Pei-Hsiu Kao

Involvement of p38 MAPK- and JNK-modulated expression of Bcl-2 and Bax in Naja nigricollis CMS-9-induced apoptosis of human leukemia K562 cells.

CMS-9, a phospholipase A(2) (PLA(2)) isolated from Naja nigricollis venom, induced apoptosis of human leukemia K562 cells, characterized by mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Moreover, an increase in intracellular Ca2+ concentration and the production of reactive oxygen species (ROS) was noted. Pretreatment with BAPTA-AM (Ca2+ chelator) and N-acetylcysteine (NAC, ROS scavenger) proved that Ca2+ was an upstream event in inducing ROS generation. Upon exposure to CMS-9, activation of p38 MAPK and JNK was observed in K562 cells. BAPTA-AM or NAC abrogated CMS-9-elicited p38 MAPK and JNK activation, and rescued viability of CMS-9-treated K562 cells. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) suppressed CMS-9-induced dissipation of mitochondrial membrane potential, Bcl-2 down-regulation, Bax up-regulation and increased mitochondrial translocation of Bax. Inactivation of PLA(2) activity reduced drastically the cytotoxicity of CMS-9, and a combination of lysophosphatidylcholine and stearic acid mimicked the cytotoxic effects of CMS-9. Taken together, our data suggest that CMS-9-induced apoptosis of K562 cells is catalytic activity-dependent and is mediated through mitochondria-mediated death pathway triggered by Ca2+/ROS-evoked p38 MAPK and JNK activation.

Membrane-damaging activity of Taiwan cobra cardiotoxin 3 is responsible for its bactericidal activity.

This study investigates the causal relationship between membrane-damaging activity and bactericidal activity of Naja naja atra (Taiwan cobra) cardiotoxin 3 (CTX3). CTX3 showed greater inhibitory activity for the growth of Staphylococcus aureus (Gram-positive bacteria) relative to that of Escherichia coli (Gram-negative bacteria). The CTX3 antibacterial activity is positively correlated with the increase in membrane permeability of bacterial cells. Morphological examination showed that CTX3 disrupted bacterial membrane integrity.CTX3 showed similar binding capability with lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and destabilization of LPS layer and inhibition of LTA biosynthesis on cell wall increased the CTX3 bactericidal effect on E. coli. and S. aureus, respectively. Compared with that of E. coli, CTX3 notably permeabilized model membrane of S. aureus. CTX3 membrane-damaging activity was inhibited by LPS and LTA, while increasing the CTX3 concentration counteracted the inhibitory action of LPS and LTA. Oxidation of Met residues on loop II of CTX3 simultaneously reduced the membrane-permeabilizing activity and bactericidal effect of CTX3. Taken together, our data indicate that CTX3 bactericidal activity depends highly on its ability to induce membrane permeability.

Differential binding to phospholipid bilayers modulates membrane-damaging activity of Naja naja atra cardiotoxins.

To address the events that modulate membrane-damaging activity of Naja naja atra cardiotoxins (CTXs), the present study was carried out. It was found that CTX isotoxins showed different activities in inducing leakage of vesicles made of egg yolk phosphatidylcholine (EYPC)/dimyristoyl phosphatidic acid (DMPA) or EYPC/egg yolk sphingomyelin (EYSM). Although CTXs had different gross conformations, the toxins showed similar binding affinity for phospholipid vesicles. Topographical contact between toxin molecules and phospholipid vesicles differed for different CTXs as evidenced by fluorescence enhancement of fluorescein-labeled phospholipid. Color transformation of phospholipid/polydiacetylene membrane assay revealed that CTX isotoxins were absorbed on lipid bilayers in different manners. Oxidation of Met residues at the tip of loop II indicated that membrane-bound conformation and orientation of CTXs played a vital role in damaging EYPC/EYSM and EYPC/DMPA vesicles, and suggested that an intact loop II was crucial for inducing leakage of EYSM-containing vesicles rather than that of DMPA-containing vesicles. Moreover, CTXs induced markedly hemolysis of cholesterol-depleted erythrocytes. Taken together, our data indicate that, in addition to membrane organization, membrane-bound conformation and interface-inserted mode of CTXs determine the potency of their membrane-damaging activity.

Interaction of Naja naja atra cardiotoxin 3 with H-trisaccharide modulates its hemolytic activity and membrane-damaging activity.

To address whether saccharide moieties of blood groups A, B and O antigens modulate hemolytic activity of Naja naja atra cardiotoxins (CTXs), the present study was carried out. Unlike other CTX isotoxins, hemolytic activity of CTX3 toward blood group O cholesterol-depleted red blood cells (RBCs) was notably lower than that of blood groups A and B cholesterol-depleted RBCs. Conversion of blood group B RBCs into blood group O RBCs by alpha-galactosidase treatment attenuated the susceptibility for hemolytic activity of CTX3, suggesting that H-antigen affected hemolytic potency of CTX3. Pre-incubation with H-trisaccharide reduced hemolytic activity and membrane-damaging activity of CTX3. Moreover, CTX3 showed a higher binding capability with H-trisaccharide than other CTXs did. CD spectra showed that the binding with H-trisaccharide induced changes in gross conformation of CTX3. Self-quenching studies revealed that oligomerization of CTX3 was affected in the presence of H-trisaccharide. Taken together, our data suggest that the binding of CTX3 with H-antigen alters its membrane-bound mode, thus reducing its hemolytic activity toward blood group O cholesterol-depleted RBCs.

