DEFINITION
PLA2G6
Phospholipase A2,Ca2+independent, group VI expressed as oligomeric complex in bone,brain and other tissues,involved in the remodeling of membranes allowing arachidonic acid to be placed in the proper position in phospholipids for stimulated release.
It has been implicated in normal phospholipid remodeling, nitric oxide-induced or vasopressin-induced arachidonic acid release and in leukotriene and prostaglandin production.
May participate in Fas mediated apoptosis and in regulating transmembrane ion flux in glucose-stimulated B-cells.
85 kDa calcium-independent phospholipase A2
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PLA2G6 gene, is located on chromosome 22 ( locus 22q13.1 ).
CHEMICAL STRUCTURE AND IMAGES
The primary structure of PLA2G6 (isoform A ) as reported on Genbank is:
1 mqffgrlvnt fsgvtnlfsn pfrvkevava dytssdrvre egqlilfqnt pnrtwdcvlv
61 nprnsqsgfr lfqleleada lvnfhqyssq llpfyesspq vlhtevlqhl tdlirnhpsw
121 svahlavelg irecfhhsri iscancaene egctplhlac rkgdgeilve lvqychtqmd
181 vtdykgetvf hyavqgdnsq vlqllgrnav aglnqvnnqg ltplhlacql gkqemvrvll
241 lcnarcnimg pngypihsam kfsqkgcaem iismdssqih skdprygasp lhwaknaema
301 rmllkrgcnv nstssagnta lhvavmrnrf dcaivllthg anadargehg ntplhlamsk
361 dnvemikali vfgaevdtpn dfgetptfla skigrlvtrk ailtllrtvg aeycfppihg
421 vpaeqgsaap hhpfsleraq pppislnnle lqdlmhisra rkpafilgsm rdekrthdhl
481 lcldgggvkg liiiqlliai ekasgvatkd lfdwvagtst ggilalailh sksmaymrgm
541 yfrmkdevfr gsrpyesgpl eeflkrefge htkmtdvrkp kvmltgtlsd rqpaelhlfr
601 nydapetvre prfnqnvnlr ppaqpsdqlv wraarssgaa ptyfrpngrf ldggllannp
661 tldamteihe ynqdlirkgq ankvkklsiv vslgtgrspq vpvtcvdvfr psnpwelakt
721 vfgakelgkm vvdcctdpdg ravdrarawc emvgiqyfrl npqlgtdiml devsdtvlvn
781 alwetevyiy ehreefqkli qlllsp
The full-length cDNA encodes an 806-amino acid protein with a lipase motif and 8 ankyrin repeats
There are two isoforms of the gene up to now described
The variant 1 of the transcript is of 3239 bp and encodes a protein of 806 aa.
The variant 2 of the transcript is of 3077 bp and encodes a protein of 752 aa.
The two isoforms have the same sequences C and N terminals, but the isoform B lacks one exon compared at the isoform A.
The isoform B is located in the cytoplasm, the isoform A is a peripheral mebran protein.
If we compare the two isoforms A and B of the protein, aligning them, you notice that the two isoforms have a homology of 99%.
The sequence ranging from nucleotide 395 to nucleotide 450, there is in the isoform A but not in the isoform B, this sequence is relative to exon missing in the isoform B.
Dashes in red represent the missing exon.
