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Volume 146, Issue 1

Monitoring the kinetics of glycoprotein synthesis and secretion in the filamentous fungus : cellobiohydrolase I (CBHI) as a model protein Free

Abstract

The authors have developed methodology to study the kinetics of protein synthesis and secretion in filamentous fungi. Production of cellobiohydrolase I (CBHI) by was studied by metabolic labelling of the proteins with [S]methionine or [C]mannose, and subsequent analysis of the labelled proteins using two-dimensional gel electrophoresis. Analysis of the different pI forms of the nascent proteins allowed monitoring of the maturation of CBHI during the transport along the biosynthetic pathway. The maturation of the pI pattern of CBHI as well as secretion into culture medium was prevented by treatment with the reducing agent DTT. The pI forms of CBHI detectable in the presence of DTT corresponded to the early endoplasmic reticulum forms of the protein. Removal of -glycans by enzymic treatment (endoglycosidase H or peptide--glycosidase F), or chemical removal of both - and -glycans, changed the pI pattern of CBHI, showing that glycan structures are involved in formation of the different pI forms of the protein. By quantifying the labelled proteins during a time course, parameters describing protein synthesis and secretion were deduced. The mean synthesis time for CBHI under the conditions used was 4 min and the minimum secretion time was 11 min. The methodology developed in this study provides tools to reveal the rate-limiting factors in protein production and to obtain information on the intracellular events involved in the secretion process.

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Keyword(s): 2D, two-dimensional , CBHI, cellobiohydrolase I , Endo H, endoglycosidase H , ER, endoplasmic reticulum , glycosylation , metabolic labelling , PNGase F, peptide-N-glycosidase F , protein transport and two-dimensional gel electrophoresis
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2000-01-01
2024-04-23
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References

