Unique Membrane Interaction Mode of Group IIF Phospholipase A2
2006; Elsevier BV; Volume: 281; Issue: 43 Linguagem: Inglês
10.1074/jbc.m606311200
ISSN1083-351X
AutoresGihani T. Wijewickrama, Alexandra Albanese, Young Jun Kim, Youn Sang Oh, Paul S. Murray, Risa Takayanagi, Takashi Tobe, Seiko Masuda, Makoto Murakami, Ichiro Kudo, David S. Ucker, Diana Murray, Wonhwa Cho,
Tópico(s)Cellular transport and secretion
ResumoThe mechanisms by which secretory phospholipases A2 (PLA2s) exert cellular effects are not fully understood. Group IIF PLA2 (gIIFPLA2) is a structurally unique secretory PLA2 with a long C-terminal extension. Homology modeling suggests that the membrane-binding surface of this acidic PLA2 contains hydrophobic residues clustered near the C-terminal extension. Vesicle leakage and monolayer penetration measurements showed that gIIFPLA2 had a unique ability to penetrate and disrupt compactly packed monolayers and bilayers whose lipid composition recapitulates that of the outer plasma membrane of mammalian cells. Fluorescence imaging showed that gIIFPLA2 could also readily enter and deform plasma membrane-mimicking giant unilamellar vesicles. Mutation analysis indicates that hydrophobic residues (Tyr115, Phe116, Val118, and Tyr119) near the C-terminal extension are responsible for these activities. When gIIFPLA2 was exogenously added to HEK293 cells, it initially bound to the plasma membrane and then rapidly entered the cells in an endocytosis-independent manner, but the cell entry did not lead to a significant degree of phospholipid hydrolysis. GIIFPLA2 mRNA was detected endogenously in human CD4+ helper T cells after in vitro stimulation and exogenously added gIIFPLA2 inhibited the proliferation of a T cell line, which was not seen with group IIA PLA2. Collectively, these data suggest that unique membrane-binding properties of gIIFPLA2 may confer special functionality on this secretory PLA2 under certain physiological conditions. The mechanisms by which secretory phospholipases A2 (PLA2s) exert cellular effects are not fully understood. Group IIF PLA2 (gIIFPLA2) is a structurally unique secretory PLA2 with a long C-terminal extension. Homology modeling suggests that the membrane-binding surface of this acidic PLA2 contains hydrophobic residues clustered near the C-terminal extension. Vesicle leakage and monolayer penetration measurements showed that gIIFPLA2 had a unique ability to penetrate and disrupt compactly packed monolayers and bilayers whose lipid composition recapitulates that of the outer plasma membrane of mammalian cells. Fluorescence imaging showed that gIIFPLA2 could also readily enter and deform plasma membrane-mimicking giant unilamellar vesicles. Mutation analysis indicates that hydrophobic residues (Tyr115, Phe116, Val118, and Tyr119) near the C-terminal extension are responsible for these activities. When gIIFPLA2 was exogenously added to HEK293 cells, it initially bound to the plasma membrane and then rapidly entered the cells in an endocytosis-independent manner, but the cell entry did not lead to a significant degree of phospholipid hydrolysis. GIIFPLA2 mRNA was detected endogenously in human CD4+ helper T cells after in vitro stimulation and exogenously added gIIFPLA2 inhibited the proliferation of a T cell line, which was not seen with group IIA PLA2. Collectively, these data suggest that unique membrane-binding properties of gIIFPLA2 may confer special functionality on this secretory PLA2 under certain physiological conditions. Phospholipase A2 (PLA 2) 3The abbreviations used are: PLA2, phospholipase A2; AA, arachidonic acid; BLPC, 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine; BLPG, 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphoglycerol; BSA, bovine serum albumin; DHPC, di-O-hexadecyl-sn-glycero-3-phosphocholine; HSPG, heparan sulfate proteoglycans; gIIAPLA2, group IIA phospholipase A2; gIIFPLA2, group IIF phospholipase A2; gVPLA2, group V phospholipase A2; gXPLA2, group X phospholipase A2; PC, phosphatidylcholine; PG, phosphatidylglycerol; PED6, N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine triethylammonium salt; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; pyrene-PG, 1-hexadecanoyl-2-(1-pyrenyldecanoyl)-sn-glycero-3-phosphoglycerol; SM, brain sphingomylein; sPLA2, secretory PLA2; NBD-cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; SPR, surface plasmon resonance; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; LUV, large unilamellar vesicle; GUV, giant unilamellar vesicles; PG, phosphatidylglycerol. 