SeqA Name Len(aa) SeqB Name Len(aa) Score
=====================================================
1) isoform_b 752 2) isoform_a 806 99%
=====================================================
isoform_b MQFFGRLVNTFSGVTNLFSNPFRVKEVAVADYTSSDRVREEGQLILFQNTPNRTWDCVLV 60
isoform_a MQFFGRLVNTFSGVTNLFSNPFRVKEVAVADYTSSDRVREEGQLILFQNTPNRTWDCVLV 60
************************************************************
isoform_b NPRNSQSGFRLFQLELEADALVNFHQYSSQLLPFYESSPQVLHTEVLQHLTDLIRNHPSW 120
isoform_a NPRNSQSGFRLFQLELEADALVNFHQYSSQLLPFYESSPQVLHTEVLQHLTDLIRNHPSW 120
************************************************************
isoform_b SVAHLAVELGIRECFHHSRIISCANCAENEEGCTPLHLACRKGDGEILVELVQYCHTQMD 180
isoform_a SVAHLAVELGIRECFHHSRIISCANCAENEEGCTPLHLACRKGDGEILVELVQYCHTQMD 180
************************************************************
isoform_b VTDYKGETVFHYAVQGDNSQVLQLLGRNAVAGLNQVNNQGLTPLHLACQLGKQEMVRVLL 240
isoform_a VTDYKGETVFHYAVQGDNSQVLQLLGRNAVAGLNQVNNQGLTPLHLACQLGKQEMVRVLL 240
************************************************************
isoform_b LCNARCNIMGPNGYPIHSAMKFSQKGCAEMIISMDSSQIHSKDPRYGASPLHWAKNAEMA 300
isoform_a LCNARCNIMGPNGYPIHSAMKFSQKGCAEMIISMDSSQIHSKDPRYGASPLHWAKNAEMA 300
************************************************************
isoform_b RMLLKRGCNVNSTSSAGNTALHVAVMRNRFDCAIVLLTHGANADARGEHGNTPLHLAMSK 360
isoform_a RMLLKRGCNVNSTSSAGNTALHVAVMRNRFDCAIVLLTHGANADARGEHGNTPLHLAMSK 360
************************************************************
isoform_b DNVEMIKALIVFGAEVDTPNDFGETPTFLASKIGR ------------------------- 395
isoform_a DNVEMIKALIVFGAEVDTPNDFGETPTFLASKIGRLVTRKAILTLLRTVGAEYCFPPIHG 420
***********************************
isoform_b ----------------------------- QLQDLMHISRARKPAFILGSMRDEKRTHDHL 426
isoform_a VPAEQGSAAPHHPFSLERAQPPPISLNNLELQDLMHISRARKPAFILGSMRDEKRTHDHL 480
:******************************
isoform_b LCLDGGGVKGLIIIQLLIAIEKASGVATKDLFDWVAGTSTGGILALAILHSKSMAYMRGM 486
isoform_a LCLDGGGVKGLIIIQLLIAIEKASGVATKDLFDWVAGTSTGGILALAILHSKSMAYMRGM 540
************************************************************
isoform_b YFRMKDEVFRGSRPYESGPLEEFLKREFGEHTKMTDVRKPKVMLTGTLSDRQPAELHLFR 546
isoform_a YFRMKDEVFRGSRPYESGPLEEFLKREFGEHTKMTDVRKPKVMLTGTLSDRQPAELHLFR 600
************************************************************
isoform_b NYDAPETVREPRFNQNVNLRPPAQPSDQLVWRAARSSGAAPTYFRPNGRFLDGGLLANNP 606
isoform_a NYDAPETVREPRFNQNVNLRPPAQPSDQLVWRAARSSGAAPTYFRPNGRFLDGGLLANNP 660
************************************************************
isoform_b TLDAMTEIHEYNQDLIRKGQANKVKKLSIVVSLGTGRSPQVPVTCVDVFRPSNPWELAKT 666
isoform_a TLDAMTEIHEYNQDLIRKGQANKVKKLSIVVSLGTGRSPQVPVTCVDVFRPSNPWELAKT 720
************************************************************
isoform_b VFGAKELGKMVVDCCTDPDGRAVDRARAWCEMVGIQYFRLNPQLGTDIMLDEVSDTVLVN 726
isoform_a VFGAKELGKMVVDCCTDPDGRAVDRARAWCEMVGIQYFRLNPQLGTDIMLDEVSDTVLVN 780
************************************************************
isoform_b ALWETEVYIYEHREEFQKLIQLLLSP 752
isoform_a ALWETEVYIYEHREEFQKLIQLLLSP 806
**************************
Protein Aminoacids Percentage
SYNTHESIS AND TURNOVER
The human calcium-independent phospholipase A2 gene
Multiple enzymes with distinct properties from a single gene, 2001
The native human 3.2-kb iPLA2 transcript was predominantly expressed in heart, brain, skeletal muscle, prostate, testis, thyroid and spinal cord, and to a lesser extent in peripheral blood leucocytes, stomach, trachea and bone marrow.
iPLA2 protein contains eight N-terminal ankyrin repeats and a GXSXG consensus lipase motif. Ankyrin repeats are suggested to participate in protein-protein interactions and could be responsible for the tetramer formation of the in-vivo active iPLA2 complex, because proteins lacking this part of the enzyme are catalytically inactive.
