Centre de Recherches sur les Macromolécules Végétales (CERMAV-UPR CNRS 5301), affiliated with the Joseph Fourier University of Grenoble, B.P. 53, 38041 Grenoble cedex 9, France
Received on March 4, 2004; revised on May 15, 2004; accepted on May 16, 2004
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Abstract |
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Key words:
13C-NMR spectroscopy
/
in vitro synthesis of (13)-ß-D-glucan
/
polysaccharide synthases
/
structural analysis of polysaccharides
/
UDP-[U-13C]glucose
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Introduction |
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Despite the importance of these polysaccharides, the mechanisms of biosynthesis of many of them remain to be elucidated. To understand these mechanisms, it is necessary to characterize the corresponding synthases, which are generally difficult to study because they are essentially membrane-bound proteins that contain transmembrane domains (see, for instance, Delmer, 1999). The isolation of these enzymes requires the use of detergents that generally yield unstable preparations. However, in vitro activities can be assayed in many cases, primarily owing to the use of radioactive substrates. The assays consist of measuring the levels of incorporation of radioactive monosaccharides into the newly synthesized polysaccharides, which can be subsequently identified by testing their sensitivity to glycoside hydrolases. This approach involves purified and highly specific hydrolytic enzymes that are either expensive or commercially unavailable. The use of enzyme preparations that are not substrate-specific or that may contain contaminating hydrolytic activities can be misleading. This problem is of particular importance for the study of synthase fractions, which contain different glycosyltransferases that use the same sugar donor to synthesize polymers with relatively subtle structural differences. For instance, cellulose and (1
3)-ß-D-glucan (callose) synthases, which are both present in plant plasma membranes, use the same substrate, UDP-glucose (Delmer, 1999
). Because commercial preparations of cellulases may be contaminated with (1
3)-ß-D-glucanases and vice versa, the distinction between the (1
3)-ß-D-glucan and cellulose synthesized in vitro is not always easy to achieve with enzymatic approaches.
Undoubtedly though, the major problem in the study of polysaccharide synthases is that very small quantities of producttypically tens or hundreds of microgramsare synthesized in vitro by these enzymes. Consequently, it is difficult to obtain a detailed structural characterization of the polysaccharide using physical and chemical techniques, which require milligram amounts of polymer. (13)-ß-D-Glucan synthases, which can be extracted from plasma membranes of various organisms in a highly active form, represent the only examples where a detailed structural characterization of the products synthesized in vitro was made possible using a series of physical and chemical methods (Bulone et al., 1995
; Lai Kee Him et al., 2001
, 2003
; Pelosi et al., 2003
). However, even in the case of these enzymes, accumulation of milligram amounts of polymer remains tedious. Research on glycan synthases would therefore benefit from a simple and sensitive method that would allow unequivocal structural characterization of enzyme products. Here we describe the development of such a method, which is based on the use of 13C-enriched substrates and 13C-nuclear magnetic resonance (NMR) spectroscopy.
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Results and discussion |
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Our strategy for the synthesis of UDP-D-[U-13C]glucose was adapted from these protocols and is summarized in Figure 1. The D-[U-13C]glucose-1-phosphate was synthesized in a five-step reaction and subsequently coupled to uridine monophosphomorpholidate under catalytic conditions (Wittmann and Wong, 1997, and references cited therein). The overall yield of UDP-D-[U-13C]glucose recovered after gel filtration was 50%.
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In vitro synthesis of (13)-ß-D-glucan and structural characterization by 13C-NMR spectroscopy
Plant (13)-ß-D-glucan synthases catalyze the repetitive transfer of glucose from UDP-glucose to form a linear polymer (Figure 2). The requirement of a primer to initiate polymerization has not been demonstrated for these enzymes and synthesis of (1
3)-ß-D-glucans occurs in vitro without adding any primer in the reaction mixture (Bulone et al., 1995
; Lai Kee Him et al., 2001
, 2003
; Pelosi et al., 2003
). In the present study, the conditions previously established in our group were used for in vitro synthesis of (1
3)-ß-D-glucan from detergent extracts of blackberry cell membranes (Lai Kee Him et al., 2003
; Pelosi et al., 2003
).
