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Lectin interactions with the variant surface glycoprotein from Trypanosoma brucei brucei incorporated into liposomes
Vol. 141, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1274-1278
December 30, 1986
LECTIN INTERACTIONS WITH THE VARIANT SURFACE GLYCOPROTEIN FROM TRYPANOSOMA BRUCEI BRUCEI INCORPORATED INTO LIPOSOMES
Brigitte Schmitz and Roger A. Klein Medical Research Council, Molteno Institute, University of Cambridge, Downing Street, CAMBRIDGE, CB2 3EE, UK Received October 22, 1986
SUMMARY: The variant specific surface glycoprotein from Trypanosoma bmcei brucei is incorporated into lipid vesicles using 8M urea as an unfolding reagent. Pronase treatment of these proteoliposomes removes most of the protein, leaving a glycophospholipopeptide which is the membrane attachment site. We show here that lectins, specific for mannose and galactose are able to recognize oligosaccharide residues on these proteoliposomes, using a straightforward aggregation assay. The relevance of these results obtained with the liposome model system to the accessibility of the surface antigens in living trypanosomes is discussed. ® 1986 AcademicPress, Inc.
Trypanosoma brucei brucei, a haemoflagellate pathogenic protozoan causing disease in cattle ('nagana') and used as a laboratory model of sleeping sickness in man, possesses an antigenic surface coat of protein, the variant specific glycoprotein, known to exist in two forms, the water-soluble form and the membrane form (1). The soluble and the membrane form are distinguished by the membrane form possessing a dimyristoylated glyceride moiety which is thought to be the membrane attachment site (2). We have recently described a procedure for preparing the C-terminal glycolipophosphopeptide from mfVSG which represents the attachment site of the protein to the plasma membrane, by incorporating mfVSG into lipid vesicles using 8M urea as the unfolding agent, followed by pronase treatment and separation of the remaining GPLP from the lipids (3). In this communication we demonstrate that GPLP shows binding to lectins that differs from binding to the intact mfVSG. Eight lectins have been tested, but only those which are specific for either D-mannose or D-galactose gave clear results. This is due to the binding to either the highmannose-type oligosaccharide, or to the cross-reacting determinant found on the VSG. The latter contains an as yet unidentified oligosaccharide composed of galactose, mannose and glucosamine (2,4). MATERIALS AND METHODS Eg~Ayolk phosphatidylcholine and cholesterol were obtained from the Sigma Chemical Company, and [~4C]iodoacetate from Amersham International, UK. Octadecylamine was purified according to the method of Klein and Ellory (7) from commercially available material.
Abbreviations: ConA, Concanavalin A; ECA, Erythrina cristagalli agglutinin; LCA, Lens culinaris agglutinin; PNA, peanut agglutinin; PSA, Pisu'm sativum agglutinm; RCA, Ricinus communis agglutinin; SBA, soybean agglutinin; WGA, wheat germ agglutinin; DTr, dith!ottE.eitol.;I ~ P E S , (hydroxyethyl)-piperazine-etfiane-sulfonicacid; MOPS, (morpnolino)-propane-sulfonic acia; sVSG, water-soluble variant specific glycoprotein; mfVSG, membrane-form variant specific glycopmtein; GPLP, glycophospholipopeptide; CRD, cross-reacting determinant. 0006-291X/86 $1.50 Copyright ~;) 1986 by Academic Press, Inc. All rights of reproduction in any jorm reserved.
