JP2002514087A - Mammalian lysophosphatidic acid acyltransferase - Google Patents

Abstract

  (57) [Summary] Disclosed are cDNA sequences and polypeptides having the activity of the enzyme lysophosphatidic acid acyltransferase (LPAAT). LPAAT is also known as 1-acyl sn-glycerol-3-phosphate acyltransferase.

Description

DETAILED DESCRIPTION OF THE INVENTION           Mammalian lysophosphatidic acid acyltransferase Field of the invention   The present invention relates to the activity of the enzyme lysophosphatidic acid acyltransferase (LPAAT). The present invention provides cDNA sequences and polypeptides having sexuality. LPAAT is 1-acyl sn-glycer Also known as roll-3-phosphate acyltransferase. The present invention Is further involved in phosphatidic acid metabolism and signaling in mammalian cells The present invention provides for the isolation and production of polypeptides, particularly the production of purified LPAAT. Background of the Invention   As originally thought as an intermediate in lipid biosynthesis (Kent, Anal. Rev. Bio. chem. 64: 315-343, 1995), phosphatidic acid (PA) and one of its precursors. One lysophosphatidic acid (LPA) can affect a wide range of biological responses It has also been identified as a quality signaling molecule (McPhail et al., Proc. Natl. Acad. Sci. USA 92: 7931-7935, 1995; Williger et al. Biol. Chem. 270: 29656-29659, 1995; M oolenaar, Curr. Opin. Cell Biol. 7: 203-210, 1995).   Cell activation in mononuclear cells and lymphoid cells is dependent on phosphatidic acid (PA) , Diacylglycerol (DAG) and glycan phosphatidylinositol (P Related to the rapid up-regulation of the synthesis of phospholipids (PL) containing I) . Phosphatidic acid (PA) is a molecular compound associated with cell activation and mitogenesis Is a second messenger of a different group of phospholipids (Singer et al., Exp. Opin. In vest. Drugs 3: 631-643, 1994). Therefore, it blocks PA production and is involved in cell activation Compounds that appear to reduce inflammatory signals may be of therapeutic interest in the field of inflammation and oncology Tasteful. Lithophilin (1- (R)-(5-hydroxyhexyl) -3,7-dimethyloxa (Singer et al., Exp. Opin. Invest. Drugs. 3: 631-643, 1994; and Rice). Proc. Natl. Acad. Sci. USA 91: 3857-3861, 1994) is an activated mononuclear cell. Lysophosphatidic acid acyltransferase (LPAAT) produced in cells Blocks the synthesis of unusual phosphatidic acid (PA), thereby enhancing cell activation Have been found to be effective inhibitors (Rice et al., Proc. Natl. Acad. Sci. . USA 91: 3857-3861, 1994). PA is phosphatidylcholine (PC) (Exton, Bioch em . Biophys. Acta 1212: 26-42, 1994) or glycan PI (Eardley et al., Science).  251: 78-81, 1991; Merida et al., DNA Cell Biol. 12: 473-479, 1993) Thus, or by phosphorylation of DAG by DAG kinase (Kanoh et al., Trends Bi ochem. Sci. 15: 47-50, 1990), or by acylating LPA at the SN2 position (Bursten et al., Am. J. Physiol. 266: C1093-C1104, 1994). Therefore Blocks PA production and reduces lipid biosynthesis and signaling involved in cell activation Potential compounds are of therapeutic interest in the fields of inflammation and tumors and obesity No.   The gene encoding LPAAT can be obtained from bacteria (Coleman, Mol. Gen. Genet. 232: 295-303, 1992), yeast (Nagiec et al., J. Biol. Chem. 268: 2). 2156-22163, 1993) and plants (Brown et al., Plant Mol. Biol. 26: 211-223, 1994). ; and Hanke et al., Eur J .; Biochem. 232: 806-810, 1995) . Cloning of the mammalian LPAAT has not been reported. Bacteria, yeast and plant L The homology between PAATs is only a few blocks of 3 or at most 4 amino acids of the entire sequence. (Brown et al., Plant Mol. Biol. 26: 211-223, 1994). ). In addition, to develop specific compounds that would inhibit this enzyme, The need to be able to screen compounds for LPAAT inhibitors is emerging in mammals. Present on source recombinant LPAAT technology. Summary of the Invention   The present invention provides cDNA sequences, polypeptides for producing isolated recombinant mammalian LPAAT. Peptide sequences and transformed cells are provided. The present invention provides two types of LPAAT Novel human polypeptides and fragments thereof are provided. Discovered herein The polypeptide is novel, called hLPAAT, and the first polypeptide found is hLPAA The second polypeptide, designated Tα and found, is called hLPAATβ. LPAAT Acyl at the sn-2 position of lysophosphatidic acid (LPA) having a fatty acid acyl chain moiety Phosphatidic acid (PA) of lysophosphatidic acid (LPA) Catalyzes the acylation to   The present invention further relates to the expression of novel LPAAT polypeptides and active fragments thereof. A nucleic acid sequence encoding The present invention further provides purified LPAATs, Lipe Antisense Oligonucleotides for Regulating Expression of Peptide-Encoding Genes Serve tide. Test compounds for their ability to inhibit LPAATs Also provided are assays for tuning.   Recombinant LPAAT is used to screen for candidate drug compounds that inhibit LPAAT activity Useful for The present invention has LPAAT activity and has SEQ ID NO: 1 or SEQ ID NO: 7. CDNA containing the DNA sequence described in Section 1 and their short fragments, or the genetic code Depending on the degeneracy, another encoding the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 8 CDNA sequences, or biologically active fragments thereof, or high stringency -Provides sequences that can hybridize to them under conditions. The present invention further relates to an amino acid having LPAAT activity, A polypeptide comprising a nucleic acid sequence or a biologically active fragment thereof.   Contains DNA sequence encoding mammalian LPAAT enzyme and is functionally linked to expression control sequences Matching vectors are also provided by the present invention. Used to make recombinant LPAAT Also provided by the present invention is a host cell transformed with such a vector. It is. The vectors and transformed cells of the invention produce mammalian LPAAT. Used for the method. The method of the present invention encodes an LPAAT enzyme and The cell line transformed with the DNA sequence operably linked to the control sequence is cultured. The present invention Is used as a host cell to express the LPAAT polypeptide, for example, a mammal. Uses many well-known cells, including cells, yeast cells, insect cells and bacterial cells be able to.   Another aspect of the invention relates to a method wherein the selected compound is an LPAAT for acylating LPA to PA. By determining whether the activity can be inhibited, a pharmaceutically active Methods are provided for identifying compounds. Compounds that enable such activity are Trilinage after cytoreductive therapy, which can indirectly inhibit SAP kinases (Trilineage) Enhancement of hematopoiesis and hypoxia and oxygen overload injury (eg, Useful for anti-inflammatory activity in inhibiting the inflammatory cascade after sepsis, trauma, ARDS, etc.) It can be a pharmaceutical compound.   The present invention further provides a transformed cell that expresses active mammalian LPAAT, Are drug candidate compounds therapeutically active by inhibiting recombinant LPAAT activity? How Means for determining whether BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the cDNA insertion DNA sequence of pZplat.11 encoding hLPAATα.   FIG. 2 shows the human LPAATα coding sequence, yeast LPAAT coding sequence, E. coli LP Amino acid sequences of AAT coding sequence and maize LPAAT coding sequence were aligned. Is shown. This comparison shows that human LPAATα is more likely to be yeast or colonic than plant LPAAT. This shows that the range of homology with bacterial LPAAT is wide.   FIG. 3 shows the DNA sequence of cDNA-inserted pSP.LPAT3 encoding hLPAATβ. cDNA chromatography Sequence analysis and restriction mapping of the 39 base pair 5 'untranslated region Region and an open library at positions 40-876 encoding a 278 amino acid polypeptide Dink frame revealed. It is also the 3 'end of the 480 base pairs of pSP.LPAT3. The translation region is also shown. The translation initiation site is at nucleotide positions 40-42, providing sufficient initiation Site requirements were met (Kozak, Critical Rev. Biochem. Mol. Biol. 27:38). 5-402, 1992).   