On the Role of Protein Disulfide Isomerase in the Retrograde Cell Transport of Secreted Phospholipases A2

Abstract

Following the finding that ammodytoxin (Atx), a neurotoxic secreted phospholipase A2 (sPLA2) 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) sPLA2. 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 sPLA2s have the same fold as Atx. The first three sPLA2s 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 sPLA2 in the Atx—hPDI complex and molecular docking of the structures. According to the generated models, mammalian GIB, GIIA and GV sPLA2s form complexes with hPDI very similar to that with Atx. The contact area between GX sPLA2 and hPDI is however different from that of the other sPLA2s. Heterologous competition of Atx binding to hPDI with GV and GX sPLA2s confirmed the model-based expectation that GV sPLA2 was a more effective inhibitor than GX sPLA2, thus validating our model. The results suggest a role of hPDI in the (patho)physiology of some snake venom and mammalian sPLA2s by assisting the retrograde transport of these molecules from the cell surface. The sPLA2–hPDI model constitutes a valuable tool to facilitate further insights into this process and into the (patho)physiology of sPLA2s in relation to their action intracellularly.

The neurotoxic secreted phospholipase A2 from the Vipera a. ammodytes venom targets cytochrome c oxidase in neuronal mitochondria

“…Rat adrenal pheochromocytoma cells, PC12, exhibit properties similar to those of neurons 19 . The fact that the sulfo-SBED-Atx derivative was able to enter the cytosol of PC12 cells rapidly qualified these cells as suitable for studying the pathway of the neurotoxic sPLA 2 from the external space, through the plasma membrane and into the cytosol 20 . As shown in this work, PC12 cells harbour a specific receptor for Atx, R25, in their mitochondrial membranes.…”

“…Sulfo-SBED-Atx was synthesized according to the published protocol 48 . PC12 cells were affinity-labelled using sulfo-SBED-Atx 20 . Intact PC12 cells, plated on 10 cm plates at 80 to 90% confluence, were treated with sulfo-SBED-Atx at a concentration of 100 nM for 1, 2, 5, 15, 30 and 60 min in HBSS in the dark at 37 °C under 5% (v/v) CO 2 .…”

“…Colocalization study on PC12 cells was performed as described previously 20 . PC12 cells on poly– L –Lys coated coverslips were incubated at 37 °C under 5% (v/v) CO 2 in the presence of 100 nM 546 Alexa–Atx for 1, 2, 5, 15, 30 and 60 min.…”

Secreted Phospholipases A2 - not just Enzymes: Revisited

“…A three-dimensional model of the complex between Atx and PDI has also been reported [20]. PDI consists of multiple domains (i.e., a, a', b, b'), and according to the model shown in Figure 2D, the Atx-binding site on PDI is situated between domains b and b'.…”

“…Despite the generally compact structure of sPLA 2 s, the flexibility of the exposed side chains of the amino acids at the IBS promotes their optimal binding to phospholipid aggregates, as their substrates, for efficient hydrolysis [62]. As indicated above, the IBS is critically involved also in the interactions of sPLA2s with the CRD-containing sPLA 2 -BPs and FXa, as also for PDI [20] and vimentin [26]. The association of the sPLA 2 IBS with a variety of different protein surfaces is most likely driven by the same principles as the association of the sPLA 2 IBS with phospholipid membranes.…”