1274
Vol. 141, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Lectins were obtained from Kem-En Tec (Henerup, Denmark) except for ConA and RCA (Sigma). Agglutination was measured as an increase in turbidity (absorbance) at 450 nm and at room temperature using a spectrophotometer (Pye-Unicam, Model SP1800). Preparation of VSG Trypanosoma brucei brucei clone MITat 1.6 were prepared by passage through rats and obtained by aortic puncture, followed by DE52 chromatography, and the sVSG was subsequently purified as previously described (5). The method of Jackson (6) was used for the preparaton of mfVSG. Incorporation of mfVSG into liposomes mfVSG was incorporated into liposomes using a modified version of the procedure described earlier (3). mfVSG (80 nmol), radio-labelled with [14C]iodoacetate after reduction with DTI', was dissolved in 1 ml of 8M urea in 10 mM Hepes (pH 7.5) and left at room temperature for 30 rain. Lipids (32 Ixmol of phosphatidylcholine:octadecylamine:cholesterol, 66:4:30) were dried under a stream of nitrogen, then 400 gl of 8M urea in 10 mM Hepes (pH 7.5) were added, followed by vortexing until the lipids were completely dispersed. This lipid suspension was added to the protein solution and incubated for 45 min at room temperature, diluted with distilled water to 30 ml and centrifuged at I00,000 gay for 1 h at 5°C using a Ti SW50.1 (Beckman) swing-out rotor. Pronase treatment The liposome pellets were resuspended in 0.5 ml 10 mM Hepes buffer (pH 7.5) containing 2 mM CaC12, and mixed by vortexing. Pronase (2% w/w) was added at the beginning of a three hour incubation at 37°C and again after 1.5 h. The proteolysis was stopped by centrifugation at 100,000 gav for 1 h at +5°C (Ti SW50.1). Lectin agglutination (8) The liposome.pellets were dissolved in 10 mM MOPS-buffer (pH 6.8), containing in the case of ConA 1 mM Caz+, Mnz+ and Mgz+. Agglutination was measured at a concentration of 1 grnol lipid/ml (corresponding to 2.5 nmol/ml of VSG). The concentration of the lectin was 100 gg/ml. Agglutination was measured either against lipid suspensions (1 gmol/ml) containing no VSG but the same amount of lectin, or in presence of non-inhibiting sugar (50 mM) against VSG(GPLP)-lipidliposomes plus inhibiting sugar (50 mM) and the same amount of lectin (100 Ixg) in both cuvettes. RESULTS AND DISCUSSION The incorporation of mfVSG into liposomes was found to be very unsatisfactory when using previously reported procedures (3,9). Unfolding of the VSG in the presence of 8 M urea yielded good results, especially when on average only one SH-group was alkylated; increasing alkylation reduced the level of incorporation. In this study negatively charged liposomes gave an incorporation of 80%. Postivively charged liposomes, however, yielded an even higher incorporation of 90-95% (detailed results not shown), probably as a result of interaction with a phosphate group on the VSG, which is known to be part of the CRD (10). Moreover, the ratio lipid:VSG could be reduced from 600:1 to 400:1. A crucial step in this procedure is the complete removal of urea by dilution and centrifugation. It is important that urea is diluted to a concentration of < 0.5 M, otherwise the liposomes would not sediment to the bottom of the tube. Other details of this method, including the identification of constituents of the membrane attachment site bound to the liposomes after pronase treatment, are described in (3). The VSG of MITat 1.6 belongs to class I VSG (11), known to contain one high-mannose-type oligosaccharide which has recently been identified by FAB-MS after Endo-H-treatment as a mixture of Man5.9GlcNAc-isomers (12). The other oligosacctlaride of unknown structure composed of galactose, mannose and glucosamine, is involved in the antigenic cross=reacting activity of different VSG strains with an antibody raised against a single VSG strain (4). In order to get more information on the structural organization of the CRD oligosaccharide, we used VSG incorporated into liposomes as a suitable model for studies of the interaction with lectins. 1275
Vol. 1 4 1 , No. 3, 1 9 8 6
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table I. Lectin interactionwith mfVSG or GPLP bound to Hposomes I.eetin
mfVSO
OPLP
Coneanavalin A
(a)
49L-4_(3)
22+10)
Peanut
Co) (a)
80+_2(3) 12,15
(~t.~4(_ 3) 68,70
Co)
48,55
Pfsum sativura Lens eulinaris Rieinus eommunis Er~darina eristagalli
115,125
+
+
Soybean Wheat germ Leetin specificity according to (14). Values for Co, A mad PNA represent the relative increase in absofl~ane¢ at 450 ran, m ~ u r e d after 30 min incubation; (a) aggregation measured against liposomes without VSG or GPLP, with I00 p.g lectin in both cuvettes; Co) measured with lipesomes plus VSG or GPLP and leetin in both euvettes, and with competing sugar in the reference euvette (methyl glucoside for ConA, galactose for PNA), and non-competing sugar (galactose or mannose respectively) in the sample cuvette. All other agglutinations were either between 1% and 20% (+) or < 1% (-) relative aggregation.