FIG. 4 shows an open reading frame of 278 amino acids of hLPAATβ. This Quer for searching homologous sequences in protein databases Used as y array. National biotechnology using the blastp program National Center for Biotechnology Informatio n) Searching the database based on (NCBI) GenBank Release 92 The proteins showed the greatest homology with yeast, bacterial and plant LPAAT.   FIG. 5 shows a coding sequence determined to be human LPAATβ of the present invention, a human LPAATα coding sequence. Sequence, yeast LPAAT coding sequence, bacteria (E. coli, H. influenzae, and S. typhimuri) um) LPAAT coding sequence and plant (L. douglassi and C. nucifera) LPAAT The sequence of the amino acid sequence of the human LPAAT is shown. Reveals much broader homology with yeast or bacterial LPAAT .   Figure 6 shows that pCE9.LPAAT1 DNA was analyzed using TLC (thin layer chromatography) analysis. Do not transfect transfected A549 cells or DNA 4 shows a comparison of LPAAT activity of cells that had been lost. These data are more detailed in Examples 3 and 4. It describes in.   Figures 7 and 8 show transfection of pCE9.LPAAT1 after stimulation with IL-1β and mouse TNF. TNF (FIG. 7) and IL-6 (FIG. 8) of the transfected A549 cells and control A549 cells. 3) shows a comparison of the production of These data are based on untransfected A5 A549 overexpressing LPAAT produces> 5 fold more TNF and> 10 fold more compared to 49 cells It shows that it produces IL-6. This indicates that LPAAT overexpression is Signal transmission response. DETAILED DESCRIPTION OF THE INVENTION   The present invention provides a novel, isolated, biologically active mammalian LPAAT enzyme. The term "isolated" refers to any LPAAT of the invention or each other polypeptide. Pass along with the peptide or gene, that is, the gene for the LPAAT polypeptide in nature. LPAAT polypeptides essentially free of other impurities that may be found Means any other gene that encodes   The present invention includes the functional polypeptide LPAAT and functional fragments thereof. Honcho The term "functional polypeptide" as used in the textbook refers to biological assays , Preferably identified by a cell-based assay to form PA species from LPA Refers to a polypeptide having a biological function, that is, activity. "Functional polynu "Nucleotide" refers to a polynucleotide that encodes a functional polypeptide. Minor changes in the primary amino acid sequence of hLPAATα may result in the sequencing described herein. A protein having substantially equivalent activity as compared to a defined hLPAATα polypeptide. Quality can be obtained. Such changes are intentionally made by site-specific mutation. It can be made or naturally occurring. The ports created by these changes As long as the acyltransferase activity of LPAAT is present, all Included in the description. This allows for smaller active molecules with greater utility Can be developed. For example, amino or carboxy unnecessary for LPAAT activity Terminal amino acids can be removed.   HLPAATα and hLPAATβ of the present invention also have a conservative mutant polypeptide sequence. Including. A “conservative variant” is one in which the residue is derived from another biologically active residue. Refers to substitution of amino acid residues. Examples of conservative variants are isoleucine, valine, Substitution of one hydrophobic residue such as isine or methionine for another, or Means arginine and lysine, glutamic acid and aspartic acid, or glutamine One polar residue, such as the substitution of asparagine and the like, with another polar residue Including the substitution of The term “conservative variant” also describes substituted polypeptides. If the antibody produced against it also immunologically reacts with the unsubstituted polypeptide, It also includes the use of a substituted amino acid in place of the unsubstituted parent amino acid.   The polypeptides of the present invention may be used commonly, such as for t-BOC or FMOC protection of the alpha amino group. It can be synthesized by the method used. Both methods start from the C-terminus of the peptide Starting with a stepwise synthesis in which one amino acid is added at each step (Colig an et al., Current Protocols in Immunology, Wiley Interscience, Unit 9, 1991 ). In addition, the polypeptides of the present invention also contain 0.1-1.0 mM amine / g polymer. Solid-phase synthesis method using copolyol (styrene-divinylbenzene) (For example, Merrifield, J. Am. Chem. Soc. 85: 2149). , 1962; and Stewart and Young, Solid Phase Peptide Synthesis, Freeman , San Francisco pp. 27-62, 1969). At the end of the chemical synthesis, at 0 ° C for about 15-60 minutes Remove the protecting groups of the polypeptide by treating with liquid HF 10% anisole. And can be cut from the polymer. After distilling off the reagents, the polymer is And then freeze-dried to obtain a crude material. This is usually A technique such as gel filtration using Sephadex G-15 using 5% acetic acid as a medium And can be purified. Freeze-drying of the appropriate fractions of the column yields a homogeneous poly Acquire peptide or polypeptide derivatives, analyze amino acids, thin-layer chromatography Fee, high performance liquid chromatography, ultraviolet absorption spectrometer, molecular rotation, solubility And standard techniques such as solid-phase Edman degradation quantification Be killed.   The present invention also provides a polynucleotide encoding the hLPAAT of the present invention. Book "Polynucleotide" as used herein, in the form of discrete fragments, Or deoxyribonucleotide or ribo as a component of a larger construct Refers to a polymer of nucleotides. The DNA encoding the polypeptide of the present invention comprises A cDNA fragment or oligonucleotide that provides a synthetic gene that can be expressed in the It may be assembled from gonucleotides. The polynucleotide sequence of the present invention may comprise DNA, R Contains NA and cDNA sequences. Preferably, the nucleotide sequence encoding hLPAAT is hL PAATα is the sequence of SEQ ID NO: 1, and LPAATβ is the sequence of SEQ ID NO: 7. D of the present invention NA sequences can be obtained in several ways. For example, c known in the art. It can be isolated using an hybridization procedure. Hive like this The redidation procedure is, for example, for detecting shared nucleotide sequences. Hybridization of probe to genome of cDNA library, normal antigen Antibodies in expression libraries to detect shared structural features such as sexual epitopes Includes screening and polymerase chain reaction (PCR) synthesis.   Hybridization procedures involve the use of labeled synthetic oligonucleotide probes. Useful for screening recombinant clones by using a mixture is there. In this case, each probe is a hybrid containing a heterogeneous mixture of denatured double-stranded DNA. Probably a perfect complement of a particular DNA sequence in the sample. In such screening, hybridization is preferably performed in one Performed on strand DNA or denatured double-stranded DNA. Hybridization is MRNA for heart polypeptides is derived from very low abundance sources It is particularly useful for detecting cDNA clones. Avoid non-specific binding The use of stringent hybridization conditions for The hybridization of the target DNA to one of the Autoradiographic visualization of specific cDNA clones as strands becomes possible ( Wallance et al. Nucl. Acid Res. 9: 879, 1981). Specific DNA sequence encoding hLPAAT Is the isolation of double-stranded DNA sequences from genomic DNA, a necessary component for the polypeptide of interest. Chemical production of DNA sequences providing don and m isolated from eukaryotic donor cells Can be formed by in vitro synthesis of double-stranded DNA sequences by reverse transcription of RNA You. In the latter case, the double-stranded DNA complement of mRNA, commonly referred to as cDNA, ultimately forms Is done. These three methods form specific DNA sequences for use in recombination procedures. Of the methods, isolation of genomic DNA isolates is less common. In the presence of introns Thus, when it is desirable to express a mammalian polypeptide with a microorganism This is especially true.   