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INTRODUCTION |
Phospholipases A2
(PLA2)1 catalyze
hydrolysis of sn-2 fatty acid substituents from
glycerophospholipid substrates to yield a free fatty acid and a
2-lysophospholipid (1-7). PLA2 is a diverse group of
enzymes, and the first well characterized members have low molecular
masses (approximately 14 kDa), require millimolar [Ca2+]
for catalytic activity, and function as extracellular secreted enzymes
(sPLA2) (3, 6). The first cloned PLA2 that is
active at [Ca2+] achieved in the cytosol of living cells
is an 85-kDa protein classified as a Group IV PLA2 and
designated cPLA2 (3, 5). This enzyme is induced to
associate with its substrates in membranes by rises in cytosolic
[Ca2+] within the range achieved in cells stimulated by
extracellular signals that induce Ca2+ release from
intracellular sites or Ca2+ entry from the extracellular
space, is also regulated by phosphorylation, and prefers substrates
with sn-2 arachidonoyl residues (5).
Recently, a second PLA2 that is active at
[Ca2+] that can be achieved in cytosol has been cloned
(8-10). This enzyme does not require Ca2+ for catalysis,
is classified as a Group VI PLA2, and is designated iPLA2 (3, 4). The iPLA2 enzymes cloned from
hamster (8), mouse (9), and rat (10) cells represent species homologs and all are 85-kDa proteins containing 752 amino acid residues with
highly homologous (approximately 95% identity) sequences. Each
contains a GXSXG lipase consensus motif and eight
stretches of a repeating motif homologous to a repetitive motif in the
integral membrane protein-binding domain of ankyrin (8-10). The
substrate preference of these iPLA2 enzymes varies with the
mode of presentation (8), but each is inhibited (8-10) by a bromoenol
lactone (BEL) suicide substrate (11, 12) that is not an effective
inhibitor of sPLA2 or cPLA2 enzymes at
comparable concentrations (4, 11-14).
Proposed functions for iPLA2 include a housekeeping role in
phospholipid remodeling that involves generation of lysophospholipid acceptors for arachidonic acid incorporation into P388D1
macrophage-like cell phospholipids (4, 15, 16). Signaling roles for
iPLA2 in generating substrate for leukotriene biosynthesis
(17) and lipid messengers that regulate ion channel activity (10, 18, 19) and apoptosis (20) have also been suggested. Recent observations with human iPLA2 suggest that the enzyme might serve
distinct functions in different cells that involve regulatory
interactions among splice variants (17, 21). Human iPLA2
cloned from B-lymphocyte lines and testis differs from
iPLA2 cloned from cells of rodent species in that it is an
88-kDa rather than an 85-kDa protein and contains a 54-amino acid
insert interrupting the eighth ankyrin repeat (21). The human
B-lymphocyte iPLA2 sequence is otherwise highly homologous
to hamster, mouse, and rat sequences and includes the seven other
ankyrin-like repeats and a GXSXG lipase sequence (21). Catalytically active iPLA2 other than the 88-kDa
isoform have not yet been observed in human cells (21).
Human B-lymphocyte lines do express truncated, inactive
iPLA2 sequences that contain the ankyrin repeat domain but
lack the catalytic domain and are thought to arise from alternative
splicing of the transcript (21). Co-expression of the truncated
sequences with full-length human iPLA2 attenuates catalytic
activity (21). Because the active form of iPLA2 is an
oligomeric complex (8, 22) that may result from subunit associations
through ankyrin repeat domains (8), this suggests that formation of
hetero-oligomeric complexes represents a means to regulate
iPLA2 activity (21). That mechanisms of iPLA2
regulation differ among human cell types is suggested by the fact that
stimuli that induce iPLA2-catalyzed arachidonate release
and leukotriene production in human granulocytes fail to induce these
events in human lymphocyte lines, even though both classes of cells
express iPLA2 and leukotriene biosynthetic enzymes (17,
21).
One human cell type in which iPLA2 may be biomedically
important is the pancreatic islet beta cell. Impaired beta cell
survival and signaling functions underlie development of types I and II diabetes mellitus, respectively; these are the most prevalent human
endocrine diseases. In rodent islets, iPLA2 has been
proposed to play a signaling role in glucose-induced insulin secretion (8, 23-25) and in experimentally induced beta cell apoptosis (26).
Human islets express a BEL-sensitive PLA2 activity that does not require Ca2+ (27, 28), but iPLA2
mRNA has not been demonstrated in human islets. We have cloned
human beta cell iPLA2 cDNA here and find that human
islets express mRNA species encoding two iPLA2 isoforms with different sizes (85 and 88 kDa) and catalytic properties. We have
also determined the human iPLA2 gene structure and its chromosomal location and find that the transcript encoding the short
isoform arises from an exon-skipping mechanism of alternative splicing.
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EXPERIMENTAL PROCEDURES |
Materials--
The compounds [32P]dCTP (3000 Ci/mmol), [35S]dATPS (1000 Ci/mmol), and
L-
-1-palmitoyl-2-[14C]arachidonoyl-phosphatidylethanolamine
(50 mCi/mmol) and ECL detection reagents were obtained from Amersham
Pharmacia Biotech, and the BEL
((E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one) iPLA2 suicide substrate was obtained from BIOMOL (Plymouth
Meeting, PA). A human placental genomic DNA library in lambda FIX II
was obtained from Stratagene (La Jolla, CA). Human promonocytic U937 cells (30) were obtained from American Type Culture Collection (Manassas, VA) and cultured as described (20, 31). Sources of other
common materials are identified elsewhere (10, 14, 23-25, 28).
