Cyst wall synthase: N-acetylgalactosaminyltransferase activity is induced to form the novel N-acetylgalactosamine polysaccharide in the Giardia cyst wall

Craig D. Karr and Edward L. Jarroll

Department of Biology, 134 Mugar Hall, Northeastern University, 360 Huntington Ave, Boston, MA 02115, USA

Correspondence
Edward L. Jarroll
e.jarroll{at}neu.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) is required in the formation of the outer filamentous wall of Giardia and is synthesized by inducible enzymes in the cytosol of encysting trophozoites. In this study, an inducible enzyme activity that is associated with a particle population isolated from encysting Giardia is reported, and this activity exclusively incorporates [1-14C]GalNAc (from UDP-[14C]GalNAc) into an ethanol precipitate with the same properties as the filamentous cyst wall of Giardia. This ethanol precipitate exhibits characteristics of Giardia cyst wall filaments in that both contain GalNAc as the only sugar moieties and are SDS-insoluble, proteinase- and alkali-resistant and acid-hydrolysable. However, since the precise chemical nature of the ethanol precipitate remains unknown, this enzyme activity is referred to tentatively as cyst wall synthase (CWS). CWS activity peaks in cells between 24 and 36 h of encystment and exhibits a high affinity and marked specificity for UDP-GalNAc as its substrate. UDP-N-acetylglucosamine, UDP-glucose, UDP-galactose, D-glucosamine and D-galactosamine were not incorporated into the ethanol precipitate. Partially purified CWS activity exhibits an apparent Km of 0·048 mM for UDP-GalNAc, a Vmax of 0·70 nmol min–1 (mg protein)–1 and a requirement for divalent cations in the following order of preference: Ca2+, Mg2+>Co2+>>>Mn2+, Zn2+. EDTA inhibits CWS activity.


Abbreviations: CWS, cyst wall synthase; GalNAc, N-acetylgalactosamine; P-fraction, particle fraction; S-fraction, soluble fraction; UDP-GalNAc, uridine-5'-diphospho-N-acetylgalactosamine


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Giardia intestinalis (syn. duodenalis, lamblia) is a major cause of enteric disease, with approximately 108 cases annually (Lane & Lloyd, 2002). In giardiasis, a trophozoite infects the proximal intestine and may cause disease. This trophozoite, in response to bile, can transform into an infectious cyst. It is as the cyst that Giardia is transmitted from one host to another.

The Giardia cyst wall is composed of an inner membranous and an outer filamentous portion (Feely et al., 1984). The outer cyst wall of Giardia is 0·3–0·5 µm thick and is composed of a network of filaments that measure 7–20 nm in diameter (Feely et al., 1984; Erlandsen et al., 1989). In mature cysts, treatment with SDS, amyloglucosidase, trypsin, chymotrypsin, papain or Pronase does not affect these filaments visibly when they are examined by electron microscopy, but they are hydrolysed in strong acid (Jarroll et al., 1989; Manning et al., 1992). Detailed biochemical analyses of these filaments revealed that they contain about 63 % carbohydrate as a {beta}(1-3)-linked N-acetylgalactosamine (GalNAc) homopolymer and about 37 % leucine-rich protein (Gerwig et al., 2002). The GalNAc present in these filaments is synthesized from endogenous glucose reserves by a pathway of inducible enzymes (Macechko et al., 1992). These enzymes include glucosamine-6-phosphate isomerase, glucosamine-6-phosphate N-acetylase, phosphoacetylglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase and UDP-N-acetylglucosamine 4'-epimerase. All these enzyme activities were detected in the cytosol of encysting trophozoites, but none of these activities could account for the incorporation of labelled carbon from uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) into cyst wall filaments. Such an enzyme activity would be expected since GalNAc is the primary constituent associated with the SDS-insoluble, proteinase-resistant, acid-hydrolysable cyst wall filaments (Gerwig et al., 2002).

In this study, we report the activity of an inducible, particle-associated enzyme which incorporates labelled carbon from UDP-[1-14C]GalNAc into an SDS-insoluble, proteinase-resistant, acid-hydrolysable material which behaves like the cyst wall polysaccharide of Giardia.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Chemicals.
All chemicals were of reagent grade and were purchased from Sigma unless otherwise indicated. UDP-N-acetyl-D-[1-14C]galactosamine (55 mCi mmol–1; 1 mCi=37 MBq), UDP-N-acetyl-D-[1-14C]glucosamine (282·8 mCi mmol–1), UDP-[U-14C]galactose (272·8 mCi mmol–1) and UDP-[U-14C]glucose (318·1 mCi mmol–1) were from New England Nuclear. [1-14C]Glucosamine (45 mCi mmol–1) was from ICN. [1-14C]Galactosamine (55·4 mCi mmol–1) was from Amersham. All radioisotopes were at least 97 % pure. Biotinylated oligosaccharides were purchased from GlycoTech, GlycoChipTM oligosaccharide arrays were purchased from GlycoMinds, and biotinylated peptides and glycopeptides were synthesized by New England Peptide and Sussex Research Laboratories, respectively. Streptavidin capture membrane was from Promega. Bovine {beta}4GalT and porcine {alpha}3GalT were from Sigma, and recombinant human {beta}3GlcNAc-T and two ppGalNAc-T activities were kindly provided by Millennium Pharmaceuticals.

Organism.
G. intestinalis (strain MR4) trophozoites were grown and encysted in axenic, asynchronous cultures as described previously (Macechko et al., 1992).

Cell fractionation and enzyme assay.
The subcellular localization of each enzyme was accomplished by differential sedimentation and isodensity centrifugation as described by Lindmark (1988). Acid phosphatase (EC 3.1.3.2), malic enzyme (EC 1.1.1.40) [marker enzymes for the particle (P) and soluble (S) fractions, respectively] and protein were assayed as described previously (Macechko et al., 1992). Cyst wall synthase (CWS) and acid phosphatase activities and protein concentrations were determined following isodensity fractionation (Lindmark, 1988) of the P-fraction from differential sedimentation, in a linear gradient with sucrose densities ranging from 1·03 to 1·3. Densities were determined by measurement in a refractometer. Peak CWS-containing fractions from isodensity gradients were pooled and the detergent dBigCHAP was added to 1·0 % (w/v). The solution was mixed for 30 min on ice and centrifuged at 105 000 g for 45 min to remove non-sedimentable proteins and detergent-extractable or soluble microsomal components. The dBigCHAP pellet was resuspended in 10 mM Tris buffer pH 7·5 with 1·0 % dBigCHAP and the process was repeated three more times. Recovery of CWS activity was typically between 100 and 125 %.

CWS activity was assayed by a modified chitin synthase assay (Duran & Cabib, 1978), and with and without 0·2 % (w/v) Triton X-100 and 1 mM DTT. The reaction mixture contained a final concentration of 75 µM (200 nCi) UDP-[1-14C]GalNAc (or appropriate amounts of other radiolabelled sugars for the determination of substrate specificity), 10 mM Ca2+, 10 mM Tris (pH 7·5) and enzyme in a total assay volume of 50 µl. With the exception of preliminary time and temperature optima characterizations, all assays were performed at 37 °C for 30 min unless otherwise noted. The reaction was stopped by the addition of 1 ml cold 66 % ethanol. The ethanol-precipitated reaction products were separated from excess radiolabelled substrate by vacuum filtration over a 2·4 cm 1·2 µm pore-size glass-fibre filter (Whatman GF-C; Polyfiltronics, 28497-641). The filters were washed once each with 0·8 M acetic acid/20 % ethanol and then with 100 % ethanol. Washed filters were transferred to liquid scintillation vials and counted.

Km and VmaxSD) were determined by non-linear regression analysis (GRAPHPAD; Prism) of data from at least three experiments. Partially purified CWS was used in these kinetics experiments. The pH optimum was determined using 0·1 M acetate/borate/cacodylate buffer as described previously (Hare et al., 1989) over the pH range 4–9. One unit of enzyme activity is defined as µmol substrate used or product formed min–1. Enzyme specific activity is either in milliunits (mU) (mg protein)–1 or in equivalent nmol min–1 (mg protein)–1 for Vmax.

