Protein glycosylation mutants of procyclic Trypanosoma brucei: defects in the asparagine-glycosylation pathway

Kuo-Yuan Hwa1,2, Alvaro Acosta-Serrano1, Kay-Hooi Khoo2, Terry W.Pearson3 and Paul T. Englund1,4

1Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA, 2Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, ROC, and 3Department of Biochemistry and Microbiology, University of Victoria, Victoria V8W 3P6, Canada

Received on May 26, 1998; revised on June 15, 1998; accepted on June 17, 1998

We employed a genetic approach to study protein glycosylation in the procyclic form of the parasite Trypanosoma brucei. Two different mutant parasites, ConA 1-1 and ConA 4-1, were isolated from mutagenized cultures by selecting cells which resisted killing or agglutination by concanavalin A. Both mutant cells show reduced concanavalin A binding. However, the mutants have different phenotypes, as indicated by the fact that ConA 1-1 binds to wheat germ agglutinin but ConA 4-1 and wild type do not. A blot probed with concanavalin A revealed that many proteins in both mutants lost the ability to bind this lectin, and the blots resembled one of wild type membrane proteins treated with PNGase F. This finding suggested that the mutants had altered asparagine-linked glycosylation. This conclusion was confirmed by studies on a flagellar protein (Fla1) and procyclic acidic repetitive protein (PARP). Structural analysis indicated that the N-glycan of wild type PARP is exclusively Man5GlcNAc2 whereas that in both mutants is predominantly a hybrid type with a terminal N-acetyllactosamine. The occupancy of the PARP glycosylation site in ConA 4-1 was much lower than that in ConA 1-1. These mutants will be useful for studying trypanosome glycosylation mechanisms and function.

Key words: concanavalin A/glycosylation mutant/glycoprotein/Trypanosoma brucei

Introduction

African trypanosomes are protozoan parasites which cause serious diseases in both humans and livestock. Trypanosoma brucei brucei, which causes the cattle disease nagana (from the Zulu, meaning poorly), is the species which has been studied most extensively in the laboratory. In its life cycle this parasite infects two different hosts. Procyclic forms reside in the tsetse fly vector whereas bloodstream forms dwell in the mammalian host. We have initiated a genetic approach for studying protein glycosylation in procyclic trypanosomes.

Procyclic trypanosomes have a number of different cell surface glycoproteins, but unlike the related parasites Leishmania and Trypanosoma cruzi, they have no abundant surface-oriented complex glycolipid molecules (such as Leishmania lipophosphoglycan and glycoinositol phospholipids; Turco and Descoteaux, 1992; McConville and Ferguson, 1993). The major and best characterized procyclic surface glycoprotein is the T.brucei procyclin, procyclic acidic repetitive protein (PARP) (Mowatt and Clayton, 1987; Roditi et al., 1987; Richardson et al., 1988). There are about 6 × 106 molecules of PARP per cell (Clayton and Mowatt, 1989, Treumann et al., 1997), and it exists in two distinct forms (Butikofer et al., 1997; Treumann et al., 1997; Mowatt et al., 1989). One form, EP-PARP, which is encoded by the A[beta], B[alpha], and B[beta] genes (each with a slightly different sequence), has 22-30 Glu-Pro (EP) repeats. The other form, GPEET-PARP, encoded by the A[alpha] gene, has six Gly-Pro-Glu-Glu-Thr (GPEET) repeats instead of EP repeats (Mowatt et al., 1989). Procyclic cells express both forms of PARP on their surface (Butikofer et al., 1997; Ruepp et al., 1997; Treumann et al., 1997). EP-PARP and GPEET-PARP are anchored to the plasma membrane by glycosyl phosphatidylinositol (GPI) anchors with similar structures (Treumann et al., 1997). These anchors have the conserved GPI core structure, ethanolamine-phospho-6Man[alpha]1-2Man[alpha]1-6-Man[alpha]1-4GlcN[alpha]1-6-PI (Field et al., 1991a,b; Treumann et al., 1997). The glycan side chain of the GPI core is unusually large, having up to 30 sugar residues organized into highly branched poly-N-acetyllactosamine chains (Ferguson et al., 1993; Treumann et al., 1997) which act as sialic acid acceptors in a reaction catalyzed by the cell surface trans-sialidase (Engstler et al., 1993; Pontes de Carvalho et al., 1993). In addition to its GPI-anchor, EP-PARP encoded by the A[beta] and B[alpha] genes has one N-glycan site in its N-terminal domain (Clayton and Mowatt, 1989) which is modified by a high mannose type oligosaccharide, exclusively Man5GlcNAc2 (Treumann et al., 1997). There is no N-linked glycosylation site on GPEET-PARP or on the EP-PARP encoded by the B[beta] gene. Interestingly, GPEET-PARP has been found to be phosphorylated, probably on the threonine residues of the repeat sequence (Butikofer et al., 1997).

To study protein glycosylation in procyclic T.brucei, our approach was to mutagenize procyclic trypanosomes and then to isolate mutants which have altered reactivity to lectins. Since concanavalin A (Con A, a lectin that binds [alpha]Man residues) killed wild type cells (Welburn et al., 1996), we were able to isolate two different mutants which were resistant to killing. These two lectin-resistant mutants have altered protein glycosylation, with both having defects in the asparagine glycosylation pathway. These are the first examples of glycosylation mutants in trypanosomes. As they have with other systems (Robbins, 1994; Stanley and Ioffe, 1995), these mutants should provide a powerful tool for study of the molecular mechanisms and functions of glycosylation in this primitive eukaryote.

Results

Isolation of Con A resistant procyclic trypanosomes

Prior to isolation of T.brucei glycosylation mutants, we tested several plant lectins for their ability to agglutinate and kill the parasite. Some lectins, including Con A, agglutinated procyclic trypanosomes, but only Con A could efficiently kill these cells. Upon addition of Con A, cells rounded up and died within 48 h without apparent disruption of the plasma membrane, consistent with a previous report (Welburn et al., 1996). We then devised two selection methods to isolate mutant trypanosomes resistant to Con A-induced killing. In one selection, using 15 µg/ml Con A, we obtained the mutant ConA 1-1. In another, using 50 µg/ml Con A in a different mutagenized culture, we obtained ConA 4-1.


Figure 1. Sensitivity of wild type and mutant trypanosomes to killing by Con A. Cells (1 ml, 2.5 × 106 cells/ml) were incubated with Con A at 27°C in SDM-79 medium. After 48 h, the percentage of surviving cells, relative to a control culture without Con A, was determined by counting in a hemocytometer. Cells were considered surviving if they were motile. Each point on the graph represents an average of three determinations, and the bars denote the SD.

