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Glycobiology Advance Access originally published online on April 7, 2004
Glycobiology 2004 14(8):713-724; doi:10.1093/glycob/cwh080
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Glycobiology vol. 14 no. 8 © Oxford University Press 2004; all rights reserved.

Factors influencing glycosylation of Trichoderma reesei cellulases. I: Postsecretorial changes of the O- and N-glycosylation pattern of Cel7A

Ingeborg Stals2, Koen Sandra2, Steven Geysens3, Roland Contreras3, Jozef Van Beeumen2 and Marc Claeyssens1,2

2 Department of Biochemistry, Physiology and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium; and 3 Department of Molecular Biomedical Research, Ghent University, Flanders Interuniversity Institute for Biotechnology (VIB), Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium

Received on November 25, 2003; revised on March 15, 2004; accepted on March 15, 2004

The glycosylation of Cel7A (CBH I) from Trichoderma reesei varies considerably when the fungus is grown under different conditions. As shown by ESI-MS and PAG-IEF analyses of both intact protein and the isolated catalytic core module, the microheterogeneity originates mainly from the variable ratio of single N-acetylglucosamine over high-mannose structures on the three N-glycosylation sites and from the presence or absence of phosphate residues. Fully N- and O-glycosylated Cel7A can only be isolated from minimal medium and probably reflects the initial complexity of the protein on leaving the glycosynthetic pathway. Extracellular activities are responsible for postsecretorial modifications in other cultivation conditions: {alpha}-(1->2)-mannosidase, {alpha}-(1->3)-glucosidase and an Endo H type activity participate in N-deglycosylation (core), whereas a phosphatase and a mannosidase are probably responsible for hydrolysis of O-glycans (linker). The effects are most prominent in corn steep liquor–enriched media, where the pH is closer to the pH optimum (5–6) of these extracellular hydrolases. In minimal medium, the low pH and the presence of proteases could explain for the absence of such activities. On the other hand, phosphodiester linkages in the catalytic module are only observed under specific conditions. The extracellular trigger is still unknown, but mannophosphorylation may be regulated intracellularly by {alpha}-(1->2)-mannosidases and phosphomannosyl transferases competing for the same intermediate in the glycosynthetic pathway.

1 To whom correspondence should be addressed; e-mail: marc.claeyssens{at}ugent.be


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