Interfacial kinetic and binding properties of mammalian group IVB phospholipase A2 (cPLA2β) and comparison with the other cPLA2 isoforms

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 46, pp. 36100–36111, November 12, 2010

Interfacial Kinetic and Binding Properties of Mammalian

Group IVB Phospholipase A₂(cPLA₂␤) and Comparison with

□

S

the Other cPLA₂Isoforms^*

Received for publication, July 17, 2010, and in revised form, August 6, 2010 Published, JBC Papers in Press, August 11, 2010, DOI 10.1074/jbc.M110.165647

Farideh Ghomashchi^‡, Gajendra S. Naika^‡, James G. Bollinger^‡, Ahmed Aloulou^‡, Matthias Lehr^§,

Christina C. Leslie^¶, and Michael H. Gelb^‡ʈ1

From the Departments of ^‡Chemistry and ^ʈBiochemistry, University of Washington, Seattle, Washington 98195, the ^§Institute of

Pharmaceutical and Medicinal Chemistry, University of Mu¨nster, Hittorfstrasse 58-62, D-48149 Mu¨nster, Germany, and the

^¶Program in Cell Biology, Department of Pediatrics, National Jewish Health, Denver, Colorado 80206

The cytosolic (group IV) phospholipase A₂(cPLA₂s) family

contains six members. We have prepared recombinant proteins

for human ␣, mouse ␤, human ␥, human ␦, human ⑀, and mouse

␨ cPLA₂s and have studied their interfacial kinetic and binding

properties in vitro. Mouse cPLA₂␤ action on phosphatidylcho-

line vesicles is activated by anionic phosphoinositides and car-

diolipin but displays a requirement for Ca^2؉only in the pres-

ence of cardiolipin. This activation pattern is explained by the

effects of anionic phospholipids and Ca^2؉on the interfacial

donic acid from the sn-2 position of membrane phospholipids

in agonist-stimulated mammalian cells for the biosynthesis of

eicosanoids. With the completion of the mouse and human

genomes, it became clear that these mammals also contain

other proteins homologous to cPLA₂␣, namely the ␤, ␥, ␦, ⑀,

and ␨ isoforms (groups IVB-F cPLA₂s) (1, 2). This study is

focused on the ␤ isoform of mouse cPLA₂(m-cPLA₂␤) and its

comparison with the other isoforms. m-cPLA₂␤ and cPLA₂␣

have a similar modular structure with an N-terminal C2

domain linked to a catalytic domain containing the active site

serine involved in the lipolytic catalysis. Human cPLA₂␤ differs

in that it contains an N-terminal truncated JmJc domain of

unknown function followed by a C2 domain and a catalytic

domain. Furthermore, human cPLA₂␤ exists as two splice

forms, human cPLA₂␤1 and human cPLA₂␤3, with the latter

being the major protein form expressed in a large variety of

human tissues (3). The differential splicing leads to a change in

a portion of the catalytic domain that is probably not part of the

active site cleft. As far as we know, m-cPLA₂␤ exists as a single

splice form (4).

binding of mouse cPLA₂␤ and its C2 domain to vesicles. Ca^2؉

-

dependent binding of mouse cPLA₂␤ to cardiolipin-containing

vesicles requires a patch of basic residues near the Ca^2؉-binding

surface loops of the C2 domain, but binding to phosphoinosit-

ide-containing vesicles does not depend on any specific cluster

of basic residues. Human cPLA₂␦ also displays Ca^2؉- and cardi-

olipin-enhanced interfacial binding and activity. The lysophos-

pholipase, phospholipase A₁, and phospholipase A₂activities of

the full set of mammalian cPLA₂s were quantified. The relative

level of these activities is very different among the isoforms, and

human cPLA₂␦ stands out as having relatively high phospho-

lipase A₁activity. We also tested the susceptibility of all cPLA₂

family members to a panel of previously reported inhibitors of

human cPLA₂␣ and analogs of these compounds. This led to the

discovery of a potent and selective inhibitor of mouse cPLA₂␤.

These in vitro studies help determine the regulation and func-

tion of the cPLA₂family members.

An intriguing property of m-cPLA₂␤, human cPLA₂␤1, and

human cPLA₂␤3 is that they display specific activities for the

hydrolysis of lysophosphatidylcholine and lysophosphati-

dylethanolamine that are ϳ100-fold higher than for the hydrol-

ysis of phosphatidylcholine and phosphatidylethanolamine (3,

4). One possibility is that the active sites of the ␤ isoforms differ

from that of cPLA₂␣ in such a way that only the lysophospho-

lipid is hydrolyzed by the former enzymes. An alternative pos-

sibility is that cPLA₂␣ can bind to the membrane interface of

phosphatidylcholine or phosphatidylethanolamine vesicles but

the ␤ isoforms cannot. In this scenario, only the former enzyme

can hydrolyze these vesicles, and the relatively high activity of

the ␤ isoforms for lysophospholipids reflects the hydrolysis of

monomeric substrate in the aqueous phase (due to the rela-

tively high solubility of lysophospholipids compared with phos-

It is well established that cytosolic phospholipase A₂-␣

(cPLA₂␣)²(also known as group IVA cPLA₂) liberates arachi-

* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth

Grants HL50040 (to M. H. G.) and HL61378 and HL34303 (to C. C. L.).

□S

The on-line version of this article (available at http://www.jbc.org) contains

supplemental Figs. 1–3 and Table 1 .

¹To whom correspondence should be addressed: Depts. of Chemistry and

Biochemistry, Campus Box 351700, University of Washington, Seattle, WA

98195. Fax: 206-685-8665; E-mail: gelb@chem.washington.edu.

line; [¹⁴C]PAPC, PAPC labeled at C-1 of the sn-2 arachidonyl with ¹⁴C;

PI(4,5)P₂, phosphatidylinositol 4,5-bisphosphate; PI, phosphatidylinositol;

PLA₁, phospholipase A₁; PLA₂, phospholipase A₂; POPA, 1-palmitoyl-2-ole-

oyl-sn-glycero-3-phosphate; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-ser-

ine; RACE, rapid amplification of cDNA ends; Ni-NTA, nickel-nitrilotriacetic

acid; POPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine; POPE, 1-palmitoyl-

2-oleoyl-phosphatidylethanolamine; POPG, 1-palmitoyl-2-oleoyl-phos-

phatidylglycerol; POPS, 1-palmitoyl-2-oleoyl-phosphatidylserine; POPI,

1-palmitoyl-2-oleoyl-phosphatidylinositol; CL, cardiolipin.

²The abbreviations used are: cPLA₂␣, cytosolic phospholipase A₂-␣ (group

IVA); h, human; h-cPLA₂␣-C2, C2 domain of h-cPLA₂␣; [¹⁴C]P-LPC, 1-[1-

¹⁴C]palmitoyl-sn-glycero-3-phosphocholine; h-cPLA₂␥, human cPLA₂

␥

isoform (group IVC); h-cPLA₂␦, human cPLA₂␦ isoform (group IVD);

h-cPLA₂⑀, human cPLA₂⑀ isoform (group IVE); h-cPLA₂␨, human cPLA₂␨

isoform (group IVF); m-cPLA₂␤, mouse cPLA₂␤ isoform (group IVB);

m-cPLA₂␤-C2, C2 domain of m-cPLA₂␤, m-cPLA₂␨, mouse cPLA₂␨ isoform

(group IVF); PAPC, 1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocho-

36100 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 46•NOVEMBER 12, 2010

Interfacial Properties of Group IVB cPLA₂␤

pholipids). This is consistent with the finding that cPLA₂␣ also 10⁶Sf9 cells in each well of a 12-well plate. Cells were incubated

displays high lysophospholipase activity (5) showing that the for 3 days post-infection, and cell lysates were analyzed by SDS-

active site of the ␣ and ␤ isoforms can accommodate lysophos- PAGE to find the optimal level of expression (the cPLA₂band

pholipids. The problem with this hypothesis is that both the ␣ was located by Coomassie Blue staining and confirmed by

and ␤ isoforms contain a C2 domain that, in the case of cPLA₂␣, Western blot using anti-His₆antiserum (Sigma)). For large

has been shown to be the basis of calcium-dependent binding to scale production, virus infection was carried out when cells

the surface of phospholipid vesicles and to cellular membranes reached a density of ϳ1.5 ϫ 10⁶/ml and then infected with the

(6, 7).

appropriate amount of virus stock. Cells were incubated at

Here, we show that m-cPLA₂␤ is as much a phospholipase A₂27 °C with stirring at ϳ90 rpm for typically 50–60 h (until

as it is a lysophospholipase in that the enzyme displays high ϳ15–25% of the cells were dead as judged by trypan blue anal-

phospholipase A₂on phosphatidylcholine vesicles that contain ysis). Cells were collected by centrifugation at 600 ϫ g at room

specific anionic phospholipids. This enzyme also has significant temperature. The cell pellet was stored at Ϫ80 °C.

phospholipase A₁(PLA₁) activity. We also compare the inter-

Frozen cell pellet from 0.5 liter of cell culture was thawed on

facial enzymatic properties of m-cPLA₂␤ to the other cPLA₂ice and resuspended in 20 ml of ice-cold buffer A (50 m^M

isoforms.

sodium phosphate, pH 8.0, 300 mM NaCl, 10 mM imidazole,

10% (v/v) glycerol and freshly added 2 m^M2-mercaptoethanol

and protease inhibitors (1 m^MPMSF, 10 ␮g/ml each of aproti-

EXPERIMENTAL PROCEDURES

Materials—The following phospholipids are from Avanti nin, leupeptin, and pepstatin A)). Cells were disrupted in a

Polar Lipids, Inc. (Alabaster, AL): cardiolipin (bovine heart, Dounce homogenizer on ice, and the suspension was ultracen-

mostly tetralinoleoyl); PAPC; PI (dioleoyl); PI(4)P (brain, trifuged at 100,000 ϫ g for 1 h at 4 °C. Ni-NTA (Qiagen) affinity

mostly sn-1-stearoyl, sn-2-arachidonyl); PI(3,4)P₂(dioleoyl); resin was washed with water and then resuspended in ice-cold

PI(3,5)P₂(dioleoyl); PI(4,5)P₂(dioleoyl); PI(3,4,5)P₃(dioleoyl); buffer A. The ultracentrifuge supernatant was added to 1 ml of

POPA; and POPS. PI(3)P (dipalmitoyl) is from Echelon Bio- packed and equilibrated Ni-NTA resin, and the suspension was

sciences, Inc. (Salt Lake City, UT). [¹⁴C]PAPC (50 mCi/mmol) gently mixed for 2 h at 4 °C and then transferred to a glass

and [¹⁴C]P-LPC (50 mCi/mmol) are from PerkinElmer Life Sci- column with the aid of some ice-cold buffer A. The column was

ences. Stock solutions of phosphoinositides were prepared in washed five times each with 1 ml of ice-cold buffer A (gravity

chloroform/methanol/water (4:4:1.2) at 0.1 mg/ml and stored flow). The column was then washed five times each with 1 ml of

at Ϫ80 °C in Teflon septum capped vials under argon. Stock ice-cold buffer A but with 25 m^Mimidazole (gravity flow).

solutions of other phospholipids were prepared in chloroform m-cPLA₂␤ was eluted with 5 ml of ice-cold cell homogenization

except for P-LPC, which was in chloroform/methanol (2:1); buffer A containing 250 m^Mimidazole. Fractions (8–10) were

these stock solutions were stored as for phosphoinositides. collected and examined for protein purity by SDS-PAGE.

