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Structure of the glycosylphosphatidylinositol membrane anchor glycan of a class-2 variant surface glycoprotein from Trypanosoma brucei 1
J. Mol. Biol. (1998) 277, 379±392
Structure of the Glycosylphosphatidylinositol Membrane Anchor Glycan of a Class-2 Variant Surface Glycoprotein from Trypanosoma brucei Angela Mehlert1, Julia M. Richardson2 and Michael A. J. Ferguson1* 1
Department of Biochemistry University of Dundee, Dundee DD1 4HN, Scotland 2
Centre for Biomolecular Science, St. Andrews University, Fife, KY16 9ST Scotland
The neutral glycan fraction of the glycosylphosphatidylinositol (GPI) membrane anchor of a class-2 variant surface glycoprotein (VSG) from Trypanosoma brucei was isolated following aqueous hydrogen ¯uoride dephosphorylation and nitrous acid deamination of the puri®ed glycoprotein. The neutral glycans were fractionated by high-pH anion exchange chromatography and gel-®ltration and six major glycan structures were solved by a combination of one and two-dimensional NMR, composition analysis, methylation linkage analysis and electrospray-mass spectrometry. The glycans were similar to those previously described for class-1 VSGs, in that they contained the linear trimannosyl sequence Mana1-2Mana1-6Man and a complex a-galactose branch of up to Gala1-2Gala1-6(Gala1-2)Gal, but most also contained an additional galactose residue attached a1-2 to the non-reducing terminal mannose residue and about one-third contained an additional galactose residue attached b1-3 to the middle mannose residue. The additional complexity of the class-2 VSG GPI glycans is discussed in terms of a biosynthetic model that explains the full range of mature GPI structures that can be expressed on different VSG classes by the same trypanosome clone. # 1998 Academic Press Limited
*Corresponding author
Keywords: trypanosome; glycosylphosphatidylinositol; VSG; glycoprotein; galactosyltransferase
Introduction The African trypanosomes belonging to the Trypanosoma brucei group are the causative agents of nagana in cattle and African sleeping sickness in humans. The parasite is protected against the alternative complement pathway by a dense surface coat of variant surface glycoprotein (VSG) and against speci®c host-immune responses by its ability to undergo antigenic variation (Pays et al., 1994; Cross, 1996; Borst et al., 1996). Antigenic variation involves the sequential expression of different VSGs from a repertoire of approximately 1000 distinct genes. The N-terminal domains of the different VSGs constitute about 75% of the mature polypeptide and have similar three-dimensional structures but Abbreviations used: CBAG, coffee bean a-galactosidase; Du, Dionex units; ES-MS, electrospraymass spectrometry; gc-ms, gas chromatography-mass spectrometry; GPI, glycosylphosphatidylinositol; Gu, glucose units; HPAEC, high-pH anion exchange chromatography; VSG, variant surface glycoprotein. 0022±2836/98/120379±14 $25.00/0/mb971600
insuf®cient primary sequence homology to allow immunological cross-reactivity (Blum et al., 1993; Carrington & Boothroyd, 1996). The VSGs can be divided into four classes based on peptide homology in their non-immunogenic C-terminal domains (Carrington et al., 1991). Most VSGs belong either to class-1 or class-2 and there are only two known examples of class-3 VSG variants and one known example of a class-4 VSG variant. The VSGs are all homodimers with monomer molecular masses of about 50 to 55 kDa. Each VSG monomer contains at least one occupied N-glycosylation site (Zamze et al., 1990, 1991) and is covalently attached to a glycosylphosphatidylinositol (GPI) membrane anchor (Ferguson et al., 1985, 1988). The GPI anchor is preassembled as a GPI precursor with the structure NH2CH2CH2PO4H-6Mana1-2Mana1-6Mana1-4GlcNa1-6myo-inositol-1-PO4H-3(sn-1,2-dimyristoylglycerol), which is attached to the mature C-terminal amino acid (Asp for class-1 VSGs, Ser for class-2 VSGs and Asn for class-3 VSGs) in exchange for a hydrophobic C-terminal GPI-addition signal peptide (Englund, 1993). The VSG-linked GPI anchors are # 1998 Academic Press Limited
380 subsequently substituted with Gal residues in a VSG class-speci®c manner. The class-1 VSG GPIs contain an average of about three Gal residues per anchor (Ferguson et al., 1988; Redman et al., 1994), the class-2 VSG GPIs signi®cantly more (Holder, 1985) and the one class-3 VSG GPI that has been analysed contains no galactose (GuÈther & Ferguson, 1993). The degree of microheterogeneity, and the precise structures of the major glycoforms, of the GPI anchors of two class-1 VSGs have been solved (Ferguson et al., 1988; Strang et al., 1993) but there is no comparable information for any class-2 VSG GPI anchor. Here we report the complete structures of the GPI glycans of a class-2 VSG (MITat.1.2) and propose a biosynthetic model to account for the range of GPI glycans that can be expressed by a single trypanosome clone.
Results Note: The residue descriptors used below (ManIIII and GalA-F) refer to the residues shown in Figure 4a and b. The class-2 VSG GPI glycans produce three core structures when digested with coffee bean a -galactosidase The sVSG MITat.1.2 GPI anchor neutral glycans were prepared by deamination/reduction and dephosphorylation (Figure 1). The deaminated/ reduced and dephosphorylated neutral glycan preparation was resolved into six peaks by Dionex HPLC, ranging from 4.2 Dionex units (Du) to 5.7 Du (Figure 2a). Each of these six peaks was further puri®ed by Bio-Gel P4 gel-®ltration, producing a set of neutral glycans between 7.0 glucose units (Gu) and 8.9 Gu in size (Table 1). In order to de®ne the structures of the underlying core-glycans, large preparations of the neutral glycans were exhaustively digested with coffee bean a-galactosidase and separated by Dionex HPLC (Figure 2b). Each of the three core-glycans was analysed by one-dimensional and two-dimensional (COSY, ROESY and TOCSY) 1H-NMR (Figure 3 and Tables 1 and 2), composition and methylation linkage analysis (Table 1) and partial acetolysis and exoglycosidase digestion (data not shown). Core-1 contained three mannose residues and one 2,5-anhydromannitol (AHM) residue (Table 1). Methylation analysis revealed the presence of partially methylated alditol acetates (PMAAs) corresponding to one terminal-Man, one 2-substitutedMan, one 6-substituted-Man and one 4-substitutedAHM residue (Table 1). Partial acetolysis of core-1 produced Man1AHM (data not shown) and, since acetolysis is selective for the Mana1-6Man glycosidic linkage, these results are consistent with the structure Mana1-2Mana1-6Mana1-4AHM (Man3AHM) (Figure 4a). These data are therefore consistent with the presence of the conserved sequence
The GPI Anchor of a Class-2 VSG
Mana1 - 2Mana1 - 6Mana1 - 4GlcNa1 - 6myo - inositol present in all GPI anchor structures characterised so far (McConville & Ferguson, 1993). Core-2 contained three mannose residues, one galactose residue and one AHM residue. Onedimensional 1H-NMR showed that this galactose residue was in the a-anomeric con®guration (Figure 3a) and methylation linkage analysis showed that the galactose was a non-reducing terminal residue (Table 1). Partial acetolysis of core-2 also produced Man1AHM (data not shown). These data, together with the rest of the methylation linkage analysis (Table 1), de®ne core-2 as Gala1-2Mana1-2Mana1-6Mana1-4AHM (Figure 4a). Since this material was resistant to coffee bean a-galactosidase, we tried to digest it with the three other a-galactosidases described in Materials and Methods but all of the digestions were unsuccessful. The signi®cance of this resistance to exoglycosidases is discussed later. Core-3 contained three mannose residues, two galactose residues and one AHM residue (Table 1). One-dimensional 1H-NMR (performed at 310 K to resolve the H1 resonances of the GalA and the ManII residues) showed that one galactose residue was in the a-anomeric con®guration (GalA) whereas the other was b (GalB; Figure 3a). Methylation linkage analysis showed that both were terminal residues (Table 1). Like core-2, core-3 was completely resistant to all four a-galactosidases and also to two b-galactosidases (data not shown) and this is discussed later. Partial acetolysis of core-3 produced Man1AHM (data not shown), suggesting the presence of the conserved Man3AHM core. Methylation linkage analysis (Table 1), which showed the presence of one 2,3-disubstituted-Man residue, one 2-substituted-Man residue and one 6-substituted-Man residue, suggested that one Gal residue was attached to the 2-position of ManIII and the other to the 3-position of either ManIII or ManII. In order to resolve the exact locations of the GalA and GalB residues, twodimensional 1H-NMR COSY and ROESY experiments were performed. The results of the ROESY experiment (Figure 3b and Table 3) showed strong, medium and weak ROEs between the H1 of GalB and the H2, H3 and H5 of ManII, respectively. Given that the ManII residue is substituted at the 2-position with the ManIII residue, these data indicate that GalB is attached to the 3-position of the Man II residue and therefore that GalA is attached to the 2-position of Man III. Thus the structure of core-3 is Gala1-2Mana1-2(Galb1-3)Mana1-6Mana14AHM (Figure 4a). The complete structures of the class-2 VSG GPI glycans Having determined the primary structures of the three underlying cores, the six individual fractions were analysed by positive ion electrospray mass spectrometry (ES-MS), composition and methylation linkage analysis (Table 1) and one-dimen-
The GPI Anchor of a Class-2 VSG
381
Figure 1. Summary of the preparation and fractionation of the sVSG MITat.1.2 GPI neutral glycans. The meanings of the Dionex unit (Du) and glucose unit (Gu) values are given in Materials and Methods. EtN, ethanolamine; P, phosphate, AHM, 2,5-anhydromannitol, Ino, myo-inositol.
sional 1H-NMR (data not shown). The ES-MS and composition data showed that the structures varied in composition from Gal3Man3AHM (fraction 1) to Gal6Man3AHM (fraction 6). Fraction 6 (Gal6Man3AHM) and fraction 5 (Gal5Man3AHM) both contained 2,3-disubstituted-Man residues and bGal, as determined by methylation linkage analysis and 1H-NMR, respectively (Table 1). The larger of these fractions (fraction 6) contained terminal-Gal, 2-substituted-Gal, 2,6-disubstituted-Gal and 3,6-disubstituted-Man, suggestive of a branched Gal4 side-chain of Gala1-2Gala16(Gala1-2)Gal attached to the 3-position of the ManI residue, similar to that previously described in the class-1 VSGs (Figure 4c). Consistent with
this, the two-dimensional COSY and TOCSY 1HNMR spectra of fraction 6 (Table 2) showed that the chemical shifts of the GalA and GalB protons were virtually identical to those in core-3, suggesting that GalA and GalB are not substituted with further Gal residues, whereas the chemical shifts of the ManI protons in fraction 6 were signi®cantly different from those in core-3, indicating substitution at this residue. A two-dimensional ROESY spectrum of fraction 6 (Figure 3c) showed a strong cross-peak between H1 of GalC and H3 of ManI and a weaker cross-peak between H1 of GalC and H4 of ManI, consistent with the substitution of the 3-position of ManI with the additional Gal residues. In addition, ROESY cross-peaks
382
Figure 2. Dionex HPAEC fractionation of sVSG MITat.1.2 GPI neutral glycans. a, The sVSG MITat.1.2 GPI neutral glycans were resolved by Dionex HPAEC into six fractions. b, After exhaustive digestion with coffee bean a-galactosidase the sVSG MITat.1.2 GPI neutral glycans were resolved into three core structures. The arrows at the top of each panel indicate the elution positions of the co-injected glucose oligomer internal standards.
