Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.
Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.
Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.
Characterization of a novel β-glucosidase from a compost microbial metagenome with strong transglycosylation activity.
Uchiyama, T., Miyazaki, K. & Yaoi, K. (2013). Journal of Biological Chemistry, 288(25), 18325-18334.
The β-glucosidase encoded by the td2f2 gene was isolated from a compost microbial metagenomic library by functional screening. The protein was identified to be a member of the glycoside hydrolase family 1 and was overexpressed in Escherichia coli, purified, and biochemically characterized. The recombinant β-glucosidase, Td2F2, exhibited enzymatic activity with β-glycosidic substrates, with preferences for glucose, fucose, and galactose. Hydrolysis occurred at the nonreducing end and in an exo manner. The order of catalytic efficiency for glucodisaccharides and cellooligosaccharides was sophorose > cellotetraose > cellotriose > laminaribiose > cellobiose > cellopentaose > gentiobiose, respectively. Intriguingly, the p-nitrophenyl-β-D-glucopyranoside hydrolysis activity of Td2F2 was activated by various monosaccharides and sugar alcohols. At a D-glucose concentration of 1000 mM, enzyme activity was 6.7-fold higher than that observed in the absence of D-glucose. With 31.3 mM D-glucose, Td2F2 catalyzed transglycosylation to generate sophorose, laminaribiose, cellobiose, and gentiobiose. Transglycosylation products were detected under all activated conditions, suggesting that the activity enhancement induced by monosaccharides and sugar alcohols may be due to the transglycosylation activity of the enzyme. These results show that Td2F2 obtained from a compost microbial metagenome may be a potent candidate for industrial applications.
Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls.
Marcus, S. E., Verhertbruggen, Y., Hervé, C., Ordaz-Ortiz, J. J., Farkas, V., Pedersen, H. L., Willats, W. G. T. & Knox, J. P. (2008). BMC Plant Biology, 8(1), 60-72.
Background: Molecular probes are required to detect cell wall polymers in-situ to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure. Results: Using a neoglycoprotein approach, in which a XXXG heptasaccharide of tamarind seed xyloglucan was coupled to BSA to produce an immunogen, we have generated a rat monoclonal antibody (designated LM15) to the XXXG structural motif of xyloglucans. The specificity of LM15 has been confirmed by the analysis of LM15 binding using glycan microarrays and oligosaccharide hapten inhibition of binding studies. The use of LM15 for the analysis of xyloglucan in the cell walls of tamarind and nasturtium seeds, in which xyloglucan occurs as a storage polysaccharide, indicated that the LM15 xyloglucan epitope occurs throughout the thickened cell walls of the tamarind seed and in the outer regions, adjacent to middle lamellae, of the thickened cell walls of the nasturtium seed. Immunofluorescence analysis of LM15 binding to sections of tobacco and pea stem internodes indicated that the xyloglucan epitope was restricted to a few cell types in these organs. Enzymatic removal of pectic homogalacturonan from equivalent sections resulted in the abundant detection of distinct patterns of the LM15 xyloglucan epitope across these organs and a diversity of occurrences in relation to the cell wall microstructure of a range of cell types. Conclusion: These observations support ideas that xyloglucan is associated with pectin in plant cell walls. They also indicate that documented patterns of cell wall epitopes in relation to cell development and cell differentiation may need to be re-considered in relation to the potential masking of cell wall epitopes by other cell wall components.
Characterization of all Family-9 glycoside hydrolase synthesized by the cellulosome-producing bacterium Clostridium cellulolyticum.
Ravachol, J., Borne, R., Tardif, C., de Philip, P. & Fierobe, H. P. (2014). Journal of Biological Chemistry, 289(11), 7335-7348.
The genome of Clostridium cellulolyticum encodes 13 GH9 enzymes that display 7 distinct domain organizations. All but one contain a dockerin module and were formerly detected in the cellulosomes, but only three of them were previously studied (Cel9E, Cel9G and Cel9M). In the present report, the ten uncharacterized GH9 enzymes were overproduced in Escherichia coli, purified and their activity pattern was investigated in the free state, or in cellulosome chimeras with key cellulosomal cellulases. The newly purified GH9 enzymes, including those that share similar organization, all exhibited distinct activity patterns, various binding capacities on cellulosic substrates, and different synergies with pivotal cellulases in minicellulosomes. Furthermore, one enzyme (Cel9X) was characterized as the first genuine endoxyloglucanase belonging to this family, with no activity on soluble and insoluble celluloses. Another GH9 enzyme (Cel9V), whose sequence is 78% identical to the cellulosomal cellulase Cel9E, was found inactive in the free and complexed states on all tested substrates. The sole non-cellulosomal GH9 (Cel9W) is a cellulase displaying a broad substrate specificity, whose engineered form bearing a dockerin can act synergistically in minicomplexes. Finally, incorporation of all GH9 cellulases in trivalent cellulosome chimera containing Cel48F and Cel9G generated a mixture of heterogeneous minicellulosomes that exhibit more activity on crystalline cellulose than the best homogenous tri-functional complex. Altogether our data emphasize the importance of GH9 diversity in bacterial cellulosomes, confirm that Cel9G is the most synergistic GH9 with the major endoprocessive cellulase Cel48F, but also identify Cel9U as an important cellulosomal component during cellulose depolymerisation.
