title: Specificity of O-glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules.
abstract: The majority of biological turnover of lignocellulosic biomass in nature is conducted by fungi, which commonly use Family 1 carbohydrate-binding modules (CBMs) for targeting enzymes to cellulose. Family 1 CBMs are glycosylated, but the effects of glycosylation on CBM function remain unknown. Here, the effects of O-mannosylation are examined on the Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase at three glycosylation sites. To enable this work, a procedure to synthesize glycosylated Family 1 CBMs was developed. Subsequently, a library of 20 CBMs was synthesized with mono-, di-, or trisaccharides at each site for comparison of binding affinity, proteolytic stability, and thermostability. The results show that, although CBM mannosylation does not induce major conformational changes, it can increase the thermolysin cleavage resistance up to 50-fold depending on the number of mannose units on the CBM and the attachment site. O-Mannosylation also increases the thermostability of CBM glycoforms up to 16 °C, and a mannose disaccharide at Ser3 seems to have the largest themostabilizing effect. Interestingly, the glycoforms with small glycans at each site displayed higher binding affinities for crystalline cellulose, and the glycoform with a single mannose at each of three positions conferred the highest affinity enhancement of 7.4-fold. Overall, by combining chemical glycoprotein synthesis and functional studies, we show that specific glycosylation events confer multiple beneficial properties on Family 1 CBMs.
authors.affiliation: Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, CO 80303; and.
authors.forename: Liqun
authors.initials: L
authors.lastname: Chen
authors.affiliation: Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, CO 80303; and.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Drake
authors.affiliation: Biosciences Center and.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Resch
authors.affiliation: Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, CO 80303; and.
authors.forename: Eric R
authors.initials: ER
authors.lastname: Greene
authors.affiliation: Biosciences Center and.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Himmel
authors.affiliation: Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, CO 80303; and.
authors.forename: Patrick K
authors.initials: PK
authors.lastname: Chaffey
authors.affiliation: National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401 gregg.beckham@nrel.gov zhongping.tan@colorado.edu.
authors.forename: Gregg T
authors.initials: GT
authors.lastname: Beckham
authors.affiliation: Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, CO 80303; and gregg.beckham@nrel.gov zhongping.tan@colorado.edu.
authors.forename: Zhongping
authors.identifier: http://orcid.org/0000-0002-9302-150X
authors.initials: Z
authors.lastname: Tan
mesh_terms: D056804:Biofuels; D044602:Cellulases / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D008031:Lignin / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D015202:Protein Engineering / Q000379:methods; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D056804:Biofuels; D011956:Receptors, Cell Surface; C055213:saccharide-binding proteins; C036909:lignocellulose; D008031:Lignin; D044602:Cellulases
keywords: biofuels; cellulase; chemical synthesis; protein engineering