Structural Characterization of N‐Linked Oligosaccharides from Cellobiohydrolase I Secreted by the Filamentous Fungus Trichoderma Reesei RUTC 30

Abstract

We have characterized the primary structures of the predominant N-linked oligosaccharides on cellobiohydrolase I from the filamentous fungus Trichoderma reesei RUTC30. Different enzymatic and chromatographic techniques were used to analyze six oligosaccharides. The combined data showed that the fungal carbohydrates have a core structure that is identical to the mammalian N-linked core. In the bulk of the N-glycans, the u-1,3 arm is extended with two mannoses and a glucose, suggesting incomplete processing of the oligosaccharides in the endoplasmic reticulum. The u-1,6 arm shows a remarkable heterogeneity: in addition to a-1,2-Man and a-1,6-Man, the presence of a terminal mannose a-1,6-phosphodiester was observed. This latter substituent has not been characterized before on mannosidase-processed N-glycan and its function and synthesis pathway are entirely unknown. The predominant Nglycans on cellobiohydrolase I can be represented as follows : GlcMan,GIcNAc,, GlcMan,GlcNAc,, Man,GlcNAc,, ManPGlcMan,GlcNAc,, GlcMan,GlcNAc, and Man,GlcNAc,.Keywords: cellobiohydrolase I ; fungus ; N-glycans ; NMR ; Trichoderma reesei.Trichoderma reesei is a filamentous fungus capable of secreting high amounts of cellulose-degrading enzymes (up to 40 g/l by manipulated strains). This remarkable secretory capacity led to the idea to use this organism as a candidate host for the production of heterologous proteins [I]. However, it is not clear whether T. reesei can be used for production of pharmaceutically important glycoproteins since post-translational modifications by the fungus, such as glycosylation, could be a major problem for application. Oligosaccharides lacking sialic acid as terminal sugar residues are easily recognized by lectins circulating in blood and present on cells of the reticulo-endothelial system. This recognition leads to fast clearance, which prevents the therapeutic proteins reaching their target organs [2, 31. Furthermore, it has been demonstrated that foreign glycosyl structures often elicit an antigenic response [4].Only few data concerning glycosylation in filamentous fungi are available. They indicate high-mannose-type glycans of limited sizes compared with these synthesized by yeast [S-121. The identification by Salovuori et al. 1121 of Man,GlcNAc, and Man,GlcNAc, as the major N-glycans on 7: reesei cellobiohydrolase I (CBH I) suggested a possible analogy between fungal and mammalian oligosaccharide structures. For identical primary structures, in vitro and eventually in vivo extension of oligosaccharides on proteins secreted from this fungus to the complex type should be possible.Correspondence to R. Contreras Enzyme. a-mannosidase (EC 3.2.1.24).During the eukaryotic secretion process, proteins receive in the endoplasmic reticulum a common precursor oligosaccharide, namely Glc,Man,GlcNAc,. This precursor is transferred by an oligosaccharyl transferase to asparagine residues in the consensus sequence Asn-Xaa-(Ser or Thr), with Xaa representing any amino acid except proline. A battery of glycosidases and gl...

Modified glycosylation of cellobiohydrolase I from a high cellulase‐producing mutant strain of Trichoderma reesei

“…A similar observation was recently reported by an independent group [12], who analysed CBHI from the related T. reesei strain QM9414. Other recent reports have also described the characterisation (but not the quantitation) of several N-glycans of CBHI which have been obtained from other related Trichoderma strains VTT-D-80133 [9] and RutC-30 [11]. In contrast to our findings and those of Klarskov et al [12], these reports describe predominately mammalian-type highmannose N-glycosylation.…”

“…It is important to note that these oligosaccharides were detected only after concentration of the released oligosaccharides by chromatography. The compositions of some of these oligosaccharides are the same as those of N-linked oligosaccharides from T. reesei strain RutC30 CBHI [11]. Edman sequencing of the 4.4 kDa peptide was heterogeneous for the N-terminal serine as was predicted by the mass spectrometric data, consequently the sequenator exhibited a constant out-of-phase cycle (Fig.…”

Trichoderma reesei RUT-C30 – thirty years of strain improvement

“…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.…”

N-Glycan Modification in Aspergillus Species

“…Far less progress has been made in glycoengineering of fungal production hosts, such as Aspergillus or Trichoderma species, although the N-glycan structures of several secreted glycoproteins have been elucidated. (1,2,17,24,28,29,34,(39)(40)(41)48) and attempts were made to modify fungal glycosylation structures by the insertion of glycan structuremodifying enzymes (17,29,32). Introduction of rabbit N-acetylglucosaminyltransferase I (GnT I) into A. nidulans has resulted in the production of an in vitro active enzyme, but no evidence for in vivo GlcNAc transfer was found (23).…”

“…In order to approach a solution for this important drawback in the possibility of using fungal heterologous glycoproteins for therapeutic applications (21,28,30), we used an approach similar to that described for Pichia pastoris and systematically engineered the glycosylation pathways of two Aspergillus species. Here, we report the introduction of heterologous fusion proteins, such as mannosidases and glycosyltransferases, as well as the deletion of a gene (algC) coding for an enzyme involved in an early step of the fungal glycosylation pathway.…”