Glycobiotogy vol. 2 no. 6 pp. 563-569, 1992
Identification and characterization of an a-mannosidase from Trypanosoma cruzi
Paul M.Swanson1, Clint E.Carter2, Cindy Hager1, Wan Joon Kim1, Sarah Obermeier1 and Thomas N.Oeltmann1'3 'Departments of Medicine and Biochemistry, and the 2Department of Biology, Vanderbilt University, Nashville, TN 37232, USA 'To whom correspondence should be addressed at: Department of Medicine/Oncology, 1956 TVC, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
Key words: a-mannosidase/lysosomal cruzi
enzyme/'Trypanosoma Results
Introduction Chagas' disease (American trypanosomiasis), which afflicts millions of people in Central and South America, is caused by the haemoflagellate protozoan Trypanosoma cruzi. Natural transmission of T. cruzi occurs via the haematophagous reduvid bugs. Trypanosoma cruzi has a complex life cycle and exhibits at least three morphologically distinct forms. Epimastigotes replicate in the insect gut and differentiate into metacyclic trypomastigotes. During the course of a blood meal, excess water is eliminated with the faeces. Metacyclic trypomastigotes are released with the faeces and can enter the skin of their mammalian host through the site of the bite, through damaged skin, or through contamination of mucous membranes. Once in the circulation, trypomastigotes can infect a variety of cell types. Inside the cell, trypomastigotes differentiate into amastigotes, replicate and then differentiate into trypomastigotes. Lysis of the infected cell releases the trypomastigotes into the circulation for reinfecting host cells. Currently, it is unknown how the parasite gains entry into the host cells and how it avoids destruction. Trypanosoma cruzi express lysosomal-like acid hydrolases on their cell surface as well as within their lysosome(s) and it is possible that cell surface hydrolases may play a role in the host-parasite interactions. Presently, little is known about the biological roles of the acid hydrolases of © Oxford University Press
The a-mannosidase was purified from the epimastigote form of T.cruzi using 4-methylumbelliferyl-a-D-mannopyranoside as substrate and the purification is summarized in Table I. As previously reported (Avila et al., 1979), the activity of a-mannosidase in crude homogenates is the highest of the acid glycosidases tested. In addition, a-mannosidase activity varied with the form of the parasite and is only slightly less than that of acid phosphatase. These findings are summarized in Table II. Extraction of a-mannosidase Epimastigotes (4-5 ml of packed cells) were harvested by centrifugation, washed three times, resuspended in 20 ml of 10 mM phosphate (pH 7.0) containing 0.4 M NaCl, 0.1% Triton X-100 and the following protease inhibitors: aprotinin (2 U/ml), leupeptin (10/tg/ml), pepstatin (5 fig/ml) and phenylmethyl sulphonyl fluoride (PMSF) (2 mM). After three freezethaw cycles, the suspension was homogenized by sonication. The mixture was centrifuged at 20 000 g for 30 min. The lowspeed supernatant was saved at 4°C and the pellet extracted two additional times by the method described above using 7.5 ml of extraction buffer. The low-speed supernatants were combined and contained - 9 0 % of the activity. The combined low-speed supernatants were diluted to 50 ml and dialysed overnight against 10 mM phosphate (pH 7.2), and then centrifuged at 150 000 g for 90 min. 563
Downloaded from http://glycob.oxfordjournals.org/ at McMaster University Library on August 7, 2015
In this report we describe the first purification and characterization of the acid a-mannosidase from the human parasite Trypanosoma cruzi. The purified enzyme exhibited a native mol. wt of 240 000 Da and is apparently composed of four identical subunits of mol. wt 58 000 Da. Each of the four subunits contains one N-linked high-mannose-type oligosaccharide. The a-mannosidase exhibited a pH optimum of 3.5 and a pi of 5.9. This low pH optimum and the ability of swainsonine to inhibit its activity suggest that the a-mannosidase is a lysosomal enzyme. Antibodies against the T.cruzi enzyme did not react with mammalian lysosomal a-mannosidase and, conversely, antibody against a rat lysosomal a-mannosidase did not react with the T. cruzi enzyme. Thus, the T. cruzi enzyme appears to be distinct from its mammalian counterpart.
