Protein structure controls the processing of the N-linked oligosaccharides and glycosylphosphatidylinositol glycans of variant surface glycoproteins expressed in bloodstream form Trypanosoma brucei

Nicole Zitzmann2,3, Angela Mehlert2, Sandra Carroué3, Pauline M. Rudd3 and Michael A. J. Ferguson1,2

2Division of Molecular Parasitology and Biological Chemistry, Department of Biochemistry, The Wellcome Trust Building, University of Dundee, Dundee DD1 5EH, UK and 3Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK

Received on June 3, 1999; revised on August 20, 1999; accepted on September 1, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The variant surface glycoproteins (VSGs) of Trypanosoma brucei are a family of homodimeric glycoproteins that adopt similar shapes. An individual trypanosome expresses one VSG at a time in the form of a dense protective mono­layer on the plasma membrane. VSG genes are expressed from one of several polycistronic transcription units (expression sites) that contain several expression site associated genes. We used a transformed trypanosome clone expressing two different VSGs (VSG121 and VSG221) from the same expression site (that of VSG221) to establish whether the genotype of the trypanosome clone or the VSG structure itself controls VSG N-linked oligosaccharide and GPI anchor glycan processing. In-gel release and fluorescent labeling of N-linked oligosaccharides and on-blot fluorescent labeling and release of GPI anchor glycans were employed to compare the carbohydrate structures of VSG121 and VSG221 when expressed individually in wild-type trypanosome clones and when expressed together in the transformed trypanosome clone. The data indicate that the genotype of the trypanosome clone has no effect on the N-linked oligosaccharide structures present on a given VSG variant and only a minor effect on the GPI anchor glycans. The latter is most likely an effect of changes in inter-VSG packing when two VGSs are expressed simultaneously. Thus, N-linked oligosaccharide and GPI anchor processing enzymes appear to be constitutively expressed in bloodstream form African trypanosomes and the tertiary and quaternary structures of the VSG homodimers appear to dictate the processing and glycoform microhetero­geneity of surface-expressed VSGs.

Key words: trypanosome/variant surface glycoprotein /N-glycosylation/glycosylphosphatidylinositol


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The bloodstream forms of Trypanosoma brucei, in common with the other African trypanosomes, express a surface coat (Vickerman and Luckins, 1969Go) composed of about 10 million copies of 50–55 kDa variant surface glycoprotein (VSG) (Cross, 1975Go) arranged as a dense monolayer of homodimers on the parasite surface (Mehlert et al., 1998bGo). The VSG coat acts as a macromolecular diffusion barrier that prevents the approach of macromolecules, such as the components of the alternative complement pathway, to the plasma membrane while allowing the free diffusion of small nutrient molecules to underlying transmembrane transporter systems (Cross and Johnson, 1976Go). The trypanosome genome contains several hundred VSG genes that encode immunologically distinct VSGs. These are expressed one at a time by individual trypanosomes but the switching of VSG gene expression by a few cells (antigenic variation) enables the parasite population to evade specific humoral immune attack (Pays et al., 1994Go; Cross, 1996Go).

VSG sequences can be classified into one of four subclasses (class-1, 2, 3, or 4), based on peptide homology and Cys residue conservation in the C-terminal domain (representing 25–30% of the mature VSG polypeptide), and into one of three subtypes (type-A, B, or C), based on conserved Cys residues and secondary structure motifs in the larger N-terminal domain (Carrington et al., 1991Go; Carrington and Boothroyd, 1996Go). Crystallography of the dimerized N-terminal domains of two type-A VSGs (ILTat.1.24 and MITat.1.2, also known as VSG221) showed these domains adopt remarkably similar tertiary structures despite minimal (16%) sequence conservation (Blum et al., 1993Go) and it is thought that all VSG N-terminal domains adopt the same protein fold.

