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Trypanosoma brucei gambiense: Partitioning of glycopeptides of bloodstream and procyclic forms in Triton X-114
EXPERIMENTAL
PARASITOLOGY
65, 290-293 (1988)
RESEARCH BRIEF Trypanosoma
brucei
Bloodstream
Partitioning of Glycopeptides and Procyclic Forms in Triton X-l 14
of
gambiense:
ANDREW E. BALBERAND LISA M. Ho Department
of Microbiology
and Immunology, P.O. Box 3010, Duke University Durham, North Carolina 27710, U.S.A.
Medical
Center,
(Accepted for publication 11 December 1987) BALBER, A. E., AND Ho., L. M. 1988. Trypanosoma brucei gambiense: Partitioning of glycopeptides of bloodstream and procyclic forms in Triton X-l 14. Experimental Parasitology 65,290-293. 0 1988 Academic Press, Inc.
Lectin blotting has recently been used to identify concanavalin A-binding (Con A) invariant glycopeptides in mammalian bloodstream (BSF) and tsetse fly midgut procyclic (PF) forms of pathogenic African trypanosomes (Frommel and Balber 1987; Frommel et al. 1987). In the experiments described here, we have examined the distribution of these glycopeptides in the aqueous and detergent phases when extracts of BSF and PF of Trypanosoma brucei gambiense are partitioned with the nonionic detergent Triton X-l 14 (TX114); proteins with hydrophobic domains permitting stable interactions with membrane lipids are recovered in the TX-114 phase (Bordier 1981). The variant surface glycoprotein (VSG) of BSF was used to monitor partitioning. VSG is anchored in the surface membrane lipid bilayer by a C-terminal glycophospholipid (reviewed Cross 1987). The hydrophobic membrane form of VSG (mfVSG) is converted by an endogenous phospholipase C (Hereld et al. 1986) to a hydrophilic form (sVSG) that migrates more slowly than mfVSG on SDS-PAGE gels (Cardoso de Almeida and Turner 1983). When VSG preparations are partitioned by TX114, mfVSG is found in the detergent phase and sVSG, in the aqueous phase (Turner et al. 1985; Ward et al. 1987). Neither VSG (Vickerman 1985) nor phospholipase C (Bulow and Overath 1985; Turner et al. 1985)is synthesized by PF. BSF and PF of the NcTat 1.36 and NcTat 1.Oclones, respectively, of the STIB 368 strain of T. b. gambiense were maintained, isolated, and washed as described previously (Frommel and Balber 1987; Frommel et al. 1987). For TX-114 extractions, PF were resuspended and washed twice by centrifugation (13OOg,5 min) in 57 mM NaCl, 2.7 mA4 KCl, 42.3 mM sodium phosphate buffer, pH 7.4 (PFB), then resuspended (1.4 X log/ml) in PFB containing 1 mM p-methylsulfonylfluoride and
1 mM iodoacetamide (PFB-PMSF-IA), transferred to microfuge tubes, incubated on ice for 15 min, collected by centrifugation (20 set, lO,OOOg,room temperature), and extracted on ice for 10 min in PFB-PMSF-IA with 1% (w/v) precycled (Bordier, 1981) TX-114, TX-114 insoluble material was collected by centrifugation (12,OOOg,4 C, 7 min), resuspended in PFB-PMSF-IA, and diluted with electrophoresis sample buffer. The TX-l 14 soluble supernatant was incubated for 10 min at 37 C to permit phase separation and centrifuged (5 min, lO,OOOg,room temperature) to partition the Tri-
a
Copyright All rights
0 1988 by Academic Press, Inc. of reproduction in any form reserved.
c
d
.--
FIG. 1. Silver-stained 10% polyacrylamide gel of procyclic form proteins following Triton X-114 partitioning. (a) TX-114 phase; (b) aqueous phase; (c) TX114insoluble extract; (d) unfractionated whole cell extract.
