Glycosylation and proteolyticprocessing of 70 kDa C-terminal recombinant polypeptides of Plasmodiumfalciparum merozoite surface protein 1 expressed in mammaliancells

Shutong Yanga, David Nikodema, Eugene A.Davidson and D.Channe Gowdab

Department of Biochemistry and Molecular Biology, GeorgetownUniversity Medical Center, Washington, DC 20007, USA

Received on April 12, 1999. revisedon June 3, 1999. accepted on June 4, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 References
 
The cDNAs that encode the 70 kDa C-terminal portionof Plasmodium falciparum merozoite surface protein1 (MSP-1), with or without an N-terminal signal peptide sequenceand C-terminal glycosylphosphatidylinositol (GPI) signal sequenceof MSP-1, were expressed in mammalian cell lines via recombinantvaccinia virus. The polypeptides were studied with respect to thenature of glycosylation, localization, and proteolytic processing.The polypeptides derived from the cDNAs that contained the N-terminalsignal peptide were modified with N-linked highmannose type structures and low levels of O-linkedoligosaccharides, whereas the polypeptides from the cDNAs that lackedthe signal peptide were not glycosylated. The GPI anchor moietyis either absent or present at a very low level in the polypeptide expressedfrom the cDNA that contained both the signal peptide and GPI signalsequences. Together, these data establish that whereas the signalpeptide of MSP-1 is functional, the GPI anchor signal is eithernonfunctional or poorly functional in mammalian cells. The polypeptidesexpressed from the cDNAs that contained the signal peptide wereproteo­lytically cleaved at their C-termini, whereas thepolypeptides expressed from the cDNAs that lacked the signal peptide wereuncleaved. While the polypeptide expressed from the cDNA containingboth the signal peptide and GPI anchor signal was truncated by ~14 kDa at the C-terminus, the polypeptidederived from the cDNA with only the signal peptide was processedto remove ~6 kDa, also from the C-terminus. Furthermore,the polypeptides derived from cDNAs that lacked the signal peptidewere exclusively localized intra­cellularly, the polypeptidesfrom cDNAs that contained the signal peptide were predominantlyintracellular, with low levels on the cell surface; none of thepolypeptides was secreted into the culture medium to a detectablelevel. These results suggest that N-glycosylationalone is not sufficient for the efficient extracellular transportof the recombinant MSP-1 polypeptides through the secretory pathway inmammalian cells.


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 References
 
The Plasmodium parasite expresses a major antigenicprotein, merozoite surface protein 1 (MSP-1), on its cell surfaceboth at the hepatic and intraerythrocytic stages (12GoHolderand Freeman, 1982; 14GoHoward et al., 1984; 27GoPirsonand Perkins, 1985). During the intraerythrocytic developmentof the parasite, MSP-1 synthesis is closely related to schizogony,and the protein is believed to have an important functional rolein the merozoite invasion of red blood cells (11GoHolder,1988). Therefore, MSP-1 is widely considered as a potentialmalaria vaccine candidate.

The structure, function, and immunologic properties of MSP-1from several Plasmodium species have been extensively studied(11GoHolder, 1988; 6GoCooper et al., 1992; 5GoCooper,1993). MSP-1 is a polymorphic protein with a significantdegree of variable amino acid sequences and varies considerablyin Mr (185,000 to 250,000) depending on the parasite species (11GoHolder, 1988; 22GoMcBride et al., 1985).The size variation is also present within different strains of asingle species; for example, MSP-1 from different P.falciparum strainshas an Mr that varies from 185 to 205 kDa (22GoMcBride et al., 1985; 11GoHolder,1988). However, MSP-1 from different species and strainsdo have several common structural features: (1) an N-terminalsignal peptide sequence, (2) a C-terminal GPI anchor signal sequence, (3)11–15 potential N-glycosylation sites;the positions of these sites, however, are not conserved even inproteins from different strains of a species, and none of the sitesis glycosylated to a significant level (11GoHolder,1988; 29GoSchofield and Hackett,1993).

MSP-1 undergoes proteolytic processing at the time of merozoitematuration (20GoLyon et al.,1986; 13GoHolder et al.,1987; 11GoHolder, 1988).The protein is cleaved into different sized N-terminal fragments(83-, 28-/30-, 38 kDa), and a 42 kDa C-terminal fragment.The cleaved fragments are held together as a noncovalent complexon the merozoite surface that is believed to serve as a receptorfor red cell recognition and/or adhesion during erythrocyticinvasion (21GoMcBride and Heidrich, 1987; 11GoHolder, 1988). Before invasion of redblood cells, the C-terminus fragment is further processed to forma 33-kDa N-terminal fragment and a 19-kDa GPI-anchored C-terminalfragment (2GoBlackman et al.,1990; 1GoBlackman and Holder, 1992).While all the N-terminal fragments are shed during invasion of redblood cells, the 19 kDa C-terminal fragment remains membrane-boundthrough the GPI anchor moiety and is carried with the invading merozoite (2GoBlackman et al., 1990),suggesting that the C-terminus of MSP-1 may have a role in the invasionprocess and/or subsequent adaptation of the parasite insidered blood cells.

Individuals living in malaria-endemic areas have anti-MSP-1 antibodies(12GoHolder and Freeman, 1982; 9GoHall et al., 1984; 11GoHolder, 1988; 28GoRiley et al., 1992). The antibodies raised against MSP-1inhibit red blood cell invasion in vitro, and providepartial or complete protection in vivo againstsubsequent challenge with parasites (15GoHuiand Siddiqui, 1987; 31GoSiddiqui et al., 1987; 11GoHolder,1988; 8GoEtlinger et al.,1991; 6GoCooper et al.,1992). Previous studies have shown that antibodies againstdisulfide bond-dependent epitopes within the 19-kDa C-terminus of MSP-1provide protective immunity to parasite infection (26GoMurphyet al., 1990; 4GoChang et al., 1992; 16GoHui et al., 1993; 17GoKumar et al., 1995; 3GoChang et al., 1996).

MSP-1 has a glycophorin recognition region (32GoSuet al., 1993). A monoclonal antibody designatedas 2B10 that is directed against an N-terminal region of glycophorinwas shown to have the same recognition determinant on human erythrocytes asMSP-1 of P.falciparum, and the binding of bothligands to the erythrocyte was dependent on sialic acid. Rabbitpolyclonal anti-idiotype antibodies raised against 2B10 recognizeboth the glycophorin binding site on 2B10 and a peptide region within MSP-1(1047–1640 amino acid C-terminus). Since 2B10 is capableof both blocking the binding of MSP-1 to human erythrocytes andinhibiting the merozoite invasion of red blood cells, we reasonthat the C-terminal polypeptide encompassing the apparent red bloodcell recognition element and the 19 kDa C-terminus would comprisea potential vaccine.