The synthesis of DNA sequences often involves the identification of amino acid residues in the desired polypeptide product. This is the preferred method when the full-length sequence is known. Desirable polypeptide amino acid It is not possible to directly synthesize a DNA sequence if the full-length sequence of the residue is not known It is desirable to synthesize a cDNA sequence. Isolation of cDNA sequences can be performed, for example, using mRNA Plasmid or plasmid containing a cDNA library, induced by reverse transcription of This can be done by forming a page. mRNA has high gene expression Are abundant in donor cells. If expression is low, PCR techniques are preferred. If most of the amino acid sequence is known, labeled single- or double-stranded DNA Or an RNA probe sequence that replicates a sequence presumed to be present in the target cDNA Is used in the DNA / DNA hybridization procedure and is Can be performed on cloned copies of cDNA (Jay et al., N ucl. Acid Res. 11: 2325, 1983).   Using antibodies specific for hLPAATα or hLPAATβ, at least one Indirectly for hLPAATα or hLPAATβ polypeptides Can be screened. Such antibodies are May be derived polyclonal or monoclonal, hLPAATα or It can be used to detect expression products that indicate the presence of hLPAAT / β cDNA.   The polynucleotide sequence can be deduced from the genetic code, Degeneracy must be considered. The polynucleotide of the present invention has a genetic code The resulting sequence contains degenerate sequences. The polynucleotide of the present invention may also comprise a gene Contains sequences that are degenerate as a result of the code. There are 20 natural amino acids, Are defined by more than one codon (3-base sequence). Therefore, hL As long as the amino acid sequence of PAATα or hLPAATβ becomes a functional polypeptide (less In the case of a sense polynucleotide chain), all degenerate nucleotide sequences are Included in the Ming. The polynucleotide sequences of hLPAATα and hLPAATβ also And a sequence complementary to the polynucleotide encoding hLPAATβ (antisense sequence Column). Antisense nucleic acids are DNs that are complementary to at least a portion of a particular mRNA molecule. A and RNA molecules (Weintraub, Sci. Amer. 262: 40, 1990). The present invention relates to hLPA Any anti-serum capable of inhibiting the production of ATα and hLPAATβ polypeptides And polynucleotides. In cells, antisense nucleic acids are converted to the corresponding mRNA. Hybridize to form double-stranded molecules. Cells produce double-stranded mRNA Because they cannot be translated, antisense nucleic acids interfere with translation of the mRNA. Nuku Antisense oligomers of about 15 leotide are easily synthesized, hLPAATα and hL May cause more problems than larger molecules when introduced into target cells that produce PAATβ It is preferable because it is stiff. The use of antisense methods to inhibit gene translation It is well known (for example, Marcus-Sakura, Anal. Biochem. 172: 289, 1988).   The ribozyme nucleotide sequences of hLPAATα and hLPAATβ are included in the present invention. It is. Ribozymes are used to convert other single-stranded RNs in a manner similar to DNA restriction endonucleases. It is an RNA molecule that has the ability to specifically cleave A. Encodes such RNA By modifying the nucleotide sequence, specific nucleotide arrangements in the RNA molecule can be The sequence can be recognized and a molecule to be cleaved can be produced (Cech, J. Amer. Med. As. sn. 260: 3030, 1988). The advantage of this method is that it is sequence-specific, That is, the mRNA having the predetermined sequence is inactivated.   Ribozymes include tetrahymena type (Hasselhoff, Nature 334: 585, 1988) and " There are two basic types of "hammer head type". Tetrahymena ribozyme Recognizes a sequence with a length of 4 bases, while the “hammerhead” ribozyme has a length of 1 Recognizes a base sequence of 1 to 18 bases. The longer the recognition sequence, the more that sequence There is a greater possibility that it is mainly present. As a result, it inactivates certain mRNA species. Therefore, hammerhead ribozymes are preferred over tetrahymena ribozymes. New   Polynucleotides encoding hLPAATα and hLPAATβ polypeptides of the invention The sequences may be expressed in prokaryotic or eukaryotic cells. Hosts are microorganisms (bacteria), yeast It can include mothers, insects and mammals. Prokaryotic eukaryotic cells or viruses Methods for expressing a DNA sequence having the sequence of a protein are well known in the art. Expression in host And plasmids with biological functions capable of replication and plasmid DN A vectors are well known in the art. Using such a vector, the DNA sequence of the present invention is used. Introduce columns. The DNA sequence encoding the polypeptide of the present invention can be prepared using the well-known trans gene. Using an infection method, the DNA can be introduced into a suitable host by introducing the DNA into the host. It can be expressed in vitro.   hLPAATα and hLPAAT / 3 DNA sequences can be inserted into recombinant expression vectors To . The term "recombinant expression vector" refers to the insertion or incorporation of a gene sequence Refers to a plasmid, virus or other messenger that has been manipulated. This Such expression vectors are used to promote efficient transcription of the inserted gene sequence into the host. Includes motor sequence. Expression vectors typically contain an origin of replication, a promoter, And specific genes that allow for phenotypic selection of transformed cells. Vectors suitable for use in the present invention include, for example, cells for expression in bacteria. Expression vector having a bacterial promoter and a ribozyme binding site (Gold, Meth. Enzymol. 185: 11, 1990), animal promoters and promoters for expression in mammalian cells. Expression vectors having an enhancer and an enhancer (Kaufman, Meth. Enzymol. 185: 487, 199). 0) and baculovirus-derived vectors for expression in insect cells (Luckow et al. , J. et al. Virol. 67: 4566, 1993). The DNA portion may be, for example, a promoter (eg, Regulatory elements such as, T7, metallothionein I or polyhedrin promoter) May be present in a vector operably linked to the vector.   Vectors are selected phenotypically to identify host cells containing the expression vector. It may include a selectable marker. Typically used for prokaryotic expression vectors Examples of markers include ampicillin (β-lactamase), tetracycline and Chloramphenicol (chloramphenicol acetyltransferase) Contains antibiotic resistance genes. This is typically used for mammalian expression vectors. Examples of markers such as adenosine deaminase (ADA), aminoglycoside Phosphotransferase (neo, G418), dihydrofolate reductase (DHFR), Igromycin-B-phosphotransferase (HPH), thymidine kinase (TK) And xanthine guanine phosphoribosyltransferase (XGPTR, gpt) Including genes.   In another preferred embodiment, the expression system used is a baculovirus polyhedrin. It is driven by a promoter. Genetics encoding polypeptides The offspring are standardized to facilitate cloning in baculovirus vectors. It can be operated by various techniques. See Ausubel et al., Supra. That. A preferred baculovirus vector is the pBlueBac vector (Invitrogen, Sorrento, CA). Spodopte is a vector that carries the polypeptide gene. La   Standard Protocol for Spodoptera frugiperda (Sf9) Cells And culturing the cells to produce the recombinant polypeptide. Process as follows. Summers et al., Baculovirus Vector Method and A Manual for Methods of Baculovirus Vector s and Insect Cell Culture Procedures, Texas Agricultural Experiment Station ( Texas Agricultural Experimental Station).   Once the full-length coding sequence of the polypeptide gene has been determined, the gene Can be inserted into the current system. Genes can be expressed in any number of different recombinant DNA expression systems. It can be expressed to produce large amounts of polypeptide. Native glycosylation Sequences and deglycosylated or unglycosylated produced by the methods described below Silylated polypeptides are included in the present invention. An example of an expression system well known to those skilled in the art is E. coli ( E. coli, etc., yeasts such as Pichia pastosis, Includes mammalian expression systems such as culoviruses and Cos or CHO cells.   