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.
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Abstract |
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Key words: trypanosome/variant surface glycoprotein /N-glycosylation/glycosylphosphatidylinositol
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Introduction |
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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 2530% 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., 1991; Carrington and Boothroyd, 1996
). 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., 1993
) 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 -carboxyl group of each monomer to a glycosylphosphatidylinositol (GPI) membrane anchor. The mature VSG GPI anchors contain the core structure of ethanolamine-HPO4-6Man
1-2Man
1-6Man
1-4GlcN
1-6PI, common to all GPI membrane anchors (McConville and Ferguson, 1993
), but this is modified in the class-1 and class-2 VSGs by galactose side chains that are unique to VSGs (Ferguson et al., 1988
; Strang et al., 1993
; Mehlert et al., 1998a
) (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., 1989
).
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Apart from providing some resistance to proteolysis (Reinwald, 1985) and being necessary for intracellular transport for some, but not most, VSGs (Ferguson et al., 1986
), 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
-helix of peptide in the VSG ILTat.1.24 structure (Blum et al., 1993
), 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, 1997; Varki, 1998
). 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., 1997
; Paturiaux-Hanocq et al., 1997
).
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.
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Results |
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Discussion |
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The structures of the GPI anchor glycans for VSG117 (Ferguson et al., 1988), VSG121 (Strang et al., 1993
), and VSG221 (Mehlert et al., 1998a
) 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., 1988
; Redman et al., 1994
) 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., 1998a
), 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., 1996), (2) in-gel release and fluorescent labeling of N-linked oligosaccharides (Küster et al., 1997
), and (3) on-blot fluorescent labeling and release of GPI anchor glycans (Zitzmann and Ferguson, 1999
). 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.
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Materials and methods |
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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., 1996), were purified from infected rat blood as previously described (Cross, 1975
) and the soluble form VSG (sVSG) was prepared according to the method of Cross (Cross, 1984
) with minor modifications (Mehlert et al., 1998a
).
In-gel PNGase F release and labeling of N-linked oligosaccharides
The samples were prepared following the method developed by Küster et al. (1997). Samples (5 µg per VSG) were applied to a 10% polyacrylamide gel and subjected to SDSPAGE. Coomassie bluestained 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 1216 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 manufacturers 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)
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 3553% A over 132 min, followed by 53100% 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., 1996
) 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 SDSPAGE. 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 manufacturers instructions. After labeling the strips for 23 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 6072 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 305395 nm, 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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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