A previous study reported the construction of cDNAs that encodea 70 kDa MSP-1 C-terminal polypeptide with or without the N-terminalsignal peptide and C-terminal GPI signal sequences, insertion ofthe cDNAs into vaccinia virus, expression of polypeptides in recombinantvirus-infected BSC-1 cells, and immune responses of denovo expressed polypeptides in mice and rabbits (34GoYanget al., 1997). The polypeptides expressed fromthe cDNAs that contained the signal peptide sequence had significantlylower Mr than expected even though they were glycosylated.The polypeptides were not detectable in the culture medium. Furthermore,the polypeptide expressed de novo from cDNAs withthe signal peptide sequence elicited an order of magnitude higherimmune response in animals compared with those derived from cDNAswithout the signal peptide sequence (34GoYanget al., 1997). In view of the potentialsignificance of these results in using MSP-1 for malaria vaccinedevelopment and to understand the biochemical basis for the observed results,the polypeptides expressed from all four cDNA constructs in mammaliancells were characterized in detail; the results are presented here.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 References
 
The construction of cDNAs that encode the 70 kDa C-terminal polypeptideof MSP-1 (amino acid residues 1047–1640) with or withouta signal peptide and a C-terminal GPI signal sequence has been described(34GoYang et al., 1997)and is shown in Figure 1. The MSP-1 signalpeptide sequence and a 108-stretch of nucleotides adjacent (downstream)to the signal peptide sequence (nucleotide residues 418–582)was added at the 3' end of the cDNA(nucleotide residues 3553–5337). An additional 2 base pairswere added between nucleotide residues 582 and 3553 to preservethe reading frame. For cDNAs lacking the GPI anchor signal sequence,a stretch of nucleotides (nucleotide residues 5280–5337)was deleted from the 5' end of the cDNAs(Figure 1). Thus, cDNA P1 contains the N-terminalsignal peptide and lacks the C-terminal GPI anchor signal; cDNAP2 has both the N-terminal signal peptide and C-terminal GPI anchorsignal; cDNA P3 is devoid of both the signal peptide and GPI anchor signal;cDNA P4 contains the C-terminal GPI anchor signal and lacks theN-terminal signal peptide (Figure 1). Theinsertion of these cDNA constructs into the thymidine kinase regionof vaccinia virus using a transfer vector (pSC65) containing a syntheticstrong early/late promoter, transfection, isolation, andproduction of high titer stocks of recombinant viruses has beenreported (34GoYang et al., 1997).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1. cDNAs that encode the 70-kDaC-terminal portion of P.falciparum MSP-1 with orwithout the MSP-1 signal peptide sequence (Si) at the N-terminalend and with or without the MSP-1 GPI anchor signal sequence (A)at the C-terminal end. P1, cDNA corresponding to the 70-kDa C-terminalportion of MSP-1 to which the signal peptide sequence plus 108 nucleotideresidues adjacent are ligated at the 5' end,and the MSP-1 GPI anchor signal sequence at the 3' endis deleted; P2, similar to cDNA P1 except that the C-terminal GPIsignal sequence at the 3' end is notdeleted; P3, similar to cDNA P1 except that the MSP-1 signal peptide sequenceis deleted; P4, similar to cDNA P2 except that the MSP-1 signalpeptide sequence is deleted. Solid triangles indicate the positionsof potential N-glycosylation sites in MSP-1.

 
Expression of polypeptides in mammalian cells andstudy their glycosylation
The polypeptides encoded by all four cDNAs (see Figure 1) were expressed in three different mammaliancells: monkey kidney CV-1 cells, and human Hu134TK andHeLa cells. The polypeptides encoded by P1 and P2 were expectedto undergo N-glycosylation because each of thepolypeptides has four potential N-glycosylationsites and the signal peptide sequence for translocation into thelumen of ER, where N-glycosylation occurs. Thepolypeptides derived from P3 and P4, however, were not expectedto be N-glycosylated since they do not contain thesignal peptide sequence. Cells infected with recombinant vacciniaviruses containing the P1, P2, P3, and P4 inserts were culturedin the presence or absence of tunicamycin. Western blot analysisof the cell lysates and the culture supernatants showed that thepolypeptides expressed from all four cDNAs were exclusively cell-associated,irrespective of whether the cells were cultured in the presenceor absence of tunicamycin (Figure 2). Inall three cells, the polypeptides from P1 and P2 were expressedas multiple bands when cells were cultured without tunicamycin.SDS–PAGE showed a ladder of five bands that were separatedfrom one another by a molecular mass of ~2000Da (Figure 2). However, when cells werecultured in the presence of tunicamycin, each of the polypeptidesfrom P1 and P2 was expressed as a single band corresponding to 64kDa and 56 kDa, respectively. The 64 kDa and 56 kDa bands corres­pondedto the lowest sized bands observed for the polypeptides obtainedfrom cells grown in the absence of tunicamycin (Figure 2). These results suggest that the polypeptidesfrom the P1 and P2 constructs contain 1–4 N-linkedoligosaccharides. The polypeptides from the P3 and P4 constructs(cDNAs that lacked the signal peptide) each gave a single band withMr 70,000 and 68,000, respectively, independent of whetherthe cells were cultured with or without added tunicamycin, suggesting that polypeptidesfrom P3 and P4 were not N-glycosylated (Figure 2).



View larger version (53K):
[in this window]
[in a new window]
 
Fig. 2. Analysis of the MSP-1 polypeptidesin cells cultured in the absence (-) or presence of tunicamycin(+, 2.5 µg/ml) byWestern blotting. Top left panel, lysates of cells expressing polypeptidefrom cDNA P1. Bottom left panel, lysates of cells expressing polypeptidefrom cDNA P2. Top right panel, lysates of CV-1 cells expressingpolypeptides from cDNAs P1, P2, P3, and P4. Lane WT, lysates ofCV-1 cells infected with wild type vaccinia virus. Bottom rightpanel, culture supernatants of HeLa cells expressing polypeptidesfrom cDNAs P1, P2, P3, and P4. Lane WT, culture supernatants ofHeLa cells infected with wild type vaccinia virus. Culture supernatantsof CV-1 and Hu134TK cells expressing polypeptidefrom cDNAs P1, P2, P3, and P4 gave similar results (not shown).

 
Western blot analysis of cell lysates also showed that polypeptidesexpressed from cDNAs P1 and P2 were differentially glycosylated(Figure 2). Although there was a batch-to-batch variationin the proportions of various glycosylated polypeptides expressedfrom P1 and P2, which could be due to minor variations in cell culturingconditions, the polypeptide from P2 was usually N-glycosylatedto a lower extent compared with the polypeptide derived from P1(Figure 2).

[3H]GlcN labeling of cells expressingthe polypeptides and analysis by SDS–PAGE and fluorographydemonstrated the presence of carbohydrate moieties only in polypeptidesfrom P1 and P2 but not in polypeptides from P3 and P4 (Figure 3).



