title: Glycosylation by Pichia pastoris decreases the affinity of a family 2a carbohydrate-binding module from Cellulomonas fimi: a functional and mutational analysis.
abstract: When produced by Pichia pastoris, three of the five Asn-Xaa-Ser/Thr sequences (corresponding to Asn-24, Asn-73 and Asn-87) in the carbohydrate-binding module CBM2a of xylanase 10A from Cellulomonas fimi are glycosylated. The glycans are of the high-mannose type, ranging in size from GlcNAc(2)Man(8) to GlcNAc(2)Man(14). The N-linked glycans block the binding of CBM2a to cellulose. Analysis of mutants of CBM2a shows that glycans on Asn-24 decrease the association constant (K(a)) for the binding of CBM2a to bacterial microcrystalline cellulose approx. 10-fold, whereas glycans on Asn-87 destroy binding. The K(a) of a mutant of CBM2a lacking all three N-linked glycosylation sites is the same when the polypeptide is produced by either Escherichia coli or P. pastoris and is approx. half that of wild-type CBM2a produced by E. coli.
authors.affiliation: The Protein Engineering Network of Centres of Excellence, PENCE Inc., National Business Centre, 750 Heritage Medical Research Centre, Edmonton, Alberta, Canada T6G 2S2.
authors.forename: A B
authors.initials: AB
authors.lastname: Boraston
authors.forename: R A
authors.initials: RA
authors.lastname: Warren
authors.forename: D G
authors.initials: DG
authors.lastname: Kilburn
mesh_terms: D000192:Actinomycetales / Q000201:enzymology*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002482:Cellulose / Q000378:metabolism; D004252:DNA Mutational Analysis; D006031:Glycosylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011135:Polysaccharides, Bacterial / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D014170:Transformation, Genetic; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D011135:Polysaccharides, Bacterial; D002482:Cellulose; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase