Department of Biology, 134 Mugar Hall, Northeastern University, 360 Huntington Ave, Boston, MA 02115, USA
Correspondence
Edward L. Jarroll
e.jarroll{at}neu.edu
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ABSTRACT |
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INTRODUCTION |
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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 0·30·5 µm thick and is composed of a network of filaments that measure 720 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
(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.
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METHODS |
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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 Vmax (±SD) 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 49. One unit of enzyme activity is defined as µmol substrate used or product formed min1. Enzyme specific activity is either in milliunits (mU) (mg protein)1 or in equivalent nmol min1 (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 0·0150 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 ml1 or control enzymes at 0·0020·010 mg protein ml1 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.
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RESULTS |
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When tested at 1·0 mg protein ml1, 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(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
4GalT, porcine
3GalT and a human
3GlcNAc-T (Millennium Pharmaceuticals) served as controls and were detectable in this system using 0·01 mg protein ml1 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 ml1 or 500-fold less total protein.
Fig. 1 shows that the CWS activity increased markedly (up to 1245-fold increase) during the first 2436 h of encystment and declined as maximal encystment was reached at 4872 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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 18 November 2003;
revised 6 February 2004;
accepted 9 February 2004.