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J Biol Chem, Vol. 275, Issue 19, 14722-14728, May 12, 2000

UDP-N-acetylglucosamine Pyrophosphorylase, a Key Enzyme in Encysting Giardia, Is Allosterically Regulated^*

Dorota A. Bulik, Peter van Ophem, James M. Manning, Zuojun Shen^§, David S. Newburg^§, and Edward L. Jarroll^¶

From the  Department of Biology, Northeastern University, Boston, Massachusetts 02115 and ^§ Program in Glycobiology, Shriver Center, Waltham, Massachusetts 02460 and Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Giardia synthesizes UDP-GalNAc during cyst wall formation (encystment) via a pathway of inducible enzymes similar to that used to synthesize chitin or peptidoglycan and that includes the UTP-requiring UDP-N-acetylglucosamine pyrophosphorylase. Although it has never been reported as a regulatory enzyme in any system studied to date, kinetic data including Hill plots demonstrate clearly that UDP-N-acetylglucosamine pyrophosphorylase activity, purified from encysting Giardia, is allosterically activated anabolically by physiological levels of glucosamine 6-phosphate (3 µM). Capillary electrophoresis demonstrates that within 24 h after trophozoites are induced to encyst, the level of glucosamine 6-phosphate increases 3-fold over that of non-encysting cells and that by 48 h into encystment the level of glucosamine 6-phosphate has decreased to non-encysting levels or below. UDP-N-acetylglucosamine pyrophosphorylase protein is present constitutively in encysting as well as non-encysting cells. UDP-N-acetylglucosamine pyrophosphorylase immunoaffinity purified from encysting and non-encysting cells exhibited the same molecular weight, amino acid composition, and circular dichroism spectra. Moreover, regardless of whether the enzyme came from encysting or non-encysting cells, the change in its circular dichroism spectra and up to a 6-fold increase in its specific activity anabolically were due to its activation with glucosamine 6-phosphate. Thus, the data support the idea that UDP-N-acetylglucosamine pyrophosphorylase is a major regulatory point in amino sugar synthesis in encysting Giardia and that its allosteric anabolic activation may shift the equilibrium of this pathway toward UDP-GalNAc synthesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Giardia lamblia, a primitive eukaryote (1), parasitizes humans worldwide causing giardiasis. The life cycle of Giardia includes cysts, by which fecal-oral transmission occurs, and vegetative trophozoites, which colonize the upper small intestine and produce cysts in response to bile (2, 3). During cyst formation (encystment), trophozoites become encased within a cyst wall, which has an inner membranous and an outer filamentous portion (4, 5). These filaments are made of a GalNAc polymer complexed with protein (6-8). The precursor for this polysaccharide, UDP-GalNAc, is synthesized from endogenous Glc by inducible enzymes, the activities of which increase during Giardia encystment from 8- to 4000-fold (9). Glucosamine-6-phosphate isomerase (GlcN-6-P isomerase), the first enzyme unique to this pathway, is transcriptionally regulated (10) and converts Fru-6-P and NH₃ to GlcN-6-P. GlcN-6-P is converted to GlcNAc-6-P and then to GlcNAc-1-P by GlcN-6-P N-acetylase and phosphoacetylglucosamine mutase, respectively (10, 11). GlcNAc-1-P in the presence of UTP is converted to UDP-GlcNAc and PP_i anabolically (the catabolic reaction is the reverse) by UDP-N-acetylglucosamine pyrophosphorylase, and UDP-GlcNAc is epimerized to UDP-GalNAc by UDP-N-acetylglucosamine 4'-epimerase (10). The UDP-GlcNAc produced in this system is also a key component of bacterial peptidoglycans (12, 13), lipopolysaccharides (14), chitin (15), and N- and O-linked glycoproteins (14, 16); however, the enzyme responsible for its synthesis, UDP-N-acetylglucosamine pyrophosphorylase, has been little studied, especially in eukaryotic systems. UDP-N-acetylglucosamine pyrophosphorylase has been partially purified from bacteria (12), yeast (17), Neurospora crassa (18), and calf liver (12); it has been purified to homogeneity from pig hepatocytes and characterized (19). Until recently, the gene for UDP-N-acetylglucosamine pyrophosphorylase had been sequenced only from prokaryotes (13, 20, 21). Mio et al. (22) cloned, sequenced, and expressed UDP-N-acetylglucosamine pyrophosphorylase from yeast and humans and partially characterized the kinetics of these enzymes. Heretofore, a regulatory role has neither been described nor proposed for UDP-N-acetylglucosamine pyrophosphorylase.

