[go: up one dir, main page]

WO2003033689A1 - Clonage et expression recombinante de la phospholipase a2 du groupe iif secretee mammifere - Google Patents

Clonage et expression recombinante de la phospholipase a2 du groupe iif secretee mammifere Download PDF

Info

Publication number
WO2003033689A1
WO2003033689A1 PCT/IB2001/002407 IB0102407W WO03033689A1 WO 2003033689 A1 WO2003033689 A1 WO 2003033689A1 IB 0102407 W IB0102407 W IB 0102407W WO 03033689 A1 WO03033689 A1 WO 03033689A1
Authority
WO
WIPO (PCT)
Prior art keywords
spla
iif
group iif
group
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2001/002407
Other languages
English (en)
Inventor
Michel Lazdunski
Gérard LAMBEAU
Emmanuel Valentin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Original Assignee
Centre National de la Recherche Scientifique CNRS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Centre National de la Recherche Scientifique CNRS filed Critical Centre National de la Recherche Scientifique CNRS
Priority to PCT/IB2001/002407 priority Critical patent/WO2003033689A1/fr
Publication of WO2003033689A1 publication Critical patent/WO2003033689A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention concerns DNA and peptide sequences encoding a novel mammalian secreted group IIF phospholipase A 2 and more particularly, a novel human group IIF phospholipase A 2 .
  • the invention also concerns the use of this enzyme in methods for screening various chemical compounds .
  • Phospholipases A 2 (PLA 2 , EC 3.1.1.4.) form a superfamily of enzymes that catalyze the hydrolysis of glycerophospholipids at the sn-2 position, producing free fatty acids and lysophospholipids [1-4].
  • sPLA 2 s Many intracellular and secreted phospholipases A 2 (sPLA 2 s) have been cloned in recent years [2,5], and several of them are involved in a variety of physiological and pathological functions including lipid digestion, cell proliferation, production of lipid mediators of inflammation, antibacterial defence, and cancer [4,6].
  • sPLA 2 s form a relatively homogenous family of enzymes, and they are characterized by the presence of several disulfides, an overall conserved three-dimensional structure and a common Ca 2+ -dependent catalytic mechanism.
  • mice sPLA 2 s Five novel mouse sPLA 2 s have been cloned during the last three years [7,8], and the mouse sPLA 2 family now comprises 8 distinct 14-16 Da sPLA 2 s called group IB, HA, lie, ID, HE, IIF, V and X, as well as otoconin-95, a sPLA 2 -like protein with peculiar structural properties [9,10].
  • genes for group HA, IIC, IID, HE, IIF, and V sPLA 2 s all map to mouse chromosome 4, suggesting the existence of a sPLA 2 gene cluster on this chromosome [8].
  • group IB, HA, IID, HE, V and X sPLA 2 s, but not group IIF have been cloned from humans [11-13].
  • group IIC sPLA 2 appears as a pseudogene in humans [14].
  • a novel human sPLA 2 with a predicted molecular mass of 55 kDa and a central domain similar to insect group III sPLA 2 s has recently been cloned [15], but it remains to be determined if this sPLA 2 is functional in the mouse species.
  • This novel human sPLA 2 is also disclosed in the international patent application N° 01/59129. All mouse and human sPLA 2 s have distinct tissue distributions, suggesting that each of them exert non redundant functions that could be related to their different enzymatic properties [6,16,17], and/or their binding properties to specific receptors [17-19].
  • each sPLA 2 is abbreviated with a lowercase letter indicating the sPLA 2 species (m, h, for mouse and human, respectively) followed by capital characters identifying the sPLA 2 group (GI, GH, GUI, GV, and GX) and subgroup (A, B, C, D, E, F).
  • the present invention concerns the cloning, tissue distribution and recombinant expression in E. coli of a novel mammalian group IIF sPLA 2 and more particularly a novel human group IIF (hGIIF) sPLA 2 .
  • This group II sPLA 2 has unique structural features including a long, proline- rich C-terminal extension with an odd cysteine, and a very low pi value. It also has a specific tissue distribution and a fairly high propensity to hydrolyse POPC versus POPG as compared to the other sPLA 2 s.
  • the gene for hGIIF sPLA 2 maps to chromosome 1 together with 5 other sPLA 2 genes to form a sPLA 2 gene cluster that spans about 300 kilobase pair (kbp) .
  • 5 of these 6 genes code for group II enzymes and share relatively high level of identity.
  • the last gene coding for group V sPLA 2 is in fact also related to group II sPLA 2 genes, as group V sPLA 2 does not contain a propeptide sequence and displays higher levels of identity to group II sPLA 2 s than to groups IB and X sPLA 2 s . It is thus likely that these 6 different genes have arisen from recent and successive gene duplication events.
