From the Department of Medical and Molecular Genetics and The Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202-5251
Phospholipase activity was first described in
pancreatic juice and cobra venom at about the turn of the century.
Phospholipase A2s
(PLA2s)1 are those
phospholipases that hydrolyze the sn-2 fatty acid acyl ester
bond of phosphoglycerides to free fatty acid and lysophospholipids. PLA2s have been divided into several groups based on
molecular weight, amino acid sequence and homology (e.g.
position of Cys residues in the low molecular weight enzymes), calcium
dependence, and cellular localization (see below). Two groups of
~14-kDa snake venom PLA2s, Group I from cobras and kraits
and Group II from rattlesnakes and vipers, are well known. Until 1989, however, the only well characterized mammalian PLA2 was the
~14-kDa enzyme from pancreatic juice, which was classified as a Group
IB enzyme (for reviews see Refs. 1 and 2). In 1989 two groups each described the gene for a ~14-kDa enzyme from synovial fluid and platelets, which is distinct from the pancreatic enzyme and was assigned to Group IIA. These publications engendered a relatively large
literature on what is thought to be Group IIA PLA2,
primarily because it is believed to be elevated in serum and exudates
in certain inflammatory diseases, suggesting its involvement in the production of lipid mediators of inflammation (see below). Also, despite conflicting data concerning its physiologic substrate, it was
recognized that Group IIA PLA2 has the potential to release arachidonic acid from membranes, which then may serve as the precursor of prostaglandins, thromboxanes, and prostacyclins via the
cyclooxygenase pathway or leukotrienes and lipoxins via the
lipoxygenase pathway (for review see Ref. 3). In 1994, our group
described the cloning of genes and expression of cDNAs for two new
mammalian ~14-kDa PLA2s (4-6). This brought the number
of well described mammalian low molecular weight PLA2 genes
and enzymes to four. This review summarizes what is now known about
mammalian ~14-kDa PLA2s, particularly those that have
been most recently described. The focus is on possible biological
functions of each of the ~14-kDa PLA2s and functional
relationships to the structurally unrelated, 85-kDa group IV cytosolic
PLA2. I also review new data that force a reevaluation of a
significant fraction of the large literature describing tissue distribution and metabolic functions of the mammalian Group IIA PLA2.
Classification of Mammalian Low Molecular Weight
PLA2 The nomenclature of PLA2 enzyme groups distinguished
on the basis of structural and other criteria has been reviewed by
Dennis (1, 2) and adopted here. Since the first review of the subject, however, two new ~14-kDa mammalian PLA2s have been
described (see below). Whereas many novel PLA2 activities
and partial amino acid sequences have been reported, we reserve group
or subgroup designations for mammalian ~14-kDa PLA2s with
characterized genes and demonstrated expression. Further, we have made
an effort to have the gene and enzyme group nomenclature correspond
with the designation of genes for both existing and newly discovered
PLA2s. Therefore, for example, the mouse PLA2
group IIA, IIC, and V genes have been designated Pla2g2a,
Pla2g2c, and Pla2g5, respectively (7).
All of the ~14-kDa PLA2s contain an even number of Cys at
characteristic positions, each of which pairs with another specific Cys
to form a disulfide bridge, thus producing a rigid three-dimensional structure. Within group I, x-ray crystallography has demonstrated that
enzymes from divergent sources such as snakes and mammals have quite
similar crystal structures (1). It is likely that all of the low
molecular weight PLA2s utilize a specific catalytic His,
assisted by an Asp, to polarize a bound H2O, which then
attacks the carbonyl group of the phospholipid substrate.
Ca2+ is required to stabilize the transition state and is
bound within the highly evolutionarily conserved "calcium binding
loop" observed in all ~14-kDa PLA2s (1, 8).
Both group IA PLA2, found only in snakes, and group IB
PLA2, which appears ubiquitously in mammals, have a
disulfide bridge connecting Cys-11 to Cys-77 and a characteristic
three-amino acid "elapid loop" composed of residues 54-56. The
mammalian group IB PLA2 has 14 Cys residues and is secreted
predominantly by the pancreas to function extracellularly in digestion
(1, 3). Group IB PLA2 is also present in some nondigestive
organs, suggesting a possible secondary role (9, 10). Group IIA
PLA2, which has been described for many mammals, and group
IIB PLA2, which has only been observed in the Gabon viper,
also contain 14 Cys residues but, in contrast to the group I enzymes,
lack the Cys-11 to Cys-77 disulfide bridge. All group II
PLA2s have a C-terminal extension of 6 amino acids that
terminates in a Cys joined to Cys-50 near the His-48 catalytic site.
