Endocytotic routes of cobra cardiotoxins depend on spatial distribution of positively charged and hydrophobic domains to target distinct types of sulfated glycoconjugates on cell surface

Abstract

Cobra cardiotoxins (CTX) are a family of three-fingered basic polypeptides known to interact with diverse targets such as heparan sulfates, sulfatides, and integrins on cell surfaces. After CTX bind to the membrane surface, they are internalized to intracellular space and exert their cytotoxicity via an unknown mechanism. By the combined in vitro kinetic binding, three-dimensional x-ray structure determination, and cell biology studies on the naturally abundant CTX homologues from the Taiwanese cobra, we showed that slight variations on the spatial distribution of positively charged or hydrophobic domains among CTX A2, A3, and A4 could lead to significant changes in their endocytotic pathways and action mechanisms via distinct sulfated glycoconjugate-mediated processes. The intracellular locations of these structurally similar CTX after internalization are shown to vary between the mitochondria and lysosomes via either dynamin2-dependent or -independent processes with distinct membrane cholesterol sensitivity. Evidence is presented to suggest that the shifting between the sulfated glycoconjugates as distinct targets of CTX A2, A3, and A4 might play roles in the co-evolutionary arms race between venomous snake toxins to cope with different membrane repair mechanisms at the cellular levels. The sensitivity of endocytotic routes to the spatial distribution of positively charged or hydrophobic domains may provide an explanation for the diverse endocytosis pathways of other cell-penetrating basic polypeptides.

Keywords: Cardiotoxin; Cholesterol; Endocytosis; Heparan Sulfate; Snake Venom; Sulfatide; X-ray Crystallography.

FIGURE 1.

Heparin and negatively charged lipid as binding targets for three CTX homologues with a well defined spatial distribution of the positively charged domain. A, primary sequences of CTX homologues show high homologies but vary in several positively charged residues at the N-terminal and loop II regions. B, vesicle leakage assay on different CTX homologues shows differences in specificity toward negatively charged lipids. Time-dependent leakage of 6-CF dyes from 50% POPS vesicles was induced by CTX (0.16 μm). The higher affinity of CTX A3 toward the negatively charged lipids, POPS, was indicated by a higher leakage content, but this was not seen for CTX A2 or A4 under the same conditions. C, Scatchard plots (R_eq/concentration (Conc.) versus R_eq) for CTX A2 (open symbols) or CTX A4 (closed symbols) in an SPR binding assay (0.15–8 μm) on immobilized heparins under different surface densities (triangle, 700 RU; circle, 350 RU; square, 200 RU) were shown. D, SPR binding of CTX homologues (0.15–20 μm) onto immobilized heparin surfaces (700 RU as representative plots) showed different retention capabilities. Significant CTX retention on immobilized heparin surfaces was observed for CTX A2 and A4 at higher concentrations but not for CTX A3.

FIGURE 2.

Structural comparisons of CTX A2, A3, and A4. A, three different views of electrostatic surfaces of the CTX homologues based on the crystal structures of CTX A2 (PDB code 4OM4 (this work)), CTX A3 (PDB code 1XT3), and CTX A4 (PDB code 4OM5 (this work)). Protein surfaces shown in blue, red, and white indicate positively charged, negatively charged, and hydrophobic regions, respectively. B, hydrophobic interface in the crystal structures of A4 dimers. Ribbon models of the CTX A4 dimer colored in cyan and green are superimposed with the CTX A2 dimer in gray. The N-terminal R1 and hydrophobic residues at the dimer interface of A4 are shown as sticks. C, the electrostatic surface of CTX A4 dimer showing a continuous patch of positively charged residues (Lys-5, Lys-12, Lys-18, and Lys-35) suitable for heparin binding. The locations of the R1 residues are indicated by arrows. de2OS, de-2-O-sulfation; de6OS, de-6-O-sulfation; deNS, de-N-sulfation.

FIGURE 3.

SPR studies for CTX A2 and A4 binding to immobilized heparin with sulfate specificity. A, effect of distinct sulfation groups on the binding of heparin mimetics to CTX A2 (open symbols) and CTX A4 (close symbols) as revealed by the SPR binding sensograms in the presence of the related desulfated heparin mimetics. The arrows emphasized the difference in K_i values for de-6-O-sulfated heparin binding to CTX A2 and A4. B, the retained CTX were dissociated from immobilized heparin surfaces by injecting heparin through competition. Insert, indicates that the competition of injected heparin was concentration-dependent, reflecting the competitive ability of injected heparin. C, effects of heparin modifications on their competitive abilities as revealed by the dissociation of the retained CTX in the presence of indicated heparin mimetics. de2OS, de-2-O-sulfation; de6OS, de-6-O-sulfation; deNS, de-N-sulfation.

