CA2291516A1 - Mammalian lysophosphatidic acid acyltransferase - Google Patents

Abstract

There is disclosed cDNA sequences and polypeptides having the enzyme lysophosphatidic acid acyltransferase (LPAAT) activity. LPAAT is also known as 1-acyl sn-glycerol-3-phosphate acyltransferase.

Description

MAMMALIAN LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE
Technical Field of the Invention This present invention provides cDNA sequences and polypeptides having the enzyme lysophosphatidic acid acyltransferase (LPAAT) activity. LPAAT is also known as 1-acyl sn-glycerol-3-phosphate acyltransferase. The present invention further provides for isolation and production of polypeptides involved in phosphatidic acid metabolism and signaling in mammalian cells, in particular, the production of purified forms of LPAAT.
Background of the Invention Originally regarded as intermediates in lipid biosynthesis {Kent, Anal. Rev.
Biochem. 64:31 S-343, 1995), phosphatidic acid (PA) and one of its precursors, lysophosphatidic acid (LPA), have also been identified as phospholipid signaling molecules that affect a wide range of biological responses (McPhail et al., Proc. Natl.
Acad. Sci. USA 92:7931-7935, 1995; Williger et al., J. Biol. Chem. 270:29656-29659, 1995; Moolenaar, Curr. Opin. Cell Biol. 7:203-210, 1995).
Cellular activation in monocytic and lymphoid cells is associated with rapid upregulation of synthesis of phospholipids (PL) that includes phosphatidic acid (PA), diacylglycerol (DAG) and glycan phosphatidylinositol (PI). Phosphatidic acids {PA) are a molecularly diverse group of phospholipid second messengers coupled to cellular activation and mitogenesis (Singer et al., Exp. Opin. Invest. Drugs 3:631-643, 1994).
Compounds that would block PA generation and hence diminish the signal involved in cell activation may therefore be of therapeutic interest in the area of inflammation and oncology. Lysofylline {1-(R)-(5-hydroxyhexyl)-3,7-dimethylxanthine) (Singer et al., Exp.
Opin. Invest. Drugs 3:631-643, 1994; and Rice et al., Proc. Natl. Acad. Sci.
USA 91:3857-3861, 1994) has been found to be an effective inhibitor of cellular activation by blocking the synthesis of a specific phosphatidic acid (PA) species produced by lysophosphatidic acid acyltransferase (LPAAT) in activated monocytic cells (Rice et al., Proc.
Natl. Acad.
Sci. USA 91:3857-3861, 1994). PA can be generated through hydrolysis of phosphatidycholine (PC) (Exton, Biochim. 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), through phosphorylation of DAG by DAG kinase (Kanoh et al., Trends Biochem.
Sci.

15:47-50, 1990) or through acylation of LPA at the SN2 position (Bursten et al., Am. J.
Physiol. 266:CI093-C1 I04, 1994). Compounds that would block PA generation and hence diminish lipid biosynthesis and the signal involved in cell activation may therefore be of therapeutic interest in the area of inflammation and oncology as well as obesity treatment.
The genes coding for LPAAT have been isolated in bacteria (Coleman, Mol. Gen.
Genet. 232:295-303, 1992), in yeast (Nagiec et al., J. Biol. Chem. 268:22156-22163, 1993) and in plants {Brown et al., Plant Mol. Biol. 26:211-223, 1994; and Hanke et al., Eur J.
Biochem. 232:806-810, 1995) using genetic complementation techniques. The cloning of a mammalian version of LPAAT has not been reported. Homology among the bacterial, yeast and plant LPAAT is only found in a very few block of three or at most four amino acids scattered throughout the sequences {Brown et al., Plant Mol. Biol.
26:211-223, 1994). Further, there is a need in the art for recombinant LPAAT from a mammalian source to enable compound screening for LPAAT inhibitors for the development of specific compounds that would inhibit this enzyme.
Summary of the Invention The present invention provides a cDNA sequence, polypeptide sequence, and transformed cells for producing isolated recombinant mammalian LPAAT. The present invention provides two novel human polypeptides, and fragments thereof, having LPAAT
activity. The polypeptides discovered herein is novel and will be called hLPAAT with the f rst one discovered designated hLPAATa and the second one discovered called hLPAAT(3. LPAAT catalyzes the acylation of lysophosphatidic acid (LPA) to phosphatidic acid (PA) by acylating the sn-2 position of LPA with a fatty acid acyl-chain moiety.
The present invention further provides nucleic acid sequences coding for expression of the novel LPAAT polypeptides and active fragments thereof. The invention further provides purified LPAATs and antisense oligonucleotides for modulation of expression of the genes coding for LPAAT polypeptides. Assays for screening test compounds for their ability to inhibit LPAATs are also provided.
Recombinant LPAAT is useful for screening candidate drug compounds that inhibit LPAAT activity. The present invention provides cDNA sequences encoding a Figure 2 shows amino acid sequence alignment of the human LPAATa coding sequence, the yeast LPAAT coding sequence, E. coli LPAAT coding sequence, and the maize LPAAT coding sequence. This comparison shows that human LPAATa has the -greatest extended homology with yeast or E. coli LPAAT than with the plant LPAAT.
Figure 3 shows the DNA sequence of the cDNA insert pSP.LPAT3 encoding hLPAAT~i. The nucleotide sequence analysis and restriction mapping of the cDNA
clone revealed a 5' untranslated region of 39 base pairs and an open reading frame encoding a 278 amino acid polypeptide that spans positions 40-876. It also shows a 3' untranslated region of 480 base pairs from pSP.LPAT3. The initiation site for translation was localized at nucleotide positions 40-42 and fulfilled the requirement for an adequate initiation site (Kozak, Critical Rev. Biochem. Mol. Biol. 27:385-402, 1992).
Figure 4 shows the sequence of the hLPAAT~3 278 amino acid open reading frame.
The amino acid sequence was used as the query sequence to search for homologous sequences in protein databases. Search of the database based on Genbank Release 92 from the National Center for Biotechnology Information (NCBI) using the blastp program showed that this protein was most homologous to the yeast, bacterial and plant LPAATs.
Figure 5 shows amino acid sequences alignment of this putative human LPAAT(3 coding sequence, human LPAATa coding, the yeast LPAAT coding sequence, the bacterial (E. coli, H. influenzae, and S. typhimurium) LPAAT coding sequences, and the plant (L. douglassi and C. nucifera) LPAAT coding sequences, revealing that the human LPAAT coding sequences have a much more extended homology with the yeast or the bacterial LPAAT than with the plant LPAAT.
Figure 6 shows a comparison of LPAAT activity in A549 cells transfected with pCE9.LPAAT 1 DNA, or no DNA using a TLC (thin layer chromatography) assay.
These data are described in more detail in examples 3 and 4.
Figures 7 and 8 show a comparison of the production of TNF (Figure 7) and IL-6 (Figure 8) between A549 cells transfected with pCE9.LPAATI and control A549 cells after stimulation with IL-1 (i and marine TNF. These data show A549 overexpressing LPAAT produces >5 fold more TNF and >10 fold more IL-6 relative to untransfected A549 cells, suggesting that overexpression of LPAAT enhances the cytokine signaling response in cells.

poIypeptide having LPAAT activity and comprising the DNA sequence set forth in SEQ
ID NO. 1 of SEQ ID NO. 7, shortened fragments thereof, or additional cDNA
sequences which due to the degeneracy of the genetic code encode a polypeptide of SEQ ID
NO. 2 0~-SEQ. ID NO. 8 or biologically active fragments thereof or a sequence capable of hybridizing thereto under high stringency conditions. The present invention further provides a polypeptide having LPAAT activity and comprising the amino acid sequence of SEQ ID NO. 2 or SEQ ID NO. 8 or biologically active fragments thereof.
Also provided by the present invention are vectors containing a DNA sequence encoding a mammalian LPAAT enzyme in operative association with an expression control sequence. Host cells, transformed with such vectors for use in producing recombinant LPAAT , are also provided with the present invention. The inventive vectors and transformed cells are employed in a process for producing recombinant mammalian LPAAT. In this process, a cell line transformed with a DNA sequence encoding on expression for a LPAAT enzyme in operative association with an expression control sequence, is cultured. The claimed process may employ a number of known cells as host cells for expression of the LPAAT polypeptide, including, for example, mammalian cells, yeast cells, insect cells and bacterial cells.
Another aspect of this invention provides a method for identifying a pharmaceutically-active compound by determining if a selected compound is capable of inhibiting the activity of LPAAT for acylating LPA to PA. A compound capable of such activity is capable of indirectly inhibiting SAPkinase and being a pharmaceutical compound useful for augmenting trilineage hematopoiesis after cytoreductive therapy and for anti-inflammatory activity in inhibiting the inflammatory cascade following hypoxia and reoxygenation injury (e.g., sepsis, trauma, ARDS, etc.).
The present invention further provides a transformed cell that expresses active mammalian LPAAT and further comprises a means for determining if a drug candidate compound is therapeutically active by inhibiting recombinant LPAAT activity.
Brief Description of the Drawings Figure 1 shows the DNA sequence of the cDNA insert of pZplat.l 1 encoding hLPAAToc.