We then devised quantitative tests to measure reactivity of these cells with Con A. In a Con A agglutination assay (see Materials and methods) we found that 80% of wild type cells could be agglutinated. Under the same conditions only 18% of ConA 1-1 cells could be agglutinated whereas ConA 4-1 cells could not be agglutinated at all. We also measured the sensitivity of the cells to killing by Con A (Figure 1). The Con A concentration required for 50% killing of wild type cells was about 1 µg/ml, that for ConA 1-1 was about 25 µg/ml, and that for ConA 4-1 was about 100 µg/ml. Next we used flow cytometry to measure binding of Con A-fluorescein to the mutant and wild type cells (Figure 2A). In this analysis the mean relative fluorescence intensity of ConA 1-1 was about 20-fold less than that of the wild type, and the intensity of ConA 4-1 was ~60-fold less. Finally we examined the cellular localization of the Con A-binding ligands by fluorescence microscopy using Con A-fluorescein as a probe (Figure 3). Consistent with the previous results, we found with wild type cells that the entire cell surface was labeled, with the most intense signal on the flagellum and within the flagellar pocket (Figure 3A). With ConA 1-1, labeling was reduced and appeared in a punctate pattern (Figure 3B), and with ConA 4-1 there was only a small amount of label, concentrated in the flagellar pocket (marked by arrows, Figure 3C).

   A
   B

Figure 2. Binding of lectins to wild type and mutant trypanosomes, as analyzed by flow cytometry. Live wild type and mutant cells (5 × 106 to 1 × 107 cells/ml, in PBSG) were incubated with Con A-fluorescein (2 µg/ml) or WGA-fluorescein (2 µg/ml) at 4°C for 30 min. Prior to flow cytometry analysis, cell samples were passed through 35 µm cell strainers to remove agglutinated cells. Flow cytometry was performed with a Coulter EPICS ELITE flow cytometer (Coulter Cytometry, Inc.), and 5000 cells were examined for each analysis. The cytometer was equipped with an argon laser with 488 nm emission. The data were recorded and analyzed as histograms with the ELITE software. Cells incubated in the absence of lectin were used as a negative control for autofluorescence. All the analyses with each lectin were done in the same experiment. (A) Cells labeled with Con A-fluorescein. (B) Cells labeled with WGA-fluorescein.

Figure 3. Analysis of Con A binding by fluorescence microscopy. Cells (50 µl, 5 × 106 cells/ml) were adhered to BIOBOND (Electron Microscopy Sciences) coated glass slides for 30 min at 4°C and then fixed with 4% paraformaldehyde, 0.5% formaldehyde, for 20 min at 4°C. They were incubated with 20 µg/ml Con A-fluorescein in TBS (137 mM NaCl, 2.7 mM KCl, 25 mM Tris-HCl, pH 7.4) containing 1 mM MgCl2, 1 mM CaCl2, and 0.1 mM MnCl2 for 30 min at 4°C and then washed with the same buffer (without Con A-fluorescein) for 30 min at 4 °C. The slides were mounted with 80% glycerol containing 2.5% 1,4-diazobicyclo-[2.2.2]-octane and were observed with a Zeiss Axioskop microscope at 1000× magnification. Photographs were taken for equal exposure times on a Zeiss MC 80 camera. (A) Wild type cells; (B) ConA 1-1 cells; (C) ConA 4-1 cells; (D) and (E) phase images of (B) and (C). Arrows in (C) indicate Con A fluorescence in the flagellar pocket.

The two ConA mutants are phenotypically different

To further compare the two ConA mutants, we used flow cytometry to examine their binding to WGA-fluorescein. WGA binds to GlcNAc and sialic acid residues. By this analysis ConA 1-1 and ConA 4-1 behaved in a strikingly different manner (Figure 2B) in that the relative fluorescence intensity of ConA 1-1 was about 8 times that of ConA 4-1 and wild type. The finding indicated that loss of the Con A ligands on the surface of ConA 1-1 was accompanied by an increase of WGA ligands, probably due to a novel glycosylation present in this mutant (see below). This differential binding of WGA-fluorescein by the two mutants, together with their differential sensitivity and binding to Con A (Figures 1, 2A, and 3), confirms that they are phenotypically distinct.

Both ConA mutants have altered asparagine glycosylation

When probing a blot containing total membrane proteins with Con A, we found that numerous membrane glycoproteins from wild type cells were bound by this lectin (Figure 4, lane 1). However, upon treatment with PNGase F, an enzyme that removes N-glycans from glycoproteins, most but not all of these proteins lost their reactivity (Figure 4, lane 2). In contrast, only a few membrane glycoproteins from ConA 1-1 and ConA 4-1 bound Con A (Figure 4, lanes 3 and 5), and PNGase F had little effect on the proteins from mutant cells (Figure 4, lanes 4 and 6). The fact that PNGase F-treated wild type proteins resemble those from the mutant cells (with or without PNGase F treatment) suggests that the mutant cells have altered asparagine glycosylation. The N-glycosylation defect in both mutants must be global, affecting many different proteins.


Figure 4. Probing of membrane glycoproteins in wild type and mutant cells with Con A. Membrane proteins (20 µl, 2 × 107 cell equivalents, prepared as described in Materials and methods) were incubated with (or without) 2 µl of Flavobacterium meningosepticum PNGase F (New England Biolabs, 500,000 U/ml) as suggested by the manufacturer. Reactions were stopped by addition of 30 µl of SDS-PAGE sample buffer and heating (100°C, 5 min). After SDS-PAGE (10% gel; 1 × 107 cell equivalents/lane), the proteins were blotted onto an Immobilon PVDF membrane (Millipore). The membrane was then incubated with 1% BSA for 2 h and then with 20 µg/ml biotin-labeled Con A in TBS buffer containing 1 mM MgCl2, 1 mM CaCl2, 0.1 mM MnCl2 at 25 °C for 1 h. After three washes with PBS, the membrane was incubated with streptavidin-alkaline phosphatase (GIBCO-BRL, diluted 1:1000) at 25°C for 1 h. The Con A binding proteins were detected by an alkaline-phosphatase assay with NBT/BCIP (Boehringer Mannheim) as substrates. Lanes contain wild type (WT), ConA 1-1, ConA 4-1, or ovalbumin (6 µg) incubated with or without PNGase F, as indicated. The wild type cells used in this experiment were from the sample frozen in 1988. Essentially identical results were obtained from the wild type cells used to generate the mutants. The strong 35 kDa band in lanes 2, 4, 6, and 8 is PNGase F, a glycoprotein that probably binds Con A through its mannosylated O-glycan (Reinhold et al., 1995). The family of bands in the 35-45 kDa region in lane 1 could be EP-PARP. Digestion of the proteins in this region by PNGase F was incomplete (lane 2), but doubling the time of digestion and amount of enzyme resulted in more extensive digestion.

Altered N-linked glycosylation of a flagellar protein, Fla1

To further characterize the defects in the two ConA mutants, we studied a specific glycoprotein, Fla1, which is localized on the parasite flagellum (Nozaki et al., 1996). Fla1 has five asparagine glycosylation sites but does not have a GPI anchor or any other known form of glycosylation in procyclic trypanosomes (Nozaki et al., 1996). As shown in a Western blot, wild type Fla1 has a Mr of 80 kDa, as previously described for this protein (Figure 5, lane 1). Fla1 from ConA 1-1 and ConA 4-1 have an Mr of about 72 and 76 kDa, respectively (Figure 5, lanes 2 and 3). Treatment of these proteins with Endo H, an enzyme that removes only high mannose and hybrid N-glycans, reduced the Mr of all three forms of Fla1 to 60 kDa (Figure 5, lanes 4-6), the size predicted for the primary translation product (Nozaki et al., 1996). These results provide strong evidence that the Fla1 protein in the mutant cells have altered N-glycosylation and that both mutants are able to synthesize high mannose and/or hybrid N-glycans which are Endo H-sensitive. Furthermore, based on the relative values of the Mr of Fla1 in wild type and mutant cells, the mutant Fla1 proteins could either contain altered N-glycans or the occupancy of the five potential N-glycosylation sites could be reduced.