between H1 of GalE and both the H1 and H2 of GalC, as well as cross-peaks between H1 of GalD and H6 of GalC, indicate that GalC is 2,6-disubstituted with residues GalE and GalD, respectively. Finally, cross-peaks between H1 of GalF and H1 and H2 of GalD indicate the linkage GalFa12GalD. Taken together, these data suggest that the principle component of fraction 6 is that shown in Figure 4b. The methylation linkage analysis of fraction 5 (Gal5Man3AHM; Table 1) is consistent with the major component of this fraction having the same structure as fraction 6, minus the GalF residue (Figure 4b). Fraction 4 (Gal5Man3AHM) did not contain a 2,3-disubstituted-Man residue or a GalB residue (Table 1). Comparison of the 1H chemical shifts of fraction 4 with those of fraction 6 (Table 2) showed that the chemical shifts of the ®ve aGal residues (GalA and GalC-F) are virtually identical whereas the chemical shifts of H2, H3 and H4 of ManII are different. These data suggest that the major com-
The GPI Anchor of a Class-2 VSG
ponent of fraction 4 has the same structure as fraction 6 minus the GalB residue (Figure 4b). Fraction 3 (Gal4Man3AHM) did not contain a 2,3-disubstituted-Man residue or a GalB residue but did contain a 2,6-disubstituted-Gal residue (Table 1), suggesting that the major component of this fraction has the same structure as fraction 6 minus the GalB and GalF residues (Figure 4b). Fraction 2 (Gal4Man3AHM) did not contain a 2,3-disubstituted-Man residue or a GalB residue and the lack of 2,6-disubstituted-Gal in the methylation data (Table 1) suggests that the major component of this fraction has the same structure as fraction 6 minus the GalB and GalE residues (Figure 4b). Fraction 1 (Gal3Man3AHM) did not contain a 2,3-disubstituted-Man residue or a GalB residue and the lack of 2,6-disubstituted-Gal and 2-substituted-Gal in the methylation data (Table 1) suggests that the major component of this fraction has the same structure as fraction 6 minus the GalB, GalE and GalF residues (Figure 4b). The structures described above relate to the major components of the fractions 1 to 6. However, the presence of small amounts of terminal-Man in the methylation analyses of several of the fractions (Table 1) suggests that some minor structures lacking the GalA residue (representing <10 mol %) are also present. Taking this into account, the proportions of core-1, core-2 and core-3 containing structures are approximately 10%, 57% and 33%, respectively. Three-dimensional structures of the class-2 VSG GPI neutral glycan cores Core-2 and core-3, obtained after exhaustive coffee bean a-galactosidase digestion, and the synthetic disaccharide Gala1-2Mana1-O-CH3 were modelled using the simulated annealing protocols described by Homans & Forster (1992) which allow distance information derived from multidimensional NMR spectra to be included in the structure calculation as restraints. Inter-residue 1 Ê , which assist in H-1H distances less than 5 A de®ning the conformation about a glycosidic linkage, were identi®ed from two-dimensional ROESY spectra. A total of eight such distances were obtained for core-3, four for core-2 and four for the synthetic disaccharide (Table 3). Each inter-residue distance was converted to a weak, medium or strong restraint. The restraints are de®ned according to the distance (R) between two protons as Ê < either weak, medium or strong when 1.8 A Ê Ê Ê Ê Ê, R < 5.0 A, 1.8 A < R < 3.3 A and 1.8 A < R < 2.7 A respectively. Initially, ten pseudo-random geometries of each glycan were computed and each geometry was subjected to simulated annealing with incorporation of the NMR-derived distance restraints. A single family of structures was obtained for each glycan. The lowest energy conformations of core-2, core-3 and Gala1-2Mana1-OCH3 are shown in Figure 5.
1
Totale NG fraction
3
3 3 3 5
ÿ 1 2
3 4 4 5 5 6
2
4-AHM
ÿ ÿ
ÿ ÿ
t-Man
ÿ
t-Gal
2-Man
ÿ ÿ ÿ
ÿ
2-Gal
ÿ
ÿ ÿ ÿ ÿ ÿ ÿ
6-Man
ÿ ÿ ÿ
6-Gal
ÿ ÿ
ÿ ÿ ÿ ÿ
2,3-Man
Methylation analysis: PMAAs corresponding toc:-
ÿ ÿ ÿ
3,6-Man
ÿ ÿ ÿ
ÿ ÿ
2,6-Gal
ÿ ÿ
ÿ ÿ ÿ ÿ
b-Gald NMR
b
Nominal mass in Daltons estimated from the [M Na] and [M H Na] pseudomolecular ions observed for the native and permethylated neutral glycans, respectively. Nearest integer values. c 4-AHM, 4-O-substituted-AHM; t-Man, terminal-Man; t-Gal, terminal-Gal; 2-Man, 2-O-substituted-Man; 2-Gal, 2-O-substituted-Gal; 6-Man, 6-O-substituted-Man; 6-Gal, 6-O-substituted-Gal; 2,3Man, 2,3-di-O-substituted-Man; 3,6-Man, 3,6-di-O-substituted-Man; 2,6-Gal, 2,6-di-O-substituted-Gal. d The presence of the bGal residue (GalB) was detected by one-dimensional NMR. e Analysis of the total mixture of GPI neutral glycans prior to fractionation by HPAEC.
a
1 1 1
3 3 3 3 3 3
Compositionb analysis AHM Man Gal
Cores (coffee bean a-galactosidase treated) 1 2.4 4.2 2 3.1 5.2 3 3.8 5.7
7.0 7.8 7.9 8.5 8.2 8.9
Massa (ES)
1 1 1 1 1 1
4.2 4.5 4.9 5.1 5.4 5.7
1 2 3 4 5 6
Size (Gu)
1138 1300 1300 1462 1462 1624
Size (Du)
Fraction Number
Table 1. Chromatographic properties and composition and methylation linkage analyses of the sVSG MITat.1.2 GPI neutral glycan fractions and core-1, core-2 and core-3
384
The GPI Anchor of a Class-2 VSG
Figure 3(a ± b) legend opposite
The GPI Anchor of a Class-2 VSG
385
Figure 3. NMR analysis of sVSG MITat.1.2 GPI neutral glycans. a, One-dimensional 1H-NMR spectra of (from top to bottom) core-1, core-2 and core-3 at 300 K and core-3 at 310 K. The resonances of the H1 (anomeric) protons of the ManI, ManII, ManIII, GalA and GalB residues are indicated. b, Detail of the two-dimensional ROESY spectrum of core-3. The resonances of the H1 anomeric protons of the ManI, ManII, ManIII, GalA and GalB residues are indicated on the right of the spectrum. The labelled boxes show the ROE cross-peaks corresponding to the through-space connectivities between ManIII-H1 and ManII-H1 (a), ManIII-H1 and ManII-H2 (b), ManIII-H1 and ManII-H3 (c), GalAH1 and ManIII-H2 (d), ManII-H1 and ManI-H6 (e), GalB-H1 and ManII-H2 (f) and GalB-H1 and ManII-H3 (g). c, Detail of the two-dimensional ROESY spectrum of fraction 6. The resonances of the H1 anomeric protons of the GalC-F residues are indicated on the right of the spectrum. The labelled boxes show the ROE cross-peaks corresponding to the through-space connectivities between GalC-H1 and GalE-H1 (a), GalC-H1 and ManI-H4 (b), GalC-H1 and ManI-H3 (c), GalE-H1 and GalC-H2 (d), GalD-H1 and GalC-H6 (e), GalF-H1 and GalD-H1 (f) and GalF-H1 and GalDH2 (g).
Discussion The VSGs can be classi®ed into four classes based on C-terminal sequence homology (Carrington et al., 1991). Most VSG variants belong to class-1 and class-2 and only one class-4 variant and two class-3 variants are known. The GPI anchor of a class-1 VSG (MITat.1.4) was the ®rst GPI structure to be solved (Ferguson et al., 1988). That study revealed microheterogeneity in the GPI neutral glycan fraction, with four major species present (Figure 4c), and identical structures were subsequently determined for another class-1 VSG (MITat.1.6; Strang et al., 1993). In contrast, the class-3 VSG MITat.1.5 does not contain any galactose and exhibits a single GPI neutral glycan with the same structure as core-1 (see Figure 4a: GuÈther & Ferguson, 1993). In this paper, we describe the structures of the GPI neutral glycans from a class-2 VSG (MITat.1.2) and show that these structures are similar to those of the class-1 VSGs except that most of them contain an additional aGal residue (GalA) attached to the 2-position of the non-reducing aMan residue (ManIII) and that about one third of these structures contain an additional bGal
residue (GalB) attached to the 3-position of the middle aMan residue (ManII). The mean Gal content of the GPI glycans of class-1 VSGs is 3.1 Gal residues per mol GPI (Ferguson et al., 1988; Redman et al., 1994), whereas this ®gure is about 4.9 Gal residues per mol GPI for the class-2 VSG (this study). This increased level of galactosylation is not only due to the presence of the GalA and GalB residues but also to the larger average size of the aGal side-chain attached to the ®rst aMan residue (ManI). The proportions of Gal2:Gal3:Gal4 side-chains are 0.18:0.47:0.35 for the class-1 VSGs and 0.06:0.37:0.57 for the class-2 VSG. The observed differences in GPI anchor galactosylation states could re¯ect differential expression of the relevant galactosyltransferases in trypanosomes expressing different VSG classes or the in¯uence of the structure of the C-terminal domain of the VSG on the access of these enzymes to the VSG GPI anchor. While VSG expression sites are associated with expression site associated genes (ESAGs; reviewed by Overath et al., 1994; Cross, 1996), none of these encode proteins with typical glycosyltransferase
386
The GPI Anchor of a Class-2 VSG
Table 2. Chemical shift assignments for core-3, fraction 6 and fraction 4 from one-dimensional and two-dimensional (COSY and TOCSY) NMR spectra A. Core-3 Residue ManI ManII ManIII GalA GalB
H1 5.17 5.25 5.52 5.27 4.64
H2 4.10 4.28 4.21 3.93 3.7
H3 3.91 4.25 4.06 4.0 3.78
H4 4.06 3.94 3.86
B. Fraction 6 Residue ManI ManII ManIII GalA GalB GalF GalD GalE GalC
H1 5.21 5.25 5.52 5.26 4.66 5.22 5.29 5.29 5.52
H2 4.40 4.30 4.21 3.93 3.70 3.95 3.97 4.08 4.08
H3 3.93 4.26 4.06 4.00 3.77 4.02 4.05 3.98
C. Fraction 4 Residue ManI ManII ManIII GalA GalF GalD GalE GalC
H1 5.21 5.23 5.49 5.22 5.22 5.29 5.29 5.52
H2 4.38 4.11 4.19 3.94 3.94 3.97 4.06 4.09
H3 3.93 4.06 4.05 4.04 4.04 4.02 3.98 4.16
H5 3.84 3.82 4.00/3.82
H6 3.83 3.91 4.00/3.82
/ /
H60 3.82 3.81
H4 4.10 3.95
H5 3.88
H6 3.83
/ /
H60 3.81
4.09 4.04 4.12 4.11 4.15
4.21
3.85
4.27 4.26 4.09
3.86 3.86 3.86
/
4.07
H4 4.09 3.78 3.83 4.09 4.10 4.10 4.13 4.19
H5
H6
/
H60
3.88 4.25 4.25 4.26
3.77 3.84 3.84 3.84
/
3.88
4.42
4.03
/
3.80
4.04
Differences between the chemical shifts of core-3 and those of fractions 4 and 6 are underlined.
