From Lilly Research Laboratory, Indianapolis, Indiana 46285
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ABSTRACT |
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Two new cloned human cDNAs encode paralogs of
the 85-kDa cytosolic phospholipase A2
(cPLA2). We propose to call these cPLA2 Enzymatic breakdown of glycerophospholipids is carried out by
numerous pathways, with the production of various bioactive lipids and
fatty acids (1-8). Diverse phospholipase A2
(PLA2)1 enzymes
hydrolyze the sn-2 bond of phospholipids, releasing
lysophospholipids and fatty acids (9-11). Often the released fatty
acid is arachidonic acid, whose further metabolism gives rise to
several types of bioactive lipids known as eicosanoids, many of which
mediate inflammation. Thus PLA2 enzymes initiate the
production of inflammatory mediators (12, 13), and for that reason they
have become targets for the development of anti-inflammatory therapies.
The 85-kDa cytosolic phospholipase A2 (reviewed in Refs.
12-15) has attracted special interest because it is the only one of numerous PLA2s that selectively releases arachidonic acid
over other fatty acids (16, 17). Recent results with transgenic mice
ablated for this enzyme (18) have demonstrated its role in allergy and
parturition. Several functional regions have been identified within its
amino acid sequence, including the C2 or calcium and lipid binding
region similar to the C2 regions of other proteins (19) such as the
calcium-dependent protein kinase C enzymes; several serine
residues capable of activation through phosphorylation (20-22); a
hydrophilic region that may have a structural role (13); and several
residues essential for catalysis, identified by mutagenesis
(23-25).
The cDNA for the 85-kDa cPLA2 shows no similarity with
the sequences of other known PLA2 enzymes, and genomic
Southern blots reveal no closely related family members (16, 26). Since
the cloning of the 85-kDa cPLA2 (cPLA2 Cloning, cDNA Expression, and
Assays--
cPLA2
cPLA2
The full-length cDNAs (spliced form for cPLA2
For Northern analysis, human multiple tissue RNA blots
(CLONTECH) were probed with 2 × 106 cpm/ml 32P random primer-labeled
cPLA2
The caspase catalytic histidine consensus motif (Fig. 1) of
cPLA2
The liposome assays for PLA2 activity were carried out as
previously published (23, 30). The enzymes were assayed in extracts of
baculovirus-infected insect cells without centrifugation but otherwise
as described (23). In this assay the activity generally seen for
purified cPLA2
Chromosomal locations were derived from nucleotide sequence matches to
STS sequences found on the NCBI Human Gene
Map.2 The genes mentioned for
chromosome 15 were listed within the region between markers D15S118 and
D15S123 [32-45 cM] or between D15S118 and D15S1016 [32-47 cM],
and those for chromosome 19 were between D19S219 and D19S418 [69-97
cM]. The calmodulin STS mapping on chromosome 19 bears the designation
stSG360 and the Swissprot accession number is P02593.
Sequence Determination and Computer Analysis--
The initial
EST (expressed sequence tag) sequences were detected using BLAST
analysis (28). The GCG (31) software was used throughout the work, as
well as Sequencher for interpretation of DNA sequencing results. All
DNA sequencing was done by either of two methods: 1) automated analysis
using an Applied Biosystems model 377 DNA sequencer, or 2) standard
radioactive analysis using Sequenase reagents from U. S. Biochemical
Corp. All sequence data reported were derived from comparison of at
least 3 times coverage in coding regions (and often more) and at least
2 times coverage in noncoding regions, all of which included data from
both strands. The exon/intron structure shown in Fig. 6 for
cPLA2
The 27-bp repeat in cPLA2 Three cPLA2s Appear Evolutionarily Related--
The EST data base
of cDNA fragment sequences has grown rapidly to include by now
cDNA fragments from a large fraction of human mRNAs.
Examination of this data base revealed two ESTs bearing significant
hints of similarity to the 85-kDa cPLA2, referred to in
this paper as cPLA2
This alignment shows that the three cPLA2 enzyme sequences
all have about 30% identity to each other and that the C2 calcium and
lipid binding domain of cPLA2 Expression, Enzyme Activity, and Chromosomal
Locations--
Northern analysis (Fig.