The human iPLA2 cDNA sequence was recently identified, as well as multiple splice variants of the iPLA2 transcript. Interestingly, two splice variants of the human iPLA2 transcript lead to the formation of iPLA2 proteins containing only the ankyrin repeats and not the active site (ankyrin-iPLA2 -1 and ankyrin-iPLA2 -2).
mRNA Synthesis
The human iPLA2 gene consists of 17 exons.
The 3'-UTR was found in exon 17 together with a poly(A) signal, AATAAA (Fig. 1A). The 5'-splice donor site sequences of the introns aligning with the defined consensus sequence GTRAGT are indicated in Fig. 1A.
Neighbouring sequences in the exons at the 5'-splice site related to the (C/A)AG consensus sequence are also indicated. Furthermore, the conserved 3'-splice acceptor site sequence, NYAG/R, is outlined in Fig. 1A. Also indicated in Fig. 1 are sequences aligning with
the consensus branch point sequence YNCTRAY for correct pre-mRNA splicing. The iPLA2 transcript has at least one noncoding exon at the 5'-end. Variants in the 5'-UTR of the iPLA2 transcript can be found within the EST database. The intron 1 might be subjected to alternative splicing. Alternatively, intron 1 might contain an intronic promotor leading to a different transcription initiation site. A schematic representation of the iPLA2
pre-mRNA and the resulting proteins are outlined in Fig. 1B, Boxes illustrate the exons. Exons 3, 9, 9a, 10a and part of exon 14 are shaded, indicating that they are subjected to alternative splicing.
Figura 1
Multiple Splice Variants of the Human Calcium-independent Phospholipase A2 and Their Effect on Enzyme Activity, 1997
The human iPLA2 splice variants were due to insertions of 52, 53 and 168 bp, respectively, not found in the iPLA2 transcript coding for the full-length protein. These insertions would introduce termination codons, thus leading to truncated proteins. The elimination of the 52- and 53-bp sequences in the iPLA2 transcript are due to skipping of exons 9a and 10a (Fig. 1B). Both these exons contain putative branch point sequences as well as a correct 3′-splice acceptor site sequence (Fig. 2A, B). Although the 5′-splice donor site of both intron sequences is well conserved, the corresponding exon sequence at this site is less conserved (Fig. 2A, B), which could explain the skipping of these exons. Furthermore, in one of the splice variants (ankyrin-iPLA2-2) there is an in-frame deletion leading to the removal of 72 amino acids between residues 70–143 in the protein. This deletion is due to skipping of the entire exon 3.
The insertion of the 168 bp sequence leads to a transcript encoding an iPLA2 protein lacking the C-terminal part found in the full-length iPLA2 protein. This 168 bp insertion is due to an alternative 3′-splice acceptor site in intron 13 (Fig. 2C). The consensus splice site sequences are indicated in Fig. 2C together with a putative branch point sequence. The branch point sequence is, however, situated more than the postulated 18–40 nucleotides away from the splice site, which might contribute to the alternative splicing of this site. This alternative splicing would give rise to an iPLA2 protein termed iPLA2-2. This form of iPLA2 would contain the active site but lack the C-terminal end.
Figura 2: The DNA sequence of the iPLA2 gene was aligned with the 52 bp (A), 53 bp (B) and 168 bp © insertion sequences found in the alternatively spliced forms of iPLA2 .
Protein synthesis and post-translational modifications
The major difference between the membran protein and the soluble forms of iPLA2 is the presence of 54 additional amino acid residues derived from exon 9. We suggest that the addition of these 54 amino acids leads to a membrane-associated protein. These results demonstrate that alternative splicing of the human iPLA2 transcript generates multiple iPLA2 isoforms with distinct tissue distribution and cellular localization.
