Azaspiracid: First evidence of protein binding in shellfish

The occurrence of azaspiracid (AZA) toxins in contaminated shellfish has been the focus of much research. The present study investigated the binding properties of these toxins in mussels of the species Mytilus edulis. The work involved extraction of proteins and AZAs from contaminated mussel hepatopancreas and examination of the extracts by isoelectric focusing (IEF), size exclusion chromatography (SEC) and sodium docecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Liquid chromatography coupled with tandem mass spectrometry analysis (LC–MS/MS) was also performed in this study to identify AZAs. Blank mussels were subjected to the same purification and analytical procedures.

AZAs were found to be weakly bound to a protein with a molecular weight of 45 kDa, in samples of contaminated mussels. This protein, which was abundant in contaminated mussels, was also present in blank mussels, albeit at much lower concentrations. It was further noted that a 22 kDa protein was also present only in contaminated mussel samples.

Proteomics Identification of Azaspiracid Toxin Biomarkers in Blue Mussels, Mytilus edulis

Azaspiracids are a class of recently discovered algae-derived shellfish toxins. Their distribution globally is on the increase with mussels being most widely implicated in azaspiracid-related food poisoning events. Evidence that these toxins were bound to proteins in contaminated mussels has been shown recently. In the present study characterization of these proteins in blue mussels, Mytilus edulis, was achieved using a range of advanced proteomics tools. Four proteins present only in the hepatopancreas of toxin-contaminated mussels sharing identity or homology with cathepsin D, superoxide dismutase, glutathione S-transferase Pi, and a bacterial flagellar protein have been characterized. Several of the proteins are known to be involved in self-defense mechanisms against xenobiotics or up-regulated in the presence of carcinogenic agents. These findings would suggest that azaspiracids should now be considered and evaluated as potential tumorigenic compounds. The presence of a bacterial protein only in contaminated mussels was an unexpected finding and requires further investigation. The proteins identified in this study should assist with development of urgently required processes for the rapid depuration of azaspiracid-contaminated shellfish. Moreover they may serve as early warning indicators of shellfish exposed to this family of toxins. Molecular & Cellular Proteomics 8: 1811-1822, 2009.