  1. Aho S., Olkkonen V., Jalava T., Paloheimo M., Bühler R., Niku-Paavola M.-L., Bamford D. H., Korhola M. 1991; Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I. Eur J Biochem 200:643–649 [CrossRef]
     [Google Scholar]
  2. Alberini C. M., Bet P., Milstein C., Sitia R. 1990; Secretion of immunoglobulin M assembly intermediates in the presence of reducing agents. Nature 347:485–487 [CrossRef]
     [Google Scholar]
  3. Archer D. B., Peberdy J. F. 1997; The molecular biology of secreted enzyme production by fungi. Crit Rev Biotechnol 17:273–306 [CrossRef]
     [Google Scholar]
  4. Basset J., Deney R. C., Jeffery G. F., Mendham J. 1987 Vogel’s Textbook of Quantitative Inorganic Analysis, 4th edn. New York: Wiley;
     [Google Scholar]
  5. Boström K., Wettesten M., Borén J., Bondjers G., Wiklund O., Olofsson S.-O. 1986; Pulse-chase studies of the synthesis and intracellular transport of apolipoprotein B-100 in Hep G2 cells. J Biol Chem 261:13800–13806
     [Google Scholar]
  6. Braakman I., HooverLitty H., Wagner K. R., Helenius A. 1991; Folding of influenza hemagglutinin in the endoplasmic reticulum. J Cell Biol 114:401–411 [CrossRef]
     [Google Scholar]
  7. Braakman I., Helenius J., Helenius A. 1992; Manipulating disulphide bond formation and protein folding in the endoplasmic reticulum. EMBO J 11:1717–1722
     [Google Scholar]
  8. Dunphy W. G., Brands R., Rothman J. E. 1985; Attachment of terminal N-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the Golgi stack. Cell 40:463–472 [CrossRef]
     [Google Scholar]
  9. Durand H., Clanet M., Tiraby G. 1988; Genetic improvement of Trichoderma reesei for large scale cellulase production. Enzyme Microb Technol 10:341–345 [CrossRef]
     [Google Scholar]
  10. Fägerstam L. G., Pettersson L. G., Engström J. Å. 1984; The primary structure of a 1,4-β-glucan cellobiohydrolase from the fungus Trichoderma reesei QM 9414. FEBS Lett 167:309–315 [CrossRef]
     [Google Scholar]
  11. Fries E., Gustafsson L., Peterson P. A. 1984; Four secretory proteins synthesized by hepatocytes are transported from endoplasmic reticulum to Golgi complex at different rates. EMBO J 3:147–152
     [Google Scholar]
  12. van Gemeren I. A., Punt P. J., Drint-Kuyvenhoven A., Broekhuijsen M. P., van’t Hoog A., Beijersbergen A., Verrips C. T., van den Hondel C. A. M. J. J. 1997; The ER chaperone encoding bipA gene of black Aspergilli is induced by heat shock and unfolded proteins. Gene 198:43–52 [CrossRef]
     [Google Scholar]
  13. Goochee C. F., Gramer M. J., Andersen D. C., Bahr J. B., Rasmussen J. R. 1991; The oligosaccharides of glycoproteins: bioprocess factors affecting oligosaccharide structure and their effect on glycoprotein properties. Bio/Technology 9:1347–1355 [CrossRef]
     [Google Scholar]
  14. Gouka R. J., Punt P. J., van den Hondel C. A. M. J. J. 1997; Efficient production of secreted proteins by Aspergillus: progress, limitations and prospects. Appl Microbiol Biotechnol 47:1–11 [CrossRef]
     [Google Scholar]
  15. Harrison M. J., Nouwens A. S., Jardine D. R., Zachara N. E., Gooley A. A., Nevalainen H., Packer N. H. 1998; Modified glycosylation of cellobiohydrolase I from a high cellulase-producing mutant strain of Trichoderma reesei. Eur J Biochem 256:119–127 [CrossRef]
     [Google Scholar]
  16. Herscovics A., Orlean P. 1993; Glycoprotein synthesis in yeast. FASEB J 7:540–550
     [Google Scholar]
  17. Hijarrubia M. J., Casqueiro J., Gutiérrez S., Fernandez F. J., Martin J. F. 1997; Characterization of the bip gene of Aspergillus awamori with an HDEL retention signal homologous to the mammalian BiP involved in polypeptide secretion. Curr Genet 32:139–146 [CrossRef]
     [Google Scholar]
  18. Hjort C. M. 1995; A fungal protein disulfide isomerase. Patent WO 95/00636
  19. Horwitz M. S., Scharff M. D., Maizel J. V. J. 1969; Synthesis and assembly of adenovirus 2. I. Polypeptide synthesis, assembly of capsomeres, and morphogenesis of the virion. Virology 39:682–694 [CrossRef]
     [Google Scholar]
  20. Ilmén M. 1997 Molecular mechanisms of glucose repression in the filamentous fungus Trichoderma reesei PhD thesis Technical Research Center of Finland;VTT Publication no. 315
     [Google Scholar]
  21. Ilmén M., Onnela M.-L., Klemsdal S., Keränen S., Penttilä M. 1996; Functional analysis of the cellobiohydrolase I promoter of the filamentous fungus Trichoderma reesei. Mol Gen Genet 253:303–314
     [Google Scholar]
  22. Ilmén M., Saloheimo A., Onnela M.-L., Penttilä M. E. 1997; Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei. Appl Environ Microbiol 63:1298–1306
     [Google Scholar]
  23. Jämsä E., Simonen M., Makarow M. 1994; Selective retention of secretory proteins in the yeast endoplasmic reticulum by treatment of cells with a reducing agent. Yeast 10:355–370 [CrossRef]
     [Google Scholar]
  24. Kajino T., Sarai K., Imaeda T., Idekoba C., Asami O., Yamada Y., Hirai M., Udaka S. 1994; Molecular cloning of a fungal cDNA encoding protein disulfide isomerase. Biosci Biotechnol Biochem 58:1424–1429 [CrossRef]
     [Google Scholar]
  25. Keizer-Gunnink I., Punt P. J., Veenhuis M., van den Hondel C. A. M. J. J. 1996; Ultrastructural studies on the localization of the secretory pathway of glucoamylase in Aspergillus niger. In Proceedings of the 3rd European Conference on Fungal Genetics p. 61 Münster; Germany:
     [Google Scholar]
  26. Keränen S., Penttilä M. 1995; Production of recombinant proteins in the filamentous fungus Trichoderma reesei. Curr Opin Biotechnol 6:534–537 [CrossRef]
     [Google Scholar]
  27. Klarskov K., Piens K., Ståhlberg J., Høj P. B., Van Beeumen J., Claeyssens M. 1997; Cellobiohydrolase I from Trichoderma reesei: identification of an active site nucleophile and additional information on sequence including the glycosylation pattern of the core protein. Carbohydr Res 304:143–154 [CrossRef]