VSG molecules are attached to the plasma membrane via a covalent linkage of the C-terminal amino acid {alpha}-carboxyl group of each monomer to a glycosylphosphatidylinositol (GPI) membrane anchor. The mature VSG GPI anchors contain the core structure of ethanolamine-HPO4-6Man{alpha}1-2Man{alpha}1-6Man{alpha}1-4GlcN{alpha}1-6PI, common to all GPI membrane anchors (McConville and Ferguson, 1993Go), but this is modified in the class-1 and class-2 VSGs by galactose side chains that are unique to VSGs (Ferguson et al., 1988Go; Strang et al., 1993Go; Mehlert et al., 1998aGo) (Figure 1). The predicted three-dimensional structure of the complete anchor of the class-1 VSG MITat. 1.4 (also known as VSG117) suggests that it forms a dense plate-like structure of carbohydrate upon which the VSG polypeptide sits (Homans et al., 1989Go).



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Fig. 1. Primary structures of class-1 VSG121 and class-2 VSG221 used in this study. The most abundant structure at each glycosylation site is shown, with the range of structures present at each glycosylation site indicated in brackets. The carbohydrate structures are taken from (Ferguson et al., 1988Go; Zamze et al., 1990, 1991; Strang et al., 1993Go; Mehlert et al., 1998aGo).

 
The N-linked oligosaccharides of several T.brucei VSGs have been solved (Zamze et al., 1990Go, 1991; Strang et al., 1993Go). Each VSG variant contains one, two, or three N-linked oligosaccharides per monomer. The class-1 VSGs, like VSG117 and MITat.1.6 (also known as VSG121), typically contain one N-glycosylation site in the C-terminal domain (about 50 residues from the mature C-terminus) that is occupied by Man9-5GlcNAc2 oligomannose structures (Figure 1). The class-2 VSGs generally contain two glycosylation sites, one in the C-terminal domain (only five or six residues from the mature C-terminus) and the other in the N-terminal domain. For the class-2 variant VSG221, the C-terminal site is principally occupied by oligomannose structures, mostly Man9-7GlcNAc2 while the N-terminal site is occupied by a variety of structures ranging from the unusual Man3GlcNAc2, GlcNAc(Man3GlcNAc2) and Gal-GlcNAc(Man3GlcNAc2) structures to the conventional biantennary complex oligo­saccharide (GalGlcNAc)2Man3GlcNAc2 (Figure 1).

Apart from providing some resistance to proteolysis (Reinwald, 1985Go) and being necessary for intracellular transport for some, but not most, VSGs (Ferguson et al., 1986Go), the role of VSG N-glycosylation is unclear. However, the first few residues of the N-linked oligosaccharides at Asn296 in the N-terminal domain of VSG221 occupy similar space to an {alpha}-helix of peptide in the VSG ILTat.1.24 structure (Blum et al., 1993Go), suggesting that, at least at this site, carbohydrate can substitute for protein within the overall VSG fold. Similarly, the N-linked oligosaccharides located in the C-terminal domains of the class-1 and class-2 VSGs, together with the GPI-anchor glycans, may compensate for differences in VSG C-terminal domain protein structure and ensure the barrier characteristics of different VSG coats.

In general, glycoprotein carbohydrate processing is governed by (1) the cell phenotype, i.e., the levels of the processing exoglycosidases and glycosyltransferases and the levels of the relevant sugar-donors and sugar-donor transporters, and (2) the structure of the protein at and around the glycosylation sites (Rudd and Dwek, 1997Go; Varki, 1998Go). The effect of the cell phenotype on VSG carbohydrate processing has been demonstrated by expressing VSG in procyclic forms of T.brucei. In this case, VSG is expressed with GPI anchor and N-linked oligosaccharide structures typical of procyclic trypanosomes (Bangs et al., 1997Go; Paturiaux-Hanocq et al., 1997Go).

