Monoclonal antibodies to rhamnogalacturonan I backbone

• Published in: Planta Vol. 231; no. 6; pp. 1373 - 1383
• Main Authors: Ralet, M.-C, Tranquet, O, Poulain, D, Moïse, A, Guillon, F
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01-05-2010; Springer-Verlag; Springer; Springer Nature B.V; Springer Verlag
• Subjects: Agriculture; Antibodies; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - immunology; Arabidopsis - chemistry; Biological and medical sciences; Biomedical and Life Sciences; Carbohydrate Sequence; Cell Wall - metabolism; Cell walls; Chromatography; Chromatography, Gel; Ecology; Enzyme-Linked Immunosorbent Assay; Epitopes; Fluorescent Antibody Technique; Forestry; Fundamental and applied biological sciences. Psychology; Galactans; Glycoproteins - biosynthesis; Haptens - biosynthesis; Haptens - immunology; Immunoglobulin Isotypes - biosynthesis; Life Sciences; Mass spectrometry; Molecular Sequence Data; Monoclonal antibodies; Oligomers; Oligosaccharides; Oligosaccharides - biosynthesis; Oligosaccharides - immunology; Original Article; Pectins - chemistry; Pectins - immunology; Plant cells; Plant Sciences; Plants; Solubility; Sugar; Sugar beets; Vegetal Biology; Water; Pectin; Immunocytochemistry; Cell wall; Pectic oligosaccharides; Oligosaccharide; Plant biology; Monoclonal antibody; PECTIC OLIGOSACCHARIDES; IMMUNOCYTOCHEMISTRY; PECTIN; IMMUNOCHIMIE; CELL WALL
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/s00425-010-1116-y

Monoclonal antibodies were raised against rhamnogalacturonan I backbone, a pectin domain, using Arabidopsis thaliana seed mucilage-derived rhamnogalacturonan I oligosaccharides—BSA conjugates. Two monoclonal antibodies, designated INRA-RU1 and INRA-RU2, selected for further characterization, were specific for the backbone of rhamnogalacturonan I, displaying no binding activity against the other pectin domains i.e. homogalacturonans, galactans or arabinans. A range of oligosaccharides was prepared by enzymatic digestion of rhamnogalacturonan I isolated from Arabidopsis thaliana seed mucilage and from sugar beet pectin, purified by low-pressure chromatography and characterized by high-performance anion-exchange chromatography and mass spectrometry. These rhamnogalacturonan I oligomers were used to characterize the binding site of the two monoclonal antibodies by competitive inhibition. Both INRA-RU1 and INRA-RU2 showed maximal binding to the [→2)-α-l-rhamnosep-(1→4)-α-d-galacturonic acid p-(1→]₇ structural motif but differed in their minimum binding requirement. INRA-RU2 required at least two disaccharide (rhamnose-galacturonic acid) repeats for the antibody to bind, while INRA-RU1 required a minimum of six disaccharide repeats. Furthermore, the binding capacity of INRA-RU1 decreased steeply as the number of disaccharide repeats go beyond seven. Each of these antibodies reacted with hairy regions isolated from sugar beet pectin. Immunofluorescence microscopy indicated that both antibodies can be readily used to detect rhamnogalacturonan I epitopes in various cell wall samples.