2) shows that messenger RNAs for all
three cPLA2 enzymes are expressed widely, although in most
tissues at a low level. The control cPLA2
Both cPLA2
For an initial survey of enzyme activity, insect cell extracts were
assayed for enzymatic activity with
1-palmitoyl-2-arachidonoyl-phosphatidylcholine liposomes. Table
I shows that while cPLA2
Fig. 4A shows the cDNA
sequence and its translation for cPLA2
BLAST analysis against the GenBankTM data base showed that
cPLA2 Sensitivity to Calcium--
In Fig. 1 we can see that
cPLA2
Although the activity of cPLA2 cPLA2 Catalytic Activity--
Table III
shows the loss of cPLA2
A similar analysis of the corresponding conserved residues of
cPLA2 A considerable variety of phospholipids is present in cells and
tissues, considering combinations of head groups, fatty acids, and
linkages (ester, ether, and alkyl). It is unlikely that a full
detailing of the catalytic repertoires and biological roles of these
three enzymes will be accomplished quickly, given such a wide range of
possible substrates. We expect that interesting functional differences
will be discovered regarding these enzymes as their enzymatic and
kinetic properties become fully characterized.
cPLA2
After much previous work (39) establishing the existence of a
calcium-insensitive PLA2 activity in heart tissue, it is
most interesting to find cPLA2
The 27-bp imperfect palindrome shown in Fig. 4A appears in
the 5'-untranslated region of cPLA2 cPLA2
It is a mystery why the mRNA for cPLA2
The intron splice sites (Fig. 6B) were seen to be all
similar to the normal GT-AG consensus sequences (36), except for intron 2. Comparison (not shown) of the intron 2 sites to other minor consensus sequences (36) showed that they were not similar to the
standard AT-AC consensus or the other known minor forms. Seven cDNA
fragments from rat cPLA2
It may be suspected that cPLA2 Phosphorylation--
cPLA2 Chromosomal Locations--
The three cPLA2s are all on
different chromosomes, 1, 15, and 19; the calcium-independent
iPLA2 can be mapped to human chromosome 22 (see
"Experimental Procedures"); and the 14-kDa secreted
cPLA2s are spread further still (10). However, as
functionally related genes are sometimes clustered together in the
genome, it is notable that within the same region of human chromosome
15 as cPLA2
The vicinity of cPLA2 Catalysis and Conservation--
As noted above, all four
catalytically essential amino acids are strictly conserved in
cPLA2
Comparison of these cPLA2 family members makes it evident
that there is an additional significant conserved region in all three
cPLA2s, lying between amino acids 350 and 400 in
cPLA2 Conclusions--
As analysis of the human genome accelerates,
protein families are rapidly expanding, and searches for possible new
members are becoming more fruitful. However, whereas estimates are that well over 70% of human genes are now represented in the EST DNA data
bases, it is likely that genes with very low levels of expression or
highly selective tissue distributions will elude discovery until even
after genomic DNA sequencing is complete. There may be additional
paralogs of cPLA2, since the present ones were only found
because their traces of homology could be recognized in the EST data
base as being significant. Exploration of gene families has yielded
much information about structure-function relationships and
understanding of molecular mechanisms and biological roles. Comparison
of these three cPLA2 paralogs is likely to advance our
understanding of lipid mediator and signaling pathways, which reach
into many important biological and disease categories.
(114 kDa) and cPLA2
(61 kDa), giving the name
cPLA2
to the well known 85-kDa enzyme.
cPLA2
mRNA is expressed more highly in cerebellum and pancreas and cPLA2
more highly in cardiac and
skeletal muscle. Sequence-tagged site mapping places
cPLA2
on chromosome 15 in a region near a
phosphoinositol bisphosphate phosphatase. The mRNA for
cPLA2
is spliced only at a very low level, and Northern blots in 24 tissues show exclusively the unspliced form.
cPLA2
has much lower activity on
2-arachidonoyl-phosphatidylcholine liposomes than either of the other
two enzymes. Its sequence contains a histidine motif characteristic of
the catalytic center of caspase proteases of the apoptotic cascade but
no region characteristic of the catalytic cysteine. Sequence-tagged
site mapping places cPLA2
on chromosome 19 near
calmodulin. cPLA2
lacks the C2 domain, which gives
cPLA2
its Ca2+ sensitivity, and accordingly
cPLA2
has no dependence upon calcium, although
cPLA2
does. cPLA2
contains a prenyl
group-binding site motif and appears to be largely membrane-bound.
cPLA2
residues activated by phosphorylation do not
appear to be well conserved in either new enzyme. In contrast, all
three previously known catalytic residues, as well as one additional
essential arginine, Arg-566 in cPLA2
, are conserved in
both new enzyme sequences. Mutagenesis shows strong dependence on these
residues for catalytic activity of all three enzymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in
this report) in 1991 (16, 26) a number of enzymes with PLA2
activity have been isolated (9). This report describes two new family
members that were molecularly defined through examination of DNA data
bases and subsequent full-length cloning. One of these new enzymes
lacks the C2 domain, but some of the other above-listed features are found in both new enzymes, called here cPLA2
and
cPLA2
. While this paper was under review a report (27)
appeared describing cPLA2
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was originally detected as the human
brain EST sequence with GenBankTM accession number R20583,
in a BLAST analysis (28). This EST was obtained from IMAGE and
sequenced. By using 5'-rapid amplification of cDNA ends (RACE) and
3'-RACE with CLONTECH's human brain "Marathon" cDNA, more fragments were obtained which showed additional regions of homology to cPLA2
and also showed evident introns,
judging by the large number of in-frame terminator codons, much greater length between homologous elements, and loss of alignment with cPLA2
. Isolation of several additional fragments by
traditional cDNA cloning from human brain and pancreas cDNA
libraries failed to yield properly spliced sequences. PCR was used to
amplify cDNA from the evidently rare mature spliced mRNA that
was represented in the same CLONTECH brain
cDNA, using the following primers bearing EcoRI and
NotI restriction sites (lowercase) and matching the termini
of our cPLA2
sequence (uppercase):
gagggaattcCTTCATGATGCCAGCTGAGCGCCGCC for the 5' end and
ggtggcggccgcCCCGGCCATCAGTGGGGCCTGCGC for the 3' end. The
5'-untranslated region of the cPLA2
mRNA was
difficult to determine, since two separate 5' fragment clones that were amplified by 5'-RACE appeared, after sequencing, to represent inverted
3' termini of unrelated mRNAs accidentally fused to the cPLA2
cDNA.