Detailed Description of the Invention The present invention provides novel, isolated, biologically active mammalian LPAAT enzymes. The term "isolated" means any LPAAT polypeptide of the present invention, or any other gene encoding LPAAT polypeptide, which is essentially free of other polypeptides or genes, respectively, or of other contaminants with which the LPAAT
polypeptide of gene might normally be found in nature.
The invention includes a functional polypeptide, LPAAT, and functional fragments thereof. As used herein, the term "functional polypeptide" refers to a polypeptide which possesses a biological function or activity which is identified through a biological assay, preferably cell-based, and which results in the formation of PA species from LPA. A
"functional polynucleotide" denotes a polynucleotide which encodes a functional polypeptide. Minor modification of the hLPAATa primary amino acid sequence may result in proteins which have substantially equivalent activity as compared to the sequenced hLPAATa polypeptide described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein as long as the acyltransferase activity of LPAAT is present. This can lead to the development of a smaller active molecule which would have broader utility. For example, it is possible to remove amino or carboxy terminal amino acids which may not be required for LPAAT
activity.
The hLPAATa and hLPAAT~3 polypeptide of the present invention also includes conservative variations of the polypeptide sequence. The term "conservative variation"
denotes the replacement of an amino acid residue by another, biologically active similar residue. Examples of conservative variations include the substitution of one hydrophobic residue, such as isoleucine, vaiine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or giutamine for asparagine, and the like. The term "conservative variation"
also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunologically react with the unsubstituted polypeptide.
Polypeptides of the present invention can be synthesized by such commonly used methods as t-BOC or FMOC protection of alpha-amino groups. Both methods involve WO 98/54303 PCTlUS97105360 step-wise syntheses whereby a single amino acid is added at each step starting from the C
terminus of the peptide (Coligan et al., Current Protocols in Immunology, Wiley lnterscience, Unit 9, 1991). In addition, polypeptide of the present invention can also be synthesized by solid phase synthesis methods (e.g., Mernfield, J. Am. Chem.
Soc. 85:2149, 1962; and Steward and Young, Solid Phase Peptide Synthesis, Freeman, San Francisco pp.
27-62, 1969) using copolyol (styrene-divinylbenzene} containing 0.1-1.0 mM
amines/g polymer. On completion of chemical synthesis, the ploypeptides can be deprotected and cleaved from the polymer by treatment with liquid HF 10% anisole for about 15-60 min at 0 °C. After evaporation of the reagents, the peptides are extracted from the polymer with 1 % acetic acid solution, which is then lyophilized to yield crude material.
This can normally be purified by such techniques as gel filtration of Sephadex G-15 using 5%
acetic acid as a solvent. Lyophiiization of appropriate fractions of the column will yield a homogeneous polypeptide or polypeptide derivatives, which are characterized by such standard techniques as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopsy, molar rotation, solubility and quantitated by solid phase Edman degradation.
The invention also provides polynucleotides which encode the hLPAAT
polypeptide of the invention. As used herein, "polynucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides in the form of a separate fragment or as a component of a larger construct. DNA encoding the polypeptide of the invention can be assembled from cDNA fragments or from oligonucleotides which provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit.
Polynucleotide sequences of the invention include DNA, RNA and cDNA sequences. Preferably, the nucleotide sequence encoding hLPAAT is the sequence of SEQ m NO. 1 for hLPAATa or SEQ ID NO. 7 for LPAAT(3. DNA sequences of the present invention can be obtained by several methods. For example, the DNA can be isolated using hybridization procedures which are known in the art. Such hybridization procedures include, for example, hybridization of probes to genomic of cDNA libraries to detect shared nucleotide sequences, antibody screening of expression libraries to detect shared structural features, such as a common antigenic epitope, and synthesis by the polymerase chain reaction (PCR).

Hybridization procedures are useful for screening of recombinant clones by using labeled mixed synthetic oligonucleotides probes, wherein each probe is potentially the complete complement of a specific DNA sequence in a hybridization sample which incIudes a heterogeneous mixture of denatured double-stranded DNA. For such screening, hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA. Hybridization is particularly useful for detection of cDNA
clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present. Using stringent hybridization conditions directed to avoid non-specific binding, it is possible to allow an autoradiographic visualization of a specific cDNA clone by the hybridization of the target DNA to that single probe in the mixture, which is its complement (Wallace et al. Nucl. Acid Res. 9:879, 1981 ). The development of specific DNA sequences encoding hLPAAT can also be obtained by isolation of double-stranded DNA sequences from the genomic DNA, chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest, and in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated for a eukaryotic donor cell In the latter case, a double-stranded DNA
complement of mRNA is eventually formed which is generally referred to as cDNA. Of these three methods for developing specific DNA sequences for use in recombinant procedures, the isolation of genomic DNA isolates is the least common. This is especially true when it is desirable to obtain the microbial expression of mammalian polypeptides due to the presence of introns.
The synthesis of DNA sequences is frequently a method that is preferred when the entire sequence of amino acids residues of the desired polypeptide product is known.
When the entire sequence of amino acid residues of the desired polypeptide is not known, direct synthesis of DNA sequences is not possible and it is desirable to synthesize cDNA
sequences. cDNA sequence isolation can be done, for example, by formation of plasmid-or phage-carrying cDNA libraries which are derived from reverse transcription of mRNA.
mRNA is abundant in donor cells that have high levels of genetic expression.
In the event of lower levels of expression, PCR techniques are preferred. When a significant portion of the amino acid sequence is known, production of labeled single or double stranded DNA
or RNA probe sequences duplicating a sequence putatively present in the target cDNA
may be employed in DNA/DNA hybridization procedures, carried out on cloned copies of the cDNA (denatured into a single-stranded form) (Jay et al., Nucl. Acid Res.
11:2325, 1983).
A cDNA expression library, such as lambda gtl l, can be screened indirectly for -hLPAATa or hLPAAT(3 polypeptide having at least one epitope, using antibodies specific for hLPAATa or hLPAAT~i. Such antibodies can be either polycionally or monoclonally derived and used to detect expression product indicative of the presence of hLPAATa or hLPAAT~i cDNA.
A polynucleotide sequence can be deduced from the genetic code, however the degeneracy of the code must be taken into account. Polynucleotides of this invention i 0 include sequences which are degenerate as a result of the genetic code.
The polynucleotides of this invention also include sequences that are degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more that one codon (a three base sequence). Therefore, as long as the amino acid sequences of hLPAATa and hLPAAT(i results in a functional polypeptide (at least, in the case of the sense polynucleotide strand), all degenerate nucleotide sequences are included in the invention. The polynucleotide sequence for hLPAATa and hLPAAT(3 also includes sequences complementary to the polynucleotides encoding hLPAATa and hLPAAT~i (antisense sequences). Antisense nucleic acids are DNA and RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Sci. Amer.
262:40, 1990). The invention embraces all antisense polynucleotides capable of inhibiting the production of hLPAATa and hLPAAT/3 polypeptide. In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule.
The antisense nucleic acids interfere with the translation of mRNA since the cell cannot translate mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target hLPAATa and hLPAAT~3-producing cell. The use of antisense methods to inhibit translation of genes is known {e.g., Marcus-Sakura, Anal. Biochem. 172:289, 1988).
In addition, ribozyme nucleotide sequences for hLPAATa and hLPAAT(3 are included in this invention. Ribozymes are RNA molecules possessing an ability to specifically cleave other single-stranded RNA in a manner analogous to DNA
restriction endonucleases. Through the modification of nucleotide sequences which encode such RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, J. Amer. Med. Assn. 260:3030, 1988}. An advantage of this approach is that only mRNAs with particular sequences are inactivated-- because they are sequence-specific.
S There are two basic types of ribozymes, tetrahymena-type (Hasselhoff, Nature 334:S8S, 1988) and "hammerhead-type". Tetrahymena-type ribozymes recognize sequences which are four bases in length, while "hammerhead-type" ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA
species.
Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species.
Polynucleotide sequences encoding the hLPAATa and hLPAATø polypeptides of the invention can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial {bacterial), yeast, insect and mammalian organisms. Methods of expressing 1 S DNA sequences having eukaryotic or viral sequences in prokaryotes are known in the art.
Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA
sequences of the invention. DNA sequences encoding the inventive polypeptides can be expressed in vitro by DNA transfer into a suitable host using known methods of transfection.
The hLPAAToc and hLPAAT(3 DNA sequences may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a plasmid, virus or other vehicle that has been manipulated by insertion or incorporation of the genetic sequences. Such expression vectors contain a promoter sequence which facilitates efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells. Vectors suitable for use in the present invention include, for example, with bacterial promoter and ribosome binding site expression vector for expression in bacteria (Gold, Meth. Enzymol. 185:11, 1990), expression vector with animal promoter and enhancer for expression in mammalian cells (Kaufrnan, Meth. Enrymol. 185:487, 1990) and baculovirus-derived vectors for expression in insect cells {Luckow et al., J. Viro1.67:4566, 1993). The DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein I, or polyhedren promoters).
The vector may include a phenotypically selectable marker to identify host cells which contain the expression vector. Examples of markers typically used in prokaryotic expression vectors include antibiotic resistance genes for ampicillin ((3-lactamases), tetracycline and chloramphenicol (chloramphenicol acetyltransferase). Examples of such markers typically used in mammalian expression vectors include the gene for adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), and i 0 xanthine guanine phosphoriboseyltransferase (XGPRT, gpt). .
In another preferred embodiment, the expression system used is one driven by the baculovirus polyhedrin promoter. The gene encoding the polypeptide can be manipulated by standard techniques in order to facilitate cloning into the baculovirus vector. See Ausubel et al., supra. A preferred baculovirus vector is the pBlueBac vector (Invitrogen, Sorrento, CA).
The vector carrying the gene for the polypeptide is transfected into Spodoptera frugiperda (Sf~) cells by standard protocols, and the cells are cultured and processed to produce the recombinant polypeptide. See Summers et al., A Manual for Methods of Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station.
Once the entire coding sequence of the gene for the polypeptides has been determined, the gene can be inserted into an appropriate expression system.
The gene can be expressed in any number of different recombinant DNA expression systems to generate large amounts of polypeptide. Included within the present invention are polypeptides having native glycosylation sequences, and deglycosylated or unglycosylated polypeptides prepared by the methods described below. Examples of expression systems known to the skilled practitioner in the art include bacteria such as E. toll, yeast such as Pichia pastoris, baculovirus, and mammalian expression systems such as in Cos or CHO cells.
The gene or gene fragment encoding the desired polypeptide can be inserted into an expression vector by standard subcloning techniques. In a preferred embodiment, an E. toll expression vector is used which produces the recombinant protein as a fusion protein, allowing rapid affinity purification of the protein. Examples of such fusion protein expression systems are the glutathione S-transferase system (Pharmacia, Piscataway, N~, the maltose binding protein system (NEB, Beverley, MA), the thiofusion system (Invotrogen, San Diego, CA), the FLAG system (IBI, New Haven, CT), and the 6xHis system (Qiagen, Chatsworth, CA). Some of these systems produce recombinant polypeptides bearing only a small number of additional amino acids, which are unlikely to affect the LPAAT
ability of the recombinant polypeptide. For example, both the FLAG system and the 6xHis system S add only short sequences, both of which are known to be poorly antigenic and which do not adversely affect folding of the polypeptide to its native conformation. Other fusion systems produce proteins where it is desirable to excise the fusion partner from the desired protein.
In a preferred embodiment, the fusion partner is linked to the recombinant polypeptide by a peptide sequence containing a specific recognition sequence for a protease.
Examples of suitable sequences are those recognized by the Tobacco Etch Virus protease (Life Technologies, Gaithersburg, MD) or Factor Xa (New England Biolabs, Beverley, MA) or enterokinase (Invotrogen, San Diego, CA).
Production of Polypeptides In a preferred embodiment, recombinant proteins are expressed in E. coli and in i 5 baculovirus expression systems. The complete gene for the polypeptide can be expressed or, alternatively, fragments of the gene encoding antigenic determinants can be produced. In a first preferred embodiment, the gene sequence encoding the polypeptide is analyzed to detect putative transmembrane sequences. Such sequences are typically very hydrophobic and are readily detected by the use of standard sequence analysis software, such as MacDNASIS
(Hitachi, San Bn.mo, CA). The presence of transmembrane sequences is often deleterious when a recombinant protein is synthesized in many expression systems, especially E. coli, as it leads to the production of insoluble aggregates which are difficult to renature into the native conformation of the polypeptide. Deletion of transmembrane sequences typically does not significantly alter the conformation of the remaining polypeptide structure.
Moreover, transmembrane sequences, being by definition embedded within a membrane, are inaccessible as antigenic determinants to a host immune system. Antibodies to these sequences will not, therefore, provide immunity to the host and, hence, little is lost in terms of immunity by omitting such sequences from the recombinant polypeptides of the invention. Deletion of transmembrane-encoding sequences from the genes used for expression can be achieved by standard techniques. See Ausubel et al., supra, Chapter 8.
For example, fortuitously-placed restriction enzyme sites can be used to excise the desired gene fragment, or the PCR can be used to amplify only the desired part of the gene.