structure (i.e. type-2 membrane proteins with small cytoplasmic domains) and, in any case, VSGs of different classes (e.g. class-1 MITat.1.4 and class-3 MITat.1.5) are known to use the same expression site (Van der Ploeg et al., 1982). On the other hand, there are a number of observations that support the notion that the GPI-modifying galactosyltransferases are constitutively expressed and that steric constraints imposed by the VSG C-terminal structure are the predominant in¯uence on the ®nal GPI glycoforms expressed. These include (a) the presence of small amounts of 2,3-disubstituted-Man residues, synonymous with the presence of the GalB residue, in the class-I VSG GPI anchor (Ferguson et al., 1988) and (b) the ability of trypanosomes expressing class-1 (MITat.1.4) and class-3 (MITat.1.5) VSGs to galactosylate excess GPI precursors in an identical fashion to yield species based on core-1 and core-3 structures (Mayor et al., 1992). Signi®cantly, whereas core-3 based structures are generated on the GPI precursors, where VSG steric in¯uences are not relevant, there are only core-1 (and a small amount of core-1 plus GalB) based structures in the ®nal VSG-linked class-1 GPI anchors (Ferguson et al., 1988). In conclusion, all of the observed mature GPI anchor glycan structures (Figure 4) can be rationalised in a biosynthetic model (Figure 6) where all of the galactosyltransferases are constitutively expressed and access of the GPI substrate is con-
strained by the three-dimensional structure of the VSG C terminus. Furthermore, the in vitro synthesis data of Mayor et al. (1992) are consistent with some of the galactosyltransferases being located in the endoplasmic reticulum while the data of Bangs et al. (1988) show that others are located later in the secretory pathway, most likely in the Golgi apparatus. These conclusions are consistent with the original suggestion Homans et al., 1989) that GPI anchor galactosylation, that is unique to African trypanosomes, is a mechanism for ®lling space close to the plasma membrane (beneath and around the VSG C terminus) and that this processing may be important in maintaining a functional protective barrier on the surface of the parasite regardless of which VSG variant is being expressed. Since the GPI-modifying galactosyltransferases are unique to these organisms, and since the repertoire of non-galactosylated class-3 VSG variants is so small, these enzymes could be considered as potential therapeutic targets. The resistance of the Gala1-2Man glycosidic bond in core-2 and core-3 to coffee bean a-galactosidase (CBAG) was surprising in view of the fact that the synthetic glycoside Gala1-2Mana1-O-CH3 is inherently sensitive to this enzyme (Brown et al., 1998). This resistance was also re¯ected by the resistance of core-2 and core-3 to a plethora of other a and b-galactosidases. Together, these results suggest unusual conformations for the GPI glycans that preclude access by the exoglycosidases. Comparison of the predicted three-dimen-
Figure 4. Structures of VSG GPI neutral glycans. a, Structures of core-1, core-2 and core-3 generated by exhaustive coffee bean a-galactosidase digestion of the sVSG MITat.1.2 GPI neutral glycans. b, Structures of the major components of fractions 1 to 6 of the sVSG MITat.1.2 GPI neutral glycans. c, Structures of major components of the class-1 sVSG MITat.1.4 GPI neutral glycan fraction (adapted from Ferguson et al., 1988).
388
The GPI Anchor of a Class-2 VSG
Table 3. Restraints calculations
used
in
simulated
H-1H distance
annealing
Molecule
1
Gala1-2Mana1-O-CH3
Gal-H1 Gal-H1 Gal-H1 Gal-H5
Core-2
ManIII-H1 to ManII-H2 ManII-H1 to ManIII-H6 ManII-H1 to ManIII-H60 GalA-H1 to ManIII-H2
Strong Medium Medium Strong
Core-3
ManIII-H1 to ManII-H2 ManIII-H1 to ManII-H3 GalA-H3 to ManIII-H3 GalA-H1 to ManIII-H1 GalA-H1 to ManIII-H2 GalB-H1 to ManII-H2 GalB-H1 to ManII-H3 GalB-H1 to ManII-H5
Medium Weak Weak Weak Strong Strong Medium Weak
to to to to
Man-H1 Man-H2 Man-H3 Man-H1
Restrainta Weak Strong Weak Weak
a The distance, R, between the two protons concerned is constrained in the simulated annealing calculation to be Ê < R < 5.0 A Ê , 1.8 < R < 3.3 A Ê , 1,8 A Ê < R < 2.7 A Ê , for weak, 1.8 A medium and strong restraints, respectively.
sional structures of Gala1-2Mana1-O-CH3 and core-2 and core-3 (Figure 5) support this and indicate that the ManI residue in core-2 and core-3
impinges on the Gala1-2Man linkage and that this is compounded in core-3 by the presence of the bGal residue. This tight packing of sugar residues is quite surprising when one considers how they were added in the ®rst place. The GalA and GalB residues are presumably added to the GPI anchor after the GPI precursor is attached to the fullyfolded VSG polypeptide, when the ethanolamine phosphate bridge and the VSG C-terminal domain can only add to the steric constraints for GalA and GalB addition. Nevertheless, in the class-2 VSGs the GalA and GalB galactosyltransferases must be able to access their highly constrained glycosylation sites on ManIII and ManII with high and medium ef®ciency, respectively. There is one notable discrepancy in the data presented here, namely the generation of signi®cant quantities of core-1 structures upon exhaustive CBAG digestion of the total GPI neutral glycan fraction (Figure 2b) when core-1 containing structures are thought to represent <10 mol % of the GPI neutral glycans and when the core-3 and core2 structures are known to be inherently resistant to this enzyme. One explanation for this discrepancy might be that the GalA residue can be slowly removed by coffee bean a-galactosidase when a large aGal branch is attached to ManI, but not once this is digested. In this model, some of core-2
Figure 5. Predicted three-dimensional structures of (a) synthetic Gala1-2Mana1-O-CH3, (b) core-2 and (c) core-3.
The GPI Anchor of a Class-2 VSG
389
Figure 6. Biosynthetic model for the galactosylation of GPI anchors on class-1, class-2 and class-3 VSGs. The model assumes that all of the galactosyltransferases are constitutively expressed, regardless of the VSG class being expressed, and that their access to the GPI substrate is controlled by the three-dimensional structure of the VSG C-terminal domain (see the text). The model incorporates data from Mayor et al. (1992) and Bangs et al. (1988) which, in the light of the structural data in Ferguson et al. (1988) and in this paper, can be interpreted in terms of the likely locations of the GPI-processing galactosyltransferases. Thus, two of the a-galactosyltransferases (one that initiates the aGal branch and one that adds the aGal to the terminal Man residue) and the single b-galactosyltransferase are probably located in the endoplasmic reticulum whereas the remaining three a-galactosytransferases (that complete the aGal branch) are probably located in the Golgi apparatus. The percentage ®gures indicate the approximate mol % of GPI glycans that contain the indicated Gal residue, all other residues are present in 100 mol % of the glycans.
(the most abundant core according to the major structures determined, Figure 4b) might be converted to core-1 before the removal of the aGal branch whereas in other molecules the prior removal of the CBAG-sensitive aGal branch prevents this. Some support for this idea can be obtained from the predicted three-dimensional structure of the class-1 VSG GPI anchor (Homans et al., 1989) that indicates that parts of the aGal
branch would lie close to the GalB residue and might therefore indirectly distort the GalA-ManIII glycosidic linkage to favour access by CBAG. Whatever the explanation, the CBAG resistance of core-2 on its own, its partial sensitivity in the presence of the aGal branch and the a- and b-galactosidase resistance of core-3 emphasise the complex steric factors that dictate whether or not broadspeci®city exoglycosidases can access their terminal
390 sugar substrates. This, in turn, emphasises the caution that must be applied to negative exoglycosidase digestion data when analysing novel glycan structures.
Materials and Methods Materials Sephacryl-S200 and QAE-Sephadex-A25 were supplied by Pharmacia, DEAE-cellulose (DE52) by Whatman and Bio-Gel P4, AG3X4(OHÿ) and AG50X12(H) by BioRad. Bovine testis b-galactosidase and coffee bean a-galactosidase were supplied by Boehringer, jack bean b-galactosidase, Escherichia coli and Aspergillus niger agalactosidases by Sigma and M. vinacea a-galactosidase by Seikagaku. Acetic anhydride and 0.5 M methanolic HCl were supplied by Supelco, 6 M HCl by Pierce, scyllo-inositol by Calbiochem, sodium borodeuteride by Fluka and sodium borotritiide (13 Ci/mmol) and En3Hance spray by DuPont-NEN. Aluminium backed HPTLC plates and all solvents (HPLC or Aristar grade) were from Merck/BDH. The synthetic glycoside Gala12Mana1-O-CH3 was prepared by Dr Jillian Brown and Dr Robert Field (St. Andrews University). Purification of the sVSG MITat.1.2 GPI anchors sVSGat.1.2 was puri®ed from bloodstream form T. brucei cells using hypotonic lysis and DE52 chromatography, as described by Cross (1975). The VSG was further puri®ed by gel ®ltration using a Sephacryl-S200 column (4 cm 90 cm) equilibrated with 0.1 M NH4HCO3. Approximately 10 mg of freeze-dried sVSG was obtained from 1010 cells. Preparation of 3H-labelled sVSG MITat.1.2 GPI neutral glycans Approximately 0.5 mg of freeze dried sVSG MITat.1.2 was deaminated by dissolving the protein in 30 ml of 0.3 M sodium acetate (pH 4.0) and adding 15 ml of freshly prepared 1 M sodium nitrite, as described by Ferguson (1992). The reaction was left for three hours in the dark and reduction was achieved by altering the pH to approximately 10.5 with 10 ml 0.8 M boric acid and 24 ml 2 M NaOH and then adding the solution to 5 ml of 36 mM NaB3H4 dissolved in 0.1 M NaOH. After 90 minutes, the reduction was completed by adding 10 ml 0.5 M NaB2H4 in water (one hour). After neutralisation with 1 M acetic acid, and freeze drying to remove tritiated water formed in the reaction, the labelled glycoprotein was redissolved in water, dialysed, freeze-dried and treated with 50 ml 50% aqueous HF on ice for 60 hours. After neutralisation and desalting (Ferguson, 1992), this procedure produced the 3H-labelled neutral glycans, containing a [1-3H]2,5-anhydromannitol (AHM) residue where the glucosamine had been. Radioactive contaminants from the sodium borotritiide reagent were removed from the labelled glycans by paper chromatography and high voltage electrophoresis, as described in Ferguson (1992). This material was used in the initial analyses of the glycans and later, when larger quantities were needed, it was used as a tracer to help follow the puri®cation of the deutero-reduced neutral glycans, the preparation of which is described below.
