Heparin Binding Stabilizes the Membrane-bound Form of Cobra Cardiotoxin

Abstract

It has been shown previously that the long chain fragments of heparin bind to the ␤-strand cationic belt of the three-finger cobra cardiotoxin (or cytotoxin, CTX) and hence enhance its penetration into phospholipid monolayer under physiological ionic conditions. By taking lysophosphatidylcholine (LPC) micelles as a membrane model, we have shown by 1 H NMR study that the binding of heparin-derived hexasaccharide (Hep-6) to CTX at the ␤-strand region can induce conformational changes of CTX near its membrane binding loops and promote the binding activity of CTX toward LPC. The Fourier-transform infrared spectra and NMR nuclear Overhauser effect of Hep-6⅐CTX and CTX⅐LPC complex in aqueous buffer also supplemented the aforementioned observation. Thus, the detected conformational change may presumably be the result of structural coupling between the connecting loops and its ␤-strands. This is the first documentation of results showing how the association of hydrophilic carbohydrate molecules with amphiphilic proteins can promote hydrophobic protein-lipid interaction via the stabilization of its membrane-bound form. A similar mechanism involving tripartite interactions of heparin, protein, and lipid molecules may be operative near the extracellular matrix of cell membranes.Cobra cardiotoxins (or cytotoxins, CTXs) 1 are highly basic, three-fingered polypeptides with the capability of inducing direct lytic effect on many cell types, including cardiac myocytes (1, 2). The available x-ray and NMR structures of CTXs are similar with a short antiparallel double-stranded ␤-sheet connected by loop I and with a long antiparallel triple-stranded ␤-sheet connected by loop II and III. Two distinct types of CTXs, i.e. P-and S-type, can be distinguished by the presence of Pro 30 and Ser 28 , respectively, near the tip of second finger (loop II) based on its binding activity toward neutral phospholipid bilayers and micelles (3). P-type CTXs, including the major CTX A3 from Taiwan cobra (Naja atra) venom, have been shown to induce a reversible formation of an extra outwardly rectifying conductance in bullfrog atrial myocytes (4) and also redistribute themselves between penetrating and peripheral binding states in phosphatidylcholine (PC) dispersion under favorable condition (5). Despite of the apparent perturbing effect of CTX toward phospholipid membranes, it is not clear how CTXs might act specifically toward different cell types.Recently, it was suggested that the sulfated polysaccharides such as glycosaminoglycans (GAGs), which are abundant in the extracellular matrix of most animal cells, are potential targets for CTX action (6 -8). Heparin not only binds to homologous CTXs, with different specificity, but also promotes its penetration into phospholipid monolayer under physiological ionic conditions (6). Analysis of binding of 10 CTX homologues to heparin reveals a new heparin-binding structural motif by involving several positive residues at the concave surface of the three-finger toxin (7). The positive residues flanking the...

Glycosphingolipid-facilitated Membrane Insertion and Internalization of Cobra Cardiotoxin

“…10A). Consistent with previous results from NMR (24), Fourier transform infrared spectroscopy (47), and x-ray structure (22), most of the electron densities assigned to the C 10 E 6 detergent surround the tips of the three hydrophobic loops that mediate peripheral binding of CTX A3 to the phospholipid membrane. However, the hydrophobic tails of the bound SGC are not facing the presumed membrane-binding surface, as expected, but are in contact with the D1 dimer from another asymmetric unit.…”

“…Barring the involvement of side chain reorientations near the tip of the loops in mediating the dimerization of CTX A3, the overall structure of CTX A3 remains unchanged after CTX-membrane interaction due to the presence of four disulfide bonds. Although in the absence of SGC, the D1 dimer can form near the membrane surface via a hydrophobic clustering of Met 24 and Met 26 ( Fig. 11C) (22), the SGC can function as a glue to effect tighter binding between monomers in the CTX A3 dimeric complex.…”

“…Extracellular heparan sulfate may be involved in the observed specificity of CTX for cardiomyocytes by mediating a cell surface retention mechanism (22)(23)(24). CTXs are also recognized as cytotoxins, with the ability to induce a general lytic effect on human erythrocytes (21), necrotic cell death in fetal rat cardiomyocytes (25), and apoptosis in cortical neurons and human leukemia cells (25,26).…”

“…Although the trivial name "sulfatides" is used as a generic term for a class of amphiphiles containing sulfate esters and carbohydrates (28), the sulfatide used in this study is 3Ј-sulfated ␤1-D-galactosylceramide (SGC) and, as sphingolipids, is composed of an 18-carbon sphingosine base that is N-amide-linked to a fatty acid ranging in length from C 14 to C 24 . In light of current interest in the role of glycosphingolipids in toxin internalization and lipid domain formation, it is important to examine whether direct interaction between CTX and SGC indeed triggers CTX-related toxicity.…”

A CREB-C/EBPβ cascade induces M2 macrophage-specific gene expression and promotes muscle injury repair

“…Tibialis anterior (TA) and quadriceps (Q) muscles of one leg were injected with cardiotoxin (CTX), while the contralateral muscles were left uninjured. CTX induces local necrosis (22), followed by an inflammatory response consisting mainly of macrophage invasion (23), and subsequent tissue reconstitution. Control and ␤⌬Cre mice developed similar necrotic injuries, as determined by histological analysis at day 2 after CTX injection ( Fig.…”