Trypanosoma brucei: Two-dimensional gel analysis of the major glycosomal proteins during the life cycle

EXPERIMENTAL PARASITOLOGY 70, 276285 (1990)

Trypanosoma

brucei: Two-Dimensional Glycosomal Proteins during

Gel Analysis of the Major the Life Cycle

MARILYN PARSONS**? AND BARBARA NIELSEN* *Seattle

Biomedical fDeparfment

Research Institute, 4 Nickerson Street, Seattle, Washington 98109-1651, of Pathobiology, University of Washington, Seattle, Washington 98195,

U.S.A., U.S.A.

and

PARSONS,M., AND NIELSEN, B. 1990. Trypanosoma brucei: Two-dimensional gel analysis of the major glycosomal proteins during the life cycle. Experimental Parasitology 70, 276285. Kinetoplastid organisms possess a unique organelle, the glycosome, which compartmentalizes the Embden-Meyerhof segment of glycolysis and several other metabolic pathways. In Trypanosoma brucei many of the enzyme activities localized to the glycosome are stage regulated. Two-dimensional gel analysis was used to examine the characteristics, expression, and biosynthesis of the major glycosomal proteins. Two-dimensional gel maps of glycosomes from slender bloodforms and late intermediate-stumpy bloodforms (the precursors of procyclic forms) were indistinguishable, while those of procyclic form glycosomes showed extensive differences. Glycosomal phosphoenolpyruvate carboxykinase and malate dehydrogenase were identified to have subunit molecular weights of 60 and 34 kDa, respectively. We detected two hitherto undescribed glycosomal proteins, one of which is found only in bloodforms. All of the major proteins, except glucose phosphate isomerase, were highly basic. Stage regulation of glycosomal enzyme activities correlated with stage regulation of specific protein biosynthesis. o 1990 Academic press, IN. INDEX DESCRIPTORSAND ABBREVIATIONS: Trypanosoma brucei brucei; Glycosomes; Microbodies; Stage regulation; Trypanosomes; Glycolytic enzymes; Adenosine-5’-triphosphate (ATP); Kilodaltons (kDa); Malate dehydrogenase (MDH); a-nicotinamide adenine dinucleotide, reduced form (NADH); Phosphoenolpyruvate carboxykinase (PEPCK); Isoelectric point (~0; Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE); Two-dimensional (2-D).

INTRODUCTION Glycosomes are specialized microbody organelles found in kinetoplastid organisms such as Trypanosoma brucei brucei. They contain the first six enzymes of glycolysis (Opperdoes and Borst 1977), other carbohydrate metabolizing enzymes (Opperdoes and Borst 1977), and enzymes involved in ether-lipid (Opperdoes 1984) and pyrimidine biosynthesis (Hammond et al. 1981). The compartmentation of glycolysis is a feature found only in species possessing glycosomes, and thus is limited to trypanosomatid (Opperdoes and Borst 1977; Hammond et al. 1981; Taylor et al. 1980) and bodonid (Opperdoes et al. 1988) protozoa. This biochemical peculiarity in such a fundamental pathway suggests that the glycosome would be a useful target for chemotherapeutic intervention. However, little is 276 0014-4894/90 $3.00

known about the specifics of glycosomal biogenesis and how it differs from microbody biogenesis in higher organisms. It is clear that, like other microbody proteins, glycosomal proteins are encoded by nuclear genes, synthesized on free polysomes, and post-translationally imported into the organelle (Borst 1986). Glycolysis is the sole source of energy in T. brucei bloodforms, while mitochondrial respiration provides energy in procyclic forms (Opperdoes 1987). Bloodforms possess higher activities of many glycolytic enzymes. This appears to result from the lack of the protein itself in the cases of the three enzymes that can be unequivocally identified by SDS-PAGE: glucose phosphate isomerase, aldolase, and phosphoglycerate kinase (Hart et al. 1984; Aman and Wang 1986).

