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
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Abstract |
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Key words: molecular modeling/N-glycosylation/trypanosome/variant surface glycoprotein
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Introduction |
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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, AC, based on Cys-residue conservation in their 350400-amino-acid N-terminal domains, and into four classes, 14, based on peptide homology and Cys-residue conservation in their 50100-amino-acid C-terminal domains (Carrington et al., 1991; Carrington and Boothroyd, 1996
). 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., 1993
).
All VSGs are glycosylphosphatidylinositol (GPI) anchored glycoproteins and all are N-glycosylated at least one site (Mehlert et al., 1998b). The structures of the N-linked oligosaccharides of three A1 VSGs (MITat.1.4 and 1.6 and ILTat.1.3) (Zamze et al., 1990
; Strang et al., 1993
; Bangs et al., 1988
), two A2 VSGs (MITat.1.1 and 1.2) (Zamze et al., 1991
), and one A3 VSG (MITat.1.5) (Zamze et al., 1991
) 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
-galactose residues (Figure 1).
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Results and discussion |
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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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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 -helices of VSG ILTat.1.24 in the lower lobe of the dumbbell (Blum et al., 1993
). (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., 1986
). 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., 1988
) and 5.5 (Mehlert et al., 1998a
) galactose residues, respectively, whereas the VSG MITat.1.5 GPI anchor has no side-chains at all (Güther et al., 1992
). 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.
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Materials and methods |
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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., 1999), based on homology to the PDB (Berman et al., 2000
) entries 1VSG (MITat.1.2) and 2VSG (ILTat.1.24; Blum et al., 1993
). 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., 1998
; Petrescu et al., 1999
) were manually oriented (using the molecular modeling program "O"; Jones et al., 1991
) 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, 1994
) 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, 1991
) and Raster3D (Merritt and Bacon, 1997
).
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Abbreviations |
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Acknowledgments |
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Footnotes |
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References |
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