Bioinspired Thermo-Responsive Xyloglucan-Cellulose Nanocrystal Hydrogels

Thermo-responsive hydrogels present unique properties, such as tunable mechanical performance or changes in volume, which make them attractive for applications including wound healing dressings, drug delivery vehicles, and implants, among others. This work reports the implementation of bio-based thermo-responsive hydrogels comprised of xyloglucan (XG) and cellulose nanocrystals (CNCs). Thermo-responsive properties were obtained by enzymatic degalactosylation of tamarind seed XG (DG-XG), which reduced the galactose residue content by ~50%, and imparted a reversible thermal transition. XG with comparable molar mass to DG-XG was achieved by ultrasonication treatment (XGu) for direct comparison of behavior. The hydrogels were prepared by simple mixing of DG-XG or XGu with CNCs in water. Phase diagrams were established to identify the ratios of DG-XG or XGu to CNCs (from 1:300 to 20:1 by mass) that yielded a viscous liquid, a phase separated mixture, a simple gel, or a thermo-responsive gel. Gelation occurred at a DG-XG or XGu to CNC ratio higher than that needed for the full surface coverage of CNCs, and required relatively high overall concentrations of both components (tested concentrations up to 20 g/L XG and 30 g/L CNCs). This is likely a result of the increase in effective hydrodynamic volume of CNCs due to the formation of XG-CNC complexes. Investigation of the adsorption behavior indicated that DG-XG formed a more rigid layer on CNCs compared to XGu. Rheological properties of the hydrogels were characterized and a reversible thermal transition was found for DG-XG/CNC gels at 35°C, where the mechanical properties of the gel could be tuned by adjusting the CNC content

Bioinspired Thermo-Responsive Xyloglucan-Cellulose Nanocrystal Hydrogels

Thermo-responsive hydrogels present unique properties, such as tunable mechanical performance or changes in volume, which make them attractive for applications including wound healing dressings, drug delivery vehicles, and implants, among others. This work reports the implementation of bio-based thermo-responsive hydrogels comprised of xyloglucan (XG) and cellulose nanocrystals (CNCs). Thermo-responsive properties were obtained by enzymatic degalactosylation of tamarind seed XG (DG-XG), which reduced the galactose residue content by ~50%, and imparted a reversible thermal transition. XG with comparable molar mass to DG-XG was achieved by ultrasonication treatment (XGu) for direct comparison of behavior. The hydrogels were prepared by simple mixing of DG-XG or XGu with CNCs in water. Phase diagrams were established to identify the ratios of DG-XG or XGu to CNCs (from 1:300 to 20:1 by mass) that yielded a viscous liquid, a phase separated mixture, a simple gel, or a thermo-responsive gel. Gelation occurred at a DG-XG or XGu to CNC ratio higher than that needed for the full surface coverage of CNCs, and required relatively high overall concentrations of both components (tested concentrations up to 20 g/L XG and 30 g/L CNCs). This is likely a result of the increase in effective hydrodynamic volume of CNCs due to the formation of XG-CNC complexes. Investigation of the adsorption behavior indicated that DG-XG formed a more rigid layer on CNCs compared to XGu. Rheological properties of the hydrogels were characterized and a reversible thermal transition was found for DG-XG/CNC gels at 35°C, where the mechanical properties of the gel could be tuned by adjusting the CNC content

Identification of two functional xyloglucan galactosyltransferase homologs BrMUR3 and BoMUR3 in brassicaceous vegetables

Xyloglucan (XyG) is the predominant hemicellulose in the primary cell walls of most dicotyledonous plants. Current models of these walls predict that XyG interacts with cellulose microfibrils to provide the wall with the rigidity and strength necessary to maintain cell integrity. Remodeling of this network is required to allow cell elongation and plant growth. In this study, homologs of Arabidopsis thaliana MURUS3 (MUR3), which encodes a XyG-specific galactosyltransferase, were obtained from Brassica rapa (BrMUR3) to Brassica oleracea (BoMUR3). Genetic complementation showed that BrMUR3 and BoMUR3 rescue the phenotypic defects of the mur3-3 mutant. Xyloglucan subunit composition analysis provided evidence that BrMUR3 and BoMUR3 encode a galactosyltransferase, which transfers a galactose residue onto XyG chains. The detection of XXFG and XLFG XyG subunits (restoration of fucosylated side chains) in mur3-3 mutants overexpressing BrMUR3 or BoMUR3 show that MUR3 from Brassica to Arabidopsis are comparable as they add Gal to the third xylosyl residue of the XXXG subunit. Our results provide additional information for functional dissection and evolutionary analysis of MUR3 genes derived from brassicaceous species.

