Patent Application: US-78110001-A

Abstract:
the present invention concerns dna and peptide sequence encoding a mammalian secreted group iii spla 2 and more particularly a human secreted group iii spla 2 . the invention also concerns the use of this secreted group iii spla 2 in methods for screening various chemical compounds .

Description:
searching for spla 2 homologs in gene databases stored at the national center for biotechnology using the tblastn sequence alignment program ( 28 ) resulted in the identification of a human genomic sequence ( pac clone dj412a9 , genbank accession number ac005005 ) of 133893 nucleotides containing several regions of homology to bee venom group iii spla 2 . this suggested that this large genomic clone contains a gene with several exons and introns coding for a novel human group iii spla 2 . the exon - intron boundaries of the human spla 2 gene were deduced according to alignment with bee venom spla 2 and exon - intron consensus sequences ( 29 ) to provide a putative cdna sequence . to demonstrate the presence of the putative cdna sequence in human tissues , a first set of rt - pcr experiments ( rt - pcr 1 in fig1 ) was performed on different human cdnas with primers flanking the ca 2 + - binding loop and the active site domain of the novel spla 2 ( sense and antisense primers correspond to nucleotides 445 to 468 and 655 to 679 , respectively , fig1 ). a dna product was amplified from human fetal lung cdna and found to have a nucleotide sequence corresponding to the putative cdna . this sequence was then used to clone the entire cdna sequence by 5 ′ and 3 ′ race - pcr experiments as previously described ( 7 ). briefly , human fetal lung poly a + rna ( 2 μg , clontech ) was reverse transcribed , and double stranded cdna was ligated to adaptors containing sequences for the universal primers sp6 and ks . pcr reactions were performed using ks primer and a specific forward or reverse primer , for 3 ′ or 5 ′ race - pcr , respectively . pcr products were subcloned into pgem - t easy vector ( promega ), and colonies were screened using an internal [ 32 p ]- labeled oligonucleotide probe . 3 ′ race - pcr experiments led to the cloning of a 1458 nucleotide sequence that contained in its 3 ′ end an in frame extension of 304 amino acids , a stop codon and a 3 ′ noncoding region of 546 nucleotides containing a putative polyadenylation site . searching in est databases resulted in the identification of an est sequence ( genbank a1282787 ), and full sequencing of this est clone revealed a 193 nucleotide sequence containing a 166 nucleotide sequence identical in its 5 ′ end to the genomic clone and a 27 nucleotide polya sequence . 5 ′ race - pcr experiments were performed with an antisense primer ( nucleotides 518 - 545 in fig1 ) and led to the cloning of a 158 nucleotide sequence , extending the 5 ′ end sequence of the rt - pcr 1 dna fragment by 20 amino acid residues . in frame with this 158 nucleotide sequence , an initiator methionine followed by a 19 amino acid sequence presenting the features of a signal peptide sequence ( 30 ) was found in the upstream genomic sequence . a primer upstream of the putative initiator methionine ( nucleotides − 254 to − 229 in fig1 ) and an antisense primer ( nucleotides 2205 to 2236 in fig1 ) derived from the above est sequence were designed and used to amplify the full - length hgiii cdna spla 2 ( rt - pcr 2 in fig1 ). this rt - pcr experiment was performed on the same human fetal lung cdna using the proofreading pwo dna polymerase and led to the cloning of a cdna fragment of 2564 nucleotides containing an open reading frame of 1530 nucleotides . to confirm that this long open reading frame resulted from a proper splicing of the hgiii spla 2 gene , exon - trapping experiments were performed . for this purpose , a genomic fragment encompassing the putative hgiii gene was amplified with the expand long template pcr system ( roche ), primers designed from the human pac clone dj412a9 ( nucleotides 36143 - 36175 and 43062 - 43092 for sense and antisense primers , respectively ), and human genomic dna as template . an expected 6 . 