Transformation of a host cell with recombinant DNA may be carried out by conventional techniques. When the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phases and subsequently treated by a CaCI, method using standard procedures.
Alternatively, MgCI= or RbCI can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.
When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate co-precipitates, conventional mechanical procedures, such as microinjection, electroporation, insertion of a plasmid encased in a liposome, or virus vectors may be used. Eukaryotic cells can also be cotransformed with DNA sequences encoding the hLPAATa and hLPAAT(3 poiypeptides of the invention, and a second foreign DNA
molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method uses a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus to transiently infect or transform eukaryotic cells and express the hLPAATa and hLPAAT~i polypeptides.
Expression vectors that are suitable for production of LPAAT polypeptides typically contain (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of transcripts, such as a transcription termination/polyadenylation sequence. LPAAT polypeptides of the present invention preferably is expressed in eukaryotic cells, such as mammalian, insect and yeast cells. Mammalian cells are especially preferred eukaryotic hosts because mammalian cells provide suitable post-translational modifications such as glycosylation.
Examples of mammalian host cells include Chinese hamster ovary cells {CHO-Kl; ATCC CCL61), rat pituitary cells (GH,; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL1548) SV40-transformed monkey kidney cells (COS-1; ATCC CRL
1650) and marine embryonic cells (NIH-3T3; ATCC CRL 1658). For a mammalian host, the transcriptional and transiational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression. Suitable transcriptional and translational regulatory sequences also can be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes.
Transcriptional regulatory sequences include a promoter region sufficient to direct the initiation of RNA synthesis. Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer et al., J. Molec. Appl. Genet.
1:273,1982); the TK
promoter of Herpes virus (McKnight, Cell 31: 355, 1982); the SV40 early promoter (Benoist et al., Nature 290:304, 1981}; the Rous sarcoma virus promoter (Gonnan et al., Proc. Nat'I.
Acid. Sci. USA 79:6777, 1982); and the cytomegalovirus promoter (Foecking et al., Gene 45:1 Ol, 1980). Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNA
I 0 polymerise promoter, can be used to control fusion gene expression if the prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et al., Mol. Cell. Biol.
10:4529, 1990;
Kaufinan et aL, Nucl. Acids Res. 19:4485, 1991 ).
An expression vector can be introduced into host cells using a variety of techniques including calcium phosphate transfection, liposome-mediated transfection, electroporation, 15 and the like. Preferably, transfected cells are selected and propagated wherein the expression vector is stably integrated in the host cell genome to produce stable transformants.
Techniques for introducing vectors into eukaryotic cells and techniques for selecting stable transformants using a dominant selectable marker are described, for example, by Ausubel and by Murray {ed.), Gene Transfer and Expression Protocols (Humana Press 1991 ).
20 Examples of mammalian host cells include COS, BHK, 293 and CHO cells.
Purification of Recombinant Polypeptides.
The polypeptide expressed in any of a number of different recombinant DNA
expression systems can be obtained in large amounts and tested for biological activity. The recombinant bacterial cells, for example E. toll, are grown in any of a number of suitable 25 media, for example LB, and the expression of the recombinant polypeptide induced by adding IPTG to the media or switching incubation to a higher temperature.
After culturing the bacteria for a further period of between 2 and 24 hours, the cells are collected by centrifugation and washed to remove residual media. The bacterial cells are then lysed, for example, by disruption in a cell homogenizer and centrifuged to separate the dense inclusion 30 bodies and cell membranes from the soluble cell components. This centrifugation can be performed under conditions whereby the dense inclusion bodies are selectively enriched by incorporation of sugars such as sucrose into the buffer and centrifugation at a selective speed.