3The abbreviations used are: PLA2, phospholipase A2; AA, arachidonic acid; BLPC, 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine; BLPG, 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphoglycerol; BSA, bovine serum albumin; DHPC, di-O-hexadecyl-sn-glycero-3-phosphocholine; HSPG, heparan sulfate proteoglycans; gIIAPLA2, group IIA phospholipase A2; gIIFPLA2, group IIF phospholipase A2; gVPLA2, group V phospholipase A2; gXPLA2, group X phospholipase A2; PC, phosphatidylcholine; PG, phosphatidylglycerol; PED6, N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine triethylammonium salt; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; pyrene-PG, 1-hexadecanoyl-2-(1-pyrenyldecanoyl)-sn-glycero-3-phosphoglycerol; SM, brain sphingomylein; sPLA2, secretory PLA2; NBD-cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; SPR, surface plasmon resonance; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; LUV, large unilamellar vesicle; GUV, giant unilamellar vesicles; PG, phosphatidylglycerol. catalyzes the hydrolysis of the sn-2 ester bond of membrane phospholipids to liberate fatty acid and lysophospholipid. One of these products, arachidonic acid (AA) is transformed into potent inflammatory lipid mediators, collectively known as eicosanoids. Multiple forms of PLA2s, including secretory PLA2s (sPLA2) (1Valentin E. Lambeau G. Biochim. Biophys. Acta. 2000; 1488: 59-70Crossref PubMed Scopus (313) Google Scholar, 2Singer A.G. Ghomashchi F. Le Calvez C. Bollinger J. Bezzine S. Rouault M. Sadilek M. Nguyen E. Lazdunski M. Lambeau G. Gelb M.H. J. Biol. Chem. 2002; 277: 48535-48549Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar) and Ca2+-dependent cytosolic PLA2s(α, β, γ, δ, ϵ, and ζ) (3Shimizu T. Ohto T. Kita Y. IUBMB Life. 2006; 58: 328-333Crossref PubMed Scopus (69) Google Scholar, 4Ohto T. Uozumi N. Hirabayashi T. Shimizu T. J. Biol. Chem. 2005; 280: 24576-24583Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) and Ca2+-independent intracellular PLA2s(β and γ) (5Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1196) Google Scholar), have been identified from mammalian tissues. Among intracellular PLA2s, group IVA cytosolic phospholipase A2 has been shown to be involved in inflammation (6Bonventre J.V. Huang Z. Taheri M.R. O'Leary E. Li E. Moskowitz M.A. Sapirstein A. Nature. 1997; 390: 622-625Crossref PubMed Scopus (755) Google Scholar, 7Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. Miyazaki J. Shimizu T. Nature. 1997; 390: 618-622Crossref PubMed Scopus (636) Google Scholar), whereas intracellular PLA2β has been implicated in spermatogenesis and insulin signaling (8Bao S. Miller D.J. Ma Z. Wohltmann M. Eng G. Ramanadham S. Moley K. Turk J. J. Biol. Chem. 2004; 279: 38194-38200Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 9Bao S. Song H. Wohltmann M. Ramanadham S. Jin W. Bohrer A. Turk J. J. Biol. Chem. 2006; 281: 20958-20973Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). However, physiological roles of other intracellular PLA2s have not been fully defined. 10 sPLA2s (groups IB, IIA, IIC, IID, IIE, IIF, III, V, X, and XII) have been identified in mammals so far (1Valentin E. Lambeau G. Biochim. Biophys. Acta. 2000; 1488: 59-70Crossref PubMed Scopus (313) Google Scholar, 10Murakami M. Kudo I. Adv. Immunol. 