Cloning cDNA Species Containing iPLA2 Sequence
from a Human Insulinoma Cell cDNA Library--
Rat islet
iPLA2 cDNA was isolated (10), labeled with
32P, and used to screen a human insulinoma cDNA library
(32) provided by Dr. Alan Permutt of Washington University. Insert
sizes in clones that hybridized with the probe were determined by
digestion with restriction endonucleases, and their sequences were
determined from the double strand (33). Two cDNA species were
obtained that contained about 1.80 and 1.59 kb, respectively, of the
3'-sequence of human iPLA2 cDNA, including the poly(A)
tail. Neither contained the 5'-end of the full coding sequence, and
RT-PCR was therefore performed with human islet RNA.
Isolation of RNA from Human Islets and Human Promonocytic U937
Cells, Reverse Transcription, and Polymerase Chain
Reactions--
Islets were isolated from human pancreata in the
Washington University Diabetes Research and Training Center (34) and
cultured as described (35). Total RNA was isolated from human islets and promonocytic U937 cells and first strand cDNA prepared by reverse transcription (RT) using standard procedures (36). PCRs were
performed under described conditions (10), and products were analyzed
by agarose gel electrophoresis (36). Primers used to generate the 5'
portion of human iPLA2 cDNA were sense
(5'-GATGCAGTTCTTTGGACGCCTGG-3'), antisense
(5'-T-CAGCATCACCTTGGGT-TTCC-3'), and nested antisense (5-AATGGCCAGGGCCAGGATG-C-3'). Two distinct cDNA fragments were obtained, subcloned, sequenced, and found to extend from the
5'-initiator codon through about 1.59 and 1.79 kb of DNA, respectively.
Preparation of cDNA Species Containing the Complete Human
Islet iPLA2 Coding Sequence--
The cDNA fragments
obtained from screening the human insulinoma cell cDNA library
overlapped at their 5'-ends with 3'-ends of cDNA fragments from
RT-PCR of human islet RNA, and the overlapping region contained an
NcoI site. The fragments were subcloned into pBluescript SK.
Fragments from RT-PCR of human islet RNA contained the
iPLA2 5'-coding sequence and were released from plasmids
with EcoRI and NcoI. Products were isolated by
agarose gel electrophoresis and ligated with a plasmid containing the
3'-end of human iPLA2 cDNA that had been treated with
NcoI. Ligation product plasmids were used to transform
bacterial host cells and sequenced. The resultant cDNA species
contained complete coding sequences of human iPLA2 isoforms
and were inserted into appropriate vectors for expression and used to
prepare 32P-labeled human iPLA2 cDNA for
genomic screening.
Bacterial Expression of Recombinant Human Islet iPLA2
Isoforms--
The cDNA species encoding full-length human islet
iPLA2 isoforms were subcloned in-frame into the
EcoRI and XhoI sites of pET-28c (Novagen). The
constructs were analyzed by restriction endonuclease digestion,
sequenced, and transformed into bacterial expression host BL21(DE3)
(Novagen). Cells transformed with pET28c without insert were negative
controls. Protein expression was induced by treating cells with 0.5 mM isopropyl-1-thio-
-D-galactopyranoside (IPTG) and assessed by SDS-polyacrylamide gel electrophoresis analyses
with Coomassie Blue staining and by immunoblotting under described
conditions (10) with a rabbit polyclonal antibody against recombinant
rat islet iPLA2.
Eukaryotic Expression of Recombinant Human Islet
iPLA2 Isoforms--
The Spodoptera frugiperda
(Sf9) insect cell-baculovirus system used to express other
PLA2 enzymes in catalytically active forms (37, 38) was
used to express human iPLA2 isoforms. The iPLA2
cDNA inserts were released from pBluescript SK plasmids by
digestion with EcoRI and XhoI and subcloned into
Eco RI and Xho I sites of pBAC-1 baculovirus
transfer plasmid (Novagen). Sf9 cells were co-transfected with
this transfer plasmid and linearized baculovirus DNA (BacVector-2000,
Novagen) to construct recombinant baculovirus with human islet
iPLA2 isoform cDNA inserts. Infection and culture were
performed under described conditions (47). At 48 h after
infection, Sf9 cells were collected by centrifugation, washed,
resuspended in buffer (250 mM sucrose, 25 mM
imidazole, pH 8.0), and disrupted by sonication. Cytosolic and
membranous fractions were prepared by sequential centrifugations
(10,000 × g for 10 min and 100,000 × g for 60 min) and used for PLA2 activity assays.
Phospholipase A2 Activity Assays--
The protein
content of Sf9 cell cytosolic and membranous fractions was
determined by Bio-Rad assay, and iPLA2 activity was measured in aliquots (approximately 20 µg of protein) added to assay
buffer (200 mM Tris-HCl, pH 7.0; total assay volume, 200 µl) containing 5 mM EGTA with or without 1 mM
ATP. Some aliquots were pretreated (2 min) with BEL (10 µM) before the assay. Reactions were initiated by
injecting substrate
(L-
-1-palmitoyl-2-[14C]arachidonoyl-phosphatidylethanolamine;
specific activity, 50 Ci/mol; final concentration, 5 µM)
in ethanol (5 µl). Assay mixtures were incubated (3 min at 37 °C),
and reactions were terminated by adding butanol (0.1 ml) and vortexing.
After centrifugation (2000 × g for 4 min), products in
the butanol layer were analyzed by silica gel G TLC in hexane/ethyl
ether/acetic acid (80:20:1). The TLC region containing free arachidonic
acid (RF, 0.58) was scraped into vials, and its
14C content was determined.
Cloning Human iPLA2 Genomic DNA Fragments,
Determination of Intron-Exon Boundaries, and Estimation of Intron
Size--
A 32P-labeled human islet iPLA2
cDNA was used to screen a human placental Lambda FIX II genomic DNA
library (Stratagene). Clones that hybridized with the probe were
isolated and plaque-purified, and the lambda DNA fragments containing
genomic DNA inserts were purified by standard procedures (36). Inserts
were excised with NotI and subcloned into a pBluescript SK
plasmid for restriction site mapping. Sequences of intron-exon
boundaries were determined by comparing sequences of genomic DNA and
cDNA. Intron sizes were estimated from lengths of PCR products from
reactions using genomic DNA as template and primers that hybridize to
sequences in adjacent exons.