290 0014-4894188$3.00
b
PHASE PARTITIONING
OF TRYPANOSOME
ton X-114 mixed micelle phase from the aqueous phase. The aqueous phase was transferred to another microfuge tube, brought to 1% TX-114, equilibrated for 10 min on ice, and recondensed and partitioned as described above. The twice-partitioned aqueous phase was brought to 2% TX-114. The two TX-114 phases were pooled and diluted with PFB-PMSF-IA to a final TX-l 14 concentration of 2%. The TX-l 14 and aqueous phases were then recondensed and centrifuged as before. The resulting purified TX-114 and aqueous phases were diluted with PFB-PMSF-IA to the same volume, vortexed, and equilibrated on ice for 10 min, diluted with electrophoresis sample buffer, boiled for 5 min, and frozen. TX-l 14 extraction and partitioning of BF was done by the same method except that 10 mM Tris-150 mM NaCI, pH 7.4, containing 1 mM each of PMSF, IA, and p-chloromecuriphenyl sulfonic acid (BFBPMSF-IA-pCMSA) was used throughout, and BF were dropped into boiling 0.4% (w/v) sodium dodecyl sulfate (SDS) in BFB-PMSF-IA-pCMSA prior to TX114 extraction to inhibit conversion of mfVSG to sVSG (Cardoso de Almeida and Turner 1983).
180)
=
b
C
291
GLYCOPROTEINS
Samples were fractionated in SDS-polyacrylamide gels, and either silver-stained to detect proteins or electrophoretically transferred to nitrocellulose and probed with horseradish peroxidase-conjugated Con A to detect glycoproteins (Frommel and Balber 1987). Mean values f standard deviation for apparent molecular masses of proteins are given below with the number of replicate experiments (n). TX-114 solubilized most proteins of both life cycle forms. The major TX-l 14 insoluble protein of PF had an apparent mass of 52.0 t 2.7 kDa (n = 5) (Fig. l), did not bind Con A (Fig. 2), and was probably tubulin (Thomashow et al. 1983). A TX-l 14 insoluble 75.3 f 2.1 kDa glycoprotein was routinely observed in PF (Fig. 2). Some mfVSG was left in the insoluble pellet of BSF (not shown). Only one silver-stained protein (Fig. 3) or Con Abinding glycoprotein (Fig. 4), mWSG (52-55 kDa), was routinely detected in the TX-114 phase of BSF extracts. The invariant glycoproteins we previously described (Frommel and Balber 1987) were all in the aqueous phase (Fig. 4). The electrophoretic mobility of mfVSG in the TX-l 14 layer was faster than that of sVSG (58-60 kDa) in the aqueous layer. The presense of sVSG means that phospholipase C was not com-
d
116)
40.5, 116,
a4-
58,
SVSC
,mf VSC 48.5,
FIG. 2. Lectin blot of 10% polyacrylamide gel of procyclic form glycoproteins following Triton X-l 14 partitioning. (a) TX-I 14 phase; (b) aqueous phase; (c) insoluble extract; (d) unfractionated whole cell extract.
FIG. 3. Silver-stained 7.5% polyacrylamide gel of bloodstream form proteins. (a) TX-114 phase; (b) whole cell extract.
292
BALBER
a
b
AND
HO
coproteins made by other cell types (Farquhar 198.5), also deserves consideration. Balber and Frommel (1988) have detected Con A-binding structures in the flagellar pocket of both Pf and BSF. Secretory products including acid hydrolases are present in the flagellar pocket (reviewed Vickerman 1985).
C
REFERENCES
BALBER,A.E., ANDFROMMEL,T.0.1988.
Trypanosoma brucei gambiense and T. b. rhodesiense: Con-
~SVSG -fVSG
canavalin A binding to the membrane and flagellar pocket of bloodstream and procyclic forms. Journal ofProtozoology 35(2), in press. BORDIER,C. 1981. Phase separation of integral membrane proteins in Triton X-114 solution. Journal of Biological Chemistry 26, 1604-1607. Buruow, R., ANDOVERATH,P. 1985. Synthesis of a hydrolase for the membrane form variant surface glycoprotein is repressed during transformation of Trypanosoma brucei. FEBS Letters 187, 105-110.