Figure 5. Analysis of the flagellar glycoprotein Fla1 in wild type and mutant cells. Membrane proteins (20 µl, 2 × 107 cell equivalents, prepared as described in Materials and methods) were incubated (lanes 4-6) or mock treated (lanes 1-3) with 2 µl of Endo-H (New England Biolabs, 500 U/ml; two additions of 1 µl at 0 and 12 h) for 24 h at 37°C, as suggested by the manufacturer. Samples were submitted to SDS-PAGE (7.5 % gel; 1 × 107 cell equivalents/lane) and transferred to a PVDF membrane as described in the Figure 4 caption. The membrane was blocked for 1.5 h with 2% BSA and then incubated with anti-Fla1 serum (diluted 1:300 in PBS/2% sodium azide) for 1 h at 25°C. After three washes with PBS, the membrane was incubated with the secondary antibody (goat anti-rat IgG conjugated with alkaline phosphatase (Sigma), diluted 1/12,500 in PBS) for 1 h at 25°C. After three washes with PBS, the anti-Fla 1 antibodies were detected as described in the Figure 4 caption. WT, 1-1 and 4-1 refer to membrane proteins from wild type, ConA 1-1, and ConA 4-1, respectively.

Analysis of EP-PARP and GPEET-PARP in the ConA mutants

To gain more insight into the nature of the defect in these mutants, we studied PARP, the major surface glycoprotein in procyclic T.brucei. Although this protein is heterogeneous and complex in structure, its abundance and ease of purification made possible the structural analysis of the mutant glycans. But first it was essential to determine whether both forms of PARP, EP-PARP, and GPEET-PARP, were present on the wild type and mutant cells. As reported previously (Treumann et al., 1997), immunofluorescence using fluorescein-tagged antibodies revealed that the wild type cells expressed both forms of PARP uniformly over their entire surfaces (unpublished observations). The same was true of ConA 1-1 and ConA 4-1 (unpublished observations). To examine quantitatively the expression of the two forms of PARP, we purified a mixture of EP- and GPEET-PARP from each cell type by organic solvent extraction followed by Octyl-Sepharose chromatography (Ferguson et al., 1993). We then performed analyses of N-terminal sequences. Interpretation of these sequences was complicated by the fact that we were analyzing a mixture of two different proteins and by the finding that there were three different N-termini for GPEET-PARP. Nevertheless, we were able to assign the sequences consistent with those predicted from the gene sequences of EP- and GPEET-PARP (Table I). The results indicated that only about 8% of the PARP in the wild type 427-60 cells was EP-PARP. In contrast, another sample of 427 cells, not passaged but stored frozen since 1988, had a much higher percent of EP-PARP, about 79%. Variation in the ratio of EP- and GPEET-PARP in a clonal line, as well as N-terminal heterogeneity of GPEET-PARP, has been observed previously (Butikofer et al., 1997; Treumann et al., 1997). The PARP molecules from the ConA 1-1 and ConA 4-1 mutants have higher percentages of EP-PARP than those of the parental wild type, 37% and 15%, respectively. These results prove that the Con A resistance of the mutants is not due to loss of expression of EP-PARP on the cell surface.

Table I. The EP-PARP/GPEET-PARP ratio in wild type and mutant trypanosomes determined from N-terminal amino acid sequences
  Number of picomolesa
Wild typeb Wild typec ConA 1-1 ConA 4-1
EP-PARP:
    AEGPEDKGL 4 54 22 16
GPEET-PARP:
    VIVKGGKGK 22 None 11 15
    KGGKGK 5 14 15 34
    GGKGK 18 None 11 44
% EP-PARP 8 79 37 15
PARP was purified by organic solvent extraction followed by Octyl-Sepharose chromatography (Ferguson et al., 1993) and N-terminal sequences were determined by automatic Edman degradation in the Protein/Peptide/DNA Laboratory, Department of Biological Chemistry, Johns Hopkins Medical School. Each sample contained 100 pmol of protein (estimated by amino acid composition analysis), and recoveries ranged from 49 to 108%. The sequences detected were a mixture, with two forms of PARP and with GPEET-PARP having three distinct N-termini. All observed sequences were in agreement with those predicted from the gene sequences.
aValues are averages of first six residues.
bWild type 427-60 clone used for generating the mutants.
cAnother 427 strain, stored frozen since 1988.

Alterations in the structure of EP-PARP in the ConA mutants

To determine whether these mutants have a defect in PARP glycosylation, we first probed an immunoblot of membrane preparations with a monoclonal antibody against GPEET-PARP. Its heterogeneity (Figure 6A) is due to variable glycosylation of its GPI anchor (Ferguson et al., 1993; Treumann et al., 1997) and possibly also to variable phosphorylation (Butikofer et al., 1997). Despite the heterogeneity, there were no detectable differences in the species of GPEET-PARP in the wild type and mutant cells (Figure 6A). Only the quantity seemed to differ, with greater amounts expressed in the wild type cells (lane 1), a result expected from the amino acid sequence analysis in Table I. Since GPEET-PARP has no N-glycosylation site, this experiment provided evidence that the mutant cells have no primary defect in assembly or modification of GPI anchors.

   A
   B

Figure 6. Electrophoretic analysis of GPEET-PARP and EP-PARP in wild type and mutant cells. (A) Analysis of GPEET-PARP. Membrane proteins of wild type and mutant cells (2 × 106 cell equivalents/lane, prepared as described in Materials and methods) were fractionated by SDS-PAGE (12% gel) and transferred onto an Immobilon PVDF membrane. The blot was incubated for 1 h at 25 °C with monoclonal antibody 9G4 (diluted 1:500 in PBS supplemented with 3% glucose and 5% FBS). The blot was washed three times (20 min, 25°C) with TBS supplemented with 0.02% Tween-20. The second antibody, goat anti-mouse IgG conjugated with alkaline phosphatase (Boehringer Mannheim, diluted 1:1000 in PBS supplemented with 3% glucose and 5% FBS) was then added. After washing, GPEET-PARP-bound antibodies were detected as in the Figure 4 caption. (B) Analysis of EP-PARP. Log phase wild type (5 × 107 cells/ml) and mutant (1 × 107 cells/ml) were harvested and incubated in 1 ml of SM medium (Cunningham, 1977) without proline but supplemented with 25 µCi/ml [3H]proline (Dupont-NEN, 44.5 Ci/mmol) and 10% FBS (dialyzed against 10 volumes of PBS, overnight, 4°C). After incubation for 3 h at 25°C, cells were washed three times with an equal volume of PBSG. Immunoprecipitation of EP-PARP, using a mixture of antibodies TBRP1/247 and TBRP1/346, was described previously (Roditi et al., 1989). Digestion of EP-PARP with Flavobacterium meningosepticum PNGase F (New England Biolabs, 500,000 U/ml) was performed as suggested by the manufacturer, with two additions of 1 µl enzyme over 48 h incubation at 37°C. Samples were analyzed by SDS-PAGE (12% gel) and fluorography, as described previously (Bangs et al., 1986).