The GPI Anchor of a Class-2 VSG Bulk preparation of sVSG MITat.1.2 GPI neutral glycans Larger amounts of the neutral glycans were needed for the composition and methylation analyses, and for NMR analysis. These were made by treating batches of 50 mg of sVSG MITat.1.2 with 1 ml 50% aqueous HF (60 hours at 0 C), and, after neutralising with saturated LiOH, precipitating the protein with 5% ice-cold trichloroacetic acid. The supernatant was desalted using a column of 3 ml AG3(OHÿ) over 4 ml QAE-Sephadex-A25. This GPI neutral glycan fraction was deaminated using 1 ml 1 M sodium acetate buffer (pH 4.0) and 1 ml freshly prepared 1 M NaNO2 (three hours at room temperature in the dark). Subsequently, the pH of the solution was adjusted to approximately 11, using 0.5 ml 0.8 M boric acid and 0.65 ml 2 M NaOH. The GPI glycans were then reduced with 1 ml 1 M NaB2H4 (one hour) and desalted, after acidi®cation with acetic acid, by passage through 3 ml AG50(H), drying and removal of boric acid by coevaporation with methanol. During the subsequent chromatography on Dionex HPAEC and Bio-Gel P4 a tracer of 106 cpm of the tritiated neutral glycans was added to assist in locating the GPI glycan peaks.
Purification of the sVSG MITat.1.2 GPI neutral glycans The tritiated neutral glycans were chromatographed by Dionex high-pH anion exchange chromatography (HPAEC) using a carbopac PA-1 column as described in (Ferguson, 1992). Sodium ions were removed from the eluate on-line using a Dionex ARRS unit and radioactivity was detected using an on-line Raytest Ramona detector with a 0.2 ml solid-scintillator X-cell. Pooled peak fractions were further desalted by passage through a small column containing AG50(H) over AG3(OHÿ) over QAE-Sephadex-A25. The size of the glycans in Dionex units (Du) was calculated by comparison to the pulsed-amperiometric detector trace of a sample of b-glucose oligomer internal standards. The individual peaks were then applied to a Bio-Gel P4 column for further puri®cation, and to enable the calculation of their size in glucose units (Gu) by comparison to the bglucose oligomer internal standards detected by refractive index. The bulk deutero-reduced GPI samples were chromatographed in a similar way, but with a trace of the tritiated material to assist location of the peaks, and several batches of the material had to be loaded onto the Dionex system.
Exoglycosidase digestions The digestions using coffee bean a-galactosidase and jack bean and bovine testis b-galactosidases were performed as described by Schneider & Ferguson (1995). For the E. coli and M. vinacea a-galactosidase digestions, one unit of enzyme in 0.1 M citrate phosphate buffer (pH 6.0) was used and for the A. niger a-galactosidase digestion 0.1 unit of enzyme in 0.1 M sodium acetate buffer (pH 4) was used. All digests were for 16 hours at 37 C and were terminated by heating to 100 C for ®ve minutes. Samples were desalted as described by Schneider & Ferguson (1995) and analysed by Dionex HPLC (Ferguson, 1992) and/or by silica HPTLC using propanol:acetone:water (10:6:5, by vol.; Schneider & Ferguson, 1995).
391
The GPI Anchor of a Class-2 VSG Inositol and carbohydrate analysis The GPI content of the protein and the recovery of GPI glycan after aqueous HF digestion was assessed by gc-ms inositol analysis. Samples were mixed with 40 pmol of [1,2,3,4,5,6-2H]myo-inositol internal standard, hydrolysed with 6 M HCl (18 hours at 110 C) and processed as described (by Ferguson, 1992). The monosaccharide contents of the puri®ed VSG MITat.1.2 neutral glycan fractions, and the coffee bean a-galactosidase treated cores, were estimated by gc-ms following methanolysis using 1 nmol scyllo-inositol as an internal standard (Ferguson, 1992). Methylation analysis Methylation analysis was carried out on the whole VSG MITat.1.2 GPI neutral glycan preparation, on the six puri®ed VSG MITat.1.2 GPI neutral glycan fractions and on the three coffee bean a-galactosidase-treated core glycans. Approximately 10 nmol of material was used for each methylation analysis and a sample of a Lacto-Ntetraose standard was analysed simultaneously. The method used is described by Ferguson (1992) and the resulting partially methylated alditol acetates (PMAAs) were analysed by gc-ms, using an SE54 Econocap column, (Alltech) and an SP2380 column (Supelco). Scylloinositol was added as an internal standard during the reduction step of the analysis. Partial acetolysis The three cores generated by exhaustive coffee bean a-galactosidase digestion were subjected to partial acetolysis, to con®rm the presence and location of the Mana1-6Man linkage. The samples were dried in 1 ml glass reactivials (Pierce) and peracetylated by heating for 30 minutes at 100 C with 40 ml of acetic anhydride: pyridine (1:1 v/v). The peracetylated glycans were dried and acetolysed by heating at 30 C for six hours with 30 ml acetic anhydride:acetic acid:conc. H2SO4 (10:10:1, by vol.). The reaction was stopped by the addition of 0.25 ml pyridine and 0.5 ml water. The acetylated products were extracted into 0.25 ml chloroform, washed three times with 0.5 ml water, dried and de-O-acetylated by heating at 37 C in 0.2 ml methanol:aq.35%NH3 (1:1, v/v) for 60 hours. The partially acetolysed neutral glycans were dried and redissolved in water for analysis by HPTLC (Schneider & Ferguson, 1995). 1
H NMR spectroscopy
The three cores generated by exhaustive coffee bean a-galactosidase digestion and the six puri®ed fractions of VSG MITat.1.2 GPI neutral glycans were analysed by one-dimensional NMR spectroscopy using a Bruker 500 MHz spectrometer. The samples were dissolved in 2 H2O, and the spectra were recorded at a probe temperature of 300 K, with the exception of the core-3 and the fraction 6 sample, which were also recorded at 310 K. The three cores generated by exhaustive coffee bean a-galactosidase digestion, and fractions 4 and 6 of the undigested glycans, were also analysed by twodimensional NMR. For these samples two-dimensional homonuclear 1H correlation spectroscopy (COSY), 1H rotating frame nuclear Overhauser spectroscopy (ROESY), and total correlation spectroscopy (TOCSY) were performed either on the Bruker spectrometer, or
on a Varian 500 MHz spectrometer. In the case of the ROESY, the transmitter offset was 5.6 ppm, the sweep width in both dimensions was 2300 Hz, and the mixing time was 500 ms, the number of experiments was 512 and the number of scans in each experiment was 64. In the TOCSY experiment, the sweep width was 1200 Hz, and the mixing time was 90 ms. Electrospray mass spectrometry A Micromass Quattro triple quadruple instrument was used in positive ion mode. The permethylated glycans were introduced into the ion source at 10 ml/min, at a concentration of 20 pmol/ml in 50% acetonitrile, 0.1% formic acid. The major pseudomolecular ions were [M Na H]2. In addition, non-derivatised glycans were also run under the same conditions, where the [M Na] pseudomolecular ions predominated; these results are shown in Table 1.
Acknowledgements This work was supported by a Programme Grant from the Wellcome Trust. J.M.R. is a Royal Society Dorothy Hodgkin Research Fellow and M.A.J.F. is a Howard Hughes International Research Scholar. We thank Dr Jillian Brown and Dr Robert Field for supplying the synthetic disaccharide Gala1-2Mana1-O-CH3 and Dr Andrei Nikolaev and Dr Janice Bramham for helpful comments.
References Bangs, J. D., Doering, T. L., Englund, P. T. & Hart, G. W. (1988). Biosynthesis of a variant surface glycoprotein of Trypanosoma brucei. J. Biol. Chem. 263, 17697± 17705. Blum, M. L., Down, J. A., Gurnett, A. M., Carrington, M., Turner, M. J. & Wiley, D. C. (1993). A structural motif in the variant surface glycoproteins of Trypanosoma brucei. Nature, 362, 603± 609. Borst, P., Rudenko, G., Taylor, M. C., Blundell, P. A., Van-Leeuwen, F., Bitter, W., Cross, M. & McCulloch, R. (1996). Antigenic variation in trypanosomes. Arch. Med. Res. 27, 379± 388. Brown, J. R., Ravindranathan, Kartha K. P., Ferguson, M. A. J. & Field, R. A. (1998). Methyl 2-O-a-D-galactopyranosyl-a-D-mannopyrannoside: Coffee bean agalactosidase sensitivity of a synthetic fragment of a Trypanosoma brucei GPI anchor. Bioorg. Med. Chem. Letters, in the press. Carrington, M. & Boothroyd, J. (1996). Implications of conserved structural motifs in disparate trypanosome surface proteins. Mol. Biochem. Parasitol. 81, 119± 126. Carrington, M., Miller, N., Blum, M., Roditi, I., Wiley, D. & Turner, M. (1991). Variant speci®c glycoprotein of Trypanosoma brucei consists of two domains each having an independently conserved pattern of cysteine residues. J. Mol. Biol. 221, 823 ± 835. Cross, G. A. M. (1975). Identi®cation, puri®cation and properties of clone-speci®c glycoprotein antigens constituting the surface coat of Trypanosoma brucei. Parasitology, 71, 393± 417.