In bloodstream form trypanosomes, VSG genes are expressed from one of several polycistronic transcription units (expression sites) that contain several expression site associated genes (ESAGs). Therefore, it could be argued that differences in VSG N-linked oligosaccharides and GPI anchors are due, either directly or indirectly, to variations in the coexpressed ESAG gene products. Alternatively, all of the carbohydrate processing machinery could be constitutively expressed and the extent of N-linked oligosaccharide and GPI glycan processing in bloodstream form trypanosomes could be controlled by the VSG polypeptide itself. In this paper, we have addressed this issue by analyzing the N-linked oligosaccharides and GPI anchor glycans from two VSG variants expressed simultaneously from the same expression site in bloodstream form trypanosomes.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
VSG N-linked oligosaccharide processing is controlled by VSG protein structure
The soluble forms of VSG121 and VSG221 were purified individually from wild-type subclones, expressing single VSG variants, and as a mixture from the genetically engineered double-expressing clone 121/221. The latter has been shown to express a VSG coat that is a mosaic of both VSG homodimers (Munoz-Jordan et al., 1996Go). The VSGs were resolved by SDS–PAGE and individual Coomassie blue–stained VSG bands (Figure 2) were excised. The N-linked oligosaccharides were released from the VSGs by in-gel PNGase F digestion, labeled with the fluorophore 2-aminobenzamide (2-AB ) by reductive amination (Küster et al., 1997Go), and analyzed by normal-phase HPLC (Guile et al., 1996Go) (Figure 3). The sensitivity and resolving power of this system is such that even relatively minor differences in N-glycosylation become evident from a comparison of HPLC oligosaccharide profiles.



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Fig. 2. Coomassie blue–stained SDS–PAGE gel of VSGs purified from wild-type clone 221 (lane 1) and the double-expressing 121/221 clone (lane 2) described in Munoz-Jordan et al., 1996Go. Molecular weight markers (kDa) are shown on the left.

 


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Fig. 3. Scheme used to obtain 2-AB labeled N-linked oligosaccharides and 2-AB labeled GPI anchor glycans from VSGs. The protocols are described in detail in (Küster et al., 1997Go) and (Zitzmann and Ferguson, 1999Go), respectively.

 
The N-linked oligosaccharide HPLC profiles for VSG221 (Figure 4a,b) and for VSG121 (Figure 4c,d) are different from each other, reflecting the known differences in N-glycosylation between these VSG variants (Zamze et al., 1990Go, 1991; Strang et al., 1993Go). Significantly, the N-linked oligosaccharide HPLC profiles were identical for each VSG variant, regardless of whether the VSG was expressed alone (Figure 4a,c) or together with the other variant (Figure 4b,d). To confirm that the HPLC profiles reflected N-linked oligosaccharides, the 2-AB labeled oligosaccharides from VSG121 were digested with jack bean {alpha}-mannosidase. The profile seen in Figure 4c was replaced with one major product eluting at 43 min (2.65 Gu), corresponding to the expected product of Man1GlcNAc2 (data not shown).



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Fig. 4. Normal-phase HPLC chromatography of 2-AB-labeled N-linked oligosaccharides. Labeled oligosaccharides from VSG221 prepared from clone 221 trypanosomes (a) and from the double-expressing (de) 121/221 clone (b) and labeled oligosaccharides from VSG121 prepared from clone 121 trypanosomes (c) and from the double-expressing 121/221 clone (d) were analyzed by normal phase HPLC. The structural assignments are based on the elution positions of the labeled peaks relative to labeled glucose oligomers (Guile et al., 1996Go; Küster et al., 1997Go) and are consistent with the deduced compositions of the unlabeled oligosaccharides, determined by matrix assisted laser desorption ionization-time of flight mass spectrometry (data not shown), and with the structures previously published for VSG221 (Zamze et al., 1991Go) and VSG121 (Zamze et al., 1990Go; Strang et al., 1993Go). The unassigned peaks marked with stars in (a) are most likely the unusual GlcNAc(Man3GlcNAc2) and GalGlcNAc(Man3GlcNAc2) structures present at Asn296 in VSG221 (Zamze et al., 1991Go).

 
VSG GPI anchor glycan processing is controlled by VSG protein structure
The same VSGs, plus VSG117, were resolved by SDS–PAGE and transferred to PVDF membrane. After staining with amido black, VSG bands were excised and the GPI anchor glycans were labeled with 2-AB by nitrous acid deamination followed by reductive amination (Zitzmann and Ferguson, 1999Go). This procedure attaches 2-AB to the aldehyde group of the 2,5-anhydromannose that is generated from the GPI GlcN residue by nitrous acid deamination. After extensive washing of the PVDF membrane to remove reagents, the 2-AB-labeled GPI glycans were released from the VSG polypeptide, and consequently from the PVDF membrane, by scission of the posphodiester linkage to the ethanolamine phosphate bridge with aqueous HF (Figure 3) (Zitzmann and Ferguson, 1999Go). Analysis of the labeled GPI glycans by Bio-Gel P4 chromatography showed that the two class-1 variants, VSG117 and VSG121, produced almost identical profiles (Figure 5a,c) whereas the class-2 variant VSG221 gave a significantly different profile of, on average, larger structures (Figure 5b). The Bio-Gel P4 profile for the 2-AB-labeled VSG117 GPI glycans produced by this "off-blot" method (Figure 5a) is identical to that produced by preparative-scale methods where the identities of the labeled glycans have been confirmed by electrospray mass spectrometry (Zitzmann and Ferguson, 1999Go).



