Glycosphingolipid-facilitated Membrane Insertion and Internalization of Cobra Cardiotoxin

Abstract

Cobra cardiotoxins, a family of basic polypeptides having lipidand heparin-binding capacities similar to the cell-penetrating peptides, induce severe tissue necrosis and systolic heart arrest in snakebite victims. Whereas cardiotoxins are specifically retained on the cell surface via heparan sulfate-mediated processes, their lipid binding ability appears to be responsible, at least in part, for cardiotoxin-induced membrane leakage and cell death. Although the exact role of lipids involved in toxin-mediated cytotoxicity remains largely unknown, monoclonal anti-sulfatide antibody O4 has recently been shown to inhibit the action of CTX A3, the major cardiotoxin from Taiwan cobra venom, on cardiomyocytes by preventing cardiotoxin-induced membrane leakage and CTX A3 internalization into mitochondria. Here, we show that anti-sulfatide acts by blocking the binding of CTX A3 to the sulfatides in the plasma membrane to prevent sulfatide-dependent CTX A3 membrane pore formation and internalization. We also describe the crystal structure of a CTX A3-sulfatide complex in a membrane-like environment at 2.3 Å resolution. The unexpected orientation of the sulfatide fatty chains in the structure allows prediction of the mode of toxin insertion into the plasma membrane. CTX A3 recognizes both the headgroup and the ceramide interfacial region of sulfatide to induce a lipid conformational change that may play a key role in CTX A3 oligomerization and cellular internalization. This proposed lipid-mediated toxin translocation mechanism may also shed light on the cellular uptake mechanism of the amphiphilic cell-penetrating peptides known to involve multiple internalization pathways.Glycosphingolipids in eukaryotic cells have important biological functions in membrane trafficking and cell signaling (1, 2). The number of disease-related proteins found to interact specifically with glycosphingolipids is also increasing (3-5). For instance, recent studies have shown that natural killer cells can be activated by glycosylceramides from the cell surface of Gram-negative bacteria that do not contain lipopolysaccharide (6, 7). There is also evidence indicating that, in response to tumor necrosis factor-␣, trafficking of ganglioside GD3 from the plasma membrane to mitochondria can trigger several cell death signaling responses (3,8,9). Although the molecular mechanisms accounting for the observed glycosphingolipid transport and glycolipidtriggered cell responses remain elusive, recent progress in determining the three-dimensional structure of protein⅐glycosphingolipid complexes has shed some light on the process (10 -13). These glycosphingolipid-binding proteins function by either extracting lipids from the membrane and forming a water-soluble protein⅐lipid complex, as in the cases of glycosphingolipid transfer protein, saposin B, and lipid-presenting CD1a (10 -12), or by localizing to a glycosphingolipid domain of the plasma membrane and eventually being internalized via the endocytic pathway, as seen for cholera and Shiga toxins (13,14). Aft...

Anionic Lipids: Determinants of Binding Cytotoxins from Snake Venom on the Surface of Cell Membranes

“…The M site (residues K5, K12, K18, Y22, R36, and C38), presents in most docking solutions, is identical to the sulphatide–binding pocket located on the convex side of the toxin molecule [17]. Indeed, the results of a chemical modification of lysine and tyrosine residues suggest that the site’s key residues, such as Y22 and K12, significantly influence the functional activity of CTs [32, 33].…”

“…Judging by the number of intermolecular hydrogen bonds and ionic contacts in the docking solutions, the M site has the best potential for forming a stable complex with the ligand. Formation of the CT A3–SGC complex in of a membrane–mimicking environment [17] indicates that other anionic components in the membrane can bind to the M site. At the same time, the superimposition of CT docking models, according to the mode by which they are embedded into the bilayer, indicates different availabilities of the sites for the PS head.…”

“…In our simulations, we used a crystallographic model of the 3D structure of CT А3 from N. atra venom (PDB code: 2BHI) [17] and NMR models of CT II in hydrated state (PDB code: 1CB9, models 1, 14 and 18) [25] and in the presence of dodecylphosphocholine (DPC) micelles (PDB code: 1FFJ, models: 1 and 10) [4]. …”

“…Based on NMR and X–ray data, a search for potential CT targets revealed a series of anionic low molecular weight ligands (heparin–derived oligosaccharides, ATP derivatives, and sulphatide) that interact with certain sites on the toxin’s surface [13–17]. Fluorescent spectroscopy studies on liposomes of various composition combined with in vivo experiments using monoclonal antibodies indicate a specific CT target—a polar head of a glycolipid sulphatide (hSGC)—located on the surface of a rat’s cardiomyocyte membrane, which mediates the toxic activity of CT А3 [17].…”

“…Fluorescent spectroscopy studies on liposomes of various composition combined with in vivo experiments using monoclonal antibodies indicate a specific CT target—a polar head of a glycolipid sulphatide (hSGC)—located on the surface of a rat’s cardiomyocyte membrane, which mediates the toxic activity of CT А3 [17]. Taking into account the fact that CTs affect various cell types and that toxins effectively lyse model membranes containing no glycolipids [5, 18, 19], one can expect CT to be able to interact nonspecifically with other anionic membrane lipids, including the most common one, phosphatidylserine (PS).…”