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. 1990 Nov;87(21):8393–8397. doi: 10.1073/pnas.87.21.8393

Molecular cloning and characterization of the immunologically protective surface glycoprotein GP46/M-2 of Leishmania amazonensis.

K L Lohman 1, P J Langer 1, D McMahon-Pratt 1
PMCID: PMC54962  PMID: 2236047

Abstract

Immunization of mice with the GP46/M-2 membrane glycoprotein has been demonstrated to elicit protection against infection with the parasitic protozoan Leishmania amazonensis. As this molecule is important for future vaccine studies of leishmaniasis, the gene encoding the GP46/M-2 surface membrane glycoprotein of Leishmania amazonensis has been cloned and sequenced. The protein sequence derived from the DNA sequence data is consistent with the known biochemical and immunochemical properties of the protein and indicates a number of structural areas of interest. A repetitive sequence (24 amino acids repeated four times) occurs within the amino-terminal portion of the molecule and constitutes approximately 22% of the total mature protein. The protease-resistant immunodominant carboxyl-terminal domain of the protein comprises approximately half of the molecule and consists of proline-rich and cysteine-rich areas of sequence; the distribution of cysteine residues is suggestive of metal binding motifs. The sequence predicts a hydrophobic leader peptide, and a putative attachment site for a glycosyl-phosphatidylinositol anchor is indicated at the carboxyl terminus, consistent with the membrane location of the protein. Southern blot analyses also indicate the presence of a GP46/M-2 gene family.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barker D. C., Gibson L. J., Kennedy W. P., Nasser A. A., Williams R. H. The potential of using recombinant DNA species-specific probes for the identification of tropical Leishmania. Parasitology. 1986;92 (Suppl):S139–S174. doi: 10.1017/s0031182000085747. [DOI] [PubMed] [Google Scholar]
  2. Berg J. M. Potential metal-binding domains in nucleic acid binding proteins. Science. 1986 Apr 25;232(4749):485–487. doi: 10.1126/science.2421409. [DOI] [PubMed] [Google Scholar]
  3. Beverley S. M., Ellenberger T. E., Cordingley J. S. Primary structure of the gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase of Leishmania major. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2584–2588. doi: 10.1073/pnas.83.8.2584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beverley S. M., Ismach R. B., Pratt D. M. Evolution of the genus Leishmania as revealed by comparisons of nuclear DNA restriction fragment patterns. Proc Natl Acad Sci U S A. 1987 Jan;84(2):484–488. doi: 10.1073/pnas.84.2.484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bishop R. P., Miles M. A. Chromosome size polymorphisms of Leishmania donovani. Mol Biochem Parasitol. 1987 Jul;24(3):263–272. doi: 10.1016/0166-6851(87)90158-7. [DOI] [PubMed] [Google Scholar]
  6. Bordier C., Etges R. J., Ward J., Turner M. J., Cardoso de Almeida M. L. Leishmania and Trypanosoma surface glycoproteins have a common glycophospholipid membrane anchor. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5988–5991. doi: 10.1073/pnas.83.16.5988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. CONVIT J., KERDEL-VEGAS F. DISSEMINATED CUTANEOUS LEISHMANIASIS; INNOCULATION TO LABORATORY ANIMALS, ELECTRON MICROSCOPY AND FLUORESCENT ANTIBODIES STUDIES. Arch Dermatol. 1965 May;91:439–447. doi: 10.1001/archderm.1965.01600110025007. [DOI] [PubMed] [Google Scholar]
  8. Champsi J., McMahon-Pratt D. Membrane glycoprotein M-2 protects against Leishmania amazonensis infection. Infect Immun. 1988 Dec;56(12):3272–3279. doi: 10.1128/iai.56.12.3272-3279.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  10. Chou P. Y., Fasman G. D. Prediction of protein conformation. Biochemistry. 1974 Jan 15;13(2):222–245. doi: 10.1021/bi00699a002. [DOI] [PubMed] [Google Scholar]