UDP-N-acetylglucosamine pyrophosphorylase, purified from G. lamblia (23), is a ~66-kDa protein with two ~33-kDa subunits. Only the UDP-N-acetylglucosamine pyrophosphorylase from pig liver has multiple subunits (~57 and ~64 kDa) for the 124-kDa enzyme (19). UDP-N-acetylglucosamine pyrophosphorylase from Giardia exhibits typical Michaelis-Menten kinetics with all substrates except GlcNAc-1-P, with which the kinetics of the enzyme suggest allosteric regulation (23). Here we examine the possible allosteric regulation of UDP-N-acetylglucosamine pyrophosphorylase in Giardia.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture and Encystment-- Giardia intestinalis (syn. lamblia, duodenalis) trophozoites (MR4) were cultured axenically in TYI-S-33 medium (24) with 1% bovine bile (0 h medium, normal growth medium for non-encysting cells) or no bile (NB medium,¹ no bile growth medium) at 37 °C (9). After 96 h in either NB or 0 h medium, cells were collected by centrifugation and induced to encyst with high bile encystment medium (48 h medium (HB), growth medium supplemented with 10 mg of bile ml¹ at pH 7.6 (3)). Encystment, which is asynchronous, typically reaches its maximum, achieving 10-50% cysts or encysting cells by 2 days in HB medium.

Purification of and Assays for UDP-N-acetylglucosamine Pyrophosphorylase-- UDP-GlcNAc pyrophosphorylase (EC 2.7.7.23) was purified from encysting Giardia trophozoites and assayed by measuring the formation of [¹⁴C]UDP-GlcNAc from [¹⁴C]UTP and GlcNAc-1-P (23) and catabolically by measuring the formation of GlcNAc spectrophotometrically at 585 nm (12) in a phosphatase-coupled assay. One unit of enzyme activity is the number of micromoles of substrate consumed or product produced min¹. Specific activity is units mg of protein¹. Protein concentration was determined (25), and enzyme purity was assessed by two-dimensional gel electrophoresis (26) (Kendrick Labs, Madison, WI) and by gel filtration chromatography (26). Isoelectric focusing was performed in glass tubes (inner diameter of 2.0 mm) using 2% BDH ampholines (Hoefer Scientific, San Francisco) with a pH range of 4.0-8.5 at 700 V for 14 h. After equilibration in buffer (0.625 M Tris-HCl, pH 6.8, 50 mM dithiothreitol, 2.3% SDS, and 10% glycerol) for 10 min, the tube gel was sealed on the top of a stacking gel overlaying a 10% polyacrylamide gel. Gel electrophoresis was performed at 12.5 mA gel¹ for 4 h. Tropomyosin (pI, 5.2 and molecular mass, 32 kDa) was used as an internal standard (1 µg). The following molecular mass standards were added to the sealing agarose: myosin (220 kDa), phosphorylase a (94 kDa), catalase (60 kDa), actin (43 kDa), carbonic anhydrase (29 kDa), and lysozyme (14 kDa). Slab gels were fixed overnight in 10% acetic acid, 50% methanol solution and stained with Coomassie Blue. Gel filtration chromatography was performed on a fast protein liquid chromatography (Amersham Pharmacia Biotech) Superose-12 size exclusion column; protein fractions were eluted isocratically in 50 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl at a flow rate of 0.4 ml min¹. The molecular mass of the native protein was calculated from its elution relative to alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12 kDa) (Bio-Rad).