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Cross, G. A. M. (1996). Antigenic variation in trypanosomes: secrets surface slowly. BioEssays, 18, 283 ± 291. Englund, P. T. (1993). The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors. Annu. Rev. Biochem. 62, 121± 138. Ferguson, M. A. J. (1992). Chemical and enzymic analysis of glycosyl-phosphatidylinositol anchors. In Lipid Modi®cation of Proteins: A Practical Approach (Turner, A. J. & Hooper, N., eds), pp. 191± 230, IRL Press, Oxford. Ferguson, M. A. J., Low, M. G. & Cross, G. A. M. (1985). Glycosyl-sn-1,2-dimyristyl phosphatidylinositol is covalently linked to Trypanosoma brucei variant surface glycoprotein. J. Biol. Chem. 260, 14547± 14555. Ferguson, M. A. J., Homans, S. W., Dwek, R. A. & Rademacher, T. W. (1988). Glycosyl-phosphatidylinositol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the membrane. Science, 239, 753± 759. GuÈther, M. L. S. & Ferguson, M. A. J. (1993). The microanalysis of glycosyl-phosphatidylinositol glycans. In Glycoprotein Analysis in Biomedicine (Hounsell, E. F., ed.), pp. 99± 117, Humana Press, UK. Holder, A. A. (1985). Glycosylation of the variant surface antigens of Trypanosoma brucei. Curr. Topics Microbiol. Immunol. 117, 57± 74. Homans, S. W. & Forster, M. (1992). Application of restrained minimization, simulated annealing and molecular dynamics simulations for the conformational analysis of oligosaccharides. Glycobiology, 2, 143± 151. Homans, S. W., Edge, C. J., Ferguson, M. A. J., Dwek, R. A. & Rademacher, T. W. (1989). Solution structure of the glycosylphosphatidylinositol membrane anchor glycan of Trypanosoma Brucei variant surface glycoprotein. Biochemistry, 28, 2881± 2887. Mayor, S., Menon, A. K. & Cross, G. A. M. (1992). Galactose-containing glycosylphosphatidylinositols in Trypanosoma brucei. J. Biol. Chem. 267, 754 ±761. McConville, M. J. & 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. Overath, P., Chaudri, M., Steverding, D. & Ziegelbauer, K. (1994). Invariant surface proteins in bloodstream forms of Trypanosoma brucei. Parasitology Today, 10(2), 53 ± 58. Pays, E., Vanhamme, L. & Berberof, M. (1994). Genetic controls for the expression of surface antigens in African trypanosomes. Annu. Rev. Microbiol. 48, 25 ± 52. Redman, C. A., Green, B. N., Thomas-Oates, J. E., Reinhold, V. N. & Ferguson, M. A. J. (1994). Analysis of glycosylphosphatidylinositol membrane anchors by electrospray ionisation mass spectrometry and collision induced dissociation. Glycoconj. J. 11, 187 ± 193. Schneider, P. & Ferguson, M. A. J. (1995). Microscale analysis of glycosylphosphatidylinositol structures. Methods Enzymol, 250, 614±630. Strang, A-M., Allen, A. K., Holder, A. A. & van Halbeek, H. (1993). The carbohydrate structures of Trypanosoma brucei brucei MITat.1.6 variant surface glycoprotein. Biochem. Biophys. Res. Commun. 196, 1430± 1439. Van der Pleog, L. H. T., Bernards, A., Rijsewijk, F. A. M. & Borst, P. (1982). Characterization of the DNA duplication-transposition that controls the expression of two genes for variant surface glycoproteins in Trypanosoma brucei. Nucl. Acids Res. 10, 593± 609. Zamze, S. E., Ashford, D. A., Wooten, E. W., Rademacher, T. W. & Dwek, R. A. (1991). Structural characterization of the asparagine-linked oligosaccharides from Trypanosoma brucei type II and type III variant surface glycoproteins. J. Biol. Chem. 266, 20244± 20261. Zamze, S. E., Wooten, E. W., Ashford, D. A., Ferguson, M. A. J., Dwek, R. A. & Rademacher, T. W. (1990). Characterisation of the asparagine-linked oligosaccharides from Trypanosoma brucei type-1 variant surface glycoproteins. Eur. J. Biochem. 187, 657 ± 663.
Edited by I. B. Holland (Received 22 September 1997; received in revised form 17 December 1997; accepted 17 December 1997)
Structure of the Glycosylphosphatidylinositol Membrane Anchor Glycan of a Class-2 Variant Surface Glycoprotein from Trypanosoma brucei Angela Mehlert1, Julia M. Richardson2 and Michael A. J. Ferguson1* 1
Department of Biochemistry University of Dundee, Dundee DD1 4HN, Scotland 2
Centre for Biomolecular Science, St. Andrews University, Fife, KY16 9ST Scotland
The neutral glycan fraction of the glycosylphosphatidylinositol (GPI) membrane anchor of a class-2 variant surface glycoprotein (VSG) from Trypanosoma brucei was isolated following aqueous hydrogen ¯uoride dephosphorylation and nitrous acid deamination of the puri®ed glycoprotein. The neutral glycans were fractionated by high-pH anion exchange chromatography and gel-®ltration and six major glycan structures were solved by a combination of one and two-dimensional NMR, composition analysis, methylation linkage analysis and electrospray-mass spectrometry. The glycans were similar to those previously described for class-1 VSGs, in that they contained the linear trimannosyl sequence Mana1-2Mana1-6Man and a complex a-galactose branch of up to Gala1-2Gala1-6(Gala1-2)Gal, but most also contained an additional galactose residue attached a1-2 to the non-reducing terminal mannose residue and about one-third contained an additional galactose residue attached b1-3 to the middle mannose residue. The additional complexity of the class-2 VSG GPI glycans is discussed in terms of a biosynthetic model that explains the full range of mature GPI structures that can be expressed on different VSG classes by the same trypanosome clone. # 1998 Academic Press Limited
*Corresponding author
Keywords: trypanosome; glycosylphosphatidylinositol; VSG; glycoprotein; galactosyltransferase
Introduction The African trypanosomes belonging to the Trypanosoma brucei group are the causative agents of nagana in cattle and African sleeping sickness in humans. The parasite is protected against the alternative complement pathway by a dense surface coat of variant surface glycoprotein (VSG) and against speci®c host-immune responses by its ability to undergo antigenic variation (Pays et al., 1994; Cross, 1996; Borst et al., 1996). Antigenic variation involves the sequential expression of different VSGs from a repertoire of approximately 1000 distinct genes. The N-terminal domains of the different VSGs constitute about 75% of the mature polypeptide and have similar three-dimensional structures but Abbreviations used: CBAG, coffee bean a-galactosidase; Du, Dionex units; ES-MS, electrospraymass spectrometry; gc-ms, gas chromatography-mass spectrometry; GPI, glycosylphosphatidylinositol; Gu, glucose units; HPAEC, high-pH anion exchange chromatography; VSG, variant surface glycoprotein. 0022±2836/98/120379±14 $25.00/0/mb971600
insuf®cient primary sequence homology to allow immunological cross-reactivity (Blum et al., 1993; Carrington & Boothroyd, 1996). The VSGs can be divided into four classes based on peptide homology in their non-immunogenic C-terminal domains (Carrington et al., 1991). Most VSGs belong either to class-1 or class-2 and there are only two known examples of class-3 VSG variants and one known example of a class-4 VSG variant. The VSGs are all homodimers with monomer molecular masses of about 50 to 55 kDa. Each VSG monomer contains at least one occupied N-glycosylation site (Zamze et al., 1990, 1991) and is covalently attached to a glycosylphosphatidylinositol (GPI) membrane anchor (Ferguson et al., 1985, 1988). The GPI anchor is preassembled as a GPI precursor with the structure NH2CH2CH2PO4H-6Mana1-2Mana1-6Mana1-4GlcNa1-6myo-inositol-1-PO4H-3(sn-1,2-dimyristoylglycerol), which is attached to the mature C-terminal amino acid (Asp for class-1 VSGs, Ser for class-2 VSGs and Asn for class-3 VSGs) in exchange for a hydrophobic C-terminal GPI-addition signal peptide (Englund, 1993). The VSG-linked GPI anchors are # 1998 Academic Press Limited
380 subsequently substituted with Gal residues in a VSG class-speci®c manner. The class-1 VSG GPIs contain an average of about three Gal residues per anchor (Ferguson et al., 1988; Redman et al., 1994), the class-2 VSG GPIs signi®cantly more (Holder, 1985) and the one class-3 VSG GPI that has been analysed contains no galactose (GuÈther & Ferguson, 1993). The degree of microheterogeneity, and the precise structures of the major glycoforms, of the GPI anchors of two class-1 VSGs have been solved (Ferguson et al., 1988; Strang et al., 1993) but there is no comparable information for any class-2 VSG GPI anchor. Here we report the complete structures of the GPI glycans of a class-2 VSG (MITat.1.2) and propose a biosynthetic model to account for the range of GPI glycans that can be expressed by a single trypanosome clone.
Results Note: The residue descriptors used below (ManIIII and GalA-F) refer to the residues shown in Figure 4a and b. The class-2 VSG GPI glycans produce three core structures when digested with coffee bean a -galactosidase The sVSG MITat.1.2 GPI anchor neutral glycans were prepared by deamination/reduction and dephosphorylation (Figure 1). The deaminated/ reduced and dephosphorylated neutral glycan preparation was resolved into six peaks by Dionex HPLC, ranging from 4.2 Dionex units (Du) to 5.7 Du (Figure 2a). Each of these six peaks was further puri®ed by Bio-Gel P4 gel-®ltration, producing a set of neutral glycans between 7.0 glucose units (Gu) and 8.9 Gu in size (Table 1). In order to de®ne the structures of the underlying core-glycans, large preparations of the neutral glycans were exhaustively digested with coffee bean a-galactosidase and separated by Dionex HPLC (Figure 2b). Each of the three core-glycans was analysed by one-dimensional and two-dimensional (COSY, ROESY and TOCSY) 1H-NMR (Figure 3 and Tables 1 and 2), composition and methylation linkage analysis (Table 1) and partial acetolysis and exoglycosidase digestion (data not shown). Core-1 contained three mannose residues and one 2,5-anhydromannitol (AHM) residue (Table 1). Methylation analysis revealed the presence of partially methylated alditol acetates (PMAAs) corresponding to one terminal-Man, one 2-substitutedMan, one 6-substituted-Man and one 4-substitutedAHM residue (Table 1). Partial acetolysis of core-1 produced Man1AHM (data not shown) and, since acetolysis is selective for the Mana1-6Man glycosidic linkage, these results are consistent with the structure Mana1-2Mana1-6Mana1-4AHM (Man3AHM) (Figure 4a). These data are therefore consistent with the presence of the conserved sequence
The GPI Anchor of a Class-2 VSG
Mana1 - 2Mana1 - 6Mana1 - 4GlcNa1 - 6myo - inositol present in all GPI anchor structures characterised so far (McConville & Ferguson, 1993). Core-2 contained three mannose residues, one galactose residue and one AHM residue. Onedimensional 1H-NMR showed that this galactose residue was in the a-anomeric con®guration (Figure 3a) and methylation linkage analysis showed that the galactose was a non-reducing terminal residue (Table 1). Partial acetolysis of core-2 also produced Man1AHM (data not shown). These data, together with the rest of the methylation linkage analysis (Table 1), de®ne core-2 as Gala1-2Mana1-2Mana1-6Mana1-4AHM (Figure 4a). Since this material was resistant to coffee bean a-galactosidase, we tried to digest it with the three other a-galactosidases described in Materials and Methods but all of the digestions were unsuccessful. The signi®cance of this resistance to exoglycosidases is discussed later. Core-3 contained three mannose residues, two galactose residues and one AHM residue (Table 1). One-dimensional 1H-NMR (performed at 310 K to resolve the H1 resonances of the GalA and the ManII residues) showed that one galactose residue was in the a-anomeric con®guration (GalA) whereas the other was b (GalB; Figure 3a). Methylation linkage analysis showed that both were terminal residues (Table 1). Like core-2, core-3 was completely resistant to all four a-galactosidases and also to two b-galactosidases (data not shown) and this is discussed later. Partial acetolysis of core-3 produced Man1AHM (data not shown), suggesting the presence of the conserved Man3AHM core. Methylation linkage analysis (Table 1), which showed the presence of one 2,3-disubstituted-Man residue, one 2-substituted-Man residue and one 6-substituted-Man residue, suggested that one Gal residue was attached to the 2-position of ManIII and the other to the 3-position of either ManIII or ManII. In order to resolve the exact locations of the GalA and GalB residues, twodimensional 1H-NMR COSY and ROESY experiments were performed. The results of the ROESY experiment (Figure 3b and Table 3) showed strong, medium and weak ROEs between the H1 of GalB and the H2, H3 and H5 of ManII, respectively. Given that the ManII residue is substituted at the 2-position with the ManIII residue, these data indicate that GalB is attached to the 3-position of the Man II residue and therefore that GalA is attached to the 2-position of Man III. Thus the structure of core-3 is Gala1-2Mana1-2(Galb1-3)Mana1-6Mana14AHM (Figure 4a). The complete structures of the class-2 VSG GPI glycans Having determined the primary structures of the three underlying cores, the six individual fractions were analysed by positive ion electrospray mass spectrometry (ES-MS), composition and methylation linkage analysis (Table 1) and one-dimen-
The GPI Anchor of a Class-2 VSG
381
Figure 1. Summary of the preparation and fractionation of the sVSG MITat.1.2 GPI neutral glycans. The meanings of the Dionex unit (Du) and glucose unit (Gu) values are given in Materials and Methods. EtN, ethanolamine; P, phosphate, AHM, 2,5-anhydromannitol, Ino, myo-inositol.
