LPAAT, also referred to as 1-acyl sn-glycerol-3-phosphate acyltransferase (EC2.31.51), is known to catalyze 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. LPA and PA, while originally identified as intermediates in lipid biosynthesis (Kent, Anal. Rev. BioChem. 64: 315-343,1995), have more recently 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). PA can be generated through hydrolysis of phosphatidycholine (PC) (Exton, Biochem. 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: C1093-C1104, 1994).

Compounds that block PA generation and hence diminish lipid biosynthesis and the signal involved in cell activation are therefore of therapeutic interest in, for example, the areas of inflammation and oncology as well as obesity treatment.

Therefore, compounds that block LPAAT activity have a similar therapeutic value.

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; Hanke et al., Eur J. Biochem. 232: 806-810,1995; Knutzon et al., Plant Physiol. 109: 999-1006,1995) using genetic complementation techniques. Two human isoforms of LPAAT, termed LPAAT-a and LPAAT-P, have been isolated (West et al., DNA Cell Biol. 6: 691-701,1997). The differences in tissue expression patterns of LPAATa and LPAATß mRNA suggest that these two genes are regulated differently and are likely to have independent functions (West et al., ibid.). Several other human LPAAT isoforms have been identified (see, e. g., US Patent No. 5,888,793).

There is a continuing need to discover new human LPAAT isoforms that are useful for the diagnosis, treatment and/or prevention of diseases and conditions in which LPAAT is implicated (e. g., cancers and autoimmune diseases).

SUMMARY OF THE INVENTION In one of its aspects, the present invention provides a novel human LPAAT isoform, hLPAATE, comprising the amino acid sequence of SEQ ID NO : 1 and enzymatically active fragments thereof.

In a second aspect, the present invention provides a nucleic acid sequence encoding a Lysophosphatidic Acid Acyltransferase (LPAAT) having a nucleotide sequence set forth in SEQ ID NO : 2, or a fragment thereof; a nucleotide sequence that encodes a polypeptide of SEQ ID NO : 1, or enzymatically active fragments of

the polypeptide; or a nucleotide sequence that hybridizes under high stringency conditions to the nucleotide sequence of SEQ ID NO: 1 or NO: 2.

In other aspects, an expression vector and host cell containing the vector is provided with a nucleic acid sequence that encodes for the polypeptide of SEQ ID NO: 1, or an enzymatically active fragment thereof.

In one of its methods aspects, the invention provides a method for producing recombinant hLPAAT s, which comprises culturing a host cell containing an expression vector carrying a nucleic acid sequence that encodes for the polypeptide of SEQ ID NO : 1, or an enzymatically active fragment thereof, under conditions for the expression of hLPAATs in detectable or recoverable amounts. Various types of cells may be used for expression of the hLPAATE polypeptide, including for example, mammalian cells, yeast cells, insect cells and bacterial cells.

In another methods aspect, nucleic acids encoding hLPAATE are detected in a biological sample using nucleic acid hybridization. In this method, a nucleic acid sequence that is complementary to a nucleotide sequence encoding a polypeptide of SEQ ID NO : 1, or enzymatically active fragments thereof, is hybridized to nucleic acids contained in the biological sample and the resulting nucleic acid hybrids are detected.

In yet another methods aspect, a screening assay is provided to detect agents that are capable of inhibiting or stimulating the activity of hLPAAT-s in a cell-free or cell-based assay. The method comprises contacting an isolated hLPAAT-s polypeptide or a cell that recombinantly expresses this polypeptide with one or more agents under assay conditions that are suitable for detecting the enzymatic activity of the polypeptide, and measuring whether the activity is inhibited or stimulated by one or more of the agents. These agents are selected from compounds and compositions, antibodies or antibody fragments, antisense sequences and ribozyme sequences. In one embodiment, the agents are selected from a combinatorial chemical library.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the DNA sequence (SEQ ID NO : 2) and the translated sequence (hLPAATs) (SEQ ID NO : 1) of the cDNA insert of pLpts 2P.

Figure 2 shows the amino acid sequence alignments between the full-length hLPAATs cDNA clone (SEQ ID NO : 1), human lysophosphatidic acid acyltransferase-8 (hLPAAT6 ; SEQ ID NO : 3) and maize LPAAT (SEQ ID NO : 4) produced using the multisequence alignment program of DNASIS v2.5software (Hitachi Software Engineering Co., Ltd., South San Francisco, California).

