Glycobiology Advance Access published online on April 7, 2004
Glycobiology, doi:10.1093/glycob/cwh081
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1 Department of Biochemistry, Physiology and Microbiology, Ghent University, Belgium
* To whom correspondence should be addressed. E-mail: marc.claeyssens{at}ugent.be.
The present study reports a systematic analysis of the N-glycosylation of the catalytic domain of cellobiohydrolase I (CBH I, Cel7A) isolated from several Trichoderma reesei strains, grown in minimal media. Using a combination of chromatographic, electrophoretic and mass spectrometric methods, the presence of previously reported glucosylated and phosphorylated oligosaccharides on the three N-glycosylation sites of Cel7A core protein (from T. reesei strains Rut-C30 and RL-P37) confirms previous findings. With N-glycans isolated from other strains, no end-capping glucose could be detected. Phosphodiester linkages were, however, found in proteins from each strain and these probably occur on both the
Revised March 16, 2004
Accepted March 18, 2004
ORIGINAL ARTICLES
Factors influencing glycosylation of Trichoderma reesei cellulases. Part two: N-glycosylation of Cel7A core protein isolated from different strains
Abstract 
1-3 and the 1-6 branch of the high mannose oligosaccharide tree. Evidence is also presented for the occurrence of mannobiosyl units on the phosphodiester linkage. Therefore, the predominant N-glycans on Cel7A can be represented as (ManP)0-1GlcMan7-8GlcNAc2 for the hyper-producing Rut-C30 and RL-P37 mutants, and as (Man1-2P)0-1-2Man5-6-7GlcNAc2 for the wild type strain and the other mutants. As shown by ESI-MS, random substitution of these structures on the N-glycosylation sites explains the heterogeneous glycoform population of the isolated core domains. PAG-IEF separates up to five isoforms, resulting from post-translational modification of Cel7A with mannosyl phosphodiester residues at the three distinct sites. This study clearly shows that post-translational phosphorylation of glycoproteins is not a-typical for Trichoderma sp. and that, in the case of the Rut-C30 and RL-P37 strains, the presence of an end-capped glucose residue at the 1-3 branch apparently hinders a second mannophoshoryl transfer.
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