The glycoforms of a Trypanosoma brucei variant surface glycoprotein and molecular modeling of a glycosylated surface coat

Angela Mehlert, Charles S. Bond and Michael A.J. Ferguson1

Division of Biological Chemistry and Molecular Microbiology, The Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, Scotland, UK

Received on May 3, 2002; revised on June 24, 2002; accepted on June 24, 2002


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Abbreviations
 Acknowledgments
 References
 
The plasma membrane of the African sleeping sickness parasite Trypanosoma brucei is covered with a dense, protective surface coat. This surface coat is a monolayer of five million variant surface glycoprotein (VSG) dimers that form a macromolecular diffusion barrier. The surface coat protects the parasite from the innate immune system and, through antigenic variation, the specific host immune response. There are several hundred VSG genes per parasite, and they encode glycoproteins that vary in primary amino acid sequence, the number of N-glycosylation sites, and the types of N-linked oligosaccharides and glycosylphosphatidylinositol membrane anchors they contain. In this study, we show that VSG MITat.1.5 is glycosylated at all three potential N-glycosylation sites, and we assign the oligosaccharides present at each site. Using the most abundant oligosaccharides at each site, we construct a molecular model of the glycoprotein to assess the role of N-linked oligosaccharides in the architecture of the surface coat.

Key words: molecular modeling/N-glycosylation/trypanosome/variant surface glycoprotein


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Abbreviations
 Acknowledgments
 References
 
The tsetse fly–transmitted protozoan parasite Trypanosoma brucei is responsible for African sleeping sickness in humans and Nagana in cattle in Sub-Saharan Africa. The bloodstream form of the parasite possesses a dense cell-surface coat (Vickerman and Luckins, 1969Go) made of variant surface glycoprotein (VSG) (Cross, 1975Go). The VSG coat, a monolayer of approximately five million VSG homodimers (Auffret and Turner, 1981Go), provides the parasite’s primary defense system against innate and specific immune responses (Cross, 1996Go). The VSG coat acts as a macromolecular sieve, preventing the approach of complement components to the otherwise sensitive plasma membrane (Ferrante and Allison, 1983Go). There are several hundred genes encoding immunologically distinct VSG variants; switching the expression of these genes allows that parasite population to evade the specific immune response through antigenic variation (Vanhamme et al., 2001Go).

The VSGs are named after the antigenically pure trypanosome clones from which they are derived. For example, VSG MITat.1.5 is VSG isolated from Molteno Institute trypanozoon antigen type 1.5. The VSGs fall into three groups, A–C, based on Cys-residue conservation in their 350–400-amino-acid N-terminal domains, and into four classes, 1–4, based on peptide homology and Cys-residue conservation in their 50–100-amino-acid C-terminal domains (Carrington et al., 1991Go; Carrington and Boothroyd, 1996Go). The available crystal structures of one A1 VSG and one A2 VSG show that despite minimal (20%) amino acid sequence similarity, the N-terminal domains adopt very similar tertiary structures (Blum et al., 1993Go).

All VSGs are glycosylphosphatidylinositol (GPI) anchored glycoproteins and all are N-glycosylated at least one site (Mehlert et al., 1998bGo). The structures of the N-linked oligosaccharides of three A1 VSGs (MITat.1.4 and 1.6 and ILTat.1.3) (Zamze et al., 1990Go; Strang et al., 1993Go; Bangs et al., 1988Go), two A2 VSGs (MITat.1.1 and 1.2) (Zamze et al., 1991Go), and one A3 VSG (MITat.1.5) (Zamze et al., 1991Go) have been determined. All these VSGs contain oligomannose-type oligosaccharides; those containing two (ILTat.1.3, MITat.1.1, and 1.2) or three (MITat.1.5) N-glycosylation sites also contain biantennary structures, some featuring N-acetyl-lactosamine units and/or terminal {alpha}-galactose residues (Figure 1).



