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Variant surface glycoproteins of Trypanosoma congolense bloodstream and metacyclic forms are anchored by a glycolipid tail
Molecular and Biochemical Parasitology, 22 (1987) 153-158 Elsevier
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MBP 00742
Variant surface glycoproteins of Trypanosoma congolense bloodstream and metacyclic forms are anchored by a glycolipid tail Carole A. Ross, M. Lucia Cardoso de Almeida* and Mervyn J. Turner** Centre for Tropical Veterinary Medicine, Easterbush, Roslin, Midlothian, Scotland and MRC Biochemical Parasitology Unit,' The Molteno Institute, Cambridge, U.K. (Received 10 June 1986; accepted 8 August 1986)
The variant surface glycoproteins (VSGs) of both metacyclic and bloodstream forms of Trypanosoma congolense are shown to be anchored to the plasma membrane through a glycolipid similar to that found in Trypanosoma brucei. Release of soluble VSG from both metacyclic and bloodstream forms is associated with the exposure of an antigenic determinant homologous to the crossreacting determinant of T. brucei VSGs. Release of soluble VSG of T. congolense can be achieved by lysates of both bloodstream and metacyclic forms of T. congolense, by lysates of T. brucei bloodstream forms, but not by lysates of procyclic forms. Key words: Trypanosoma brucei; Trypanosoma congolense; Cross-reacting determinant; Phosphatidylinositol phospholipase C; Myristic acid
Introduction
Variant surface glycoproteins (VSGs) of African trypanosomes are anchored to the plasma membrane through a glycolipid covalently bound to the C-terminal amino acid [1-4]. Release of dimyristylglycerol from the glycolipid on activation of an endogenous phospholipase releases the surface coat in soluble form [2,3]. The substrate and product of this enzyme are referred to as mfVSG * Present address: Escola Paulista de Medicina, Rua Botucatu, No. 862, 8° And., 04023 Sao Paulo, S.P., Brazil. ** Present address: Merck Sharp & Dohme Research Laboratories, P.O. Box 2000, Rahway, NJ 07065, U.S.A. Correspondence address: C.A. Ross, Centre for Tropical Veterinary Medicine, Easterbush, Roslin, Midlothian EH25 9RG, Scotland. Abbreviations: mfVSG and sVSG, membrane form and soluble form of variant surface glycoproteins; CRD, cross-reacting determinant of the VSG; VAT, variant antigen type; mVAT, metacyclic VAT; TX-100, Triton X-100; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; TLCK, n-tosyl-l-lysyl-chloromethylketone; PBS, phosphatebuffered saline.
and sVSG, respectively. In mfVSG, the dimyristylglycerol is phosphodiester-linked to myo-inositol phosphate, which in turn is coupled to the reducing terminus of the oligosaccharide component of the glycolipid through an O-glycosidic bond to glucosamine [1]. Conversion of mfVSG to sVSG can be monitored through the exposure of a conserved antigenic determinant (the cross-reacting determinant or CRD) within the C-terminal oligosaccharide of sVSG [2], or by monitoring the release of radiolabeled myristate [5], and also by monitoring for the slight change in mobility on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) [2]. The phospholipase is present in bloodstream forms of the parasite, but although it is probably activated immediately on induction of differentiation to the procyclic trypomastigote form, which lacks the surface coat, the activity is rapidly depressed and is absent from fully transformed procyclics [6,7]. It is not known at what stage in the life cycle the activity of the enzyme reappears. The phospholipase and its mfVSG substrate have been largely characterized in Trypanosoma brucei. There are reports indicating that VSGs of Trypanosoma congolense isolated from bloodstream stages may share the
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T. brucei CRD [8], but no structural information on the CRD in T. congolense is available, although the N-linked oligosaccharide has been sequenced in at least one instance [9]. A number of novel methods have been reported for the isolation of T. congolense VSG to avoid problems associated with extensive proteolysis [10-12], but the mechanism of release has not been characterized. We wished to establish whether a similar linkage and release mechanism is employed in T. congolense and to use the in vitro cultivation system developed for T. congolense to determine whether metacyclic trypomastigotes share the same release mechanism with bloodstream forms. Materials and Methods
T. congolense stocks. TREU 1627 is a cloned derivative of a Gambian isolate, Kantong Kunda/ 77/LUMP/1794, isolated and maintained in culture [13]. TREU 1457, TREU 1881 and TREU 1885 cultured trypanosomes were derived respectively from cloned stocks of a Nigerian isolate Zaria/67/LUMP/69 and the Zambian isolates TRPZ 105 and DA/ZM/81/TRPZ 132. Cultivation of parasites. Infective metacyclic, procyclic and bloodstream forms of T. congolense stocks were grown, maintained and harvested from axenic and bovine aorta endothelium feeder layer-supplemented cultures respectively using methods of Gray et al. [14-16]. Bloodstream forms were harvested from cultures within one month of their initiation from metacyclic trypanosomes, when metacyclic variant antigen types (mVATs) still predominate in the population [17]. Antisera. Female New Zealand white rabbits were given an intradermal injection of 105 cultured metacyclic forms of T. congolense stocks. Stockspecific antiserum against each metacyclic trypanosome population was collected at weekly intervals for 4 weeks.