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In this report we describe a 2-D gel analysis of glycosomal proteins from slender and stumpy bloodforms, and from procyclic forms, of T. brucei brucei. We have been able to separate proteins which comigrate on SDS-PAGE and have resolved and identified most of the major glycosomal constituents. These studies show that stage-regulated activity reflects the relative abundance of specific protein species. Analysis of biosynthetically labeled proteins demonstrates that stage regulation occurs at the level of protein biosynthesis. While major differences in the 2-D protein maps of procyclic and bloodform glycosomes exist, no clear differences are observed between slender and stumpy bloodforms. In procyclic as well as bloodform glycosomes, the major proteins, including the integral membrane proteins, are very basic. METHODS Trypanosomes and glycosomes. Bloodforms and midlog phase procyclic culture forms of T. brucei brucei EATRO 164 and TREU 667 were grown, harvested, and characterized as previously described (Parsons and Hill 1989). Biosynthetic labelings were performed as described (Parsons and Hill 1989). For the first glycosomes purifications, cells were lysed by grinding with silicon carbide (Aman and Wang 1986); however, we subsequently moved to a gentler homogenization procedure which increased yields (Dovey et al. 1988). In the latter, each homogenization step was followed by a 20-set vortexing. Glycosomes were prepared according to published procedures (Aman and Wang 1986; Aman et al. 1985), except that two successive sucrose step gradients (35,45,50,55, and 60%) were used. Glycosomes are found at the interface of the 50 and 55% sucrose steps and were stored in TEDS at - 70°C. Gel analysis. SDS-PAGE (Laemmli 1970) used 10% acrylamide/0.26% bisacrylamide gels. 2-D gel analysis was performed using nonequilibrium pH gradient gel electrophoresis with an enriched basic ampholine mixture followed by SDS-PAGE (Parsons and Hill 1989). When purified proteins were examined, carbamylated standards (Pharmacia) were included to facilitate alignment with control gels. Gels were silver stained (Amess and Sprang 1986). Gels of biosynthetically labeled cells were fluorographed using Entensify (New England Nuclear) and autoradiographed. Protein purification and enzyme assays. Partially

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purified glycosomes (45,OOOg pellet in the scheme above) from strain EATRO 164 were solubilized in the column starting buffer plus 0.1% Triton X-100 and 0.15 M NaCI. For hydrophobic interaction chromatography, the proteins were loaded onto a phenyl column (TSK spherogel 5PW, 7.5 mm x 7.5 cm) in column buffer A (25 mM Tris-HCI, pH 7.6, 5 mM EDTA, 1 mM NaN,, 1 mM dithiothreitol, 2 PM leupeptin) plus 1 M (NH&SO,. Buffer B was the same as A, except that it contained 10% ethylene glycol. Samples were chromatographed at 1 ml/min. After a 15-min wash in buffer A, bloodform glycosomal proteins were eluted using 5-min steps of 24,26,50,70, and 100% buffer B. Phosphofructokinase and hexokinase activity peaks eluted 35 and 36 min after injection. Glycerol kinase eluted at 45 min. Procyclic glycosomal proteins were eluted with 7-min steps of 20, 30, 50, 70, and 100% buffer B. Phosphoenolpyruvate carboxykinase activity eluted at 30 min and malate dehydrogenase at 50 min. For ion-exchange chromatography, an analytical weak cation exchange column (BakerBond CBX COOH) was employed. The starting buffer was 10 mM sodium phosphate, pH 7.4, 1 mM dithiothreitol, 1 mM EDTA, 1 PM leupeptin, and 5% ethylene glycol. After 15 min, proteins were eluted with a linear gradient of (M.5 M NaCl over 1 hr. Peak MDH activity eluted at 62 min. Enzymes were spectrophotometrically assayed in 100 mMTris-HCI, pH 7.5, except as noted. Glycolytic enzymes were assayed by published procedures (Misset and Opperdoes 1984), except that phosphofructokinase was assayed in 10 mM KCl, 1.7 mM MgCI,, 1.6 mM ATP, 0.3 mM NADH, 2.2 mM phosphoenolpyruvate, and 25 +g/ml each pyruvate kinase and lactate dehydrogenase. MDH was assayed using 0.3 mM NADH and 5 mM oxaloacetate. PEPCK (ATP) was assayed in 100 mM MOPS, pH 6.6, 1 mM MnSO,, 1.25 mM ADP, 50 mM NaHCO,, 0.3 mM NADH, 17 U/ml MDH, and 1.25 mM phosphoenolpyruvate. Enzymes and substrates were purchased from Sigma Chemical co. Integral glycosomal membrane proteins were prepared by sodium carbonate extraction at pH 11.5 (Fujiki et al. 1982). After extraction the membrane pellets were rinsed once in the carbonate solution before analysis. RESULTS