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Fig. 5. BioGel P4 gel-filtration of 2-AB labeled GPI anchor glycans. Labeled GPI glycans were prepared from VSG117 (a), VSG221 (b), and VSG121 (c) using trypanosome clones 117, 221 and 121, respectively. Labeled GPI glycans were also prepared from a mixture of VSG121 and VSG221 (d), and from individual VSG221 (e) and VSG121 (f) bands using the double-expressing (de) clone 121/221. The numbers at the top of (a) and (b) indicate the positions of coinjected glucose oligomers. The Gal numbers indicate the numbers of Gal residues attached to the common Man3-AHM-2-AB core.

 
Analysis of the mixture of VSG121 and VSG221 from the double-expressing 121/221 clone produced a profile consistent with a mixture of VSG121-type and VSG221-type GPI glycan structures (Figure 5d). The GPI glycans of the individual VSGs from the double-expressing 121/221 clone, prepared by excision of individual VSG bands from the PVDF membrane, were also analyzed by BioGel P4 chromatography. The VSG221 band gave a profile almost identical to that of singly expressed VSG221 (compare Figure 5e with 5b) and the VSG121 band gave a profile similar, but not identical, to that of singly-expressed VSG121 (compare Figure 5f with 5c).


    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
VSG221 contains two N-glycosylation sites, one at Asn428, only 5 residues from the mature C-terminus and GPI membrane anchor, and the other in the N-terminal domain at Asn263. The former is occupied principally by a mixture of conventional Man9GlcNAc2 to Man7GlcNAc2 oligomannose structures while the latter is occupied by highly-processed structures ranging from Man3GlcNAc2 to (GalGlcNAc)2Man3GlcNAc2 (Zamze et al., 1991Go) (Figure 1). Thus, variant 221 trypanosomes clearly contain sufficient processing machinery both to truncate their N-linked oligosaccharides and to produce complex biantennary N-linked oligosaccharides. The coexpression of a second VSG does not appear to affect this in any significant way (compare Figures 4a and 4b). On the other hand, VSG121 contains a single N-glycosylation site at Asn432 in the C-terminal domain that is occupied by conventional Man9GlcNAc2 to Man5GlcNAc2 oligomannose structures (Zamze et al., 1990Go; Strang et al., 1993Go), see Figure 4c. This could be due to a lack of processing enzymes in the 121 trypanosome clone or to steric constraints on N-linked oligosaccharide processing imposed by the environment around the VSG glycosylation site. Since the glycosylation profile of VSG121 does not change when this VSG is expressed in a clone 221 background (Figure 4d), we can conclude that the latter is the case. We can also conclude that inter-VSG dimer interactions have little or no effect on N-linked oligosaccharide processing for VSG121 and VSG221.

The structures of the GPI anchor glycans for VSG117 (Ferguson et al., 1988Go), VSG121 (Strang et al., 1993Go), and VSG221 (Mehlert et al., 1998aGo) have been determined. The structures for the two class-1 VSGs (VSG117 and VSG121) are identical and are made up of predominantly Gal4-, Gal3-, and Gal2-containing structures. The ratio of these structures (1.7:2.0:1.0) has been determined for VSG117 (Ferguson et al., 1988Go; Redman et al., 1994Go) but not for VSG121. Here we show, by direct comparison, that the ratio of these structures is very similar for VSG121, i.e., 1.5:2.0:1.0 (compare Figure 5a and 5c). In contrast, the class-2 VSG221 contains mostly Gal6-, Gal5-, and Gal4-containing structures in the ratio 1.0:2.1:1.4 (Mehlert et al., 1998aGo), consistent with the Bio-Gel P4 profile in (Figure 5b).