View larger version (86K):
[in this window]
[in a new window]
 
Fig. 3. SDS–PAGE/fluorographicanalysis of [3H]GlcN-labeled recombinant70 kDa C-terminal polypeptides of MSP-1 expressed in CV-1 cellsinfected with recombinant vaccinia virus containing P1, P2, P3,and P4 cDNA inserts, and with wild type (WT) vaccinia virus. ThecDNAs expressed in Hu134TK cells and HeLa cellsgave similar results (not shown).

 
Characterization of N-linked oligosaccharides inthe recombinant polypeptides
To study the type of N-linked oligosaccharidespresent in polypeptides derived from cDNAs P1 and P2, cell lysateswere treated with either N-glycanase or endoglycosidaseH, and then analyzed by Western blotting. Both enzymes effectively removedthe oligosaccharide chains causing disappearance of the multipleband patterns from both P1 and P2; in each case a single polypeptideband was observed (Figure 4). These results weresimilar in all three cell lines studied. The polypeptides treatedwith endoglycosidase H electrophoresed slightly slower than thosetreated with N-glycanase due to the presence ofa single GlcNAc left after the action of the former enzyme (comparelanes B and C in Figure 4). The completeremoval of N-linked oligosaccharides by endoglycosidaseH suggest that the polypeptides of both P1 and P2 contain almostexclusively high mannose type N-linked oligosaccharides,independent of the cell lines in which they were expressed.



View larger version (73K):
[in this window]
[in a new window]
 
Fig. 4. Analysis of the recombinantMSP-1 polypeptides for N-linked oligosaccharides.Top panel, lysates of cells infected with vaccinia virus containingcDNA P1 insert; Bottom panel, lysates of cells infected with vacciniavirus containing cDNA P2 insert. In both panels: lane A, untreatedcell lysate; lane B, cell lysate treated with endoglycosidase H;lane C, cell lysate treated with N-glycanase.

 
Analysis of O-linked oligosaccharides in the recombinant polypeptides
The polypeptides of P1 and P2 expressed in CV-1 and Hu134TK cellswere then analyzed for the presence of O-linked carbo­hydrates.Carbohydrate compositional analysis showed that the polypeptidesfrom both P1 and P2 contain low levels of O-linked carbohydratesas evident from the presence of low proportions of D-galactosamine(GalN) (Table Go). The polypeptides fromboth P1 and P2 also contained very small amounts of N-acetylneuraminicacid (Table Go). To determine whether N-acetylneuraminic acid is present in O-and/or N-linked oligo­saccharides, thecell lysates were treated with N-glycanase, andthe polypeptides were immunoprecipitated, electrophoresed on SDS–polyacrylamidegels and transferred to PVDF membranes. Carbohydrate compositionalanalysis of the polypeptide bands showed no noticeable decreasein the content of sialic acid on treatment with N-glycanase,suggesting that the sialic acid is present in the O-linkedcarbohydrate moieties of the polypeptides. Based on the ratios ofGalN and GlcN, the contents of O-linked carbohydratecorrespond to about 1 oligosaccharide chain per several moleculesfor the polypeptide derived from P2 and almost none for the polypeptidefrom P1. These results further suggest that a small pool of polypeptidesderived from cDNAs P1 and P2 entered the Golgi.


View this table:
[in this window]
[in a new window]
 
Table I. Carbohydrate compositional analysis (Mol proportion) of [3H]GlcN-labeledrecombinant MSP-1 C-terminal polypeptides expressed in CV-1 and Hu134TKcells
 
Analysis for GPI anchor moiety in the polypeptidefrom P2
If the GPI anchor signal sequence of MSP-1 is functional in mammaliancells, then the polypeptide from P2 is expected to be modified withGPI anchor moieties. Treatments of the [3H]GlcN-labeledpolypeptide of P2 on PVDF membranes with Pronase released all theradiolabeled carbohydrate moieties from the membranes. The releasedradioactivity should represent N-and O-linkedoligosaccharides plus the carbohydrates of the GPI anchor moieties,if the latter modification is present. Upon partitioning of thePronase-released components between water and water-saturated 1-butanol,radiolabel was present almost quantitatively in the aqueous phase(Figure 5). This suggests that the polypeptidefrom P2 is either not modified with a GPI anchor moiety or it containsvery low levels of GPI anchor. The cell lysates that contained thepolypeptide were treated with N-glycanase, electrophoresedon SDS–PAGE, and transferred to PVDF membranes. The membraneswere divided into two portions and one portion was treated with HNO2.HPLC analysis of the acid hydrolysates indicated that the GlcN contentin HNO2-treated polypeptide was similar to that of polypeptidesnot treated with HNO2 (data not shown). Furthermore,neither PI-PLC nor PI-PLD released a detectable level of polypeptidefrom CV-1 or Hu134TK cells expressing polypeptidefrom cDNA P2 (data not shown). Together these data suggested thata GPI moiety is either absent or present only at very low levelsin the polypeptide from cDNA P2.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5. Analysis of the recombinantMSP-1 polypeptides for GPI anchor moiety. The lysates of [3H]GlcN-labeledCV1 cells were electrophoresed on SDS–polyacrylamide, theprotein bands were transferred to PVDF membranes, and the recombinantMSP-1 polypeptide bands were analyzed for GPI anchor moiety as outlined.

 
Analysis for proteolytic processing of the polypeptides
The observed Mr, 64,000 and 56,000, respectively,of the non-N-glycosylated polypeptides from P1and P2 expressed in cells cultured in the presence of tunicamycinand of N-deglycosylated (N-glycanase-treated)polypeptides expressed in cells in the absence of tunicamycin weresignificantly lower than the expected Mr of 70,000 forthe non-N-glycosylated poly­peptides (seeFigure 3). This suggests that the polypeptides fromP1 and P2 were truncated differentially either at the N- or C-termini.Upon Western blotting, the 5.2 monoclonal antibody that specificallyrecognizes a peptide epitope at the C-terminal end of MSP-1 reactedwith the polypeptides derived from P3 and P4 but not with the polypeptidesfrom P1 and P2 (Figure 6). These resultsindicate that the observed lower Mr of the polypeptidesfrom P1 and P2 compared with the expected value of 70,000 is dueto differential proteolytic processing at the C-termini by eitherER or Golgi protease(s).



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 6. Western blot analysis of therecombinant MSP-1 polypeptides expressed in CV-1 cells for proteolyticprocessing.

 
The observed Mr of the polypeptide expressed fromP3 was comparable to the expected value of 70,000. However, the electrophoreticmobility of the polypeptide derived from P4 corresponded to an Mr of68,000, which is 2000 lower than the expected value of 70,000. Thereason for the observed lower molecular weight of the polypeptidefrom P4 is not clear.

