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Characterization and modification of two carbohydrate-binding modules Boraston, Alisdair Bennett

Abstract

The C-terminal carbohydrate-binding module of Xylanase 10A CBM2a, from Cellulomonas fimi was produced and secreted by the methylotrophic yeast Pichia pastoris. The polypeptide was highly N-glycosylated. MALDI-TOF mass spectrometry combined with protease digests and site-directed mutation located the N-linked glycans to three of five potential N-linked glycosylation sites. The glycans were of the high mannose type ranging in size from (GlcNAC)₂-(Man)₈ to (GlcNAC)₂-(Man)₁₄. A small proportion of the N-linked glycans had increased masses and negative charge consistent with the presence of phosphate groups. There was also a low level of O-glycosylation on the CBM. Construction of an N-glycosylation negative mutant allowed characterization of the O-glycosylation. O-linked glycans were composed entirely of mannose in a ratio of one mole of mannose to four moles of protein. The overall distribution of mannose on the O-glycosylated C BM mutant ranged from one to nine mannose residues with the oligosaccharide sizes ranging from (Man)₁ to (Man)₄. An extension of the fluorophore-assisted carbohydrate electrophoresis technique allowed the identification of α 1 - 2, α1-3, and α1-6 linkages in the O-linked glycans. MALDI-TOF mass spectrometry mapping isolated the glycosylation to three regions of the polypeptide with each region having a maximum of four mannose residues attached to each. The glycans inhibit the binding of CBM2a to cellulose. Removal of glycosylation sites by mutation allowed production of CBM2a mutants N-glycosylated at single sites. Glycans on N87 drastically impaired (200-300 fold decrease in K[sub a] ) the binding of CBM2a to bacterial - microcrystalline cellulose (BMCC). In contrast, glycans on N24 decreased the K[sub a] for BMCC only ten-fold. A CBM2a mutant without N-glycosylation sites had only a 2-3 fold lower binding affinity than CBM2a produced by E. coli. Although N-glycosylation did not affect the thermal or chemical stability of CBM2a significantly, UV resonance enhanced Raman spectroscopy and fluorescence spectroscopy of the glycosylated CBM2a indicated an alteration in the environment of one or more internal tryptophan residues and a change in the hydrogen-bonding pattern of one or more surface tryptophans. The C-terminal carbohydrate-binding module of xylanase 10A, CBM13, from Streptomyces lividans belongs to the family 13 carbohydrate-binding modules. CBM13 binds to the insoluble polysaccharides xylan, holo-cellulose, pachyman, and lichenan. It also binds soluble xylan, arabino-galactan, and laminarin. The association constant for binding to soluble xylan is ~6 x 10³ per mole of xylan polymer. Site-directed mutation was used to demonstrate the presence of three functional sites involved in the binding of CBM13. These binding sites are similar in sequence and predicted to be similar in structural organization to the α, β, and ƴ sites in ricin toxin B-chain (RTB). Fluorescence spectrophotometric titrations were used to quantify the binding of saccharides to CBM13. The binding specificity was very low being restricted only by the requirement for pyranose sugars. The association constants for binding to small sugars were also low (~1 x 10² M⁻¹ to 1 x 10³ M⁻¹). This is the first bacterial family 13 C BM to be characterized in detail and the first C BM shown to be multivalent.

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