Figure 3 shows the relative LPAAT activity, assessed by the conversion of NBD-LPA to NBD-PA, in ECV304 cell extracts from, respectively, cells overexpressing LPAAT-a or LPAAT-, five putative LPAAT-e expressing clones, and cells containing the control vector, pCivIrPu.

DETAILED DESCRIPTION OF THE INVENTION This invention provides a novel human isoform of lysophosphatidic acid acyltransferase, hLPAAT s, and polynucleotides encoding this activity. The invention further provides expression vectors, host cells containing these vectors, antisense oligonucleocides and ribozymes, methods for producing purified polypeptides having hLPAATs activity and methods for detecting polynucleotides encoding these polypeptides. hLPAATs is useful for diagnostic, therapeutic and screening applications.

For example, hLPAATs can be included in a pharmaceutical composition as a therapeutic agent, and can be used for the preparation of immunodiagnostic and immunotherapeutic agents. hLPAAT E can also be used in pharmaceutical screening assays to detect agents that modulate LPAAT activity. Nucleic acid sequences encoding hLPAATE have utility in diagnostic tests (e. g., tests to detect the presence or expression of this particular LPAAT isoform in relation to certain diseases and conditions). These sequences can be used in disease prevention and

treatment (e. g., vaccines and gene therapy). Antisense oligonucleotides and ribozymes are useful for altering the expression of hLPAAT-s in vivo.

The term"hLPAAT s"is used herein to mean isolated, biologically active LPAAT-E polypeptides from any species of organism, preferably mammalian, most preferably human. It should be understood that the invention is not limited to hLPAATs derived from natural sources, but encompasses as well hLPAATs that is prepared by chemical synthesis or by recombinant DNA technology. The term"biologically active"refers to an activity of the protein in a biological system. This activity may be regulatory, structural, immunologic, or catalytic (i. e., enzymatic).

Preferably, biologically active hLPAAT E (and biologically active fragments thereof) will be enzymatically active. As used herein, the term "enzymatically active"in the context of LPAAT activity refers to the ability to catalyze the acylation of lysophosphatidic acid (LPA) to phosphatidic acid (PA) by acylating the sn-2 position LPA with a fatty acid acyl-chain moiety. In this context, the term"polypeptide"or"polypeptide sequence"or"amino acid sequence"is used herein to denote a peptide, oligopeptide, polypeptide or protein sequence having LPAAT activity. The term"isolated", in this context, denotes a polypeptide or polynucleotide essentially free of other polypeptides or nucleic acid sequences, respectively, or of other contaminants normally found in nature.

In one embodiment of the invention, the polypeptide comprises the amino acid sequence of SEQ ID NO : 1, as shown in Figure 1. The hLPAAT is 364 amino acids in length. As shown in Figure 2, hLPAATE has chemical and structural homology with human hLPAATB. In particular, hLPAATs and hLPAATB share 6.3% overall amino acid match.

The present invention contemplates modifications of the hLPAATE polypeptide sequence. 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, provided that the modified polypeptide is biologically active, preferably enzymatically active.

For example, the present invention contemplates the deletion of one or more amino acids from the polypeptide sequence of the hLPAATs to create deletion variants. This deletion can be of one or more amino or carboxy terminal amino acids or one or more internal amino acids.

The present invention further contemplates one or more amino acid substitutions to the polypeptide sequence of hLPAATE to create substitutional variants. The present invention contemplates that such substitutional variants may show altered properties e. g., changes in stability to proteolysis or temperature, without loss of their biological activity, preferably enzymatic activity.

Substitutions are preferably 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; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine 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.

The present invention further contemplates the insertion of one or more amino acids into the polypeptide sequences of hLPAATE to create an insertional variant. Examples of such insertional variants include fusion proteins such as those used to facilitate rapid purification of the polypeptide and also can include hybrid polypeptides containing sequences from other proteins and polypeptides that 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, for example, to disrupt a protease cleavage site.

Polypeptides of the present invention can be synthesized by chemical methods using, for example, step-wise addition of suitably protected amino acids starting from the carboxy terminus of the peptide (Coligan et al., Current Protocols in Immunology, Wiley Interscience, Unit 9,1991). Solid phase synthesis methods are well-known in the art (e. g., Merrifield, J. Am. Chem. Soc.

85: 2149,1962; and Steward and Young, Solid Phase Peptide Synthesis, Freeman, San Francisco pp. 27-62,1969). Polypeptide modifications can be introduced during synthesis.