2001; 77: 163-194Crossref PubMed Scopus (134) Google Scholar). Many sPLA2s have been shown to induce or augment cellular AA release and eicosanoid biosynthesis when overexpressed in or exogenously added to mammalian cells. However, it is not clear whether or not these sPLA2s are directly involved in AA production and inflammation under physiological conditions. Among various sPLA2s, group V PLA2 (gVPLA2) has been implicated in inflammation by a recent gene knock-out study (11Satake Y. Diaz B.L. Balestrieri B. Lam B.K. Kanaoka Y. Grusby M.J. Arm J.P. J. Biol. Chem. 2004; 279: 16488-16494Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). It has been reported that sPLA2s can exert cellular effects through different mechanisms (12Wijewickrama G.T. Kim J.H. Kim Y.J. Abraham A. Oh Y. Ananthanarayanan B. Kwatia M. Ackerman S.J. Cho W. J. Biol. Chem. 2006; 281: 10935-10944Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Based on the earlier finding that the level of sPLA2 was elevated in inflammatory exudates (13Vadas P. Stefanski E. Pruzanski W. Life Sci. 1985; 36: 579-587Crossref PubMed Scopus (101) Google Scholar, 14Vadas P. Pruzanski W. Lab. Invest. 1986; 55: 391-404PubMed Google Scholar), it was generally thought that sPLA2s are released to the extracellular medium in response to specific stimuli and act on different target cells by a transcellular or paracrine mechanism. However, Kudo and co-workers found that many basic sPLA2s, including group IIA PLA2 (gIIAPLA2) and gVPLA2, remained bound to their parent cells after secretion due to their high affinity for cell surface heparan sulfate proteoglycans (HSPG) and were reinternalized to augment the stimulus-dependent AA release (15Suga H. Murakami M. Kudo I. Inoue K. Eur. J. Biochem. 1993; 218: 807-813Crossref PubMed Scopus (70) Google Scholar, 16Murakami M. Kudo I. Inoue K. J. Biol. Chem. 1993; 268: 839-844Abstract Full Text PDF PubMed Google Scholar, 17Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 18Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 19Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 20Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 21Murakami M. Yoshihara K. Shimbara S. Lambeau G. Singer A. Gelb M.H. Sawada M. Inagaki N. Nagai H. Kudo I. Biochem. Biophys. Res. Commun. 2002; 292: 689-696Crossref PubMed Scopus (48) Google Scholar). This HSPG affinity has been shown to be important for the entry of different types of sPLA2s into mammalian cells (12Wijewickrama G.T. Kim J.H. Kim Y.J. Abraham A. Oh Y. Ananthanarayanan B. Kwatia M. Ackerman S.J. Cho W. J. Biol. Chem. 2006; 281: 10935-10944Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 22Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). More recently, it was reported that gIIAPLA2 and group X (gXPLA2) could also induce the cellular AA release during the secretory process (23Mounier C.M. Ghomashchi F. Lindsay M.R. James S. Singer A.G. Parton R.G. Gelb M.H. J. Biol. Chem. 2004; 279: 25024-25038Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Among known sPLA2s, gVPLA2 (24Han S.-K. Yoon E.T. Cho W. Biochem. J. 1998; 331: 353-357Crossref PubMed Scopus (52) Google Scholar, 25Han S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar) and gXPLA2 (26Hanasaki K. Ono T. Saiga A. Morioka Y. Ikeda M. Kawamoto K. Higashino K. Nakano K. Yamada K. Ishizaki J. Arita H. J. Biol. Chem. 1999; 274: 34203-34211Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 27Bezzine S. Koduri R.S. Valentin E. Murakami M. Kudo I. Ghomashchi F. Sadilek M. Lambeau G. Gelb M.H. J. Biol. Chem. 2000; 275: 3179-3191Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) can effectively bind and hydrolyze zwitterionic phosphatidylcholine (PC) that is rich in the external leaflet of mammalian plasma membranes. As a result, these sPLA2s are able to directly act on mammalian cells and catalyze the hydrolysis of cell surface phospholipids. Lastly, some sPLA2s have been reported to exert cellular effects through the binding to cell surface receptors (28Lambeau G. Lazdunski M. Trends Pharmacol. Sci. 1999; 20: 162-170Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Group IIF PLA2 (gIIFPLA2) is unique