Chromosomal Mapping of Human iPLA2 Gene by
Fluorescence in Situ Hybridization--
A human iPLA2
genomic DNA clone was biotinylated with dATP (40, 41) and used as a
probe to map the chromosomal location of the human iPLA2
gene. Fluorescence in situ hybridization (FISH) detection of
the locus of hybridization of the fluorescent probe with chromosomal
DNA was performed by See DNA Biotech Inc. (Downsview, Ontario, Canada)
using described methods (40, 41). Human blood lymphocytes were cultured
in
-minimal essential medium supplemented with 10% fatal calf serum
and phytohemagglutinin at 37 °C for 68-72 h. Bromodeoxyuridine
(0.18 mg/ml, Sigma) was used to synchronize the cell populations, which
were then washed three times with serum-free medium to release the
block and recultured (37 °C, 6 h) in
-minimal essential
medium with thymine (2.5 µg/ml, Sigma). Cells were harvested and
slides were prepared by standard procedures, including hypotonic
treatment, fixation, and air-drying (40, 41), and the slides were baked
(55 °C, 1 h). After RNase treatment, slides were denatured in
70% formamide and dehydrated with ethanol. Probes were denatured
(75 °C, 5 min) in a hybridization mixture (50% formamide, 10%
dextran sulfate, and human cot I DNA). After incubation (15 min,
37 °C) to suppress repetitive sequences, probes were loaded on
denatured chromosomal slides, which were incubated overnight, washed,
and subjected to detection and amplification procedures. FISH signals
and DAPI banding patterns were recorded in separate photographs.
Assignment of FISH mapping data with chromosomal bands was achieved by
superimposing FISH signals with DAPI banding pattern on the chromosomes
(40, 41).
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RESULTS |
Characterization of iPLA2 cDNA from Human
Islets--
To determine whether human pancreatic islet beta cells
express mRNA species encoding iPLA2, a human insulinoma
cell cDNA library (32) was screened with a 32P-labeled
rat iPLA2 cDNA (10) probe. Two clones (INS-C1 and INS-C2) of about 1.59 and 1.80 kb in length, respectively, hybridized to the probe and were sequenced. Both clones contained identical 3'-sequences that included a presumptive polyadenylation sequence and a
poly(A) tail, and their sequences were identical except for additional
5'-sequence in the longer clone not contained in the shorter clone.
Alignment with the rat iPLA2 cDNA sequence indicated
that the clones contained the 3'-end of human iPLA2 cDNA, but neither contained the full 5'-coding sequence (Fig. 1). RNA from human islets was therefore
used as template in RT-PCRs with primers designed from the 5'-sequence
of rat iPLA2 cDNA and from sequence in INS-C1 and
INS-C2 clones. The primers were designed to amplify cDNA from the
initiator methionine codon at the 5'-end through the region of sequence
at the 3'-end recognized by primers designed from INS-C1 and INS-C2
sequences. A nested primer approach was employed in 3'-end primers to
verify specificity of amplification products. When used with the same
5'-primer, one of the 3'-primers was expected to yield a longer product
than the other.

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Fig. 1.
Summary of cDNA fragments used to
construct cDNA species containing the complete coding sequences of
human islet iPLA2 isoforms. The two cDNA clones
obtained by screening the human insulinoma cell cDNA library that
contain the 3'-sequence of the human islet iPLA2 cDNA
are designated INS-C1 and INS-C2. The RT-PCR products obtained using
human islet RNA as template that contain the 5'-end of the human islet
iPLA2 coding sequence (see Fig. 2) are designated human
islet PCR long fragment and human islet PCR short fragment.
Arrows indicate the location of the recognition site for the
restriction endonuclease NcoI that is contained in the
region of overlap between the insulinoma cell cDNA fragments and
the human islet RNA RT-PCR products. The cDNA species containing
the complete coding sequence of the long and short isoforms of human
iPLA2 are designated LH-iPLA2 and
SH-iPLA2, respectively. These cDNA species were
prepared by NcoI digestion and ligation of the insulinoma
cell cDNA fragment and one of the two RT-PCR products derived from
human islet RNA. R-iPLA2, rat islet
iPLA2 cDNA. The region of the 162-bp insert that
distinguishes LH-iPLA2 from SH-iPLA2 is
indicated by the black bar. The remainder of the coding
sequence is indicated by shaded bars. The lighter
shading represents human iPLA2 coding
sequence; the darker shading represents rat
iPLA2 coding sequence.
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RT-PCRs with a given set of these primers using human islet RNA as
template yielded two products (Fig.
2A). The experiments shown in
lanes 1-4 of the agarose gel electrophoretic analysis of
the RT-PCR products (Fig. 2A) were performed with a primer set expected to yield a product of 1.65 kb in length based on the rat
iPLA2 cDNA sequence. Lanes 1 and
3 represent PCRs performed without an RT step to exclude
contamination with genomic DNA, and no amplification products were
observed. Lanes 2 and 4 represent RT-PCRs
performed with two different preparations of human islet RNA, which
both yielded two products. The more intensely staining product
exhibited the 1.65-kb length expected from the rat iPLA2 cDNA sequence. There was also a less intensely staining band at 1.85 kb. Lanes 5 and 6 represent RT-PCRs
performed with human RNA as template, the same 5'-end primer used in
lanes 1-4, and a nested 3'-end primer expected to yield a
shorter product than that obtained with the 3'-end primer used in the
experiments shown in lanes 1-4. Two products were again
observed. The more intensely staining band exhibited the length
expected based on the rat iPLA2 cDNA sequence, and it
was accompanied by another band about 0.2 kb longer.

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Fig. 2.