The polypeptides thus obtained can be purified by standard chromatographic techniques such as preparative HPLC (see, e. g., Chiez and Regnier, Meth. Enzymol. 182 : 392 (1990). Sequencing, amino acid analysis and spectroscopic techniques can be used to confirm the primary structures of the synthetic polypeptides. See, e. g., Stolowitz, Curr. Opin. Biotechnol. 4: 9-13 (1993); Jensen, Methods Mol. Biol. 112: 571-88 (1999).

The invention also provides nucleotide sequences that encode the hLPAATE polypeptides of the invention. These nucleotide sequences were first identified in an EST cDNA clone (GenBank# ai248500). A consensus sequence, SEQ ID NO : 2, was derived from the following overlapping and/or extended nucleic acid sequences: IMAGE EST clones (GenBank# aa056538, and GenBank# ai248500)).

The term"consensus sequence"is used herein to mean a nucleic acid sequence that has been assembled from the overlapping sequences of more than one cDNA clone using a computer program for fragment assembly, such as DNASIS (Hitachi Genetic System, Alameda, CA), or a nucleic acid sequence that has been resequenced to resolve uncalled bases, extended in the 5'and/or 3'

direction using, for example, XL-PCR (Perkin Elmer, Norwalk, Conn.) or BIO- X-ACT (Bioline, Reno, NV), and resequenced, or a combination of these methods.

As used herein, the term"nucleic acid sequence"or"nucleotide sequence" or"polynucleotide"or"polynucleotide sequence"refers to a single stranded or double stranded genomic or synthetic DNA, cDNA, RNA, or DNA-like or RNA- like molecules, such as deoxyribonucleotide or ribonucleotide polymers having a backbone comprising non-phosphodiester linkages.

Polynucleotide sequences are"complementary"to one another if they are capable of base-pairing with one another to form a hydrogen-bonded complex.

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 or 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).

As used herein, the term"hybridization"refers to the formation of double- stranded nucleic acid molecules by sequence-specific base pairing of complementary single strands. Wahl et al. Meth. Enzymol. 152: 399 (1987).

Hybridization procedures are useful for screening of recombinant clones by using labeled mixed synthetic oligonucleotide probes, wherein each probe is potentially the complete complement of a specific DNA sequence in a hybridization sample which includes 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 mRNA sequences relating to the polypeptide of interest are present in extremely low amounts. Using stringent hybridization conditions, it is possible to detect 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).

As used herein, the term"stringency"refers to hybridization or posthybridization wash conditions that can be adjusted to discriminate hybrids formed between related but nonidentical sequences from hybrids formed between identical polynucleotide sequences. The term"stringent hybridization conditions" as used herein refers to conditions that permit hybridization to occur between the claimed polynucleotide sequences and polynucleotide sequences that preferably show at least about 80%, more preferably at least about 90%, and most preferably at least about 95% sequence identity to the claimed polynucleotide sequence of SEQ ID NO: 2. Stringent conditions preferably include high stringency conditions. Stringency can be increased by reducing the salt concentration, increasing the concentration of formamide (or other helix destabilizing agent) and/or raising the temperature. See, for example, Maniatis et al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, pages 387-389, 1982; Wahl et al., Meth. Enzymol. 152: pp. 399-407 (1987). Exemplary high stringency conditions include 4x-6x SSC at a temperature in the range of 65° to 70°C in the absence of formamide and in the range of 40°C to 45°C in the presence of 50% formamide. Variations on these conditions are well known in the art.

Specific DNA sequences encoding hLPAAT can also be obtained by isolation of double-stranded DNA sequences from the genomic DNA, by chemical synthesis and by in vitro synthesis of a double-stranded cDNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. These methods are well known in the art. Mutations or codon variations may be introduced into a DNA sequence during chemical synthesis to optimize expression of the polypeptide of interest by a desired host cell, for example, without altering the amino acid sequence of the polypeptide. 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 desired to obtain the microbial expression of mammalian polypeptides due to the presence of introns in the genomic DNA.

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 gtll, can be screened for hLPAAT£ polypeptide using antibodies specific for the protein. Such antibodies can be either polyclonally or monoclonally derived.

The polynucleotides of this invention include sequences that are degenerate as a result of the genetic code. The genetic code is described as degenerate because more than one nucleotide triplet, called a codon, can code for a single amino acid. The present invention contemplates the degeneracy of the genetic code and includes all degenerate nucleotide sequences which encode hLPAATs.

