Molecular and Biochemical Parasitology, 56 (1992) 161 168 ~) 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851./92/$05.00

161

MOLBIO 01833

Identification and partial characterization of a lipophosphoglycan from a pathogenic strain of Entamoeba histolytica A l o k B h a t t a c h a r y a a, R a m a s a r e P r a s a d a a n d D a v i d L. Sacks b ~School of Life Sciences, Jawaharlal Nehru University, New Delhi, India and bLaboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA (Received 12 March 1992; accepted 28 July 1992)

An acidic glycoconjugate could be extracted from a delipidated residue fraction of [3H]galactose, [3H]mannose or [32p]orthophosphate metabolically labeled Entamoeba histolytica with water/ethanol/diethylether/pyridine/NH4OH (15:15:5:1:0.017). The radioactively labeled glycoconjugate comprised 50-55% of the total [3H]galactose label incorporated into macromolecules. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the radiolabeled glycoconjugate showed two diffuse smears centering around 110 kDa and 45 kDa. Similar profiles were observed for both [3H]galactose- and [32p]orthophosphate-labeled glycoconjugate. No such bands were visible in [35S]methionine-labeled material. The hydrophobic nature of this glycoconjugate was inferred from its chromatographic behavior on phenyl-Sepharose. The molecule was rendered hydrophilic after digestion with phosphatidylinositol-specific phospholipase C. It was also sensitive to deamination by nitrous acid. Mild acid hydrolysis led to its fragmentation into smaller molecules as revealed by Sepharose 4B chromatography. Paper chromatographic analysis of the depolymerized [3H]galactose- and [3H]mannose-labeled fragments revealed that each was sensitive to alkaline phosphatase. The major dephosphorylated fragment migrated as an apparent galactose and mannose containing disaccharide which migrated identically to the Galfll-4Man disaccharide derived from the lipophosphoglycan of Leishmania donovani. The above data support the existence of a major acidic glycoconjugate in E. histolytica bearing striking structural similarities to the lipophosphoglycan of Leishmania. Key words: Entamoeba histolytica; Lipophosphoglycan; Glycoconjugate

Introduction

Only a few surface molecules of Entamoeba histolytica have been described to date, namely, the 170 kDa galactose/N-acetyl galactosamine-binding lectin [1], the l l2-kDa surface adhesin [2], and the 220-kDa N-acetyl glucosamine-binding lectin [3]. These are presumably involved in attachment to target epithelial cells as revealed by in vitro assays. It is not yet clear whether these molecules, individually or Correspondence address: David Sacks, Laboratory of Parasitic Diseases, NIAID, Building 4, Room 126, National Institutes of Health, Bethesda, MD 20892, USA. Abbreviations: PI-PLC, phosphotidylinositol-specificphospholipase C; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; LPG, lipophosphoglyean.

collectively, are involved in attachment to the target cells in vivo. A few other surface molecules, namely the 125-kDa and 29-kDa molecules with as yet unknown functions, have also been described [4,5]. Existence of a glycocalyx coat and surface molecules rich in carbohydrates have been demonstrated by electron microscopic and lectin-binding studies [6,7]. Some of these studies have shown that species and strains of E. histolytica can be distinguished from each other on the basis of their surface carbohydrates. The chemical extraction of a lipopeptidophosphoglycan has been reported in a preliminary communication [8], and most recently, the extraction of a phosphorylated, lipid-containing, surface glycoconjugate involved in adhesion has been described [9]. In this report, we present

162

evidence for the presence of a lipophosphoglycan in a virulent strain of E. histolytica.