Whole cell lysates. Trypanosomes (usually in quantities of 5 × 107 to 108 parasites) were lysed by several methods so that properties of the VSGs prepared under different conditions could be compared. All buffers used in lysis procedures
contained 0.25 mM n-tosyl-l-lysyl-chloromethylketone (TLCK) to minimize proteolysis of VSGs. SDS lysates were produced by resuspending trypanosomes at 5 x 108 m1-1 in phosphatebuffered saline (PBS) containing 2% SDS at 100°C. After cooling, 2.5% Triton X-100 (TX100) in PBS was added to give a final concentration of 2% TX-100 and 0.4% SDS. Alternatively, metacyclics were resuspended in PBS/TLCK at a concentration of 4 x 10s m1-1 and dioxan lysis cocktail [12,18] added to 5% (v/v). After incubation for 2 h at room temperature with occasional gentle stirring, trypanosomes were spun at 200000 x g for 30 min in a Beckman centrifuge. Both supernatant and pellet were then prepared for SDS-PAGE. Hypotonic lysates were prepared by resuspending trypanosomes at 2 x 10s m1-1 in PBS/TLCK and then lysed by adding 20 volumes of 0.25 mM TLCK in water, followed by three cycles of freeze-thaw in liquid nitrogen. The lysed cells were then spun at 200000 × g for 30 min and supernatant fractions freeze dried before analysis by electrophoresis. Lysates of T. congolense metacyclic and procyclic forms and T. brucei MITat 1.4 bloodstream forms were prepared and tested for their ability to change the electrophoretic mobility of iodinated T. congolense metacyclic surface antigens. Trypanosomes previously washed in PBS/TLCK at 4 × 108 m1-1 (T. congolense) and 1 x 107 (T. brucei) were resuspended in 0.1% TX-100/TLCK.
Surface iodination of trypanosomes. Surface proteins were radiolabeled with 125I (Amersham) by a modification of a method used by Lanar and Manning [19]. Cultured metacyclic or bloodstream forms were washed in PBS pH 8.0 containing 1% (w/v) glucose (PSG) and resuspended to 5 x 107 ml-1 in PSG. The suspension (600 Ixl) was aliquoted into glass bijou bottles containing 20 Ixg iodogen, previously dried down from a chloroform solution. The labelling was carried out for 10 min in the presence of 300 IxCi Na[125I] either at 4°C for bloodstream forms or at 20°C for metacyclic forms. After this time, cells were normal and motile when observed under the microscope. Following several washes with PSG/TLCK, cells were lysed according to the protocols outlined above. In SDS-PAGE analysis, samples
155 containing 50000-100000 counts min -1 were applied to each track.
radiolabel visualized by autoradiography at -70°C using Fuji RX X-ray film.
Biosynthetic labelling of trypanosomes. The ra-
PAGE and fluorography. Cell lysates containing the equivalent of 2 x 107 metacyclic forms or 1 × 107 bloodstream forms were solubilized in SDS and mercaptoethanol and analyzed on 7-20% polyacrylamide gels in the presence of 0.1% SDS using the buffer system of Laemmli [20]. Some gels contained 4 M urea. Fluorography was performed on gels containing [3H]myristic acid labelled samples and stained with Coomassie Brilliant Blue R. After destaining, gels were incubated for 1 h at 20°C in 100 ml Amplify (Amersham), dried and exposed to Kodak X-Omat film at -70°C.
diolabel [9,10(n)-3H]myristic acid in toluene (Amersham) was prepared for incorporation into trypanosomes by drying off the solvent with nitrogen gas followed by solubilization in ethanol at 2 mCi m1-1. After harvesting and washing in PBS/TLCK, 2 x 107 cultured trypanosomes were resuspended in 1.0 ml of Minimum Essential Medium with Earle's salts (Flow Laboratories) supplemented with 1 mg m1-1 fraction V bovine albumin, essentially fatty acid free (Sigma). The radiolabel was then added, in three or four small portions to a final concentration of 100 p~Ci m1-1 (5% ethanol) and the suspension incubated at 28°C for 1 h. Following washing with PBS/TLCK, SDS, hypotonic or dioxan lysates were prepared. Metacyclic trypanosomes, which had incorporated [3H]myristic acid and which were to be used for immunoprecipitation experiments, were lysed by resuspension at 2 x 108 parasites ml -a in PBS/TLCK containing 1% n-octyl glucoside at 100°C. In SDS-PAGE analysis, samples containing either 2 x 107 metacyclic forms with [3H]myristic acid label or 1 x 107 bloodstream forms were applied to each track.
Immunoprecipitation. The ability of antisera from rabbits infected with cultured metacyclic populations to recognize T. congolense proteins labelled with [125I]iodine or [3H]myristic acid was tested by immunoprecipitation. Aliquots of labelled material were incubated with 20 Ixl of antisera. The immune complexes were precipitated by adding 100 Ixl of a 50% suspension of protein-A Sepharose (Sigma) and the products released by boiling in SDS-gel sample buffer.