Cellular localization of the polypeptides
Polypeptides from all four cDNAs were not detectable on cell surfacesupon immunostaining using peroxidase-conjugated secondary antibody(Figure 7). However, upon permeabilization, cellsexpressing all four polypeptides were strongly stained (Figure 7). Previously, indirect immunofluorescencestaining suggested that the polypeptides from P1 and P2 were expressedon cell surfaces, whereas the polypeptides from P3 and P4 were exclusivelylocalized inside the cells (34GoYang etal., 1997). The detection of polypeptides from P1and P2 on cell surfaces of intact cells by immunofluorescence staining(34GoYang et al., 1997)but not by immunoperoxidase staining (this study) suggests thatthese polypeptides are present on cell surfaces at levels belowthe detection limits of immunoperoxidase staining. Thus, these resultsdemonstrate that polypeptides from P3 and P4 are exclusively localizedinside the cells, whereas those from P1 and P2 are predominantlylocalized intracellularly with very low levels on cell surfaces.



View larger version (112K):
[in this window]
[in a new window]
 
Fig. 7. Immunostaining of CV-1 cellsexpressing the recombinant MSP-1 polypeptides. (A)Permeabilized cells expressing polypeptide from cDNA P1; (B)intact cells expressing polypeptide from cDNA P1; (C)permeabilized cells expressing polypeptide from cDNA P2; (D)intact cells expressing polypeptide from cDNA P2; (E)permeabilized cells expressing polypeptide from cDNA P3; (F)permeabilized cells expressing polypeptide from cDNA P4. Cells infectedwith wild type vaccinia virus (both permeabilized and intact) werenot stained (not shown).

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 References
 
In this study, four cDNA constructs that encode the 70-kDa C-terminalpolypeptide of P.falciparum MSP-1, with or without MSP-1N-terminal signal peptide and C-terminal GPI anchor signal sequences,were expressed in three different mammalian cell lines. The expressedproteins were characterized with respect to the recognition of theN-terminal signal peptide and C-terminal GPI anchor signal sequencesof MSP-1, proteolytic processing, N- and O-glycosylation,and GPI anchor modification in mammalian cells. The key findingsare as follows. (1) The MSP-1 signal peptide sequence functionsnormally in mammalian cells. (2) All four N-glycosylationsites of the MSP-1 70 kDa C-terminal polypeptide are functional,i.e., they serve as acceptor sites for the dolichol phosphate oligosaccharyltransferaseof mammalian cells. (3) The MSP-1 GPI anchor signal functions verypoorly in mammalian cells. (4) The addition of N-linked oligosaccharidesdoes not facilitate the extracellular transport of the MSP-1 polypeptidesexpressed in mammalian cells. (5) The MSP-1 polypeptides expressedfrom cDNAs that contain an N-terminal signal peptide undergo proteolyticprocessing at their C-termini in mammalian cells. (6) The polypeptides encodedby cDNAs lacking the signal peptide were neither glycosylated norproteolytically processed, and were exclusively localized insidethe cells, apparently in the cytoplasm.

This study demonstrates that the P.falciparum MSP-1signal peptide is efficiently recognized by the mammalian signalpeptide recognition particles, allowing for the translocation ofMSP-1 polypeptides to the lumen of the ER membranes. The signal peptidesequences of P.falciparum and P.vivax MSP-1are also recognized in Sf9 insect cells (26GoMurphyet al., 1990; 19GoLongacreet al., 1994).

All four N-glycosylation sites of the P.falciparum MSP-1 70-kDaC-terminal portion are functional in the mammalian cells since majorportions of the polypeptides derived from P1 and P2 constructs contain1–4 N-linked oligosaccharide chains withfully glycosylated polypeptides predominating in the polypeptidefrom P1 (see Figures 2, 3).The N-glycosylation sites of 42-kDa P.falciparum and P.vivax MSP-1 C-terminal sequences are also glycosylatedwhen expressed in baculovirus-infected Sf9 cells (26GoMurphyet al., 1990; 19GoLongacreet al., 1994). The N-glycosylationcapability of P.falciparum and P.vivax polypeptidesexpressed in mammalian and insect cells argue that the absence ornegligible N-glycosylation of native MSP-1 is notbecause of nonfunctional N-glycosylation sitesbut is due to very low levels of dolichol phosphate oligosaccharide donorsand/or oligosaccharyltransferase activity in the parasite,as was previously shown (7GoDieckmann-Schuppert et al., 1992).

The polypeptide derived from the cDNA P1 that contain the signalpeptide sequence but lack the GPI anchor signal sequence is expectedto be secreted if it normally passes through the secretory pathwayin the mammalian cells. However, the expressed polypeptide was retainedmainly intracellularly with a very low level on the cell surface.Furthermore, the polypeptide contained almost exclusively the highmannose-type N-linked oligosaccharides. These datasuggest that all or a major portion of the polypeptide has not traffickedthrough the Golgi and was likely retained in the Golgi or the pre-Golgi compartments.In contrast, it has been previously shown that the 42 kDa C-terminalpolypeptides of P.falciparum and P. vivax,and 19 kDa C-terminal polypeptide of P.vivax MSP-1 thatcontained the signal peptides expressed in Sf9 insect cells wereefficiently secreted (26GoMurphy etal., 1990; 19GoLongacre et al., 1994). In Sf9 insect cells, N-glycosylationapparently facilitated passage through the secretory pathway sincethe inhibition of glycosylation with tunicamycin caused a markedreduction of extracellular secretion of the polypeptides (19GoLongacre et al., 1994).

The data also demonstrate that the P.falciparum MSP-1GPI anchor signal sequence is either nonfunctional or very poorly functionalin mammalian cells. Several lines of evidence support this finding.(1) Partitioning of radioactive components obtained by the Pronasedigestion of [3H]GlcN-labeled polypeptideencoded by P2 showed the absence of lipid-bound carbohydrate moiety.(2) The expressed polypeptide could not be released by either PI-PLCor PI-PLD. (3) GlcN (non-N-acetylated) residueswere not detectable in the [3H]GlcN-labeled, N-glycanase-treated polypeptide encoded by P2.(4) Immuno­fluorescence (34GoYanget al., 1997) and immunoperoxidase staining studiesshowed that the polypeptide is predominantly localized intracellularlywith only a very low level expression on the surfaces of mammaliancells. These data agree with the finding that the GPI anchor signalsequence of the P.falciparum circumsporozite proteinis poorly recognized by mammalian cells and that the requirementsfor the GPI anchor attachment are not identical in mammalian cellsand the malaria parasite (25GoMoran and Caras,1994). Although the P.falciparum MSP-1 GPIanchor signal is nonfunctional in mammalian cells, the P.vivax MSP-1GPI anchor signal appears to be functional in insect cells. Thus,significant amounts of the 19 kDa and 42 kDa C-terminalpolypeptide of P.vivax MSP-1 expressed in insectcells have been shown to be GPI anchored (19GoLongacreet al., 1994).