A major difference between the human enzyme and that of other species is the insertion of a 54-amino acid proline-rich sequence in the eighth ankyrin motif.
The human iPLA2 transcript is known to be subjected to alternative splicing. However, it is highly likely that the various iPLA2 transcripts originate from the iPLA2 gene and that this gene results in multiple transcripts encoding iPLA2 proteins with distinct tissue distribution and functions.
Figura 3: the tissue distribution of the human PLA2 mRNA, as well as the existence of several distinct tissue-specific PLA2 transcripts
Expression of the human iPLA2 mRNA transcript was investigated using various multiple tissue Northern blots. Using full-length native iPLA2 cDNA as a probe at high stringency conditions, four distinct transcripts with approximate sizes of 1.8, 2.0, 3.2 and 4.2 kb, respectively, were detected (Fig.3). The 3.2-kb transcript is likely to represent the human form of native iPLA2 and was abundantly expressed in heart, brain, skeletal muscle, prostate, testis, thyroid and spinal cord, and to a lesser extent in PBL, stomach, trachea and bone marrow. The 1.8-kb transcript was only observed in heart, skeletal muscle and to a low extent in bone marrow. The 2.0-kb band was expressed ubiquitously. The majority of the tissues examined expressed the 4.2-kb band, although it was found to a greater extent in kidney, pancreas, ovary and thyroid. As a control, the same blots were also analysed using a β-actin cDNA probe (Fig.3).
CELLULAR FUNCTIONS
Cellular localization
iPLA2 splice variants have different functions and tissue distribution.
Phospholipases A2 are a rapidly growing family of diverse enzymes that hydrolyze fatty acids at the sn-2 position of phospholipids. PLA2 enzymes can be subdivided into two classes, extracellular or intracellular, depending on the enzymes localization during catalysis. The intracellular PLA2s can be further categorized into calcium-dependent, best exemplified by the cytosolic phospholipase A2 (cPLA2) , and calcium-independent forms (iPLA2), which tend to be quite diverse and have until recently been less characterized at the molecular level. The calcium-independent PLA2s have a wide tissue distribution and have been purified from human myocardium, bovine brain, P388D1 murine macrophages, and rabbit kidney. They all have distinct molecular masses, indicating the diversity of iPLA2s. Recently, an 85-kDa iPLA2 was purified and cloned from CHO cells, and its sequence was found to be analogous to the 85-kDa iPLA2 from P388D1 cells.
As can be seen from experiments of RT-PCR, iPLA2 is also expressed in human B cells
RT-PCR analysis of total RNA from BL-41 E95A and Raji cell lines using iPLA2 primers 1 and 2; At least three distinct amplified products were observed. The most abundant product was the expected 217-bp fragment, whose identity was confirmed by sequencing, as well as two additional fragments with the apparent sizes of 330 and 380 bp, respectively.
The results from the RT-PCR indicated that iPLA2 is at least one of the more significant PLA2 enzymes in B-cells.
Biological function
PLA2G6, ctalyzes the release of fatty acids from phospholipids. It has been implicated in normal phospholipid
remodeling, nitric oxide-induced or vasopressin-induced arachidonic acid release and in leukotriene and prostaglandin production. May participate in fas mediated apoptosis and in regulating transmembrane ion flux in glucose-stimulated B-cells. Has a role in cardiolipin (CL) deacylation.
Isoform ankyrin-iPLA2-1 and isoform ankyrin-iPLA2-2, which lack the catalytic domain, are probably involved in the negative regulation of iPLA2 activity.
The amino acid sequence indicated the presence of eight ankyrin repeats and the GXSXG conserved catalytic sequence, as found in other lipases. Although there were apparent differences in ATP sensitivity among the enzymes, the biochemical, immunological, and sequence data indicate that these three enzymes are likely to be species variants of the same protein. In both P388D1 cells and in rat pancreatic islets it is thought that iPLA2 has a function in membrane phospholipid remodeling. It has been postulated that the rat islet iPLA2 may be involved in arachidonic acid release leading to activation of β-cell ion channels.
Multiple Splice Variants of the Human Calcium-independent Phospholipase A2 and Their Effect on Enzyme Activity, 1997
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The CHO cell -derived iPLA2 amino acid sequence was used to perform a TBLASTN data base search of GenBank.
