1887

Abstract

In this paper, the identification and functional analysis of a fatty acyl-CoA-binding protein (ACBP) gene from the opportunistic protist are described. The gene encodes a protein of 268 aa that is three times larger than typical ACBPs (i.e. ∼90 aa) of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (∼90 aa) and a C-terminal ankyrin repeat sequence (∼170 aa). The entire ORF was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl-CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 is palmitoyl-CoA. RT-PCR, Western blotting and immunolabelling analyses clearly showed that the gene is mainly expressed during the intracellular developmental stages and that the level increases during parasite development. Immunofluorescence microscopy showed that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in lipid remodelling in the PVM, or in the transport of fatty acids across the membrane.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.28944-0
2006-08-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/152/8/2355.html?itemId=/content/journal/micro/10.1099/mic.0.28944-0&mimeType=html&fmt=ahah

References

  1. Abrahamsen M. S, Schroeder A. A. 1999; Characterization of intracellular Cryptosporidium parvum gene expression. Mol Biochem Parasitol 104:141–146 [CrossRef]
    [Google Scholar]
  2. Abrahamsen M. S, Templeton T. J, Enomoto S. 17 other authors 2004; Complete genome sequence of the apicomplexan, Cryptosporidium parvum . Science 304:441–445 [CrossRef]
    [Google Scholar]
  3. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254 [CrossRef]
    [Google Scholar]
  4. Burton M, Rose T. M, Faergeman N. J, Knudsen J. 2005; Evolution of the acyl-CoA binding protein (ACBP). Biochem J 392:299–307 [CrossRef]
    [Google Scholar]
  5. Cai X, Herschap D, Zhu G. 2005; Functional characterization of an evolutionarily distinct phosphopantetheinyl transferase in the apicomplexan Cryptosporidium parvum . Eukaryot Cell 4:1211–1220 [CrossRef]
    [Google Scholar]
  6. Chao H, Martin G. G, Russell W. K, Waghela S. D, Russell D. H, Schroeder F, Kier A. B. 2002; Membrane charge and curvature determine interaction with acyl-CoA binding protein (ACBP) and fatty acyl-CoA targeting. Biochemistry 41:10540–10553 [CrossRef]
    [Google Scholar]
  7. Chappell C. L, Okhuysen P. C. 2002; Cryptosporidiosis. Curr Opin Infect Dis 15:523–527 [CrossRef]
    [Google Scholar]
  8. Chen X. M, Keithly J. S, Paya C. V, LaRusso N. F. 2002; Cryptosporidiosis. N Engl J Med 346:1723–1731 [CrossRef]
    [Google Scholar]
  9. Chen X. M, Huang B. Q, Splinter P. L, Cao H, Zhu G, McNiven M. A, LaRusso N. F. 2003; Cryptosporidium parvum invasion of biliary epithelia requires host cell tyrosine phosphorylation of cortactin via c-Src. Gastroenterology 125:216–228 [CrossRef]
    [Google Scholar]
  10. Chye M. L, Huang B. Q, Zee S. Y. 1999; Isolation of a gene encoding Arabidopsis membrane-associated acyl-CoA binding protein and immunolocalization of its gene product. Plant J 18:205–214 [CrossRef]
    [Google Scholar]
  11. Frolov A, Schroeder F. 1998; Acyl coenzyme A binding protein. Conformational sensitivity to long chain fatty acyl-CoA. J Biol Chem 273:11049–11055 [CrossRef]
    [Google Scholar]
  12. Geisbrecht B. V, Zhang D, Schulz H, Gould S. J. 1999; Characterization of PECI, a novel monofunctional Delta(3), Delta(2)-enoyl-CoA isomerase of mammalian peroxisomes. J Biol Chem 274:21797–21803 [CrossRef]
    [Google Scholar]