  • group IIA, IIC, IID, HE and V sPLA 2 s are all basic enzymes while group IIF is very acidic.
  • group IB and X sPLA 2 s appear more divergent in sequence and are located on different chromosomes [13]. Both contain a propeptide sequence and the group I specific disulfide bond between cysteines 11 and 77. Whether group IB, X or one of the group H-like sPLA 2 s is more related to the sPLA 2 ancestor gene of the group I/II/V/X sPLA 2 collection [5] remains to be determined.
  • the invention concerns a novel mammalian secreted group IIF sPLA 2 wherein said enzyme is Ca 2+ - dependent, maximally active at pH 7-8, and hydrolyzes phosphatidylglycerol versus phosphatidylcholine with a 15- fold preference.
  • the invention concerns more particularly a mammalian secreted group IIF sPLA 2 constituted by or comprising the sequence of amino acids in the list of sequences under the number SEQ ID N°2. More particularly, the mammalian secreted group IIF sPLA 2 is a human secreted group IIF sPLA 2 .
  • the invention concerns a nucleic acid molecule comprising or constituted of an encoding nucleic sequence for a mammalian secreted group IIF sPLA 2 or for a fragment of a mammalian secreted group IIF sPLA 2 whose amino acid sequence is represented in the list of sequences in the appendix under the number SEQ ID N°2.
  • the invention relates more particularly to a nucleic acid molecule constituted by or comprising the sequence in the list of sequences in the appendix under the number SEQ ID N°l.
  • the invention also concerns nucleotide sequences derived from the above sequence, for example, from the degeneracy of the genetic code or by the suppression or insertion of nucleotides (such as introns ) , and which encode for proteins presenting characteristics and properties of group IIF SPLA 2 .
  • Another aim of the present invention is polyclonal or monoclonal antibodies directed against one secreted group IIF sPLA 2 of the invention, a derivative or a fragment of these.
  • These antibodies can be prepared by the methods described in the literature. According to prior art techniques, polyclonal antibodies are formed by the injection of proteins, extracted from animal tissues or produced by genetic transformation of a host, into animals, and then recuperation of antiserums and antibodies from the antiserums for example by affinity chromatography.
  • the monoclonal antibodies can be produced by fusing myeloma cells with spleen cells from animals previously immunised using the proteins of the invention. These antibodies are useful in the search for new secreted mammalian group IIF sPLA 2 or the homologues of this enzyme in other mammals or again for studying the relationship between the secreted group IIF sPLA 2 of different individuals or species.
  • the invention also concerns a vector comprising at least one molecule of nucleic acid above, advantageously associated with adapted control sequences, together with a production or expression process in a cellular host of a mammalian group IIF sPLA 2 of the invention or a fragment thereof.
  • the preparation of these vectors as well as the production or expression in a protein host of the invention can be carried out by molecular biology and genetic engineering techniques well known to the professional.
  • An encoding nucleic acid molecule for a mammalian secreted group IIF sPLA 2 or a vector according to the invention can also be used to transform animals and establish a line of transgenic animals.
  • the vector used is chosen in function of the host into which it is to be transferred; it can be any vector such as a plasmid.
  • the invention also relates to cellular hosts expressing mammalian secreted group IIF sPLA 2 obtained in conformity with the preceding processes.
  • the invention also relates to nucleic and oligonucleotide probes prepared from the molecules of nucleic acid according to the invention. These probes, marked advantageously, are useful for hybridisation detection of similar group IIF sPLA 2 in other individuals or species. According to prior art techniques, these probes are put into contact with a biological sample. Different hybridisation techniques can be used, such as Dot-blot hybridisation or replica hybridisation (Southern technique) or other techniques (DNA chips). Such probes constitute the tools making it possible to detect similar sequences quickly in the encoding genes for group IIF sPLA 2 which allow study of the presence, origin and preservation of these proteins.
  • the oligonucleotide probes are useful for PCR experiments, for example to search for genes in other species or with a diagnostic aim.
