Differentiation Between the Complement Modulating Effects of an Arabinogalactan-Protein from Echinacea purpurea and Heparin

Abstract

Due to the important physiological role of the complement system, complement modulation, either inhibition or stimulation, is an interesting target for drug development. Several plant polysaccharides are known to exhibit complement modulating activities. Sometimes these effects are described as complement inhibition, although the basic mechanism is a stimulation of the complement activation. This misinterpretation is due to the observed reduced haemolysis in the widely used haemolytic complement assay, which does not allow to differentiate between complement activators and inhibitors, when it is performed in the classical manner. The aim of the presented study was to demonstrate that by simple modifications of the classical procedure this assay becomes an efficient tool to distinguish between real complement inhibitors and complement activating compounds without performing expensive, molecular mechanistic investigations. As practical examples heparin with proven complement inhibiting activity and AGP, a new arabinogalacatan-protein type II isolated from pressed juice of the aerial parts of Echinacea purpurea, as a potential complement activating compound were included in the study. By means of varying the preincubation time of the test compound with complement, AGP was clearly identified as a stimulator of both the classical and alternative pathway of complement activation. These findings correspond to the results of molecular mechanistic investigations. Selective removal of the arabinose side chains of AGP resulted in considerably reduced activity. Therefore, the three-dimensional structure of the polysaccharide, i. e., a backbone branched by side chains, is supposed to be important for the interactions with the complement system. The complement activating effects of AGP may contribute to the well-established immunostimulating effects of the pressed juice from Echinacea purpurea.

Abbreviations

AGP:arabinogalactan-protein

AGP-hydr.:hydrolysed arabinogalactan-protein

AP-CA:haemolytic complement assay for the alternative
pathway

CP-CA:haemolytic complement assay for the classical
pathway

EGTA-VB:veronal buffered saline containing EGTA and Mg²⁺

HPS:human pooled serum

RT:room temperature

LPS:lipopolysaccharide

RaE:rabbit erythrocytes

RT:room temperature

ShE(A):(sensitised) sheep erythrocytes

VB:veronal buffered saline containing Ca²⁺ and Mg²⁺

Key words

Arabinogalactan-protein - Echinacea purpurea - Asteraceae - heparin - haemolytic complement assay - complement modulation - polysaccharides

References

• 1 Morgan B P. Physiology and pathophysiology of complement: progress and trends.  Critical Reviews in Clinical Laboratory Sciences. 1995;  32 265-98
• 2 Marsh J E, Pratt J R, Sacks S H. Targeting the complement system.  Current Opinion in Nephrology and Hypertension. 1999;  8 557-62
• 3 Figueroa J E, Densen P. Infectious diseases associated with complement deficiencies.  Clinical Microbiology Reviews. 1991;  4 359-95
• 4 Saxen H, Reima I, Makela P H. Alternative complement pathway activation by Salmonella O polysaccharide as a virulence determinant in the mouse.  Microbial Pathogenesis. 1987;  2 15-28
• 5 Levy N J, Kasper D L. Surface-bound capsular polysaccharide of type Ia group B Streptococcus mediates C1 binding and activation of the classic complement pathway.  Journal of Immunology. 1986;  136 4157-62
• 6 Schifferle R E, Wilson M E, Levine M J, Genco R J. Activation of serum complement by polysaccharide-containing antigens of Porphyromonas gingivalis .  Journal of Periodeontal Research. 1993;  28 248-54
• 7 Alvarez D, Merino S, Tomas J M, Benedi V J, Alberti S. Capsular polysaccharide is a major complement resistance factor in lipopolysaccharide O side chain-deficient Klebsiella pneumoniae clinical isolates.  Infection and Immunity. 2000;  68 953-5
• 8 Edens R E, Linhardt R J, Weiler J M. Heparin is not just an anticoagulant anymore: six and one-half decades of studies on the ability of heparin to regulate complement activity. In: Cruse JM, Lewis RE Jr, editors Complement Profiles. Basel; Karger 1993: 96-120
• 9 Yamada H, Nagai T, Cyong J C, Otsuka Y, Tomoda M, Shimizu N, Gonda R. Relationship between chemical structure and activating potencies of complement by an acidic polysaccharide, Plantago-mucilage A, from the seed of Plantago asiatica .  Carbohydrate Research. 1986;  156 137-45
• 10 Yamada H. Pectic polysaccharides from Chinese herbs: structure and biological activity.  Carbohydrate Polymers. 1994;  25 269-76
• 11 Michaelsen T E, Gilje A, Samuelsen A B, Hogasen K, Paulsen B S. Interaction between human complement and a pectin type polysaccharide fraction, PMII, from the leaves of Plantago major L.  Scandavian Journal of Immunology. 2000;  52 483-90
• 12 Shin K S, Kwon K S, Yang H C. Screening and characteristics of anti-complementary polysaccharides from Chinese medicinal herbs.  Han’guk Nonghwa Hakhoechi. 1992;  35 42-50
• 13 Samuelsen A B, Lund I. Djahromi JM, Paulsen BS, Wold JK, Knutsen SH. Structural features and anti-complementary activity of some heteroxylan polysaccharide fractions from the seeds of Plantago major L.  Carbohydrate Polymers. 1999;  38 133-43
• 14 Zvyagintseva T N, Shevchenko N M, Nazarova I V, Scobun A S, Luk’yanov P A, Elyakova L A. Inhibition of complement activation by water-soluble polysaccharides of some far-eastern brown seaweeds.  Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 2000;  126C 209-15
• 15 Classen B, Witthohn K, Blaschek W. Characterization of an arabinogalactan-protein isolated from pressed juice of Echinacea purpurea by precipitation with the b-glucosyl Yariv reagent.  Carbohydrate Research. 2000;  327 497-504
• 16 Bauer R. Echinacea drugs-effects and active ingredients.  Zeitschrift für Ärztliche Fortbildung. 1996;  90 111-5
• 17 Yariv J, Rapport M M, Graf L. The interaction of glycosides and saccharides with antibody to the corresponding phenylazo glycosides.  Biochemical Journal. 1962;  85 383-8
• 18 Aspinall G O. Carbohydrate Polymers of Plant Cell Walls. In: Loewus F, editor Biogenesis of Plant Cell Wall Polysaccharides. New York; Academic Press 1973: 95
• 19 Klerx J P, Beukelman C J, Van Dijk H, Willers J M. Microassay for colorimetric estimation of complement activity in guinea pig, human and mouse serum.  Journal of Immunological Methods. 1983;  63 215-20
• 20 Beukelman C J, Rademaker P M, van Dijk H, Aerts P C, Berrens L, Willers J M. House dust allergen activates the classical complement pathway in mouse serum.  Immunological Letters. 1986;  13 159-64
• 21 Yamada H, Kiyohara H, Cyong J C, Otsuka Y. Studies on polysaccharides from Angelica acutiloba-IV. Characterization of an anti-complementary arabinogalactan from the roots of Angelica acutiloba Kitagawa.  Molecular Immunology. 1985;  22 295-304
• 22 Diallo D, Paulsen B S, Liljeback T HA. Michaelsen TE. Polysaccharides from the roots of Entada africana Guill. et Perr., Mimosaceae, with complement fixing activity.  Journal of Ethnopharmacology. 2001;  74 159-71
• 23 Odenthal K P, Schwarz T, Witthohn K, Loos M. Bioassay-guided identification of immunomodulating constituents in Echinacin®. International Congress and 48th Annual Meeting of the Society for Medicinal Plant Research Zürich; 3. - 7.9.2000: P4B/16
• 24 Lachmann P J, Hughes-Jones N C. Initiation of complement activation.  Springer Seminars in Immunopathology. 1984;  7 143-62
• 25 Kirschfink M. The clinical laboratory: Testing the complement system. In: Rother K, Till GO, Hänsch GM, editors The complement system. Berlin; Springer 1998: 522-4