A gene or gene fragment encoding the desired polypeptide can be It can be inserted into an expression vector by a cloning technique. In a preferred manner Uses an E. coli expression vector that produces a recombinant protein as a fusion protein. When used as a protein, rapid affinity purification of proteins is possible. this An example of a fusion protein expression system is the glutathione S-transferase system (Pharmacia). , Piscataway, NJ), maltose binding protein system (NEB, Beverley, MA), Fusion (Invotrogen, San Diego, CA), FLAG (IBI, New Haven, C) T) and the 6xHis system (Qiagen, Chatsworth, CA). Some of these systems Only a small number of alternatives that may not affect the LPAAT capacity of the recombinant polypeptide To produce a recombinant polypeptide that forms For example, FLAG and 6x The His system adds very short sequences, both of which are well known to have low antigenicity and remain unchanged. Does not adversely affect polypeptide folding on sexual conformation . Other fusion systems require removal of the fusion partner from the desired protein. Produce new proteins. In a preferred embodiment, the fusion partner is a protein Recombinant polypeptide by peptide sequence with recognition sequence specific for ase Join. An example of a suitable sequence is Tobacco Etch Virus Protease (Tobacco Etch).  Virus pro tease) (Life Technologies, Gaithersburg, MD) or Factor Xa (New England) Biolabs, Beverley, MA) or enterokinase (Invotrogen, San Diego, CA) ).Production of polypeptide   In a preferred embodiment, the recombinant protein is E. coli and baculo. And expressed in the rovirus expression system. Even if the full-length gene of the polypeptide is expressed Alternatively, fragments of the gene encoding the antigenic determinant may be generated. First In a preferred embodiment, the gene sequence encoding the polypeptide is analyzed to Detect putative sequences. Such sequences are typically very hydrophobic , MacDNASIS (Hitachi, San Bruno, CA) and other standard sequence analysis software Is easily detected by using. Recombinant proteins can be used in many expression systems Especially when synthesized in E. coli, the presence of the transmembrane sequence Insoluble aggregation makes restoration to the native polypeptide conformation difficult They are often harmful because they form things. Deleting the transmembrane sequence typically results in Does not significantly affect the conformation of the remaining polypeptide structure. In addition, transmembrane sequences, by definition, are embedded in the membrane, and thus, Not accepted as antigenic determinant. Therefore, antibodies against these sequences Such sequences are removed from the recombinant polypeptides of the present invention because they do not primarily confer immunity. Even so, there is little loss in immunity. Transmembrane from genes used for expression Excluding the transcoding sequence can be performed by standard techniques. Oath See Ausubel et al., Supra, Chapter 8. For example, an accidentally placed restriction enzyme The desired gene fragment may be excised using elementary sites, or using PCR Only the desired parts of the gene may be amplified.   Transformation of host cells with recombinant DNA can be performed by conventional techniques. Wear. If the host is a prokaryotic cell, such as E. coli, use standard procedures. Sample after the exponential growth phase_TwoFrom cells treated with the method Ability cells that can take up NA can be prepared. Or MgCl_TwoAlso Alternatively, RbCl can be used. Forming protoplasts of host cells Or by electroporation. You.   If the host is a eukaryotic cell, calcium phosphate co-precipitation, microinjection Conventional methods such as electrophoresis, electroporation, and insertion of liposome-encapsulated plasmids Mechanical procedures or DNA transfection methods such as viral vectors Can be used. Eukaryotic cells contain the hLPAATα and hLPAATβ polypeptides of the invention. Selection of DNA sequences encoding tides and herpes simplex thymidine kinase gene Can be co-transformed with a second foreign DNA molecule encoding an alternative phenotype. Wear. Other methods include Simian virus 40 (SV40) or bovine papilloma virus. Which eukaryotic virus vector is used to temporarily infect or shape eukaryotic cells To express hLPAATα and hLPAATβ polypeptides.   Expression vectors suitable for producing LPAAT polypeptides are typically (1) 2.) to provide for the origin of bacterial replication and expression vector propagation and selection in bacterial hosts. Prokaryotic DNA elements encoding various antibiotic resistance markers; (2) promoters, etc. Eukaryotic DNA elements that control the initiation of transcription; and (3) transcription termination / polyadenylation DNA elements that control the processing of the transcript, such as transcription sequences. LPAAT polypep of the present invention The tide is preferably found in eukaryotic cells such as mammalian cells, insect cells and yeast cells. Is expressed. Because mammalian cells provide suitable post-translational regulation, such as glycosylation, In particular, mammalian cells are particularly preferred eukaryotic host cells. An example of a mammalian host cell is cha Chinese hamster ovary cells (CHO-K1; ATCC CCL61), rat pituitary cells (GH₁; A TCC CCL82), HeLaS3 cells (ATCC CCL2.2), rat hepatocellular carcinoma cells (H-4-II-E; ATC C CRL1548), SV40-transformed monkey kidney cells (COS-1; ATCC CRL1650) and mice Includes fetal cells (NIH-3T3; ATCC CRL 1658). In mammalian hosts, transcription and translation Regulatory signals are those in which the regulatory signal is bound to a specific gene that is highly expressed. Viruses such as denovirus, bovine papilloma virus, simian virus, etc. Can be derived from a source. Suitable transcription and translation control sequences include actin, Obtained from mammalian genes such as the lagen, myosin and metallothionein genes You can also.   Transcription regulatory sequences include a promoter region sufficient to initiate RNA synthesis. A suitable eukaryotic promoter is the mouse metallothionein I gene (Hamer et al., JM olec. Appl. Genet. 1: 273, 1982); Herpesvirus TK promoter (McKn i ght, Cell 31: 355, 1982); SV40 early promoter (Benoist et al., Nature 290: 30). Rous sarcoma virus promoter (Gorman et al., Proc. Natl. Acad. Sc. i. USA 79: 6777, 1982); and the cytomegalovirus promoter (Foesking). Gene 45: 101, 1980). Alternatively, the prokaryotic polypeptide is an eukaryotic pro- If regulated by a motor, bacteriophage T3 RNA polymerase Use prokaryotic polypeptides such as promoters to control fusion gene expression (Zhou et al., Mol. Cell. Biol. 10: 4529, 1990; Kaufman et al., Nucl. Acids Res. 19: 4485, 1991).   Calcium phosphate transfection, liposome-mediated transfection The expression vector can be harbored using various techniques, including electroporation, electroporation, etc. It can be introduced into the main cell. The expression vector is stably integrated into the host cell genome. Transfected cells that produce stable and stable transformants are selected And preferably proliferate. Techniques and preferences for introducing vectors into eukaryotic cells Techniques for selecting stable transformants using various selectable markers include, for example, Ausubel and Murray (eds.), Gene transfer and expression Protocol (Gene Transfer and Expression Protocols) (Humana Press 199 It is described in 1). Examples of mammalian host cells include COS, BHK, 293 and CHO cells. No.Purification of recombinant polypeptide   Large amounts of polypeptides expressed in any of a number of different recombinant DNA expression systems Quantities can be obtained and tested for biological activity. For example, E. coli (E.col) Recombinant bacteria such as i) can be grown in any of a number of suitable media such as, for example, LB. The expression of the recombinant polypeptide can be determined by adding IPTG to the culture medium or incubating at higher temperatures. Induced by transferring the cubation. Bacteria for another 2 to 24 hours After culturing, collect cells by centrifugation and wash to remove residual medium . For example, lyse bacterial