FIGURE 4.

Distinct heparinase I and III sensitivity to CTX A2/A4 retention and endocytosis indicating the involvement of different HS domains. A, retentions of different CTX on immobilized cell surfaces were dependent on surface GAG and were sensitive to different heparinase treatments as revealed by the reduced length of the blots (three individual repeats to show retained CTX). B, endocytotic CTX A2 and CTX A4 inside the H9C2 cells was located at the lysosome (as indicated by LysoTracker) but not at the mitochondria (as indicated by MitoTracker). Bar = 20 μm. C, endocytosis of different CTX into H9C2 or CHO cells was dependent on surface GAG and was sensitive to different heparinase treatments. Bar = 5 μm. D, CTX A2 and A4 induced lysosomal membrane permeabilization. CTX A2 or A4 decreased the red fluorescence of acridine orange (upper panels; green fluorescence was from LysoTracker Green DND-26) and the green fluorescence of LysoSensor DND-189 (middle panels) as compared with naphthazarin (lysosomal destabilizer, 5 μm). Treatment with naphthazarin released cathepsin D into the cytosol, but this was not observed for CTX A2 orA4 (intact spots remained). Data were mean ± S.E. (n > 10); **, p < 0.01.

FIGURE 5.

Dynasore effect on the internalization of CTX homologues. A, CTX A2 and A4 showed dynamin-dependent endocytosis as evidenced by dynasore treatment, whereas CTX A3 endocytosis was dynamin-independent. Bar = 20 μm. B, CTX-induced cytotoxicities were performed in the absence and presence of dynasore, a specific blocker of dynamin-dependent endocytosis. Dynasore inhibited CTX A2/A4-induced cytotoxicity but had no effect on that of CTX A3. Data were mean ± S.E. (n = 5); **, p < 0.01. C, confocal images show partial colocalization of rhodamine-labeled CTX A2 and fluorescin-labeled CTX A4 in H9C2 cells. The percentage of colocalization was calculated as 67.0% (n = 5). Bar = 20 μm. D, colocalization experiments show possible endocytotic mechanisms of CTX A2 and A4. Fluorescence-labeled albumin (Alb), transferrin (Tfn), and dextran (Dex) were indicators for caveolae-mediated, clathrin-mediated, and marcopinocytosis pathways, respectively. The percentage of colocalization with transferrin, albumin, and dextran distinguished possible mechanisms of endocytosis (n ≦ 5). Bar = 20 μm. Data were mean ± S.E. (n = 5); **, p < 0.01.

FIGURE 6.

Effect of membrane cholesterol concentration on the internalization and cytotoxicity of CTX homologues. A and B, confocal images and quantifications of CTX endocytosis upon cholesterol (chol.) depletion (10 mm MβCD) or cholesterol enrichment (0.2 mm cholesterol/2 mm MβCD). Filipin, the cholesterol chelator, is used to reflect the decrease or increase in cholesterol concentration as a result of the depletion or enrichment process. Bar = 20 μm. Data were mean ± S.E. (n = 5); *, p < 0.05; **, p < 0.01. C, CTX A3-induced cytotoxicity and endocytosis as a result of cholesterol depletion and incorporation. D, inhibition of CTX A3-induced leakage of 6-CF-loaded vesicles by elevated cholesterol content in sulfated glycosphingolipid (SGC)-containing vesicles.

FIGURE 7.

Extracellular Ca²⁺-mediated membrane repair mechanism to hinder CTX A3-induced cytotoxicity. A, extracellular Ca²⁺ influx into Fluo-3-loaded NIH3T3 cells was induced by CTX A3-specific (but not by CTX A2 and A4) pore formations within 1 min. B, depletion of extracellular Ca²⁺ enhanced CTX A3 internalization. C, plasma membrane repair of CTX A3-permeabilized NIH3T3 cells was Ca²⁺-dependent. The blue color for Hoechst staining indicates the nuclear location; propidium iodide influx, stained red, indicates that the cells failed to reseal. D, endocytosis of fluorescence-labeled dextran (4 kDa) was significantly enhanced by CTX A3 under Ca²⁺-free condition. Bar = 20 μm. Data were mean ± S.E. (n = 5); **, p < 0.01.