HK1068636B - Polypeptides encoded by a human lipase-like gene, compositions and methods - Google Patents

Description

polypeptides encoded by human lipase-like gene and compositions and methods utilizing same

The present application is a divisional application of Chinese patent application No. 97180348, applied on 5/12/1997, and X "polypeptide encoded by human lipase-like gene and compositions and methods using the same".

The present application claims the rights of two co-pending provisional applications 60/032,254 and 60/032,783 filed on 6.12.1996.

Technical Field

The present invention relates to polypeptides of the triacylglycerol lipase family, nucleic acids encoding the polypeptides, antisense sequences derived from the nucleic acids and antibodies directed against the polypeptides. The invention also relates to the recombinant production of said polypeptide and to the use of said polypeptide for screening for agonists and/or antagonists thereof. The invention also relates to methods of using these polypeptides and nucleic acid sequences encoding these polypeptides in pharmaceutical compositions for the treatment of disorders of lipid and lipoprotein metabolism, including gene therapy.

Background

A) Lipids

Lipids are water-insoluble organic biomolecules that are essential components of a variety of biological functions including energy storage, transport and metabolism, and membrane structure and mobility. In humans and animals, lipids come from two sources: some lipids are ingested in the form of dietary fats and oils, and others are biosynthesized by humans and animals. At least 10% of the body weight of mammals is lipids, the majority of which are present in the form of triacylglycerols.

Triacylglycerols, also known as triglycerides and triacylglycerides, consist of three fatty acids esterified to glycerol. Dietary triacylglycerides are stored in adipose tissue as an energy source or are hydrolyzed in the digestive tract by triacylglycerol lipases, the most important of which is pancreatic lipase. Triacylglycerols are transported between tissues in the form of lipoproteins.

Lipoproteins found in plasma are micellar-like clusters, containing various proportions of different types of lipids and proteins (called apoproteins). There are five major plasma lipoprotein classes, the primary function of which is lipid transport. In order of increasing density, these species are chylomicrons, Very Low Density Lipoproteins (VLDL), Intermediate Density Lipoproteins (IDL), Low Density Lipoproteins (LDL), and High Density Lipoproteins (HDL). Although each lipoprotein was found to be associated with multiple types of lipids, each transports predominantly one type of lipid: the triacylglycerols mentioned above are transported in chylomicrons, VLDL and IDL; while phospholipids and cholesteryl esters are transported in HDL and LDL, respectively.

Phospholipids are di-fatty acid esters of phosphoglycerol and also contain a polar group coupled to the phosphate. Phospholipids are important structural components of cell membranes. Phospholipids are hydrolyzed by phospholipases. Phosphatidylcholine is a typical phospholipid which is a major component of most eukaryotic cell membranes.

Cholesterol is a metabolic precursor of steroid hormones and bile acids, and is also an essential constituent of cell membranes. In humans and other animals, cholesterol can be ingested via the diet, and can also be synthesized by the liver and other tissues. Cholesterol can be transported between tissues in the form of cholesteryl esters in LDL and other lipoproteins.

Membranes surround each living cell and act as barriers between intracellular and extracellular compartments. The membrane also surrounds the eukaryotic nucleus, forms the endoplasmic reticulum, and serves a specific function in, for example, the myelin sheath surrounding axons. Typical membranes contain about 40% lipid and 60% protein, but there are also large differences. The main lipid components are phospholipids, in particular phosphatidylcholine, phosphatidylethanolamine and cholesterol, the physiochemical characteristics of the membrane, such as fluidity, can be altered by adjusting the fatty acid composition or the cholesterol content of the phospholipids. Modulating the composition and composition of membrane lipids also modulates membrane-dependent cellular functions such as receptor activity, endocytosis and cholesterol efflux.

B) Enzyme

Triacylglycerol lipases are a family of enzymes that play several key roles in lipid metabolism in the body. Three members of the human triacylglycerol lipase family have been described: pancreatic lipase, lipoprotein lipase and hepatic lipase (Goldberg, I.J., Le, N., Ginsberg, H.N., Krauss, R.M. and Lindgren, F.T. (1988) J.Clin.81, 561-. Pancreatic lipase is primarily responsible for the hydrolysis of dietary lipids. Various pancreatic lipases have been described, but their physiological role has not been established (Giller, T., Buchwald, P., Blum-Kaelin, D., and Hunziker, W. (1992) J. Biochem., 267, 16509-16516). Lipoprotein lipase is the major enzyme responsible for triacylglycerol distribution and utilization in vivo. Lipoprotein lipase hydrolyses triacylglycerides in chylomicrons and VLDL. Hepatic lipase hydrolyses triacylglycerides in IDL and HDL and is responsible for lipoprotein reconstitution. Hepatic lipase also functions as a phospholipase and hydrolyzes phospholipids in HDL.

Phospholipases play an important role in the catabolism and remodeling of the phospholipid component of lipoproteins and membrane phospholipids. Phospholipases play a role in the release of arachidonic acid and the subsequent formation of prostaglandins, leukotrienes and other lipids involved in various inflammatory processes.

The lipase polypeptides encoded by these lipase genes are about 450 amino acids long and have a leader signal peptide that promotes secretion. Lipases consist of two major domains (Winkler, k., D' Arcy, a. and huntziker, W. (1990) nature, 343, 771-. The amino-terminal domain contains a catalytic site and the carboxyl domain is thought to be responsible for substrate binding, cofactor binding and interaction with cellular receptors (Wong, H., Davis, R.C., Nikazy, J., Seebart, K.E., and Schott, M.C. (1991) Proc. Natl. Acad. Sci. USA 88, 11290-11294; van Tilbeurgh, H., Roussel, A., Lalouel, J., M. and Camblilau, C. (1994) J. Biochem 269, 4626-4633; Wong, H., Davis, R.C., Thuren, T., Goers, J.W., Nikazy, J., Waite, M. and schott, M.C. (1994) J. Biochem. 269, 1039-10323; Chappell, D.A., Ine, I.j., G.L., L. D.D.D.D.D.201., Stroal. D.D.D.D.D.D.S.D.S.D.S.D.S.S.D.S.S.S.D.S.S.H.S.S.S.S.D. 27, S.S.S.J.S.H.H.H.S.S.A. 27. H.A. acide.A. A. acide. acide, H. A. acide.A. No. 27, and Strand Stroal.S. 27. acide.S. acide.H. acide.A..

Naturally occurring lipoprotein lipase proteins are glycosylated and glycosylation is essential for LPL enzymatic activity (Semenkovich, C.F., Luo, C. -C., Nakanishi, M.K., Chen, S. -H., Smith, L.C., and Chan L. (1990) J. Biochem. chem., 265, 5429-. In addition, four pairs of cysteine-forming disulfide bonds are necessary to maintain structural integrity of the enzyme activity (Lo, J. -Y., Smith, L.C., and Chan, L. (1995) biophysical and biochemical communications 206, 266-.

Members of the triacylglycerol esterase family share many common conserved structural features. One such feature is the "GXSXG" motif, in which the central serine residue is one of the three residues that make up the "catalytic triad" (Winkler, K., D' Arcy, A. and Hunziker, W. (1990) Nature 343, 771-72; Faustinella, F., Smith, L.C. and Chan, L. (1992) biochemistry 31, 7219-7223). Conserved aspartic acid and histidine residues constitute the balance of the catalytic triad. A short stretch of 19-23 amino acids ("cap") forms an amphipathic helix and covers the catalytic pocket of the enzyme (Winkler, K., D' Arcy, A. and Hunziker, W. (1990) Nature 343, 771-774). This region differs among members of the family, and it was recently established that this segment confers enzyme substrate specificity (Dugi, K.A., Dichek H.L., and Santamalina-Fojo, S. (1995) J. Biochem. 270, 25396-25401). Comparison between hepatic lipase and lipoprotein lipase has shown differences in triacylglycerol lipases, and that the phospholipase activity of the enzymes is regulated by this cap region (Dugi, K.A., Dichek H.L., and Santamarina-Fojo, S. (1995) J. biochem. 270, 25396-25401).