If the recombinant polypeptide is expressed in the inclusion, these can be washed in any of several solutions to remove some of the contaminating host proteins, then solubilized in solutions containing high concentrations of urea (e.g., 8 M) or chaotropic agents such as guanidine hydrochloride in the presence of reducing agents such as 13-mercaptoethanol or DTT (dithiothreitol). At this stage it may be advantageous to incubate the polypeptide for several hours under conditions suitable for the polypeptide to undergo a refolding process into a conformation which more closely resembles that of the native polypeptide. Such conditions generally include low polypeptide (concentrations less than 500 mg/ml), low levels of reducing agent, concentrations of urea less than 2 M and often the presence of reagents such as a mixture of reduced and oxidized glutathione which facilitate the interchange of disulphide bonds within the protein molecule. The refolding process can be monitored, for example, by SDS-PAGE or with antibodies which are specific for the native molecule. Following refolding, the polypeptide can then be purified further and separated from the refolding mixture by chromatography on any of several supports including ion exchange resins, gel permeation resins or on a variety of affinity columns.
Isolation and purif cation of host cell expressed polypeptide, or fragments thereof may be carried out by conventional means including, but not limited to, preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
These polypeptides may be produced in a variety of ways, including via recombinant DNA techniques, to enable large scale production of pure, active hLPAATa and hLPAAT(3 useful for screening compounds for trilineage hematopoietic and anti-inflammatory therapeutic applications, and developing antibodies for therapeutic, diagnostic and research use.
The hLPAATa and hLPAAT(3 polypeptides of the present invention are useful in a screening methodology for identifying compounds or compositions which affect cellular signaling of an inflammatory response. This method comprises incubating the hLPAATa and hLPAAT(3 polypeptides or a cell transfected with cDNA encoding hLPAATa and hLPAAT(3 under conditions sufficient to allow the components to interact, and then measuring the effect of the compound or composition on hLPAATa and hLPAAT(3 activity. The observed effect on hLPAATa and hLPAAT[3 may be either inhibitory or stimulatory.

hLPAATa Search of the Genbank database of expressed sequence tag (dbest) using either the yeast or plant LPAAT protein sequences as probe came up with several short stretches of cDNA sequences with homology to the yeast or plant LPAAT protein sequence.
These cDNA sequences of interest were derived from single-run partial sequencing of random - human cDNA clones projects carried out by either the WashU-Merck EST or the Genexpress-Genethon program. An example of the amino acids sequence homology between the yeast LPAAT and a human cDNA clone (dbest#102250) is shown below by comparing SEQ ID NO. 3 (top amino acid sequence) with SEQ ID NO 4 (bottom amino IO acid sequence):
PFKKGAFHLAQQGKIPIVPVVVSNTSTLVSPKYGVFNRGCMIVRILKPIST
E
* ****** * **** * * * * * ** * * **
PSNCGAFHLAVQAQVPIVPIVMSSYQDFYCKKERRFTSGQCQVRVLPPVPT
IS E
The top line refers to the yeast LPAAT sequence from amino acids 169 to 220 and the bottom line refers to the homologous region from the dbest clone#102250.
Identical amino acids between these two sequences are shown in block letters with asterisks in between 20 Accordingly, a synthetic oligonucleotide (o.BLPAT.2R), S'-TGCAAGATGGAAGGCGCC-3' (SEQ ID NO. 5), was made based on the complement sequence of the conserved amino acids region, GAFHLA (SEQ >D NO. 6), of clone#102250. o.BPLAT.2R was radiolabeled at its 5'-end using Y-32p-ATP and T4 polynucleotide kinase as a probe in screening a ,zap human brain cDNA library 25 (Stratagene).
Screening of the cDNA library was accomplished by filter hybridization using standard methods (Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1995). Duplicate filters containing DNA derived from ~, phage plagues were prehybridized at 60 °C for 2 hr in 6X SSC (I X SSC is 0.15 M NaCI, 0.015 M sodium 30 citrate, pH 7.0), SX Denhardt's solution (1X Denhardt's solution is 0.02%
Ficoll, 0.02%
bovine serum albumin, and 0.02% polyvinyl-pyrrolidone), 0.1% sodium dodecyl sulfate (SDS), 50 mg/ml sonicated and denatured salmon sperm DNA. Hybridization was carried out in the same buffer as used for prehybridzation. After hybridization, the filters were washed in 6 X SSC at 42 °C, and autoradiographed.
Of the approximately 1 X 106 clones from the human brain cDNA library that were screened, twelve clones were identified that hybridized with the probe in duplicate filters. Eleven out twelve clones were enriched and recovered after a secondary screen.
Ten enriched phage samples were then converted to plasmid transformed cells by co-infecting E. coli XLl-Blue with the helper phage 8408 using Stratagene's recommended procedure. Colony filter hybridization was performed and identified those colonies that "lit up" with the probe. Seven out of the ten pools of colonies contained positive clones.
Two out of these seven clones, pZlpat.l0 and pZlpat.l l, contained inserts >2 kb.
Restriction mapping using a combination of Sst I, Pst I and BamHI digests showed these two clones contained many common fragments with respect to each other.
Nucleotide sequencing of the cDNA inserts in pZlpat.l0 and pZlpat.l 1 was i 5 performed. Figure 1 shows the DNA sequence of the cDNA insert of pZplat. l l . The nucleotide sequence analysis and restriction mapping of the cDNA clone revealed a 5'-untranslated region of >300 bp, an open reading frame capable of encoding a 283 amino acid polypeptide, and a 3'-untranslated region of >800 bp. The initiation site for translation was localized at nucleotide positions 319-321 and fulfilled the requirement for an adequate initiation site according to Kozak (Kozak, Critical Rev. Biochem.
Mol. Biol.
27:385-402, 1992). There was another upstream ATG at positions 131-133 with an in-phase stop codon at positions 17b-178. Except with a shorter S'-untranslated region, the cDNA insert of pZplat.10 has the same DNA sequence as that of pZplat. l l .
The sequence of the 283 amino acid open reading frame in pZplat. l I was used as the query sequence to search for homologous sequences in protein databases.
Search of the database based on Genbank Release 90 from the National Center for Biotechnology Information (NCBI) using the blastp program showed that the protein encoded by pZplat.l 1 was most homologous to the yeast and bacterial LPAATs. Figure 2 shows amino acid sequences alignment of the putative human LPAATa coding sequence, the yeast LPA.AT coding sequence, the E. toll LPAAT coding sequence, and the maize LPAAT coding sequence, revealing that human LPAATa has a much more extended homology with the yeast or the E. toll LPAAT than with the plant LPAAT.

hLPAAT
Search of the Genbank database (Boguski, et al., Science 265:1993-1994, 1994) of expressed sequence tag (dbEST) using either the yeast or plant LPAAT protein sequences as probe came up with several short stretches of cDNA sequences with homology to the yeast or plant LPAAT protein sequence. These cDNA sequences of interest were derived from single-run partial sequencing of random human cDNA clones projects carried out mainly by LM.A.G.E. Consortium [LLNL] cDNA clones program. An example of the amino acids sequence homology between the yeast LPAAT and a human cDNA clone (dbEST#363498) is shown below:

QQGKIPIVPVWSNTSTLVSPKYGVFNRGCMIVRILKPISTENLTKDKIGEFAEKVRDQM
VRENVPIVPVVYSSFSSFYNTKKKFFTSGTVTVQVLEAIPTSGLTAADVPALRGTPATGP

The top line refers to the yeast LPAAT sequence from amino acids 171 to 230 (SEQ ID NO. 9) and the bottom line refers to the homologous region from the dbest clone#363498 using the +1 reading frame (SEQ ID NO. 10). Identical and conserved amino acids between these two sequences are shown with double dots and single dot, respectively, in between. In order to find out if such cDNA clones with limited homology to yeast LPAAT sequence indeed encode human LPAAT[3 sequence, it was necessary to isolate the full-length cDNA clone, insert it into an expression vector, and to test if cells transformed or transfected with the cDNA expression vector produced more LPAAT
activity.
Accordingly, two synthetic oligonucleotides, 5'-CCTCAAAGTG
TGGATCTATC-3' (o.LPAT3.F) {SEQ ID NO. 11 ) and S'-GGAAGAGTAC
ACCACGGGGA C-3' (o.LPAT3.R), {SEQ ID NO. 12) were ordered (Life Technologies, Gaithersburg, MD) based on, respectively, the coding and the complement sequence of clone#363498. o.LPAT3.R was used in combination with a forward vector primer (o.sport.l), 5'- GACTCTAGCC TAGGCTTTTG C-3'(SEQ ID NO. 13) for amplification of the 5'-region, while o.LPAT3.F was used in combination with a reverse vector primer (o.sport.lR), 5'-CTAGCTTATA ATACGACTCA C-3' (SEQ ID NO. 14), for amplification of the 3'-region of potential LPAAT[3 sequences from a pCMV.SPORT
human leukocyte cDNA library (Life Technologies, Gaithersburg, MD). A 700 by PCR

