Glycosylation by Pichia pastoris decreases the affinity of a family 2a carbohydrate-binding module from Cellulomonas fimi: a functional and mutational analysis

When produced by Pichia pastoris, three of the five Asn-Xaa-Ser/Thr sequenc es (corresponding to Asn-24, Asn-73 and Asn87) in the carbohydrate-binding module CBM2a of xylanase 10A from Cellulomonas fimi are glycosylated. The g lycans are of the high-mannose type, ranging in size from GlcNAc(2)Man(2) t o GlcNAc(2)Man(14), The N-linked glycans block the binding of CBM2a to cell ulose. Analysis of mutants of CBM2a shows that glycans on Asn-24 decrease t he association constant (K-a) for the binding of CBM2a to bacterial microcr ystalline cellulose approx. 10-fold, whereas glycans on Asn-87 destroy bind ing. 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 co li or P. pastoris and is approx. half that of wild-type CBM2a produced by E . coli.