The present invention also includes polynucleotide sequences complementary to the polynucleotides encoding hLPAAT. Specifically, the present invention includes antisense polynucleotides. An antisense polynucleotide is a DNA or RNA molecule 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 expression of hLPAATE.

In a cell, the antisense polynuycleotides are thought to inhibit transcription or translation by binding to complementary cellular nucleic acids. For example, the hybridization of an antisense polynucleotide to a corresponding mRNA would form a double-stranded molecule that cannot be translated by the cell. 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 hLPAAT s-producing cell. The use of antisense methods to inhibit translation of genes is known (e. g., Marcus-Sakura, Anal. Biochem. 172: 289,1988).

The present invention further includes allelic variations in the polynucleotide sequences encoding hLPAATs, i. e., naturally-occurring base changes in a species population which may or may not result in an amino acid change in the polypeptide. The inventive polynucleotide sequences further comprise those sequences which hybridize under high stringency conditions to the coding regions or to the complement of the coding regions of hLPAATs. One such high stringency 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 high stringency hybridization condition is in 50% formamide, 4 X SSC at 42°C.

In addition, ribozyme nucleotide sequences that cleave hLPAATs 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 transcribe such RNAs, it is possible to engineer ribozymes that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, J. Amer.

Med. Assn. 260: 3030,1988).

There are two basic types of ribozymes, tetrahymena-type (Hasselhoff, Nature 334: 585,1988) and"hammerhead-type". Tetrahymena-type ribozymes

recognize sequences that 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.

In the practice of this invention, sequence information for any of the embodiments may be obtained with the use of generally available art-recognized DNA sequencing methods.

Production of Polypeptides Polynucleotide sequences encoding hLPAAT £ polypeptides of the invention can be expressed in either prokaryotes or eukaryotes. Hosts can include, but are not limited to, microbial (bacterial), yeast, insect cells, animal cells and plant cells. Methods of expressing DNA sequences inserted downstream of prokaryotic or viral regulatory sequences in prokaryotes are known in the art (Makrides, Microbio. Rev. 60: 512,1996). Biologically functional viral and plasmid DNA vectors capable of expression and replication in a eukaryotic host are known in the art (Cachianes, Biotechniques 15: 255,1993). 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. hLPAAT s 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 inserting or incorporating 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 and a promoter, as well as specific genes which allow phenotypic selection of the transformed cells. The DNA segment can be present in the vector, operably linked to regulatory

elements, for example, a promoter (e. g., T7, metallothionein I, or polyhedrin promoters). Vectors suitable for use in the present invention, include, for example, bacterial expression vectors, with bacterial promoter and ribosome binding sites for expression in bacteria (Gold, Meth. Enzymol. 185: 11,1990), expression vectors with animal promoter and enhancer for expression in mammalian cells (Kaufman, Meth. Enzymol. 185: 487,1990) and baculovirus- derived vectors for expression in insect cells (Luckow et al., J. Virol. 67: 4566, 1993).

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 (P-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 xanthine guanine phosphoribosyltransferase (XGPRT, gpt).

In another preferred embodiment, the expression system used is one driven by the baculovirus polyhedrin promoter. The polynucleotide encoding LPAAT 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 a polynucleotide encoding LPAAT is transfected into Spodoptera frugiperda (Sf9) 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.

The polynucleotides of the present invention can be expressed in any number of different recombinant DNA expression systems to generate large

amounts of polypeptide. Included within the present invention are LPAAT 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. coli, yeast such as Pichia pastoris, baculovirus, and mammalian expression systems such as in Cos or CHO cells.

The polynucleotides of the present invention can be inserted into an expression vector by standard subcloning techniques. In a preferred embodiment, an E. coli 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, NJ), the maltose binding protein system (NEB, Beverley, MA), the thiofusion system (Invitrogen, San Diego, CA), the Strep-tag II system (Genosys, Woodlands, TX), 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 activity of the recombinant polypeptide.

For example, both the FLAG system and the 6xHis system 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 (Invitrogen, San Diego, CA).

The invention encompasses hLPAATs polynucleotides from which one or more transmembrane sequences are deleted. In certain expression systems, e. g.,

E. coli, the presence of transmembrane sequences may result in the production of insoluble aggregates that are difficult to renature into the native conformation of the polypeptide. Such sequences are typically very hydrophobic and are readily detected by the use of standard sequence analysis software, such as MacDNASIS (Hitachi, San Bruno, CA).

