(~) INSTITUT PASTEUR/ELSEVIEr: Paris 1990
Res. Immunol. 1990, 141, 743-755
GLYCOLIPID ANCHORAGE OF P L A S M O D I U M F A L C I P A R U M SURFACE ANTIGENS C. Braun Breton
(1),
T.L. Rosenberry
(2) (*)
and L.H. Pereira da Silva (1)
Unit o f Experimental Parasitology (]), Unit of Membra:Te Biology (2), Institut Pasteur, 75624 Paris Cedex 15
SUMMARY
Human red blood cells (RBC) were infected with the malarial parasite Plaso modium falciparum, the anchoring of schizont proteins to RBC membranes by glycoinositol phospholipids was demonstrated by three criteria: (1) metabolic incorporation of 3H-ethanolamine and 3H-myristate into the protein; (2) release of 35S-methionine-labelled protein into the supernatant after incubation with phosphatidylinositol-specific phospholipase C; and (3) the exposure of a glycoinositol phosphate epitope on the methioninelabelled protein following phospholipase C cleavage. Labelled proteins were analysed by immunoprecipitation, polyacrylamide gel electrophoresis in sodium dodecylsulphate and gel fluorography. Several candidate proteins were observed when each criteria was investigated. Among these, 3 proteins which met all three criteria were identified by immunoprecipitation with monospecific sera or monoclonal antibodies. These included 3 possible vaccine candidates, the p190 major surface antigen, the p76 serine protease and the p71 protein which is thought to be a member of the family of heat-shock Hsp70 proteins. KEY-WORDS: Plasmodium falciparum, Malaria, Glycolipid, Surface antigen; Phospholipids, Proteins, RBC anchoring, Vaccination. INTRODUCTION
Parasite surface proteins play potential roles in metabolite transport and cell-cell interaction and offer preferential targets for the host protective
Submitted July 11, 1990, accepted September 28, 1990. (*) Permanent address: Dept. of Pharmacology, Case Western Reserve University, Cleveland, OH 44106 (USA).
744
C. B R A U N B R E T O N E T A L .
response (Scaife, 1988). During its intraerythrocytic cycle, the malarial parasite Piasmodium f a t d p a r u m extensively modifies the erythrocyte plasma membrane by inserting parasite proteins such as metabolite transporters (Ginsburg el al., 1983), electron-dense knob-associated proteins (Hadley et al., 1983) and a putative transferrin receptor (Jungery and Rodrigues-Lopez, 1986). Merozoite-membrane-associated proteins may also be involved in the process of red blood cell (RBC) invasion. We have recently reported that one of them, a 76-kDa polypeptide (p76), exhibits proteolytic activity following its release by phosphatidylinositol-specific phospholipase C (PI-PLC) (Braun Breton et al., 1986, 1988). Cleavage by PI-PLC implies that a membrane protein is anchored in the membrane by a glycosyl phospholipid moiety (GPI). GPI anchors have been reported for a diverse group of eukaryotic membrane proteins (Low and Saltiel, 1988; Cross, 1987; Ferguson and Williams, 1988), including variant surface glycoprotein (VSG) from Trypanosoma brucei (Ferguson et al., .1988), acetylcholinesterase (Roberts et al., 1988), decay-accelerating factor (Medof et al., 1986) and rat Thy-1 protein (Homans et al., 1988). All these proteins contain an inositol phosgholipid connected through an oligosaccharide to an ethanolamine in amide linkage to the protein COOH-terminal amino acid. They can be labelled biosynthetically with 3H-ethanolamine (Medof et al., 1986; Rifkin and Fairlamb, 1985; Fatemi et al., 1987) and 3H-myristate (Ferguson et al., 1985) as well as other anchor components (Howard et al., 1987; Takami et al., 1988). Many GPI-anchored proteins are cleaved by PI-PLC (Low and Saltiel, 1988), including p63 Leishmania protease (Bordier et al., 1986). This cleavage may be detected either as a release of the protein from intact cells (Low and Finean, 1977) or membrane fractions (Futerman et al., 1985) or as the removal of radiolahelled fatty a c i d f r n m t h o n r n t o l n ~ ~ r t l a e~,SdAo~ 8l ,w.,,'~,voxr n l l~Ll l ~ r t ~ l e n n o t al., 1985) or an alkylglycerol (Roberts et al., 1987, 1988a). However, certain proteins known to have GPI anchors are resistant to cleavage by PI-PLC (Low and Finean, 1977; Roberts et al., 1988b). Cleavage of the GPI anchor on VSG by PI-PLC unmasks an epitope, a cross-reacting determinant (CRD), on the residual anchor glycoinositol phosphate which produces antisera that cross-react with the s31uble forms of several VSG classes (Cardoso de Almeida and Turner, 1983). These antisera have been used to detect GPI anchors in several other proteins (Zamze et al., 1988). . . . . . . . . . . . . . . . . . . . .