Figure 1 shows a 2-D gel analysis of glycosomes isolated from bloodforms and from procyclic forms of the pleiomorphic strain TREU 667. Proteins which have been identified as to activity are indicated. Proteins of glycosomal origin (Hart et al. 1984; Aman and Wang 1986), but of unknown

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FIG. 1. Two-dimensional gel analysis of glycosomal proteins. Glycosomal protein (7.5 kg) from strain TREU 667 was analyzed by 2-D gel analysis. The first dimension was nonequilibrium pH gradient gel electrophoresis and the second dimension was SDS-PAGE. The gels were silver stained. The positions of molecular weight markers at 94,67,45, and 30 kDa are indicated by hash marks. Proteins were identified as described in the text: ALD (aldolase), GAPDH (glyceraldehyde phosphate dehydrogenase), GDH (glycerol phosphate dehydrogenase), GK (glycerol kinase), HK (hexokinase), MDH (malate dehydrogenase), PEPCK (phosphoenolpyruvate carboxykinase), PFK (phosphofructokinase), PGK (phosphoglycerate kinase), PGI (glucose phosphate isomerase), TPI (triose phosphate isomerase). Proteins of unknown function are indicated by their molecular weights. (A) Glycosomes from slender bloodforms (70% slender, 25% intermediate, 5% stumpy). (B) Glycosomes from stumpy bloodforms (65% stumpy, 30% intermediate, 5% slender). (C) Glycosomes from procyclic forms.

function, are identified by their subunit molecular weights. Since the SDS-PAGE migration patterns of many bloodfotm glycosomal proteins have been described (Misset et al. 1988), it was possible to identify many

of the glycosomal proteins by virtue of their migration in the second dimension. These proteins include glucose phosphate isomerase, phosphoglycerate kinase, aldolase, glyceraldehyde phosphate dehydrogenase,

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glycerol phosphate dehydrogenase, and triose phosphate isomerase. The evidence for the identification of other known proteins is presented below. Certain nonglycosomal proteins were observed on occasion in these preparations. In bloodform glycosome preparations the variant surface glycoprotein was sometimes observed. The other major contaminant is an acidic doublet at approximately 74 kDa, a component of the cytoskeleton that frequently contaminates glycosome preparations (Kueng et al. 1989). In addition to the major proteins, several minor low molecular weight spots were occasionally observed. However, they were not resistant to externally added protease (unpublished results), suggesting they may be contaminants. TREU 667 trypanosomes differentiate naturally in vivo from slender to intermediate to stumpy forms. The intermediate or stumpy forms are thought to be the progenitors of procyclic forms. When glycosomes from late intermediate-stumpy forms are examined (Fig. IB), they are undistinguishable from those from slender forms (Fig. 1A). 2-D gel patterns of glycosomes from the monomorphic strain EATRO 164, which does not differentiate in vivo, are very similar to their pleiomorphic counterparts. The major difference was that the glucose phosphate isomerase spot is very much decreased in the pleiomorphic bloodform glycosomes. This probably reflects the high solubility of glucose phosphate isomerase and the low latency of the TREU 667 bloodform glycosomes. A second difference was that TREU 667 shows a double spot in the triose phosphate isomerase region, while EATRO 164 shows a single spot (compare Figs. 1 and 2A). This was seen in all life cycle stages examined. We propose that this represents a genetic polymorphism at the triose phosphate isomerase locus. This locus is genetically polymorphic, with at least one restriction site polymorphism within the coding region of the gene (Gibson et al. 1985).