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Interestingly, solid-state 13C-NMR spectroscopy, which usually requires a minimum of tens of milligrams of sample, could be used to characterize the in vitro product synthesized from UDP-D-[U-13C]glucose (Figure 3B). The chemical shifts for the C atoms have similar values as those of well-characterized (13)-ß-D-glucans (Saito et al., 1989
), and the spectrum is typical of dried native (1
3)-ß-D-glucans (Pelosi et al., 2003
). The possibility of using solid-state NMR spectroscopy for characterization of products synthesized in vitro is particularly valuable for the analysis of polymers that have an organized 3D structure. For instance, 13C solid-state NMR spectroscopy allows the distinction between different allomorphs of (1
3)-ß-D-glucans (Chuah and Sarko, 1983
; Pelosi et al., 2003
). Also, in the case of cellulose, it may be used to estimate the degree of crystallinity of the polymer as well as the proportions of the I
and Iß allomorphs in a given sample (Atalla and VanderHart, 1984
; Heux et al., 1999
; Lennholm et al., 1994
). Another application of solid-state NMR spectroscopy is the determination of the degree of acetylation of chitin and chitosan (Heux et al., 2000
), which can also be synthesized in vitro using cell-free extracts (Gay et al., 1993
; Ruiz-Herrera and Bartnicki-Garcia, 1974
; Ruiz-Herrera et al., 1975
). In the spectrum in Figure 3B, the signal corresponding to C3 is very broad and contains two parts reciprocally centered at 85.6 and 89.8 ppm. This result suggests that this sample contains two allomorphs of (1
3)-ß-D-glucan. Indeed, the part of the signal centered at 89.8 ppm has been proposed to be characteristic of the occurrence of disordered chains in (1
3)-ß-D-glucan crystals, whereas the part of the peak centered at 85.6 ppm has been described to be more specifically related to an organization of the glucan chains in triple helices (Chuah and Sarko, 1983
). The spectrum shown in Figure 3B suggests that the two allomorphs of (1
3)-ß-D-glucan are present in similar proportions in the sample.
Advantages of the use of 13C-enriched substrates
Undoubtedly, the main advantage of the method presented here is sensitivity. Numerous polysaccharide synthases of interest, such as cellulose synthases, xylan and arabinan synthases, or mixed (13, 1
4)-ß-D-glucan synthases do not yield in vitro as much product as the plant (1
3)-ß-D-glucan synthases (Gibeaut and Carpita, 1993
; Kerry et al., 2001
; Kudlicka et al., 1995
, 1996
; Kudlicka and Brown, 1997
; Lai Kee Him et al., 2002
). Our method should now make possible the chemical characterization of the polysaccharides synthesized in vitro by these enzymes. Its sensitivity is well illustrated by the relative ease in obtaining solid-state 13C-NMR spectra with the same amount of in vitro product (100 µg) as required for liquid 13C-NMR spectroscopy. Because solid-state NMR spectroscopy is a nondestructive technique that preserves the 3D structure of the polymer analyzed, the same sample can be successively studied by this method and by liquid NMR spectroscopy. In addition, solid-state 13C-NMR spectroscopy allows the direct structural characterization of the different polysaccharides present in a given preparation. This is not always possible by liquid 13C-NMR spectroscopy, especially when the sample contains some polymers that are poorly or not soluble in common solvents used for NMR analysis.
The possibility of using both liquid and solid-state 13C-NMR spectroscopy for structural analysis of polysaccharides synthesized in vitro is of great interest for the study of plant glucan synthase preparations. In particular, it has been shown that active cellulose synthase preparations can be isolated from various plant species, but that the reaction mixtures recovered after in vitro synthesis always contain (13)-ß-D-glucan and cellulose (Colombani et al., forthcoming; Kudlicka et al., 1995
, 1996
; Kudlicka and Brown, 1997
; Lai Kee Him et al., 2002
). As opposed to cellulose, (1
3)-ß-D-glucans are soluble in solvents like dimethyl sulfoxide and aqueous NaOH, which are commonly employed for sample handling in liquid NMR spectroscopy. Thus this last technique can be used to specifically identify (1
3)-ß-D-glucans after solubilization in one of these solvents, whereas cellulose can be recovered in an insoluble form and characterized by solid-state 13C-NMR spectroscopy. Such experiments are under way in our laboratory using enzyme preparations from hybrid aspen (Colombani et al., forthcoming).