Activation of UDP-N-acetylglucosamine Pyrophosphorylase-- The effect of the pathway intermediates and their analogs on UDP-N-acetylglucosamine pyrophosphorylase activity was measured anabolically and catabolically; Fru-6-P, Gal-6-P, Glc-6-P, GlcN-6-P, GalNAc, GlcNAc, GlcNAc-6-P, Man-6-P, UDP-GalNAc, and UDP-GlcNAc were tested at concentrations from 0.001 to 0.5 mM, after verifying purity by high pressure liquid chromatography. Each experiment was performed in duplicate and replicated three times.

Optimum activator concentrations for UDP-N-acetylglucosamine pyrophosphorylase from encysting cells were determined by testing potential activators at concentrations from 0.025 µM to 10 mM, whereas the substrate (UTP and GlcNAc-1-P) concentrations were at either their K_m values or greater than 5 times their respective K_m values. Activation kinetics were determined by keeping activator concentration optimal and varying GlcNAc-1-P concentrations from 0.111 to 12 mM. Experiments were performed in duplicate and replicated three to five times.

The reversibility of enzyme activation was assessed by dialyzing UDP-N-acetylglucosamine pyrophosphorylase and a mixture of UDP-N-acetylglucosamine pyrophosphorylase (0.119 mg ml¹) with activator overnight against 20 mM Tris-HCl (pH 7.5) and comparing their activities with that of undialyzed UDP-N-acetylglucosamine pyrophosphorylase. To determine whether preincubation of the enzyme with activator alone or with the activator and either of the substrates affects activation, UDP-N-acetylglucosamine pyrophosphorylase from 48-h encysting cells was incubated with activator alone, activator plus UTP (6.7 mM), or activator plus GlcNAc-1-P (6.7 mM) at room temperature for 30 min prior to starting the assays.

Capillary Electrophoresis and the Quantitation of GlcN-6-phosphate-- Using a Hewlett-Packard three-dimensional capillary electrophoresis apparatus, conditions were developed for the capillary zone electrophoretic resolution of a mixture of hexosamine phosphates: -D-Man-1-P, -D-Man-6-P, -D-GalN-1-P, -D-Fru-6-P, -D-Gal-1-P, -D-Gal-6-P, UDP-GlcNAc, UDP-GalNAc, -D-GlcNAc-1-P, -D-GlcNAc-6-P, -D-GlcN-1-P, -D-GlcN-6-P, -D-Glc-1-P, and -D-Glc-6-P (Sigma). Capillary electrophoresis was performed at 30 kV normal polarity (sample loaded at the anode and detected at the cathode) in running buffer (30 mM sodium borate, pH 9.2) at 20 °C. The capillary had an effective length of 56 cm and an interior diameter of 50 µm with an extended light path geometry at the detector. Samples were injected into the column at 50 millibars for 2 s loading ~1 nl. Electropherogram peaks were identified by co-migration with authentic standards. The presence of phosphate was confirmed by loss of the peak after alkaline phosphatase treatment.

The identity of the Giardia peak that ran at the identical time as the GlcN-6-P standard was confirmed by its co-elution with varying amounts of standard GlcN-6-P added to the cells before extraction of the sugars. Increments of exogenously added GlcN-6-P yielded values that were linearly related to the amount added. In Giardia, the difference in area of the GlcN-6-P peak before and after digestion with calf intestinal alkaline phosphatase (New England BioLabs) was used to calculate the amount of GlcN-6-P.