CARDOSO DE ALMEIDA, M.L., AND TURNER,M.J. FIG. 4. Lectin blot at 7.5% polyacrylamide gel of bloodstream form glycoproteins following Triton TX114 partitioning. (a) whole cell extract; (b) aqueous phase; (c) TX-l 14 phase. pletely inhibited during sample preparation; other TX114 soluble proteins bearing phospholipase C sensitive bonds could also have been converted into forms that partition into the aqueous phase. Two Con A-binding glycoproteins of Pf, Gp44p and Gp57p (apparent masses of 44 * 2.3 and 57 4 1.9 kDa, respectively; n = 10; see Frommel et al. 1987 for terminology) , partitioned exclusively into the TX- 114 phase (Fig. 2). TX-114 extraction routinely resolved these proteins from other glycoproteins with similar, but not identical, electrophoretic migrations that were present in the aqueous phase. Although Gp44p and Gp57p were readily detected with lectin blotting, they were difficult to detect with silver staining (Fig. 1). Thus, these components may be heavily glycosylated. Not all membrane proteins partition into the TX-114 phase (Bordier 1981), but proteins in the detergent are likely to be components of surface or internal membranes. These two putative membrane glycopeptides do not have the same apparent molecular masses as the major PF surface proteins identified by Gardiner et al. (1983). Cully et al. (1987) did not detect expression of a 46 kDa BSF glycoprotein in Pf. Except for mfVSG, Gp44p, and Gp57p, all the Con A-binding, glycoproteins of BSF and PF partitioned in the aqueous phase. Some may be membrane proteins that do not form mixed micelles with TX-114 efficiently under the conditions we used. The possibility that some of these glycoproteins are acid hydrolases or secretory proteins, major classes of water soluble gly-
1983. The membrane form of variant specific glycoproteins of Trypanosoma brucei. Nature (London) 302, 349-352.
CROSS,G. A. M. 1987. Eucaryotic protein moditication and membrane attachment via phosphatidylinositol. Cell 48, 179-181.
CULLY, D.F., GIBBS,C.P., AND CROSS,G. A.M. 1987. Identification of proteins encoded by variant specific glycoprotein expression site-associated genes in Trypanosoma brucei. Molecular and Biochemical Parasitology
21, 189-197.
FARQUHAR, M. G. 1985. Progress in unraveling pathways of Golgi traffic. Annual Reviews of Cell Biology 1, 447-488.
FROMMEL,T.O.,ANDBALBER,A.E. 1987. Trypanosoma brucei brucei, T. brucei gambiense, and T. brucei rhodesiense: Common glycoproteins and gly-
coprotein oligosaccharide heterogeneity identified by lectin affinity blotting and endoglycosidase H treatment. Experimental Parasitology 63, 3241.
FROMMEL,T.O.,KOHLER,M., AND BALBER,A. E. 1987. Trypanosoma brucei brucei and Trypanosoma gambiense: Stage specific differences in wheat germ
agglutinin binding and in endoglycosidase H sensitivity of glycoprotein oligosaccharides. Experimental Parasifology 64, 104-110.
GARDINER,P. R., FINERTY, J. F., AND DWYER, D. M. 1983. Iodination and identification of surface membrane antigens in procyclic Trypanosoma rhodesiense. Journal
of Immunology
131, 454-457.
HERELD, D., KRAKOW, J. L., BANGS, J. D., HART, G. W., ANDENGLUND,P. T. 1986. A phospholipase c from Trypanosoma brucei which selectively cleaves the glycolipid on the variant surface glycoprotein. Journal ofBiological Chemistry 261, 1381313819.
PHASE
PARTITIONING
OF TRYPANOSOME
GLYCOPROTEINS
293
THOMASOW, L. S., MILHALJSER, M., RUTTER, W. J., AND AGABIAN, N. 1983. Tubulin genes are tandemly linked and clustered in the genome of Trypanosoma brucei. Cell 32, 3543. TURNER, M. J., CARDOSO DE ALMEIDA, M., GURNETT, A. M., RAPER, J., AND WARD, J. 1985. Bio-
VICKERMAN, K. 1985. Developmental cycles and biology of pathogenic trypanosomes. British Medical Bulletin 41, 105-114. WARD, J., CARDOSO DE ALMEIDA, M. L., TURNER, M. J., ETGES, R., AND BORDIER, C. 1987. The assay of membrane bound Tvpanosoma brucei phospho-
synthesis, attachment and release of variant surface glycoproteins of the African trypanosomes. Current
lipase using an integral membrane protein substrate and detergent phase separation. Molecular and Biochemical Parasitology 23, l-7.
