progress in nucleic acid research and molecular biology [vol 67] - k. moldave (ap, 2001) ww

Structural analyses reveal that iPLA2~ contains unique structural features that include a serine lipase consensus motif GXSXG, a putative ATP-binding domain, an ankyrin-repeat domain, a

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The Molecular Biology of the

Group VIA Ca2+-Independent

Phospholipase A2

Z H O N G M I N MA 1 AND J O H N TURK

Division of Endocrinolof~y, Diabetes, and Metabolism

Department of Medicine Washington University School of Medicine

St Louis, Missouri 63110

I Introduction 2

II Classification and Nomenclature 3

iii Sequence and Structural Characteristics 4

A Lipase Consensus Motif GXSXG 5

B ATP-Binding Domain S C Ankyrin-Repeat Domain 10

D Bipartite Nuclei Localization Signal 14

E Caspase-3 Cleavage Site 15

F Proline-Rieh Region of Hnman Long Group VIA PLA2 lsoform 15

G Other Features 16

IV Gene Structure, Alternative Splicing, and Chromosomal Localization 17

V Tissue Distribution and Expression 20

VI Enzymology of Group VIA PLA2 20

A Phospholipase A2 and Phospholipase AI Aeti~dties of Group VIA PLA2 20 B Selectivity of Group VIA PLA2 for Phospholipids 20

C Lysophospholipase, PAF Aeetylhydrolase, and Transaeylase Activities of Group VIA PLA~ 21

VII Potential Celhdar Functions 22

A Signaling Function in Insulin-Secreting Cells 22

B Apoptosis 24

(2 Membrane Phospholipid Remodeling 25

D Membrane Homeostasis and Other Functions "26 VIII Future Perspectives 28

References 29

The group VIA PLA2 is a member of the PLAg superfamily This enzyme, which is cytosolie and Cag+-independent, has been designated iPLA2fl to distinguish it from another recently eloned Ca2+-independent PLA2 Features

of iPLA2/3 moleeular strueture offer some insight into possible eellular funetions

of the enzyme At least two catalytically active iPLAzfi/isoforms and additional

1To whom eorrespondenee should he addressed

Progress in Nucleic Acid Research Copyright O 2001 by Academic Press and Molecular Biology, Vo] 67 l All rights of reproduction m any fonn reserved

2 ZHONGMIN MA AND JOHN TURK

splicing variants are derived from a single gene that consists of at least 17 ex- ons located on human chromosome 22q13.1 Potential tumor suppressor genes also reside at or near this locus Structural analyses reveal that iPLA2~ contains unique structural features that include a serine lipase consensus motif (GXSXG),

a putative ATP-binding domain, an ankyrin-repeat domain, a caspase-3 cleav- age motif DVTD138y/N, a bipartite nuclear localization signal sequence, and

a proline-rich region in the human long isoform, iPLA2~ is widely expressed among mammalian tissues, with highest expression in testis and brain, iPLA2~ prefers to hydrolyze fatty acid at the sn-2 fatty acid substituent but also exhibits phospholipase AI, lysophospholipase, PAF acetylhydrolase, and transacylase ac- tivities, iPLA~/3 may participate in signaling, apoptosis, membrane phospholipid remodeling, membrane homeostasis, arachidonatc release, and exocytotic mem- brane fusion Structural features and the existence of multiple splicing variants ofiPLAg/~ suggest that iPLAg/3 may be subject to complex regulatory mechanisms that differ among cell types Further study of its regulation and interaction with other proteins may yield insight into how its structural features are related to its

f u n c t i o n © 2001 Academic Press

I Introduction

In response to cellular stimulation, membrane phospholipids are often hy- drolyzed to generate intraeellular and intercellular messengers Phospholipase

A-2 (PLA2) enzymes catalyze hydrolysis ofsn-2 fatty acid substituents from glyc-

erophospholipid substrates to yield a free fatty acid and a 2-1ysophospholipid (1) This group of enzymes has been intensively studied because they play cru- cial roles in diverse cellular responses, including phospholipid digestion and metabolism, host defense and signal transduction, and production of proinflam- matory mediators, such as prostaglandins and leukotrienes, through the release

of arachidonie acid (AA) from membrane phospholipids (2, 3) The lysophospho- lipid generated in PLA2 hydrolysis serves as a precursor for the proinflammatory molecule platelet-activating factor (PAF), and lysophosphatidie acid is a potent mitogen (4)

PLA2 enzymes are a rapidly growing superfamily of diverse enzymes that have been classified into at least 11 groups (5) Recent advances in DNA and protein databases that permit BLAST analyses and EST searches have permitted cloning of new PLA2 species This chapter summarizes the molecular biology

of a recently cloned intracellular Ca2+-independent PLA2 that has been clas- sified as group VIA PLA2 (5) and is designated iPLA2fl here to distinguish it from another recently cloned Ca2+-independent PLA2 (6) iPLA2/~ was first pu- rified from the nmrine P338D1 macrophage-like cells as an 80-kDa protein on sodium doeeeyl sulfate-polyaerylamide gel electrophoresis (SDS-PAGE) (7) The enzyme was subsequently isolated from chinese hamster ovary (CHO) cells

(8), which led to the cloning of its cDNAs from several sources (8-12) Analyses

GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 3

of its primary sequence have revealed structural characteristics that may pro- vide clues about the roles of the enzyme in cellular processes Determination of the structure of the human iPLA2~ gene has yielded insight into the geneses of multiple iPLA,2~ splice variants (11, 12), and the gene has been found to reside

in a chromosomal location that contains loci for genes associated with human diseases

II Classification and Nomenclature

Based on their dependence on Ca 2+ for their enzymatic activity, PLA2 en- zymes can be dMded into Ca'2+-dependent and Ca2+-independent classes The former includes several groups of secretory PLA2s (sPLA2), which require mil- limolar Ca '2+ concentrations for catalytic activity, and group IV Ca2+-dependent cytosolie PLA,2 isoenzymes (cPLA2~ and -fl), which require submieromolar Ca 2+ concentrations to associate with membrane substrates The Ca'2+-independent PLA2s appear to represent a diverse group of enzymes that can be further sub- divided into several categories: group VIA intraeellular Ca2+-independent PLA,2 (iPLA2fl) (8-12), membrane-associated Ca%-independent PLA.)(iPLA2F) (6), 61-kDa group IV cytosolic PLA2F (ePLA2F) (13, 14), and PAF ace@hydro- lases (1.5, 16) A common feature of these Ca2+-independent PLA.2s is the pres- ence of the lipase consensus motif GXSXG These enzymes exhibit no other similarities

iPLAafi was initially identified and purified from murine P388DI maerophage-like cells (7, 17) and classified as group VI PLA2 (1, 18) and sub- sequently as group VIA PLA,2 (5) In the remainder of this chapter, the group VIA PLA,~ will be designated iPLA.2fl for simplicity, unless otherwise indicated The eDNA encoding this enzyme was first cloned from CHO cells (8) and sub- sequently from other sources (9-12) iPLA2fi has two recognized enzymatically active isoforms (12) and exhibits lysophospholipase activity in addition to PLA2 activity (7, 8, 19) Sequence analyses reveal that iPLA.~fl contains several inter- esting structural features that may be related to its functions ill cells

Analyses of a predicted 40-kDa protein identified by the human genome project and a TBLASTN database search of GenBank led to the cloning of a novel Ca2+-independent, membrane-associated PLA.2 that has been designated iPLA.~ F (6) The deduced amino acid sequence fi'om this transcript showed no homology to known Ca2+-independent PLA9 enzymes except the putative ATP- binding and GXSXG lipase consensus motifs that also occur in iPLA2fl Both of these motifs also exist in a 40-kDa enzyme from potato with Ca'e+-independent phospholipase A2 activity (20, 21) that has been designated iPLA,)oe (6) The classification sehelne of Six and Dennis designates iPLA,~fl as group VIA and iPLA_gF as group VIB PLA2, respectively (5) iPLA2F also contains a C-terminal

peroxisomal targeting sequence (SKL) (22, 23) Because iPLA2F is tightly bound

to membrane fractions in cell homogenates, it may be that the major subcellular location of iPLA2F is in the peroxisomal matrix enclosed within the peroxisomal membrane (6)

By combined BLAST and EST database searches and 5'-RACE methods, a largely membrane-bound PLA2 with a calculated molecular mass of 60.9 kDa ho- mologous to ePLA20t (group IVA PLA2) was cloned (13, 14) This protein, which exhibits Ca'2+-independent PLA2 activity, has been designated cPLA2F (13, 14)

According to the scheme of Six and Dennis, this enzymes is classified as group VIC PLA2 (5) The deduced amino acid sequence indicates that the cPLA.2F protein lacks the C2 domain of cPLA2a, and accordingly has no dependence upon Ca ~+ for membrane association or catalytic activity This enzyme is thus a Ca'2+-independent PLA2 cPLA2F protein contains a pren~lation motif(-CCLA)

(24) at the C terminus (13) The isoprenoid precursor [ H]mevalonolactone is incorporated into the prenylation motif ofcPLA2F when expressed in COS cells, and the mutagenesis of CCLA to SSLA at the C terminus of cPLA_gF prevents the ['3H]mevalonolactone incorporation, suggesting that the consensus prenyla- tion site is indeed utilized This may account for the membrane localization of cPLA2F (13)

Platelet-activating factor (PAF) aeetylhydrolases are also Ca2+-independent PLA2 (16) PAF acetylhydrolases are structurally diverse isoenzymes that cat- alyze hydrolysis of the sn-2 aeyl group of choline glycerolipids containing an sn-1

alkyl ether linkage and a short-chain or oxidized sn-2 substituent (16) The clas- sification scheme of Six and Dennis (5) places these enzymes into two groups The group VII enzymes have molecular masses of 40-45 kDa and include both secreted isozymes found in plasma (group VIIA) (25) and intracellular, myris- toylated enzymes found in lung and kidney (group VIIB) (26) The group VIII enzymes are intraeeIlular, have molecular mass of 29-30 kDa, and are found in brain (16, 27)