The effects of freezing and thawing, divalent cations and chelating agents on CWS activity were examined. P-fractions were subjected to 10 cycles of freezing in a dry ice/ethanol bath followed by thawing at 37 °C. The effect of divalent cations was examined by adding Ca2+, Mg2+, Co2+, Mn2+ and Zn2+ over a range of 0·01–50 mM (final concentration) to the assay mixture; the effect of EDTA was studied by adding from 0·001 to 10 mM EDTA (final concentration) to the reaction mixture.

The washed precipitate from the enzyme assay was either left untreated or subjected to the following treatments from Manning et al. (1992): 5 % SDS at 100 °C for 5 min; 2 M HCl at 100 °C for 3 h; 1 M KOH at 100 °C for 1 h; proteinase K (EC 3.4.21.14; Sigma P-0390) (in 50 mM Tris, pH 8·0, 60 °C for 2 h); papain (EC 3.4.22.2; Sigma P-4762); Pronase E (EC number not assigned; Sigma P-6911); and trypsin (EC 3.4.21.4; Sigma T-8128). The treated precipitates were collected by centrifugation at 15 000 g for 2 min and scintillation counting was performed. The entire CWS reaction mixture using a P-fraction from encysting or from non-encysting trophozoites as enzyme, and the washed ethanol precipitate when P-fraction from encysting trophozoites was used as enzyme, were subjected to SDS-PAGE. SDS-PAGE was performed as described previously (Laemmli, 1970); gels were stained with Coomassie blue R-250, photographed and subjected to autoradiography. For autoradiograms, gels were treated with EnhanceTM before drying and exposed to X-Omat AR5 film for 2 weeks.

Determination of CWS activity and control enzyme activity on 0·1 mM synthetic biotinylated oligosaccharides, peptides or glycopeptides was accomplished by incubating CWS at 1·0 mg protein ml–1 or control enzymes at 0·002–0·010 mg protein ml–1 with 0·1 mM (200 nCi) of UDP-[14C]sugar substrates in 10 µl volumes. For solid phase arrays, 0·2 ml of reaction mixtures were added to each slide and covered with a silanized-cover slip. Solution phase assays were transferred to streptavidin capture membrane, then both the membranes and the solid array slides were washed three times with PBS, dried and exposed to a phosphorimaging screen overnight. Detection was accomplished using a Fuji Medical Systems BAS2500 phosphorimaging system (Stanford, CT) and quantification of radiolabelling was done by using the manufacturer's software.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
CWS activity increased with increasing protein as well as with increasing assay time for up to 60 min. This activity exhibited a temperature optimum of 30–37 °C; the activity dropped dramatically above 40 °C. The optimal pH was 7·5, but CWS activity was at least 50 % of the maximal activity over the range of pH 5·5–9·0. CWS activity exhibited a requirement for divalent cations. Ca2+ or Mg2+ was preferred and CWS activity increased up to about 11-fold with the addition of 10 mM Ca2+ or Mg2+ over the level of activity when no exogenous Ca2+ or Mg2+ was added. Co2+ stimulated activity by up to about sevenfold at 10 mM, but Mn2+ and Zn2+ stimulated activity by only about twofold above activity without any added divalent cation. EDTA, at a final concentration of 1 mM or above, inhibited CWS activity by about 99 % of control values; inhibition of CWS activity by EDTA can be overcome by adding exogenous Mg2+. DTT was not required for maximal activity and 0·2 % Triton X-100 inhibited the assay.

When tested at 1·0 mg protein ml–1, partially purified CWS did not incorporate [1-14C]GalNAc into a panel of 109 synthetic monovalent biotinylated oligosaccharide acceptors, including the highly relevant disaccharide GalNAc({beta}1-3)GalNAc, as determined following streptavidin membrane capture and phosphorimaging detection. Incorporation of [1-14C]GalNAc by CWS was also below the level of detection when synthetic oligosaccharides were tested in a solid phase polyvalent array. Bovine {beta}4GalT, porcine {alpha}3GalT and a human {beta}3GlcNAc-T (Millennium Pharmaceuticals) served as controls and were detectable in this system using 0·01 mg protein ml–1 or 100-fold less total protein.