To determine whether encysting Giardia exhibit an increase in the amount of GlcN-6-P above non-encysting levels, cells were harvested from NB medium (non-encysting, 7.5 × 10⁹ cells) and at 24 and 48 h from HB medium (encysting, 2.1 × 10⁹ and 1.1 × 10¹⁰ cells, respectively). Cells were suspended in 8 ml of ice-cold distilled water and divided into two equal samples, one of which was treated with 70 µl of phosphatase inhibitor (Phosphatase Inhibitor Cocktail 2, Sigma) and brought up to 7 ml with ice-cold distilled water. Cells were homogenized and centrifuged at 80,000 × g for 1 h, and the supernatant was passed through a 10,000 molecular weight cut-off filter (Filtron Technology). The ultrafiltrate was lyophilized and redissolved, and an aliquot of each sample that had not been treated with phosphatase inhibitor was subjected to digestion by alkaline phosphatase. Aliquots of phosphatase inhibitor mixture-treated samples were treated identically but with water substituted for the enzyme. Capillary electrophoresis was performed on each of the six samples, and the difference between the GlcN-6-P peak areas of the phosphatase inhibitor mixture-treated material and those of the identical material treated with alkaline phosphatase was used to calculate the GlcN-6-P content of the cells under each experimental condition.

Circular Dichroism (CD)-- CD analyses were performed at room temperature in a Jasco J-715 spectropolarimeter at a  of 260-200 nm and a speed of 10 nm min¹ using a 300-µl quartz cell containing 10 mM phosphate buffer at pH 7.5. CD spectra of purified UDP-N-acetylglucosamine pyrophosphorylase (2.6 µM) from encysting and non-encysting cells were recorded for the enzyme alone and for the enzyme in the presence of 5 µM GlcN-6-P.

Amino Acid Analysis-- Amino acid analysis was performed by a modified method of Moore and Stein (27). UDP-GlcNAc pyrophosphorylase, affinity purified from NB medium and HB medium cell homogenates (50 µl of 3.2 µM), was hydrolyzed in 6 N HCl for 20 h at 120 °C. Subsequent amino acid composition analysis was performed on a Beckman 6300 amino acid analyzer.

Antibody Production, Western Blot, and Immunoaffinity Column-- Monospecific, polyclonal antibody was raised against UDP-N-acetylglucosamine pyrophosphorylase in a New Zealand White rabbit using purified protein excised from SDS-polyacrylamide gel electrophoresis gels and allowed to diffuse into 10 mM phosphate buffer, pH 7.0, for 48 h at 4 °C. Preimmune rabbit serum collected prior to immunization served as a control in immunodetection experiments. TiterMax® (Sigma) adjuvant mixed with ~70 µg (1:1) of the pure protein was injected subcutaneously in the rabbit. IgG fractions of rabbit serum were purified on a Protein A affinity column (ImmunoPure® IgG purification kit, Pierce); purification was monitored by Western blot analysis (28). Cells grown in NB, 0 h, and HB media were harvested, lysed, and electrophoresed in 10% polyacrylamide gel electrophoresis gels under reducing or nonreducing conditions. Proteins were transferred to a membrane and screened with the antibody (1:500). Peroxidase-conjugated goat anti-rabbit IgG was used as a secondary antibody (1:3000).

Purification of UDP-N-acetylglucosamine Pyrophosphorylase by Immunoaffinity Column-- An immunoaffinity column was constructed by immobilizing anti-UDP-N-acetylglucosamine pyrophosphorylase IgG onto an rProtein A column for one-step purification of UDP-N-acetylglucosamine pyrophosphorylase from cytosolic (S) fractions from either non-encysting or encysting trophozoites (23, 29). Purified anti-UDP-N-acetylglucosamine pyrophosphorylase IgG (~6 mg) was covalently immobilized on the rProtein A matrix (ImmunoPure® rProtein A IgG Plus orientation kit, Pierce). The column binding capacity for UDP-N-acetylglucosamine pyrophosphorylase was ~160 µg. S fractions from non-encysting cells, diluted 1:1 with binding buffer (50 mM Tris-HCl, pH 7.8), were applied to the column that had been equilibrated with the binding buffer. The column was then washed with at least 6 column volumes of binding buffer. UDP-N-acetylglucosamine pyrophosphorylase was eluted with 50 mM Tris-HCl buffer (pH 7.8) containing 1 M NaCl; 2-ml fractions were collected until base-line absorbance at 280 nm was reached. To verify that the protein eluted was the pyrophosphorylase, fractions were assayed for UDP-N-acetylglucosamine pyrophosphorylase activity and then assessed for purity by SDS-polyacrylamide gel electrophoresis.

