Yi-Ling Chiou

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.

Quercetin modulates activities of Taiwan cobra phospholipase A2 via its effects on membrane structure and membrane-bound mode of phospholipase A2.

The goal of the present study is to elucidate the mechanism of quercetin on modulating Naja naja atra phospholipase A2 (PLA2) activities. Sphingomyelin inhibited PLA2 enzymatic activity and membrane-damaging activity against egg yolk phosphatidylcholine (EYPC), while cholesterol and quercetin abrogated the sphingomeyelin inhibitory effect. Quercetin incorporation led to a reduction in PLA2 enzymatic activity and membrane-damaging activity toward EYPC/sphingomyelin/cholesterol vesicles. Both cholesterol and quercetin increased detergent resistance and reduced membrane fluidity of EYPC/sphingomyelin vesicles. Quercetin reduced detergent insolubility but increased ordered lipid packing of EYPC/sphingomyelin/cholesterol vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that quercetin altered the membrane-bound mode of PLA2 differently upon absorption onto the membrane bilayers of different lipid compositions. However, 8-anilinonaphthalene sulphonate-binding assay revealed that quercetin marginally affected the interaction between active site of PLA2 with phospholipid vesicles. Collectively, our data indicate that membrane-inserted quercetin modulates PLA2 interfacial activity and membrane-damaging activity via its effects on membrane structure and membrane-bound mode of PLA2.

Lipid domain formation modulates activities of snake venom phospholipase A2 enzymes

The goal of the present study is to elucidate the effect of lipid domain formation on activities of Naja naja atra and Bungarus multicinctus phospholipase A(2) (PLA(2)) enzymes. Sphingomyelin inhibited enzymatic activity and membrane-damaging activity of PLA(2) against egg yolk phosphatidylcholine (EYPC), while cholesterol and cholesterol sulfate abrogated the inhibitory effect of sphingomyelin. The ability of cholesterol and cholesterol sulfate to abolish the inhibitory effect of sphingomyelin was closely related to their capacity to induce domain formation in EYPC/sphingomyelin vesicles. Laurdan fluorescence measurement revealed that membrane packing of EYPC/sphingomyelin vesicles was differently affected by cholesterol and cholesterol sulfate. Unlike cholesterol, cholesterol sulfate was unable to promote domain formation in dipalmitoylphosphatidylcholine (DPPC) vesicles. Cholesterol increased but cholesterol sulfate reduced PLA(2) activity against DPPC. Self-quenching studies and trinitrophenylation of Lys residues revealed that PLA(2) enzymes adopted different membrane-bound mode upon absorption onto the membrane bilayers comprised of different lipid compositions. Collectively, our data indicate that lipid domain formation regulates PLA(2) activity, and suggest that the physical state of membrane bilayers changes the interactive mode of PLA(2) with phospholipids.

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.

Effect of cholesterol on membrane-damaging activity of Naja nigricollis toxin γ toward phospholipid vesicles.

Membrane-damaging activity of Naja nigricollis toxin gamma on phospholipid vesicles was attenuated by incorporation of cholesterol into phospholipid vesicles. Deprivation of cholesterol from erythrocyte membrane enhanced notably hemolytic activity of toxin gamma, while the hemolytic activity of toxin gamma on cholesterol-depleted erythrocytes decreased when cholesterol was restored to membrane. Phospholipid-binding capability and oligomeric assembly upon binding with lipid vesicles were modestly affected in the presence of cholesterol. Time-resolved fluorescence and Fourier transform infrared spectra showed that phospholipid-bound toxin gamma and cholesterol/phospholipid-bound toxin gamma did not adopt the same conformation. Moreover, geometrical arrangement of toxin gamma in contact with phospholipid vesicles was different from that with cholesterol/phospholipid vesicles as evidenced by N-(fluorescein-5-thiocarbamoyl)-1, 2-dihexadecanoyl-phosphatidylethanolamine fluorescence enhancement and color transformation of phospholipid/polydiacetylene membrane assay. Taken together, our data show that ordered phospholipid phase arising from incorporation of cholesterol affects conformation and topographical arrangement of toxin gamma on water-lipid interface, thus attenuating its membrane-damaging activity against phospholipid vesicles.

