Molecular and Biochemical Parasitology, 56 (1992) 279-288 © 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00
279
MOLBIO 01848
Recognition of Entamoeba histolytica lipophosphoglycan by a strain-specific monoclonal antibody and human immune sera R a m a s a r e Prasad a, M o n i k a Tola a, Sudha B h a t t a c h a r y a b, M.P. S h a r m a c and A l o k B h a t t a c h a r y a a aSchool of Life Sciences and bSchool of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India, and CDepartment of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India (Received 18 May 1992; accepted 7 August 1992)
Western blot analysis showed that the monoclonal antibody 2D7.10 recognized lipophosphoglycan (LPG) from Entamoeba histolytica HM-I:IMSS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of [3H]galactose-labeled LPG and Western blot analysis of total lysate of E. histolytica with 2D7.10 revealed patterns similar to that of LPG with 2D7.10. This antibody could also immunoprecipitate purified LPG from the strain HM-I:IMSS after biosynthetically labeling with [3H]galactose and [32p]orthophosphate. However, no immunoprecipitation was observed when 2D7.10 was incubated with [32p]orthophosphate-labeled purified LPG from strain 200:NIH. Sera from patients suffering from invasive amoebiasis also immunoprecipitated 32p-labeled, purified LPG and could immunostain this molecule in Western blots. The human immune sera recognized carbohydrate epitopes but not the associated polypeptides of LPG, as evidenced by sensitivity to periodate digestion, mild acid hydrolysis but not to pronase treatment. It was earlier shown that 2D7.10 binds a carbohydrate epitope in a subset of axenized pathogenic strains of E. histolytica and that this epitope undergoes changes when cultured along with bacteria. These observations suggest that the E. histolytica LPG contains a strain-specific, variable epitope and that LPG is immunogenic in human. Key words: Monoclonal antibody; Surface antigen; Lipophosphoglycan; Entamoeba histolytica
Introduction
Cell-surface molecules play a very important role in pathophysiological mechanisms of Entamoeba histolytica [for review see ref. 1]. Isolation and characterization of these molecules is important in order to understand hostparasite relationship in amoebiasis. Recently, the presence of lipophosphoglycan (LPG) has been demonstrated in E. histolytica [2]. LPG Correspondence address: Alok Bhattacharya, School of Life Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi 110067, India. Fax: (91) 11-686-5886. Abbreviations: ELISA, enzyme-linked immunosorbent assay; LPG, lipophosphoglycan; McAb, monoclonal antibody; SDSPAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.
had earlier been identified on promastigotes of Leishmania species as a major cell-surface glycoconjugate [for review see ref. 3]. Recent studies have shown polymorphic forms of LPG in Leishmania [4]. Besides interspecies differences, qualitative and quantitative variations have been observed during metacyclogenesis [5]. These changes in LPG may be responsible for altered pathogenic properties of these cells [6]. It is speculated that LPG may also be involved in cytopathogenic properties of E. histolytica [2]. We have reported earlier that monoclonal antibody (McAb) 2D7.10 recognizes only some of the axenized strains of E. histolytica [7]. Further, the 2D7.10 epitope was only present in axenized strains and could not be detected in xenic strains [8]. These observations suggested
280
that 2D7.10 epitope is polymorphic, changing in response to culture conditions. In the present report we demonstrate that the 2D7.10 epitope resides in LPG. We also present data to suggest that LPG stimulates antibody response in amoebiasis patients. It is, therefore, likely that amoebic LPG might also be polymorphic like LPG from Leishmania.
Materials and Methods
Cultures. E. histolytica strains, HM-l:IMSS clone 6 and 200:NIH clone 2, were used in this study and were maintained in TYI-S-33 medium at 36°C [9]. Sera. H u m a n immune sera were obtained from clinically proven amoebiasis patients from New Delhi, India. These patients were diagnosed by attending consulting clinicians and confirmed by either stool examination or by serology. Healthy control serum was obtained in the United States from a person with no known history of amoebiasis. Polyclonal antibody aEhM raised against hydrophobic membrane components has been described earlier [8]. Rabbit antibody against pronase-digested E. histolytica LPG (aLPG.HM1) has also been used in this study. Generation of this antibody and its characterization will be described elsewhere. Antigen preparation. Antigens were prepared essentially as described [7]. Cells were harvested by chilling cultures in ice-water for 10 min and were collected by centrifugation for 7 min. Cells were lysed by resuspending the pellet in lysis buffer (20 m M Tris-HC1, pH 7.5; 2 m M phenylmethylsulfonyl fluoride; 5 m M p-hydroxymercuribenzoate; 10 #g ml 1 leupeptin) for 30 min at 4°C followed by homogenization in a glass-teflon homogenizer. Enzyme-linked immunosorbent assay. ELISA was carried out according to published procedures [7]. Either LPG or total cell lysate of HM-1 strain were used to coat each well of microtiter plates (Costar, USA). Non-specific
sites were blocked with 3% (w/v) gelatin in Tris-buffered saline (TBS, 20 mM Tris-HC1, pH 7.5, 500 m M NaC1). All incubations with antibodies were carried out for 2 h at room temperature in 1% gelatin-TBS. Bound antibodies were detected using appropriate alkaline phosphatase-labeled second antibody using p-nitrophenylphosphate as substrate. Sandwich ELISA was essentially carried out as above except the first antibody was dried on the plate at 37°C instead of coating. Wells were washed thoroughly before blocking.