We next analyzed the effect of the mutations on EP-PARP. We metabolically labeled the cells with [3H]proline, immunoprecipitated the EP-PARP with a mixture of monoclonal antibodies, and analyzed the precipitates by SDS-PAGE. We found that the wild type EP-PARP (which contains a GPI anchor and, in two of its three forms, one N-glycosylation site) migrates as a smear, predominantly between 40 and 44 kDa (Figure 6B, lanes 1 and 7). As with GPEET-PARP, this electrophoretic behavior had been observed previously (Butikofer et al., 1997; Richardson et al., 1988; Treumann et al., 1997). When compared to the wild type protein, the mutant EP-PARPs had significantly higher electrophoretic mobility. EP-PARP from ConA 1-1 (lane 3) formed a smear with an Mr in the range of 33-37 kDa. ConA 4-1 (lane 5) consistently formed a bimodal distribution. The upper band had an Mr near that of wild type EP-PARP, and the lower band had an Mr ranging from 30-35 kDa. Identical results were obtained with EP-PARP labeled in its GPI anchor with [3H]palmitate (data not shown).

Because of the results presented earlier (Figures 4, 5, and 6A), we thought it likely that the mobility differences of EP-PARP from wild type and mutant cells could be attributed to alterations in N-glycosylation. To test this hypothesis, we treated the proteins with PNGase F. As predicted, the entire population of wild type EP-PARP when treated with this enzyme (Figure 6B, lane 2) migrated faster than untreated protein (lane 1). The entire population of EP-PARP from ConA 1-1 cells (lane 4) also migrated faster than untreated protein (lane 3), and considerably faster than wild type EP-PARP treated with PNGase F (lane 2). As for ConA 4-1, the lower band (lane 5) already migrated similar to that of EP-PARP from ConA 1-1 treated with PNGase F cells (lane 4) and its mobility was unaffected by PNGase F treatment (lane 6). This finding implies that the lower band (lane 5) has no N-glycan. In contrast the upper band of EP-PARP from ConA 4-1 (lane 5) merges with the lower band on treatment with PNGase F (lane 6), implying that it is N-glycosylated.

In summary these results indicate that most molecules of wild type EP-PARP contain an N-glycan which is removed by PNGase F. The same is true of EP-PARP from ConA 1-1 cells. In contrast, a large fraction of the EP-PARP from ConA 4-1 cells appears to contain no N-glycan. One unexpected finding, which will be addressed in the Discussion, was that EP-PARP from wild type cells when treated with PNGase F had a larger average Mr than those from the mutants treated with PNGase F. To determine whether the alteration in N-glycan structure between the wild type and mutant EP-PARPs could account for their difference in electrophoretic mobility, it was necessary to examine these structures, as described in the following section.

Structural analysis on N-glycans of EP-PARP

We used PNGase F to release N-glycans from wild type and mutant PARP and then studied the structure of these oligosaccharides using FAB-MS and GC-MS. The data are presented in Figure 7 and Table II, and the proposed partial structures are shown in Figure 8.


Figure 7. FAB-MS analysis of permethylated N-glycans. Wild type and mutant PARP N-glycans were permethylated and analyzed by FAB-MS as described in Materials and methods. The major signals at m/z 1579 (A) and 1824 (B and C) correspond to [M + Na]+ of Hex5HexNAc2 and Hex5HexNAc3, respectively. Other minor molecular ion signals observed include [M + Na]+ of Hex4HexNAc2 (m/z 1375), Hex4HexNAc3 (m/z 1620), and [M + H]+ of Hex5HexNAc2 (m/z 1557). The signals at 1280 and 1525 correspond respectively to A-type oxonium fragment ions (Hex5)HexNAc+ and (Hex5HexNAc1)HexNAc+, derived from cleavage at the N,N[prime]-diacetyl chitobiose cores of the two major components identified (Dell et al., 1993). (A) Wild type; (B) ConA 1-1; (C) ConA 4-1.

Figure 8. Proposed structures of the N-linked oligosaccharides present in wild type and mutant EP-PARP. (A) Wild type. (B) ConA 1-1 and ConA 4-1. The structures for the mutants were proposed from the experimental data described in the text and from the structure described previously for wild type (Treumann et al., 1997). The exact position of the terminal N-acetyllactosamine group present in both mutant N-glycans is unknown. Anomeric configurations in the mutant glycans have been inferred by comparison with conventional N-glycan structures.

For the wild type oligosaccharide, analysis of the permethylated glycans by FAB-MS revealed a component that produced an [M + Na]+ pseudomolecular ion at m/z 1579 (Figure 7A), suggesting a composition of Hex5HexNAc2. GC-MS monosaccharide compositional analysis revealed only Man and GlcNAc (data not shown), and the GC-MS methylation analysis revealed a terminal Man residue, a 3,6-di-O-substituted Man and a 4-O-substituted GlcNAc (Table II). Taken together, these data suggest that wild type EP-PARP contains a Man5GlcNAc2 high mannose N-glycan (Figure 8) in complete agreement with the structure previously described for the same molecule (Treumann et al., 1997).

For the mutant N-glycans, analysis of the permethylated glycans by FAB-MS revealed, in both spectra (Figure 7B,C), a major component that produced an [M + Na]+ pseudomolecular ion at m/z 1824 and another minor component at m/z 1375 (the latter was barely detectable in EP-PARP from ConA 4-1). These components correspond to Hex5HexNAc3 and Hex4HexNAc2 structures, respectively. Other minor species are discussed in the Figure 7 caption. Interestingly, the GC-MS monosaccharide compositional analysis revealed for glycans from both mutants GlcNAc, Man, and Gal, in a ratio of about 1.0:1.3:0.5, respectively (data not shown). We detected no sialic acid in the N-glycan of either mutant. The GC-MS methylation analysis of the mutant glycans revealed a terminal Man residue, a terminal Gal residue, a 2-O-substituted Man residue, a 3-O-substituted Man residue, a 3,6-di-O-substituted Man residue, and a 4-O-substituted GlcNAc (Table II). Taken together, these data suggest that EP-PARP from both mutants contain, as the major component, a hybrid type N-linked oligosaccharide terminated by an N-acetyllactosamine (the latter indicated by the terminal Gal and the 4-O-substituted GlcNAc). See proposed structure in Figure 8. This species differs strikingly from the high mannose type N-glycan present in wild type EP-PARP, also shown in Figure 8 (Treumann et al., 1997). We tentatively assigned the minor peak at m/z 1375 (Figure 7B and possibly in Figure 7C) as Man4GlcNAc2.

Although the major EP-PARP N-glycan species in both mutants are identical, they differ strikingly in occupancy of the single N-glycosylation site. ConA 1-1 has a high occupancy, and ConA 4-1 has a low occupancy. This conclusion is based on our finding that during purification of N-glycans the recovery from ConA 4-1 PARP was lower than that from ConA 1-1 PARP, as indicated by GC-MS sugar composition analysis and phenol/sulfuric acid colorimetric assay (data not shown). Also, as mentioned above, only a small fraction of ConA 4-1 EP-PARP was susceptible to PNGase F (Figure 6B, lane 6), undergoing a mobility shift on SDS-PAGE. In contrast, most of the ConA 1-1 EP-PARP was susceptible to this enzyme (Figure 6B, lane 4).