III Sequence and Structural Characteristics

The iPLA2fl cDNAs have been cloned from several sources (8-12) Rodent iPLA2fi and the human short isoform of iPLA2fi eDNA species encode a sin- gle 752-amino acid protein with calculated molecular mass of about 85 kDa The long isoform of human iPLA2fi eDNA encodes an 807-amino acid protein which has a 55-amino acid residue insertion at position 395 (Fig 1) (11, 12) The iPLA2fl enzymes share no sequence similarity with other known PLA2 enzymes Among the consensus structural features of sPLA2 enzymes are a Ca2+-binding

loop with the typical glyclne-neh sequence Y ' -G-C-X-C-G-X-G-G-X-X-X-P (the number of amino acid residues is based on Type I PLA2) and the residue Asp 49, and an active site His 48 (28) Asp 49 is located adjacent to the catalytic

GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 ,5

Eight Ankyrin-repeats domain [ ] Bipartite nuclear localization signal

FIG 1 Schematic representation of the structure of iPLA:lfi The upper bar represents the rodent or human short isoform ofiPLA2fl, and the lower bar represents the human long isofnrm of iPLA2/~ The position of the eight an lg, r in repeats, the putative ATP-binding domain, the bipartite nuclear localization signal, the proline-rich region of HL-iPLA2/~, the caspase-3 cleavage site, and the lipase consensus motif are shown

His 4s, forming the so-called His/Asp dyad Mechanistic studies indicate that the sPLA.2 do not form a classic aeyl enzyme intermediate that is characteristic

of serine esterases Instead, they utilize the catalytic site His, assisted by Asp,

to polarize a bound water molecule that then attacks the substrate earbonyl group The Ca '2+ ion, bound in the conserved Ca2+-binding loop, stabilizes the transition state Serine esterases such as iPLA,2 employ a mechanism for catalysis that is different from that of sPLA2 enzymes The group IVA cytosolic Ca'2+-dependent PLA.2 (ePLA2ot) has a Ca2+-dependent lipid-binding (CaLB) domain at its N terminus that is responsible for transloeation of cPLA2 from cytosol to membranes in response to rises in cytosolic [Ca 2+] induced by ex-

homology with the C2 domains in proteins such as protein kinase C, GTPase- activating protein (GAP), synaptotagmin, and phospholipase C Such domains

not yet certain what t<actors govern association of iPLA0fi with membrane phos- pholipids, but this is a potentially important control point because iPLA2fl is a eytosolie protein in resting cells Analyses ofiPLA2/3 structural features may yield some clues about potential mechanisms for regulating membrane association of the enzyme

A Lipase Consensus Motif GXSXG

The amino acid sequence of iPLA2f contains a lipase consensus motif GXS465XG (Ser 619 in long isoform of iPLA2/3) (Fig 1) that is commonly found

an iPLA2/3 mutant protein expressed in CHO cells with an iPLA2/3 eDNA

containing a mutation a t Ser 465 to Ala completely abolished the catalytic ac- tivity of iPLA2/~, supporting the participation of Ser 465 in catalysis (8) In the case of cPLA2ot, mutation of Sel 2es to Ala at GLS22SGS, which is similar to but slightly different from the classical serine lipase consensus, also completely eliminates both PLA2 and lysophospholipase activities of ePLA2~ without sig-

ited by arachidonyl trifluoromethyl ketone (AATFMK, also referred to as

AACOCF3) and methyl arachidonyl fluorophosphonate (MAFP) (17, 18), per- haps because both the cPLA2a and iPLA2/~ appear to use a central Ser in similar catalytic mechanisms The activities of iPLA2/3 and cPLA2a are dif- ferentially affected by a bromoenol lactone (BEL) suicide substrate at a con- centration as low as 1 #M At such low concentrations, BEL inhibits iPLA2/~

covalently to iPLA2/3 (17), although the site of attachment has not been de- termined This covalent modification inactivates the enzyme irreversibly Iden- tification of the BEL binding site could be useful in relating iPLA2]3 struc- tural features to its catalytic activities A partial lipase consensus GPS'252GF also occurs in hamster iPLA2/~ (8) A similar sequence in cPLA2ot contains the active site serine, but site-directed mutagenesis Ser 2'52 to Ala in iPLA2/~ does not alter catalytic activity when the enzyme is expressed in CHO cells (8) The Ser 25'2 in hamster iPLA.2/~ is also not conserved in iPLA2]3 cloned from other species

cPLA2F (group IVC) contains a GvsS'2GS motif that also occurs in cPLA2~

(13, 14) Mutagenesis of Ser "22s, Arg a°°, Asp 549, or Arg 566 also abolishes cPLA,)c~

duces cPLA2F activity, indicating that Arg 54, Asp 3s5, and Arg 4°2 are required for

Analysis of the deduced amino acid sequence of iPLA2F (group VIB PLA2) revealed that this novel enzyme, like iPLAeJ3, also contains a Gxs4SaXG motif (6) This conserved sequence motif occurs in two translational variants with apparent molecular masses of 77 and 63 kDa These proteins may arise from use

is likely that the GXSXG consensus motif contains the active serine required for catalysis There is also a partial lipase consensus sequence (GDS2°3FY) in the deduced amino acid sequence of iPLA2F (6) This motif is probably included

in the 77-kDa protein but not in the 63-kDa protein based on the proposed translation initiation sites Further studies by site-directed mutagenesis will be required to determine whether the Gxs4S3XG or GDS2°3Fy sequences contain

an active site Ser that is required for catalysis by iPLA2F

lar isoform II (26) Group VIII includes the/3 and F subunits of intracellular

GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A,~ 7' isoform Ib (16, 37, 38) The cloning of a eDNA encoding the plasma form of PAF aeetylhydrolase was reported in 1995 (25), and the deduced amino acid sequence contained a GHS 273FG motif (Table I) Using site-directed mutage- nesis, Set 273 was found to be essential for catalysis Both Asp 296 and His 351 are also essential for catalytic activity (25) The orientation and spacing of these cat- alytic residues are consistent with an ot/fl hydrolase conformation that has been observed in other lipases (25) The deduced amino acid sequence ofisoform II

of PAF acetylhydrolases (group VIIB) exhibits 41% identity with that of plasma PAF acetylhydrolase (group VIIA) and also has a G H S F G consensus motif that occurs in plasma PAF aeetylhydrolase (group VIIA) The group VIIB enzyme

is inactivated by diisopropyl fluorophosphate consistent with involvement of a Ser residue in catalysis, and the substrate specificity of the group VIIB enzyme

is similar to that of plasma PAF aeetylhydrolase (39) The I3 and y subunits of isoform Ib (group VIII) exhibit 63.2% identity in deduced amino acid sequence overall There is 86% identity in the catalytic and PAF-receptor-homologous domains There is no overall homology with other PAF aeetylhydrolases (38)

This group of enzymes contains a GXSXV motif instead of GXSXG (Table I)

CtIARACTERISTICS OF THE CLONED Ca- -INDEPENDENT PHOSPI1OLIPASE A2

Name Location Source Size (kDa) Active site References iPLA2fl (group VIA PLA2) Intraeelhdar Islet, CHO, P338D, 85 and 88 GTSTG 8-12

B lymphoe~es Patatins and Patatin-like Intraeelhdar Potato tubes 40 GTSTG (5, 20, 21

(iPLA2a)

iPLA2F (group VIB PLA,2) Membrane Human heart and 88.5, 77, GVSTG 6

-associated skeletal nmscle and 63 ePLA2y (group IVC PLA,2) Iutracellular Brain EST 61 GVSGS 13, 14

PAF acetylhydrolase Ib, Intraeellular Brain 29 GDSMV 16, 27

F subunit (group VIIIB)

8 ZHONGMIN MA AND JOHN TURK indicate that the central serine in this motif participates in catalysis in these enzymes (Table I)

B ATP-Binding Domain

When the cloned rat islet iPLA2fi eDNA is transiently expressed in COS-7

or CHO cells, its actMty is stimulated about 2-4-fold by adenosine triphosphate (ATP) (9) This is similar to the effect of ATP on the iPLA2fl activity isolated from P388D1 macrophage-like cells (7) Furthermore, iPLA2fl overexpressed in

S frugiperda (Sfg) cells adsorbs to both ATP-agarose and ealmodulin-agarose matrices, and this can be exploited in affinity chromatographic purification of

the enzyme (40, 41) Recently, Yang et al (42) reported the purification of an

80-kDa Ca2+-independent PLA2 from rat brain using the same strategy Amino acid sequencing of several peptides from tryptie digests of the purified rat brain iPLA2 protein indicated that it corresponds to the iPLA2fl cloned from rat pan-

ereatie islets by Ma et al (9) These observations suggest that an ATP-binding

motif exists within the iPLA2fi molecule

Surveys of the protein kinase family reveal that their catalytic domains ex- hibit a highly conserved G-X-G-X-X-G sequence motif that appears to par-

tieipate in ATP binding (43) The consensus motif the G-X-G-X-X-G is also

found in many nueleotide binding proteins in addition to the protein kinases

(43, 44) Alignment of the iPLA2fl sequence with the ATP-binding domain of protein kinases revealed that the sequence from amino acid residue 431 to 457

of iPLA2fl exhibits similarity to the region of protein kinases involved in ATP

binding (Fig 2) A model for the ATP-binding site of the protein kinase v-src,

based on the three-dimensional structures from other nucleotide-binding pro- teins, shows that the G-X-G-X-X-G residues form an elbow around ATR with the first glyeine in contact with the ribose moiety and the second glyeine ly- ing near the terminal pyrophosphate Mutagenesis analyses of the ATP-binding site of cAMP-dependent protein kinase indicated that replacing the third Gly 5'5 had minimal effects on steady-state kinetic parameters, whereas replacement

of either Gly 5° or Gly '52 had major effects on both K,,, and kc~t values con- sistent with the predicted importance of the tip of the glyeine-rieh loop for

catalysis (45) A nearly invariant Val residue lies within subdomain I of v-src

(Fig g) and is located just two positions on the carboxyl-terminal side of the G-X-G-X-X-G consensus This residue may contribute to the positioning of con-

served glyeines (43) In addition, several amino acid residues in subdomain II of v-src, such as alanine '29s, lysine 3°°, and leucine 3°2, are represented at analogous

positions in the sequence ofiPLA2fi Lys 3°° in v-src appears to be directly involved

in the phospho-transfer reaction (46) Since iPLA2CJ adsorbs to ATP-agarose and

is desorbed by ATR it is possible that ATP directly binds to the ATP-binding do- main of iPLA.2JL Sueh binding may modulate the function of iPLA~, although

GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 9

at the top of the alignment Residues conserved in all or nearly dl protein kinases are enclosed in black boxes

this possibility has not been verified experimentally Interestingly, analyses of hmnan iPLA2fl gene strneture revealed that the ATP-binding domain and lipase

suggests that these two domains eould interact to regulate the iPLA2fi fimetion ATP could affect iPLA2fl function by at least three mechanisms First, ATP could bind to the ATP-binding domain of iPLA2fi to stimulate its intrinsie enzymatic aetivity Second, ATP could induce association of iPLA2fl monomers to form muhimerie aggregates Third, ATP could stimulate transloeation ofiPLA2fl from eytosol to membranes, where its substrates are located In many eases, iPLA2fi activity has been found to be stimulated by ATE as well as other di- and triphos-

to stimulate activity of purified or partially purified iPLA2fl Interestingly, activity

affected by ATR but activity of the long isoform of human iPLA2fi increases about

ATE stabilizes iPLA2fl and protects it from denaturation In cells, iPLA2fi might interact with other proteins that negatively regulate its activity ATP may induce dissociation of iPLA2fi from such negative modulators and result in the activa- tion of iPLA2fi Nevertheless, the faet that iPLA2fi can bind to an ATP-affinity column suggests that ATP might interact with iPLA.~fi in vivo; this possibility

10 ZHONGMIN MA AND JOHN TURK iPLA2F, like iPLA2fl, contains an ATP-binding motif at amino acid sequence 449GGGTRGW457 All glycines in this motif are conserved between iPLA2/3 and iPLA2F, as is the Val residue (Ile in human iPLA.2fl) two positions after Gly 4'55

In contrast to iPLA2fi, both the ATP-binding motif and the lipase consensus sequence are encoded by two adjacent exons in the iPLA2F gene (6)

(47) reported an "~33-residue repeating motif in the sequence of two yeast cell- cycle regulators, SWi6 and cdcl0, and in the Notch and LIN-12 developmental

quently, the discovery of 24 copies of this sequence in the cytoskeletal protein

repeat-containing proteins perform a wide variety of biological functions and have been detected in organisms ranging from viruses to humans The motif has now been recognized in >400 proteins, and the number of repeats within any

ankyrin proteins that link integral membrane proteins to cytoskeletal elements

GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 11

(48, 50), developmental regulators in Drosophila and C elegans, cell-cycle con- trol proteins in yeast, transcriptional factors, toxins, and viral proteins (Table II)

(49-5l) Recently, a novel family of postsynaptic-density proteins, Shank, has been reported to contain seven ankyrin repeats at the N terminus (52) These molecules are present in the nucleus, cytoplasm, and membranes, as well as the extracellular milieu (49)

The role of ankyrin repeats in mediating protein-protein interactions is well documented, and their presence is often interpreted as an indicator of a sin> ilar filnction in otherwise uncharacterized systems The presence of ankyrin repeats in iPLA2fl suggests that intra- or intermolecular protein-protein interac- tions may regulate its function Ankyrin is a linker molecule between membrane and cytoskeletal proteins (50) Its C-terminal domain binds to cytoskeletal pro- teins such as spectrin and tubulin, while its N-terminal 89-kDa ankyrin-repeat domain binds to integral membrane proteins, such as ion channels and cell adhesion/signaling molecules (Table III) (50) The ion channels that ankyrin- repeat domains bind include the Na+,K+-ATPase of renal basolateral men> branes, a renal amiloride-sensitive Na + channel, the red cell anion exchanger, a cerebellar inositol triphosphate receptor, and voltage-dependent Na + channel in myelinated neurons (50) The binding of ankyrin repeats to integral membrane proteins raises two possible roles for ankyrin repeats in the iPLA2 protein

TABLE II PROTEINS WITII ANKYRIN REPEATS

SWI4, SWI6" (yeast)

Latrotoxin/latroinsectotoxin (black widow spider) 19

Vaeeina 32-1d (vaeeinia vires) 3 Cowpox HRP (cowpox virus)

Fowlpox 47-kd (fbwlpox virus)

Group VI PLA2 (iPLA,2fl) 8

12 ZHONGMIN MA AND JOHN TURK

TABLE III INTEGRAL MEMBRANE PROTEINS THAT BIND TO ANKYRIN REPEAT

Ion channels

Bed cell anion exchanger (AE1)

AE1 (kidney isofbrm)

IP3 receptor (270 kDa)

Cell adhesion/signaling molecules

Lymphocyte adhesion antigen CD44

(putative hyaluronic acid receptor)

GP116 (CD44-1ike endothelial protein)

Ankyrin-binding glycoprotein 205 (AB-GP205)

Erythroid ankyrin (Ankl) Epithelial anlgwin(s) Brain ankyrin (Ank2) Brain ankyrin (Ank2)

Epithelial ankyrin(s) Epithelial ankyrin(s) Lymphocyte ankyrin Erythroid ankyrin (Ankl) Brain ankyrin (Ank2)

(1) Although cPLA2 has a CaLB domain that shares homology with the C2 domain in the conventional isoforms of PKC, phospholipase CF, synaptotamin, and so forth (29, 30), iPLA,213 has no similar sequence that might mediate associ- ation with membrane phospholipids The translocation iPLA213 from cytosol to membrane is likely to be important for its function in cells because its phospho- lipid substrates are in membranes It is possible that iPLA213 is able to associate with membrane phospholipids through the binding of its ankyrin-repeat domain

to integral membrane proteins Recently, we demonstrated that iPLA213 can be induced to associate with a membrane fraction of INS-1 insulinoma cells upon cell stimulation (manuscript in preparation) The long isoform of human iPLA.213 was also found to be associated with membrane when overexpressed in COS-7 cells (53) Interestingly, Western blot analysis of iPLA213 in adult rat ventrieular myocytes revealed that full-length iPLA213 is detected only in the membrane fraction (54) (2) Regulation of ionic fluxes is critical to the function of pan- creatic islet 13 cells, neurons, and muscle cells, and proteins containing ankyrin repeats associate with a number of ion transporters (Table III) Arachidonic acid (AA) and other polyunsaturated fatty acids affect many ion channels (55) The concentrations of free AA and other polyunsaturated acids within cells are very low It is possible that, upon activation, iPLA213 might transloeate to associate with membrane ion channels via its ankyrin-repeat domain Hydrolysis of AA and other polyunsaturated acids from phospholipids catalyzed by iPLA213 could then yield high regional concentrations of polyunsaturated acids that could affect ion channel functions It was reported that Na + flux induced by angiotension II

GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 13 required the activation of a BEL-sensitive iPLA2 in LLC-PK1 cells (56), consis- tent with the possibility that iPLAefl might interact with ion channels

The first three-dimensional structure of an ankyrin-repeat-containing mol- ecule was determined by X-ray crystal structural analysis of 53BP2 bound to the p53 cell-cycle tumor suppressor (57) Subsequently, the ankyrin-repeat struc- tures of several proteins, including cyclin-dependent kinase (CDK) inhibitors, GABPa/fl, myotrophin, IKBa-NF4cB, Swi6, and PYK2, were determined by X-ray and/or NMR methods (Table II) (58, 59) The ankyrin repeat consists of pairs of antiparallel a helices stacked side-by-side and connected by a series

of intervening fl-hairpin motifs The extended fl sheet projects away from the helical pairs almost at right angles to them, resulting in a characteristic L-shaped cross section This assembled structure has been likened to a cupped hand: the

fi hairpins form the fingers, and the concave, inner surface of the anlcyrin groove, which is made up of solvent-exposed residues from the a-helical bundle, Ibrms the palm (,58, 59)

The crystal structure analyses revealed that ankyrin repeats play a critical role in forming fimctional complexes It has been reported that iPLA2 exists as a multimerie complex of 270-350 kDa (8) Deletion of the N-terminal 150-amino acid residues plus the eight ankyrin repeats of iPLA,2fl results in loss of catalytic actMty (8) This could indicate that the ankyrin-repeat domain is important for formation of a multimeric complex of iPLA2fl and that this is the catalytic active ibrm A recent report by Larsson et al (11) demonstrated that cells cotrans- feeted with full-length iPLA2fl and with a deletion mutant that contained the ankyrin-repeat domain but not the catalytic domain (ankyrin-iPLA2-1) exhib- ited decreased activity compared to cells transfected with full-length iPLA2fl alone This suggests that multimerie complexes of iPLA,2/? represent the func- tional forms The fact that there is residual iPLA2fi activity in the cotransfectants could reflect the existence of a subpopulation of homomultimeric complexes Alternatively, heteromultimeric complexes might retain some activity In either case, these results suggest that the ankyrin-repeat domain participates in forma- tion of fimctional of iPLA2/? complexes

Inflamed tissue expresses a host of proteins that are not normally expressed Many of the genes encoding such proteins are activated by NF-x B, a transcrip- tional factor that is normally in an inactive form in the cytoplasm NFKB can be activated by a variety of proinflammatory and noxious stimuli (60, 61) Under resting conditions, NF-gB is tightly associated with IKBs, a class of specific in- hibitory proteins that prevent nuclear transloeation and DNA binding of NF-K B The structural hallmark of the various IicB proteins is an ankyrin-repeat domain containing six or seven closely adjacent repeats (6,2) Crystal structure analyses

of the bc B/p65/p50 complex indicate a fundamental role of ankyrin repeats in the formation of inactive IKB-NF4cB complexes The ankyrin-repeat domain

