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Proteome Analysis of Aspergillus fumigatus Total Membrane Proteins Identifies Proteins Associated with the Glycoconjugates and Cell Wall Biosynthesis Using 2D LC-MS/MS

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An Erratum to this article was published on 12 February 2010

Abstract

We attempted to identify membrane proteins associated with the glycoconjugates and cell wall biosynthesis in the total membrane preparations of Aspergillus fumigatus. The total membrane preparations were first run on 1D gels, and then the stained gels were cut and submitted to in-gel digestion followed by 2D LC-MS/MS and database search. A total of 530 proteins were identified with at least two peptides detected with MS/MS spectra. Seventeen integral membrane proteins were involved in N-, O-glycosylation or GPI anchor biosynthesis. Nine membrane proteins were involved in cell wall biosynthesis. Eight proteins were identified as enzymes involved in sphingolipid synthesis. In addition, the proteins involved in cell wall and ergosterol biosynthesis can potentially be used as antifungal drug targets. Our method, for the first time, clearly provided a global view of the membrane proteins associated with glycoconjugates and cell wall biosynthesis in the total membrane proteome of A. fumigatus.

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References

  1. Denning, D. W., Anderson, M. J., Turner, G., Latge, J. P., & Bennett, J. W. (2002). Sequencing the Aspergillus fumigatus genome. The Lancet Infectious Diseases, 2(4), 251–253.

    Article  CAS  Google Scholar 

  2. Casadevall, A., & Pirofski, L. A. (1999). Host-pathogen interactions: Redefining the basic concepts of virulence and pathogenicity. Infection and Immunity, 67(8), 3703–3713.

    CAS  Google Scholar 

  3. Denning, D. W. (1998). Invasive aspergillosis. Clinical Infectious Diseases, 26(4), 781–803.

    Article  CAS  Google Scholar 

  4. Greenberger, P. A. (2002). Allergic bronchopulmonary aspergillosis. Journal of Allergy and Clinical Immunology, 110(5), 685–692.

    Article  Google Scholar 

  5. Kukuruzinska, M. A., Bergh, M. L., & Jackson, B. J. (1987). Protein glycosylation in yeast. Annual Review of Biochemistry, 56, 915–944.

    Article  CAS  Google Scholar 

  6. Bernard, M., & Latge, J. P. (2001). Aspergillus fumigatus cell wall: Composition and biosynthesis. Medical Mycology, 39(Suppl 1), 9–17.

    CAS  Google Scholar 

  7. Bruneau, J. M., Magnin, T., Tagat, E., Legrand, R., Bernard, M., Diaquin, M., et al. (2001). Proteome analysis of Aspergillus fumigatus identifies glycosylphosphatidylinositol-anchored proteins associated to the cell wall biosynthesis. Electrophoresis, 22(13), 2812–2823.

    Article  CAS  Google Scholar 

  8. Le, S. H., Galve-Roperh, I., Peterson, C., Milstien, S., & Spiegel, S. (2002). Sphingosine-1-phosphate phosphohydrolase in regulation of sphingolipid metabolism and apoptosis. Journal of Cell Biology, 158(6), 1039–1049.

    Article  CAS  Google Scholar 

  9. Nierman, W. C., Pain, A., Anderson, M. J., Wortman, J. R., & Kim, H. S. (2005). Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature, 438(7071), 1151–1156.

    Article  CAS  Google Scholar 

  10. Kniemeyer, O., Lessing, F., Scheibner, O., Hertweck, C., & Brakhage, A. A. (2006). Optimisation of a 2-D gel electrophoresis protocol for the human-pathogenic fungus Aspergillus fumigatus. Current Genetics, 49(3), 178–189.

    Article  CAS  Google Scholar 

  11. Link, A. J., Eng, J., Schieltz, D. M., Carmack, E., Mize, G. J., Morris, D. R., et al. (1999). Direct analysis of protein complexes using mass spectrometry. Nature Biotechnology, 17(7), 676–682.

    Article  CAS  Google Scholar 

  12. Washburn, M. P., Wolters, D., & Yates, R., Jr. (2001). Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nature Biotechnology, 19(3), 242–247.

    Article  CAS  Google Scholar 

  13. Abruzzo, G. K., Gill, C. J., Flattery, A. M., Kong, L., Leighton, C., Smith, J. G., et al. (2000). Efficacy of the echinocandin caspofungin against disseminated aspergillosis and candidiasis in cyclophosphamide-induced immunosuppressed mice. Antimicrobial Agents and Chemotherapy, 44(9), 2310–2318.