Deletion of transmembrane sequences typically does not significantly alter the conformation or activity of the remaining polypeptide structure. However, one can determine whether the deletion of one or more of the transmembrane sequences has affected the enzymatic activity of the hLPAATs protein so modified, for example, by assaying its activity and comparing this activity to that of unmodified hLPAAT s protein. Assaying hLPAAT s activity can be accomplished, for example, by contacting the hLPAATs protein of interest with the substrates LPA and fatty acyl-CoA and measuring the generation of PA or free CoA. Such assays for determining LPAAT activity are described in more detail below in the section describing screening assays.

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 generating monoclonal or polyclonal antibodies by omitting such sequences from the recombinant polypeptides of the invention.

Deletion of transmembrane-encoding sequences from the polynucleotide used for expression can be achieved by standard techniques. See Ausubel et al., supra, Chapter 8. For example, appropriately-placed restriction enzyme sites can be used to excise the desired gene fragment, or 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 that are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by a CaCk

method using standard procedures. Alternatively, MgCk or RbCl 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, methods of transfection of DNA, such as calcium phosphate co-precipitates, conventional mechanical procedures, (e. g., microinjection), electroporation, liposome-encased plasmids, or virus vectors may be used. Eukaryotic cells can also be cotransformed with DNA sequences encoding hLPAAT polypeptides 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 hLPAAT E polypeptides.

Expression vectors that are suitable for production of LPAAT polypeptides preferably 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 are 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-K1 ; ATCC CCL61), rat pituitary cells (GHl ; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548) SV40- transformed monkey kidney cells (COS-1 ; ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658). For a mammalian host, the transcriptional and translational 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 (Gorman et al., Proc. Nat'l. Acad. Sci. USA 79: 6777, 1982); and the cytomegalovirus promoter (Foecking et al., Gene 45 : 101,1980).

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNA polymerase 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; Kaufman 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, 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).

Examples of mammalian host cells include COS, BHK, 293 and CHO cells.

Purification of Recombinant Polypeptides.

The LPAAT polypeptide expressed in any of a number of different recombinant DNA expression systems can be obtained in large amounts and tested for biological activity.

Recombinant bacterial cells, for example E. coli, are grown in any of a number of suitable 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 bodies and cell membranes from the soluble cell components. The content of dense inclusion bodies can be selectively enriched by incorporating sugars such as sucrose into the buffer and centrifuging at a selective speed. If the recombinant polypeptide is expressed in the inclusion bodies, contaminating host proteins can be removed by washing the bodies in any of several solutions prior to solubilization of the bodies 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 p-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 a low polypeptide concentration (i. e., 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 that facilitate the interchange of disulphide bonds within the protein molecule. The refolding process can be monitored, for example, by polyacrylamide gel electrophoresis or with antibodies that 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.

Alternatively, proteins may be produced in a secretion system. Expression vectors containing DNA encoding hLPAATs can be engineered to contain signal sequences for directing the secretion of the polypeptide from the cell.

Isolation and purification 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 using monoclonal or polyclonal antibodies.

A variety of techniques including recombinant DNA technology can be used to enable the large scale production of pure, biologically active hLPAATs for use in drug discovery screening assays and for developing antibodies for therapeutic, diagnostic and research use. See, e. g., Yarranton, Curr. Opin.

Biotechnol. 2: 133-40 (1990); Sudbery, Curr. Opin. Biotechnol, 7: 517-24 (1996); Hannig & Makrides, Trends Biotechnol. 16: 54-60 (1998).

ScreeningAssays The hLPAAT£ polypeptides of the present invention can be used in a screening methodology for identifying putative pharmaceutical agents that affect LPAAT activity. Such compounds or compositions to be tested can be selected from a combinatorial chemical library or any other suitable source (Hogan, Jr., Nat. Biotechnology 15: 328,1997).

This method comprises, for example, incubating an hLPAATs polypeptide, or a cell transfected with a nucleotide sequence encoding hLPAATs, with one or more of the agents to be tested under conditions sufficient to allow the polypeptide to interact with the test agent or agents, and then measuring the effect on hLPAATs activity. The hLPAATs proteins used in the assay can either be purified prior to incubation or can be contained in extracts from a cell line or cell lines (for example, Sf9, ECV304, A549) transfected with cDNA encoding these polypeptides (West et al., DNA Cell Biol. 16: 691,1997).