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Although the glycosomal profiles of slender and stumpy bloodforms are quite similar, that of procyclic forms is dramatically different. Nevertheless, all of the major glycosomal proteins in procyclic forms, like those in bloodforms, show high apparent pZs. This is most clearly seen in Fig. 2A, where all of the procyclic glycosomal proteins are clearly much more basic than the standard protein at pZ 8.3. Several of the major glycosomal proteins could not be identified simply by virtue of their migration on the second dimension, either because subunit molecular weights were unknown (i.e., malate dehydrogenase and phosphoenolpyruvate carboxykinase) or because a second protein of similar molecular weight was revealed on the 2-D gels (i.e., glycerol kinase, hexokinase, and phosphofructokinase). Therefore, partial purifications were undertaken. On SDSPAGE, hexokinase and phosphofructokinase comigrate at approximately 50 kDa (Misset et al. 1988). On 2-D gels, two protein species were resolved. Following separation of bloodform glycosomal proteins by hydrophobic interaction chromatography, fractions enriched for each enzyme were analyzed by 2-D gel analysis (not shown). From these experiments it appeared that the less basic protein is hexokinase, while the more basic is phosphofructokinase. This was confirmed by analysis of hexokinase and phosphofructokinase further purified by ion-exchange chromatography (kindly provided by Dr. C. C. Wang, University of California at San Francisco). These results, obtained under denaturing conditions, are in contrast with those obtained under nondenaturing conditions where phosphofructokinase shows a lower pZ (Misset et al. 1988). The levels of both of these proteins are reduced in procyclic forms (Fig. l), resulting in the reduced enzyme activities. Glycerol kinase has a subunit molecular weight of approximately 53 kDa (Misset et al. 1988). In the corresponding region on

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FIG. 2. Identification of glycosomal proteins. Partially purified glycosomal proteins are displayed following 2-D gel analysis and silver staining. The two trails of protein spots are carbamylated creatine phosphokinase (40 kDa, pI range 49-7.1) and glyceraldehyde phosphate dehydrogenase (36 kDa, pl range 4.7-8.3). The triangles mark the most basic spot in each trail, with apparent pZs of 7.1 and 8.3. Abbreviations for proteins are as in Fig. 1. (A) Procyclic glycosomes (EATRO 164). (B) Partially purified glycerol kinase. (C) Partially purified PEPCK. (D) Partially purified glycosomal membrane proteins.

2-D gels, two protein species are detected, one slightly larger and more basic than the other. Glycerol kinase from bloodform glycosomes was partially purified by hydrophobic interaction chromatography. In addition to glycerol kinase activity, the preparation also contained glucose phosphate isomerase activity. On 2-D gels a major spot migrating at 53 kDa is observed, as

well as a minor spot at 62 kDa (Fig. 2B). These correspond to the respective subunit molecular weights of glycerol kinase and glucose phosphate isomerase. The trail of spots from the acidic side is formed by carbamylated standards included for alignment of gels. As expected, when fractions containing glucose phosphate isomerase but not glycerol kinase activity were analyzed

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on 2-D gels, only the 62-kDa spot was observed (not shown). Thus the 53-kDa spot represents glycerol kinase. The identity of the more basic 5%kDa protein remains unknown. For convenience we will term it ~55. The levels of glycerol kinase and ~55 are not stage regulated. Glycosomes from procyclic forms show increased levels of MDH and PEPCK (ATP) (Opperdoes and Cottem 1982; Broman et al. 1983). PEPCK was purified from procyclic glycosomes by hydrophobic interaction chromatography. SDS-PAGE analysis indicated a subunit molecular weight of 60 kDa (Fig. 3, lane 3). Comparison of the 2-D gel pattern obtained with this protein indicates that it corresponds to the major 60-kDa protein observed in procyclic glycosomes (Fig. 2C). This protein is present in all stages examined, but levels are higher in procyclic forms. The gene encoding glycosomal ~60, a procyclic“specific” glycosomal protein, has been cloned and sequenced (Kueng et al. 1989). We have recently shown that this sequence is highly homologous to yeast PEPCK

1234

.FIG. 3. Identification of MDH. Partially purified proteins were separated by SDS-PAGE and silver stained. Lane 1, molecular weight standards (94, 67, 45, 30 kDa). Lane 2, partially purified procyclic glycosomes (45,OOOg pellet from glycosome purification scheme). Lane 3, partially purified PEPCK. Lane 4, partially purified MDH obtained by hydrophobic interaction chromatography. Lane 5, partially purified MDH obtained by ion-exchange chromatography.

