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Identification of Collagen-Binding Proteins from the Cytoplasmic Membrane of Entamoeba histolytica
Archives of Medical Research 31 (2000) S149–S150
Identification of Collagen-Binding Proteins from the Cytoplasmic Membrane of Entamoeba histolytica Ma. de Lourdes Muñoz, Saúl Ramírez-Mejía and Rosalinda Tovar Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del I.P.N. (Cinvestav), Mexico City, Mexico
Key Words: Entamoeba histolytica, Membrane, Collagen, Triton X-114, Receptors.
Introduction An enteric illness, amebiasis, is caused by the parasite Entamoeba histolytica, and is characterized by the capacity of trophozoites to destroy human tissues. This has been attributed mainly to the production of cytolytic or hydrolytic substances including metalloproteinase-like collagenases that digest human collagen type I (1). Collagen is one of the major components of the extracellular matrix (ECM) in human tissues. Consequently, collagenase associated with electrondense granules (EDG) from E. histolytica has been considered an important factor in the pathogenicity of this parasite. Interaction of the parasite with collagen type I in the presence of Ca2⫹ produces increased adhesion and tyrosine phosphorylation, activation of pp125FAK and p42MAPK, resulting in cytoskeleton organization and release of EDGs in a strictly time-dependent fashion (2). The collagen receptors that generate this response are unknown; therefore, we describe here the membrane proteins that bind collagen under different conditions.
drophobic phases were stored at ⫺20⬚C until use (4). The protein content was determined by Bradford’s method with bovine serum albumin as a standard. Affinity chromatography. A total of 800 g of protein/mL from the hydrophilic phase was loaded onto a 10-mL collagen-sepharose column preequilibrated with 0.1 M TrisHCl and 0.002 M pHMB, pH 7.5, and incubated at different temperatures for 1 h. The column was extensively washed at the same temperature until absorbance at 280 nm reached baseline values. Finally, the bound protein was eluted with the same buffer containing 2 M NaCl. The effluent was monitored at 280 nm, and 0.5-mL fractions were collected. Fractions 5–13 containing the proteins bound to collagen were stored at ⫺20⬚C. Gel electrophoresis. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemli’s method using a 4% stacking gel and 10% resolving gel. Proteins were visualized by Coomassie Blue staining.
Materials and Methods
Results and Discussion
Parasites. E. histolytica HM-1:IMSS trophozoites were cultured axenically in TYI-S-33 medium (1) at 37⬚C. All experiments were carried out using trophozoites harvested after 48 h of culture.
Initially, type I collagen coupled to Sepharose was used to purify collagen-binding proteins from trophozoite plasma membranes. To elucidate whether binding of these proteins to collagen depends on temperature, collagen columns with the hydrophilic membrane fraction were incubated overnight at 4⬚C and for 1 h at 20 or 37⬚C. Results showed that at 4⬚C binding was very poor, increasing at 20⬚C. Maximal binding was observed at 37⬚C (Figure 1A). SDS-PAGE of the eluted proteins gave approximately seven bands. The presence of 220-, 128-, 114-, 98-, 69-, 59-, and 51-kDa proteins (Figure 1B) was consistent with other column runs. The polypeptides of 220, 114, 98, and 59 kDa were the most prominent. It is interesting to note that an earlier report showed that polypeptides with apparent molecular weights of 105, 56, and 30 kDa from total trophozoite extract bind to collagen-type I (5). The peptides of 105 and 56 may be similar to our polypeptides of 98 and 51, respec-
Separation of proteins in Triton X-114. The crude extract of plasma membranes isolated as described previously (3) was suspended in 1% Triton X-114; after 5 min at 4⬚C, the solubilized membrane was incubated at 37⬚C for 5 min and centrifuged at 600 ⫻ g for 5 min. The hydrophilic and hy-
Address reprint requests to: Ma. de Lourdes Muñoz, Departamento de Genética y Biología Molecular Av. Instituto Politécnico Nacional #2508, Col. San Pedro Zacatenco, 07360 México, D.F., México. Tel.: (⫹525) 7473800, ext. 5380; FAX: (⫹525) 747-7100. Presenting author: Ma. de Lourdes Muñoz.