Topics in Microbiology
and Immunology
117,23-54.
PARASITOLOGY
65, 290-293 (1988)
RESEARCH BRIEF Trypanosoma
brucei
Bloodstream
Partitioning of Glycopeptides and Procyclic Forms in Triton X-l 14
of
gambiense:
ANDREW E. BALBERAND LISA M. Ho Department
of Microbiology
and Immunology, P.O. Box 3010, Duke University Durham, North Carolina 27710, U.S.A.
Medical
Center,
(Accepted for publication 11 December 1987) BALBER, A. E., AND Ho., L. M. 1988. Trypanosoma brucei gambiense: Partitioning of glycopeptides of bloodstream and procyclic forms in Triton X-l 14. Experimental Parasitology 65,290-293. 0 1988 Academic Press, Inc.
Lectin blotting has recently been used to identify concanavalin A-binding (Con A) invariant glycopeptides in mammalian bloodstream (BSF) and tsetse fly midgut procyclic (PF) forms of pathogenic African trypanosomes (Frommel and Balber 1987; Frommel et al. 1987). In the experiments described here, we have examined the distribution of these glycopeptides in the aqueous and detergent phases when extracts of BSF and PF of Trypanosoma brucei gambiense are partitioned with the nonionic detergent Triton X-l 14 (TX114); proteins with hydrophobic domains permitting stable interactions with membrane lipids are recovered in the TX-114 phase (Bordier 1981). The variant surface glycoprotein (VSG) of BSF was used to monitor partitioning. VSG is anchored in the surface membrane lipid bilayer by a C-terminal glycophospholipid (reviewed Cross 1987). The hydrophobic membrane form of VSG (mfVSG) is converted by an endogenous phospholipase C (Hereld et al. 1986) to a hydrophilic form (sVSG) that migrates more slowly than mfVSG on SDS-PAGE gels (Cardoso de Almeida and Turner 1983). When VSG preparations are partitioned by TX114, mfVSG is found in the detergent phase and sVSG, in the aqueous phase (Turner et al. 1985; Ward et al. 1987). Neither VSG (Vickerman 1985) nor phospholipase C (Bulow and Overath 1985; Turner et al. 1985)is synthesized by PF. BSF and PF of the NcTat 1.36 and NcTat 1.Oclones, respectively, of the STIB 368 strain of T. b. gambiense were maintained, isolated, and washed as described previously (Frommel and Balber 1987; Frommel et al. 1987). For TX-114 extractions, PF were resuspended and washed twice by centrifugation (13OOg,5 min) in 57 mM NaCl, 2.7 mA4 KCl, 42.3 mM sodium phosphate buffer, pH 7.4 (PFB), then resuspended (1.4 X log/ml) in PFB containing 1 mM p-methylsulfonylfluoride and
1 mM iodoacetamide (PFB-PMSF-IA), transferred to microfuge tubes, incubated on ice for 15 min, collected by centrifugation (20 set, lO,OOOg,room temperature), and extracted on ice for 10 min in PFB-PMSF-IA with 1% (w/v) precycled (Bordier, 1981) TX-114, TX-114 insoluble material was collected by centrifugation (12,OOOg,4 C, 7 min), resuspended in PFB-PMSF-IA, and diluted with electrophoresis sample buffer. The TX-l 14 soluble supernatant was incubated for 10 min at 37 C to permit phase separation and centrifuged (5 min, lO,OOOg,room temperature) to partition the Tri-
a
Copyright All rights
0 1988 by Academic Press, Inc. of reproduction in any form reserved.
c
d
.--
FIG. 1. Silver-stained 10% polyacrylamide gel of procyclic form proteins following Triton X-114 partitioning. (a) TX-114 phase; (b) aqueous phase; (c) TX114insoluble extract; (d) unfractionated whole cell extract.