Triacylglycerol lipases have varying degrees of heparin binding activity. Lipoprotein lipase has the highest affinity for heparin and this binding activity has been localized to the positively charged region of the amino-terminal domain (Ma, y., Henderson, h.e., Liu, m. -s., Zhang, h., Forsythe, i.j., Clarke-Lewis, l., Hayden, m.r., and Brunzell, j.d. journal of lipid research 35, 2049-. The localization of lipoprotein lipase on the surface of endothelial cells (Cheng, C.F., Oosta, G.M., Benladoun, A. and Rosenberg, R.D. (1981) J. Biochem.256, 12893-. This binding activity serves as a bridge between LDL and cell surface to accelerate LDL uptake (Mulder, M., Lombardi, P., Jansen, H., van Berkel T.J., FrantsR.R., and Havekes, L.M. (1992) biophysical and biochemical communications 185, 582-.

Lipoprotein lipase and hepatic lipase are known to act in conjunction with coactivator proteins: for lipoprotein lipase, apolipoprotein CII; and pancrelipase is a co-lipase.

The gene sequences encoding human pancreatic lipase, hepatic lipase and lipoprotein lipase have been reported (GenBank accession # M93285, # J03540 and # M15856, respectively). Messenger RNAs for human hepatic lipase and pancreatic lipase were about 1.7 and 1.8kb, respectively. Two mRNA transcripts of 3.6 and 3.2kb were synthesized from the human lipoprotein lipase gene. These two transcripts use alternating polyadenylation signals and their translational efficiencies are different (Ranganathan, G., Ong, J.M., Yukht, A., Saghizadeh, M., Simsolo, R.B., Pauer, A. and Kem, P.A. (1995) J.Biochem., 270, 7149-.

C) Physiological process

Lipid metabolism involves the interaction of lipid apoproteins, lipoproteins and enzymes.

Hepatic lipase and lipoprotein lipase are multifunctional proteins that mediate the binding, uptake, catabolism and remodeling of lipoproteins and phospholipids. Lipoprotein lipase and hepatic lipase function when bound to the luminal surface of peripheral tissues and liver endothelial cells, respectively. Both enzymes are involved in reverse cholesterol transport, i.e., the movement of cholesterol from peripheral tissues to the liver for excretion or recirculation from the body. Genetic defects in hepatic lipase and lipoprotein lipase are known to be responsible for familial disorders of lipoprotein metabolism. Defects in lipoprotein metabolism lead to serious metabolic disorders including hypercholesterolemia, hyperlipidemia, and atherosclerosis.

Atherosclerosis is a complex multigenic disease, defined histologically as the deposition of lipids and other blood derivatives (lipid or fibrolipid plaques) on the walls of blood vessels, particularly the aorta (aorta, coronary arteries, carotid arteries). Depending on the extent to which the atherosclerotic process proceeds, these plaques, which are more or less calcified, may be associated with lesions and with an accumulation in the blood vessels of fatty deposits mainly consisting of cholesterol esters. These plaques are accompanied by thickening of the vessel wall, hypertrophy of the smooth muscle, appearance of foam cells (lipid-filled cells produced by unrestrained uptake of cholesterol by aggregated macrophages) and accumulation of fibrous tissue. The atheromatous plaque clearly protrudes from the vessel wall, proving that it causes stenosis, responsible for atheromatous, thrombotic or embolic vessel occlusion in those patients most susceptible, which lesions may lead to serious cardiovascular pathologies such as infarction, sudden death, cardiac insufficiency and stroke.

The role of triacylglycerol lipases in vascular pathologies such as atherosclerosis has been a field of intense research (reviewed by Olivecrona, G., and Olivecrona, T. (1995) in the modern lipid review 6, 291-. Overall, the effect of triacylglycerol lipases is believed to be anti-atherogenic, as these enzymes lower serum triacylglycerol levels and promote HDL formation. Transgenic animals expressing human lipoprotein lipase or hepatic lipase have reduced plasma triacylglycerols and increased High Density Lipoprotein (HDL) levels (Shimada, M., Shimano, H., Gotoda, T., Yamamoto, K., Kawamura, M., Inaba, T., Yazaki, T., and Yamada, N. (1993) J. Biochem.268, 17924. 17929; Liu, M. -S., Jirik, F.R., LeBoeuf, R.C., Henderson, H., Castellani, L.W., Lusis, A.J., ma, Y., Forsycok, I.J., Zhang, H., Kirk, E., Brunnezen, J.D. and Hayden, M.R. (J. chem.269, 1994). It has been found that people with genetic defects that result in a reduced level of lipoprotein lipase activity suffer from hypertriglyceridemia and thus do not have an increased risk of coronary heart disease. This is reported to be due to lack of medium size; atherogenic lipoproteins that can accumulate in the subendothelial cell region (Zilversmit, D.B, (1972) cycle study, 33, 633-638).

However, it is speculated that an elevated level of lipase activity accelerates the atherogenic process in the region of the distribution of atherosclerosis (Zilversmit, D.B. (1995) clinical chemistry 41, 153-. This may be due to lipase mediated lipoprotein increase in vascular tissue binding and uptake (Eisenberg, S., Sehayek, E., Olivecrona, T.Vlodavsky, I. (1992) J. Clin. Res.90, 2013-Ascidin 2021; Tabas, I., Li, I., Brocia R.W., Xu, S.W., Swenson T.L. and Williams, K.J. (1993) J. Biochemical (268, 20419-Ascidin 20432; Nordestgaard, B.G. and Nielsen, A.G. (1994) modern lipid review 5, 252-Ascidin 257; Williams, K.J. and Tabas, I. (1995) Art.Thromb.and K.Biol.15, 551. Sc.561.) in addition, high levels of lyso-active phosphatidylcholine precursors that can lead to the synthesis of lysophosphatidylcholine and fatty acid in arterial lesions.

Despite the advances in understanding the role of lipase activity in lipid homeostasis, there remains a need in the art to identify other genes encoding proteins that regulate lipid metabolism.

Summary of The Invention

The present invention relates to the discovery of lipase-like genes (LLG), polypeptide products expressed therefrom, and compositions and methods utilizing the same. LLG polypeptides bind to heparin, are homologous to human lipoprotein lipase and hepatic lipase and contain a 39kD catalytic domain of the triacylglycerol lipase family. In a further embodiment, the polypeptide has phospholipase A activity.

The present invention provides a polypeptide comprising the sequence SEQ ID NO: 10.

The invention further provides a polypeptide comprising the sequence SEQ ID NO: 8 and presenting on a 10% SDS-PAGE gel an isolated polypeptide having a molecular weight of about 55kD or 68 kD.

The invention also provides a polypeptide comprising SEQ ID NO: 6 and presents an isolated polypeptide with a molecular weight of about 40kD on a 10% SDS-PAGE gel.

The invention further provides antigenic fragments of the LLG polypeptides.

Another aspect of the invention is an isolated nucleic acid encoding a polypeptide having the foregoing sequence.

Another aspect of the invention is a vector comprising the aforementioned nucleic acid encoding the polypeptide operably linked to a regulatory region such as a promoter.

Another aspect of the present invention is a recombinant cell comprising the above-described vector.

Another aspect of the invention is a method of producing a polypeptide comprising culturing a recombinant cell containing a nucleic acid encoding the polypeptide under conditions permitting expression of the polypeptide.