Phospholipid concentrations in stock solutions were deter- Appropriate fractions were pooled, and the sample was concen-

mined by inorganic phosphate assay using the ammonium trated in an Amicon Ultra-15 ultrafilter at 4 °C, and buffer was

molybdate procedure on acid-degraded samples (8). Phospho- exchanged to 25 m^MTris-HCl, pH 7.5, 150 mM NaCl, 1 mM

inositides were analyzed for loss of phosphate due to hydrolysis EDTA, 2 mM fresh DTT by three rounds of addition and con-

by negative ion electrospray mass spectrometry (Bruker centration. Glycerol was added to give 30% (v/v), and the sam-

Esquire Ion Trap); no hydrolysis was detected. Purity of phos- ple was stored at Ϫ20 °C. The protein concentration was esti-

phoinositides is stated to be Ͼ99% by Avanti Polar Lipids, Inc. mated with the Bradford dye binding assay (Bio-Rad) using

h-cPLA₂␣, h-cPLA₂␥, and m-cPLA₂␨ (Ͼ90% pure) were pre- bovine serum albumin as a standard. The enzyme was judged to

pared as described previously (4, 9, 10). Bovine serum albumin be ϳ80% pure by SDS-PAGE (7.5% gel) with Coomassie Blue

is fatty acid-free (Sigma catalog no. A6003). Wyeth-1 (also staining. The specific activity of the purified m-cPLA₂␤ using

known as ecopladib), Wyeth-2 (also known as giripladib), the radiometric [¹⁴C]P-LPC assay (see below) was ϳ350 (nmol/

Wyeth-3, pyrrolidine-1, and pyrrolidine-2 (also known as pyr- min)/mg (Table 1). The yield of purified protein is ϳ5 mg/liter

rophenone) were prepared as described previously (11, 12). The of Sf9 cell culture.

other inhibitors listed in supplemental Table 1 were prepared as

described previously (13–15).

RACE Analysis of the h-cPLA₂⑀ Gene—A commercial human

cDNA multiple tissue panel (Clontech catalog no. PT3158-1)

Production of m-cPLA₂␤—The recombinant baculovirus was used in PCRs with the primers 5Ј-ccatctacctcaccatcaatgtc

expressing m-cPLA₂␤ bearing an N-terminal His₆-tagged has and 5Ј-aactcctccgaggtgtgtgacag to determine which tissues

been described previously (4). This protein contains the express a relatively high level of h-cPLA₂⑀. Based on this, we

sequence MSPIDPMGHHHHHHGRRRASVAAGILVPRGSP- selected lung tissue and used commercial RACE-ready cDNA

GLDGICSRNSGLLAFKL fused to the N-terminal Met of made from human lung (Clontech catalog no. 639308). RACE

m-cPLA₂␤. The baculovirus was plaque-purified and amplified. PCR was carried out according to the manufacturer’s instruc-

Protein was produced in Sf9 cells in 1 liter of complete supple- tions using the following primer pair in the first round of PCR:

ment Grace’s medium (Invitrogen) containing 10% heat-inac- gene-specific primer 5Ј-gtcctttaccacatgtctccgagcc and the

tivated FBS and 1% lipid concentrate (Invitrogen) and penicil- adapter primer 5Ј-ccatcctaatacgactcactatagggc. In the second

lin/streptomycin/fungizone (100-fold dilution of Invitrogen round of PCR using the first round PCR product as template,

catalog no. 15240-062) in a 3-liter spinner flask. For each virus the primers used were gene-specific primer 5Ј-gcagccccagcag-

stock, a small scale expression test was carried out using 0.5 ϫ gtggccatac and adapter primer 5Ј-actcactatagggctcgagcggc.

NOVEMBER 12, 2010•VOLUME 285•NUMBER 46

JOURNAL OF BIOLOGICAL CHEMISTRY 36101

Interfacial Properties of Group IVB cPLA₂␤

The PCR product was gel-purified, cloned into the pGEM-T Coomassie Blue staining. The yield is about 5 mg of purified

Easy vector after A tailing according to the manufacturer’s protein/liter of bacterial culture. h-cPLA₂␣-C2 was produced

directions (Promega catalog no. A-1360), and submitted to as above using the plasmid described previously (17). Mutants

DNA sequencing.

of m-cPLA₂␤-C2 were prepared using the QuikChange kit

Production of h-cPLA₂␦ and h-cPLA₂⑀—Synthetic genes cod- (Stratagene), and the full-length coding regions were se-

ing for these enzymes were prepared by Genscript (Piscataway, quenced to confirm the products.

NJ) (the DNA sequences are given in the supplemental mate-

Radiometric Assay of m-cPLA₂␤ with [¹⁴C]P-LPC—This

rial). The genes was subcloned into the baculovirus transfer assay was routinely used to quantify m-cPLA₂␤. The 100-␮l

plasmid pAc-HLT (BD Biosciences) using the flanking restric- assay mixture in a polypropylene microcentrifuge tube con-

tion sites EcoRI and NotI. Baculoviruses were prepared using tained 25 ␮M P-LPC, 120,000 dpm [¹⁴C]P-LPC in 10 mM

the BaculoGold kit (BD Biosciences). Proteins were expressed MOPS, pH 7.2, 0.5 mM EGTA, 100 mM KCl, 4 mM CaCl₂, and 0.5

in Sf9 cells as for m-cPLA₂␤ (see above). h-cPLA₂␦ was purified mg/ml bovine serum albumin. After adding enzyme, the mix-

as for m-cPLA₂␤. The yield is 5–7 mg/liter of cell culture. ture was incubated at 37 °C for 20 min. The mixture was ana-

h-cPLA₂⑀ was purified as for m-cPLA₂␤ with the following lyzed for released fatty acid as described previously (18).

exception. After washing the Ni-NTA gel with buffer A, a

Because the addition of diglycerides was found to increase

10–250 m^Mimidazole gradient in buffer A (total volume 15 ml) the activity of h-cPLA₂⑀ acting on P-LPC, inhibition studies

was run at 0.25 ml/min, collecting 4-min fractions. The yield of with this enzyme were carried out with 30 ␮M [¹⁴C]P-LPC

h-cPLA₂⑀ is 0.25 mg/liter of cell culture. Estimated purities (45,000 dpm per assay), 10 ␮M 1,2-dioleoyl-sn-glycerol (Avanti

(supplemental Fig. 1) are Ͼ90% for h-cPLA₂␦ and ϳ80% for Polar Lipids) in 80 ␮l of 50 mM HEPES, pH 7.4, 150 mM NaCl, 1

h-cPLA₂⑀.

m^MEGTA, 5 mM CaCl₂without albumin for 30 min at 37 °C.

Production of m-cPLA₂␤-C2—The C2 domain was identified Reactions were analyzed for free fatty acid analysis as described

based on the x-ray structure of the C2 domain from h-cPLA₂␣ above.

(residues 1–130 for cPLA₂␤-C2) (16). The gene coding for

Vesicle Binding Studies—Interfacial binding of m-cPLA₂␤ to

m-cPLA₂␤-C2 was amplified using PCR and the full-length 0.2-␮m sucrose-loaded vesicles was measured as described pre-

gene in pAcHLT (4) as template. The primers used are as fol- viously (18). Vesicles were extruded through 0.2-␮m mem-

lows: 5Ј-CCG CAT ATG CAG GCA AAG GTG CCA GAG branes in 10 m^MMOPS, pH 7.2, 176 mM sucrose, prespun,

ACA-3Ј and 5Ј-CCT CTC GAG TCA TGT CAG AGT CTG quantified, and diluted in 10 m^MMOPS, pH 7.2, 100 mM KCl,

TAG CCG-3Ј, which introduce an NdeI site immediately 0.5 m^MEGTA (18). Extruded vesicles were prepared fresh daily.

upstream of the ATG start codon and a XhoI site immediately m-cPLA₂␤ binding reactions were prepared in 1.5-ml polyallo-

downstream of the stop codon. The PCR product was digested mar microcentrifuge tubes and contained 10 m^MMOPS, pH

with NdeI/XhoI, and the product was ligated into the pET15b 7.2, 100 m^MKCl, 0.5 mM EGTA with 0.5 mg/ml bovine serum

expression vector (Novagen), which encodes a 20-residue albumin and various amounts of free calcium in the range 0–20

N-terminal extension MGSSHHHHHHSSGLVPRGSH.