95 kilobase pair genomic fragment was amplified and subcloned into the exon trapping pet01 vector ( mobitech ), partially sequenced , and the resulting plasmid was transfected into cos cells . three days after transfection , total rna was prepared , reverse transcribed with oligodt , and submitted to pcr with primers flanking the hgiii spla 2 open reading frame . a pcr fragment of 1530 nucleotides was amplified , cloned into pgem - t easy vector ( promega ), and found to encode for the full - length hgiii open reading frame . no amplification was observed with cdna from cos cells transfected with the parent exon - trapping vector . a human northern blot ( clontech catalog # 7780 - 1 ) was probed with a [ 32 p ]- labeled riboprobe corresponding to the nucleotide sequence 445 to 679 of hgiii spla 2 ( fig1 ) in ultrahyb hybridization buffer ( ambion , catalog # 8670 ) for 18 h at 70 ° c . high - sensitivity stripable antisense riboprobe was synthesized using the strip - ez rna ambion kit ( catalog # 1360 ). the blot was washed to a final stringency of 0 . 1 × ssc ( 30 mm nacl , 3 mm trisodium citrate , ph 7 . 0 ) in 0 . 1 % sds at 70 ° c . and exposed to kodak biomax ms films with a transcreen - he intensifying screen . ii . 3 recombinant expression of hgiii spla 2 in cos cells . the full - length cdna sequence coding for hgiii spla 2 was subcloned into the expression vector prc / cmvneo ( invitrogen ) and a consensus kozak sequence was added to enhance protein expression as previously described ( 6 ). the dna construct was sequenced after subcloning and transiently transfected into cos cells using deae - dextran ( 7 ). five days after transfection , cell medium was collected and partially purified on an anion exchange column . briefly , cos cell culture medium ( 9 ml ) was loaded at 1 ml / min onto a 10 ml column of q - sepharose fast flow ( pharmacia ) previously equilibrated in 25 mm tris , ph 8 . 0 at 4 ° c . after washing with equilibration buffer to remove unbound protein , the solvent program was started ( 10 min in equilibration buffer followed by a linear gradient of nacl from 0 to 1 m nacl over 40 min ). hgiii spla 2 enzymatic activity was detected using the fluorimetric assay with 1 - palmitoyl - 2 -( 1 0 - pyrenedecanoyl )- sn - glycero - 3 - phosphomethanol as described ( 8 ). the pool of hgiii - containing fractions was concentrated approximately 10 - fold by centrifugal ultrafiltration ( ym - 10 membrane , amicon ) at 4 ° c ., and the concentrate was stored at − 20 ° c . using this assay , no phospholipase a 2 activity was detected in culture medium from cos cells transfected with the parent expression vector . studies to measure the initial rate of hydrolysis of small unilamellar vesicles of phosphatidylglycerol ( 1 - palmitoyl - 2 -([ 9 , 10 [ 3 h ])- palmitoyl - sn - glycero - 3 - phosphoglycerol in 1 - palmitoyl - 2 - oleoyl - sn - glycero - 3 - phosphoglycerol at 50 ci / mol ) and phosphatidylcholine ( 1 - palmitoyl - 2 -([ 9 , 10 [ 3 h ])- palmitoyl - sn - glycero - 3 - phosphocholine , 50 ci / mol ) were carried as described ( 8 ) using q - sepharose purified hgiii spla 2 . initial rates were calculated from 3 time points in the linear portion of the product versus time curve . ph - rate profiles for the hydrolysis of phosphatidylcholine were obtained as described ( 8 ). the ca 2 + dependency of phospholipid hydrolysis was carried out with the fluorimetric assay ( described above ) with 10 μm egta ( no ca 2 + ) or with cacl 2 in excess of egta to give 10 - 650 μm ca 2 + . screening of mammalian nucleic sequence databases with various venom spla 2 s led us to identify a large human genomic fragment of 133893 nucleotides presenting several regions of homology with bee venom group iii spla 2 . this suggested that the genomic clone contains a complete gene with several exons and introns coding for a putative human group iii ( hgiii ) spla 2 . a first set of sense and antisense primers was designed from the genomic sequences homologous to bee venom spla 2 and used for rt - pcr experiments ( rt - pcr 1 in fig1 a ) on human cdnas from brain , pancreas , spleen , skeletal muscle , and fetal lung . a dna fragment was amplified from fetal lung cdna and its sequence was found to correspond to the expected spliced exons from the genomic sequence . 