cells by disruption with a cell homogenizer and centrifuge. Separation separates dense inclusion bodies and cell membranes from soluble cellular components. This far For centrifugation, add sugar, such as sucrose, to the buffer and centrifuge at the selected speed. Can be performed under conditions that selectively enrich dense inclusion bodies . If the recombinant polypeptides are expressed in inclusion bodies, Of solution Wash with any of them to remove some host protein impurities Chaotropic reagents such as urea or guanidine hydrochloride at high concentrations (for example, 8M) Add β-mercaptoethanol or DTT (dithiothreitol) to the solution containing the drug. Dissolves in the presence of a reducing agent such as At this stage, native polypeptide The folding process into a conformation more similar to the Incubate the polypeptide for several hours under conditions suitable for the peptide to undergo May be advantageous. Such conditions are generally associated with low concentrations of poly. Peptide (less than 500 mg / ml), low concentration of reducing agent, urine less than 2M Reduction, which often involves the exchange of disulfide bonds in protein molecules Reagents, such as a mixture of oxidized and oxidized glutathione. The folding process is like For example, by SDS-PAGE or using an antibody specific for the native molecule Can be. After folding, several supports including ion exchange resin, Kell permeable resin Chromatography on any of the various affinity columns Allows the polypeptide to be further purified and separated from the folding mixture. Wear.   Isolation and purification of the polypeptide or fragment thereof expressed by the host cell can be accomplished by preparative chromatography. Immunological components, including monoclonal and polyclonal antibodies It can be implemented by conventional means, including but not limited to separation.   Compounds for trilineage hematopoietic and anti-inflammatory therapeutic applications Useful for cleaning and forming antibodies for therapeutic, diagnostic and research applications To enable large-scale production of pure and active hLPAATα and hLPAATβ, These polypeptides can be produced in a variety of ways, including recombinant DNA techniques. You.   The hLPAATα and hLPAATβ polypeptides of the present invention are used for cell signaling of inflammatory response. When screening for methods to identify compounds or compositions that have an effect Useful for This method is based on hLPAATα and hLPAATβ polypeptides or hLPAAT Cells transfected with cDNAs encoding α and hLPAATβ Incubate under conditions sufficient for interaction, then hLPAATα and measuring the effect of the compound or composition on hLPAATβ activity. hLPAAT The observed effects on α and hLPAATβ may be inhibitory or stimulatory. No.hLPAAT α   Expressed using yeast or plant LPAAT protein sequence as probe Searching the Genbank database for sequence tags (dbest) Extracts several short strands of the cDNA sequence with homology to the plant LPAAT protein sequence. I found it. These cDNA sequences of interest can be obtained from WashU-Merck EST or Genexpress- Random human cDNA clone project implemented by Genethon program Derived from single-run partial sequencing. Yeast LPAAT and human cDNA An example of amino acid sequence homology with Lone (dbest # 102250) is SEQ ID NO: 3 (Upper amino acid sequence) and SEQ ID NO: 4 (lower amino acid sequence) According to:   The upper row is amino acids 169-220 of the yeast LPAAT sequence, and the lower row is the phase of dbest clone # 102250. Refers to the same area. The same amino acid in these two sequences is indicated by an asterisk Display in block characters with.   Therefore, the complementary sequence of the conserved amino acid region of clone # 102250, GAFHL A (SEQ ID NO: 6), a synthetic oligonucleotide (o.BPLAT.2R), 5'-TGC AAGATGGAAGGCGCC-3 ′ (SEQ ID NO: 5) was prepared. o.BPLAT.2R is λzap human brain cD As a probe for screening NA library (Stratagene), γ-³²P -The 5'-end was radiolabeled using ATP and T4 polynucleotide kinase.   Screening of cDNA libraries is performed using standard methods Performed by hybridization (Current Protocols in Molecular Biolo gy, John Wiley & Sons, Inc., 1995). Contains DNA derived from λ phage amplification 6XSSC (1XSSC is 0.15M NaCl, 0.015M sodium citrate) M , PH 7.0), a solution of 5X Denhardt (1D Denhardt) ) Solution is 0.02% Ficoll, 0.02% bovine serum albumin and 0.02% polyvinyl-pyro 0.1% sodium dodecyl sulfate (SDS), sonicated 50mg / ml Pre-hybridization in denatured salmon sperm DNA at 60 ° C for 2 hours Was. Hybridization is the same as that used for prehybridization. Performed in the same buffer. After hybridization, filter at 42 ° C Washed in 6XSSC and autoradiographed.   About 1 × 10 of human brain cDNA library screened⁶Double of clones Twelve clones that hybridized to the probe in the filter were identified. After the second screening, 11 of the 12 clones were enriched and recovered. Was. The colon was then purified using the procedure recommended by Stratagene. By co-infection of E. coli XL 1-Blue with helper phage R408, Ten concentrated phage samples were converted into plasmid-transformed cells. Colony Perform filter hybridization and “lit up” the probe. Colonies were identified. Seven of the ten colony pools contained positive clones. I had. Two of these seven clones, pZplat.10 and pZplat.11, had> 2 kb inserts. The contents were contained. Restriction mapping using a combination of Sst I, Pst I and BamHI digestion Pinging confirms that these two clones contain many common fragments with each other. Indicated.   Perform sequencing of pZplat.10 and pZplat.1lc DNA insert nucleotide sequences. gave. FIG. 1 shows the DNA sequence of the cDNA insert of pZplat.11. cDNA clone Reotide sequence analysis and restriction mapping were performed for> 300 bp 5'-untranslated region, amino acids Open reading frame that can encode 283 polypeptides And the 3'-untranslated region of> 800 bp were revealed. Translation start site is 319-321 Localization at the nucleotide position and the requirement for a suitable start site by Kozak (Kozak, Critical Rev. Biochem. Mol. Biol. 27: 385-402, 1992). ). Another upstream ATG exists at positions 131-133, and an interface (inp hase) There is a stop codon. PZplat.10 except that the 5'-untranslated region is shorter. The cDNA insert has the same DNA sequence as pZplat.11.   query sequence of the open reading frame of 283 amino acids of pZplat.1l Column Was used to search for homologous sequences in the protein database. blastp blog Ram using the National Center for Biotechnology Information (National  Center for Biotechnology Information (NCBI) Genbank Release (G enbank Release) 90, a database search by pZplat.1l Proteins show highest homology with yeast and bacterial LPAAT did. Figure 2 shows the putative human LPAATα coding sequence, the yeast LPAAT coding sequence. , The E. coli LPAAT coding sequence and the maize LPAAT coding sequence. Shows a sequence of amino acid sequences, where human LPAATα is more yeast or cerebral than plant LPAAT. It demonstrates that homology with bacterial LPAAT is even more extensive.hLPAAT β   Expression sequence marker using yeast or plant LPAAT protein sequence as probe (DBEST) Genbank database (Boguski, et.al) , Science 265: 1993-1994, 1994), and found that yeast or plant LPAAT proteins Several short cDNA sequence strands with homology to the protein sequence were found. Interested These cDNA sequences correspond to I. M. A. G. E. Consortium [LLNL] cDN A random human cDNA cloning program primarily performed by the A cloning program. Derived from single-run partial sequencing of the project. Yeast LPAAT and human An example of amino acid sequence homology with the cDNA clone (dbEST # 363498) is shown below. You:   The upper row refers to amino acids 171-230 of the yeast LPAAT sequence (SEQ ID NO: 9), Column refers to homologous region of dbest clone # 363498 using +1 reading frame (SEQ ID NO: 10). Identical, conserved amino acids between these two sequences Is indicated by a double dot and a single dot. Low homology with yeast LPAAT sequence Whether a cDNA Clone Like Really Encodes the Human LPAATβ Sequence To isolate a full-length cDNA clone, insert it into an expression vector and Vector Transformed or transfected cells produce stronger LPAAT activity It was necessary to test whether or not.   