␮M (measured with a Ca^2ϩ-sensing dye (18)), phospholipid

The C2 domain was expressed as a soluble protein in Esche- (200 ␮M total phospholipid), and 62 ng of m-cPLA₂␤ in a total

richia coli BL21(DE3) (Novagen). Cells were grown at 37 °C in 1 volume of 0.5 ml. Samples were centrifuged at 100,000 ϫ

liter of Luria broth containing 100 ␮g/ml ampicillin, and pro-

g_averageat 21 °C for 1 h, and two 90-␮l aliquots of supernatant

tein was induced with 0.5 m^Misopropyl 1-thio-␤-D-galactopy- were submitted to duplicate radiometric m-cPLA₂␤ assays by

ranoside at 25 °C for 12 h when the A₆₀₀reached ϳ0.5. Cells adding 10 ␮l of P-LPC stock solution (250 ␮M P-LPC contain-

were harvested by centrifugation at 4000 ϫ g at 4 °C, and the ing 1,200 dpm [¹⁴C]P-LPC per ␮l, 10 mM MOPS, pH 7.2, 0.5 mM

pellets were stored at Ϫ80 °C. Frozen pellets were thawed and EGTA, 100 mM KCl, 40 mM CaCl₂). The results were compared

resuspended in ice-cold 50 m^MNaH₂PO₄buffer, pH 8.0, 300 with a standard curve of [¹⁴C]P-LPC hydrolysis versus the

m^MNaCl, 10 mM imidazole, 10% (v/v) glycerol, and freshly amount of m-cPLA₂␤ (0–64 ng, linear response obtained, data

added 5 ␮M 2-mercaptoethanol and protease inhibitors (1 mM not shown) to obtain the amount of enzyme in the supernatant.

PMSF, 10 ␮g/ml each of aprotinin, leupeptin, and pepstatin A). See above for other details of the radiometric [¹⁴C]P-LPC assay.

The suspension was sonicated on ice to disrupt cells, and the The amount of m-cPLA₂␤ was compared with the amount

mixture was centrifuged at 14,000 ϫ g for 10 min at 4 °C. The measured in an ultracentrifuged mixture containing all of the

supernatant was gently shaken with 2 ml of Ni-NTA resin (50% above components except vesicles to obtain the percentage of

slurry) (Qiagen) at 4 °C for 1 h, and the resin was then packed total enzyme remaining in the supernatant above pelleted ves-

into a column. The resin was washed with 10 ml of extraction icles. Recovery of m-cPLA₂␤ from this ultracentrifuged sample

buffer containing 20 m^Mimidazole at 4 °C. The protein was lacking vesicles was ϳ100%, showing minimal binding of

eluted with 10 ml of extraction buffer containing 120 m^Mimid- enzyme to the tube wall. Ultracentrifugation of vesicles doped

azole at 4 °C. The protein solution was concentrated to ϳ10 with a small amount of [¹⁴C]PAPC and scintillation counting of

mg/ml in a centrifugal ultrafilter (Millipore, 5-kDa cutoff), and the supernatant showed that virtually all of the phospholipid

buffer was exchanged into 20 m^MTris-HCl, pH 7.5, 150 mM was pelleted.

NaCl for storage at Ϫ20 °C. Protein concentration was esti-

Binding of m-cPLA₂␤-C2 to vesicles was measured as

mated using the Bradford dye binding assay (Bio-Rad) using described previously (19). Reaction mixtures (500 ␮l) contained

bovine serum albumin as a standard. The purified protein was 5 ␮g of m-cPLA₂␤-C2 and 200 ␮M phospholipid as vesicles in

estimated to be 90% pure for m-cPLA₂␤-C2 by SDS-PAGE with 10 m^MMOPS, pH 7.2, 100 mM KCl, 0.5 mM EGTA with 0.5

36102 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 46•NOVEMBER 12, 2010

Interfacial Properties of Group IVB cPLA₂␤

mg/ml bovine serum albumin and various amounts of free cal- fatty acid was quantified as for the kinetic studies described

cium in the range 0–20 ␮M (18). After incubation for 10 min at above. Inhibition studies with h-cPLA₂⑀ (1 ␮g per assay) were

37 °C, the mixture was centrifuged at 100,000 ϫ g at 21 °C for carried out with the [¹⁴C]P-LPC/1,2-dioleoyl-sn-glycerol assay

1 h, and the pellet was submitted to SDS-PAGE on a 15% gel to (as described above) because the activity of this isoform on

estimate the amount of vesicle-bound protein. The same pro- [¹⁴C]PAPC was very low.

cedure was used to measure binding of h-cPLA₂␦ to vesicles.

RESULTS

Kinetic Studies—Extruded vesicles were prepared as

described above and diluted in 80 ␮l of 10 mM MOPS, pH 7.2,

Preparation of cPLA₂Proteins—All cPLA₂proteins were pre-

100 m^MKCl, 0.5 mM EGTA containing various concentrations pared as recombinant proteins using the baculovirus/Sf9 cell

of free calcium (18). Reactions were carried out in polypro- expression system. All proteins bear an N-terminal His₆tag

pylene microcentrifuge tubes at 37 °C for 10 min. Vesicle com- except for h-cPLA₂␣, which bears a C-terminal His₆tag. A

position and concentration are given in the figure and table complete cDNA sequence for h-cPLA₂⑀ was not available in the

legends. Reaction workup to determine the amount of released public databases, so we obtained the full-length sequence by

radiolabeled arachidonic acid was carried out as described pre- RACE PCR analysis of mRNA obtained from human lung. We

viously (18).

used either native genes or synthetic genes (with optimized

Phospholipids used for studying the PLA₁activity using mass codons for expression in Sf 9 cells, see under “Experimental Pro-

spectrometry were purified as follows to remove trace amounts cedures” and supplemental material). The purity of all proteins

of lysophospholipids. POPC, POPA, POPS, POPE, and POPG was Ͼ90% as determined by SDS-PAGE analysis (supplemental

(5 mg, Avanti Polar Lipids) were individually injected onto a Fig. 1 ) except for m-cPLA₂␤ and h-cPLA₂⑀ (ϳ80% pure).

normal phase HPLC column (Varian MonoChrom 5 ␮m, Si,

Kinetic Properties of m-cPLA₂␤—When human cPLA₂␤ was

1 ϫ 25 cm, catalog no. A0501-250-X100) in 1:1 solvent A (30: first cloned, it was shown using crude lysates from baculovirus-

40, v/v, n-hexane/isopropyl alcohol)/solvent B (43:47:10, v/v, infected insect cells that it had a very low specific activity on

n-hexane/isopropyl alcohol, 100 m^Mammonium formate in PAPC vesicles compared with cPLA₂␣ (21). We previously

water). The solvent program is as follows: 0–5 min, 25% B; 5–35 reported using purified human cPLA₂␤ that it displays a spe-

min, 25–70% B; 35–36 min, 70–99% B, 36–45 min, 99% B; cific activity on vesicles composed of 75% PAPC and 25% 1,2-

45–46 min, 99 to 25% B at 3 ml/min. A splitter was used to dioleoyl-sn-glycerol that is about 30-fold lower than that of

divert a portion of the column eluant (0.05 ml/min) to the mass h-cPLA₂␣ and that human cPLA₂␤ is about 15-fold more active

spectrometer; the main stream was connected to a fraction col- on [¹⁴C]P-LPC (presumably as micelles) than on [¹⁴C]PAPC

lector. Phospholipid and lysophospholipid molecular species (sonicated liposomes) (3). We also reported that purified

were monitored by mass spectrometry as described previously m-cPLA₂␤ is about 25-fold more active on [¹⁴C]P-LPC micelles

(20) except that full scan (400–900 atomic mass units) mass than on vesicles of 75% PAPC and 25% 1,2-dioleoyl-sn-glycerol

spectrometry was carried out instead of tandem mass spec- (4). Furthermore, the specific activity of human cPLA₂␤ on

trometry. POPI (Avanti) was not purified because no lysophos- [¹⁴C]PAPC vesicles was about 30-fold lower than that of

pholipid contaminant was detected by mass spectrometry. h-cPLA₂␣ (3). These initial studies suggested that the human

Reaction mixtures contained extruded phospholipid vesicles and mouse ␤ isoforms behave more as a lysophospholipase than

(50 ␮M total phospholipid) in 10 mM MOPS, pH 7.2, 100 mM as a phospholipase.

KCl, 0.5 mM EGTA, and sufficient CaCl₂to give ϳ100 ␮M free

Table 1 gives the specific activities of m-cPLA₂␤ and

Ca^2ϩ(monitored using Calcium Green^TM-2, Invitrogen). Reac- h-cPLA₂␣ acting on various substrates. Specific activities are

tions were started by adding 2 ␮g of each cPLA₂and incubated based on initial reaction velocities because reactions were

for 10 min at 37 °C. Reactions were extracted for lysophospho- quenched after 10 min, which is in the linear part of the product

lipid analysis as described previously (20) (because the sample versus time curve for both enzymes (supplemental Fig. 2). In the

did not contain plasmalogens, the two-stage extraction proce- absence of Ca^2ϩ, m-cPLA₂␤ is 1500-fold more active on [¹⁴C]P-

dure was not used; rather the sample was acidified and LPC than on [¹⁴C]PAPC (Table 1, entries 1 and 2). These values

extracted with organic solvent). The 1-palmitoyl-lysophospho- do not change significantly if there is 20 ␮M free Ca^2ϩin the

lipids and the 2-oleoyl-lysophospholipids were quantified by buffer (Table 1, entries 1 and 2). By comparison, h-cPLA₂␣ is

liquid chromatography/tandem mass spectrometry as de- slightly less active on [¹⁴C]P-LPC versus [¹⁴C]PAPC and shows

scribed previously (20).

a clear increase in activity on [¹⁴C]PAPC in the presence of

Inhibition Studies—Reaction mixtures contained [¹⁴C]PAPC Ca^2ϩ(Table 1, entries 13 and 14).

vesicles (4.9 ␮M, 58 Ci/mol) in 80 ␮l of 10 mM MOPS, pH 7.2,

The lack of phospholipase A₂activation by Ca^2ϩis surprising

100 m^MKCl, 0.5 mM EGTA, 1.5 mM CaCl₂, and 0.5 mg/ml fatty because in an earlier study it was reported that 1 m^MCa^2ϩ

acid-free bovine serum albumin. Vesicles were prepared by activates human cPLA₂␤ by 5–8-fold when acting on sonicated

adding [¹⁴C]PAPC in DMSO (2 ␮l per 80-ml reaction) and mix- liposomes of [¹⁴C]PAPC vesicles containing 30 mol % 1,2-di-

ing with a vortexer immediately after dilution into buffer (to oleoyl-sn-glycerol (21), and in a later study an ϳ6-fold activation

form unilamellar vesicles of nonuniform size). Reactions also of human cPLA₂␤ by 4 mM CaCl₂was reported when acting on

contained 1–2 ␮M inhibitor (added in 1 ␮l of DMSO). Reactions [¹⁴C]1-palmitoyl-2-arachidonyl-phosphatidylethanolamine vesi-

were started by addition of cPLA₂(10 ng of h-cPLA₂␣, 100 ng of cles containing 30 mol % 1,2-dioleoyl-sn-glycerol (3). There are

m-cPLA₂␤, 400 ng of h-cPLA₂␥, 1 ␮g of h-cPLA₂␦, or 10 ng of no earlier data on the Ca^2ϩdependence of m-cPLA₂␤. We also

m-cPLA₂␨) and were incubated at 37 °C for 30 min. Released tested our preparation of m-cPLA₂␤ using the exact sub-