1""
~,,A~:~t.~o,~.p~
I..¢
Two P. falciparum proteins, the 190-kDa major surface antigen (p 190) and the 102-kDa putative transferrin receptor, have been shown previously to con-
CRD GPI mAb PBS PI-PLC
-= = -=
cross-reacting determinant. glycosyl phosphatidylinositol. monoclonal antibody. phosphate-buffered saline. phosphatidylinositol-specific phospholipase C.
RBC = red blood cell. SDS-PAGE = sodium dodecyl sulphate/polyacrylamide gel electrophoresis. VSG = variant surface glycoprotein.
GPI ANCHORING
OF MALARIA
SURFACE ANTIGENS
745
tain 1,2-sn-diacylglycerol and thus, potentially, to be membrane-associated by a G P I anchor (Haldar et al., 1985, 1986). M o r e recently, we have demonstrated that the p76 serine protease o f P. falciparum is a substrate for P I - P L C (Braun Breton et a!., 1988). In the present communication we describe P. falciparum proteins that meet 3 basic criteria for attachment to a GPI anchor: metabolic labelling with 3Hethanolamine and 3H-myristate, release from membranes by P I - P L C and exhibition o f the C R D epitope following P I - P L C cleavage.
MATERIALS AND METHODS Parasite culture.
The Palo Alto FUP strain of P. falciparum was cultivated as described (Braun Breton et aL, 1986). Synchronization was performed by incubation of cells in 2 volumes of 0.3 M alanine, 10 mM Hepes for 3 min at 37°C. Infected RBC were purified on P.ercoll-sorbitol gradients as described (Aley et al., 1984). Metabolic labelling.
Purified schizonts were incubated in culture medium at 1 % haematocrit in the presence of 250 t.tCi/ml 35S-methionine (1,100 Ci/mmol, Radiochemical Center, Amersham), 100 ~.Ci/ml 3H-ethan-l-ol-2-amine hydrochloride (5-30 Ci mmol-I, Amersham) or 100 ~Ci/ml 3H-myristic acid (40-60 Ci/mmol, Amersham) for 2 h at 37°C. Maturation of the cullures was followed in Giemsa-stained smears of the cultures. Mature schizonts (> 8 nuclei) were harvested and further processed for cell fractionation or resuspended in fresh culture medium until merozoite release. Merozoites were purified from the culture supernatant as described (Mrema et al., i982). Cell fractionation.
Infected RBC were lysed by osmotic shock ~Braun Breton et al., 1986) and the lysate was centrifuged at 10,000 g for 30 min at 4°C. The supernatant was defined as the soluble fraction. The pellet, washed 5 times in water and 5 times in PBS (phosphate buffered saline), was termed the men:brane fraction and contained 3 membrane compartments: the red cell plasma membrane, the parasitophorous vacuole membrane and the parasite membrane. Proteins released by Pl-PLC treatment.