5 ′ and 3 ′ race - pcr experiments followed by a second round of rt - pcr ( rt - pcr 2 in fig1 a ) on human fetal lung cdna led to the cloning of a cdna fragment of 2564 nucleotides containing a large open reading frame of 1530 nucleotides ( see fig1 and experimental procedures for details ). screening of est databases resulted in the identification of a single human est sequence ( genbank a1282787 ) of 193 nucleotides containing a polya tail , suggesting that this est sequence corresponds to the 3 ′ end of the hgiii spla 2 mrna ( fig1 a ). comparison of the 2564 nucleotide cdna sequence with the pac genomic sequence indicated that the hgiii spla 2 gene is composed of at least 7 exons and 6 introns spanning about 7 kilobase pairs ( fig1 a ). exon - trapping experiments were performed and found to confirm the exon - intron structure and the sequence of the complete hgiii spla 2 open reading frame of 1530 nucleotides ( see experimental procedures ). the pac clone dj412a9 ( genbank ac005005 ) containing the hgiii spla 2 gene was generated by the sequencing program of human chromosome 22 ( 31 ), indicating that the hgiii spla 2 gene maps to this chromosome between the genethon markers d22s1150 and d22s273 . the location of the hgiii gene is thus distinct from those of genes for human group ib , iia , iid , v and x spla 2 s ( 8 , 9 ). similar to other mammalian spla 2 s , the open reading frame of hgiii spla 2 begins with a signal peptide of 19 amino acids ( 30 ), indicating that the novel enzyme could be secreted . in contrast to other mammalian spla 2 s ( 117 to 148 amino acids ), the hgiii open reading frame codes for a much larger protein of 490 amino acids ( calculated molecular mass 55 . 3 kda , calculated pi 9 . 1 ) containing five putative n - glycosylation sites ( fig1 b ). this protein is made up of a central spla 2 domain ( 141 residues ) flanked by n - and c - terminal regions ( 130 and 219 residues , respectively ). based on the alignment with venom group iii spla 2 s ( fig2 ), the spla 2 domain comprises 141 amino acids ( calculated molecular mass 16 kda , calculated pi 5 . 4 ) and displays the typical features of group iii spla 2 s including the 10 cysteines specific for group iii spla 2 s and the key residues of the ca 2 + - loop and catalytic site . the spla 2 domain contains 2 putative n - glycosylation sites which are not conserved with that of bee venom spla 2 located at position 15 in fig2 . however , one of them is located only 4 residues downstream of the glycosylation site in bee venom spla 2 . interestingly , the hgiii domain is more similar to venom group iii spla 2 s identified from vertebrates . indeed , higher levels of identity are found with the isoforms pa - 2 and pa - 5 ( 43 and 46 %, respectively ) purified from the lizard gila monster ( 27 ), while lower levels are observed with venom group iii spla 2 s from honey bee , bumble bee and the scorpion pandinus imperator ( fig2 ). no protein database entries with significant homology to the n - and c - terminal regions flanking the spla 2 domain of the hgiii spla 2 gene could be found . they are both basic ( calculated pi 9 . 1 and 11 . 3 for n - and c - terminal regions , respectively ) and contain 4 and 8 cysteines , suggesting that they may fold separately from the spla 2 domain . the function of these two domains are completely unknown at present . one possibility is that these domains could be involved in the maturation of hgiii spla 2 during or after its secretion from cells . although the maturation processing of hgiii spla 2 clearly remains to be elucidated , the presence of a basic doublet kr at the end of the n - terminal domain ( fig1 b ) suggests that this domain could serve as a long propeptide that can be cleaved by subtilisin - like protein convertase in the golgi apparatus ( 32 ). interestingly , the mature protein sequence of bee venom spla 2 is preceded by an arginine residue ( 20 ) and a short propeptide sequence ending with an arginine doublet has been found in human group x spla 2 ( 6 ). the c - terminal region also contains several basic residues including basic doublets , which may be involved in protein maturation as well . in addition , the c - terminal domain contains numerous prolines and a pentapeptide rrlar similar to that found in imperatoxin i from pandinus imperator venom ( 22 ). in this regard , it is not yet clear whether some venom group iii spla 2 s also have such large n - and c - terminal regions , since only mature protein sequences and partial cdna sequences have been determined so far ( 20 , 23 , 25 - 27 ), except for the pandinus imperator venom spla 2 s ( 22 , 24 ). a second possibility may be that the n - and c - terminal domains are involved in spla 2 dimerization , cell targeting or interaction with cellular proteins possibly including spla 2 receptors ( 4 ). the last possibility may be that these domains play a role in regulating hgiii