The mammalian group IIA has been reported to occur in relatively small
amounts in mast cells, macrophages, and diverse tissues such as liver
and spleen (3). It is also reported to occur in greater amounts in
fluid from arthritic synovia and serum from patients with inflammatory diseases such as acute pancreatitis and sepsis (11). The group IIA
enzyme is frequently referred to as "secreted PLA2,"
but this term lacks precision because the enzyme has also been
localized within mitochondria (12), and other "secreted"
PLA2s are now known. As described below, reports of
expression of group IIA PLA2 in various cell types will
require detailed reevaluation in light of new data that indicate some
methods used for its detection also detect the more recently described
group V PLA2.
The group IIC PLA2 gene has been characterized in rat and
mouse (5, 13). It encodes a mature enzyme, with a calculated molecular
mass of 14.8 kDa, which does not contain the Cys-11 to Cys-77 disulfide
bridge or elapid loop characteristic of group I but does contain the
6-amino acid C-terminal extension characteristic of group II. The group
IIC enzyme is distinguished from group IIA and group IIB enzymes in
that it contains 16 Cys residues. Further, the group IIC enzyme from
mouse and rat contains only 17 of the 18 amino acids that had been
thought to be invariantly conserved in low molecular weight
PLA2s (14, 15), Ile-9 being replaced by Val. Rodent group
IIC PLA2 is highly expressed in adult but not prepubescent
testis (5). In situ hybridization of testis tissue sections
indicates that the group IIC gene is expressed mainly in pachytene
spermatocytes, secondary spermatocytes, and round spermatids but not in
spermatogonia, elongating spermatids, or Sertoli cells (13). The
N-terminal portion of exon III is absent in the human group IIC
PLA2 gene, and about 16% of alleles also exhibit a common
nonsense mutation in exon II. Significantly, all other parts of the
human group IIC gene appear potentially functional and highly
homologous to the functional rodent genes, but there is no evidence for
group IIC gene expression in human tissues. Thus, we conclude that the
group IIC gene has recently evolved into a pseudogene in humans (7). It
is not known whether there is compensatory activity of one of the other
PLA2 genes in human testis or other tissues.
The group V PLA2 gene and its product have been
characterized in human (4), rat (6), and mouse
(16).2 The mature enzyme, with a calculated
molecular mass of 13.6 kDa, contains neither the elapid loop of group I
nor the 6-amino acid carboxyl extension of group II. Further, it
contains only 12 of the Cys found in group I and II PLA2s.
Thus, this second new ~14-kDa PLA2 has been placed into a
new group known as group V3 (6). The group
V PLA2 gene is expressed highly in heart, placenta, and, to
a lesser extent, lung and liver (4, 6). Further, group V, rather than
group IIA as was previously believed, appears to be the primary
~14-kDa PLA2 expressed by P388D1
macrophage-like cells and mast cells (see below). As is the case for
the group IIA and IIC genes, the group V gene product is expressed
initially as a prepeptide with the first 20 amino acids probably
representing a signal peptide that is subsequently cleaved (4).
Some distinguishing properties of the ~14-kDa PLA2s are
summarized in Table I. In addition, there are common
features such as pH 7-9 activity optima and a requirement for about
1-10 mM Ca2+ for maximal activity (4-6). This
latter property stands in contrast with the 85-kDa group IV enzyme,
which is activated by Ca2+ concentrations in the micromolar
range (for review see Ref. 1). However, there is one report that under
certain conditions group IIA enzyme, but not group IB, achieves 50% of
maximal response with 0.5 µM Ca2+ (17). It is
also important to recognize that the demonstration of substrate
preferences for each of the ~14-kDa PLA2s
(e.g. Refs. 4-6) was merely intended to distinguish between
the various groups of PLA2s at a particular assay pH and
Ca2+ concentration. Because of inherent variation in the
presentation of lipid substrate (1), such assay data should not be
generalized to characterize activity in vivo.
Table I.
