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• W2090178694 abstract "The Ca2+-dependent lipid binding domain of the 85-kDa cytosolic phospholipase A2 (cPLA2) is a homolog of C2 domains present in protein kinase C, synaptotagmin, and numerous other proteins involved in signal transduction. NH2-terminal fragments of cPLA2 spanning the C2 domain were expressed as inclusion bodies in Escherichia coli, extracted with solvent to remove phospholipids, and refolded to yield a domain capable of binding phospholipid vesicles in a Ca2+-dependent manner. Unlike other C2 domains characterized to date, the cPLA2 C2 domain bound preferentially to vesicles comprised of phosphatidylcholine in response to physiological concentrations of Ca2+. Binding of the cPLA2 C2 domain to vesicles in the presence of excess Ca2+ chelator was induced by high concentrations of salts that promote hydrophobic interactions. Despite the selective hydrolysis of arachidonyl-containing phospholipid vesicles by cPLA2, the cPLA2 C2 domain did not discriminate among phospholipid vesicles containing saturated or unsaturated sn-2 fatty acyl chains. Moreover, the cPLA2 C2 domain bound to phospholipid vesicles containing sn-1 and -2 ether linkages and sphingomyelin at Ca2+ concentrations that caused binding to vesicles containing ester linkages, demonstrating that the carbonyl oxygens of the sn-1 and-2 ester linkage are not critical for binding. These results suggest that the cPLA2C2 domain interacts primarily with the headgroup of the phospholipid. The cPLA2 C2 domain displayed selectivity among group IIA cations, preferring Ca2+ approximately 50-fold over Sr2+ and nearly 10,000-fold over Ba2+ for vesicle binding. No binding to vesicles was observed in the presence of greater than 10 mm Mg2+. Such strong selectivity for Ca2+ over Mg2+ reinforces the view that C2 domains link second messenger Ca2+ to signal transduction events at the membrane. The Ca2+-dependent lipid binding domain of the 85-kDa cytosolic phospholipase A2 (cPLA2) is a homolog of C2 domains present in protein kinase C, synaptotagmin, and numerous other proteins involved in signal transduction. NH2-terminal fragments of cPLA2 spanning the C2 domain were expressed as inclusion bodies in Escherichia coli, extracted with solvent to remove phospholipids, and refolded to yield a domain capable of binding phospholipid vesicles in a Ca2+-dependent manner. Unlike other C2 domains characterized to date, the cPLA2 C2 domain bound preferentially to vesicles comprised of phosphatidylcholine in response to physiological concentrations of Ca2+. Binding of the cPLA2 C2 domain to vesicles in the presence of excess Ca2+ chelator was induced by high concentrations of salts that promote hydrophobic interactions. Despite the selective hydrolysis of arachidonyl-containing phospholipid vesicles by cPLA2, the cPLA2 C2 domain did not discriminate among phospholipid vesicles containing saturated or unsaturated sn-2 fatty acyl chains. Moreover, the cPLA2 C2 domain bound to phospholipid vesicles containing sn-1 and -2 ether linkages and sphingomyelin at Ca2+ concentrations that caused binding to vesicles containing ester linkages, demonstrating that the carbonyl oxygens of the sn-1 and-2 ester linkage are not critical for binding. These results suggest that the cPLA2C2 domain interacts primarily with the headgroup of the phospholipid. The cPLA2 C2 domain displayed selectivity among group IIA cations, preferring Ca2+ approximately 50-fold over Sr2+ and nearly 10,000-fold over Ba2+ for vesicle binding. No binding to vesicles was observed in the presence of greater than 10 mm Mg2+. Such strong selectivity for Ca2+ over Mg2+ reinforces the view that C2 domains link second messenger Ca2+ to signal transduction events at the membrane. Much of the interest in the 85-kDa cytosolic PLA2(cPLA2) 1The abbreviations used are: cPLA2, cytosolic phospholipase A2; C2, second conserved; PLC, phospholipase C; CHO, Chinese hamster ovary; GST, glutathione S-transferase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GndHCl, guanidine