was discovered in a BLAST analysis as the human
brain EST with GenBankTM accession number N56796 encoding a
sequence similar to that just C-terminal to the Asp-549 catalytic
region of cPLA2
. Using 5'-RACE with the
CLONTECH brain cDNA, the additional 5' sequence obtained had homology with cPLA2
and was identical to
another human brain EST with GenBankTM accession number
R61677. Further sequencing of R61677 revealed that this IMAGE clone
contained the entire coding region for cPLA2
.
) were
subcloned and introduced into baculovirus DNA by recombination as
described (23). Significant amounts of recombinant proteins were
produced (Fig. 3). Mutant forms of cPLA2 cDNAs,
including those for the His-tagged cPLA2s, were each
constructed and confirmed by sequence analysis also as described (23).
The His-tagged cPLA2 forms were modified at the C or N
terminus as follows (last or first 3 amino acids of each
cPLA2 are in lowercase): cPLA2
, ...
rphHHHHH, and cPLA2
, MHHHHHHAmgs. . . . . Western
analysis of those proteins was done as described (23) using the
penta-His antibody purchased from Qiagen.
, cPLA2
, cPLA2
, or
-actin cDNA probes, using the procedure recommended by the
manufacturer (CLONTECH).
was found by comparison of the sequence to the
Prosite data set with the GCG program Findpatterns. The pattern match is nearly perfect, matching 6 of the 7 specified residues exactly, and
having alanine (A) where the consensus (27) calls for a large
hydrophobic [LIVMF]: cPLA2
= ...
HKLSDQREALSHG ...
consensus = H-X(2,4)-[SC]-X(4)-[LIVMF](2)-[ST]-H-G,
using notation in which parentheses indicate a number or range of
repeated residues; X represents any residue; and square
brackets indicate alternative residues for one position. During the
writing of this paper, we completed the analysis of one additional EST
cDNA clone (GenBankTM accession number R25833) which
provided an extension of the 5'-untranslated region of
cPLA2
, showing by comparison that the other
5'-untranslated region clones we had analyzed contained a 98-bp
unspliced intron. The spliced sequence revealed a continuation of the
open reading frame for 99 aa before the initiator methionine of Fig. 1,
suggesting that the full length of cPLA2
may properly be
1012 aa, particularly in light of the improved Kozak consensus (cagccATGG) at this initiator methionine codon. This
information is included in the DNA sequences filed in the listed
GenBankTM accession numbers AF065215 and AF065216 for
cPLA2
. The added amino acids increase the predicted
protein size to 114,120 daltons, but the protein we expressed here for
most experiments (Fig. 3) should be 103 kDa as stated above. The
additional 5' intron's splice sites also conform quite well to the
splice consensus sequences, leaving as unconventional only intron 2 (see "Results and Discussion"), which should subsequently be called
intron 3.
is 2.2 µmol/min/mg protein. To compare this value qualitatively with those of Table I, we estimate that the
insect cells produce a minimum of 1% of their total protein as cPLA2
(perhaps a bit less for cPLA2
), thus predicting a
specific activity of at least 20 nmol/min/mg total protein in the
lysates for cPLA2
. Thus the values in Table I show that
cPLA2
and cPLA2
are about 2 and 3 orders
of magnitude lower, respectively, than cPLA2
in apparent
specific activity.
was derived from direct comparison of the
sequences of the 8.5- and the 3.0-kb mRNAs.
cDNA was discovered by
comparison of three distinct cDNA clones containing from 1 to 3 tandemly repeated copies of the sequence, with one small variation.
Each clone contained the sequence CTCCGCACCGGATTCCGGAGCACAAG
as shown in Fig. 4; however, in the clones bearing two and three copies of this sequence, the first (or first two) copies, reading from 5' to
3', differed at one position (underlined):
CCTCCGCACCGGATTCCGGAGCCCAAG, bringing them one base closer
to the potassium channel sequence (shown as 27 ic* in Fig.
