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In vitro biosynthesis and in vivo processing of the major microneme antigen ofSarcocystis muris cyst merozoites

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Abstract

The cDNA clone pSM/1.6 encoding the 26.5-kDa precursor molecule of the 16/17-kDa microneme antigen ofSarcocystis muris cyst merozoites was expressed in a cell-free translation/translocation system to study translocation of the protein across membranes. The antigen was found to be translocated across heterologous endoplasmic reticulum membranes. Translocation was accompanied by cleavage of a signal peptide to create a 23-kDa polypeptide that was completely protected from digestion with proteinase K. Pulse-chase analysis of [35S]-methionine-labeledS. muris cyst merozoites demonstrated that the 16/17-kDa antigen derived from a 23-kDa precursor molecule and that its processing occurred at between a few minutes and 2 h after biosynthesis. This leads to the conclusion that the native microneme antigen is secreted from the parasite cell via the endoplasmic reticulum. Sorting into micronemes might occur during transition through a Golgi-like structure, involving cleavage of the hydrophilic propeptide to create the mature 16/17-kDa protein.

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Abbreviations

aa:

Amino acids

BFA:

brefeldin A

bp:

base pairs

ER:

endoplasmic reticulum

FCS:

fetal calf serum

HEPES N:

(2-hydroxyethyl)piperazine-.N′-(2-ethanesulfonic acid)

mAb(s):

monoclonal antibodies

NP-40:

Nonidet P-40

ORF:

open reading frame

pSM/1.6:

plasmid carrying the cDNA insert

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electro-phoresis

SRP:

signal recognition particle

References

  • Achbarou A, Mercereau-Puijalon O, Autheman JM, Fortier B, Camus D, Dubremetz JF (1991) Characterization of microneme proteins ofToxoplasma gondii. Mol Biochem Parasitai 47: 223–234

    Article  CAS  Google Scholar 

  • Adams JH, Sim BKL, Dolan SA, Fang X, Kaslow DC (1992) A family of erythrocyte binding proteins of malaria parasites. Proc Natl Acad Sci USA 89:7085–7089

    Article  PubMed  CAS  Google Scholar 

  • Aikawa M (1971)Plasmodium: the fine structure of malaria parasites. Exp Parasitai 30:284–320

    Article  CAS  Google Scholar 

  • Bermudes D, Dubremetz JF, Achbarou A, Joiner KA (1994) Cloning of a cDNA encoding the dense granule protein GRA3 fromToxoplasma gondii. Mol Biochem Parasitai 68:247–257

    Article  CAS  Google Scholar 

  • Blobel G, Dobberstein B (1975a) Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains membrane-bound ribosomes of murine myeloma. J Cell Biol 67:835–851

    Article  PubMed  CAS  Google Scholar 

  • Blobel G, Dobberstein B (1975b) Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol 67:852–862

    Article  CAS  Google Scholar 

  • Brown HJ, Coppel RL (1991) Primary structure of aPlasmodium falciparum rhoptry antigen. Mol Biochem Parasitai 49:99–110

    Article  CAS  Google Scholar 

  • Burg JL, Perelman D, Kasper LH, Ware PL, Boothroyd JC (1988) Molecular analysis of the gene encoding the major surface antigen ofToxoplasma gondii. J Immunol 141:3584–3591

    PubMed  CAS  Google Scholar 

  • Cesbron-Delauw MF (1994) Dense-granule organelles ofToxoplasma gondii: their role in the host-parasite relationship. Parasitai Today 10:293–296

    Article  CAS  Google Scholar 

  • Crary JL, Haldar K (1992) Brefeldin A inhibits protein secretion and parasite maturation in the ring stage ofPlasmodium falciparum. Mol Biochem Parasitai 53:185–192

    Article  CAS  Google Scholar 

  • Elmendorf HG, Haldar K (1993a) Identification and localization of ERD2 in the malaria parasitePlasmodium falciparum: separation from sites of sphingomyelin synthesis and implications for organization of the Golgi. EMBO J 12:4763–4773

    PubMed  CAS  Google Scholar 

  • Elmendorf HG, Haldar K (1993b) Secretory transport inPlasmodium. Parasitai Today 9:98–102

    Article  CAS  Google Scholar 

  • Elmendorf HG, Haldar K (1994)Plasmodium falciparum exports the Golgi marker sphingomyelin synthase into a tubovesicular network in the cytoplasm of mature erythrocytes. J Cell Biol 124:449–462

    Article  PubMed  CAS  Google Scholar 

  • Entzeroth R, König A, Dubremetz JF (1991) Monoclonal antibodies identify micronemes and a new population of cytoplasmic granules cross-reacting with micronemes of cystozoites ofSarcocystis muris. Parasitai Res 77:59–64

    Article  CAS  Google Scholar 

  • Entzeroth R, Kerckhoff H, König (1992) Microneme secretion in Coccidia: confocal laser scanning and electron microscopy ofSarcocystis muris in cell culture. Eur J Cell Biol 59:405–413

