Molecular Cloning of Two New Human Paralogs of 85-kDa Cytosolic Phospholipase A2*

R. Todd Pickard, Beth A. Strifler, Ruth M. Kramer, and John D. SharpDagger

From Lilly Research Laboratory, Indianapolis, Indiana 46285

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Two new cloned human cDNAs encode paralogs of the 85-kDa cytosolic phospholipase A2 (cPLA2). We propose to call these cPLA2beta (114 kDa) and cPLA2gamma (61 kDa), giving the name cPLA2alpha to the well known 85-kDa enzyme. cPLA2beta mRNA is expressed more highly in cerebellum and pancreas and cPLA2gamma more highly in cardiac and skeletal muscle. Sequence-tagged site mapping places cPLA2beta on chromosome 15 in a region near a phosphoinositol bisphosphate phosphatase. The mRNA for cPLA2beta is spliced only at a very low level, and Northern blots in 24 tissues show exclusively the unspliced form. cPLA2beta 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 cPLA2gamma on chromosome 19 near calmodulin. cPLA2gamma lacks the C2 domain, which gives cPLA2alpha its Ca2+ sensitivity, and accordingly cPLA2gamma has no dependence upon calcium, although cPLA2beta does. cPLA2gamma contains a prenyl group-binding site motif and appears to be largely membrane-bound. cPLA2alpha 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 cPLA2alpha , 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

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 (cPLA2alpha 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 cPLA2beta and cPLA2gamma . While this paper was under review a report (27) appeared describing cPLA2gamma .

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning, cDNA Expression, and Assays-- cPLA2beta 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 cPLA2alpha 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 cPLA2alpha . 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 cPLA2beta sequence (uppercase): gagggaattcCTTCATGATGCCAGCTGAGCGCCGCC for the 5' end and ggtggcggccgcCCCGGCCATCAGTGGGGCCTGCGC for the 3' end. The 5'-untranslated region of the cPLA2beta 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 cPLA2beta cDNA.

cPLA2gamma 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 cPLA2alpha . Using 5'-RACE with the CLONTECH brain cDNA, the additional 5' sequence obtained had homology with cPLA2alpha 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 cPLA2gamma .

The full-length cDNAs (spliced form for cPLA2beta ) 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): cPLA2beta ,  ... rphHHHHH, and cPLA2gamma , MHHHHHHAmgs. . . . . Western analysis of those proteins was done as described (23) using the penta-His antibody purchased from Qiagen.

For Northern analysis, human multiple tissue RNA blots (CLONTECH) were probed with 2 × 106 cpm/ml 32P random primer-labeled cPLA2alpha , cPLA2beta , cPLA2gamma , or beta -actin cDNA probes, using the procedure recommended by the manufacturer (CLONTECH).

The caspase catalytic histidine consensus motif (Fig. 1) of cPLA2beta 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]: cPLA2beta  =  ... 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 cPLA2beta , 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 cPLA2beta 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 cPLA2beta . 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.

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 cPLA2alpha 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 cPLA2gamma ), thus predicting a specific activity of at least 20 nmol/min/mg total protein in the lysates for cPLA2alpha . Thus the values in Table I show that cPLA2gamma and cPLA2beta are about 2 and 3 orders of magnitude lower, respectively, than cPLA2alpha in apparent specific activity.

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 cPLA2beta was derived from direct comparison of the sequences of the 8.5- and the 3.0-kb mRNAs.

The 27-bp repeat in cPLA2gamma 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

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 cPLA2alpha . The EST for cPLA2beta included the region aligning with the catalytic Ser-228 of cPLA2alpha (23), whereas the EST for cPLA2gamma 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 cPLA2alpha , cPLA2beta , and cPLA2gamma 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 cPLA2alpha 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 cPLA2alpha C2 (37). Five of those are preserved in cPLA2beta or replaced with another oxygen-bearing side chain, as in the case of Asp-40 of cPLA2alpha , replaced by Thr. The caspase catalytic histidine motif (see "Experimental Procedures") is underlined in cPLA2beta 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 cPLA2alpha , known to be phosphorylated (22), and Ser-537 of cPLA2gamma . There is some additional N-terminal sequence (99 aa) to cPLA2beta , not shown here (see "Experimental Procedures").

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 cPLA2alpha has a counterpart in cPLA2beta but not in the shorter cPLA2gamma . Fig. 1 also shows that there is total conservation, among all three sequences, of the three previously published catalytically essential cPLA2alpha 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 cPLA2alpha 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 cPLA2beta or cPLA2gamma , and the alignment in that region is weak, suggesting that the phosphorylation activation pathway demonstrated for cPLA2alpha (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 cPLA2alpha .