Alternatively, hLPAAT protein can be purified from transfected cells, and the protein, being a transmembrane protein, can then be reconstituted in a lipid

bilayer to form liposomes for delivery into cells (Weiner, Immunomethods 4: 201, 1994). Other methods are known to be useful for introducing biologically active proteins into cells. For example, the protein can be expressed either as a fusion protein with the sequence at the N-terminal end derived from the 38kD Herpes- Simplex 1 structural protein VP22 (Invitrogen, Carlsbad, CA), which has the unique ability to translocate from the cytoplasm of the producing cells to the nuclei of the surrounding cells. Alternatively, the protein can be expressed as a fusion protein with the sequence at the N-terminal end derived from the protein transduction domain from the human immunodeficiency virus TAT protein (Schwarze et al., Science 285: 1569-1572 (1999)).

The effect of a compound or composition on hLPAATs activity can be determined, for example, by TLC methods described below in Examples 2 and 3.

Alternatively, LPAAT activity can be assayed by detecting the formation of free CoA in reaction. CoA, which contains a free sulfhydryl-group, can be measured either by, for example, colorimetric or fluorescence methods with sulfhydryl- specific reagents, such as, 5,5'-dithiobis- (2-nitrobenzoic acid) (DTNB) or ThioGlo (Covalent Associates, Woburn, MA). The observed effect on hLPAATs may be either inhibitory or stimulatory.

Peptide Sequencing 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 protein prior to sequencing. This can be achieved by using glycosidase enzymes. Preferably, glycosidase F (Boehringer-Mannheim, Indianapolis, IN) 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.

Antibodies Directed to LPAAT Antibodies to human LPAAT 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 Polyclonal 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 chromatograph. 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-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 al., 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 murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine 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. Nat'l 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.

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.

Use of hLPAAT e polvnucleotides for diagnostic purposes Altered expression of hLPAAT may be associated with inflammation, autoimmune disorders, cancer and obesity. The availability of purified hLPAATg and polynucleotides encoding this polypeptide can be applied to the diagnosis of diseases and conditions in which the expression of the enzyme is abnormal.

Probes for the detection of the polynucleotide sequences encoding the polypeptide sequence of SEQ ID NO: 1 can be made, for example, to a specific region of the coding sequence of SEQ ID NO: 2. The probes may be RNA or DNA and may be chemically or enzymatically synthesized in vitro, or produced by recombinant techniques using commercially available vectors. Reporter groups (e. g., enzymes, radioisotopes, fluorophores or chemiluminescent moieties) can be introduced into the probes, if desired, by conventional methods. Alternatively, the assays may be carried out with the use of unlabeled probes and labeled secondary detection molecules.

Diagnosis can be carried out by hybridization in various formats using body fluids, cells and tissues, cell extracts and the like as the source of nucleic acids for probing. The hybridization reaction can be performed, for example, with test nucleic acids and probes both in solution, or with immobilized nucleic acids or probes. For immobilization, nucleic acids can be attached to beads or other solid substrate with or without prior separation (e. g., Northern blots,

Southern blots, dot blots, slot blots and the like) prior to exposure to the probe.

Alternatively, the probes may be immobilized (e. g., in microarrays for high throughput screening). The nucleic acids may be probed in biological tissues by fluorescence in situ hybridization (FISH). Diagnosis can also be carried out by PCR-based tests, using oligonucleotide primers based on the hLPAATs polynucleotide sequence.

In order to further illustrate the present invention and advantages thereof, the following specific examples are given but are not meant to limit the scope of the claim in any way.

EXAMPLES Example 1. Isolation of human LPAATs A. Identification of hLPAAT s coding sequences The human LPAAT6 protein sequence, which was previously found by the inventors to have LPAAT activity (see Example 2 below), was used as a probe to search the Genbank database (Boguski, et al., Science 265: 1993-1994,1994; Altschul, et al., Nucleic Acids Res. 25: 3389-3402,1997) of expressed sequence tag (dbEST). Several short stretches of human cDNA sequences were identified that showed homology to the hLPAATB protein sequence, but were distinct from it as well as from other known LPAAT sequences. These cDNA sequences of interest were derived from single-run partial sequencing of random human cDNA clones projects carried out by the I. M. A. G. E. Consortium [LLNL] cDNA clones program. An example of the amino acids sequence homology between the human LPAAT s and a human cDNA clone (GenBank#ai248500) is shown below:

140 150 160 170 180 LPAAT-8 IIGWMWYFTEM--VFCSRKWEQDRKTVATSLQHLRDYPEKYFFLIHCEGTRF---TEKKH ai248500 LPLYGCYFAQHGGIYVKRSAKFXEKCkNKI. QSYVDAGTPMYLVIFPEGTRYNPEQTXVL 10 20 30 40 50 60 190 200 210 Z20 230 <BR> <BR> LPAAT-õEiSMQVARAKGLPRLKHHLLPRTKGFAITVRSLRNWSAVYDCTLNF------ -RNNENP<BR> ai249500SASQAFAAQRGLAVLKHVLTPRIRATHVAPDCIIYLDAIYDVTVVYEGKDDG GQRRESP 70 80 90 100 110 120 240 250 260 270 280 290 LPAAT-6 TLLGVLNGKKYHADLYVRRIPLEDIPEDDDECSAMLHKLTfQEKDAFQEEYYRTGTF ai248500TMTEFLCKECPKIHIHIDRIDKKBVPEEQEHHRRMLHERFEIKOKMLIEFYE SPDP 130 140 150 160 170 The top lines refer to the human LPAAT-8 sequence from amino acids 131 to 294 (SEQ ID: NO 3) and the bottom lines refer to the homologous region from the dbEST clone with GenBank# ai248500 (SEQ ID: No 5). Identical and conserved amino acids between these two sequences are shown as double dots and single dots, respectively, in the rows in between.

The DNA sequence from dbEST was used as the query sequence to search for overlapping sequences in the dbEST. A human EST, GenBank# aa056538, was identified that extended the cDNA sequence by approximately 300 bp further into the 3'end, when compared to the sequence from GenBank# ai248500.

B. Generation of expression plasmids To assemble the full-length human LPAAT-s cDNA clone for expression, a 1130 Xho I-Sfu I fragment from the dbEST clone with GenBank# ai248500 and a 400 bp Sfu I-Not I fragment from the dbEST clone with GenBank# aa056538 were inserted into, an expression vector, pCivIrPu, between the Xho I and Not I sites to generate the plasmid pLpts 2P. Such expression plasmids can be used to test if cells transformed or transfected with these cDNA expression vectors produced more LPAAT activity. pCivIrPu is derived from the plasmids pIRESpuro (Clonetech, Palo Alto, CA) and pCIneo (Promega, Madison, WI).

Specifically, the 980 bp Spe I-Not I fragment from pCIneo was inserted into the cloning vector pSL1180 (Amersham Pharmacia, Piscataway, NJ) between the

restriction sites Spe I and Not I to generate pSLCI. The 1000 bp Spe I-Nsi I fragment from pSLCI was then isolated for insertion into the vector pIRESpuro between the restriction sites Spe I and Nsi I to generate pCivIrPu.

C. The DNA and translated sequence of the cDNA insert of pLpts 2P Figure 1 shows the DNA and the translated sequence (LPAAT-s) of the cDNA insert of pLptg_2P. Nucleotide sequence analysis and restriction mapping of the cDNA clone revealed a 5'-untranslated region of 274 bp containing one upstream ATG and with stop codons in all three reading frames, an open reading frame capable of encoding a 364 amino acids polypeptide that spans nucleotide positions 275 to 1369 and a 3'-untranslated region of 139 bp. The initiation site for translation was localized at nucleotide positions 275-277 and fulfilled the requirement for an adequate initiation site (Kozak, Critical Rev. BioChem. Mol.

Biol. 27: 385-402,1992). The 5'-untranslated region of LPAAT-E was found to be highly GC-rich with a base composition of 80% GC. Since GC-rich 5'- untranslated region has been found to have an inhibitory effect on the translation of certain mRNAs (Kauppinen, FEBS Lett 365: 61-5,1995), an additional expression plasmid for LPAAT-s was constructed by removing most the 5'- untranslated region of LPAAT-g. Specifically, the 1270 bp Sst I-Not I fragment from pLpts_2P was inserted between the Sst I and Not I sites of the vector pSL1180 (Amersham Pharmacia, Piscataway, NJ). The 1280 bp Xho I- Not I fragment derived from the resultant plasmid was then inserted into the expression vector, pCivIrPu, between the Xho I and Not I sites to generate the plasmid pLpte IP.

D. Amino acid sequence comparisons of hLPAAT g with several known homologs Figure 2 shows the amino acid sequence alignments for the full-length hLPAATs cDNA clone, hLPAATB and a homologous LPAAT sequence from maize produced using the multisequence alignment program of DNASIS

v2.5software (Hitachi Software Engineering Co., Ltd., South San Francisco, California). Amino acids identical in at least two sequences are highlighted.