hydrochloride; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; dansyl-PE,N-(5-dimethylaminonaphthalene-1-sulfonyl)-1-palmitoyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine; PO-PC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PO-PE, -phosphoethanolamine; PO-PA, -phosphate; PO-PS, -phosphoserine; -PG, phospho-rac-(1-glycerol); mixed acyl-PI, phosphatidylinositol containing a mixture of acyl chains; PP-PC, 1-palmitoyl-2-palmitoyl-sn-glycero-3-phosphocholine; PA-PC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; PL-PC, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine;sn-1 and -2 ether-linked phospholipid, 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine; PIPES, 1,4-piperazinediethanesulfonic acid. stems from its ability to release arachidonic acid from membranes selectively, thus initiating the biosynthesis of prostaglandins, leukotrienes, and platelet-activating factor (for review, see Ref. 1Clark J.D. Schievella A.R. Nalefski E.A. Lin L.-L. J. Lipid Mediators Cell Signalling. 1995; 12: 83-118Crossref PubMed Scopus (422) Google Scholar). Maximal activation of cPLA2 in intact cells requires phosphorylation by a member of the mitogen-activated protein kinase family on Ser-505 (2Lin L.-L. Lin A.Y. Knopf J.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6147-6151Crossref PubMed Scopus (519) Google Scholar, 3Lin L.-L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1659) Google Scholar, 4Kramer R.M. Roberts E.F. Strifler B.A. Johnstone E.M. J. Biol. Chem. 1995; 270: 27395-27398Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 5Qui Z.H. Leslie C.C. J. Biol. Chem. 1994; 269: 19480-19487Abstract Full Text PDF PubMed Google Scholar). However, in the absence of an increase in cytosolic Ca2+, even stoichiometrically phosphorylated cPLA2 fails to release arachidonic acid because Ca2+ is obligatory for binding to the membrane substrate (3Lin L.-L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1659) Google Scholar, 4Kramer R.M. Roberts E.F. Strifler B.A. Johnstone E.M. J. Biol. Chem. 1995; 270: 27395-27398Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 5Qui Z.H. Leslie C.C. J. Biol. Chem. 1994; 269: 19480-19487Abstract Full Text PDF PubMed Google Scholar, 6Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1465) Google Scholar, 7Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-18249Abstract Full Text PDF PubMed Google Scholar, 8Channon J.Y. Leslie C.C. J. Biol. Chem. 1990; 265: 5409-5413Abstract Full Text PDF PubMed Google Scholar). In cells, the increase in Ca2+ results in the selective translocation of cPLA2 to the membranes of the nuclear envelope and endoplasmic reticulum (9Peters-Golden M. McNish R.W. Biochem. Biophys. Res. Commun. 1993; 196: 147-153Crossref PubMed Scopus (177) Google Scholar, 10Glover S. Bayburt T. Jonas M. Chi E. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 11Schievella A. Regier M.K. Smith W.L. Lin L.-L. J. Biol. Chem. 1995; 270: 30749-30754Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar), resulting in the colocalization of cPLA2 with the downstream enzymes responsible for metabolizing arachidonic acid to prostaglandins and leukotrienes. We have shown previously that the domain responsible for the membrane association is encoded at the NH2 terminus of cPLA2 (6Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1465) Google Scholar, 7Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-18249Abstract Full Text PDF PubMed Google Scholar) and serves to bring a Ca2+-independent catalytic domain to the membrane substrate in response to increases in second messenger Ca2+. This Ca2+-dependent lipid binding domain is homologous to the regulatory C2 domain originally described in the classical isoforms of protein kinase C (12Coussens L. Parker P.J. Rhee L. Yang-Feng T.L. Chen E. Waterfield M.D. Franke U. Ullrich A. Science. 1986; 233: 859-866Crossref PubMed Scopus (762) Google Scholar) but now recognized to be present in numerous proteins, and it may serve as a paradigm to explain both the features common to these domains as well as those that provide specificity among the domains. Homologs of the protein kinase C C2 domain have been identified in at least four classes of eukaryotic proteins that carry out critical functions at cellular membranes (1Clark J.D. Schievella A.R. Nalefski E.A. Lin L.