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Fig. 1. Summary of published glycosylation site data for VSGs. The VSG classifications are taken from (Carrington et al., 1991Go). Only the most abundant oligosaccharides at each site are shown (Zamze et al., 1990Go, 1991; Bangs et al., 1988Go; Strang et al., 1993Go). Square brackets indicate that the precise position of a glycosylation site is unknown (MITat.1.7) or that the glycosylation status of predicted N-glycosylation sited is unknown (MITat.1.5).

 
In this article, we took a mass spectrometric approach to investigate which of the three potential N-glycosylation sites in VSG MITat.1.5 are occupied, and we mapped the relative proportions of each of the 11 different N-linked oligosaccharide structures found in this VSG to each occupied site. These data were used to build a molecular model of an A3 VSG, variant VSG MITat.1.5, to assess the role of N-glycosylation in trypanosome surface coat architecture.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Abbreviations
 Acknowledgments
 References
 
Mapping N-linked oligosaccharides to the three glycosylation sites of soluble VSG (sVSG) MITat.1.5
Reduction and S-alkylation of soluble VSG118 with iodoacetamide was confirmed by matrix-assisted laser desorption and ionization–time of flight (MALDI-TOF) analysis of the glycoprotein before and after treatment. An increase in the centroid of the glycoprotein envelope from 50,840 Da to 51,720 Da was observed, consistent with the alkylation of, on average, 12 out of the 14 Cys residues (data not shown).

MALDI-TOF spectra of the reduced and alkylated sVSG118 tryptic peptides/glycopeptides before and after PNGase-F digestion are shown in Figure 2, and the identities of the various ions are indicated in Table I. The appearance of the ions at 1480.64, 2219.46 and 2851.03 only after PNGase-F digestion (Figure 2A) suggests that all three potential Asn-Xaa-Ser/Thr N-glycosylation sites in VSG MITat.1.5 are occupied.



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Fig. 2. Positive ion MALDI-TOF mass spectra of sVSG MITat.1.5 tryptic digests after (A) and before (B and C) digestion with PNGase-F. The groups of ions corresponding to the peptides containing the N-terminal (I36–K55), middle (G56–K82) and C-terminal (E302–R313) N-glycosylation sites are indicated in C. The ions corresponding to the same deglycosylated peptides are indicated in A.

 

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Table I. List of ions and corresponding tryptic peptides and glycopeptides
 
Analysis of the glycopeptide region of the total sVSG digest prior to PNGase-F digestion (Figure 2B,C), together with the published data on the pool of N-linked oligosaccharides present in sVSG MITat.1.5 (Zamze et al., 1991Go), allowed us to assign individual oligosaccharides to individual glycosylation sites (Figure 3). The sites at Asn52 and Asn307 carry a mixture of oligomannose and biantennary complex oligosaccharides (Man9–5GlcNAc2 and Gal2–4GlcNAc2Man3GlcNAc2), whereas the site at Asn73 carries only biantennary complex oligosaccharides.



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Fig. 3. Assignment of oligosaccharides to individual glycosylation sites. The assignments are made on the basis of the glycopeptide masses (Table I) and the known repertoire of VSG MITat.1.5 N-linked oligosaccharides (Zamze et al., 1991Go). The presence of a particular oligosaccharide structure is indicated by +. The most abundant structure at each site is indicated by a circle. The glycosylation sequons NAT, NNT, and NVS are indicated in bold.