The results presented here show that the polypeptides derivedfrom P1 and P2 cDNAs were differentially truncated at their C-termini,apparently by proteases. Previous studies have shown that polypeptideswith uncleaved GPI anchor signals were retained inside the cellsand localized in the ER-Golgi intermediate compartments (24GoMoran and Caras, 1992). In agreement withthis finding, the polypeptide expressed from P2, which containsa noncleavable GPI anchor signal sequence, was found to be predominantlylocalized inside the cells. The polypeptide derived from cDNA P1,which lacks the GPI anchor signal, is also proteolytically processedand predominantly retained inside the cells. Accordingly, the intracellularretention and processing of the polypeptide derived from cDNA P2may not entirely be due to the presence of the uncleaved GPI anchor signalsequence. The small amount of polypeptide from P2 found on the cellsurface may be due to a low level of GPI anchor modification orthe inefficient transport of a membrane-bound truncated form ofP2. A similar low level cell surface localization of the polypeptidefrom P1 suggests the latter possibility. The 19- and 42-kDa P.vivax MSP-1C-terminal polypeptides expressed in Sf9 cells were not proteolytically processed(19GoLongacre et al., 1994)indicating differences between the protein secretory pathways ofthe mammalian and insect cells.

Although the reason for the differential processing of polypeptidesfrom P1 and P2 is not known, N-glycosylation haslittle or no contribution since the molecular weights of polypeptidesof P1 and P2 expressed in the presence of tunicamycin were similarto those synthesized in the absence of tunicamycin after treatmentwith N-glycanase (see Figures 2, 4).

The results of the present study demonstrate that the MSP-1 C-terminalpolypeptides lacking the GPI anchor modification remain primarilyintracellular when expressed in mammalian cells. However, the polypeptidesexpressed from cDNAs that are engineered to contain mammalian GPIanchor signal sequences at their C-termini are expected to be modifiedwith GPI anchor moieties (25GoMoran and Caras,1994), and the GPI-anchored polypeptides are likely tobe transported to the cell surfaces. The GPI anchored proteins canelicit a far more effective immune response compared with correspondingproteins that lack GPI anchor moieties (30GoSchofieldet al., 1999). Thus, cDNAs correspondingto the MSP-1 C-terminal polypeptides that are engineered to containa GPI anchor signal sequence functional in mammalian cells may proveto be effective immunogens.


    Material and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 References
 
Materials
Eagle’s minimum essential medium, Dulbecco’smodified minimum essential medium, and fetal calf serum were from LifeTechnologies, Inc. (Gaithersburg, MD). D-[6-3H]gluco­saminehydrochloride (3H-GlcN) (40–60 Ci/mmol)was from American Radiolabeled Chemicals (St. Louis, MO). EN3HANCEfluorographic spray was from Dupont-New England Nuclear ResearchProducts (Boston, MA). Endoglycosidase H and N-glycanasewere from New England Biolabs (Beverly, MA). Alkaline phosphatase–conjugatedgoat antimouse IgG and the Western blue stabilized substrate foralkaline phosphatase were from Promega (Madison, WI). AmplifyTM fluoro­graphicsolution and peroxidase-conjugated goat antimouse IgG were fromAmersham Corp. (Arlington Heights, IL). Bacillus thuringiensis phosphatidylinositol-specificphos­pholipase C (PI-PLC) (1000 U/mg) was fromOxford Glycosystems (Rosedale, NY). Tunicamycin, dianisidine hydro­chloride, and Bacillus cereus PI-PLC (3000 units/mg),were from Sigma Chemical Co. (St. Louis, MO). Polyvinylidene difluoride(PVDF) membranes were from Millipore Corp. (Bedford, MA); Centricon30 microconcentrators were from Amicon (Danvers, MA). Monkey kidneyBSC-1 and CV-1 cells, and human Hu134TK andHeLa cells were obtained from the American Type Culture Collection(Rockville, MD). Antimouse polyclonal IgG against the C-terminal70 kDa polypeptide of MSP-1 expressed in E.coli wasfrom the previous study (34GoYang etal., 1997). The monoclonal antibody 5.2 (IgG) thatspecifically recognizes a peptide epitope within the 14-kDa C-terminusof the MSP-1 was a gift from Dr. Sandra Chang, University of Hawaii.

Preparation of recombinant vaccinia virus
Vaccinia viruses containing cDNA inserts corresponding to 70 kDaC-terminal polypeptide of MSP-1, with or without the peptide andGPI anchor signal sequences, were prepared as reported previously(34GoYang et al., 1997).The plaque purified recombinant viruses were used for the preparationof high-titer stocks in HeLa S3 cells. All virus stocks were purifiedby ultracentrifugation through a 36% sucrose cushion. Viruseswere titered on monolayers of BSC-1 cells. Recombinant vaccinia virusesand wild type vaccinia virus (Western Reserve strain) were culturedin CV-1, HeLa, and Hu134TK cells.

Cell culture
The BSC-1 and Hu134TKcells were culturedin Eagle’s minimum essential medium supplemented with 10% fetalbovine serum. The CV-1 and HeLa cells were grown in Dulbecco’smodified minimum essential medium supplemented with 10% fetal bovineserum. Cells were cultured as monolayers at 37°Cin a humidified incubator.

Expression and analysis of the polypeptides incells infected with recombinant viruses
Confluent monolayers of cells in six-well plates, each well containing1 ml medium with 1% fetal bovine serum (in some experiments0.1% fetal bovine serum was used to avoid interferenceby serum albumin on the mobility of polypeptides during SDS–PAGE),were infected with recombinant vaccinia virus at a multiplicityof 10 PFU. After 20 h, the medium from each well was removed, thecell layers were gently rinsed with fresh medium, and 50 µlof 0.5% SDS and 1% 2-mercaptoethanol in waterwas added to each well. The cell lysates were analyzed by Westernblotting either directly or after treatment with endoglycosidaseH or N-glycanase (see below). The spent mediumfrom each well was concentrated to ~50 µl in Centricon 30 microconcentratorsand 10 µl was analyzed by Western blotting.

Inhibition of N-linked glycosylation with tunicamycin
Confluent monolayers of cells cultured in six-well plates were incubatedfor 4 h with medium containing 2.5 µgtunicamycin/ml. The cells were then infected separatelywith each recombinant vaccinia virus and wild type vaccinia virusat a multiplicity of 10 PFU. After 20 h, the medium was removed,the cell layers were rinsed with fresh medium, and 50 µl0.5% SDS and 1% 2-mercaptoethanol in water wasadded to each well. The lysates were mixed with 50 µlof 100 mM Tris–HCl, pH 6.8, 200 mM dithiothreitol, 4% SDS,0.2% bromophenol blue and 20% glycerol. The solutionswere electrophoresed (Laemmli, 1970), and detected by antibody affinityblotting (see below).