The results of lectin binding to the VSG and to the GPLP bound to liposomes are shown in Table 1 and Figure 1. From all the lectins tested, only those specific for either mannose or galactose showed clearly positive results. The highest aggregation was found for PNA with the GPLP when measured in the presence of non-competing/competing sugar in the sample and reference cuvette respectively, whereas much less binding was found for VSG. ConA binding was, as expected, higher for VSG than for GPLP.
120
d 100 to
== 80'
60 o} >, t~
t~
40 ¸
20 ¸
lb
~o
~o
~o
rain
Figure 1. Time course for the agglutination of VSG and GPLP incorporated into liposomes. Conditions are as described in the legend to Table 1 under (b). CortA plus (a) VSG, Co) GPLP; PNA plus (c) VSG and (d) GPLP. 1276
Vol. 141, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
As liposomes aggregate in the presence of lectins even if they do not carry any VSG (not shown), values of aggregation measured against liposomes on their own are always lower than those measured against liposomes carrying VSG (or GPLP) in the presence of competing sugar because control sample aggregation is prevented. With all the other lectins tested, some show a weak effect (< 20% relative aggregation) with VSG or GPLP, but they all are known to bind to mannose (PSA, LCA) or galactose (ECA, RCA). Their weaker binding is probably due to specific structural requirements. SBA and WGA show no activity at all in agreement with none of the sugars, for which they are specific, occuring in VSGs. Lectins (ConA, LCA) have been used by several workers for the purification of VSG with varying success (5). The oligosaccharides of VSGs are not exposed at the surface of the trypanosome, as ConA binding can only be observed after trypsin digestion of living trypanosomes (11), and the oligosaccharide of the CRD is probably prevented from interaction with lectins in a similar way as it is from binding to CRD-antibodies.VSG molecules are tightly packed on trypanosomes; an area of 16.5 nm2/VSG molecule has been estimated based upon electron microscopy and size distribution analysis (6). In our experiments, with a ratio of lipids:VSG of 400:1, an approximate area of 32-48 nm2/ VSG is calculated, if one takes into account that the liposomes prepared by the urea method contain on average 5 bilayers and that the VSG is only incorporated into the outer layer (3). For this reason lectins will have access to the high-mannose-type oligosaccharide of the VSG which is about 50 amino acids from the C-terminus. The binding of ConA to liposomes carrying the GPLP is to be expected as the CRD contains mannose too, which although part of a complex oligosaccharide structure, can also bind to ConA (13). It is unlikely that GPLP contains the highmannose-type oligosaccharide because pronase treatment releases 85-90% of the radiolabelled mfVSG incorporated into liposomes (3). The strong binding of PNA to GPLP incorporated into liposomes is interpreted as binding to a galactose residue of the CRD, which is probably only possible because of the less tight packing of VSG in liposomes making a lectin interaction close to, or at the interface of the liposomes more likely. The differences in accessibility of the oligosaccharide residues to lectins in the liposome model system and the living trypanosome seem to result from the effects of molecular packing in the liposomes and in the plasma membrane. This should be considered in the context of the difference in binding for CRD-antibodies to soluble VSG and membrane-bound VSG, where the shielding effects of the membrane attachment site may also play a role (11). ACKNOWLEDGEMENTS We should like to acknowledge the help and assistance of both Imogen Duncan and Salih Eresh. REFERENCES 1. Cardoso de Almeida, M.L. and Turner, M.J. (1983) Nature 302: 349-352. 2. Ferguson, M.A.J., Halgar, K. and Cross, G.A.M. (1985) J. Biol. Chem. 260: 4963-4968. 3. Schmitz, B., Klein, R.A., Egge, H. and Peter-Katalinic, J. (1986) Mol. Biochem. Parasitol. 20: 191197. 4. Holder, A.A. and Cross, G.A.M. (1981) Mol. Biochem. Parasitol. 2: 135-150. 5. Cross, G.A.M. (1984) J. Cell Biochem. 24: 79-90. 6. Jackson, D.G., Owen, M.J. and Voorheis, H.P. (1985) Biochem. J. 230: 195-202. 7. Klein, R.A. and Ellory, J.C. (1980) J. Membr. Biol. 55: 123-131. 1277