spla 2 activity . unlike group i and ii spla 2 s which contain a hydrogen bond network linking the n - terminus to catalytic residues , the x - ray structure of bee venom spla 2 shows that the n - terminus does not form part of the active site structure ( 11 ). indeed , recombinant bee venom spla 2 expressed as an n - terminal fusion protein exhibits the same catalytic activity as the cleaved fusion or the native enzyme ( 33 ). this suggests that the presence of the n - terminal extension ( and presumably the c - terminal region which is also not part of the catalytic site ( 11 )) would not interfere with the catalytic activity of hgiii spla 2 . full - length or partially cleaved hgiii spla 2 may thus be catalytically active and n - and c - terminal domains may participate to the hgiii enzymatic properties . further studies are clearly needed to elucidate the maturation process of the hgiii spla 2 protein and the role of these additional n - and c - terminal regions . the tissue distribution of hgiii spla 2 was analyzed by hybridization at high stringency to a human northern blot ( fig3 ). the hgiii spla 2 is expressed as a single transcript of 4 . 4 kilobase which is abundant in kidney , heart , liver and skeletal muscle , and is also present at low levels in placenta and peripheral blood leukocytes . little , if any , expression was detected in brain , colon , thymus , spleen , small intestine and lung . the pattern of expression of hgiii spla 2 is distinct from that of other human spla 2 s , suggesting that this novel enzyme has specific function ( s ). notably , hgiii spla 2 is expressed in kidney while no expression was previously detected in this tissue for human group ib , iia , iid , v and x spla 2 s ( 6 , 9 ). on the other hand , hgiii spla 2 is co - expressed in heart with human group iia and v spla 2 s , and in liver and skeletal muscle with human group iia spla 2 ( 6 ). i . 3 recombinant expression of hgiii spla 2 and enzymatic properties . when the hgiii spla 2 cdna was transiently transfected in cos cells , spla 2 activity accumulated in the culture medium , indicating that the hgiii spla 2 cdna codes for a secreted active enzyme . the level of pla 2 activity measured after washing the cells with high salt buffer containing 1 m nacl and in cell lysate was low , suggesting that hgiii spla 2 is not tightly bound to the cell surface and is efficiently secreted . the hgiii spla 2 was partially purified by chromatography on a q - sepharose fast flow column and the eluted spla 2 fraction was used to analyze the enzymatic properties . like all mammalian spla 2 s that have been kinetically characterized ( 7 , 8 , 34 , 35 ), hgiii spla 2 is considerably more active ( 11 - fold based on initial velocities ) on anionic phosphatidylglycerol vesicles versus zwitterionic phosphatidylcholine vesicles ( not shown ). further studies with pure hgiii spla 2 in larger quantities are required to determine if this rate difference is due to an increased fraction of enzyme bound to the anionic versus zwitterionic interface , a lower value of the interfacial km for phosphatidylglycerol versus phosphatidylcholine , or both . as shown in fig4 a , the rate of phosphatidylmethanol vesicle hydrolysis by hgiii is completely ca 2 + - dependent with a kd of 6 ± 0 . 8 μm . the kd for ca 2 + of 6 μm for the action of hgiii spla 2 on phosphatidylmethanol vesicles is considerably lower than the sub - millimolar to millimolar values reported for other spla 2 s . however , the kd value measured in this study is an apparent value . for spla 2 s , phospholipid binding to the active site is ca 2 + dependent , and thus the observed apparent kd for ca 2 + depends on the affinity of enzyme &# 39 ; s active site for phospholipid substrate ( 36 ). kd for ca 2 + is also modulated by the affinity of the enzyme for the vesicle interface since interfacial binding is a prerequisite for the binding of long - chain phospholipids to the enzyme &# 39 ; s active site . in this context , it may be noted that human group iia spla 2 binds ca 2 + with millimolar affinity in the absence of substrate ( 37 , 38 ), but the kd for ca 2 + in the presence of phosphatidylglycerol ( which supports tight interfacial and active site binding ) is in the low micromolar range ( 39 ). once large amounts of recombinant hgiii spla 2 are available , it will be possible to use spectroscopic methods to measure the affinity of the enzyme for ca 2 + in the absence