1 Department of Biology, Northeastern University, Boston, MA 02115, USA
2 Program in Glycobiology, Shriver Center at University of Massachusetts Medical School, 200 Trapelo Road, Waltham, MA 02452, USA
Correspondence
David S. Newburg
david.newburg{at}umassmed.edu
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ABSTRACT |
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Present address: Anhui Provincial Center for Clinical Laboratories, Anhui Medical University, Hefei, P. R. China.
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INTRODUCTION |
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Giardia's life cycle includes trophozoites and cysts. The vegetative trophozoite infects the proximal small intestine and may cause disease. In response to bile, the trophozoite can encyst (elaborate a cyst wall), and after being shed via the faeces into the environment, cysts remain viable for several months in cool moist conditions and resist chlorination (deRegnier et al., 1989; Farthing, 1996
). It is as cysts that Giardia survives outside of the host and can thus be transmitted from one host to the next.
Giardia's cyst wall is composed of an inner membranous and an outer filamentous portion (Feely et al., 1984). The outer filamentous wall is made up of proteins (37 %) and carbohydrate filaments (63 %) composed mainly of a [D-galnac-
(1-3)-D-GalNAc]n homopolymer (Gerwig et al., 2002
). The precursor for the GalNAc homopolymer is UDP-GalNAc, which is synthesized from glucose by a pathway of inducible enzymes and amino sugar phosphate intermediates shown in Fig. 1
. The enzymes involved in the synthesis of UDP-GalNAc include glucosamine-6-phosphate isomerase, glucosamine-6-phosphate N-acetylase, phosphoacetylglucosamine mutase, UDP-GlcNAc pyrophosphorylase and UDP-GlcNAc 4'-epimerase. All of these enzymes have been localized to the cytosol of encysting Giardia (Macechko et al., 1992
). UDP-GalNAc is then converted into a unique
(1-3)-D-GalNAc homopolysaccharide by the action of a particle-associated
(1-3)GalNAc transferase, tentatively termed cyst wall synthase (CWS) (Jarroll et al., 2001
). When trophozoites are induced to encyst, the first five of these enzymes are transcriptionally activated (Lopez et al., 2003
) and the specific activities of all these enzymes increase (Macechko et al., 1992
). Although CWS, which catalyses the ultimate step of the pathway, has not yet been cloned, we hypothesize that its transcription could also be induced, as it is part of the same pathway and its activity also increases during encystment. However, changes in levels of enzyme expression provide only an indirect measure of potential metabolic changes and control in an organism; changes in enzyme activity are often more relevant towards understanding the proximate basis of fine metabolic control. In this context, Bulik et al. (2000)
showed that the GlcN 6-P concentration increased threefold during encystment and that GlcN 6-P can allosterically activate UDP-GlcNAc pyrophosphorylase in the direction of UDP-GlcNAc synthesis. However, changes in enzyme activity, measured in isolated enzymes in vitro, may not reflect their true activity in intact cells. Thus a more direct means of understanding the control of a metabolic pathway requires simultaneous measurement of levels of metabolic intermediates for each of the steps of the pathways under different physiological states. A long-standing difficulty with such simultaneous measurements of sugar phosphate intermediates in any metabolic pathway is their low concentrations in cells (Bessman, 1974
).
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METHODS |
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Amino sugar phosphate extraction.
Cells from each culture were resuspended in 30 ml ice-cold ultrapure water and divided into three equal aliquots. Each aliquot was treated as follows before homogenization. Two aliquots were made 1 % with phosphatase inhibitor (Phosphatase Inhibitor Cocktail 2; Sigma), and into one of the two, a mixture of authentic sugar phosphate standards containing GlcNAc 6-P, GlcN 1-P, GalN 1-P, GlcNAc 1-P, GlcN 6-P, UDP-GalNAc, UDP-GlcNAc and UDP-Glc (Sigma) was added. The first of these aliquots was used to quantify each peak that corresponded to an amino sugar phosphate of interest. The second with authentic standards was used to confirm the identity of each peak by co-elution with, and to calculate the recovery of, each of the added standards. The third aliquot, without inhibitor, was used to measure the organic phosphate peaks in the absence of phosphatase inhibitor, both with and without treatment with exogenous phosphatase. The three aliquots were homogenized on ice for 4 min using a Polytron homogenizer (model PT 10/35; Brinkman Instruments) at setting no. 4, with a 9 mm microprobe. The homogenates were centrifuged on a swinging bucket rotor (SW28) in a Beckman ultracentrifuge at 100 000 g for 90 min. Supernatants were transferred to 10 000 MWCO centrifugal ultrafilters (Filtron; Gelman) and filtered by centrifugation at 4900 g for 39 h. After measuring filtrate volumes, the filtrates were lyophilized and redissolved in 200 µl ultrapure water. From the third sample an 80 µl aliquot was removed for treatment with alkaline phosphatase as follows: into an 80 µl aliquot, 10 units (10 µl) shrimp alkaline phosphatase (New England BioLabs) and 10 µl 10x alkaline phosphatase buffer (New England BioLabs) were added and the solution was digested at 25 °C for 1 h. Disappearance of the peaks in this sample confirmed that the peaks co-eluting with the amino sugar phosphate standards were also themselves phosphorylated. As positive controls for the phosphatase digestion, a mixture of pure authentic standards at concentrations similar to those found in our samples was incubated with alkaline phosphatase. All samples were analysed by capillary electrophoresis.