Agarose gel electrophoretic analyses of
products of RT-PCRs performed with human islet RNA or U937 cell RNA as
template and primers designed to amplify the 5'-end of human
iPLA2 cDNA. RT-PCRs were performed with RNA
isolated from human islets from two different donors (A) or
from two different preparations of human promonocytic U937 cell RNA
(B). In A, experiments shown in lanes 1, 2, and 5 were performed with RNA from islets from donor
1, and experiments shown in lanes 3, 4, and 6 were performed with RNA from islets from donor 2. Reverse transcriptase
was omitted from the reactions analyzed in lanes 1 and
3 to exclude contamination from genomic DNA in the human
islet RNA preparations. In reactions analyzed in lanes 1-4
(A), a set of PCR primers was used that was expected to
yield a single 1.65-kb product, based on the rat islet
iPLA2 cDNA sequence. In reactions analyzed in
lanes 5 and 6 (A), the same 5'-primer
was used as in the reactions analyzed in lanes 1-4, but a
different 3'-primer was used that was expected to yield a shorter
product, based on the rat iPLA2 cDNA sequence. The
sequences of the 5'-primer and of the two 3'-primers used in these
reactions are specified under "Experimental Procedures." In
B, experiments shown in lanes 1 and 2 were performed with RNA from U936 cell preparation 1, and experiments
shown in lanes 3 and 4 were performed with RNA
from U937 cell preparation 2. Reverse transcriptase was omitted from
the reactions analyzed in lanes 1 and 3 (B). In reactions analyzed in lanes 1-4
(B), the set of PCR primers was the same as that in
lanes 1-4 of A. Both of the RT-PCR products
visualized in lanes 2 and 4 (B) were
subcloned and sequenced, and the results were identical to those for
the products in lanes 2 and 4 of
A.
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The 1.65- and 1.85-kb human islet RT-PCR products in Fig. 2A,
lanes 2 and 4, were subcloned and sequenced. Each
contained a 5'-coding sequence that specified an amino acid sequence
highly homologous to the N-terminal portion of rat iPLA2.
The nucleotide sequences of the two human islet RT-PCR products were
identical except for a 162-bp insert in the longer product that was not contained in the shorter product. This insert did not interrupt the
reading frame and encoded a 54-amino acid insert in the eighth ankyrin
repeat of the iPLA2 amino acid sequence. Similar RT-PCRs using RNA from human U937 promonocytic cells as template and the primer
set employed in Fig. 2A, lanes 1-4, indicated that U937 cells also express two distinct iPLA2 mRNA species
(Fig. 2B), and the lengths of the two RT-PCR products
corresponded exactly to those from human islet RNA. The relative
intensities of the two products differed, however, and staining of the
band for the longer product was more intense than that for the shorter
product when U937 cell RNA was used as template. The converse was true with human islet RNA. The U937 cell RT-PCR products were subcloned and
sequenced and were identical to the products from human islet RNA.
RT-PCRs in Fig. 2 are analogous to competitive PCR (10, 66, 67) and
involve amplification of two distinct cDNA species from the same
primer set in the same reaction mixture. As with competitive RT-PCR,
relative abundances of reaction products in Fig. 2 may reflect the
relative abundances of different cDNA species in the original
reaction mixture.
These findings indicate that some human cells express mRNA species
that encode two distinct isoforms of iPLA2. While these experiments were in progress, cloning of human iPLA2 from
lymphocyte lines and testis was reported (21). That report identified
only one human mRNA species that encoded a full-length
iPLA2 sequence, and it corresponded to the longer isoform
predicted from our results. No mRNA species corresponding to the
shorter human iPLA2 isoform predicted from our results was
observed in human B-lymphocyte lines or human testis, but two mRNA
species, thought to represent alternative splicing products, were
observed that encoded truncated iPLA2 variants that
contained the ankyrin repeat region but lacked the catalytic domain
(21). We sought evidence for expression mRNA encoding these
truncated iPLA2 variants in human islets and human U937
promonocytic cells and observed them in U937 cells but not in islets
(not shown), suggesting that there is heterogeneity among human cells
in expression of products of the iPLA2 gene.
Fig. 3 illustrates nucleotide and deduced
amino acid sequences predicted from our results for cDNAs encoding
the long and short isoforms of human iPLA2. The predicted
amino acid sequence for the long isoform differs from that for the
short isoform only by the presence of a 54-amino acid insert
interrupting the eighth ankyrin repeat. The short isoform is highly
homologous to the hamster, mouse, and rat iPLA2 sequences,
all of which also lack the 54-amino acid insert (8-10). 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
Caenorhabditis elegans (42), which is most closely related
(42) to mammalian Smad4 (43). The proline-rich middle linker region of
Smad4 shares a
PX5PX8HHPX12NX4Q
motif with the corresponding region of DAF-3 and the proline-rich
insert in the long human iPLA2 isoform. In Fig.
4, residues that are identical among the
three sequences are indicated by dark boxes, and residues
with chemically similar side chains are indicated by light
boxes. The Smad4 middle linker domain mediates protein
interactions with signaling partners (43), 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
(54). The proline-rich insert in the long iPLA2 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.


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Fig. 3.
Nucleotide and deduced amino acid sequences
of cDNA species encoding the long and short isoforms of human
iPLA2. Species of cDNA containing the full coding
region and the 3'-untranslated region for transcripts encoding
LH-iPLA2 and SH-iPLA2 were constructed with
cDNA fragments from the INS-C1 and INS-C2 clones and from the long
and short fragments obtained from RT-PCRs with human islet RNA, as
illustrated schematically in Fig. 1. The resultant cDNA species
were subcloned and sequenced. The figure displays the nucleotide
(top row) and deduced amino acid sequences (bottom
row) for the LH-iPLA2 cDNA. The sequences of the
forward primer, the reverse primer, and the nested reverse primer used
in the RT-PCRs with human islet RNA are indicated, as is the location
of the NcoI restriction endonuclease site. The 162-bp insert
that is present in LH-iPLA2 cDNA but absent from
SH-iPLA2 cDNA is underlined. The stop codon
TGA is indicated by an asterisk. The presumptive
polyadenylation signal sequence is indicated in boldface
type.