Materials and Methods

Cells'. E. histolytica strain HM-I:IMSS, clone 6, was used throughout this study. The cells were grown in TY1-S-33 medium at 36°C [10]. The cells were harvested by chilling at 4°C for 7 min followed by centrifugation at 275 x g for 7 min. The cells were washed 3 times at 4°C using phosphate-buffered saline No. 8 [11]. Metabolic labeling and extraction. 2-5 x l 0 6 cells ml -~ in TY1-S-33 base were incubated with either [3H]galactose (100-200 ~Ci ml-1), [3H]mannose (200 /~Ci ml-1), [3SS]methionine (100-200 /~Ci ml 1), or [32p]orthophosphate (0.5 mCi m l - l ) for 3 h at 36°C. The cells were then washed with PBS 3 times at 4°C and delipidated by extracting sequentially with chloroform/methanol/water (3:2:1); 5 parts of chloroform/methanol/water (10:10:3), and one part of chloroform/methanol (1:1); and chloroform/methanol/water (10:10:3). The pellet was then extracted with solvent E (water/ethanol/ diethylether/pyridine/ammonium hydroxide, 15:15:5:1:0.017) as described [12,13]. Hydrophobic chromatography. Crude glycolipids were resuspended in 0.1 M NaCl/0.1 M acetic acid and passed through phenyl-Sepharose CL 4B (Pharmacia, Sweden). The columns were washed sequentially with 0.1 M acetic acid containing 0.1 M NaC1/0.1 M acetic acid, water, and solvent E. Fractions (0.5 ml) were collected and radioactivity determined. Enzyme digestion, nitrous acid deamination, and mild acid treatment. All enzyme treatments were carried out for 16 h at 37°C. Glycoconjugates were resuspended in 200 #1 25 mM Hepes, pH 7.4/0.1% Chaps/5 mM EDTA, and incubated with 0.1 unit of phosphotidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis. Alkaline phosphatase (Worthington Chemicals) digestion was done using 0.1~).3 units in 1 mM Tris, pH 8.

For nitrous acid deamination, glycoconjugate was resuspended in 0.1 M sodium acetate buffer, pH 4.0, containing 0.25 M sodium nitrite (NaNO2) and incubated for 16 h at room temperature. Mild acid conditions of 0.02 M HC1, 5 min at 100°C, were used to cleave sugar-l-phosphate bonds. Following acid hydrolysis, the sample was dried under a stream of nitrogen.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis. Radiolabeled samples were analyzed by discontinuous SDS-PAGE under reducing conditions according to Laemmli [14]. The separating gel was 10% acrylamide. After electrophoresis, the gels were subjected to fluorography for 3H or 35S according to Laskey and Mills [15]. Gels containing 32p_ labeled materials were dried and exposed to Xray films in the presence of intensifying screens. Gel filtration and paper chromatograph),. PIPLC-treated, [3H]galactose-labeled glycoconjugates were purified on phenyl-Sepharose. The dried delipidated glycoconjugate was suspended in 40 mM NHaOH/1 mM EDTA, and applied to a column of Sepharose-CL 4B equilibrated in the same buffer. Approximately 0.7 ml fractions were collected and radioactivity determined. In order to determine the effect of mild acid treatment, the PI-PLCtreated glycoconjugate was subjected to mild acid hydrolysis, dried, and chromatographed as described above. For analysis of phosphorylated saccharide fragments, PI-PLC treated [3H]galactose and [3H]mannose-labeled glycoconjugate was hydrolyzed with mild acid and subsequently by alkaline phosphatase for 16 h, and the fragments analyzed by paper chromatography in n-butyl alcohol/pyridine/water (6:4:3) for 72 h. Radioactivity was determined by cutting the paper strips into 1-cm segments, soaking the segments in 0.6 ml of 1% SDS, and counting in a vial containing scintillation fluid. For comparison, de~hosphorylated oligosaccharides from [- H]mannose-labeled L. donovani lipophosphoglycan (LPG), kindly supplied by S. Turco, University of Ken-

163

tucky, were also run. Standard sugars were detected by staining with alkaline silver nitrate [16].

Results

Purification of glycoconjugate from E. histolytica. Glycoconjugate from a 'pathogenic' E. histolytica strain, H M - I : I M S S , clone 6, was extracted using the protocol devised for extracting L P G from Leishmania [13]. The cells were metabolically radiolabeled with either [3H]galactose, [3H]mannose, or [32p]orthophosphate prior to extraction. In some experiments, [35S]methionine-labeled cells were also used for extraction. The material extracted by solvent E accounted for 50-55% of the total [3H]galactose label incorporated into macromolecules. The extracted acidic glycoconjugate was further purified by hydrophobic chromatography on phenylSepharose. About 50% of the labeled material bound to the column and could be eluted by solvent E. All subsequent experiments were

carried out with the organic solvent-elutable material. The [3H]galactose-, [32p]orthophosphate-, and [35S]methionine-labeled extracts were resolved in a 10% polyacrylamide gel and located by fluorography/autoradiography, respectively. Both [3H]galactoseand [- 2P]orthophosphate-labeled materials resolved into 2 distinct broad bands of average sizes 110 and 45 kDa (Fig. 1, lanes a and b). The relative proportion of the 2 bands varied with labeling conditions. When labeled with [3H]galactose, there was more radioactivity in the 45-kDa band. No such bands were visible in [35S]methionine-labeled material (lane c); instead, autoradiograms revealed radiolabeled material near the dye front. The data suggest that the glycoconjugates contain carbohydrate (galactose) and phosphate, but no peptides. Peptides which may be co-purified with the glycoconjugate appear to be dissociated under denaturing conditions. ~CPM * n-PROPANOL (%) 60

800 700 ~

a

b

c

t

/

50

I

600 ~

....

