On the Role of Protein Disulfide Isomerase in the Retrograde Cell Transport of Secreted Phospholipases A ₂

Abstract

Following the finding that ammodytoxin (Atx), a neurotoxic secreted phospholipase A ₂ (sPLA ₂) in snake venom, binds specifically to protein disulfide isomerase (PDI) in vitro we show that these proteins also interact in living rat PC12 cells that are able to internalize this group IIA (GIIA) sPLA ₂. Atx and PDI co-localize in both differentiated and non-differentiated PC12 cells, as shown by fluorescence microscopy. Based on a model of the complex between Atx and yeast PDI (yPDI), a three-dimensional model of the complex between Atx and human PDI (hPDI) was constructed. The Atx binding site on hPDI is situated between domains b and b’. Atx interacts hPDI with an extensive area on its interfacial binding surface. The mammalian GIB, GIIA, GV and GX sPLA ₂s have the same fold as Atx. The first three sPLA ₂s have been detected intracellularly but not the last one. The models of their complexes with hPDI were constructed by replacement of Atx with the respective mammalian sPLA ₂ in the Atx—hPDI complex and molecular docking of the structures. According to the generated models, mammalian GIB, GIIA and GV sPLA ₂s form complexes with hPDI very similar to that with Atx. The contact area between GX sPLA ₂ and hPDI is however different from that of the other sPLA ₂s. Heterologous competition of Atx binding to hPDI with GV and GX sPLA ₂s confirmed the model-based expectation that GV sPLA ₂ was a more effective inhibitor than GX sPLA ₂, thus validating our model. The results suggest a role of hPDI in the (patho)physiology of some snake venom and mammalian sPLA ₂s by assisting the retrograde transport of these molecules from the cell surface. The sPLA ₂–hPDI model constitutes a valuable tool to facilitate further insights into this process and into the (patho)physiology of sPLA ₂s in relation to their action intracellularly.