Metabolic labeling and extraction. 2-5 × 10 6 cells/ml in TY1-S-33 base were incubated with either [3H]galactose (100-200 /~Ci ml 1), or [32p]orthophosphate (0.5 mCi ml 1) for 3 h at 36°C. Cells were washed with PBS 3 times at 4°C and LPG was extracted as described [10]. Briefly, labeled cells were extracted sequentially with chloroform/methanol (3:2) 5 parts and 1 part of 4 m M MgCI2, 5 parts of chloroform/methanol/water (10:10:3) and 1 part of chloroform/methanol (1 : 1), and chloroform/methanol/water (10:10:3). The pellet is then extracted with solvent E (water/ethanol/ diethylether/pyridine/ammonium hydroxide, 15:15:5:1:0.017) as described [10]. Hydrophobic chromatography. Crude glycolipids were resuspended in 0.1 M NaC1, 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. 0.5-ml fractions were collected and radioactivity determined. The relative amount of glycolipid was ascertained by titrating in Western blots using McAb 2D7.10. The amount of LPG was also estimated on the basis of total hexose [11] and total phosphate [12]. Mild acid treatment. Mild acid conditions of 0.02 M HC1, 5 min at 100°C were used to fragment the molecule as phosphorylated saccharide repeat units from the carbohydrate core region.
281
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Radiolabeled samples were analyzed by discontinuous SDS-PAGE under reducing conditions according to Laemmli [13]. The separating gel was 10% acrylamide. After electrophoresis the ~els were subjected to fluorography (for ~H) according to Laskey and Mills [14]. Gels containing 32p-labeled materials were dried and exposed to X-ray films in the presence of intensifying screens. Pronase digestion. Immobilized pronase CB (35 #g, Pierce, USA) was incubated with LPG for 30 min at 37°C. Digested preparation was solubilized in SDS-PAGE sample buffer and subjected to Western blotting as described later. Periodate treatment. Periodate treatment was carried out using 20 mM sodium periodate for 30 min as described before [7]. Western blotting. Prewarmed total cell lysate was treated with preheated 2 x SDS-PAGE sample buffer [13]. Polyacrylamide gels (10%) were used for all separations. Typically 0.5 #g of LPG (based on hexose) and 100 #g protein of total cell lysate were used for each analysis. After separation, the molecules were transferred to nitrocellulose membrane electrophoretically [15]. After transfer the efficiency and amount bound to nitrocellulose membrane were routinely checked by staining with ponceau S (Sigma, USA). No detectable band(s) was observed with LPG (data not shown). In some experiments, Immobilon P membranes (Millipore, USA) were used for Western blotting. The immunoreactive molecules were located using specific antibodies and alkaline phosphatase-conjugated anti-mouse immunoglobulins (Bio-Rad, USA). Color development used nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrate. Immunoprecipitation. Immunoprecipitation from detergent-solubilized amoebae was carried out exactly as previously described [16].
Radiolabeled and purified LPG was incubated with 2D7.10 or control ascites (1 /tl) for 2 h at 4°C. The immune complexes were separated by incubation with protein A-Sepharose 4B (Pharmacia, Sweden). Resin was washed extensively as described before [16]. The bound material was eluted by SDS-PAGE sample buffer and analyzed on a 10% acrylamide gels. Gels were fluorographed and exposed to X-ray films [14].
