Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques
Summary
This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).
Abstract
This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.
Introduction
Heteroxylans are a family of polysaccharides that are the predominant non-cellulosic polysaccharides of the primary walls of grasses and the secondary walls of all angiosperms3-6. The xylan backbones differ in their types and patterns of substitution with glycosyl (glucuronic acid (GlcA), arabinose (Araf)) and non-glycosyl (O-acetyl, ferulic acid) residues depending upon tissue type, developmental stage and species7.
Walls from wheat (Triticum aestivum L.) endosperm are composed primarily of arabinoxylans (AXs) (70%) and (1→3)(1→4)-β-D-glucans (20%) with minor amounts of cellulose and heteromannans (2% each)8. The xylan backbone may be variously un-substituted and predominantly mono-substituted (primarily O-2 position and to a lesser extent O-3 position) and di-substituted (O-2 and O-3 positions) with α-L-Araf residues9. The reducing end (RE) of heteroxylans from dicots (for example, Arabidopsis thaliana)10 and gymnosperms (for example, spruce (Picea abies))11 contains a characteristic tetrasaccharide glycosyl sequence; -β-D-Xylp-(1→3)-α-L-Rhap-(1→2)-α-D-GalpA-(1→4)-D-Xylp. To understand heteroxylan biosynthesis and function (biological and industrial), it is important to fully sequence the xylan backbone to understand the types and the patterns of substitutions as well as the sequence of the reducing end (RE).
Specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans are described in this manuscript. The techniques rely on fluorophore tagging (with 2 aminobenzamide (2AB)) the reducing end (RE) of the heteroxylan chain prior to enzymatic (endoxylanase) hydrolysis. This approach, particularly for the RE sequencing, was first reported by the York laboratory10,12-13 but is now extended to include the internal region sequencing and is a combination of established techniques that is equally adaptable to all heteroxylans independent of their source of isolation. This approach can also be applied to other classes of polysaccharides using (where available) the appropriate endo-hydrolases.
In the present study, de-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR) and sequentially extracted with water (W-sol Fr) and 1M KOH containing 1% NaBH4 (KOH-sol Fr) as described in Ratnayake et al. (2014)2. The released oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, ESI-QTOF-MS-coupled with HPLC with the online chromatographic separation using a RP C-18 column and ESI-MSn. Endoxylanase digested KOH-sol AXs was also characterized by nuclear magnetic resonance (NMR).
Protocol
Representative Results
Discussion
Most matrix phase cell wall polysaccharides have seemingly randomly substituted backbones (with both glycosyl and non-glycosyl residues) that are highly variable depending upon the plant species, developmental stage and tissue type3. Since polysaccharides are secondary gene products their sequence is not template derived and there is therefore no single analytical approach, such as exists for nucleic acids and proteins, for their sequencing. The availability of purified linkage-specific hydrolytic enzymes has …
Declarações
The authors have nothing to disclose.
Acknowledgements
This project was supported by funds from Commonwealth Scientific and Research Organisation Flagship Collaborative Research Program, provided to the High Fibre Grains Cluster via the Food Futures Flagship. AB also acknowledges the support of an Australia Research Council (ARC) grant to the ARC Centre of Excellence in Plant Cell Walls (CE110001007).
Materials
| 2 aminobenzamide (2AB) | Sigma-Aldrich (www.sigmaaldrich.com) | A89804 | |
| sodium borohydride (NaBH4) | Sigma-Aldrich (www.sigmaaldrich.com) | 247677 | Hazardous, handle with care |
| sodium cyanoborohydride (NaBH3CN) | Sigma-Aldrich (www.sigmaaldrich.com) | 156159 | Hazardous, handle with care |
| endo-1,4-β-Xylanase M1 (from Trichoderma viride) (120101a) | Megazyme (www.megazyme.com) | E-XYTR1 | |
| Deuterium Oxide (D2O) | Sigma-Aldrich (www.sigmaaldrich.com) | 151882 | |
| Freeze dryer (CHRIST-ALPHA 1-4 LD plus) | |||
| RP C18 Zorbax eclipse plus column | Agilent | (2.1×100 mm; 1.8 µm bead size) | |
| MicroFlex MALDI-TOF MS (Model – MicroFlex LR) | (Bruker Daltonics, Germany) | ||
| (ESI) -(QTOF) MS (Model # 6520) | (Agilent, Palo Alto, CA ) | ||
| ESI-MSn - ion-trap (Model # 1100 HCT) | (Agilent, Palo Alto, CA). | ||
| Bruker Avance III 600 MHz -NMR | Bruker Daltonics, Germany | ||
| Topspin (version 3.0)-Biospin- software | Bruker | ||
| GC-MS (Model # 7890B) | Agilent |
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