CWS, also, did not incorporate [1-14C]GalNAc into a panel of 12 mucin-type biotinylated peptides and two biotinylated glycopeptides, as assessed by streptavidin membrane capture and phosphorimaging detection. Two novel recombinant ppGalNAc-T activities (Millennium Pharmaceuticals) served as controls and were detectable in this system using 0·002 mg protein ml–1 or 500-fold less total protein.

Fig. 1 shows that the CWS activity increased markedly (up to 1245-fold increase) during the first 24–36 h of encystment and declined as maximal encystment was reached at 48–72 h. However, since these protozoan cultures were neither growing nor encysting synchronously, the times at which a given culture exhibited maximal CWS activity and reached peak encystment varied, but in all of our trials the pattern of increase in CWS activity and in vitro formed cysts was similar to that shown in Fig. 1. Furthermore, radiolabelled GalNAc from the CWS reaction products does not appear to enter the SDS-PAGE gel, suggesting that the CWS product is a very large molecule whose properties preclude gel entry.



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Fig. 1. Representative graph depicting changes in CWS activity and percentage of encystment of in vitro encysting G. intestinalis. Incorporation of [1-14C]GalNAc by CWS was monitored quantitatively by liquid scintillation counting, as described in Methods. Specific activity on the left refers to the CWS activity in cell homogenates; maximal encystment equalled 68 % at 36 h post-induction. The time-course plot of induction of CWS activity ({bullet}) and encystment ({circ}) is superimposed on an autoradiograph corresponding to an SDS-PAGE gel in which the remaining assay mixture (100 µg protein per assay per lane) was loaded after scintillation counting aliquots were taken. Incorporation of [1-14C]GalNAc by CWS was monitored qualitatively by SDS-PAGE/autoradiography, as described in Methods. Radiolabelled CWS reaction products appear too large to enter the SDS-PAGE gel (e.g. the arrow points to the top of the stacking gel in the 20 h sample). For SDS-PAGE, all samples were mixed with sample buffer containing DTT, and electrophoresed through a 5 % stacking and 10 % running gel. The gel was stained with Coomassie blue R-250, and the dried gel was sprayed with EnhanceTM and exposed to autoradiographic film for 2 weeks at –70 °C.

 
Fig. 2(a) depicts a typical differential sedimentation of homogenates of 30 h encysting trophozoites. From these results, it is clear that the activity of CWS is in the P-fraction, suggesting that it is particle associated, and the activity did not exhibit latency after freezing and thawing for 10 cycles. Fig. 2(b) depicts the pattern of CWS activity when the P-fraction shown in Fig. 2(a) was subjected to isodensity centrifugation. In this representative fractionation, the peak CWS activity corresponded to a particle population with a density in sucrose of 1·16 g ml–1. Lysosome-like organelles had a peak density in sucrose of 1·18 g ml–1, as determined by acid phosphatase activity (Fig. 2b). The densities of encystation-specific vesicles and lysosome-like vesicles vary slightly from encystment to encystment since cells are asynchronously encysting.



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Fig. 2. (a) Representative distribution of enzymes after differential centrifugation of a G. intestinalis homogenate. Relative specific activity (RSA) of enzymes was plotted against cumulative percentage of protein recovered in each fraction. In the graph, the direction from left to right corresponds to increasing centrifugal force. The far right-hand block represents the final supernatant. Shaded area designates the fraction subjected to isodensity centrifugation (b). Recoveries were 71 % for acid phosphatase (AP), 77 % for malic enzyme (ME), 94 % for protein and 243 % for CWS activity. (b) Representative distribution of CWS activity, acid phosphatase and protein after isodensity centrifugation of the P-fraction represented by the shaded areas in (a). Density/frequency plots are shown. Recoveries were 34 % for CWS, 87 % for acid phosphatase and 84 % for protein.

