Generation of Monoclonal Antibodies against Plant Cell-Wall Polysaccharides (I. Characterization of a Monoclonal Antibody to a Terminal [alpha]-(1->2)-Linked Fucosyl-Containing Epitope

Abstract

Monoclonal antibodies (McAbs) generated against rhamnogalacturonan I (RC-I) purified from suspension-cultured sycamore maple (Acer pseudoplafanus) cells fall into three recognition groups. Four McAbs (group I) recognize an epitope that appears to be immunodominant and is present on RC-I from maize and sycamore maple, pectin and polygalacturonic acid from citrus, gum tragacanth, and membrane glycoproteins from suspension-cultured cells of maize, tobacco, parsley, bean, and sycamore maple. A second set of McAbs (group II) recognizes an epitope present in sycamore maple RC-I but does not bind to any of the other polysaccharides or glycoproteins recognized by group 1. Lastly, one McAb, CCRC-M1 (group III), binds to RC-I and more strongly to xyloglucan (XC) from sycamore maple but not to maize RC-I, citrus polygaladuronic acid, or to the plant membrane glycoproteins recognized by group 1. lhe epitope to which CCRC-M1 binds has been examined in detail. Ligand competition assays using a series of oligosaccharides derived from or related to sycamore maple XC demonstrated that a terminal a-(142)-linked fucosyl residue constitutes an essentia1 part of the epitope recognized by CCRC-M1. Oligosaccharides containing this structural motif compete with intact sycamore maple XC for binding to the antibody, whereas structurally related oligosaccharides, which do not contain terminal fucosyl residues or in which the terminal fucosyl residue is linked a-(1+3) to the adjacent glycosyl residue, do not compete for the antibody binding site. lhe ligand binding assays also indicate that CCRC-M1 binds to a conformationally dependent structure of the polysaccharide. Other results of this study establish that some of the carbohydrate

Group III-AXTHGenes of Arabidopsis Encode Predominant Xyloglucan Endohydrolases That Are Dispensable for Normal Growth

“…S9B) and the xth31-1/xth32-1 mutant (Supplemental Fig. S9B) using the Complex Carbohydrate Research Center-Monoclonal1 (CCRC-M1) antibody, which recognizes a(1-2)-linked fucosyl residues present in xyloglucan and rhamnogalacturonan-I (Puhlmann et al, 1994), revealed no significant differences in microscopic cell wall composition in the mutant line. CCRC-M1 staining was observed throughout the roots, including the root hairs, except at the root tip, similar to earlier reports (Freshour et al, 1996).…”

Deciphering the Responses of Root Border-Like Cells of Arabidopsis and Flax to Pathogen-Derived Elicitors

“…1, D-G). These include xyloglucan epitopes, as revealed by the monoclonal antibodies (mAbs) LM15 (Marcus et al, 2008) and CCRCM1 (Puhlmann et al, 1994;Fig. 1, D and E), and pectins including homogalacturonan and xylogalacturonan, as revealed by the mAbs LM19 (Verhertbruggen et al, 2009) and LM8 (Willats et al, 2004), respectively ( Fig.…”

“…The monoclonal antibodies specific for cell wall polysaccharides used in this study were LM15 (Marcus et al, 2008), CCRC-M1 (Puhlmann et al, 1994), LM19 (Verhertbruggen et al, 2009), LM8 (Willats et al, 2004), and LM1 (Smallwood et al, 1995). Roots were fixed for 30 min in 4% (w/v) paraformaldehyde in 50 mM PIPES, 1 mM CaCl 2 , pH 7, and immunolabeled according to Willats et al (2001).…”

Developmental and Tissue-Specific Structural Alterations of the Cell-Wall Polysaccharides of Arabidopsis thaliana Roots

“…Studies of the temporal, spatial, and developmental dynamics of plant cell-wall glycoconjugates have lagged behind corresponding animal studies because of the more limited collection of monoclonal antibodies against cell-wall carbohydrate epitopes that is available and because of the difficulty in characterizing the epitopes recognized by the available antibodies. Previous studies of plant cell-wall dynamics have focused on cell surface glycoproteins, since arrays of monoclonal antibodies have been generated against two types of such complex glycoconjugates, the arabinogalactan-proteins (Anderson et al, 1984;Brewin et al, 1985;Norman et al, 1986;Villanueva et al, 1986;Hahn et al, 1987;Knox et al, 1989Knox et al, ,1991Pennell et al, 1989Pennell et al, ,1991Horsley et al, 1993;Puhlmann et al, 1994;Kreuger and Van Holst, 1995) and Hyp-rich glycoproteins (Smallwood et al, 1994(Smallwood et al, , 1995Knox et al, 1995). Immunohistochemical studies have documented subcellular (Herman and Lamb, 1992;Rae et al, 1992;Van Aelst and Van Went, 1992;Horsley et al, 1993;Sherrier and VandenBosch, 1994) and cell-type-specific (Knox et al, ,1991Stacey et al, 1990;Van Aelst and Van Went, 1992;Benfey et al, 1993;Schindler et al, 1995) distribution of arabinogalactan epitopes.…”

“…We have generated a small pane1 of monoclonal antibodies that recognize carbohydrate epitopes in plant cell-wall polysaccharides (Puhlmann et al, 1994). The epitopes of two of these antibodies were characterized in some detail.…”