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 cPLA2alpha mRNA was expressed more evenly than the others, with heart and pancreas having higher expression (Fig. 2A). cPLA2beta mRNA is expressed strongly in pancreas (Fig. 2B) and most strongly in cerebellum (Fig. 2D), whereas cPLA2gamma mRNA is expressed most strongly in skeletal muscle and heart (Fig. 2C). The beta -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), beta -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 cPLA2alpha , cPLA2beta , and cPLA2gamma 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 cPLA2alpha (A); cPLA2beta (B); cPLA2gamma (C); again cPLA2beta (D). Controls (E and F) were probed with beta -actin cDNA.

Both cPLA2beta and cPLA2gamma proteins were produced in insect cells as done previously for cPLA2alpha , 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 cPLA2beta and 60 kDa for cPLA2gamma , agree well with the sizes predicted from the cDNA coding regions (102,984 daltons for beta  and 60,948 for gamma ). Because there is a baculovirus protein produced in the infected insect cells that migrate with cPLA2gamma at 60 kDa, we used immunoblotting of cPLA2beta and cPLA2gamma bearing His-tag additions (see "Experimental Procedures") to confirm that the increased density at 60 kDa was due to cPLA2gamma (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 cPLA2beta and -gamma proteins. The "full-length" 1012-aa cPLA2beta 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 cPLA2beta proteins; and E is Western-blotted mutant cPLA2gamma 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, cPLA2alpha -HT; lane 2, cPLA2beta -HT; lane 3, infected Sf-9 cell control; lane 4, cPLA2beta -FL (1012 aa; see "Experimental Procedures"); lane 5, cPLA2gamma -HT; for C and D, lane 1, cPLA2beta ; lane 2, cPLA2beta histidine-tagged; lane 3, cPLA2beta R764A; lane 4, cPLA2beta D747A; lane 5, cPLA2beta H560A; lane 6, cPLA2beta S467A; lane 7, non-recombinant AcMNPV (baculovirus); lane 8, uninfected Sf-9 insect cells; lane 9, cPLA2gamma -HT; and for E (all cPLA2gamma -HT except as noted): lane 1, R54A; lane 2, S82A; lane 3, D385A; lane 4, R402A; lane 5, wild type; lane 6, cPLA2alpha -HT; lane 7, uninfected Sf9; and lane 8, Sf9 infected with non-recombinant baculovirus.

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 cPLA2beta and cPLA2gamma are catalytically active their enzymatic activity is several orders of magnitude lower than that of cPLA2alpha (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 cPLA2gamma 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 cPLA2beta the activity, although clearly significantly above the assay background, was also very low (Table I).

                              
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Table I
PLA2 activity of cPLA2beta and cPLA2gamma
The proteins indicated were produced in baculovirus-infected insect cells, and extracts were prepared and assayed (see "Experimental Procedures"). All three cPLA2 forms typically represent about 1% of total cell protein (see Fig. 3, A and B). The figure listed for cPLA2alpha 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.

Fig. 4A shows the cDNA sequence and its translation for cPLA2gamma . 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 cPLA2gamma , apparently a result of alternative mRNA splicing.


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Fig. 4.   A, cDNA for cPLA2gamma and its translation. cPLA2gamma 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 cPLA2gamma . 27 bp represents the sequence from cPLA2gamma , 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.

BLAST analysis against the GenBankTM data base showed that cPLA2beta and cPLA2gamma both have exact matches in STS sequence entries, corresponding to mapped locations on human chromosomes 15 (STS WI-13757 matches cPLA2beta ) and 19 (STS WI-14200 matches cPLA2gamma ), respectively. Thus since cPLA2alpha 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.

Sensitivity to Calcium-- In Fig. 1 we can see that cPLA2alpha and cPLA2beta have a significant alignment in the C2 region. The figure also shows that cPLA2gamma 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 cPLA2alpha (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 cPLA2gamma . Baculovirus-infected insect cell lysates producing cPLA2gamma 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.

Although the activity of cPLA2beta 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+.

                              
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Table II
Calcium dependence of cPLA2beta
Baculovirus-infected insect cell lysates producing cPLA2gamma 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. beta -HT is the His-tag form of cPLA2beta 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.

cPLA2beta Splicing Control-- The majority of EST and other cDNA fragments obtained for cPLA2beta included segments that were obviously introns, both by comparison to rarer spliced fragments and to the sequence of cPLA2alpha . In order to obtain a completely spliced cDNA for cPLA2beta , 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 cPLA2beta 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 cPLA2beta 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 cPLA2beta .