In addition, even in the case where a given sample contains a mixture of (13)-ß-D-glucan and cello-oligosaccharides that are soluble in dimethyl sulfoxide, the distinction between (1
3)-ß and (1
4)-ß linkages can be readily made from the analysis of the spectra obtained by liquid 13C-NMR spectroscopy. In particular, spectra obtained from solutions of cello-oligosaccharides are characterized by a signal at 80 ppm (Gast et al., 1980
), which is not present in the spectra corresponding to (1
3)-ß-D-glucans (Bulone et al., 1995
; Kogan et al., 1988
; Lai Kee Him et al., 2001
; Saito et al., 1979
). Conversely, a resonance signal at 86.3 ppm, which is characteristic of the C-3 involved in the glycosidic linkages of (1
3)-ß-D-glucans (Kogan et al., 1988
; Saito et al., 1979
), is not present in the spectra recorded from solutions of cello-oligosaccharides (Gast et al., 1980
). Also, even though the method was developed to facilitate the structural characterization of polysaccharides synthesized in vitro and not to assay glycosyltransferases, the ratios of different types of linkages in a given preparation can be estimated by NMR analysis. For instance, ratios of (1
3) to (1
4) linkages can be deduced from integrals corresponding to the C-3 and C-4 signals (Dais and Perlin, 1982
).
As opposed to enzyme preparations from most plants, detergent extracts from several members of the Poaceae family are able to synthesize mixed (13, 1
4)-ß-D-glucans in vitro, in addition to (1
3)-ß-D-glucans and cellulose (Gibeaut and Carpita, 1993
; Meikle et al., 1991
). The (1
3, 1
4)-ß-D-glucans can be distinguished from cellulose not only by their solubility in dimethyl sulfoxide but also from the analysis of the corresponding solid-state 13C-NMR spectra. More precisely, spectra of crystalline cellulose are characterized by resonance signals at 65 and 90 ppm (Atalla and VanderHart, 1984
; Atalla 1999
), which are not present in the spectra of mixed (1
3, 1
4)-ß-D-glucans (Morgan et al., 1999
). Instead, the latter contain a signal of a very weak intensity at 92.2 ppm (Morgan et al., 1999
). (1
3, 1
4)-ß-D-Glucans and (1
3)-ß-D-glucans can be easily distinguished by liquid 13C-NMR spectroscopy analysis. The spectra of (1
3, 1
4)-ß-D-glucans are more complex than those of (1
3)-ß-D-glucans (Cui et al., 2000
; Dais and Perlin, 1982
). They are characterized by several signals at about 80 ppm arising from the interglycosidic C-4, which are not present in spectra corresponding to (1
3)-ß-D-glucans (Cui et al., 2000
; Dais and Perlin, 1982
; Kogan et al., 1988
; Saito et al., 1979
).
Overall, these observations show that the method presented is very powerful, especially when both liquid and solid-state 13C-NMR spectroscopy are combined, because it allows distinction between polysaccharides that contain subtle structural differences. In most cases however, the situation is much simpler than for cell-free extracts from Poaceae, and the use of one of the two NMR techniques is usually sufficient for product characterization.
In addition to the advantages presented, the use of 13C-enriched substrates demonstrates that the polymer analyzed was indeed synthesized de novo and not arising from the cell wall or the membrane preparation used for detergent extraction of the synthase. This is particularly important when enzymes that form major cell wall polysaccharides are studied. Also, unlike conventional biochemical approaches based on the use of radioactive substrates and specific glycoside hydrolases, the method described in the present work does not require any specific environment to perform the experiments and does not generate radioactive waste.