4B). The iPLA2 mapping was done using BLAST on
GenBankTM accession number MMU88624, the iPLA2
cDNA, which showed highly significant matches in the 98-kb
chromosome 22 genomic fragment AL022322.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. The EST for cPLA2
included the region aligning with the catalytic Ser-228 of
cPLA2
(23), whereas the EST for cPLA2
aligned with Arg-566, which we also knew was required for enzyme
activity.3 Sequencing of
these original ESTs and isolation of overlapping cDNA clones (see
"Experimental Procedures") revealed cDNAs encoding the two
protein sequences compared in Fig. 1.
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Fig. 1.
Amino acid alignment of cPLA2
family members cPLA2 ,
cPLA2
, and
cPLA2
produced by Pileup from the
GCG software. Amino acids conserved in two of the sequences are
indicated with lowercase letters above the
alignment, and uppercase letters indicate residues conserved
in all three sequences. Amino acids essential for cPLA2
enzyme activity are indicated: Arg-200, Ser-228, Asp-549, and Arg-566
(see text). Ser-505 is phosphorylated for activation (20-22). The C2
domain is boxed, and asterisks indicate the 6 residues contacting Ca2+ through side chain oxygen atoms in
the recently solved structure for the cPLA2
C2 (37).
Five of those are preserved in cPLA2
or replaced with
another oxygen-bearing side chain, as in the case of Asp-40 of
cPLA2
, replaced by Thr. The caspase catalytic histidine
motif (see "Experimental Procedures") is underlined in
cPLA2
with the catalytic His corresponding to position
560. Small clusters of basic residues (Lys or Arg) are also
underlined near the C termini of all three proteins. Shown
in bold are the aligned Ser-727 of cPLA2
,
known to be phosphorylated (22), and Ser-537 of cPLA2
.
There is some additional N-terminal sequence (99 aa) to
cPLA2
, not shown here (see "Experimental
Procedures").
has a counterpart in
cPLA2
but not in the shorter cPLA2
. Fig.
1 also shows that there is total conservation, among all three
sequences, of the three previously published catalytically essential
cPLA2
amino acids Arg-200, Ser-228, and Asp-549 (23,
24), as well as Arg-566, which also appears to be essential for
catalysis, since all enzyme activity is lost upon changing Arg-566 to
alanine.3 cPLA2
contains a
mitogen-activating protein kinase motif PXSP, and the
Ser-505 in this motif is phosphorylated, activating the enzyme
(20-22). The PXSP motif is not found in the sequences of either cPLA2
or cPLA2
, and the alignment
in that region is weak, suggesting that the phosphorylation activation
pathway demonstrated for cPLA2
(20-22) may not be
operating for the new enzymes. There are concentrations of basic
residues (underlined) at the C termini of all three protein sequences.
In addition to the two catalytic regions, a third region of salient
conservation, and devoid of gaps, is seen between residues 346 and 396 of cPLA2
.
mRNA was
expressed more evenly than the others, with heart and pancreas having
higher expression (Fig. 2A). cPLA2
mRNA
is expressed strongly in pancreas (Fig. 2B) and most
strongly in cerebellum (Fig. 2D), whereas
cPLA2
mRNA is expressed most strongly in skeletal
muscle and heart (Fig. 2C). The
-actin control (Fig. 2,
E and F) shows that similar amounts of mRNA
were loaded into each lane of the gels. As has been well documented
(32),
-actin has higher expressed alternatively spliced forms in
heart and skeletal muscle, which are seen in Fig. 2E, lanes
1 and 6.
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Fig. 2.
Northern analysis of
cPLA2 ,
cPLA2
, and
cPLA2
cDNA with human
mRNA. Numbers to left indicate
approximate size of transcripts in kb. The tissue sources of the
mRNA are numbered as follows: lane 1, heart; lane
2, brain; lane 3, placenta; lane 4, lung;
lane 5, liver; lane 6, skeletal muscle;
lane 7, kidney; lane 8, pancreas; lane
9, amygdala; lane 10, caudate nucleus; lane
11, corpus callosum; lane 12, hippocampus; lane
13, whole brain; lane 14, substantia nigra; lane
15, subthalamic nucleus; lane 16, thalamus; lane
17, cerebellum; lane 18, cerebral cortex; lane
19, medulla; lane 20, occipital pole; lane
21, frontal lobe; lane 22, termporal lobe; lane 23,
putamen; and lane 24, spinal cord, lanes
9-23, are all from regions of brain. cDNAs used as probes for
each panel are cPLA2
(A);
cPLA2
(B); cPLA2
(C); again cPLA2
(D). Controls
(E and F) were probed with
-actin cDNA.
and cPLA2
proteins were
produced in insect cells as done previously for cPLA2
,
and robust expression was obtained for each enzyme (see "Experimental
Procedures"), producing clearly enhanced bands among the total set of
stained proteins upon SDS electrophoresis (Fig.
