Carbohydrate-binding module O-mannosylation alters binding selectivity to cellulose and lignin

Improved understanding of the effect of protein glycosylation is expected to provide the foundation for the design of protein glycoengineering strategies. In this study, we examine the impact of O-glycosylation on the binding selectivity of a model Family 1 carbohydrate-binding module (CBM), which has been shown to be one of the primary sub-domains responsible for non-productive lignin binding in multi-modular cellulases. Specifically, we examine the relationship between glycan structure and the binding specificity of the CBM to cellulose and lignin substrates. We find that the glycosylation pattern of the CBM exhibits a strong influence on the binding affinity and the selectivity between both cellulose and lignin. In addition, the large set of binding data collected allows us to examine the relationship between binding affinity and the correlation in motion between pairs of glycosylation sites. Our results suggest that glycoforms displaying highly correlated motion in their glycosylation sites tend to bind cellulose with high affinity and lignin with low affinity. Taken together, this work helps lay the groundwork for future exploitation of glycoengineering as a tool to improve the performance of industrial enzymes.

Improved understanding of the effect of protein glycosylation is expected to provide the foundation for the design of protein glycoengineering strategies.

The cell walls of terrestrial plants primarily comprise the polysaccharides cellulose, hemicellulose, and pectin, as well as the heterogeneous aromatic polymer, lignin. In nature, carbohydrates derived from plant polysaccharides provide a massive carbon and energy source for biomass-degrading fungi, bacteria, and archaea, which together are the primary organisms that recycle plant matter and are a critical component of the global carbon cycle. Across the various environments in which these microbes break down lignocellulose, a few known enzymatic and chemical systems have evolved to deconstruct polysaccharides to soluble sugars.^1–6 These natural systems are, in several cases, being evaluated for industrial use to produce sugars for further conversion into renewable biofuels and chemicals.From an industrial perspective, overcoming biomass recalcitrance to cost-effectively produce soluble intermediates, including sugars for further upgrading remains the main challenge in biomass conversion. Lignin, the evolution of which in planta provided a significant advantage for terrestrial plants to mitigate microbial attack, is now widely recognized as a primary cause of biomass recalcitrance.⁷ Chemical and/or biological processing scenarios of lignocellulose have been evaluated⁸ and several approaches have been scaled to industrial biorefineries to date. Many biomass conversion technologies overcome recalcitrance by partially or wholly removing lignin from biomass using thermochemical pretreatment or fractionation. This approach enables easier polysaccharide access for carbohydrate-active enzymes and/or microbes. There are however, several biomass deconstruction approaches that employ enzymes or microbes with whole, unpretreated biomass.^9,10 In most realistic biomass conversion scenarios wherein enzymes or microbes are used to depolymerize polysaccharides, native or residual lignin remains.^11,12 It is important to note that lignin can bind and sequester carbohydrate-active enzymes, which in turn can affect conversion performance.¹³Therefore, efforts aimed at improving cellulose binding selectivity relative to lignin have emerged as major thrusts in cellulase studies.^14–25 Multiple reports in the past a few years have made exciting new contributions to our collective understanding of how fungal glycoside hydrolases, which are among the most well-characterized cellulolytic enzymes given their importance to cellulosic biofuels production, bind to lignin from various pretreatments.^15,17 Taken together, these studies have demonstrated that the Family 1 carbohydrate-binding modules (CBMs) often found in fungal cellulases are the most relevant sub-domains for non-productive binding to lignin,^15,17,20,26 likely due to the hydrophobic face of these CBMs that is known to be also responsible for cellulose binding (Fig. 1).²⁷Open in a separate windowFig. 1Model of glycosylated CBM binding the surface of a cellulose crystal. Glycans are shown in green with oxygen atoms in red, tyrosines known to be critical to binding shown in purple, and disulfide bonds Cys8–Cys25 and Cys19–Cys35 in yellow.Furthermore, several studies have been published recently using protein engineering of Family 1 CBMs to improve CBM binding selectivity to cellulose with respect to lignin. Of particular note, Strobel et al. screened a large library of point mutations in both the Family 1 CBM and the linker connecting the catalytic domain (CD) and CBM.^21,22 These studies demonstrated that several mutations in the CBM and one in the linker led to improved cellulose binding selectivity compared to lignin. The emerging picture is that the CBM-cellulose interaction, which occurs mainly as a result of stacking between the flat, hydrophobic CBM face (which is decorated with aromatic residues) and the hydrophobic crystal face of cellulose I, is also likely the main driving force in the CBM-lignin interaction given the strong potential for aromatic–aromatic and hydrophobic interactions.Alongside amino acid changes, modification of O-glycosylation has recently emerged as a potential tool in engineering fungal CBMs, which Harrison et al. demonstrated to be O-glycosylated.^28–31 In particular, we have revealed that the O-mannosylation of a Family 1 CBM of Trichoderma reesei cellobiohydrolase I (TrCel7A) can lead to significant enhancements in the binding affinity towards bacterial microcrystalline cellulose (BMCC).^30,32,33 This observation, together with the fact that glycans have the potential to form both hydrophilic and hydrophobic interactions with other molecules, led us to hypothesize that glycosylation may have a unique role in the binding selectivity of Family 1 CBMs to cellulose relative to lignin and as such, glycoengineering may be exploited to improve the industrial performance of these enzymes. To test this hypothesis, in the present study, we systematically probed the effects of glycosylation on CBM binding affinity for a variety of lignocellulose-derived cellulose and lignin substrates and investigated routes to computationally predict the binding properties of different glycosylated CBMs.     