Another aspect of the invention is an antibody capable of specifically binding to and/or neutralizing the biological activity of a polypeptide according to the invention. Indeed, the polypeptides of the invention are further characterized by specific combinations of antibodies of the invention, i.e., antibodies specific for LLG polypeptides.

Another aspect of the invention is a composition comprising a polypeptide, nucleic acid, vector, antisense nucleic acid or antibody according to the invention and a pharmaceutically acceptable carrier.

Another aspect of the invention is a method of screening for agonists and antagonists of the enzymatic activity exhibited by a polypeptide of the invention, comprising contacting a potential agonist or antagonist with the polypeptide and its substrate and measuring the ability of the potential agonist or antagonist to enhance or inhibit the activity.

Another aspect of the invention is a method for the enzymatic hydrolysis of a phosphatidylcholine ester comprising contacting the phosphatidylcholine ester with a polypeptide of the invention.

Another aspect of the invention is a therapeutic method for improving the serum lipid profile of a human or other animal having an undesirable lipid profile comprising administering thereto an effective amount of a composition according to the invention.

Another aspect of the invention is a method of treating or preventing atherosclerosis in a human or other animal comprising administering thereto an effective amount of a composition according to the invention.

Other aspects and advantages of the present invention are further described in the accompanying drawings and the following detailed description of the preferred embodiments.

Brief Description of Drawings

FIG. 1 shows the primer sequences (SEQ ID Nos: 17-31) used in the exemplified PCR amplification.

FIG. 2 shows the nucleic acid sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of a differential display RT-PCR product containing a lipase-like gene cDNA. The sequences corresponding to the two primers used in the amplification are underlined. The stop codon and polyadenylation signal are boxed. The GAATCC motif and flanking sequences are from the pCRII vector in which the product was cloned.

FIG. 3 shows the nucleic acid sequence (SEQ ID NO: 3) and the deduced amino acid sequence (SEQ ID NO: 4) of the 5' RACE extension of LLG cDNA. The sequences corresponding to the two primers used in the amplification are underlined. The GAATCC motif and flanking sequences are from the pCRII vector in which the product was cloned.

FIG. 4 shows the cDNA sequence (SEQ ID NO: 7) containing the complete open reading frame of the lipase-like gene LLGXL. The start codon (ATG) and the stop codon (TGA) are boxed. The DraI site (TTTAAA) and SrfI site (GCCCGGGC) used in the construction of the expression vector are underlined.

FIG. 5 shows the deduced amino acid sequence of the LLGXL protein (SEQ ID NO: 8). The predicted signal sequence is underlined.

FIG. 6 shows the protein sequence matching of members of the triacylglycerol lipase gene family (SEQ ID Nos: 13-15). The shaded residues are identical to the LLGXL protein (SEQ ID NO: 8). Gaps were introduced in the sequence to maximize the match value using the CLUSTAL program.

FIG. 7 shows Northern analysis of LLG mRNA in THP-1 cells. Cells were stimulated with either PMA or PMA and oxidized LDL (PMA + oxLDL). The numbers on the left indicate the position of the RNA standard (in kilobases).

FIG. 8 shows Northern analysis of mRNA from various human tissues probed with LLG, lipoprotein lipase (LPL) and human beta actin cDNA. The position of the 4.4kb RNA standard is shown on the left side of the LLG and LPL panels.

FIG. 9 shows Northern analysis of LLG and LPL expression in cultured human endothelial cells and THP-1 cells. The cells are either unstimulated (not in contact with PMA) or stimulated with PMA.

FIG. 10 shows the sequence of the immunizing peptide (SEQ ID NO: 16) and its relationship to the LLGXL protein sequence. Peptides are indicated in shaded boxes. The terminal cysteine was introduced to facilitate coupling of the peptide to a carrier protein.

FIG. 11 shows a Western analysis of heparin-Sepharose concentrated protein in conditioned media from cultured endothelial cells. The blot was probed with anti-LLG antiserum. The numbers on the left represent the positions of the protein standards in kilodaltons.

FIG. 12 shows a Western analysis of heparin-Sepharose binding protein in conditioned media from COS-7 cells transiently transfected with either cDNA or DNA-free expression vectors containing LLGN or LLGXL (mock). Proteins from PMA-stimulated endothelial cells (HCAEC + PMA) were included as size references. The numbers on the left represent the apparent molecular weight of the major immunoreactive protein as determined by comparison to protein standards.

FIG. 13 shows the sequence of the rabbit LLG PCR product (RLLG. SEQ, SEQ ID NO: 12) and the alignment between the rabbit LLG PCR product and the corresponding human cDNA sequence (LLG 7742A). Identical nucleotides are shaded.

FIG. 14 shows phospholipase A activity of human LPL, LLGN and LLGXL using phosphatidylcholine as a substrate.

FIG. 15 shows the triacylglycerol lipase activities of human LPL, LLGN and LLGXL using triolein as a substrate.

FIG. 16 shows the hybridization of LLG and LPL probes to genomic DNA of different species.

Detailed Description

The present invention relates to the discovery of lipase-like genes (LLG) and polypeptide products expressed therefrom. These polypeptide products are members of the triacylglycerol lipase family, comprising a catalytic region of about 39kD of the triacylglycerol lipase family, such as a polypeptide having the sequence of SEQ ID NO: 10. one embodiment of the invention is a LLGN polypeptide of 354 amino acids. A second embodiment of the invention is a LLGXL polypeptide of 500 amino acids having 43% similarity to human lipoprotein lipase and 37% similarity to human hepatic lipase. The LLGXL polypeptide has phospholipase A activity.

The present inventors isolated partial cDNA from mRNA of THP-1 cells contacted with phorbol ester and oxidized LDL. After 5' RACE extension of this partial cDNA, smaller alternatively spliced cDNAs were isolated. A second, larger cDNA was isolated from a human placental cDNA library.

Northern analysis showed that LLG gene was expressed in endothelial cells. Antisera raised against polypeptides predicted from the open reading frame of the cDNA detected proteins with predicted sizes of LLGN and LLGXL in the conditioned medium of cultured endothelial cells. Treatment of endothelial cells with phorbol esters results in elevated levels of LLG mRNA and protein. This was the first found member of the family of triacylglycerol lipases expressed by endothelial cells.

A) Definition of

The terms defined below are used in this specification to aid in understanding the scope and practice of the invention.

A "polypeptide" is a macromolecular compound comprising covalently linked amino acids. Amino acids have the following general structure:

amino acids are classified into seven classes according to side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxyl group (OH), (3) side chains containing a sulfur atom, (4) side chains containing an acidic or amide gene, (5) side chains containing a basic gene, (6) side chains containing an aromatic ring, and (7) proline, in which the side chains are bonded to an amino group.

"protein" refers to a polypeptide that plays a structural or functional role in a living cell.

The polypeptides and proteins of the invention may be glycosylated or non-glycosylated.

"homology" refers to the similarity between sequences that reflect a common evolutionary origin. A polypeptide or protein is said to have homology or similarity if a significant number of the amino acids in them are (1) identical or (2) have chemically similar R side chains. Nucleic acids are said to be homologous if a significant number of nucleotides in the nucleic acid are identical.

An "isolated polypeptide" or "isolated protein" refers to a polypeptide or protein that is substantially separated from compounds with which it is normally associated in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids). "isolated" does not exclude artificial or synthetic mixtures with other compounds; or the presence of impurities which do not affect biological activity, which may be due to, for example, incomplete purification, the addition of stabilizers, or compounding into pharmaceutically acceptable preparations.

A molecule is "antigenic" if it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor. An antigenic polypeptide contains at least about 5, preferably about 10 amino acids. The antigenic portion of the molecule may be that which is immunodominant for antibody or T cell receptor recognition, or that which is used to raise antibodies to the molecule by coupling the antigenic portion to a carrier molecule. The antigenic molecule itself need not be immunogenic, i.e., capable of eliciting an immune response in the absence of a carrier.