 
Using partially purified enzyme (Table 1) resulting from the isodensity gradient fraction described above, CWS activity exhibited an apparent Km of 0·048±0·003 mM for UDP-GalNAc and a Vmax of 0·70±0·085 nmol UDP-GalNAc min–1 (mg protein)–1 incorporated into the ethanol precipitate (Fig. 3).


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Table 1. Partial purification of CWS from encysting Giardia

These data are representative of at least three experiments, which were performed as described in Methods. One unit of enzyme activity is defined as µmol substrate used or product formed min–1.

 


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Fig. 3. Determination of the apparent kinetic properties of CWS partially purified from the microsomal fraction of encysting G. intestinalis. Assays were performed in triplicate as described in Methods under optimal reaction conditions. Michaelis–Menten and Lineweaver–Burke (inset) analyses were produced using GRAPHPAD 3.0 (Prism) software over a range of UDP-GalNAc concentrations, from 10 to 800 µM.

 
CWS activity is quite specific for UDP-GalNAc (Table 2). There was no detectable incorporation of any UDP-N-acetylglucosamine, UDP-galactose, UDP-glucose, galactosamine or glucosamine. When S-fractions were used as enzyme there was no appreciable incorporation of any of these sugars, including UDP-GalNAc, into the ethanol precipitate.


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Table 2. Substrate specificity of CWS activity in encysting Giardia

These assays were performed in triplicate as described in Methods using particles from the P-fraction or the cytosolic fraction (S-fraction) as enzyme. All sugars were 14C-labelled as described in Methods. Results are expressed as nmol min–1 (mg protein)–1SD).

 
Fig. 4 shows the results of SDS-PAGE analysis of the CWS reaction mixture and washed ethanol precipitate. While it is clear from the SDS-PAGE gel that some proteins entered the PAGE matrix, all of the incorporated label from UDP-GalNAc remains in the sample-well and does not migrate into these gels. In Lane 1, representing the electrophoresis of the entire reaction mixture, there is a band that has entered only the stacking gel. This band could represent a precursor and/or a degradation product of the material or the enzyme itself. There was no detectable incorporation of radiolabelled UDP-GalNAc (or other sugars) into products when a P-fraction from non-encysting cells was used as enzyme.



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Fig. 4. Representative SDS-PAGE gel (a) and autoradiogram (b) of G. intestinalis CWS reaction product. CWS was assayed with radiolabelled UDP-GalNAc as described in Methods. Lanes contained total assay mixture (1), washed ethanol precipitate only (2) using 90 µg protein (P-fraction) from encysting cells as enzyme, and total assay mixture (3) using 90 µg protein (P-fraction) from non-encysting cells as enzyme. All samples were mixed with sample buffer containing DTT, and electrophoresed through a 5 % stacking and 10 % running gel. Gels were stained with Coomassie blue R-250 (left); the dried gel was sprayed with EnhanceTM and exposed to autoradiographic film (right) for 2 weeks at –70 °C. Molecular masses are given in kDa.

 
The effects of various treatments on the [1-14C]GalNAc-containing ethanol precipitate are shown in Table 3. The level of radiolabel from UDP-GalNAc incorporated into the precipitate was virtually unaffected by SDS, trypsin and Pronase E treatments, and was only partially degraded by papain, proteinase K and KOH. However, CWS product was, as were the cyst wall filaments themselves, only hydrolysed completely by acid hydrolysis.