NOVEMBER 12, 2010•VOLUME 285•NUMBER 46

JOURNAL OF BIOLOGICAL CHEMISTRY 36103

Interfacial Properties of Group IVB cPLA₂␤

TABLE 1

Interfacial kinetic properties of m-cPLA₂␤ and h-cPLA₂␣

Entry

Enzyme

Substrate

Specific activity (0 ␮M Ca^2؉

)

Specific activity (20 ␮M Ca^2؉

)

nmol/min/mg protein

1

m-cPLA₂␤

h-cPLA₂␣

͓

¹⁴C͔P-LPC

381, 365^a

0.24, 0.25

0.25, 0.25

0.6, 0.6

394, 336

0.28, 0.31

1.0, 0.7

0.9, 1.1

0.36, 0.37

1.8, 1.4

4.0, 4.4

5.5, 6.4

6.0, 6.0

3.7, 4.0

3.7, 3.7

11.0, 11.4

129, 122

230^b

2

¹⁴C͔PAPC

3

¹⁴C͔PAPC, 10% POPS

¹⁴C͔PAPC, 10% POPA

¹⁴C͔PAPC, 10% PI

4

5

0.44, 0.38

1.4, 1.6

6

¹⁴C͔PAPC, 10% PI(3)P

¹⁴C͔PAPC, 10% PI(4)P

¹⁴C͔PAPC, 10% PI(3,4)P₂

¹⁴C͔PAPC, 10% PI(3,5)P₂

¹⁴C͔PAPC, 10% PI(4,5)P₂

¹⁴C͔PAPC, 10% PI(3,4,5)P₃

¹⁴C͔PAPC, 10% CL

¹⁴C͔P-LPC

7

4.3, 4.5

8

2.9, 2.9

9

5.9, 6.4

10

11

12

13

14

15

16

17

18

3.5, 3.6

3.6, 3.5

0.93, 0.9

135, 154

1.2, 1.0

¹⁴C͔PAPC

¹⁴C͔PAPC, 10% POPS

¹⁴C͔PAPC, 10% POPA

¹⁴C͔PAPC, 10% PI(4,5)P₂

¹⁴C͔PAPC, 10% CL

1.5, 1.6

220, 200

400, 390

1,030²

1.6, 2.0

11.2, 10.6

2.1, 1.9

200, 205

^aDuplicate assay values are given. Reactions with ͓¹⁴C͔P-LPC contained 25 ␮M P-LPC (100,000 dpm ͓¹⁴C͔P-LPC per assay) in 100 ␮l of 10 mM MOPS, pH 7.2, 0.5 mM EGTA, 100

m^MKCl, 0.5 mg/ml bovine serum albumin, a free Ca^2ϩconcentration of 0 or 20 ␮M, and 100 ng of enzyme at 37 °C. Reactions with vesicles contained 200 ␮M total phospholipid

(as extruded vesicles) in 80 ␮l of the above buffer at 37 °C and 0.8–1 ␮g of m-cPLA₂␤ or 250 ng of h-cPLA₂␣.

^bData were taken from our previous studies using identical reaction conditions as for m-cPLA₂␤ (18).

strate composition ([¹⁴C]1-palmitoyl-2-arachidonyl-phos- (Table 1, entries 3 and 4). In the presence of 10% POPS,

phatidylethanolamine/1,2-dioleoyl-sn-glycerol-sonicated vesi- m-cPLA₂␤ showed a 4-fold increase in specific activity when

cles) and buffer conditions reported previously (3) and found a [Ca^2ϩ] goes from 0 to 20 ␮M; the effect of calcium when POPA

3-fold activation by 4 m^MCa^2ϩ(data not shown). When this is present is only 1.6-fold. Addition of POPS had no effect on

assay was repeated in the buffer used throughout this study in the specific activity of h-cPLA₂␣, and the addition of POPA

the presence of 0 versus 20 ␮M free Ca^2ϩ, the specific activities increased the activity by 2-fold when calcium was present

in duplicate measurements were 2.0 and 2.2 (nmol/min)/mg of (Table 1, entries 15 and 16). PI(4,5)P₂activates h-cPLA₂␣ about

protein in the absence of Ca^2ϩand 2.4 and 2.5 (nmol/min)/mg 8-fold (Table 1, entry 17) as reported previously (18). A number

in the presence of 20 ␮M free Ca^2ϩ. When sonicated vesicles of of phosphoinositides activate m- cPLA₂␤ when present in

[¹⁴C]PAPC/1,2-dioleoyl-sn-glycerol were used, the specific PAPC vesicles at 10 mol % (Table 1, entries 5–11). The most

activities were 0.7 and 0.5 (nmol/min)/mg in the absence of 20 effective is PI(3,5)P₂, which increases the specific activity by

␮M free Ca^2ϩand 0.7 and 0.5 (nmol/min)/mg in the presence of ϳ25-fold, but other phosphoinositides are almost as effective.

20 ␮M free Ca^2ϩ. Thus, we conclude that m-cPLA₂␤ is not PI, which contains no phosphate monoesters, activates

activated by physiologically relevant concentrations of Ca^2ϩm-cPLA₂␤ by only ϳ1.5-fold. PI(3)P and especially PI(4)P are

when these substrate preparations are used. It may be noted better activators. PI(3,4,5)P₃is a slightly worse activator than

that cPLA₂␣ shows a phase of Ca^2ϩactivation in the 0–20 ␮M PI(3,5)P₂. In all cases with phosphatidylinositols, there was no

range and a second phase of activation if the concentration of effect of adding 20 ␮M calcium on the specific activity.

Ca^2ϩis increased into the millimolar range (for example see h-cPLA₂␣ shows activation by PI(4,5)P₂anionic phospholipids

Ref. 22). The first phase is due to Ca^2ϩ-dependent binding of (Table 1, entry 17). In all cases, and in marked contrast to

the C2 domain of h-cPLA₂␣ to vesicles in vitro and to cellular m-cPLA₂␤, h-cPLA₂␣ shows a high dependence on the pres-

membranes in vivo (18, 23). The origin of the effect of the supra- ence of Ca^2ϩ

physiological Ca^2ϩlevel on cPLA₂␣ activity is not known but

Next we tested the dependence of the specific activity of

.

perhaps is due to an effect of this cation on the physical state of m-cPLA₂␤ on the mol % of an activating phosphatidylinositol,

the substrate aggregate. It is interesting to note that addition of PI(3,4,5)P₃, in [¹⁴C]PAPC, and results are shown in Fig. 1.

the Ca^2ϩionophore A23187 to Sf9 cells expressing the ␨ iso- Again, essentially no effect of calcium was seen (Fig. 1). The

form of cPLA₂, a highly calcium-dependent enzyme, causes an results show that most of the activation occurs in the 4–6

ϳ10-fold increase in arachidonic acid release, whereas calcium mol % range, and the activation plateaus as the amount of

ionophore treatment increases arachidonate release in Sf9 cells PI(3,4,5)P₃approaches 10 mol %. Based on these data, the phos-

expressing m-cPLA₂␤ by only ϳ2-fold (4). All together, the phoinositide species were retested at 6 rather than 10 mol % to

data show that m-cPLA₂␤ is not activated by low micromolar see if the phosphoinositide species activation selectivity

concentrations of Ca^2ϩin the presence of phosphatidylcholine increases when they are present at subsaturating concentra-

and phosphatidylethanolamine vesicles, and previous data on tions in vesicles. A similar phosphoinositide activation specific-

modest Ca^2ϩactivation of this enzyme is probably the result of ity is seen with 6 mol % anionic phospholipid in [¹⁴C]PAPC

an effect of millimolar Ca^2ϩconcentration on vesicle structure. vesicles as that seen with 10 mol % (data not shown).

Next, we examined the effect of anionic phospholipids on the

The effect of calcium and PI(3,4,5)P₃on the activity of

specific activity of m-cPLA₂␤ acting on extruded [¹⁴C]PAPC m-cPLA₂␤ was also tested on mixed phospholipid vesicles

vesicles. The addition of 10 mol % POPS or POPA to composed of 23 mol % POPC, 40 mol % POPE, and 30 mol %

[¹⁴C]PAPC vesicles had a modest effect on the specific activity POPS (the approximate composition of the inner leaflet or

36104 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 46•NOVEMBER 12, 2010

Interfacial Properties of Group IVB cPLA₂␤

FIGURE 3. Specific activity of m-cPLA₂␤ on [¹⁴C]PAPC vesicles as a func-

tion of the mol % of CL in vesicles. Total phospholipid concentration was

200 ␮M. All reactions contained 20 ␮M Ca^2ϩand 2 ␮g of m-cPLA₂␤ at 37 °C for

20 min.

FIGURE 1. Specific activity of m-cPLA₂␤ on [¹⁴C]PAPC vesicles as a func-

tion of the mol % of PI(3,4,5)P₃(200 ␮M total phospholipids). Filled circles,

0 ␮M Ca^2ϩ; open circles, 2 ␮M Ca^2ϩ; filled diamonds, 20 ␮M Ca^2ϩ

.

lyzed. A specific activity was not calculated because it is prob-

able that POPC, POPE, and POPS compete with [¹⁴C]PAPC for

binding to the active site of the enzyme; thus, the specific activ-

ity was not comparable with that measured with vesicles com-

posed only of [¹⁴C]PAPC vesicles.

Next, we examined the effect of addition of 10 mol % CL to

[¹⁴C]PAPC vesicles. Among the anionic phospholipids tested,

CL was the best activator of m-cPLA₂␤ (Table 1, entry 12).

Remarkably, the enzyme now becomes highly Ca^2ϩ-dependent

when CL is present in [¹⁴C]PAPC vesicles (Table 1, entry 12).

The specific activity with [¹⁴C]PAPC, 10 mol % CL vesicles in

the presence of 20 ␮M Ca^2ϩis 35-fold higher than with

[¹⁴C]PAPC vesicles. In contrast, CL only activates h-cPLA₂␣ by

2-fold and only in the absence of Ca^2ϩ

.