fragment derived from o.sport.l and o.LPAT3.R amplification was cut with EcoR
I before inserting in between the Sma I and EcoR I of pBluescript(II)SK(-) (Stratagene, LaJolla, CA) to generate pLPAT3.5'. A 900 by PCR fragment derived from o.sport.lR and o.LPAT3.F amplification was cut with Xba I before inserting in between the Sma I and Xba I of pBluescript(II)SK(-) (Stratagene, LaJolla, CA) to generate pLPAT3.3'.
Nucleotide sequencing analysis of the cDNA inserts from these two plasmids showed they contained overlapping sequences with each other, sequences that matched with the dbEST#363498 as well as extensive homology with the yeast LPAAT amino acids sequence (Nagiec et al., .l. Biol. Chem. 268:22156-22163, 1993). To assemble the two halves of the cDNA into a full-length clone, the 560 by Nco I - Nar I fragment from pLPAT3.5' and the 780 by Nar I - Xba I fragment from pLPAT3.3' were inserted into the Nco I l Xba I vector prepared from pSP-luc+ (promega, Madison, WI) via a three-part ligation to generate pSP.LPAT3.
Figure 3 shows the DNA sequence ID of the cDNA insert of pSP.LPAT3. The I S nucleotide sequence analysis and restriction mapping of the cDNA clone revealed a S'-untranslated region of 39 bp, an open reading frame capable of encoding a 278 amino acids polypeptide that spans nucleotide positions 40 to 876 and a 3'-untranslated region of 480 by (Figure 3). The initiation site for translation was localized at nucleotide positions 40-42 and fulfilled the requirement for an adequate initiation site according to Kozak (Kozak, Critical Rev. Biochem. Mol. Biol. 27:385-402, 1992).
The sequence of the 278 amino acid open reading frame (Figure 4) was used as the query sequence to search for homologous sequences in protein databases. Search of the database based on Genbank Release 92 from the National Center for Biotechnology Information (NCBI) using the blastp program showed that this protein was most homologous to the yeast, bacterial and plant LPAATs. Figure 5 shows amino acid sequences alignment of this putative human LPAAT~i coding sequence, human LPAATa.
coding, the yeast LPAAT coding sequence, the bacterial (E. toll, H. in, fluenzae, and S.
typhimurium) LPAAT coding sequences, and the plant (L. douglassi and C.
nucifera) LPAAT coding sequences, revealing that the human LPAAT coding sequences have a much more extended homology with the yeast or the bacterial LPAAT than with the plant LPAAT.
Characterization of the Invention i8 Accordingly, human LPAATa is characterized by the 283 amino acids of SEQ ID
NO. 2. The present invention further includes allelic variations {naturally-occurnng base changes in the species population which may or may not result in an amino acid change) of the DNA sequences herein encoding active LPAAT polypeptides and active fragments thereof. The inventive DNA sequences further comprise those sequences which hybridize under stringent conditions (see, for example, Maniatis et al, Molecular Coining (A
Laboratory Manual), Cold Spring Harbor Laboratory, pages 387-389, 1982) to the coding region (e.g., nucleotide #319 to nucleotide #1167). One such stringent hybridization condition is, for example, 4 X SSC at 65 °C, followed by washing in 0.1 X SSC at 65 °C
for thirty minutes. Alternatively, another stringent hybridization condition is in 50%
formamide, 4 X SSC at 42 °C. The present invention further includes DNA
sequences which code for LPAAT polypeptides having LPAAT activity but differ in codon sequence due to degeneracy of the genetic code. Variations in the DNA sequences which are caused by point mutations or by induced modifications of the sequence of SEQ ID NO.
l, which enhance the activity of the encoded polypeptide or production of the encoded LPAAT
polypeptide are also encompassed by the present invention.
Definitions In the description that follows, a number of terms are utilized extensively.
Definitions are provided to facilitate understanding of the invention.
The term "isolated" applied throughout the specification to polypeptides refers to that level of purity in which the polypeptide is sufficiently free of other materials endogenous to the host from which the polypeptide is isolated such that any remaining materials do not materially affect the biological properties of the polypeptide.
The term "derived" as used throughout the specification in relation to the polypeptides of the present invention, encompasses polypeptides obtained by isolation and purification from host cells, as well as polypeptides obtained by manipulation and expression of nucleotide sequences prepared from host cells. It also encompasses nucleotide sequences including genomic DNA, mRNA, cDNA synthesized from mRNA, and synthetic oligonucleotides having sequences corresponding to the inventive nucleotide sequences. It further encompasses synthetic polypeptide antigens prepared on the basis of the known amino acid sequences of the proteins of the present invention.
The term "expression product" as used throughout the specification refers to materials produced by recombinant DNA techniques.
Peptide seauencing of polypeptides Purified polypeptides prepared by the methods described above can be sequenced using methods well known in the art, for example using a gas phase peptide sequencer (Applied Biosystems, Foster City, CA). Because the proteins of the present invention may be glycosylated, it is preferred that the carbohydrate groups are removed from the proteins prior to sequencing. This can be achieved by using glycosidase enzymes.
Preferably, glycosidase F (Boehringer-Mannheim, Indianapolis, Il~ is used. To determine as much of the polypeptide sequence as possible, it is preferred that the polypeptides of the present invention be cleaved into smaller fragments more suitable for gas-phase sequence analysis.
This can be achieved by treatment of the polypeptides with selective peptidases, and in a particularly preferred embodiment, with endoproteinase lys-C (Boehringer). The fragments so produced can be separated by reversed-phase HPLC chromatography.
Substitutional variants typically contain the exchange of one amino acid for another 1 S at one or more sites within the protein, and are designed to modulate one or more properties of the polypeptides such as stability against proteolytic cleavage.
Substitutions preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge.
Conservative substitutions are well known in the art and include, for example, the changes of alanine to serine; arginine to lysine; asparigine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparigine; glutamate to aspartate; glycine to praline; histidine to asparigine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Insertional variants contain fusion proteins such as those used to allow rapid purification of the polypeptide and also can include hybrid polypeptides containing sequences from other proteins and polypeptides which are homologues of the inventive polypeptide. For example, an insertional variant could include portions of the amino acid sequence of the polypeptide from one species, together with portions of the homologous polypeptide from another species. Other insertional variants can include those in which additional amino acids are introduced within the coding sequence of the polypeptides. These typically are smaller insertions than the fusion proteins described above and are introduced, far example, to disrupt a protease cleavage site.
Antibodies to human LPAAT protein can be obtained using the product of an LPAAT expression vector or synthetic peptides derived from the LPAAT coding sequence coupled to a carrier (Pasnett et al., J. Biol. Chem. 263:1728, 1988) as an antigen. The preparation of polyclonal antibodies is well-known to those of skill in the art. See, for example, Green et al., "Production of Polycional Antisera," in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992). Alternatively, an LPAAT antibody of the present invention may be derived from a rodent monoclonal antibody (MAb).
Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature 256:495, 1975, and Coligan et al. (eds.), Current Protocols in Immunology, 1:2.5.1-2.6.7 (John Wiley &
Sons 1991).
Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography.
See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et al., "Purification of Immunoglobulin G (IgG)," in Methods in Molecular Biology, 10:79-104 Humana Press, Inc. 1992. An LPAAT antibody of the present invention may also be derived from a subhuman primate antibody. General techniques for raising therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et a1, international patent publication No. WO 91/11465 (1991), and in Losman et al., Int. J. Cancer 46:310, 1990.
Alternatively, a therapeutically useful LPAAT antibody may be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then, substituting human residues in the framework regions of the marine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of marine constant regions. General techniques for cloning marine immunoglobulin variable domains are described, for example, by the publication of Orlandi et al., Proc. Nat'l. Acad. Sci. USA 86:3833, 1989.
Techniques for producing humanized MAbs are described, for example, by Jones et al., Nature 321:522, 1986, Riechmann et al., Nature 332:323, 1988, Verhoeyen et al., Science 239:1534, 1988, Carter et al., Proc. ~Vat'I Acad. Sci. USA 89:4285, 1992, Sandhu, Crit. Rev.
Biotech. 12: 437, 1992, and Singer et al., J. Immun. 150:2844, 1993, each of which is hereby incorporated by reference.
I O As an alternative, an LPAAT antibody of the present invention may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library.
See, for example, Barbas et al., METHODS. A Companion to Methods in Enzymology 2:119 1991, and Winter et al., Ann. Rev. Immunol. 12:433, 1994, which are incorporated by reference.
Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA). In addition, an LPAAT antibody of the present invention may be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic challenge.
