Isolation and purification of a neutral [alpha](1,2)-mannosidase from Trypanosoma cruzi

Pedro Bonay1 and Manuel Fresno

Centro de Biologia Molecular "Severo Ochoa," Universidad Autonoma de Madrid, Cantoblanco, Madrid 28049, Spain

Received on May 14, 1998; revised on October 1, 1998; accepted on October 11, 1998

Trypanosoma cruzi is an obligatory intracellular protozoan parasite that causes Chagas' disease in humans. Although a fair amount is known about the biochemistry of certain trypanosomes, very little is known about the enzymic complement of synthesis and processing of glycoproteins and/or functions of the subcellular organelles in this parasite. There have been very few reports on the presence of acid and neutral hydrolases in Trypanosoma cruzi. Here we report the first purification and characterization of a neutral mannosidase from the epimastigote stage of Trypanosoma cruzi. The neutral mannosidase was purified nearly 800-fold with an 8% recovery to apparent homogeneity from a CHAPS extract of epimastigotes by the following procedures: (1) metal affinity chromatography on Co+2-Sepharose, (2) anion exchange, and (3) hydroxylapatite. The purified enzyme has a native molecular weight of 150-160 kDa and is apparently composed of two subunits of 76 kDa. The purified enzyme exhibits a broad pH profile with a maximum at pH 5.9-6.3. It is inhibited by swainsonine (Ki, 0.1 µM), d-mannono-[part]-lactam (Ki, 20 µM), kifunensine (Ki, 60µM) but not significantly by deoxymannojirimycin. The enzyme is activated by Co2+ and Ni2+ and strongly inhibited by EDTA and Fe2+.The purified enzyme is active against p-nitrophenyl [alpha]-d-mannoside (km = 87 µM). High-mannose Man9GlcNAc substrate was hydrolyzed by the purified enzyme to Man7GlcNAc at pH 6.1. The purified enzyme does not show activity against [alpha]1,3- or [alpha]1,6-linked mannose residues. Antibodies against the recently purified lysosomal [alpha]-mannosidase from T.cruzi did not react with the neutral mannosidase reported here.

Key words: T.cruzi/Chagas' disease/mannosidase/purification

Introduction

The intracellular protozoan parasite, Trypanosoma cruzi is the causative agent of American Trypanosomiasis or Chagas' disease, a chronic and debilitating multisystemic disorder that affects about 25 million people in Latin America (Organization, 1990). From a clinical point of view, the disease is characterized by an acute phase with high parasitemia and strong immunosuppresion (Brener, 1980; Beltz and Kierszenbaum, 1987), followed by a chronic phase with an autoimmune pathology (Hudson, 1985; Kierszenbaum, 1986).

The parasite life cycle can be divided into four stages (Brener, 1973; de-Souza, 1984); the parasite is taken in the bloodmeal of the insect as trypomastigote, which differentiates into the epimastigote that multiplies extracellularly in the midgut of reduviid insects. In the hind gut, epimastigotes transform into infective nondividing metacyclic trypomastigotes, which are released in the feces. Metacyclic trypomastigotes are able to invade a wide variety of host mammalian cells, phagocytic and nonphagocytic (de-Souza, 1984; Pereira, 1990). Once inside the cells, the metacyclic forms escape from endocytic vacuoles to the cytoplasm where they transform into amastigotes, which multiply intracellularly. Upon rupture of host cells, they differentiate into trypomastigotes that circulate in the blood until they encounter appropriate target cells and then go through another intracellular cycle or are taken up by the insect again. This complex developmental cycle requires the mutual recognition between the parasite and the host cell prior to adhesion as an essential requisite for parasite penetration (Piras et al., 1983; Pereira, 1990; Ortega-Barria and Pereira, 1991). There is relevant evidence pointing to the surface glycans in both the parasite and the host cell as the prospective "ligands" for target cell receptors (Villalta and Kierszenbaum, 1983, 1985a,b, 1987; Bonay and Fresno, 1995). Although, some aspects of the glycobiology of Trypanosoma cruzi are known, there are no data on the enzymes involved in the biosynthesis and modification of Trypanosoma cruzi glycoproteins. With the sole exception of F9 teratocarcinoma cells (Romero and Herscovics, 1986), T.cruzi and other trypanosomatid protozoa are the only eukaryotic cells known so far to transfer in vivo unglucosylated oligosaccharides (Man9GlcNAc2; Parodi, 1993a,b). Experiments performed with intact cells showed that high mannose oligosaccharides present in mature processed proteins have the compositions Man5-9GlcNAc2 (Parodi et al., 1983), and the structures of the main isomers of Man7-8GlcNAc2 were identical with those found in mammalian cells, suggesting that the endoplasmic reticulum and Golgi apparatus of Trypanosoma cruzi may contain [alpha](1-2) mannosidases, although no such enzyme has yet been characterized. While some cell surface hydrolases described previously, such as the trans-sialidase (Schenkman et al., 1991, 1994), play a role in the host-parasite interactions, very little is known about other acid or neutral glyco-hydrolases. Avila et al. (Avila et al., 1979) reported the presence of 12 acid hydrolases, including a [alpha]-mannosidase, alkaline phosphatase, and four neutral hydrolases. Recently, the purification and partial characterization of an acid mannosidase from Trypanosoma cruzi has been reported (Swanson et al., 1992), and Xavier et al. (Xavier et al., 1994) presented evidence for the presence in microsomes from Trypanosoma cruzi epimastigotes of broad specificity ([alpha]1,2, [alpha]1,3, [alpha]1,6) mannosidase(s) allegedly not involved in protein-linked oligosaccharide processing based on the in vivo effect of glycoprotein processing enzymes inhibitors like swainsonine and deoxymannojirimycin compared with cell-free assays. Here we report the identification and biochemical characterization of a neutral [alpha](1-2) mannosidase from Trypanosoma cruzi epimastigotes able to act on Man9GlcNAc2 to form Man7GlcNAc2.