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(ATP) but has no detectable homology with other vertebrate PEPCKs which all require GTP (Parsons and Smith 1989). In similar separations, fractions containing MDH activity showed the presence of two proteins, with subunit molecular weights of 90 and 34 kDa (Fig. 3). Since other MDHs, including Leishmania mexicunu MDH, have subunit molecular weights of approximately 35 kDa (Banaszak and Bradshaw 1975; Mottram and Coombs 1985) the 34-kDa protein was the best candidate for T. brucei glycosomal MDH. Attempts to further separate these two proteins in the partially purified fractions failed because the MDH activity was very unstable. However, using ionexchange chromatography, we fractionated total glycosomal proteins and separated p34 and ~90. p90 eluted before the MDH activity, while the 34-kDa protein elution pattern paralleled that of the MDH activity. Lane 5 of Fig. 3 shows the proteins in the fraction containing the peak of MDH activity. In addition to ~34, glycerol phosphate dehydrogenase was found in this fraction, but no p90 was observed here or in the remaining fractions containing MDH activity. The data indicate that the 34-kDa protein is glycosomal MDH. Glycosomes from bloodforms completely lack the MDH protein. Glycosomes possess two major integral membrane proteins of subunit molecular weights 24 and 26 kDa (Aman and Wang 1988). 2-D gels of total glycosomal proteins did not reveal clearly resolved spots in the 25-kDa region. Therefore, the membrane proteins were purified by carbonate extraction (Fujiki et al. 1982) and analyzed by 2-D gels. Two species were observed (Fig. 2D). The larger, corresponding to 26 kDa, was very basic and diffuse. The smaller species migrated as a streak in the first dimension, suggesting that it is poorly soluble in the gel (which contains both NP-40 and 8 M urea). Since these gels are loaded on the acidic side and since the streak extends well past the standard with a pZ of 8.3, membrane

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protein p24 is not acidic. Neither of these proteins appears to be stage regulated. In addition to detecting the constituitively expressed glycosomal protein ~55, this study revealed a stage-regulated 71kDa protein in glycosomal preparations of both slender and stumpy forms, but not in procyclic forms. Two major proteins which are found only in procyclic glycosomes, p90 and ~110, and the constituitively expressed ~28 remain unidentified as to function. To study the biosynthesis of glycosomal proteins we metabolically labeled bloodforms and procyclic forms from the strains and stages described above. The labeled proteins were separated by 2-D electrophoresis and were revealed by fluorography and autoradiography (Fig. 4). Many of the spots in the basic region of the gel are glycosomal proteins and were easily identified by performing 2-D gel electrophoresis on mixtures of glycosomes and metabolically labeled lysates. The autoradiogram was then simply overlaid onto the silver-stained gel. We could not easily identify the lower molecular weight glycosomal proteins or

glucose phosphate isomerase in this way because there were many more proteins in those regions of the gel. For comparative purposes, the positions of key proteins are marked even if a spot is not evident. The major stage-regulated proteins pIlO (only faintly visible), ~90, ~71, phosphoglycerate kinase, aldolase, glycerol phosphate dehydrogenase, and malate dehydrogenase show a corresponding regulation in their biosynthesis. Hexokinase and phosphofructokinase biosynthesis also appears stage regulated, although the spots are somewhat obscured by the heavily labeled nonglycosomal protein between them. Upon overexposing the gels, however, most if not all of these proteins appear to be synthesized in all stages examined. Although the figure shows the pattern obtained after a 30-min labeling, identical glycosomal protein patterns were obtained using labelings as short as 2 min and as long as 2 hr. The glycosomal biosynthesis patterns of slender and stumpy bloodforms and monomorphic bloodforms appear qualitatively the same (not shown). PF

l-NEPHGE-

FIG. 4. Biosynthesis of glycosomal proteins. EATRO 164 bloodforms (BF) and procyclic forms (PF) were metabolically labeled with [3sS]methionine, lysed, and analyzed by 2-D gel electrophoresis. The positions of major glycosomal proteins are indicated as in Fig. 1. Hash marks indicate 67, 45, and 30 kDa.