40

-~66 500

400

30

- ~ 116 -.~29

300 20 200

--~20

I0 100 0 ~

F

'

I

10 20 FRACTION NUMBER

Fig. 1. Gel profile of radiolabeled extracted and purified glycoconjugate. E. histolytica cells were labeled with different isotopes, delipidated and extracted with solvent E, and further purified over a phenyl-Sepharose column. The purified materials (approximately 8000-10000 cpm) were subjected to 10% SDS-PAGE followed by autoradiography or fluorogra~hy. Lane a, [3H]galactose-labeled material; lane b, [3 P]orthophosphate-labeled material; lane c, [35S]methionine-labeled material. Arrows designate positions of molecular weight standards.

' 30

Fig. 2. Phenyl-Sepharose chromatography of labeled glycoconjugate from E. histolytica. Glycolipids were extracted from cells, labeled with [3H]galactose, and purified on a phenyl-Sepharose column as described. The purified material, in 0.1 M NaCl/0.1 M acetic acid/5% npropanol (v/v), was allowed to bind to the phenylSepharose column. The bound material was eluted with a gradient of n-propanol (5-50%) in 0.1 M NaCl/0.1 M acetic acid.

164

The purity of extracted material was further examined by hydrophobic chromatography on phenyl-Sepharose using an increasing gradient of n-propanol for elution. It was found to elute as a single species between 30-35% n-propanol (v/v) (Fig. 2). This confirms the purity and hydrophobicity of extracted glycoconjugate, and establishes an elution pattern similar to that described for the L P G of L. major [17], as well as the amebic glycoconjugate previously described [9].

retained on the column and only eluted with solvent E. After digestion with PI-PLC, most of the material (80%) no longer bound to the column, but eluted in the acetic acid/NaCl eluent. Similar results were obtained for both [3H]galactose- and [32p]orthophosphate-labeled glycoconjugates. This suggests that PIPLC digestion removes a lipid moiety attached to the glycoconjugate possibly by a GPI linkage.

Deamination of glycosamine residues. Glucosamine-inositol linkage is highly sensitive to deamination by nitrous acid, which results in cleavage of the hydrophobic tail [18]. Both [3H]galactose- and [32p]orthophosphate-labeled glycoconjugates were treated with nitrous acid and analyzed by phenyl-Sepharose chromatography (Fig. 3). After deamination, labeled glycoconjugate failed to bind to the column and eluted in aqueous buffer. This suggests that E. histolytica glycoconjugate contains a glucosamine-inositol linkage simi-

E. histolytica glycoconjugate contains a lipid moiety linked by glycophosphoinositol. Chromatography over phenyl-Sepharose columns suggested that the purified glycoconjugate of E. histolytica has a hydrophobic character, possibly due to an attached lipid. The possibility that the lipid may be a glycophosphoinositol was explored by treating the radiolabeled material with PI-PLC and reapplication to the phenyl-Sepharose column (Fig. 3). Purified, untreated glycoconjugate was 2000-

I

E

untreated

~

ib ic

I0o0,

nitrous acid

~

~ ic

ooO°°oo1°°1,. ooOO°° Oi

0 2

U~ 4~a

1200 0

1000

3 4

Pl - P L C

5

6 7

8

9 loll 12131415

iblC

1 2

3 4

5

OO]lid0oo c

6

7 8

910

112131415

c

800 6OO 400

500

200 0

2 3 4 5 6 7 8 9 I011 12131415

Fraction

1 2 3 4 5 6 7 8 9 I01112131415

number

Fig. 3. Phenyl-Sepharosechromatography of amebic glycoconjugatesafter different treatments. Purified [3H]galactose-labeled glycoconjugatewas run untreated or subjectedto either PI-PLC digestion, mild acid treatment, and nitrous acid dearnination before chromatography over phenyl-Sepharose. Columns were equilibrated and washed with 0.1 M NaC1/0.1 M acetic acid. Column washes with 0.1 M acetic acid, water, and solvent E, are indicated by a, b, and c, respectively.

