1887

Abstract

Industrially important extracellular enzymes from filamentous fungi are often -mannosylated. The structure and function of the () gene encoding the protein --mannosyltransferase of were characterized. The disruptant, designated AaPMTA, was constructed by homologous recombination. The strain AaPMTA exhibited fragile cell morphology with respect to hyphal extension, as well as swollen hyphae formation and conidia formation in potato dextrose medium. Moreover, the disruptant showed increased sensitivity to high temperature and Congo red. Thus, the AaPmtA protein is involved in the formation of the normal cell wall. The strain AaPMTA could grow well in liquid synthetic medium and secrete glucoamylase I (GAI-AaPMTA) to a similar extent to the wild-type strain (GAI-WT). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the GAIs revealed that approximately 33 mannose moieties of GAI were absent in strain AaPMTA. This result indicates that the AaPmtA protein is responsible for the transfer of mannose to GAI. Structural analysis of the -linked oligosaccharides of GAI also demonstrated that the disruption resulted in a reduction of the amounts of -linked oligosaccharides, such as -mannose and -1,2-mannotriose, in GAI-AaPMTA. However, the amount of -1,2-mannobiose was comparable between GAI-WT and GAI-AaPMTA. The result suggests the presence of a compensatory mechanism in the synthetic pathway of -mannosylation in .

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.28088-0
2005-11-01
2019-11-22
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/11/3657.html?itemId=/content/journal/micro/10.1099/mic.0.28088-0&mimeType=html&fmt=ahah

References

  1. Barratt, R. W., Johnson, G. B. & Ogata, W. N. ( 1965; ). Wild-type and mutant stocks of Aspergillus nidulans. Genetics 52, 233–246.
    [Google Scholar]
  2. Beltran-Valero de Bernabe, D., Currier, S., Steinbrecher, A. & 14 other authors ( 2002; ). Mutations in the O-mannosyltransferase gene POMT1 give rise to the severe neuronal migration disorder Walker–Warburg syndrome. Am J Hum Genet 71, 1033–1043.[CrossRef]
    [Google Scholar]
  3. Bourdineaud, J. P., van der Vaart, J. M., Donzeau, M., de Sampaio, G., Verrips, C. T. & Lauquin, G. J. ( 1998; ). Pmt1 mannosyl transferase is involved in cell wall incorporation of several proteins in Saccharomyces cerevisiae. Mol Microbiol 27, 85–98.[CrossRef]
    [Google Scholar]
  4. Burda, P. & Aebi, M. ( 1999; ). The dolichol pathway of N-linked glycosylation. Biochim Biophys Acta 1426, 239–257.[CrossRef]
    [Google Scholar]
  5. Chen, H. M., Ford, C. & Reilly, P. J. ( 1994; ). Substitution of asparagine residues in Aspergillus awamori glucoamylase by site-directed mutagenesis to eliminate N-glycosylation and inactivation by deamidation. Biochem J 301, 275–281.
    [Google Scholar]
  6. Fontaine, T., Simenel, C., Dubreucq, G., Adam, O., Delepierre, M., Lemoine, J., Vorgias, C. E., Diaquin, M. & Latge, J. P. ( 2002; ). Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall. J Biol Chem 275, 27594–27607.
    [Google Scholar]
  7. Gemmill, T. R. & Trimble, R. B. ( 1999; ). Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim Biophys Acta 1426, 227–237.[CrossRef]
    [Google Scholar]
  8. Gentzsch, M. & Tanner, W. ( 1996; ). The PMT gene family: protein O-glycosylation in Saccharomyces cerevisiae is vital. EMBO J 15, 5752–5759.