3, A and C). The
molecular sizes estimated from these gels, 100 kDa for
cPLA2
and 60 kDa for cPLA2
, agree well
with the sizes predicted from the cDNA coding regions (102,984 daltons for
and 60,948 for
). Because there is a baculovirus protein produced in the infected insect cells that migrate with cPLA2
at 60 kDa, we used immunoblotting of
cPLA2
and cPLA2
bearing His-tag additions
(see "Experimental Procedures") to confirm that the increased
density at 60 kDa was due to cPLA2
(Fig. 3B). The Western analysis of Fig. 3B shows that the appropriate
60- and 100-kDa bands are reactive with the His-tag antibody (as well as some apparent hydrolysis products, ensuring that those bands truly
represent the cPLA2
and -
proteins. The
"full-length" 1012-aa cPLA2
form (see
"Experimental Procedures") in lane 4 of Fig.
3A, shows a band near the expected 114 kDa, although it produced only about the same activity as the 103-kDa form (data not
shown).
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Fig. 3.
Detection of cPLA2
proteins. Sf-9 insect cells were infected with recombinant
baculovirus encoding the proteins indicated, lysed, and the
supernatants analyzed on SDS gels, either by Coomassie staining or by
Western blotting. Numbers to the left indicate
sizes of protein standards in kDa. A and B are
stained and Western-blotted, respectively, of wild-type proteins;
C and D are stained and Western-blotted,
respectively, of mutant cPLA2 proteins; and E
is Western-blotted mutant cPLA2
proteins. The Western
blots are probed with anti-His antibody. The proteins in each numbered
lane are as follows: for A and B, lane
1, cPLA2
-HT; lane 2, cPLA2
-HT; lane 3, infected Sf-9 cell control;
lane 4, cPLA2
-FL (1012 aa; see
"Experimental Procedures"); lane 5, cPLA2
-HT; for C and D, lane
1, cPLA2
; lane 2, cPLA2
histidine-tagged; lane 3, cPLA2
R764A;
lane 4, cPLA2
D747A; lane 5, cPLA2
H560A; lane 6, cPLA2
S467A; lane 7, non-recombinant AcMNPV (baculovirus);
lane 8, uninfected Sf-9 insect cells; lane 9, cPLA2
-HT; and for E (all
cPLA2
-HT except as noted): lane 1, R54A;
lane 2, S82A; lane 3, D385A; lane 4, R402A; lane 5, wild type; lane 6, cPLA2
-HT; lane 7, uninfected Sf9; and
lane 8, Sf9 infected with non-recombinant
baculovirus.
and cPLA2
are catalytically active their enzymatic
activity is several orders of magnitude lower than that of
cPLA2
(see "Experimental Procedures"), despite the
fact that the proteins were present in similar amounts (Fig. 3,
A and B, lanes 1, 2, and 5). In Fig.
3B we see that the overexposed main bands represent the
majority of the cPLA2s in their full-length forms, although
several apparently degraded forms are also seen. For
cPLA2
we know that most of the enzyme protein remained
bound to membranes due to a lipid anchor (27), which may have
restricted access to the labeled liposomes. For cPLA2
the activity, although clearly significantly above the assay
background, was also very low (Table I).
PLA2 activity of cPLA2 and cPLA2
is estimated from
the well established value for the purified form (17, 18, 24). The
Negative control was expressing MAO-B, a protein not known to have
phospholipase A2 activity. The Vector control was Sf9
cells infected with non-recombinant baculovirus. Values are averages
from duplicate assays of two independently infected samples of
Sf9 insect cells.
. An interesting
feature of this cDNA sequence is a 27-bp imperfect palindrome found
before the initiator codon, indicated in Fig. 4A by
underlining. This 27-bp segment was found repeated 2 or 3 times in tandem in separate additional cDNA clones encoding cPLA2
, apparently a result of alternative mRNA
splicing.
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Fig. 4.
A, cDNA for cPLA2 and its
translation. cPLA2
is predicted to be a 541-aa protein
encoded by a mRNA of at least 2472 nucleotides. The translation is
shown above the DNA sequence. Single underlining
denotes the 27-bp partial palindrome (see text), the initiator ATG
codon, and the AATAAA polyadenylation signal. Double
underlining shows the CAAX box at the C terminus of the
protein sequence. B, repeated palindrome from the
5'-untranslated region of cPLA2
. 27 bp
represents the sequence from cPLA2
, and 27 ic
is its inverted complement, aligned to show its palindromic property.