In this technique, elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994, and Taylor et al., Int. Immun. 6:579, 1994.
Example 1 This example illustrates an experiment to determine if the human LPAATa clone encodes a protein with LPAAT activity, an E. coli vector expressing the human LPAATa.
as a fusion protein with (3-galactosidase was transformed into a LPAAT minus strain ofE.
toll to see if it would complement the defect in E. toll. Specifically, the 840 by Bgl II-Nco I fragment, which spans the coding region of human LPAATcc from amino acid 68 to beyond the stop codon, derived from pZplat. l l was inserted into a Bgl II l Nco I digested cloning vector pLitmus28 (Evans et al., BioTechniques 19:130-I35, 1995) to generate the plasmid p28BgN. This plaslilid is expected to express the human LPAATa as a fusion protein containing the first 16 amino acids of ~i-galactosidase and the last 216 residues of the human LPAATa coding sequence using the lac promoter in pLitmus28. This plasmid was transformed into the E. coli strain JC201 (obtained from Dr. Jack Coleman, Louisiana State University). JC201 (Coleman, Mol. Gen. Genet. 232:295-303, 1992; Nagiec et al., J.
Biol. Chem. 268:22156-22163, 1993; and Brown et al., Plant Mol. Biol. 26:211-223, 1994) is deficient in LPAAT activity due to mutation in the plsC locus. This mutation leads to a temperature-sensitive phenotype that causes JC201 to grow slowly at 37 °C, almost not at all at 42 °C, and not at all at 44 °C. JC201 transformed with p28BgN was able to grow normally at 44 °C when compared to the wild type strain JC200 {pisC+), while JC201 transformed with pLitmus28 vector was not able to support growth at 44 °C. These data suggest that the putative human LPAATa cDNA isolated here does possess LPAAT
activity, as the Iast 216 amino acids of this cDNA is sufficient to complement the defective LPAAT gene (plsC) in JC201.
Example 2 To see if the putative human LPAAT(3 clone encodes a protein with LPAAT
activity, an E. coli vector expressing this human LPAAT/3 as a direct product was transformed into a LPAAT minus strain of E. coli to see if it would complement the defect in E. coli. Specifically, the 1350 by Nco I - Xba I fragment from pSP.LPAT3, which spans the entire coding region from amino acid 1 to beyond the stop codon, was inserted into a Nco I l Xba I digested cloning vector pKK388-1 (Clontech, Palo Alto, CA) to generate the plasmid pTrc.LPAT3. This plasmid was transformed into the E. coli strain JC201 (obtained from Dr. Jack Coleman, Louisiana State University). JC201 (Coleman, Mol.
Gen. Genet. 232:295-303, 1992) is deficient in LPAAT activity due to mutation in the plsC locus. This mutation leads to a temperature-sensitive phenotype that causes JC201 to grow slowly at 37 °C, almost not at all at 42 °C, and not at all at 44 °C. JC201 transformed with pTrc.LPAT3 was able to grow normally at 44 °C when compared to the wild type strain JC200 (plsC+), while JC201 transformed with pKK388-1 vector was not able to support growth at 44 °C. These data suggest that the putative human LPAAT(3 WO 98/54303 PCTlUS97/05360 cDNA isolated here does possess LPAAT activity, as the putative protein product of this cDNA is able to complement the defective LPAAT gene (plsC) in JC201.
Example 3 This example illustrates a group of experiments to see if overexpression of this human LPAATa, would have any effect on mammalian cells. The entire cDNA insert (2,300 bp) from pZplat.l l was cleaved with Asp718 I and Xho I for insertion into the mammalian expression vector pCE9 to generate pCE9.LPAAT 1. pCE9 was derived from pCE2 with two modifications. The 550 by BstY I fragment within the elongation factor-1 a (EF-1 a) intron of pCE2 was deleted. The multiple cloning region of pCE2 between the Asp718 I and BamH I site was replaced with the multiple cloning region spanning the Asp718 I and Bgl II sites from pLitmus28. The plasmid pCE2 was derived from pREP7b (Leung, et al., Proc. Natl. Acad. Sci. USA, 92: 4813-4817, 1995) with the RSV
promoter region replaced by the CMV enhancer and the elongation factor-1 a (EF-1 a) promoter and intron. The CMV enhancer came from a 380 by Xba I-Sph I fragment produced by PCR
from pCEP4 (Invitrogen, San Diego, CA) using the primers 5'-GGCTCTAGAT
ATTAATAGTA ATCAATTAC-3' and 5'-CCTCACGCAT GCACCATGGT AATAGC-3'. The EF-1 a promoter and intron (LJetsuki, et al., J. Biol. Chem., 264:
5791-5798, 1989) came from a 1200 by Sph I-Asp718 I fragment produced by PCR from human genomic DNA using the primers 5'-GGTGCATGCG TGAGGCTCCG GTGC-3' and 5'-GTAGTTTTCA CGGTACCTGA AATGGAAG-3'. These 2 fragments were ligated into a Xba I/Asp718 I digested vector derived from pREP7b'to generate pCE2.
pCE9.LPAATI DNA was transfected into several mammalian cell lines, including A549 cells, ECV304 cells (American Type Culture Collection, Rockville, MD), two human cell line that would produce IL-6 and TNF upon stimulation with IL-Ib and murine TNF and 293-EBNA cells (Invitrogen, San Diego, CA). pCE9.LPAAT1 was digested with BspH I before electroporating into these cell lines with a Cell-PoratorTM
(Life Technologies, Gaithersburg,1VID) using conditions described previously (Cachianes, et al., Biotechniques 15:255-259, 1993). After adherence of the transfected cells 24 hours later, the cells were grown in the presence of 200 pg / ml Hygromycin B (Hyg) (Calbiochem, La Jolla, CA) to select for cells that had incorporated both plasmids. Hyg-resistant clones that expressed LPAAT mRNA at a level more than 20 fold higher relative to untranfected cells based on Northern Blot analysis (Kroczek, et al., Anal. Biochem. 184: 90-95, 1990) were selected for further study.
Figure 6 compares the LPAAT activity in A549 cells and in A549 cells transfected with pCE9.LPAAT1 DNA using aTLC assay. This screening assay for LPAAT activity in cell extracts was based on a fluorecent assay using fluorecent lipid substrates (Ella, et al., Anal. Biochem. 218: 136-142, 1994). Instead of using the PC-substrate, BPC
(Molecular Probes, Eugene, OR), a synthetic PC that contains an ether linkage at the SN1 position with a fluorescent Bodipy moiety incorporated into the end of the alkyl-chain at the SN1 position, BPC was converted to Bodipy-PA using cabbage phospholipase D (Sigma, St.
Louis, MO). Bodipy-PA was then converted to Bodipy-LPA using snake venom phospholipase A2. The Bodipy-LPA obtained was purif ed by preparative TLC for use in the LPAAT assay. The assay was carried out in total cell extracts resuspended in lysis buffer (Ella, et al., Anal. Biochem. 218: 136-142, 1994) supplemented with 0.5 mM ATP, 0.3 mM MgClz, 100 pM oleoyl-CoA and 10 uM Bodipy LPA. The samples were incubated for 30 min before loading onto TLC plates.
Lane 1 refers to Bodipy LPA incubated with buffer only without any cell extract added. Lane 9 refers to BPC treated with cabbage phospholipase D for generateing a Bodipy-PA marker. Lanes 2 and 4 refer to to Bodipy LPA incubated with control cell extracts with or without lipid A, respectively. Lanes 3 and 5 refer to Bodipy LPA
incubated with A549 cell extracts transfected with pCE9.LPAAT1 DNA with or without lipid A, respectively. Figure 3 shows A549 cells transfected with the LPAAT
cDNA
(lanes 3 and 5) contain much more LPAAT activity than those of control cells (lanes 2 and 4) as evidenced by the increased conversion of Bodipy-LPA to Bodipy-PA.
Addition of lipid A to the cell extracts has little effect on LPAAT activity (lanes 2 vs 4 and 3 vs 5).
A549 cell extract also contains a phosphohydrolase activiity that converts Bodipy-LPA to Bodipy-monoalkylglycerol (lanes 2 to S). Interestingly, A549 cells overexpressing LPAAT (lanes 3 and 5) have less of this activiity compared to control cells (lanes 2 and 4), suggesting this phosphohydrolase prefers LPA to PA as substrate. There is also an increase of DAG in transfected cells (lanes 3 and 5) compared to control cells (lanes 2 and 4) possibly due to partial conversion of the PA formed to DAG from this endogenous phosphohydrolase.
Example 4 To see if the expressed LPAAT cDNA clone described here would also use other glycerol-lipids that contain a free-hydroxyl group at the SN2 position, the cell extracts were incubated with the substrates NBD-IysoPC (lanes 6 and 7} and NBD-monoacylglycerol (MAG) (lanes 10 and I I) to see if there is increased conversion to lysoPC and DAG, respectively. Lane 8 and 12 refer, respectively, to NBD-lysoPC
and NBD-MAG incubated with buffer only without any cell extract added. TLC
analysis shows little difference in the lipid profile between the transfected and control cells (lanes 7 vs 6, lanes I lvs 10), suggesting the cloned LPAAT enzyme uses LPA as the preferred substrate. It is likely that the acyltransferases for lysoPC (Fyrst, et al., Biochem. J.
306:793-799, 1995) and for MAG (Bhat, et al., Biochemistry 34: 11237-11244, 1995) represent different enzymes from the LPAAT described here.
Example 5 pCE9.LPAAT1 DNA was transfected into A549 cells (American Type Culture Collection, Rockville, MD), a human cell line that would produce IL-6 and TNF
upon stimulation with IL-1 (3 and marine TNF. pCE9.LPAAT 1 was digested with BspH I
before electroporating into A549 cells with a Cell-PoratorTM (Life Technologies, Gaithersburg, MD) using conditions described previously (Cachianes, et al., Biotechniques 15:255-259, 1993). After adherence of the transfected cells 24 hours later, the cells were grown in the presence of 200 ug/ml Hygromycin B (Hyg) (Calbiochem, La Jolla, CA) to select for cells that had incorporated both plasmids. A Hyg-resistant clone that expressed LPAAT mRNA
at a level more than 20 fold higher relative to untranfected A549 cells based on Northern Blot analysis (Kroczek et al., Anal. Biochem. 184:90-95, 1990) was selected for further study.
A comparison of the production of TNF (Figure 7) and IL-6 (Figure 8) between A549 cells transfected with pCE9.LPAAT1 and control A549 cells after stimulation with IL-1~3 and marine TNF shows A549 overexpressing LPAAT produces >5 fold more TNF

