Harrison MJ, Nouwens AS, Jardine DR, Zachara NE, Gooley AA, Nevalainen H, Packer NH Modified glycosylation of cellobiohydrolase I from a high cellulase-producing mutant strain of Trichoderma reesei European Journal of Biochemistry256 (1998)
119-127
Publication DOI:10.1046/j.1432-1327.1998.2560119.x Journal NLM ID:0107600 Publisher: Oxford, UK: Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies Correspondence: nicolle.packermq.edu.au Institutions: Macquarie University Centre for Analytical Biotechnology, School of Biological Sciences and School of Chemistry, Macquarie University, Sydney, Australia
Cellobiohydrolase I is an industrially important exocellulase secreted in high yields by the filamentous fungus Trichoderma reesei. The nature and effect of glycosylation of CBHI and other cellulolytic enzymes is largely unknown, although many other structural and mechanistic aspects of cellulolytic enzymes are well characterised. Using a combination of liquid chromatography, electrospray mass spectrometry, solid-phase Edman degradation, and monosaccharide analysis we have identified every site of glycosylation of CBHI from a high cellulase-producing mutant strain of T. reesei, ALKO2877, and characterised each site in terms of its modifying carbohydrate and site-specific heterogeneity. The catalytic core domain comprises three N-linked glycans which each consist of a single N-acetylglucosamine residue. Within the glycopeptide linker domain, all eight threonines are variably glycosylated with between at least one, and up to three, mannose residues per site. All serines in this domain are at least partially glycosylated with a single mannose residue. This linker region has also been shown to be sulfated by a combination of ion chromatography and collision-induced dissociation electrospray mass spectrometry. The sulfate is probably mannose-linked. The biological significance of N-linked single N-acetylglucosamine in the catalytic core, and mannose sulfation in the linker region, is not known.
N-acetylglucosamine, cellulase, cellobiohydrolase I, Trichoderma, fungal glycosylation
Methods: ESI-MS, HPAEC, HPLC, enzymatic digestion, reduction, HPLC-MS, alkylation, ion chromatography, size-exclusion chromatography, SEC-ESI-MS, Edman degradation Comments, role: is attached to Asn45, Asn270, and Asn384 of cellobiohydrolase I
Harrison MJ, Nouwens AS, Jardine DR, Zachara NE, Gooley AA, Nevalainen H, Packer NH Modified glycosylation of cellobiohydrolase I from a high cellulase-producing mutant strain of Trichoderma reesei European Journal of Biochemistry256 (1998)
119-127
/Variants 0/-Thr
/Variants 0/ is:
Man-(1-3)-
OR (exclusively)
Man-(1-?)-Man-(1-3)-
OR (exclusively)
Man-(1-?)-Man-(1-?)-Man-(1-3)-
Publication DOI:10.1046/j.1432-1327.1998.2560119.x Journal NLM ID:0107600 Publisher: Oxford, UK: Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies Correspondence: nicolle.packermq.edu.au Institutions: Macquarie University Centre for Analytical Biotechnology, School of Biological Sciences and School of Chemistry, Macquarie University, Sydney, Australia
Cellobiohydrolase I is an industrially important exocellulase secreted in high yields by the filamentous fungus Trichoderma reesei. The nature and effect of glycosylation of CBHI and other cellulolytic enzymes is largely unknown, although many other structural and mechanistic aspects of cellulolytic enzymes are well characterised. Using a combination of liquid chromatography, electrospray mass spectrometry, solid-phase Edman degradation, and monosaccharide analysis we have identified every site of glycosylation of CBHI from a high cellulase-producing mutant strain of T. reesei, ALKO2877, and characterised each site in terms of its modifying carbohydrate and site-specific heterogeneity. The catalytic core domain comprises three N-linked glycans which each consist of a single N-acetylglucosamine residue. Within the glycopeptide linker domain, all eight threonines are variably glycosylated with between at least one, and up to three, mannose residues per site. All serines in this domain are at least partially glycosylated with a single mannose residue. This linker region has also been shown to be sulfated by a combination of ion chromatography and collision-induced dissociation electrospray mass spectrometry. The sulfate is probably mannose-linked. The biological significance of N-linked single N-acetylglucosamine in the catalytic core, and mannose sulfation in the linker region, is not known.
N-acetylglucosamine, cellulase, cellobiohydrolase I, Trichoderma, fungal glycosylation
Methods: ESI-MS, HPAEC, HPLC, enzymatic digestion, reduction, HPLC-MS, alkylation, ion chromatography, size-exclusion chromatography, SEC-ESI-MS, Edman degradation Comments, role: is attached to Thr residues (in particular, Thr462) of cellobiohydrolase I
Harrison MJ, Nouwens AS, Jardine DR, Zachara NE, Gooley AA, Nevalainen H, Packer NH Modified glycosylation of cellobiohydrolase I from a high cellulase-producing mutant strain of Trichoderma reesei European Journal of Biochemistry256 (1998)
119-127
Publication DOI:10.1046/j.1432-1327.1998.2560119.x Journal NLM ID:0107600 Publisher: Oxford, UK: Blackwell Science Ltd. on behalf of the Federation of European Biochemical Societies Correspondence: nicolle.packermq.edu.au Institutions: Macquarie University Centre for Analytical Biotechnology, School of Biological Sciences and School of Chemistry, Macquarie University, Sydney, Australia
Cellobiohydrolase I is an industrially important exocellulase secreted in high yields by the filamentous fungus Trichoderma reesei. The nature and effect of glycosylation of CBHI and other cellulolytic enzymes is largely unknown, although many other structural and mechanistic aspects of cellulolytic enzymes are well characterised. Using a combination of liquid chromatography, electrospray mass spectrometry, solid-phase Edman degradation, and monosaccharide analysis we have identified every site of glycosylation of CBHI from a high cellulase-producing mutant strain of T. reesei, ALKO2877, and characterised each site in terms of its modifying carbohydrate and site-specific heterogeneity. The catalytic core domain comprises three N-linked glycans which each consist of a single N-acetylglucosamine residue. Within the glycopeptide linker domain, all eight threonines are variably glycosylated with between at least one, and up to three, mannose residues per site. All serines in this domain are at least partially glycosylated with a single mannose residue. This linker region has also been shown to be sulfated by a combination of ion chromatography and collision-induced dissociation electrospray mass spectrometry. The sulfate is probably mannose-linked. The biological significance of N-linked single N-acetylglucosamine in the catalytic core, and mannose sulfation in the linker region, is not known.
N-acetylglucosamine, cellulase, cellobiohydrolase I, Trichoderma, fungal glycosylation
Methods: ESI-MS, HPAEC, HPLC, enzymatic digestion, reduction, HPLC-MS, alkylation, ion chromatography, size-exclusion chromatography, SEC-ESI-MS, Edman degradation Comments, role: is attached to Ser residues (in particular, Ser464) of cellobiohydrolase I
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