Modified glycosylation of cellobiohydrolase I from a high cellulase-producing mutant strain of Trichoderma reesei.
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.
Harrison
M J
MJ
Macquarie University Centre for Analytical Biotechnology, School of Biological Sciences, Macquarie University, Sydney, Australia.
Nouwens
A S
AS
Jardine
D R
DR
Zachara
N E
NE
Gooley
A A
AA
Nevalainen
H
H
Packer
N H
NH
D000117:Acetylglucosamine / Q000032:analysis; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D002480:Cellulase / Q000737:chemistry*; D043366:Cellulose 1,4-beta-Cellobiosidase; D006020:Glycopeptides / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D013463:Sulfuric Acid Esters / Q000737:chemistry; D014242:Trichoderma / Q000201:enzymology*
D006020:Glycopeptides; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D013463:Sulfuric Acid Esters; C025363:mannoproteins; D001216:Asparagine; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase; D000117:Acetylglucosamine