Results

Purification of neutral mannosidase

The neutral mannosidase was purified from the epimastigote stage of Trypanosoma cruzi by serial column chromatography using pNPM as substrate.

We decided to use cobalt-chelating chromatography due to the activating effect of cobalt ions on the T.cruzi mannosidase (data not shown), and also based on several reports showing similar effects (Mathur and Balasubramanian, 1984; Bonay and Hughes, 1991; Grard et al., 1994).

The cell extract from 4-5 ml packed cells (15-18 ml) was applied to a column (1.5 × 20cm) of cobalt-Sepharose (chelating-Sepharose preloaded with CoCl2) equilibrated in buffer MEAC as described in Materials and methods. The active imidazol-eluted fractions were pooled as shown in Figure 1a. This chromatographic step allows the separation of at least two forms of mannosidase. An unbound form summing about 60-70% of total pNPM [alpha]-mannosidase activity and the imidazol-eluted fraction (Fraction C) that contains the remaining [alpha] mannosidase activity with an enrichment relative to the homogenate of 5- to 7-fold (Table I). While the two fractions are active against pNPM as substrate, their sensitivity to some effectors and reactivity toward oligosaccharide substrates were different (see below). The mannosidase present on the unbound fraction is currently under biochemical characterization (Bonay and Fresno, unpublished observations). The imidazol-eluted fraction was further purified by several chromatographic steps including anion exchange on Q-Sepharose, Hydroxylapatite chromatography and gel-filtration chromatography. The purification procedure was quite reproducible and the neutral [alpha]-mannosidase was purified at least 800 fold over the crude extract with a yield of ~8%. A summary of a representative purification is shown in Table I. The enzymatic activity of the purified mannosidase against pNPM was quite stable (50% activity loss over 6 months at 4°C but only 10% when stored at -20°C or -70°C). However the activity against Man9-7GlcNAc2 oligosaccharide was drastically reduced under the same conditions unless Co+2 ions and glycerol up to 10% were present.


Figure 1. Elution profiles of the column chromatographic steps during the purification of the neutral [alpha]-mannosidase from T.cruzi. (A) Metal affinity chromatography. The soluble extract of T.cruzi epimastigotes was applied to a Co-Sepharose column (1.5 × 20cm). After washing the column with buffer MEAC, it was eluted with elution buffer at the fraction indicated by the arrow. Fractions of 4 ml were collected. (B) Anion exchange chromatography. The pooled active fractions from the MEAC column (fraction C, Table I) were adjusted to pH 8.3 and applied to a Q-Sepharose (1.5 × 15 cm) equilibrated in buffer Q. After washing with the same buffer until A280 was below 0.05 the column was eluted with a linear gradient of NaCl 50-500 mM in buffer Q (dotted line) collecting fractions of 2.5 ml. (C) Hydroxylapatite chromatography. The active [alpha]-mannosidase-containing fractions from the Q-Sepharose (Fraction Q, Table I) were directly applied to a HTP column (0.75 × 10 cm) equilibrated in buffer HTP. After washing the column with buffer HTP (15 ml), the column was eluted with a linear gradient of phosphate, 10-500 mM in buffer HTP (dotted line). Fractions of 1 ml were collected. All collected fractions were analyzed for protein by BCA (A590; open circles) and for neutral [alpha]-mannosidase activity against pNPM (A405; solid circles).

Biochemical Properties of the purified mannosidase

The fraction G (Table I) gave a single protein band upon native electrophoresis in a 7.5% polyacrylamide gel (Figure 2a).

An unstained gel run side by side was used to determine whether the single protein band exhibited [alpha]-mannosidase activity by cutting the gel into equal slices. Each slice was homogenized in extraction buffer (100 µl) and assayed for [alpha]-mannosidase activity against Man9GlcNAc2. As shown in Figure 2c, the activity comigrated with the protein band. An identical track of the native gel was incubated at 37°C with fluorogenic substrate 4-methylumbelliferyl [alpha]-d-mannopyranoside, showing a single fluorescent band comigrating with the protein band (Figure 2b). Analysis of the purified mannosidase by SDS-PAGE 10% polyacrylamide gel under reducing conditions showed a single band migrating in a position corresponding to about 70 kDa (Figure 2d). Similar values were obtained when 7.5% or 12% polyacrylamide gel were used (data not shown). The apparent molecular weight of the native enzyme was determined by gel filtration on a calibrated gel filtration column (two Superose 12 HR 10/30) run under nondenaturing conditions (Figure 3). Peak fractions of [alpha]-mannosidase activity eluted with an apparent molecular mass of 150-160 kDa (Figure 3, inset), suggesting a dimeric structure for the native enzyme composed of two identical or very similar subunits.

Table I. Summary of purification of neutral [alpha]-mannosidase from Trypanosoma cruzi
Fraction Total protein Total activity Specific activity Recovery Purification
  (mg) (Units) (Units/mg) (%) (-fold)
Soluble extract 645 875 1.35 100 1
Co2+-Sepharose (Fraction C) 34.8 306 8.79 35 6.5
Q-Sepharose FF (Fraction Q) 2.49 175 70.20 20 52
Hydroxylapatite (Fraction H) 0.27 122 441.45 14 327
Gel filtration (Fraction G) 0.064 70 1093.75 8 809
The soluble fraction was prepared from 5-6 ml of packed epimastigotes of T.cruzi collected at mid-log growth phase. The [alpha]-mannosidase activity from the different purification steps was assayed against pNPM as described in Material and methods. One unit of enzyme is defined as the amount of enzyme catalyzing the release of 1 nmol p-nitrophenol/min under the conditions of the standard assay described.

The pH optimum for the neutral [alpha]-mannosidase was 5.9-6.3 with half-maximal activity at pH 7.5 and pH 4. The activity decreased more rapidly on the basic side of the pH profile (Figure 4a) clearly distinct to the acid mannosidase described previously (Swanson et al., 1992). The unbound fraction showed a pH profile similar to the bound (purified) mannosidase (data not shown). Stability assays in which aliquots of purified enzyme were incubated at different pH in the absence of substrate and then assayed for activity after readjustment the pH to the optimum value confirmed the greater stability of the enzyme to more acidic pH values (Figure 4b).