-L. J. Lipid Mediators Cell Signalling. 1995; 12: 83-118Crossref PubMed Scopus (422) Google Scholar, 6Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1465) Google Scholar, 13Brose N. Hofmann K. Hata Y. Südhof T.C. J. Biol. Chem. 1995; 270: 25273-25280Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 14Ponting C.P. Parker P.J. Protein Sci. 1996; 5: 162-166Crossref PubMed Scopus (155) Google Scholar, 15Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (691) Google Scholar). These proteins include lipid-modifying enzymes (e.g. cPLA2, phosphoinositide-specific phospholipase C (PLC), yeast phosphatidylserine decarboxylase-2, several isoforms of the catalytic subunit of phosphatidylinositol 3-kinase, and a plant phospholipase D), protein kinases (e.g. α, β, γ, δ, ε, η, and θ isoforms of protein kinase C and certain related protein kinase C), GTPase-activating proteins (ras-GTPase-activating protein and its relatives), and regulators of vesicle transport (e.g. synaptotagmin, rabphilin, DOC2, UNC-13, and perforin). Although the functions of the proteins that contain C2 domains are in many cases well defined, the roles that C2 domains play in these proteins are poorly understood. The generalized function of the C2 domain appears to be membrane association; the ligands for C2 domains identified to date comprise various components of cellular membranes, including phospholipids, inositol polyphosphates, and other membrane-associated proteins (for review in detail, see Ref. 15Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (691) Google Scholar). Interactions between C2 domains and these ligands are predicted to regulate the various protein-specific biochemical activities. For example, the C2 domain of PLC-δ1 has been proposed to orient or “fix” the catalytic domain of the enzyme to the membranes after it has been “tethered” by the binding of the pleckstrin homology domain to phosphatidylinositol bisphosphate (16Essen L.-O. Perisic O. Katan M. Williams R.L. Nature. 1996; 380: 595-602Crossref PubMed Scopus (519) Google Scholar). To study the ligand specificity of the cPLA2 C2 domain without the effects of lipid binding to the catalytic domain of cPLA2, we have expressed the cPLA2 C2 domain initially as a fusion protein and subsequently as a polypeptide refolded from inclusion bodies free of lipid contamination. This domain binds zwitterionic phospholipid vesicles composed of phosphatidylcholine preferentially over anionic vesicles in the presence of physiological levels of Ca2+. This domain is highly selective for Ca2+ over Sr2+, Ba2+, and Mg2+ with respect to vesicle binding. In addition, this domain binds to phospholipid vesicles lackingsn-1 or -2 carbonyl oxygens and is insensitive to changes in the length and degree of saturation of the sn-2 fatty acyl chain, suggesting that the specific interactions with the ligand are limited to the headgroup of the phospholipid. Based on these results, we compare the properties of the cPLA2 C2 domain with other C2 domains and other proteins that bind phospholipids in a Ca2+-dependent manner. Constructions were carried out using conventional protocols (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) to generate DNA encoding proteins that could be expressed in mammalian COS or Chinese hamster ovary (CHO) cells or in Escherichia coli either as fusion proteins with glutathione S-transferase (GST) or as independent polypeptides in inclusion bodies. DNA fragments encoding cPLA2 residues 1–126, 1–138, 1–155, and 1–178 were generated by polymerase chain reaction (PCR) using full-length human cPLA2 cDNA as a template and antisense primers incorporating a 3′-stop codon and EcoRI/SalI restriction sites. For construction of GST fusion proteins, 5′-PCR sense primers incorporated a BglII restriction site, codons for Gly and Ser residues as part of a thrombin cleavage site, and the first seven codons of cPLA2. cPLA2 PCR fragments generated from these PCRs were