Western blotting. Following SDS-PAGE [20], transfer of proteins to nitrocellulose membranes was performed using a modification of the method of Towbin et al. [21]. The paper, after blocking with excess protein, was incubated overnight with anti-CRD antibody, purified as described in Cardoso de Almeida and Turner [2]. Antibody bound to the nitrocellulose was detected using ~25I-labelled donkey anti-rabbit immunoglobulin and
Lysate mixtures. Iodinated metacyclic forms, which had been lysed in SDS/TX-100, were incubated with 50 txl of whole cell extracts of T. congolense metacyclic or procyclic forms or T. brucei bloodstream forms previously lysed in 0.1% TX-100. Control incubations for each different trypanosome form containing inactivated TX-100 lysate were also performed. After 1 h at 30°C, the mixtures were boiled in SDS-gel sample buffer and subjected to SDS-PAGE in the presence of 4 M urea. Results
Surface iodination of cultured metacyclic and bloodstream forms of T. congolense was used to identify VSGs. Surface iodination labelled a single band or group of bands of molecular weight ca. 54000 in both metacyclic and bloodstream forms. Conversion of mfVSG to sVSG in T. brucei is associated with exposure of the cross-reacting determinant. As shown in Fig. 1, anti-CRD raised against T. brucei VSG recognized nothing in an SDS lysate of T. congolense metacyclic forms or in the pellet produced following hypotonic lysis. These are the conditions under which the higher mobility mfVSG is retained. However, the supernatant fraction produced on hypotonic lysis, which contains the lower mobility sVSG, reacts with the anti-CRD. The same result was reproduced with bloodstream trypomastigotes.
156
1 2 3 4 5 6
92-,,* 67..~
m
45,,~
31--~ 21-.~
conversion and for the presence of the converting phosphatidylinositol-specific phospholipase C [2,6,22]. As shown in Fig. 3, this assay provides evidence for the presence of the converting enzyme in both bloodstream and metacyclic forms of T. congolense, and its absence from the respective procyclic forms. Also, the lysate of bloodstream T. brucei which contains the active phospholipase can act upon the T. congolense substrate. Thus, tracks 1 and 2 of Fig. 3 show the change in electrophoretic mobility when VSGs of metacyclic trypomastigotes are extracted either with SDS or by hypotonic lysis. When an SDS lysate containing the higher mobility mfVSG is incubated with a T. brucei lysate, the mobility change is apparent only if the T. brucei lysate has not been heat-inactivated. A similar result is obtained when the metacyclic mfVSG is incubated with a heterologous lysate of metacyclic trypomastigotes which, again, only produces conversion if the lysate is not heat-inactivated. Lysates
Fig. 1. Western blot analysis of metacyclic and bloodstream form lysates, after SDS-PAGE in the presence of 4 M urea. This autoradiograph shows the pattern of recognition of T. congolense antigens by antibody to T. brucei cross reacting determinant (anti-CRD). Samples analyzed were: (1) TREU 1627 metacyclic forms lysed in SDS; (2) 200000 x g supernatant and (3) pellet of metacyclic forms after hypotonic lysis; (4) T R E U 1627 bloodstream forms lysed in SDS; (5) 200000 x g supernatant and (6) pellet of bloodstream forms after hypotonic lysis.
VSGs of T. brucei incorporate [3H]myristate into mfVSG, but the radiolabel, as expected, is lost following conversion to sVSG [5]. As shown in Fig. 2, T. congolense bloodstream and metacyclic forms incorporate [3H] myristate into protein under the same conditions used for labelling of T. brucei VSGs and the radiolabel is detectable only in SDS lysates and in the pellet produced following either hypotonic lysis or extraction with dioxan. The absence of myristate from the pellet produced after hypotonic lysis of bloodstream forms (Fig. 2, track 8) is puzzling, and may reflect proteolysis. The change in electrophoretic mobility on conversion of mfVSG to sVSG has been used in our laboratory as a simple but effective test for
Fig. 2. Myristic acid incorporation by cultured TREU 1627 metacyclic and bloodstream forms. Samples were analyzed by SDS-PAGE in the absence of urea, followed by fluorography of this gel. (1) Metacyclic forms lysed in SDS; (2) 14000 x g supernatant and (3) pellet of metacyclics after hypotonic lysis; (4) 14 000 x g supernatant and (5) pellet of metacyclics treated with dioxan; (6) bloodstream forms lysed in SDS; (7) 14000 x g supernatant and (8) pellet of bloodstream forms after hypotonic lysis.
157 1
2
3
4
5
6
7
8
92-'~ 67-'~
45--*
31--* 21-~
Fig. 3. Change in electrophoretic mobility of iodinated metacyclic antigens after incubation with whole cell lysates of T. congolense and T. brucei trypanosomes. Reactions were carried out as described in Methods and samples were analysed by SDS-PAGE in the presence of urea. This autoradiograph of the gel shows: track 1, iodinated metacyclic forms lysed in SDS; track 2, 200000 x g supernatant of hypotonically lysed metacyclic forms; tracks 3 and 4, iodinated SDS lysate incubated with T. brucei MITat 1.4 bloodstream forms lysed in TX100 at 100°C and 20°C, respectively; tracks 5 and 6, iodinated SDS lysate incubated with T. congolense TREU 1457 metacyclic forms lysed in TX-100 at 100°C and 20°C, respectively; tracks 7 and 8, iodinated SDS lysate incubated with T. congolense TREU 1885 procyclic forms lysed in TX-100 at 100°C and 20°C, respectively.
from procyclic trypomastigotes of T. congolense were incapable of effecting conversion (track 7,
8). Discussion
The attachment and release of VSGs represents one of the most interesting areas of research in antigenic variation. The phosphatidylinositol-specific phospholipase C m a y be a target for c h e m o t h e r a p y , as m a y the assembly of the glycolipid and its transfer onto the nascent V S G polypeptide. It has been established in T. brucei that the lipase is present, though quiescent, in bloodstream stages, but probably becomes activated when differentiation to procyclic trypomastigotes is initiated and loss of coat observed.