If the processing of the class-1 VSGs was limited by the repertoire or levels of GPI glycan processing enzymes then we would expect a mixture of VSG121 and VSG221 expressed in a variant 221 background to produce only VSG221-type GPI glycans. This is not the case (Figure 5d). Analysis of the individual VSGs made by the double-expressing 121/221 clone showed that the VSG221 GPI glycan profile was unaffected by coexpression with VSG121 (Figure 5e) whereas the VSG121 GPI glycan profile was slightly changed, i.e., the ratio of Gal4-:Gal3-containing structures was increased (Figure 5f). The possibility that this subtle change in profile (compare Figures 5c and 5f) might be due to the cross-contamination of the VSG121 band with some VSG221 can be ruled out. A synthetic mixture of individually purified VSG121 and VSG221, processed in parallel, produced a profile for the VSG121 band identical to that shown in (Figure 5c) (data not shown). Significantly, there is no evidence for the production of Gal5- and Gal6-containing GPI glycans (typical of VSG221) on the clone 121/221 coexpressed VSG121 (Figure 5f). These data suggest that the primary factor controlling GPI anchor glycan processing is the VSG protein structure adjacent to the GPI anchor but that inter-VSG packing may also influence the degree of GPI galactosylation such that copackaging of VSG121 and VSG221 in the secretory pathway leads to the slightly modified galactosylation profile seen in (Figure 5f).

In summary, we have applied three relatively new procedures to establish whether the VSG expression site or the VSG structure itself controls VSG N-linked oligosaccharide and GPI anchor glycan processing. These procedures are (1) the stable transformation of bloodstream form African trypanosomes (Munoz-Jordan et al., 1996Go), (2) in-gel release and fluorescent labeling of N-linked oligosaccharides (Küster et al., 1997Go), and (3) on-blot fluorescent labeling and release of GPI anchor glycans (Zitzmann and Ferguson, 1999Go). The data suggest that the N-linked oligosaccharide and GPI anchor glycan processing enzymes are constitutively expressed in bloodstream form African trypanosomes and that the tertiary and quaternary structure of the VSG homodimers dictate the processing and the microheterogeneity of the surface-expressed VSGs. We hope to obtain crystal structures of intact VSGs to help us understand what structural features at and around the N-glycosylation and GPI attachment sites dictate VSG-specific processing.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
General reagents were purchased from BDH-Merck (Poole, Dorset, UK) and Sigma (Poole, Dorset, UK). Dowex AG50X12(H+), 200–400 mesh, and AG3X4(OH-), 200–400 mesh ion exchange resins were from Bio-Rad (Hemel Hempstead, UK). The BioGel P4 system (RAAM 1000 GlycoSequencer), the GlycoSep-N column and the Signal 2-AB labeling kits were from Oxford GlycoSciences (Abingdon, UK). The PVDF membrane was from Amersham and the semi-dry blotting apparatus was from Hoefer Scientific Instruments. PNGase F (recombinant, glycerol-free) was purchased from Boehringer Mannheim (Mannheim, Germany) and dialyzed against 20 mM NaHCO3 prior to use.

Preparation of trypanosomes and VSG
Trypanosome subclones MITat 1.4 (clone 117), MITat 1.6 (clone 121), and MITat 1.2 (clone 221) of T.brucei strain 427, as well as the double-expressing trypanosome clone 121/221 (Munoz-Jordan et al., 1996Go), were purified from infected rat blood as previously described (Cross, 1975Go) and the soluble form VSG (sVSG) was prepared according to the method of Cross (Cross, 1984Go) with minor modifications (Mehlert et al., 1998aGo).