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Fig. 6.   A, introns and exons of the major and minor forms of cPLA2beta mRNA. Distances, marked below in kb, were derived by comparing the assembly of major cDNA fragments sequenced to the minor PCR fragment that encodes cPLA2beta . 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 cPLA2alpha . B, intron borders for human cPLA2beta , 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 cPLA2beta 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.

Catalytic Activity-- Table III shows the loss of cPLA2beta activity seen when the Ser, Arg, or Asp residues aligned with Ser-228, Asp-549, or Arg-566 of cPLA2alpha , 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 cPLA2alpha (23, 24),3 and this result suggests they serve the same function for cPLA2beta . It is of interest to note a motif (Fig. 1, around His-560, underlined) in cPLA2beta 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 cPLA2beta to alanine left the cPLA2 activity intact (Table III), demonstrating that this particular histidine residue is not essential for PLA2 activity.

                              
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Table III
Test for essential catalytic center elements
The cPLA2beta and -gamma mutant proteins indicated were produced in baculovirus-infected insect cells, and extracts were prepared and assayed (see "Experimental Procedures"). The cPLA2gamma 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.

A similar analysis of the corresponding conserved residues of cPLA2gamma shows (Table III) that all four aligned conserved residues Arg-54, Ser-82, Asp-285, and Arg-402 are essential for cPLA2gamma activity just as they were shown earlier for cPLA2alpha (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

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.

cPLA2gamma -- 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 cPLA2gamma 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).

After much previous work (39) establishing the existence of a calcium-insensitive PLA2 activity in heart tissue, it is most interesting to find cPLA2gamma 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 cPLA2gamma , 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 cPLA2gamma . Since cPLA2gamma 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.

The 27-bp imperfect palindrome shown in Fig. 4A appears in the 5'-untranslated region of cPLA2gamma 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.

cPLA2beta -- The C2 domain has been shown to mediate calcium sensitivity in cPLA2alpha (20-22, 38). This C2, or calcium and lipid binding, domain is widely distributed among diverse proteins (35). Five of the six cPLA2alpha side chain oxygen atoms now known to be in contact with calcium (37) are indicated in Fig. 1 by asterisks and are aligned in cPLA2beta , as explained in the figure legend. Consequently, the degree of alignment seen in Fig. 1 may well signify that cPLA2beta 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 cPLA2beta (i.e. with the extra N-terminal 99 aa), and it showed no higher activity in our assay than the 913-aa form.

It is a mystery why the mRNA for cPLA2beta 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 cPLA2beta cDNA (depicted in Fig. 6) encoding the cPLA2beta 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 cPLA2beta 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 cPLA2beta .

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 cPLA2beta 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 cPLA2beta 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 cPLA2beta mRNA.

It may be suspected that cPLA2beta 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 cPLA2beta . In addition, conservation of cPLA2beta is high between human and rat. Seven rat cPLA2beta cDNA fragments covering 530 aa had 85% identity (not shown) to the human cPLA2beta 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-alpha , interleukin-2, and interleukin-1beta (42-44). However, in these cases splicing regulation is not nearly as stringently restricted as we see for cPLA2beta . 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.

Phosphorylation-- cPLA2alpha has been shown to have several sites of phosphorylation, which alter the activity of the enzyme (20-22). For cPLA2alpha , 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 cPLA2alpha is aligned with Ser-537 of cPLA2gamma , albeit in a region of weak conservation, positioned immediately before the C-terminal CAAX box in cPLA2gamma . We conclude that if there are phosphorylation sites in cPLA2beta or cPLA2gamma , they are probably specific to each enzyme and not a conserved feature within the family.

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 cPLA2beta (see "Experimental Procedures"), there are 14 mapped known genes, including the one encoding 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase beta 2. This phosphodiesterase beta 2 has the ability to produce and to degrade PIP2, the compound shown (46, 47) to activate cPLA2alpha by about 40-fold. This nearby chromosomal location to cPLA2beta suggests a relationship, for example possibly PIP2 can activate cPLA2beta . It will be interesting to explore this possibility.

The vicinity of cPLA2gamma 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 cPLA2gamma 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 cPLA2gamma 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 cPLA2gamma and calmodulin also suggests the possibility of a functional relationship between these two proteins.

Catalysis and Conservation-- As noted above, all four catalytically essential amino acids are strictly conserved in cPLA2beta and cPLA2gamma . The subtilase aspartate motif surrounding Asp-549 of cPLA2alpha is conserved quite well in cPLA2gamma and in cPLA2beta 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).

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 cPLA2alpha (see Fig. 1), a region also noticeably conserved among cPLA2alpha 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 cPLA2alpha 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.

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.

    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.

    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 cPLA2beta spliced and unspliced cDNA, respectively, and AF065214 for cPLA2gamma cDNA.

Dagger 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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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