2 Bijvoet Center, Department of Bio-Organic Chemistry, Section of Glycoscience and Biocatalysis, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands; and 3 Department of Biology, Northeastern University, Boston, MA 02115, USA
Received on March 5, 2002; revised on April 2, 2002; accepted on April 3, 2002
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Abstract |
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Key words: cyst wall/Giardia /1H NMR spectroscopy/mass spectrometry/polysaccharide
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Introduction |
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For the study of the cytodifferentiation from trophozoite to cyst as well as for our study of the biogenesis of the Giardia cyst wall, detailed information of the molecular structure of the cyst wall material is a prerequisite. It is widely accepted that many protozoan cyst walls contain chitin, but there is little sound biochemical evidence supporting this assumption. Indeed, a series of histochemical and biochemical studies suggested that the cyst wall of Giardia was composed of chitin (reviewed in Jarroll and Paget, 1995). However, in a series of studies (Jarroll et al., 1989
; Manning et al., 1992
; Macechko et al., 1992
) focused on G. intestinalis and G. muris, it was demonstrated that the filamentous cyst walls contain carbohydrate and protein and that the major monosaccharide constituent is N-acetylgalactosamine (GalNAc). Subsequently, these investigators have shown that GalNAc is synthesized de novo from endogenous glucose (Macechko et al., 1992
) via a pathway of inducible enzymes, which are transcriptionally as well as allosterically regulated (Van Keulen et al., 1998
; Bulik et al., 2000
), and that GalNAc is fixed into an insoluble polysaccharide by the action of "cyst wall synthase" (Jarroll and Paget, 1995
). Considering the insolubility of the cyst wall and the abundance of GalNAc, it was suggested that the filaments are composed of a carbohydrate(polysaccharide)protein complex. With respect to the carbohydrate component, this report presents results demonstrating conclusively that the Giardia cyst wall filaments are not composed of chitin but of a previously undescribed (ß1-3)-linked GalNAc homopolymer. Furthermore, insight is provided into conformational aspects of this polysaccharide.
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Results |
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Gas-liquid chromatography (GLC) analysis after methanolysis of CWM and subsequent derivatization (re-N-acetylation, then trimethylsilylation) revealed D-GalNAc to be the only monosaccharide constituent. The total carbohydrate content amounted 63% (w/w). GLC analysis after acid hydrolysis of CWM and subsequent derivatization (trimethylsilylation) indicated the presence of galactosamine (GalN). Protein determination of CWM revealed a protein content of 36% (w/w). Taking into account the carbohydrate and protein content of CWM and the fact that CWM is hydrolyzable under the methanolysis/hydrolysis conditions used (Kamerling and Vliegenthart, 1989), a high percentage of GalN as native monosaccharide constituent in CWM can be ruled out. The identity of the N-acyl group as N-acetyl was demonstrated by 1H nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) (vide infra). Therefore it can be assumed that the carbohydrate part of CWM is a polysaccharide built up (nearly completely) from GalNAc residues.
To generate soluble material, the highly insoluble CWM was partially hydrolyzed (PH) by repeated short treatments with 0.5 M trifluroacetic acid (TFA). The mixture obtained (CWM PH) was fractionated by gel filtration chromatography (Figure 1) yielding five fractions, denoted CWM PH 15, which were rechromatographed to obtain better defined fractions. Fraction CWM PH 5 contained free GalNAc according to monosaccharide and 1H NMR analysis. MALDI-TOF-MS analysis of fraction CWM PH 4 (Figure 2) only showed the presence of the pseudomolecular ion [M+Na]+ of HexNAc2 at m/z 446.9. After reduction (CWM PH 4R), monosaccharide analysis revealed the occurrence of GalNAc and GalNAc-ol in the molar ratio of 0.9:1.0, whereas methylation analysis demonstrated the presence of terminal GalNAc and 3-substituted GalNAc-ol.