14 ZHONGMIN MA AND JOHN TURK

of IK Ba forms a slightly bent cylinder with five loops protruding from the packed arrangement of stacked a helices The loops between the repeats contain residues that specifically recognize NF-KB The appearance of IxB in the NF-xB com- plex is reminiscent of a backbone lying between two lungs, where each ankyrin repeat is a vertebra (62-64) Interestingly, ankyrin repeats 1 and 2 interact with sequences encompassing the nuclear localization signal (NLS) of p65 (62) Re- peats 3 to 5 bind tightly over a large surface to the C-terminal Ig-fike domains

of both p50 and p65 Rel homology domains (RHDs) (62) Structural analyses ofiPLA2fi reveal that the enzyme contains a bipartite nuclear localization signal sequence (NLS), as discussed below This raises the possibility that the ankyrin- repeat domain might bind the NLS of iPLA.2/~ intramolecularly to regulate the translocation of iPLA2/3 from cytoplasm to nucleus This would be analogous to the binding of the ankyrin repeats of IKBot to the NLS of NF-KB in IxB-NF-KB complexes

D Bipartite Nuclei Localization Signal

Using the ExPaSy (Expert Protein Analysis System) profileScan to scan the iPLA2/3 amino acid sequence against protein profile databases (including PROSITE), only two domains in iPLA2J3 yielded significant matches with con- sensus domains in the databases These are the ankyrin-repeat domain and a bipartite nuclear localization signal sequence (Fig 1) The two best defined NLSs are that of SV40 large T antigen (SV40TAg), which has a simple basic NLS (PKKKRKV) sequence (65), and that of nncleoplasmin (66), which has

a bipartite basic NLS sequence (KRPAATKKAGQAKKKK), in which two in- terdependent clusters of basic amino acids are separated by a flexible spacer

(66, 67) Usually, the first two adjacent basic amino acids (Arg or Lys) in this sequence are followed by a spacer region of any ten residues and at least three basic residues (Arg or Lys) in the five positions after the spacer region (67) Pro- teins containing NLS are transported into the nucleus in a process that involves NLS binding to the nuclear import receptor importin/~ together with members

of the importin a family (68) The sequence511KREFGEHTKMTDV KKPK 52'

of rodent iPLA2fl (or 565KREFGEHTKMTDV ff-KPK TM of the long is~orm of human iPLA2/3) perfectly matches the bipartite nuclear localization signal in nu- cleoplasmin (66), suggesting that iPLA2fl might have the ability to translocate to the nucleus Recently, we found that iPLA2fl protein can be identified immuno- chemically in nuclei isolated from iPLA2fl-overexpressing IN S-1 insulinoma cells (manuscript in preparation) We found that a small percentage of iPLA2fi was detected in nuclei compared with cytosol under resting conditions We are ex- ploring the possibility that the translocation ofiPLA2fl to nuclei can be stimulated under some conditions As suggested by crystal structures of IKB-NF-xB com- plexes (63, 64), we propose that the ankyrin-repeat domain of iPLA2fi might

GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 15

regulate nuclear translocation through its binding with the NLS of iPLA2fi in- tramolecularly The ankyrin repeats might form a slightly bent cylinder like those

of IxBce The NLS might fold back to contact the ankyrin-repeat domain and thereby block the NLS of iPLA2fl and prevent recognition by nuclear import receptors Upon stimulation, modulatory factors might interact with the ankyrin- repeat domain to release the NLS of iPLA0fi and lead to nuclear translocation

E Caspase-3 Cleavage Site

Recently, Atsumi et al (69, 70) reported that treatment of human promono- eytie U937 cells with apoptosis-indncing agents, such as anti-Fas antibody or TNFa/cyeloheximide (CHX), was accompanied by a time-dependent increase

in [3H]araehidonie acid (AA) release from prelabeled cells The time-dependent [3H]AA release paralleled the accumulation of apoptotie cells By immunoblot- ting analyses of U937 cells with anti-iPLA2j~ antibody, this group observed that, in addition to an intact 85-kDa iPLA2J? protein, another immunoreaetive band with

an estimated molecular mass of 70 kDa became visible 6-12 h after treatment with TNFot/CHX During this time period, TNFo~/CHX-indueed AA release and easpase-3 activity increased significantly (70), suggesting that the 70-kDa immunoreactive band might be produced by caspase-3 action Indeed, a po-

ls3 tential caspase-3 cleavage site, DXXDIX (71), occurs in iPLAafl around Asp ', which is located near the N-terminal end of the first ankyrin repeat Moreover;

ifiPLA2fl is cleaved at this site (DVTD Y in humans, DVTD N in rodents), the predicted size of the resulting C-terminal fragment would be consistent with the size of the cleaved fragment observed in this study (70) Caspase-3, one of the key executioners of apoptosis, participates in proteolytic cleavage of many key proteins, such as the nuclear enzyme poly(ADP-ribose} polymerase (PARP), during apoptosis ( 72} A survey of many protein substrates of easpase-3 indicates that each contains a common cleavage motif DXXDSX (71) The fact that iPLA2fl is a substrate for caspase-3 was further confirmed by eotransfection ofcaspase-3 and iPLA2fi Cleavage at Asp is'3 resulted in the activation ofiPLA2fi activity {70} cPLA2ol also contains a caspase-3 cleavage motif DELD5225A, and cleavage of cPLA2a at Asp 5~2 leads to inactivation (69), snggesting that cPLA2a and iPLA2~ activities are differentially modulated in apoptosis

F Proline-Rich Region of Human Long

Group VIA PLA2 Isoform

Cloning of human islet iPLA2fl eDNA species from pancreatic islets and insulinoma cells (12) revealed two isoforms of different lengths (Fig 1) The short iPLA2/3 isoform (SH-iPLA,2fi) corresponds to rodent iPLA.2]~, and the long iPLA2/3 isoform (LH-iPLA2fl) corresponds to that cloned from human B-lymphocyte-derived cell lines (11) The amino acid sequence for the long

16 ZHONGMIN MA AND JOHN TURK

LH-iPLA2~

P H N~N G ] H I L I Q ~ P J M ~ P I P [H P GIH Y ~ V H ~ A Smad4

p x ¢ _G ¢ P Q n x ¢ ¢ I:! !t P F_ x , 1~ ¢ Q_p _p ¢ ¢ S N L ¢ Q Consensus Fie, 4 Alignment of proline-rich region of LH-iPLA2 with the proline-rich middle linker domains of the Smad proteins DAF-3 and Smad4 Identical residues are enclosed in black boxes and conservative changes are enclosed in open boxes In the consensus sequence, amino acid residues that are identical between at least two of the three sequences are indicated by underlined capihdized letters Conservative changes are based on functional groups of amino acids Basic residues (H, K, and R) by/~; hydrophobie residues (A, F, I, L, M, P, V, and W) by qb; polar residues (C, G, N, Q,

S, T, and Y) by n Positions at which there is no similarity are denoted by dots [Reprinted with permission from Z Ma, X Wang, W Nowatzke, S Ramanadham, and J Turk, J Biol Chem 274, 9607-9616 (1999).]

isoform differs from the short isoform by the presence of a 54-amino acid in- sert in the region of the eighth ankyrin repeat This insert corresponds exactly

to the amino acid sequence encoded by exon 8 of the human iPLA2fl gene

(12, 53) This insert is proline-rich, and a BLAST search revealed similarities

to the proline-rich middle linker domain of the DAF-3 Smad protein from

C elegans (73), which is most closely related to mammalian Smad4 (Fig 4)

(74) Smad4 is a Mad-related protein and has been identified as the product of the tumor suppressor gene dpc4 This gene is deleted or mutated in a propor- tion of human pancreatic (75), breast, ovarian (76), and co!orectal tumors (77)

The tumor suppressor activity of Smad4 is probably attributable to its participa- tion in the signaling pathway of a family of cytokines that includes TGF-fl (78)

The proline-rich middle linker region of Smad4 shares a PXsPXsHHPX12NX4Q motif with the corresponding region of DAF-3 and the proline-rich region in the long human iPLA2fl isoform The Smad4 middle linker domain mediates pro- tein interactions with signaling partners (74), is located near the center of the protein, and separates an N-terminal MH1 domain with DNA binding activity from a C-terminal MH2 domain with transcriptional activity (79) The proline- rich region in the long iPLA2fl isoform is also located near the center of the protein and separates an N-terminal domain with protein binding activity from

a C-terminal catalytic domain (12) Smad proteins participate in controlling cell proliferation and apoptosis and form heterooligomers with signaling partners, via the proline-rich middle linker domain in the case of Smad4 (79)

G Other Features

Another feature of iPLA2/8 is its ability to bind calmodulin, a regulatory pro- tein involved in a variety of cellular calcium-dependent signaling pathways Both iPLA~fl expressed in Sf9 cells from the rat eDNA and native iPLA2fl in rat brain

GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 17 can be purified by ealmodulin-affinity column (41, 42) These results suggest that iPLA~2~ is able to bind calmodulin in a Ca2+-dependent manner Removal

of Ca 2+ leads to the dissociation of iPLA2fi from ealmodulin-affinity matrices

In the presence of Ca ")+, the aetivi~ of iPLA2fi is inhibited by calmodulin ill

a concentration-dependent manner (41), suggesting that Ca '2+ and ealmodulin negatively regulate iPLA2~ activity Cahnodulin is a protein capable of recog- nizing positively charged, amphiphilie oe-helical peptides rather than a clearly defined amino acid sequence motif (80); thus, it is relatively difficult to identify cahnodulin-binding domains from sequence analyses The amino acid sequences

of a number of cahnodulin-binding proteins have been determined, and in many cases the loeations of the binding domains have been mapped by deletion nm- tagenesis or chemical methods (80) Similar studies might permit identification

of the eahnodulin-binding domain of iPLA2~

Phosphorylation is an important posttranslational modification for regulat- ing the function of proteins To date, no phosphorylation of iPLA2~ has been reported, although PROSCAN results indicate that iPLA.2~ contains eonsensns phosphorylation sites for calcium/calmodulin-dependent protein kinase II, pro- tein kinase A, protein kinase C, protein kinase G, and casein kinase I!

