Functionalization of Cellulose-Based Hydrogels with Bi-Functional Fusion Proteins Containing Carbohydrate-Binding Modules

Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. This functionalization can deliver tailored cellulose-based materials with enhanced physical and chemical properties and control of biological interactions that match specific applications. One of the foundations for the success of such biomaterials is to efficiently control the capacity to combine relevant biomolecules into cellulose materials in such a way that the desired functionality is attained. In this context, our main goal was to develop bi-functional biomolecular constructs for the precise modification of cellulose hydrogels with bioactive molecules of interest. The main idea was to use biomolecular engineering techniques to generate and purify different recombinant fusions of carbohydrate binding modules (CBMs) with significant biological entities. Specifically, CBM-based fusions were designed to enable the bridging of proteins or oligonucleotides with cellulose hydrogels. The work focused on constructs that combine a family 3 CBM derived from the cellulosomal-scaffolding protein A from Clostridium thermocellum (CBM3) with the following: (i) an N-terminal green fluorescent protein (GFP) domain (GFP-CBM3); (ii) a double Z domain that recognizes IgG antibodies; and (iii) a C-terminal cysteine (CBM3C). The ability of the CBM fusions to bind and/or anchor their counterparts onto the surface of cellulose hydrogels was evaluated with pull-down assays. Capture of GFP-CBM3 by cellulose was first demonstrated qualitatively by fluorescence microscopy. The binding of the fusion proteins, the capture of antibodies (by ZZ-CBM3), and the grafting of an oligonucleotide (to CBM3C) were successfully demonstrated. The bioactive cellulose platform described here enables the precise anchoring of different biomolecules onto cellulose hydrogels and could contribute significatively to the development of advanced medical diagnostic sensors or specialized biomaterials, among others.

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Ubiquitous Carbohydrate Binding Modules Decorate 936 Lactococcal Siphophage Virions

Viruses ◽

10.3390/v11070631 ◽

2019 ◽

Vol 11 (7) ◽

pp. 631 ◽

Cited By ~ 7

Author(s):

Stephen Hayes ◽

Jennifer Mahony ◽

Renaud Vincentelli ◽

Laurie Ramond ◽

Arjen Nauta ◽

...

Keyword(s):

Fluorescent Protein ◽

Bioinformatic Analysis ◽

Infection Process ◽

Ecological Niches ◽

Carbohydrate Binding ◽

Binding Assays ◽

Protein Fusion ◽

Carbohydrate Binding Modules ◽

Structural Assembly ◽

Green Fluorescent

With the availability of an increasing number of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and functionality of phage adhesion devices. In the current study, we examined 113 members of the 936 group of lactococcal siphophages, and identified a number of Carbohydrate Binding Modules (CBMs) in the neck passage structure and major tail protein, on top of evolved Dit proteins, as recently reported by us. The binding ability of such CBM-containing proteins was assessed through the construction of green fluorescent protein fusion proteins and subsequent binding assays. Two CBMs, one from the phage tail and another from the neck, demonstrated definite binding to their phage-specific host. Bioinformatic analysis of the structural proteins of 936 phages reveals that they incorporate binding modules which exhibit structural homology to those found in other lactococcal phage groups and beyond, indicating that phages utilize common structural “bricks” to enhance host binding capabilities. The omnipresence of CBMs in Siphophages supports their beneficial role in the infection process, as they can be combined in various ways to form appendages with different shapes and functionalities, ensuring their success in host detection in their respective ecological niches.