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Monosaccharide analysis of fraction CWM PH 1 demonstrated a carbohydrate (GalNAc) content of 40% (w/w), whereas methylation analysis again showed only terminal GalNAc and 3-substituted GalNAc. The protein determination of CWM PH 1 showed a protein content of 60% (w/w). In the MALDI-TOF-MS spectrum of CWM PH 1 (Figure 2) a series of pseudomolecular ions [M+Na]+ of oligosaccharides ranging from HexNAc5 up to HexNAc23 (m/z 4710.2) was observed. Because chain cleavage occurred during partial hydrolysis, the degree of polymerization in the native cyst wall material may be much higher.
The 1D 1H-NMR spectrum of CWM PH 1 (carbohydrate region) is shown in Figure 3B, and the included proton assignments were obtained through analysis of 2D TOCSY and nuclear Overhauser effect spectroscopy (NOESY) NMR spectra. According to the various data, it can be concluded that the carbohydrate moiety of CWM is composed of a (ß1-3)-linked GalNAc homopolymer. The structural analysis has been performed on GalNAc oligosaccharides released by partial acid hydrolysis, but in view of the high protein content in CWM PH 1, it is likely that a covalent linkage between protein and GalNAc oligomers occurs in CWM.
Conformational analysis of the carbohydrate moiety
To gain insight into the conformational aspects of the GalNAc polysaccharide, especially in relation to its insolubility, molecular dynamics (MD) calculations with the constituting disaccharide GalNAc(ß1-3)GalNAc were performed. The results were used to evaluate possible conformations of polysaccharide structures.
To explore the preferred conformations of the glycosidic linkages (O5'-C1'-O3-C3) and
(C1'-O3-C3-C2) (IUPAC-IUB Joint Commission on Biochemical Nomenclature. , 1983
), a relaxed energy map of the methyl glycoside of the disaccharide in vacuum was constructed with the use of the CHEAT force field (Grootenhuis and Haasnoot, 1993
; Kouwijzer and Grootenhuis, 1995
). This map (Figure 4A) showed two conformational regions with a low energy, one (
,
= 60, 120) is denoted region I, and the other (
,
= 60, 60) is denoted region II. Region I represents the global lowest energy. Next, minimal energy conformations selected from regions I and II were minimized again, this time with fully relaxed interglycosidic dihedral angles.
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To assess the value of the computer simulation results, the 1H NMR data of fraction CWM PH 1 were used. The NOESY spectra contained several medium to strong cross-peaks that in many cases were intraresidual. The H1'-H4 interaction would be detectable for the regions Ib (strong) and II (weak). Its absence however, demonstrated the dominant role of the Ia conformation. Signal overlap prevented a straightforward quantitative analysis of the NOEs.
To determine the population distribution of conformations more precisely, the method of adaptive umbrella sampling of the potential of mean force (Hooft et al., 1992) was used. With this method, free energy differences are calculated between conformations in relation to the interglycosidic dihedral angle
. From this information, the population distribution of the conformations can be determined and this result is shown in Figure 4B. Inspection of this distribution profile shows that the GalNAc disaccharide only occurs in the global lowest energy conformation (region I). This energy minimum can be subdivided into two subminima, which are populated in a ratio of 4:1. Although the two conformations do not differ a great deal with respect to the interglycosidic dihedral angles, this small difference can have a significant effect on the total conformation of the GalNAc polysaccharide.
To get an impression of the conformation of this polysaccharide in solution, some polysaccharide models with a size of 30 monosaccharide units were constructed from disaccharides and linkage information obtained from the MD runs. Polysaccharide models constructed from disaccharide residues collected from region Ia form a right-handed helical structure. The average size of a helical turn is approximately seven residues. In contrast, polymer models constructed from disaccharide residues and linkages collected from region Ib gave a left-handed helical structure with an average helical-turn size of about 8.4 residues. A polysaccharide model chain, constructed from disaccharide structures and linkages selected from both regions in a ratio of Ia:Ib = 4:1, showed a random coil with a much more folded (less rodlike) conformation than observed for the two previous polysaccharide models (Figure 5).