Catalytic activity-independent pathway is involved in phospholipase A2-induced apoptotic death of human leukemia U937 cells via Ca2+-mediated p38 MAPK activation and mitochondrial depolarization

In view of the controversial role of catalytic activity on the cytotoxicity of phospholipase A(2) (PLA(2)), the present study is conducted to explore whether PLA(2) induces apoptotic process of human leukemia U937 cells through catalytic activity-independent pathway. Modification of His-48 (according to the sequence alignment with porcine pancreatic PLA(2)) with p-bromophenacyl bromide (BPB) caused over 99.9% drop in enzymatic activity Naja naja atra PLA(2). It was found that BPB-PLA(2)-induced apoptotic death of U937 cells was associated with mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Upon exposure to BPB-PLA(2), elevation of intracellular Ca(2+) levels and p38 MAPK activation were observed in U937 cells. Pretreatment with BAPTA-AM (Ca(2+) chelator) and nifedipine (L-type Ca(2+) channel blocker) abrogated Ca(2+) increase and p38 MAPK activation, and rescued viability of BPB-PLA(2)-treated U937 cells. BPB-PLA(2)-induced dissipation of mitochondrial membrane potential and down-regulation of Bcl-2 were suppressed by SB202190 (p38MAPK inhibitor). Although PLA(2) mutants in which His-48 and Asp-49 were substituted by Ala and Lys, respectively, did not display detectable PLA(2) activity, they induced death of U937 cells. The signaling pathway of PLA(2) mutants in inducing cell death was indistinguishable from that of BPB-PLA(2). Taken together, our data indicate that catalytic activity-independent pathway is involved in PLA(2)-induced apoptotic death of human leukemia U937 cells via mitochondria-mediated death pathway triggering by Ca(2+)-mediated p38 MAPK activation.

Antibacterial and membrane-damaging activities of β-bungarotoxin B chain

This study investigates whether the B chain of β‐bungarotoxin exerted antibacterial activity against Escherichia coli (Gram‐negative bacteria) and Staphylococcus aureus (Gram‐positive bacteria) via its membrane‐damaging activity. The B chain exhibited a growth inhibition effect on E. coli but did not show a bactericidal effect on S. aureus. The B‐chain bactericidal action on E. coli positively correlated with an increase in membrane permeability in the bacterial cells. Lipopolysaccharide (LPS) layer destabilization and lipoteichoic acid (LTA) biosynthesis inhibition in the cell wall increased the B‐chain bactericidal effect on E. coli and S. aureus. The B chain induced leakage and fusion in E. coli and S. aureus membrane‐mimicking liposomes. Compared with LPS, LTA notably suppressed the membrane‐damaging activity and fusogenicity of the B chain. The B chain showed similar binding affinity with LPS and LTA, whereas LPS and LTA binding differently induced B‐chain conformational change as evidenced by the circular dichroism spectra. Taken together, our data indicate that the antibacterial action of the B chain is related to its ability to induce membrane permeability and suggest that the LPS‐induced and LTA‐induced B‐chain conformational change differently affects the bactericidal action of the B chain. Copyright

Membrane-bound conformation of Naja nigricollis toxin γ affects its membrane-damaging activity

To address whether the conformational events associated with the absorption of Naja nigricollis toxin gamma on water-lipid interface plays a vital role in its membrane-damaging activity, the present study is carried out. Membrane-damaging activity of toxin gamma on 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC)/1, 2-dimyristoyl-phosphatidic acid (DMPA) vesicles was approximately 13-fold of that on 1, 2-dipalmitoyl-phosphatidylcholine (DPPC)/DMPA vesicles, while the binding affinity of toxin gamma for POPC/DMPA was twofold of that for DPPC/DMPA. Time-resolved fluorescence, acrylamide quenching and Fourier transform infrared spectra showed that POPC/DMPA-bound toxin gamma and DPPC/DMPA-bound toxin gamma did not adopt the same conformation. Moreover, geometrical arrangement of toxin gamma in contact with POPC/DMPA vesicles was different from that with DPPC/DMPA vesicles as evidenced by N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-phosphatidylcholine fluorescence enhancement and cross-linking of membrane-bound toxin gamma. Taken together, our data show that different membrane packing densities arising from phospholipid acyl chain affect membrane-bound conformation of toxin gamma, thus changing its membrane-damaging activity.