and > 10 fold more IL-6 relative to untransfected A549 cells, suggesting that overexpression of LPAAT would enhance the cytokine signaling response in cells.
Development of compounds that would modulate LPAAT activity should therefore be of therapeutic interest in the field of inflammation.
SEQUENCE LISTING
(1) GENERAL
INFORMATION:

(i) APPLICANTS: Leung, David W.

West, James Tompkins, Christopher (ii) TITLE OF INVENTION: MAMMALIAN LYSOPHOSPHATIDIC ACID

ACYL TRANSFERASE

(iii ) NUMBER OF SEQUENCES: 18 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Cell Therapeutics, Inc.

{B) STREET: 201 Elliott Avenue West (C) CITY: Seattle (D) STATE: Washington (E) COUNTRY: U.S.A.

(F) ZIP 98119 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5" disk, 1.44Mb, double side, high density (B) COMPUTER: PC Clone (486 microprocessor) (C) OPERATING SYSTEM: MS-DOS Version 6.1, Windows 3.1 (D) SOFTWARE: WORD 6.0 (vi) CURRENT APPLICATION DATA .

(A) APPLICATION NUMBER:

(B) FILING DATE: 15-Dec-1995 {vii i) ATTORNEY/AGENT INFORMATION:

(A) NAME: Oster, Jeffrey B.

(B) REGISTRATION NUMBER: 32,585 {C) REFERENCE/DOCKET NUMBER:1801 (ix} TELECOMMUNICATION INFORMATION:

(A) TELEPHONE:(206)282-7100 (B) TELEFAX:(206)284-6206 (2) INFORMATION
FOR SEQ
ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH:2242 (B) TYPE: nucleic acid (C) STRANDEDNESS: double stranded (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA

(iii) HYPOTHETICAL:
no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE:

(vi} ORIGINAL SOURCE:

{A) ORGANISM: homo sapien (B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

( E ) HAPLOTYPE

(F) TISSUE TYPE: brain (G) CELL TYPE: -(H) CELL LINE

(I) ORGANELLE:

( ix) FEATURE

(A) NAME/KEY: hLPAATa (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

1266 GTCTCCCCTCTCCCCACTTATTCTCCTCTTTGGAATCTTCA.ACTTCTGAA

(2) INFORMATION
FOR SEQ
ID N0:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH:283 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (iii ) HYPOTHETICAL: no (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE:

(vi) ORIGINAL SOURCE:

(A) ORGANISM: homo sapien (B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE: brain (G) CELL TYPE:

(H) CELL LINE:

(I) ORGANELLE:

(ix) FEATURE:

(A} NAME/KEY: hLPAATa (B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:

1 Met Asp Leu Trp Pro Gly Ala Trp Met Leu Leu Leu Leu Leu Phe 16 Leu Leu Leu Leu Phe Leu Leu Pro Thr Leu Trp Phe Cys Ser Pro 31 Ser Ala Lys Tyr Phe Phe Lys Met Ala Phe Tyr Asn Gly Trp Ile -46 Leu Phe Leu Ala Val Leu Ala Ile Pro Val Cys Ala Val Arg Gly 61 Arg Asn Val Glu Asn Met Lys Ile Leu Arg Leu Met Leu Leu His 76 Ile Lys Tyr Leu Tyr Gly Ile Arg Val Glu Val Arg Gly Ala His 91 His Phe Pro Pro Ser Gln Pro Tyr Val Val Val Ser Asn His Gln 106 Ser Ser Leu Asp Leu Leu Gly Met Met Glu Val Leu Pro Gly Arg 121 Cys Val Pro Ile Ala Lys Arg Glu Leu Leu Trp Ala Gly Ser Ala 136 Gly Leu Ala Cys Trp Leu Ala Gly Val Ile Phe Ile Asp Arg Lys 151 Arg Thr Gly Asp Ala Ile Ser Val Met Ser Glu Val Ala Gln Thr 166 Leu Leu Thr Gln Asp Val Arg Val Trp Val Phe Pro Glu Gly Thr 181 Arg Asn His Asn Gly Ser Met Leu Pro Phe Lys Arg Gly Ala Phe 196 His Leu Ala Val Gln Ala Gln Val Pro Ile Val Pro Ile Val Met 211 Ser Ser Tyr Gln Asp Phe Tyr Cys Lys Lys Glu Arg Arg Phe Thr 226 Ser Gly Gln Cys Gln Val.Arg Val Leu Pro Pro Val Pro Thr Glu 241 Gly Leu Thr Pro Asp Asp Val Pro Ala Leu Ala Asp Arg Val Arg 256 His Ser Met Leu Thr Val Phe Arg Glu Ile Ser Thr Asp Gly Arg 271 Gly Gly Gly Asp Tyr Leu Lys Lys Pro Gly Gly Gly Gly ***
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:52 (B) TYPE: AMINO acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: yeast (B) STRAIN:
(C) INDIVIDUAL ISOLATE:
{D) DEVELOPMENTAL STAGE:
(E} HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
{H) CELL LINE
(I) ORGANELLE:
( ix) FEATURE
(A} NAME/KEY: LPAAT fragment (B) LOCATION:169-220 (C) IDENTIFICATION METHOD:
(J) PUBLICATION DATE:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3: _ (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:52 (B) TYPE : amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (iii) HYPOTHETICAL: no 1$ (iv) ANTI-SENSE: no (v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapien (B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
{E} HAPLOTYPE:
{F) TISSUE TYPE: placenta {G) CELL TYPE:
{H) CELL LINE:
(I) ORGANELLE:
( ix ) FEATURE
(A) NAME/KEY: dbest clone #102250 (B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:

(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH:18 (B) TYPE: nucleotide (C) STRANDEDNESS: single {D} TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide fragment (iii) HYPOTHETICAL:
no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE:

(vi) ORIGINAL SOURCE:

(A) ORGANISM:

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE:

(H) CELL LINE

( I ) ORGANELLE

( ix) FEATURE

(A) NAME/KEY: o.BLPAT.2R

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:

{2) INFORMATION
FOR SEQ
ID N0:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH:6 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide fragment (iii ) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v} FRAGMENT TYPE:

(vi) ORIGINAL SOURCE:

(A) ORGANISM:

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE:

(H) CELL LINE:

(I) ORGANELLE:

(ix) FEATURE:

(A) NAME/KEY: o.BLPAT.2R

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:

(2) INFORMATION
FOR SEQ
ID N0:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH:1373 (B) TYPE: nucleic acid (C) STRANDEDNESS: double stranded (D} TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA

(iii ) HYPOTHETICAL: no (iv) ANTI-SENSE: no {v) FRAGMENT TYPE:

(vi) ORIGINAL SOURCE:

(A) ORGANISM: homo sapien (B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE:

(H) CELL LINE:

(I) ORGANELLE:

(ix) FEATURE:

(A) NAME/KEY: hLPAAT~i (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:

GGCGGCGCCG
TCGGGCGCCG
GGCCGGGCCA
TGGAGCTGTG

1351 AAA..1373 {2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:274 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE:
{vi) ORIGINAL SOURCE:
{A) ORGANISM: homo sapien (B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
{F) TISSUE TYPE:
(G) CELL TYPE
(H) CELL LINE:
(I) ORGANELLE:
( ix) FEATURE
(A) NAME/KEY: hLPAAT~i {B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
1 Met Glu Leu Trp Cys Leu Ala Ala Ala Leu Leu Leu Leu Leu Leu 16 Leu Val Gln Ser Arg Ala Ala Glu Phe Tyr AIa Lys Val Ala Leu WO 98!54303 PCT/US97/05360 31 Tyr Cys Leu Cys Phe ThrVal Ser Ala Val Ala Ser Leu Val Cys 46 Leu Cys His Gly Gly ArgThr Val Glu Asn Met Ser Ile Ile Gly -61 Trp Phe Val Arg Ser PheLys Tyr Phe Tyr Gly Leu Arg Phe Glu 76 Val Arg Asp Pro Arg ArgLeu Gln Glu Ala Arg Pro Cys Val Ile 91 Val Ser Asn His Gln SerIle Leu Asp Met Met Gly Leu Met 10Glu 106 Val Leu Pro Glu Arg CysVal Gln Ile Ala Lys Arg Glu Leu Leu 121 Phe Leu GIy Pro Val GlyLeu Ile Met Tyr Leu Gly Gly Val Phe 15136 Phe Ile Asn Arg Gln ArgSer Ser Thr Ala Met Thr Val Met Ala 151 Asp Leu Gly Glu Arg MetVal Arg Glu Asn Leu Lys Val Trp Ile 166 Tyr Pro Glu Gly Thr ArgAsn Asp Asn Gly Asp Leu Leu Pro 20Phe 181 Lys Lys Gly Ala Phe TyrLeu Ala Val Gln Ala Gln Val Pro Ile 196 Val Pro Val Val Tyr SerSer Phe Ser Ser Phe Tyr Asn Thr Lys 25211 Lys Lys Phe Phe Thr SerGly Thr Val Thr Val Gln Val Leu Glu 226 Ala Ile Pro Thr Ser GlyLeu Thr Ala Ala Asp Val Pro Ala Leu 241 Val Asp Thr Cys His ArgAla Met Arg Thr Thr Phe Leu His 30Ile 256 Ser Lys Thr Pro Gln GluAsn Gly Ala Thr Ala Gly Ser Gly Val 271 Gln Pro Ala Gln *** 274 35 (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE
CHARACTERISTICS:

(A) LENGTH:60 (B) TYPE: amino acid (C) STRANDEDNESS: single 40 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: NO

(v) FRAGMENT
TYPE:

45 (vi ) ORI GINAL SOURCE

(A) ORGANISM: yeast (B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

50 (E) HAPLOTYPE:

(F) TISSUE TYPE:

{G) CELL TYPE:

(H) CELL LINE:

(I) ORGANELLE:

55 ( ix) FEATURE

(A) NAME/KEY: LPAAT fragment (B) LOCATION:171-230 (C) IDENTIFICATION METHOD:

WO 98!54303 PCT/US97/05360 (J) PUBLICATION DATE:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
I
QQGKIPIVPVWSNTSTLVSPKYGVFNRGCMIVRILKPISTENLTKDKIGEFAEKVRDQM~
(2) INFORMATION
FOR SEQ
ID NO:IO:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH:60 10 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (iii} HYPOTHETICAL:
no 15 (iv} ANTI-SENSE: no (v) FRAGMENT TYPE:

(vi) ORIGINAL SOURCE:

(A) ORGANISM: homo sapien (B) STRAIN:

20 (C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE:

25 (H) CELL LINE:

(I) ORGANELLE:

(ix) FEATURE:

(A) NAME/KEY: dbest clone #363498 (B) LOCATION:

30 (C} IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:

VRENVPIVPVVYSSFSSFYNTKKKFFTSGTVTVQVLEAIPTSGLTAADVPALRGTPATGP

(2) INFORMATION
FOR SEQ
ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH:20 40 (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide fragment (iii ) HYPOTHETICAL: no 45 (iv) ANTI-SENSE: no (v) FRAGMENT TYPE:

(vi) ORIGINAL SOURCE:

(A) ORGANISM:

(B) STRAIN:

50 (C} INDIVIDUAL ISOLATE:

(D} DEVELOPMENTAL STAGE:

(E} HAPLOTYPE:

(F) TISSUE TYPE:

(G} CELL TYPE:

55 (H} CELL LINE:

(I) ORGANELLE:

(ix) FEATURE:

(A) NAME/KEY: o. LPAT.3F

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: -(2) INFORMATION
FOR SEQ
ID N0:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH:21 (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide {iii ) HYPOTHETICAL: no {iv) ANTI-SENSE: no (v) FRAGMENT TYPE:

(vi) ORIGINAL SOURCE:

(A) ORGANISM:

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

( E ) HAPLOTYPE

(F) TISSUE TYPE:

{G) CELL TYPE:

(H) CELL LINE:

(I) ORGANELLE:

(ix) FEATURE:

(A) NAME/KEY: o.LPAT3.R

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:

(2) INFORMATION
FOR SEQ
ID N0:13:

(i) SEQUENCE CHARACTERISTICS:

{A) LENGTH:21 (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide ( i i i ) HYPOTHET
I CAL
: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE

(vi ) ORIGINAL SOURCE

(A) ORGANISM:

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE:

(H) CELL LINE

(I) ORGANELLE:

(ix) FEATURE:

(A) NAME/KEY: o.sport.l (B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:

(2) INFORMATION
FOR SEQ
ID N0:14:

S (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH:21 (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) HYPOTHETICAL:
no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE:

{vi) ORIGINAL SOURCE:

(A) ORGANISM:

(B) STRAIN:

{C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

{F) TISSUE TYPE:

(G) CELL TYPE:

(H) CELL LINE:

(I) ORGANELLE:

(ix) FEATURE:

(A) NAME/KEY: o.sport.lR

{B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:

(2) INFORMATION
FOR SEQ
ID NO:15:

{i) SEQUENCE CHARACTERISTICS:

(A) LENGTH:29 {B) TYPE: nucleotide (C) STRANDEDNESS: single {D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii ) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE:

(vi) ORTGINAL SOURCE:

(A) ORGANISM:

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE:

(H) CELL LINE:

(I) ORGANELLE:

(ix) FEATURE:

(A) NAME/KEY:

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:

(2) INFORMATION
FOR SEQ
ID N0:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH:26 (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii ) HYPOTHETICAL: no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE:

(vi) ORIGINAL SOURCE:

(A) ORGANISM:

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E) HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE:

(H) CELL LINE:

( I ) ORGANELLE

( i x ) FEATURE

{A} NAME/KEY:

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:

GCACCATGGT
AATAGC

(2) INFORMATION
FOR SEQ
ID N0:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH:24 (B) TYPE: nucleotide {C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) HYPOTHETICAL:
no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE:

(vi) ORIGINAL SOURCE:

{A) ORGANISM:

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

(E} HAPLOTYPE:

(F) TISSUE TYPE:

(G) CELL TYPE:

(H) CELL LINE

{I) ORGANELLE:

( ix) FEATURE

(A) NAME/KEY:

{B) LOCATION:

SS (C) IDENTIFICATION METHOD:

{D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:

(2) INFORMATION
FOR SEQ
ID N0:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH:28 (B) TYPE: nucleotide (C) STRANDEDNESS: single (D) TOPOLOGY: linear i0 (ii) MOLECULE TYPE: oligonucleotide (iii) HYPOTHETICAL:
no (iv) ANTI-SENSE: no (v) FRAGMENT TYPE:

(vi) ORIGINAL SOURCE:

(A) ORGANISM:

(B) STRAIN:

(C) INDIVIDUAL ISOLATE:

(D) DEVELOPMENTAL STAGE:

( E ) HAPLOTYPE

(F) TISSUE TYPE:

(G) CELL TYPE

(H) CELL LINE

(I) ORGANELLE:

(ix) FEATURE:

(A) NAME/KEY:

(B) LOCATION:

(C) IDENTIFICATION METHOD:

(D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:

CGGTACCTGA
AATGGAAG

Claims (4)

We claim:

1. A nucleic acid sequence coding on expression for an LPAAT enzyme selected from the group consisting of:
(a) a DNA sequence set forth in SEQ ID NO. 1, SEQ ID NO. 7, and shortened fragments thereof;
(b) a cDNA sequence which, due to the degeneracy of the genetic code, encodes a polypeptide of SEQ ID NO. 2, SEQ ID NO. 8, and enzymatically active fragments thereof;
and (c) a cDNA sequence capable of hybridizing to the cDNA of (a) or (b) under high stringency conditions and which encodes a polypeptide having LPAAT activity.

2. An LPAAT enzyme selected from the group consisting of an amino acid set forth in SEQ ID NO. 2, SEQ ID NO. 8, and enzymatically active fragments thereof.

3. A method for screening drug candidate compounds having activity as antiinflammatory agents, for increasing hematopoiesis, and preventing reoxygenation injury following cytoreductive therapy, comprising:
(a) obtaining an LPAAT polypeptide according to claim 2, having LPAAT
enzymatic activity;
(b) contacting the LPAAT polypeptide with different concentrations of the drug candidate and a control sample; and (c) measuring LPAAT activity with and without different concentrations of the drug candidate.

4. The method of claim 3 wherein the drug candidate can be a pool of compounds from combinatorial library expression.