  11. Cowman A. F., Saint R. B., Coppel R. L., Brown G. V., Anders R. F., Kemp D. J. Conserved sequences flank variable tandem repeats in two S-antigen genes of Plasmodium falciparum. Cell. 1985 Apr;40(4):775–783. doi: 10.1016/0092-8674(85)90337-x. [DOI] [PubMed] [Google Scholar]
  12. Dale R. M., McClure B. A., Houchins J. P. A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA. Plasmid. 1985 Jan;13(1):31–40. doi: 10.1016/0147-619x(85)90053-8. [DOI] [PubMed] [Google Scholar]
  13. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Evans D. A., Lanham S. M., Baldwin C. I., Peters W. The isolation and isoenzyme characterization of Leishmania braziliensis subsp. from patients with cutaneous leishmaniasis acquired in Belize. Trans R Soc Trop Med Hyg. 1984;78(1):35–42. doi: 10.1016/0035-9203(84)90168-8. [DOI] [PubMed] [Google Scholar]
  15. Ferguson M. A., Low M. G., Cross G. A. Glycosyl-sn-1,2-dimyristylphosphatidylinositol is covalently linked to Trypanosoma brucei variant surface glycoprotein. J Biol Chem. 1985 Nov 25;260(27):14547–14555. [PubMed] [Google Scholar]
  16. Ferguson M. A., Williams A. F. Cell-surface anchoring of proteins via glycosyl-phosphatidylinositol structures. Annu Rev Biochem. 1988;57:285–320. doi: 10.1146/annurev.bi.57.070188.001441. [DOI] [PubMed] [Google Scholar]
  17. Frankel A. D., Berg J. M., Pabo C. O. Metal-dependent folding of a single zinc finger from transcription factor IIIA. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4841–4845. doi: 10.1073/pnas.84.14.4841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Giannini S. H., Schittini M., Keithly J. S., Warburton P. W., Cantor C. R., Van der Ploeg L. H. Karyotype analysis of Leishmania species and its use in classification and clinical diagnosis. Science. 1986 May 9;232(4751):762–765. doi: 10.1126/science.3961502. [DOI] [PubMed] [Google Scholar]
  19. Grimaldi G., Jr, David J. R., McMahon-Pratt D. Identification and distribution of New World Leishmania species characterized by serodeme analysis using monoclonal antibodies. Am J Trop Med Hyg. 1987 Mar;36(2):270–287. doi: 10.4269/ajtmh.1987.36.270. [DOI] [PubMed] [Google Scholar]
  20. Handman E., Mitchell G. F. Immunization with Leishmania receptor for macrophages protects mice against cutaneous leishmaniasis. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5910–5914. doi: 10.1073/pnas.82.17.5910. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hendricks L. D., Wood D. E., Hajduk M. E. Haemoflagellates: commercially available liquid media for rapid cultivation. Parasitology. 1978 Jun;76(3):309–316. doi: 10.1017/s0031182000048186. [DOI] [PubMed] [Google Scholar]
  22. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  23. Kahl L. P., McMahon-Pratt D. Structural and antigenic characterization of a species- and promastigote-specific Leishmania mexicana amazonensis membrane protein. J Immunol. 1987 Mar 1;138(5):1587–1595. [PubMed] [Google Scholar]
  24. Kornfeld R., Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985;54:631–664. doi: 10.1146/annurev.bi.54.070185.003215. [DOI] [PubMed] [Google Scholar]
  25. Kreutzer R. D., Semko M. E., Hendricks L. D., Wright N. Identification of Leishmania spp. by multiple isozyme analysis. Am J Trop Med Hyg. 1983 Jul;32(4):703–715. doi: 10.4269/ajtmh.1983.32.703. [DOI] [PubMed] [Google Scholar]
  26. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Lainson R. The American leishmaniases: some observations on their ecology and epidemiology. Trans R Soc Trop Med Hyg. 1983;77(5):569–596. doi: 10.1016/0035-9203(83)90185-2. [DOI] [PubMed] [Google Scholar]