Analysis of the polypeptides by Western blotting
Cells expressing polypeptides from cDNAs P1, P2, P3, and P4 were culturedin the absence (-) or presence of tunicamycin (+, 2.5 µg/ml), cell lysates preparedas described above, and 10 µl aliquots analyzedon 8% SDS–polyacrylamide gels (Laemmli, 1970). Theprotein bands on gels were electrotransferred to PVDF membranes.The membranes were blocked with 5% BSA in 200 mM Tris–HCl,pH 7.5, for 2 h at room temperature and then treated with a 1:500dilution of antimouse serum against the 70-kDa C-terminal MSP-1polypeptide. After 2 h incubation, the membranes were washed andtreated with 1:7500 diluted alkaline phosphatase-conjugated goatantimouse IgG in Tris–HCl, pH 7.5, for 90 min at room temperature.The protein bands on membranes were visualized with Western bluestabilized substrate for alkaline phosphatase according to manufacturer’s instructions.

Metabolic labeling of the polypeptides with [3H]GlcNand analysis by SDS–PAGE/fluorography
Confluent monolayers of cells in six-well plates, each well containing1 ml medium, were separately infected with recombinant vacciniavirus and wild type virus at a multiplicity of 10 PFU.After 45 min, the medium in each well was replaced with 1 ml mediumcontaining 0.05% glucose and 50 µCi [3H]-GlcN,and further cultured for 18 h. The medium was removed, the cellswere rinsed three times with fresh medium, and 100 µl of50 mM Tris–HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenolblue and 10% glycerol added. About 10 µl celllysates were electrophoresed on 8% SDS–polyacrylamide gels(Laemmli, 1970). The gels were treated with MeOH, water, glacialHOAc (50:40:10, v/v) for 1 h, washed with water for 5 min,soaked in Amplify fluorographic solution for 1 h, dried, and exposedto x-ray film at –80°C.

Analysis of N-glycanase– and endoglycosidaseH–treated polypeptides by Western blotting
Lysates of CV-1, Hu134TK and HeLa cellsexpressing polypeptides from cDNAs P1 and P2 (see above) were heated at100°C for 10 min, cooled, and mixedwith equal volumes of 100 mM sodium phosphate, 2% NP-40,pH 7.5, and 10 µl of the solution incubatedwith 1 IUB milliunit of N-glycanase at 37°Cfor 2 h. For digestion with endoglycosidase H, the lysates weremixed with equal volumes of 100 mM sodium citrate, pH 5.5,and 10 µl of this solution incubatedwith 1 IUB unit of endoglycosidase H at 37°Cfor 2 h. Untreated and enzyme-treated cell lysates were eletrophoresedon 8% SDS–polyacryl­amide gels, the proteinbands were electrotransferred onto PVDF membranes and detected withmouse antiserum against the 70 kDa recombinant MSP-1 polypeptide.Alkaline phosphatase–conjugated goat antimouse IgG wasused as the secondary antibody.

Carbohydrate analysis
Cells expressing polypeptides from cDNAs P1 and P2 were metabolicallylabeled with [3H]GlcN and then lysedwith 50 µl of water containing 0.5% SDS,1% 2-mercaptoethanol (see above). Portions of lysates weretreated with N-glycanase. The untreated and N-glycanase-treatedcell lysates were electrophoresed on SDS–polyacrylamidegels, transferred onto PVDF membranes, and stained with Coomassieblue. In some experiments, untreated and N-glycanase-treatedcell lysates were diluted to 0.5 ml with 50 mM Tris–HCl,150 mM NaCl, pH 7.5, and the polypeptides immunoprecipitatedwith antimouse IgG against the 70-kDa MSP-1 C-terminal polypeptide. Theimmunoprecipitated polypeptides were recovered by protein A affinityabsorption, analyzed by SDS–PAGE, and transferred to PVDFmembranes.

The PVDF membranes were thoroughly washed with water, the membranescontaining polypeptide bands excised, and hydrolyzed with 400 µl of 3.5 M HCl at 100°Cfor 6 h. For sialic acid analysis, the membranes containing polypeptide bandswere hydrolyzed with 400 µl of 2 M HOAcat 80°C for 4 h. The hydrolysates wererecovered and dried in a Speed-Vac. The residues were dissolvedin water, mixed with appropriate standard sugars, and analyzed byhigh-pH anion-exchange chromatography with a Dionex BioLC HPLC coupledto pulsed amperometric detection using a CarboPac PA1 column (4 x 250 mm) (10GoHardyand Townsend, 1994). The eluents were: (1) 20 mM NaOHat a flow rate of 0.8 ml/min for hexosamines, (2) 100 mMNaOH, 150 mM NaOAc at a flow rate of 0.8 ml/min for sialicacids. Elution of radioactivity was monitored by liquid scintillationcounting of fractions collected at 0.3–0.4 min intervals. [3H]-labeledsugars were identified by either coelution or comparison of elutiontime with standard sugars. Nonradioactive standard sugars were detectedby pulsed amperometric detection.

Analysis of the polypeptide for GPI anchor moieties
Monolayers of CV-1 cells infected with recombinant vaccinia viruscontaining cDNA P2 inserts were metabolically labeled with [3H]GlcN.The cell lysates were electrophoresed on 8% polyacrylamidegels under reducing conditions and the protein bands were transferredon PVDF membranes. The radiolabeled protein band corresponding tothe polypeptide from P2 was excised and analyzed for GPI moietiesby the following methods.

The [3H]GlcN-labeled polypeptidebands (5000–6000 c.p.m.) on PVDF membranes were suspendedin 500 µl of 100 mM Tris–HCl,1 mM CaCl2, pH 8.0, containing 0.05% SDS and 0.5% NP-40,and incubated with pronase (1.0 mg, added 0.5 mg aliquotsat 0 and 24 h) at 55°C for 48 h. Theenzyme digests were centrifuged, the membranes washed with water (3 x 100 µl),and the combined supernatants and washings dried in a Speed Vac.The residues were dissolved in water (0.5 ml) and extractedwith water-saturated 1-butanol (see Figure 5).The aqueous and organic phases were separately dried in a rotaryevaporator and dissolved in water, and the radioactivity was measuredby liquid scintillation counting.

The [3H]GlcN-labeled polypeptideband (derived from cDNA P2, ~10,000 c.p.m.) on PVDF membrane wassuspended in 400 µl of 0.2M NaOAc, pH 3.8, containing 1 M NaNO2 (28). After 18h at room temperature, the pH of the solution was adjusted to 7with 2 M NaOH, centrifuged and the membrane washed with water. Thecombined supernatant and washings was measured for radioactivityby liquid scintillation counting. The membrane was hydrolyzed with400 µl of 3.5 M HCl at 100°Cfor 6 h. The polypeptide band not treated with HNO2 wassimilarly analyzed as a control. The hydrolysates were dried ina Speed Vac and analyzed by high-pH anion-exchange chromatography(10GoHardy and Townsend, 1994).