    Article  CAS  Google Scholar 

  14. Fontaine, T., Magnin, T., Melhert, A., Lamont, D., Latge, J. P., & Ferguson, M. A. (2003). Structures of the glycosylphosphatidylinositol membrane anchors from Aspergillus fumigatus membrane proteins. Glycobiology, 13(3), 169–177.

    Article  CAS  Google Scholar 

  15. Gharahdaghi, F., Weinberg, C. R., Meagher, D. A., Imai, B. S., & Mische, S. M. (1999). Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity. Electrophoresis, 20(3), 601–605.

    Article  CAS  Google Scholar 

  16. Sun, W., Li, F., Wang, J., Zheng, D., & Gao, Y. (2004). AMASS: Software for automatically validating the quality of MS/MS spectrum from SEQUEST results. Molecular & Cellular Proteomics, 3(12), 1194–1199.

    Article  CAS  Google Scholar 

  17. Li, F., Sun, W., Gao, Y., & Wang, J. (2004). RScore: A peptide randomicity score for evaluating tandem mass spectra. Rapid Communications in Mass Spectrometry, 18(14), 1655–1659.

    Article  CAS  Google Scholar 

  18. Sirover, M. A. (1999). New insights into an old protein: The functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase. Biochimica et Biophysica Acta, 1432(2), 159–184.

    CAS  Google Scholar 

  19. Sulli, C., Fang, Z., Muchhal, U., & Schwartzbach, S. D. (1999). Topology of Euglena chloroplast protein precursors within endoplasmic reticulum to Golgi to chloroplast transport vesicles. Journal of Biological Chemistry, 274(1), 457–463.

    Article  CAS  Google Scholar 

  20. Seeger, M., & Payne, G. S. (1992). A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast. EMBO Journal, 11(8), 2811–2818.

    CAS  Google Scholar 

  21. Egner, R., Thumm, M., Straub, M., Simeon, A., Schuller, H. J., & Wolf, D. H. (1993). Tracing intracellular proteolytic pathways. Proteolysis of fatty acid synthase and other cytoplasmic proteins in the yeast Saccharomyces cerevisiae. Journal of Biological Chemistry, 268(36), 27269–27276.

    CAS  Google Scholar 

  22. Burre, J., & Volknandt, W. (2007). The synaptic vesicle proteome. Journal of Neurochemistry, 101(6), 1448–1462.

    Article  CAS  Google Scholar 

  23. Walton, P. A., Wendland, M., Subramani, S., Rachubinski, R. A., & Welch, W. J. (1994). Involvement of 70-kD heat-shock proteins in peroxisomal import. Journal of Cell Biology, 125(5), 1037–1046.

    Article  CAS  Google Scholar 

  24. Tisdale, E. J., Kelly, C., & Artalejo, C. R. (2004). Glyceraldehyde-3-phosphate dehydrogenase interacts with Rab2 and plays an essential role in endoplasmic reticulum to Golgi transport exclusive of its glycolytic activity. Journal of Biological Chemistry, 279(52), 54046–54052.

    Article  CAS  Google Scholar 

  25. Yan, A., & Lennarz, W. J. (2005). Unraveling the mechanism of protein N-glycosylation. Journal of Biological Chemistry, 280(5), 3121–3124.

    Article  CAS  Google Scholar 

  26. Lehle, L., Strahl, S., & Tanner, W. (2006). Protein glycosylation, conserved from yeast to man: A model organism helps elucidate congenital human diseases. Angewandte Chemie, 45(41), 6802–6818. (International ed. in English).

    Article  CAS  Google Scholar 

  27. Knauer, R., & Lehle, L. (1999). The oligosaccharyltransferase complex from yeast. Biochimica et Biophysica Acta, 1426(2), 259–273.

    CAS  Google Scholar 

  28. Kelleher, D. J., & Gilmore, R. (2006). An evolving view of the eukaryotic oligosaccharyltransferase. Glycobiology, 16(4), 47R–62R.

    Article  CAS  Google Scholar 

  29. Munro, S. (2001). What can yeast tell us about N-linked glycosylation in the Golgi apparatus? FEBS Letters, 498(2–3), 223–227.

    Article  CAS  Google Scholar 

  30. Leitao, E. A., Bittencourt, V. C., Haido, R. M., Valente, A. P., Peter-Katalinic, J., Letzel, M., et al. (2003). Beta-galactofuranose-containing O-linked oligosaccharides present in the cell wall peptidogalactomannan of Aspergillus fumigatus contain immunodominant epitopes. Glycobiol, 13(10), 681–692.

    Article  CAS  Google Scholar 

  31. Goto, M. (2007). Protein O-glycosylation in fungi: Diverse structures and multiple functions. Bioscience, Biotechnology, and Biochemistry, 71(6), 1415–1427.

    Article  CAS  Google Scholar 

  32. Lussier, M., Sdicu, A. M., & Bussey, H. (1999). The KTR and MNN1 mannosyltransferase families of Saccharomyces cerevisiae. Biochimica et Biophysica Acta, 1426(2), 323–334.

    CAS  Google Scholar 

  33. Li, H., Zhou, H., Luo, Y., Ouyang, H., Hu, H., & Jin, C. (2007). Glycosylphosphatidylinositol (GPI) anchor is required in Aspergillus fumigatus for morphogenesis and virulence. Molecular Microbiology, 64(4), 1014–1027.

    Article  CAS  Google Scholar 

  34. Pittet, M., & Conzelmann, A. (2007). Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae. Biochimica et Biophysica Acta, 1771(3), 405–420.

    CAS  Google Scholar 

  35. Maxwell, S. E., Ramalingam, S., Gerber, L. D., Brink, L., & Udenfriend, S. (1995). An active carbonyl formed during glycosylphosphatidylinositol addition to a protein is evidence of catalysis by a transamidase. Journal of Biological Chemistry, 270(33), 19576–19582.

    Article  CAS  Google Scholar 

  36. Levery, S. B., Momany, M., Lindsey, R., Toledo, M. S., Shayman, J. A., Fuller, M., et al. (2002). Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc:ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth. FEBS Letters, 525(1–3), 59–64.

    Article  CAS  Google Scholar 

  37. Do, J. H., Park, T. K., & Choi, D. K. (2005). A computational approach to the inference of sphingolipid pathways from the genome of Aspergillus fumigatus. Current Genetics, 48(2), 134–141.

    Article  CAS  Google Scholar 

  38. Mouyna, I., Fontaine, T., Vai, M., Monod, M., Fonzi, W. A., Diaquin, M., et al. (2000). Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. Journal of Biological Chemistry, 275(20), 14882–14889.

    Article  CAS  Google Scholar 

  39. Mouyna, I., Morelle, W., Vai, M., Monod, M., Lechenne, B., Fontaine, T., et al. (2005). Deletion of GEL2 encoding for a beta(1–3)glucanosyltransferase affects morphogenesis and virulence in Aspergillus fumigatus. Molecular Microbiology, 56(6), 1675–1688.

    Article  CAS  Google Scholar 

  40. Schmidt, M., Strenk, M. E., Boyer, M. P., & Fritsch, B. J. (2005). Importance of cell wall mannoproteins for septum formation in Saccharomyces cerevisiae. Yeast, 22(9), 715–723.

    Article  CAS  Google Scholar 

  41. Veen, M., & Lang, C. (2005). Interactions of the ergosterol biosynthetic pathway with other lipid pathways. Biochemical Society Transactions, 33(Pt 5), 1178–1181.

    CAS  Google Scholar 

  42. Lupetti, A., Danesi, R., Campa, M., Del, T. M., & Kelly, S. (2002). Molecular basis of resistance to azole antifungals. Trends in Molecular Medicine, 8(2), 76–81.

    Article  CAS  Google Scholar 

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Acknowledgments

This project was supported by the State “863” High-tech Project (2007AA02Z164), the National Basic Research Program of China (2006CB504400), and the Youth Fund from Institute of Microbiology, Chinese Academy of Sciences (0654041005).

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Correspondence to Yuanming Luo or Cheng Jin.

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An erratum to this article can be found at http://dx.doi.org/10.1007/s12033-010-9253-x

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Ouyang, H., Luo, Y., Zhang, L. et al. Proteome Analysis of Aspergillus fumigatus Total Membrane Proteins Identifies Proteins Associated with the Glycoconjugates and Cell Wall Biosynthesis Using 2D LC-MS/MS. Mol Biotechnol 44, 177–189 (2010). https://doi.org/10.1007/s12033-009-9224-2

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