Factors influencing glycosylation of Trichoderma reesei cellulases. I: Postsecretorial changes of the O- and N-glycosylation pattern of Cel7A
| Autor: | Marc Claeyssens, Koen Sandra, Roland Contreras, Ingeborg Stals, Steven Geysens, Jozef Van Beeumen |
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| Rok vydání: | 2004 |
| Předmět: |
Mannosidase
Spectrometry Mass Electrospray Ionization Glycosylation Cellulase Biochemistry Serine chemistry.chemical_compound N-linked glycosylation Polysaccharides Catalytic Domain Hydrolase Cellulose 1 4-beta-Cellobiosidase Threonine Trichoderma reesei Trichoderma biology Extracellular Fluid Hydrogen-Ion Concentration biology.organism_classification Culture Media carbohydrates (lipids) chemistry biology.protein Isoelectric Focusing |
| Zdroj: | Glycobiology. 14:713-724 |
| ISSN: | 1460-2423 |
| Popis: | The glycosylation of Cel7A (CBH I) from Trichoderma reeseivaries considerably when the fungus is grown under differentconditions. As shown by ESI-MS and PAG-IEF analyses ofboth intact protein and the isolated catalytic core module, themicroheterogeneity originates mainly from the variable ratioof single N-acetylglucosamine over high-mannose structureson the three N-glycosylation sites and from the presence orabsence of phosphate residues. Fully N- and O-glycosylatedCel7A can only be isolated from minimal medium and prob-ablyreflectstheinitialcomplexityoftheproteinonleavingtheglycosynthetic pathway. Extracellular activities are responsi-ble for postsecretorial modifications in other cultivation con-ditions: a-(1!2)-mannosidase, a-(1!3)-glucosidase and anEndo H type activity participate in N-deglycosylation (core),whereas a phosphatase and a mannosidase are probablyresponsible for hydrolysis of O-glycans (linker). The effectsare most prominent in corn steep liquor–enriched media,where the pH is closer to the pH optimum (5–6) of theseextracellular hydrolases. In minimal medium, the low pH andthepresenceofproteasescouldexplainfortheabsenceofsuchactivities. On the other hand, phosphodiester linkages in thecatalytic module are only observed under specific conditions.The extracellular trigger is still unknown, but manno-phosphorylationmayberegulatedintracellularlybya-(1!2)-mannosidases and phosphomannosyl transferases competingfor the same intermediate in the glycosynthetic pathway.Key words: Cel7A/endoglycosidase/N- andO-glycosylation/postsecretorial modifications/Trichoderma reeseiIntroductionCellulases belonging to different glycosyl hydrolase familiesvery often exhibit a multidomain structure (Coutinhoand Henrissat, 1999): a catalytic domain (core) and acarbohydrate-binding module are separated by a linkerpeptide rich in proline, serine, and threonine. Fungalcellulases carry posttranslational modifications on bothdomains: Whereas the linker peptide is highly O-glycosylated, N-glycosylation seems to be restricted to thecore. Filamentous fungi typically synthesize short-chain O-and N-glycans, resembling the mammalian high-mannosetype rather than the hyperglycosylated yeast structures.The glycosylation of cellobiohydrolase I (CBH I, Cel7A),a cellulase abundantly expressed by most Trichodermareesei strains, has been studied particularly well(Table I). Trichoderma cellulases appear in several isoformswith similar catalytic and adsorption properties (Medveet al., 1998), and it has been shown that both N- andO-glycans account for the many isoforms of Cel7A(Pakula et al., 2000).Detailed structural investigations (Harrison et al., 1998;Klarskov et al., 1997) revealed the N-glycosylation inT. reesei Cel7A from respectively strains QM9414 andALKO2877 (derived from QM9414): Single GlcNAc resi-dues in three (Asn45, Asn270, and Asn384) out of fourpotential sites were characterized in the catalytic domain.More complex N-glycan structures were observed withCel7A from the hyperproducing mutant Rut-C30 strain(Maras et al., 1997). The majority are monoglucosylatedhigh-mannose glycans (GlcMan |
| Databáze: | OpenAIRE |
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