Protein determination. All protein concentrations were determined with Bicinchoninic acid reagent (Pierce Chemical Company, USA). Results
2D7.10 recognizes LPG in Western blots. The nature of the molecule recognized by 2D7.10 was investigated by Western-blot analysis of a
b
1
2
1
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-'~ 66
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279
MOLBIO 01848
Recognition of Entamoeba histolytica lipophosphoglycan by a strain-specific monoclonal antibody and human immune sera R a m a s a r e Prasad a, M o n i k a Tola a, Sudha B h a t t a c h a r y a b, M.P. S h a r m a c and A l o k B h a t t a c h a r y a a aSchool of Life Sciences and bSchool of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India, and CDepartment of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India (Received 18 May 1992; accepted 7 August 1992)
Western blot analysis showed that the monoclonal antibody 2D7.10 recognized lipophosphoglycan (LPG) from Entamoeba histolytica HM-I:IMSS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of [3H]galactose-labeled LPG and Western blot analysis of total lysate of E. histolytica with 2D7.10 revealed patterns similar to that of LPG with 2D7.10. This antibody could also immunoprecipitate purified LPG from the strain HM-I:IMSS after biosynthetically labeling with [3H]galactose and [32p]orthophosphate. However, no immunoprecipitation was observed when 2D7.10 was incubated with [32p]orthophosphate-labeled purified LPG from strain 200:NIH. Sera from patients suffering from invasive amoebiasis also immunoprecipitated 32p-labeled, purified LPG and could immunostain this molecule in Western blots. The human immune sera recognized carbohydrate epitopes but not the associated polypeptides of LPG, as evidenced by sensitivity to periodate digestion, mild acid hydrolysis but not to pronase treatment. It was earlier shown that 2D7.10 binds a carbohydrate epitope in a subset of axenized pathogenic strains of E. histolytica and that this epitope undergoes changes when cultured along with bacteria. These observations suggest that the E. histolytica LPG contains a strain-specific, variable epitope and that LPG is immunogenic in human. Key words: Monoclonal antibody; Surface antigen; Lipophosphoglycan; Entamoeba histolytica
Introduction
Cell-surface molecules play a very important role in pathophysiological mechanisms of Entamoeba histolytica [for review see ref. 1]. Isolation and characterization of these molecules is important in order to understand hostparasite relationship in amoebiasis. Recently, the presence of lipophosphoglycan (LPG) has been demonstrated in E. histolytica [2]. LPG Correspondence address: Alok Bhattacharya, School of Life Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi 110067, India. Fax: (91) 11-686-5886. Abbreviations: ELISA, enzyme-linked immunosorbent assay; LPG, lipophosphoglycan; McAb, monoclonal antibody; SDSPAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.
had earlier been identified on promastigotes of Leishmania species as a major cell-surface glycoconjugate [for review see ref. 3]. Recent studies have shown polymorphic forms of LPG in Leishmania [4]. Besides interspecies differences, qualitative and quantitative variations have been observed during metacyclogenesis [5]. These changes in LPG may be responsible for altered pathogenic properties of these cells [6]. It is speculated that LPG may also be involved in cytopathogenic properties of E. histolytica [2]. We have reported earlier that monoclonal antibody (McAb) 2D7.10 recognizes only some of the axenized strains of E. histolytica [7]. Further, the 2D7.10 epitope was only present in axenized strains and could not be detected in xenic strains [8]. These observations suggested
280
that 2D7.10 epitope is polymorphic, changing in response to culture conditions. In the present report we demonstrate that the 2D7.10 epitope resides in LPG. We also present data to suggest that LPG stimulates antibody response in amoebiasis patients. It is, therefore, likely that amoebic LPG might also be polymorphic like LPG from Leishmania.
Materials and Methods
Cultures. E. histolytica strains, HM-l:IMSS clone 6 and 200:NIH clone 2, were used in this study and were maintained in TYI-S-33 medium at 36°C [9]. Sera. H u m a n immune sera were obtained from clinically proven amoebiasis patients from New Delhi, India. These patients were diagnosed by attending consulting clinicians and confirmed by either stool examination or by serology. Healthy control serum was obtained in the United States from a person with no known history of amoebiasis. Polyclonal antibody aEhM raised against hydrophobic membrane components has been described earlier [8]. Rabbit antibody against pronase-digested E. histolytica LPG (aLPG.HM1) has also been used in this study. Generation of this antibody and its characterization will be described elsewhere. Antigen preparation. Antigens were prepared essentially as described [7]. Cells were harvested by chilling cultures in ice-water for 10 min and were collected by centrifugation for 7 min. Cells were lysed by resuspending the pellet in lysis buffer (20 m M Tris-HC1, pH 7.5; 2 m M phenylmethylsulfonyl fluoride; 5 m M p-hydroxymercuribenzoate; 10 #g ml 1 leupeptin) for 30 min at 4°C followed by homogenization in a glass-teflon homogenizer. Enzyme-linked immunosorbent assay. ELISA was carried out according to published procedures [7]. Either LPG or total cell lysate of HM-1 strain were used to coat each well of microtiter plates (Costar, USA). Non-specific
sites were blocked with 3% (w/v) gelatin in Tris-buffered saline (TBS, 20 mM Tris-HC1, pH 7.5, 500 m M NaC1). All incubations with antibodies were carried out for 2 h at room temperature in 1% gelatin-TBS. Bound antibodies were detected using appropriate alkaline phosphatase-labeled second antibody using p-nitrophenylphosphate as substrate. Sandwich ELISA was essentially carried out as above except the first antibody was dried on the plate at 37°C instead of coating. Wells were washed thoroughly before blocking.