Discussion

We have studied two Con A resistant mutants of T.brucei, ConA 1-1 and ConA 4-1, which we isolated from independently mutagenized cultures. Both mutants resisted agglutination and killing by Con A, with ConA 4-1 being more resistant to this lectin (Figure 1). The two mutants differed in their binding to WGA (Figure 2B). Wild type and ConA 4-1 bound much lower levels of WGA than did ConA 1-1, confirming that the two mutants are phenotypically different.

Table II. GC-MS linkage analysis of N-linked glycan of wild type and mutant PARP
Peak no. Fragment ions PMAA Assignment Wild type ConA 1-1 ConA 4-1
1 101, 117, 129, 145, 161, 205 1,5-Di-O-acetyl-2,3,4,6-tetra-O-methylmannitol Terminal Man + + +
2 101, 117, 129, 145, 161, 205 1,5-Di-O-acetyl-2,3,4,6-tetra-O-methylgalacitol Terminal Gal - + +
3 101, 129, 161, 189 1,2,5-Tri-O-acetyl-3,4,6-tri-O-methyl-mannitol 2-O-Substituted Man - + +
4 87, 101, 117, 131, 161, 233 1,3,5-Tri-O-acetyl-2,4,6-tri-O-methyl-mannitol 3-O-Substituted Man - + +
5 87, 117, 129, 189, 233 1,3,5,6-Tetra-O-acetyl-di-O-2,4-methyl-mannitol 3,6-Di-O-Substituted Man + + +
6 88, 116, 158, 233 1,4,5-Tri-O-acetyl-2,3,6-tri-O-methyl-N-acetyl-glucosaminitol 4-O-Substituted GlcNAc + + +
PARP N-glycans were permethylated, acid hydrolyzed, and acetylated (Dell et al., 1993). The partially permethylated alditol acetates (PMAA) were analyzed by GC-MS as described in Materials and methods.
+ Indicates presence of PMAA; - indicates PMAA not detectable.

What is the molecular effect of the two mutations? Analysis of a blot of total membrane proteins probed with Con A indicated that both mutants are severely deficient in Con A-binding proteins (Figure 4). This observation indicated that the mutations affected glycosylation of many proteins, rather than expression of a single major Con A-binding glycoprotein. Furthermore, experiments with PNGase F showed that most of the Con A-binding was to N-linked oligosaccharides. From the Con A-probed blot we could conclude that the two mutants either synthesize reduced levels of N-glycans or they synthesize these glycans with an altered structure (with lower affinity for Con A). However, based on studies with the flagellar protein Fla1, it was clear that both mutants can in fact synthesize at least some N-glycans which are sensitive to cleavage by Endo H (Figure 5).

Our most informative studies were conducted on PARP, the major surface glycoprotein of procyclic T.brucei. The reduced binding to Con A was not due to reduced synthesis of EP-PARP, the form of PARP which contains a N-glycosylation site. In fact, the mutants contain considerably higher levels of EP-PARP than the parental wild type cells (Table I). This change in expression of EP-PARP is likely unrelated to the mutations as changes in the ratio of EP- to GPEET-PARP are known to occur over time in a clonal strain of parasite (Butikofer et al., 1997; Treumann et al., 1997). The GPEET-PARP was unaffected in both mutants in its mobility during SDS-PAGE (Figure 6A). Since this protein contains no N-glycosylation site, this result provides evidence that there is no direct effect of either mutation on the glycosylation or any other modification of the GPI anchor (although SDS-PAGE might not detect small effects). Consistent with that conclusion, we also found that the two mutants synthesize PP1, the precursor of the PARP GPI anchor (Field et al., 1991), at levels comparable to that of wild type (unpublished observations).

The situation was different with EP-PARP. In this case there were significant increases in electrophoretic mobility of EP-PARP in the two mutants, relative to that of wild type (Figure 6B). Furthermore, structural analysis of the N-glycans from the two mutants clearly demonstrated that they differed from that of the wild type (Figure 7, Table II). In the case of wild type we found a single species of N-glycan, Man5GlcNAc2, in precise agreement with a previous report (Treumann et al., 1997). In contrast, both mutants have a major species which is a hybrid type N-glycan, terminated by N-acetyllactosamine. ConA 1-1, and probably ConA 4-1, have a minor species of N-glycan on EP-PARP which is probably Man4GlcNAc2. The EP-PARPs from the two mutants differ most significantly in occupancy of the N-glycosylation site. Based primarily on the shift in SDS-PAGE mobility following PNGase F treatment (Figure 6B), the EP-PARP from ConA 1-1 has a glycosylation site with a high occupancy, whereas that from ConA 4-1 has a low occupancy. Because PARP is a major surface glycoprotein in procyclic cells, the low occupancy seen in ConA 4-1 might contribute to its greater resistance to Con A killing (Figure 1).

The fact that EP-PARP molecules in both ConA 1-1 and ConA 4-1 are modified by the same major N-glycan raises the possibility that the two mutants have defects in the same gene. Although not yet proven rigorously, we think this is not the case. In an unpublished collaborative study with George Quellhorst, Jessica O'Rear, and Sharon Krag (Johns Hopkins University), we have found that ConA 1-1, but not ConA 4-1 parasites, apparently has a reduced level of polyprenol reductase, a key enzyme in synthesis of the oligosaccharide-lipid precursor molecule. Further studies, now underway, will be needed to determine how a defect in this enzyme could ultimately account for the alteration in the N-glycan structure in EP-PARP and other glycoproteins. We are also in the process of identifying the enzymatic defect in ConA 4-1.

The identification of the structures of the EP-PARP N-glycan in the two mutants raises the question of why the hybrid oligosaccharide on the two mutants is not modified by sialylation. The poly-N-acetyllactosamine chains which form the side chain on the PARP GPI anchor are efficiently sialylated by a cell surface trans-sialidase (Engstler et al., 1993; Ferguson et al., 1993; Pontes de Carvalho et al., 1993; Treumann et al., 1997). Therefore, it is surprising that the N-acetyllactosamine on the N-glycan is also not sialylated. It is unlikely that we selectively lost sialylated N-glycans during their purification as the same purification procedure used for other glycoproteins resulted in efficient recovery of sialylated N-glycans (unpublished observations). One possibility for the absence of sialylation is that conformational constraints of EP-PARP prevented the N-glycan from gaining access to the trans-sialidase. For example, the long EP repeat sequence which is C-terminal to the N-glycosylation site may position it too far from the cell surface where the trans-sialidase is thought to be located (this hypothesis is consistent with a conformational model proposed for EP-PARP (Roditi et al., 1989)).