Amino sugar phosphate measurement.
Capillary zone electrophoresis conditions were developed for the resolution of the mixture of amino sugar phosphate standards listed above: 30 kV normal polarity (sample loaded in the anode and detected at the cathode) in 30 mM sodium borate running buffer, pH 9·0 at 19 °C. The capillary had an effective length of 56 cm and an interior diameter of 50 µm with extended light path geometry at the detector. The hexosamine phosphates and nucleotide sugars were detected by their absorbance at 200 nm. This is the first technique capable of measuring all of the known intermediates of amino sugar phosphate metabolism as found naturally in Giardia and other tissues (D. S. Newburg and others, unpublished).
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RESULTS |
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This technique allowed the simultaneous measurement of amino sugar phosphates in Giardia as its trophozoite at 0 h, at 24 h after exposure to bile (peak of encystment) and at 48 h after exposure (when encystment is approaching completion). The data in Table 1 and Fig. 3
demonstrate that during encystment, the levels of all of the amino sugar phosphates in the UDP-GalNAc synthetic pathway rise above those levels seen in the trophozoite at 0 h in a co-ordinated fashion, and that after encystment, they return towards non-encysting levels. Such an increase is not seen in amino sugar phosphates that are not part of this UDP-GalNAc synthetic pathway, such as the GalN 1-P and GlcN 1-P shown at the bottom of Table 1
.
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In all of our measurements of amino sugar phosphate intermediates in Giardia, there are higher levels of UDP-GalNAc than of its precursor, UDP-GlcNAc (Fig. 3). If the synthesis of UDP-GalNAc from the apparent rate-controlling substrate UDP-GlcNAc were controlled by the withdrawal of the UDP-GalNAc for synthesis of the cell wall, the amount of the latter would have been less than that of the former. However, because the amount is greater, it implies that this ultimate conversion step of the pathway is through an active synthetic process that favours the production of UDP-GalNAc from its immediate precursor, UDP-GlcNAc.
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DISCUSSION |
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The co-ordinated increase and decrease through Giardia's encystment process was specific to the metabolic intermediates from GlcN 6-P through UDP-GalNAc, and not observed in similar compounds that are not part of this pathway, such as GlcN 1-P and GalN 1-P. This provides additional confidence that the method correctly measures the compounds of interest, and that the pathway shown in Fig. 1 correctly describes metabolic changes in Giardia that are specific to encystment.
These changes in the level of the sugar phosphate intermediates suggest co-ordinated metabolic control of UDP-GalNAc synthesis associated with encystment. At 24 h all of the pathway intermediates increased in concert when encystment was at its peak, and most of them returned to non-encysting levels by 48 h, when encystment approached completion. These in vivo findings are consistent with the findings of Macechko et al. (1992) that the activity of pathway enzymes in vitro increased by 24 h and sharply decreased by 48 h.
The increase in pathway intermediates was greatest in absolute amount for GlcN 6-P, which is a positive allosteric effector in the synthetic direction for the pathway's putative rate-controlling enzyme UDP-GlcNAc pyrophosphorylase (Bulik et al., 2000). This tends to push the pathway in favour of cyst wall synthesis. In this pathway, the UDP-GlcNAc pyrophosphorylase converts GlcNAc 1-P to UDP-GlcNAc. The proposed regulatory role of this enzyme in this pathway, both with regard to being rate-limiting and of being allosterically activated during encystment, is supported by our finding a ninefold increase (highest of any of the sugar phosphate intermediates) in UDP-GlcNAc during encystment.
In vitro, the equilibrium of the epimerase reaction UDP-GlcNAc to UDP-GalNAc results in higher levels of UDP-GlcNAc than UDP-GalNAc, which suggested that in Giardia the reaction might be pulled in the direction of synthesis of UDP-GalNAc by depletion of UDP-GalNAc as it is used in cyst wall synthesis. However, we detected more UDP-GalNAc at 24 h than UDP-GlcNAc, its direct precursor. This suggests that there is a shift in the preferred direction of synthesis by the UDP-GlcNAc 4'-epimerase, the enzyme immediately following the pyrophosphorylase in this pathway. These results suggest that in the cytoplasm of encysting Giardia there are kinetic pressures (such as increased amounts of UDP-GalNAc) forcing the reaction in the direction that supports (pushes) cell wall synthesis. Because this major distinction between in vitro and our in vivo observations is based on differences in measurements of very small quantities, it warrants confirmation by an independent form of analysis. These control points of metabolic pathways essential for encystment in Giardia may be candidate targets for specific inhibitors that may be of therapeutic value against Giardia.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 10 November 2003;
revised 30 January 2004;
accepted 30 January 2004.