Arabinopentaose

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.

Plant cell walls are constructed from a diversity of polysaccharide components. Molecular probes directed to structural elements of these polymers are required to assay polysaccharide structures in situ, and to determine polymer roles in the context of cell wall biology. Here, we report on the isolation and the characterization of three rat monoclonal antibodies that are directed to 1,5-linked arabinans and related polymers. LM13, LM16 and LM17, together with LM6, constitute a set of antibodies that can detect differing aspects of arabinan structures within cell walls. Each of these antibodies binds strongly to isolated sugar beet arabinan samples in ELISAs. Competitive-inhibition ELISAs indicate the antibodies bind differentially to arabinans with the binding of LM6 and LM17 being effectively inhibited by short oligoarabinosides. LM13 binds preferentially to longer oligoarabinosides, and its binding is highly sensitive to arabinanase action, indicating the recognition of a longer linearized arabinan epitope. In contrast, the binding of LM16 to branched arabinan and to cell walls is increased by arabinofuranosidase action. The presence of all epitopes can be differentially modulated in vitro using glycoside hydrolase family 43 and family 51 arabinofuranosidases. In addition, the LM16 epitope is sensitive to the action of β-galactosidase. Immunofluorescence microscopy indicates that the antibodies can be used to detect epitopes in cell walls, and that the four antibodies reveal complex patterns of epitope occurrence that vary between organs and species, and relate both to the probable processing of arabinan structural elements and the differing mechanical properties of cell walls.

The importance of the gut microbiota in human health has led to an increased interest to study probiotic bacteria. Fermented food is a source of already established probiotics, but it also offers an opportunity to discover new taxa. Four strains of Weissella sp. isolated from Indian fermented food have been genome sequenced and classified into the species W. cibaria based on whole-genome phylogeny. The genome of W. cibaria strain 92, known to utilise xylooligosaccharides and produce lactate and acetate, was analysed to identify genes for oligosaccharide utilisation. Clusters including genes involved in transportation, hydrolysis and metabolism of xylooligosaccharides, arabinooligosaccharides and β-glucosides were identified. Growth on arabinobiose and laminaribiose was detected. A 6-phospho-β-glucosidase clustered with a phosphotransferase system was found upregulated during growth on laminaribiose, indicating a mechanism for laminaribiose utilisation. The genome of W. cibaria strain 92 harbours genes for utilising the phosphoketolase pathway for the production of both acetate and lactate from pentose and hexose sugars but lacks two genes necessary for utilising the pentose phosphate pathway. The ability of W. cibaria strain 92 to utilise several types of oligosaccharides derived from dietary fibres, and produce lactate and acetate makes it interesting as a probiotic candidate for further evaluation.

Human milk (HM) supports the healthy development of neonates and exerts many of its beneficial effects via contained free human milk oligosaccharides (HMOS). These HMOS exhibit a complexity and structural diversity that pose a significant analytical challenge. A detailed characterization of HMOS is essential as every individual structure may have a different function/activity. Certain HMOS isomers may even fundamentally differ in their biological function, and especially their characterization by LC or LC-MS is often impaired by co-elution phenomena. Thus, more efficient analytical methodologies with enhanced structural selectivity are required. Therefore, we developed a negative ion mode LC-ESI-MS² approach featuring straightforward sample preparation, environmentally friendly EtOH gradient elution, and enhanced, semiquantitative characterization of distinct native HMOS by multiple reaction monitoring (MRM). Our MRM-LC-MS setup takes advantage of highly selective, glycan configuration-dependent collision-induced dissociation (CID) fragments to identify individual neutral and acidic HMOS. Notably, many human milk oligosaccharide isomers could be distinguished in a retention time-independent manner. This contrasts with other contemporary MRM approaches relying on rather unspecific MRM transitions. Our method was used to determine the most abundant human milk tri-, tetra-, penta-, and hexaoses semiquantitatively in a single LC-MS assay. Detected HMO structures included fucosyllactoses (e.g., 2′-FL), lacto-N-difucotetraose (LDFT), lacto-N-tetraoses (LNTs), lacto-N-fucopentaoses (e.g., LNFP I, LNFP II and III), lacto-N-difucohexaoses (LNDFHs) as well as sialyllactoses (SLs) and tentatively assigned blood group A and B tetrasaccharides from which correct human milk type assignment could be also demonstrated. Correctness of milk typing was validated for milk groups I-IV by high pressure anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (HPAEC-PAD).