Amino acid sequence and alignment of human iPLA2 and the various isoforms with the CHO sequence. A, the amino acid sequence of human and CHO cell iPLA2 as well as the various iPLA2 isoforms were compared using the Genetics Computer Group program GAP. The seven ankyrin repeats in the human sequence areoverlined (I–VII), and the eighth repeat, which is underlined in the CHO sequence, is interrupted by an insertion in the human sequence. The active site (GTSTG) is indicated in bold, and termination is indicated by anasterisk. The EST 30643 sequence is the partial sequence from the EST clone. Shading denotes identity, and thedots indicate the presence of gaps in the sequence.B, a diagrammatic representation of the iPLA2isoforms. The 54-amino acid insertion into the eighth ankyrin repeat of the human sequences and the 72-amino acid deletion in ankyrin-iPLA2-2 are illustrated. The active site serine is indicated for both the CHO and human sequence. The C-terminal hatched region in ankyrin-iPLA2-2 denotes nonidentical sequence, and the number of amino acids in each sequence is also shown.
The major difference between the human sequence and the other species is the insertion of an additional 54 amino acids that would interrupt the last putative ankyrin repeat as defined in the hamster and rat iPLA2 sequences. This sequence is also present in the other human iPLA2 isoforms.
A diagrammatic representation of the iPLA2 isoforms is illustrated in box B; the ankyrin-iPLA2-1 sequence is identical to the human iPLA2 up to the last three C-terminal amino acids and terminates about 40 residues before the active site serine. In contrast, although the ankyrin-iPLA2-2 sequence has the same iPLA2ankyrin repeats, it also contains more structural and sequence variation than does ankyrin-iPLA2-1.
REGULATION
The presence of these iPLA2 isoforms indicates that the iPLA2sequence is subjected to a significant amount of alternative splicing.
The ankyrin structural motif appears to have a function in the formation of various types of protein-protein interactions. Deletion of the ankyrin repeats of the CHO cell iPLA2results in the loss of lipase activity, suggesting that this structure is required for enzyme activity.
The alternative splicing of the iPLA2 pre-mRNA can result in the production of regulatory subunits that can modify iPLA2 in vivo activity.
The characterization of the human iPLA2 gene, and the alternative splicing of the resulting transcript generates multiple transcripts encoding iPLA2 enzymes with distinct tissue distributions and functions.
DISEASES AND DIAGNOSTIC USE
PLA2G6-Associated Neurodegeneration, 2012
The location of the iPLA2 gene on chromosome 22q13.1.
Interestingly, a putative loci for schizophrenia (schizophrenia disorder) has been mapped to the same region on chromosome 22 (q11-13) as the human iPLA2 gene and increased PLA2 activity can indeed be observed in certain form of schizophrenia.
Furthermore, PLA2 enzymes have been implicated in atherogenic processes and PLA2 enzymes have been found within atherosclerotic lesions. The proliferation of smooth muscle cells is suggested to be involved in the pathogenesis of atherosclerosis and PLA2 enzymes have been
demonstrated to play a role in this process. The presence iPLA2 in vascular smooth muscle
cells. However, the involvement of iPLA2 in pathological processes remains to be determined.
In summary, we report that the various alternatively spliced iPLA2 transcripts are derived from one gene. This single gene can give rise to multiple iPLA2 proteins having distinct properties. The soluble iPLA2 form (lacking exon 9) and the membrane-associated iPLA2 form (containing
exon 9) should be referred to as different subgroups (A and B) of group VI according to the group-type numbering of PLA2 enzymes.
The iPLA2 gene should be referred to as Pla2g6.
Infantile neuroaxonal dystrophy: What's most important for the diagnosis?, 2008
Catalytic function of PLA2G6 is impaired by mutations associated with infantile neuroaxonal dystrophy but not dystonia-parkinsonism, 2010
PLA2G6 mutations and other rare causes of Neurodegeneration with Brain Iron Accumulation, 2012
PLA2G6 Mutation Underlies Infantile Neuroaxonal Dystrophy, 2007