subcloned into theBamHI/EcoRI polylinker site of the vector pGEX-4T-1 (Pharmacia Biotech Inc.) 3′ of the GST gene. Consequently, cPLA2 polypeptides liberated from GST fusion proteins by cleavage with thrombin contain additional NH2-terminal Gly-Ser residues. For construction of cPLA2 polypeptides produced in inclusion bodies, cPLA2 cDNA that had been modified previously by substitution of several 5′-codons with codons preferentially utilized in bacteria served as template DNA for the PCR; 5′-PCR primers incorporated these silent changes and anAflIII restriction site; 3′-PCR primers were the same as those used in GST fusion protein constructions. These PCR products were subcloned into the NcoI/EcoRI sites of the vector pTrcHisB (Invitrogen), removing the 5′-codons encoding the NH2 terminus of the polyHis fusion protein. Consequently, the pTrc constructs reported here utilize the trc promoter and initiator ATG codon normally employed by His fusion proteins, yet they encode cPLA2 residues exclusively when expressed inE. coli. DNA was transformed into the bacterial strain HB101. All DNA constructs were sequenced and shown to be correct. Details of constructions may be obtained from the authors. Bacteria expressing the appropriate constructs were fermented to late log phase and induced with 1 mm isopropyl β-d-thiogalactopyranoside at 25 °C for GST constructs and 37 °C for inclusion body proteins. For isolation of GST fusion proteins, bacterial cell pellets were lysed by nitrogen cavitation. Supernatant was adsorbed to glutathione-Sepharose beads (Pharmacia), and fusion proteins were removed from beads by cleavage with thrombin (Sigma). Protein was passed over a Mono Q column (Pharmacia) and eluted with approximately 100 mm salt. Protein was dialyzed, concentrated, and stored in buffer (20 mm Tris (pH 7.4), and 0.1 mmEDTA) at 4 °C. Protein was greater than 90% pure, as judged by SDS-PAGE analysis (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). For protein expressed in inclusion bodies, bacterial cell pellets were lysed by microfluidization. Inclusion bodies were harvested, washed, and extracted with chloroform:methanol (2:1) to remove lipids. Inclusion bodies were solubilized in 6m guanidine HCl (GndHCl), passed over a TSK3000 gel filtration column (Toso-Haas) run in 8 m urea, 20 mm Tris (pH 7.4), 5 mm EDTA, and 5 mm dithiothreitol at room temperature. The appropriate peak was passed over a Mono Q column at room temperature and eluted with 400–500 mm NaCl. Protein was refolded by diluting the desired Mono Q peak into a solution of 0.5 m arginine HCl, 25 mm Tris (pH 8.0), and 5 mm dithiothreitol while stirring slowly at 4 °C. Refolded protein was diluted slowly 2-fold into buffer (25 mm Tris (pH 8.0) and 5 mm dithiothreitol) and again 2-fold into a solution of 2m (NH4)2SO4, 20 mm Tris (pH 8.0), 5 mm dithiothreitol, and 5 mm EDTA. Refolded protein was adsorbed to a Toyo-phenyl 650S column (Toso-Haas) equilibrated with buffer containing 1m (NH4)2SO4 and eluted in a single step with salt-free buffer. Eluted protein was concentrated by vacuum dialysis and passed over a Mono Q column. Correctly folded protein eluted with approximately 100 mm NaCl, whereas incorrectly folded protein, as judged by fluorescence spectroscopy (see below), eluted with 400–500 mm NaCl. Correctly folded protein was passed over a phenyl-5PW column (Toso-Haas) and eluted with a linear gradient of decreasing (NH4)2SO4. Correctly folded protein eluted with approximately 100 mm(NH4)2SO4, whereas incorrectly folded protein eluted during the final column wash in salt-free buffer. Protein was concentrated by vacuum dialysis and dialyzed against storage buffer. Protein purity was evaluated by SDS-PAGE on 4–20% and 14% Tris-glycine and 10% Tricine gels (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). The Ca2+-dependent binding of recombinant proteins to natural membranes isolated from CHO cells was determined as described in detail previously (6Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1465) Google Scholar, 7Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-18249Abstract Full Text PDF