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. 1990 Sep;10(9):4506–4517. doi: 10.1128/mcb.10.9.4506

Characterization of a cDNA encoding a cysteine-rich cell surface protein located in the flagellar pocket of the protozoan Trypanosoma brucei.

M G Lee 1, B E Bihain 1, D G Russell 1, R J Deckelbaum 1, L H Van der Ploeg 1
PMCID: PMC361037  PMID: 1697030

Abstract

We have characterized a cDNA encoding a cysteine-rich, acidic integral membrane protein (CRAM) of the parasitic protozoa Trypanosoma brucei and Trypanosoma equiperdum. Unlike other membrane proteins of T. brucei, which are distributed throughout the cell surface, CRAM is concentrated in the flagellar pocket, an invagination of the cell surface of the trypanosome where endocytosis has been documented. Accordingly, CRAM also locates to vesicles located underneath the pocket, providing evidence of its internalization. CRAM has a predicted molecular mass of 130 kilodaltons and has a signal peptide, a transmembrane domain, and a 41-amino-acid cytoplasmic extension. A characteristic feature of CRAM is a large extracellular domain with a roughly 66-fold acidic, cysteine-rich 12-amino-acid repeat. CRAM is conserved among different protozoan species, including Trypanosoma cruzi, and CRAM has structural similarities with eucaryotic cell surface receptors. The most striking homology of CRAM is to the human low-density-lipoprotein receptor. We propose that CRAM functions as a cell surface receptor of different trypanosome species.

