 150 RU of dimeric Con A (active surface). A flow cell containing 150 RU of denatured dimeric Con A was used as a control surface; denaturation was carried out on the chip using 3 x 40 µl 6 M guanidinium chloride (pH 1.5). The experiments were performed in running buffer at a flow rate of 5 µl/min, as already mentioned. Oligosaccharide fractions, at concentrations between 6.25 and 100 µmol/L, were injected for 3 min and left to dissociate for a further 3 min. Saturation of the surface was achieved by injecting 1 mM of each isolated oligosaccharide fraction across the surface.
Association and dissociation rate constants (ka and kd, respectively), and the equilibrium association constant (KA) were calculated by nonlinear fitting of the primary sensorgram (BIAevaluation program version 3.0, 1997) data using the BIAevaluation 3.0 software (Pharmacia).
For the preparation of the LTA lectin surface, a similar protocol was used, injecting the lectin solution across the surface for 4 min (20 µl). The resulting response was 10,000 RU for each of the four surfaces. Regeneration was accomplished by using 2 mM (40 µl) and 10 mM (40 µl) methyl
-L-fucopyranoside.
HPLC profiling
Samples collected during the SPR experiments were directly applied to a ultrafree MC centrifugal Eppendorf filter (5000 nmwl), and centrifuged at 8000 x g for 7 min. Subsequently, the filters were washed with 3 x 100 µl double distilled water and centrifuged. The pooled effluents were lyophilized, redisolved in 100 µl starting HPLC buffer, and profiled. The HPLC system used for the profiling consisted of a Waters (Milford, CT) 2690 XE module equipped with an in-line degasser, a temperature control unit (maintained at 30°C throughout the experiments) and a 474 scanning fluorescence detector. The system was controlled via a LAC/E interface using Waters Millennium 32 software. The GlycosepN, normal phase HPLC column (4.6 x 100 mm) was obtained from Oxford Glycosciences. The column was calibrated in GU with a standard mixture of glucose oligomers.
Normal phase HPLC was carried out using the following gradient conditions using 50 mM ammonium formate buffer (pH 4.4; solvent A) and acetonitrile (solvent B): gradient 1 (Figure 2), solvent A and 20% solvent A in solvent B (solvent C) at a flow rate of 0.8 ml/min. Following injection, samples were eluted with a linear gradient of 6.544% A over 100 min, followed by a linear gradient of 44100% A over the next 3 min; gradient 2 (Figure 4), solvent A and solvent B at a flow rate of 0.4 ml/min. Following injection, samples were eluted with a linear gradient of 3553% A over 92 min, followed by a linear gradient of 53100% A over the next 3 min. In both cases, the flow rate was increased to 1 ml/min with 100% A over the next 2 min and then for a further 5 min. The system was then reequilibrated to 6.5% A (Figure 2) or 35% A (Figure 4). The total run time was 140 min.
For normal phase HPLC profiling of milk oligosaccharides and O-glycans, the following gradient conditions were used (gradient 3): solvent A was 50 mM ammonium formate (pH 4.4), solvent B was acetonitrile, and the flow rate was 0.4 ml/min. Following injection, samples were eluted by a linear gradient of 2047.5% A over 120 min, followed by a linear gradient of 47.5100% A over the next 3 min. Using 100% A the flow rate was then increased to 1 ml/min over the next 2 min, then the column was eluted with 100% A for 5 min and subsequently reequilibrated in 20% A before injection of the next sample. The total run time was 160 min.
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Acknowledgements |
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Footnotes |
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1 Present address: Municipal Institute of Medical Research, Department of Experimental and Health Sciences, University Pompeu Fabra, c/o Doctor Aiguader 80, 08003-Barcelona, Spain
2 Present address: CBS Porton Down, DSTL, Salisbury SP4 0JQ, United Kingdom
3 These authors contributed equally to this article.
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Abbreviations |
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References |
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