165

lar to other GPI-anchored macromolecules.

Mild acid hydrolysis of the glycoconjugate. Glycoconjugates can be characterized on the basis of lability to acid treatment [19]. A salient feature of the lipophosphoglycan of Leishmania is its extreme sensitivity to mild acid hydrolysis which depolymerizes the molecule into phosphorylated saccharide repeat units. Mild acid-treated [3H]galactoselabeled amebic glycoconjugate failed to bind to the phenyl-Sepharose column (Fig. 3), suggesting that the molecule underwent fragmentation, separating the lipid moiety from carbohydrate residues. The [3H]carbohydrate fragments released by PI-PLC and mild acid hydrolysis were subjected to gel filtration on a Sepharose-4B column. The elution profile of the glycoconjugate treated only with PI-PLC shows a broad band with some of the material eluting near the column void volume (Fig. 4). After mild acid treatment, all of the labeled material eluted in a sharp peak at the column retention volume, suggesting that the glycoconjugate of E. histolytica is depolymerized by mild acid treatment into smaller saccharidecontaining fragments. Alkaline phosphatase treatment and paper chromatography of mild acid hydrolysis fragments. The phosphorylated saccharide-con-

rur

S00~

I=¢

8jl

mltl~

400300200"

1000 500.

2O

lO

B

400.

~. ~

200 ~00

\

G

=00

~

2D

IO

c

~

2000

dO

III

1200 1000 800 600 2O 4OO

fraction

number

2OO 0

40

60

80 volume (ml)

100

Fig. 4. Sepharose 4B chromatography of [3H]galactoselabeled glycoconjugate after mild acid treatment. PI-PLCdigested [3H]galactose-labeled material was chromatographed on a phenyl-Sepharose column. Unbound lipid released material was further subjected to mild acid treatment. D, untreated; II, mild acid treated.

Fig. 5. Paper chromatography of the fragments generated by mild acid hydrolysis of the PI-PLC treated [3H]galactose or [3H]mannose-labeled glycoconjugate. (A) [3H]galactoselabeled amebic glycoconjugate without alkaline phosphatase treatment. (B) After alkaline phosphatase treatment. (C) De~olymerized and dephosphorylated saccharides from [ H]mannose-labeled L. donovani LPG. (D) [3H]Mannose-labeled, dephosphorylated amebic glycoconjugate. Standards: raf, raffinose (trisaccharide); lac, lactose (disaccharide); gal, galactose (monosaccharide); man, mannose (monosaccharide).

166

taining fragments generated by mild acid hydrolysis were further analyzed by descending paper chromatography in a non-polar solvent. In the absence of alkaline phosphatase treatment, all of the [3H]galactose-labeled material remained at the origin (Fig. 5), suggesting that each fragment is phosphorylated. Pretreatment of the material with alkaline phosphatase resulted in a major fragment migrating just ahead of the lactose disaccharide standard. Lesser amounts of material ran as a larger fragment just behind the raffinose trisaccharide standard. Virtually no material remained at the origin. The major dephos~horylated saccharide fragment of [~H]mannose-labeled glycoconjugate also migrated as an apparent disaccharide, with some material migrating with mannose, the monosaccharide standard. The [3H]galactose- and [3H]mannose-labeled disaccharides in each case migrated identically with the Gal/~l4Man disaccharide derived from L. donovani LPG.

Discussion

In recent years, much attention has been focused on various surface molecules of E. histolytica involved in recognition and killing of target cells. It is presumed that these molecules, whose functional involvement has mostly been demonstrated in vitro, also participate in cytopathic activity associated with amebic virulence. Among these molecules, the 170-kDa galactose/N-acetyl galactosaminebinding lectin has been characterized in detail [1,20]. A few other surface molecules whose functions are not yet understood, namely the 125-kDa and 29-30-kDa molecules, have also been identified [5,21,22]. The data presented in this paper provide evidence that E. histolytica expresses a major acidic glycoconjugate which is strikingly similar to the well-described LPG from Leishmania. Leishmanial LPG, which is the major surface glycoconjugate of these parasites, is a polymer of phosphorylated oligosaccharide repeats linked via a phosphosaccharide