T. cruzi. Reports of purified acid hydrolases from parasites are rare. Three acid phosphatases have been purified to homogeneity from Leishmania donovani (Remaley et al., 1985). Acid phosphatase has only been demonstrated, but not purified or characterized in T.cruzi (Letelier et al., 1985, 1986; Nagakura et al., 1985). This activity has been shown to exist both intracellularly as well as on the plasma membrane. In addition, this cell surface-associated activity was shown to be altered with the morphological transformation in T. cruzi (Nagakura et al., 1985). Avila et al. (1979) reported the presence of 12 acid hydrolases, including an a-mannosidase, four near neutral hydrolases and alkaline phosphatase in T. cruzi. No fractionation of these enzymes was attempted and only the pH optimum for each was reported. More recently, Bontempi et al. (1989) reported that the a-mannosidase required detergent (1.5 mg/ml digitonin) for complete solubilization and suggest that it is lysosomal, but no purification was reported. Thus, there has been no reported purification of T.cruzi acid glycosidases. In this report, we have identified the acid a-mannosidase activity, purified and characterized the a-mannosidase, and demonstrate that it is immunologically distinct from the corresponding host enzyme. Utilizing antibodies against the purified a-mannosidase, it will now be possible to investigate the biosynthetic, processing and sorting pathway for this enzyme in particular and other intracellular hydrolases of T.cruzi in general.
P.M.Swanson et aJ.
Table I. Summary of purification of a-mannosidase Step
Total units'
Total protein (mg)
Specific activity (U/mg)
Yield (%)
Fold purification
Crude homogenate DEAE-cellulose Sepharose 4B IEF S-200 Con A
115 80 72 67 59 49
130 40 22 3 1.7 0.05
0.88 1 98 3.20 22 00 39 00 980.00
_ 69 62 58 51 42
— 2.25 3.63 25.00 44.00 1113.6
'Units are /imol 4-methylumbelliferyl-a-D-mannopyranoside hydrolysed per hour at 37°C and pH 3.5.
ed sharply at pH 5.9. The fractions which contained the a-mannosidase activity were pooled and made 0.3 M in NaCl to displace the ampholytes, and concentrated to 2 ml by vacuum dialysis. Sephacryl S-200 gel filtration The pooled, concentrated a-mannosidase activity from the preparative IEF step was applied to a Sephacryl S-200 column (1.5 x 90 cm) equilibrated with 10 mM phosphate (pH 7.2). The a-mannosidase eluted at the void volume (Figure 3). The fractions (37-49) containing the activity were pooled. Concanavalin A—agarose affinity purification
Table n . Acid hydrolase content of T.cruzi at different stages
Acid phosphatase /3-Hexosaminidase £-Fucosidase a-Glucosidase /J-Glucosidase a-Mannosidase ^-Mannosidase /3-Glucuronidase a-Galactosidase /3-Galactosidase
Trypomastigote
Epimastigote
Amastigote
1.03 0 03 0.04
Identification and characterization of an a-mannosidase from Trypanosoma cruzi
Paul M.Swanson1, Clint E.Carter2, Cindy Hager1, Wan Joon Kim1, Sarah Obermeier1 and Thomas N.Oeltmann1'3 'Departments of Medicine and Biochemistry, and the 2Department of Biology, Vanderbilt University, Nashville, TN 37232, USA 'To whom correspondence should be addressed at: Department of Medicine/Oncology, 1956 TVC, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
Key words: a-mannosidase/lysosomal cruzi
enzyme/'Trypanosoma Results
Introduction Chagas' disease (American trypanosomiasis), which afflicts millions of people in Central and South America, is caused by the haemoflagellate protozoan Trypanosoma cruzi. Natural transmission of T. cruzi occurs via the haematophagous reduvid bugs. Trypanosoma cruzi has a complex life cycle and exhibits at least three morphologically distinct forms. Epimastigotes replicate in the insect gut and differentiate into metacyclic trypomastigotes. During the course of a blood meal, excess water is eliminated with the faeces. Metacyclic trypomastigotes are released with the faeces and can enter the skin of their mammalian host through the site of the bite, through damaged skin, or through contamination of mucous membranes. Once in the circulation, trypomastigotes can infect a variety of cell types. Inside the cell, trypomastigotes differentiate into amastigotes, replicate and then differentiate into trypomastigotes. Lysis of the infected cell releases the trypomastigotes into the circulation for reinfecting host cells. Currently, it is unknown how the parasite gains entry into the host cells and how it avoids destruction. Trypanosoma cruzi express lysosomal-like acid hydrolases on their cell surface as well as within their lysosome(s) and it is possible that cell surface hydrolases may play a role in the host-parasite interactions. Presently, little is known about the biological roles of the acid hydrolases of © Oxford University Press