The structural analysis of the mutant EP-PARP N-glycans, and especially the finding of low occupancy in the N-glycosylation site of EP-PARP from ConA 4-1, clarifies the Con A binding specificity (although we do not know if similar alterations are present on other surface glycoproteins in the mutant cells). It is known that the Man5GlcNAc2 on wild type EP-PARP binds Con A with high affinity (Treumann et al., 1997), whereas the hybrid oligosaccharide, present on both mutants, might bind with a lower affinity due in part to the N-acetyllactosamine group capping a terminal [alpha]Man residue. Another important cause of low Con A binding (indicated by the presence of a 3-O-substituted Man in the mutant glycans; Table II) is the absence of the [alpha]1-6-linked Man residue which, in the wild type glycan, is linked to the Man[alpha]1-6Man[beta]1- arm of the tri-mannosyl core (see Figure 8). A similar explanation for low Con A affinity has been presented for a CHO Con A-resistant cell line (Hunt, 1982). Other studies have also demonstrated that the [alpha]1-6 linked Man residue makes a major contribution to the free energy of Con A binding (Carver and Brisson, 1984). Why is the [alpha]1-6 Man residue missing in the mutants? It is probably also missing in the Hex4HexNAc2 (observed in Figure 7B, and possibly in 7C), which we assume is Man4GlcNAc2 (an oligosaccharide which also has a low affinity for Con A; Hunt, 1982). We assume that this species is an intermediate in synthesis of the N-acetyllactosamine-capped species present in both mutants (Figure 8). We have preliminary evidence that the oligosaccharide-lipid precursor in the mutant cells is smaller than that of wild type, and although we have not yet determined its structure it may also be missing the branch initiated by the [alpha]1-6 linked Man. The absence of this branch would result in a high mannose core containing four Man residues (as in the mutants) instead of five (as in the wild type). As for the WGA binding specificity, the N-acetyllactosamine present on the mutants provides a strong ligand for this lectin because of the presence of the C-4 substituted GlcNAc (Goldstein and Poretz, 1986). For ConA 4-1, the low occupancy of the EP-PARP N-glycosylation site would reduce WGA binding.

There is a dilemma concerning the alteration of the electrophoretic migration of EP-PARP in the mutants relative to wild type (Figure 6B). It would be surprising if the slight difference in size of the N-glycan structure between wild type and ConA 1-1 (Hex5HexNAc2 versus Hex5HexNAc3) could explain the difference in electrophoretic mobility between the EP-PARPs from these two cell lines (compare lanes 1 and 3). Furthermore, these differences persisted after PNGase treatment in that the enzyme-treated EP-PARP from the mutants migrated faster than that of the wild type (compare lanes 2, 4, and 6). Therefore, we think that there may be differences between the EP-PARPs in the wild type and mutant cells in addition to the altered N-glycosylation. These differences could be secondary effects, dictated by the primary alteration in EP-PARP's N-glycan structure. Alternatively, the differences could result from altered N-glycosylation of proteins involved in the posttranslational modification of EP-PARP. The differences could reside in modifications of the GPI anchor, in the length of the polypeptide chain (different EP-PARP gene products differ in the number of EP repeats, ranging from 22 to 30 copies), or in some other unknown modification. We are currently trying to identify these differences.

The mechanism by which Con A kills procyclic T.brucei is not well understood although it has been reported that this cell death resembles apoptosis (Welburn et al., 1996). We have found that the killing does not depend upon the tetravalency of Con A, as the divalent lectin, succinyl Con A (Fraser et al., 1976), also killed the trypanosomes at a concentration comparable to that required for native Con A (unpublished data). In contrast, the two monoclonal antibodies recognizing polypeptide epitopes on EP-PARP or GPEET-PARP did not kill the parasite (unpublished observations), suggesting that the trigger for this process is a carbohydrate-dependent phenomenon.

There recently have been significant advances in trypanosome molecular biology, and genetic complementation has been achieved with this parasite (Sommer et al., 1996). Using these techniques, we are anxious to identify the defective genes in these mutants. This knowledge should provide another very powerful tool for investigating glycosylation mechanisms in this ancient eukaryotic cell. The mutants should also be useful for studying the biological properties of this parasite, for example its interaction with its insect vector.

Materials and methods

Procyclic trypanosomes

Wild type procyclic T.brucei brucei (strain 427-60) and another 427 strain frozen 6 years earlier (in 1988) were gifts of Dr. Mary G.-S.Lee (New York University). These and the Con A-resistant cells generated in this study were grown at 25-27°C in SDM-79 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Life Technologies) and 20 µg/ml gentamicin (Life Technologies), as described (Brun and Shonenberger, 1979). In this article SDM-79 refers to the supplemented medium. The cells were maintained at 5 × 105 to 2 × 107 cells/ml. The standard protocol for centrifugation of cells was 2000 × g for 15 min at 4°C.

Lectins and antibodies

Con A, Con A-biotin, wheat germ agglutinin (WGA), and WGA-fluorescein were from Sigma. Con A-fluorescein was from Calbiochem. Monoclonal antibody TBRP 1/247 (IgG[gamma]1) specifically recognizes the dipeptidyl repeats of EP-PARP (Richardson et al., 1988). Monoclonal antibody TBRP 1/346 (IgG1) specifically recognizes the N-terminal domain of EP-PARP (Richardson et al., 1988). Monoclonal antibody 9G4 (IgG1) was generated against a recombinant protein containing the pentapeptidyl repeat sequence of GPEET-PARP linked to the C-terminus of glutathione-S-transferase. All monoclonal antibodies were purified from mouse ascites fluids (Richardson et al., 1988). Anti-Fla1 antibody (Nozaki et al., 1996) was provided by Drs. Tomo Nozaki and George Cross (Rockefeller University).

Mutagenesis of procyclic trypanosomes

The protocol for mutagenesis was modified from that of King and Turco (King and Turco, 1988). Late log-phase wild type cells (250 ml, 1.2-1.4 × 107 cells/ml) were harvested by centrifugation. The cell pellet was resuspended in 50 ml of freshly made SDM-79 medium containing 4 µg/ml of N-methyl-3-nitro-1-nitrosoguanidine (Sigma) and the suspension was incubated at room temperature for 3-4 h. After mutagenesis, cells were centrifuged and washed once with PBSG (PBS supplemented with 3% glucose). Since the mutagenesis resulted in the death of about 90% of the cells, conditioned medium was used to support cell growth at a low density. Conditioned medium was made by mixing equal volumes of fresh SDM-79 and filtered SDM-79 in which a trypanosome culture had been grown to about 1 × 107 cells/ml. The washed cells were harvested by centrifugation, resuspended in 500 ml of conditioned medium, and grown for an additional 7-10 days to allow expression of mutant phenotypes.

Selection of Con A-resistant mutant cells

For selection of the mutant ConA 1-1, the 500 ml culture after 10 days of growth was harvested by centrifugation and resuspended in 250 ml of SDM-79 medium containing 15 µg/ml Con A. After 24 h at 27°C, most parasites had rounded up, an abnormal morphology characteristic of dying cells (Welburn et al., 1996). After 2 days, 200 ml of medium containing 15 µg/ml Con A were added to the flask. After four days, the cells were harvested and resuspended in 200 ml of conditioned medium containing 15 µg/ml Con A. Dividing cells were visible after 10 days of selection with Con A. The surviving cells were then counted and cloned by limiting dilution.