However, the lipase activity is absent from fully differentiated procyclic forms [2,6,7]. Transcription of V S G genes also stops very rapidly when this differentiation step is induced, and VSGs are absent from the trypanosome until the metacyclic trypomastigotes develop in the salivary glands of the tsetse fly [23,24]. We were interested to determine whether the activity of the phospholipase was coordinated with V S G gene expression and the data presented here show that enzyme activity is present in metacyclic and bloodstream stages but not in procyclic trypomastigotes. Epimastigote forms were not tested. Furthermore, the assay used is qualitative and actual enzyme levels should be measured at different stages of the life cycle. The corollary to this finding is that metacyclic VSGs seem to use a m e m b r a n e anchor indistinguishable from that on bloodstream VSGs. An advantage in working with T. congolense is the ability to complete the entire life cycle in culture and in such a way that relatively large amounts of each stage are available for analysis. This is particularly true for the metacyclic trypomastigotes, which in all other systems are available in only very small numbers. Thus, with T. congolense, experiments consuming more than 108 metacyclic trypomastigotes were possible and this simply cannot be done at present with T. brucei. Acknowledgements
We wish to thank Dr. Scott Crowe (Wellcome Laboratories) for his initial interest in this project, Dr. J.D. Ansell of the Dept. of Zoology, University of Edinburgh for allowing us to use equipment in his laboratory, and Iain F r a m e who isolated the Z a m b i a n stocks of T. congolense into culture. C . A . R . is supported by funds from the Overseas D e v e l o p m e n t Administration. M.L.C. de A. is a research fellow of St. John's College, Cambridge. M.J.T. received support from the U N D P / W o r l d B a n k / W H O Special Program for Research and Training in Tropical Diseases and from the International L a b o r a t o r y for Research on Animal Diseases, Nairobi.
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References 1 Ferguson, M.A.J., Low, M. and Cross, G.A.M. (1985) Glycosyl-sn-l,2-dimyristyl phosphatidylinositol is covalently linked to Trypanosorna brucei variant surface glycoprotein. J. Biol. Chem. 260, 14547-14555. 2 Cardoso de Almeida, M.L. and Turner, M.J. (1983) The membrane form of variant surface glycoproteins of Trypanosoma brucei. Nature 302, 349-352. 3 Ferguson, M.A.J., Haldar, K. and Cross, G.A.M. (1985) Trypanosoma brucei variant surface glycoprotein has a sn1,2-dimyristylglycerol membrane anchor at its COOH terminus. J. Biol. Chem. 260, 4963-4968. 4 Holder, A.A. (1983) Carbohydrate is linked through ethanolamine to the C-terminal amino acid of Trypanosoma brucei variant surface glycoprotein. Biochem. J. 209, 261-262. 5 Ferguson, M.A.J. and Cross, G.A.M. (1984) Myristylation of the membrane form of Trypanosoma brucei variant surface glycoprotein. J. Biol. Chem. 259, 3011-3015. 6 Cardoso de Almeida, M.L., LePage R.W.F. and Turner, M.J. (1984) The release of variant surface glycoproteins of Trypanosoma brucei. In: Proceedings of the 3rd John Jacob Abel Symposium on Drug Development: Molecular Parasitology (August, T., ed.), pp. 19-31, Allen Press, Kansas City. 7 Bulow, R. and Overath, P. (1985) Synthesis of a hydrolase for the membrane-form variant surface glycoprotein is repressed during transformation of Trypanosoma brucei. FEBS Lett. 187, 105-110. 8 Barbet, A.F. and McGuire T.C. (1978) Cross reacting determinants in variant specific surface antigens of African trypanosomes. Proc. Natl. Acad. Sci. USA 75, 1989-1993. 9 Savage, A., Geyer, R., Stirum, S., Reinwald, E. and Risse, H.J. (1984) Structural studies on the major oligosaccharides in a variant surface glycoprotein of Trypanosoma congolense. Mol. Biochem. Parasitol. 4, 129-138. 10 Onodera, M., Rosen, N.L., Lifter, J., Hotez, P.J., Bogucki, M.S., Davis G., Patton, C.L., Konigsberg, W.H. and Richards, F.F. (1981) Trypanosoma congolense: surface glycoproteins of two early bloodstream variants. II. Purification and partial chemical characterization. Exp. Parasitol. 52, 42%434. 11 Reinwald, E., Rautenberg, P. and Risse, H.J. (1979) Trypanosoma congolense: mechanical removal of the surface coat in vitro. Exp. Parasitol. 48, 384-397. 12 Reinwald, E., Rautenberg, P. and Risse, H.J. (1981) Purification of the variant antigens of Trypanosoma congolense. A new approach to the isolation of glycoproteins. Biochim. Biophys. Acta 668, 119-131. 13 Ross, C.A., Gray, M.A., Taylor, A.M. and Luckins, A.G.