sional 1H-NMR (data not shown). The ES-MS and composition data showed that the structures varied in composition from Gal3Man3AHM (fraction 1) to Gal6Man3AHM (fraction 6). Fraction 6 (Gal6Man3AHM) and fraction 5 (Gal5Man3AHM) both contained 2,3-disubstituted-Man residues and bGal, as determined by methylation linkage analysis and 1H-NMR, respectively (Table 1). The larger of these fractions (fraction 6) contained terminal-Gal, 2-substituted-Gal, 2,6-disubstituted-Gal and 3,6-disubstituted-Man, suggestive of a branched Gal4 side-chain of Gala1-2Gala16(Gala1-2)Gal attached to the 3-position of the ManI residue, similar to that previously described in the class-1 VSGs (Figure 4c). Consistent with
this, the two-dimensional COSY and TOCSY 1HNMR spectra of fraction 6 (Table 2) showed that the chemical shifts of the GalA and GalB protons were virtually identical to those in core-3, suggesting that GalA and GalB are not substituted with further Gal residues, whereas the chemical shifts of the ManI protons in fraction 6 were signi®cantly different from those in core-3, indicating substitution at this residue. A two-dimensional ROESY spectrum of fraction 6 (Figure 3c) showed a strong cross-peak between H1 of GalC and H3 of ManI and a weaker cross-peak between H1 of GalC and H4 of ManI, consistent with the substitution of the 3-position of ManI with the additional Gal residues. In addition, ROESY cross-peaks
382
Figure 2. Dionex HPAEC fractionation of sVSG MITat.1.2 GPI neutral glycans. a, The sVSG MITat.1.2 GPI neutral glycans were resolved by Dionex HPAEC into six fractions. b, After exhaustive digestion with coffee bean a-galactosidase the sVSG MITat.1.2 GPI neutral glycans were resolved into three core structures. The arrows at the top of each panel indicate the elution positions of the co-injected glucose oligomer internal standards.
between H1 of GalE and both the H1 and H2 of GalC, as well as cross-peaks between H1 of GalD and H6 of GalC, indicate that GalC is 2,6-disubstituted with residues GalE and GalD, respectively. Finally, cross-peaks between H1 of GalF and H1 and H2 of GalD indicate the linkage GalFa12GalD. Taken together, these data suggest that the principle component of fraction 6 is that shown in Figure 4b. The methylation linkage analysis of fraction 5 (Gal5Man3AHM; Table 1) is consistent with the major component of this fraction having the same structure as fraction 6, minus the GalF residue (Figure 4b). Fraction 4 (Gal5Man3AHM) did not contain a 2,3-disubstituted-Man residue or a GalB residue (Table 1). Comparison of the 1H chemical shifts of fraction 4 with those of fraction 6 (Table 2) showed that the chemical shifts of the ®ve aGal residues (GalA and GalC-F) are virtually identical whereas the chemical shifts of H2, H3 and H4 of ManII are different. These data suggest that the major com-
The GPI Anchor of a Class-2 VSG
ponent of fraction 4 has the same structure as fraction 6 minus the GalB residue (Figure 4b). Fraction 3 (Gal4Man3AHM) did not contain a 2,3-disubstituted-Man residue or a GalB residue but did contain a 2,6-disubstituted-Gal residue (Table 1), suggesting that the major component of this fraction has the same structure as fraction 6 minus the GalB and GalF residues (Figure 4b). Fraction 2 (Gal4Man3AHM) did not contain a 2,3-disubstituted-Man residue or a GalB residue and the lack of 2,6-disubstituted-Gal in the methylation data (Table 1) suggests that the major component of this fraction has the same structure as fraction 6 minus the GalB and GalE residues (Figure 4b). Fraction 1 (Gal3Man3AHM) did not contain a 2,3-disubstituted-Man residue or a GalB residue and the lack of 2,6-disubstituted-Gal and 2-substituted-Gal in the methylation data (Table 1) suggests that the major component of this fraction has the same structure as fraction 6 minus the GalB, GalE and GalF residues (Figure 4b). The structures described above relate to the major components of the fractions 1 to 6. However, the presence of small amounts of terminal-Man in the methylation analyses of several of the fractions (Table 1) suggests that some minor structures lacking the GalA residue (representing <10 mol %) are also present. Taking this into account, the proportions of core-1, core-2 and core-3 containing structures are approximately 10%, 57% and 33%, respectively. Three-dimensional structures of the class-2 VSG GPI neutral glycan cores Core-2 and core-3, obtained after exhaustive coffee bean a-galactosidase digestion, and the synthetic disaccharide Gala1-2Mana1-O-CH3 were modelled using the simulated annealing protocols described by Homans & Forster (1992) which allow distance information derived from multidimensional NMR spectra to be included in the structure calculation as restraints. Inter-residue 1 Ê , which assist in H-1H distances less than 5 A de®ning the conformation about a glycosidic linkage, were identi®ed from two-dimensional ROESY spectra. A total of eight such distances were obtained for core-3, four for core-2 and four for the synthetic disaccharide (Table 3). Each inter-residue distance was converted to a weak, medium or strong restraint. The restraints are de®ned according to the distance (R) between two protons as Ê < either weak, medium or strong when 1.8 A Ê Ê Ê Ê Ê, R < 5.0 A, 1.8 A < R < 3.3 A and 1.8 A < R < 2.7 A respectively. Initially, ten pseudo-random geometries of each glycan were computed and each geometry was subjected to simulated annealing with incorporation of the NMR-derived distance restraints. A single family of structures was obtained for each glycan. The lowest energy conformations of core-2, core-3 and Gala1-2Mana1-OCH3 are shown in Figure 5.
1
Totale NG fraction
3
3 3 3 5
ÿ 1 2
3 4 4 5 5 6
2
4-AHM
ÿ ÿ
ÿ ÿ
t-Man
ÿ
t-Gal
2-Man
ÿ ÿ ÿ
ÿ
2-Gal
ÿ
ÿ ÿ ÿ ÿ ÿ ÿ
6-Man
ÿ ÿ ÿ
6-Gal
ÿ ÿ
ÿ ÿ ÿ ÿ
2,3-Man
Methylation analysis: PMAAs corresponding toc:-
ÿ ÿ ÿ
3,6-Man
ÿ ÿ ÿ
ÿ ÿ
2,6-Gal
ÿ ÿ
ÿ ÿ ÿ ÿ
b-Gald NMR
b
Nominal mass in Daltons estimated from the [M Na] and [M H Na] pseudomolecular ions observed for the native and permethylated neutral glycans, respectively. Nearest integer values. c 4-AHM, 4-O-substituted-AHM; t-Man, terminal-Man; t-Gal, terminal-Gal; 2-Man, 2-O-substituted-Man; 2-Gal, 2-O-substituted-Gal; 6-Man, 6-O-substituted-Man; 6-Gal, 6-O-substituted-Gal; 2,3Man, 2,3-di-O-substituted-Man; 3,6-Man, 3,6-di-O-substituted-Man; 2,6-Gal, 2,6-di-O-substituted-Gal. d The presence of the bGal residue (GalB) was detected by one-dimensional NMR. e Analysis of the total mixture of GPI neutral glycans prior to fractionation by HPAEC.
a
1 1 1
3 3 3 3 3 3
Compositionb analysis AHM Man Gal
Cores (coffee bean a-galactosidase treated) 1 2.4 4.2 2 3.1 5.2 3 3.8 5.7
7.0 7.8 7.9 8.5 8.2 8.9
Massa (ES)
1 1 1 1 1 1
4.2 4.5 4.9 5.1 5.4 5.7
1 2 3 4 5 6
Size (Gu)
1138 1300 1300 1462 1462 1624
Size (Du)
Fraction Number
Table 1. Chromatographic properties and composition and methylation linkage analyses of the sVSG MITat.1.2 GPI neutral glycan fractions and core-1, core-2 and core-3
384
The GPI Anchor of a Class-2 VSG
Figure 3(a ± b) legend opposite
The GPI Anchor of a Class-2 VSG
385
Figure 3. NMR analysis of sVSG MITat.1.2 GPI neutral glycans. a, One-dimensional 1H-NMR spectra of (from top to bottom) core-1, core-2 and core-3 at 300 K and core-3 at 310 K. The resonances of the H1 (anomeric) protons of the ManI, ManII, ManIII, GalA and GalB residues are indicated. b, Detail of the two-dimensional ROESY spectrum of core-3. The resonances of the H1 anomeric protons of the ManI, ManII, ManIII, GalA and GalB residues are indicated on the right of the spectrum. The labelled boxes show the ROE cross-peaks corresponding to the through-space connectivities between ManIII-H1 and ManII-H1 (a), ManIII-H1 and ManII-H2 (b), ManIII-H1 and ManII-H3 (c), GalAH1 and ManIII-H2 (d), ManII-H1 and ManI-H6 (e), GalB-H1 and ManII-H2 (f) and GalB-H1 and ManII-H3 (g). c, Detail of the two-dimensional ROESY spectrum of fraction 6. The resonances of the H1 anomeric protons of the GalC-F residues are indicated on the right of the spectrum. The labelled boxes show the ROE cross-peaks corresponding to the through-space connectivities between GalC-H1 and GalE-H1 (a), GalC-H1 and ManI-H4 (b), GalC-H1 and ManI-H3 (c), GalE-H1 and GalC-H2 (d), GalD-H1 and GalC-H6 (e), GalF-H1 and GalD-H1 (f) and GalF-H1 and GalDH2 (g).