Samples of 35S-methionine-labelled schizont membrane fractions were suspended in PBS for 30 min at 37°C in the presence of 1-5 unit/ml of Staphylococcus aureus PI-PLC (a kind gift of Dr. Martin Low) or 1-5 unit/ml of T. brucei PI-PLC (a kind gift of Dr. Peter Overath). The membranes were then pelleted by 15-min centrifugation at 10,000 g and 4°C, and the supernatant was kept. The membrane pellet and the supernatant were resuspended in 1 volume of 2 % Triton-X 100, 1 M NaCI, 20 mM Tris-HCl, 20 mM EDTA pH 7.5.
746
C. B R A U N B R E T O N
ET AL.
lmmunochemical analysis. Immunoprecipitation was performed as described (Braun Breton et al., 1986). The following sera and monoclonal antibodies (mAb) were employed: R5, a rabbit serum raised against purified p190 (Perrin et al., 1984); LI and L2, rabbit sera raised against the P. falciparum membrane fraction; 419, a polyspecific monkey serum that is protective on passive transfer to P. falciparum-infected monkeys (Gysin et al., 1982); R34, a mouse serum raised against recombinant R34 fusion protein that contains part of the sequence of the p71 antigen (Ozaki et al., 1987); Hb31cl3, a mouse mAb against the p76 serine protease (Perrin and Dayal, 1982); XIV-7, a mouse mAb against the p90 heat-shock protein that is not GPI-anchored, and is thus a control antibody for the precipitation of GPl-anchored proteins (Braun Breton et al., 1986; Jendoubi and Bonnefoy, 1988). Rabbit sera were raised against the soluble forms of two different T. brucei VSG variants (a kind gift of Dr. Michael Ferguson).
RESULTS
Metabolic labelling with 3SS-methionine, 3H-ethanolamine and 3H-myristic acid. Schizonts of the P. f a l c i p a r u m FUP strain were metabolically labelled with 35S-methionine, 3H-ethanolamine or 3H-myristate. The distribution of proteins labelled with each of the 3 precursors in total schizont extracts is shown in figure IA. The labelling pattern observed with 35S-methionine (lane 1) differed significantly from those observed with the two other precursors. The 3H-ethanolamine- or 3H-myristate-labelled proteins were detected only in the membrane fraction and not in the soluble fraction (fig. iA). Several membrane proteins appeared to be labelled by both 3H-ethanolamine and 3H-myristate (lanes 2 and 4). Note that labelling with 3H-ethanolamine and 3H-myristate was much less efficient than with 35S-methionine. Tc identify individual polypeptides labelled with 3H-ethanolamine, immunoprecipitation was performed with monospecific sera or mAb (fig. 1B). Two proteins, labelled by ethanolamine, were identified by monospecific antibodies: the p190 polymorphic major surface antigen (lane 1 ; Perrin et al., 1984; Holder and Freeman, 1982; Hall et al., 1984) and the p71 antigen described as showing some homology to grp78, which is related to heat-shock Hsp70 proteins (lane 5; Mattei et al., 1988). The second protein (50-kDa band) in lane 5 might be a degradation product of p71 since this protein was also identified en Wcstcrn blots by several different anti-p71 antibodies. The high molecular weight band in this immunoprecipitate was only detected by immunoprecipitation and appears to coprecipitate with p71. A third minor protein was also faintly labelled (compared to control in lane 2) and is the previously described p76 serine-protease (lane 3; Braun Breton et al., 1988). Labelling of this protein is consistent with it being a substrate for PI-PLC. These were not the only proteins labelled by ethanolamine as a polyclonal polyspecific serum identified several other proteins (lane 4).