Quantitation of UDP-N-acetylglucosamine Pyrophosphorylase in Non-encysting and Encysting Trophozoites-- [³⁵S]Met was added to the growth or encystment medium, and trophozoites were grown for 24 or 48 h in the presence of [³⁵S]Met (0.5 µCi ml¹) and then either harvested or induced to encyst for 24 h without the radioisotope in the encystment medium. Alternatively, cells were grown in the growth medium for 24 or 48 h and transferred to encystment medium containing [³⁵S]Met. Each experiment was performed at least twice.

Trophozoites were harvested, washed three times in 20 mM phosphate buffer, pH 7.2, and homogenized by 10 freeze-thaw cycles. Cell homogenates were applied to the anti-UDP-N-acetylglucosamine pyrophosphorylase immunoaffinity column equilibrated with 50 mM Tris buffer, pH 7.8, and the enzyme was eluted with 50 mM Tris, 1 M NaCl buffer, pH 7.8. Eluates containing UDP-N-acetylglucosamine pyrophosphorylase activity were pooled and concentrated, and radioactivity and protein concentrations were determined. The void volumes were reapplied to the affinity column to ensure that all of the UDP-N-acetylglucosamine pyrophosphorylase was collected.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Enzyme Purity-- UDP-N-acetylglucosamine pyrophosphorylase was purified, and it migrated as a single spot with a pI of 7.3 on a two-dimensional gel (Fig. 1A) and appeared as a single peak with the same specific activity along the peak (Fig. 1B) in gel filtration fast protein liquid chromatography.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Purity of UDP-N-acetylglucosamine pyrophosphorylase. A, SDS-polyacrylamide gel electrophoresis of purified UDP-N-acetylglucosamine pyrophosphorylase. B, two-dimensional polyacrylamide gel of purified UDP-N-acetylglucosamine pyrophosphorylase (7.5 µg) (open arrowhead) compared with tropomyosin (1 µg), an internal standard (closed arrowhead). C, chromatogram of gel filtration fast protein liquid chromatography showing the elution pattern for purified UDP-N-acetylglucosamine pyrophosphorylase.

Activation of UDP-N-acetylglucosamine Pyrophosphorylase-- Only GlcN-6-P showed any effect on UDP-N-acetylglucosamine pyrophosphorylase activity. GlcN-6-P enhanced the activity of the pyrophosphorylase by ~3-fold (Fig. 2), but in some experiments activation of up to 6-fold was observed. Notably, this activation was detected only in the anabolic direction; neither GlcN-6-P nor any of the other pathway sugars examined had any effect on UDP-N-acetylglucosamine pyrophosphorylase catabolically at concentrations up to 1 mM.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Effect of varying concentrations of GlcN-6-P on UDP-N-acetylglucosamine pyrophosphorylase specific activity. Data points represent the means of at least three experiments performed in duplicate. Error bars represent standard errors.

The binding kinetics of UDP-N-acetylglucosamine pyrophosphorylase for GlcNAc-1-P in the presence or absence of GlcN-6-P gave sigmoidal curves (Fig. 3), indicating binding cooperativeness in both cases. Furthermore, the velocity of enzymatic activity increased 5-fold in the presence of 3 µM GlcN-6-P. Hill plots (Fig. 4) for this enzyme with GlcNAc-1-P as substrate yielded Hill coefficients of ~2 in the absence of and 1.4 in the presence of GlcN-6-P, indicating a greater degree of positive cooperativity in substrate binding of the enzyme alone than in the presence of GlcN-6-P.