among sPLA2sintwo respects. First, gIIFPLA2 was shown to induce or augment the cellular AA and eicosanoid formation when overexpressed in mammalian cells despite having extremely low HSPG affinity (29Murakami M. Yoshihara K. Shimbara S. Lambeau G. Gelb M.H. Singer A.G. Sawada M. Inagaki N. Nagai H. Ishihara M. Ishikawa Y. Ishii T. Kudo I. J. Biol. Chem. 2002; 277: 19145-19155Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and low activity on PC vesicles (2Singer A.G. Ghomashchi F. Le Calvez C. Bollinger J. Bezzine S. Rouault M. Sadilek M. Nguyen E. Lazdunski M. Lambeau G. Gelb M.H. J. Biol. Chem. 2002; 277: 48535-48549Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). This suggests that gIIFPLA2 might have a unique mode of cellular action. Second, gIIFPLA2 is structurally unique in that it has an unusually long, proline-rich C-terminal extension (30Valentin E. Ghomashchi F. Gelb M.H. Lazdunski M. Lambeau G. J. Biol. Chem. 1999; 274: 31195-31202Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 31Valentin E. Singer A.G. Ghomashchi F. Lazdunski M. Gelb M.H. Lambeau G. Biochem. Biophys. Res. Commun. 2000; 279: 223-228Crossref PubMed Scopus (62) Google Scholar) (see Fig. 1A). To elucidate the mechanism by which gIIFPLA2 acts on mammalian cells, we built a model tertiary structure of gIIFPLA2 by homology modeling and measured the interactions of wild type and selected mutants of gIIFPLA2 with various model membranes and mammalian cells. Results show that due to its unique structural and membrane binding properties, gIIFPLA2 has an unprecedented ability to traverse the plasma membrane of mammalian cells, which is independent of binding to cell surface HSPG or phospholipid hydrolysis on the outer plasma membrane. These unique properties of gIIFPLA2 may allow this sPLA2 to perform some unusual functions under certain physiological conditions. Materials—1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol and brain sphingomyelin (SM) were from Avanti Polar Lipids, Inc. (Alabaster, AL), and 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine (DHPC) was from Sigma. 5-Carboxyfluorescein, 1-hexadecanoyl-2-(1-pyrenyldecanoyl)-sn-glycero-3-phosphoglycerol (pyrene-PG), N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-2(4,4-difluro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (PED6), Texas Red™ C2-maleimide, and 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol (NBD-cholesterol) were purchased from Invitrogen. 1,2-Bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (BLPC) and -glycerol (BLPG) were synthesized, and polymerized mixed vesicles (100 nm in diameter) were prepared as described previously (32Wu S.-K. Cho W. Biochemistry. 1993; 32: 13902-13908Crossref PubMed Scopus (35) Google Scholar, 33Wu S.-K. Cho W. Anal. Biochem. 1994; 221: 152-159Crossref PubMed Scopus (23) Google Scholar). Phospholipid concentrations were determined by a modified Bartlett analysis (34Kates M. Techniques of Lipidology. 2nd Ed. Elsevier, Amsterdam1986: 114-115Google Scholar). Dulbecco's modified Eagle's medium (DMEM) and inactivated fetal bovine serum (FBS) were from Invitrogen. Human embryonic kidney cell line HEK293 and Zeocin™ were from Invitrogen. Recombinant human gIIAPLA2 (35Snitko Y. Koduri R. Han S.-K. Othman R. Baker S.F. Molini B.J. Wilton D.C. Gelb M.H. Cho W. Biochemistry. 1997; 36: 14325-14333Crossref PubMed Scopus (109) Google Scholar) and gVPLA2 (25Han S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar) were expressed in Escherichia coli and purified as described. Mutagenesis and Protein Expression—The cDNA of full-length mouse gIIFPLA2 was cloned from the mouse testis cDNA library (Clontech) and subcloned into the pET-21a(+) vector (Novagen, Madison, WI) between the restriction sites NdeI and XhoI. Site-directed mutagenesis was carried out by the overlap extension PCR. All mutant constructs were transformed into DH5α cells for plasmid isolation, and their DNA sequences were verified. E. coli strain BL21 (DE3) was used as a host for the protein expression. 