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Selected References

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

  1. BONE G. J., STEINERT M. Isotopes incorporated in the nucleic acids of Trypanosoma mega. Nature. 1956 Aug 11;178(4528):308–309. doi: 10.1038/178308a0. [DOI] [PubMed] [Google Scholar]
  2. Basu S. K., Goldstein J. L., Anderson G. W., Brown M. S. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3178–3182. doi: 10.1073/pnas.73.9.3178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Black S., Vandeweerd V. Serum lipoproteins are required for multiplication of Trypanosoma brucei brucei under axenic culture conditions. Mol Biochem Parasitol. 1989 Nov;37(1):65–72. doi: 10.1016/0166-6851(89)90103-5. [DOI] [PubMed] [Google Scholar]
  4. Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Borst P. Discontinuous transcription and antigenic variation in trypanosomes. Annu Rev Biochem. 1986;55:701–732. doi: 10.1146/annurev.bi.55.070186.003413. [DOI] [PubMed] [Google Scholar]
  6. Brown M. S., Goldstein J. L. A receptor-mediated pathway for cholesterol homeostasis. Science. 1986 Apr 4;232(4746):34–47. doi: 10.1126/science.3513311. [DOI] [PubMed] [Google Scholar]
  7. Brown R. C., Evans D. A., Vickerman K. Changes in oxidative metabolism and ultrastructure accompanying differentiation of the mitochondrion in Trypanosoma brucei. Int J Parasitol. 1973 Sep;3(5):691–704. doi: 10.1016/0020-7519(73)90095-7. [DOI] [PubMed] [Google Scholar]
  8. Brun R., Schönenberger Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop. 1979 Sep;36(3):289–292. [PubMed] [Google Scholar]
  9. Catterall C. F., Lyons A., Sim R. B., Day A. J., Harris T. J. Characterization of primary amino acid sequence of human complement control protein factor I from an analysis of cDNA clones. Biochem J. 1987 Mar 15;242(3):849–856. doi: 10.1042/bj2420849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Clayton C. E., Mowatt M. R. The procyclic acidic repetitive proteins of Trypanosoma brucei. Purification and post-translational modification. J Biol Chem. 1989 Sep 5;264(25):15088–15093. [PubMed] [Google Scholar]
  12. Coppens I., Baudhuin P., Opperdoes F. R., Courtoy P. J. Receptors for the host low density lipoproteins on the hemoflagellate Trypanosoma brucei: purification and involvement in the growth of the parasite. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6753–6757. doi: 10.1073/pnas.85.18.6753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Coppens I., Opperdoes F. R., Courtoy P. J., Baudhuin P. Receptor-mediated endocytosis in the bloodstream form of Trypanosoma brucei. J Protozool. 1987 Nov;34(4):465–473. doi: 10.1111/j.1550-7408.1987.tb03216.x. [DOI] [PubMed] [Google Scholar]
  14. Davis C. G., van Driel I. R., Russell D. W., Brown M. S., Goldstein J. L. The low density lipoprotein receptor. Identification of amino acids in cytoplasmic domain required for rapid endocytosis. J Biol Chem. 1987 Mar 25;262(9):4075–4082. [PubMed] [Google Scholar]
  15. DiScipio R. G., Chakravarti D. N., Muller-Eberhard H. J., Fey G. H. The structure of human complement component C7 and the C5b-7 complex. J Biol Chem. 1988 Jan 5;263(1):549–560. [PubMed] [Google Scholar]
  16. Fairlamb A. H., Weislogel P. O., Hoeijmakers J. H., Borst P. Isolation and characterization of kinetoplast DNA from bloodstream form of Trypanosoma brucei. J Cell Biol. 1978 Feb;76(2):293–309. doi: 10.1083/jcb.76.2.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Frevert U., Reinwald E. Endocytosis and intracellular occurrence of the variant surface glycoprotein in Trypanosoma congolense. J Ultrastruct Mol Struct Res. 1988 May;99(2):137–149. doi: 10.1016/0889-1605(88)90049-3. [DOI] [PubMed] [Google Scholar]
  18. Giannini S. H., D'Alesandro P. A. Isolation of protective antigens from Trypanosoma lewisi by using trypanostatic (ablastic) immunoglobulin G from the surface coat. Infect Immun. 1984 Feb;43(2):617–621. doi: 10.1128/iai.43.2.617-621.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Gray A., Dull T. J., Ullrich A. Nucleotide sequence of epidermal growth factor cDNA predicts a 128,000-molecular weight protein precursor. Nature. 1983 Jun 23;303(5919):722–725. doi: 10.1038/303722a0. [DOI] [PubMed] [Google Scholar]
  21. Haefliger J. A., Tschopp J., Nardelli D., Wahli W., Kocher H. P., Tosi M., Stanley K. K. Complementary DNA cloning of complement C8 beta and its sequence homology to C9. Biochemistry. 1987 Jun 16;26(12):3551–3556. doi: 10.1021/bi00386a045. [DOI] [PubMed] [Google Scholar]