290 0014-4894188$3.00
b
PHASE PARTITIONING
OF TRYPANOSOME
ton X-114 mixed micelle phase from the aqueous phase. The aqueous phase was transferred to another microfuge tube, brought to 1% TX-114, equilibrated for 10 min on ice, and recondensed and partitioned as described above. The twice-partitioned aqueous phase was brought to 2% TX-114. The two TX-114 phases were pooled and diluted with PFB-PMSF-IA to a final TX-l 14 concentration of 2%. The TX-l 14 and aqueous phases were then recondensed and centrifuged as before. The resulting purified TX-114 and aqueous phases were diluted with PFB-PMSF-IA to the same volume, vortexed, and equilibrated on ice for 10 min, diluted with electrophoresis sample buffer, boiled for 5 min, and frozen. TX-l 14 extraction and partitioning of BF was done by the same method except that 10 mM Tris-150 mM NaCI, pH 7.4, containing 1 mM each of PMSF, IA, and p-chloromecuriphenyl sulfonic acid (BFBPMSF-IA-pCMSA) was used throughout, and BF were dropped into boiling 0.4% (w/v) sodium dodecyl sulfate (SDS) in BFB-PMSF-IA-pCMSA prior to TX114 extraction to inhibit conversion of mfVSG to sVSG (Cardoso de Almeida and Turner 1983).
180)
=
b
C
291
GLYCOPROTEINS
Samples were fractionated in SDS-polyacrylamide gels, and either silver-stained to detect proteins or electrophoretically transferred to nitrocellulose and probed with horseradish peroxidase-conjugated Con A to detect glycoproteins (Frommel and Balber 1987). Mean values f standard deviation for apparent molecular masses of proteins are given below with the number of replicate experiments (n). TX-114 solubilized most proteins of both life cycle forms. The major TX-l 14 insoluble protein of PF had an apparent mass of 52.0 t 2.7 kDa (n = 5) (Fig. l), did not bind Con A (Fig. 2), and was probably tubulin (Thomashow et al. 1983). A TX-l 14 insoluble 75.3 f 2.1 kDa glycoprotein was routinely observed in PF (Fig. 2). Some mfVSG was left in the insoluble pellet of BSF (not shown). Only one silver-stained protein (Fig. 3) or Con Abinding glycoprotein (Fig. 4), mWSG (52-55 kDa), was routinely detected in the TX-114 phase of BSF extracts. The invariant glycoproteins we previously described (Frommel and Balber 1987) were all in the aqueous phase (Fig. 4). The electrophoretic mobility of mfVSG in the TX-l 14 layer was faster than that of sVSG (58-60 kDa) in the aqueous layer. The presense of sVSG means that phospholipase C was not com-
d
116)
40.5, 116,
a4-
58,
SVSC
,mf VSC 48.5,
FIG. 2. Lectin blot of 10% polyacrylamide gel of procyclic form glycoproteins following Triton X-l 14 partitioning. (a) TX-I 14 phase; (b) aqueous phase; (c) insoluble extract; (d) unfractionated whole cell extract.
FIG. 3. Silver-stained 7.5% polyacrylamide gel of bloodstream form proteins. (a) TX-114 phase; (b) whole cell extract.
292
BALBER
a
b
AND
HO
coproteins made by other cell types (Farquhar 198.5), also deserves consideration. Balber and Frommel (1988) have detected Con A-binding structures in the flagellar pocket of both Pf and BSF. Secretory products including acid hydrolases are present in the flagellar pocket (reviewed Vickerman 1985).
C
REFERENCES
BALBER,A.E., ANDFROMMEL,T.0.1988.
Trypanosoma brucei gambiense and T. b. rhodesiense: Con-
~SVSG -fVSG
canavalin A binding to the membrane and flagellar pocket of bloodstream and procyclic forms. Journal ofProtozoology 35(2), in press. BORDIER,C. 1981. Phase separation of integral membrane proteins in Triton X-114 solution. Journal of Biological Chemistry 26, 1604-1607. Buruow, R., ANDOVERATH,P. 1985. Synthesis of a hydrolase for the membrane form variant surface glycoprotein is repressed during transformation of Trypanosoma brucei. FEBS Letters 187, 105-110.