Vol. 141, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
8. Juliano, R.L. and Stamp, D. (1976) Nature 261: 235-237. 9. Racker, E.(ed.) (1985) Reconstitution of Transporters, Receptors and Pathological States, Academic Press, London. 10. Ferguson, M.A.J., Low, M.G. and Cross, G.A.M. (1985) J. Biol. Chem. 260: 14547-14555. 11. Tumer, M.J. (1985) Ann. Inst. Pasteur Immunol. 136C: 41-49. 12. Egge, H., Gunawan, J., Peter-Katalinic, J., Schmitz, B., Duncan, I.A. and Klein, R.A. (1986) in "Topics in Lipid Research - from Structural Elucidation to Biological Function", pp. 141-151, eds. R.A. Klein and B. Schmitz, Royal Society of Chemistry, London. 13. Debray, H., Decout, D., Strecker, G., Spik, G. and Montreuil, J. (1981) Eur. J. Biochem. 117: 4155. 14. Goldstein, I.J. and Hayes, C.E. (1978) Adv. Carbohydr. Chem. Biochem. 35: 127-340.
1278
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1274-1278
December 30, 1986
LECTIN INTERACTIONS WITH THE VARIANT SURFACE GLYCOPROTEIN FROM TRYPANOSOMA BRUCEI BRUCEI INCORPORATED INTO LIPOSOMES
Brigitte Schmitz and Roger A. Klein Medical Research Council, Molteno Institute, University of Cambridge, Downing Street, CAMBRIDGE, CB2 3EE, UK Received October 22, 1986
SUMMARY: The variant specific surface glycoprotein from Trypanosoma bmcei brucei is incorporated into lipid vesicles using 8M urea as an unfolding reagent. Pronase treatment of these proteoliposomes removes most of the protein, leaving a glycophospholipopeptide which is the membrane attachment site. We show here that lectins, specific for mannose and galactose are able to recognize oligosaccharide residues on these proteoliposomes, using a straightforward aggregation assay. The relevance of these results obtained with the liposome model system to the accessibility of the surface antigens in living trypanosomes is discussed. ® 1986 AcademicPress, Inc.
Trypanosoma brucei brucei, a haemoflagellate pathogenic protozoan causing disease in cattle ('nagana') and used as a laboratory model of sleeping sickness in man, possesses an antigenic surface coat of protein, the variant specific glycoprotein, known to exist in two forms, the water-soluble form and the membrane form (1). The soluble and the membrane form are distinguished by the membrane form possessing a dimyristoylated glyceride moiety which is thought to be the membrane attachment site (2). We have recently described a procedure for preparing the C-terminal glycolipophosphopeptide from mfVSG which represents the attachment site of the protein to the plasma membrane, by incorporating mfVSG into lipid vesicles using 8M urea as the unfolding agent, followed by pronase treatment and separation of the remaining GPLP from the lipids (3). In this communication we demonstrate that GPLP shows binding to lectins that differs from binding to the intact mfVSG. Eight lectins have been tested, but only those which are specific for either D-mannose or D-galactose gave clear results. This is due to the binding to either the highmannose-type oligosaccharide, or to the cross-reacting determinant found on the VSG. The latter contains an as yet unidentified oligosaccharide composed of galactose, mannose and glucosamine (2,4). MATERIALS AND METHODS Eg~Ayolk phosphatidylcholine and cholesterol were obtained from the Sigma Chemical Company, and [~4C]iodoacetate from Amersham International, UK. Octadecylamine was purified according to the method of Klein and Ellory (7) from commercially available material.