We studied the dependence of the specific activity of

m-cPLA₂␤ on the mol % of CL in [¹⁴C]PAPC vesicles and the

effect of calcium in more detail. As can be seen in Fig. 3, the

activity shows a sigmoidal dependence on mol % CL with max-

imal activation seen at around 25 mol % CL (all in the presence

of 20 ␮M Ca^2ϩ). The specific activity of m-cPLA₂␤ approaches

that of the highly active h-cPLA₂␣ acting on pure [¹⁴C]PAPC

vesicles. With [¹⁴C]PAPC, 10 mol % CL vesicles, the specific

activity increases ϳ8-fold as the concentration of calcium

increases from 0 to 100 ␮M with no evidence of saturation (Fig.

4, top panel). However, when the mol % CL is increased to 30%,

the increase in specific activity saturates when [Ca^2ϩ] reaches

about 10 ␮M (Fig. 4, bottom panel). This response to calcium is

near the physiologically relevant range of intracellular calcium

FIGURE 2. Activity of m-cPLA₂␤ on vesicles containing 23 mol % POPC, 40

mol % POPE, and 30 mol % POPS (200 ␮M total phospholipids) without

PI(3,4,5)P₃or vesicles containing 20 mol % POPC, 37 mol % POPE, 27

mol % POPS, and 10 mol % PI(3,4,5)P₃in the absence or presence of 20

␮M Ca^2؉. Vesicles also contained a trace of [¹⁴C]PAPC (100,000 dpm). Activity

is expressed as percent of [¹⁴C]PAPC hydrolyzed.

erythrocyte plasma membranes (24)). As with [¹⁴C]PAPC ves- variation.

icles, the activity of m-cPLA₂␤ was calcium-independent

We studied whether m-cPLA₂␤ displays any PLA₁activity.

(Fig. 2). In these studies, the mixed phospholipid vesicles were Vesicles of POPC were used, and liquid chromatography com-

doped with a small amount of [¹⁴C]PAPC, and the activity of bined with electrospray tandem mass spectrometry was used to

m-cPLA₂␤ was reported as the % of total [¹⁴C]PAPC hydro- measure the amount of P-LPC (PLA₂) and 2-oleoyl-sn-glycero-

NOVEMBER 12, 2010•VOLUME 285•NUMBER 46

JOURNAL OF BIOLOGICAL CHEMISTRY 36105

Interfacial Properties of Group IVB cPLA₂␤

TABLE 3

PLA₁versus PLA₂activities of cPLA₂(mixed phospholipid vesicles)

PLA₁, specific

PLA₂, specific

Phospholipid

species

Entry

Enzyme

activity (100 ␮M

Ca^2؉

)

Ca^2؉

)

nmol/min/mg protein nmol/min/mg protein

1

m-cPLA₂␤

POPC

POPE

POPS

POPI

0.13, 0.05, 0.06

0.00, 0.00

0.04, 0.01

0.03, 0.04

0.00, 0.02

0.00, 0.01

0.00, 0.00

0.01, 0.01

0.00, 0.01

0.00, 0.03

0.00, 0.00

7.59, 5.10

1.4, 1.45

0.00, 0.00, 0.00

0.03, 0.04

0.13, 0.12

0.03, 0.08

0.24, 0.14

0.25, 0.23

0.33, 0.27

0.06, 0.04

0.08, 0.04

0.07, 0.07

0.06, 0.02

0.15, 0.05

0.00, 0.03

0.00, 0.00

0.00, 0.06

0.00, 0.00

1.13, 0.85

0.24, 0.28

0.16, 0.21

0.00, 0.10

0.07, 0.05

0.14, 0.04

0.00, 0.00

0.00, 0.01

0.00, 0.00

0.66, 0.67

0.23, 0.21

0.28, 0.22

0.15, 0.12

0.21, 0.09

0.28, 0.27

2

3

4

5

POPG

POPA

POPC

POPE

POPS

POPI

6

7

h-cPLA₂␣

h-cPLA₂␥

h-cPLA₂␦

h-cPLA₂⑀

m-cPLA₂␨

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

POPG

POPA

POPC

POPE

POPS

POPI

POPG

POPA

POPC

POPE

POPS

POPI

1.05, 1.25

0.55, 0.63

0.62, 0.36

0.54, 0.60

0.25, 0.26

0.03, 0.05

0.00, 0.00

0.00, 0.05

0.00, 0.00

0.04, 0.04

0.48, 0.31

0.22, 0.24

0.08, 0.09

0.00, 0.00

0.07, 0.00

0.24, 0.14

POPG

POPA

POPC

POPE

POPS

POPI

POPG

POPA

POPC

POPE

POPS

POPI

POPG

POPA

3-phosphocholine (PLA₁) formation. All studies were carried

out with 100 ␮M Ca^2ϩ. Results summarized in Table 2 show

that m-cPLA₂␤ displays a PLA₁/PLA₂-specific activity ratio of

1.3 showing that it is a dual PLA₁/PLA₂enzyme (Table 1, entry

1). In contrast, h-cPLA₂␣ is mainly a PLA₂, displaying a PLA₁/

PLA₂ratio of 0.1 (Table 1, entry 2). We also prepared mixed

phospholipid vesicles composed of 50 mol % POPC and 10

mol % each of POPG, POPA, POPE, POPS, and POPI. We mon-

itored the formation of palmitoyl-containing sn-1 lysophos-

pholipid species (PLA₂) and oleoyl-containing sn-2 lysophos-

pholipid species (PLA₁), and the results are summarized in

Table 3. Interestingly, m-cPLA₂␤ preferentially utilized anionic

phospholipids (POPA, POPS, and POPG) over zwitterionic

phospholipids (POPC and POPE) in mixed phospholipid vesi-

cles, and the activity on anionic phospholipids is mainly as a

PLA₂and not as a PLA₁(Table 3, entries 1–6). The activity of

m-PLA₂␤ on the POPC in these vesicles is mainly as a PLA₁,

whereas in pure PAPC vesicles it displays both PLA₁and PLA₂

activities (Table 2, entry 1). h-cPLA₂␣ displays a high PLA₂/

PLA₁ratio on all phospholipids in the mixed vesicles and pre-

fers POPC (Table 3, entries 7–12).

FIGURE 4. Specific activity of m-cPLA₂␤ on [¹⁴C]PAPC/CL vesicles as a

function of [Ca^2؉]. Top panel, vesicles contain 10 mol % CL; bottom panel,

vesicles contain 30 mol % CL. In both cases reactions contained 200 ␮M total

phospholipid and 1 ␮g of m-cPLA₂␤ at 37 °C for 10 min.

TABLE 2

PLA₁versus PLA₂activities of cPLA₂(POPC vesicles)

PLA₁, specific

PLA₂, specific

PLA₁/PLA₂

activity

ratio

Entry

Enzyme

activity (100 ␮M

Ca^2؉

)

Ca^2؉

)

Interfacial Binding Properties of m-cPLA₂␤—We studied the

binding of m-cPLA₂␤ to vesicles loaded with sucrose, which

can be pelleted by ultracentrifugation. With 200 ␮M PAPC ves-

icles in the presence or absence of 20 ␮M Ca^2ϩ, no binding of

m-cPLA₂␤ was observed (Fig. 5). When PAPC vesicles con-

tained 10 mol % PI(3,4,5)P₃, about 50% of the enzyme was

bound in the presence or absence of 20 ␮M Ca^2ϩ(Fig. 5).

nmol/min/mg protein nmol/min/mg protein

1

2

3

4

5

6

m-cPLA₂␤

h-cPLA₂␣

h-cPLA₂␥

h-cPLA₂␦

h-cPLA₂⑀

m-cPLA₂␨

0.08

0.36

0.02

6.0

0.06

1.3

0.1

0.2

5.3

4.4

0.10

1.13

0.00^a

1.07

0.02

0.65

0.6

^aAlthough no activity was detected by mass spectrometry, h-cPLA₂⑀ has detectable

PLA₂activity when measured using the more sensitive radiometric assay (Table 1).

36106 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 46•NOVEMBER 12, 2010

Interfacial Properties of Group IVB cPLA₂␤

FIGURE 6. Interfacial binding to vesicles. Top panel, SDS-PAGE analysis of

m-cPLA₂␤-C2 in the pelleted vesicle fraction: lane 1, PAPC vesicles ϩ 0 ␮M

Ca^2ϩ; lane 2, PAPC vesicles ϩ 20 ␮M Ca^2ϩ; lane 3, PAPC, /10 mol % PI(3,4,5)P₃

vesiclesϩ0␮M Ca^2ϩ;lane4, PAPC, 10mol %PI(3,4,5)P₃vesiclesϩ20␮M Ca^2ϩ

.

Bottom panel, SDS-PAGE analysis of h-cPLA₂␣-C2 in the pelleted vesicle frac-

tion: lane 1, PAPC vesicles ϩ 0 ␮M Ca^2ϩ; lane 2, PAPC/10 mol % PI(3,4,5)P₃

vesicles ϩ 0 ␮M Ca^2ϩ; lane 3, PAPC vesicles ϩ 20 ␮M Ca^2ϩ; lane 4, PAPC/10

mol % PI(3,4,5)P₃vesicles ϩ 20 ␮M Ca^2ϩ. Data shown are representative of

two independent SDS-PAGE analyses.

FIGURE5. Bindingofm-cPLA₂␤tovesicles. Bindingreactionscontained200

␮M total phospholipid as vesicles and 62 ng of enzyme in buffer with or with-

out 20 ␮M free Ca^2ϩ. See “Experimental Procedures” for more information.

Plotted is the amount of enzyme in the supernatant above pelleted vesicles

expressed as a percentage of the amount of enzyme measured in a binding

reaction that lacked vesicles. See “Experimental Procedures” for other details.

Because there is no binding to PAPC vesicles in the absence of

PI(3,4,5)P₃, it cannot be determined whether this phosphoino-

sitide activates the enzyme bound to the interface. However, it

is certainly the case that PI(3,4,5)P₃enhances the binding of

enzyme to vesicles, and this explains at least part of the activa-

tion by this phosphoinositide. The lack of an effect of Ca^2ϩon

the extent of interfacial binding of enzyme is consistent with the

lack of Ca^2ϩdependence on the enzymatic activity.