Analysis for GPI anchor moieties in the polypeptidesby treatment of cells with PI-PLC and phosphatidylinositol-specific phospholipaseD (PI-PLD)
The monolayers of cells (in a 6-well plate) expressing the polypeptidesfrom P2 were gently rinsed with 25 mM HEPES, 150 mM NaCl, pH 7.4,and then treated with 1 unit of PI-PLC in 300 µlof the above buffer at 37°C for 1 h.Equivalent control cells were incubated in parallel under the similarconditions but without the enzyme. The buffers were carefully removed, centrifugedat 4000 r.p.m. to remove any detached cells, concentrated on Centricon30 tubes and analyzed by Western blotting as described above. ForPI-PLD treatment, the cell layers were incubated in the above buffercontaining 2 mM CaCl2 and 10 µlof fresh rabbit serum (as source of PI-PLD) (23GoMenon,1994). The supernatants were analyzed by Western blotting.

Analysis of the polypeptides for proteolytic processing
Lysates of cells expressing polypeptides from cDNAs P1, P2, P3,and P4 were electrophoresed on 8% SDS–polyacrylamide gels.The protein bands on gels were electrotransferred onto PVDF membranes.Detection of polypeptides on the membranes using 5.2 monoclonalantibody specific to the C-terminal region of MSP-1 was carriedout as described above. Alkaline phosphatase–conjugatedgoat antimouse IgG was the secondary antibody and Western blue stabilizedsubstrate for alkaline phosphatase was the color developing reagent.

Immunostaining of cell expressing the polypeptides
Confluent monolayers of cells in six-well plates, each well containing1 ml medium with 2% bovine fetal serum, were infected withrecombinant vaccinia virus at a multiplicity of 10 PFU.After 20 h, the medium from each well was removed and the cell layerswere gently rinsed with fresh medium. Immunostaining using peroxidase-conjugatedsecondary antibody was carried out according to Sutter etal., 1994. The cells were fixed with acetone,methanol (1:1 v/v) for 2 min, washed with PBS, pH 7.4,and then incubated at room temperature with 1:200 diluted mousepolyclonal antiserum against 70 kDa MSP-1 polypeptide. After 1 h,the cells were washed three times with 1 ml PBS, pH 7.4, and thenincubated at room temperature with 1:1000 diluted peroxidase-conjugatedgoat antimouse IgG in PBS, pH 7.4, containing 2% fetalbovine serum. After 45 min, the plates were incubated with 0.5 mlof PBS, pH 7.4, containing 80 µg/mldianisidine hydrochloride and 0.03% hydrogen peroxide atroom temperature until color developed.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 References
 
We thank Dr. Sandra Chang, University of Hawaii, Hawaii, for providing5.2 monoclonal antibody. This work was supported by the U.S. Departmentof Defense Grant N00014–90-J-2032.


    Abbreviations
 
MSP-1, merozoite surface protein 1; GPI, glycosylphos­phatidylinositol;PFU, plaque-forming units; GlcN, glucos­amine; GlaN, galactosamine;PVDF, polyvinylidene difluoride; PI-PLC, phosphatidylinositol-specificphospholipase C; PI-PLD, phosphatidylinositol-specific phospholipaseD.


    Footnotes
 
a The authors Shutong Yang and DavidNikodem contributed equally to this study. Back

b Towhom correspondence should be addressed at: Department of Biochemistryand Molecular Biology, Georgetown University Medical Center, 3900Reservoir Road, NW, Washington, DC 20007 Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 References
 
1 Blackman,M.J. andHolder,A.A. (1992) Secondary processing of the Plasmodium falciparum merozoitesurface protein-1 (MSP-1) by a calcium-dependent membrane-boundserine protease: shedding of MSP-133 as a noncovalently associatedcomplex with other fragments of the MSP1. Mol. Biochem. Parasitol., 50, 307–315.[ISI][Medline]

2 Blackman,M.J.,Heidrich,H.-G., Donachie,S., McBride,J.S. and Holder,A.A. (1990)A single fragment of a malaria merozoite surface protein-1 remainson the parasite during red cell invasion and is the target of invasion-inhibiting antibodies. J.Exp. Med. 172, 379–382.[Abstract]

3 Chang,S.P.,Case,S.E., Gosnell,W.L., Hashimoto,A., Kramer,K.J., Tam,L.Q., Hashiro,C.Q.,Nikaido,C.M., Gibson,H.L., Lee-Ng,C.T., Barr,P.J., Yokota,B.T. andHui,G.S.N. (1996) A recombinant baculovirus 42-kilodalton C-terminalfragment of Plasmodium falciparum merozoite surfaceprotein 1 protects Aotus monkeys against malaria. Infect. Immun., 64, 253–261.[Abstract]

4 Chang,S.P.,Gibson,H.L., Lee-Ng,C.T., Barr,P.J. and Hui,G.S.N. (1992)A carboxyl-terminal fragment of Plasmodium falciparum gp195expressed by a recombinant baculovirus induces antibodies that completelyinhibit parasite growth. J. Immunol., 149, 548–555.[Abstract/Free Full Text]

5 Cooper,J.A. (1993)Merozoite surface antigen-1 of Plasmodium. Parasitol. Today, 9, 39–43.

6 Cooper,J.A.,Cooper,L.T. and Saul,A. (1992) Mapping of the regionpredominantly recognized by antibodies to the Plasmodiumfalciparum merozoite surface antigen MSA 1. Mol. Biochem.Parasitol., 51, 301–312.

7 Dieckmann-Schuppert,A.,Bender,S., Odenthal-Schnittler,M., Bause,E. and Schwarz,R.T. (1992)Apparent lack of N-glycosylation inthe asexual intraerythrocytic stage of Plasmodium falciparum. Eur.J. Biochem., 205, 815–825.[Abstract]

8 Etlinger,H.M.,Caspers,P., Matile,H., Schoenfeld,H.-J., Stueber,D. and Takacs,B.(1991) Ability of recombinant or native proteins toprotect monkeys against heterologous challenge with Plasmodiumfalciparum. Infect. Immun. 59, 3498–3503.[ISI][Medline]

9 Hall,R.,Osland,A., Hyde,J.E., Simmons,D.L., Hope,I.A. and Scaife,J.G. (1984)Processing, polymorphism and biological significance of P190, a majorsurface antigen of the erythrocytic forms of Plasmodiumfalciparum. Mol. Biochem. Parasitol., 11, 61–80.[ISI][Medline]

10 Hardy,M.R. andTownsend,R.R. (1994). High-pH anion-exchange chromatography ofglycoprotein-derived carbohydrates. Methods Enzymol., 230, 208–225.[ISI][Medline]