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Fig. 4.
Comparison of the amino acid sequence of the
proline-rich insert in the long form of human iPLA2 to
sequences in the proline-rich middle linker domains of the Smad
proteins DAF-3 and Smad4. The deduced amino acid sequence between
residues 412-448 for the long isoform of human iPLA2 is
designated H-iPLA2 in the figure. A BLAST search
indicated similarities between this sequence and that of residues
400-434 of the C. elegans DAF-3 Smad protein, which falls
within the proline-rich middle linker domain of that protein. DAF-3 is
most closely related to the mammalian protein Smad4, and residues
275-312 within the proline-rich middle linker domain of the Smad4
sequence are illustrated in the figure. Amino acid residues that are
contained in the iPLA2 sequence and one or both of the
other sequences are illustrated by dark boxes, and residues
with chemically similar side chains are illustrated by light
boxes. In the consensus sequence, residues that are identical
among the three sequences are indicated by underlined,
capitalized Roman letters. Residues that are common to the
iPLA2 sequence and to one but not both of the other
sequences are indicated by capitalized Roman letters that
are not underlined. Positions at which residues with chemically similar
side chains are observed in two or three of the sequences are
designated with Greek letters. Acidic residues (Asp and Glu)
are denoted by ; basic residues (His, Lys, and Arg) by ; neutral,
nonpolar residues (Ala, Phe, Ile, Leu, Met, Pro, Val, and His) by ;
and neutral, polar residues (Gly, Asn, Gln, Ser, Thr, and Tyr) by .
Positions at which there is no similarity among the three sequences are
denoted with an x.
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Fig. 1 summarizes relationships among the human beta cell
iPLA2 cDNA fragments obtained from screening the
insulinoma cell cDNA library and from RT-PCRs with human islet RNA
relative to the predicted sequences for the two full-length
iPLA2 isoforms. The 5'-fragments obtained from RT-PCR
overlap the 3'-fragments obtained from library screening, and within
the region of overlap is a NcoI restriction endonuclease
site. There are no other NcoI sites in the sequences. To
obtain cDNA species with the full coding sequences for the long
(LH-iPLA2) and the short (SH-iPLA2) human islet
iPLA2 isoforms, appropriate 5'- and 3'-fragments were
digested with NcoI, and ligation reactions were performed.
The resultant plasmids were used to transform bacterial host cells and
sequenced. The cDNA species so obtained contained the full coding
sequences of human iPLA2 isoforms; they were inserted into
vectors for expression in bacteria and in Sf9 insect cells and
used to prepare 32P-labeled human iPLA2
cDNA to generate a probe for genomic screening.
Bacterial Expression of Recombinant Human Islet iPLA2
Isoforms--
To demonstrate that the human islet iPLA2
isoform cDNA species encoded proteins of expected sizes, they were
subcloned into expression vector pET-28c, and the resultant constructs
were used to transform bacterial host BL21(DE3). Expression of proteins encoded by cDNA inserts was induced by IPTG treatment, and proteins were analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 5). In IPTG-treated cells, proteins of
the expected sizes, 85 (lane 2) or 88 (lane 4)
kDa, were produced from cDNA for SH-iPLA2 or
LH-iPLA2, respectively, in much greater abundance than in
non-IPTG-treated cells (lanes 1 and 3). Both
proteins were recognized by a polyclonal antibody against rat
iPLA2 (not shown).

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Fig. 5.
Bacterial expression of the long isoform and
short isoform human islet iPLA2 proteins. The cDNA
species containing the complete coding regions of the short isoform
(lanes 1 and 2) or the long isoform (lanes
3 and 4) of human islet iPLA2 were
subcloned into the bacterial expression vector pET-28c (Novagen). The
pET28-iPLA2 constructs were then used to transform the
bacterial expression host BL21(DE3) (Novagen). Expression of proteins
encoded by the cDNA inserts was induced by treating the cells with
IPTG, and proteins expressed by induced (lanes 2 and
4) and noninduced (lanes 1 and 3)
cells were compared by SDS-polyacrylamide gel electrophoresis analyses
with Coomassie Blue staining. The expected molecular mass of the short
isoform of human islet iPLA2 is 85 kDa (lane 2),
and that of the long isoform of human islet iPLA2 is 88 kDa
(lane 4).
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Eukaryotic Expression of Recombinant Human Islet iPLA2
Isoforms--
To determine whether human islet iPLA2
isoform cDNA species encoded catalytically active enzymes, a
baculovirus vector-Sf9 cell system was used in which other
PLA2 enzymes have been expressed (38, 39). Recombinant
baculovirus that contained inserts encoding LH-iPLA2 or
SH-iPLA2 were used for infection, and subcellular fractions
from infected cells were assayed for iPLA2 activity. Uninfected Sf9 cells exhibited no detectable iPLA2
activity, but such activity was observed in cytosolic and membranous
fractions of cells infected with baculovirus that contained cDNA
inserts encoding LH-iPLA2 or SH-iPLA2 (Fig.
6). The iPLA2 activities
expressed in cells infected with baculovirus that contained cDNA
encoding either human islet iPLA2 isoform were inhibited by
the iPLA2 suicide substrate (4, 8-12) BEL (Fig. 6). We
believe this to be the first demonstration that recombinant human
iPLA2 is inhibited by BEL, as this issue was not examined
in a recent report on human iPLA2 cloned from lymphocyte
lines and testis (21). Activities of recombinant LH-iPLA2
and SH-iPLA2 were affected differently by 1 mM
ATP (Fig. 6). ATP exerted a stimulatory effect on LH-iPLA2 activities in cytosol or membranes but did not affect
SH-iPLA2 activities. ATP has been reported to stimulate
iPLA2 activities from rat islets (10) and murine P388D1
cells (9) but not to affect the iPLA2 activity of Chinese
hamster ovary cells (8). These findings indicate that
cDNA species for both LH-iPLA2 and SH-iPLA2
encode catalytically active enzymes and that catalytic properties of
the two human iPLA2 isoforms differ. The experiments also
suggest that the ratio of membranous to cytosolic activity may differ
somewhat for LH-iPLA2 and SH-iPLA2 under
these assay conditions (Fig. 6).