FIGURE 7. Basic residues of m-cPLA₂␤-C2. Basic residues mutated to aspar-

agine are as follows: Arg-17/Arg-55 (red); Lys-24/Arg-49/Lys-52 (yellow);

Arg-14/Arg-107/Lys-117 (magenta); Lys-78 (orange); His-44/His-82 (green);

Arg-66/Arg-69/Arg-125 (blue); His-64/His-68 (gray); Lys-72 (pink). The Ca^2ϩ

-

We also studied interfacial binding of m-cPLA₂␤-C2 to ves-

icles. In the presence of 200 ␮M PAPC vesicles with 0 or 20 ␮M

Ca^2ϩ, no binding of m-cPLA₂␤-C2 was observed when the pel-

leted vesicles were subjected to SDS-PAGE analysis to detect

the protein (Fig. 6, top panel). When 10 mol % PI(3,4,5)P₃was

added to PAPC vesicles, interfacial binding of m-cPLA₂␤-C2

was observed, and the extent of binding was not modulated

by addition of 20 ␮M Ca^2ϩ(Fig. 6, top panel). Based on the gel

band intensity relative to that from a known amount of

m-cPLA₂␤-C2 loaded on to the gel, we estimate that about 50%

of the m-cPLA₂␤-C2 is bound to PAPC/PI(3,4,5)P₃vesicles.

When 10 mol % POPS or POPA was added to PAPC vesicles, no

increase in binding of m-cPLA₂␤-C2 was seen in the absence or

presence of 20 ␮M Ca^2ϩ(data not shown). As shown in Fig. 6

(bottom panel), h-cPLA₂␣-C2 binds to PAPC only in the pres-

ence of Ca^2ϩ(20 ␮M), and binding in the presence and absence

of Ca^2ϩis not modulated by the presence of 10 mol %

PI(3,4,5)P₃in the PAPC vesicles. All of these data are consistent

with the kinetic studies of m-cPLA₂␤, which shows that

PI(3,4,5)P₃but not POPA or POPS activates the enzyme in a

Ca^2ϩ-independent manner. Binding studies with h-cPLA₂␣-C2

binding surface loops are shown at the top panel. The left image is rotated

ϳ180° along the vertical axis to give the right image.

phosphoinositides induce the binding of full-length cPLA₂␣ to

PAPC vesicles (18) (Fig. 6, bottom).

Next, we prepared a number of m-cPLA₂␤-C2 mutants in

which surface cationic residues (arginine, lysine, and histidine)

were mutated to the neutral residue asparagine to gauge the

importance of cationic residues in supporting PI(3,4,5)P₃

dependence of the binding of the protein to vesicles. The circu-

lar dichroism spectra of all mutants were similar to that from

wild type m-cPLA₂␤ (data not shown) suggesting that all pro-

teins are properly folded. Also, all mutants were expressed in

E. coli as soluble proteins. The location of the mutants on the

structure of m-cPLA₂␤-C2 (homology model based on the

x-ray structure of other C2 domains) is shown in Fig. 7.

Remarkably, all mutants bound to PAPC, 10 mol % PI(3,4,5)P₃

vesicles to the same extent as the wild type m-cPLA₂␤-C2 as

judged by the protein band intensities after the same type of

SDS-PAGE analysis as shown in Fig. 6 (data not shown). Thus,

enhanced binding of m-cPLA₂␤-C2 to PAPC vesicles caused by

are consistent with our early studies showing that Ca^2ϩbut not addition of PI(3,4,5)P₃cannot be attributed to any single cluster

NOVEMBER 12, 2010•VOLUME 285•NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 36107

Interfacial Properties of Group IVB cPLA₂␤

substrates and additives used to study m-cPLA₂␤, and the

results are summarized in Table 4. h-cPLA₂␥ displays lysophos-

pholipase activity (Table 4, entry 1) on [¹⁴C]P-LPC comparable

with that of m-cPLA₂␤ and h-cPLA₂␣, but it displays relatively

low PLA₂activity on [¹⁴C]PAPC vesicles in the absence and

presence of anionic phospholipids (Table 4, entries 2–12). Spe-

cific activities with 0 and 20 ␮M Ca^2ϩare essentially identical,

consistent with the fact that this cPLA₂lacks a C2 domain but

rather contains a C-terminal farnesyl-cysteine methyl ester (9).

h-cPLA₂␦ displays lysophospholipase A₂activity comparable

with the other isoforms (Table 4, entry 13) and relatively low

PLA₂activity (entries 14–24). Among the anionic additives,

only the addition of 10% CL to [¹⁴C]PAPC shows activation

(8-fold) (Table 4), and the increase is Ca^2ϩ-dependent (Table 4,

entry 24). The latter result may be explained by the fact that

h-cPLA₂␦ has a C2 domain. To explore this further, we studied

the binding of h-cPLA₂␦ to vesicles. As shown in Fig. 8 (bottom

panel), no binding of this enzyme to PAPC was seen in the

absence or presence of 20 ␮M Ca^2ϩ. Binding to PAPC, 10% CL

vesicles was enhanced by the presence of 20 ␮M Ca^2ϩ. These

binding data explain at least part of the activation of h-cPLA₂␦

seen by CL and Ca^2ϩ(Table 4).

h-cPLA₂⑀ has very low specific activity as a lysophospho-

lipase on [¹⁴C]P-LPC and as a PLA₂on [¹⁴C]PAPC vesicles with

or without anionic phospholipid additives (Table 4, entries

25–36), although some of the phosphoinositides (especially

PI(3,4)P₂, PI(3,5)P₂, and PI(3,4,5)P₃) activate up to ϳ10-fold

(Table 4, entries 32, 33, and 35). Specific activities in the

absence and presence of 20 ␮M Ca^2ϩare similar.

FIGURE 8. Effect of CL and basic residue mutations on interfacial binding

of m-cPLA₂␤-C2 to vesicles. Top panel, SDS-PAGE analysis of the pelleted

vesicle fraction: lane 1, m-cPLA₂␤-C2 (5 ␮g) in the absence of vesicles; lane 2,

PAPC, 10 mol % CL vesicles ϩ 0 ␮M Ca^2ϩ; lane 3, PAPC, 10 mol % CL vesicles ϩ

20 ␮M Ca^2ϩ; lane 4, PAPC, 20 mol % CL vesicles ϩ 0 ␮M Ca^2ϩ; lane 5, PAPC, 20

mol % CL vesicles ϩ 20 ␮M Ca^2ϩ; lane 6, PAPC, 30 mol % CL vesicles ϩ 0 ␮M

Ca^2ϩ; lane 7, PAPC, 30 mol % CL vesicles ϩ 20 ␮M Ca^2ϩ. Middle panel, lane 1,

wild type m-cPLA₂␤-C2 (5 ␮g) in the absence of vesicles; lane 2, vesicle pellet

fraction using PAPC, 30 mol % CL vesicles with wild type m-cPLA₂␤-C2 ϩ 0 ␮M

Ca^2ϩ; lane 3, same as lane 2 but ϩ 20 ␮M Ca^2ϩ; lane 4, same as lane 2 but with

K24N/R49N/K52N mutant of m-cPLA₂␤-C2 ϩ 0 ␮M Ca^2ϩ; lane 5, same as lane

4 but ϩ 20 ␮M Ca^2ϩ; lane 6, same as lane 2 but with the H44N/H82N mutant of

m-cPLA₂␤-C2 ϩ 0 ␮M Ca^2ϩ; lane 7, same as lane 6 but ϩ 20 ␮M Ca^2ϩ;lane 8,

same as lane 2 but with the K78N mutant of m-cPLA₂␤-C2 ϩ 0 ␮M Ca^2ϩ; lane

9, same as lane 8 but with 20 ␮M Ca^2ϩ. Bottom panel, binding of h-cPLA₂␦ to

vesicles (SDS-PAGE analysis of the pelleted vesicle fraction): lane 1, PAPC ves-

icles ϩ 0 ␮M Ca^2ϩ; lane 2, PAPC vesicles ϩ 20 ␮M Ca^2ϩ; lane 3, PAPC, 10% CL

m-cPLA₂␨ displays relatively high lysophospholipase activity

on [¹⁴C]P-LPC (Table 4, entry 37) and PLA₂activity on

[¹⁴C]PAPC similar to that of h-cPLA₂␣ (entries 38–48). The

PLA₂activity but not the lysophospholipase A₂is Ca^2ϩ-depen-

dent (as is the case for h-cPLA₂␣). m-cPLA₂␨ displays signifi-

cant activation when most of the anionic phospholipids are

added to [¹⁴C]PAPC vesicles as follows: 3-fold by POPA; up to

ϳ2-fold by most of the phosphoinositides, and 3-fold by CL. In

vesicles ϩ 0 ␮M Ca^2ϩ; lane 4, PAPC, 10% CL vesicles ϩ 20 ␮M Ca^2ϩ

.

of basic residues mutated in this study (all basic residues pre- all cases, the PLA₂activity is Ca^2ϩ-dependent consistent with

dicted to be on the protein surface of the homology model).

the presence of a C2 domain.