Discussion The VSGs can be classi®ed into four classes based on C-terminal sequence homology (Carrington et al., 1991). Most VSG variants belong to class-1 and class-2 and only one class-4 variant and two class-3 variants are known. The GPI anchor of a class-1 VSG (MITat.1.4) was the ®rst GPI structure to be solved (Ferguson et al., 1988). That study revealed microheterogeneity in the GPI neutral glycan fraction, with four major species present (Figure 4c), and identical structures were subsequently determined for another class-1 VSG (MITat.1.6; Strang et al., 1993). In contrast, the class-3 VSG MITat.1.5 does not contain any galactose and exhibits a single GPI neutral glycan with the same structure as core-1 (see Figure 4a: GuÈther & Ferguson, 1993). In this paper, we describe the structures of the GPI neutral glycans from a class-2 VSG (MITat.1.2) and show that these structures are similar to those of the class-1 VSGs except that most of them contain an additional aGal residue (GalA) attached to the 2-position of the non-reducing aMan residue (ManIII) and that about one third of these structures contain an additional bGal
residue (GalB) attached to the 3-position of the middle aMan residue (ManII). The mean Gal content of the GPI glycans of class-1 VSGs is 3.1 Gal residues per mol GPI (Ferguson et al., 1988; Redman et al., 1994), whereas this ®gure is about 4.9 Gal residues per mol GPI for the class-2 VSG (this study). This increased level of galactosylation is not only due to the presence of the GalA and GalB residues but also to the larger average size of the aGal side-chain attached to the ®rst aMan residue (ManI). The proportions of Gal2:Gal3:Gal4 side-chains are 0.18:0.47:0.35 for the class-1 VSGs and 0.06:0.37:0.57 for the class-2 VSG. The observed differences in GPI anchor galactosylation states could re¯ect differential expression of the relevant galactosyltransferases in trypanosomes expressing different VSG classes or the in¯uence of the structure of the C-terminal domain of the VSG on the access of these enzymes to the VSG GPI anchor. While VSG expression sites are associated with expression site associated genes (ESAGs; reviewed by Overath et al., 1994; Cross, 1996), none of these encode proteins with typical glycosyltransferase
386
The GPI Anchor of a Class-2 VSG
Table 2. Chemical shift assignments for core-3, fraction 6 and fraction 4 from one-dimensional and two-dimensional (COSY and TOCSY) NMR spectra A. Core-3 Residue ManI ManII ManIII GalA GalB
H1 5.17 5.25 5.52 5.27 4.64
H2 4.10 4.28 4.21 3.93 3.7
H3 3.91 4.25 4.06 4.0 3.78
H4 4.06 3.94 3.86
B. Fraction 6 Residue ManI ManII ManIII GalA GalB GalF GalD GalE GalC
H1 5.21 5.25 5.52 5.26 4.66 5.22 5.29 5.29 5.52
H2 4.40 4.30 4.21 3.93 3.70 3.95 3.97 4.08 4.08
H3 3.93 4.26 4.06 4.00 3.77 4.02 4.05 3.98
C. Fraction 4 Residue ManI ManII ManIII GalA GalF GalD GalE GalC
H1 5.21 5.23 5.49 5.22 5.22 5.29 5.29 5.52
H2 4.38 4.11 4.19 3.94 3.94 3.97 4.06 4.09
H3 3.93 4.06 4.05 4.04 4.04 4.02 3.98 4.16
H5 3.84 3.82 4.00/3.82
H6 3.83 3.91 4.00/3.82
/ /
H60 3.82 3.81
H4 4.10 3.95
H5 3.88
H6 3.83
/ /
H60 3.81
4.09 4.04 4.12 4.11 4.15
4.21
3.85
4.27 4.26 4.09
3.86 3.86 3.86
/
4.07
H4 4.09 3.78 3.83 4.09 4.10 4.10 4.13 4.19
H5
H6
/
H60
3.88 4.25 4.25 4.26
3.77 3.84 3.84 3.84
/
3.88
4.42
4.03
/
3.80
4.04
Differences between the chemical shifts of core-3 and those of fractions 4 and 6 are underlined.
structure (i.e. type-2 membrane proteins with small cytoplasmic domains) and, in any case, VSGs of different classes (e.g. class-1 MITat.1.4 and class-3 MITat.1.5) are known to use the same expression site (Van der Ploeg et al., 1982). On the other hand, there are a number of observations that support the notion that the GPI-modifying galactosyltransferases are constitutively expressed and that steric constraints imposed by the VSG C-terminal structure are the predominant in¯uence on the ®nal GPI glycoforms expressed. These include (a) the presence of small amounts of 2,3-disubstituted-Man residues, synonymous with the presence of the GalB residue, in the class-I VSG GPI anchor (Ferguson et al., 1988) and (b) the ability of trypanosomes expressing class-1 (MITat.1.4) and class-3 (MITat.1.5) VSGs to galactosylate excess GPI precursors in an identical fashion to yield species based on core-1 and core-3 structures (Mayor et al., 1992). Signi®cantly, whereas core-3 based structures are generated on the GPI precursors, where VSG steric in¯uences are not relevant, there are only core-1 (and a small amount of core-1 plus GalB) based structures in the ®nal VSG-linked class-1 GPI anchors (Ferguson et al., 1988). In conclusion, all of the observed mature GPI anchor glycan structures (Figure 4) can be rationalised in a biosynthetic model (Figure 6) where all of the galactosyltransferases are constitutively expressed and access of the GPI substrate is con-
strained by the three-dimensional structure of the VSG C terminus. Furthermore, the in vitro synthesis data of Mayor et al. (1992) are consistent with some of the galactosyltransferases being located in the endoplasmic reticulum while the data of Bangs et al. (1988) show that others are located later in the secretory pathway, most likely in the Golgi apparatus. These conclusions are consistent with the original suggestion Homans et al., 1989) that GPI anchor galactosylation, that is unique to African trypanosomes, is a mechanism for ®lling space close to the plasma membrane (beneath and around the VSG C terminus) and that this processing may be important in maintaining a functional protective barrier on the surface of the parasite regardless of which VSG variant is being expressed. Since the GPI-modifying galactosyltransferases are unique to these organisms, and since the repertoire of non-galactosylated class-3 VSG variants is so small, these enzymes could be considered as potential therapeutic targets. The resistance of the Gala1-2Man glycosidic bond in core-2 and core-3 to coffee bean a-galactosidase (CBAG) was surprising in view of the fact that the synthetic glycoside Gala1-2Mana1-O-CH3 is inherently sensitive to this enzyme (Brown et al., 1998). This resistance was also re¯ected by the resistance of core-2 and core-3 to a plethora of other a and b-galactosidases. Together, these results suggest unusual conformations for the GPI glycans that preclude access by the exoglycosidases. Comparison of the predicted three-dimen-
Figure 4. Structures of VSG GPI neutral glycans. a, Structures of core-1, core-2 and core-3 generated by exhaustive coffee bean a-galactosidase digestion of the sVSG MITat.1.2 GPI neutral glycans. b, Structures of the major components of fractions 1 to 6 of the sVSG MITat.1.2 GPI neutral glycans. c, Structures of major components of the class-1 sVSG MITat.1.4 GPI neutral glycan fraction (adapted from Ferguson et al., 1988).
388
The GPI Anchor of a Class-2 VSG
Table 3. Restraints calculations
used
in
simulated
H-1H distance
annealing
Molecule
1
Gala1-2Mana1-O-CH3
Gal-H1 Gal-H1 Gal-H1 Gal-H5
Core-2
ManIII-H1 to ManII-H2 ManII-H1 to ManIII-H6 ManII-H1 to ManIII-H60 GalA-H1 to ManIII-H2
Strong Medium Medium Strong
Core-3
ManIII-H1 to ManII-H2 ManIII-H1 to ManII-H3 GalA-H3 to ManIII-H3 GalA-H1 to ManIII-H1 GalA-H1 to ManIII-H2 GalB-H1 to ManII-H2 GalB-H1 to ManII-H3 GalB-H1 to ManII-H5
Medium Weak Weak Weak Strong Strong Medium Weak
to to to to
Man-H1 Man-H2 Man-H3 Man-H1
Restrainta Weak Strong Weak Weak
a The distance, R, between the two protons concerned is constrained in the simulated annealing calculation to be Ê < R < 5.0 A Ê , 1.8 < R < 3.3 A Ê , 1,8 A Ê < R < 2.7 A Ê , for weak, 1.8 A medium and strong restraints, respectively.
sional structures of Gala1-2Mana1-O-CH3 and core-2 and core-3 (Figure 5) support this and indicate that the ManI residue in core-2 and core-3
impinges on the Gala1-2Man linkage and that this is compounded in core-3 by the presence of the bGal residue. This tight packing of sugar residues is quite surprising when one considers how they were added in the ®rst place. The GalA and GalB residues are presumably added to the GPI anchor after the GPI precursor is attached to the fullyfolded VSG polypeptide, when the ethanolamine phosphate bridge and the VSG C-terminal domain can only add to the steric constraints for GalA and GalB addition. Nevertheless, in the class-2 VSGs the GalA and GalB galactosyltransferases must be able to access their highly constrained glycosylation sites on ManIII and ManII with high and medium ef®ciency, respectively. There is one notable discrepancy in the data presented here, namely the generation of signi®cant quantities of core-1 structures upon exhaustive CBAG digestion of the total GPI neutral glycan fraction (Figure 2b) when core-1 containing structures are thought to represent <10 mol % of the GPI neutral glycans and when the core-3 and core2 structures are known to be inherently resistant to this enzyme. One explanation for this discrepancy might be that the GalA residue can be slowly removed by coffee bean a-galactosidase when a large aGal branch is attached to ManI, but not once this is digested. In this model, some of core-2
Figure 5. Predicted three-dimensional structures of (a) synthetic Gala1-2Mana1-O-CH3, (b) core-2 and (c) core-3.
The GPI Anchor of a Class-2 VSG
389
Figure 6. Biosynthetic model for the galactosylation of GPI anchors on class-1, class-2 and class-3 VSGs. The model assumes that all of the galactosyltransferases are constitutively expressed, regardless of the VSG class being expressed, and that their access to the GPI substrate is controlled by the three-dimensional structure of the VSG C-terminal domain (see the text). The model incorporates data from Mayor et al. (1992) and Bangs et al. (1988) which, in the light of the structural data in Ferguson et al. (1988) and in this paper, can be interpreted in terms of the likely locations of the GPI-processing galactosyltransferases. Thus, two of the a-galactosyltransferases (one that initiates the aGal branch and one that adds the aGal to the terminal Man residue) and the single b-galactosyltransferase are probably located in the endoplasmic reticulum whereas the remaining three a-galactosytransferases (that complete the aGal branch) are probably located in the Golgi apparatus. The percentage ®gures indicate the approximate mol % of GPI glycans that contain the indicated Gal residue, all other residues are present in 100 mol % of the glycans.