Thus, based on the coding sequence and the complementary sequence of clone # 363498, respectively. , Two synthetic oligonucleotides, 5'-CCTCAAAGTGTGGATCTATC-3 '(o. LPAT3.F) (SEQ ID NO: 11) and 5'-GGAAGAGTACACCACGGGGAC-3 '(o. LPAT3.R) (SEQ ID NO: No .: 12) (Life Technologies, Gaithersburg, MD). o. LPAT3. R is a forward vector primer (o.sport.1) to amplify the 5'-side region , 5'-GACTCTAGCCTAGGCTTTTGC-3 '(SEQ ID NO: 13) . LPAT 3.F is pCMV. SPORT human leukocyte cDNA library (Life Technologies, Ga ithersburg, MD) to amplify the 3'-region of the putative LPAATβ sequence. Vector primer (o.sport.1R), 5'-CTAGCTTATAATACGACTCAC-3 ' Used in combination with column number: 14). o. sport. 1 and o. From LPAT3.R amplification The induced 700 bp PCR fragment was prepared using pBluescript (II) SK (-) (Stratagene, LaJoll a, CA) between EcoR I and Sma I to create pLPAT3.5 '. Cut. o. sport.1R and o. The 900 bp PCR fragment derived from LPAT3.F amplification , PBluescript (II) SK (-) (Stratagene, LaJolla, CA) between Sma I and Xba I And cut with XbaI before making pLPAT3.3 '. These two pluses Nucleotide sequence analysis of mid cDNA inserts shows that they overlap with each other, Has the same sequence as dbEST # 363498 and extensive homology with the amino acid sequence of yeast LPAAT (Nagiec et al., J. Biol. Chem. 268: 22156-22163, 1993). cDNA To assemble the two halves into a full-length clone, pLPAT3.5 '560 bp Nco I-Nar The I fragment and the 780 bp Nar I Xba I fragment of pLPAT3.3 'were subjected to 3-part ligation. And inserted into the Nco I / Xba I vector created from pSP-luc + (Promega, Madison, WI). To make pSP.LPAT3.   FIG. 3 shows the DNA sequence ID of the cDNA insert of pSP.LPAT3. Nucleotide of cDNA clone Peptide sequence analysis and restriction mapping revealed a 39 bp 5'-untranslated region, nucleotides Encoding a 278 amino acid polypeptide spanning positions 40-876 is an open source The coding frame and the 480 bp 3′-side untranslated region were revealed (FIG. 3). Transliteration The translation start site is localized at nucleotide positions 40-42 and is derived from Kozak, Criz. ti cal Rev. Biochem. Mol. Biol. 27: 385-402, 1992) Was satisfied.   The open reading frame sequence of 278 amino acids (Fig. 4) To search for homologous sequences in protein databases. blastp blog National Biotechnology Information Center using rum (NationaI  Center for Biotechnology Information (NCBI) Genbank Release (G enbank Release) 92, a search of the database indicates that this protein is yeast, It showed the greatest homology with bacterial and plant LPAAT. FIG. 5 shows human LPAATβ Putative coding sequence, human LPAATα code, yeast LPAAT coding sequence, bacteria (E. coli, H. influenzae and S. typhimurium) LPAAT coding sequence and plants (L. douglassi and C. nucifera) The amino acid sequence of the LPAAT coding sequence was aligned. Indicate that the human LPAAT coding sequence is more compatible with yeast or bacterial LPAAT than plant LPAAT. It reveals that homosexuality is even more widespread.Characteristic analysis of the present invention   Thus, human LPAATα is characterized by 283 amino acids of SEQ ID NO: 2. You. The invention further provides for encoding active LPAAT polypeptides and active fragments thereof. Allelic forms of the DNA described herein (with or without Alteration of naturally occurring bases in a population of species). DN of the present invention The A sequence can also be used under stringent conditions (eg, Maniatis et.al. , Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laborator y, pages 387-389, 1982), coding regions (e.g., nucleotides # 319-nucleotides) Sequence # 1167). Such a stringent One of the hybridization conditions is, for example, 4XSSC at 65 ° C, then 65 ° C. For 30 minutes with 0.1XSSC. Or another stringent high Hybridization conditions are at 42 ° C, 50% formamide, 4XSSC. The present invention is Furthermore, they encode an LPAAT polypeptide having LPAAT activity, but have a reduced gene code. Includes DNA sequences whose coding sequence differs depending on the weight. Point mutation or induction of the sequence of SEQ ID NO: 1 The activity of the encoded polypeptide or the encoded LPAA resulting from the genetic modification Alterations in DNA sequence that increase the production of a T polypeptide are also included in the invention.Definition   In the description that follows, a number of terms are used extensively. Understanding the invention Provide definitions to facilitate   As used herein, the term "isolated" as applied to a polypeptide refers to a peptide. Any material other than tide does not materially affect the biological properties of the polypeptide As such, the polypeptide may provide other material endogenous to the host from which the polypeptide is isolated. It is the degree of purity that is almost not included.   The term "induced" as used herein in connection with the polypeptides of the invention The term refers to a polypeptide obtained by isolation and purification from a host cell, And the manipulation and expression of nucleotide sequences prepared from host cells. Polypeptides. It also has a sequence corresponding to the nucleotide sequence of the present invention. Genomic DNA, mRNA, cDNA synthesized from mRNA and synthetic oligonucleotides Includes nucleotide sequences. In addition, the known amino acid sequence of the protein of the invention Include synthetic polypeptide antigens made based on the sequence.   As used herein, the term "expression product" is produced by recombinant DNA technology. Material to be used.Peptide sequencing of polypeptides   The purified polypeptide produced by the above method is, for example, a gas phase peptide peptide. Technology (Applied Biosystems, Foster City, CA) Sequencing is possible using well-known methods. The protein of the present invention Because it may be lycosylated, It is preferred to remove carbohydrate groups. It uses a glycosidase enzyme Can be implemented. Preferably, glycosidase F (Boehringe r-Mannheim, Indianapolis, IN) is used. As many polypeptides as possible To determine, the polypeptides of the invention are smaller than those suitable for gas phase sequencing. It is preferred to cut into fragments. This is a selective peptidase with polypeptide In a particularly preferred embodiment. Use Rotinase lys-C (Boehringer). The fragment prepared in this way is reverse-phase HPL It can be separated by C chromatography.   Substitution variants are typically one at one or more sites in the protein. Including the exchange of one amino acid for another, including stability against protein cleavage Are made to improve the properties of one or more of the polypeptides. Substitution is preferably Is conservative. That is, one amino acid is an amino acid with the same shape and charge Replace with Conservative substitutions are well known in the art, and include, for example, alanine to serine; Nin to Lysine; Asparagine to Glutamine or Histidine; Asparagine Acid to glutamic acid; cysteine to serine; glutamine to asparagine; Glutamic acid to aspartic acid; glycine to proline; histidine to aspa Lagin or glutamine; isoleucine to leucine or valine; leucine or Valine or isoleucine; lysine to arginine, glutamine or gluta Minic acid; leucine or isoleucine from methionine; Rosin, leucine or methionine; serine to threonine; Phosphorus; tryptophan to tyrosine; tyrosine to tryptophan or phenyl And the change from valine to isoleucine or leucine. Insertion Input mutants, such as those used to allow rapid purification of polypeptides Other proteins that are homologs of the polypeptides of the invention, including fusion proteins of And hybrid polypeptides containing polypeptide-derived sequences. Good. For example, an insertional variant is an amino acid sequence portion of a polypeptide from one species. And a homologous polypeptide portion from another species. Other insertional variants are Includes additional amino acids incorporated within the coding sequence of the repeptide . These are typically smaller inserts than the fusion proteins described above, for example, Incorporated to interrupt the protease cleavage site.   