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Discussion |
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In our conformational studies, two preferential structures could be identified for GalNAc(ß1-3)GalNAc. A 4:1 combination of these two conformations resulted in a flexible, random-coil model for the GalNAc polymer. From the conformational studies, it is clear that the highly insoluble nature of the cyst wall does not stem from the conformational properties of a single GalNAc polysaccharide chain but must be due to strong interchain interactions. The potential covalent linkage between the GalNAc polymers and the detected protein could also play a role. This is currently the subject of further study.
Of biological interest here is the fact that the cyst wall filaments of Giardia are not composed of chitin but rather of a novel polysaccharide for which no known degradative enzyme has been found as yet. Because many of the microorganisms that inhabit the intestines where these cysts are formed can produce chitinase, there could be a clear evolutionary advantage to Giardia not having its protective cyst wall composed of chitin. It is also interesting to speculate that since the cyst wall of Giardia is made of this novel GalNAc homopolymer, then there must be some hydrolase(s) capable of degrading it. Whether this hydrolase exists and is involved in the cytodifferentiation called excystation (trophozoites emerging from the cyst) is unknown. It is possible that excystation might occur simply by proteolysis of the protein present in the cyst wall as suggested by Ward et al. (1997). However, it is also likely that there exists a hydrolase capable of degrading this polymer, otherwise the amount of this material in the ecosystem would tend to accumulate irreversibly.
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Materials and methods |
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The cysts were concentrated by centrifugation at 12,000 x g for 5 min (as in subsequent steps), and the supernatant was discarded. The cyst pellet was washed in 10 ml distilled water five times until there were no visible signs of the detergent. The resulting cyst pellet was resuspended in 3 ml of amyloglucosidase buffer (20 mM acetate buffer, pH 4.5), transferred to a 20-ml glass screw-capped scintillation vial with a flat bottom and a stir bar. One hundred units of amyloglucosidase (EC 3.2.1.3, Sigma A-3514, St. Louis, MO) was added to the vial that was placed on a stir plate in a 55°C warm air incubator for 60 min. This procedure was repeated once more, and each time the supernatant was discarded.
After the two amyloglucosidase treatments, the SDS treatment was performed once again. The resulting cyst pellet was washed in 10 ml of freshly made papain buffer (100 mM phosphate buffered saline [PBS], pH 7.2, with 5 mM cysteine and 5 mM ethylenediamine tetra-acetic acid [EDTA]). The pellet was resuspended in 3 ml papain buffer and again placed in a glass scintillation vial with a stir bar. The sample was stirred with 50 U of papain (EC 3.4.22.2, Sigma P-4762) for 3 h at 60°C. This procedure was repeated once more, and each time the supernatant was discarded. Again the resulting cyst pellet was collected by centrifugation and washed, as in step one, with 10% SDS and water.
The pellet resulting from the papain treatment was subjected to DNase (EC 3.1.21.1, Sigma DN-25) and RNase (EC 3.1.27.5, Sigma R-4875) treatments. The pellet was resuspended in 3 ml of XNase buffer (0.1 M PBS, pH 7.2, with 5 mM MgCl2) in a glass scintillation vial with a stir bar. The sample was incubated with 500 Kunitz units of DNase and 5 Kunitz units of RNase stirring for 2 h at 37°C. This treatment was repeated once more and the pellet washed with amyloglucosidase buffer. At this point, all subsequent centrifugation steps were carried out at 44,000 x g for 10 min at 10°C. Following the DNase/RNase treatment, the pellet was subjected to another amyloglucosidase treatment as described and then to a proteinase K (EC 3.4.21.14, Sigma P-0390) treatment. The pellet was washed once in proteinase K buffer (50 mM Tris, pH 8.0, 0.2% Triton X-100, 1 mM CaCl2). The pellet was resuspended in the proteinase K buffer, placed in a glass scintillation vial with 50 U proteinase K, and stirred for 2 h at 60°C. This procedure was repeated once more, and the pellet was washed with an equal volume of 10% SDS containing 100 mM dithiothreitol (DTT) for 5 min at 100°C. The pellet was then washed five times in distilled water to remove the detergent and DTT. The resulting CWM was lyophilized for 48 h, yielding approximately 150 mg dry fine-powdered white material.