LPAAT-s and LPAAT-8 have an overall amino acid match of 6.3% with respect to each other and an overall amino acid match of 9.8% with respect to the maize LPAAT sequence.

Example 2. Expression of hLPAATS in mammalian cells.

A. Preparation of plasmid pCE2 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-la (EF-la) promoter and intron. The CMV enhancer came from a 380 bp Xba I-Sph I fragment produced by PCR from pCEP4 (Invitrogen, San Diego, CA) using the primers 5'-GGCTCTAGAT ATTAATAGTA ATCAATTA-3'and 5'-CCTCACGCAT GCACCATGGT AATAGC-3'. The EF-la promoter and intron (Uetsuki, et al., J. Biol. Chem., 264: 579 1-5798,1989) came from a 1200 bp 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.

B. Construction of pC2LPT8 Genbank # H18562 was identified by the inventors of the present application as an hLPAATB clone. For construction of the expression vector pC2LPT8, PCR primers were designed and used to amplify an 1100 bp Acc65 I- Xba I fragment from the template #H18562. The fragment generated was then inserted into a Acc65 I/Nhe I digested pCE2 (West, et al., DNA Cell Biol. 6: 691-701,1997) expression vector to make pC2LPT6.

C. Expression of hLPAAT8 in mammalian cells Plasmid pC2LPT8 was stably transfected into endothelial ECV304 cells (American Type Culture Collection, Rockville, MD). Specifically, pC2LPT8 was digested with BspH I before electroporating into these cell lines with a Cell- PoratorTM (Life Technologies, Gaithersburg, MD). After adherence of the transfected cells 24 hours later, the cells were grown in the presence of 500 pg/ml Hygromycin B (Hyg) (Calbiochem, La Jolla, CA) to select for cells that had incorporated plasmids. Hyg-resistant clones that expressed LPAAT-8 mRNA at a level more than 10 fold higher than that of cells transfected with pCE2 vector, based on Northern Blot analysis (Kroczek, et al., Anal. Biochem. 184: 90-95, 1990), were assayed for LPAAT activity and compared with cells transfected with the control vector alone, as follows.

The screening assay for LPAAT activity was based on the conversion of ['4C] oleoyl-CoA to ['4C] PA by total cell extracts as determined by TLC. The assay was carried out with total cell extracts resuspended in lysis buffer (Ella, et al., Anal. Biochem. 218: 136-142,1994) supplemented with 50 uM [l4C] oleoyl-CoA and 200 uM LPA. The samples were incubated for 10 min, extracted from chloroform, before loading onto TLC plates.

The results showed that ECV304 cells transfected with LPAAT-8 cDNA contain about 2.5 times more LPAAT activity than those of control cells (i. e., transfected with vector alone), as evidenced by the increased conversion of [l4C] oleoyl-CoA to ['4C] PA.

Example 3. Expression of hLPAATs in mammalian cells To determine whether the hLPAAT-s cDNA sequence encodes a protein with LPAAT activity, the plasmid pLptE-1P was stably transfected into EVC304 cells (American Type Culture Collection, Richmond, VA) to generate puromycin- resistant clones. Specifically, pLpt s IP was digested with BspH I before electroporating into these cell lines 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 0.5 pg/ml puromycin to select for cells that had incorporated the plasmid. Five puromycin-resistant clones were randomly selected to check for LPAAT activity using a TLC assay that measures the conversion of NBD-LPA to NBD-PA (West, et al., DNA Cell Biol. 6: 691- 701,1997). The assay was carried out in total ECV304 cell extracts resuspended in lysis buffer (Ella, et al., Anal. Biochem,. 218: 136-142,1994) supplemented with 100 M oleoyl-CoA and 10 uM NBD-LPA. The samples were incubated for 30 min before loading onto TLC plates. Figure 3 shows that two of the five clones screened for LPAAT activity (i. e., lanes 3 and 5) exhibit more LPAAT activity (i. e., generate more NBD-PA) than that of the control cells containing pCivIrPu vector alone (control lane). The substrate lane (shown on the right side of the figure) refers to NBD-LPA incubated with buffer only without any cell extract added. The apparently weaker activity of hLPAATs towards the substrate NBD-LPA by comparison with ECV304 cell extracts overexpressing hLPAATa or hLPAATß (shown in left two lanes of Figure 3) suggests that LPAATE may acylate other substrates that are yet to be determined more effectively.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

All of the publications, patent applications and patents cited in this application are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.