 
Molecular modeling of VSG118
The similar folding of two dissimilar VSG N-terminal domain sequences led to the hypothesis that all VSG N-terminal domains adopt a similar shape (Blum et al., 1993Go). This hypothesis has received support from studies on the T. brucei transferrin receptor. The latter is a heterodimer of the products of the ESAG6 and ESAG7 genes that encode two similar subunits (one with a GPI anchor and the other without) (Steverding et al., 1994Go, 1995) with significant sequence similarity (21% identity and 41% similarity) to the N-terminal domain of VSG MITat.1.5. Modeling studies using the predicted ESAG6p and ESAG7p amino acid sequences superimposed on the VSG MITat.1.2 N-terminal domain crystal structure (assisted by aligning regions of predicted secondary structure) allowed the successful prediction of residues involved in transferrin binding and the design of functional (transferrin-binding) ESAG6/7-VSG MITat.1.5 chimeras (Salmon et al., 1997Go). Taking a similar approach, we modeled the N-terminal domain of VSG MITat.1.5 based on the crystal structure of the N-terminal domain of VSG MITat.1.2 and ILTat.1.24. The model predicts a surface location for the three glycosylation sites (Asn52, Asn73, and Asn307), and we attached the energy-minimized structures (Woods et al., 1998Go; Petrescu et al., 1999Go) of the most abundant oligosaccharide for each site to their respective Asn residues (Figure 3A). Other nuclear magnetic resonance (NMR) and modeling studies have shown that there are no unique solutions to glycoprotein structures, due to mobility within the oligosaccharide chains themselves and the conformational flexibility of the N-glycosidic linkage (Rudd and Dwek, 1997Go). In this case, orientation of the N-linked oligosaccharide structures by minimizing steric clashes with other atoms in the VSG dimer led to a model with dimensions that are consistent with predicted inter-VSG steric constraints that can be deduced from the average cell surface area of T. brucei (165 µm2) (Jackson et al., 1985) and number of VSG homodimers per cell (5 x 106). Thus, two of the N-linked oligosaccharides (at Asn73 and Asn307) of VSG MITat.1.5 appear to project into the hollow of the VSG peptide dumbbell. The third (at Asn52) appears to project down below the dumbbell, adjacent to the (structurally undefined) C-terminal domain (Figure 4A). The effect of N-glycosylation of VSG MITat.1.5, with respect to the density of the surface coat, can be seen by comparing Figure 4B and C, where it appears that the oligosaccharides occupy a significant amount of inter-VSG space.



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Fig. 4. Molecular models of (A) VSG MITat.1.5 with attached , energy-minimised, N-linked oligosaccharides and (B and C) the cell surface coat, viewed from the side and above without (right) and with (left) attached N-linked oligosaccharides.

 
It is noteworthy that VSG MITat.1.5 (a class A3 VSG) has three N-glycosylation sites and that they all lie in the N-terminal domain. The class A1 VSGs typically have one or two occupied N-glycosylation sites within the C-terminal domain, and the A2 VSGs typically have one occupied N-glycosylation site extremely close to the C-terminal GPI anchor and another in the N-terminal domain (Figure 1). Thus, in the absence of structural data on the C-terminal domains, VSG MITat.1.5 is the only VSG variant for which all of the N-linked carbohydrate can be built into a molecular model.

From the data available, it would appear that T. brucei is quite flexible in the range, location (Figure 1) and function of N-linked oligosaccharides in its VSG repertoire. For example: (1) The unusual Man3GlcNAc2 and Man4GlcNAc2 structures found at Asn263 of VSG MITat.1.2 appear to occupy the same space as one of the short {alpha}-helices of VSG ILTat.1.24 in the lower lobe of the dumbbell (Blum et al., 1993Go). (2) The N-glycosylation of VSG MITat.1.5 is essential for efficient transport of this VSG to the cell surface, whereas this is not the case for VSGs MITat.1.4 or MITat.1.2 (Ferguson et al., 1986Go). Furthermore, it is possible that the inter-VSG space occupied by the three N-terminal domain N-linked oligosaccharides of the A3 VSG MITat.1.5 (Figure 4) is compensated for being closer to the membrane in A1 and A2 VSGs by the C-terminal domain N-linked oligosaccharides and GPI anchor side-chains present in these variants. In this context, it is worth noting that the GPI anchors of A1 and A2 VSGs contain side-chains of, on average, 3.5 (Ferguson et al., 1988Go) and 5.5 (Mehlert et al., 1998aGo) galactose residues, respectively, whereas the VSG MITat.1.5 GPI anchor has no side-chains at all (Güther et al., 1992Go). It will be interesting to compare models of glycosylated VSGs of all subclasses once 3D data on the C-terminal domains become available. However, these domains have thus far been refractory to crystallographic and NMR analyses.


    Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Abbreviations
 Acknowledgments
 References
 
Purification of sVSG118
sVSG MITat.1.5 (also known as variant 118) was purified from bloodstream-form T. brucei strain 427 variant MITat.1.5 cells using hypotonic lysis and DE52 chromatography, as described in Cross (1975)Go. The VSG was further purified by gel filtration using a Sephacryl S200 column (4 x 90 cm) equilibrated with 0.1 M NH4HCO3. Typically, 10 mg freeze-dried sVSG was obtained from 1010 cells.

Reduction, alkylation, tryptic digestion, and analysis of sVSG118
sVSG118 (1 mg) was dissolved in 0.25 ml 4 M guanidine-HCl and reduced with 20 mM dithiothreitol (1 min, 100°C), cooled, and alkylated (in the dark) with 50 mM iodoacetamide for 15 min. The molecular weight of the reduced and alkylated sVSG was checked by MALDI-TOF (Voyager DE-STR) in linear mode after dialyzing a small aliquot against water and mixing 1:1 with 10 mg/ml sinapinic acid matrix. The remaining reduced and alkylated sVSG solution was dialyzed, made 10 mM with respect to ammonium bicarbonate, and digested (16 h, 37°C) with 10 µg trypsin (Roche, Basel, Switzerland, modified sequence grade). Aliquots (1 µl) of the digestion products were mixed 1:1 with dihydroxybenzoic acid matrix and analysed by MALDI-TOF in reflectron mode. The remaining digest was subjected to PNGase-F digestion using 1 µl enzyme solution (Roche) per 10 µg protein (16 h, 37°C) and the products analysed by MALDI-TOF using dihydroxybenzoic acid as matrix.

Molecular modeling
A model of the N-terminal domain of a single subunit of MITat.1.5 was produced with the aid of SWISSPDBVIEWER and the SWISSMODEL server (Guex et al., 1999Go), based on homology to the PDB (Berman et al., 2000Go) entries 1VSG (MITat.1.2) and 2VSG (ILTat.1.24; Blum et al., 1993Go). Superposition of this model onto both monomers of the 1VSG structure yielded a model of the MITat.1.5 dimer. Using the predicted positions of the relevant Asn glycosylation sites as anchors, minimized average NMR structures of the N-linked oligosaccharides (Woods et al., 1998Go; Petrescu et al., 1999Go) were manually oriented (using the molecular modeling program "O"; Jones et al., 1991Go) to minimize clashes with other atoms while preserving the symmetry of the dimer. In the absence of structural data for the VSG C-terminal domain, a simplified representation, as shown in Figure 4, was used. Copies of the model were placed on a hexagonal grid of spacing 5.7 nm (Ferguson, 1994Go) with a random displacement of up to 0.5 nm and a random rotation perpendicular to the membrane to produce the arrangement shown in Figure 4. Molecular graphics were prepared using MOLSCRIPT (Krulis, 1991Go) and Raster3D (Merritt and Bacon, 1997Go).


    Abbreviations
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Abbreviations
 Acknowledgments
 References
 
GPI, glycosylphosphatidylinositol; MALDI-TOF, matrix-assisted laser desorption and ionization–time of flight; MIT, Molteno Institute trypanozoon; NMR, nuclear magnetic resonance; sVSG, soluble-form VSG; VSG, variant surface glycoprotein.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Abbreviations
 Acknowledgments
 References
 
This work was supported by a Wellcome Trust Programme Grant (054491). CSB is a BBSRC David Philips Research Fellow. We thank Mark Wormald, Oxford Glycobiology Institute, for providing us with oligosaccharide coordinates.


    Footnotes
 
1 To whom correspondence should be addressed; E-mail: m.a.j.ferguson@dundee.ac.uk Back


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