  • the secreted phospholipases A 2 are Ca 2+ - dependent, disulfide-rich, 14-18 kDa enzymes that catalyze the hydrolysis of phospholipids at the sn-2 position to release fatty acids and lysophospholipids.
  • sPLA 2 s are also ligands that bind to a collection of soluble and membrane bound proteins which are likely to play a role in the biological functions of these enzymes.
  • sPLA 2 s are structurally distinct mammalian sPLA 2 s, and it has become clear that these sPLA 2 s are expressed in a variety of tissues under both normal and pathological conditions (including inflammatory diseases, cancers, cardiac and brain ischemia, etc.), and are involved in a myriad of physiological and pathological roles.
  • sPLA 2 s In mammalian cells stimulated with proinflammatory agonists, a subset of sPLA 2 s play a role in the release of arachidonic acid for eicosanoid production.
  • sPLA 2 s are also involved in cell proliferation, cell migration, angiogenesis, cell contraction, apoptosis, neurosecretion, blood coagulation, adipogenesis , lipid metabolism (digestion, skin lipid barrier and lung surfactant formation, lipoprotein metabolism, etc.), spermatogenesis, fecondation, and embryogenesis . They also play a role in host defense and have antiviral and antibacterial properties against viruses like HIV-1 and various Gram- positive and Gram-negative bacterial strains. They also have antitumoral properties. They are also involved in various pathological conditions such as acute lung injury, acute respiratory distress syndrome, Crohn's disease, and various types of cancers where sPLA 2 s can act as gene suppressors.
  • the invention concerns pharmaceutical compositions comprising as active agent at least an encoding nucleic acid molecule for a mammalian secreted group IIF sPLA 2 , or one molecule for a mammalian secreted group IIF sPLA 2 or a derivative of this protein.
  • These pharmaceutical compositions can be used to treat or prevent viral and bacterial infections. They also can be used to treat or prevent cancers .
  • the present invention can also be useful in methods for identifying biologically active compounds with anti-inf lammatory properties or more generally for identifying compounds that modulate sPLA 2 biological activities as listed above .
  • Such biologically active compounds can be identified by determining if a selected compound is capable of inhibiting the catalytic activity of sPLA 2 in cleaving a phospholipid to release fatty acids and lysophospholipids in a mixed micelle assay, a liposome assay, a system utilizing natural membranes, or in whole cells overexpressing this enzyme.
  • a compound capable of inhibiting sPLA 2 catalytic activity may have anti- inflammatory or may behave as an antagonist of sPLA 2 in the sPLA 2 biological activities listed above.
  • screening of compounds for potential anti-inflammatory activity can be performed with the novel sPLA 2 enzymes of this invention, purified to homogeneity from cell sources or produced recombinantly or synthetically.
  • a selected compound may be added to a sPLA 2 enzyme of this invention in a mixed micelle assay, a liposome assay, or an assay system utilizing natural membranes and analyzed for inhibition of sPLA 2 activity.
  • a selected compound may be added to whole cells which overexpress the sPLA 2 and the cells examined for inhibition of release of fatty acids or lysophospholipids.
  • normal cells and cells overexpressing sPLA 2 can be cultured in labelled arachidonic acid.
  • Signal is measured between the secreted products of both the normal and overexpressing cells to provide a baseline of sPLA 2 expression.
  • a selected compound is then added to cultures and the cultures are grown in labelled arachidonic acid. If there is a difference in the signal (e.g., the amount of arachidonic acid produced) in the cells in the presence of the compound, this compound inhibits sPLA 2 activity and may be a potential anti- inflammatory compound.
  • Biologically active compounds can also be identified by screening the selected compounds for their binding properties to sPLA 2 receptors that bind group IIF sPLA 2 s of this invention. These receptors include the family of N-type and M-type receptors which are likely to be involved in several biological activities of sPLA 2 s including HIV-1 antiviral properties. For example, radioactively or fluorescently labelled sPLA 2 s can be used in competition binding assays and selected compounds can be screened for inhibition of sPLA 2 binding.
  • Biologically active compounds can also be identified by screening the selected compounds for modulation of a sPLA 2 biological effect such as those listed above.
  • sPLA 2 s of this invention may be added to cells in the presence or absence of a selected compound and cells may be assayed for cell proliferation, cell migration, cell contraction or apoptosis.
