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.
A polysaccharide with 40% mono-O-methylated monosaccharides from the bark of Cola cordifolia (Sterculiaceae), a medicinal tree from Mali (West Africa).
Togola, A., Naess, K. H., Diallo, D., Barsett, H., Michaelsen, T. E. & Paulsen, B. S. (2008). Carbohydrate Polymers, 73(2), 280-288.
A novel type polysaccharide was isolated from the bark of Cola cordifolia (Cav.) R. Br. (Sterculiaceae), a plant used in traditional medicine in Mali (West Africa). The polysaccharide was isolated from the water extract by ion exchange chromatography. Structural studies showed that this was a highly complex new type polysaccharide containing 20% of 2,3- and 2,4-linked rhamnose, 24% of galacturonic acid mostly 4-linked, 15% of terminal, 3- and 4-linked galactose, 20% of terminal and 3-linked 2-O-methyl galactose, 18% of 4-O-methyl glucuronic acid which was also terminally linked, and 2% of terminal 2-O-methyl fucose. This paper reports in addition to structural features, physical property and complement fixating activity using human serum as target of this novel polysaccharide. This is a first report of a plant polysaccharide containing such a diverse composition and quantity of natively methylated monosaccharides.
Cloning, characterisation and expression analysis of α-glucuronidase from the thermophilic fungus Talaromyces emersonii.
Heneghan, M. N., McLoughlin, L., Murray, P. G. & Tuohy, M. G. (2007). Enzyme and Microbial Technology, 41(6), 677-682.
The aguA gene encoding α-glucuronidase was isolated from the thermophilic fungus Talaromyces emersonii by degenerate PCR. AguA has no introns and consists of an open reading frame of 2511 bp, encoding a putative protein of 837 amino acids. The N-terminus of the protein contains a putative signal peptide of 17 amino acids yielding a mature protein of 820 amino acids with a predicted molecular mass of 91.6 kDa. Twenty putative N-glycosylation sites and four O-glycosylation were identiﬁed. The T. emersonii α-glucuronidase falls into glycosyl hydrolase family 67, showing approximately 63% identity to similar enzymes from other fungi. Analysis of the aguA promoter revealed several possible regulatory motifs including two XlnR and a CreA binding site. Enzyme activity was optimal at 50°C and pH 5. Enzyme production was investigated on a range of carbon sources and showed induction on beechwood, oat spelt and birchwood xylan, and repression by glucose or glucuronic acid.
UDP-xylose-stimulated glucuronyltransferase activity in wheat microsomal membranes: characterization and Role in glucurono(arabino)xylan biosynthesis.
Zeng, W., Chatterjee, M. & Faik, A.(2008). Plant Physiology, Vol. 147.
Microsomal membranes from etiolated wheat (Triticum aestivum) seedlings cooperatively incorporated xylose (Xyl), arabinose, and glucuronic acid residues from their corresponding uridine 5′-diphosphosugars into an ethanol-insoluble glucurono(arabino)xylan (GAX)-like product. A glucuronyltransferase activity that is enhanced by the presence of UDP-Xyl was also identified in these microsomes. Wheat glucuronyltransferase activity was optimal at pH 7 and required manganese ions, and several lines of evidence suggest its involvement in GAX-like biosynthesis. The GAX characteristics of the 14C-product were confirmed by digestion with a purified endo-xylanase from Aspergillus awamori (endo-xylanase III) and by total acid hydrolysis, resulting in a Xyl:arabinose:glucuronic acid molar ratio of approximately 105:34:1. Endo-xylanase III released only three types of oligosaccharides in addition to free Xyl. No radiolabel was released as xylobiose, xylotriose, or xylotetraose, indicating the absence of long stretches of unbranched Xyl residues in the nascent GAX-like product. High-pH anion exchange chromatography analysis of the resulting oligosaccharides along with known arabinoxylan oligosaccharide standards suggests that a portion of the nascent GAX-like product has a relatively regular structure. The other portion of the [14C]GAX-like polymer was resistant to proteinase K, endo-polygalacturonase, and endo-xylanase III (GH11 family) but was degraded by Driselase, supporting the hypothesis that the xylan backbone in this portion of the product is most likely highly substituted. Size exclusion chromatography indicated that the nascent GAX-like polymer had an apparent molecular mass of approximately 10 to 15 kD; however, mature GAXs from wheat cell walls had larger apparent molecular masses (>66 kD).
The Structural Basis for Catalysis and Specificity of the Pseudomonas cellulosa α-Glucuronidase, GlcA67A.
Nurizzo, D., Nagy, T., Gilbert, H. J. & Davies, G. J. (2002). Structure, 10(4), 547-556.
α-glucuronidases, components of an ensemble of enzymes central to the recycling of photosynthetic biomass, remove the α-1,2 linked 4-O-methyl glucuronic acid from xylans. The structure of the α-glucuronidase, GlcA67A, from Pseudomonas cellulosa reveals three domains, the central of which is a (β/α)8 barrel housing the catalytic apparatus. Complexes of the enzyme with the individual reaction products, either xylobiose or glucuronic acid, and the ternary complex of both glucuronic acid and xylotriose reveal a “blind” pocket which selects for short decorated xylooligosaccharides substituted with the uronic acid at their nonreducing end, consistent with kinetic data. The catalytic center reveals a constellation of carboxylates; Glu292 is poised to provide protonic assistance to leaving group departure with Glu393 and Asp365 both appropriately positioned to provide base-catalyzed assistance for inverting nucleophilic attack by water.