View larger version (9K): [in this window] [in a new window]   Fig. 3.   The kinetics of UDP-N-acetylglucosamine pyrophosphorylase in the presence () of GlcN-6-P (3 µM) and absence () of GlcN-6-P.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Hill plots of the kinetics data. Hill coefficients representing the slopes of the lines are 1.97 (top) and 1.38 (bottom). Open circles represent the absence of GlcN-6-P (3 µM), and closed circles represent the presence of GlcN-6-P (3 µM).

GlcN-6-P activation of UDP-N-acetylglucosamine pyrophosphorylase is reversible. Dialysis of the enzyme and the activator against 20 mM Tris-HCl (pH 7.5) returned the activity of the enzyme to that prior to the addition of the activator (data not shown). Preincubation of GlcN-6-P with either of its substrates plus the enzyme or with the enzyme alone had no effect on the activation kinetics (data not shown).

Quantitation of GlcN-6-P-- The GlcN-6-P peak was resolved from other related compounds (Fig. 5B). The entire peak produced by the standard GlcN-6-P (Fig. 5C) was eliminated by alkaline phosphatase digestion (Fig. 5D) as were most of the unknown peaks from Giardia (data not shown).

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Hexosamine analysis by capillary electrophoresis. A, sugar phosphate peaks of Giardia grown in the absence of bile (non-encysting). B, sugar phosphate peaks of authentic standards: 1, GlcNAc-6-P; 2, -D-GlcN-1-P; 3, -D-GalN-1-P; 4, GlcNAc-1-P; 5, -D-Glcn-6-P; 6, UDP-GalNAc. Other standards were detected after the first 10 min of the electrophoresis shown here. C, authentic GlcN-6-P standard peak. D, authentic GlcN-6-P standard after treatment with alkaline phosphatase.

A GlcN-6-P peak (Fig. 5A) is apparent in Giardia extracts whether or not they are grown in the presence of bile. Non-encysting Giardia exhibited a small amount of GlcN-6-P (220 amol cell¹). At 24 h after trophozoites were induced to encyst, the amount of GlcN-6-P increased ~3-fold to 710 amol cell¹, and by 48 h into the encystment process this concentration had dropped to 140 amol cell¹.

Western Blot Analysis and Purification of UDP-N-acetylglucosamine Pyrophosphorylase by Immunoaffinity Column-- Fig. 6 shows the results of Western blot analysis using IgG fractions of the monospecific, polyclonal anti-UDP-N-acetylglucosamine pyrophosphorylase. This antibody recognized a single band on the native gel (arrow). Some slight proteolysis of enzyme appears in 48 h and NB media S fractions. Preimmune serum showed no reactivity (data not shown). S fractions from NB, 0 h, or 48 h media encysting cells all contain UDP-N-acetylglucosamine pyrophosphorylase. Whereas the lanes contain equal amounts of S fraction proteins and whereas band intensities suggest that the amount of enzyme for each medium condition is approximately equal, the specific activity of UDP-N-acetylglucosamine pyrophosphorylase was markedly different for each. The results of [³⁵S]methionine labeling confirm that the amount of UDP-N-acetylglucosamine pyrophosphorylase protein remains relatively constant in cells even during encystment.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Western blot showing the presence of UDP-N-acetylglucosamine pyrophosphorylase in Giardia grown in different media. A native gel of S fractions of Giardia grown in media containing no bile (NB, non-encysting), low bile (0 h, non-encysting), and high bile (48 h, encysting) was transblotted to nitrocellulose and probed with anti-UDP-N-acetylglucosamine pyrophosphorylase. The arrow indicates the detected UDP-N-acetylglucosamine pyrophosphorylase (PPylase). Each lane of the original gel contained 75 µg of S fraction protein lane1. UDP-N-acetylglucosamine pyrophosphorylase specific activity in S fractions is as follows: 0.042 units mg of protein1 in 48 h encysting medium cells, 0.012 units mg of protein1 in 0 h medium cells, and 0.008 units mg of protein1 in NB medium cells.