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Differential Evolution of α-Glucan Water Dikinase (GWD) in Plants

Plants ◽

10.3390/plants9091101 ◽

2020 ◽

Vol 9 (9) ◽

pp. 1101

Author(s):

Muyiwa S. Adegbaju ◽

Olanrewaju B. Morenikeji ◽

Eli J. Borrego ◽

André O. Hudson ◽

Bolaji N. Thomas

Keyword(s):

Plant Species ◽

Chemical Properties ◽

Starch Degradation ◽

Carbohydrate Binding ◽

Interacting Proteins ◽

Amino Acid Residues ◽

Phosphate Content ◽

Deleterious Mutations ◽

Carbohydrate Binding Modules ◽

High Phosphate

The alpha-glucan water dikinase (GWD) enzyme catalyzes starch phosphorylation, an integral step in transitory starch degradation. The high phosphate content in stored starch has great industrial value, due to its physio–chemical properties making it more versatile, although the phosphate content of stored starch varies depending on the botanical source. In this study, we used various computational approaches to gain insights into the evolution of the GWD protein in 48 plant species with possible roles in enzyme function and alteration of phosphate content in their stored starch. Our analyses identified deleterious mutations, particularly in the highly conserved 5 aromatic amino acid residues in the dual tandem carbohydrate binding modules (CBM-45) of GWD protein in C. zofingiensis, G. hirsutum, A. protothecoides, P. miliaceum, and C. reinhardtii. These findings will inform experimental designs for simultaneous repression of genes coding for GWD and the predicted interacting proteins to elucidate the role this enzyme plays in starch degradation. Our results reveal significant diversity in the evolution of GWD enzyme across plant species, which may be evolutionarily advantageous according to the varying needs for phosphorylated stored starch between plants and environments.

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Ubiquitous Carbohydrate Binding Modules Decorate 936 Lactococcal Siphophage Virions

10.20944/preprints201907.0044.v1 ◽

2019 ◽

Author(s):

Stephen Hayes ◽

Jennifer Mahony ◽

Renaud Vincentelli ◽

Laurie Ramond ◽

Arjen Nauta ◽

...

Keyword(s):

Fluorescent Protein ◽

Bioinformatic Analysis ◽

Infection Process ◽

Ecological Niches ◽

Carbohydrate Binding ◽

Binding Assays ◽

Protein Fusion ◽

Carbohydrate Binding Modules ◽

Structural Assembly ◽

Green Fluorescent

Abstract: With the availability of an increasing number of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and functionality of phage adhesion devices. In the current study, we examined 113 members of the 936 group of lactococcal siphophages, and identified a number of Carbohydrate Binding Modules (CBMs) in the neck passage structure and major tail protein, on top of evolved Dit proteins as recently reported by us. The binding ability of such CBM-containing proteins was assessed through the construction of green fluorescent protein fusion proteins and subsequent binding assays. Two CBMs, one from the phage tail and another from the neck, demonstrated definite binding to their phage-specific host. Bioinformatic analysis of the structural proteins of 936 phages reveals that they incorporate binding modules which exhibit structural homology to those found in other lactococcal phage groups and beyond, indicating that phages utilize common structural “bricks” to enhance host binding capabilities. The omnipresence of CBMs in Siphophages supports their beneficial role in the infection process, as they can be combined in various ways to form appendages with different shapes and functionalities, ensuring their success in host detection in their respective ecological niches.

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The most abundant cyst wall proteins ofAcanthamoeba castellaniiare cellulose-binding lectins from three gene families that localize to distinct structures in cyst walls

10.1101/496307 ◽

2018 ◽

Cited By ~ 1

Author(s):

Pamela Magistrado-Coxen ◽

Yousuf Aqeel ◽

Angelo Lopez ◽

John R. Haserick ◽

Breeanna R. Urbanowicz ◽

...

Keyword(s):

Cyst Wall ◽

Fluorescent Protein ◽

Acanthamoeba Castellanii ◽

Gene Families ◽

Carbohydrate Binding ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Carbohydrate Binding Modules ◽