The a-mannosidase was purified from the epimastigote form of T.cruzi using 4-methylumbelliferyl-a-D-mannopyranoside as substrate and the purification is summarized in Table I. As previously reported (Avila et al., 1979), the activity of a-mannosidase in crude homogenates is the highest of the acid glycosidases tested. In addition, a-mannosidase activity varied with the form of the parasite and is only slightly less than that of acid phosphatase. These findings are summarized in Table II. Extraction of a-mannosidase Epimastigotes (4-5 ml of packed cells) were harvested by centrifugation, washed three times, resuspended in 20 ml of 10 mM phosphate (pH 7.0) containing 0.4 M NaCl, 0.1% Triton X-100 and the following protease inhibitors: aprotinin (2 U/ml), leupeptin (10/tg/ml), pepstatin (5 fig/ml) and phenylmethyl sulphonyl fluoride (PMSF) (2 mM). After three freezethaw cycles, the suspension was homogenized by sonication. The mixture was centrifuged at 20 000 g for 30 min. The lowspeed supernatant was saved at 4°C and the pellet extracted two additional times by the method described above using 7.5 ml of extraction buffer. The low-speed supernatants were combined and contained - 9 0 % of the activity. The combined low-speed supernatants were diluted to 50 ml and dialysed overnight against 10 mM phosphate (pH 7.2), and then centrifuged at 150 000 g for 90 min. 563
Downloaded from http://glycob.oxfordjournals.org/ at McMaster University Library on August 7, 2015
In this report we describe the first purification and characterization of the acid a-mannosidase from the human parasite Trypanosoma cruzi. The purified enzyme exhibited a native mol. wt of 240 000 Da and is apparently composed of four identical subunits of mol. wt 58 000 Da. Each of the four subunits contains one N-linked high-mannose-type oligosaccharide. The a-mannosidase exhibited a pH optimum of 3.5 and a pi of 5.9. This low pH optimum and the ability of swainsonine to inhibit its activity suggest that the a-mannosidase is a lysosomal enzyme. Antibodies against the T.cruzi enzyme did not react with mammalian lysosomal a-mannosidase and, conversely, antibody against a rat lysosomal a-mannosidase did not react with the T. cruzi enzyme. Thus, the T. cruzi enzyme appears to be distinct from its mammalian counterpart.
T. cruzi. Reports of purified acid hydrolases from parasites are rare. Three acid phosphatases have been purified to homogeneity from Leishmania donovani (Remaley et al., 1985). Acid phosphatase has only been demonstrated, but not purified or characterized in T.cruzi (Letelier et al., 1985, 1986; Nagakura et al., 1985). This activity has been shown to exist both intracellularly as well as on the plasma membrane. In addition, this cell surface-associated activity was shown to be altered with the morphological transformation in T. cruzi (Nagakura et al., 1985). Avila et al. (1979) reported the presence of 12 acid hydrolases, including an a-mannosidase, four near neutral hydrolases and alkaline phosphatase in T. cruzi. No fractionation of these enzymes was attempted and only the pH optimum for each was reported. More recently, Bontempi et al. (1989) reported that the a-mannosidase required detergent (1.5 mg/ml digitonin) for complete solubilization and suggest that it is lysosomal, but no purification was reported. Thus, there has been no reported purification of T.cruzi acid glycosidases. In this report, we have identified the acid a-mannosidase activity, purified and characterized the a-mannosidase, and demonstrate that it is immunologically distinct from the corresponding host enzyme. Utilizing antibodies against the purified a-mannosidase, it will now be possible to investigate the biosynthetic, processing and sorting pathway for this enzyme in particular and other intracellular hydrolases of T.cruzi in general.
P.M.Swanson et aJ.
Table I. Summary of purification of a-mannosidase Step
Total units'
Total protein (mg)
Specific activity (U/mg)
Yield (%)
Fold purification
Crude homogenate DEAE-cellulose Sepharose 4B IEF S-200 Con A
115 80 72 67 59 49
130 40 22 3 1.7 0.05
0.88 1 98 3.20 22 00 39 00 980.00
_ 69 62 58 51 42
— 2.25 3.63 25.00 44.00 1113.6
'Units are /imol 4-methylumbelliferyl-a-D-mannopyranoside hydrolysed per hour at 37°C and pH 3.5.
ed sharply at pH 5.9. The fractions which contained the a-mannosidase activity were pooled and made 0.3 M in NaCl to displace the ampholytes, and concentrated to 2 ml by vacuum dialysis. Sephacryl S-200 gel filtration The pooled, concentrated a-mannosidase activity from the preparative IEF step was applied to a Sephacryl S-200 column (1.5 x 90 cm) equilibrated with 10 mM phosphate (pH 7.2). The a-mannosidase eluted at the void volume (Figure 3). The fractions (37-49) containing the activity were pooled. Concanavalin A—agarose affinity purification
Table n . Acid hydrolase content of T.cruzi at different stages
Acid phosphatase /3-Hexosaminidase £-Fucosidase a-Glucosidase /J-Glucosidase a-Mannosidase ^-Mannosidase /3-Glucuronidase a-Galactosidase /3-Galactosidase
Trypomastigote
Epimastigote
Amastigote
1.03 0 03 0.04
