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

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 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.

Trichoderma reesei RUT-C30 – thirty years of strain improvement

“…The detected frameshift mutation in gls2a was also found in NG14, the parent strain of RUT-C30, but was not present in QM9414 (Geysens et al, 2005). Furthermore, the N-glycan profile of secreted proteins of NG14 was found to be very similar to that of RUT-C30, whereas N-glycans from QM9414 were significantly different and were not monoglucosylated, a finding consistent with previous research (García et al, 2001;Stals et al, 2004). Consequently, the abnormal glycosylation of secreted proteins resulting from the mutant gls2a was confirmed as being specific to RUT-C30, its parent strain NG14 and probably other strains derived from it, such as RL-P37 (Stals et al, 2004;Geysens et al, 2005).…”

“…The N-glycans on the main secreted cellulase of T. reesei RUT-C30, CBHI, were found to carry an unusual a-1,3-glucose residue (De Bruyn et al, 1997;Maras et al, 1997a;Hui et al, 2001;Stals et al, 2004), indicating incomplete glycan processing. The unusual glycosylation profile was also found on CBHI secreted by T. reesei RL-P37, a mutant strain derived from NG14 from which RUT-C30 was obtained.…”

“…The acetylxylan esterase from RUT-C30 was also found to exhibit an unusual glycosylation profile in which the N-glycan was both phosphorylated and highly mannosylated, and the linker peptide was heavily O-glycosylated and also sulphated (Harrison et al, 2002). In comparison, CBHI of T. reesei QM9414, a strain from a separate mutation line derived from QM6a by irradiation using a linear accelerator (Mandels et al, 1971), carried fully trimmed glycans closely resembling those on CBHI of the wild-type QM6a (Stals et al, 2004).…”

“…However, it was not until the glucosidase II alpha subunit gene (gls2a) was isolated and characterized that a frameshift mutation was revealed (Geysens et al, 2005). A transformed strain of RUT-C30 with a repaired gls2a gene produced extracellular proteins with fewer monoglucosylated N-glycans and more high mannose Nglycans than those typical of the secreted glycoproteins of the wild-type QM6a and strains from other mutagenesis lines (Stals et al, 2004). The detected frameshift mutation in gls2a was also found in NG14, the parent strain of RUT-C30, but was not present in QM9414 (Geysens et al, 2005).…”

Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

“…There is little sequence conservation in linkers, which is a common feature of intrinsically disordered proteins, and they typically contain significant glycine, proline, serine, and threonine content. Moreover, the serine and threonine residues often exhibit O-glycosylation (25)(26)(27), which imparts protease resistance (28). Taken together, the general consensus for linkers in lignocellulose-degrading enzymes is that they are flexible connectors between ordered, functional domains, with glycosylation to prevent proteolysis, but with little function beyond domain connection.…”