Figure 2. Electrophoretic analysis of the purified [alpha]-mannosidase. (A) PAGE of purified mannosidase (4 µg) under nondenaturing conditions in a 7% gel and Coomassie blue staining. (B) An identical track run side-by-side was cut in 1 mm slices, and each slice was suspended in buffer 6.9 with an homogenizer and assayed for mannosidase activity using [3H] Man9GlcNAc as substrate during 4 h at pH 6.1. (C) Another track was equilibrated after the electrophoresis in buffer 6.1 for 10 min with two buffer exchanges after which it was overlaid with substrate solution (5 mM 4-methylumbelliferyl-[alpha]-d-mannopyranoside in buffer 6.1) and incubated in a humid chamber at 37°C for 2 h before photographing the gel under UV light. (D) SDS-PAGE of purified [alpha]-mannosidase under reducing conditions in a 12% gel and silver stained. Molecular weight markers (Bio-Rad prestained MW markers) are indicated by the numbers at left. 1, Myosin, 203 kDa; 2, [beta]-galactosidase, 118 kDa; 3, BSA, 86 kDa; 4, OVA, 51.6 kDa; 5, carbonic anhydrase, 34.1 kDa; 6, soybean trypsin inhibitor, 29 kDa.


Figure 3. Molecular size determination of the neutral [alpha]-mannosidase. A sample of the purified [alpha]-mannosidase (Fraction G, 80 µg, Table I) was rechromatographed on a calibrated gel filtration system (two coupled Superose 12 HR 10/30 columns) equilibrated in buffer G at 200 µl/min. Fractions of 300 µl were collected and assayed for mannosidase activity as described and for radioactivity by scintillation counting. The molecular weight standards used were as follows. 1, [beta]-amylase, 205 kDa; 2, alcohol dehydrogenase, yeast, 150 kDa; 3, BSA, 66 kDa; 4, carbonic anhydrase, 29 kDa; 5, cyt c, 12,4 kDa. Vo, Blue dextran elution volume.


Figure 4. Effect of pH on [alpha]-mannosidase activity of T.cruzi. [alpha]-Mannosidase activity against pNPM was measured at each indicated pH for 2 h at 37°C as described in Materials and methods. Aliquots of purified mannosidase (Fraction G, 2 µg) were incubated in the absence of substrate at the stated pH values for 30 min at 37°C, the pH was then adjusted to pH 6.1 and supplemented with Co2+ and the reaction carried out against pNPM for 4 h at 37°C.

The purified [alpha]-mannosidase activity against pNPM was strongly inhibited by 1 mM Fe+2 (80% inhibition, Figure 5) whereas DTT caused a 50% inhibition. Other metal ions like Zn2+, Cu2+, Co2+, Ni2+, and Fe3+ acted like activators or stabilizers for the enzyme activity (Figure 5). It was found that purified enzyme preparations containing Co2+ or Fe3+ could be stored longer at either 4°C or -20°C without appreciable loss of activity compared to similar preparations stored in the absence of the cations. Other divalent cations like Ca2+, Mg2+, Mn2+, or Li2+ had no effect on the mannosidase activity and EDTA only had a marginal effect (10-15% inhibition, Figure 5). By contrast the unbound [alpha]-mannosidase activity was insensitive to the effect of ions Fe3+ and was strongly inhibited by Cu2+ (Figure 5). In general it was noticed an inhibitory or neutral effect of the metal ions tested (Zn2+, Co2+, Ni2+) toward the unbound fraction compared to the stimulatory effect seen in the purified mannosidase. Those results clearly demonstrate that the two fractions are distinct enzyme species and not only different forms of the same enzyme.


Figure 5. Effectors of neutral [alpha]-mannosidase activity. Each compound was incubated with either purified [alpha]-mannosidase (3 µg, white bars) or with unbound-fraction (5 µg, solid bars) for 1 h at room temperature in buffer 6.1. Mannosidase activity was assayed against pNPM as described in Materials and methods. Activity is expressed as percentage relative to the control value (no effector) and are the means of at least three independent determinations from two different mannosidase preparations.

Swainsonine had a very strong inhibitory effect on the purified [alpha]-mannosidase activity towards pNPM (Figure 6a), with 90% inhibition at 2 µM (Ki < 0.1µM), similar to the values reported for the acid mannosidase of T.cruzi (Swanson et al., 1992) and other lysosomal or neutral Golgi [alpha]-mannosidase II or higher eukaryotes (Daniel et al., 1994; Moremen et al., 1994). Another alkaloid, kifunensine, was found to be also a potent inhibitor (Ki = 60 µM) of the purified T.cruzi neutral [alpha]-mannosidase. Kifunensine has been reported to be a potent inhibitor of Golgi mannosidase I (Elbein et al., 1990, 1991) but not ER/soluble mannosidases (Weng and Spiro, 1996). The mannose analogue, 1-deoxymannojirimycin, also a potent inhibitor of Golgi mannosidase I (Bischoff and Kornfeld, 1984; Bischoff et al., 1986), had no significant effect on the purified [alpha]-mannosidase toward pNPM (50% inhibition at 0.8-1 mM). Similarly it has no effect on lysosomal mannosidase or Golgi mannosidase II. A novel inhibitor, mannono-[part]-lactam, was also tested against the purified [alpha]-mannosidase activity, and proved to be a better inhibitor than kifunensine but weaker than swainsonine (50% inhibition at 2-3 µM, Figure 6). As far as we know, there are no reports on the use of this particular inhibitor to distinguish processing mannosidases. Mannostatin was also tested and shown to be a poor inhibitor like deoxymannojirimycin. The pattern of inhibition was almost identical regardless of the substrate used for the analysis as shown in Figure 6 (A: pNPM; B: [H3]-Man9GlcNAc2 substrate). Those results, taken together, indicate the enzyme reported here is distinct to any previously described mannosidase (Daniel et al., 1994; Moremen et al., 1994).


Figure 6. Effect of swainsonine, 1-deoxymannojirimycin, kifunensine, mannono-[part]-lactam, and mannostatin on the neutral [alpha]-mannosidase activity of T.cruzi. Purified neutral [alpha]-mannosidase (5 µg, Fraction G) was preincubated at 37°C for 30 min in buffer 6.1 with the different inhibitors at the final concentrations indicated. Either pNPM at 4 mM final concentration (A) or [3H] Man9GlcNAc (8000 c.p.m.). (B) were added and the release of p-nitrophenol or unbound [3H] mannose was assayed after incubation at 37°C for 4 h as described in Materials and methods. The results are shown as percentage of control experiments without inhibitor. Symbols are as follows: solid circles, 1-deoxymannojirimycin; open circles, swainsonine; open squares, kifunensine; solid triangles, mannono-[part]-lactam; and solid squares, mannostatin.

The purified enzyme was subjected to N-terminal amino acid sequencing. It is noteworthy to mention that in each sequencing cycle just one single amino acid peak was released, thus further supporting the data on purity of the enzyme shown in Figure 2.

Table II. Substrate specificity of the purified neutral [alpha]-mannosidase of T.cruzi
Substrate Activity (unit) N° of residues cleaved
[H3]-Man9GlcNAc 20.7 ± 1.8 1.88
[H3]-Man8GlcNAc mixturea 11.2 ± 0.9 0.93
[H3]-Man8GlcNAc isomer D1 D3 12.5 ± 1.9 1.03
[H3]-Man8GlcNAc isomer D1 D2 2.2 ± 0.7 0.18
[H3]-Man7GlcNAc mixtureb 2.6 ± 0.5 0.18
[H3]-Man6GlcNAc 2.1 ± 1.8 0.12
[H3]-Man5GlcNAcd 2.2 ± 0.4 0.11
GlcNAc-[H3]-Man5GlcNAcd 1.1 ± 0.04 0.05
[H3]-ManGlcNAcc 0.09 ± 0.04 -
Purified [alpha]-mannosidase (5 µg; Fraction G, Table I) were incubated in buffer 6.9 supplemented with 0.2mM CoCl2 at 37°C during 8 h with ~10,000 c.p.m. of the high-mannose and hybrid oligosaccharides [3H]-mannose-labeled Man9-5GlcNAc and the reaction products separated and quantitated by paper chromatography as described in Material and methods. One activity unit is defined as the release of 1% of the total radioactivity. D1 refers to the terminal [alpha]1,2 mannose on the inner [alpha]1,3 branch. D2 refers to the terminal [alpha]1,2 mannose on the [alpha]1,3 branch of the inner [alpha]1,6 branch. D3 refers to the terminal [alpha]1,2 mannose on the [alpha]1,6 branch of the inner [alpha]1,6 branch.
aThe proportion of Man8GlcNAc isomers is: 28% D1 D2; 72% D1 D3.
bThe proportion of Man7GlcNAc isomers is: 53% D1 and D3; 47% D2.
cThis substrate only contains a [beta]1,4-mannose cleavable.
dThose substrates only contain [alpha]1,3- and [alpha]1,6-mannose residues cleavable.

The partial N-terminal sequence from the purified enzyme was MAPPELEPLT, which shows some similarities to the mannosidase Type I family (Daniel et al., 1994; Moremen et al., 1994), but insufficient sequence has been defined to allow a family designation.

Kinetics and substrate specificity

Under standard assay conditions the rate of hydrolysis of pNPM was directly proportional to the amount of purified enzyme (Fraction G, Table I) over a range of 0.6-10 µg. When 0.8 µg of enzyme was used, the reaction was linear with respect of time up to 4 h at 37°C and pH 6.1. The kinetic data could be fitted to a typical Michaelis-Menten plot (data not shown), and the apparent km for pNPM was calculated to be about 87 µM. This value is in the range reported for the Tris-eluted or Cobalt-bound form of the soluble [alpha]-mannosidase from rat liver (72 µM; Grard et al., 1994).


Figure 7. Kinetics of appearance of products of hydrolysis of [3H]-Man9GlcNAc by the purified mannosidase. (A) Purified neutral mannosidase (3 µg) was incubated with [3H]-Man9GlcNAc (600,000 c.p.m.) at pH 6.1. At the indicated times, aliquots were removed and subjected to paper chromatography on solvent A for 36 h. Radioactive components were located by eluting 1cm strips with water and counting in scintillation cocktail. The migration of [3H]-Man9-7GlcNAc standard oligosaccharides is indicated by the numbers at the top. The analysis of a reaction mixture heated at zero time is shown (open circles). (B) The peaks obtained from several experiments after paper chromatography of the reaction mixtures removed at different times were integrated and the percentage of total radioactivity present in individual peaks are plotted as a function of incubation time. Solid circles, [3H]-Man9GlcNAc; open circles, [3H]-Man8GlcNAc; solid squares, [3H]-Man7GlcNAc.

