A putative link between phagocytosis-induced apoptosis and hemocyanin-derived phenoloxidase activation
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Apoptosis and phagocytosis are crucial processes required for developmental morphogenesis, pathogen deterrence and immunomodulation in metazoans. We present data showing that amebocytes of the chelicerate, Limulus polyphemus, undergo phagocytosis-induced cell death after ingesting spores of the fungus, Beauveria bassiana, in vitro. The observed biochemical and morphological modifications associated with dying amebocytes are congruent with the hallmarks of apoptosis, including: extracellularisation of phosphatidylserine, intranucleosomal DNA fragmentation and an increase in caspase 3/7-like activities. Previous studies have demonstrated that phosphatidylserine is a putative endogenous activator of hemocyanin-derived phenoloxidase, inducing conformational changes that permit phenolic substrate access to the active site. Here, we observed extracellular hemocyanin-derived phenoloxidase activity levels increase in the presence of apoptotic amebocytes. Enzyme activity induced by phosphatidylserine or apoptotic amebocytes was reduced completely upon incubation with the phosphatidylserine binding protein, annexin V. We propose that phosphatidylserine redistributed to the outer plasma membrane of amebocytes undergoing phagocytosis-induced apoptosis could interact with hemocyanin, thus facilitating its conversion into a phenoloxidase-like enzyme, during immune challenge.
KeywordsPhosphatidylserine Apoptosis Phagocytosis Innate immunity Limulus polyphemus Damage-associated patterns
This work was supported by the University of Stirling. With thanks to Alex Mühlhölzl, C.T.O., Marine Biotech Limited, for providing access to L. polyphemus. We are grateful to Prof. Seamus J. Martin and Conor M. Henry (Molecular Cell Biology Laboratory, Trinity College Dublin) for kindly providing the pProEx.Htb.annexin V plasmid.
Conflict of interest
The authors declare no financial or other potential conflicts of interest.
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