  • Novel pharmaceutical compositions may contain a therapeutically effective amount of a compound identified by an above method of this invention. These pharmaceutical compositions may be employed in methods for treating disease states or disorders involving group IIF sPLA 2 s of this invention.
  • the figure 1 represents the alignment of the amino acid sequences of human sPLA 2 s. Sequences of full- length sPLA 2 proteins are shown. A consensus sequence for the 7 group I/II/V/X human sPLA 2 s is presented.
  • the figure 2 represents a schematic diagram of the organisation of the human chromosome lp35 sPLA 2 gene cluster.
  • the total length between hGIIE gene and hGIIC pseudogene is about 300 kbp.
  • the PAC clone GenBank n° AL358253 is not yet fully sequenced and the hatched bars indicate the different contigs of this PAC clone.
  • the orientation and exon-intron bondaries of the different sPLA 2 genes are schematically shown.
  • the possible presence of 5' non coding exons in the hGIIC, hGHD, hGIIE and hGIIF genes remain to be determined.
  • the orientation and exact positions of the hGIIE and hGIIA genes are unknown.
  • the figure 3 represents the tissue distribution of the human sPLA 2 s, as determined by RT-PCR on human adult cDNA panels. The amplified products were analysed by Southern blot as described in materials and methods . No amplification was observed when cDNA was omitted in the PCR reaction (control lane).
  • the figure 4 represents the enzymatic properties of recombinant hGIIF sPLA 2 .
  • A Ca 2+ -dependence of the hydrolysis of phosphatidylcholine vesicles;
  • B pH- dependence of the hydrolysis of phosphatidylglycerol vesicles;
  • C Initial velocities for the hydrolysis of the indicated phospholipid vesicles. Full experimental details are provided in materials and methods. I. Materials and methods.
  • a set of oligonucleotides was designed from this genomic sequence (sense primer 5'- ATGAAGAAGTTCTTCACCGTGGCCA-3' (SEQ ID N°3 in the list of sequences in the appendix) and reverse primer 5'- ACCCTCCTCCCGCTCTCTCTCAAA-3 ' ( SEQ ID N°4 in the list of sequences in the appendix) ) and used in RT-PCR experiments on different human cDNAs .
  • a DNA product of the expected size was amplified from human cDNAs from spleen, heart, and fetal lung. Sequencing of the DNA fragments revealed complete identity with the genomic sequence after its appropriate splicing according to consensus exon-intron boundaries [21].
  • the preparation of a truncated GST hGIIF sPLA 2 construct, bacterial induction and preparation of sulfonated protein from inclusion bodies were carried out as previously described for mouse group IID sPLA 2 [7].
  • the hGIIF fusion protein was refolded by a rapid dilution method as follows.
  • Sulfonated protein was dissolved to 10 mg/ l in 4 ml of 6 M guanidine-HCl, 50 mM Tris-HCl, pH 8.0, and added dropwise (-1 drop per second) to 2 liters of refolding buffer (50 mM Tris-HCl, pH 8.0, 0.9 M guanidine- HCl, 10 mM CaCl 2 , 5 mM freshly added cysteine, 30% acetonitrile) with constant stirring at room temperature. Stirring was continued for a few minutes, and then the solution was allowed to sit without stirring at room temperature for -2-3 days. The sPLA 2 enzymatic activity was monitored with the fluorimetric assay [16] until the activity increase starts to level off.
  • reaction mixture was directly loaded at 3 ml/min on a Vydac 218 TP1010 C18 reverse phase column equilibrated with solvent A (20% acetonitrile, 0.1% trifluoroacetic acid, 1 mM methionine). Elution was performed at 3 ml/min using a linear gradient (0-6.3% B over 2 min, followed by 6.3-27.5% B over 42 in) of solvent B (100% acetonitrile, 0.1% trifluoroacetic acid, 1 mM methionine) .
  • HPLC purified hGIIF sPLA 2 was neutralized with 2 M Tris base, 5 mM lauryl sulfobetaine was added and the sample was concentrated in a Centriprep-10 (Amicon) .
  • the protein was then dialyzed against 10 mM Tris pH 8.0, 0.1 mM DTT, at 4 °C for 1 cycle to cleave the disulfide between the cysteine in the C-terminal extension of the hGIIF sPLA 2 and free cysteine from the refolding buffer, and then against 10 mM Tris-HCl, pH 8.0 for two cycles.
  • the approximate yield of final product per liter of E. coli culture is 3.7 mg. Concentrations of recombinant hGIIF sPLA 2 were determined by OD at 280 nm using an extinction coefficient of 10.37 calculated from the amino acid sequence .
  • the Ca 2+ and pH dependencies of hGIIF sPLA 2 were measured with POPC vesicles containing l-palmitoyl-2-[ 1- 3 H]palmitoyl-sn-glycero-3-phosphocholine vesicles and POPG vesicles containing l-palmitoyl-2- [ 1- 3 H ]palmitoyl-sn- glycero-3-phosphoglycerol, respectively [7].
