BRIEF REPORT |
Monoclonal Antibodies to Plant Cell Wall Xylans and Arabinoxylans
Centre for Plant Sciences, University of Leeds, Leeds, UK
Correspondence to: Dr. Paul Knox, University of Leeds, Centre for Plant Sciences, Faculty of Biological Sciences, Leeds, LS2 9JT, United Kingdom. E-mail: j.p.knox{at}leeds.ac.uk
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Summary |
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(J Histochem Cytochem 53:543546, 2005)
Key Words: arabinoxylan cell wall glucuronoarabinoxylan monoclonal antibody polysaccharide xylan
XYLANS ARE major noncellulosic polysaccharides of plant cell walls and are especially abundant in secondary cell walls. Xylans are chains of ß-1,4-linked D-xylopyranosyl residues that can be substituted with arabinosyl, glucuronosyl (and its 4-O-methyl ether derivative), or acetyl residues (Ebringerová and Heinze 2000). Glucuronoxylans occur in angiosperm secondary cell walls, whereas Commelinoid monocotyledon primary cell walls have abundant glucuronoarabinoxylans (GAXs), and cereal grains have neutral arabinoxylans (Carpita 1996
; Ebringerová and Heinze 2000
). Structural features of xylans can vary during development (Obel et al. 2002
; Suzuki et al. 2000
). All xylans are thought to cross-link cellulose microfibrils and contribute to cell mechanical properties. Commercially, arabinoxylans are important for the functionality of flour and the nutritional value of animal feed.
To understand the functional roles of cell wall polymers and their various substituted forms, antibodies are required to enable the location of specific structural features of polymers in the context of cells and tissues. Such probes are also useful for the analysis of differences in cell walls of mutants and the characterization of polymers during their isolation (Willats and Knox 2003). Antisera to high- and low-substituted forms of xylans and a monoclonal antibody specific to arabinoxylans have been described (Suzuki et al. 2000
; Ordaz-Ortiz et al. 2004
; Guillon et al. 2004
). Here, we describe the generation and use of two monoclonal antibodies for the analysis of xylans and arabinoxylans in planta.
A neoglycoprotein (X5-BSA) was prepared by coupling xylopentaose (X5, Megazyme, Bray, Ireland) to BSA by reductive amination (Roy et al. 1983). X5 BSA (10 mg) was dissolved in 0.5 ml of 0.2 M sodium borate buffer pH 7.3. This was followed by the addition of 15 mg BSA and then 16 mg of sodium cyanoborohydride, and the tube was maintained in a water bath at 50C for 48 hr with occasional mixing. After 48 hr the pH was adjusted to pH 4.0 by the addition of 5.5 µl of 80% (v/v) acetic acid. The solution was then dialyzed extensively against distilled water with several changes over 72 hr. The coupling efficiency was determined following a phenol-sulphuric acid assay of carbohydrate content and indicated an average of 14 X5 oligosaccharides per BSA molecule.
Rat immunization, hybridoma preparation, and cloning procedures were performed as described previously (Willats et al. 1998). Two male Wistar rats were challenged with approximately 100 µg X5 BSA in complete Freund's adjuvant, administered subcutaneously on day 0, with the same amount administered with incomplete Freund's adjuvant on days 34 and 64. On day 115, a selected rat was given a prefusion boost of 100 µg X5 BSA in 1 ml PBS by intraperitoneal injection, and the spleen was taken three days later for isolation of lymphocytes and fusion with rat myeloma cell line IR983F (Bazin 1982
). Antibodies were selected by ELISA using birch wood xylan and wheat arabinoxylan as antigens. Subsequent characterization involved a combination of immunodot assays (IDAs) and competitive-inhibition ELISAs as described by Willats et al. (1998)
. Two monoclonal antibodies, designated LM10 (rat immunoglobulin class IgG2c) and LM11 (class IgM), were selected for full characterization.
Birch wood xylan (98% Xyl), 4-O-methyl-D-glucurono-D-xylan (80% Xyl, 18% Me-GlcA) were obtained from Sigma (UK). Wheat flour arabinoxylan (61% Xyl, 37% Ara) and xylooligosaccharides (X2X6) were obtained from Megazyme International (Bray, Ireland). Soluble oat spelt xylan (93% Xyl, 6% Ara) was provided by Professor H.J. Gilbert (University of Newcastle-upon-Tyne). Maize GAX (48% Xyl, 38% Ara, 8% GlcA) and sorghum GAX (41% Xyl, 46% Ara, 10% GlcA) were provided by Dr. H.A. Schols (Wageningen University, The Netherlands). Other plant cell wall polysaccharides were obtained and prepared as described elsewhere (McCartney et al. 2004).
For immunolabeling experiments, plant materials were excised and immediately fixed in PEM buffer (50 mM PIPES (piperazine-N-N'-bis[2-ethane-sulfonic acid]), 5 mM EGTA (ethylene glycol bis(ß-aminoethylether)-N,N,N'N'-tetraacetic acid), 5 mM MgSO4, pH 6.9) containing 4% paraformaldehyde. Wheat grains were imbibed overnight and then were cut into small cubes of approximately 8 mm3 to include the aleurone layer and were immediately fixed as described previously. Procedures for embedding of plant material in Steedman's wax, sectioning, immunocytochemistry, and use of cellulose-binding Calcofluor White M2R fluorochrome (Sigma-Aldrich; Poole, Dorset, UK) are described elsewhere (McCartney et al. 2003).
Monoclonal antibodies LM10 and LM11 were selected because of their binding to series of soluble polysaccharides. The specificity of LM10 and LM11 is clearly demonstrated by immunodot assay, as shown in Figure 1. They detected approximately 50 ng xylan/dot. LM10 bound only to no- or low-substituted xylans such as birch wood and oat spelt xylans and did not bind to any other samples, including arabinoxylan or GAX. LM11, in contrast, bound strongly to wheat arabinoxylan and, to a lesser extent, to maize GAX in addition to the low-substitution xylans (Figure 1). Neither monoclonal antibody bound to the most substituted sorghum GAX nor to any other of a range of cell wall polysaccharides including glucans and pectins when applied at up to 1 µg/dot.
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Acknowledgments |
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We thank Dr. Christine Andème-Onzighi for the preparation of flax hypocotyls and Dr. Henk Schols and Professor Harry Gilbert for xylans.
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Footnotes |
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Literature Cited |
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