IV Gene Structure, Alternative Splicing, and

Chromosomal Localization

Recently, we reported the cloning of the human iPLA2~ gene by screening

a human Lambda FIX II genomic library and determination of its structure

by combining sequencing and PCR approaches (12) Subsequently, Larsson

et al (53) reported the analysis of human iPLA2fi gene from two genomie clones (H $228A9 and H $447C4, accession numbers AL022322 and AL021977, Sanger Center, Hinxton, Cambridgeshire CB10 1SA, UK) The human iPLA2fi gene spans about 70 kb and consists of at least 17 exons, ranging from 74 to

811 bp in size, and 16 introns, ranging from 0.2 kb to 23 kb (Fig 5) The 5'-untranslated region was identified as exon la and part of lb Exon la is con- tained in done HS447C4, while exon lb and the rest of the exons are contained

in clone HS228A9 The translational stop codon of iPLA2~, the 3'-untranslated region, and the polyadenylation signal were located in exon 16 Analysis of the exon/intron boundary sequences indicated that the 5'-donor and 3~-aceeptor sequences at splicing sites conform to the generally recognized consensus se- quences (12, 53)

Human islets express mRNA speeies encoding two iPLA2]~ isoforms, as do human U937 promonocytie cells (12) The 162-bp in-frame insertion in the

GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 19 eighth ankyrin repeat of S H-iPLA2/3 corresponds exactly to exon 8 of the human iPLA2/~ gene This indicates that mRNA encoding the SH-iPLA2/3 isoform arises

variants of human iPLA2/3 have been identified from EST clones and reflect insertions of 52, 53, and 168 bp, respectively, that do not occur in the transcripts encoding LH- and S H-iPLA2/~ isoforms (11) Indeed, these insertions arise from introns and are designated E8b, E9b, and E13a, accordingly Analyses of the exon/intron boundary sequences of these alternative-splice sites demonstrate

alternative splicing events may yield three putative transcripts, as elucidated in Fig 5 These putative transcripts contain a polyadenylation signal encoded by exon 16 and therefore acquire a poly(A) tail required for translational compe- tence The iPLA2-2 transcript includes E13a and encodes a C- terminally trun- cated protein due to the introduction of a premature translational stop codon x~thin E13a This variant retains the GXSXG sequence The ankyrin-iPLAe-1 transcript includes E9a and encodes a C-terminally trnncated protein due to the introduction of a premature translational stop eodon within Ega The tran- script ankyrin-iPLA2-2 results from skipping of exon 2 and inclusion of ESa and E9a This transcript encodes a truncated protein that has a deletion close to the

the E9a Neither variant ankyrin-iPLA2-1 nor -2 contains GXSXG sequence, and both are catalytic inactive

Since iPLA2/~ cDNAs cloned from rodents are similar to human SH-iPLA2¢I

(8-10, 12) a question of interest is whether a long iPLA2/~ isoform also exists

taining a sequence corresponding to exon 8 of the hmnan iPLA2/~ gene from the total RNA of rat vascular smooth muscle cells and concluded that the pres- ence or absence of the exon 8 is a tissue-specific and not a species-specific feature

We have mapped the human iPLA2/~ gene to chromosome 22q13.1 us-

clones HS228A9 and HS447C4 represent the DNA fragments from chromo-

Allelic losses of chromosome arm 22q are frequently observed in human menin- giomas and carcinomas of the colon, ovary, and breast Recent studies of loss

of heterozygosity (LOH) in human breast carcinomas revealed that 40-66% of these tumors were associated with LOH on chromosome 22q13.1, suggesting that one or more tumor suppressor genes associated with breast cancer reside

also indicated the possibility that tumor suppressor genes reside on human chro-

a candidate for a tumor suppressor gene located on chromosome 22q13.1

20 ZHONGMIN MA AND JOHN TURK

V Tissue Distribution and Expression

The mRNA encoding iPLA2fl has been found in all human, mouse, and rat tissues studied, with different relative abundance (8-12, 53) The tissue distri- bution in rodents has been examined by Northern blot analysis using mouse/rat multiple tissue blot (8) The data obtained from this study revealed that a single 3.2-kb iPLA2fl transcript could be identified in all examined tissues, with high- est expression in testis followed by liver and kidney Analyses of human tissues identified multiple bands of different sizes (53) Among these, a 3.2-kb band, which might represent the full-length human iPLA,2fl transcripts, was abun- dantly expressed in testis, brain, spinal cord, and thyroid On the other hand, the 3.2-kb band may contain multiple transcripts that include alternative splicing variants because the "-~200-bp differences among these variants are difficult to distinguish by Northern blots

Characterization of the regional distribution of various iPLA2fi enzymes in organs has been reported (42, 85) Although several of groups of PLA,2 are ex- pressed in rat brain, Ca2+-independent PLA2 activity accounts for the dominant PLA2 activity in all brain regions, including cerebral cortex, cerebellum, hip- pocampus, hypothalamus, and striatum (42) Northern blot analyses indicated that mRNA species of iPLA2fl, iPLA,2F, and cPLA2F are expressed in brain

(42) Because iPLA2F and cPLA2F are largely associated with membranes, the Ca2+-independent PLA2 activity measured in the cytosolic fraction of each brain region probably represents the iPLA2fl activity (42)

VI Enzymology of Group VIA PLA2

A Phospholipase A2 and Phospholipase A1 Activities

of Group VIA PLA2

Enzymatic activities of iPLA2fl have been characterized with the protein purified from cultured cells (7, 8) and tissue (42) and with recombinant enzyme expressed from iPLA2fl eDNA in various host cells (40, 41 ) Using singly labeled

(1-pa mitoyl-2-[1- C]palmitoyl-sn-glycerol-3-phosphorylcholine) and doubly labeled substrates (1,2-[1-14C]palmitoyl-sn-glycerol-3-phosphorylcholine), pu- rified iPLA2fl has been demonstrated to exhibit both phospholipase A2 and A1 activities The enzyme preferentially hydrolyzes sn-2 over sn-1 fatty acid sub- stituents by a factor of 5 for 1,2-dipalmitoyl phosphatidylcholine (PC) in mixed micelles (7, 8)

B Selectivity of Group VIA PLA2 for Phospholipids

The fatty acid selectivity of iPLA2fl was evaluated by using different sub- strates When purified iPLA2fl from P388D1 macrophage-like cells was assayed

GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A:~ 21 using mixed micelles of 100 #M phospholipid and 400/zM Triton X-100, the enzyme showed no preference for either sn-2 arachidonic acid- or sn-1 alkyl ether-containing phospholipids (7) Under the same conditions, purified rat brain iPLA2/~ revealed the following substrate preference toward the fatty acid chain in the sn-2 position of PC: lionleoyl > palmit@ > oleoyl > arachidonoyl

(42) In the case ofiPLA2fl purified from CHO cells (8), the enzyme also failed

to exhibit any significant preference for particular fatty acid sn-2 substituents, although different rates of hydrolysis of unsaturated t~atty acids were observed

in the assay with different Triton X-100 concentrations When the activity assay was performed with 50 #M or 500 #M Triton X-100, different hydrolysis rates were observed with some substrates The overall rates of hydrolysis were, at least for some substrates, sensitive to lipid presentation, e.g., 1,2-dipalmitoyl PC was hydrolyzed four times more rapidly when substrate was dispersed in 500

vs 50 #M Triton, whereas 1-hexadecyl-2-arachidonyl-PC was hydrolyzed eight times more rapidly when substrate was presented in 50 vs 500 #M Triton These findings indicate that the apparent fatty acid selectivity ofiPLA.~fl depends upon substrate presentation (8)

Rat brain iPLA2fl has been reported to hydrolyze PC substrates with an sn-2

linoleate residue five times more rapidly these with an sn-2 arachidonate sub- stituent (42) Yang et al (42) have proposed that iPLA2fl could be an important enzyme in linoleate metabolism

Purified recombinant iPLA2/~ exhibits little preference between substrates containing choline vs ethanolamine head groups (7, 8, 40), although choline substrates are hydrolyzed more rapidly than other head-group classes under some assay conditions (8) Purified rat brain iPLA2fl also showed no significant head-group preference (42)

C Lysophospholipase, PAF Acetylhydrolase,

and Transacylase Activities ol" Group VIA PLA2

Purified iPLA2fl from 388D1 cells and recombinant iPLA2fl expressed from the CHO cell cDNA exhibited detectable lysophospholipase activity in a Triton X-100 mixed-micelle assay (7, 8) The kinetic studies byWolfand Gross (40) using the purified recombinant iPLA2fl overexpressed in Sf9 cells from CHO cDNA using L-or 1-palmitoyl-2- [ 1-14C]arachidonyl-PC as substrate demonstrated that the recombinant protein exhibited calcium-independent phospholipase A1/A2 and lysophospholipase activities at similar levels when assayed in the absence

of Triton X-100 Assay conditions therefore have an important impact on the apparent expression of various lipase activities of iPLAefl With the combina- tion of 50 #M Triton X-100 and 50% glycerol, the lysophospholipase activity

of iPLA2fl appeared equivalent to its PLA2 activity (19) The iPLA2fl activity varied with Triton-X 100 concentration, and optimal activity was observed at a Triton/phospholipid molar ratio of 4:1 (7)

22 ZHONGMIN MA AND JOHN TURK The sn-1 ether-linked substrate PAF can be hydrolyzed by iPLA2/3 in assays employing 500/zM or 50/zM Triton X-100, indicating that the phospholipase A2 activity of iPLA2/3 is not restricted to long-chain fatty acids (7, 8) The ob- served PAF acetylhydrolase activity was about 5% of the corresponding to the iPLA2 activity (7, 8) Like the lysophospholipase activity of iPLA2/3, the ap- parent PAF acetylhydrolase activity was also influenced by assay conditions, suggesting that the enzyme activity is strongly dependent on substrate presen- tation (19) The iPLA2/3 also exhibited both lysophospholipid/transacylase and phospholipid/transacylase activities that were susceptible to inhibition by BEL (19)