14
15
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18
19
20
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22
23
24
(1985) In vitro cultivation of Trypanosoma congolense: establishment of infective mammalian forms in continuous culture after isolation from the blood of infected mice. Acta Trop. 42, 113-122. Gray, M.A., Cunningham, I., Gardiner, P.R., Taylor, A.M. and Luckins, A.G. (1981) Cultivation of infective forms of Trypanosoma congolense at 28°C from trypanosomes in the proboscis of Glossina morsitans. Parasitology 92, 81-95. Gray, M.A., Ross, C.A., Taylor, A.M. and Luckins, A.G. (1984) In vitro cultivation of Trypanosoma congolense: the production of infective metacyclic trypanosomes in cultures initiated from cloned stocks. Acta Trop. 41,343-353. Gray, M.A., Ross, C.A., Taylor, A.M., Tetley, L. and Luckins, A.G. (1985) In vitro cultivation of Trypanosoma congolense: the production of infective forms from metacyclic trypanosomes cultured on bovine endothelial cell monolayers. Acta Trop. 42, 99-111. Luckins, A.G., Frame, I.A., Gray, M.A., Crowe, J.S. and Ross, C.A. (1986) Analysis of trypanosome variable antigen types in cultures of metacyclic and mammalian forms of Trypanosoma congolense. Parasitology 93, 99-109. Cook, G.A., Honigberg, B.M. and Zimmerman, R.A. (1985) Isolation and cell free synthesis of variant surface glycoproteins from Trypanosoma congolense. Mol. Biochem. Parasitol. 15,281-294. Lanar, D.E. and Manning, S.E. (1984) Major surface proteins and antigens on the different in vivo and in vitro forms of Trypanosoma cruzi. Mol. Biochem. Parasitol. 11, 119-131. Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227,680-685. Towbin, H., Staehlin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedures and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354. Cardoso de Almeida, M.L., Allan, L.M. and Turner M.J. (1984) Purification and properties of the membrane form of variant surface glycoproteins (VSGs) from Trypanosoma brucei. J. Protozool. 31, 53-60. Barry, J.D., Hajduk, S.L., Vickerman, K. and LeRay, D. (1979) Detection of multiple variable antigen types in metacyclic populations of Trypanosoma brucei. Trans. R. Soc. Trop. Med. Hyg. 34, 53-64. Crowe, J.S., Barry, J.D., Luckins, A.G., Ross, C.A. and Vickerman, K. (1983) All metacyclic variable antigen types of Trypanosoma congolense identified using monoclonal antibodies. Nature 306,389-391.
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Variant surface glycoproteins of Trypanosoma congolense bloodstream and metacyclic forms are anchored by a glycolipid tail Carole A. Ross, M. Lucia Cardoso de Almeida* and Mervyn J. Turner** Centre for Tropical Veterinary Medicine, Easterbush, Roslin, Midlothian, Scotland and MRC Biochemical Parasitology Unit,' The Molteno Institute, Cambridge, U.K. (Received 10 June 1986; accepted 8 August 1986)
The variant surface glycoproteins (VSGs) of both metacyclic and bloodstream forms of Trypanosoma congolense are shown to be anchored to the plasma membrane through a glycolipid similar to that found in Trypanosoma brucei. Release of soluble VSG from both metacyclic and bloodstream forms is associated with the exposure of an antigenic determinant homologous to the crossreacting determinant of T. brucei VSGs. Release of soluble VSG of T. congolense can be achieved by lysates of both bloodstream and metacyclic forms of T. congolense, by lysates of T. brucei bloodstream forms, but not by lysates of procyclic forms. Key words: Trypanosoma brucei; Trypanosoma congolense; Cross-reacting determinant; Phosphatidylinositol phospholipase C; Myristic acid
Introduction
Variant surface glycoproteins (VSGs) of African trypanosomes are anchored to the plasma membrane through a glycolipid covalently bound to the C-terminal amino acid [1-4]. Release of dimyristylglycerol from the glycolipid on activation of an endogenous phospholipase releases the surface coat in soluble form [2,3]. The substrate and product of this enzyme are referred to as mfVSG * Present address: Escola Paulista de Medicina, Rua Botucatu, No. 862, 8° And., 04023 Sao Paulo, S.P., Brazil. ** Present address: Merck Sharp & Dohme Research Laboratories, P.O. Box 2000, Rahway, NJ 07065, U.S.A. Correspondence address: C.A. Ross, Centre for Tropical Veterinary Medicine, Easterbush, Roslin, Midlothian EH25 9RG, Scotland. Abbreviations: mfVSG and sVSG, membrane form and soluble form of variant surface glycoproteins; CRD, cross-reacting determinant of the VSG; VAT, variant antigen type; mVAT, metacyclic VAT; TX-100, Triton X-100; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; TLCK, n-tosyl-l-lysyl-chloromethylketone; PBS, phosphatebuffered saline.