  13. Gornicki P. 2003; Apicoplast fatty acid biosynthesis as a target for medical intervention in apicomplexan parasites. Int J Parasitol 33:885–896 [CrossRef]
    [Google Scholar]
  14. Gossett R. E, Frolov A. A, Roths J. B, Behnke W. D, Kier A. B, Schroeder F. 1996; Acyl-CoA binding proteins: multiplicity and function. Lipids 31:895–918 [CrossRef]
    [Google Scholar]
  15. Guidotti A, Forchetti C. M, Corda M. G, Konkel D, Bennett C. D, Costa E. 1983; Isolation, characterization, and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors. Proc Natl Acad Sci U S A 80:3531–3535 [CrossRef]
    [Google Scholar]
  16. Knudsen J, Neergaard T. B, Gaigg B, Jensen M. V, Hansen J. K. 2000; Role of acyl-CoA binding protein in acyl-CoA metabolism and acyl-CoA-mediated cell signaling. J Nutr 130:294S–298S
    [Google Scholar]
  17. Kuo M. R, Morbidoni H. R, Alland D. 14 other authors 2003; Targeting tuberculosis and malaria through inhibition of enoyl reductase: compound activity and structural data. J Biol Chem 278:20851–20859 [CrossRef]
    [Google Scholar]
  18. Leung K. C, Li H. Y, Xiao S, Tse M. H, Chye M. L. 2005; Arabidopsis ACBP3 is an extracellularly targeted acyl-CoA-binding protein. Planta1–11
    [Google Scholar]
  19. Li H. Y, Chye M. L. 2003; Membrane localization of Arabidopsis acyl-CoA binding protein ACBP2. Plant Mol Biol 51:483–492 [CrossRef]
    [Google Scholar]
  20. Madern D, Cai X, Abrahamsen M. S, Zhu G. 2004; Evolution of Cryptosporidium parvum lactate dehydrogenase from malate dehydrogenase by a very recent event of gene duplication. Mol Biol Evol 21:489–497
    [Google Scholar]
  21. Millership J. J, Cai X, Zhu G. 2004a; Functional characterization of replication protein A2 (RPA2) from Cryptosporidium parvum . Microbiology 150:1197–1205 [CrossRef]
    [Google Scholar]
  22. Millership J. J, Waghela P, Cai X, Cockerham A, Zhu G. 2004b; Differential expression and interaction of transcription co-activator MBF1 with TATA-binding protein (TBP) in the apicomplexan Cryptosporidium parvum . Microbiology 150:1207–1213 [CrossRef]
    [Google Scholar]
  23. Milne K. G, Ferguson M. A. 2000; Cloning, expression, and characterization of the acyl-CoA-binding protein in African trypanosomes. J Biol Chem 275:12503–12508 [CrossRef]
    [Google Scholar]
  24. Milne K. G, Guther M. L, Ferguson M. A. 2001; Acyl-CoA binding protein is essential in bloodstream form Trypanosoma brucei . Mol Biochem Parasitol 112:301–304 [CrossRef]
    [Google Scholar]
  25. Priest J. W, Mehlert A, Arrowood M. J, Riggs M. W, Ferguson M. A. 2003; Characterization of a low molecular weight glycolipid antigen from Cryptosporidium parvum . J Biol Chem 278:52212–52222 [CrossRef]
    [Google Scholar]
  26. Ralph S. A, D'Ombrain M. C, McFadden G. I. 2001; The apicoplast as an antimalarial drug target. Drug Resist Updat 4:145–151 [CrossRef]
    [Google Scholar]
  27. Rasmussen J. T, Borchers T, Knudsen J. 1990; Comparison of the binding affinities of acyl-CoA-binding protein and fatty-acid-binding protein for long-chain acyl-CoA esters. Biochem J 265:849–855
    [Google Scholar]
  28. Rasmussen J. T, Faergeman N. J, Kristiansen K, Knudsen J. 1994; Acyl-CoA-binding protein (ACBP) can mediate intermembrane acyl-CoA transport and donate acyl-CoA for beta-oxidation and glycerolipid synthesis. Biochem J 299:165–170
    [Google Scholar]
  29. Rider S. D., Jr, Cai X, Smith A. T, Radke J, White M, Zhu G, Sullivan W. J., Jr. 2005; The protozoan parasite Cryptosporidium parvum possesses two functionally and evolutionarily divergent replication protein A large subunits. J Biol Chem 280:31460–31469 [CrossRef]