VII Potential Cellular Functions

A Signaling Function in Insulin-Secreting Cells

Stimulation of islets with glucose leads to insulin secretion and hydrolysis

of arachidonate from/3-cell membrane phospholipids (86, 87) Nonesterified arachidonate may participate as a second messenger in glucose-stimulated in- sulin secretion (see Refs 88-91) The mechanism of glucose-stimulated arachi- donate release from the membrane phospholipids is incompletely understood Many studies (92-101) have demonstrated that PLAz activation is involved

in insulin secretion since glucose and other secretagogues such as carbachol and CCK-8 stimulate PLA2 activation in islets Inhibition of PLA2 activities suppresses insulin secretion, release of AA, and accumulation of lysophospho- lipids Recent studies (102, 103) in insulin-resistant mice indicate that islet P LA2 activation is potentiated in these animals, providing evidence that islet PLA2 ac- tivation occurs in hyperinsulinemic mice Studies in isolated islets, insulinoma cells, and whole animals thus suggest that activation of PLA,2 is involved in secretagogue-induced insulin secretion

Several PLA2s have been identified in islets (9, 104-107) with various sen- sitivities to PLA2 inhibitors Any individual or multiple PLA2 enzymes might play signaling roles in islet/3 cells The suicide substrate BEL inhibits iPLA2/3 at concentrations that do not inhibit Ca2+-dependent sPLA2s or cPLA2 activities

(17, 35) BEL is thus a useful tool for distinguishing iPLA2/3 from other PLA2s

(18), although BEL also inhibits the MgZ+-dependent phosphatidic acid phos- phohydrolase (PAPH-1) (108) and iPLA2F (group VIB PLA2) (6) Interestingly, glucose-induced release of arachidonate from islets requires glucose to be me- tabolized ( 88, 109, 11 O) but can occur without calcium influx (86, 88), suggesting that the PLA2 responsible for AA release in islet/3 cells may be activated by glu- cose metabolism and is Ca2+-independent Treatment of isolated islets or insuli- noma cells with BE L suppresses both hydrolysis of arachidonate from membrane phospholipids and glucose-stimulated insulin secretion (111-116), suggesting

GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 23 that iPLA2 may be responsible for glucose-stimulated AA release These obser- vations have led to the hypothesis that iPLA2 in fi cells may participate in glucose- stimulated insulin secretion by releasing AA upon glucose stimulation (88)

Characterization of islet iPLA2 activity led to the cloning of iPLA2fi from rat and human islets (9, 12) BEL also inhibits the Mg2+-dependent PAPH-1 activ-

ity (108) To circumvent this problem, Balsinde et al (117) have used antisense oligonucleotides to suppress iPLA2~ expression in P388D1 cells; however, sim- ilar approaches have not been successful in insulinoma cells, perhaps because

of the high level of iPLA2fl expression in these cells (118) Nevertheless, the PAPH-1 inhibitor propranolol failed to suppress glucose-induced arachidonate release, as measured by isotope dilution gas chromatography/mass spectrometry (GC/MS), from isolated islets under conditions where such release was effec- tively suppressed by BEL (118) This indicates that PAPH-1 is not the BEL- sensitive target involved in glucose-stimulated arachidonate release and insulin secretion, but that iPLA2fl might be Substantial evidence indicates that PLA2

is involved in secretagogue-induced insulin secretion and AA release, and the studies with BEL suggest that iPLA2fl participates in these processes In order

to examine the role of iPLA2fi further, we have achieved stable overexpression

of iPLAefl in INS-1 cells, an insulinoma cell line (119) We found that iPLA2fl- overexpressing INS-1 cells exhibit increased secretory responses to insulin see- retagogues compared to parent IN S-1 cells or INS-1 cells transfected with vector only (119a) This enhanced insulin secretory response is inhibited by BEL but not by propranolol These data support the hypothesis that iPLA2/~ participates

in glucose-stimulated insulin secretion Islet PLA2 activation also has been re- ported to be involved in insulin secretion induced by cholecystokinin-8 (CCK-8)

(95, 98) CCK-8 was found to induce accumulation of lysophosphatidylcholine (LPC) and AA in a Ca2+-independent manner Inhibition of islet iPLA2 activity with BEL reduced CCK-8-induced AA release and insulin secretion, suggest- ing that iPLA2 contributes to the insulinotropie action of eholecystokinin-8 in rat islets (99)

It is also possible that PLA2 enzymes in addition to the group VIA PLA2s are also involved in insulin secretion Our hypothesis is that secretagogue-induced activation of iPLA2 in islet fl ceils results in accumulation of nonesterified AA and that this amplifies the glucose-induced rise in cytosolic [Ca2+] This could result in activation of cPLA2, which is also expressed by islet fl cells, and cPLA2 could further amplify the release of AA Islets also express group IB sPLA2 in their secretory granules, and this enzyme might participate in the process of fusion of insulin secretory granules and plasma membranes in the final steps

in exocytosis (120) It is not known whether islets express the recently cloned membrane-associated iPLA2F (group VIB PLA2) (6), but this enzyme is also BEL-sensitive and is another candidate for the BEL-sensitive target(s) that participate in glucose-induced insulin secretion

24 ZHONGMIN MA AND JOHN TURK

B Apoptosis

Apoptosis, the process of programmed cell death, is associated with changes

in glycerophospholipid metabolism In some cells induced to undergo apoptosis, arachidonate release parallels the reduction in cell viability and DNA fragmenta- tion (121,122) cPLA2 has been implicated in AA release during TNFc~-induced apoptosis (123-125) Fas-induced apoptosis of human promonocytic U937 cells

is also accompanied by fatty acid release Recently, it was reported that both Fas- induced AA and oleic acid release from U937 cells are mediated by iPLA2 but not by cPLA2 (69) In this study, it was observed that, during apoptosis, cPLA2a was converted to a 78-kDa fragment with concomitant loss of catalytic activity Cleavage of cPLA2a correlated with increased caspase-3-1ike protease activity

in apoptotic cells Neither cPLA20t nor secretory PLA2 inhibitors suppressed AA release, but inhibitors of iPLA2 suppressed AA release and delayed cell death induced by Fas, suggesting that Fas-induced AA release is mediated by iPLA2

In a subsequent study (70), these investigators provided additional evidence that iPLA2 mediates enhanced release of fatty acids from apoptotic cells Proteolytic cleavage of iPLA2fl by the action of caspase-3 results in loss of the N terminus through the first ankyrin repeat and in production of a form of iPLA2fl that is more active than the uncleaved form In fact, iPLAzfl does contain a caspase-3 cleavage consensus site DVTDlS3Y

Apoptosis in islet fl cells is induced by stimuli that induce Ca2+-store de- pletion This process requires hydrolysis of AA from membrane phospholipids and its conversion to 12-tipoxygenase metabolites by a mechanism that does not

require a rise in cytosolic [Ca ] (126) Ca -store-depletion-induced hydrolysis

of arachidonate from islet and smooth muscle cell phospholipids also does not require a rise in cytosolic [Ca 2+] and is mediated by a BEL-sensitive PLA2

(41,127) The PAPH-1 inhibitor propranolol does not block Ca2+-store-deple - tion-induced release of arachidonate from islet phospholipids (41) Activation

of iPLA2 during Ca2+-store depletion may therefore participate in Ca2+-store - depletion-induced apoptosis of fi cells

Some observations raise the possibility that iPLAz may participate in inter- leukin-lfi (IL-lfi) actions, including induction ofapoptosis IL-I~ induces apop- tosis of human islet ~ cells through Fas-mediated events (128) It was found that IL-I~ induces nitric oxide (NO) production, inducible nitric oxide syn- thase (iNOS) expression, and accumulation of nonesterified arachidonate and

a 12-1ipoxygenase product in islets (129) by a BEL-sensitive mechanism (106)

Inhibition of iPLA2 by BEL attenuates prostaglandin generation induced by IL-lfi in renal mesangial cells (130) Recently, it was reported that iPLA2 reg- ulates iNOS induction in cardiac myocytes through its product lysophospha- tidic acid (131) When neonatal ventricular myocytes were treated with BEL, IL-l~-induced PGE2 production, AA release, NO production, and iNOS expres- sion were all inhibited In addition, iPLA2 has been reported to be involved

GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 25

in potassium-regulated IL-1/~ processing (132) Both IL-1/3 maturation and for- mation of glycerophosphocholine were blocked by BEL, suggesting that iPLA,2

is involved in IL-1/3 processing

Several features of iPLA2fl fuel interest in the possible participation of the enzyme in apoptosis These include the presence of the caspase-3 cleavage site consensus sequence DVTD18a(y/N) and the fact that the cleavage at this site removes 183 amino acid residues from the N terminus of iPLA2fl and yields a protein with enhanced catalytic activity (70) In addition, the human long isoform ofiPLA2fl contains a proline-rich region encoded by exon 8 with homology to the proline-rich middle linker domain of Smad4 (Fig 4) Smad4 is the product of a tumor suppressor gene, participates in apoptosis in some settings, and interacts with its signaling partners via the proline-rich middle linker domain that exhibits homology to the region encoded by exon 8 in human iPLA2/3 Finally, the location

of the human iPLA2fl gene on chromosome 22q13.1 occurs at a site known to contain human tumor suppressor gene(s)

C Membrane Phospholipid Remodeling

A role ofiPLA2/~ in membrane phospholipid remodeling has been proposed

by Dennis and colleagues using the routine P388D1 macrophage-like cell line

as a model system (15, 117, 133) There are two pathways for incorporation of fatty acids into cellular phospholipids One is the deacylation/reaeylation cycle

of membrane phospholipid remodeling, and the other is the de novo synthesis pathway Most araehidonate in cellular phospholipids is incorporated via a deacy- lation/reaeylation cycle Macrophages and maerophage cell lines exhibit the abil- ity to incorporate AA into their membrane phospholipids in a Ca2+-independent manner (134, 135) In P388Dt maerophage-like cells, BEL was found to inhibit

AA esterifieation in a dose-dependent and saturatable manner, and the decrease

in AA incorporation was associated with inhibition of cellular iPLA2 activity and

a reduction in LPC content (133) Based on this study, it was proposed that iPLA2 acts to generate LPC aceeptors for arachidonate incorporation into mem- brane PC and therefore plays a housekeeping role in phospholipid remodeling