We also studied the effect of CL on binding of m-cPLA₂␤-C2

We also studied the PLA₁versus PLA₂activity of these addi-

domains to PAPC vesicles. As shown in Fig. 8 (top panel), tional cPLA₂isoforms, and the results with POPC vesicles are

increasing amounts of CL in PAPC vesicles increases the summarized in Table 2. h-cPLA₂␥ is a better PLA₂than a PLA₁

amount of m-cPLA₂␤-C2 bound to vesicles, and the binding is (Table 2, entry 3). h-cPLA₂␦ has much higher PLA₁activity

enhanced in the presence of Ca^2ϩ. In the presence of 30 mol % than PLA₂activity (Table 2, entry 4). Again, h-cPLA₂⑀ has very

CL and 20 ␮M Ca^2ϩabout 50% of the m-cPLA₂␤-C2 is vesicle- low PLA₂activity, and its PLA₁activity is also relatively low

bound (Fig. 8, top panel). We studied the binding of the (Table 2, entry 5). The PLA₁and PLA₂activities of m-cPLA₂␨

m-cPLA₂␤-C2 mutants to PAPC, 30 mol % CL vesicles in the are comparable (Table 2, entry 6). With the mixed phospholipid

presence and absence of 20 ␮M Ca^2ϩ. As shown in Fig. 8 (middle vesicles, h-cPLA₂␥ displays low PLA₁and PLA₂activity on all

panel, lanes 4 and 5), the triple mutant K24N/R49N/K52N does phospholipid species (Table 4, entries 13–18). h-cPLA₂␦ dis-

not show Ca^2ϩ-enhanced binding to PAPC/30 mol % CL vesi- plays a high PLA₁/PLA₂ratio and hydrolyzes all of the phos-

cles. The H44N/H82N mutant (Fig. 8, middle panel, lanes 6 and pholipid species in the mixed vesicles with comparable effi-

7) and the K78N mutant (Fig. 8, middle panel, lanes 8 and 9) ciency (Table 4, entries 19–24) (note the higher values for

show some Ca^2ϩ-enhanced binding, but it is less than that for POPC hydrolysis is probably due to the fact that POPC is pres-

the wild type C2 domain. All other basic residue mutants show ent at a 5-fold higher concentration than the other phospho-

the same Ca^2ϩ-enhanced binding to PAPC, 30 mol % CL vesi- lipid species in these mixed phospholipid vesicles). h-cPLA₂⑀

cles as does the wild type C2 domain (data not shown).

displays low PLA₁and PLA activities on all phospholipid spe-

Kinetic Properties of Other cPLA₂Isoforms—We measured cies except for POPC in which it acts mainly as a PLA₁(Table 4,

the specific activities of the other cPLA₂isoforms using the entries 25–30). m-cPLA₂␨ displays comparable PLA₁and PLA₂

36108 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 46•NOVEMBER 12, 2010

Interfacial Properties of Group IVB cPLA₂␤

TABLE 4

Lipolysis by cPLA₂isoforms

Entry

Enzyme

Substrate

Specific activity (0 ␮M Ca^2؉

)

Specific activity (20 ␮M Ca^2؉

)

nmol/min/mg protein

1

h-cPLA₂␥

h-cPLA₂␦

h-cPLA₂⑀

m-cPLA₂␨

͓

¹⁴C͔P-LPC

61, 55^a

0.3, 0.3

0.2, 0.3

0.4, 0.4

0.3, 0.3

0.4, 0.4

0.6, 0.6

0.7, 0.7

0.6, 0.7

0.2, 0.2

23, 22

52, 48

0.3, 0.3

0.2, 0.3

0.4, 0.4

0.3, 0.3

0.4, 0.4

0.6, 0.6

0.5, 0.5

0.7, 0.7

0.6, 0.6

0.2, 0.3

33, 35

2

¹⁴C͔PAPC

3

¹⁴C͔PAPC, 10% POPS

¹⁴C͔PAPC, 10% POPA

¹⁴C͔PAPC, 10% PI

4

5

6

¹⁴C͔PAPC, 10% PI(3)P

¹⁴C͔PAPC, 10% PI(4)P

¹⁴C͔PAPC, 10% PI(3,4)P₂

¹⁴C͔PAPC, 10% PI(3,5)P₂

¹⁴C͔PAPC, 10% PI(4,5)P₂

¹⁴C͔PAPC, 10% PI(3,4,5)P₃

¹⁴C͔PAPC, 10% CL

¹⁴C͔P-LPC

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

¹⁴C͔PAPC

0.06, 0.06

0.06, 0.04

0.1, 0.2

0.09, 0.08

0.2, 0.2

0.3, 0.3

0.2, 0.3

0.3, 0.3

0.1, 0.1

0.22, 0.20

0.02, 0.1

0.05, 0.1

0.06, 0.12

0.06, 0.05

0.14, 0.06

0.26, 0.31

0.4, 0.4

0.5, 0.4

0.5, 0.5

0.05, 0.09

375, 359

2.3, 2.6

2.7, 2.6

3.3, 4.2

3.0, 2.8

4.9, 5.4

8.8, 8.1

8.3, 6.6

8.0, 8.2

7.8, 8.1

8.2, 8.4

5.6, 5.8

0.1, 0.1

0.2, 0.1

0.2, 0.3

0.1, 0.1

0.2, 0.2

0.3, 0.3

0.4, 0.4

0.3, 0.3

0.8, 0.7

0.15, 0.14

0.05, 0.03

0.2, 0.05

0.08, 0.12

0.02, 0.02

0.06, 0.5

0.3, 0.3

0.5, 0.5

0.5, 0.6

0.5, 0.5

0.5, 0.6

0.05, 0.06

391, 348

61, 59

¹⁴C͔PAPC, 10% POPS

¹⁴C͔PAPC, 10% POPA

¹⁴C͔PAPC, 10% PI

¹⁴C͔PAPC, 10% PI(3)P

¹⁴C͔PAPC, 10% PI(4)P

¹⁴C͔PAPC, 10% PI(3,4)P₂

¹⁴C͔PAPC, 10% PI(3,5)P₂

¹⁴C͔PAPC, 10% PI(4,5)P₂

¹⁴C͔PAPC, 10% PI(3,4,5)P₃

¹⁴C͔PAPC, 10% CL

¹⁴C͔P-LPC

¹⁴C͔PAPC

¹⁴C͔PAPC, 10% POPS

¹⁴C͔PAPC, 10% POPA

¹⁴C͔PAPC, 10% PI

¹⁴C͔PAPC, 10% PI(3)P

¹⁴C͔PAPC, 10% PI(4)P

¹⁴C͔PAPC, 10% PI(3,4)P₂

¹⁴C͔PAPC, 10% PI(3,5)P₂

¹⁴C͔PAPC, 10% PI(4,5)P₂

¹⁴C͔PAPC, 10% PI(3,4,5)P₃

¹⁴C͔PAPC, 10% CL

¹⁴C͔P-LPC

¹⁴C͔PAPC

¹⁴C͔PAPC, 10% POPS

¹⁴C͔PAPC, 10% POPA

¹⁴C͔PAPC, 10% PI

62, 62

159, 159

85, 94

¹⁴C͔PAPC, 10% PI(3)P

¹⁴C͔PAPC, 10% PI(4)P

¹⁴C͔PAPC, 10% PI(3,4)P₂

¹⁴C͔PAPC, 10% PI(3,5)P₂

¹⁴C͔PAP, 10% PI(4,5)P₂

¹⁴C͔PAPC, 10% PI(3,4,5)P₃

¹⁴C͔PAPC, 10% CL

73, 69

94, 90

89, 103

104, 96

93, 117

88, 99

192, 190

^aDuplicate assay values are given.

activities on all species except for POPI and POPG, where it made use of the assay with [¹⁴C]P-LPC, 1,2-dioleoyl-sn-glyc-

shows mainly PLA₂activity (Table 4, entries 31–36). erol because this enzyme has almost no PLA₂activity. Pyr-

Inhibition Studies—A number of highly potent h-cPLA₂␣ rolidine-1 and -2 and Wyeth-1, -2, and -3 display high

inhibitors were tested on the full set of cPLA₂isoforms. The inhib- potency on h-cPLA₂␣ and m-cPLA₂␨ and are relatively inac-

itors include pyrrolidine-1 and pyrrolidine-2 developed by tive against all other cPLA₂isoforms. A subset of the elec-

Shionogi & Co., Ltd. (25, 26), which contain an electrophilic trophilic ketone inhibitors was potent on h-cPLA₂␣; some

ketone group that mayform ahemiketal adductwiththeactivesite show intermediate potency, and some are not active. The

serine residue (working hypothesis) (structures of all inhibitors are structure-activity data for these compounds against

given in supplemental Fig. 3 ). Wyeth-1, -2, and -3 are indole-based h-cPLA₂␣ and m-cPLA₂␨ are very similar. Among all the

h-cPLA₂␣inhibitors developedby Wyeth(12). Compoundsinthis inhibitors tested, only a subset of the electrophilic ketone

series are undergoing clinical trials for various inflammatory dis- inhibitors was active on m-cPLA₂␤, and Lf-80 emerges as a

orders. All of the other compounds studied are analogs of the orig- specific and reasonably potent inhibitor of this isoform. All

inal h-cPLA₂␣ inhibitor, arachidonyltrifluoromethyl ketone (27), of the inhibitors tested are relatively ineffective at blocking

and contain an electrophilic ketone group (15).

the activity of h-cPLA₂␥, h-cPLA₂␦, and h-cPLA₂⑀. We also

The supplemental Table 1 gives the percent inhibition of tested pyrrolidine-1 and -2 and Wyeth-1, -2, and -3 on

each cPLA₂isoform by each inhibitor present at 1 ␮M (pyrrol- m-cPLA₂␤ using [¹⁴C]PAPC vesicles containing 30 mol %

idine-1 and -2, and Wyeth-1, -2, and -3) or 2 ␮M (remaining CL (optimal activity conditions for this isoform), and again

compounds). All studies were done with [¹⁴C]PAPC vesicles in we found no inhibition by these compounds (data not

the presence of Ca^2ϩexcept for studies with h-cPLA₂⑀, which shown).

NOVEMBER 12, 2010•VOLUME 285•NUMBER 46

JOURNAL OF BIOLOGICAL CHEMISTRY 36109

Interfacial Properties of Group IVB cPLA₂␤

DISCUSSION

cles. Thus, the low activity suggests that the phospholipids

present are not optimal substrates for this enzyme.