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Table 3. Effects of various treatments on the ethanol precipitates from the CWS assay

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
An enzyme activity, which exclusively incorporates radiolabelled [1-14C]GalNAc into an ethanol precipitate chemically resembling Giardia cyst wall filaments, is induced when Giardia trophozoites encyst. Appearance of this activity in encysting cells coincides with that of the other recently described inducible enzymes of Giardia, which collectively synthesize the UDP-GalNAc from endogenous glucose reserves (Macechko et al., 1992). Furthermore, its appearance coincides with the appearance of encystation-specific vesicles (Faubert et al., 1991) and with the appearance of intact Giardia cysts in vitro. This enzyme activity exhibits a very low Km for UDP-GalNAc, suggesting that this enzyme has a high affinity for UDP-GalNAc. Moreover, substrate-specificity studies show that, under the conditions tested, UDP-GalNAc is the only substrate which is fixed into the ethanol precipitate. We describe a novel enzyme activity that specifically incorporates GalNAc into an ethanol precipitate which is SDS-insoluble, acid-hydrolysable, resistant to complete proteinase degradation, has a high molecular mass and chemically resembles the recently described {beta}(1-3)-linked GalNAc homopolymer known to make up about 63 % of the Giardia filamentous cyst wall. The fact that a small amount of GalNAc-labelled material enters an SDS-PAGE gel and the fact that there is partial degradation of the ethanol precipitate by papain and proteinase K suggest that the ethanol-precipitable product may contain some glycoproteins or that the enzyme itself with substrate attached may have been degraded. This suggestion is in line with the results of Manning et al. (1992) and Gerwig et al. (2002), who stated that the Giardia cyst wall filaments represented a carbohydrate–peptide complex. However, it should be noted that the ethanol precipitate from the CWS reaction (i) does not necessarily represent mature cyst wall filaments and thus may not react to these treatments in exactly the same fashion as the filaments in mature cysts, and (ii) does not necessarily represent the product of a single enzyme operating in these relatively crude preparations.

Since non-encysting Giardia trophozoites do not exhibit CWS activity, and since mature Giardia cysts have GalNAc (Jarroll et al., 1989) in their outer cyst wall filaments, which are SDS-insoluble, acid-hydrolysable and proteinase-resistant (Manning et al., 1992; Gerwig et al., 2002), it is suggested that this enzyme activity is involved in the formation of a product that is a major component of the outer cyst wall filaments. However, until the exact chemical nature of the ethanol precipitate and the cyst wall filaments can be completely elucidated, we choose to refer to this enzyme activity tentatively as CWS.

Unlike the other enzymes in the GalNAc synthetic pathway, all of which are found in the cytosolic portion of the encysting trophozoite (Macechko et al., 1992), CWS activity is associated with a particle population whose equilibrium density ranges from 1·15 to 1·18 in sucrose (in 30 h encysting cells). Since the peak activity of acid phosphatase, the lysosome-like organelle marker, is distinctly at 1·18 and the peak activity of CWS is at 1·16, it is suggested that the CWS activity is associated with different particles than acid phosphatase. Because of the apparent particle association of CWS activity, it is possible that CWS is associated with encystation-specific vesicles (ESVs) (Faubert et al., 1991) since these vesicles do sediment with a P-fraction under the organelle-preserving conditions of our cellular fractionation procedure (E. L. Jarroll, unpublished observations). Samples tested in the laboratory of Dr Ted Nash, NIH, demonstrated that CWS-containing vesicles react positively with monoclonal antibodies 5-3C, 8G8 and 7D2, which specifically recognize the developmentally induced ESV-associated cyst wall proteins CWP1 and CWP2 (Lujan et al., 1995b; Mowatt et al., 1995; Boone et al., 1999). However, generation of antibodies to CWS for co-localization studies will confirm this. The fact that CWS does not show latency could suggest that there is a transmembrane portion of the enzyme or simply the particles with which the activity is associated are somewhat perforated by the fractionation process, exposing the enzyme active site(s).

Das & Gillin (1996) reported encystment-activated N-acetylglucosamine and GalNAc transferase activities in Giardia. Although we have been unable to find incorporation of UDP-N-acetylglucosamine into a precipitable product, it is likely that the UDP-GalNAc fixing activity Das & Gillin (1996) reported represents our CWS activity. Lujan et al. (1995a) also described a GalNAc transferase activity from encysting Giardia and suggested that this activity was likely associated with a developmentally regulated Golgi-like organelle system. Similar to CWS, GalNAc transferase activity was undetectable in non-encysting cells and was localized to discrete particle populations upon isopycnic centrifugation. Unlike CWS activity, Lujan et al. (1995a) followed incorporation of radiolabelled GalNAc into trichloroacetic acid precipitates using ovalbumin as an exogenous glycoprotein acceptor. We have been unable to show that CWS was positively or negatively affected by adding exogenous ovalbumin or other potential protein acceptors. However, since Lujan et al. (1995a) only estimated the density of the microsomal fractions it is not possible for us to compare directly the localization of CWS and their reported GalNAc transferase activity.