Antibodies to the human LPAAT protein can be obtained from the product or carrier of the LPAAT expression vector. Synthetic peptide derived from the LPAAT coding sequence linked to the peptide (Pasnett et al., J. Bio. l. Chem. 263: 1728, 1988) as an antigen. Polycloth The production of internal antibodies is well known to those skilled in the art. For example, an immunochemistry protocol (Immu nochemical Protocols) (Manson, eds.), pages 1-5 (Humana Press 1992) Green et al., “Production of polyclonal antiserum (Product ion of Polyclonal Antisera). Alternatively, the LPAAT antibody of the present invention Rodent Can be derived from a class of monoclonal antibodies (MAbs). Against a specific antigen Rodent monoclonal antibodies can be obtained by methods well known to those skilled in the art. . See, for example, Kohler and Milstein, Nature 256: 4. 95, 1975, and Coligan et al. (Eds.), Current Protocols in Immuno. logy, 1: 2.5.1-2.6.7 (John Wiley & Sons 1991). Briefly explain Then, the mouse is injected with the composition containing the antigen, and a serum sample is collected to Confirm the presence of somatic production, remove the spleen to obtain B-lymphocytes, A hybridoma is prepared by fusing with a myeloma cell, and the hybridoma is cloned. And select a positive clone producing an antibody against the antigen. Culturing body-producing clones and isolating antibodies from hybridoma cultures Thus, a monoclonal antibody can be obtained.   Isolating MAbs from hybridomas by a variety of well-established techniques; It can be purified. Such isolation techniques use Protein-A Sepharose. Affinity chromatography, molecular sieve chromatography and ion Including exchange chromatography. For example, Coligan's 2.7.1-2.7.12 See pages and pages 2.9.1-2.9.3. Also, Methods in Molecular Bi ology, 10: 79-104 Listed in Humana Press, Inc., 1992 Baines et al., "Purification of Immunoglobulin G (IgG)" munoglobulin G) ". The LPAAT antibody of the present invention is derived from a human primate antibody. May be guided. A general procedure for producing therapeutically useful antibodies in baboons Techniques are described, for example, in Goldenberg et al., WO 91/11465. No. (1991) and Losman et al., Int. J. Cancer 46: 310, 1990 Can be served.   Alternatively, therapeutically useful LPAAT antibodies are derived from "humanized" monoclonal antibodies Can be guided. Humanized monoclonal antibody is used for mouse immunoglobulin Mouse heavy chain and light chain variable regions are introduced into human variable regions. By substituting human residues in the framework region of the mouse counterpart, To be produced. Use antibody components derived from humanized monoclonal antibodies Avoids potential problems associated with the immunogenicity of the constant part of the mouse You Will be. General for cloning the variable region of mouse immunoglobulin Techniques are described, for example, in the report of Orlandi et al., Proc. Nat'l. Acad. Sc i. USA 86: 3833, 1989. Techniques for producing humanized MAbs include, for example, Jones et al., Nat, each of which is incorporated herein by reference. ure 321: 522, 1986; Riechmann et al., NaLure 332: 323, 1988; -Verhoeyen et al., Science 239: 1534, 1988; Carter et al. Proc. Nat'l. Acad. Sci. USA 89: 4285, 1992, Sandhu, Crit. R ev. Biotech. 12: 437, 1992 and Singer et al. Immun. 150: 2844 , 1993.   Alternatively, the LPAAT antibodies of the invention can be used in combination immunoglobulin libraries. May be derived from a human antibody fragment isolated from a human antibody. For example, included as a reference Barbas et al., Methods: A Companion to Methos in Enzym ology 2: 119 1991 and Winter et al., Ann. Rev. Immunol. 12: 433 , 1994. To create a human immunoglobulin phage library Cloning and expression vectors useful for STRATAGENE cloning Available from Tem (LaJolla, CA). In addition, the LPAAT antibody of the present invention And monoclonal antibodies. Such antibodies can be Transgenes that are "engineered" to produce specific human antibodies in response to Obtained from a mouse. This technique involves elements of the human heavy and light chain loci Are derived from embryonic stem cell lines and contain target fragments of endogenous heavy and light chain loci To be introduced into the mouse species. Transgenic mice are human specific for human antigens Antibodies can be synthesized and the mouse is used to hybridize Redomas can be made. Obtaining human antibodies from transgenic mice A method for this is described in Green et al., Nature Genet. 7:13, 1994; Romberg ( Lonberg) et al., Nature 368: 856, 1994, and Taylor et al., Int. Immu n. 6: 579, 1994.                                 Example 1   This example demonstrates that the human LPAATα clone encodes a protein having LPAAT activity. Shows an experiment to determine whether or not to perform a fusion protein with β-galactosidase When Escherichia coli (E. coli) vector expressing human LPAATα Transform into PAAT-deficient strain, and the vector complements the defective part of E. coli I asked whether to do it. Specifically, it is derived from pZplat. An 840 bp BglII-NcoI fragment of the coding region spanning the amino acid to the stop codon was o The cloning vector pLitmus28 (Ivans et al., BioTechniques 19:13) 0-135, 1995) to produce plasmid p28BgN. This plasmid is called pLi Using the lac promoter in tmus28, the first 16 members of β-galactosidase were used. Fusion proteins having amino acids and the last 216 residues of the human LPAATα coding sequence Is expected to express human LPAATα. This plasmid was used in E. coli (E. coli) JC201 strain (Dr. Jack Coleman, obtained from Louisiana State University) Transformation. JC201 (Coleman, Mol. Gen. Genet. 232: 295-303, 1992; Nagiec J. et al. Biol. Chem. 268: 22156-22163, 1993; and Brown et al., Plant Mol. Biol . 26: 211-223, 1994) found that LPAAT activity was lost due to a mutation in the plsC locus. I have. This mutation forms a temperature-sensitive phenotype, and JC201 is slow at 37 ° C. It grows individually, hardly grows at 42 ° C, and does not grow at 44 ° C at all. Form with p28BgN Transformed JC201 is wild-type JC200 (plsC⁺Grows normally at 44 ° C compared to strain JC201 transformed with the pLitmus28 vector grew at 44 ° C. Could not support. These data are based on the human LPAATa isolated here. The putative cDNA is that the last 216 amino acids of this cDNA are the LPAAT residue of JC201. It has enough LPAAT activity to complement the gene (plsC) deficiency. Suggests.                                 Example 2   A putative human LPAATβ clone encodes a protein with LPAAT activity. Express this human LPAATβ as a direct product to determine Transformed E. coli vector into LPAAT-deficient E. coli strain We investigated whether it complements the E. coli deficiency. In particular Derived from pSP.LPAT3, spanning the entire coding region from the first amino acid to the stop codon The 1350 bp Nco I-Xba I fragment was cloned into Nco I / Xba I cloning vector pK Insert into K388-1 (Clontech, Palo Alto, CA) to create plasmid pTrc.LPAT3 You. This Of E. coli JC201 strain (Dr. Jack Coleman, Louisiana Stat) e-University). JC201 (Coleman, Mol. Gen. Genet. 