Phospholipase A2 activity-dependent and -independent fusogenic activity of Naja nigricollis CMS-9 on zwitterionic and anionic phospholipid vesicles

CMS-9, a phospholipase A(2) (PLA(2)) from Naja nigricollis venom, induced the death of human breast cancer MCF-7 cells accompanied with the formation of cell clumps without clear boundaries between cells. Annexin V-FITC staining indicated that abundant phosphatidylserine appeared on the outer membrane of MCF-7 cell clumps, implying the possibility that CMS-9 may promote membrane fusion via anionic phospholipids. To validate this proposition, fusogenic activity of CMS-9 on vesicles composed of zwitterionic phospholipid alone or a combination of zwitterionic and anionic phospholipids was examined. Although CMS-9-induced fusion of zwitterionic phospholipid vesicles depended on PLA(2) activity, CMS-9-induced fusion of vesicles containing anionic phospholipids could occur without the involvement of PLA(2) activity. Membrane-damaging activity of CMS-9 was associated with its fusogenicity. Moreover, CMS-9 induced differently membrane leakage and membrane fusion of vesicles with different compositions. Membrane fluidity and binding capability with phospholipid vesicles were not related to the fusogenicity of CMS-9. However, membrane-bound conformation and mode of CMS-9 depended on phospholipid compositions. Collectively, our data suggest that PLA(2) activity-dependent and -independent fusogenicity of CMS-9 are closely related to its membrane-bound modes and targeted membrane compositions.

Sphingomyelin modulates interfacial binding of Taiwan cobra phospholipase A2

The goal of the present study is to elucidate the effect of sphingomyelin on interfacial binding of Taiwan cobra phospholipase A(2) (PLA(2)). Substitution of Asn-1 with Met caused a reduction in enzymatic activity and membrane-damaging activity of PLA(2) toward phospholipid vesicles, while sphingomyelin exerted an inhibitory effect on the biological activities of native and mutated PLA(2). Incorporation of sphingomyelin reduced membrane fluidity of phospholipid vesicles as evidenced by Laurdan fluorescence measurement. The results of self-quenching studies, binding of fluorescent probe, trinitrophenylation of Lys residues and fluorescence energy transfer between protein and lipid revealed that sphingomyelin altered differently membrane-bound mode of native and mutated PLA(2). Moreover, it was found that PLA(2) and N-terminally mutated PLA(2) adopted different conformation and geometrical arrangement on binding with membrane bilayer. Nevertheless, the binding affinity of PLA(2) and N-terminal mutant for phospholipid vesicles was not greatly affected by sphingomyelin. Together with the finding that mutation on N-terminus altered the gross conformation of PLA(2), our data indicate that sphingomyelin modulates the mode of membrane binding of PLA(2) at water/lipid interface, and suggest that the modulated effect of sphingomyelin depends on inherent structural elements of PLA(2).

Guanidination of notexin alters its membrane-damaging activity in response to sphingomyelin and cholesterol

To elucidate the contribution of phospholipase A2 (PLA2) activity of notexin to its ability to perturb membranes, comparative studies on the interaction of notexin and guanidinated notexin (Gu-notexin) with egg yolk phosphatidylcholine (EYPC), EYPC/egg yolk sphingomyelin (EYSM) and EYPC/EYSM/cholesterol vesicles were conducted. EYSM notably reduced the membrane-damaging activity of notexin against EYPC vesicles, but had an insignificant influence on that of Gu-notexin. Unlike the effects noted with notexin, inactivation of PLA2 activity by EDTA led to a reduction in the ability of Gu-notexin to induce EYPC/EYSM vesicle leakage and to increase Gu-notexin-induced membrane permeability of EYPC/EYSM/cholesterol vesicles. The geometrical arrangement of notexin and Gu-notexin in contact with either EYPC/EYSM vesicles or EYPC/EYSM/cholesterol vesicles differed. Moreover, global conformation of notexin and Gu-notexin differed in either Ca2+-bound or metal-free states. These results indicate that notexin and Gu-notexin could induce membrane permeability without the involvement of PLA2 activity, and suggest that guanidination alters the membrane-bound mode of notexin on damaging phospholipid vesicles containing sphingomyelin and cholesterol.