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Fig. 6.
Catalytic activities of recombinant long and
short isoforms of human islet iPLA2 expressed in Sf9
cells. Recombinant baculovirus that contained cDNA inserts
encoding either the long or short isoforms of human islet
iPLA2 were prepared and used to infect Sf9 cells. At
48 h after infection, subcellular fractions were prepared from the
Sf9 cells and assayed for iPLA2 activity with a
radiolabeled phospholipid substrate. Uninfected Sf9 cells
exhibited no detectable iPLA2 activity in either cytosol
(Cont.-Cyt.) or membranes (Cont.-Mem.). Activity
was observed in both cytosolic (S-Cyt. and
L-Cyt.) and membranous (S-Mem. and
L-Mem.) fractions of cells infected with baculovirus that
contained cDNA inserts encoding LH-iPLA2 or
SH-iPLA2, and activity was also observed in both
subcellular fractions when cells were co-infected with baculovirus
mixtures that contained both the LH-iPLA2 and
SH-iPLA2 cDNA inserts (S+L-Cyt. and
S+L-Mem.). The iPLA2 activities expressed in
cells infected with baculovirus that contained cDNA encoding either
human islet iPLA2 isoform were susceptible to inhibition by
the iPLA2 suicide substrate BEL (filled bars).
ATP (1 mM) was included in some assay solutions
(hatched bars). Open bars, no ATP.
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Characterization of the Human iPLA2 Gene--
To
explore the basis for producing human islet mRNA species that
encode the two distinct iPLA2 isoforms, the structure of the human iPLA2 gene was determined. A
32P-labeled LH-iPLA2 cDNA was used as probe
to screen a human Lambda FIX II genomic DNA library. Eight genomic
clones with overlapping regions of sequence were isolated and analyzed
by Southern blotting and restriction endonuclease digestion. Fig.
7 is a schematic representation of the
human iPLA2 gene structure. The cloned sequence spans over
60 kb of DNA and includes 16 exons representing 5'-untranslated region,
the entire coding sequence, and 3'-untranslated region of the
LH-iPLA2 transcript. Intron sizes were estimated from the length of PCR fragments produced by using genomic DNA as template and
primers that hybridize with sequences in adjacent exons. The sequences
of intron-exon boundaries were determined by comparing the sequences of
genomic DNA and cDNA. Table I
summarizes the sequences at the 3'-acceptor sites and the 5'-donor
sites at these boundaries. In each case, the intron sequence at the
5'-boundary of the exon ended in the dinucleotide AG and that at the
3'-boundary of the exon began with the dinucleotide GT, conforming to
recognized rules for sequences at such junctions (29).

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Fig. 7.
Schematic representation of the structure of
the human iPLA2 gene and its restriction endonuclease
map. The line at the top of the diagram
indicates the scale in kb. There is an interruption in the scale
between 0 and 25 kb because of the long length of the first intron. The
locations of cleavage sites for restriction endonucleases are
illustrated beneath the scale line. Below the summary of endonuclease
sites, the lines designated HG6, HG5, HG4, HG7, HG3, and
HG8 represent the regions of sequence contained in separate
genomic clones (HGn) obtained from screening a human genomic library
with 32P-labeled cDNA containing the full coding
sequence of the long isoform of human islet iPLA2 and the
3'-untranslated region of its transcript. The genomic clones span over
60 kb of DNA and contain 16 exons represented in the cDNA for the
long isoform of human iPLA2 that include 5'-untranslated
region, the entire coding region, and 3'-untranslated region. The
line below the genomic clone lines represents the locations
of exons, which are identified by black rectangles, and the
approximate lengths of the intervening introns are indicated by the
lengths of the lines between the exons. The dark portions of
the rectangles representing the exons indicate coding regions, and the
unshaded portions of the rectangles representing exons 1 and
16 indicate untranslated regions. In the lower portion of
the diagram, the region of the gene that includes exons 7-10 and the
intervening introns is represented on an expanded scale, and the number
of base pairs in each exon in this region is indicated. The regions of
the gene that are included in four recognized splice-variant products
iPLA2 gene are illustrated schematically in the
bottom four lines. The human islet LH-iPLA2
isoform transcript contains exons 1-16 but not the alternate exons 8b
and 9b. The human islet SH-iPLA2 isoform transcript
contains exons 1-7 and 9-16 but not exon 8 or alternate exons 8b or
9b. Two iPLA2 splice variants have been reported by others
in human B-lymphocyte lines (21). The transcripts for these variants
contain intron sequences that result in premature stop codons and
encode truncated forms of iPLA2 that contain the ankyrin
repeat domain but lack the catalytic domain. These variants are
designated human B-lymphocyte ankyrin-iPLA2-1
and human B-lymphocyte ankyrin-iPLA2-2. The
open rectangles reflect the sites of the intron sequences
that are contained in these truncation variants, and the location of
these intron sequences in the iPLA2 gene are designated by
sites 8b and 9b.
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Table I
Exon-intron boundary sequences of the human iPLA2 gene
The sequences of exon-intron boundaries were determined by comparing
the sequences of genomic DNA and of human islet LH-iPLA2
cDNA. The table displays the 10 nucleotides at the 5'-end and at
the 3'-end of each identified exon in the human iPLA2 gene and
that portion of the nucleotide sequences of the immediately adjacent
introns that form junctions with the exons. Alternate exons 8b and 9b
are not observed in the transcripts for human islet LH-iPLA2 or
SH-iPLA2 but are observed in transcripts encoding catalytically
inactive, truncated iPLA2 variants in human lymphoma cell lines
(21). Exon sequence is represented by upper case and intron sequence by
lower case letters.