Green Fluorescent ◽

Cellulose Binding

AbstractAcanthamoeba castellanii, cause of keratitis and blindness, is an emerging pathogen because of its association with contact lens use. The cyst wall contributes to pathogenesis as cysts are resistant to sterilizing reagents in lens solutions and to antibiotics applied to the eye. Here we used structured illumination microscopy (SIM) and probes for glycopolymers to show that purified cyst walls ofA. castellaniiretain endocyst and ectocyst layers and conical structures (ostioles) that connect them. Mass spectrometry showed candidate cyst wall proteins (CWPs) are dominated by three families of lectins (named here Luke, Leo, and Jonah), because each binds to microcrystalline cellulose +/- chitin. Luke lectins contain two or three carbohydrate-binding modules (CBM49), which were first identified in a tomato cellulase. Leo lectins have two unique domains with eight Cys residues each (8-Cys) +/- a Thr-, Lys-, and His-rich spacer. Jonah lectins contain one or three choice-of-anchor A (CAA) domains previously of unknown function. Representative members of each family were tagged with green fluorescent protein (GFP) and expressed under their own promoters in transfected parasites. A representative Jonah lectin with one CAA domain is made early during encystation and localizes to the ectocyst layer. In contrast, Leo and Luke lectins are made later and localize to the endocyst layer and ostioles. Probes for CWPs (anti-GFP antibodies) and for glycopolymers (maltose-binding protein-fusions with CWPs) suggest Jonah lectin and the glycopolymers to which it binds are accessible in the ectocyst layer, while Luke and Leo lectins and their epitopes are mostly inaccessible in the ectocyst layer and ostioles. In summary, the most abundantA. castellaniiCWPs are three sets of lectins, which have conserved (CBM49s of Luke), newly characterized (CAA of Jonah), or unique carbohydrate-binding modules (8-Cys of Jonah).Author summaryFifty years ago, the cyst wall ofAcanthamoeba castellaniiwas shown to contain cellulose and have an ectocyst layer, an endocyst layer, and conical ostioles that attach them. The goals here were to identify abundant cyst wall proteins (CWPs) and begin to determine how the wall is assembled. We used wheat germ agglutinin to show cyst walls also contain chitin fibrils. When trophozoites are starved of nutrients, they become immotile and make CWPs and glycopolymers in dozens of small vesicles. The primordial cyst wall is composed of a single, thin layer containing cellulose, chitin, and an abundant CWP we called Jonah. The primordial wall also has small, flat ostioles that contain another abundant CWP we called Luke. Jonah (the best candidate for diagnostic antibodies) is accessible in the ectocyst layer of mature cyst walls, while Luke and a third abundant CWP we termed Leo are present but mostly inaccessible in the endocyst layer and ostioles. WhileA. castellaniicyst walls contain cellulose (like plants) and chitin (like fungi), the glycopolymers are made in vesicles rather than at the plasma membrane, and the CWPs (Luke, Leo, and Jonah lectins) are unique to the protist.

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The use of fluorescent protein-tagged carbohydrate-binding modules to evaluate the influence of drying on cellulose accessibility and enzymatic hydrolysis

RSC Advances ◽

10.1039/d0ra05333c ◽

2020 ◽

Vol 10 (45) ◽

pp. 27152-27160

Author(s):

Drake Mboowa ◽

Vinay Khatri ◽

Jack N. Saddler

Keyword(s):

Enzymatic Hydrolysis ◽

Fluorescent Protein ◽

Carbohydrate Binding ◽

Fiber Structure ◽

Micro Scale ◽

Cellulose Accessibility ◽

Carbohydrate Binding Modules

Drying restricts cellulose accessibility at macro- and micro-scale of fiber structure with restriction at macro-fiber significantly limiting accessibility to micro-fibrils.

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Expression, purification, crystallization and preliminary X-ray analysis of CttA, a putative cellulose-binding protein fromRuminococcus flavefaciens

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15008249 ◽

2015 ◽

Vol 71 (6) ◽

pp. 784-789 ◽

Cited By ~ 1

Author(s):

Immacolata Venditto ◽

Pedro Bule ◽

Andrew Thompson ◽

Juan Sanchez-Weatherby ◽

James Sandy ◽

...