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DISCUSSION

Glycosomes are organelles which house pivotal biochemical pathways in African trypanosomes, Leishmania spp., and T. cruzi. The enzyme activities in isolated glycosomes differ according to the life cycle stage of the parasite (Hart et al. 1984; Aman and Wang 1986). We have developed 2-D gel maps of the glycosomal proteins from different T. brucei life cycle stages and identified the major stage-regulated proteins. We have shown that all of the major proteins in procyclic glycosomes are highly basic, like those in bloodform glycosomes. This supports the hypothesis that a high p1 is important for glycosomal function, whether in import of protein into the organelle as has been proposed (Wierenga et al. 1987; Borst 1986), in subsequent assembly, or in neutralization of highly charged substrates. At least one T. brucei glycosomal protein, glucose phosphate isomerase, is not particularly basic (Misset et al. 1988), nor is the glycosomal phosphoglycerate kinase of Crithidia fusciculutu (Swinkels et al. 1988). Glucose is not readily available to procyclic forms in vivo (Vickerman 1985). Nevertheless, procyclic forms possess detectable levels of all of the glycolytic enzymes, which in most other organisms function in gluconeogenesis as well as in glycolysis. This is somewhat paradoxical since trypanosomes lack a key enzyme for gluconeogenesis, fructose-l ,6-diphosphatase, and synthesis of ribose-5-phosphate appears to occur cytoplasmically (Cronin et al. 1989). Although not systematically studied, variations in glycosomal phosphoglycerate kinase levels in procyclic culture forms have been proposed to correlate to a switch from growth on amino acids to growth on glucose as cells approach stationary phase (Misset and Opperdoes 1987). The significance of PEPCK in bloodform glycosomes is also moot. PEPCK, together with glycosomal MDH, is thought to be important in

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maintaining intraglycosomal ATP and NAD+ levels in procyclic forms (Opperdoes 1987; Broman et al. 1983). However, in bloodforms, phosphoenolpyruvate is metabolized to pyruvate and glycosomal MDH is absent. The physiological role of these stage-regulated proteins during the life cycle deserves further study. At least three proteins, glycosomal MDH, ~90, and ~110, appear to be completely absent from slender bloodform glycosomes. Since they are absent in late intermediate-stumpy glycosomes it is unlikely that these proteins are required immediately after ingestion by the fly. Indeed, stumpy forms display no preadaptations for differentiation into procyclic forms at the level of glycosomal proteins. These findings are in contrast with the preadaptations which occur in mitochondrial morphology (Vickerman 1965) and transcripts (Feagin et al. 1986) in stumpy forms. Our data indicate that stage regulation of glycosomal enzyme activity primarily reflects regulation of enzyme biosynthesis and not some subsequent step. Transcripts encoding several of these proteins, including phosphoglycerate kinase (Osinga et al. 1985) aldolase (Clayton 1985), and PEPCK (Kueng et al. 1989), are stage regulated (Osinga et al. 1985; Clayton 1985; Kueng et al. 1989) but the abundance of the proteins do not strictly reflect the abundance of the transcripts (Clayton 1985; Parsons and Hill 1989). The stage-regulated transformation in glycosome protein composition appears to resemble in part the transition from glyoxysomes to peroxisomes which occurs during greening of cotyledons in plants. In this microbody transition the resulting altered protein composition does not reflect a differential ability of microbodies of different stages to import specific proteins, but rather a difference in the synthesis of specific proteins in the cytoplasm (Sautter et al. 1988). This appears to be combined with an intraorganellar protein degradation sys-

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tern which is active during the transition process (Mori and Nishimura 1989). Whether the latter phenomenon is important in the stage regulation of glycosomal enzymes remains to be seen. It is intriguing to note that proteolytic activity associated with the glycosome has been described (Aman and Wang 1988).

We thank Teresa Hill for excellent technical assistance and Drs. Keith Alexander, C. C. Wang, and Jessica Krakow for helpful discussions. Koral MassieLavelle provided valued secretarial assistance. This investigation was supported in part by NIH A122635, the UNDPiWorld Bank/WHO Special Programme for Research and Training in Tropical Diseases, and the Murdock Charitable Trust.

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