Metabolic labeling and extraction. 2-5 × 10 6 cells/ml in TY1-S-33 base were incubated with either [3H]galactose (100-200 /~Ci ml 1), or [32p]orthophosphate (0.5 mCi ml 1) for 3 h at 36°C. Cells were washed with PBS 3 times at 4°C and LPG was extracted as described [10]. Briefly, labeled cells were extracted sequentially with chloroform/methanol (3:2) 5 parts and 1 part of 4 m M MgCI2, 5 parts of chloroform/methanol/water (10:10:3) and 1 part of chloroform/methanol (1 : 1), and chloroform/methanol/water (10:10:3). The pellet is then extracted with solvent E (water/ethanol/ diethylether/pyridine/ammonium hydroxide, 15:15:5:1:0.017) as described [10]. Hydrophobic chromatography. Crude glycolipids were resuspended in 0.1 M NaC1, 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. 0.5-ml fractions were collected and radioactivity determined. The relative amount of glycolipid was ascertained by titrating in Western blots using McAb 2D7.10. The amount of LPG was also estimated on the basis of total hexose [11] and total phosphate [12]. Mild acid treatment. Mild acid conditions of 0.02 M HC1, 5 min at 100°C were used to fragment the molecule as phosphorylated saccharide repeat units from the carbohydrate core region.
281
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Radiolabeled samples were analyzed by discontinuous SDS-PAGE under reducing conditions according to Laemmli [13]. The separating gel was 10% acrylamide. After electrophoresis the ~els were subjected to fluorography (for ~H) according to Laskey and Mills [14]. Gels containing 32p-labeled materials were dried and exposed to X-ray films in the presence of intensifying screens. Pronase digestion. Immobilized pronase CB (35 #g, Pierce, USA) was incubated with LPG for 30 min at 37°C. Digested preparation was solubilized in SDS-PAGE sample buffer and subjected to Western blotting as described later. Periodate treatment. Periodate treatment was carried out using 20 mM sodium periodate for 30 min as described before [7]. Western blotting. Prewarmed total cell lysate was treated with preheated 2 x SDS-PAGE sample buffer [13]. Polyacrylamide gels (10%) were used for all separations. Typically 0.5 #g of LPG (based on hexose) and 100 #g protein of total cell lysate were used for each analysis. After separation, the molecules were transferred to nitrocellulose membrane electrophoretically [15]. After transfer the efficiency and amount bound to nitrocellulose membrane were routinely checked by staining with ponceau S (Sigma, USA). No detectable band(s) was observed with LPG (data not shown). In some experiments, Immobilon P membranes (Millipore, USA) were used for Western blotting. The immunoreactive molecules were located using specific antibodies and alkaline phosphatase-conjugated anti-mouse immunoglobulins (Bio-Rad, USA). Color development used nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrate. Immunoprecipitation. Immunoprecipitation from detergent-solubilized amoebae was carried out exactly as previously described [16].
Radiolabeled and purified LPG was incubated with 2D7.10 or control ascites (1 /tl) for 2 h at 4°C. The immune complexes were separated by incubation with protein A-Sepharose 4B (Pharmacia, Sweden). Resin was washed extensively as described before [16]. The bound material was eluted by SDS-PAGE sample buffer and analyzed on a 10% acrylamide gels. Gels were fluorographed and exposed to X-ray films [14].
Protein determination. All protein concentrations were determined with Bicinchoninic acid reagent (Pierce Chemical Company, USA). Results
2D7.10 recognizes LPG in Western blots. The nature of the molecule recognized by 2D7.10 was investigated by Western-blot analysis of a
b
1
2
1
"~NO
200~,"-
974:=='6 8 ~'--
~t7.4
-'~ 66
~88
"