4 liters of Luria broth medium containing 100 μg/ml ampicillin was inoculated with 100 ml of the overnight culture from a freshly transformed single colony. The culture was grown at 37 °C. When the optical density of the culture at 600 nm reached 0.8-1.0, the culture was induced by 1mm isopropyl-1-thio-β-d-galactopyranoside (Research Products, Mount Prospect, IL). After incubation for 4 h at 37 °C, cells were harvested at 5000 × g for 10 min at 4 °C and frozen at -20 °C. The cells were resuspended in the CelLytic B-11 (Sigma) bacterial cell lysis extraction reagent (5 ml/g of cell paste), and deoxyribonuclease was added to a final concentration of 5 μg/ml to reduce the viscosity of the suspension. The extraction suspension was shaken at room temperature for 15 min and centrifuged at 25,000 × g for 15 min. Pellets were dissolved in CelLytic B-11 diluted 20-fold in water, incubated, and centrifuged as described above, and these steps were repeated twice to obtain clear inclusion body pellets. Inclusion bodies were solubilized in 10 ml of 50 mm Tris buffer, pH 8.0, containing 6 m guanidinium chloride, 1 mm EDTA and stirred overnight at 4 °C. Any insoluble matter was removed by centrifugation at 50,000 × g for 40 min at 4 °C. The supernatant was loaded to a Superdex G-200 column (Amersham Biosciences) equilibrated with 50 mm Tris buffer, pH 8.0, containing 3 m guanidinium chloride and 5 mm EDTA. Fractions corresponding to the protein peak were pooled and added dropwise to 50 ml of 50 mm Tris, pH 8.0, containing 5 mm EDTA, 20 mm reduced glutathione, and 10 mm oxidized glutathione over 3 h. The solution was kept at room temperature for 20 h. The refolded protein solution was dialyzed against 4 liters of 25 mm Tris buffer, pH 8.0, containing 1 m urea for 4 h at 4°C and against 4 liters of 25 mm Tris buffer, pH 8.0, containing 0.5 m urea, 0.1 mm dithiothreitol for 2 h at 4°C, and finally against 25 mm Tris, pH 8.0, containing 0.5 m urea for 4 h at 4°C. The protein solution was centrifuged at 50,000 × g for 40 min to remove insoluble matter, and the clear solution was loaded to a phenyl-Sepharose column (Amersham Biosciences) that was attached to an AKTA FPLC system (Amersham Biosciences) and equilibrated with 25 mm Tris, pH 7.4, containing 1 m ammonium sulfate. The column was eluted with a linear gradient of ammonium sulfate from 1 to 0 m and then with a linear gradient of 0-30% (v/v) acetonitrile in the same buffer. Fractions corresponding to the major protein peak were pooled and dialyzed against 25 mm Tris, pH 7.4, containing 160 mm NaCl, and stored at 4 °C. The purity of protein (>90%) was confirmed by SDS-PAGE. Protein concentration was determined by the bicinchoninic acid method (Pierce) using bovine serum albumin (BSA) as a standard. PLA2 Activity Assay—The PLA2-catalyzed hydrolysis of polymerized mixed vesicles (0.1 μm pyrene-PG inserted in 9.9 μm BLPC or BLPG) was carried out at 37 °C in 2 ml of 10 mm Tris buffer, pH 7.4, containing 0.16 m KCl, 1 mm CaCl2, and 2 μm BSA (32Wu S.-K. Cho W. Biochemistry. 1993; 32: 13902-13908Crossref PubMed Scopus (35) Google Scholar, 33Wu S.-K. Cho W. Anal. Biochem. 1994; 221: 152-159Crossref PubMed Scopus (23) Google Scholar). The progress of hydrolysis was monitored as an increase in fluorescence emission at 378 nm using a Hitachi F4500 Fluorescence spectrophotometer with excitation wavelength set at 345 nm, and spectral bandwidth was set at 10 nm for both excitation and emission. The PLA2-catalyzed hydrolysis of PED6 in the mixed vesicles of POPS/cholesterol/POPG/PED6 (107:31:20:1) was carried out at 37 °C in 2 ml of 10 mm HEPES, pH 7.4, containing 0.16 m KCl, 1 mm Ca2+. The progress of hydrolysis was monitored as an increase in fluorescence emission at 520 nm with the excitation wavelength set at 488 nm. Spectral bandwidth was set at 10 nm for both excitation and emission. Values of specific activity were determined from the initial rates of hydrolysis. Surface Plasmon Resonance Analysis—Kinetics of vesicle-PLA2 binding was measured by the surface plasmon resonance (SPR) analysis using a BIAcore X biosensor system (Biacore AB) and the L1 chip as described previously (36Stahelin R.V. Cho W. Biochemistry. 