    PubMed  CAS  Google Scholar 

  • Eschenbacher KH, Sommer I, Meyer HE, Mehlhorn H, Rüger W (1992) Cloning and expression inEscherichia coli of cDNAs encoding a 31-kilodalton surface antigen ofSarcocystis muris. Mol Biochem Parasitai 53:159–168

    Article  CAS  Google Scholar 

  • Eschenbacher KH, Klein H, Sommer I, Meyer HE, Entzeroth R, Mehlhorn H, Rüger W (1993) Characterization of cDNA clones encoding a major microneme antigen ofSarcocystis muris (Apicomplexa) cyst merozoites. Mol Biochem Parasitai 62:27–36

    Article  CAS  Google Scholar 

  • Farquhar MG, Palade GE (1981) The Golgi apparatus (complex) — (1954-1981) — from artifact to center stage. J Cell Biol 91:77s-103s

    Article  PubMed  CAS  Google Scholar 

  • Günther K, Tümmler M, Arnold HH, Ridley R, Goman M, Scaife JG, Lingelbach K (1991) An exported protein ofPlasmodium falciparum is synthesized as an integral membrane protein. Mol Biochem Parasitai 46:149–158

    Article  Google Scholar 

  • Hirschberg CB, Snider MD (1987) Topography of glycosylation in the rough endoplasmic reticulum and Golgi apparatus. Annu Rev Biochem 56:63–87

    Article  PubMed  CAS  Google Scholar 

  • Hortsch M, Meyer D (1986) Transfer of secretory proteins through the membrane of the endoplasmic reticulum. Int Rev Cytol 102:215–242

    Article  PubMed  CAS  Google Scholar 

  • Jackson RC, Blobel G (1977) Posttranslational cleavage of presecretory proteins with an extract of dog pancreas rough microsomes containing signal peptidase activity. Proc Natl Acad Sci USA 74:5598–5602

    Article  PubMed  CAS  Google Scholar 

  • Klein H, Mehlhorn H, Rüger W (1995) Characterization of genomic clones encoding two microneme antigens of Sarcocystis muris (Apicomplexa). Parasitai Res (in press)

  • Knapp B, Hundt E, Nau U, Küpper HA (1989) Molecular cloning, genomic structure and localization of a blood stage antigen ofPlasmodium falciparum characterized by a serine stretch. Mol Biochem Parasitai 32:73–84

    Article  CAS  Google Scholar 

  • Kozak M (1981) Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes. Nucleic Acids Res 9:5233–5252

    Article  PubMed  CAS  Google Scholar 

  • Kozak M (1989) The scanning model for translation: an update. J Cell Biol 108:229–241

    Article  PubMed  CAS  Google Scholar 

  • Kumar N, Zheng H (1992) Nucleotide sequence of aPlasmodium falciparum stress protein with similarity to mammalian 78-kDa glucose-regulated protein. Mol Biochem Parasitai 56:353–356

    Article  CAS  Google Scholar 

  • Kumar N, Koski G, Harada M, Aikawa M, Zheng H (1991) Induction and localization ofPlasmodium falciparum stress proteins related to the heat shock protein 70 family. Mol Biochem Parasitai 48:47–58

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Lecordier L, Mercier C, Torpier G, Tourvieille B, Darcy F, Liu JL, Maes P, Tartar A, Capron A, Cesbron-Delauw MF (1993) Molecular structure of aToxoplasma gondii dense granule antigen (GRA 5) associated with the parasitophorous vacuole membrane. Mol Biochem Parasitai 59:143–154

    Article  CAS  Google Scholar 

  • Lecordier L, Moleon-Borodowsky I, Dubremetz JF, Tourvieille B, Mercier C, Deslee D, Capron A, Cesbron-Delauw MF (1995) Characterization of a dense granule antigen ofToxoplasma gondii (GRA6) associated to the network of the parasitophorous vacuole. Mol Biochem Parasitai 70:85–94

    Article  CAS  Google Scholar 

  • Lingelbach KR (1993)Plasmodium falciparum: a molecular view of protein transport from the parasite into the host erythrocyte. Exp Parasitai 76:318–327

    Article  CAS  Google Scholar 

  • Lipp J, Dobberstein B (1986) The membrane-spanning segment of invariant chain (Iy) contains a potentially cleavable signal sequence. Cell 46:1103–1112

    Article  PubMed  CAS  Google Scholar 

  • Lippincott-Schwartz J, Yuan LC, Bonifacino JS, Klausner RD (1989) Rapid redistribution of Golgi proteins into the ER in cells treated with Brefeldin A: evidence for membrane cycling from Golgi to ER. Cell 56:801–813

    Article  PubMed  CAS  Google Scholar 

  • Mehlhorn H (1988) Cellular organization of the protozoa. In: Mehlhorn H (ed) Parasitology in focus. Springer, Berlin Heidelberg New York, pp 161–188

    Google Scholar 

  • Mehlhorn H, Heydorn AO (1978) The Sarcosporidia (Protozoa, Sporozoa): life cycle and fine structure. Adv Parasitai 16:43–91

    Article  CAS  Google Scholar 

  • Mercier C, Lecordier L, Darcy F, Deslee D, Murray A, Tourvieille B, Maes P, Capron A, Cesbron-Delauw MF (1993) Molecular characterization of a dense granule antigen (Gra 2) associated with the network of the parasitophorous vacuole inToxoplasma gondii. Mol Biochem Parasitai 58:71–82