Amino Acid Analysis-- The amino acid composition of UDP-N-acetylglucosamine pyrophosphorylase purified from Giardia encysting (HB medium) and non-encysting trophozoites (NB medium) have the same amino acid composition (Table I), further confirming that these enzymes are identical.

The Effect of GlcN-6-P on Affinity-purified UDP-N-acetylglucosamine Pyrophosphorylase from Encysting and Non-encysting Giardia Trophozoites-- Fig. 7 demonstrates that despite constant levels of UDP-N-acetylglucosamine pyrophosphorylase in Giardia trophozoites during encystment, the specific activity of the enzyme increases with the presence of bile in the growth medium. Furthermore, the affinity-purified enzyme, regardless of the status of the trophozoite from which it is isolated, increases its activity 4-5-fold with the addition of GlcN-6-P.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   The effect of GlcN-6-P on affinity-purified UDP-N-acetylglucosamine pyrophosphorylase from encysting and non-encysting Giardia trophozoites. UDP-N-acetylglucosamine pyrophosphorylase was purified from the S fractions of encysting (48 h medium) and non-encysting trophozoites (0 h and NB media) by immunoaffinity column as described under "Experimental Procedures." The effect of GlcN-6-P on UDP-N-acetylglucosamine pyrophosphorylase was measured in the anabolic direction under the standard assay conditions in the presence of 5 µM GlcN-6-P.

Circular Dichroism-- Fig. 8 shows CD analyses of affinity-purified UDP-N-acetylglucosamine pyrophosphorylase from non-encysting and encysting cells in the presence or absence of GlcN-6P. The spectra of UDP-N-acetylglucosamine pyrophosphorylase from encysting (48 h encysting medium) and non-encysting (NB medium) cells in the absence of GlcN-6-P appear similar, suggesting the presence of the same enzyme in either condition. In both enzymes the addition of GlcN-6-P induces similar spectral changes, suggesting a conformational change in the protein due to activator binding.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Circular dichroism analyses of UDP-N-acetylglucosamine pyrophosphorylase. Curve 1, spectrum of purified UDP-N-acetylglucosamine pyrophosphorylase from non-encysting cells (NB medium); curve 2, same as curve 1 but in the presence of 5 µM GlcN-6-P; curve 3, spectrum of purified UDP-N-acetylglucosamine pyrophosphorylase from encysting cells (48 h); curve 4, same as curve 3 but in the presence of 5 µM GlcN-6-P.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

UDP-N-acetylglucosamine pyrophosphorylase has been recognized for years as a key enzyme in the formation of UDP-GlcNAc for the synthesis of peptidoglycan, chitin, and glycoconjugates in a variety of prokaryotic and eukaryotic systems (12, 14-16). In recent years, we have shown that it is a key enzyme in the synthesis of GalNAc for formation of the Giardia cyst wall filaments (9). In this report, we demonstrate that a UDP-N-acetylglucosamine pyrophosphorylase is a regulatory point in the pathway leading to the synthesis of UDP-GlcNAc or UDP-GalNAc in Giardia. We conclude that purified Giardia UDP-N-acetylglucosamine pyrophosphorylase in the presence of physiological levels of GlcN-6-P can be activated allosterically more than 3-fold in anabolic activity with no detectable effect on the catabolic activity of the enzyme. This conclusion is based on the following three considerations. First, exposure to increasing amounts of GlcN-6-P, but not other pathway intermediates or their analogs, increases enzyme specific activity in a sigmoidal fashion. Second, Hill coefficients indicate cooperative binding of GlcNAc-1-P and GlcN-6-P by UDP-N-acetylglucosamine pyrophosphorylase. Third, the V_max of UDP-N-acetylglucosamine pyrophosphorylase increased in the presence of GlcN-6-P by ~5-fold with respect to GlcNAc-1-P.