of substrate . as shown in fig4 b , hgiii spla 2 is optimally active on phosphatidylcholine vesicles at ph 8 . the ph - rate profile of hgiii is similar to most spla 2 s ( 12 ). the increase in rate up to ph 8 probably reflects deprotonation of the active site histidine so that it can function as a general base for the attack of a water molecule on the substrate ester carbonyl group ( 13 ). over the past few years , the molecular biology approach has revealed the presence of a diversity of spla 2 s in mammals ( 5 - 9 ). the mammalian spla 2 family comprises eight members of 14 - 17 kda including a group 1 , 5 group ii , a group v and a group x spla 2 s . it also includes otoconin - 95 , a major protein of the extracellular otoconial complex of inner ear , which consists of a large secreted protein of 469 residues containing two spla 2 - like domains ( 40 , 41 ). all these spla 2 s have a conserved primary structures , have in common various disulfide , and several have a similar genomic organization . these spla 2 s are thus structurally - related enzymes that fall within the same set of proteins , namely the i / ii / v / x spla 2 collection . it should be noted however that they all have distinct tissue distribution and function . the mammalian spla 2 family now also comprises the human group iii spla 2 which does not belong to the i / ii / v / x spla 2 collection . hgiii spla 2 has a distinct spla 2 primary sequence from the above spla 2 s , contains extra n - and c - terminal regions , and has a different genomic organization . together , this indicates that mammals can express spla 2 s of the group i / ii / v / x collection and of the distinct group iii collection . interestingly , the same can be observed in reptiles , since spla 2 s found in snake venoms are group i or ii enzymes while those found in lizard venoms belong to group iii ( 15 ). in addition , as previously pointed out ( 15 ), it is likely that a single snake species can express several spla 2 s from different groups which are present in various tissues other than the venom gland . finally , while most spla 2 s reported so far in the venom of invertebrates appear to be group iii enzymes ( 20 , 22 - 25 ), scanning of nucleic databases indicates that invertebrates also express spla 2 s from the group i / ii / v / x collection in other tissues . in short , this makes likely that both vertebrates and invertebrates express a variety of spla 2 s of the group i / ii / v / x collection and of group iii , and that these spla 2 s are present in various tissues to deserve specific functions . lastly , based on the current spla 2 s found in mammals , it is tempting to speculate that vertebrates have “ chosen ” to generate a spla 2 diversity from the group i / ii / v / x collection and not from the group iii collection . it remains however to determine if more than one group iii spla 2 is expressed in mammals , and if reptiles and invertebrates have made the same “ choice ” to make their own spla 2 diversity . in conclusion , a novel human spla 2 that clearly belongs to group iii was cloned . this spla 2 seems to have a number of distinct structural features compared to the known venom group iii spla 2 s , suggesting that hgiii spla 2 may not be the structural “ equivalent ” of these venom spla 2 s ( 4 ). its tissue distribution appears non redundant with other human spla 2 s , suggesting particular function ( s ). our initial survey indicate a strong expression of hgiii spla 2 in heart , kidney , liver and skeletal muscle , but a more extensive analysis in a wide variety of tissues , cell types and extracellular fluids under both normal and pathological conditions could emphasize unsuspected spla 2 functions . so far , spla 2 s have been found in many tissues and cells , and their functions are only slowly being discovered . some of them have been implicated as potent mediators of inflammation and their levels are elevated in numerous inflammatory diseases and after challenge by proinflammatory cytokines and endotoxins ( 3 , 4 , 9 , 42 ). levels of spla 2 s are also increased in cancer and spla 2 s have been proposed to play a role in cell proliferation and cancer ( 3 , 4 , 9 ). spla 2 s are also increased after ischemia ( 3 , 43 ) and they may play a role in neurotransmission ( 44 ). finally , spla 2 s have been involved in host defense mechanisms against different bacterial strains ( 45 - 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