In conclusion, we have developed an efficient and sensitive method that allows a direct and unequivocal structural characterization of products synthesized in vitro by glycosyltransferases, by combining the use of 13C-enriched sugar donors with 13C-NMR spectroscopy analyses of the newly synthesized carbohydrates. The method described may be applied to the study of any glycosyltransferase, provided that the corresponding 13C-enriched substrate can be obtained with significantly high yields by chemical or enzymatic synthesis. It is noteworthy that several protocols are already available for the synthesis of various nucleotide sugars, such as UDP-galactose (Hanessian et al., 1998; Wittmann and Wong, 1997
), GDP-mannose and GDP-fucose (Wittmann and Wong, 1997
), UDP-glucosamine and UDP-galactosamine (Rao and Mendicino, 1978
), UDP-galacturonic acid (Basu et al., 2000
), UDP-rhamnose (Barber and Behrman, 1991
), and UDP-arabinose (Zhang and Liu, 2001
). This makes the method highly suitable for the study of many glycosyltransferases from various sources.
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Materials and methods |
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Preparation of UDP-D-[U-13C]glucose
1H- and 13C-NMR spectra were recorded at 298 K for product analysis with a Bruker AC-300 (300 MHz for 1H and 75.5 MHz for 13C), Avance 400 (400 MHz for 1H and 160 MHz for 31P) or Varian Unity (500 MHz for 1H and 200 MHz for 31P) spectrometer. Solutions of the products were prepared either in CDCl3, using residual CHCl3 (1H, 7.24) and CDCl3 (13C,
77.0) as internal standards or in D2O. Mass spectra were recorded by ESI-MS in the negative mode.
Triethylammonium -D-[U-13C]glucose-1-phosphate was prepared from D-[U-13C]glucose by modification of the procedure of Sabesan and Neira (1992)
. The method consisted of acetylation (Ac2O/pyridine), anomeric deacylation (H2N:NH2.AcOH/DMF), phosphorylation (N,N-dimethylaminopyridine/ClPO[OPh]2/CH2Cl2), hydrogenolysis [H2 (12 atm)/PtO2], and transesterification (NH3/MeOH/H2O) (Figure 1). The reaction product was lyophilized to yield a hygroscopic, off-white-colored powder (70% yield, five steps). The triethylammonium
-D-[U-13C]glucose-1-phosphate was characterized by NMR spectroscopy [13C-NMR (300 MHz, D2O)
94.3 (d, J(C,C) 42.5 Hz, C-1), 73.271.2, 69.768.6 (2 m, C-2, C-3, C-4, C-5), 60.3 (d, J(C,C) 42.5 Hz, C-6)] and TLC using commercially available aluminum plates precoated with silica gel 60 F254 (Merck, Darmstadt, Germany). The compound was visualized by charring with 5% sulfuric acid in MeOH/water. It was indistinguishable (Rf of 0.092 in CH3CN/NH4HCO3 [0.1 M], 3:1) from commercially available
-D-glucose-1-phosphate.
The purified D-[U-13C]glucose-1-phosphate (107 mg, 0.29 mmol) was then dissolved in water (2 ml), coevaporated with pyridine (3 x 10 ml) and further dried under vacuum for 2 h. 4-Morpholine-N,N'-dicyclohexylcarboxamidinium uridine 5'-monophosphomorpholidate (300 mg, 0.44 mmol) and 1H-tetrazole (61 mg, 0.87 mmol) were dried separately for 1 h before being dissolved in pyridine (2 ml). They were then added to the D-[U-13C]glucose-1-phosphate and the mixture was stirred at 25°C for 7 days. Water (2 ml) was added to the mixture and the organic solvent evaporated under reduced pressure. The residue was dissolved in aqueous NH4HCO3 (0.1 M), stored as a lyophilized powder, and purified by gel filtration chromatography (BioGel P2) using NH4HCO3 solution (0.1 M) as the elution solvent.
The fractions containing the nucleotide sugar were pooled and lyophilized to obtain a white powder (88 mg, 50%). The final product was characterized by NMR spectroscopy [13C-NMR (300 MHz, D2O) 96.0 (d, J(C,C) 43.0 Hz, C-1), 73.871.4, 69.969.0 (2 m, C-2, C-3, C-4, C-5), 60.7 (d, J(C,C) 42.5 Hz, C-6)] and expected ions for UDP-D-[U-13C]glucose ([M-H], m/z 571 and [M-2H + Na], m/z 593) were obtained by ESI-MS in the negative mode. The purified product was also analyzed by TLC in the conditions described. It was indistinguishable (Rf of 0.260 in CH3CN/NH4HCO3 [0.1 M], 3:1) from commercially available UDP-glucose.