    [Google Scholar]
  30. Roberts C. W, McLeod R, Rice D. W, Ginger M, Chance M. L, Goad L. J. 2003; Fatty acid and sterol metabolism: potential antimicrobial targets in apicomplexan and trypanosomatid parasitic protozoa. Mol Biochem Parasitol 126:129–142 [CrossRef]
    [Google Scholar]
  31. Roos D. S, Crawford M. J, Donald R. G, Fraunholz M, Harb O. S, He C. Y, Kissinger J. C, Shaw M. K, Striepen B. 2002; Mining the Plasmodium genome database to define organellar function: what does the apicoplast do?. Philos Trans R Soc Lond B Biol Sci 357:35–46 [CrossRef]
    [Google Scholar]
  32. Rosendal J, Ertbjerg P, Knudsen J. 1993; Characterization of ligand binding to acyl-CoA-binding protein. Biochem J 290:321–326
    [Google Scholar]
  33. Schroeder F, Jolly C. A, Cho T. H, Frolov A. 1998; Fatty acid binding protein isoforms: structure and function. Chem Phys Lipids 92:1–25 [CrossRef]
    [Google Scholar]
  34. Thompson R. C, Olson M. E, Zhu G, Enomoto S, Abrahamsen M. S, Hijjawi N. S. 2005; Cryptosporidium and cryptosporidiosis. Adv Parasitol 59:77–158
    [Google Scholar]
  35. Tzipori S, Widmer G. 2000; The biology of Cryptosporidium . Contrib Microbiol 6:1–32
    [Google Scholar]
  36. van Aalten D. M, Milne K. G, Zou J. Y, Kleywegt G. J, Bergfors T, Ferguson M. A, Knudsen J, Jones T. A. 2001; Binding site differences revealed by crystal structures of Plasmodium falciparum and bovine acyl-CoA binding protein. J Mol Biol 309:181–192 [CrossRef]
    [Google Scholar]
  37. Wadum M. C, Villadsen J. K, Feddersen S, Moller R. S, Neergaard T. B, Kragelund B. B, Hojrup P, Faergeman N. J, Knudsen J. 2002; Fluorescently labelled bovine acyl-CoA-binding protein acting as an acyl-CoA sensor: interaction with CoA and acyl-CoA esters and its use in measuring free acyl-CoA esters and non-esterified fatty acids. Biochem J 365:165–172 [CrossRef]
    [Google Scholar]
  38. Waller R. F, Ralph S. A, Reed M. B, Su V, Douglas J. D, Minnikin D. E, Cowman A. F, Besra G. S, McFadden G. I. 2003; A type II pathway for fatty acid biosynthesis presents drug targets in Plasmodium falciparum . Antimicrob Agents Chemother 47:297–301 [CrossRef]
    [Google Scholar]
  39. Zhu G. 2004; Current progress in the fatty acid metabolism in Cryptosporidium parvum . J Eukaryot Microbiol 51:381–388 [CrossRef]
    [Google Scholar]
  40. Zhu G, Keithly J. S, Philippe H. 2000a; What is the phylogenetic position of Cryptosporidium ?. Int J Syst Evol Microbiol 50:1673–1681 [CrossRef]
    [Google Scholar]
  41. Zhu G, Marchewka M. J, Woods K. M, Upton S. J, Keithly J. S. 2000b; Molecular analysis of a Type I fatty acid synthase in Cryptosporidium parvum . Mol Biochem Parasitol 105:253–260 [CrossRef]
    [Google Scholar]
  42. Zhu G, LaGier M. J, Stejskal F, Millership J. J, Cai X, Keithly J. S. 2002; Cryptosporidium parvum : the first protist known to encode a putative polyketide synthase. Gene 298:79–89 [CrossRef]
    [Google Scholar]
  43. Zhu G, Li Y, Cai X, Millership J. J, Marchewka M. J, Keithly J. S. 2004; Expression and functional characterization of a giant Type I fatty acid synthase (CpFAS1) gene from Cryptosporidium parvum . Mol Biochem Parasitol 134:127–135 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.28944-0
Loading
/content/journal/micro/10.1099/mic.0.28944-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error