The inverted complement 27 ic* (1-base variant, see
"Experimental Procedures") aligns with the forward orientation of
the (identical) initiator regions from human and mouse potassium
channel protein cDNAs, listed as their GenBankTM
accession numbers U04270 and AF012868, respectively.
and cPLA2
both have exact matches
in STS sequence entries, corresponding to mapped locations on human
chromosomes 15 (STS WI-13757 matches cPLA2
) and 19 (STS
WI-14200 matches cPLA2
), respectively. Thus since
cPLA2
has been mapped to human chromosome 1, it is clear
that these three related genes cannot lie within a gene cluster, as
some related genes do.
and cPLA2
have a significant
alignment in the C2 region. The figure also shows that
cPLA2
lacks the C2 domain and therefore might be
expected to have no Ca2+ requirement for catalytic
activity. Accordingly, Fig. 5 shows that
this enzyme does not have the calcium requirement displayed by
cPLA2
(30, 33, 34, and reviewed in Ref. 35) activity
levels in all samples being somewhat raised, rather than lowered, when calcium is chelated by the presence of 5 mM EDTA. Since our
assays were done in crude extracts, the higher activity with EDTA may not necessarily be observed with purified enzyme.
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Fig. 5.
Calcium independence of
cPLA2 . Baculovirus-infected
insect cell lysates producing cPLA2
were prepared as
described ("Experimental Procedures") and tested in the liposome
assay with or without EDTA to chelate the available free calcium. The
indicated amounts of total cell lysate protein were assayed in a total
volume of 200 µl. The curves are as labeled. The No enzyme
curves had 5 mM EDTA (triangles) and 1 mM Ca2+ (circles). Concentrated
extracts are inhibitory, allowing greater specific activity to be seen
at the lower concentrations.
was low, it was possible
to demonstrate (Table II) its calcium
sensitivity, presumably due to its C2-like domain. In 5 mM
EDTA the activity, after subtracting the control values, is decreased
by 5- or 10-fold relative to that seen in 1 mM
Ca2+.
Calcium dependence of cPLA2
were prepared and assayed as described (see "Experimental
Procedures") using the liposome assay with or without EDTA to chelate
the available free calcium. Between 30 and 46 µg of cell extract
protein was assayed in 200 µl.
-HT is the His-tag form of
cPLA2
described under "Experimental Procedures." Values
shown, with vector control subtracted (see Table I), are the average of
two measurements, and semicolons separate independently prepared cell
lysates.
Splicing Control--
The majority of EST and
other cDNA fragments obtained for cPLA2
included
segments that were obviously introns, both by comparison to rarer
spliced fragments and to the sequence of cPLA2
. In order to obtain a completely spliced cDNA for cPLA2
, it
was necessary to screen using PCR amplification (see "Experimental
Procedures"). Consistent with this observation, in Northern blots
(Fig. 2, B and D) the mRNA for
cPLA2
is seen exclusively in the unspliced form (8.5 kb)
rather than in its mature form (3 kb). Fig.
6 compares the major mRNA form and
the PCR-amplified spliced form of cPLA2
mRNA. The
intron/exon structure is shown in Fig. 6A, and the intron boundary sequences are shown in Fig. 6B. Most of the splice
junctions conform to the normal consensus for donor and acceptor splice sites (36), but both donor and acceptor sites are poorly conserved for
intron 2. Intron 2 consists of 98 nucleotides that are highly homologous to part of an Alu repetitive sequence. No analysis of
genomic DNA has been done for cPLA2
.
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Fig. 6.
A, introns and exons of the major and
minor forms of cPLA2 mRNA. Distances, marked below
in kb, were derived by comparing the assembly of major cDNA
fragments sequenced to the minor PCR fragment that encodes
cPLA2
. The letters i1 to i4 are
placed over the introns. The uppercase letters indicate the
exons that encode the C2 domain and the amino acids that align (Fig. 1)
with the catalytically essential Arg-200 and Ser-228 (R and
S) and Asp-549 and Arg-566 (D and R)
of cPLA2
. B, intron borders for human
cPLA2
, showing alignment with consensus donor and acceptor sequences
for the predominant human U2 (GT-AG) splice sites (36), shown in
uppercase letters in the first line. Intron
borders are in lowercase, and exon borders are in
uppercase letters. Conventional ambiguity codes used here
for nucleotides are K = G or T; R = A
or G; Y = C or T. Hyphens indicate splice
sites. The human cPLA2
borders are labeled i1, i2, i3,
and i4. Asterisks below the border
sequences indicate agreement with the consensus. A rat sequence
(labeled rat i2) for the acceptor site of intron 2 is also
shown.
activity seen when the Ser, Arg,
or Asp residues aligned with Ser-228, Asp-549, or Arg-566 of
cPLA2
, respectively, are changed to alanine. The loss of
activity is not due to failure to produce the mutant proteins, since
they are all produced in normal amounts (Fig. 3C). These are
three of the four known catalytically essential residues of
cPLA2
(23, 24),3 and this result suggests
they serve the same function for cPLA2
. It is of
interest to note a motif (Fig. 1, around His-560,
underlined) in cPLA2
which includes the
catalytic histidine identified in caspase proteases of the apoptosis
pathway (29).4 It is not
clear what the function of this motif might be, especially in light of
the fact that there is no semblance of the cysteine-containing motif
also essential for the proteolytic activity of caspases. Changing
His-560 of cPLA2
to alanine left the cPLA2
activity intact (Table III), demonstrating that this particular
histidine residue is not essential for PLA2 activity.