"LLGN polypeptide" and "LLGN protein" refer to a polypeptide comprising the sequence SEQ ID NO: 6, which polypeptide is glycosylated or non-glycosylated.

"LLGXL polypeptide" and "LLGXL protein" refer to a polypeptide comprising the sequence SEQ ID NO: 8, which polypeptide is glycosylated or non-glycosylated.

"LLG polypeptide" generally describes LLGN polypeptides and LLGXL polypeptides.

The LLG polypeptides or proteins of the invention include analogs, fragments, derivatives or mutants derived from LLG polypeptides that retain at least one biological property of the LLG polypeptide. Various variants of LLG polypeptides exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequence of the structural gene encoding this protein or may involve differential splicing or post-translational modifications. The skilled artisan is able to prepare variants having single or multiple amino acid substitutions, deletions, additions or substitutions. These variants may include, in particular, (a) variants in which one or more amino acid residues are substituted with a conserved or non-conserved amino acid, (b) variants in which one or more amino acids are added to the LLG polypeptide, (c) variants in which one or more amino acids comprise a substituent, and (d) variants in which the LLG polypeptide is fused to another polypeptide, such as serum albumin. Other LLG polypeptides of the invention include substitutions of amino acid residues at conserved or non-conserved positions of one species with corresponding residues of another species. In another embodiment, an amino acid residue at a non-conserved position is substituted with a conserved or non-conserved residue. Techniques for obtaining such variants include genetic (inhibition, deletion, mutation, etc.), chemical and enzymatic techniques, which are well known to those of ordinary skill in the art.

It is within the scope of the present invention if such allelic variations, analogs, fragments, derivatives, mutants, and modifications, including mRNA splice forms and post-translational modifications, result in LLG polypeptide derivatives that retain any of the biological properties of the LLG polypeptide.

Nucleic acids are macromolecular compounds comprising covalently linked subunits called nucleotides. Nucleic acids include polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), which may be single-stranded or double-stranded. DNA includes cDNA, genomic DNA, synthetic DNA and semisynthetic DNA. The nucleotide sequence encoding the protein is referred to as the sense sequence.

An "antisense nucleic acid" is a nucleotide sequence that is complementary to a sense sequence. Antisense nucleic acids can be used to down-regulate or block expression of a polypeptide encoded by the sense strand.

An "isolated nucleic acid" refers to a nucleic acid that is substantially free of compounds with which it is normally associated in its natural state. "isolated" does not exclude artificial or synthetic mixtures with other compounds or the presence of impurities which do not affect the biological activity, which may be present, for example, as a result of incomplete purification, addition of stabilizers or complexation into pharmaceutically acceptable preparations.

The phrase "nucleic acid that hybridizes under high stringency conditions" refers to a nucleic acid that hybridizes to resist washing under high stringency conditions. One example of highly stringent washing conditions for DNA-DNA hybridization is 0.1 XSSC, 0.5% SDS at 68 ℃. Other highly stringent wash conditions are well known to those of ordinary skill in the art.

"regulatory region" refers to a nucleic acid sequence that regulates the expression of a nucleic acid. Regulatory regions may include sequences that are naturally responsible for expression of a particular nucleic acid (homologous regions) or include sequences of different origins of replication (responsible for expression of different proteins or even synthetic proteins). In particular, the sequence may be a eukaryotic or viral gene sequence or derivative sequence which can stimulate or inhibit gene expression in a specific or non-specific manner and in an inducible or non-inducible manner. Regulatory regions include origins of replication, RNA splice sites, enhancers, transcription termination sequences, signal sequences that direct the polypeptide into the secretory pathway in the target cell, and promoters.

A "heterologous" regulatory region is a regulatory region not naturally associated with the nucleic acid to be expressed. Heterologous regulatory regions include regulatory regions from different species, regulatory regions from different genes, hybrid regulatory sequences, and regulatory sequences not naturally occurring and designed by one of ordinary skill in the art.

A "vector" is any method of introducing a nucleic acid according to the invention into a host cell. The term "vector" includes viral and non-viral methods for introducing nucleic acids into prokaryotic or eukaryotic cells in vitro, ex vivo or in vivo. Non-viral vectors include plasmids, liposomes, charged lipids (cytofectins), DNA-protein complexes and biopolymers. Viral vectors include retroviral, adeno-associated virus, poxvirus, baculovirus, vaccinia, herpes simplex, Epstein-Barr virus and adenoviral vectors. In addition to the nucleic acids according to the invention, the vectors may also comprise one or more regulatory regions and/or selection markers for selecting, determining and monitoring the result of the nucleic acid transfer (to which tissue, time of expression, etc.).

A "recombinant cell" is a cell that contains a nucleic acid that does not naturally occur in the cell. "recombinant cells" include higher eukaryotic cells such as mammalian cells, lower eukaryotic cells such as yeast cells, prokaryotic cells, and archaeal cells.

"pharmaceutically acceptable carriers" include diluents and fillers for pharmaceutically acceptable administration, which are sterile and may be liquid or oily suspensions made with suitable dispersing or wetting agents and suspending agents. The particular pharmaceutically acceptable carrier and the ratio of active compound to carrier will be determined by the solubility and chemical characteristics of the composition, the particular mode of administration and standard pharmaceutical practice.

"Lipase" is a protein that cleaves a lipid substrate.

"phospholipases" are proteins that cleave phospholipid substrates.

"triacylglycerol lipases" are proteins which cleave triacylglycerol substrates.

"Phosphatidylcholine" is a glycerophospholipid having the following structure:

r and R' are hydrocarbon side chains of fatty acids. Phosphatidylcholine is also known as lecithin.

"lipid profile" refers to the concentration of cholesterol, triacylglycerols, lipoprotein cholesterol and other lipids in a human or other animal body.

An "undesirable lipid profile" refers to a condition in which the concentration of cholesterol, triacylglycerol, or lipoprotein cholesterol is outside of a reference range for age and gender adjustment. In general, a total cholesterol concentration >200mg/d1, a plasma triacylglyceride concentration >200mg/dl, an LDL cholesterol concentration >130mg/d1, an HDL cholesterol concentration >39mg/dl, or a ratio of total to HDL cholesterol >4.0 is considered an undesirable lipid profile. Undesirable lipid profiles are associated with a variety of pathological conditions including hyperlipidemia, diabetic hypercholesterolemia, atherosclerosis, and other forms of coronary artery disease.

B) Polypeptides

The present invention provides a triacylglycerol lipase family member polypeptide comprising a catalytic domain of 39kD of the triacylglycerol lipase family, such as a polypeptide having the sequence of SEQ ID NO: 10. One embodiment of the invention is a polypeptide comprising the sequence SEQ ID NO: 6 and on a 10% SDS-PAGE gel to express an isolated LLG polypeptide having a molecular weight of about 40 kD. Another embodiment of the invention is a polypeptide comprising the sequence SEQ ID NO: 8 and on a 10% SDS-PAGE gel to express an isolated LLG polypeptide having a molecular weight of about 55kD or 68 kD.

The polypeptides and proteins of the invention may be recombinant, natural or synthetic polypeptides, and may be derived from human, rabbit or other animals. These polypeptides are characterized by reproducible single and/or multiple molecular weights, chromatography and elution profiles, amino acid composition and sequence, and biological activity.

The polypeptides of the invention can be isolated from natural sources such as placental extract, human plasma or conditioned medium of cultured cells such as macrophages or endothelial cells by purification methods well known to those skilled in the art.