Functionalized nanoparticles for liquid atmospheric pressure matrix-assisted laser desorption/ionization peptide analysis

Nanoparticles for the extraction of peptides and subsequent analysis using atmospheric pressure matrix-assisted laser desorption/ionization (APMALDI) have been evaluated. The atmospheric pressure source allows for particles to be directly introduced in the liquid matrix, minimizing sample loss and analysis time. Described in this work are two sample preparation procedures for liquid APMALDI analysis: a C18 functionalized silica nanoparticle for hydrophobic extractions, and an aptamer functionalized magnetite core nanoparticle for rapid, affinity extractions. The C18 particles provide a non-selective support for rapid profiling applications, while the aptamer particles are directed towards reducing the complexity in biological samples. The aptamer functionalized particles provide a more selective analyte-nanoparticle interaction whereby the tertiary structure of the analyte becomes more critical to the extraction. In both cases, the liquid APMALDI matrix provides a support for ionization, and acts as the releasing agent for the analyte-particle interaction. Additionally, analyte enrichment was possible due to the large surface-to-volume ratio of the particles. The experiments conducted with functionalized nanoparticles, in an atmospheric pressure liquid matrix, present a basis for further methodologies and utilities of silica nanoparticles to be developed.     

Natural anti-HIV agents. Part 3: Litseaverticillols A-H, novel sesquiterpenes from Litsea verticillata

Bioassay directed-fractionation led to the identification of litseaverticillols A-H (1-8) from the leaves and twigs of Litsea verticillata Hance. These new sesquiterpenes possess a unique skeleton that was recently designated as ‘litseane’. The structures of these compounds were determined by spectroscopic means including 1D and 2D NMR data. Structural configurations were determined by ROESY experiments. Mosher ester reactions and optical rotation measurements established the sesquiterpenes 1-8 as racemates. Isolates 1-8 inhibited HIV-1 replication in HOG.R5 cells with IC₅₀ values ranging from 2 to 15 μg/ml (8-58 μM) while affecting the growth of HOG.R5 at concentrations 2-3-fold higher. Based on this data, structure-activity relationships can be discerned, suggesting compounds of this class are good candidates for analog production.     

Full-wave analysis of radiation effect of microstrip transmission lines

A full-wave analysis of radiation effects produced by discontinuities in microstrip and buried microstrip transmission lines is presented. Beginning with the dyadic Green's function for a dielectric slab, with an embedded source, an integral equation is formulated. This equation is then solved by the method of moments to obtain the current distributions along the transmission line, in particular, near the discontinuities. Employing these results, the near- and far-zone fields, as well as radiation patterns are computed. The results from our method showed good agreement with those of previous publications in complex reflection and transmission coefficients, and equivalent capacitance values. It is found that under resonance conditions the radiation efficiency of a simple structure can exceed 41%, which may cause a potential problem in electromagnetic compatibility. Our analytic result also shows that the maximum radiation occurs when the source is located at the height of from the bottom ground plane, which should be prevented.     

Lattice-BGK approach to simulating granular flows

Many continuum theories for granular flow produce an equation of motion for the fluctuating kinetic energy density (granular temperature) that accounts for the energy lost in inelastic collisions. Apart from the presence of an extra dissipative term, this equation is very similar in form to the usual temperature equation in hydrodynamics. It is shown how a lattice-kinetic model based on the Bhatnagar-Gross-Krook (BGK) equation that was previously derived for a miscible two-component fluid may be modified to model the continuum equations for granular flow. This is done by noting that the variable corresponding to the concentration of one species follows an equation that is essentially analogous to the granular temperature equation. A simulation of an unforced granular fluid using the modified model reproduces the phenomenon of clustering instability, namely the spontaneous agglomeration of particles into dense clusters, which occurs generically in all granular flows. The success of the continuum theory in capturing the gross features of this basic phenomenon is discussed. Some shear flow simulations are also presented.     

Layered image coding using the DCT pyramid

A block-based subband image coder that exploits the ability to perform decimation in the discrete cosine transform (DCT) domain to effect a pyramidal data structure is described. The proposed "DCT pyramid" has a distinct feature of improved image rendition properties without the associated blocking artifacts at low bit-rates.     

In Situ Topochemical Transformation of ZnIn2S4 for Efficient Photocatalytic Oxidation of 5-Hydroxymethylfurfural to 2,5-Diformylfuran

Photocatalytic selective oxidation of 5-hydroxymethylfurfural (HMF) coupled H₂ production offers a promising approach to producing valuable chemicals. Herein, an efficient in situ topological transformation tactic is developed for producing porous O-doped ZnIn₂S₄ nanosheets for HMF oxidation cooperative with H₂ evolution. Aberration-corrected high-angle annular dark-field scanning TEM images show that the hierarchical porous O-ZIS-120 possesses abundant atomic scale edge steps and lattice defects, which is beneficial for electron accumulation and molecule adsorption. The optimal catalyst (O-ZIS-120) exhibits remarkable performance with 2,5-diformylfuran (DFF) yields of 1624 µmol h⁻¹ g⁻¹ and the selectivity of >97%, simultaneously with the H₂ evolution rate of 1522 µmol h⁻¹ g⁻¹. Mechanistic investigations through theoretical calculations show that O in the O-ZIS-120 lattice can reduce the oxidation energy barrier of hydroxyl groups of HMF. In situ attenuated total reflection surface-enhanced infrared absorption spectroscopy (ATR-SEIRAS) results reveal that DFF^* (C₄H₂(CHO)₂O^*) intermediate has a weak interaction with O-ZIS-120 and desorb as the final product. This study elucidates the topotactic structural transitions of 2D materials simultaneously with electronic structure modulation for efficient photocatalytic DFF production.