Factors influencing glycosylation of Trichoderma reesei cellulases. II: N-glycosylation of Cel7A core protein isolated from different strains

Abstract

A systematic analysis of the N-glycosylation of the catalytic domain of cellobiohydrolase I (Cel7A or CBH I) isolated from several Trichoderma reesei strains grown in minimal media was performed. Using a combination of chromatographic, electrophoretic, and mass spectrometric methods, the presence of 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 alpha1-3 and the alpha1-6 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)(0-1)GlcMan(7-8)GlcNAc2 for the hyperproducing Rut-C30 and RL-P37 mutants and as (Man(1-2)P)(0-1-2)Man(5-6-7)GlcNAc2 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 posttranslational modification of Cel7A with mannosyl phosphodiester residues at the three distinct sites. This study clearly shows that posttranslational phosphorylation of glycoproteins is not atypical 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 alpha1-3 branch apparently hinders a second mannophoshoryl transfer.

Small Angle Neutron Scattering Reveals pH-dependent Conformational Changes in Trichoderma reesei Cellobiohydrolase I

“…In Cel7A, these domains have 436, 36, and 24 amino acid residues, respectively. The enzyme is highly glycosylated (5)(6)(7)(8)(9), with sites on the catalytic core and linker.…”

“…The glycosylation of the enzyme is variable, resulting in a range of pI values for the holoenzyme. Isoforms having pI values between 3.5 and 4.2 are found depending on the growth conditions and the specific strain of the fungus (8,9).…”

Trichoderma reesei: A Fungal Enzyme Producer for Cellulosic Biofuels

“…As a consequence of the N-overglycosylation a reduced activity and increased non-productive binding on cellulose was observed (Jeoh et al, 2008). Even when produced by T. reesei, the glycosylation pattern is not uniform and depends on the fungal strain, the fermentation medium and pH, and the secretion of other carbohydrate modifying activities into the medium (Stals et al, 2004a;Stals et al, 2004b). In addition to the impact of glycosylation in the catalytic domain on enzyme activity, the O-glycosylation of the linker region connecting catalytic domain and CBM was examined.…”

Promotion of Extracellular Activity of Cellobiohydrolase I from Trichoderma reesei by Protein Glycosylation Engineering in Saccharomyces cerevisiae

“…A glycosyl chain unit modified on Tr-Cel7A is Man 5 -8 GlcNAc 2 formed from the Man 9 GlcNAc 2, a same core structure as it in S. cerevisiae [13,14]. But during its secretion process, unlike in S. cerevisiae, all of the three α-1, 2-mannosyl residues are digested by α-1,2-mannosidase (Tr-Mds1p) in the ER and/or outside of the cell in T. reesei [15][16][17].…”