Alternatively, the polypeptides of the invention may be prepared by recombinant DNA techniques, including recombining the nucleic acid encoding the polypeptide into a suitable vector, inserting the resulting vector into a suitable host cell, recovering the polypeptide synthesized by the resulting host cell and purifying the recovered polypeptide.

C) Nucleic acids

The present invention provides isolated nucleic acids encoding LLG polypeptides.

The invention also provides antisense nucleic acids that can be used to negatively regulate or block LLG polypeptide expression in vitro, ex vivo, or in vivo.

Recombinant DNA techniques are well known to those of ordinary skill in the art. General techniques for cloning and representing recombinant molecules are described in Maniatis (molecular cloning, Cold spring harbor laboratory, 1982) and Ausubel modern molecular biology methods, Wiley and Sons, 1987, which are incorporated herein by reference.

The nucleic acids of the invention may be linked to one or more regulatory regions. Selection of suitable regulatory or other regions is routine and within the level of ordinary skill in the art. Regulatory regions include promoters, and may also include enhancers, repressors, and the like.

Promoters useful in the present invention include constitutive promoters and regulatable (inducible) promoters. Depending on the host, the promoter may be prokaryotic or eukaryotic. Prokaryotic (including phage) promoters useful in the practice of the present invention are lacI, lacZ, T₃，T₇，λP_r，P_IAnd the Trp promoter. Eukaryotic (including viral) promoters useful in the practice of the present invention are ubiquitous promoters (e.g., HPRT, vimentin, actin, tubulin), intermediate fiber promoters (e.g., desmin, neurofilament, keratin, GFAP), therapeutic gene promoters (e.g., MDR-type, CFTR, factor VIII), tissue-specific promoters (e.g., actin promoter in smooth muscle cells, or Flt and Flk promoters active in endothelial cells), promoters preferentially activated in dividing cells, promoters responsive to stimuli (e.g., steroid hormone receptors, retinoic acid receptors), tetracycline-regulated transcriptional regulators, cytomegalovirus immediate-early, retroviral LTR, metallothionein, SV-40, E1a, and MLP promoters. Tetracycline-regulated transcriptional regulators and CMV promoters are described in WO96/01313, U.S. Pat. No. 5,168,062 and 5,385,839, the contents of which are incorporated herein by reference.

Preferably, viral vectors for gene therapy are replication-defective, that is, they are unable to replicate autonomously in the target cell. Typically, the genome of the replication deficient viral vectors used within the scope of the present invention lacks at least one region essential for replication of the virus in the infected cell. These regions may be removed (in whole or in part) to render them non-functional by any technique known to those skilled in the art. These techniques include total removal, substitution (with other sequences, in particular by inserting nucleic acids), partial deletion or addition of one or more bases in the necessary region (for replication). Such techniques may be carried out in vitro (on isolated DNA) or in situ by genetic manipulation techniques or by mutagenic treatment.

Preferably, the replication-defective virus retains its genomic sequence essential for encapsidation of the viral particle.

Retroviruses are integrating viruses that infect dividing cells. The retrovirus includes two LTRs, a encapsidation sequence and three coding regions (gag, pol and env). The construction of recombinant retroviral vectors has been described: see in particular EP453242, EP178220, Bernstein et al, genetic engineering 7(1985) 235; McCormick, Biotechnology 3(1985)689, et al. In recombinant retroviral vectors, the gag, pol and env genes are typically deleted in whole or in part and replaced with a heterologous nucleic acid sequence of interest. These vectors can be derived from different types of retroviruses such as MoMuLV ("murine moloney leukemia virus"); MSV ("murine moloney sarcoma virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("rous sarcoma virus") and florode virus construction.

Generally, to construct a recombinant retrovirus containing sequences encoding an LLG according to the invention, a plasmid containing the LTR, encapsidation sequences and coding sequences is constructed. This construct was used to transfect packaging cell lines that provide plasmid-deficient retroviral functions in trans. Typically, packaging cell lines express the gag, pol and env genes. Such packaging cell lines, in particular the cell line PA317 (U.S. Pat. No. 4,861,719); PsiCRIP cell lines (WO90/02806) and GP + envAm-12 cell lines (WO89/07150) are described in the prior art. In addition, recombinant retroviral vectors may be modified at the LTR to suppress transcriptional activity and a number of encapsidation sequences, which may comprise part of the gag gene (Bender et al, J. Pneumatology 61(1987) 1639). Recombinant retroviral vectors are purified by standard techniques well known to those of ordinary skill in the art.

Adeno-associated viruses (AAV) are relatively small DNA viruses that integrate in a stable and site-specific manner into the genome of the cells they infect. They are capable of infecting a broad spectrum of cells without inducing any effect on cell growth, morphology or differentiation and have not been found to be involved in human pathology. AAV genomes have been cloned, sequenced and characterized. It comprises an Inverted Terminal Repeat (ITR) region of about 4700 bases and 145 bases at each end that serves as the origin of viral replication. The remainder of the genome is divided into two essential regions with encapsidation functions: the left part of the genome, comprising the rep gene involved in viral replication and viral gene expression; the right part of the genome contains the cap gene encoding the viral capsid protein.

The use of vectors derived from AAV for transferring genes in vitro and in vivo has been described (see WO 91/18088; WO 93/09239; U.S. Pat. No. 4,797,368, U.S. Pat. No. 5,139,941, EP488,528). These publications describe various AAV-derived constructs in which the rep and/or cap genes are deleted and replaced by a gene of interest and the use of these constructs for transferring a gene of interest in vitro (into cultured cells) or in vivo (directly into an organism). A replication-defective recombinant AAV according to the invention can be prepared by co-transfecting a plasmid containing the gene of interest flanked by two AAV Inverted Terminal Repeat (ITR) regions and a plasmid carrying AAV encapsidation genes (rep and cap genes) into a cell line infected with a human helper virus (e.g., adenovirus). The AAV recombinants produced are then purified by standard techniques. Thus, the invention also relates to AAV-derived recombinant viruses whose genome comprises sequences encoding LLG polypeptides flanked by AAV ITRs. The invention also relates to a plasmid comprising a sequence encoding an LLG polypeptide flanked by two ITRs from AAV. Such plasmids can be used for transferring LLG sequences, and if appropriate, can be incorporated into liposome vectors (pseudoviruses).

In a preferred embodiment, the vector is an adenoviral vector.

Adenoviruses are eukaryotic DNA viruses that have been modified to efficiently transfer nucleic acids of the invention into a variety of cell types.

Various serotypes of adenovirus exist. Of these serotypes, within the scope of the invention, preference is given to using adenoviruses of human or animal origin of type 2 or 5 (Ad2 or Ad5) (see WO 94/26914). Within the scope of the present invention, animals that may be used include adenoviruses of canine, bovine, murine (e.g., MavI, Beard et al, virology 75(1990)81), ovine, porcine, avian, and simian (e.g., SAV) origin. Preferably, the animal-derived adenovirus is a canine adenovirus, more preferably, a CAV2 adenovirus (e.g., Manhattan or strain A26/61 (ATCC VR-80)).

Preferably, the replication-defective adenoviral vector of the invention comprises an ITR, a encapsidation sequence and a nucleic acid of interest. More preferably, at least the E1 region of the adenoviral vector is non-functional. The deletion of the E1 region preferably extends from nucleotide 455 to nucleotide 3329 of the Ad5 adenoviral sequence. Other regions may also be modified, in particular the E3 region (WO95/02697), the E2 region (WO94/28938), the E4 region (WO94/28152, WO94/12649 and WO95/02697) or within any of the late genes L1-L5. Defective retroviral vectors are disclosed in WO 95/02697.

In a preferred embodiment, the adenoviral vector has deletions in the E1 and E4 regions. In another embodiment, the adenoviral vector has a deletion in the E1 region, into which the E4 region and the sequence encoding LLG have been inserted (see FR 9413355).