    [Google Scholar]
  9. Gentzsch, M. & Tanner, W. ( 1997; ). Protein-O-glycosylation in yeast: protein-specific mannosyltransferases. Glycobiology 7, 481–486.[CrossRef]
    [Google Scholar]
  10. Girrbach, V. & Strahl, S. ( 2003; ). Members of the evolutionarily conserved PMT family of protein O-mannosyltransferases form distinct protein complexes among themselves. J Biol Chem 278, 12554–12562.[CrossRef]
    [Google Scholar]
  11. Girrbach, V., Zeller, T., Priesmeier, M. & Strahl-Bolsinger, S. ( 2000; ). Structure–function analysis of the dolichyl phosphate-mannose: protein O-mannosyltransferase ScPmt1p. J Biol Chem 25, 19288–19296.
    [Google Scholar]
  12. Goto, M., Ekino, K. & Furukawa, K. ( 1997; ). Expression and functional analysis of a hyperglycosylated glucoamylase in a parental host, Aspergillus awamori var. kawachi. Appl Environ Microbiol 63, 2940–2943.
    [Google Scholar]
  13. Goto, M., Tsukamoto, M., Kwon, I., Ekino, K. & Furukawa, K. ( 1999; ). Functional analysis of O-linked oligosaccharides in threonine/serine-rich region of Aspergillus glucoamylase by expression in mannosyltransferase-disruptants of yeast. Eur J Biochem 260, 596–602.[CrossRef]
    [Google Scholar]
  14. Goto, M., Shinoda, N., Oka, T., Sameshima, Y., Ekino, K. & Furukawa, K. ( 2004; ). Thr/Ser-rich domain of Aspergillus glucoamylase is essential for secretion. Biosci Biotechnol Biochem 68, 961–963.[CrossRef]
    [Google Scholar]
  15. Gunnarsson, A., Svensson, B., Nilsson, B. & Svensson, S. ( 1984; ). Structural studies on the O-glycosidically linked carbohydrate chains of glucoamylase G1 from Aspergillus niger. Eur J Biochem 145, 463–467.[CrossRef]
    [Google Scholar]
  16. Haltiwanger, R. S. & Lowe, J. B. ( 2004; ). Role of glycosylation in development. Annu Rev Biochem 73, 491–537.[CrossRef]
    [Google Scholar]
  17. Harty, C., Strahl, S. & Romisch, K. ( 2001; ). O-Mannosylation protects mutant alpha-factor precursor from endoplasmic reticulum-associated degradation. Mol Biol Cell 12, 1093–1101.[CrossRef]
    [Google Scholar]
  18. Hayashida, S., Kuroda, K., Ohta, K., Kuhara, S., Fukuda, K. & Sakaki, Y. ( 1989; ). Molecular cloning of the glucoamylase I gene of Aspergillus awamori var. kawachi for localization of the raw-starch-affinity site. Agric Biol Chem 53, 923–929.[CrossRef]
    [Google Scholar]
  19. Hui, J. P., White, T. C. & Thibault, P. ( 2002; ). Identification of glycan structure and glycosylation sites in cellobiohydrolase II and endoglucanases I and II from Trichoderma reesei. Glycobiology 12, 837–849.[CrossRef]
    [Google Scholar]
  20. Janik, A., Sosnowska, M., Kruszewska, J., Krotkiewski, H., Lehle, L. & Palamarczyk, G. ( 2003; ). Overexpression of GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae corrects defects in dolichol-linked saccharide formation and protein glycosylation. Biochim Biophys Acta 1621, 22–30.[CrossRef]
    [Google Scholar]
  21. Kobayashi, I., Yasuno, S., Hashimoto, H., Ikura, K., Nakano, H. & Kitahara, S. ( 2002; ). Analysis of positional isomers of α-linked glucooligosaccharides by p-aminobenzoic acid ethyl ester-conversion method. J Appl Glycosci 49, 461–468.[CrossRef]
    [Google Scholar]
  22. Kruszewska, J. S., Butterweck, A. H., Kurzatkowski, W., Migdalski, A., Kubicek, C. P. & Palamarczyk, G. ( 1999; ). Overexpression of the Saccharomyces cerevisiae mannosylphosphodolichol synthase-encoding gene in Trichoderma reesei results in an increased level of protein secretion and abnormal cell ultrastructure. Appl Environ Microbiol 65, 2382–2387.
