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. chemical synthesis | cellulase | biofuels | protein engineering

Chemically Precise Glycoengineering Improves Human Insulin

“…58 After synthesis, the correct folding, identity, and homogeneity of the synthetic products were confirmed by a combination of circular dichroism (CD, Figure 3), ultraperformance liquid chromatography mass-spectrometry, and Glu-C digestion (Supporting Information). 30,33,52 …”

“…60,61 This cleavage also causes an easily detectable change in molecular mass, and therefore each insulin glycoform’s half-life toward α -chymotrypsin degradation can be calculated by monitoring the first-order exponential decay of the full-length molecule. 30,60 Quantitative Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-ToF MS) was used to measure the concentration over time. 62 Any change in proteolytic stability introduced by the O -linked glycans on human insulin could then be established by comparing the half-lives of glycoforms 2–6 with that of the unglycosylated insulin 1 .…”

Modification of paper properties using carbohydrate-binding module 3 from the Clostridium thermocellum CipA scaffolding protein produced in Pichia pastoris: elucidation of the glycosylation effect

“…3. Glycans may interfere with the binding of CBM3 to cellulose likely through a steric hindrance mechanism, since long glycans may hamper the interactions between the cellulose surface and the amino acid residues at the binding site (Bager et al 2013;Chen et al 2014). Indeed, large high mannose-type N-linked glycans (added by P. pastoris) near the binding face of a CBM2 were reported to have a detrimental effect on cellulose affinity (Boraston et al 2001b).…”

“…Indeed, large high mannose-type N-linked glycans (added by P. pastoris) near the binding face of a CBM2 were reported to have a detrimental effect on cellulose affinity (Boraston et al 2001b). Still, it is worth of noting that glycans have been reported to produce different effects on CBMs: they may not affect (CBM2; Boraston et al 2003), or they can either decrease (CBM2; Boraston et al 2001a, b) or increase (CBM1; Chen et al 2014;Taylor et al 2012) the binding affinity of CBMs to cellulose, depending on the glycan localization and/or amount.…”

Characterization of Aspergillus niger endo-1,4-β-glucanase ENG1 secreted from Saccharomyces cerevisiae using different expression vectors

“…Olinked glycosylation is important for enzyme stabilization (Chen et al, 2014). It is likely that the O-glycosylated form of ENG1 retains more stable activity during continuous cultivation of recombinant yeasts than the form that is only N-glycosylated.…”