2 32: 295-303, 1992) found that LPAAT activity was deficient due to a mutation in the plsC locus. You. This mutation results in the formation of a temperature-sensitive phenotype, with JC201 gradually increasing at 37 ° C. And grows little at 42 ° C, and does not grow at all at 44 ° C. in pTrc.LPAT3 The transformed JC201 is wild-type JC200 (plsC⁺) Normal growth at 44 ° C compared to strain However, JC201 transformed with the pKK388-1 vector increased at 44 ° C. The breeding could not be supported. These data are based on the human LPAA isolated here. The putative Tβ cDNA is the putative protein product of this cDNA, JC2 It has LPAAT activity because it can complement the LPAAT gene (plsC) deletion in 01 Suggests that                                 Example 3   This example demonstrates that overexpression of the human LPAATα of the present invention has some effect on mammalian cells. An example of an experimental group for examining whether or not it has an effect is shown. Inserted into mammalian expression vector pCE9 To insert, the entire cDNA insert (~ 2,300 bp) from pZplat. By cutting with I, pCE9.LPAAT1 was prepared. pCE9 is derived by adding two modifications to pCE2 did. The 550 bp BStY I fragment in the elongation factor-1a (EF-1a) intron of pCE2 was deleted. I was The multiple cloning region between Asp718 I and BamHI site of pCE2 was The mus28 was replaced with a multiple cloning region from Asp718I to BglII. Plastic Smid pCE2 binds the RSV promoter with the CMV enhancer and the elongation factor-1a (EF-1a) promoter. PREP7b (Leung, et al., Proc. Natl. Acad. Sci.) . USA, 92: 4813-4817, 1995). CMV enhancer is primer 5 ' -GGCTCTAGATATTAATAGTAATCAATTAC-3 'and 5'-CCTCACGCATGCACCATGGTAATAGC-3' Was used to generate 380 from pCEP4 (Invitrogen, San Diego, CA) by PCR. It was made from a bp Xba I-Sph I fragment. EF-1a promoter and intron (Uets uki, et al. Biol. Chem., 264: 5791-5798, 1989) is a primer 5'-GGTGCATGCG PCR using TGAGGCTCCGGTGC-3 ′ and 5′-GTAGTTTTCACGGTACCTGAAATGGAAG-3 ′ Made from a 1200 bp Sph I-Asp718 I fragment made from human genomic DNA . These two fragments were ligated into an XbaI / Asp718I deletion vector derived from pREP7b and Make CE2 Made.   A549 cells, ECV304 cells (American Type Culture Collection, Rockville, MD ), Two human cell lines that are thought to produce IL-6 and TNF when stimulated with IL-1b And mouse TNF and 293-EBNA cells (Invitrogen, San Diego, CA). Several mammalian cell lines were transfected with pCE9.LPAAT1 DNA. before Use the conditions described (Cachianes, et al., Biotechniques 15: 255-259, 1993). Cell-Porator (Life Technologies, Gaith) erberg, MD) prior to electroporation of pCE9.LPAAT1 with BspH I did. After 24 hours, fix the transfected cells, and then Growing cells in the presence of glomycin B (Hyg) (Calbiochem, La Jolla, CA) Then, cells into which both plasmids had been introduced were selected. Northern blot analysis (Kroc zek, et. al., Anal. Biochem. 184: 90-95, 1990). Expressed LPAAT mRNA at levels 20 times greater than in non-transfected cells Hyg-resistant clones were selected for further study.   Figure 6 shows LPAAT activity of A549 cells compared to pCE9.LPAAT1 DNA using TLC assay. The transfected A549 cells are compared with LPAAT activity. LPAAT of cell extract This screening assay for activity is compatible with fluorescent assays using fluorescent liquid substrates. (Ella, et al., Anal. Biochem. 218: 136-142, 1994). Use PC-substrate Instead of using BPCs (Molecular Probes, Eugene, OR), And a fluorescent Bodipy moiety is introduced at the end of the alkyl chain at SN1 position. BPC, which is a synthetic PC, and cabbage phospholipase D (Sigma, St. Louis, MO) Used and converted to Bodipy-PA. Then using snake venom phospholipase A2 Bodipy-PA was converted to Bodipy-LPA. Obtained for use in LPAAT assays Bodipy-LPA was purified by preparative TLC. 0.5mMATP, 0.3mM MgCl_Two, 100 μM ole Lysis buffer supplemented with oil-CoA and 10 μM Bodipy LPA (Ella, et al., Anal. Bi. ochem. 218: 136-142, 1994). Sample After incubation for 30 minutes, they were loaded on TLC plates.   Lane 1 shows the incubation of Bodipy LPA with buffer alone without any cell extract. Shows what has been Lane 9 is for making Bodipy-PA marker. BP C shows a treatment of cabbage phospholipase D with C. Lanes 2 and 4 are Control A549 cell extract and Bodipy LPA with or without lipid A Shown after incubation. Lanes 3 and 5 contain lipid A, respectively. A549 cell extract transformed with or without pCE9.LPAAT1 DNA, with or without Shown is the incubation of Bodipy LPA. Figure 3 shows the transfection of LPAAT cDNA. The transfected A549 cells (lanes 3 and 5) bind Bodipy-LPA to Bodipy-PA. Than control cells (lanes 2 and 4), as evidenced by increased conversion 9 shows that it contains much stronger LPAAT activity. Addition of lipid A to cell extract Has little effect on LPAAT activity (lanes 2 and 4 and lanes 3 and 5). A549 Cell extracts also convert Bodipy-LPA to Bodipy-monoalkylglycerol It also has phosphohydrolase activity (lanes 2-5). Interestingly, LPAAT Overexpressed A549 cells (lanes 3 and 5) compared to control cells (lanes 2 and 4) This phosphohydrolase prefers LPA to PA as a substrate. And suggests. It is also probably formed by this endogenous phosphohydrolase. Control cells (lanes 2 and 4) due to the partial conversion of the converted PA to DAG In comparison, transfected cells (lanes 3 and 5) also have increased DAG. You.                                 Example 4   The expression of the LPAAT cDNA clone described herein resulted in a free release at the SN2 position. To determine whether other glycerol lipids with hydroxyl groups are also used, Extract the substrate NBD-lysoPC (lanes 6 and 7) and NBD-monoacylglycerol (MAG) (lane 10 and lane 11) and incubated with lysoP It was determined whether conversion to C and DAG was increasing. Lanes 8 and 12 are In each case, NBD-lysoPC and NBD-MAG were combined with buffer only, without adding any cell extract. Shown after incubation. TLC analysis was performed on transfected cells. Shows that there is little difference in the lipid profile between the vesicles and the control cells (lane 7: 6, lanes 11:10), where the cloned LPAAT enzyme uses LPA as the preferred substrate Suggest to use as. lysoPC (Fyrst, et al., Biochem. J. 306: 793). -799, 1995) and MAG (Bhat, et al., Biochemistry 34: 11237-11244, 1995). Syltransferase may represent an enzyme different from LPAAT described herein. Ability is there.                                 Example 5   A549 cells (American Type Culture Collection, Rockville, MD), IL-1β Human cell lines and mouse TNF that are likely to produce IL-6 and TNF when stimulated Was transfected with pCE9.LPAAT1 DNA. The conditions listed previously (C achianes, et al., Biotechniques 15: 255-259, 1993) and registered in A549 cells Electroporation with the trademark Cell-Porator (Life Technologies, Gaitherberg, MD) Prior to digestion, pCE9.LPAATI was digested with BspH I. 24 hours later, transfection After fixation of the modified cells, 200 μg / ml hygromycin B (Hyg) (Calbioche m, La Jolla, CA), cells are grown and both plasmids are introduced Cells were selected. Northern blot analysis (Kroczek, et.al., Anal. Biochem. 18). 4: 90-95, 1990) and compared to untransfected A549 cells Hyg-resistant clones that expressed LPAAT mRNA at 20-fold higher levels Selected to do.   Transfection with pCE9.LPAAT1 due to stimulation with IL-1β and mouse TNF Of TNF (FIG. 7) and IL-6 (FIG. 8) production between In comparison, A549 cells that overproduce LPAAT were untransfected A549 cells. Shows that it produces> 5-fold TNF and> 10-fold IL-6 as compared to vesicles. This is the LPAAT Overexpression suggests that the cytokine signaling response in the cell is increased. Therefore, the development of compounds that would modulate LPAAT activity would be Should be interesting.

────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Tompkins Christopher Kay.             United States Bothell, Washington             86th Avenue N. E. 17660

Claims (1)

[Claims] 1. (a) DNA sequences described in SEQ ID NO: 1 and SEQ ID NO: 7 and their truncations Fragments,     (B) Due to the degeneracy of the genetic code, the polypeptides of SEQ ID NO: 2 and SEQ ID NO: 8 CDNA sequences encoding the enzyme and its fragments having enzymatic activity; and     (C) hybridizing to the cDNA of (a) or (b) under high stringency conditions A cDN encoding a polypeptide that can be iserized and has LPAAT activity A nucleic acid sequence encoding an LPAAT enzyme selected from the group consisting of the A sequences. 2. selected from the group consisting of the amino acid sets set forth in SEQ ID NO: 2 and SEQ ID NO: 8 LPAAT enzyme and enzymatically active fragments thereof. 3. Anti-inflammatory drugs to increase hematopoiesis and prevent oxygen load injury after cytoreductive therapy A method for screening a drug candidate compound having an activity as   (A) obtaining an LPAAT polypeptide according to claim 2, which has LPAAT enzyme activity; ,   (B) contacting the LPAAT polypeptide with different concentrations of the candidate drug and a control sample And   (C) Measure LPAAT activity with and without different concentrations of candidate drugs And a method comprising steps. 4. The candidate drug may be a pool of compounds expressed in a combinatorial library 3. The method described.