In-gel PNGase F release and labeling of N-linked oligosaccharides
The samples were prepared following the method developed by Küster et al. (1997)Go. Samples (5 µg per VSG) were applied to a 10% polyacrylamide gel and subjected to SDS–PAGE. Coomassie blue–stained bands of interest were excised and transferred to screw-top Eppendorf tubes. The gel pieces were washed twice with 1 ml 20 mM NaHCO3 for 30 min to replace the gel running buffer. The wash was discarded and replaced by 300 µl of fresh 20 mM NaHCO3. To this solution 20 µl of 45 mM dithiothreitol was added and the protein was reduced at 65°C for 30 min. After cooling to room temperature, 20 µl of freshly prepared 100 mM iodoacetamide was added and the protein was alkylated for 30 min at room temperature in the dark. The reagent solution was removed and the gel pieces were washed with 1 ml acetonitrile/20 mM NaHCO3 (1:1, v/v) for 1 h. The gel pieces were cut into smaller pieces of about 1 mm2, completely dried in a SpeedVac and rehydrated in 30 µl 20 mM NaHCO3 containing PNGase F (100U/ml). An additional 100 µl of 20 mM NaHCO3 was added and after 12–16 h at 37°C, released N-glycans were recovered by combining the incubation buffer with three changes of 200 µl water with sonication for 30 min each. The combined extracts were incubated with 30 µl AG50(H+) for 5 min, filtered through a 0.22 µm filter, and dried. Dry samples were labeled with 2-AB using the Signal 2-AB labeling kit following the manufacturer’s instructions. Labeled N-glycans were redissolved in 100 µl of 80% acetonitrile and applied onto a normal-phase (Glycosep-N) HPLC column. The glycans were eluted following the method devised by Guile et al. (1996)Go using the following gradient: solvent A was 50 mM ammonium formate, pH 4.4, and solvent B was acetonitrile. Initial conditions were 20% A at a flow rate of 0.4 ml/min, followed by a linear gradient of 35–53% A over 132 min, followed by 53–100% over the next 3 min. The flow rate was then increased to 1 ml/min over the next 2 min and the column was washed in 100% A for 5 min before reequilibration in the starting solvent. The total run time was 180 min and the column temperature was 30°C. N-glycans were detected using a Waters FP-474 fluorescence detector. For identification (Guile et al., 1996Go) the elution positions of the glycans were determined in glucose units by comparison with the elution positions of a standard mixture of 2-AB-labeled glucose oligomers.

On-blot labeling and chemical release of GPI anchor glycans
Samples (5 µg per VSG) were applied to a 10% polyacrylamide gel and subjected to SDS–PAGE. Proteins were transferred to a PVDF membrane (40 mA per gel, 1 h) and stained with amido-black. Bands of interest were cut out and transferred into screw-top Eppendorf tubes. The samples were deaminated by submerging the blot strips in 50 µl of 0.3 M sodium acetate buffer, pH 4.0, and 50 µl of freshly prepared NaNO2 (2 h at 37°C). The strips were then washed three times with water, transferred into 0.5 ml Eppendorf tubes, and dried. The Signal® 2-AB labeling reagent was prepared following the manufacturer’s instructions. After labeling the strips for 2–3 h at 65°C with 15 µl 2-AB reagent in a heating block, they were washed three times with 10 ml of 50% acetonitrile, transferred to screw-top Eppendorf tubes and dried. Ice-cold 48% aqueous HF (40 µl) was added to each tube and the samples were dephosphorylated for 60–72 h on ice-water. The aqueous HF was removed by freeze-drying. Water (100 µl) was added to each tube, and the samples were freeze-dried again. The tubes and strips were then washed five times with 100 µl water, and the combined washings were filtered through a prewashed 0.2 µm microcentrifuge filter and dried. The samples were redissolved in 100 µl water and applied to the Bio-Gel P4 gel filtration column of a RAAM 1000 Glycosequencer. Samples were eluted using the constant-flow program and detected using a Gilson 121 fluorometer fitted with an excitation filter (bandpass 305–395 nm, {lambda}max 365 nm), an emission filter (bandpass 40 nm, center wavelength 450 nm) and a 9 µl flow cell. The elution positions of the GPI glycans were determined in glucose units by comparison with the elution positions of a standard mixture of glucose oligomers that were detected by refractive index.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We are grateful to Jorge Munoz-Jordan, Rockefeller University, for supplying the transformed double-expressing trypanosome clone and to George Cross for his input in conceiving this study, for helpful discussions and for comments on the manuscript. We also thank David Neville for helpful discussions. This work was supported by a Programme Grant (054491) from the Wellcome Trust. Nicole Zitzmann thanks the Wellcome Trust for a Prize Studentship.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
2-AB, 2-aminobenzamide; ESAG, expression site associated gene; GPI, glycosylphosphatidylinositol; PNGase F, peptide N-glycosidase F; VSG, variant surface glycoprotein.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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