  29. Lathe R. Synthetic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations. J Mol Biol. 1985 May 5;183(1):1–12. doi: 10.1016/0022-2836(85)90276-1. [DOI] [PubMed] [Google Scholar]
  30. Matsumura M., Signor G., Matthews B. W. Substantial increase of protein stability by multiple disulphide bonds. Nature. 1989 Nov 16;342(6247):291–293. doi: 10.1038/342291a0. [DOI] [PubMed] [Google Scholar]
  31. Miller L. H., Howard R. J., Carter R., Good M. F., Nussenzweig V., Nussenzweig R. S. Research toward malaria vaccines. Science. 1986 Dec 12;234(4782):1349–1356. doi: 10.1126/science.2431481. [DOI] [PubMed] [Google Scholar]
  32. Ozaki L. S., Svec P., Nussenzweig R. S., Nussenzweig V., Godson G. N. Structure of the plasmodium knowlesi gene coding for the circumsporozoite protein. Cell. 1983 Oct;34(3):815–822. doi: 10.1016/0092-8674(83)90538-x. [DOI] [PubMed] [Google Scholar]
  33. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  34. Perlman D., Halvorson H. O. A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides. J Mol Biol. 1983 Jun 25;167(2):391–409. doi: 10.1016/s0022-2836(83)80341-6. [DOI] [PubMed] [Google Scholar]
  35. Petersen E. A., Neva F. A., Oster C. N., Bogaert Diaz H. Specific inhibition of lymphocyte-proliferation responses by adherent suppressor cells in diffuse cutaneous leishmaniasis. N Engl J Med. 1982 Feb 18;306(7):387–392. doi: 10.1056/NEJM198202183060702. [DOI] [PubMed] [Google Scholar]
  36. Peterson D. S., Wrightsman R. A., Manning J. E. Cloning of a major surface-antigen gene of Trypanosoma cruzi and identification of a nonapeptide repeat. Nature. 1986 Aug 7;322(6079):566–568. doi: 10.1038/322566a0. [DOI] [PubMed] [Google Scholar]
  37. Peterson M. G., Coppel R. L., McIntyre P., Langford C. J., Woodrow G., Brown G. V., Anders R. F., Kemp D. J. Variation in the precursor to the major merozoite surface antigens of Plasmodium falciparum. Mol Biochem Parasitol. 1988 Jan 15;27(2-3):291–301. doi: 10.1016/0166-6851(88)90049-7. [DOI] [PubMed] [Google Scholar]
  38. Rogers W. O., Wirth D. F. Kinetoplast DNA minicircles: regions of extensive sequence divergence. Proc Natl Acad Sci U S A. 1987 Jan;84(2):565–569. doi: 10.1073/pnas.84.2.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Russell D. G., Alexander J. Effective immunization against cutaneous leishmaniasis with defined membrane antigens reconstituted into liposomes. J Immunol. 1988 Feb 15;140(4):1274–1279. [PubMed] [Google Scholar]
  40. Samaras N., Spithill T. W. Molecular karyotype of five species of Leishmania and analysis of gene locations and chromosomal rearrangements. Mol Biochem Parasitol. 1987 Oct;25(3):279–291. doi: 10.1016/0166-6851(87)90092-2. [DOI] [PubMed] [Google Scholar]
  41. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Scott P., Pearce E., Natovitz P., Sher A. Vaccination against cutaneous leishmaniasis in a murine model. I. Induction of protective immunity with a soluble extract of promastigotes. J Immunol. 1987 Jul 1;139(1):221–227. [PubMed] [Google Scholar]
  43. Shaw J. J., Lainson R. Leishmaniasis in Brazil: X. Some observations of intradermal reactions to different trypanosomatid antigens of patients suffering from cutaneous and mucocutaneous leishmaniasis. Trans R Soc Trop Med Hyg. 1975;69(3):323–335. doi: 10.1016/0035-9203(75)90127-3. [DOI] [PubMed] [Google Scholar]
  44. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  45. Stenflo J., Fernlund P. Amino acid sequence of the heavy chain of bovine protein C. J Biol Chem. 1982 Oct 25;257(20):12180–12190. [PubMed] [Google Scholar]
  46. Wallis A. E., McMaster W. R. Identification of Leishmania genes encoding proteins containing tandemly repeating peptides. J Exp Med. 1987 Dec 1;166(6):1814–1824. doi: 10.1084/jem.166.6.1814. [DOI] [PMC free article] [PubMed] [Google Scholar]

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