0188-4409/00 $–see front matter. Copyright © 2000 IMSS. Published by Elsevier Science Inc. PII S0188-4409(00)00 2 5 0 - 2
S150
Muñoz et al./ Archives of Medical Research 31 (2000) S149–S150
Figure 1. Purification of collagen-binding proteins by collagen-Sepharose affinity chromatography. (A) Purified trophozoite plasma membranes were solubilized with Triton X-114, and the hydrophilic proteins were loaded onto a collagen-Sepharose and incubated at 20⬚C (panel 1) or 37⬚C (panel 2). Collagenbinding proteins were eluted with buffer containing 2 M NaCl; and (B) SDS-PAGE analysis of proteins eluted from the affinity column at 37⬚C. Lane 1, Coomassie Blue staining of total hydrophilic proteins; lane 2–4 proteins obtained from collected fractions 5–7, respectively. Position of molecular weight standards is given on the right side of the panel.
tively, although they are difficult to compare, because these authors purify the proteins from total extracts. Polyclonal antibodies specific for each of these proteins were raised in mice. The 98 kDa protein was the most antigenic protein. Immunofluorescence assays using these antibodies showed enrichment of the collagen-binding proteins on the membrane of trophozoites activated by collagen-type I and Ca2⫹ for 3 h. It can be concluded that E. histolytica trophozoites have at least seven plasma membrane proteins that bind to collagen at 37⬚C (Figure 1). The hydrophilic proteins that were collagen affinity-purified were obtained by Triton X-114 phase partitioning. Therefore, it is more likely that these proteins are exposed to the extracellular medium, and are probably responsible for cell adhesion to collagen. Consequently, one of these proteins may be the true collagen receptor(s).
Acknowledgments This work was supported by grant no. 211085-5 30581-M from Conacyt (Mexico).
References 1. Muñoz ML, Lamoyi E, León G, Tovar R, Pérez-García J, De la Torre M, Murueta E, Bernal R. Antigens in electron-dense granules from Entamoeba histolytica as possible markers for pathogenicity. J Clin Microbiol 1992;28:2418. 2. Muñoz ML, Tovar R, Moreno M, León G, Das P. Cytoskeleton of Entamoeba histolytica during electron dense granule secretion. In: Ozcel AM, editor. New dimensions in parasitology. Acta Parasitol Turcica 1996:20:193. 3. Aley SB, Scott WA, Cohn ZA. Plasma membrane of Entamoeba histolytica. J Exp Med 1980;152:391. 4. Bordier C. Phase separation of integral proteins in Triton X-114 solution. J Biol Chem 1981;256:1604. 5. Rosales-Encina JL, Campos-Salazar MS, Rojkind M. Entamoeba histolytica collagen binding proteins. Arch Invest Med 1992;23:109.
Identification of Collagen-Binding Proteins from the Cytoplasmic Membrane of Entamoeba histolytica Ma. de Lourdes Muñoz, Saúl Ramírez-Mejía and Rosalinda Tovar Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del I.P.N. (Cinvestav), Mexico City, Mexico
Key Words: Entamoeba histolytica, Membrane, Collagen, Triton X-114, Receptors.
Introduction An enteric illness, amebiasis, is caused by the parasite Entamoeba histolytica, and is characterized by the capacity of trophozoites to destroy human tissues. This has been attributed mainly to the production of cytolytic or hydrolytic substances including metalloproteinase-like collagenases that digest human collagen type I (1). Collagen is one of the major components of the extracellular matrix (ECM) in human tissues. Consequently, collagenase associated with electrondense granules (EDG) from E. histolytica has been considered an important factor in the pathogenicity of this parasite. Interaction of the parasite with collagen type I in the presence of Ca2⫹ produces increased adhesion and tyrosine phosphorylation, activation of pp125FAK and p42MAPK, resulting in cytoskeleton organization and release of EDGs in a strictly time-dependent fashion (2). The collagen receptors that generate this response are unknown; therefore, we describe here the membrane proteins that bind collagen under different conditions.
drophobic phases were stored at ⫺20⬚C until use (4). The protein content was determined by Bradford’s method with bovine serum albumin as a standard. Affinity chromatography. A total of 800 g of protein/mL from the hydrophilic phase was loaded onto a 10-mL collagen-sepharose column preequilibrated with 0.1 M TrisHCl and 0.002 M pHMB, pH 7.5, and incubated at different temperatures for 1 h. The column was extensively washed at the same temperature until absorbance at 280 nm reached baseline values. Finally, the bound protein was eluted with the same buffer containing 2 M NaCl. The effluent was monitored at 280 nm, and 0.5-mL fractions were collected. Fractions 5–13 containing the proteins bound to collagen were stored at ⫺20⬚C. Gel electrophoresis. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemli’s method using a 4% stacking gel and 10% resolving gel. Proteins were visualized by Coomassie Blue staining.