11 Holder,A.A. (1988)The precursor to major surface antigens: structure and role in immunity. Prog. Allergy, 41, 72–97.

12 Holder,A. andFreeman,R.R. (1982) Biosynthesis and processing ofa Plasmodium falciparum schizont antigen recognizedby immune serum and a monoclonal antibody. J. Exp. Med., 156, 1528–1538.[Abstract]

13 Holder,A.A.,Sandhu,J.S., Hillman,Y., Davey,L.S., Nicholls,S.C., Cooper,H. andLockyer,M.J. (1987) Processing of the precursor tothe major merozoite surface antigens of Plasmodium falciparum. Parasitology, 94, 199–208.[ISI][Medline]

14 Howard,R.,Lyon,J., Diggs,C., Haynes,J., Leech,J., Barnwell,J., Aley,S., Aikawa,M.and Miller,L. (1984) Localization of the major Plasmodium falciparum glycoproteinon the surface of mature intraerythrocytic trophozoites and schizonts. Mol.Biochem. Parasitol., 11, 349–362.[ISI][Medline]

15 Hui,G.S.N. andSiddiqui,W.A. (1987) Serum from Pf195 protected Aotus monkeysinhibit Plasmodium falciparum growth invitro. Exp. Parasitol., 64, 519–522.[ISI][Medline]

16 Hui,G.S.N.,Hashiro,C., Nikaido,C., Case,S.E., Hashimoto,A., Gibson,H., Barr,P.J.and Chang,S.P. (1993) Immunological cross-reactivityof the C-terminal 42-kilodalton fragment of Plasmodiumfalciparum merozoite surface protein 1 expressed in baculovirus. Infect.Immun., 61, 3404–3411.

17 Kumar,S.,Yadava,A., Keister,D.B., Tian,J.H., Ohl,M., Perdue-Greenfield,K.A.,Miller,L.H. and Kaslow,D. (1995) Immunogenicity and in vivo efficacy of recombinant Plasmodiumfalciparum merozoite surface protein-1 in Aotus monkeys. Mol.Med., 1, 325–332.[ISI][Medline]

18 Laemmli,U.K. (1970)Cleavage of structural proteins during the assembly of the headof bacteriophage T4. Nature, 227, 680–685.[ISI][Medline]

19 Longacre,S.,Medis,K.N., and David,P.H. (1994) Plasmodiumvivax merozoite surface protein 1 C-terminal recombinant proteinsin baculovirus. Mol. Biochem. Parasitol., 64, 191–205.[ISI][Medline]

20 Lyon,J.A.,Geller,R.H., Haynes,J.D., Chulay,J.D. and Weber,J.L. (1986)Epitope map and processing scheme for the 195,000-dalton surfaceglycoprotein of Plasmodium falciparum merozoitesdeduced from cloned overlapping segments of the gene. Proc.Natl Acd. Sci. USA, 83, 2989–2993.

21 McBride,J.S. andHeidrich,H.-G. (1987). Fragments of the polymorphicMr 185,000 glycoprotein from the surface of isolated Plasmodium falciparum merozoites form an antigeniccomplex. Mol. Biochem. Parasitol., 23, 71–84.[ISI][Medline]

22 McBride,J.S.,Newbold,C.I. and Anand,R. (1985) Polymorphism of ahigh molecular weight schizont antigen of the human malaria parasite Plasmodium falciparum. J. Exp. Med., 161, 160–180.[Abstract]

23 Menon,A.K. (1994)Structural analysis of glycosylphosphatidylinositol anchors. MethodsEnzymol., 230, 418–442.

24 Moran,P. andCaras,I.W. (1992) Proteins containing an uncleavedsignal for glycophosphatidylinositol membrane anchor attachmentare retained in a post-ER compartment. J. Cell Biol., 119, 763–772.[Abstract]

25 Moran,P. andCaras,I.W. (1994) Requirements for glycosylphosphatidylinositol attachmentare similar but not identical in mammalian cells and parasitic protozoa. J.Cell Biol., 125, 333–343.[Abstract]

26 Murphy,V.F.,Rowan,W.C., Page,M.J. and Holder,A.A. (1990) Expressionof hybrid malaria antigens in insect cells and their engineeringfor correct folding and secretion. Parasitology, 100, 177–183.[ISI][Medline]

27 Pirson,P.J. andPerkins,M.E. (1985) Characterization with monoclonalantibodies of a surface antigen of Plasmodium falciparum merozoites. J. Immunol., 134, 1946–1951.[Abstract/Free Full Text]

28 Riley,E.M.,Allen,S.J., Wheeler,J.G., Blackman,M.J., Bennett,S., Takacs,B., Schoenfeld,H.-J.,Holder,A.A. and Greenwood,B.M. (1992) Naturally acquiredcellular and humoral immune responses to the major merozoite surfaceantigen (PfMSP1) of Plasmodium falciparum are associated with reduced malaria morbidity. ParasiteImmunol., 14, 321–337.

29 Schofield,L. andHackett,F. (1993) Signal transduction in host cellsby glycosylphosphatidylinositol toxin of malaria parasites. J.Exp. Med., 177, 145–153.[Abstract]

30 Schofield,L.,McConville,M.J., Hansen,D., Campbell,A.S., Fraser-Reid,B., Grusby,M.J.and Tachado,S.D. (1999) Cd1d-restricted immunoglobulinG formation to GPI-anchored antigens mediated by NKT cells. Science, 283, 225–229.[Abstract/Free Full Text]

31 Siddiqui,W.A.,Tam,L.Q., Kramer,K.J., Hui,G.S.N., Case,S.E., Yamaga,K.M., Chang,S.P.,Chan,E.B.T. and Kan,S.-C. (1987) Merozoite surfacecoat precursor protein completely protects Aotus monkeys against Plasmodium falciparum malaria. Proc. Natl.Acad. Sci. USA, 84, 3014–3018.[Abstract]

32 Su,S.,Sanadi,A.R., Ifon,E. and Davidson,E.A. (1993) A monoclonalantibody capable of blocking the binding of Pf200 (MSA-1) to humanerythrocytes and inhibiting the invasion of Plasmodiumfalciparum merozoites into human erythrocytes. J. Immunol., 151, 2309–2317.[Abstract/Free Full Text]

33 Wyatt,L.S.,Wyatt,L.S., Foley,P.L., Bennink,J.R. and Moss,B. (1994)A recombinant vector derived from the host range-restricted andhighly attenuated MVA strain of vaccinia virus stimulates protectiveimmunity in mice to influenza virus. Vaccine, 12, 1032–1039.[ISI][Medline]

34 Yang,S.,Carroll,M.W., Torres-Duarte,A.P., Moss,B. and Davidson,E.A. (1997) Additionof the MSA1 signal and anchor sequences to the malaria merozoite surfaceantigen 1 C-terminal region enhances immunogenicity when expressedby recombinant vaccinia virus. Vaccine, 15, 1303–1313.[ISI][Medline]