  22. Hajduk S. L., Moore D. R., Vasudevacharya J., Siqueira H., Torri A. F., Tytler E. M., Esko J. D. Lysis of Trypanosoma brucei by a toxic subspecies of human high density lipoprotein. J Biol Chem. 1989 Mar 25;264(9):5210–5217. [PubMed] [Google Scholar]
  23. Herz J., Hamann U., Rogne S., Myklebost O., Gausepohl H., Stanley K. K. Surface location and high affinity for calcium of a 500-kd liver membrane protein closely related to the LDL-receptor suggest a physiological role as lipoprotein receptor. EMBO J. 1988 Dec 20;7(13):4119–4127. doi: 10.1002/j.1460-2075.1988.tb03306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Iovannisci D. M., Kaur K., Young L., Ullman B. Genetic analysis of nucleoside transport in Leishmania donovani. Mol Cell Biol. 1984 Jun;4(6):1013–1019. doi: 10.1128/mcb.4.6.1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Johnson D., Lanahan A., Buck C. R., Sehgal A., Morgan C., Mercer E., Bothwell M., Chao M. Expression and structure of the human NGF receptor. Cell. 1986 Nov 21;47(4):545–554. doi: 10.1016/0092-8674(86)90619-7. [DOI] [PubMed] [Google Scholar]
  26. Kemp D. J., Coppel R. L., Stahl H. D., Bianco A. E., Corcoran L. M., McIntyre P., Langford C. J., Favaloro J. M., Crewther P. E., Brown G. V. The Wellcome Trust lecture. Genes for antigens of Plasmodium falciparum. Parasitology. 1986;92 (Suppl):S83–108. doi: 10.1017/s0031182000085711. [DOI] [PubMed] [Google Scholar]
  27. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Langreth S. G., Balber A. E. Protein uptake and digestion in bloodstream and culture forms of Trypanosoma brucei. J Protozool. 1975 Feb;22(1):40–53. doi: 10.1111/j.1550-7408.1975.tb00943.x. [DOI] [PubMed] [Google Scholar]
  31. Le Blancq S. M., Swinkels B. W., Gibson W. C., Borst P. Evidence for gene conversion between the phosphoglycerate kinase genes of Trypanosoma brucei. J Mol Biol. 1988 Apr 5;200(3):439–447. doi: 10.1016/0022-2836(88)90534-7. [DOI] [PubMed] [Google Scholar]
  32. Le Trant N., Meshnick S. R., Kitchener K., Eaton J. W., Cerami A. Iron-containing superoxide dismutase from Crithidia fasciculata. Purification, characterization, and similarity to Leishmanial and trypanosomal enzymes. J Biol Chem. 1983 Jan 10;258(1):125–130. [PubMed] [Google Scholar]
  33. Lee M. G., Van der Ploeg L. H. Frequent independent duplicative transpositions activate a single VSG gene. Mol Cell Biol. 1987 Jan;7(1):357–364. doi: 10.1128/mcb.7.1.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Livneh E., Glazer L., Segal D., Schlessinger J., Shilo B. Z. The Drosophila EGF receptor gene homolog: conservation of both hormone binding and kinase domains. Cell. 1985 Mar;40(3):599–607. doi: 10.1016/0092-8674(85)90208-9. [DOI] [PubMed] [Google Scholar]
  35. Longacre S., Hibner U., Raibaud A., Eisen H., Baltz T., Giroud C., Baltz D. DNA rearrangements and antigenic variation in Trypanosoma equiperdum: multiple expression-linked sites in independent isolates of trypanosomes expressing the same antigen. Mol Cell Biol. 1983 Mar;3(3):399–409. doi: 10.1128/mcb.3.3.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mahley R. W., Innerarity T. L., Pitas R. E., Weisgraber K. H., Brown J. H., Gross E. Inhibition of lipoprotein binding to cell surface receptors of fibroblasts following selective modification of arginyl residues in arginine-rich and B apoproteins. J Biol Chem. 1977 Oct 25;252(20):7279–7287. [PubMed] [Google Scholar]
  37. Mowatt M. R., Clayton C. E. Developmental regulation of a novel repetitive protein of Trypanosoma brucei. Mol Cell Biol. 1987 Aug;7(8):2838–2844. doi: 10.1128/mcb.7.8.2838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Olenick J. G., Wolff R., Nauman R. K., McLaughlin J. A flagellar pocket membrane fraction from Trypanosoma brucei rhodesiense: immunogold localization and nonvariant immunoprotection. Infect Immun. 1988 Jan;56(1):92–98. doi: 10.1128/iai.56.1.92-98.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Porter A. G., Barber C., Carey N. H., Hallewell R. A., Threlfall G., Emtage J. S. Complete nucleotide sequence of an influenza virus haemagglutinin gene from cloned DNA. Nature. 1979 Nov 29;282(5738):471–477. doi: 10.1038/282471a0. [DOI] [PubMed] [Google Scholar]
  41. Richardson J. P., Beecroft R. P., Tolson D. L., Liu M. K., Pearson T. W. Procyclin: an unusual immunodominant glycoprotein surface antigen from the procyclic stage of African trypanosomes. Mol Biochem Parasitol. 1988 Dec;31(3):203–216. doi: 10.1016/0166-6851(88)90150-8. [DOI] [PubMed] [Google Scholar]