Abbreviations: ConA, Concanavalin A; ECA, Erythrina cristagalli agglutinin; LCA, Lens culinaris agglutinin; PNA, peanut agglutinin; PSA, Pisu'm sativum agglutinm; RCA, Ricinus communis agglutinin; SBA, soybean agglutinin; WGA, wheat germ agglutinin; DTr, dith!ottE.eitol.;I ~ P E S , (hydroxyethyl)-piperazine-etfiane-sulfonicacid; MOPS, (morpnolino)-propane-sulfonic acia; sVSG, water-soluble variant specific glycoprotein; mfVSG, membrane-form variant specific glycopmtein; GPLP, glycophospholipopeptide; CRD, cross-reacting determinant. 0006-291X/86 $1.50 Copyright ~;) 1986 by Academic Press, Inc. All rights of reproduction in any jorm reserved.
1274
Vol. 141, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Lectins were obtained from Kem-En Tec (Henerup, Denmark) except for ConA and RCA (Sigma). Agglutination was measured as an increase in turbidity (absorbance) at 450 nm and at room temperature using a spectrophotometer (Pye-Unicam, Model SP1800). Preparation of VSG Trypanosoma brucei brucei clone MITat 1.6 were prepared by passage through rats and obtained by aortic puncture, followed by DE52 chromatography, and the sVSG was subsequently purified as previously described (5). The method of Jackson (6) was used for the preparaton of mfVSG. Incorporation of mfVSG into liposomes mfVSG was incorporated into liposomes using a modified version of the procedure described earlier (3). mfVSG (80 nmol), radio-labelled with [14C]iodoacetate after reduction with DTI', was dissolved in 1 ml of 8M urea in 10 mM Hepes (pH 7.5) and left at room temperature for 30 rain. Lipids (32 Ixmol of phosphatidylcholine:octadecylamine:cholesterol, 66:4:30) were dried under a stream of nitrogen, then 400 gl of 8M urea in 10 mM Hepes (pH 7.5) were added, followed by vortexing until the lipids were completely dispersed. This lipid suspension was added to the protein solution and incubated for 45 min at room temperature, diluted with distilled water to 30 ml and centrifuged at I00,000 gay for 1 h at 5°C using a Ti SW50.1 (Beckman) swing-out rotor. Pronase treatment The liposome pellets were resuspended in 0.5 ml 10 mM Hepes buffer (pH 7.5) containing 2 mM CaC12, and mixed by vortexing. Pronase (2% w/w) was added at the beginning of a three hour incubation at 37°C and again after 1.5 h. The proteolysis was stopped by centrifugation at 100,000 gav for 1 h at +5°C (Ti SW50.1). Lectin agglutination (8) The liposome.pellets were dissolved in 10 mM MOPS-buffer (pH 6.8), containing in the case of ConA 1 mM Caz+, Mnz+ and Mgz+. Agglutination was measured at a concentration of 1 grnol lipid/ml (corresponding to 2.5 nmol/ml of VSG). The concentration of the lectin was 100 gg/ml. Agglutination was measured either against lipid suspensions (1 gmol/ml) containing no VSG but the same amount of lectin, or in presence of non-inhibiting sugar (50 mM) against VSG(GPLP)-lipidliposomes plus inhibiting sugar (50 mM) and the same amount of lectin (100 Ixg) in both cuvettes. RESULTS AND DISCUSSION The incorporation of mfVSG into liposomes was found to be very unsatisfactory when using previously reported