Properties of ~14-kDa mammalian PLA2s
Characteristics
Group
IBHuman
IIAHuman
IICRat
VHuman
No. of amino acids in mature protein
126
124
130
118
No. of cysteines in mature protein
14
14
16
12
Pre/propeptide
+/+
+/
+/?
+/
Level of mRNA
expression
Pancreas
lung
Placenta > synovia = platelets
Testis
Heart > placenta > lung > liver
Low Molecular Weight PLA2 Genomics and
Evolution
The human group IB PLA2 gene has been shown to reside on chromosome 12 (18), and the human groups IIA and V genes and group IIC pseudogene are tightly linked on chromosome 1p34-p36.1. Consistent with the human localization, the mouse group IIA, IIC, and V genes are also tightly linked and located on the distal region of chromosome 4, which is known to be syntenic with human 1p34-36.1. The data from radiation hybrids suggest that the human group IIA and group V genes are very close whereas the group IIC pseudogene is located about 1 centimorgan toward the centromere (7). The clustering of PLA2 genes invites speculation about possible complex coordinate regulation of expression as is the case for mammalian globin genes.
The amino acid coding regions of each of the ~14-kDa
PLA2s are interrupted by 3 introns, which are almost
identically positioned when the enzymes are compared in homologous
alignment shown in Fig. 1 (4-6, 19-21). In addition,
the human and rat group V (4, 6) and the mouse group IIA (22) gene have
one upstream noncoding exon, and the rat group IIC gene has three
upstream noncoding exons. The first two exons in the rat group IIC gene
are alternatively transcribed in testis and brain mRNA (5). This
amino acid alignment clearly suggests the common evolutionary origin of
these genes. We propose the evolutionary scheme of ~14-kDa
PLA2 gene duplication events shown in Fig.
2, which is a modification of Davidson and Dennis (14)
in light of the subsequent discovery of groups IIC and
V.4 Group V PLA2s exhibit 12 Cys residues that are identical in position to the 12 of 14 Cys
residues common to Groups I and II. This suggests that group V is the
progenitor to groups I and II. Group III PLA2s, which are
found only in bees and some lizards, have 10 Cys residues and may have
diverged from the common ancestral PLA2 before the divergence of invertebrates and vertebrates (14). The divergence of
groups I and II may have occurred simultaneously or at different times.
Group IIC PLA2 has 16 Cys residues, 14 of which are shared with group IIA and 12 of which are identical to all 12 Cys residues of
group IIB (14). Therefore, we suggest that Group IIC diverged from a
common group II ancestor with 14 Cys residues and that Groups IIA and
IIB diverged at a later time.
Revised View of PLA2 Gene Expression in Cell
Signaling
Although it is well documented that individual cells of different types contain multiple PLA2s (23-25), some understanding of how these different enzymes cooperate in receptor-coupled cellular activation has only recently emerged. In certain cell types, agonist-induced PLA2 activity can be either short term, long term, or biphasic, depending on the agonist or combination of agonists. These patterns are reflected in the spatial and temporal kinetics of arachidonic acid release and the subsequent production of eicosanoids. For example, in P388D1 macrophage-like cells and mast cells arachidonic acid release in response to certain agonist combinations can be shown to be biphasic and dependent on the activities of both group IV cytosolic PLA2 and a low molecular weight PLA2 that was, until recently, believed to be group IIA. Balsinde et al. (25) showed that P388D1 mouse cells stimulated with bacterial lipopolysaccharide and platelet-activating factor release arachidonic acid in two phases, an initial rapid accumulation inside of the cell within the first few minutes and a subsequent sustained phase of accumulation in the culture medium. It was subsequently shown that group IV PLA2 acting intracellularly is responsible for the initial, rapid release of arachidonic acid whereas a ~14-kDa PLA2 acting on the outer surface of the cell was responsible for the greater, mostly extracellular, sustained release of arachidonic acid (26). The data also suggested that intracellular and extracellular arachidonic acid arise from different phospholipid pools within the cell. Most recently, Balboa et al. (16) showed that the ~14-kDa enzyme in P388D1 cells associated with the sustained release of arachidonic acid is group V and not group IIA as was previously believed. Whereas there was no detectable mRNA for PLA2 groups IIA or IIC in either resting or activated cells, the group V mRNA was abundant in both. Antisense oligonucleotides for the highly conserved Ca2+-binding domain of rat group IIA mRNA, which had previously been used in experiments that were interpreted as implicating group IIA (27), were shown to act in a nonspecific way on group V. However, more specific antisense oligonucleotides against a unique exon region of mouse group V PLA2 blocked expression by about 60-70%, whereas the control sense oligonucleotide was without effect. Interestingly, a polyclonal antiserum against human synovial fluid PLA2, presumably group IIA, was used to successfully detect expression of the P388D1 cell surface PLA2. This result indicates that this antiserum, and such antisera in general, may not be able to distinguish between PLA2 groups IIA and V (see below).