(15, 133) In addition, studies using antisense inhibition of iPLA2fl expression

in the same system supported this proposal (117) In this model, the function of iPLA2fl is to regulate the deaeylation/reacylation cycle, through which the cells incorporate AA and other unsaturated fatty acids into their membrane phos- pholipids, by providing the lysophospholipid aeeeptor employed in the acylation reaction By regulating AA esterifieation through generation of LPC acceptor molecules, iPLA2 might also play a major role in regulating AA turnover in cells

2 6 ZHONGMIN MA AND JOHN "lURK

of [3H]AA into phospholipids is not suppressed, but rather is enhanced This is also true for INS-1 insulinoma cells Under these conditions, the PAPH-1 in- hibitor propranolol does not influence [3H]AA incorporation into either islet or insulinoma cell phospholipids, indicating that the effects of BEL on [3H]AA in- corporation do not reflect PAPH-1 inhibition Measurement of islet LPC levels

by electrospray ionization mass spectrometry (ESI/MS) with an internal stan- dard indicates that treatment of islets with BEL induces only a 25% decline in LPC content This modest reduction in islet LPC levels does not limit arachi- donate incorporation into islet PC and does not interfere with the subsequent transfer of arachidonate to phosphatidylenthanolamine (PE) (118) Moreover, ESI/MS analyses of incorporation of arachidonate mass into PC of INS-1 in- sulinoma cells indicate that inhibition of iPLA2 with BEL does not retard, but slightly accelerates this process (118) This is also the case in differentiated hu- man U937 promonocytic cells (137) We have also observed that incorporation of neither [3H]AA nor arachidonate mass, as assessed by ESI/MS, into PC is accel- erated in INS-1 insulinoma cells that overexpress iPLA2/~ after stable transfec- tion with rat iPLA2/~ cDNA (119a) Treatment of such iPLA2/~-overepxressing cells with BEL also fails to suppress arachidonate incorporation into PC under these conditions These observations imply that iPLA2/~ does not play a general role in arachidonate incorporation into cellular PC, although it appears to do

so in murine P388D1 macrophage-like cells (117, 133) These cells, however, exhibit some atypical features of arachidonate incorporation and contain only about 3% arachidonate in their phospholipids (135) compared to 25% for natural macrophages (138, 139) and 27% for differentiated U937 promonocytic cells, which are also of monocyte-macrophage lineage The paucity of arachidonate in the phospholipids of murine P388D1 macrophage-like cells suggests that these cells may be deficient in the arachidonate incorporation mechanisms that are employed by cells capable of maintaining a high content of arachidonate in their phospholipids In contrast, islets exhibit among the highest arachidonate content

in phospholipids of any known tissue (112, 113, 140)

D Membrane Homeostasis and Other Functions

iPLA2 has been suggested to participate in membrane homeostasis by reg- ulation of PC catabolism (141) PC is the most abundant phospholipid in mam- malian cell membranes and is essential for cell viability The cellular levels of this lipid are tightly controlled Changes in the rate of PC synthesis are generally bal- anced by changes in PC catabolism CDP:phosphocholine cytidylyltransferase (CCT) is the rate-limiting enzyme in PC biosynthesis Overexpression of CCT

in Hela cells (141) and CHO cells (142) results not only in increased rates

of PC synthesis but also in increased rates of PC breakdown to its catabolite glycerophosphocholine There is thus little increase in net PC accumulation

GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 27

(141) The increased PC breakdown under these conditions was associated with increased levels of iPLA2 activity and in iPLA2fl immunoreactive protein (142)

Treatment with BEL blocks the accumulation of glycerophosphocholine (141)

These observations suggest that upregulation of iPLA2fl occurred as compen- satory response to overexpression of CCT in order to maintain a homeostatic relationship between PC biosynthesis and degradation

iPLA2 has been implicated in AA release and eicosanoid synthesis It has been reported that iPLA2 is involved in the protein kinase C-dependent AA release induced by zymosan in P388D1 macrophage-like cells (143) In this system, inhibition of iPLA2 with either BEL or an antisense oligonucleotide resulted in a decrease in zymosan-induced prostaglandin E2 (PGE2) genera- tion (143) Others subsequently reported that zymosan can induce arachidonate release from a P388D1 subclone without the participation of iPLA2 (144) In isolated ductal cells of rat submandibular gland, activation of iPLA2 by P2X7 agonists ATP or the ATP analog Bz-ATP [2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate] was observed (145) ATP or Bz-ATP induces the release of [3H]AA to the extracellular medium in a time- and dose-dependent manner In the absence of extracellular calcium, the release of [3H]AA in response to the purinergic agonists was completely blocked by BEL In addition, ATP and Bz- ATP stimulated Ca2+-independent secretion of the serine protease kallikrein from these cells, and this secretory event was also blocked by BEL These investigators concluded that the P2X7 receptor in ductal cells is coupled to kallikrein secretion through iPLA2 activation (145) In human granulocytes, BEL was found to inhibit leukotriene synthesis induced by 0.1-0.15 #M of calcium ionophore A23187 or opsonized zymosan (146) Treatment with pro- pranolol did not attenuate leukotriene synthesis under these conditions, indi- cating that PAPH-1 was not involved in leukotriene generation (146) A role for iPLA2fl in prostaglandin generation has been suggested in the iPLA2fl- overexpressing human embryonic kidney 293 cells (147, 148) iPLA2/3 over- expression resulted in increased spontaneous fatty acids release (147) and in the A23187-induced fatty acid release (148) iPLA2fl-derived AA was not me- tabolized to PGE2 When cotransfected with hamster iPLA2fl cDNA and hu- man cyclooxygenase-1 (COX-l) cDNA, the transfectants released an amount of PGE2 that was substantially higher than that released by cells cotransfected with iPLA2fl cDNA and cyclooxygenase 2 (COX-2), suggesting that A23187-induced

AA release by iPLA2/~ is preferentially metabolized by COX-1 compared to COX-2 (148)

Production oflysophosphatidic acid (LPA) by iPLA2 has also been suggested

to play several important roles in cells It was reported that iPLA2 is required for ot2-adrenergic-indueed preadipocyte spreading (•49) BEL specifically blocks this response without affecting spreading induced by LPA or serum The effect

of BEL to inhibit c~2-adrenergic agonist-induced preadipocyte spreading was

28 ZHONGMIN MA AND JOHN TURK reversed by LPA but not by AA and other fatty acids These observations suggest that iPLA2 is involved in ot2-adrenergic control of preadipocyte spreading via its product LPA (149)

Studies in parotid acinar cells (150) suggest that iPLA2 might also be involved

in exocytotic membrane fusion In these cells, exocytosis of amylase stimulated

by isoproterenol was dose-dependently inhibited by BEL but not by AACOCF3

at concentrations up to 30/zM BEL also inhibited amylase release evoked by forskolin and membrane-permeable cAMP analogs, but it did not inhibit cAMP- dependent protein kinase activity in parotid acinar cells (150)

VIII Future Perspectives

Emerging evidence suggests that iPLA2fl could play a variety of roles in different cell types Salient structural features of the enzyme include a GXSXG serine lipase consensus motif in which the central serine is required for catal- ysis, an ATP-binding domain, an ankyrin-repeat domain that probably medi- ates homomultimer formation and could mediate heteromultimer formation with regulatory proteins, and a bipartite nuclear localization signal sequence In addition, multiple splicing variants of the iPLA2fl exist, and the long isoform

of human iPLA2fl contains a region encoded by a distinct exon of the human iPLA2fl gene This region shares homology with the proline-rich middle linker domain of Smad4, and this domain mediates interaction of Smad4 with other proteins That iPLA2fl activity can be regulated by formation ofheterooligomefic complexes is suggested by the fact that coexpression of truncated, catalytically inactive splicing variants with full-length iPLA2 results in alternation of iPLA2fl activity These observation suggest that iPLA,2fl may be subject to complex regu- latory mechanisms that differ among cell types Further studies are required to determine how iPLA2fl is activated by extracellular signals and how it is regulated within the cell

Protein evolution demands conservation of key residues to maintain struc- tural integrity but allows for sequence variation that provides functional speci- ficity The existence of an ankyrin-repeat domain in iPLA2fl suggests that the enzyme could interact with other proteins, such as integral membrane proteins

or cytosolic proteins Such binding could regulate iPLA2fl function either pos- itively or negatively This structural feature is unique to iPLA2fl among the PLA2 family but is common in some regulatory proteins such as transcriptional factors and cell-cycle regulators This suggests that iPLA2~ may be involved

in similar functions, such as regulating nuclear membrane homeostasis during the cell cycle Such a role would be consistent with the presence of a bipar- tite nuclear localization signal sequence within the iPLA2fl molecule Changes

in cell nuclei are hallmarks of apoptosis Nuclear membrane degradation re- sulting from activation of iPLA2fl could explain its suggested involvement in

GROUP VIA Ca2+-INDEPENDENT PHOSPHOLIPASE A2 29

apoptosis D e t e r m i n i n g w h e t h e r iPLA2fl t r a n s l o c a t e s f r o m cytosol to n u c l e u s

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CooA: A Heme-Containing

Regulatory Protein That Serves

as a Specific Sensor of Both

Carbon Monoxide and

t Department of Biochemistry and Molecular Biology

Program in Macromolecular Structure University of California-Irvine Irvine, California 92697

I Introduction 36

II CO Oxidation by RhodospiriUum rubrum and Other Microorganisms 36 III The coo Genes and Their Regulation 38

IV General Behavior of CooA as a Transcriptional Activator Responding to

the Redox State and the Presence of CO 40

V The Structure of CooA and Its Implications 42

A Comparison of CooA Structure to That of CRP 42

B The H e m e Region of CooA 47

C Model for Activation of CooA by CO 50

VI CooA as a Redox Sensor 52 VII CooA as a CO Sensor 53 VIII Cooperativity of Ligand Binding 54