Keyword(s):

Plant Cell Wall ◽

Binding Protein ◽

Scaffolding Protein ◽

Carbohydrate Binding ◽

Bacterial Cells ◽

X Ray ◽

Anaerobic Microorganisms ◽

Carbohydrate Binding Modules ◽

Genes Encoding ◽

Cellulose Binding

A number of anaerobic microorganisms produce multi-modular, multi-enzyme complexes termed cellulosomes. These extracellular macromolecular nanomachines are designed for the efficient degradation of plant cell-wall carbohydrates to smaller sugars that are subsequently used as a source of carbon and energy. Cellulolytic strains from the rumens of mammals, such asRuminococcus flavefaciens, have been shown to have one of the most complex cellulosomal systems known. Cellulosome assembly requires the binding of dockerin modules located in cellulosomal enzymes to cohesin modules located in a macromolecular scaffolding protein. Over 220 genes encoding dockerin-containing proteins have been identified in theR. flavefaciensgenome. The dockerin-containing enzymes can be incorporated into the primary scaffoldin (ScaA), which in turn can bind to adaptor scaffoldins (ScaB or ScaC) and subsequently to anchoring scaffoldin (ScaE), thereby attaching the whole complex to the cell surface. However, unlike other cellulosomes such as that fromClostridium thermocellum, theRuminococcusspecies lack a specific carbohydrate-binding module (CBM) on ScaA which recruits the entire complex onto the surface of the substrate. Instead, a cellulose-binding protein, CttA, comprising two putative tandem novel carbohydrate-binding modules and a C-terminal X-dockerin module, which can bind to the cohesin of ScaE, may mediate the attachment of bacterial cells to cellulose. Here, the expression, purification and crystallization of the carbohydrate-binding modular part of the CttA fromR. flavefaciensare described. X-ray data have been collected to resolutions of 3.23 and to 1.61 Å in space groupsP3121 orP3221 andP21, respectively. The structure was phased using bound iodide from the crystallization buffer by SAD experiments.

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Characterization of Thermobifida fusca Cutinase-Carbohydrate-Binding Module Fusion Proteins and Their Potential Application in Bioscouring

Applied and Environmental Microbiology ◽

10.1128/aem.00896-10 ◽

2010 ◽

Vol 76 (20) ◽

pp. 6870-6876 ◽

Cited By ~ 29

Author(s):

Yao Zhang ◽

Sheng Chen ◽

Meng Xu ◽

Artur Cavoco-Paulo ◽

Jing Wu ◽

...

Keyword(s):

Cotton Fiber ◽

Potential Application ◽

Fusion Proteins ◽

Textile Industry ◽

Enzyme Characterization ◽

Binding Activity ◽

Thermobifida Fusca ◽

Carbohydrate Binding ◽

Carbohydrate Binding Modules ◽

Fusion Enzymes

ABSTRACT Cutinase from Thermobifida fusca is thermally stable and has potential application in the bioscouring of cotton in the textile industry. In the present study, the carbohydrate-binding modules (CBMs) from T. fusca cellulase Cel6A (CBMCel6A) and Cellulomonas fimi cellulase CenA (CBMCenA) were fused, separately, to the carboxyl terminus of T. fusca cutinase. Both fusion enzymes, cutinase-CBMCel6A and cutinase-CBMCenA, were expressed in Escherichia coli and purified to homogeneity. Enzyme characterization showed that both displayed similar catalytic properties and pH stabilities in response to T. fusca cutinase. In addition, both fusion proteins displayed an activity half-life of 53 h at their optimal temperature of 50Ã¯Â¿Â½C. Compared to T. fusca cutinase, in the absence of pectinase, the binding activity on cotton fiber was enhanced by 2% for cutinase-CBMCel6A and by 28% for cutinase-CBMCenA, whereas in the presence of pectinase, the binding activity was enhanced by 40% for the former and 45% for the latter. Notably, a dramatic increase of up to 3-fold was observed in the amount of released fatty acids from cotton fiber by both cutinase-CBM fusion proteins when acting in concert with pectinase. This is the first report of improving the scouring efficiency of cutinase by fusing it with CBM. The improvement in activity and the strong synergistic effect between the fusion proteins and pectinase suggest that they may have better applications in textile bioscouring than the native cutinase.

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