Glucocerebrosidases catalyze a transgalactosylation reaction that yields a newly-identified brain sterol metabolite, galactosylated cholesterol

β-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and nonlysosomal GBA2 possess not only GlcCer hydrolase activity, but also transglucosylation activity to transfer the glucose residue from GlcCer to cholesterol to form β-cholesterylglucoside (β-GlcChol) in vitro. β-GlcChol is a member of sterylglycosides present in diverse species. How GBA1 and GBA2 mediate β-GlcChol metabolism in the brain is unknown. Here, we purified and characterized sterylglycosides from rodent and fish brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of rat brain sterylglycosides revealed the presence of galactosylated cholesterol (β-GalChol), in addition to β-GlcChol. Analyses of brain tissues from GBA2-deficient mice and GBA1- and/or GBA2-deficient Japanese rice fish (Oryzias latipes) revealed that GBA1 and GBA2 are responsible for β-GlcChol degradation and formation, respectively, and that both GBA1 and GBA2 are responsible for β-GalChol formation. Liquid chromatography–tandem MS revealed that β-GlcChol and β-GalChol are present throughout development from embryo to adult in the mouse brain. We found that β-GalChol expression depends on galactosylceramide (GalCer), and developmental onset of β-GalChol biosynthesis appeared to be during myelination. We also found that β-GlcChol and β-GalChol are secreted from neurons and glial cells in association with exosomes. In vitro enzyme assays confirmed that GBA1 and GBA2 have transgalactosylation activity to transfer the galactose residue from GalCer to cholesterol to form β-GalChol. This is the first report of the existence of β-GalChol in vertebrates and how β-GlcChol and β-GalChol are formed in the brain.

GtcA is required for LTA glycosylation in Listeria monocytogenes serovar 1/2a and Bacillus subtilis

ABSTRACTThe cell wall polymers wall teichoic acid (WTA) and lipoteichoic acid (LTA) are often modified with glycosyl and D-alanine residues. Recent studies have shown that a three-component glycosylation system is used for the modification of LTA in several Gram-positive bacteria including Bacillus subtilis and Listeria monocytogenes. In the L. monocytogenes 1/2a strain 10403S, the cytoplasmic glycosyltransferase GtlA is thought to use UDP-galactose to produce the C55-P-galactose lipid intermediate, which is transported across the membrane by an unknown flippase. Next, the galactose residue is transferred onto the LTA backbone on the outside of the cell by the glycosyltransferase GtlB. Here we show that GtcA is necessary for the glycosylation of LTA in L. monocytogenes 10403S and B. subtilis 168 and we hypothesize that these proteins act as C55-P-sugar flippases. With this we revealed that GtcA is involved in the glycosylation of both teichoic acid polymers in L. monocytogenes 10403S, namely WTA with N-acetylglucosamine and LTA with galactose residues. These findings indicate that the L. monocytogenes GtcA protein can act on different C55-P-sugar intermediates. Further characterization of GtcA in L. monocytogenes led to the identification of residues essential for its overall function as well as residues, which predominately impact WTA or LTA glycosylation.GRAPHICAL ABSTRACT

Biochemical and structural investigation of two paralogous glycoside hydrolases fromZobellia galactanivorans: novel insights into the evolution, dimerization plasticity and catalytic mechanism of the GH117 family

The family 117 glycoside hydrolase (GH117) enzymes have exo-α-1,3-(3,6-anhydro)-L-galactosidase activity, removing terminal nonreducing α-1,3-linked 3,6-anhydro-L-galactose residues from their red algal neoagarose substrate. These enzymes have previously been phylogenetically divided into clades, and only the clade A enzymes have been experimentally studied to date. The investigation of two GH117 enzymes, Zg3615 and Zg3597, produced by the marine bacteriumZobellia galactanivoransreveals structural, biochemical and further phylogenetic diversity between clades. A product complex with the unusual β-3,6-anhydro-L-galactose residue sheds light on the inverting catalytic mechanism of the GH117 enzymes as well as the structure of this unique sugar produced by hydrolysis of the agarophyte red algal cell wall.

Extraction and Partial Characterisation of Hydrocolloid Gums from Some African Legumes

In an attempt to understand the potential valorisation of local African legumes, hydrocolloids of five legumes (Corchorus olithorus, Triumfetta cordifolia, Cerathoteca sesamoides, Adansona digitata, and Bridelia thermifolia) were extracted and characterised as polysaccharides. All the gum extracted were rich in galactose residue (31-62 percent), suggesting a galactan backbone for the polysaccharides structure. The other sugar residues of the polysaccharides were arabinose (22-30 percent) in T. cordifolia and B. thermifolia, glucose (22-36 percent) in B. thermofolia, A. digitata and C. olithorus, and mannose (32.9 percent) in C. sesamoides. The intrinsic viscosity measurements showed that gums from T. cordifolia, B. thermifolia, C sesamoides and C. olithorus are high molecular weight polymers, while A. digitata contains low molecular weight polymers. The gum extracts also showed oil/water emulsion activity and were able to keep 60-90 percent of the emulsion stable on heating.

Thiolactosides: Scaffolds for the Synthesis of Glycolipids in Animal Cells

Various glycolipids were synthesized using thiolactosides as scaffolds for glycosylation in animal cells. The basic building blocks, n-dodecyl β-D-thiolactoside (β-LacSC12) and n-dodecyl α-D-thiolactoside (α-LacSC12), were chemically synthesized in 2 steps: glycosylation followed by deacylation. The thiolactosides were administered to animal cells in culture and served as substrates for cellular enzyme-catalyzed glycosylation. Incubation of mouse melanoma B16 cells in the presence of β-LacSC12 or α-LacSC12 resulted in sialylation of the terminal galactose residue and gave a GM3-type ganglioside. Administration of β-Lac SC12 in Madin-Darby canine kidney (MDCK) cells likewise gave a GM3-type ganglioside. On the other hand, introduction of β-LacSC12 in African green monkey kidney (Vero) cells gave Gb3- and Gb4-type glycolipids aside from GM3-type ganglioside. In the course of the study, significant changes in B16 cell morphology and elevated secretion of melanin were also observed.