Monosaccharide analysis and protein determination
Monosaccharide analysis after methanolysis, re-N-acetylation, and trimethylsilylation, including absolute configuration determination using (-)-2-butyl glycosides, was performed by GLC-MS as described (Gerwig et al., 1979; Kamerling and Vliegenthart, 1989
). Protein determinations were carried out using a Micro BCATM Protein Assay Reagent Kit (Pierce).
Acid hydrolysis
Dried CWM (1 mg) and GalNAc (1 mg) were each treated with 1 ml of 2 M HCl for 3 h at 100°C and with 1 ml of 4 M TFA for 5 h at 100°C. After lyophilization, the residues were dissolved in 1 ml H2O. Aliquots of 25 µl of the four samples were taken, lyophilized, and trimethylsilylated with 25 µl pyridine/hexamethyldisilazane/chlorotrimethylsilane 5:1:1 (v/v) for 30 min at room temperature. Of each sample, 1 µl was analyzed by GLC (Chrompack CP 9002 gaschromatograph with CP Sil 5CB column, 25 m x 0.32 mm); temperature program: 140240°C at 4°C/min).
Partial acid hydrolysis
Dried CWM (8 mg) was treated with 0.5 ml of 0.5 M TFA for 15 min at 100°C. After centrifugation, the supernatant was collected and the sediment was treated again with 0.5 ml of 0.5 M TFA for 15 min at 100°C. This procedure was repeated several times until no sediment was left. Then, the total supernatant was concentrated by a stream of N2 and lyophilized twice. The residue was suspended in 2 ml H2O and filtered through a 0.22-µm Millex-GS filter (Millipore); the filtrate was fractionated on a column (68 x 1.6 cm) of Bio-Gel P-2 (Bio-Rad), using bidistilled H2O as eluent (22 ml/h) and UV monitoring at 206 nm. Definite fractions (Figure 1) were rechromatographed on the same column.
Methylation analysis
Permethylation was performed essentially as described (Ciucanu and Kerek, 1984). Dried sample (0.11.0 mg) was dissolved in 1 ml dry dimethyl sulfoxide by ultrasonication for 30 min at 40°C. Freshly powdered NaOH (~10 mg) was added under an inert atmosphere, and the mixture was sonicated for 20 min at room temperature. After cooling to 0°C, 400 µl methyl iodide was added, and sonication was continued for 30 min at a temperature not exceeding 20°C. The latter step was repeated with a second addition of 400 µl methyl iodide. Then, 1 ml H2O containing a few crystals of sodium thiosulfate was added, and the methylated product was isolated by extraction with chloroform (3 x 0.5 ml). The organic phase was washed with water (3 x 0.5 ml) and concentrated under a stream of N2. The residue was hydrolyzed with 1 ml of 4 M TFA (4 h, 100°C) and, after coevaporation with methanol, reduced with NaBD4 in water (2 h, room temperature). The excess of reductant was destroyed by the addition of 2 M acetic acid. Boric acid was removed by repeated evaporations with methanol. The sample was finally acetylated with 0.5 ml pyridine/acetic anhydride 1:1 (v/v) for 30 min at 100°C. The solvent was evaporated by a gentle stream of N2 with intermediate addition of one drop of toluene, and the residue was redissolved in dichloromethane. The partially methylated alditol acetates were analyzed by GLC-MS (Kamerling and Vliegenthart, 1989
).