The purified enzyme was tested against [3H]-mannose-labeled oligosaccharides containing 5-9 mannose residues and the hybrid oligosaccharide GlcNAcMan5GlcNAc as well as the core glycan containing [beta]-linked mannose residue ManGlcNAc. As shown in Table II, the enzyme was able to remove mannose residues from Man8-9GlcNAc at similar rates and the end product in each case was an octasaccharide migrating at the position of Man7GlcNAc. No significant mannose removal from Man7-5GlcNAc substrate was observed even after new additions of fresh enzyme during long-term incubations. The specificity of the enzyme was shown when using two different isomers of Man8GlcNAc (D1D3 and D1D2). As shown in Table II, the enzyme is able to remove just one mannose residue from the isomer D1D3 but no significant mannose release was observed from the isomer D1D2, thus providing further insight on the substrate specificity of this novel mannosidase. Furthermore, the purified mannosidase is not active on the product of the GlcNAc-transferase I, GlcNAcMan5GlcNAc (Table II), clearly distinguishing this enzyme from the Golgi mannosidase(s) II described so far (Moremen et al., 1994). Moreover, the enzyme does not exhibit [beta]-mannosidase activity when assayed against ManGlcNAc (Table II) or p-nitrophenyl-[beta]-mannopyranoside (data not shown). Thus, the activity of this enzyme on oligosaccharide substrates is in marked contrast with the properties reported for the acid mannosidase of T.cruzi (Swanson et al., 1992). To further confirm the substrate specificity, samples of purified neutral mannosidase were incubated with [H3]-Man9GlcNAc at 37°C and pH 6.1. After various times of incubation up to 12 h the products of hydrolysis were separated by paper chromatography. As shown in Figure 7A, the purified neutral mannosidase is able to sequentially hydrolyze [H3]-Man9GlcNAc to [H3]-Man7GlcNAc via [H3]-Man8GlcNAc as an intermediate product with no further breakdown. The data from several similar experiments were quantified by integration of each peak separated by paper chromatography. The results shown in Figure 7B confirm the hydrolysis of [H3]-Man9GlcNAc first to [H3]-Man8GlcNAc, which slightly accumulates by 4 h of hydrolysis before further breakdown to [H3]-Man7GlcNAc that remains as the end-product of hydrolysis.


Figure 8. Order of mannose removal from Man9GlcNAc. [3H]Man9GlcNAc (8 × 105 c.p.m.) was incubated with the purified neutral [alpha]-mannosidase (3 µg) and the reaction products separated by paper chromatography on solvent A. The peaks of radioactivity identified by comigrating with standards were eluted and treated as described in Materials and methods. Acetolysis products were separated by paper chromatography in solvent A for 4 h. The numbers indicate the position of standards as follows. 1, Man4GlcNAcol; 2, mannotriose; 3, mannobiose; 4, mannose. The structures compatible with the acetolysis fragmentation are shown to the right.

The next step in the characterization was to stablish the order of mannose removal from Man9GlcNAc by the purified mannosidase in order to structurally identify intermediates and to further correlate the data with substrate specificity (Table II). Aliquots of a large reaction mixture were taken at different time points and the products separated by paper chromatography. Each oligosaccharide (ranging from Man9GlcNAc to Man7GlcNAc) was eluted from the paper, reduced, and subjected to acetolysis, a reaction that selectively cleaves [alpha]1-6 linkages. The acetolysis products were separated by paper chromatography and their composition deduced from the migration of standards (Figure 8). The acetolysis products (Man4GlcNAcol and mannobiose in a proportion 1:2, where GlcNAcol is N-acetylglucosaminitol) of the first intermediate product generated by the neutral [alpha]-mannosidase on Man9GlcNAc, are consistent with the removal of a single [alpha]1-2 mannose residue in the middle branch giving the Man8GlcNAc isomer D1D3 shown in Figure 8b, which as shown in Table II is a preferred substrate for the enzyme. The acetolysis pattern of the end product of the digestion shows Man4GlcNAcol, mannobiose, and mannose in equal proportions. This pattern is consistent with either the release of the Man[alpha]1-3 next to the previously released or the Man[alpha]1-2 in the [alpha]1-6 branch (Figure 8c).

The fact that the purified enzyme is not active on [alpha]1-3 or [alpha]1-6-linked mannose residues as shown in Table II clearly indicates the latter structure as the one produced by the neutral [alpha]-mannosidase. Those results taken together suggest a very specific and ordered mannose cleavage by the newly described T.cruzi neutral mannosidase.

Discussion

Here, we report the purification and kinetic and biochemical characterization of a neutral [alpha]-mannosidase from the protozoan parasite Trypanosoma cruzi that in vitro is able to act on high-mannose oligosaccharides.

Several [alpha]-mannosidase have been described in mammalian cells involved in both the biosynthesis and catabolism of N-linked glycans and have been classified into two groups first according to their inhibition by mannose analogs and biochemical properties (like size, cation requirements, etc.; Daniel et al., 1994) and on the basis of protein sequence homologies (Moremen et al., 1994). Thus, the first group or Class I mannosidase is comprised of [alpha]1-2 mannosidases from mammals and yeast with encoded polypeptides of 65-75 kDa. Most of them are Ca2+-dependent enzymes located in the ER and/or Golgi which are inhibited by substrate mimics like kifunensine and deoxymannojirimycin. Their specificity is restricted to [alpha](1-2) linkages, and they are mostly inactive towards artificial substrates like aryl mannosides. Those in Class II are more heterogeneous and include Golgi mannosidase II, lysosomal mannosidases, and the mammalian cytosolic/ER [alpha]-mannosidase and some previously unclassified mannosidases from rat brain (Tulsiani and Touster, 1985), sperm (Tulsiani et al., 1989), and liver microsomes (Bonay and Hughes, 1991). These enzymes are 105-140 kDa, have a broader substrate specificity (able to act on [alpha]1-2/1-3/1-6 linked mannose residues), and also are active toward aryl mannosides. They are inhibited to some extent by the alkaloid swainsonine and other transition state analogues resembling the conformation of the mannosyl cation (Winchester and Fleet, 1992; Daniel et al., 1994). Notwithstanding their varied intracellular localization and function, they share some degree of sequence homology (Moremen et al., 1994).