    Article  CAS  Google Scholar 

  • Mevelec MN, Chardes T, Mercereau-Puijalon O, Bourguin I, Achbarou A, Dubremetz JF, Bout D (1992) Molecular cloning of GRA 4, aToxoplasma gondii dense granule protein, recognized by mucosal IgA antibodies. Mol Biochem Parasitai 56: 227–238

    Article  CAS  Google Scholar 

  • Meyer DI, Krause E, Dobberstein B (1982) Secretory protein translocation across membranes — the role of the “docking protein”. Nature 297:647–650

    Article  PubMed  CAS  Google Scholar 

  • Montag T, Löschner B, Pörtner M, Strobel J, Hiepe T, Otto A, Klein H, Rüger W, Entzeroth R (1995) Immunomodulation by lectins isolated from Sarcosporidia. Zentralbl Bakteriol Mikro-biol Hyg [A] 282:184

    Google Scholar 

  • Ossorio PN, Schwartzman JD, Boothroyd JC (1992) AToxoplasma gondii rhoptry protein associated with host cell penetration has unusual charge asymmetry. Mol Biochem Parasitai 50:1–16

    Article  CAS  Google Scholar 

  • Paterson BM, Rosenberg M (1979) Efficient translation of prokaryotic mRNAs in a eukaryotic cell-free system requires addition of a cap structure. Nature 279:692–696

    Article  PubMed  CAS  Google Scholar 

  • Perkins ME (1992) Rhoptry organelles of apicomplexan parasites. Parasitai Today 8:28–32

    Article  CAS  Google Scholar 

  • Ragge K, Arnold HH, Tümmler M, Knapp B, Hundt E, Lingelbach K (1990) In vitro biosynthesis and membrane translocation of the serine rich protein ofPlasmodium falciparum. Mol Biochem Parasitai 42:93–100

    Article  CAS  Google Scholar 

  • Ridley RG, Takacs B, Lahm HW, Delves CJ, Goman M, Certa U, Matile H, Woollett GR, Scaife JG (1990) Characterization and sequence of a protective rhoptry antigen fromPlasmodium falciparum. Mol Biochem Parasitai 41:125–134

    Article  CAS  Google Scholar 

  • Roger N, Dubremetz JF, Delplace P, Fortier B, Tronchin G, Vernes A (1988) Characterization of a 225 kDa rhoptry protein ofPlasmodium falciparum. Mol Biochem Parasitai 27:135–142

    Article  CAS  Google Scholar 

  • Rogers WO, Rogers MD, Hedstrom RC, Hoffman SL (1992) Characterization of the gene encoding sporozoite surface protein 2, a protectivePlasmodium yoelii sporozoite antigen. Mol Biochem Parasitai 53:45–52

    Article  CAS  Google Scholar 

  • Sadak A, Taghy Z, Fortier B, Dubremetz JF (1988) Characterization of a family of rhoptry proteins ofToxoplasma gondii. Mol Biochem Parasitol 29:203–211

    Article  PubMed  CAS  Google Scholar 

  • Sommer I, Mehlhorn H, Rüger W (1991) Biochemical and immunological characterization of major surface antigens ofSarcocystis muris andS. suicanis cyst merozoites. Parasitol Res 77:204–211

    Article  PubMed  CAS  Google Scholar 

  • Tomley FM, Clarke LE, Kawazoe U, Dijkema R, Kok JJ (1991) Sequence of the gene encoding an immunodominant microneme protein ofEimeria tenella. Mol Biochem Parasitol 49:277–288

    Article  PubMed  CAS  Google Scholar 

  • Von Heinje G (1985) Signal sequences: the limits of variation. J Mol Biol 184:99–105

    Article  Google Scholar 

  • Von Heinje G (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14:4683–4690

    Article  Google Scholar 

  • Walter P, Blobel G (1981) Translocation of protein across the endoplasmic reticulum. III. Signal recognition protein (SRP) causes signal sequence-dependent and site-specific arrest of chain elongation that is released by microsomal membranes. J Cell Biol 91:557–561

    Article  PubMed  CAS  Google Scholar 

  • Walter P, Ibrahimi I, Blobel G (1981) Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein. J Cell Biol 91:545–550

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Fakultät für Biologie, Arbeitsgruppe Molekulare Genetik, Ruhr-Universität Bochum, Universitätsstr. 150, D-44780, Bochum, Germany

    H. Klein & W. Rüger

  2. Lehrstuhl für Zoomorphologie, Zellbiologie und Parasitologie der Heinrich-Heine Universität, Düsseldorf, Germany

    H. Mehlhorn

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  1. H. Klein
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  2. H. Mehlhorn
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  3. W. Rüger
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Correspondence to W. Rüger.

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Klein, H., Mehlhorn, H. & Rüger, W. In vitro biosynthesis and in vivo processing of the major microneme antigen ofSarcocystis muris cyst merozoites. Parasitol Res 82, 468–474 (1996). https://doi.org/10.1007/s004360050146

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