  42. Richardson J. P., Jenni L., Beecroft R. P., Pearson T. W. Procyclic tsetse fly midgut forms and culture forms of African trypanosomes share stage- and species-specific surface antigens identified by monoclonal antibodies. J Immunol. 1986 Mar 15;136(6):2259–2264. [PubMed] [Google Scholar]
  43. Roditi I., Carrington M., Turner M. Expression of a polypeptide containing a dipeptide repeat is confined to the insect stage of Trypanosoma brucei. Nature. 1987 Jan 15;325(6101):272–274. doi: 10.1038/325272a0. [DOI] [PubMed] [Google Scholar]
  44. Russell D. W., Schneider W. J., Yamamoto T., Luskey K. L., Brown M. S., Goldstein J. L. Domain map of the LDL receptor: sequence homology with the epidermal growth factor precursor. Cell. 1984 Jun;37(2):577–585. doi: 10.1016/0092-8674(84)90388-x. [DOI] [PubMed] [Google Scholar]
  45. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  46. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  47. Spindler K. R., Rosser D. S., Berk A. J. Analysis of adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichia coli. J Virol. 1984 Jan;49(1):132–141. doi: 10.1128/jvi.49.1.132-141.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stanley K. K., Kocher H. P., Luzio J. P., Jackson P., Tschopp J. The sequence and topology of human complement component C9. EMBO J. 1985 Feb;4(2):375–382. doi: 10.1002/j.1460-2075.1985.tb03639.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Stanley K. K., Page M., Campbell A. K., Luzio J. P. A mechanism for the insertion of complement component C9 into target membranes. Mol Immunol. 1986 May;23(5):451–458. doi: 10.1016/0161-5890(86)90108-2. [DOI] [PubMed] [Google Scholar]
  50. Stanley K. K. The molecular mechanism of complement C9 insertion and polymerisation in biological membranes. Curr Top Microbiol Immunol. 1989;140:49–65. doi: 10.1007/978-3-642-73911-8_5. [DOI] [PubMed] [Google Scholar]
  51. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Ullrich A., Coussens L., Hayflick J. S., Dull T. J., Gray A., Tam A. W., Lee J., Yarden Y., Libermann T. A., Schlessinger J. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. 1984 May 31-Jun 6Nature. 309(5967):418–425. doi: 10.1038/309418a0. [DOI] [PubMed] [Google Scholar]
  53. Van der Ploeg L. H., Bernards A., Rijsewijk F. A., Borst P. Characterization of the DNA duplication-transposition that controls the expression of two genes for variant surface glycoproteins in Trypanosoma brucei. Nucleic Acids Res. 1982 Jan 22;10(2):593–609. doi: 10.1093/nar/10.2.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Van der Ploeg L. H. Control of variant surface antigen switching in trypanosomes. Cell. 1987 Oct 23;51(2):159–161. doi: 10.1016/0092-8674(87)90140-1. [DOI] [PubMed] [Google Scholar]
  55. Vickerman K. Developmental cycles and biology of pathogenic trypanosomes. Br Med Bull. 1985 Apr;41(2):105–114. doi: 10.1093/oxfordjournals.bmb.a072036. [DOI] [PubMed] [Google Scholar]
  56. Vickerman K. The fine structure of Trypanosoma congolense in its bloodstream phase. J Protozool. 1969 Feb;16(1):54–69. doi: 10.1111/j.1550-7408.1969.tb02233.x. [DOI] [PubMed] [Google Scholar]
  57. Ward C. W., Dopheide T. A. Primary structure of the Hong Kong (H3) haemagglutinin. Br Med Bull. 1979 Jan;35(1):51–56. doi: 10.1093/oxfordjournals.bmb.a071542. [DOI] [PubMed] [Google Scholar]
  58. Webster P., Grab D. J. Intracellular colocalization of variant surface glycoprotein and transferrin-gold in Trypanosoma brucei. J Cell Biol. 1988 Feb;106(2):279–288. doi: 10.1083/jcb.106.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Weisgraber K. H., Innerarity T. L., Mahley R. W. Role of lysine residues of plasma lipoproteins in high affinity binding to cell surface receptors on human fibroblasts. J Biol Chem. 1978 Dec 25;253(24):9053–9062. [PubMed] [Google Scholar]
  60. Yamamoto T., Davis C. G., Brown M. S., Schneider W. J., Casey M. L., Goldstein J. L., Russell D. W. The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA. Cell. 1984 Nov;39(1):27–38. doi: 10.1016/0092-8674(84)90188-0. [DOI] [PubMed] [Google Scholar]
  61. von Heijne G. Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem. 1983 Jun 1;133(1):17–21. doi: 10.1111/j.1432-1033.1983.tb07424.x. [DOI] [PubMed] [Google Scholar]

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