and sVSG, respectively. In mfVSG, the dimyristylglycerol is phosphodiester-linked to myo-inositol phosphate, which in turn is coupled to the reducing terminus of the oligosaccharide component of the glycolipid through an O-glycosidic bond to glucosamine [1]. Conversion of mfVSG to sVSG can be monitored through the exposure of a conserved antigenic determinant (the cross-reacting determinant or CRD) within the C-terminal oligosaccharide of sVSG [2], or by monitoring the release of radiolabeled myristate [5], and also by monitoring for the slight change in mobility on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) [2]. The phospholipase is present in bloodstream forms of the parasite, but although it is probably activated immediately on induction of differentiation to the procyclic trypomastigote form, which lacks the surface coat, the activity is rapidly depressed and is absent from fully transformed procyclics [6,7]. It is not known at what stage in the life cycle the activity of the enzyme reappears. The phospholipase and its mfVSG substrate have been largely characterized in Trypanosoma brucei. There are reports indicating that VSGs of Trypanosoma congolense isolated from bloodstream stages may share the
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T. brucei CRD [8], but no structural information on the CRD in T. congolense is available, although the N-linked oligosaccharide has been sequenced in at least one instance [9]. A number of novel methods have been reported for the isolation of T. congolense VSG to avoid problems associated with extensive proteolysis [10-12], but the mechanism of release has not been characterized. We wished to establish whether a similar linkage and release mechanism is employed in T. congolense and to use the in vitro cultivation system developed for T. congolense to determine whether metacyclic trypomastigotes share the same release mechanism with bloodstream forms. Materials and Methods
T. congolense stocks. TREU 1627 is a cloned derivative of a Gambian isolate, Kantong Kunda/ 77/LUMP/1794, isolated and maintained in culture [13]. TREU 1457, TREU 1881 and TREU 1885 cultured trypanosomes were derived respectively from cloned stocks of a Nigerian isolate Zaria/67/LUMP/69 and the Zambian isolates TRPZ 105 and DA/ZM/81/TRPZ 132. Cultivation of parasites. Infective metacyclic, procyclic and bloodstream forms of T. congolense stocks were grown, maintained and harvested from axenic and bovine aorta endothelium feeder layer-supplemented cultures respectively using methods of Gray et al. [14-16]. Bloodstream forms were harvested from cultures within one month of their initiation from metacyclic trypanosomes, when metacyclic variant antigen types (mVATs) still predominate in the population [17]. Antisera. Female New Zealand white rabbits were given an intradermal injection of 105 cultured metacyclic forms of T. congolense stocks. Stockspecific antiserum against each metacyclic trypanosome population was collected at weekly intervals for 4 weeks.
Whole cell lysates. Trypanosomes (usually in quantities of 5 × 107 to 108 parasites) were lysed by several methods so that properties of the VSGs prepared under different conditions could be compared. All buffers used in lysis procedures
contained 0.25 mM n-tosyl-l-lysyl-chloromethylketone (TLCK) to minimize proteolysis of VSGs. SDS lysates were produced by resuspending trypanosomes at 5 x 108 m1-1 in phosphatebuffered saline (PBS) containing 2% SDS at 100°C. After cooling, 2.5% Triton X-100 (TX100) in PBS was added to give a final concentration of 2% TX-100 and 0.4% SDS. Alternatively, metacyclics were resuspended in PBS/TLCK at a concentration of 4 x 10s m1-1 and dioxan lysis cocktail [12,18] added to 5% (v/v). After incubation for 2 h at room temperature with occasional gentle stirring, trypanosomes were spun at 200000 x g for 30 min in a Beckman centrifuge. Both supernatant and pellet were then prepared for SDS-PAGE. Hypotonic lysates were prepared by resuspending trypanosomes at 2 x 10s m1-1 in PBS/TLCK and then lysed by adding 20 volumes of 0.25 mM TLCK in water, followed by three cycles of freeze-thaw in liquid nitrogen. The lysed cells were then spun at 200000 × g for 30 min and supernatant fractions freeze dried before analysis by electrophoresis. Lysates of T. congolense metacyclic and procyclic forms and T. brucei MITat 1.4 bloodstream forms were prepared and tested for their ability to change the electrophoretic mobility of iodinated T. congolense metacyclic surface antigens. Trypanosomes previously washed in PBS/TLCK at 4 × 108 m1-1 (T. congolense) and 1 x 107 (T. brucei) were resuspended in 0.1% TX-100/TLCK.
Surface iodination of trypanosomes. Surface proteins were radiolabeled with 125I (Amersham) by a modification of a method used by Lanar and Manning [19]. Cultured metacyclic or bloodstream forms were washed in PBS pH 8.0 containing 1% (w/v) glucose (PSG) and resuspended to 5 x 107 ml-1 in PSG. The suspension (600 Ixl) was aliquoted into glass bijou bottles containing 20 Ixg iodogen, previously dried down from a chloroform solution. The labelling was carried out for 10 min in the presence of 300 IxCi Na[125I] either at 4°C for bloodstream forms or at 20°C for metacyclic forms. After this time, cells were normal and motile when observed under the microscope. Following several washes with PSG/TLCK, cells were lysed according to the protocols outlined above. In SDS-PAGE analysis, samples
155 containing 50000-100000 counts min -1 were applied to each track.
radiolabel visualized by autoradiography at -70°C using Fuji RX X-ray film.