Trypanosoma cruzi has considerable medical importance as the causative agent of Chagas' disease, a chronic and debilitating disorder that affects 20 million people in Latin America. Trypanosomatid protozoa are characterized by several biochemical features unique to them as are the miniexon addition, the presence of an unusual mitochondrial DNA (kinetoplast), and the existence of a glycosome among others. Regarding N-glycosylation, Trypanosomatids also exhibit several rather unique features like the presence of shorter polyprenols (dolichol) than the mammalian, fungal, or plant counterparts (i.e., T.cruzi dolichol monophosphate derivatives is 10-13 isoprene units; Parodi and Quesada-Allue, 1982). Together with the F9 teratocarcinoma cells (Romero and Herscovics, 1986), they are the only wild-type eukaryotic cells known to transfer to protein in vivo unglucosylated oligosaccharides, being Man9GlcNAc2 in Trypanosoma cruzi epimastigotes and intracellular amastigotes but Man7GlcNAc2 and Man9GlcNAc2 in infective trypomastigotes (Engel and Parodi, 1985; Doyle et al., 1986). The synthesis of unglucosylated oligosaccharide is due to a defective formation of dolichol-P-Glc (Parodi and Quesada-Allue, 1982; de la Canal and Parodi, 1987). Interestingly, the oligosaccharyltransferase from trypanosomatids is able to transfer equally Man6-9GlcNAc2 and Glc1-3Man9GlcNAc2 (Bosch et al., 1988a,b). Furthermore, glycan analysis performed in intact cells has shown that Trypanosoma cruzi presumably generates Glc1Man7-9GlcNAc2 (by transient reglucosylation) as mammalian cells in the endoplasmic reticulum, and that the isomer composition of the high-mannose oligosaccharides found in mature glycoproteins are identical to those found in other eukaryotic cells (Parodi et al., 1983). Regarding the next steps in the synthesis and processing of N-glycans (mannose trimming and complex chains formation) in Trypanosoma cruzi, it is known that complex-type oligosaccharides from Trypanosoma cruzi have in addition to N-acetylglucosamine and mannose, [beta]1-4 and [alpha]1-3-linked galactose, fucose, and sialic acid residues (Couto et al., 1987, 1990), it has been assumed that this process occurs similarly to mammalian cells (Parodi, 1993a,b), but there are no studies on the enzymes involved or their subcellular localization.

There are several reports on acid hydrolases from Trypanosoma cruzi. Thus, Avila et al. (Avila et al., 1979) identified 12 acid hydrolases including an [alpha]-mannosidase that seems to require detergent for complete solubilization (Bontempi et al., 1989), suggesting that it is a lysosomal enzyme; and Swanson et al. (Swanson et al., 1992) reported the purification of a soluble acid [alpha]-mannosidase from T.cruzi epimastigotes distinct from the host enzyme. More recently, Xavier et al. (Xavier et al., 1994) presented evidence for membrane-bound broad specificity [alpha]-mannosidase(s) in T.cruzi microsomes that were inhibited in vitro by swainsonine, exhibiting a neutral pH optimum, not activated by calcium nor inhibited by EGTA. However, there was some discrepancy between their results of inhibition in the in vitro and in vivo assays.

The enzyme reported in this work is clearly distinct from the acid [alpha]-mannosidase reported by Swanson et al. (1992). It is a dimer with a subunit molecular weight of about 70 kDa compared with the tetrameric structure (subunit of 58 kDa) of the acid mannosidase. There are more differences between these two enzymes including the pH optimum and the substrate specificity (the acid mannosidase is unable to act on high-mannose oligosaccharides). In addition, antibodies generated against the acid mannosidase do not cross-react with the neutral mannosidase (T. Oeltmann, Vanderbilt University School of Medicine, Nashville, TN, personal communication).

Despite sharing with Class I mannosidases the subunit molecular weight (57-73 kDa) and the specificity for [alpha]1-2 linked mannose residues, the neutral mannosidase can be distinguished by the fact that it is able to act on aryl-mannosides and its resistance to deoxymannojirimycin (potent inhibitor of all Class I mannosidases described so far). Further distinction from other [alpha]1-2 mannosidases like the rat (Tabas and Kornfeld, 1979; Tulsiani and Touster, 1988; Velasco et al., 1993) and mouse (Herscovics et al., 1994; Lal et al., 1994) Golgi mannosidase IA and IB is inferred from the high susceptibility to the alkaloid swainsonine and the relative poor effect of EDTA.

The neutral T.cruzi [alpha]-mannosidase can also be distinguished from the Golgi [alpha]-mannosidase II by its inability to act on the hybrid oligosaccharide GlcNAcMan5GlcNAc2 (Table II); this fact also distinguishes it from the broad specificity [alpha]-mannosidases described in rat brain (Tulsiani and Touster, 1985) and sperm (Tulsiani et al., 1989). The immunological data suggest strongly that another broad specificity [alpha]-mannosidase, described in rat microsomes (Bonay and Hughes, 1991), is clearly distinct from the [alpha]-mannosidase described here, besides the different effects of swainsonine on the two enzymes.(P. Bonay and M. Fresno, unpublished observations). The distinctive feature of the neutral [alpha]-mannosidase from T.cruzi is its substrate specificity, quite similar to the rat ER-mannosidase I (Bischoff and Kornfeld, 1983; Weng and Spiro, 1993, 1996) and the strict order of mannose removal (Figure 8) generating the isomer B of Man8GlcNAc2. The enzyme described here shares some other properties with that enzyme, like the sensitivity to kifunensine and resistance to deoxymannojirimycin; however, it can distinguished by its resistance to swainsonine (Daniel et al., 1994). Finally, it should be emphasized that the preliminary immunological data gathered by the use of antibodies directed against the ER/cytosolic [alpha]-mannosidase and the Golgi [alpha]-mannosidase II clearly indicate the non-identity of those mannosidases to the enzyme reported here (P. Bonay and M. Fresno, unpublished observations).

Regarding the functional role of this newly described enzyme, it is important to mention that the substrate specificity and the order of mannose removal catalyzed by the neutral [alpha]-mannosidase described here is consistent with the two possible ways for generating the glucosylated-high mannose oligosaccharides (demannosylation of GlcMan9-8GlcNac2 or, alternatively, by reglucosylation of Man8-7GlcNAc2) present in mature T.cruzi glycoproteins (Parodi, 1993a,b). However, whether or not this fact reflects a functional role of the enzyme in the biosynthetic glycoprotein processing or by contrary may suggest a role in the regulated catabolism and/or demannosylation previous to remodeling in the endo/exocytic pathway remains to be elucidated. Experiments testing the effect both in vitro and in vivo of kifunensine and mannostatin on the oligosaccharide composition of mature Trypanosoma cruzi glycoproteins are currently being carried on and will provide insight necessary in order to assign a functional role to this enzyme in the N-glycan metabolism of Trypanosoma cruzi.