(the most abundant core according to the major structures determined, Figure 4b) might be converted to core-1 before the removal of the aGal branch whereas in other molecules the prior removal of the CBAG-sensitive aGal branch prevents this. Some support for this idea can be obtained from the predicted three-dimensional structure of the class-1 VSG GPI anchor (Homans et al., 1989) that indicates that parts of the aGal
branch would lie close to the GalB residue and might therefore indirectly distort the GalA-ManIII glycosidic linkage to favour access by CBAG. Whatever the explanation, the CBAG resistance of core-2 on its own, its partial sensitivity in the presence of the aGal branch and the a- and b-galactosidase resistance of core-3 emphasise the complex steric factors that dictate whether or not broadspeci®city exoglycosidases can access their terminal
390 sugar substrates. This, in turn, emphasises the caution that must be applied to negative exoglycosidase digestion data when analysing novel glycan structures.
Materials and Methods Materials Sephacryl-S200 and QAE-Sephadex-A25 were supplied by Pharmacia, DEAE-cellulose (DE52) by Whatman and Bio-Gel P4, AG3X4(OHÿ) and AG50X12(H) by BioRad. Bovine testis b-galactosidase and coffee bean a-galactosidase were supplied by Boehringer, jack bean b-galactosidase, Escherichia coli and Aspergillus niger agalactosidases by Sigma and M. vinacea a-galactosidase by Seikagaku. Acetic anhydride and 0.5 M methanolic HCl were supplied by Supelco, 6 M HCl by Pierce, scyllo-inositol by Calbiochem, sodium borodeuteride by Fluka and sodium borotritiide (13 Ci/mmol) and En3Hance spray by DuPont-NEN. Aluminium backed HPTLC plates and all solvents (HPLC or Aristar grade) were from Merck/BDH. The synthetic glycoside Gala12Mana1-O-CH3 was prepared by Dr Jillian Brown and Dr Robert Field (St. Andrews University). Purification of the sVSG MITat.1.2 GPI anchors sVSGat.1.2 was puri®ed from bloodstream form T. brucei cells using hypotonic lysis and DE52 chromatography, as described by Cross (1975). The VSG was further puri®ed by gel ®ltration using a Sephacryl-S200 column (4 cm 90 cm) equilibrated with 0.1 M NH4HCO3. Approximately 10 mg of freeze-dried sVSG was obtained from 1010 cells. Preparation of 3H-labelled sVSG MITat.1.2 GPI neutral glycans Approximately 0.5 mg of freeze dried sVSG MITat.1.2 was deaminated by dissolving the protein in 30 ml of 0.3 M sodium acetate (pH 4.0) and adding 15 ml of freshly prepared 1 M sodium nitrite, as described by Ferguson (1992). The reaction was left for three hours in the dark and reduction was achieved by altering the pH to approximately 10.5 with 10 ml 0.8 M boric acid and 24 ml 2 M NaOH and then adding the solution to 5 ml of 36 mM NaB3H4 dissolved in 0.1 M NaOH. After 90 minutes, the reduction was completed by adding 10 ml 0.5 M NaB2H4 in water (one hour). After neutralisation with 1 M acetic acid, and freeze drying to remove tritiated water formed in the reaction, the labelled glycoprotein was redissolved in water, dialysed, freeze-dried and treated with 50 ml 50% aqueous HF on ice for 60 hours. After neutralisation and desalting (Ferguson, 1992), this procedure produced the 3H-labelled neutral glycans, containing a [1-3H]2,5-anhydromannitol (AHM) residue where the glucosamine had been. Radioactive contaminants from the sodium borotritiide reagent were removed from the labelled glycans by paper chromatography and high voltage electrophoresis, as described in Ferguson (1992). This material was used in the initial analyses of the glycans and later, when larger quantities were needed, it was used as a tracer to help follow the puri®cation of the deutero-reduced neutral glycans, the preparation of which is described below.
The GPI Anchor of a Class-2 VSG Bulk preparation of sVSG MITat.1.2 GPI neutral glycans Larger amounts of the neutral glycans were needed for the composition and methylation analyses, and for NMR analysis. These were made by treating batches of 50 mg of sVSG MITat.1.2 with 1 ml 50% aqueous HF (60 hours at 0 C), and, after neutralising with saturated LiOH, precipitating the protein with 5% ice-cold trichloroacetic acid. The supernatant was desalted using a column of 3 ml AG3(OHÿ) over 4 ml QAE-Sephadex-A25. This GPI neutral glycan fraction was deaminated using 1 ml 1 M sodium acetate buffer (pH 4.0) and 1 ml freshly prepared 1 M NaNO2 (three hours at room temperature in the dark). Subsequently, the pH of the solution was adjusted to approximately 11, using 0.5 ml 0.8 M boric acid and 0.65 ml 2 M NaOH. The GPI glycans were then reduced with 1 ml 1 M NaB2H4 (one hour) and desalted, after acidi®cation with acetic acid, by passage through 3 ml AG50(H), drying and removal of boric acid by coevaporation with methanol. During the subsequent chromatography on Dionex HPAEC and Bio-Gel P4 a tracer of 106 cpm of the tritiated neutral glycans was added to assist in locating the GPI glycan peaks.
Purification of the sVSG MITat.1.2 GPI neutral glycans The tritiated neutral glycans were chromatographed by Dionex high-pH anion exchange chromatography (HPAEC) using a carbopac PA-1 column as described in (Ferguson, 1992). Sodium ions were removed from the eluate on-line using a Dionex ARRS unit and radioactivity was detected using an on-line Raytest Ramona detector with a 0.2 ml solid-scintillator X-cell. Pooled peak fractions were further desalted by passage through a small column containing AG50(H) over AG3(OHÿ) over QAE-Sephadex-A25. The size of the glycans in Dionex units (Du) was calculated by comparison to the pulsed-amperiometric detector trace of a sample of b-glucose oligomer internal standards. The individual peaks were then applied to a Bio-Gel P4 column for further puri®cation, and to enable the calculation of their size in glucose units (Gu) by comparison to the bglucose oligomer internal standards detected by refractive index. The bulk deutero-reduced GPI samples were chromatographed in a similar way, but with a trace of the tritiated material to assist location of the peaks, and several batches of the material had to be loaded onto the Dionex system.
Exoglycosidase digestions The digestions using coffee bean a-galactosidase and jack bean and bovine testis b-galactosidases were performed as described by Schneider & Ferguson (1995). For the E. coli and M. vinacea a-galactosidase digestions, one unit of enzyme in 0.1 M citrate phosphate buffer (pH 6.0) was used and for the A. niger a-galactosidase digestion 0.1 unit of enzyme in 0.1 M sodium acetate buffer (pH 4) was used. All digests were for 16 hours at 37 C and were terminated by heating to 100 C for ®ve minutes. Samples were desalted as described by Schneider & Ferguson (1995) and analysed by Dionex HPLC (Ferguson, 1992) and/or by silica HPTLC using propanol:acetone:water (10:6:5, by vol.; Schneider & Ferguson, 1995).
391
The GPI Anchor of a Class-2 VSG Inositol and carbohydrate analysis The GPI content of the protein and the recovery of GPI glycan after aqueous HF digestion was assessed by gc-ms inositol analysis. Samples were mixed with 40 pmol of [1,2,3,4,5,6-2H]myo-inositol internal standard, hydrolysed with 6 M HCl (18 hours at 110 C) and processed as described (by Ferguson, 1992). The monosaccharide contents of the puri®ed VSG MITat.1.2 neutral glycan fractions, and the coffee bean a-galactosidase treated cores, were estimated by gc-ms following methanolysis using 1 nmol scyllo-inositol as an internal standard (Ferguson, 1992). Methylation analysis Methylation analysis was carried out on the whole VSG MITat.1.2 GPI neutral glycan preparation, on the six puri®ed VSG MITat.1.2 GPI neutral glycan fractions and on the three coffee bean a-galactosidase-treated core glycans. Approximately 10 nmol of material was used for each methylation analysis and a sample of a Lacto-Ntetraose standard was analysed simultaneously. The method used is described by Ferguson (1992) and the resulting partially methylated alditol acetates (PMAAs) were analysed by gc-ms, using an SE54 Econocap column, (Alltech) and an SP2380 column (Supelco). Scylloinositol was added as an internal standard during the reduction step of the analysis. Partial acetolysis The three cores generated by exhaustive coffee bean a-galactosidase digestion were subjected to partial acetolysis, to con®rm the presence and location of the Mana1-6Man linkage. The samples were dried in 1 ml glass reactivials (Pierce) and peracetylated by heating for 30 minutes at 100 C with 40 ml of acetic anhydride: pyridine (1:1 v/v). The peracetylated glycans were dried and acetolysed by heating at 30 C for six hours with 30 ml acetic anhydride:acetic acid:conc. H2SO4 (10:10:1, by vol.). The reaction was stopped by the addition of 0.25 ml pyridine and 0.5 ml water. The acetylated products were extracted into 0.25 ml chloroform, washed three times with 0.5 ml water, dried and de-O-acetylated by heating at 37 C in 0.2 ml methanol:aq.35%NH3 (1:1, v/v) for 60 hours. The partially acetolysed neutral glycans were dried and redissolved in water for analysis by HPTLC (Schneider & Ferguson, 1995). 1
H NMR spectroscopy
The three cores generated by exhaustive coffee bean a-galactosidase digestion and the six puri®ed fractions of VSG MITat.1.2 GPI neutral glycans were analysed by one-dimensional NMR spectroscopy using a Bruker 500 MHz spectrometer. The samples were dissolved in 2 H2O, and the spectra were recorded at a probe temperature of 300 K, with the exception of the core-3 and the fraction 6 sample, which were also recorded at 310 K. The three cores generated by exhaustive coffee bean a-galactosidase digestion, and fractions 4 and 6 of the undigested glycans, were also analysed by twodimensional NMR. For these samples two-dimensional homonuclear 1H correlation spectroscopy (COSY), 1H rotating frame nuclear Overhauser spectroscopy (ROESY), and total correlation spectroscopy (TOCSY) were performed either on the Bruker spectrometer, or
on a Varian 500 MHz spectrometer. In the case of the ROESY, the transmitter offset was 5.6 ppm, the sweep width in both dimensions was 2300 Hz, and the mixing time was 500 ms, the number of experiments was 512 and the number of scans in each experiment was 64. In the TOCSY experiment, the sweep width was 1200 Hz, and the mixing time was 90 ms. Electrospray mass spectrometry A Micromass Quattro triple quadruple instrument was used in positive ion mode. The permethylated glycans were introduced into the ion source at 10 ml/min, at a concentration of 20 pmol/ml in 50% acetonitrile, 0.1% formic acid. The major pseudomolecular ions were [M Na H]2. In addition, non-derivatised glycans were also run under the same conditions, where the [M Na] pseudomolecular ions predominated; these results are shown in Table 1.
Acknowledgements This work was supported by a Programme Grant from the Wellcome Trust. J.M.R. is a Royal Society Dorothy Hodgkin Research Fellow and M.A.J.F. is a Howard Hughes International Research Scholar. We thank Dr Jillian Brown and Dr Robert Field for supplying the synthetic disaccharide Gala1-2Mana1-O-CH3 and Dr Andrei Nikolaev and Dr Janice Bramham for helpful comments.
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Edited by I. B. Holland (Received 22 September 1997; received in revised form 17 December 1997; accepted 17 December 1997)