Test for essential catalytic center elements
and -
mutant proteins indicated were produced
in baculovirus-infected insect cells, and extracts were prepared and
assayed (see "Experimental Procedures"). The cPLA2
mutants were all made in the His-tag (HT) form. Values shown, with
vector control subtracted (see Table I), are the average of duplicates
of two independently infected samples of Sf9 insect cells.
shows (Table III) that all four aligned conserved
residues Arg-54, Ser-82, Asp-285, and Arg-402 are essential for
cPLA2
activity just as they were shown earlier for
cPLA2
(24).3 The mutant proteins were
produced in very similar amounts as shown in Fig. 3E.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
In light of extensive studies (19, 35,
37, 38) to characterize the C2 domain of cPLA2 as giving to
the enzyme its sensitivity to Ca2+, it is satisfying to see
in cPLA2
an additional confirmation that in this
naturally occurring enzyme, whose mRNA is expressed highly in
skeletal and heart muscle, the Ca2+ sensitivity is absent
without the C2 domain (Fig. 5).
mRNA selectively
expressed in heart and skeletal muscle (Fig. 2C). The enzyme
iPLA2 (see Ref. 40 and reviewed in Ref. 41), which has no
sequence similarity whatever to cPLA2
, is the only
calcium-insensitive cPLA2 previously known to be expressed
in heart. It seems likely that some of the enzyme activity seen in
heart tissue may be due to cPLA2
. Since
cPLA2
appears to be largely membrane-bound through a
lipid anchor motif at its C terminus (Fig. 4A; see also Ref.
27), there may well be separate cell compartments and biological roles
for these two cytosolic PLA2s in cardiac tissue.
cDNA, and in
other clones analyzed this 27-bp segment appeared two or three times as
concatenated repeats (not shown), presumably through alternative
splicing. A search of GenBankTM, now just over 1 billion
nucleotides, revealed that this exact sequence does not appear
elsewhere in known genes. Interestingly, the closest match found was 23 of 27 bases, surrounding the initiator methionine codon of a human
potassium channel protein, perfectly preserved in its mouse homolog
(Fig. 4B), making a stronger case for a possible functional
relationship to translational regulation by these closely related
sequence segments.
--
The C2 domain has been shown to mediate
calcium sensitivity in cPLA2
(20-22, 38). This C2, or
calcium and lipid binding, domain is widely distributed among diverse
proteins (35). Five of the six cPLA2
side chain oxygen
atoms now known to be in contact with calcium (37) are indicated in
Fig. 1 by asterisks and are aligned in cPLA2
,
as explained in the figure legend. Consequently, the degree of
alignment seen in Fig. 1 may well signify that cPLA2
has
a functional C2 domain. Despite its low activity in our assay system,
the data in Table II support this concept. This activity was also
tested (not shown) in the full-length form of cPLA2
(i.e. with the extra N-terminal 99 aa), and it showed no
higher activity in our assay than the 913-aa form.
is not fully
processed in the 24 tissues we examined, and one should be cautious in interpreting this fact. Nevertheless, it should be noted that the
spliced cPLA2
cDNA (depicted in Fig. 6) encoding the
cPLA2
protein (shown in Fig. 1) was not derived from a
prediction but was amplified by reverse transcriptase-PCR from human
brain tissue, demonstrating that it is spliced in cells at a low level.
The major (unspliced) mRNA form for cPLA2
is not
necessarily identical to the genomic sequence (i.e. it may
represent a stable, partially spliced product); we have not examined
chromosomal DNA encoding cPLA2
.
cDNA were also sequenced
(not shown) and allowed comparison to introns 3-4, and notably the
acceptor site of intron 2 (the latter shown in Fig. 6B),
which all had normal GT-AG consensus sequences. Since splicing
generally has been observed to follow no requisite order, it does not
seem likely that one unusual intron splice site could prevent the
normal splicing of other introns. In addition, the introns in the seven
rat cPLA2
cDNA clones were mostly unspliced, despite
the normal consensus sequence at the acceptor site for intron 2. We
must conclude that there is no obvious explanation for the inverted
ratio of spliced to unspliced cPLA2
mRNA.
is a pseudogene; however,
this does not seem likely, since most pseudogenes have accumulated premature terminator codons randomly within their coding regions, over
much shorter spans than the >100 kDa of cPLA2
. In
addition, conservation of cPLA2
is high between human
and rat. Seven rat cPLA2
cDNA fragments covering 530 aa had 85% identity (not shown) to the human cPLA2
sequence, far higher than would be expected for a nonfunctional
pseudogene (the introns and 3'-untranslated regions were not detectably
conserved). It is possible that in some biological setting the mRNA
becomes fully spliced, as a form of activation. For example this
process may follow the form of splicing regulation seen dramatically
for several genes in the inflammatory cytokine pathway, such as tumor
necrosis factor-
, interleukin-2, and interleukin-1
(42-44).