The agonist-mediated activation of mast cells includes degranulation
and release of ligands such as serotonin and histamine and is similar
in several key respects to the biphasic response observed in
P388D1 cells. Bone marrow-derived mouse mast cells stimulated by antigen aggregation of high affinity IgE receptors on the
cell surface (28) or with c-kit ligand, IL-10, and IL-1 or by priming with c-kit ligand and IL-10 followed by IgE
and antigen activation (29) exhibit an immediate phase of arachidonic acid release during the first 10-20 min followed by a delayed phase
from hours 2 to 7. These phases of arachidonic acid release are
reflected by early and late phases of prostaglandin D2
(PGD2) synthesis, which are mediated by the constitutive
prostaglandin synthase 1 (PGS1) and the inducible prostaglandin
synthase 2 (PGS2), respectively (28, 29). However, there is controversy
as to which PLA2s provide the arachidonic acid for early
and late PGD2 synthesis. The results from one group
indicate that a ~14-kDa PLA2 coupled to PGS1 is
responsible for early phase PGD2 synthesis whereas the
group IV PLA2 coupled to PGS2 is responsible for late phase
PGD2 synthesis (30). In contrast, a second group's data suggest the reverse, i.e. a ~14-kDa PLA2
coupled with PGS2 is responsible for late phase PGD2
production (31). These apparently contradictory conclusions may be a
consequence of differences in experimental methodologies. Both groups,
however, clearly demonstrate that one phase of PGD2
synthesis is a consequence of the activity of a low molecular weight
PLA2 that was initially believed to be group IIA. As
described below, we now know that Group IIA PLA2 is
not involved in mast cell activation.
The involvement of specific ~14-kDa PLA2s in mast cell
activation has been clarified by the discovery of a naturally occurring mutation in the group IIA PLA2 gene of many inbred mouse
strains. The "murine intestinal neoplasia" or
ApcMin gene is the ortholog of the human
APC gene, which has been shown to be mutated in a hereditary
form of colon cancer known as familial adenomatous polyposis coli. The
number of intestinal tumors is increased in mouse strains that also
carry the Mom1 (modifier of Min-1) mutation that is likely a
frameshift mutation in the gene for group IIA PLA2, such
that Mom1 homozygotes (Pla2g2a/
genotype) express little or no group IIA mRNA or protein (22, 32).
Bingham et al. (31) demonstrated that mast cells from Mom1 homozygotes exhibit a normal biphasic response to
ligand stimulation and that one phase of this response, previously
attributed to group IIA PLA2, must therefore be mediated by
another PLA2 that has some properties in common with the
group IIA enzyme. Contemporaneously, Reddy et al. (33)
demonstrated that mast cells from both Mom1 homozygotes and
normal mice exhibit biphasic responses to activation and that both
early and delayed PGD2 production and ~14-kDa
PLA2 secretion into the medium are similar in both genotypes. Neither Mom1 homozygotes nor normal cells
exhibited any group IIA or group IIC mRNA as determined by Northern
blotting and the more sensitive technique of reverse
transcriptase/polymerase chain reaction amplification. However, cells
of both genotypes exhibited Group V mRNA, and PLA2
activity was secreted into the medium as determined by assay, binding
to monoclonal antibody directed against recombinant human group IIA
PLA2, and inhibition by a drug (SB203347) developed as an
inhibitor of the group IIA enzyme (33). Also, it was shown that the
PLA2 secreted from both Pla2g2a+/+
and Pla2g2a
/
mast cells could release
arachidonic acid in distal mouse Swiss 3T3 cells, which then utilized
this arachidonic acid for prostaglandin synthesis (33, 34). These data
clearly implicate involvement of the group V PLA2 in one
phase of mast cell immune activation.