2001; 40: 4672-4678Crossref PubMed Scopus (141) Google Scholar). All measurements were performed at 23 °C in 5 mm HEPES buffer, pH 7.4, containing 160 mm NaCl and 0.1 mm EDTA. The first flow cell was used as a control cell and was coated with 5400 resonance units of BSA. The second flow cell contained the surface coated with vesicles with varying lipid compositions at 5400 resonance units. After lipid coating, 30 μl of 50 mm NaOH was injected at 100 μl/min three times to wash out loosely bound lipids. Typically, no further decrease in SPR signal was observed after one wash cycle. After coating, the drift in signal was allowed to stabilize below 0.3 resonance units/min before any binding measurements, which were performed with a flow rate of 30 μl/min. 90 μl of protein sample was injected for an association time of 3 min, and the dissociation was then monitored for 10 min in running buffer. After each measurement, the lipid surface was typically regenerated with a 10-μl pulse of 50 mm NaOH. The regeneration solution was passed over the immobilized vesicle surface until the SPR signal reached the initial background value before protein injection. When needed, the entire lipid surface was removed with a 5-min injection of 40 mm CHAPS followed by a 5-min injection of 40 mm octyl glucoside at 5 μl/min, and the sensor chip was recoated for the next set of measurements. All data were analyzed using BIAevaluation 3.0 software (Biacore). Vesicle Leakage Experiments—Appropriate amounts of lipids in chloroform were mixed, and the solvent was gently evaporated under a steam of dry N2 to obtain the thin lipid film at bottom of a small thick-walled glass tube. To the dry lipid samples, 500 μl of 5 mm HEPES buffer, pH 7.4, containing 50 mm 5-carboxyfluorescein was added, and the mixture was vortexed. Large unilamellar vesicles (LUVs) were prepared by repeated extrusion through 100-nm polycarbonate filters using a Liposofast extruder (Avestin, Ottawa, Canada). Vesicles were separated from nonencapsulated 5-carboxyfluorescein by gel filtration using a Sephadex G-50 column eluted with 5 mm HEPES buffer, pH 7.4, containing 160 mm NaCl and 0.1 mm EDTA. 150 nm (final concentration) sPLA2 proteins were added to 300 nm (final concentration) 5-carboxyfluorescein-containing vesicles in 2.0 ml of 5 mm HEPES buffer, pH 7.4, containing 160 mm NaCl and 0.1 mm EDTA, and the release of 5-carboxyfluorescein was measured using a Hitachi F4500 spectrofluorometer with excitation and emission wavelengths set at 430 and 520 nm, respectively. After each leakage measurement, 20 μl of Triton X-100 (Pierce) was added to the mixture to achieve 100% release of 5-carboxyfluorescein. The percentage of leakage was calculated as (F - F0)/(Fmax - F0) × 100, where F0 is the fluorescence emission intensity before adding sPLA2, and F and Fmax represent the final fluorescence values after adding sPLA2 and Triton X-100, respectively. All measurements were performed at 25 °C. Monolayer Measurements—Surface pressure (π) of solution in a circular Teflon trough (4-cm diameter × 1-cm depth) was measured using a Wilhelmy plate attached to a computer-controlled Cahn electrobalance (model C-32) as described previously (37Medkova M. Cho W. J. Biol. Chem. 1998; 273: 17544-17552Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). 