core to a GPI anchor [23]. The amebic glycoconjugate also contains galactose, mannose and is phosphorylated, as indicated by its metabolic labeling characteristics. In addition, it is hydrophobic in nature, as revealed by its elution profile on phenyl-Sepharose in the presence of 30-35% n-propanol. Its sensitivity to PI-PLC digestion and to nitrous acid deamination strongly suggests the presence of an unacetylated hexosamine and a phosphatidylinositol lipid anchor, the particular derivative of which is not yet known. In all of these characteristics, the molecule is also similar to the phosphorylated lipid-containing glycoconjugate from E. histolytica recently described by Stanley et al. [9]. In the current studies, the similarity of the amebic glycoconjugate to the leishmanial LPG was conclusively supported by its solubility properties in solvent E, and, most importantly, by its extreme lability to mild acid treatment, suggesting that it too contains sugar-l-phosphate linkage(s) which can be hydrolyzed by mild acid conditions to release phosphorylated oligosaccharide s u b units, The mild acid generated fragments of the amebic glycoconjugate were each found to be phosphorylated, as evidenced by their mobility in a non-polar solvent only after treatment with alkaline phosphatase. The major dephosphorylated fragment ran as a galactose and mannose containing disaccharide, suggesting that the salient structural feature of leishmanial LPG, which bears a backbone structure formed by phosphodiester linked galactosyl-/%mannose disaccharide repeats, might be remarkably similar in amebic LPG. Analysis of the radiolabeled products by SDS-PAGE showed that the molecule contains sugar and phosphate, but no polypeptides. It is not possible to rule out that small polypeptides may be associated with these molecules. Indeed, there were some small [35S]methionine-labeled products which appeared to be copurified with the amebic LPG, as has also been reported for the leishmanial LPG [24]. The shift in molecular size of the glycoconjugate reported by Stanley et al. [9] after pronase treatment might be

167

explained by these LPG associated peptides. A feature apparent from our studies, but not from those of Stanley et al., is the possibility that there may be two different types of LPG in amebae with a high degree of heterogeneity. Experiments are in progress to examine in detail various structural features, for example, the nature of the lipid moiety and the number and composition of the sugar-phosphate repeating units present in these molecules. If the amebic LPG is a component of the cell surface, then it may have an important role in parasite-host cell interactions involved in amebic virulence. The LPG-like glycoconjugate described by Stanley et al. was shown to be a cell surface molecule involved in adhesion based on the ability of a monoctonal antibody to inhibit amebic adhesion to a Chinese hamster ovary cell line. We have recently generated a monoclonal antibody, 2D7.10, which recognizes a surface antigen of E. histolytica in a strain-specific manner [25]. This antibody recognizes the LPG described in this report (manuscript in preparation), and studies are currently underway to determine the role of the polymorphic forms of the antigen in E. histolytica adhesion and cytotoxicity.

Acknowledgements We thank Drs. L.S. Diamond, M.J. McConville, and especially S. Turco for helpful discussions during the course of the work, and Sheryl Rathke for help with the preparation of the manuscript. This work was partly supported by a Rockefeller Career Development Fellowship (A.B.) and a research grant from the Department of Science and Technology, India (A.B.).

References 1 Petri, W.A., Jr., Chapman, M.D., Snodgrass, J., Mann, B.J., Broman, J. and Ravdin, J.I. (1989) Subunit structure of the galactose and N-acetyl-D-galactosamine inhibitable adherence lectin of Entamoeba histolytica. J. Biol. Chem. 264, 3007-3012.