7
MWM ,200
_
100
-
925
-69
-46
-30
123 FIG. 1. -
45
Identification
of P. falciparum
2 proteins
labelled
3 biosynthetically
with
A) SDS-PAGE
analysis of schizont-infected red cell proteins following !abe!!ing with “S-methionine (lane l), “H-ethanolamine (lanes 2 and 3) and 3H-myristate (lanes 4 and 5) as outlined in “Materials and Methods”. Lane 1 = total 1 % Triton-XI00 extract; lane 2 = membrane fraction ; lane 3 = soluble fraction ; lane 4 = membrane fraction ; lane 5 = soluble fraction. Radiomethylated protein standards are indicated by the corresponding molecular masses. Due to poor incorporation of 3H-ethanolamine and myristic acid, lanes 2,3,4 and 5 were exposed 30 times longer than lane 1. B) SDS-PAGE analysis of immunoprecipitates of 3H-ethanolamine-labelled schizont proteins. Infected RBC were extracted with 1 % Triton-X100 and the following immunoreactions were performed with different sera on samples of this total cell extract serum RS (lane 1); mAb XIV-7 (lane 2); mAb Hb31c13 (lane 3); serum 419 (lane 4); serum R34 (lane 5). Several bands with intensities significantly greater than those observed with the control antibody in lane 2 are indicated by a dot on the fluorograph and an arrow corresponding to their apparent mass in kDa.
To compensate for the incomplete release by PI- LC and the subsequent hioninedifficulty in detecting the released products, only the readily 3 C. labelled membrane preparation was used as a substrate for
748
C. B R A U N B R E T O N E T A L .
The proteins released from the membrane fraction into the supernatant by treatment with highly purified S. aureus PI-PLC (from Dr. M. Low) were investigated in figure 2A. Immunoprecipitates of supernatants of untreated control membrane fractions revealed no labelled proteins with the 4 antibody preparations tested (lanes 1-4). Following treatment with S. aureus PI-PLC, p76 and 41-kDa protein release to the supernatant was indicated by immunoprecipitation with mAb Hb31cl3 (lane 7), and faint p71 release was revealed by monospecific serum R34 (lane 8). Immunoprecipitation with polyspecific sera demonstrated the release of p190 (lane 6) as well as p76 and the 41-kDa protein (lane 5).
A •
200
•
t
B
4IlD,.,'-,~
-
..
Res. Immunol. 1990, 141, 743-755
GLYCOLIPID ANCHORAGE OF P L A S M O D I U M F A L C I P A R U M SURFACE ANTIGENS C. Braun Breton
(1),
T.L. Rosenberry
(2) (*)
and L.H. Pereira da Silva (1)
Unit o f Experimental Parasitology (]), Unit of Membra:Te Biology (2), Institut Pasteur, 75624 Paris Cedex 15
SUMMARY
Human red blood cells (RBC) were infected with the malarial parasite Plaso modium falciparum, the anchoring of schizont proteins to RBC membranes by glycoinositol phospholipids was demonstrated by three criteria: (1) metabolic incorporation of 3H-ethanolamine and 3H-myristate into the protein; (2) release of 35S-methionine-labelled protein into the supernatant after incubation with phosphatidylinositol-specific phospholipase C; and (3) the exposure of a glycoinositol phosphate epitope on the methioninelabelled protein following phospholipase C cleavage. Labelled proteins were analysed by immunoprecipitation, polyacrylamide gel electrophoresis in sodium dodecylsulphate and gel fluorography. Several candidate proteins were observed when each criteria was investigated. Among these, 3 proteins which met all three criteria were identified by immunoprecipitation with monospecific sera or monoclonal antibodies. These included 3 possible vaccine candidates, the p190 major surface antigen, the p76 serine protease and the p71 protein which is thought to be a member of the family of heat-shock Hsp70 proteins. KEY-WORDS: Plasmodium falciparum, Malaria, Glycolipid, Surface antigen; Phospholipids, Proteins, RBC anchoring, Vaccination. INTRODUCTION
Parasite surface proteins play potential roles in metabolite transport and cell-cell interaction and offer preferential targets for the host protective
Submitted July 11, 1990, accepted September 28, 1990. (*) Permanent address: Dept. of Pharmacology, Case Western Reserve University, Cleveland, OH 44106 (USA).
