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Glycobiology Advance Access published online on April 7, 2004
Glycobiology, doi:10.1093/glycob/cwh080
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Received November 25, 2003
Revised March 15, 2004
Accepted March 15, 2004

ORIGINAL ARTICLES

Factors influencing glycosylation of Trichoderma reesei cellulases. Part one: Post-secretorial changes of the O- and N-glycosylation pattern of Cel7A

Ingeborg Stals 1, Koen Sandra 1, Steven Geysens 2, Roland Contreras 2, Jozef Van Beeumen 1, Marc Claeyssens 1*

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

* To whom correspondence should be addressed. E-mail: marc.claeyssens{at}ugent.be.


  Abstract

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 upon 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 EndoH type activity participate in N-deglycosylation (core), while 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.

Key words: Trichoderma reesei, Cel7A, N- and O-glycosylation, endoglycosidase, post-secretorial modifications


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