In addition to exogenous glycoprotein acceptors, we have not been able to demonstrate CWS activity above the level of detection using synthetic exogenous oligosaccharide or peptide acceptors. While these studies do not preclude CWS from using specific acceptor ligands, they do suggest that an acceptor is not absolutely required for activity. If so, then CWS may be functionally related to Pasteurella hyaluronan synthase (HAS), which has been purified to homogeneity from a recombinant system lacking HAS activity and demonstrated to be active without endogenous or exogenous acceptor present (DeAngelis, 1999).

We report in Giardia an inducible enzyme activity which incorporates radiolabelled carbon from UDP-GalNAc into an ethanol precipitate that is SDS-insoluble, proteinase- and alkaline-resistant and acid-hydrolysable, as are the cyst wall filaments of Giardia. The activity, tentatively called CWS, is associated with subcellular particles and exhibits very high affinity and specificity for its substrate, UDP-GalNAc.


   ACKNOWLEDGEMENTS
 
We would like to thank Drs Ted Nash and Hugo Lujan at the NIH, Bethesda, MD for providing and testing the cyst wall protein-specific monoclonal antibodies described in the text. Supported by the Ohio Board of Reagents Academic and Research Challenge Programs in Molecular Parasitology and by NIH grant AI29591.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Boone, J. H., Wilkins, T. D., Nash, T. E., Brandon, J. E., Macias, E. A., Jerris, R. C. & Lyerly, D. M. (1999). TechLab and Alexon Giardia enzyme-linked immunosorbent assay kits detect cyst wall protein 1. J Clin Microbiol 37, 611–614.[Abstract/Free Full Text]

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Gerwig, G. J., van Kuik, J. A., Leeflang, B. R., Kamerling, J. P., Vliegenthart, J. F., Karr, C. D. & Jarroll, E. L. (2002). The Giardia intestinalis filamentous cyst wall contains a novel {beta}(1-3)-N-acetyl-D-galactosamine polymer: a structural and conformational study. Glycobiology 12, 499–505.[Abstract/Free Full Text]

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Lujan, H. D., Marotta, A., Mowatt, M. R., Sciaky, N., Lippincott-Scwartz, J. & Nash, T. E. (1995a). Developmental induction of Golgi structure and function in the primitive eukaryote Giardia lamblia. J Biol Chem 270, 4612–4618.[Abstract/Free Full Text]

Lujan, H. D., Mowatt, M. R., Conrad, J. T., Bowers, B. & Nash, T. E. (1995b). Identification of a novel Giardia lamblia cyst wall protein with leucine-rich repeats. Implications for secretory granule formation and protein assembly into the cyst wall. J Biol Chem 270, 29307–29313.[Abstract/Free Full Text]

Macechko, P. T., Steimle, P. A., Lindmark, D. G., Erlandsen, S. L. & Jarroll, E. L. (1992). Galactosamine-synthesizing enzymes are induced when Giardia encyst. Mol Biochem Parasitol 56, 301–310.[CrossRef][Medline]

Manning, P., Erlandsen, S. L. & Jarroll, E. L. (1992). Carbohydrate and amino acid analyses of Giardia muris cysts. J Protozool 39, 290–296.[Medline]

Mowatt, M. R., Lujan, H. D., Cotten, D. B., Bowers, B., Yee, J., Nash, T. E. & Stibbs, H. H. (1995). Developmentally regulated expression of a Giardia lamblia cyst wall protein gene. Mol Microbiol 15, 955–963.[Medline]

Received 18 November 2003; revised 6 February 2004; accepted 9 February 2004.