The replication deficient recombinant adenovirus vectors according to the present invention may be prepared by techniques well known to those skilled in the art (Levrero et al, Gene 101(1991)195, EP 185573; Graham EMBO J.3(1984) 2917). In particular, they can be prepared by homologous recombination between the adenovirus and, in particular, the plasmid carrying the DNA sequence of interest. Homologous recombination is achieved after co-transfection of the adenovirus and plasmid into a suitable cell line. Preferably, the cell line employed should (i) be transformable by the element and (ii) preferably comprise in integrated form sequences capable of complementing parts of the replication-defective adenovirus genome to avoid the risk of recombination. Examples of alternative cell lines are the human embryonic kidney cell line 293(Graham et al, J. Gen. Virol. 36(1977)59) comprising the left-hand part (12%) of the Ad5 adenovirus genome integrated into its genome and cell lines complementing the functions of E1 and E4 as described in applications WO94/26914 and WO 95/02697. The recombinant adenovirus can be recovered and purified using standard molecular biology techniques well known to those of ordinary skill in the art.

Negative regulation of gene expression using antisense nucleic acids can be achieved at the translational or transcriptional level. Preferred antisense nucleic acids of the invention are nucleic acid fragments that specifically hybridize to all or part of a nucleic acid encoding LLG or the corresponding messenger RNA. These antisense nucleic acids can be synthetic oligonucleotides that are selectively modified to improve their stability and selectivity. They can also be DNA sequences which are expressed in the cell to produce RNA complementary to all or part of the LLG mRNA. As described in EP140308, the expression of a polypeptide selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7 or SEQ ID NO: 11, or a portion thereof, to produce an antisense nucleic acid. Any length of antisense sequence is suitable for the practice of the invention, so long as it can down-regulate or block the expression of LLG. Preferably, the antisense sequence is at least 20 nucleotides in length. The preparation and use of antisense nucleic acids, DNA encoding antisense RNA and the use of oligo-and genetic antisense is disclosed in WO92/15680, the contents of which are incorporated herein by reference.

D) Antibodies

The present invention provides antibodies against LLG polypeptides. These antibodies may be monoclonal or polyclonal. The invention includes chimeric, single chain and humanized antibodies as well as Fab fragments and products of Fab expression libraries.

Polyclonal antibodies can be raised against antigenic fragments of the LLG polypeptide as described in example 4A. Antibodies can also be raised against the entire LLG protein or polypeptide, or against fragments, derivatives or epitopes of such protein or polypeptide. Antibodies can be obtained by administering the protein, polypeptide, fragment, derivative or epitope to an animal using techniques and methods known in the art.

Monoclonal antibodies can be prepared by the method described by Mishell, B.B.et al, methods of cellular immunological selection (edited by W.H.Freeman) San Francisco (1980). Briefly, Balb/C mouse splenocytes were immunized with a polypeptide of the invention. Fusing the immunized spleen cells with myeloma cells. Fused cells containing characteristics of spleen and myeloma cells are screened by growth on HAT medium which kills both parental cells but allows the fusion product to survive and grow.

The monoclonal antibodies of the invention may be "humanized" to prevent the host from generating an immune response to the antibody. A "humanized antibody" is an antibody in which the Complement Determining Regions (CDRs) and/or other portions of the light and/or heavy chain variable region frameworks are derived from a non-human immunoglobulin and the remainder of the molecule is derived from one or more human immunoglobulins. Humanized antibodies also include antibodies characterized by a humanized heavy chain associated with an unmodified donor or acceptor light chain or a chimeric light chain, and vice versa. Humanization of antibodies can be achieved by methods known in the art (see, e.g., G.E.Mark and E.A.Padlan, "Chapter 4: humanization of monoclonal antibodies," A.C. handbook of Experimental pharmacy, Vol.113, Springer-Verlag, New York, 1994). Transgenic animals can be used to express humanized antibodies.

Techniques for making single chain antibodies well known in the art can be adapted to make single chain antibodies to the immunogenic polypeptides and proteins of the invention.

anti-LLG antibodies are useful in assays to detect or quantify LLG levels. In one embodiment, these experiments provide a method for the clinical diagnosis and assessment of LLG and monitoring of the efficacy of treatment in various disease conditions.

E) Method for screening agonists or antagonists

The present invention provides methods for screening agonists (enhancers or coactivators, including proteinaceous coactivators) or antagonists (inhibitors) of LLGXL activity from small molecule libraries or natural product sources. Contacting a potential agonist or antagonist with a LLGXL protein and a LLGXL substrate, and determining the ability of the potential agonist or antagonist to enhance or inhibit LLGXL activity.

The LLGXL protein used in this method can be prepared in a variety of host cells including mammalian cells (as shown in example 7), baculovirus infected insect cells, yeast and bacteria. The expression of LLG in stably transfected CHO cells can be optimized by MTX amplification of the cells. The LLGXL protein may also be purified from natural sources such as human plasma, placental extract or conditioned medium of cultured endothelial cells, THP-1 cells or macrophages.

Optimization of experimental parameters including pH, ionic concentration, temperature, substrate concentration and emulsification conditions are determined empirically by one of ordinary skill in the art.

The fatty acid substituents of the substrate may vary in chain length, degree and location of unsaturation. These substrates may be radiolabeled at any one of several positions. Phospholipid substrates such as phosphatidylcholine can be radiolabeled at, for example, Sn-1 or Sn-2 fatty acid sites or in glycerol, phosphate or polar head genes (choline in phosphatidylcholine).

As an alternative to radiolabelling the substrate, other kinds of labelled substrates, such as fluorescent substrates or sulphur-containing substrates, may also be used in the screening method.

Fluorescent substrates are particularly effective in screening experiments because enzymatic catalysis can be continuously measured by measuring fluorescence intensity without physically separating (extracting) the product from the substrate. An example of a fluorescent phosphatidylcholine substrate is C₆NBD-PC { 1-acyl-2- [6- (nitro-2, 1, 3-benzodiazole-2-oyl) amino]Hexanoyl phosphatidylcholine }.

Sulfur-containing substrates include 1, 2-bis (hexanoylthio) -1, 2-dideoxy-sn-glycero-3-phosphocholine (L.J.Reynolds, W.N.Washburm, R.A.Deems and E.A.Dennis, 1991, methods in enzymology 197: 3-23; L.Yu and E.A.Dennis, 1991, methods in enzymology 197: 65-75; L.A.Wittenauer, K.Shirai, R.L.Jackson and J.D.Johnson, 1984, communications for biochemical and biophysical studies 118: 894-.

F) Hydrolysis of phosphatidylcholine esters

The present invention provides a method for enzymatic hydrolysis of phosphatidylcholine esters, e.g. for use in industrial or food processing or laundry detergents the polypeptides of the invention may be used to hydrolyse phosphatidylcholine esters in solution; or the enzyme may be bound to a solid support and then contacted with the substrate. The process can be used to prepare lysophospholipids and free fatty acids.

G) Composition comprising a metal oxide and a metal oxide

The present invention provides compositions comprising the polypeptides, nucleic acids, vectors, and antibodies of the invention in a biologically compatible (biocompatible) solution. A biologically compatible solution is one in which the polypeptide, nucleic acid, vector or antibody of the invention remains in an active form, e.g., in a form that enables biological activity. For example, a polypeptide of the invention will have phospholipase activity and the nucleic acid will be capable of replication, translation information, or hybridization to a complementary nucleic acid; the vector will be capable of transfecting the target cell; the antibody will bind to the polypeptide of the invention. Typically, such biologically compatible solutions are typically aqueous buffers containing salt ions such as Tris, phosphate or HEPES buffers. Typically the concentration of the salt ion is similar to physiological levels. In a particular embodiment, the biocompatible solution is a pharmaceutically acceptable composition. Biologically compatible liquids may include stabilizers and preservatives.