The behavior of the C2 domain of m-cPLA₂␤ is unusual

among previously reported C2 domains. Interfacial binding of

this C2 domain and the activity of the full-length protein are

promoted by phosphoinositides, especially highly phosphory-

lated species, but Ca^2ϩis not needed. Presumably, cationic

amino acids are required for forming favorable electrostatic

interactions with the anionic phosphoinositides, but after

mutating all cationic patches on the surface of the C2 domain,

we were not able to locate a patch that is critical for interfacial

binding. The only hypotheses we can put forward is that the C2

domain of m-cPLA₂␤ binds to anionic phosphoinositides in

lipid vesicles either in multiple orientations, making use of dif-

ferent clusters of cationic residues, or that many more than 2–3

basic residues form electrostatic interactions with the interface

such that mutating only a few of them is insufficient to see a

reduction in interfacial binding. These results are surprising

because the amino acid sequence of this C2 domain, when

aligned with other Ca^2ϩ-responsive C2 domains, appears to

have all the ligands that interact with Ca^2ϩions. In fact the C2

domain of m-cPLA₂␤ is responsive to Ca^2ϩwhen the anionic

phospholipid CL is present in zwitterionic phosphatidylcholine

vesicles. The concentration of Ca^2ϩrequired for maximal acti-

It has been previously reported that cPLA₂␣ displays PLA₁,

PLA₂, and lysophospholipase activities (see for example Refs. 4,

5). This is in marked contrast to secreted phospholipases A₂,

which are strict PLA₂s. The x-ray structure of phospholipid

analogs bound to secreted PLA₂s shows how the sn-3 phos-

phate coordinates to the active site of Ca^2ϩto position the sn-2

ester close to the catalytic residues required for ester hydrolysis

(28). There are no x-ray structures on phospholipid analogs

bound to the active site of cPLA₂s, so it is not possible to under-

stand why these enzymes have multiple phospholipase activi-

ties; this degree of multiple substrate tolerance is unusual for

enzymes. Our studies of the full set of mammalian isoforms

show that they all display PLA₁and PLA₂activities, but the

relative amount of each is dramatically different among the

isoforms. m-cPLA₂␤ is both a PLA₁and a PLA₂, and the ratio of

these activities depends dramatically on the headgroup of the

phospholipid structure. It is preferentially a PLA₁on POPC but

is preferentially a PLA₂on anionic phospholipids (POPS, POPI,

POPG, and POPA). m-cPLA₂␨ displays comparable PLA₂and

PLA₁activities on phospholipids with different headgroups, an

additional feature that distinguishes it from h-cPLA₂␣. Among

the cPLA₂isoforms, h-cPLA₂␦ displays the strongest PLA₁ver-

vation of m-cPLA₂␤ depends on the mole fraction of CL in sus PLA₂bias, and this extends to phospholipids with different

PAPC vesicles; higher mol % CL leads to a lower Ca^2ϩrequire-

ment. This suggests that CL and Ca^2ϩwork synergistically

together to promote interfacial binding of this cPLA₂isoform

to the membrane interface. It will be interesting to see where

m-cPLA₂␤ traffics in mammalian cells. One possibility sug-

gested by the data of this study is that when intracellular Ca^2ϩis

low, the enzyme associates with a membrane surface that is rich

in phosphoinositides, but when intracellular Ca^2ϩrises, the

enzyme may move to a membrane rich in CL such as mitochon-

drial membranes.

The PLA₂activity of m-cPLA₂␤ approaches its lysophospho-

lipase activity only when a vesicle composition is used that sup-

ports a high level of interfacial enzyme binding (i.e. PAPC ϩ

CL ϩ Ca^2ϩ). PAPC plus Ca^2ϩis sufficient to support a high

level of h-cPLA₂␣ interfacial binding. Like h-cPLA₂␣,

m-cPLA₂␨ displays activity on PAPC vesicles in the presence of

Ca^2ϩthat approaches its lysophospholipase A₂activity. How-

ever, m-cPLA₂␨ and h-cPLA₂␣ behave differently in that phos-

phoinositides activate only the latter (mainly by increasing the

catalytic activity of the vesicle-bound enzyme rather than pro-

moting interfacial binding of enzyme (18)). The relatively small

amount of activation of m-cPLA₂␨ and h-cPLA₂␣ by CL distin-

guishes these enzymes from the behavior of m-cPLA₂␤.

h-cPLA₂␥ and h-cPLA₂␦ display high lysophospholipase activ-

ity and low PLA₂/PLA₁activities at least among the substrates

tested in this study. h-cPLA₂⑀ displays much lower lysophos-

pholipase activity relative to the other isoforms studied, and its

PLA₁and PLA₂activities are also low. In the case of h-cPLA₂␦,

some increase in phospholipase activity was seen when CL was

present in vesicles and Ca^2ϩwas added, but the specific activity

still remains relatively low. Interfacial binding studies show that

CL and Ca^2ϩpromote interfacial binding of h-cPLA₂␦ to vesi-

headgroups.

With the realization that several cPLA₂isoforms are present

in mammals and in some cells several cPLA₂s are present (see

for example Ref. 4) comes the concern that previously reported

h-cPLA₂␣ inhibitors may not be specific to a single isoform. We

have already shown that previously reported h-cPLA₂␣ inhibi-

tors block arachidonate release in mouse lung fibroblasts that

lack h-cPLA₂␣, a result attributed to inhibition of m-cPLA₂␨

(4). In this study, we have tested a large set of previously

reported potent h-cPLA₂␣ inhibitors, and we failed to find a

compound that can well distinguish h-cPLA₂␣ from m-cPLA₂␨.

The overlap in structure-activity data for this large set of inhib-

itors is remarkable. Whether or not some of these compounds

also inhibit human cPLA₂␨ remains to be determined. As of yet,

we have not been able to express this human enzyme despite

attempts using several expression systems. The inhibitors stud-

ied here do not significantly inhibit h-cPLA₂␥, h-cPLA₂␦, and

h-cPLA₂⑀, but a subset inhibits m-cPLA₂␤. Among the latter,

we discovered a few specific inhibitors of m-cPLA₂␤, which

should prove useful in cell studies to study the function of this

enzyme.

Acknowledgment—We acknowledge the technical help of Robyn

Loper for production of baculoviruses.

REFERENCES

1. Ohto, T., Uozumi, N., Hirabayashi, T., and Shimizu, T. (2005) J. Biol.

Chem. 280, 24576–24583

2. Ghosh, M., Tucker, D. E., Burchett, S. A., and Leslie, C. C. (2006) Prog.

Lipid Res. 45, 487–510

3. Ghosh, M., Loper, R., Gelb, M. H., and Leslie, C. C. (2006) J. Biol. Chem.

281, 16615–16624

4. Ghosh, M., Loper, R., Ghomashchi, F., Tucker, D. E., Bonventre, J. V.,

36110 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 46•NOVEMBER 12, 2010

Interfacial Properties of Group IVB cPLA₂␤

Gelb, M. H., and Leslie, C. C. (2007) J. Biol. Chem. 282, 11676–11686

5. de Carvalho, M. G., Garritano, J., and Leslie, C. C. (1995) J. Biol. Chem.

270, 20439–20446

18. Tucker, D. E., Ghosh, M., Ghomashchi, F., Loper, R., Suram, S., John, B. S.,

Girotti, M., Bollinger, J. G., Gelb, M. H., and Leslie, C. C. (2009) J. Biol.

Chem. 284, 9596–9611

6. Nalefski, E. A., and Falke, J. J. (1996) Protein Sci. 5, 2375–2390

7. Evans, J. H., Spencer, D. M., Zweifach, A., and Leslie, C. C. (2001) J. Biol.

Chem. 276, 30150–30160

19. Davletov, B., Perisic, O., and Williams, R. L. (1998) J. Biol. Chem. 273,

19093–19096

20. Bollinger, J. G., Ii, H., Sadilek, M., and Gelb, M. H. (2010) J. Lipid Res. 51,

440–447

8. Eaton, B. R., and Dennis, E. A. (1976) Arch. Biochem. Biophys. 176,

604–609

21. Pickard, R. T., Strifler, B. A., Kramer, R. M., and Sharp, J. D. (1999) J. Biol.

Chem. 274, 8823–8831

9. Tucker, D. E., Stewart, A., Nallan, L., Bendale, P., Ghomashchi, F., Gelb,

M. H., and Leslie, C. C. (2005) J. Lipid Res. 46, 2122–2133

10. Hixon, M. S., Ball, A., and Gelb, M. H. (1998) Biochemistry 37, 8516–8526

11. Degousee, N., Ghomashchi, F., Stefanski, E., Singer, A., Smart, B. P., Bor-

regaard, N., Reithmeier, R., Lindsay, T. F., Lichtenberger, C., Reinisch, W.,

Lambeau, G., Arm, J., Tischfield, J., Gelb, M. H., and Rubin, B. B. (2002)

J. Biol. Chem. 277, 5061–5073

22. Clark, J. D., Milona, N., and Knopf, J. L. (1990) Proc. Natl. Acad. Sci. U.S.A.

87, 7708–7712

23. Leslie, C. C. (1997) J. Biol. Chem. 272, 16709–16712

24. Zwaal, R. F., Roelofsen, B., Comfurius, P., and van Deenen, L. L. (1975)

Biochim. Biophys. Acta 406, 83–96

25. Seno, K., Okuno, T., Nishi, K., Murakami, Y., Watanabe, F., Matsuura, T.,

Wada, M., Fujii, Y., Yamada, M., Ogawa, T., Okada, T., Hashizume, H., Kii,

M., Hara, S., Hagishita, S., Nakamoto, S., Yamada, K., Chikazawa, Y., Ueno,

M., Teshirogi, I., Ono, T., and Ohtani, M. (2000) J. Med. Chem. 43,

1041–1044

12. McKew, J. C., Tam, S. Y., Lee, K. L., Chen, L., Thakker, P., Sum, F.-W.,

Behnke, M., Hu, B., and Clark, J. D. (2004) U. S. Patent 6,797,708

13. Lehr, M., and Ludwig, J. (2009) U.S. patent 7608633

14. Ludwig, J., Bovens, S., Brauch, C., Elfringhoff, A. S., and Lehr, M. (2006)

J. Med. Chem. 49, 2611–2620

26. Seno, K., Ohtani, M., Watanabe, F., and Tamauchi, H. (1998) Japan Patent

WO98/33797

27. Street, I. P., Lin, H. K., Laliberte´, F., Ghomashchi, F., Wang, Z., Perrier, H.,

Tremblay, N. M., Huang, Z., Weech, P. K., and Gelb, M. H. (1993) Bio-

chemistry 32, 5935–5940

15. Forster, L., Ludwig, J., Kaptur, M., Bovens, S., Elfringhoff, A. S., Holtfre-

rich, A., and Lehr, M. (2010) Bioorg. Med. Chem. 18, 945–952

16. Perisic, O., Fong, S., Lynch, D. E., Bycroft, M., and Williams, R. L. (1998)

J. Biol. Chem. 273, 1596–1604

17. Ball, A., Nielsen, R., Gelb, M. H., and Robinson, B. H. (1999) Proc. Natl. 28. Scott, D. L., White, S. P., Otwinowski, Z., Yuan, W., Gelb, M. H., and Sigler,

Acad. Sci. U.S.A. 96, 6637–6642

P. B. (1990) Science 250, 1541–1546

NOVEMBER 12, 2010•VOLUME 285•NUMBER 46

JOURNAL OF BIOLOGICAL CHEMISTRY 36111