The mutant ConA 4-1 was selected from a different mutagenized culture as follows. Parasites were centrifuged and resuspended in 200 ml of SDM-79 medium containing 50 µg/ml Con A. Cells were then shaken gently for 30 min at room temperature. Some of the cells agglutinated, whereas others did not. The cell suspension was centrifuged to deplete agglutinated cells (40 × g, 25°C, 15 min) and those remaining in the supernatant were selected for 5 additional days with 50 µg/ml Con A in conditioned medium. The surviving cells were cloned by limiting dilution.

After cloning, both ConA 1-1 and ConA 4-1 were grown in the absence of Con A and they maintained their Con A resistance for at least 6 months. However, upon longer cultivation (~18 months), some revertants to Con A sensitivity were found. Therefore, all the experiments were conducted either on cells stored in liquid nitrogen soon after the original cloning or on recloned cells.

Con A agglutination assay

Wild type or mutant cells (1-1.3 × 107 cells/ml) in SDM-79 medium were treated with 10 µg/ml Con A. After 30 min at 25 °C, agglutinated cells were centrifuged (14 × g, 25°C, 5 min, Sorvall 24S Microspin). Cells remaining in the medium were counted, and the percentage agglutination was calculated.

Preparation of trypanosome membrane proteins

Log phase cells (1 × 108) were washed in PBS and then lysed in 1 ml of 5 mM EDTA by two cycles of freeze (dry ice-ethanol) and thaw (37°C). Crude membranes were centrifuged (15 min, 4°C, 12,000 × g, Fisher Microfuge) and washed in 5 mM EDTA. The pellet, which we refer to as 'membrane proteins," was then dissolved in 100 µl of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% SDS and stored at -80°C until use.

Structural analysis of PARP glycans

Total PARP, purified from 1010 wild type or mutant cells using organic extraction and Octyl-Sepharose chromatography (Ferguson et al., 1993), was dried and then resuspended by sonication in 100 µl of 50 mM ammonium bicarbonate, pH 8.4. The solution was then treated with 2 µl of Flavobacterium meningosepticum PNGase F (500,000 units/ml, New England Biolabs) at 37°C for 48 h. Then samples were loaded onto a C18 Sep-Pack column (Waters) and eluted with 5% acetic acid (Dell et al., 1993). For fast atom bombardment mass spectrometry (FAB-MS), the glycans were permethylated using the NaOH/dimethyl sulfoxide slurry method (Dell et al., 1993). FAB mass spectra of the permethylated samples were obtained on an Autospec OA-TOF mass spectrometer (Micromass, UK) fitted with a cesium ion gun operated at 25 kV. Samples were dissolved in methanol for loading onto the probe tip coated with monothioglycerol as matrix. For linkage analysis, partially methylated alditol acetates (PMAA) for GC-MS analysis were generated from permethyl derivatives (Dell et al., 1993) by hydrolysis (2 M TFA, 121°C, 2 h), reduction (10 mg/ml NaBH4, 25°C, 2 h), and acetylation (acetic anhydride, 100°C, 1 h). GC-MS was carried out using a Hewlett-Packard Gas Chromatograph 6890 connected to a HP 5973 Mass Selective Detector. Sample was dissolved in hexane prior to splitless injection into a HP-5MS with fused silica capillary column (30 m, 0.25 mm I.D., HP). The column head pressure was maintained at around 8.2 psi to give a constant flow rate of 1 ml/min using helium as carrier gas. Initial oven temperature was held at 60°C for 1 min, increased to 90°C in 1 min, and then to 290°C in 25 min. For monosaccharide composition analysis, released N-glycans were methanolyzed with 0.5 M methanolic-HCl (Supelco) at 80°C for 16 h, re-N-acetylated with 500 µl of methanol, 10 µl of pyridine, and 50 µl of acetic anhydride, and then treated with the Sylon HTP TMS-derivatizing reagent (Supelco) for 20 min at room temperature, dried down, and redissolved in hexane. GC-MS analysis of the TMS derivatives was performed on the same HP system using a temperature gradient of 60°C to 140°C at 25°C/min, increased to 250°C at 5°C/min, and then increased to 300°C at 10°C/min.

Acknowledgments

We thank Sharon Krag, Stephen Beverley, Michael Ferguson, Mary Lee, Gerald Hart, Albert Descoteaux, Hans-Jurgen Hoppe, Kenneth Milne, Yasu Morita, and James Morris for helpful discussions. We also appreciate assistance from Holly Berkovitz, Karen Chadwick, Jennifer Senft, Denise Wenzel, Kuei-Ching Fan, Robert Beecroft, and Joseph Margolick in various aspects of this project. We thank Mary Lee for the wild type trypanosomes and Tomo Nozaki and George Cross for providing the Fla1 antibody. This work was supported by an NIH grant (AI27608) to P.T.E., an Academia Sinica biotechnology grant to K.H.K., a Natural Sciences and Engineering Research Council grant to T.W.P., an Academia Sinica Postdoctoral Fellowship to K.Y.H., and a postdoctoral fellowship from CONICIT (Venezuela) to A.A.S.

Abbreviations

PARP, procyclic acidic repetitive protein (procyclin); EP-PARP, a form of PARP with glu-pro repeats; GPEET-PARP, a form of PARP with gly-pro-glu-glu-thr repeats; GPI, glycosyl phosphatidylinositol; FAB-MS, fast atom bombardment mass spectrometry; GC-MS, gas chromatography-mass spectrometry; PMAA, permethylated alditol acetate; Con A, concanavalin A; WGA, wheat germ agglutinin; PBS, phosphate-buffered saline; PBSG, PBS containing 3% glucose; FBS, fetal bovine serum; PNGase F, peptide N4-( N-acetyl-[beta]-glucosaminyl) asparagine amidase F; EndoH, endo-[beta]-N-acetylglucosaminidase H; SDM-79, a culture medium for trypanosomes; SM medium, a culture medium for trypanosomes; PVDF, polyvinylidene difluoride; BCIP, 5-bromo-4-chloro-3-indolylphosphate; NBT, tetranitroblue tetrazolium.

References

Bangs ,J.D., Andrews,N.W., Hart,G.W. and Englund,P.T. (1986) Posttranslational modification and intracellular transport of a trypanosome variant surface glycoprotein. J. Cell Biol., 103, 255-263. MEDLINE Abstract

Brun ,R. and Shonenberger,M. (1979) Cultivation and in vitro cloning of procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Acta Tropica, 36, 289-292. MEDLINE Abstract

Butikofer ,P., Ruepp,S., Boschung,M. and Roditi,I. (1997) `GPEET' procyclin is the major surface protein of procyclic culture forms of Trypanosoma brucei brucei strain 427. Biochem. J., 326, 415-423. MEDLINE Abstract

Carver ,J.P. and Brisson,J.-R. (1984) The three-dimensional structure of N-linked oligosaccharides. In Ginsburg,V. and Robbins,P.W. (eds.), Biology of Carbohydrates, Vol. 2. John Wiley, New York, pp. 289-331.