Putative arabinanase (PcARA) was cloned from cDNA of Phanerochaete chrysosporium. The gene sequencing indicated that PcARA consisted of 939 nucleotides that encodes for 312 amino acid arabinanase-polypeptide chain, including a signal peptide of 19 amino acids. Three-dimensional homology indicated that this enzyme is a five-bladed β-propeller, belonging to glycosidase family 43 and its secondary structure is consisted of 24 β-sheets. The PcARA-cDNA was expressed in Pichia pastoris using pPICZαC. SDS-PAGE of purified arabinanase showed a single band of 33 kDa that is very close to theoretical molecular mass of 33.9 kDa calculated by its amino acid content. Recombinant arabinanase (rPcARA) exhibited maximum activity at pH and temperature of 5.0 and 60°C, respectively. End-product analysis of debranched arabinan hydrolysis by thin-layer chromatography indicated that rPcARA acted as endo-type. The synergistic action of rPcARA with recombinant xylanase resulted in 72 and 9.3% release of total soluble sugar of arabinoxylan and NaOH-pretreated barley straw, respectively.

Arabinoxylan arabinofuranohydrolases (AXAHs) are family GH51 enzymes that have been implicated in the removal of arabinofuranosyl residues from the (1,4)-β-xylan backbone of heteroxylans. Five genes encoding barley AXAHs range in size from 4.6 kb to 7.1 kb and each contains 16 introns. The barley HvAXAH genes map to chromosomes 2H, 4H, and 5H. A small cluster of three HvAXAH genes is located on chromosome 4H and there is evidence for gene duplication and the presence of pseudogenes in barley. The cDNAs corresponding to barley and wheat AXAH genes were cloned, and transcript levels of the genes were profiled across a range of tissues at different developmental stages. Two HvAXAH cDNAs that were successfully expressed in Nicotiana benthamiana leaves exhibited similar activities against 4-nitrophenyl α-L-arabinofuranoside, but HvAXAH2 activity was significantly higher against wheat flour arabinoxylan, compared with HvAXAH1. HvAXAH2 also displayed activity against (1,5)-α-L-arabinopentaose and debranched arabinan. Western blotting with an anti-HvAXAH antibody was used to define further the locations of the AXAH enzymes in developing barley grain, where high levels were detected in the outer layers of the grain but little or no protein was detected in the endosperm. The chromosomal locations of the genes do not correspond to any previously identified genomic regions shown to influence heteroxylan structure. The data are therefore consistent with a role for AXAH in depolymerizing arabinoxylans in maternal tissues during grain development, but do not provide compelling evidence for a role in remodelling arabinoxylans during endosperm or coleoptile development in barley as previously proposed.

A neoglycoprotein (a heptasaccharide of (1→5)-α-L-linked-arabinosyl residues linked to bovine serum albumin) has been used to generate a rat monoclonal antibody specific to a linear chain of (1→5)-α-L-arabinan which is a structural feature of the side chains of pectins. The antibody, designated LM6, detected 100 ng of debranched sugar beet arabinan in an immunodot binding assay and 1 µg of commercial citrus pectin in a similar assay. Hapten inhibition studies indicated that the antibody recognized 5–6 Ara residues and 50% inhibition of antibody binding in a competitive inhibition ELISA was achieved with ca. 2 ng (21 nM) of (1→5)-α-L-Arabinohexaose. The antibody will be useful for the localization of arabinans in plant tissue and will have uses in the analyses of pectin structure. We report here on the localization of the arabinan epitope in lemon fruits using tissue printing.