5-10 μl of phospholipid solution in ethanol/hexane (1:9 (v/v)) was spread onto 10 ml of subphase (25 mm Tris, pH 7.4, containing 0.16 m KCl and either 0.1 mm EGTA or 0.1 mm CaCl2) to form a monolayer with a given initial surface pressure (π0). Once the surface pressure reading of monolayer had been stabilized (after ∼5 min), the protein solution (typically 40 μl) was injected into the subphase through a small hole drilled at an angle through the wall of the trough, and the change in surface pressure (Δπ) was measured as a function of time at 23 °C. Typically, the Δπ value reached a maximum after 30 min. The maximal Δπ value at a given π0 depended on the protein concentration and reached a saturation value when [sPLA2] was ≥2 μg/ml. Protein concentrations in the subphase were therefore maintained above such values to ensure that the observed Δπ represented a maximal value. The critical surface pressure (πc) was determined by extrapolating the Δπ versus π0 plot to the x axis (38Cho W. Bittova L. Stahelin R.V. Anal. Biochem. 2001; 296: 153-161Crossref PubMed Scopus (111) Google Scholar). Fluorescence Labeling of sPLA2s—Purified mouse gIIFPLA2 wild type and mutant proteins (H47Q and Y115A/F116A/V118A/Y119A) were dialyzed against 25 mm Tris, pH 7.2, containing 0.5 m guanidinium chloride for 4 h at 4 °C. Proteins were treated with a 10-fold molar excess of Texas Red™ C2-maleimide for 3 h at room temperature. The reaction was quenched by incubating the mixture with an excess amount (10-fold excess of maleimide) of cysteine for 30 min. The solution of labeled protein was dialyzed against 25 mm Tris, pH 7.2, containing 15% ammonium sulfate for 2 h at 4°C to remove excess reagents. The labeled proteins were purified using a phenyl-Sepharose column (Amersham Biosciences) as described above. Labeled protein fractions were collected and dialyzed against 25 mm Tris, pH 7.4, 160 mm NaCl for 24 h at 4 °C and then stored at -20 °C. W79C human gVPLA2 was purified and labeled as described previously (22Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 39Kim Y.J. Kim K.P. Rhee H.J. Das S. Rafter J.D. Oh Y.S. Cho W. J. Biol. Chem. 2002; 277: 9358-13174Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Microscopy Measurements on Giant Unilamellar Vesicles (GUVs)—GUVs were prepared by the electroformation method using a home-built device as described previously (40Bagatolli L.A. Gratton E. Biophys. J. 1999; 77: 2090-2101Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 41Gokhale N.A. Abraham A. Digman M.A. Gratton E. Cho W. J. Biol. Chem. 2005; 280: 42831-42840Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Briefly, GUVs were grown in deionized water at 60 °C for 30 min by spreading ∼3 μl of the lipid stock with various compositions on platinum wires. During GUV growth, the platinum wires were connected to a function generator (Hewlett-Packard, Santa Clara, CA) for 30 min, and a low frequency AC field (sinusoidal wave function with a frequency of 10 Hz and an amplitude of 3 V) was applied. After 45 min, the temperature was lowered to 40 °C, and the frequency generator was switched off after the system attained this temperature. All subsequent measurements were carried out at 40 °C in deionized water. All microscopy measurements were carried out using a custom-built combination laser-scanning and multiphoton microscope that was described previously (42Stahelin R.V. Digman M.A. Medkova M. Ananthanarayanan B. Rafter J.D. Melowic H.R. Cho W. J. Biol. Chem. 2004; 279: 29501-29512Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Briefly, a 920-nm ultrafast pulsed beam from a tunable Tsunami laser, set up for femtosecond operation (Spectra Physics, Mountain View, CA) was spatially filtered and launched into the scan head. The beam was directed toward the primary dichroic mirror (Chroma Technology, Brattleboro, VT) and then toward the XY scan mirrors (model 6350, Cambridge Technologies, Cambridge, MA). A Prairie Technologies scan lens (Middleton, WI) was used to focus the laser light, collimated by the ×1 Zeiss tube lens and directed toward a ×40 water-corrected 1.2 numerical aperture Zeiss objective, mounted on a Zeiss 200 M platform (Carl Zeiss Inc., Thornwood, NY). Light excited by a 920-nm ultrafast pulse was collected on a nondescanned pathway by the Peltier-cooled 1477P style Hamamatsu photomultiplier tubes. The light was reflected and filtered using appropriate optics. I
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