744
C. B R A U N B R E T O N E T A L .
response (Scaife, 1988). During its intraerythrocytic cycle, the malarial parasite Piasmodium f a t d p a r u m extensively modifies the erythrocyte plasma membrane by inserting parasite proteins such as metabolite transporters (Ginsburg el al., 1983), electron-dense knob-associated proteins (Hadley et al., 1983) and a putative transferrin receptor (Jungery and Rodrigues-Lopez, 1986). Merozoite-membrane-associated proteins may also be involved in the process of red blood cell (RBC) invasion. We have recently reported that one of them, a 76-kDa polypeptide (p76), exhibits proteolytic activity following its release by phosphatidylinositol-specific phospholipase C (PI-PLC) (Braun Breton et al., 1986, 1988). Cleavage by PI-PLC implies that a membrane protein is anchored in the membrane by a glycosyl phospholipid moiety (GPI). GPI anchors have been reported for a diverse group of eukaryotic membrane proteins (Low and Saltiel, 1988; Cross, 1987; Ferguson and Williams, 1988), including variant surface glycoprotein (VSG) from Trypanosoma brucei (Ferguson et al., .1988), acetylcholinesterase (Roberts et al., 1988), decay-accelerating factor (Medof et al., 1986) and rat Thy-1 protein (Homans et al., 1988). All these proteins contain an inositol phosgholipid connected through an oligosaccharide to an ethanolamine in amide linkage to the protein COOH-terminal amino acid. They can be labelled biosynthetically with 3H-ethanolamine (Medof et al., 1986; Rifkin and Fairlamb, 1985; Fatemi et al., 1987) and 3H-myristate (Ferguson et al., 1985) as well as other anchor components (Howard et al., 1987; Takami et al., 1988). Many GPI-anchored proteins are cleaved by PI-PLC (Low and Saltiel, 1988), including p63 Leishmania protease (Bordier et al., 1986). This cleavage may be detected either as a release of the protein from intact cells (Low and Finean, 1977) or membrane fractions (Futerman et al., 1985) or as the removal of radiolahelled fatty a c i d f r n m t h o n r n t o l n ~ ~ r t l a e~,SdAo~ 8l ,w.,,'~,voxr n l l~Ll l ~ r t ~ l e n n o t al., 1985) or an alkylglycerol (Roberts et al., 1987, 1988a). However, certain proteins known to have GPI anchors are resistant to cleavage by PI-PLC (Low and Finean, 1977; Roberts et al., 1988b). Cleavage of the GPI anchor on VSG by PI-PLC unmasks an epitope, a cross-reacting determinant (CRD), on the residual anchor glycoinositol phosphate which produces antisera that cross-react with the s31uble forms of several VSG classes (Cardoso de Almeida and Turner, 1983). These antisera have been used to detect GPI anchors in several other proteins (Zamze et al., 1988). . . . . . . . . . . . . . . . . . . . .
1""
~,,A~:~t.~o,~.p~
I..¢
Two P. falciparum proteins, the 190-kDa major surface antigen (p 190) and the 102-kDa putative transferrin receptor, have been shown previously to con-
CRD GPI mAb PBS PI-PLC
-= = -=
cross-reacting determinant. glycosyl phosphatidylinositol. monoclonal antibody. phosphate-buffered saline. phosphatidylinositol-specific phospholipase C.
RBC = red blood cell. SDS-PAGE = sodium dodecyl sulphate/polyacrylamide gel electrophoresis. VSG = variant surface glycoprotein.
GPI ANCHORING
OF MALARIA
SURFACE ANTIGENS
745
tain 1,2-sn-diacylglycerol and thus, potentially, to be membrane-associated by a G P I anchor (Haldar et al., 1985, 1986). M o r e recently, we have demonstrated that the p76 serine protease o f P. falciparum is a substrate for P I - P L C (Braun Breton et a!., 1988). In the present communication we describe P. falciparum proteins that meet 3 basic criteria for attachment to a GPI anchor: metabolic labelling with 3Hethanolamine and 3H-myristate, release from membranes by P I - P L C and exhibition o f the C R D epitope following P I - P L C cleavage.