IX Transcriptional Activation by CooA 56

X D N A Recognition Properties of CooA 58

XI Future Direction and Open Questions 59 References 60

CooA, the heme-containing carbon monoxide (CO) sensor from the bac-

sion o f certain genes in response to CO As with other h e m c proteins, CooA is

1To whom correspondence should be addressed

Progress in Nucleic Acid Research Cop~,Tight O 2001 by Academic Press and Molecular Biology, Vol 67 35 All rights of reproduction in any form reserved

36 GARY E ROBERTS ET AL

unable to bind CO w h e n the Fe h e m e is oxidized, consistent with the fact that some o f the regulated gene products are oxygen-labile Upon reduction, there is

an unusual switch o f protein ligands to the six-coordinate h e m e and the reduced heine is able to bind CO CO binding stabilizes a conformation of the dimeric protein that allows sequence-specific DNA binding, and transcription is activated through contacts b e t w e e n CooA and RNA polymerase CooA is therefore a novel redox sensor as well as a specific CO sensor CooA is a homolog o f catabolite responsive protein (CRP), whose transcriptionally active conformation has been known for some time The recent solution o f the crystal structure o f the CO-free (transcriptionally inactive) form o f CooA has allowed insights into the mechanism

by which both proteins respond to their specific small-molecule effectors © 2001

Rhodospirillum rubrum can oxidize CO to CO2, and the expression of genes involved in that process are regulated by CooA (CO-oxidation activator) This heine-containing dimer functions both as a redox sensor and as a specific CO sensor Part of this process involves a highly unusual switch of protein ligands upon reduction of the heme, as well as the utilization of proline as an axial li- gand, which has not previously been seen (19) As a member of the CRP/FNR superfamily of regulatory proteins, many of which respond to small-molecule effectors, the solution of its structure in the effector-free form has provided in- sight into the mechanism by which CooA, and presumably some other members

of this family, become activated in response to their effector molecules The combination of structural information with the results of a variety of mutagenic, spectroscopic, and functional analyses has also revealed important features that underlie its sensing of redox and CO The analysis of CooA provides an impor- tant addition to the understanding of the selectivity in such sensing systems, as well as suggesting the mechanism by which many members of the CRP/FNR superfamily become active for DNA binding

II CO Oxidation by Rhodospirillum rubrum

and Other Microorganisms

CO is found throughout the environment and is the product of chem- ical decomposition, biological processes, atmospheric reactions, and human

CooA: A CO-SENSING TRANSCRIPTIONAL FACTOR 37 activity (20) In aquatic environments, the most significant source of CO is

thought to be abiotic photooxidation of biological molecules (21, 22), and direct

measurements indicate its presence throughout the water column at nanomo- far concentrations (21, 23) The removal of CO is through oxidation by radical

chemistry in the upper atmosphere or through microbial metabolism, with the latter processes encompassing adventitious mechanisms in which CO is consid- ered a fortuitous substrate, or biochemical systems in which the metabolism of

CO (often as an enzyme-bound intermediate) is the connection between one- and two-carbon metabolites of an anabolic or catabolic process These types

of metabolism depend on one of two distinct biochemical mechanisms (24)

Aerobic, "carboxydotrophic," organisms exclusively elaborate a molybdenum- containing hydroxylase that catalyzes the catabolic oxidation of CO and water to CO2 plus reducing equivalents, which ultimately yield H2 through the activity of

a hydrogenase Expression of these systems, such as the 12-gene cox cluster of Oligotr~rpha carboxidovorans, requires the presence of CO, and the cox genes are

found adjacent to cbb genes that encode components of the Calvin cycle (25) As

yet, the regulatory mechanism of the cox (and homologous) systems has not been

described It will be interesting to compare this CO-dependent regulation, which enables aerobic transcription, with CooA, which controls a low-redox process Anaerobic CO-oxidizing microbes span a tremendous diversity and encom- pass methanogenic, acetogenic, and sulfate-reducing organisms The oxygen- labile enzymatic oxidation of CO to CO2 routinely assayed in vitro is catalyzed

by a Ni-containing enzyme that performs, in vivo, the fundamental steps in

the interconversion of single-carbon intermediates and acetyl-CoA While the level of the enzyme may be modestly influenced by the particular carbon sub- strate upon which the organism is cultivated, its expression does not require the presence of exogenous CO (24)

The observation that purple nonsulfur bacteria can anaerobically metabolize

CO was first made by Uffen with an organism that is now known as Rubrivivax gelatinosus (26), although R rubrum is now the best characterized example of

the group Phototrophs express a "hybrid" system: the central Ni-containing enzyme catalyzing CO oxidation is termed carbon monoxide dehydrogenase

(abbreviated CODH), and the R rubrum enzyme is certainly evolutionarily re-

lated to those of strict anaerobes, based on predicted primary protein sequences

(27) Yet the R rubrum enzyme, like that of aerobic carboxydotrophs, solely

catalyzes CO oxidation to CO,2 and reducing equivalents, which in turn yield

He via electron carriers and a specific hydrogenase The 12-gene coo regulon,

aside from that encoding the regulatory protein, is transcriptionally dependent upon the presence of CO Carboxydothermus hydrogenoformans, a nonpho-

totrophic anaerobe isolated from a volcanic swamp, probably contains a similar CO-oxidation system (28)

Perhaps aside from organisms isolated from burning coal piles (29) or vol-

canic environments (28) where exceptional levels of CO can be expected, the

38 GARY E ROBERTS ET AL biological niche for most CO-utilizing aerobes and anaerobes is obscure: Mea- sured bulk CO levels, normally in the nanomolar range in aqueous environments and in the range of parts per billion in the atmosphere, are generally well be-

low the Km for CODHs of isolated organisms (30) This is also the case for the

R rubrum coo system It does not appear to be for "detoxification," both because

mutants lacking the coo system are highly tolerant of CO under lab conditions and because R rubrum is surprisingly capable of coupling the thermodynam-

ically poor anaerobic conversion of CO to CO2 plus H2 to grow at a rate only

20% slower than phototrophic growth in the same medium (31) This complex, specific, and regulated metabolism implies either that R rubrum naturally ex-

periences periods of exceptional CO exposure or that its native environments are so energy-limiting as to make this system advantageous

III The coo Genes and Their Regulation

All known coo genes fall into three contiguous transcripts on the R rubrum chromosome (27, 32, 33), as depicted in Fig 1 coos encodes the CODH activity and cooF encodes a ferredoxin that forms a tight complex with CooS and pro-

motes its association with the membrane This membrane association apparently

supports efficient electron transfer to the products of the cooMKLXUH operon Based on sequence similarity and some biochemical characterization (32), the

products of the first five genes of this operon form a membrane-associated com- plex that passes reductant to CooH In the course of this electron transfer, useful energy is generated, presumably by the generation of a proton gradient,

that supports growth on CO as sole energy source (31) CooH is an unusual NiFe hydrogenase in that it is highly tolerant of CO (34), consistent with its

role in this CO-oxidizing system, and that it lacks a cleavable C terminus This

C terminus is removed in many Ni-containing hydrogenases upon Ni insertion, but in CooH, the site of normal cleavage is replaced by a stop codon; it is es-

sentially "precleaved" (32) The CooCTJ products are apparently involved in Ni

co

/

~ - - - - - C o o A active ~ C i n a c t i v e

cooM I( L X U H cooF S C T J c o o A nadC B

F[c 1 Transcriptional organization of the coo regu]on in R mbrum The dark horizontal arrows

indicate the direction of transcription, nadBC, ORF, and ahpC are not involved in CO oxidation

CooA: A CO-SENSING TRANSCRIPTIONAL FACTOR 39 processing for CooS based on mutant phenotype, similarity to other genes, and substantial biochemical and physiological characterization (33, 35, 36) Both the

of CooA, the CO sensor that is the focus of this review, but cooA has a separate low-level promoter so that it is present in the cell under all growth conditions examined (Y He and G P Roberts, unpublished data)

The expression of the coo genes is under the control of CooA, which activates transcription only when it is reduced and binds CO (17, 37, 38) This regulation

of coo gene expression can be rationalized in light of our knowledge of the regulated gene products For example, CO is the only known substrate of CooS, the C O D H encoded by this system (39) [although CooS can also run the reverse reaction (40)], and the regulation of coo expression in the absence of CO appears

to be extremely tight in R rubrum, with C O D H activity, C O D H antigen, and

cooFSCTJ mRNA being undetectable in the absence of CO In the presence of low levels of CO, however, the genes are highly expressed (41), consistent with the potential utilization of this system as sole energy source in the cell The affinity

of CooA (K,I ~ 4 # M ) for CO (M V Thorsteinsson et al., unpublished data) is

in the same range as the Km of CooS ("~32 #M) (42), so that CooA causes Coos

to be synthesized when CO levels allow a reasonable level of enzymatic activity CooS has also been found to be O2-sensitive, as has CooH, so it is not sur- prising that the coo genes are also expressed only under anaerobic conditions Less obviously, Coos is maximally active at a redox poise of - 3 0 0 mV and below (J Heo and P W Ludden, personal communication), and the behavior of CooA is consistent with this fact CooA is competent to bind CO only when its heme is re- duced, and the reduction and oxidation midpoint potentials have been reported

to be - 3 2 0 and - 2 6 0 mV, respectively (43), reflecting the different initial CooA species present in each redox titration Although this comparison is certainly simplistic and we have not determined the internal redox state of R rubrum cells expressing the coo genes, it serves to suggest that regulation of CooA activ- ity might be sufficient to explain the observation that reducing culture conditions are necessary for consistent high-level coo expression in R rubrum (31)

In the absence of CO or in the presence of oxidizing conditions, only cooA

is expressed However, under reducing conditions in the presence of CO,

being expressed at a level approximately fivefold higher than the latter Oddly,

in the presence of CO, some portion of the cooFSCTJ transcript appears to read through into cooA, and Western analysis has shown an increase in CooA accumulation in R rubrum in the presence of CO (Y He and G P Roberts, un- published data) The physiological reason for this readthrough, if any, remains unknown

Given the ability of the coo system to provide the sole energy source for the cell, one might expect its expression to be regulated by the energy status of the