Identification and Use of Fluorescent Dyes for Plant Cell Wall Imaging Using High-Throughput Screening.
Anderson, C. T. & Carroll, A. (2014). Plant Chemical Genomics Methods in Molecular Biology, 1056, 103-109.
Plant cell walls define cell shape during development and are composed of interlaced carbohydrate and protein networks. Fluorescent dyes have long been used to label plant cell walls, enabling optical microscopy-based interrogation of cell wall structure and composition. However, the specific cell wall components to which these dyes bind are often poorly defined. The availability of fluorescent compound libraries provides the potential to screen for and identify new fluorescent compounds that interact with specific plant cell wall components, enabling the study of cell wall architecture in intact, living tissues. Here, we describe a technique for screening fluorescent compound libraries for enhanced fluorescence upon interaction with plant cell walls, a secondary screening method to identify which cell wall components interact with a given dye, and a protocol for staining and observing Arabidopsis seedlings using a fluorescent cell wall-labeling dye. These methods have the potential to be applied to screening for differences in cell wall structure and composition among genetically diverse plant varieties or species.
Real-time imaging of cellulose reorientation during cell wall expansion in Arabidopsis roots.
Anderson, C. T., Carroll, A., Akhmetova, L. & Somerville, C. (2010). Plant Physiology, 152(2), 787-796.
Cellulose forms the major load-bearing network of the plant cell wall, which simultaneously protects the cell and directs its growth. Although the process of cellulose synthesis has been observed, little is known about the behavior of cellulose in the wall after synthesis. Using Pontamine Fast Scarlet 4B, a dye that fluoresces preferentially in the presence of cellulose and has excitation and emission wavelengths suitable for confocal microscopy, we imaged the architecture and dynamics of cellulose in the cell walls of expanding root cells. We found that cellulose exists in Arabidopsis (Arabidopsis thaliana) cell walls in large fibrillar bundles that vary in orientation. During anisotropic wall expansion in wild-type plants, we observed that these cellulose bundles rotate in a transverse to longitudinal direction. We also found that cellulose organization is significantly altered in mutants lacking either a cellulose synthase subunit or two xyloglucan xylosyltransferase isoforms. Our results support a model in which cellulose is deposited transversely to accommodate longitudinal cell expansion and reoriented during expansion to generate a cell wall that is fortified against strain from any direction.
Isolation and characterization of two types of xyloglucanases from a phytopathogenic fungus, Verticillium dahliae.
Sato, S., Ohta, K., Kojima, K., Kozeki, T., Ohmachi, T. & Yoshida, T. (2016). Journal of Applied Glycoscience, 63(1), 13-18.
Xyloglucan is a major hemicellulosic component in plant cell walls. Phytopathogenic fungi secrete cell wall-degrading enzymes on their infection to hosts, while the nature of the cell wall-lytic enzymes of such fungi are yet to be fully understood. Verticillium dahliae is a soil-borne fungus that causes vascular wilt diseases in a variety of commercially important crops worldwide. We purified two types of xyloglucanases, XEG12A and XEG74B, from the culture of naturally isolated Verticillium dahliae strain 2148. XEG12A showed a molecular size of 23 kDa with its maximal activity at pH 7.5. XEG12A specifically hydrolyzed xyloglucan with no activity on other β-glucans. XEG74B had a molecular size of 110 kDa with its optimum pH at 6.0. XEG74B primarily hydrolyzed xyloglucan, with a slight activity on β-1,3-1,4-glucan. Analysis of hydrolytic products of xyloglucanooligasaccharide (XXXGXXXG) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that the both enzymes cleaved β-1,4-glucosidic linkage at the position of unbranched chain, while XEG74B showed a little fluctuation with the cleavage site. Both enzymes did not hydrolyzed xyloglucanoheptasaccharide (XXXG) at all. N-Terminal and internal amino acid sequencing of the enzymes revealed that XEG12A and XEG74B belonged to Glycoside Hydrolase (GH) Families 12 and 74, respectively. Based on these results we concluded that V. dahliae XEG12A and XEG74B were xyloglucan-specific endo-β-1,4-glucanases (EC 188.8.131.52).