  • Substrate specificity studies were carried out using a slightly modified assay with the fatty acid binding protein [7].
  • Reaction mixtures contained 30 ⁇ M POPC, POPG, or POPS large unilamellar vesicles (0.1 ⁇ m, prepared by extrusion as described [22]) in Hanks' balanced salt solution with 1 mM Ca 2+ , 1 mM Mg + , 9.7 ⁇ g fatty acid binding protein, and 1 ⁇ M 11-dansyl-undecanoic acid at 37 °C. Assays were calibrated by adding a known amount of oleic acid to the complete assay in the absence of enzyme.
  • the hGIIF mature protein sequence (calculated molecular mass 15,598 Da) is the most acidic sPLA 2 identified so far in mammals, with a calculated pi of 4.51.
  • the 23 amino acid C-terminal extension of hGIIF also appears relatively acidic, as it contains 3 glutamic acid residues and no basic residues. Furthermore, one third (8 out 23) of the residues of this C-terminal extension are proline residues. Interestingly, these specific features appear to be conserved among species, as the mouse group IIF C-terminal sequence is also acidic and proline-rich.
  • the odd cysteine residue found in the mGIIF sPLA 2 C- terminal extension is also conserved in the hGIIF sPLA 2 sequence.
  • hGIIF sPLA 2 contains the different residues which are conserved in all catalytically active sPLA 2 s and is particularly well- conserved with other human sPLA 2 s in the Ca 2+ loop and the active site domains. hGIIF sPLA 2 however shows low levels of identity with other human sPLA 2 s, and the most closely related sPLA 2 is hGIID with only 41% identity (Table I), indicating that hGIIF sPLA 2 is not an isoform of the previously cloned human sPLA 2 s. It should be noted that the highest level of identity between any two sPLA 2 s is observed between GIIA and GIIE (55% of identity in human species (Table I) and 51% in mouse species [8].
  • hGIIF sP A gene maps to chromosome 1 and belongs to a sPLA 2 gene cluster.
  • sPLA 2 gene cluster The organisation of the sPLA 2 gene cluster is presented in Fig. 2.
  • the human PAC clone dJl69023 (GenBank n° AL158172) of 141,865 bp that contains the hGIIF gene was generated by the sequencing program of human chromosome 1 , assigning the hGIIF gene to this chromosome.
  • this PAC clone contains also the full- length genes for h GV, h GIID , as well as the hGIIC pseudogene.
  • the hGIIA and hGIIE genes were found to be localized on the overlapping PAC clone dJ169023 (GenBank n° AL358253) in the telomeric direction (Fig. 2).
  • the available sequence of this PAC clone is composed of 49 unordered contigs of different lengths .
  • the overlapping sequence between AL158172 and AL358253 is estimated to be about 28 kbp.
  • the relative orientation of hGIIA and hGIIE with the other sPLA 2 genes and the exact distances between hGIIA and hGIIE genes, and hGIIA and hGV genes are unknown.
  • hGIIF and other human sPLA 2 s were analysed by RT-PCR experiments using commercial human cDNA panels. As shown in Fig. 3, hGIIF sPLA 2 is expressed at high levels in placenta, testis, thymus , and at lower levels in heart, kidney, liver and prostate. Very low signals are observed in skeletal muscle, pancreas, small intestine and spleen. In the mouse species, mGIIF transcripts were detected mostly in testis but also in several other tissues [8]. In the future, it will be interesting to analyse the expression of hGIIF sPLA 2 in embryos, since high levels of mGIIF transcripts were observed at different stages of embryonic development [8].
  • the fusion protein contains a factor Xa recognition site adjacent to the N-terminal residue of mature hGIIF which could be efficiently cleaved by using Factor Xa and trysin.
  • Cleaved hGIIF sPLA 2 was purified to homogeneity by chromatography on a C18 reverse phase column, and the overall yield of purified hGIIF sPLA 2 is 3.7 mg per liter of E. coli culture (data not shown).
  • the interfacial enzymatic properties of hGIIF sPLA 2 are summarized in Fig. 4.
  • the hydrolysis of phospholipid vesicles by hGIIF sPLA 2 is strictly Ca 2+ dependent, as expected for a typical sPLA 2 .
  • Fig. 4A shows that the rate of hydrolysis of phosphatidylglycerol vesicles by hGIIF sPLA 2 increases with pH in the range 5-7, as expected from the deprotonation of the active site histidine residue, and then decreases slightly at pH above 7.
  • the relative rates for the hydrolysis of POPG, POPS, and POPC vesicles by hGIIF sPLA 2 are compared in Fig. 4C.
  • hGIIF appears more similar to hGV and hGX sPLA 2 s, which are 3- and 10-fold more active on POPG versus POPC vesicles, respectively [16]. Whether exogenous hGIIF sPLA 2 , like hGX sPLA 2 , is able to efficiently release arachidonic acid from adherent cells will be interesting to analyse [16].

