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⁺

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

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