Modulated mechanism of phosphatidylserine on the catalytic activity of Naja naja atra phospholipase A2 and Notechis scutatus scutatus notexin.

Phosphatidylserine (PS) externalization is a hallmark for apoptotic death of cells. Previous studies showed that Naja naja atra phospholipase A2 (NnaPLA2) and Notechis scutatus scutatus notexin induced apoptosis of human cancer cells. However, NnaPLA2 and notexin did not markedly disrupt the integrity of cellular membrane as evidenced by membrane permeability of propidium iodide. These findings reflected that the ability of NnaPLA2 and notexin to hydrolyze membrane phospholipids may be affected by PS externalization. To address that question, this study investigated the membrane-interacted mode and catalytic activity of NnaPLA2 and notexin toward outer leaflet (phosphatidylcholine/sphingomyelin/cholesterol, PC/SM/Chol) and inner leaflet (phosphatidylserine/phosphatidylethanolamine/cholesterol, PS/PE/Chol) of plasma membrane-mimicking vesicles. PS incorporation promoted enzymatic activity of NnaPLA2 and notexin on PC and PC/SM vesicles, but suppressed NnaPLA2 and notexin activity on PC/SM/Chol and PE/Chol vesicles. PS incorporation increased the membrane fluidity of PC vesicles but reduced membrane fluidity of PC/SM, PC/SM/Chol and PE/Chol vesicles. PS increased the phospholipid order of all the tested vesicles. Moreover, PS incorporation did not greatly alter the binding affinity of notexin and NnaPLA2 with phospholipid vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that membrane-bound mode of notexin and NnaPLA2 varied with the targeted membrane compositions. The fine structure of catalytic site in NnaPLA2 and notexin in all the tested vesicles showed different changes. Collectively, the present data suggest that membrane-inserted PS modulates PLA2 interfacial activity via its effects on membrane structure and membrane-bound mode of NnaPLA2 and notexin, and membrane compositions determine the effect of PS on PLA2 activity.

Guanidination of notexin promotes its phospholipase A2 activity-independent fusogenicity on vesicles with lipid-supplied negative curvature

To address the requirement of phospholipase A(2) (PLA(2)) activity in membrane fusion events and membrane perturbation activity of notexin and guanidinated notexin (Gu-notexin), the present study was conducted. Notexin and Gu-notexin did not show PLA(2) activity after the removal of Ca(2+) with EDTA. Metal-free notexin and Gu-notexin were found to induce membrane leakage and fusion of phospholipid vesicles. Fusogenic activity of native and modified notexin correlated positively with their membrane-damaging activity underlying the deprivation of PLA(2) activity. Compared with Ca(2+)-bound Gu-notexin, fusogenicity of metal-free Gu-notexin was notably increased by incorporation of cholesterol, cholesterol sulfate, phosphatidylethanolamine, α-tocopherol and phosphatidic acid that supplied negative curvature into phospholipid bilayer. The ability of Gu-notexin to induce membrane fusion of vesicles with lipid-supplied negative curvature was higher than that of notexin regardless of the absence or presence of Ca(2+). Consistently, metal-free Gu-notexin markedly induced membrane fusion of red blood cells (RBCs) compared with metal-free notexin, and fusion activity of metal-free Gu-notexin on cholesterol-depleted RBCs notably reduced. Compared with notexin, Gu-notexin highly induced uptake of calcein-loaded phosphatidylcholine (PC)/cholesterol and PC/cholesterol sulfate vesicles by K562 cells in the presence of EDTA. Taken together, our data suggest that notexin and Gu-notexin could induce vesicle leakage and fusion via a PLA(2) activity-independent mechanism, and guanidination promotes PLA(2) activity-independent fusogenicity of notexin on vesicles with lipid-supplied negative curvature.