Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry

Hui, Joseph P. M.; Lanthier, Patricia; White, Theresa C.; McHugh, Sylvia G.; Yaguchi, Makoto; Roy, René; Thibault, Pierre

doi:10.1016/s0378-4347(00)00373-x

Cited by 100 publications

(93 citation statements)

References 34 publications

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“…As shown in Fig. S9, peaks separated by 57 Da are peptides with either 0 or 1 glycine at the N terminus, as observed previously (25,27). Binding affinity measurements were conducted for TrCel7A proteins because this enzyme has a significantly shorter linker than TrCel6A, and thus if the linker contributes to the binding affinity in TrCel7A, it is likely that the much larger linker from TrCel6A will contribute even more binding affinity.…”

Section: Binding Affinity Measurements Indicate That Linkers Contributementioning

confidence: 68%

“…The CBM was taken from the NMR structure (40). N-glycans on the CD were constructed as Man 5 GlcNAc 2 units on Asn-45, Asn-270, and Asn-384, as described in several studies characterizing glycosylation on the TrCel7A CD (25,27), which is known to be heterogeneous. The O-glycans were taken from a detailed mass spectrometry study from Harrison et al (26) and were α-O-linked to serine or threonine (47).…”

Section: Methodsmentioning

confidence: 99%

“…The Family 1 CBM in both enzymes was initially bound on the hydrophobic face of cellulose (12). The glycosylation pattern of the TrCel7A CD, TrCel7A linker, and the TrCel6A CD were adopted from previous work (25)(26)(27). O-glycosylation on the TrCel6A linker was assumed as two mannose units per glycosylation site (26).…”

Section: Simulations Predictmentioning

confidence: 99%

“…There is little sequence conservation in linkers, which is a common feature of intrinsically disordered proteins, and they typically contain significant glycine, proline, serine, and threonine content. Moreover, the serine and threonine residues often exhibit O-glycosylation (25)(26)(27), which imparts protease resistance (28). Taken together, the general consensus for linkers in lignocellulose-degrading enzymes is that they are flexible connectors between ordered, functional domains, with glycosylation to prevent proteolysis, but with little function beyond domain connection.…”

mentioning

confidence: 99%

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Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

Payne

Resch

Chen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

160

147

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Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls. biofuels | cellulase | post-translational modification | carbohydrate recognition

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Section: Binding Affinity Measurements Indicate That Linkers Contributementioning

confidence: 68%

Section: Methodsmentioning

confidence: 99%

Section: Simulations Predictmentioning

confidence: 99%

mentioning

confidence: 99%

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Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

Payne

Resch

Chen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

160

147

View full text Add to dashboard Cite

show abstract

“…To date, few studies have been conducted to examine glycosylation in secreted fungal enzymes to determine the extent and factors that control it, including growth conditions and extracellular glycan-trimming enzymes. Catalytic domains can exhibit both N-and O-linked glycans (18,19), whereas the linkers connecting enzymatic domains to CBMs are decorated with O-linked glycosylation, which has long been attributed to protease protection (20) and more recently implicated in substrate binding (21). For TrCel7A, Harrison et al (22) published the original characterization of the glycosylation pattern on the TrCel7A linker.…”

mentioning

confidence: 99%

Specificity of O -glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

Chen

Drake

Resch

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

154

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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. chemical synthesis | cellulase | biofuels | protein engineering

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Capillary electrophoresis of proteins 1999-2001

Dolnı́k¹,

Hutterer²

2001

Electrophoresis

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This review article with 223 references describes recent developments in capillary electrophoresis (CE) of proteins and covers papers published during last two years, from the previous review (V. Dolnik, Electrophoresis 1999, 20, 3106-3115) through Spring 2001. It describes the topics related to CE of proteins including modeling of the electrophoretic properties of proteins, sample pretreatment, wall coatings, improving selectivity, detection, special electrophoretic techniques, and applications.

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Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry

Cited by 100 publications

References 34 publications

Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

Specificity of O -glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

Capillary electrophoresis of proteins 1999-2001

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