    [Google Scholar]
  23. Lommel, M., Bagnat, M. & Strahl, S. ( 2004; ). Aberrant processing of the WSC family and Mid2p cell surface sensors results in cell death of Saccharomyces cerevisiae O-mannosylation mutants. Mol Cell Biol 24, 46–57.[CrossRef]
    [Google Scholar]
  24. Lussier, M., Sdicu, A. M., Bussereau, F., Jacquet, M. & Bussey, H. ( 1997; ). The Ktr1p, Ktr3p, and Kre2p/Mnt1p mannosyltransferases participate in the elaboration of yeast O- and N-linked carbohydrate chains. J Biol Chem 13, 15527–15531.
    [Google Scholar]
  25. Lussier, M., Sdicu, A. M. & Bussey, H. ( 1999; ). The KTR and MNN1 mannosyltransferase families of Saccharomyces cerevisiae. Biochim Biophys Acta 1426, 323–334.[CrossRef]
    [Google Scholar]
  26. Manya, H., Chiba, A., Yoshida, A., Wang, X., Chiba, Y., Jigami, Y., Margolis, R. U. & Endo, T. ( 2004; ). Demonstration of mammalian protein O-mannosyltransferase activity: coexpression of POMT1 and POMT2 required for enzymatic activity. Proc Natl Acad Sci U S A 101, 500–505.[CrossRef]
    [Google Scholar]
  27. Mochizuki, S., Hamato, N., Hirose, M., Miyano, K., Ohtani, W., Kameyama, S., Kuwae, S., Tokuyama, T. & Ohi, H. ( 2001; ). Expression and characterization of recombinant human antithrombin III in Pichia pastoris. Protein Expr Purif 23, 55–65.[CrossRef]
    [Google Scholar]
  28. Momany, M., Westfall, P. J. & Abramowsky, G. ( 1999; ). Aspergillus nidulans swo mutants show defects in polarity establishment, polarity maintenance and hyphal morphogenesis. Genetics 151, 557–567.
    [Google Scholar]
  29. Mrsa, V. & Tanner, W. ( 1999; ). Role of NaOH-extractable cell wall proteins Ccw5p, Ccw6p, Ccw7p and Ccw8p (members of the Pir protein family) in stability of the Saccharomyces cerevisiae cell wall. Yeast 15, 813–820.[CrossRef]
    [Google Scholar]
  30. Nakatsukasa, K., Okada, S., Umebayashi, K., Fukuda, R., Nishikawa, S. & Endo, T. ( 2004; ). Roles of O-mannosylation of aberrant proteins in reduction of the load for endoplasmic reticulum chaperones in yeast. J Biol Chem 279, 49762–49772.[CrossRef]
    [Google Scholar]
  31. Oka, T., Hamaguchi, T., Sameshima, Y., Goto, M. & Furukawa, K. ( 2004; ). Molecular characterization of protein O-mannosyltransferase and its involvement in cell-wall synthesis in Aspergillus nidulans. Microbiology 150, 1973–1982.[CrossRef]
    [Google Scholar]
  32. Pazur, J. H., Tominaga, Y., Forsberg, L. S. & Simpson, D. L. ( 1980; ). Glycoenzymes: an unusual type of glycoprotein structure for a glucoamylase. Carbohydr Res 84, 103–114.[CrossRef]
    [Google Scholar]
  33. Proszynski, T. J., Simons, K. & Bagnat, M. ( 2004; ). O-Glycosylation as a sorting determinant for cell surface delivery in yeast. Mol Biol Cell 15, 1533–1543.[CrossRef]
    [Google Scholar]
  34. Romero, P. A., Lussier, M., Veronneau, S., Sdicu, A. M., Herscovics, A. & Bussey, H. ( 1999; ). Mnt2p and Mnt3p of Saccharomyces cerevisiae are members of the Mnn1p family of α-1,3-mannosyltransferases responsible for adding the terminal mannose residues of O-linked oligosaccharides. Glycobiology 9, 1045–1051.[CrossRef]
    [Google Scholar]
  35. Sauer, J., Sigurskjold, B. W., Christensen, U., Frandsen, T. P., Mirgorodskaya, E., Harrison, M., Roepstorff, P. & Svensson, B. ( 2000; ). Glucoamylase: structure/function relationships, and protein engineering. Biochim Biophys Acta 1543, 275–293.[CrossRef]
    [Google Scholar]
  36. Semimaru, T., Goto, M., Furukawa, K. & Hayashida, S. ( 1995; ). Functional analysis of the threonine- and serine-rich Gp-I domain of glucoamylase I from Aspergillus awamori var. kawachi. Appl Environ Microbiol 61, 2885–2890.