Such compositions can be formulated for administration by topical, oral, parenteral, intranasal, subcutaneous, and intraocular routes. Parenteral administration includes intravenous injection, intramuscular injection, intraarterial injection, or infusion techniques. The compositions may be administered parenterally in dosage unit formulations containing standard well-known non-toxic physiologically acceptable carriers, adjuvants and vehicles.

Preferred sterile injectable preparations may be solutions or suspensions in non-toxic parenterally acceptable solvents or diluents. Examples of pharmaceutically acceptable carriers are saline, buffered saline, isotonic saline (e.g., monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride; or mixtures of these salts), ringer's solution, dextrose, water, sterile water, glycerol, ethanol, and combinations thereof. 1, 3-butanediol and sterile, stable oils are convenient solvents or suspending media for use. Any stable oil including synthetic mono-or diglycerides can be used. Fatty acids such as oleic acid find use in the preparation of injectables.

The composition medium may be a hydrogel prepared from any biocompatible or non-cytotoxic (homo-or hetero-) polymer, such as a hydrophilic polyacrylic polymer that can be used as a drug-absorbing sponge. Such multimers are described, for example, in application WO93/08845, the entire contents of which are incorporated herein by reference. Some of them, such as in particular those obtained from ethylene and/or propylene oxide, are available from commercial sources. The hydrogel may be placed directly on the surface of the tissue to be treated, for example, during surgical intervention.

Another preferred embodiment of the present invention relates to a pharmaceutical composition comprising a replication deficient recombinant virus and a polyhydroxyalkylene. More specifically, the present invention relates to compositions comprising a replication-deficient recombinant virus comprising a nucleic acid encoding a LLG polypeptide and a polyhydroxyalkylene. The preferred polyhydroxyene is polyhydroxyene 407, which is commercially available (BASF, Parsippany, NJ) and is a non-toxic biocompatible polyol, with the most preferred. The polyhydroxylated hydrocarbon impregnated with the recombinant virus may be placed directly on the surface of the treated tissue, for example during surgical intervention. Despite the lower viscosity, the polyhydroxyalkylenes have essentially the same advantages as the hydrogels.

H) Method of treatment

The present invention provides methods of treatment comprising administering to a human or other animal an effective amount of a composition of the present invention.

The effective amount will vary with age, the type and severity of the condition to be treated, body weight, length of treatment, method of administration and other parameters. The effective amount is determined by a physician or other qualified medical professional.

The polypeptide according to the invention is generally administered in a dose of about 0.01mg to about 100mg, preferably about 0.1mg to about 50mg, more preferably about 1mg to about 10mg per kg body weight per day.

Recombinant viruses according to the invention are generally present at about 10⁴To about 10¹⁴Doses of pfu were formulated and administered. For AAV and adenovirus, it is preferable to use about 10⁶To about 10¹¹Dose of pfu. The term pfu ("plaque forming unit") corresponds to the infectivity of a virosome suspension, determined by infecting an appropriate cell culture and determining the number of plaque formations. Techniques for determining pfu titer of a viral solution are documented in the prior art.

The present invention provides methods for treating atherosclerosis caused by excessive, abnormal, or insufficient expression of LLG polypeptide activity.

The present invention further provides methods of treating humans or other animals having an undesirable lipid profile resulting from abnormally high or insufficient expression of LLG polypeptide activity.

The invention further provides methods of treating diabetes, hyperlipidemia, intrahepatic cholestasis, or other metabolic disorders resulting from abnormally high or insufficient expression of LIG polypeptide activity.

1) Treatment of undesirable lipid profiles associated with elevated LLG polypeptide expression

Methods of reducing the expression of LLG polypeptides to correct the condition of diseases or disorders associated with undesirable lipid profiles in which LLG polypeptide activity plays a role include, but are not limited to: administering a composition comprising an antisense nucleic acid, administering a composition comprising an intracellular binding protein such as an antibody, administering a composition comprising an LLGN polypeptide or other fragment of LLG, and administering a composition comprising a nucleic acid encoding an LLGN polypeptide or other fragment of LLG.

In one embodiment, the composition comprising an antisense nucleic acid is used to down-regulate or block the expression of LLG. In a preferred embodiment, the nucleic acid encodes an antisense RNA molecule. In this embodiment, the nucleic acid is operably linked to signals that allow expression of the nucleic acid sequence and is preferably introduced into a cell using a recombinant vector construct that, once introduced into the cell, expresses the antisense nucleic acid. Suitable vectors include plasmids, adenoviruses, adeno-associated viruses, retroviruses and herpes viruses. Preferably, the vector is an adenovirus. Most preferably, the vector is a replication-defective adenovirus comprising a deletion in the viral E1 and/or E3 regions.

In another embodiment, expression of a nucleic acid sequence encoding an intracellular binding protein capable of selectively interacting with LLG down-regulates or blocks the expression of LLG. WO94/29446 and WO94/02610, the contents of which are incorporated herein by reference, disclose cell transfection with genes encoding intracellular binding proteins. Intracellular binding proteins include any protein that can selectively interact with or bind to LLG within the cell in which it is expressed and neutralize the function of the bound LLG. Preferably, the cell binding protein is an antibody or a fragment of an antibody. More preferably, the intracellular binding protein is a single chain antibody.

WO94/02610 discloses the preparation of antibodies and the identification of nucleic acids encoding particular antibodies. Specific monoclonal antibodies are prepared from LLG or fragments thereof by techniques known to those skilled in the art. Vectors which subsequently comprise a nucleic acid encoding an intracellular binding protein or portion thereof and which are capable of expression in a host cell are prepared for use in the methods of the invention.

Alternatively, LLG activity can be blocked by administration of neutralizing antibodies to the circulatory system. The neutralizing antibody can be administered directly as a protein or expressed from a vector (with a secretion signal).

In another embodiment, the LLGXL activity is inhibited by administering a composition comprising an LLGN polypeptide or other fragment of LLG. The composition may be administered in a convenient manner, such as by oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The composition can be administered directly or encapsulated (e.g., in a lipid system, in an amino acid sequence microsphere, or in a spherical dendrimer). In some cases, the polypeptide can bind to another polymer, such as serum albumin or polyvinylpyrrolidone.

In another embodiment, the LLGXL activity is inhibited by using a small molecular weight compound that blocks its enzymatic properties or prevents the correct recognition of its cellular binding site.

In another embodiment, LLGXL activity is inhibited by the use of gene therapy, that is, by administering a composition comprising a nucleic acid encoding and directing the expression of an LLGN polypeptide or another fragment of LLG.

In a particular embodiment, the LLG gene of the invention also has affinity for heparin. Binding of LLG polypeptides to extracellular heparin within the vascular lumen allows LLG to bind to LDL and act as a bridge between LDL and extracellular heparin to accelerate LDL uptake. It is speculated that elevated levels of lipase activity accelerate the atherogenic process in localized regions of atherosclerotic lesions (Zilversmit, D.B. (1995) clinical chemistry 41, 153-. This may be due to a lipase-mediated increase in the binding and uptake of lipoproteins by vascular tissues (Eisenberg, S., Sehayek, E., Olivecrona, T.Vlodavsky, I. (1992) J. Clin clinical research 90, 2013-Asn 2021; Tabas, I., Li, I., Brocia R.W., Xu, S.W., Wwenson T.L., Williams K.J. (1993) J. Biochem 268, 20419-Asn 20432; Nordestgaard, B.G., and Nielsen, A.G. (1994) modern lipid reviews 5, 252-Asn 257; Williams, K.J., and Tabas, I. (1995) Art.Thromb.and Asn, biol.15, 551. 561. in addition, a local increase in the level of lipase activity in arterial lesions may lead to an inhibition of the production of lipoproteins in vivo by a specific lipoprotein-producing cholesterol-producing genetic activity in a subject (e.g.) such as a lipase-induced by an increase in the level of lipoprotein-producing cholesterol-producing a lipid precursor protein in vivo, such as a lipid-producing a lipid-like (LLG) in a lipid-producing lipid-lipid And (6) accumulating.

