Factors influencing glycosylation of Trichoderma reesei cellulases. II: N-glycosylation of Cel7A core protein isolated from different strains.
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.
Stals
Ingeborg
I
Department of Biochemistry, Physiology and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium.
Sandra
Koen
K
Devreese
Bart
B
Van Beeumen
Jozef
J
Claeyssens
Marc
M
D002240:Carbohydrate Sequence; D020134:Catalytic Domain; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000302:isolation & purification / Q000378:metabolism*; D002853:Chromatography, Liquid; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011134:Polysaccharides / Q000032:analysis; D011499:Protein Processing, Post-Translational; D013045:Species Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
D011134:Polysaccharides; D043366:Cellulose 1,4-beta-Cellobiosidase