Biosynthetic labelling of trypanosomes. The ra-
PAGE and fluorography. Cell lysates containing the equivalent of 2 x 107 metacyclic forms or 1 × 107 bloodstream forms were solubilized in SDS and mercaptoethanol and analyzed on 7-20% polyacrylamide gels in the presence of 0.1% SDS using the buffer system of Laemmli [20]. Some gels contained 4 M urea. Fluorography was performed on gels containing [3H]myristic acid labelled samples and stained with Coomassie Brilliant Blue R. After destaining, gels were incubated for 1 h at 20°C in 100 ml Amplify (Amersham), dried and exposed to Kodak X-Omat film at -70°C.
diolabel [9,10(n)-3H]myristic acid in toluene (Amersham) was prepared for incorporation into trypanosomes by drying off the solvent with nitrogen gas followed by solubilization in ethanol at 2 mCi m1-1. After harvesting and washing in PBS/TLCK, 2 x 107 cultured trypanosomes were resuspended in 1.0 ml of Minimum Essential Medium with Earle's salts (Flow Laboratories) supplemented with 1 mg m1-1 fraction V bovine albumin, essentially fatty acid free (Sigma). The radiolabel was then added, in three or four small portions to a final concentration of 100 p~Ci m1-1 (5% ethanol) and the suspension incubated at 28°C for 1 h. Following washing with PBS/TLCK, SDS, hypotonic or dioxan lysates were prepared. Metacyclic trypanosomes, which had incorporated [3H]myristic acid and which were to be used for immunoprecipitation experiments, were lysed by resuspension at 2 x 108 parasites ml -a in PBS/TLCK containing 1% n-octyl glucoside at 100°C. In SDS-PAGE analysis, samples containing either 2 x 107 metacyclic forms with [3H]myristic acid label or 1 x 107 bloodstream forms were applied to each track.
Immunoprecipitation. The ability of antisera from rabbits infected with cultured metacyclic populations to recognize T. congolense proteins labelled with [125I]iodine or [3H]myristic acid was tested by immunoprecipitation. Aliquots of labelled material were incubated with 20 Ixl of antisera. The immune complexes were precipitated by adding 100 Ixl of a 50% suspension of protein-A Sepharose (Sigma) and the products released by boiling in SDS-gel sample buffer.
Western blotting. Following SDS-PAGE [20], transfer of proteins to nitrocellulose membranes was performed using a modification of the method of Towbin et al. [21]. The paper, after blocking with excess protein, was incubated overnight with anti-CRD antibody, purified as described in Cardoso de Almeida and Turner [2]. Antibody bound to the nitrocellulose was detected using ~25I-labelled donkey anti-rabbit immunoglobulin and
Lysate mixtures. Iodinated metacyclic forms, which had been lysed in SDS/TX-100, were incubated with 50 txl of whole cell extracts of T. congolense metacyclic or procyclic forms or T. brucei bloodstream forms previously lysed in 0.1% TX-100. Control incubations for each different trypanosome form containing inactivated TX-100 lysate were also performed. After 1 h at 30°C, the mixtures were boiled in SDS-gel sample buffer and subjected to SDS-PAGE in the presence of 4 M urea. Results
Surface iodination of cultured metacyclic and bloodstream forms of T. congolense was used to identify VSGs. Surface iodination labelled a single band or group of bands of molecular weight ca. 54000 in both metacyclic and bloodstream forms. Conversion of mfVSG to sVSG in T. brucei is associated with exposure of the cross-reacting determinant. As shown in Fig. 1, anti-CRD raised against T. brucei VSG recognized nothing in an SDS lysate of T. congolense metacyclic forms or in the pellet produced following hypotonic lysis. These are the conditions under which the higher mobility mfVSG is retained. However, the supernatant fraction produced on hypotonic lysis, which contains the lower mobility sVSG, reacts with the anti-CRD. The same result was reproduced with bloodstream trypomastigotes.
156
1 2 3 4 5 6
92-,,* 67..~
m
45,,~
31--~ 21-.~
conversion and for the presence of the converting phosphatidylinositol-specific phospholipase C [2,6,22]. As shown in Fig. 3, this assay provides evidence for the presence of the converting enzyme in both bloodstream and metacyclic forms of T. congolense, and its absence from the respective procyclic forms. Also, the lysate of bloodstream T. brucei which contains the active phospholipase can act upon the T. congolense substrate. Thus, tracks 1 and 2 of Fig. 3 show the change in electrophoretic mobility when VSGs of metacyclic trypomastigotes are extracted either with SDS or by hypotonic lysis. When an SDS lysate containing the higher mobility mfVSG is incubated with a T. brucei lysate, the mobility change is apparent only if the T. brucei lysate has not been heat-inactivated. A similar result is obtained when the metacyclic mfVSG is incubated with a heterologous lysate of metacyclic trypomastigotes which, again, only produces conversion if the lysate is not heat-inactivated. Lysates
Fig. 1. Western blot analysis of metacyclic and bloodstream form lysates, after SDS-PAGE in the presence of 4 M urea. This autoradiograph shows the pattern of recognition of T. congolense antigens by antibody to T. brucei cross reacting determinant (anti-CRD). Samples analyzed were: (1) TREU 1627 metacyclic forms lysed in SDS; (2) 200000 x g supernatant and (3) pellet of metacyclic forms after hypotonic lysis; (4) T R E U 1627 bloodstream forms lysed in SDS; (5) 200000 x g supernatant and (6) pellet of bloodstream forms after hypotonic lysis.