PubMed Google Scholar). In short, at least 2 μg of test protein was mixed with CHO cell membranes in the presence of 150 mm NaCl, 20 mm HEPES (pH 7.4), and Ca2+/EGTA buffers to maintain the desired free Ca2+ concentration and incubated for 15 min at 30 °C. After centrifugation at 100,000 × g, equal proportions of supernatant and pellet fractions were subjected to SDS-PAGE on 4–20% Tris-glycine gels. Gels were blotted onto nitrocellulose, and test proteins appearing in supernatant and pellet fractions were detected by immunostaining with cPLA2 antisera and visualized by chemiluminescence. The binding of the cPLA2 C2 domain, which acts as the fluorescence donor, to small unilamellar phospholipid vesicles containing the fluorescent probe dansylphosphatidylethanolamine (dansyl-PE) (Molecular Probes, Eugene, OR), the fluorescence acceptor, was measured as described previously (7Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-18249Abstract Full Text PDF PubMed Google Scholar) with modification. A total of 25–50 μg of test protein was diluted into a 2-ml solution containing 60 μg of test liposomes (composed of 5–10% dansyl-PE) in 150 mm NaCl, 20 mm HEPES (pH 7.4), and 1 mm EGTA in 3-ml quartz cuvettes. In experiments comparing different divalent cations, binding reactions were carried out in buffer containing 100 mm KCl, 20 mm HEPES (pH 7.4), and 1 mm EDTA. KCl was diluted from a Ca2+-free Ca2+ electrode-filling solution (Orion Research Incorporated, Boston). Reaction mixtures were stirred continuously, maintained at 20 °C, and illuminated with 284 nm wavelength light; emission of dansyl-PE was recorded at a wavelength of 520 nm. Levels of free divalent cations were raised by the addition of concentrated stocks of cations: for most experiments, a Ca2+ atomic absorption standard (VHG Laboratories, Manchester, NH) was used. Free Ca2+ levels were calculated using the Chelator program (19Schoenmakers T.J.M. Visser G.J. Flik G. Theuvenet A.P.R. BioTechniques. 1992; 12: 870-879PubMed Google Scholar) taking into account the pH of the binding reactions, measured separately. For experiments testing different divalent cations, the chloride salt of the metal ions (Fluka, puriss grade) was used; free cation levels were calculated according to Raaflaub (20Raaflaub J. Methods Biochem. Anal. 1960; 3: 301-325Crossref Google Scholar). The fluorescence emission of dansyl-PE in the presence of EGTA or EDTA (I 0) was subtracted from that in the presence of added metal ion (I) to determine energy transfer induced by the added metal ion, which was normalized to give (I −I 0)/I 0, expressed as a percentage. Vesicle compositions were varied according to the experiment. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PO-PC), -phosphoethanolamine (PO-PE), -phosphate (PO-PA), -phosphoserine (PO-PS) and -phospho-rac-(1-glycerol) (PO-PG),l-α-phosphatidylinositol (mixed acyl-PI), 1-palmitoyl-2-palmitoyl-sn-glycero-3-phosphocholine (PP-PC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PA-PC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PL-PC), 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine (assn-1 and -2 ether-linked phospholipid) and brain sphingomyelin were purchased from Avanti Polar Lipids (Alabaster, AL). In experiments testing the effect of salt on vesicle binding, reactions were carried out in buffer containing 20 mm HEPES (pH 7.4) and 5 mm EGTA to chelate potential contaminating Ca2+. A solution containing vesicles and protein was reciprocally diluted by a like solution containing concentrated salts to increase the salt concentration and to keep the concentration of vesicles and protein constant. For each salt tested, a parallel series of experiments was carried out using stock solutions free of protein. Fluorescence emission of dansyl-PE in binding reactions free of protein (I 0) was subtracted from that containing protein (I) to determine energy transfer due to protein at each salt concentration, which was normalized to give (I −I 0)/I 0, expressed as a percentage. NaCl, (NH4)2SO4, and NH4Cl were greater than 99.5% pure (Fluka), and Na2SO4 was greater than 99% pure (Aldrich). All salts were specified by the manufacturer to contain less than 0.001% Ca2+. Binding of the C2 domain to synthetic vesicles was tested additionally as described above for natural membranes with modification. Synthetic phospholipid vesicles were prepared by brief sonication and collected by centrifugation at 100,000 × g. 10 μg of C2 domain was mixed with a 10 mm concentration of the resuspended phospholipid vesicles in the presence of 100 mm KCl, 20 mm PIPES (pH 7.0), and 1 mm EDTA or 1 mm EDTA plus 1.1 mm CaCl2 (to maintain 100 μm free Ca2+) in 100 μl for 5 min at room temperature. After centrifugation, pellet fractions were washed once with reaction buffer and resuspended in buffer containing 0.2% Triton X-100. Equal proportions of supernatant and pellet fractions were subjected to SDS-PAGE on 15% gels. Gels were stained with Coomassie Brilliant Blue R-250, and the amount of protein was quantified by scanning densitometry. Results were expressed as the ratio of the amount of protein present in the pellet fraction to the total amount in pellet and supernatant fractions. Total phosphate analysis of the resuspended vesicles verified that equivalent amounts of PO-PC, -PA, -PS, and -PG and mixed acyl-PI were present in the assay. The amounts of PO-PE which could be collected by centrifugation were very low; therefore, PO-PE was not used in the experiment. A total of 2 μg of the C2 domain, diluted into a 1-ml solution, with or without denaturant, containing 150 mm NaCl, 20 mm Tris (pH 7.4), and 5 mm EDTA, was excited with 280 nm wavelength light in a 1.5-ml quartz cuvette at room temperature. Fluorescence emission wavelengths were scanned from 300 to 400 nm. Excitation and emission slit widths were both 5 nm. Similar emission maxima (but of lower intensity) were recorded when 294 nm wavelength excitation light was used. For renaturation experiments, a concentrated stock solution of protein prepared in a high concentration of denaturant was serially diluted into buffer without denaturant. The NH2-terminal 178 residues of cPLA2(cPLA2(1–178)) contain a C2 domain, a sequence motif that functions as a Ca2+-dependent lipid binding domain (6Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1465) Google Scholar, 7Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-18249Abstract Full Text PDF PubMed Google Scholar). Previously, cPLA2(1–178) was expressed as an independent polypeptide in COS cells and bound natural membranesin vitro at Ca2+ concentrations that activated enzymatic activity of cPLA2 (7Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-18249Abstract Full Text PDF PubMed Google Scholar). In addition, these residues conferred upon a heterologous fusion protein the ability to bind to natural membranes and synthetic phospholipid vesicles in the presence of physiological levels of Ca2+ in vitro (7Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-18249Abstract Full Text PDF PubMed Google Scholar). We sought to define better the minimal residues of the C2 domain. Based on the structure of the first C2 domain of synaptotagmin (21Sutton R.B. Davletov B.A. Berghuis A.M. Südhof T.C. Sprang S.R. Cell. 1995; 80: 929-938Abstract Full Text PDF PubMed Scopus (606) Google Scholar, 22Shao X. Davletov B.A. Sutton R.B. Südhof T.C. Rizo J. Science. 1996; 273: 248-251Crossref PubMed Scopus (293) Google Scholar), the cPLA2 C2 domain was predicted to terminate prior to the exon boundary at Gln-126 (Fig.1). However, sequence alignment based on the report of a second C2 domain topological fold, that of PLC-δ1 (16Essen L.-O. Perisic O. Katan M. Williams R.L. Nature. 