CARDOSO DE ALMEIDA, M.L., AND TURNER,M.J. FIG. 4. Lectin blot at 7.5% polyacrylamide gel of bloodstream form glycoproteins following Triton TX114 partitioning. (a) whole cell extract; (b) aqueous phase; (c) TX-l 14 phase. pletely inhibited during sample preparation; other TX114 soluble proteins bearing phospholipase C sensitive bonds could also have been converted into forms that partition into the aqueous phase. Two Con A-binding glycoproteins of Pf, Gp44p and Gp57p (apparent masses of 44 * 2.3 and 57 4 1.9 kDa, respectively; n = 10; see Frommel et al. 1987 for terminology) , partitioned exclusively into the TX- 114 phase (Fig. 2). TX-114 extraction routinely resolved these proteins from other glycoproteins with similar, but not identical, electrophoretic migrations that were present in the aqueous phase. Although Gp44p and Gp57p were readily detected with lectin blotting, they were difficult to detect with silver staining (Fig. 1). Thus, these components may be heavily glycosylated. Not all membrane proteins partition into the TX-114 phase (Bordier 1981), but proteins in the detergent are likely to be components of surface or internal membranes. These two putative membrane glycopeptides do not have the same apparent molecular masses as the major PF surface proteins identified by Gardiner et al. (1983). Cully et al. (1987) did not detect expression of a 46 kDa BSF glycoprotein in Pf. Except for mfVSG, Gp44p, and Gp57p, all the Con A-binding, glycoproteins of BSF and PF partitioned in the aqueous phase. Some may be membrane proteins that do not form mixed micelles with TX-114 efficiently under the conditions we used. The possibility that some of these glycoproteins are acid hydrolases or secretory proteins, major classes of water soluble gly-
1983. The membrane form of variant specific glycoproteins of Trypanosoma brucei. Nature (London) 302, 349-352.
CROSS,G. A. M. 1987. Eucaryotic protein moditication and membrane attachment via phosphatidylinositol. Cell 48, 179-181.
CULLY, D.F., GIBBS,C.P., AND CROSS,G. A.M. 1987. Identification of proteins encoded by variant specific glycoprotein expression site-associated genes in Trypanosoma brucei. Molecular and Biochemical Parasitology
21, 189-197.
FARQUHAR, M. G. 1985. Progress in unraveling pathways of Golgi traffic. Annual Reviews of Cell Biology 1, 447-488.
FROMMEL,T.O.,ANDBALBER,A.E. 1987. Trypanosoma brucei brucei, T. brucei gambiense, and T. brucei rhodesiense: Common glycoproteins and gly-
coprotein oligosaccharide heterogeneity identified by lectin affinity blotting and endoglycosidase H treatment. Experimental Parasitology 63, 3241.
FROMMEL,T.O.,KOHLER,M., AND BALBER,A. E. 1987. Trypanosoma brucei brucei and Trypanosoma gambiense: Stage specific differences in wheat germ
agglutinin binding and in endoglycosidase H sensitivity of glycoprotein oligosaccharides. Experimental Parasifology 64, 104-110.
GARDINER,P. R., FINERTY, J. F., AND DWYER, D. M. 1983. Iodination and identification of surface membrane antigens in procyclic Trypanosoma rhodesiense. Journal
of Immunology
131, 454-457.
HERELD, D., KRAKOW, J. L., BANGS, J. D., HART, G. W., ANDENGLUND,P. T. 1986. A phospholipase c from Trypanosoma brucei which selectively cleaves the glycolipid on the variant surface glycoprotein. Journal ofBiological Chemistry 261, 1381313819.
PHASE
PARTITIONING
OF TRYPANOSOME
GLYCOPROTEINS
293
THOMASOW, L. S., MILHALJSER, M., RUTTER, W. J., AND AGABIAN, N. 1983. Tubulin genes are tandemly linked and clustered in the genome of Trypanosoma brucei. Cell 32, 3543. TURNER, M. J., CARDOSO DE ALMEIDA, M., GURNETT, A. M., RAPER, J., AND WARD, J. 1985. Bio-
VICKERMAN, K. 1985. Developmental cycles and biology of pathogenic trypanosomes. British Medical Bulletin 41, 105-114. WARD, J., CARDOSO DE ALMEIDA, M. L., TURNER, M. J., ETGES, R., AND BORDIER, C. 1987. The assay of membrane bound Tvpanosoma brucei phospho-
synthesis, attachment and release of variant surface glycoproteins of the African trypanosomes. Current
lipase using an integral membrane protein substrate and detergent phase separation. Molecular and Biochemical Parasitology 23, l-7.
Topics in Microbiology
and Immunology
117,23-54.