A biphasic response implicating both the group IV and a ~14-kDa
PLA2 is also observed in cytokine-stimulated rat mesangial cells (35, 36) and human endothelial cells (37). Clearly, the precise
identity of the ~14-kDa PLA2 will have to be reevaluated in light of the above data from P388D1 and mast cells. High
levels of ~14-kDa PLA2, believed to be group IIA, have
been described in serum and tissue exudates from patients with a
variety of inflammatory diseases including arthritis, pancreatitis,
adult respiratory distress syndrome, and septic shock (20, 38, 39,
41-45). The identification of group IIA PLA2 in these
diseases has most frequently been based on enzymatic assays, use of
inhibitors developed against group IIA PLA2, or immunologic
detection (cf. Ref. 46). The data of Balboa et
al. (16), Bingham et al. (31), and Reddy et
al. (33) clearly indicate that these criteria are inadequate for
distinguishing between the group IIA and group V PLA2s.
Thus, an entire literature will require reevaluation. Furthermore,
existing and new PLA2 inhibitors will require evaluation
with regard to their activity against both the group IIA and group V
enzymes (47). A recent publication by Tseng et al. (48)
suggests that it may be possible to design specific peptide inhibitors
for each of the ~14-kDa PLA2s based on their individual
-loop pentapeptide sequences.
Knowledge of at least three (humans) or four (rodents) related genes encoding distinct ~14-kDa PLA2s that are expressed in a tissue-specific manner begs the question of whether or not there are differences among the PLA2s that relate to function in vivo. A first step toward answering this question will be determination of the expression of each PLA2 in a wide variety of cell types and under different circumstances (e.g. activation). This, in turn, may require technical innovations such as more specific inhibitors or antibodies for immunocytochemistry. Naturally occurring (e.g. Mom1) or targeted gene knockouts could also be useful to this end, especially if, as has been the case to date (33), there is no compensatory activation of another PLA2 gene. One should be cautious, however, in postulating a molecular basis for a phenotype in knockout mice. For example, cyclooxygenase-2 gene knockout mice or mice treated with specific cyclooxygenase-2 inhibitors exhibit a reduced level of ApcMin-related intestinal polyposis (49). One might have anticipated the opposite result from what is known about Mom1 mice, given that arachidonic acid is the substrate for cyclooxygenase-2. Mom1 mice, which exhibit increased intestinal polyposis, may have reduced levels of arachidonic acid release in intestinal cells, which clearly express the group IIA enzyme in wild-type mice (16). Also, our consideration of phospholipid metabolism should include possible functions of newly discovered PLA2-like genes, such as PLA2L mapped to human chromosome 8q24 (50).
Finally, the gene encoding a receptor for PLA2 has been cloned from cow (51), rabbit (52), and human (53). These homologous receptors are composed of about 1460 amino acids and are structurally related to the macrophage mannose receptor (51). Whereas the cow and rabbit receptors bind porcine group IB PLA2 with high affinity and the rabbit receptor binds group IIA PLA2 with high but somewhat lesser affinity, the human kidney receptor has only weak affinity for group IB and little affinity for group IIA (53). The human PLA2 receptor is highly expressed in diverse tissues such as kidney, lung, placenta, and skeletal muscle, a pattern that is different from rabbit. Humans also exhibit a soluble form of the receptor not reported for the other species (53), in addition to the membrane-bound form. The initial view based on the research in cow was that the PLA2 receptor mediated extrapancreatic group IB PLA2-induced DNA synthesis, contraction, eicosanoid production, and chemokinetic cell migration (see Ref. 51), but the lack of receptor affinity for group IB PLA2 in humans does not support this idea (53). The critical region(s) in PLA2 for binding to the receptor appears to reside within the Ca2+-binding domain (40), which exhibits relatively high homology among all ~14-kDa PLA2s (1, 15). There are, as yet, no reports of experiments testing the binding of group IIC or group V PLA2 to this receptor. It is possible that either one or both of these enzymes will prove to be a natural ligand for the PLA2 receptor in a particular species, suggesting novel functions for these enzymes. The PLA2 receptor has also been implicated in the induction of group IIA PLA2 mRNA transcription by the group IB pancreatic enzyme in rat mesangial cells (36). Perhaps the PLA2 receptor also mediates cross-talk between additional ~14-kDa PLA2s in a cell type- or species-specific manner.
I thank Michelle V. Winstead for Fig. 1 and helpful comments, and Drs. E. A. Dennis and H. R. Herschman for critically reading the manuscript.