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Alternatively Spliced mRNA Species Encoding Long and Short
Isoforms of Human iPLA2--
The 54-amino acid insert
interrupting the last ankyrin repeat in the LH-iPLA2
isoform corresponds exactly to the amino acid sequence encoded by exon
8 of the human iPLA2 gene. This indicates that mRNA
encoding the SH-iPLA2 isoform arises from an exon-skipping mechanism of alternative splicing (29) in transcription of the iPLA2 gene. Different mechanisms of alternative splicing
are involved in producing iPLA2 truncation variants
observed in human B-lymphocyte lines (21), as illustrated in Fig. 7.
The variants contain additional sequence arising from introns that
results in premature stop codons, and they encode truncated proteins
that contain the ankyrin repeat domain but lack the iPLA2
catalytic domain. The locations within the human iPLA2 gene
of intron sequences in the transcripts for the truncation variants were
determined from PCR experiments using primers designed from the
identified exon sequences and from the published (21) sequences of
cDNA species encoding the truncation variants. The truncation
variant human B-lymphocyte ankyrin-iPLA2-1 contains
sequence from the intron between exons 9 and 10. The truncation variant
human B-lymphocyte ankryin-iPLA2-2 contains sequence from
two intron regions. The first resides between exons 8 and 9 and the
second between exons 9 and 10. The second region of intron sequence
occurs in transcripts encoding each of the truncation variants (Fig.
7). Table I indicates the sequences at the intron-exon junctions for
these alternate exons.
Chromosomal Localization of Human iPLA2 Gene--
To
determine the location of the iPLA2 gene on human
chromosomes, a clone identified in screening the human genomic DNA
library with LH-iPLA2 cDNA was biotinylated to generate
a probe for FISH experiments with human lymphocyte chromosomes (40,
41). Using this probe, 91 of 100 examined mitotic figures exhibited
fluorescent signals on one pair of chromosomes (Fig.
8), indicating a hybridization efficiency
of 91%. The human chromosomes were identified by their DAPI banding
patterns (40, 41), and these patterns were correlated with the site of
fluorescent signal from biotinylated probe. Such comparisons indicated
that the iPLA2 gene resides on human chromosome 22. A
detailed positional assignment achieved from analyses of 10 photographs
indicated that the iPLA2 gene resides in region q13.1 of
chromosome 22 (Fig. 8). No other loci of hybridization of the probe
were observed.

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Fig. 8.
Localization of the human iPLA2
gene to chromosome 22q13.1 by fluorescence in situ hybridization.
A genomic clone identified in screening the human genomic DNA library
with the iPLA2 cDNA was biotinylated to generate a
probe for FISH experiments with human lymphocyte chromosomes. Using
this probe, 91 of 100 examined mitotic figures exhibited fluorescent
signals on one pair of chromosomes. The white arrowhead in
the left panel indicates the location of the two intense
fluorescent spots reflecting hybridization of the probe with each
member of the chromosome pair. The center panel illustrates
the DAPI staining pattern of the same mitotic figure and identifies the
chromosome as number 22. The diagram in the right panel
illustrates regions of human chromosome 22 determined by DAPI banding
patterns. These patterns were correlated with the site of fluorescent
signal from the biotinylated iPLA2 genomic clone. A
detailed positional assignment was achieved from analyses of 10 photographs from different preparations. The black circles
indicate the location of the probe observed in each of the 10 experiments. In each case, the probe localized to region q13.1 of human
chromosome 22. No other loci of hybridization of the biotinylated probe
were observed.
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DISCUSSION |
Our results indicate that human pancreatic islets express mRNA
species encoding two distinct, catalytically active isoforms of
iPLA2 that are distinguishable by size and by their
susceptibility to activation by ATP. These two mRNA species are
also observed in human U937 promonocytic cells. These two human
iPLA2 isoforms differ only by the presence of a 54-amino
acid insert in the longer isoform that is absent from the shorter
isoform, and the deduced amino acid sequences of the two isoforms are
otherwise identical. This insert is encoded in its entirety by exon 8 of the human iPLA2 gene, which resides in region q13.1 of
human chromosome 22, indicating that the mRNA species encoding the
short iPLA2 isoform arises from an exon-skipping mechanism
of alternative splicing. The mRNA encoding the short
iPLA2 isoform has not previously been identified in human
cells, and we believe our studies are the first to demonstrate
expression of iPLA2 mRNA by human islets or U937 cells
and to examine effects of BEL and ATP on activities of recombinant
human iPLA2 enzymes.
The demonstration that both human islets and U937 cells express
iPLA2 mRNA species that encode BEL-sensitive enzymes is
of interest in the context of recent reports suggesting that
iPLA2 may participate in apoptosis in both U937 cells (20)
and in islet beta cells (26). U937 cells express the protein Fas on their surfaces (20), and ligation of Fas molecules with the protein Fas
ligand or with agonistic anti-Fas antibodies induces a cell death
program that involves activation of caspase intracellular proteases
(44-46). U937 cells also express a PLA2 activity distinct from cPLA2 or sPLA2 that does not require
Ca2+ and exhibits a profile of sensitivity to inhibitors
such as BEL and methyl arachidonylfluorophosphate that is similar to
that of iPLA2 (20). Our demonstration that U937 cells
express iPLA2 mRNA indicates that this PLA2
activity may reside in the iPLA2 protein. Anti-Fas
antibodies induce U937 cell apoptosis and hydrolysis of arachidonic
acid from cell membranes (20). During this process, cPLA2
is proteolytically inactivated by caspases, but iPLA2
activity is preserved (20). Inhibitors of iPLA2 both
suppress Fas antibody-induced arachidonate release from U937 cells and
retard cell death (20), suggesting that iPLA2 may
participate in apoptosis.