VSGs of T. brucei incorporate [3H]myristate into mfVSG, but the radiolabel, as expected, is lost following conversion to sVSG [5]. As shown in Fig. 2, T. congolense bloodstream and metacyclic forms incorporate [3H] myristate into protein under the same conditions used for labelling of T. brucei VSGs and the radiolabel is detectable only in SDS lysates and in the pellet produced following either hypotonic lysis or extraction with dioxan. The absence of myristate from the pellet produced after hypotonic lysis of bloodstream forms (Fig. 2, track 8) is puzzling, and may reflect proteolysis. The change in electrophoretic mobility on conversion of mfVSG to sVSG has been used in our laboratory as a simple but effective test for
Fig. 2. Myristic acid incorporation by cultured TREU 1627 metacyclic and bloodstream forms. Samples were analyzed by SDS-PAGE in the absence of urea, followed by fluorography of this gel. (1) Metacyclic forms lysed in SDS; (2) 14000 x g supernatant and (3) pellet of metacyclics after hypotonic lysis; (4) 14 000 x g supernatant and (5) pellet of metacyclics treated with dioxan; (6) bloodstream forms lysed in SDS; (7) 14000 x g supernatant and (8) pellet of bloodstream forms after hypotonic lysis.
157 1
2
3
4
5
6
7
8
92-'~ 67-'~
45--*
31--* 21-~
Fig. 3. Change in electrophoretic mobility of iodinated metacyclic antigens after incubation with whole cell lysates of T. congolense and T. brucei trypanosomes. Reactions were carried out as described in Methods and samples were analysed by SDS-PAGE in the presence of urea. This autoradiograph of the gel shows: track 1, iodinated metacyclic forms lysed in SDS; track 2, 200000 x g supernatant of hypotonically lysed metacyclic forms; tracks 3 and 4, iodinated SDS lysate incubated with T. brucei MITat 1.4 bloodstream forms lysed in TX100 at 100°C and 20°C, respectively; tracks 5 and 6, iodinated SDS lysate incubated with T. congolense TREU 1457 metacyclic forms lysed in TX-100 at 100°C and 20°C, respectively; tracks 7 and 8, iodinated SDS lysate incubated with T. congolense TREU 1885 procyclic forms lysed in TX-100 at 100°C and 20°C, respectively.
from procyclic trypomastigotes of T. congolense were incapable of effecting conversion (track 7,
8). Discussion
The attachment and release of VSGs represents one of the most interesting areas of research in antigenic variation. The phosphatidylinositol-specific phospholipase C m a y be a target for c h e m o t h e r a p y , as m a y the assembly of the glycolipid and its transfer onto the nascent V S G polypeptide. It has been established in T. brucei that the lipase is present, though quiescent, in bloodstream stages, but probably becomes activated when differentiation to procyclic trypomastigotes is initiated and loss of coat observed.
However, the lipase activity is absent from fully differentiated procyclic forms [2,6,7]. Transcription of V S G genes also stops very rapidly when this differentiation step is induced, and VSGs are absent from the trypanosome until the metacyclic trypomastigotes develop in the salivary glands of the tsetse fly [23,24]. We were interested to determine whether the activity of the phospholipase was coordinated with V S G gene expression and the data presented here show that enzyme activity is present in metacyclic and bloodstream stages but not in procyclic trypomastigotes. Epimastigote forms were not tested. Furthermore, the assay used is qualitative and actual enzyme levels should be measured at different stages of the life cycle. The corollary to this finding is that metacyclic VSGs seem to use a m e m b r a n e anchor indistinguishable from that on bloodstream VSGs. An advantage in working with T. congolense is the ability to complete the entire life cycle in culture and in such a way that relatively large amounts of each stage are available for analysis. This is particularly true for the metacyclic trypomastigotes, which in all other systems are available in only very small numbers. Thus, with T. congolense, experiments consuming more than 108 metacyclic trypomastigotes were possible and this simply cannot be done at present with T. brucei. Acknowledgements
We wish to thank Dr. Scott Crowe (Wellcome Laboratories) for his initial interest in this project, Dr. J.D. Ansell of the Dept. of Zoology, University of Edinburgh for allowing us to use equipment in his laboratory, and Iain F r a m e who isolated the Z a m b i a n stocks of T. congolense into culture. C . A . R . is supported by funds from the Overseas D e v e l o p m e n t Administration. M.L.C. de A. is a research fellow of St. John's College, Cambridge. M.J.T. received support from the U N D P / W o r l d B a n k / W H O Special Program for Research and Training in Tropical Diseases and from the International L a b o r a t o r y for Research on Animal Diseases, Nairobi.
158
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