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des séquences d'ADN et des séquences peptidiques codant pour une nouvelle phospholipase A2 du groupe IIF sécrétée mammifère. Cette enzyme est dépendante de Ca2+, présente une activité maximale à un pH 7-8, et hydrolyse le phosphatidylglycérol vis à vis de la phosphatidylcholine avec une préférence de l'ordre de 15. Plus spécifiquement, l'invention concerne une nouvelle phospholipase A2 du groupe IIF humain. L'invention concerne également l'utilisation de cette enzyme dans des procédés de criblage de différents composés chimiques.
PCT/IB2001/002407 2001-10-12 2001-10-12 Clonage et expression recombinante de la phospholipase a2 du groupe iif secretee mammifere Ceased WO2003033689A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/IB2001/002407 WO2003033689A1 (fr) 2001-10-12 2001-10-12 Clonage et expression recombinante de la phospholipase a2 du groupe iif secretee mammifere

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IB2001/002407 WO2003033689A1 (fr) 2001-10-12 2001-10-12 Clonage et expression recombinante de la phospholipase a2 du groupe iif secretee mammifere

Publications (1)

Publication Number Publication Date
WO2003033689A1 true WO2003033689A1 (fr) 2003-04-24

Family

ID=11004228

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2001/002407 Ceased WO2003033689A1 (fr) 2001-10-12 2001-10-12 Clonage et expression recombinante de la phospholipase a2 du groupe iif secretee mammifere

Country Status (1)