Naja naja atra and Naja nigricollis cardiotoxins induce fusion of Escherichia coli and Staphylococcus aureus membrane-mimicking liposomes

Our previous studies showed that the bactericidal effect of Naja naja atra cardiotoxin 3 (CTX3) and Naja nigricollis toxin γ was associated with their membrane-damaging activity. To elucidate the mechanism responsible for CTX3- and toxin γ-induced membrane permeability, we investigated the interacted mode of CTX3 and toxin γ with model membrane of Escherichia coli (phosphatidylethanolamine (PE)/phosphatidylglycerol (PG), mol/mol, 75/25) and Staphylococcus aureus (PG/cardiolipin, mol/mol, 60/40) in this study. Membrane-damaging activity of toxin γ on PE/PG and PG/cardiolipin vesicles were similar, while CTX3-induced leakage of PG/cardiolipin vesicles was notably higher than that of PE/PG vesicles. Noticeably, fusogenic activity of CTX3 and toxin γ on the phospholipid vesicles correlated positively with their membrane-damaging activity. Unlike toxin γ, CTX3 induced increasingly leakage and fusion of phospholipid vesicles with increased cardiolipin content. Changes in membrane fluidity and lipid packing occurred with the binding of CTX3 and toxin γ with vesicles, reflecting the penetration of toxin molecules into membrane bilayers. Consistent with the finding that PE/PG and PG/cardiolipin vesicles induced differently conformational changes of CTX3 and toxin γ, CTX3 and toxin γ adopted different membrane bound-mode upon absorption onto either PE/PG or PG/cardiolipin vesicles. Taken together, our data indicate that membrane-bound mode and membrane-perturbing effect of CTX3 and toxin γ in concert with targeted membrane compositions determine their fusogenicity and membrane-damaging activity, and suggest a causal relationship between bactericidal activity and fusogenicity of CTX3 and toxin γ.

Roles of lysine residues and N-terminal α-amino group in membrane-damaging activity of Taiwan cobra cardiotoxin 3 toward anionic and zwitterionic phospholipid vesicles.

In contrast to a slight increase in activity toward phosphatidylcholine (EYPC)/dimyristoyl phosphatidic acid (DMPA) vesicles, guanidination of Naja naja atra cardiotoxin 3 (CTX3) and selective trinitrophenylation of N-terminal alpha-amino group enhanced notably membrane-damaging activity on EYPC/egg yolk sphingomyelin (EYSM) vesicles. Chemically modified CTX3 showed a reduction in its hemolytic activity and cytotoxicity. These reflected that membrane-damaging activity of CTX3 was affected by phospholipid compositions. Phospholipid-binding capability and oligomeric assembly upon binding with lipid vesicles did not closely correlate with membrane-damaging potency of native and modified CTX3. Moreover, different topographical contacts and distinctive modes for the binding of CTX3 and its modified derivatives with anionic phospholipid vesicles (EYPC/DMPA) and zwitterionic phospholipid vesicles (EYPC/EYSM) were observed. Compared with in the case of EYPC/DMPA, the interaction between CTX molecules and EYPC/EYSM was drastically reduced by increasing salt concentration and heparin. Taken together, our data indicate that guanidination of Lys residues and trinitrophenylation of alpha-amino group alter differently the interacted modes upon absorption on anionic phospholipid vesicles and zwitterionic phospholipid vesicles. The findings also suggest that positively charged residues of CTX3 play a distinctive role in damaging anionic and zwitterionic phospholipid vesicles.

Bovine serum albumin with glycated carboxyl groups shows membrane-perturbing activities

The aim of the present study aimed to investigate whether glycated bovine serum albumin (BSA) showed novel activities on the lipid-water interface. Mannosylated BSA (Man-BSA) was prepared by modification of the carboxyl groups with p-aminophenyl α-d-mannopyranoside. In contrast to BSA, Man-BSA notably induced membrane permeability of egg yolk phosphatidylcholine (EYPC)/egg yolk sphingomyelin (EYSM)/cholesterol (Chol) and EYPC/EYSM vesicles. Noticeably, Man-BSA induced the fusion of EYPC/EYSM/Chol vesicles, but not of EYPC/EYSM vesicles. Although BSA and Man-BSA showed similar binding affinity for lipid vesicles, the lipid-bound conformation of Man-BSA was distinct from that of BSA. Moreover, Man-BSA adopted distinct structure upon binding with the EYPC/EYSM/Chol and EYPC/EYSM vesicles. Man-BSA could induce the fusion of EYPC/EYSM/Chol vesicles with K562 and MCF-7 cells, while Man-BSA greatly induced the leakage of Chol-depleted K562 and MCF-7 cells. The modified BSA prepared by conjugating carboxyl groups with p-aminophenyl α-d-glucopyranoside also showed membrane-perturbing activities. Collectively, our data indicate that conjugation of carboxyl groups with monosaccharide generates functional BSA with membrane-perturbing activities on the lipid-water interface.