Further work is needed to determine the subcellular localization and biological function of this new [alpha]-mannosidase and to characterize the unbound mannosidase, as more precise localization will point to a functional role.

Materials and methods

Buffers

The following buffers were used. Extraction buffer: 25 mM HEPES, pH 7.5; 100 mM NaCl; 0.2% CHAPS; 2% glycerol; 1 mM CaCl2; and protease inhibitor cocktail. Buffer 6.1: 50 mM MES, pH 6.1; 10% glycerol; 0.05% CHAPS; 50 mM NaCl. Buffer 3.9: 50 mM sodium citrate, pH 3.9; 10% glycerol; 0.05% CHAPS; 50 mM NaCl. Buffer MEAC: 50 mM HEPES, pH 7.6.0; 250 mM NaCl; 10% glycerol; 1mM CaCl2; 0.1% CHAPS; protease inhibitor cocktail. Elution buffer: 100 mM imidazol, pH 6.5; 50 mM NaCl; 5% glycerol; 0.1% CHAPS; 0.1 mM CaCl2; protease inhibitor cocktail. Buffer Q: 50 mM Tris/HCl pH 8.3; 50 mM NaCl; 10% glycerol; 1 mM CaCl2; 0,5 mM CoCl2; 0,1 µM dithiothreitol; 0.1% CHAPS; protease inhibitor cocktail. Buffer HTP: 10 mM Na2HPO4, pH 7,2; 50 mM NaCl; 2% glycerol; 0.1 mM CoCl2; 0.1% CHAPS. Protease inhibitor cocktail contained pepstatin (0.5mg), leupeptin (5 mg), aprotinin (5 mg), E-64 (1 mg) in 10 ml H2O used at a 1:100 dilution and phenylmethylsulfonyl fluoride (86 mg/ml) in ethanol used at a 1:1000 dilution.

Antibodies

Antibodies raised against the ER-cytosolic mannosidase (Bischoff and Kornfeld, 1986) and Golgi [alpha]-mannosidase II (Moremen and Touster, 1985, 1986) were kindly provided by Dr. Joyce Bischoff (Whitehead Institute, Cambridge, MA) and Dr. Keley Moremen (CCRC, USA), respectively.

Parasites

The strain of T.cruzi used was originally obtained from a patient with Chagas' disease at the Instituto Nacional de la Salud, Madrid, Spain. It was cloned and named strain G (Alcina and Fresno, 1988). Epimastigotes were continuously cultured in liver infusion-tryptose (LIT) medium supplemented with 10% fetal calf serum (FCS) as described previously (Alcina and Fresno, 1988). Metacyclics trypomastigotes were obtained by metacyclogenesis induced by incubating late-log epimastigotes in TAU3AAG medium plus 0.035% sodium carbonate for 96 h (Goldenberg et al., 1987). Transformation of the parasite was assessed by resistance to complement lysis using horse serum. After 96 h in metacyclogenesis medium, the parasites were washed with PBS and resuspended to 1 × 108 per ml in PBS. Then, 500 µl samples of parasites were mixed with equal volumes of 70% (v/v) fresh serum and incubated 1 h at 37°C. The mixture was washed in PBS and the surviving parasites (metacyclics) counted in a hemocytometer.

Enzyme purification

The cell extract from 4-5 ml packed cells (15-18 ml) was applied to a column (1.5 × 20 cm) of cobalt-Sepharose (chelating-Sepharose preloaded with CoCl2) equilibrated in buffer MEAC. The column was washed (25 ml/h) with buffer MEAC until the OD280 was below 0.05 and then eluted with elution buffer (10 ml/h). Fractions of 4 ml were collected and analyzed for neutral [alpha]-mannosidase activity against pNPM. The active imidazol-eluted fractions were pooled as shown in Figure 1a. The imidazol-eluted pool (Fraction C, Table I) was adjusted to pH 8.3 with buffer Q and applied to a Q-Sepharose column (1.5 × 15 cm) equilibrated in buffer Q. Under such conditions 80-90% of the [alpha]-mannosidase activity binds to the column (Figure 1b). The column was washed with buffer Q (80 ml) collecting fractions of 2.5 ml at 25 ml/h and then eluted with a linear gradient of 0.05-0.5 M NaCl in buffer Q (Figure 1b). The active fractions against pNPM eluted at about 150-175 mM NaCl. Those fractions were pooled (Fraction Q, Table I) as shown in Figure 1b representing a purification of ~8-fold. Fraction Q was directly applied at 5 ml/h to a small HTP column (0.75 × 10 cm) equilibrated in buffer HTP. The column was washed until OD280 was zero and then eluted with a linear gradient of 10-500 mM phosphate in buffer HTP collecting fractions of 1 ml. The fractions eluted at 100-175 mM phosphate containing neutral [alpha]-mannosidase activity were combined as shown in Figure 1c and concentrated to 0.2-0.5 ml by using Centricon 30 microconcentrators (Fraction H, Table I). Fraction H was further purified by Gel filtration on a calibrated Superose system (two Superose 12 HR 10/30 columns) equilibrated in extraction buffer. Peak fractions (0.5 ml) containing [alpha]-mannosidase activity were pooled and concentrated to 0.5 ml (Fraction G, Table I).

Enzyme assays

Microplate mannosidase assays were setup using 2 mM p-nitro-[alpha]-d-mannopyranoside (pNPM) in either Buffer 6.1 or Buffer 3.9 in a total volume of 75 µl at 37°C. The reactions were stopped by the addition of Stop solution (133 mM glycine; 67 mM NaCl; 83 mM Na2CO3, pH 10.7; 150 µl) and the OD at 405 nm determined. Enzyme and substrate controls were run with each assay.