Clayton ,C.E. and Mowatt,M.R. (1989) The procyclic acidic repetitive proteins of Trypanosoma brucei. Purification and post-translational modification. J. Biol. Chem., 264, 15088-15093. MEDLINE Abstract

Cunningham ,I. (1977) New culture medium for maintenance of tsetse tissues and growth of trypanosomatids. J. Protozool., 24, 325-329. MEDLINE Abstract

Dell ,A., Khoo,K.-H., Panico,M., McDowell,R.A., Etienne,A.T., Reason,A.J. and Morris,H.R. (1993) In Fukuda,M. and Kobata,A. (eds.), Glycobiology, A Practical Approach. Oxford University Press, New York, pp. 187-222.

Engstler ,M., Reuter,G. and Schauer,R. (1993) The developmentally regulated trans-sialidase from Trypanosoma brucei sialylates the procyclic acidic repetitive protein. Mol. Biochem. Parasitol., 61, 1-13. MEDLINE Abstract

Ferguson ,M.A.J., Murray,P., Rutherford,H. and McConville,M.J. (1993) A simple purification of procyclic acidic repetitive protein and demonstration of a sialylated glycosyl-phosphatidylinositol membrane anchor. Biochem. J., 291, 51-55. MEDLINE Abstract

Field ,M.C., Menon,A.K. and Cross,G.A.M. (1991) Developmental variation of glycosylphosphatidylinositol membrane anchors in Trypanosoma brucei. Identification of a candidate biosynthetic precursor of the glycosylphosphatidylinositol anchor of the major procyclic stage surface glycoprotein. J. Biol. Chem., 266, 8392-8400. MEDLINE Abstract

Field ,M.C., Menon,A.K. and Cross,G.A.M. (1991) A glycosylphosphatidylinositol protein anchor from procyclic stage Trypanosoma brucei: lipid structure and biosynthesis. EMBO J., 10, 2731-2739. MEDLINE Abstract

Fraser ,A.R., Hemperly,J.J., Wang,J.L. and Edelman,G.M. (1976) Monovalent derivatives of concanavalin A. Proc. Natl. Acad. Sci. USA, 73, 790-794. MEDLINE Abstract

Goldstein ,I.J. and Poretz,R.D. (1986) Isolation, physicochemical characterization, and carbohydrate-binding specificity of lectins. In Liener,I.E., Sharon,N. and Goldstein,I.J. (eds.), The Lectins: Properties, Functions, and Applications in Biology and Medicine. Academic Press, Orlando, pp. 33-247.

Hunt ,L.A. (1982) Lectin affinity chromatography of glycopeptides and oligosaccharides from normal and lectin-resistant Chinese-hamster ovary cells. Biochem. J., 205, 623-630. MEDLINE Abstract

King ,D.L. and Turco,S.J. (1988) A ricin agglutinin-resistant clone of Leishmania donovani deficient in lipophosphoglycan. Mol. Biochem. Parasitol., 28, 285-293. MEDLINE Abstract

McConville ,M.J. and Ferguson,M.A.J. (1993) The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes. Biochem. J., 294, 305-324. MEDLINE Abstract

Mowatt ,M.R. and Clayton, C.E. (1987) Developmental regulation of a novel repetitive protein of Trypanosoma brucei. Mol. Cell Biol., 7, 2838-2844. MEDLINE Abstract

Mowatt ,M.R., Wisdom,G.S. and Clayton,C.E. (1989) Variation of tandem repeats in the developmentally regulated procyclic acidic repetitive proteins of Trypanosoma brucei. Mol. Cell Biol., 9, 1332-1335. MEDLINE Abstract

Nozaki ,T., Haynes,P.A. and Cross,G.A.M. (1996) Characterization of the Trypanosoma brucei homologue of a Trypanosoma cruzi flagellum-adhesion glycoprotein. Mol. Biochem. Parasitol., 82, 245-255. MEDLINE Abstract

Pontes de Carvalho ,L.C., Tomlinson,S., Vandekerckhove,F., Bienen,E.J., Clarkson,A.B., Jiang,M.-S., Hart,G.W. and Nussenzweig,V. (1993) Characterization of a novel trans-sialidase of Trypanosoma brucei procyclic trypomastigotes and identification of procyclin as the main sialic acid acceptor. J. Exp. Med., 177, 465-474. MEDLINE Abstract

Reinhold ,B.B., Hauer,C.R., Plummer,T.H. and Reinhold,V.N. (1995) Detailed structural analysis of a novel, specific O-linked glycan from the prokaryote Flavobacterium meningosepticum. J. Biol. Chem., 270, 13197-13203. MEDLINE Abstract

Richardson ,J.P., Beecroft,R.P., Tolson,D.L., Liu,M.K. and Pearson,T.W. (1988) Procyclin: an unusual immunodominant glycoprotein surface antigen from the procyclic stage of African trypanosomes. Mol. Biochem. Parasitol., 31, 203-216. MEDLINE Abstract

Robbins ,P.W. (1994) The ALG (asparagine linked glycosylation) genes. In Rothblatt,J., Novick,P. and Stevens,T. (eds.), Guidebook to the Secretory Pathway. Oxford University Press, Oxford, pp. 57-59.

Roditi ,I., Carrington,M. and Turner,M. (1987) Expression of a polypeptide containing a dipeptide repeat is confined to the insect stage of Trypanosoma brucei. Nature, 325, 272-274. MEDLINE Abstract

Roditi ,I., Schwarz,H., Pearson,T.W., Beecroft,R.P., Liu,M.K., Richardson,J.P., Buhring,H.J., Pleiss,J., Bulow,R., Williams,R.O. and Overath,P. (1989) Procyclin gene expression and loss of the variant surface glycoprotein during differentiation of Trypanosoma brucei. J. Cell. Biol., 108, 737-746. MEDLINE Abstract

Ruepp ,S., Furger,A., Kurath,U., Renggli,C.K., Hemphill,A., Brun,R. and Roditi,I. (1997) Survival of Trypanosoma brucei in the tsetse fly is enhanced by the expression of specific forms of procyclin. J. Cell Biol., 137, 1369-1379. MEDLINE Abstract

Sommer ,J.M., Hua,S., Li,F., Gottesdiener,K.M. and Wang,C.C. (1996) Cloning by functional complementation in Trypanosoma brucei. Mol. Biochem. Parasitol., 76, 83-89. MEDLINE Abstract

Stanley ,P. and Ioffe,E. (1995) Glycosyltransferase mutants: key to new insights in glycobiology. FASEB J., 9, 1436-1444. MEDLINE Abstract

Treumann ,A., Zitzmann,N., Hulsmeier,A., Prescott,A.R., Almond,A., Sheehan,J. and Ferguson,M.A. (1997) Structural characterisation of two forms of procyclic acidic repetitive protein expressed by procyclic forms of Trypanosoma brucei. J. Mol. Biol., 269, 529-547. MEDLINE Abstract

Turco ,S.J. and Descoteaux,A. (1992) The lipophosphoglycan of Leishmania parasites. Annu. Rev. Microbiol., 46, 65-94. MEDLINE Abstract

Welburn ,S.C., Dale,C., Ellis,D., Beecroft,R. and Pearson,T.W. (1996) Apoptosis in procyclic Trypanosoma brucei rhodesiense in vitro. Cell Death Differ., 3, 229-236.


4To whom correspondence should be addressed at: Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA


This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 6 Feb 1999
Copyright©Oxford University Press, 1999.