procedures (3,9). Unfolding of the VSG in the presence of 8 M urea yielded good results, especially when on average only one SH-group was alkylated; increasing alkylation reduced the level of incorporation. In this study negatively charged liposomes gave an incorporation of 80%. Postivively charged liposomes, however, yielded an even higher incorporation of 90-95% (detailed results not shown), probably as a result of interaction with a phosphate group on the VSG, which is known to be part of the CRD (10). Moreover, the ratio lipid:VSG could be reduced from 600:1 to 400:1. A crucial step in this procedure is the complete removal of urea by dilution and centrifugation. It is important that urea is diluted to a concentration of < 0.5 M, otherwise the liposomes would not sediment to the bottom of the tube. Other details of this method, including the identification of constituents of the membrane attachment site bound to the liposomes after pronase treatment, are described in (3). The VSG of MITat 1.6 belongs to class I VSG (11), known to contain one high-mannose-type oligosaccharide which has recently been identified by FAB-MS after Endo-H-treatment as a mixture of Man5.9GlcNAc-isomers (12). The other oligosacctlaride of unknown structure composed of galactose, mannose and glucosamine, is involved in the antigenic cross=reacting activity of different VSG strains with an antibody raised against a single VSG strain (4). In order to get more information on the structural organization of the CRD oligosaccharide, we used VSG incorporated into liposomes as a suitable model for studies of the interaction with lectins. 1275
Vol. 1 4 1 , No. 3, 1 9 8 6
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table I. Lectin interactionwith mfVSG or GPLP bound to Hposomes I.eetin
mfVSO
OPLP
Coneanavalin A
(a)
49L-4_(3)
22+10)
Peanut
Co) (a)
80+_2(3) 12,15
(~t.~4(_ 3) 68,70
Co)
48,55
Pfsum sativura Lens eulinaris Rieinus eommunis Er~darina eristagalli
115,125
+
+
Soybean Wheat germ Leetin specificity according to (14). Values for Co, A mad PNA represent the relative increase in absofl~ane¢ at 450 ran, m ~ u r e d after 30 min incubation; (a) aggregation measured against liposomes without VSG or GPLP, with I00 p.g lectin in both cuvettes; Co) measured with lipesomes plus VSG or GPLP and leetin in both euvettes, and with competing sugar in the reference euvette (methyl glucoside for ConA, galactose for PNA), and non-competing sugar (galactose or mannose respectively) in the sample cuvette. All other agglutinations were either between 1% and 20% (+) or < 1% (-) relative aggregation.
The results of lectin binding to the VSG and to the GPLP bound to liposomes are shown in Table 1 and Figure 1. From all the lectins tested, only those specific for either mannose or galactose showed clearly positive results. The highest aggregation was found for PNA with the GPLP when measured in the presence of non-competing/competing sugar in the sample and reference cuvette respectively, whereas much less binding was found for VSG. ConA binding was, as expected, higher for VSG than for GPLP.