2 Arroyo, R. and Orozco, E. (1987) Localization and identification of an Entamoeba histolytica adhesin. Mol. Biochem. Parasitol. 23, 151 158. 3 Meza, I., Cazares, F., Rosales-Encina, J.L.. TalamasRohana, P. and Rajkind, M. (1987) Use of antibodies to characterize a 220-kilodalton surface protein from Entamoeba histolytica. J. Infect. Dis. 156, 790 805. 4 Edman, U., Meraz, M.A., Rausser, S., Agabian, N. and Meza, I. (1990) Characterization of an immunodominant variable surface antigen from pathogenic and nonpathogenic Entamoeba histolytica. J. Exp. Med. 172, 879 888. 5 Vinayak, V.K. and Shandil, R.K. (1990) Recognition of 29 kDA surface associated adherence molecules of Entamoeba histolytica by monoclonal antibodies. FEMS Microbiol. Immunol. 2, 169 177. 6 0 r o z c o , E., Hernandez, F. and Rodriguez, M.A. (1988) Virulence-related properties in Entamoeba histolytica. In: Amebiasis: human infection by Entamoeba histolytica (Ravdin, J.I., ed.), pp. 314 325. Wiley, New York. 7 Lushbaugh, W.B. and Miller, J.H. (1988) The morphology of Entamoeba histolytica. In: Amebiasis: Human Infection by Entamoeba histolytica (Ravdin, J.I., ed.), pp. 41 68. Wiley, New York. 8 Isibasi, A., Cruz, M.S., Cottlieb, M. and Kumate, J. (1986) Purification of the polysaccharide portion of the lipopeptidophosphoglycan extracted from Trophozoites of Entamoeba histolytica. Arch. Invest. Med. (Mex.) 17 (Suppl.), 73 79. 9 Stanley, S.L., Huizenga, H. and Li, E. (1992) Isolation and partial characterization of a surface glycoconjugate of Entamoeba histolytica. Mol. Biochem. Parasitol. 50, 127 138. 10 Diamond, L.S., Harlow, D.R. and Cunnick, C. (1978) A new medium for axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R. Soc. Trop. Med. Hyg. 72, 431~32. 11 Bhattacharya, A., Bhattacharya, S., Sharma, M.P. and Diamond, L.S. (1990) Metabolic labeling of Entamoeba histolytica antigens: Characterization of a 28-kDa major intracellular antigen. Exp. Parasitol. 70, 255 263. 12 Sacks, D.L., Brodin, T.N. and Turco, S.A. (1990) Developmental modification of the lipophosphoglycan from Leishmania major promastigotes during metacyclogenesis. Mol. Biochem. Parasitol. 42, 225 234. 13 Turco, S.J., Wilkerson, M.A. and Clawson, D.R. (1984) Expression of an unusual acidic glucoconjugate in Leishmania donovani. J. Biol. Chem. 259, 3883-3889. 14 Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 277, 68(~685. 15 Laskey, R.A. and Mills, A.D. (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335 342. 16 Anet, E.F. and Reynolds, T.M. (1954) Isolation of mucic acid from fruits. Nature 174, 930 932. 17 McConville, M.J., Bacic, A., Mitchell, G.F. and Handman, E. (1987) Lipophosphoglycan of Leishmania major that vaccinates against cutaneous leishmaniasis contains an alkylglycerophophoinositol lipid anchor. Proc. Natl. Acad. Sci. USA 84, 8941 8945. 18 Orlandi, P.A. and Turco, S.J. (1987) Structure of the lipid moiety of the Leishmania donovani. J. Biol. Chem. 262, 10384~10391.

168 19 Leloir, L.F. and Cardini, C.E. (1957) Characterization of phosphorous compounds by acid lability. Methods Enzymol. 3, 840-850. 20 Tannich, E., Ebert, F. and Horstmann, R.D. (1991) Primary structure of the 170-kDa surface lectin of pathogenic Entarnoeba histolytica. Proc. Natl. Acad. Sci. USA 88, 1849 1953. 21 Blakely, P., Sargeaunt, P.G. and Reed, S.L. (1990) An immunogenic 30-kDa surface antigen of pathogenic clinical isolates of Entarnoeba histolytica. J. Infect. Dis. 162, 949-954. 22 Torian, B.E., Flores, B.M., Stroeher, V.L., Hagen, F.S. and Stamm, W.E. (1990) cDNA sequence analysis of a 29-kDa cysteine rich surface antigen of pathogenic

Entamoeba histolytica. Proc. Natl. Acad. Soc. USA 87, 6358 6362. 23 Turco, S.J. (1990) The Le&hman& lipophosphoglycan: a multifunctional molecule. Exp. Parasitol. 70, 241 245. 24 Jardim, A., Tolson, D.L., Turco, S.J., Pearson, T.W. and Olafson, R.W. (1991) The Le&hmania donovani lipophosphoglycan T lymphocyte-reactive component is a tightly associated protein complex. J. Immunol. 147, 3538-3544. 25 Bhattacharya, A., Ghildyal, R., Bhattacharya, S. and Diamond, L.S. (1990) Characterization of a monoclonal antibody that selectively recognizes a subset of Entamoeba histolytica isolates. Infect. Immun. 58, 3458 3461.