MATERIALS AND METHODS Parasite culture.
The Palo Alto FUP strain of P. falciparum was cultivated as described (Braun Breton et aL, 1986). Synchronization was performed by incubation of cells in 2 volumes of 0.3 M alanine, 10 mM Hepes for 3 min at 37°C. Infected RBC were purified on P.ercoll-sorbitol gradients as described (Aley et al., 1984). Metabolic labelling.
Purified schizonts were incubated in culture medium at 1 % haematocrit in the presence of 250 t.tCi/ml 35S-methionine (1,100 Ci/mmol, Radiochemical Center, Amersham), 100 ~.Ci/ml 3H-ethan-l-ol-2-amine hydrochloride (5-30 Ci mmol-I, Amersham) or 100 ~Ci/ml 3H-myristic acid (40-60 Ci/mmol, Amersham) for 2 h at 37°C. Maturation of the cullures was followed in Giemsa-stained smears of the cultures. Mature schizonts (> 8 nuclei) were harvested and further processed for cell fractionation or resuspended in fresh culture medium until merozoite release. Merozoites were purified from the culture supernatant as described (Mrema et al., i982). Cell fractionation.
Infected RBC were lysed by osmotic shock ~Braun Breton et al., 1986) and the lysate was centrifuged at 10,000 g for 30 min at 4°C. The supernatant was defined as the soluble fraction. The pellet, washed 5 times in water and 5 times in PBS (phosphate buffered saline), was termed the men:brane fraction and contained 3 membrane compartments: the red cell plasma membrane, the parasitophorous vacuole membrane and the parasite membrane. Proteins released by Pl-PLC treatment.
Samples of 35S-methionine-labelled schizont membrane fractions were suspended in PBS for 30 min at 37°C in the presence of 1-5 unit/ml of Staphylococcus aureus PI-PLC (a kind gift of Dr. Martin Low) or 1-5 unit/ml of T. brucei PI-PLC (a kind gift of Dr. Peter Overath). The membranes were then pelleted by 15-min centrifugation at 10,000 g and 4°C, and the supernatant was kept. The membrane pellet and the supernatant were resuspended in 1 volume of 2 % Triton-X 100, 1 M NaCI, 20 mM Tris-HCl, 20 mM EDTA pH 7.5.
746
C. B R A U N B R E T O N
ET AL.
lmmunochemical analysis. Immunoprecipitation was performed as described (Braun Breton et al., 1986). The following sera and monoclonal antibodies (mAb) were employed: R5, a rabbit serum raised against purified p190 (Perrin et al., 1984); LI and L2, rabbit sera raised against the P. falciparum membrane fraction; 419, a polyspecific monkey serum that is protective on passive transfer to P. falciparum-infected monkeys (Gysin et al., 1982); R34, a mouse serum raised against recombinant R34 fusion protein that contains part of the sequence of the p71 antigen (Ozaki et al., 1987); Hb31cl3, a mouse mAb against the p76 serine protease (Perrin and Dayal, 1982); XIV-7, a mouse mAb against the p90 heat-shock protein that is not GPI-anchored, and is thus a control antibody for the precipitation of GPl-anchored proteins (Braun Breton et al., 1986; Jendoubi and Bonnefoy, 1988). Rabbit sera were raised against the soluble forms of two different T. brucei VSG variants (a kind gift of Dr. Michael Ferguson).