MALDI-TOF MS
Positive-ion MALDI-TOF mass spectra were obtained on a Voyager-DE (PerSeptive Biosystems) instrument operating at an accelerating voltage of 21 kV (grid voltage 95%, ion guide wire voltage 0.05%) and equipped with a VSL-337ND-N2 laser. The samples were dissolved in H2O (1 µg/µl) and mixed with 2,5-dihydroxybenzoic acid (10 mg/ml) in a ratio of 1:2. Recorded data were processed using GRAMS/386 software (v. 3.04, Galactic Industries).
1H NMR spectroscopy
Samples were exchanged twice in D2O (99.9 atom% D, Isotec) with intermediate lyophilization. Finally, the material was dissolved in 0.5 ml of D2O (99.96 atom% D, Isotec). One-dimensional resolution-enhanced 500-MHz 1H-NMR spectra were recorded on a Bruker AMX-500 spectrometer at a probe temperature of 300 K. Chemical shifts are expressed in ppm by reference to internal acetone ( 2.225) (Vliegenthart et al., 1983
). 2D TOCSY spectra with mixing times of 12, 25, 41, 50, and 125 ms were recorded at 500 MHz on a Bruker DRX-500 instrument. The spectra were processed using locally developed NMR software (Van Kuik, Bijvoet Center, Utrecht University). 2D ROESY experiments were carried out at 280 K with mixing times of 100, 200, 300, and 500 ms. The HOD signal was presaturated for 1 s during the relaxation delay.
Minimum energy calculations
Minimum energy calculations in vacuo were performed with CHEAT (Grootenhuis and Haasnoot, 1993; Kouwijzer and Grootenhuis, 1995
), a CHARMm-based force field for carbohydrates in which hydroxyl groups are represented by extended atoms to prevent the formation of intramolecular hydrogen bonds and whereby energies are independent of the hydroxyl group orientation. A relaxed energy map was generated for the GalNAc(ß1-3)GalNAc disaccharide by changing the interresidual torsion angles in steps of 10° resulting in a 36 x 36 grid. For each grid point calculation, three intraglycosidic dihedral angles per monosaccharide ring and both interglycosidic angles are kept fixed by setting constraints during a first minimization. Next, all constraints in the ring were removed and the molecule was minimized a second time. Contour levels were plotted in steps of 1 kcal/mol from the global minimum.
Molecular dynamics calculations
Molecular dynamics simulations were carried out using the GROMOS program package (Van Gunsteren, 1987) and the updated carbohydrate force field for GROMOS (Spieser et al., 1999
) on local PentiumII class computers running Linux. Each molecule was surrounded by SPC/E (Berendsen et al., 1987
) water molecules and placed in a truncated octahedral periodic box. All bond lengths were kept fixed using the SHAKE procedure (Ryckaert et al., 1977
). A cut-off radius of 0.8 nm and a time step of 2 fs were used. Simulations were performed with loose coupling to a pressure bath at 1 atm and a temperature bath at 300 K (Berendsen et al., 1984
) with time constants of 0.5 and 0.1 ps, respectively. To confirm calculated data, (ß1-3)-linked GalNAc oligosaccharide fragments with DP ~5 and DP >20 were analyzed by 1H NMR spectroscopy.
Potential of mean force calculations on the GalNAc-GalNAc glycosidic linkage
To calculate the free energy differences of the conformations around the GalNAc(ß1-3)GalNAc glycosidic angle, potential-of-mean-force calculations (Hooft et al., 1992
) were run for the methyl ß-glycoside of the disaccharide with the GROMOS force field. All simulations were divided into jobs of 10 ps. The
values were collected into 72 classes, each with a width of
= 5°. The derivatives were fitted to a 12-term Fourier series. The first 0.2 ps of each job were discarded.
Persistence length calculations
The persistence length calculations of the GalNAc polymer were performed with data selected from appropriated parts of the MD simulations of the GalNAc(ß1-3)GalNAc disaccharide (Kroon-Batenburg et al., 1997). For each calculation, 1000 frames were selected and used to create chains of 300 residues.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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