1996; 380: 595-602Crossref PubMed Scopus (519) Google Scholar), in which the β-strand that corresponds topologically to the first β-strand of synaptotagmin is located at the COOH terminus of the PLC-δ1 C2 domain, predicted that the cPLA2 C2 domain terminates at the next exon boundary Val-138 (15Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (691) Google Scholar). Thus, cPLA2 was COOH-terminally truncated at Gln-126 and Val-138, two exon boundaries, to ascertain whether they demarcated minimal functional boundaries. cPLA2 was also truncated in a polar stretch of the polypeptide at Lys-155 to test the importance of residues between 138 and 178. These polypeptides were expressed in COS or CHO cells as independently expressed polypeptides, or they were expressed in E. coli as fusion partners of GST or as inclusion body proteins that could be refolded. Recombinant fragments containing, at a minimum, residues 1–155, were expressed in both COS and CHO cells as soluble polypeptides that bound cellular membranes in a Ca2+-dependent manner (data not shown). In contrast, both cPLA2(1–126) and (1–138) were expressed as insoluble polypeptides in COS cells. We turned to expression of recombinant protein in E. coli, reasoning that expression of cPLA2(1–126) and (1–138) in bacteria grown at lower temperatures in fusion to a highly soluble partner (GST) might overcome the solubility problems associated with protein overexpression in COS cells cultured at 37 °C. Indeed, recombinant fragments containing, at a minimum, residues 1–126 attached to the COOH terminus of GST were expressed as soluble fusion proteins in E. coli. However, GST-cPLA2(1–126) was considerably more insoluble than the other three fusion proteins; the little soluble cPLA2(1–126) that could be liberated from GST-cPLA2(1–126) bound in a Ca2+-independent manner to cellular membranes (data not shown). GST-cPLA2(1–138), (1–155), and (1–178) were fully functional, binding in a Ca2+-dependent manner in vitro to cellular membranes (data not shown). When liberated from GST-fusion proteins by thrombin cleavage and purified, cPLA2(1–138) and (1–155) reversibly bound to phosphatidylcholine vesicles (Fig. 2) and cellular membranes (data not shown) at low micromolar Ca2+levels. The additional Gly-Ser residues attached to the NH2terminus of cPLA2(1–138) and (1–155) to create a thrombin cleavage site (see “Experimental Procedures”) thus do not hamper binding to phospholipid vesicles. Although cPLA2(1–138) represented the minimal, fully functional C2 domain, we chose to characterize cPLA2(1–155) initially because of its higher yields. cPLA2(1–155) contains several aromatic residues, including a single tryptophan, Trp-71, which serves as a useful spectroscopic probe. Excitation of cPLA2(1–155) at 280 nm resulted in a strong fluorescence emission wavelength maximum at approximately 325 nm (Fig. 3 A), which is characteristic of tryptophan buried in a relatively nonpolar environment (23Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Press, New York1986: 345-350Google Scholar). When diluted into high concentrations of urea or GndHCl, this fluorescence wavelength maximum red-shifted to approximately 348 nm, characteristic of tryptophan exposure to polar solvent. This indicates that Trp-71 of native cPLA2 is buried in a relatively nonpolar environment and that upon denaturation it becomes exposed to solvent. The spectroscopic properties of Trp-71 in cPLA2 are consistent with the location of the homologous residues Phe-206 of synaptotagmin I (21Sutton R.B. Davletov B.A. Berghuis A.M. Südhof T.C. Sprang S.R. Cell. 1995; 80: 929-938Abstract Full Text PDF PubMed Scopus (606) Google Scholar) and Trp-684 of PLC-δ1 (16Essen L.-O. Perisic O. Katan M. Williams R.L. Nature. 1996; 380: 595-602Crossref PubMed Scopus (519) Google Scholar), which are buried in the β5- and β4-strands of type I and II C2 domains, respectively (see Fig. 1). Titration of cPLA2(1–155) with urea or GndHCl revealed a single major transition in unfolding (Fig. 3 B). When cPLA2(1–155) was first denatured in urea or GndHCl and then serially diluted into buffer without denaturant, the fluorescence wavelength maximum blue-shifted back to approximately 325 nm (Fig.3 C), revealing that denaturation by these chaotro" @default.
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• W2090178694 date "1998-01-01" @default.
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• W2090178694 title "Independent Folding and Ligand Specificity of the C2 Calciumdependent Lipid Binding Domain of Cytosolic Phospholipase A2" @default.
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