RESULTS
Metabolic labelling with 3SS-methionine, 3H-ethanolamine and 3H-myristic acid. Schizonts of the P. f a l c i p a r u m FUP strain were metabolically labelled with 35S-methionine, 3H-ethanolamine or 3H-myristate. The distribution of proteins labelled with each of the 3 precursors in total schizont extracts is shown in figure IA. The labelling pattern observed with 35S-methionine (lane 1) differed significantly from those observed with the two other precursors. The 3H-ethanolamine- or 3H-myristate-labelled proteins were detected only in the membrane fraction and not in the soluble fraction (fig. iA). Several membrane proteins appeared to be labelled by both 3H-ethanolamine and 3H-myristate (lanes 2 and 4). Note that labelling with 3H-ethanolamine and 3H-myristate was much less efficient than with 35S-methionine. Tc identify individual polypeptides labelled with 3H-ethanolamine, immunoprecipitation was performed with monospecific sera or mAb (fig. 1B). Two proteins, labelled by ethanolamine, were identified by monospecific antibodies: the p190 polymorphic major surface antigen (lane 1 ; Perrin et al., 1984; Holder and Freeman, 1982; Hall et al., 1984) and the p71 antigen described as showing some homology to grp78, which is related to heat-shock Hsp70 proteins (lane 5; Mattei et al., 1988). The second protein (50-kDa band) in lane 5 might be a degradation product of p71 since this protein was also identified en Wcstcrn blots by several different anti-p71 antibodies. The high molecular weight band in this immunoprecipitate was only detected by immunoprecipitation and appears to coprecipitate with p71. A third minor protein was also faintly labelled (compared to control in lane 2) and is the previously described p76 serine-protease (lane 3; Braun Breton et al., 1988). Labelling of this protein is consistent with it being a substrate for PI-PLC. These were not the only proteins labelled by ethanolamine as a polyclonal polyspecific serum identified several other proteins (lane 4).
7
MWM ,200
_
100
-
925
-69
-46
-30
123 FIG. 1. -
45
Identification
of P. falciparum
2 proteins
labelled
3 biosynthetically
with
A) SDS-PAGE
analysis of schizont-infected red cell proteins following !abe!!ing with “S-methionine (lane l), “H-ethanolamine (lanes 2 and 3) and 3H-myristate (lanes 4 and 5) as outlined in “Materials and Methods”. Lane 1 = total 1 % Triton-XI00 extract; lane 2 = membrane fraction ; lane 3 = soluble fraction ; lane 4 = membrane fraction ; lane 5 = soluble fraction. Radiomethylated protein standards are indicated by the corresponding molecular masses. Due to poor incorporation of 3H-ethanolamine and myristic acid, lanes 2,3,4 and 5 were exposed 30 times longer than lane 1. B) SDS-PAGE analysis of immunoprecipitates of 3H-ethanolamine-labelled schizont proteins. Infected RBC were extracted with 1 % Triton-X100 and the following immunoreactions were performed with different sera on samples of this total cell extract serum RS (lane 1); mAb XIV-7 (lane 2); mAb Hb31c13 (lane 3); serum 419 (lane 4); serum R34 (lane 5). Several bands with intensities significantly greater than those observed with the control antibody in lane 2 are indicated by a dot on the fluorograph and an arrow corresponding to their apparent mass in kDa.
To compensate for the incomplete release by PI- LC and the subsequent hioninedifficulty in detecting the released products, only the readily 3 C. labelled membrane preparation was used as a substrate for
748
C. B R A U N B R E T O N E T A L .
The proteins released from the membrane fraction into the supernatant by treatment with highly purified S. aureus PI-PLC (from Dr. M. Low) were investigated in figure 2A. Immunoprecipitates of supernatants of untreated control membrane fractions revealed no labelled proteins with the 4 antibody preparations tested (lanes 1-4). Following treatment with S. aureus PI-PLC, p76 and 41-kDa protein release to the supernatant was indicated by immunoprecipitation with mAb Hb31cl3 (lane 7), and faint p71 release was revealed by monospecific serum R34 (lane 8). Immunoprecipitation with polyspecific sera demonstrated the release of p190 (lane 6) as well as p76 and the 41-kDa protein (lane 5).
A •
200
•
t
B
4IlD,.,'-,~
-
..