For the determination of oligosaccharide mannosidase activity with radiolabeled substrates, a modification of the assay described by Kang et al. (Kang et al., 1989) in microplates was used. Briefly, the assays were carried out in a final volume of 0.1 ml of sample and buffer (6.1 or 3.9), which contained [3H]-mannose labeled oligosaccharide (5 × 103 counts/min). After incubation at 37°C the reaction mixture was stopped by the addition of 20 µl of glacial acetic acid. 150 µl of concanavalin A-Sepharose in buffer A (75 µl packed gel) were added and the mixture incubated for 1 h at RT. The microplate was then spun at 750 × g for 5 min. An aliquot of supernatant (125 µl) was taken for scintillation counting. Appropriate controls were run in parallel.

Kinetic properties of the purified mannosidase

[alpha]-Mannosidase activity towards increasing concentrations of p-nitrophenyl [alpha]-d-mannopyranoside (0.005-10mM) were measured as described above.

Sequencing of purified mannosidase

The purified mannosidase was electroblotted onto Immobilon P, detected by Coomassie (Matsudaira, 1987) or fluorescamine (Vandekerckhove et al., 1985) staining, excised, and placed into the sequencer cartridge. Automated sequencing was done on a model 473A sequencer (Applied Biosystems, Foster City, CA).

Glycan analysis

BHK RicR -14 cells were metabolically labeled at equilibrium with [3H]-mannose (50 µCi/ml, Amersham, UK) in glucose-free DMEM-5% dialyzed FCS for 16 h as described previously (Monis et al., 1987; Bonay and Hughes, 1991). The isomer composition of the oligomers was determined as described before (Animasahaun and Hughes, 1989). Hybrid oligosaccharide GlcNAcMan5GlcNAc was prepared from the major monoantennary hybrid glycopeptide fraction of RicR 15 cells labeled with [3H]-mannose as described previously (Monis et al., 1987); (Hughes and Feeney, 1986; Bonay and Hughes, 1991). The structural analysis by acetolysis was carried out as previously described (Monis et al., 1987; Bonay and Hughes, 1991). Analysis of anionic oligosaccharides was carried out essentially as described previously (Varki and Kornfeld, 1983). The oligosaccharide [3H]ManGlcNAc was prepared by exhaustive [alpha]-mannosidase (jack bean and Aspergillus satoi, from Oxford Glycosystems, Oxford, UK) digestion of the [3H]Man5GlcNAc glycan obtained from BHK RicR 14 cells and further purification by paper chromatography. The structure of the glycan was confirmed by [beta]-mannosidase (Helix pomatia; Oxford Glycosystems, Oxford, UK) digestion and separation by paper chromatography.

Acetolysis

It was carried out essentially as described previously (Bonay and Hughes, 1991). [3H]-Mannose-labeled oligosaccharides (up to 105 counts/min) were freeze-dried and reduced by treatment with 0.2 ml of 1 M Na BH4/0.2 M borate buffer, pH 9.8 for 2 h at 37°C. In order to eliminate residual NaBH4, 10 µl glacial acetic acid was added followed 2-3 min later by 1 ml methanol containing a drop of glacial acetic acid. The solution was dried three times with methanol (1 ml) containing a drop of glacial acetic acid. Finally, the residue in the tube was redissolved in 1 ml H2O and desalted on a 2 ml column Amberlite MB-3 (mixed bed resin, 16-50 mesh, H+, OH-) column. The material, eluted with 9 ml H2O, containing the oligosaccharides alditols was dried and subjected to acetolysis (Varki and Kornfeld, 1983). The acetolysis fragments were deacetylated by incubation in 0.2 ml 0.2 NaOH at 37°C for 45 min, desalted by passage over a column of Amberlite MB-3 mixed bed resin (3ml) in water, freeze-dried, and dissolved in H2O, and aliquots containing approximately 104 counts/min were examined by paper chromatography. Standards included Man[alpha]1-2Man[alpha]1-3Man[beta]1-4GlcNAcOH, Man[alpha]1-3Man[beta]1-4GlcNAcOH, Man[alpha]1-2Man, and mannose.

Paper chromatography

Descending paper chromatography was performed on Whatman 3MM paper in Solvent A: ethylacetate/pyridine/acetic acid/H2O (5:5:1:3, by vol.) for 1-4 days at RT. Sample tracks after chromatography were cut into 1 cm strips, eluted with 1 ml H2O and aliquots were counted for radioactivity in scintillation counter.

Solubilization and extraction

Epimastigotes at mid-log growth were harvested by centrifugation (4-6 ml packed cells), washed three times, and resuspended in 12 ml of extraction buffer. The cells were disrupted by three cycles of freeze-thaw and sonicating the suspension at 4°C in a Branson sonifier (position 4, 65 W) for three cycles of 30 s with 10 s rest. The cell homogenate was centrifuged at 150,000 × g for 1 h at 4°C in a Beckman TL100 ultracentrifuge (TL100-3 rotor). The pellet was extracted again using 5 ml of extraction buffer and the supernatants containing more than 95% of the neutral [alpha]-mannosidase activity against pNPM were combined.

Abbreviations

BHK, baby hamster kidney cells; BHK-RicR, baby hamster kidney cells-ricin resistant; CHAPS, 3-[(3-cholamido propyl)-dimethyl ammonio]-1-propane sulfonate; DTT, dithiothreitol; EDTA, ethylenediaminetetra-acetic acid; EGTA, ethylene glycol-bis([beta]-aminoethyl ether) N,N,N[prime],N[prime],tetra acetic acid; ER, endoplasmic reticulum; GlcNAc, 2-acetamido-2-deoxy-d-glucose (N-acetylglucosamine); HEPES, N-2-hydroxyethyl piperazine-N[prime]-2-ethane sulfonic acid; HTP, hydroxylapatite; Man, mannose; MEAC, metal affinity chromatography; pNPM, p-nitro-phenyl [alpha]-d-mannopyranoside.

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