    [Google Scholar]
  37. Shaw, B. D. & Momany, M. ( 2002; ). Aspergillus nidulans polarity mutant swoA is complemented by protein O-mannosyltransferase pmtA. Fungal Genet Biol 37, 263–270.[CrossRef]
    [Google Scholar]
  38. Strahl-Bolsinger, S. & Scheinost, A. ( 1999; ). Transmembrane topology of Pmt1p, a member of an evolutionarily conserved family of protein O-mannosyltransferases. J Biol Chem 274, 9068–9075.[CrossRef]
    [Google Scholar]
  39. Strahl-Bolsinger, S., Gentzsch, M. & Tanner, W. ( 1999; ). Protein O-mannosylation. Biochim Biophys Acta 1426, 297–307.[CrossRef]
    [Google Scholar]
  40. Wallis, G. L., Swift, R. J., Hemming, F. W., Trinci, A. P. & Peberdy, J. F. ( 1999; ). Glucoamylase overexpression and secretion in Aspergillus niger: analysis of glycosylation. Biochim Biophys Acta 1472, 576–586.[CrossRef]
    [Google Scholar]
  41. Willer, T., Valero, M. C., Tanner, W., Cruces, J. & Strahl, S. ( 2003; ). O-Mannosyl glycans: from yeast to novel associations with human disease. Curr Opin Struct Biol 13, 621–630.[CrossRef]
    [Google Scholar]
  42. Willer, T., Prados, B., Falcon-Perez, J. M. & 10 other authors ( 2004; ). Targeted disruption of the Walker–Warburg syndrome gene Pomt1 in mouse results in embryonic lethality. Proc Natl Acad Sci U S A 101, 14126–14131.[CrossRef]
    [Google Scholar]
  43. Woo, P. C., Chong, K. T., Leung, A. S., Wong, S. S., Lau, S. K. & Yuen, K. Y. ( 2003; ). AFLMP1 encodes an antigenic cell wall protein in Aspergillus flavus. J Clin Microbiol 41, 845–850.[CrossRef]
    [Google Scholar]
  44. Yelton, M. M., Hamer, J. E. & Timberlake, W. E. ( 1984; ). Transformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci U S A 81, 1470–1474.[CrossRef]
    [Google Scholar]
  45. Yip, C. L., Welch, S. K., Klebl, F., Gilbert, T., Seidel, P., Grant, F. J., O'Hara, P. J. & MacKay, V. L. ( 1994; ). Cloning and analysis of the Saccharomyces cerevisiae MNN9 and MNN1 genes required for complex glycosylation of secreted proteins. Proc Natl Acad Sci U S A 91, 2723–2727.[CrossRef]
    [Google Scholar]
  46. Yoko-o, T., Roy, S. K. & Jigami, Y. ( 1998; ). Differences in in vivo acceptor specificity of two galactosyltransferases, the gmh3+ and gma12+ gene products from Schizosaccharomyces pombe. Eur J Biochem 257, 630–637.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.28088-0
Loading
/content/journal/micro/10.1099/mic.0.28088-0
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