Materials and Methods
Results and Discussion
Parasites. E. histolytica HM-1:IMSS trophozoites were cultured axenically in TYI-S-33 medium (1) at 37⬚C. All experiments were carried out using trophozoites harvested after 48 h of culture.
Initially, type I collagen coupled to Sepharose was used to purify collagen-binding proteins from trophozoite plasma membranes. To elucidate whether binding of these proteins to collagen depends on temperature, collagen columns with the hydrophilic membrane fraction were incubated overnight at 4⬚C and for 1 h at 20 or 37⬚C. Results showed that at 4⬚C binding was very poor, increasing at 20⬚C. Maximal binding was observed at 37⬚C (Figure 1A). SDS-PAGE of the eluted proteins gave approximately seven bands. The presence of 220-, 128-, 114-, 98-, 69-, 59-, and 51-kDa proteins (Figure 1B) was consistent with other column runs. The polypeptides of 220, 114, 98, and 59 kDa were the most prominent. It is interesting to note that an earlier report showed that polypeptides with apparent molecular weights of 105, 56, and 30 kDa from total trophozoite extract bind to collagen-type I (5). The peptides of 105 and 56 may be similar to our polypeptides of 98 and 51, respec-
Separation of proteins in Triton X-114. The crude extract of plasma membranes isolated as described previously (3) was suspended in 1% Triton X-114; after 5 min at 4⬚C, the solubilized membrane was incubated at 37⬚C for 5 min and centrifuged at 600 ⫻ g for 5 min. The hydrophilic and hy-
Address reprint requests to: Ma. de Lourdes Muñoz, Departamento de Genética y Biología Molecular Av. Instituto Politécnico Nacional #2508, Col. San Pedro Zacatenco, 07360 México, D.F., México. Tel.: (⫹525) 7473800, ext. 5380; FAX: (⫹525) 747-7100. Presenting author: Ma. de Lourdes Muñoz.
0188-4409/00 $–see front matter. Copyright © 2000 IMSS. Published by Elsevier Science Inc. PII S0188-4409(00)00 2 5 0 - 2
S150
Muñoz et al./ Archives of Medical Research 31 (2000) S149–S150
Figure 1. Purification of collagen-binding proteins by collagen-Sepharose affinity chromatography. (A) Purified trophozoite plasma membranes were solubilized with Triton X-114, and the hydrophilic proteins were loaded onto a collagen-Sepharose and incubated at 20⬚C (panel 1) or 37⬚C (panel 2). Collagenbinding proteins were eluted with buffer containing 2 M NaCl; and (B) SDS-PAGE analysis of proteins eluted from the affinity column at 37⬚C. Lane 1, Coomassie Blue staining of total hydrophilic proteins; lane 2–4 proteins obtained from collected fractions 5–7, respectively. Position of molecular weight standards is given on the right side of the panel.
tively, although they are difficult to compare, because these authors purify the proteins from total extracts. Polyclonal antibodies specific for each of these proteins were raised in mice. The 98 kDa protein was the most antigenic protein. Immunofluorescence assays using these antibodies showed enrichment of the collagen-binding proteins on the membrane of trophozoites activated by collagen-type I and Ca2⫹ for 3 h. It can be concluded that E. histolytica trophozoites have at least seven plasma membrane proteins that bind to collagen at 37⬚C (Figure 1). The hydrophilic proteins that were collagen affinity-purified were obtained by Triton X-114 phase partitioning. Therefore, it is more likely that these proteins are exposed to the extracellular medium, and are probably responsible for cell adhesion to collagen. Consequently, one of these proteins may be the true collagen receptor(s).
Acknowledgments This work was supported by grant no. 211085-5 30581-M from Conacyt (Mexico).
References 1. Muñoz ML, Lamoyi E, León G, Tovar R, Pérez-García J, De la Torre M, Murueta E, Bernal R. Antigens in electron-dense granules from Entamoeba histolytica as possible markers for pathogenicity. J Clin Microbiol 1992;28:2418. 2. Muñoz ML, Tovar R, Moreno M, León G, Das P. Cytoskeleton of Entamoeba histolytica during electron dense granule secretion. In: Ozcel AM, editor. New dimensions in parasitology. Acta Parasitol Turcica 1996:20:193. 3. Aley SB, Scott WA, Cohn ZA. Plasma membrane of Entamoeba histolytica. J Exp Med 1980;152:391. 4. Bordier C. Phase separation of integral proteins in Triton X-114 solution. J Biol Chem 1981;256:1604. 5. Rosales-Encina JL, Campos-Salazar MS, Rojkind M. Entamoeba histolytica collagen binding proteins. Arch Invest Med 1992;23:109.