Monoclonal Antibodies against an Arabinogalactan-Protein from Pressed Juice of Echinacea purpurea

Abstract

Pressed juices of the aerial parts of Echinacea purpurea are used as non-specific immunostimulants, and arabinogalactan-proteins (AGPs) have been shown to be part of the active principle. Monoclonal antibodies against an AGP from pressed juice of Echinacea purpurea with complement-stimulating activity [1] have been established by means of hybridoma techniques [2]. To test the specificity of the antibodies, several other arabinogalactan-proteins from suspension cultures of Echinacea purpurea, the roots of Echinacea pallida, the aerial parts of Rudbeckia hirta, the roots of Baptisia tinctoria and gum arabic as well as an arabinogalactan from larch wood were tested in a competitive ELISA for cross reactivities. Chemical modifications at the periphery of the AGP molecules either by reduction of uronic acids or by dearabinosylation had no influence on the reactivity of the molecules towards the antibodies. For further characterization of the epitope, different Ara-Gal-oligosaccharides were used as antigens. A hexasaccharide consisting of a backbone of four molecules of 6-linked β-D-Galp, the second and the fourth of them branched at O-2 to an α-L-Araf residue [3] showed weak but reproducible cross reactivity, indicating that the antibodies may be at least in part directed to the carbohydrate moiety of the AGP. Testing of anti-AGP antibodies JIM 8 and LM 2 revealed good reactivity of LM 2 with the Echinacea AGP, whereas Jim 8 showed only very weak interaction.

Echinacea purpurea - Asteraceae - monoclonal antibodies - arabinogalactan-proteins - ELISA

References

• 1 Alban S, Classen B, Brunner G, Blaschek W. Differentiation between the complement modulating effects of an arabinogalactan-protein from Echinacea purpurea and heparin.  Planta Medica. 2002;  68 1118-24
• 2 Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity.  Nature. 1975;  256 495-7
• 3 Csávás M, Borbás A, Jánossy L, Batta G, Lipták A. Synthesis of the α-L-Araf-(1→2)-β-D-Galp-(1→6)-β-D-Galp-(1→6)-[α-L-Araf-(1→2)]-β-D-Galp-(1→6)-D-Gal hexasaccharide as a possible repeating unit of the cell-cultured exudates of Echinacea purpurea arabinogalactan.  Carbohydr Res. 2001;  336 107-15
• 4 Bauer R. The Echinacea Story-The scientific development of an herbal immunostimulant. In: Prendergast HDV, Etkin NL, Harris DR, Houghton PJ, editors Royal Botanic Gardens Kew;; 1998: pp 317-32
• 5 Classen B, Witthohn K, Blaschek W. Characterization of an arabinogalactan-protein isolated from pressed juice of Echinacea purpurea by precipitation with the ß-glucosyl Yariv reagent.  Carbohydr Res. 2000;  327 497-504
• 6 Gaspar Y, Johnson K L, McKenna J A, Bacic A, Schultz C J. The complex structures of arabinogalactan-proteins and the journey towards function.  Plant Mol Biol. 2001;  47 161-176
• 7 Nothnagel E A. Proteoglycans and Related Components in Plant Cells. In: Jeon KW, editor Academic Press San Diego; 1997: pp 195-291
• 8 Knox J P. The use of antibodies to study the architecture and developmental regulation of plant cell walls.  Int J Cytol. 1997;  171 79-120
• 9 Egert D, Bodinet C, Beuscher N. Adaption of an ELISA of immunoreactive glycoproteins/polysaccharides in the medicinal plants Baptisia and Echinacea for quality control.  Planta Medica. 1989;  55 637
• 10 Yariv J, Rapport M M, Graf L. The interaction of glycosides and saccharides with antibody to the corresponding phenylazo glycosides.  Biochem J. 1962;  85 383-8
• 11 Kreuger M, van Holst G J. Arabinogalactan-proteins are essential in somatic embryogenesis of Daucus carota L.  Planta. 1993;  189 243-8
• 12 Kreuger M, van Holst G J. Arabinogalactan-protein epitopes in somatic embryogenesis of Daucus carota L.  Planta. 1995;  197 135-41
• 13 Blakeney A B, Harris P J, Henry R J, Stone B A. A simple and rapid preparation of alditol acetates for monosaccharide analysis.  Carbohydr Res. 1983;  113 291-9
• 14 Taylor R L, Conrad H E. Stoichiometric depolymerization of polyuronides and glycosaminoglycuronans to monosaccharides following reduction of their carbodiimide-activated carboxyl groups.  Biochemistry. 1972;  11/8 1383-8
• 15 Gleeson P A, Clarke A E. Structural studies on the major component of Gladiolus style mucilage, an arabinogalactan-protein.  Biochem J. 1979;  181 607-21
• 16 Harris P J, Henry R J, Blakeney A B, Stone B A. An improved procedure for the methylation analysis of oligosaccharides and polysaccharides.  Carbohydr Res. 1984;  127 59-73
• 17 Csávás M, Borbás A, Szilágyi L, Lipták A. Successful combination of (methoxydimethyl)methyl (MIP) and (2-naphthyl)methyl (NAP) ether for the synthesis of arabinogalactan-type oligosaccharides. Synlett 2002: 887-90
• 18 Csávás M, Borbás A, Jánossy L, Lipták A. Synthesis of an arabinogalactan-type octa- and two isomeric nonasaccharides. Suitable tuning of protecting groups.  Tetrahedron Lett. 2003;  44 631-5
• 19 Knox J P. The use of antibodies to study the architecture and developmental regulation of plant cell walls.  Int J Cytol. 1997;  171 79-120
• 20 Smallwood M, Yates E A, Willats W GT, Martin H, Knox J P. Immunochemical comparison of membrane-associated and secreted arabinogalactan-proteins in rice and carrot.  Planta. 1996;  198 452-9
• 21 Aspinall G O. The Polysaccharides. In: Aspinall GO, editor Academic Press New York; 1982