UDP-N-acetylglucosamine pyrophosphorylase is detectable even in non-encysting Giardia cells, indicating that it is a constitutive enzyme. Additionally, Western blot analysis and [³⁵S]Met incorporation revealed that equivalent amounts of UDP-N-acetylglucosamine pyrophosphorylase are present in encysting and non-encysting cells. UDP-N-acetylglucosamine pyrophosphorylase immunoaffinity purified from non-encysting and encysting cells appears to be the same enzyme, based on the following data. First, these proteins exhibit the same immunoreactivity with monospecific, polyclonal antibody against UDP-N-acetylglucosamine pyrophosphorylase. Second, these proteins behave in the same fashion with respect to GlcN-6-P, which in both cases enhances the velocity of the enzymatic reaction 3-5-fold. Third, circular dichroism analysis shows the same spectra for the enzymes alone and similar shifts in spectra in the presence of GlcN-6-P. Fourth, amino acid analysis shows that the enzymes have essentially the same amino acid composition. Notably, the specific activity of UDP-N-acetylglucosamine pyrophosphorylase in non-encysting cells is very low and increases during the course of encystment and/or in the presence of GlcN-6-P, reaching peak activity by 48 h into the encystment process (9). The amount of GlcN-6-P increases more than 3-fold in Giardia cells during encystment. Based on [³⁵S]Met incorporation, it appears that the level of translation is virtually the same in non-encysting cells, increasing only slightly in encysting cells. The fact that the GlcN-6-P concentration increases by 24 h within encysting cells and falls back to non-encysting levels by 48 h may coincide with the peak utilization of UDP-N-acetylglucosamine for the formation of the cyst wall polysaccharide at 24 h. At 48 h, however, encystment is approaching completion in these cultures. This is consistent with the putative role of GlcN-6-P as an activator of the key allosteric branch point enzyme UDP-N-acetylglucosamine pyrophosphorylase in Giardia.

In bacterial and yeast systems, GlcN-6-P isomerase is regarded as an enzyme that operates catabolically (30, 31) in the degradation of amino sugars. However, an in vivo anabolic role for GlcN-6-P isomerase has been proposed in Drosophila, Musca domestica, and Homo sapiens and in Escherichia coli mutants lacking glucosamine synthase (L-glutamine:D-fructose-6-phosphate aminotransferase, EC 2.6.1.16) activity (32-35). Despite the fact that the isomerase from Giardia more closely resembles the catabolic isomerase than the anabolic ones and despite the fact that it has a greater affinity for GlcN-6-P than for Fru-6-P and NH₄Cl, Steimle et al. (11) proposed an anabolic role for GlcN-6-P isomerase in Giardia because its anabolic activity is greater than its deaminase activity and because Giardia lacks glutamine synthase. These investigators (11) suggested further that the equilibrium of the GlcN-6-P isomerase reaction in vivo might be shifted toward GlcN-6-P production by the increased activity of the subsequent acetylase and mutase. Whereas our preliminary evidence suggests that GlcN-6-P has no activating effect on either the acetylase or mutase,² GlcN-6-P does exhibit a marked effect on UDP-N-acetylglucosamine pyrophosphorylase, which might actually be the enzyme that shifts the equilibrium of this pathway in the anabolic direction, thus promoting synthesis of UDP-GalNAc and eventually the cyst wall filaments.

Based on the current evidence, we propose that during Giardia encystment signal transduction events occur that cause the transcription of GlcN-6-P isomerase, which in turn produces GlcN-6-P. This phosphorylated amino sugar then interacts allosterically with constitutive UDP-N-acetylglucosamine pyrophosphorylase to activate this enzyme, shifting the equilibrium of this pathway toward UDP-GalNAc synthesis.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 617-373-2260; Fax: 617-373-3724; E-mail: ejarroll@nunet.neu.edu.

² D. A. Bulik and E. L. Jarroll, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: NB medium, no bile growth medium; HB medium, high bile encystment medium..

	REFERENCES

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