120
d 100 to
== 80'
60 o} >, t~
t~
40 ¸
20 ¸
lb
~o
~o
~o
rain
Figure 1. Time course for the agglutination of VSG and GPLP incorporated into liposomes. Conditions are as described in the legend to Table 1 under (b). CortA plus (a) VSG, Co) GPLP; PNA plus (c) VSG and (d) GPLP. 1276
Vol. 141, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
As liposomes aggregate in the presence of lectins even if they do not carry any VSG (not shown), values of aggregation measured against liposomes on their own are always lower than those measured against liposomes carrying VSG (or GPLP) in the presence of competing sugar because control sample aggregation is prevented. With all the other lectins tested, some show a weak effect (< 20% relative aggregation) with VSG or GPLP, but they all are known to bind to mannose (PSA, LCA) or galactose (ECA, RCA). Their weaker binding is probably due to specific structural requirements. SBA and WGA show no activity at all in agreement with none of the sugars, for which they are specific, occuring in VSGs. Lectins (ConA, LCA) have been used by several workers for the purification of VSG with varying success (5). The oligosaccharides of VSGs are not exposed at the surface of the trypanosome, as ConA binding can only be observed after trypsin digestion of living trypanosomes (11), and the oligosaccharide of the CRD is probably prevented from interaction with lectins in a similar way as it is from binding to CRD-antibodies.VSG molecules are tightly packed on trypanosomes; an area of 16.5 nm2/VSG molecule has been estimated based upon electron microscopy and size distribution analysis (6). In our experiments, with a ratio of lipids:VSG of 400:1, an approximate area of 32-48 nm2/ VSG is calculated, if one takes into account that the liposomes prepared by the urea method contain on average 5 bilayers and that the VSG is only incorporated into the outer layer (3). For this reason lectins will have access to the high-mannose-type oligosaccharide of the VSG which is about 50 amino acids from the C-terminus. The binding of ConA to liposomes carrying the GPLP is to be expected as the CRD contains mannose too, which although part of a complex oligosaccharide structure, can also bind to ConA (13). It is unlikely that GPLP contains the highmannose-type oligosaccharide because pronase treatment releases 85-90% of the radiolabelled mfVSG incorporated into liposomes (3). The strong binding of PNA to GPLP incorporated into liposomes is interpreted as binding to a galactose residue of the CRD, which is probably only possible because of the less tight packing of VSG in liposomes making a lectin interaction close to, or at the interface of the liposomes more likely. The differences in accessibility of the oligosaccharide residues to lectins in the liposome model system and the living trypanosome seem to result from the effects of molecular packing in the liposomes and in the plasma membrane. This should be considered in the context of the difference in binding for CRD-antibodies to soluble VSG and membrane-bound VSG, where the shielding effects of the membrane attachment site may also play a role (11). ACKNOWLEDGEMENTS We should like to acknowledge the help and assistance of both Imogen Duncan and Salih Eresh. REFERENCES 1. Cardoso de Almeida, M.L. and Turner, M.J. (1983) Nature 302: 349-352. 2. Ferguson, M.A.J., Halgar, K. and Cross, G.A.M. (1985) J. Biol. Chem. 260: 4963-4968. 3. Schmitz, B., Klein, R.A., Egge, H. and Peter-Katalinic, J. (1986) Mol. Biochem. Parasitol. 20: 191197. 4. Holder, A.A. and Cross, G.A.M. (1981) Mol. Biochem. Parasitol. 2: 135-150. 5. Cross, G.A.M. (1984) J. Cell Biochem. 24: 79-90. 6. Jackson, D.G., Owen, M.J. and Voorheis, H.P. (1985) Biochem. J. 230: 195-202. 7. Klein, R.A. and Ellory, J.C. (1980) J. Membr. Biol. 55: 123-131. 1277
Vol. 141, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
8. Juliano, R.L. and Stamp, D. (1976) Nature 261: 235-237. 9. Racker, E.(ed.) (1985) Reconstitution of Transporters, Receptors and Pathological States, Academic Press, London. 10. Ferguson, M.A.J., Low, M.G. and Cross, G.A.M. (1985) J. Biol. Chem. 260: 14547-14555. 11. Tumer, M.J. (1985) Ann. Inst. Pasteur Immunol. 136C: 41-49. 12. Egge, H., Gunawan, J., Peter-Katalinic, J., Schmitz, B., Duncan, I.A. and Klein, R.A. (1986) in "Topics in Lipid Research - from Structural Elucidation to Biological Function", pp. 141-151, eds. R.A. Klein and B. Schmitz, Royal Society of Chemistry, London. 13. Debray, H., Decout, D., Strecker, G., Spik, G. and Montreuil, J. (1981) Eur. J. Biochem. 117: 4155. 14. Goldstein, I.J. and Hayes, C.E. (1978) Adv. Carbohydr. Chem. Biochem. 35: 127-340.
1278