Country Link
WO (1) WO2003033689A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001085956A2 (fr) * 2000-05-11 2001-11-15 Incyte Genomics, Inc. Enzymes du metabolisme lipidique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001085956A2 (fr) * 2000-05-11 2001-11-15 Incyte Genomics, Inc. Enzymes du metabolisme lipidique

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL 18 January 2001 (2001-01-18) *
DATABASE EMBL 25 October 2000 (2000-10-25), XP002197143 *
DATABASE EMBL 4 November 1999 (1999-11-04) *
DATABASE EMBL 5 October 2000 (2000-10-05), XP002197142 *
VALENTIN EMMANUEL ET AL: "Cloning and recombinant expression of human group IIF-secreted phospholipase A2.", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 279, no. 1, 9 December 2000 (2000-12-09), pages 223 - 228, XP002197140, ISSN: 0006-291X *
VALENTIN EMMANUEL ET AL: "On the diversity of secreted phospholipases A2: Cloning, tissue distribution, and functional expression of two novel mouse group II enzymes.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 44, 29 October 1999 (1999-10-29), pages 31195 - 31202, XP002197141, ISSN: 0021-9258 *

Similar Documents

Publication Publication Date Title
Valentin et al. Cloning and recombinant expression of human group IIF-secreted phospholipase A2
Monti et al. Molecular cloning and characterization of NEU4, the fourth member of the human sialidase gene family
Valentin et al. Cloning and recombinant expression of a novel mouse-secreted phospholipase A2
US5702936A (en) Cyclic GMP-binding, cyclic GMP-specific phosphodiesterase materials and methods
Nagai et al. An alternative splicing form of phosphatidylserine-specific phospholipase A1 that exhibits lysophosphatidylserine-specific lysophospholipase activity in humans
US5759811A (en) Mutant human hedgehog gene
Xin et al. Molecular cloning and characterization of a venom phospholipase A2 from the bumblebee Bombus ignitus
Ponce-Soto et al. Determination of primary structure of two isoforms 6-1 and 6-2 PLA2 D49 from Bothrops jararacussu snake venom and neurotoxic characterization using in vitro neuromuscular preparation
US6812017B2 (en) Mammalian secreted group IIF phospholipase A2
US6797816B2 (en) Identification and cloning of a new subfamily of sulfatases and functional embryonic techniques for characterization of such proteins
JPH09509319A (ja) Ｒｎａ修正酵素およびその使用方法
EP0206200B1 (fr) Protéines d'origine humaine du type cholinestérase et leur production
JP2003523723A (ja) ヘルマンスキー−パドラック症候群タンパク質相互作用タンパク質およびその使用の方法
EP1254239B1 (fr) Phospholipase a2 secretee mammalienne du groupe iii
US20020119139A1 (en) Cloning and recombinant expression of mammalian group XII secreted phospholipase A2
WO1995030008A1 (fr) Nouvelles tyrosine phosphatases a densite renforcee
US20040253221A1 (en) Novel pla1
WO2003033689A1 (fr) Clonage et expression recombinante de la phospholipase a2 du groupe iif secretee mammifere
CZ9902005A3 (cs) Nové calpainy, jejich výroba a použití
Braren et al. Use of the EST database resource to identify and clone novel mono (ADP-ribosyl) transferase gene family members
KR20010023282A (ko) 신규 조직 특이적 칼파인, 그의 제조 방법 및 용도
JPH10201479A (ja) フォスフォリパーゼａ１ 及びそれをコードする核酸
KR100636587B1 (ko) 스핑고지질 세라마이드 디아실라제 유전자
JP4169846B2 (ja) マウス新規分泌型ホスホリパ−ゼａ２をコードする遺伝子
Bazan et al. MONO (ADP-RIBOSYL) TRANSFERASE GENE

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BY BZ CA CH CN CO CR CU CZ DE DM DZ EC EE ES FI GB GD GE GH HR HU ID IL IN IS JP KE KG KP KR LC LK LR LS LT LU LV MA MD MG MN MW MX MZ NO NZ PH PL PT RO SD SE SG SI SK SL TJ TM TR TT TZ UG UZ VN YU ZA

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZW AM AZ BY KG KZ MD TJ TM AT BE CH CY DE DK ES FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP