Anticomplementary activity of fucoidan: determination of the targeted proteins and of their interactions

International audienceThe fucoidan extracted from brown seaweed like Ascophyllum nodosum is a sulfated polysaccharide whose structure is based on a a-(10/3)- and (10/4)-linked fucose backbone. The fucoidan is one of the most potent inhibitor of the activation of complement system (IC50 9 and 6 mg/ml on the classical and alternative pathway, respectively) by mechanisms that so far remain to be unraveled.Our goal is to identify which proteins of the cascade are specifically concerned by the fucoidan action and to characterize their interactions. For that purpose gel co-affinity electrophoresis and a new affinity capillary electrophoresis (ACE) method have been implemented to characterize fucoidan-complement protein complexes. Concerning the classical pathway fucoidan mainly interferes at the first steps of the activation, by inhibiting the formation of the complex C1 (3 mg of fucoidan leads to a 60% inhibition of the complex formation) and of the classical C3 convertase. We demonstrated by affinity capillary electrophoresis and haemolytic assays that fucoidan interacts with proteins C1q and C4. Specific labelling of lysine residues of C1q resulted in the inhibition of its haemolytic activity. The presence of fucoidan during the labelling reaction preserved the functional activity of C1q, likely through the protection of the essential lysines. Hence, we assumed that fucoidan interacts with the essential lysines residues of C1q in such a manner that its functionality is impaired resulting in the inhibition of the classical pathway activation. The interaction of fucoidan with C1q was ascertained by taking advantage of the binding properties of C1q toward DNA and of an emerging technique allowing the molecular combing of DNA strands and its observation by singlemolecule spectroscopy. This approach allowed us to implement an analytical tool useful to investigate the binding of fucoidan to C1q through competition experiments between DNA and the fucoidan. We have addressed the question of whether a C1q inhibitor (fucoidan) and a C1q activator (DNA) are able to bind to the same region of the protein by using not only native C1q but also the isolated domains of CLR and of the globular regions (GR). Concerning the alternative pathway, we demonstrated that fucoidan did not prevent the cleavage of Factor B, but may interfere in the discrimination between activating surfaces

ANTICOMPLEMENTARY ACTIVITY OF FUCOIDAN : DETERMINATION OF THE TARGETED PROTEINS AND OF THEIR INTERACTIONS

Conférence du 08 au 12 Juin 2003. Communication Orale