     [Google Scholar]
  28. Lee B. R., Yamada O., Kitamoto K., Takahashi K. 1996; Cloning, characterization and overexpression of a gene (pdiA) encoding protein disulfide isomerase of Aspergillus oryzae. J Ferment Bioeng 82:538–543 [CrossRef]
     [Google Scholar]
  29. Lodish H. F., Kong N., Snider M., Strous G. J. A. M. 1983; Hepatoma secretory proteins migrate from rough endoplasmic reticulum to Golgi at characteristic rates. Nature 304:80–83 [CrossRef]
     [Google Scholar]
  30. Loftfield R. B., Eigner E. A. 1958; The time required for the synthesis of a ferritin molecule in rat liver. J Biol Chem 231:925–943
     [Google Scholar]
  31. Malpricht S., Thamm A., Khanh N. Q. 1996; Cloning of cDNA for the protein disulfide isomerase from Aspergillus niger strain NNRL3 using PCR. Biotechnol Lett 18:445–450 [CrossRef]
     [Google Scholar]
  32. Maras M., De Bruyn A., Schraml J., Herdewijn P., Claeyssens M., Fiers W., Contreras R. 1997a; Structural characterization of N-linked oligosaccharides from cellobiohydrolase I secreted by the filamentous fungus Trichoderma reesei RUTC 30. Eur J Biochem 245:617–625 [CrossRef]
     [Google Scholar]
  33. Maras M., Saelens X., Laroy W., Piens K., Claeyssens M., Fiers W., Contreras R. 1997b; In vitro conversion of the carbohydrate moiety of fungal glycoproteins to mammalian-type oligosaccharides. Evidence for N-acetylglucosaminyltransferase-I-accepting glycans from Trichoderma reesei. Eur J Biochem 249:701–707 [CrossRef]
     [Google Scholar]
  34. Matlin K. S., Simons K. 1983; Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation. Cell 34:233–243 [CrossRef]
     [Google Scholar]
  35. Montenecourt B. S., Eveleigh D. E. 1979; Selective screening methods for the isolation of high yielding cellulase mutants of Trichoderma reesei. Adv Chem Ser 181:289–301
     [Google Scholar]
  36. Nevalainen H., Penttilä M. 1995; Molecular biology of cellulolytic fungi. In The Mycota II: Genetics and Biotechnology pp. 303–319Edited by Kück U. Berlin & Heidelberg: Springer;
     [Google Scholar]
  37. Ngiam C., Jeenes D. J., Archer D. B. 1997; Isolation and characterization of a gene encoding protein disulfide isomerase, pdiA, from Aspergillus niger. Curr Genet 31:133–138 [CrossRef]
     [Google Scholar]
  38. Novick P., Ferro S., Schekman R. 1981; Order of events in the yeast secretory pathway. Cell 25:461–469 [CrossRef]
     [Google Scholar]
  39. Nykänen M., Saarelainen R., Raudaskoski M., Nevalainen K. M. H., Mikkonen A. 1997; Expression and secretion of barley cysteine endopeptidase B and cellobiohydrolase I in Trichoderma reesei. Appl Environ Microbiol 63:4929–4937
     [Google Scholar]
  40. Nyyssönen E., Keränen S. 1995; Multiple roles of the cellulase CBHI in enhancing production of fusion antibodies by the filamentous fungus Trichoderma reesei. Curr Genet 28:71–79 [CrossRef]
     [Google Scholar]
  41. Penttilä M. 1998; Heterologous protein production in Trichoderma. In Trichoderma & Gliocladium pp. 365–382Edited by Harman G. E., Kubicek C. P. London: Taylor & Francis;
     [Google Scholar]
  42. Penttilä M., Teeri T. T., Nevalainen H., Knowles J. K. C. 1991; The molecular biology of Trichoderma reesei and its application to biotechnology. In Applied Molecular Genetics of Fungi pp. 85–102Edited by Peberdy J. F., Caten C. E., Ogden J. E., Bennett J. W. Cambridge: Cambridge University Press;
     [Google Scholar]
  43. St Leger R. J., Staples R. C., Roberts D. W. 1991; Changes in translatable mRNA species associated with nutrient deprivation and protease synthesis in Metarhizium anisopliae. J Gen Microbiol 137:807–815 [CrossRef]
     [Google Scholar]
  44. Saloheimo M., Lund M., Penttilä M. 1999; The protein disulphide isomerase gene of the fungus Trichoderma reesei is induced by endoplasmic reticulum stress and regulated by the carbon source. Mol Gen Genet 262:35–45 [CrossRef]
     [Google Scholar]
  45. Salovuori I., Makarow M., Rauvala H., Knowles J., Kääriäinen L. 1987; Low molecular weight high-mannose type glycans in a secreted protein of the filamentous fungus Trichoderma reesei. Bio/Technology 5:152–156 [CrossRef]
     [Google Scholar]
  46. Teeri T. T., Penttilä M., Keränen S., Nevalainen H., Knowles J. K. C. 1992; Structure, function, and genetics of cellulases. In Biotechnology of Filamentous Fungi. Technology and Products pp. 417–445Edited by Finkelstein D. B., Ball C. Boston: Butterworth-Heinemann;
     [Google Scholar]
  47. Thompson S. A., Golightly E. J., Yaver D. S. 1995; Nucleotide sequence of the Aspergillus niger srpA gene. Gene 167:337–338 [CrossRef]
     [Google Scholar]
  48. Veldhuisen G., Saloheimo M., Fiers M. A., Punt P. J., Contreras R., Penttilä M., van den Hondel C. A. M. J. J. 1997; Isolation and analysis of functional homologues of the secretion-related SAR1 gene of Saccharomyces cerevisiae from Aspergillus niger and Trichoderma. Mol Gen Genet 256:446–455
     [Google Scholar]
  49. Wösten H. A., Moukha S. M., Sietsma J. H., Wessels J. G. 1991; Localization of growth and secretion of proteins in Aspergillus niger. J Gen Microbiol 137:2017–2023 [CrossRef]
     [Google Scholar]
  50. Yeo K.-T., Parent J. B., Yeo T.-K., Olden K. 1985; Variability in transport rates of secretory glycoproteins through the endoplasmic reticulum and Golgi in human hepatoma cells. J Biol Chem 260:7896–7902
     [Google Scholar]
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Monitoring the kinetics of glycoprotein synthesis and secretion in the filamentous fungus Trichoderma reesei: cellobiohydrolase I (CBHI) as a model protein
Microbiology 146, 223 (2000); https://doi.org/10.1099/00221287-146-1-223
/content/journal/micro/10.1099/00221287-146-1-223
/content/journal/micro/10.1099/00221287-146-1-223
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