Suspension cultures of R. fruticosus cells and preparation of active (13)-ß-D-glucan synthase fractions
Cells from R. fruticosus were grown as previously described (Lai Kee Him et al., 2002) and harvested in exponential phase after 1216 days of culture. Cell disruption, isolation of microsomal membranes, and solubilization of (1
3)-ß-D-glucan synthase were essentially performed as indicated by Pelosi et al. (2003)
, except that taurocholate (0.3% final concentration) was used as a detergent instead of 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate. Typically, the culture of about 50 g of fresh cells (12 bottles containing 160 ml culture medium) allows the preparation of 25 ml detergent extract containing 12 mg protein per ml.
In vitro synthesis of (13)-ß-D-glucan
For in vitro synthesis of (13)-ß-D-glucan, the active synthase present in the taurocholate extract (6 ml) was incubated in a total volume of 24 ml of a reaction mixture containing 100 mM MOPS/NaOH buffer (pH 6.8), 8 mM CaCl2, 20 mM cellobiose, and 1 mM UDP-D-[U-13C]glucose (final concentrations). The in vitro product was recovered by low-speed centrifugation after 1 h incubation at 25°C.
To determine the yield of (13)-ß-D-glucan, UDP-D-[U-13C]glucose was replaced by 1 mM UDP-glucose and 1.9 µM UDP-D-[U-14C]glucose (final concentrations). The reaction was stopped after 1 h incubation at 25°C by the addition of five volumes of absolute ethanol. The product was precipitated overnight at 20°C and filtered through glass-fiber filters. It was successively washed with 4 ml water and 4 ml absolute ethanol. The amount of radioactive polysaccharide retained on the filter was determined by scintillation counting, using 4 ml scintillation cocktail.
Even though in vitro experiments performed in the conditions described have previously shown that (13)-ß-D-glucan is the only product synthesized in the reaction mixture (Lai Kee Him et al., 2003
), a control that consisted of testing the sensitivity of the radioactive polymer to glycoside hydrolases was prepared. The in vitro product was completely hydrolyzed by a specific endo-(1
3)-ß-D-glucanase (laminarinase from mollusk, Sigma) under the conditions described by Pelosi et al. (2003)
, confirming it to be the only polysaccharide synthesized by the detergent-extracted glucan synthase.
Structural analysis of the in vitro product by 13C-NMR spectroscopy
A total amount of 100 µg 13C-enriched in vitro product was used for both liquid- and solid-state NMR spectroscopy. This amount was calculated from the quantitative in vitro synthesis experiments performed with radioactive substrate instead of UDP-D-[U-13C]glucose, in the conditions described. The in vitro product recovered by low-speed centrifugation as indicated in the previous section was purified as previously described (Pelosi et al., 2003). It was then lyophilized and either used directly for solid-state NMR spectroscopy or dissolved in (CD3)2SO for liquid NMR spectroscopy.
Analysis by liquid 13C-NMR spectroscopy was performed with a Bruker AC 300 spectrometer operated at 295 K and 75.5 MHz. The central peak of the (CD3)2SO multiplet was used as a reference.
For solid-state NMR spectroscopy, the spectra were recorded with a Bruker Avance spectrometer operated at a 13C frequency of 100 MHz, using the combined techniques of proton dipolar decoupling, magic angle spinning, and cross-polarization. The cross-polarization transfer was achieved using a ramped amplitude sequence for an optimized total contact time of 1 ms (Peersen et al., 1993). The spinning speed was set at 8 kHz. A sweep width of 50,000 Hz and a recycled delay of 4 s were selected. A typical number of 1000 scans was acquired. Chemical shifts were referred to tetramethylsilane after external calibration with the carbonyl signal of glycine at 176.03 ppm.
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Acknowledgements |
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Footnotes |
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1 Present address: Equipe Organisation et Dynamique des Membranes Biologiques, UMR CNRS 5013, Bâtiment Chevreul 4ème étage, Université Claude Bernard-Lyon I, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne cedex, France
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Abbreviations |
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References |
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