Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei) Cel7B catalytic module.
The catalytic module of Hypocrea jecorina (previously Trichoderma reesei) Cel7B was homologously expressed by transformation of strain QM9414. Post-translational modifications in purified Cel7B preparations were analysed by enzymatic digestions, high performance chromatography, mass spectrometry and site-directed mutagenesis. Of the five potential sites found in the wild-type enzyme, only Asn56 and Asn182 were found to be N-glycosylated. GlcNAc(2)Man(5) was identified as the predominant N-glycan, although lesser amounts of GlcNAc(2)Man(7) and glycans carrying a mannophosphodiester bond were also detected. Repartition of neutral and charged glycan structures over the two glycosylation sites mainly accounts for the observed microheterogeneity of the protein. However, partial deamidation of Asn259 and a partially occupied O-glycosylation site give rise to further complexity in enzyme preparations.
Eriksson
Torny
T
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, Sweden.
Stals
Ingeborg
I
Collén
Anna
A
Tjerneld
Folke
F
Claeyssens
Marc
M
Stålbrand
Henrik
H
Brumer
Harry
H
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry*; D044602:Cellulases / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry; D006031:Glycosylation; D006868:Hydrolysis; D035901:Hypocrea / Q000378:metabolism*; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D014242:Trichoderma / Q000378:metabolism*; D043323:alpha-Mannosidase / Q000737:chemistry
D006023:Glycoproteins; D010455:Peptides; D020033:Protein Isoforms; D011506:Proteins; D001216:Asparagine; D000469:Alkaline Phosphatase; D044602:Cellulases; D006026:Glycoside Hydrolases; D043323:alpha-Mannosidase; D002480:Cellulase; D010450:Endopeptidases