Similarly, stimuli that induce Ca2+ store depletion in
islet beta cells induce apoptosis by a mechanism that does not require a rise in cytosolic [Ca2+] but that does require
hydrolysis of arachidonic acid from membrane phospholipids and its
conversion to 12-lipoxygenase metabolites (26). Ca2+ store
depletion-induced hydrolysis of arachidonic acid from islet phospholipids also does not require a rise in cytosolic
[Ca2+] and is mediated by a BEL-sensitive phospholipase
(47), such as iPLA2. Although phosphatidate
phosphohydrolase is also inhibited by BEL (48), the phosphatidate
phosphohydrolase inhibitor propranolol (49) does not block
Ca2+ store depletion-induced release of arachidonate from
islet phospholipids (47), suggesting that iPLA2 may mediate
this phenomenon. Interleukin-1 also induces accumulation of
non-esterified arachidonate and its 12-lipoxygenase products in islets
by a BEL-sensitive mechanism (50), and interleukin-1 induces apoptosis
of human islet beta cells through Fas-mediated events (51). In the
context of these observations, our findings that human islets express
iPLA2 mRNA raise the possibility that iPLA2
might participate in Fas-mediated apoptosis in human beta cells in a
manner similar to that in U937 cells (20). Beta cell apoptosis is
thought to contribute to development of diabetes mellitus (52).
The amino acid sequence of the insert that distinguishes the long and
short isoforms of human iPLA2 is of interest in the context
of the potential involvement of iPLA2 in apoptosis. This insert shares a
PX5PX8HHPX12NX4Q
consensus motif with the proline-rich middle linker domains of the
C. elegans Smad protein DAF-3 (42) and the mammalian protein
Smad4 (43). Smad proteins participate in controlling cell proliferation
and apoptosis and form hetero-oligomers with signaling partners (54),
via the proline-rich middle linker domain in the case of Smad 4 (43).
Smad4 and Smad2 are products of tumor-suppressor genes that are deleted
or mutated in some human carcinomas (54-58). Studies of allelic losses
in human breast and head and neck carcinomas indicate that a tumor
suppressor gene(s) resides on human chromosome 22q13.1 (59, 60), which is the chromosomal location of the human iPLA2 gene. If
iPLA2 does participate in apoptosis (20, 26), it might
exert tumor suppressor activity.
The active form of iPLA2 appears to be an oligomer of
interacting protein subunits. Radiation inactivation studies of
iPLA2 activity in crude cytosol indicate a size of 337 kDa
for the active complex (22).The iPLA2 activity in crude
cytosol also migrates with an apparent molecular mass of 250-350 kDa
on gel filtration chromatography (8, 22, 61), and this is also the case
for the iPLA2 activity of the purified 85-kDa
iPLA2 protein (8). This has been taken to suggest that the
active form of iPLA2 is an oligomer of 85-kDa subunits and
that the subunits may associate with each other via their ankyrin
repeat regions (8) because such ankyrin repeats participate in many
other protein-protein interactions (53). Consistent with this
possibility is the observation that iPLA2 deletion mutants
lacking the ankyrin repeat domain but retaining the catalytic domain
are catalytically inactive (8). In the long isoform of human
iPLA2, the last iPLA2 ankyrin repeat is
interrupted by a proline-rich insert with some similarities to the
Smad4 domain that mediates interactions with signaling partners (43).
This raises the possibility that the proline-rich insert in human
iPLA2 might allow it to interact with proteins not
recognized by the short isoform of iPLA2.
That the long isoform of human iPLA2 can form
hetero-oligomers with altered catalytic properties is suggested by the
finding that activity of this protein is reduced when it is
co-expressed with truncated iPLA2-like proteins that retain
the ankyrin repeat domain but lack the catalytic domain (21). These
truncated iPLA2 variants arise from alternatively spliced
transcripts that contain intron sequences that result in a premature
stop codon, and these transcripts are expressed in human lymphoma cell
lines (21). These lymphoma cell lines do not express mRNA species
encoding the catalytically active short isoform of iPLA2
observed in human islets that arises from another mechanism of
alternative splicing of the transcript from the iPLA2 gene.
Human islets do not express mRNA species encoding the truncated
iPLA2 variants observed in human lymphoma cells. In
contrast, human U937 promonocytic cells express mRNA species
encoding both the long and short isoforms of catalytically active
iPLA2 and also express mRNA species encoding an
iPLA2 truncation variant. This indicates that there is
heterogeneity among human cells in expression of iPLA2 gene
products that arise from alternative splicing.
The presence of two distinct domains that might mediate protein-protein
interactions in the long isoform of human iPLA2 could cause
it to interact with a variety of other proteins. Various participants
in hetero-oligomeric complexes with iPLA2 have been suggested to alter iPLA2 catalytic properties (21, 47,
61-64). These include calmodulin (63), which physically interacts with and negatively modulates the activity of recombinant 85-kDa
iPLA2 cloned from Chinese hamster ovary cells (64) and rat
islets (47). This has been offered as one explanation of why
Ca2+ store depletion activates iPLA2 (65).
Although the mechanism underlying this effect may be complex, the
effect itself has occurs in vascular myocytes (64), pancreatic islet
beta cells (47), and human granulocytes (17). Ca2+ store
depletion also activates hydrolysis of arachidonate from phospholipids
in differentiated human U937 promonocytic cells by a mechanism that
does not require a rise in cytosolic [Ca2+] (31). Our
demonstration that U937 cells express mRNA species encoding
iPLA2 isoforms suggest that iPLA2 is one
candidate for mediating Ca2+ store depletion-induced
arachidonate release in those cells. The differences in products of the
iPLA2 gene expressed in specific human cells suggest that
iPLA2 regulation might be complex, as also indicated by the
fact that stimuli that induce iPLA2-catalyzed arachidonate
release and leukotriene production in granulocytes fail to induce these
events in lymphocytes, even though both classes of cells express
iPLA2 and leukotriene biosynthetic enzymes (17, 21).