However, in these cases splicing regulation is not nearly as
stringently restricted as we see for cPLA2
. Viruses are
known (45) to exert very stringent temporal mRNA splicing control.
However, to our knowledge such stringent splicing control has not been
previously seen in cellular mRNAs.
has been shown to have
several sites of phosphorylation, which alter the activity of the
enzyme (20-22). For cPLA2
, the pattern PXSP
seen around Ser-505 has been most studied, but in addition
phosphorylation has been detected on serine at positions 437, 454, and
especially 727 (22). Comparison of the sequences for the three enzymes
(Fig. 1) reveals that none of these serines is preserved in the
alignment, with the lone exception that Ser-727 of cPLA2
is aligned with Ser-537 of cPLA2
, albeit in a region of
weak conservation, positioned immediately before the C-terminal CAAX box in cPLA2
. We conclude that if there
are phosphorylation sites in cPLA2
or
cPLA2
, they are probably specific to each enzyme and not
a conserved feature within the family.
(see "Experimental Procedures"), there
are 14 mapped known genes, including the one encoding
1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase
2. This
phosphodiesterase
2 has the ability to produce and to degrade
PIP2, the compound shown (46, 47) to activate
cPLA2
by about 40-fold. This nearby chromosomal location
to cPLA2
suggests a relationship, for example possibly
PIP2 can activate cPLA2
. It will be
interesting to explore this possibility.
on chromosome 19 (see
"Experimental Procedures") contains 20 known mapped genes, notable
among them being apolipoprotein E and calmodulin. While certainly
apolipoprotein E is prominent in many areas, particularly
cardiovascular biology (48, 49), the precise connection with
cPLA2
may not be immediately evident. However, the
relationship postulated between calmodulin and another
calcium-independent cytosolic PLA2 (50) makes this chromosomal location intriguing. Those authors postulated that iPLA2 may achieve calcium responsiveness and membrane
access through association with calmodulin. Although
cPLA2
likely achieves its membrane access through a
lipid anchor suggested by the prenyl group-binding site motif (27),
also called a "CAAX box," at its C terminus, the close
chromosomal location for cPLA2
and calmodulin also
suggests the possibility of a functional relationship between these two proteins.
and cPLA2
. The subtilase aspartate motif surrounding Asp-549 of cPLA2
is conserved quite
well in cPLA2
and in cPLA2
at least as
well as in the yeast orthologs (compare Fig. 1 here and Fig. 1 of Ref.
24). Mutation studies support the idea that these four residues are
essential for catalysis in all three of these paralogs (Ref. 24 and
Table III).
(see Fig. 1), a region also noticeably conserved
among cPLA2
orthologs of six species (Fig. 6 of Ref.
24). Although several other regions of the sequence have been defined
as to their function, this one has not. Mosior et al. (47)
recently made a striking observation investigating the discovery of
Leslie and Channon (46) that PIP2 can activate
cPLA2
to a remarkable extent, providing a direct link
between the phospholipase C and cPLA2 signaling pathways.
It is tempting to suspect that this conserved region might mediate the
PIP2 response, and it will be interesting to see whether
the two new enzymes also have this property.
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge the outstanding help of Grace Chiou for enthusiastic participation at an early stage of this work; Rebecca Little and the Lilly DNA sequencing group; and the continued support and encouragement of Neal Roehm. We also thank Ed Johnstone for technical consultation and for the use of the human brain cDNA library and Chris Burge for sharing information about splice consensus sequences prior to publication.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF065215 and AF065216 for cPLA2 spliced and
unspliced cDNA, respectively, and AF065214 for cPLA2
cDNA.
To whom correspondence should be addressed: Millennium
Biotherapeutics, 620 Memorial Drive, Cambridge, MA 02139. Tel.:
617-679-7065; Fax: 617-374-7653; E-mail: sharp{at}mpi.com.
2 NCBI Human Gene Map available on-line at the following address: http://www.ncbi.nlm.nih.gov/science96/.
3 X. G. Chiou, unpublished observations.
4 See also entry PDOC00864 at the PROSITE internet page at http://www.expasy.ch/sprot/prosite.html.
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ABBREVIATIONS |
---|
The abbreviations used are: PLA2, phospholipase A2; cPLA2, cytosolic PLA2; iPLA2, calcium independent PLA2; HT, histidine-tagged proteins; STS, sequence tagged sites for chromosomal location; bp, base pair; kb, kilobase pair; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; aa, amino acid(s); PIP2, phosphatidylinositol-4,5-bisphosphate; EST, expressed sequence tag.
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REFERENCES |
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