2) Treatment of undesirable lipid profiles associated with LLG polypeptide deficiencies

Methods of increasing the expression of LLG to correct the condition of diseases or disorders associated with undesirable lipid profiles in which LLG polypeptide activity plays a role include, but are not limited to: administering a composition comprising an LLGXL polypeptide and administering a composition comprising a nucleic acid encoding an LLGXL polypeptide.

In another embodiment, the level of LLGXL activity is increased by administering a composition comprising an LLGXL polypeptide. The composition may be administered in a convenient manner, such as by oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The composition can be used as is or encapsulated (e.g., in a lipid system, in an amino acid microsphere, or in a spherical dendrimer). In some cases, the polypeptide can bind to another polymer, such as serum albumin or polyvinylpyrrolidone.

In another embodiment, the level of LLGXL is increased by using a small molecular weight compound that is capable of positively regulating LLGXL expression at the transcriptional, translational or post-translational level.

In another embodiment, the level of LLGXL is increased by the use of gene therapy, that is, by administering a composition comprising a nucleic acid encoding and directing the expression of an LLGXL polypeptide.

Intrahepatic cholestasis may be characterized by elevated serum cholesterol and phospholipid levels. Recently, it has been described that the paradoxin drug-induced intrahepatic cholestasis model in rats shows a significant increase in serum cholesterol and phospholipid levels (Ishizaki, k., Kinbara, s., Miyazawa, n., Takeuchi, y., hirabayayayayash, n., Kasai, h., and Araki, T. (1997), toxicol, letters 90, 29-34). The products of the invention are useful for treating patients with intrahepatic cholestasis with increased serum cholesterol and/or phospholipids. In addition, this rat model also showed a severe decrease in the rate of bile cholesterol secretion.

Intrahepatic cholestasis is also characterized by a weakening of bile flow from the liver. Recently, the loci of progressive familial intrahepatic cholestasis (PFIC or Byler disease) and Benign Recurrent Intrahepatic Cholestasis (BRIC) were located at 18q21-q22 (Carlton, V.E.H., Knisely, A.S. and Freemer, N.B. (1995) human molecular genetics, 4, 1049-. Because the LLG gene is localized to the chromosomal region 18q21, the LLG gene or product of the invention can be used to treat patients suffering from intrahepatic cholestasis caused by mutation or defective expression of the PFIC/BRIC disease gene.

In another embodiment, the LLG gene or polypeptide product of the invention can be used to treat a patient suffering from intrahepatic cholestasis not caused by a defect in the PFIC/BRIC disease gene at 18q21-q 22. Recent studies have shown that another locus located outside the 18q21-q22 region can also produce the PFIC phenotype (Strantnieks, S.S., Kagalwalla, A.F., Tanner, M.S., Gardiner, R.M., and Thompson, R.J, (1996) J. Med. Genet 33, 833-. However, administration of LLG polypeptides, either directly or by gene therapy, can alleviate this form of disease.

In gene therapy, one or more nucleic acids encoding a polypeptide and regulatory regions controlling its expression are transferred to target cells in humans or other animals. This transfer is performed ex vivo by transferring the nucleic acid into cells in a laboratory and then administering the modified cells to a human or other animal; alternatively, in vivo, by direct transfer of the nucleic acid into cells in humans or other animals.

Non-viral vectors can be transferred by any method known in the art, including calcium phosphate co-precipitation, lipofection (synthetic anionic and cationic liposomes), receptor-mediated gene transfer, naked DNA injection, electroporation, and biolistic or particle acceleration.

Examples

The following examples illustrate the invention. These examples are illustrative only and do not limit the scope of the present invention.

EXAMPLE 1 identification of differentially expressed cDNAs

A) RNA preparation

Human monocytic THP-1 cells (Smith, p.k., Krohn, r.i., Hermanson, g.t., Mallia, a.k., Gartner, f.h.provenzano, m.d., Fujimoto, E.K, Goeke, n.m., Olson, b.j., and Klenk, d.c. (1985) biochem 150, 76-85) were cultured in RPMI-1640 medium (GIBCO) containing 25mM HEPES, 10% fetal bovine serum, 100 units/ml penicillin G sodium salt and 100 units/ml streptomycin sulfate. Cells were plated at 1.5X 10⁷Cells/plate on 15cm tissue culture dish and treat 48 h with 40ng/ml of 12-myristate 13-acetate phorbol (sigma)To induce cell differentiation. Human Low Density Lipoprotein (LDL) was purchased from Calbiochem and dialyzed thoroughly against PBS at 4 ℃. LDL was then diluted to 500. mu.g/ml and plated at 37 ℃ with 5. mu.M CuSO₄Was dialyzed against PBS for 16 hours. To stop oxidation, LDL was dialyzed thoroughly against 150mM NaCl, 0.3mM EDTA, then filter sterilized. Protein concentrations were determined by the BCA method (Schuh, J.Fairclough, G.F., and Haschemeyer, R.H. (1978) Proc. Natl. Acad. Sci. USA 75, 3173-317) (Pierce). The oxidation procedure was determined by TBARS (chomczynski, P. (1993) Biotechnology 15, 532-. Differentiated THP-1 cells were contacted with 50. mu.g/ml oxidized LDL or NaCl-EDTA buffer in RPMI medium containing 10% lipoprotein-deficient fetal bovine serum (sigma) for 24 hours. To harvest RNA, the plates were washed with 10ml PBS and then 14ml TRIZOL (Liang, P. and Pardee, A.B. (1992) science 257, 967-. The solution was pipetted several times to mix, then the samples were collected into centrifuge tubes and 3ml of chloroform was added to each plate and mixed. The tubes were centrifuged at 12000Xg for 15 minutes. After centrifugation, the supernatant was transferred to a new centrifuge tube, and 7.5ml of isopropanol was added to each plate and mixed well. The tube was centrifuged at 12000Xg for 20 minutes, and the pellet was washed with ice-cold 70% ethanol and dried at room temperature. The pellet was suspended in 500. mu.l TE (Tris-EDTA) and treated with 200 units of RNase-free DNAseI and 200 units of RNase inhibitor placental RNase inhibitor (Promega) at 37 ℃ for 30 minutes. RNA was purified by extracting RNA with phenol, phenol/chloroform/isoamyl alcohol (25: 24: 1) and chloroform/isoamyl alcohol (24: 1) in this order, followed by ethanol precipitation.

B) cDNA Synthesis

cDNA synthesis and PCR amplification were performed using the differential display kit, version 1.0(display systems Biotechnology, Inc). This system is based on the technology originally described by Liang and Pardee (Mead, d.a., Pey, N.K, Herrnstadt, c., Marcil, r.a., and Smith, L.M (1991) bio/technology 9, 657-. The primer pair for generating the cDNA fragment containing the initial information of the lipase-like gene is the downstream primer 7 and the upstream primer 15. Using RNA from PMA-treated THP-1 cells contacted with buffer or oxidized LDL, SynthesiscDNA for amplification: mu.l of 25. mu.M of the reverse primer 7 and 7.5. mu.l of Diethylpyrocarbonate (DEPC) -treated water were added to 300ng (3.0. mu.l) of RNA from either THP-1RNA sample. Heated at 70 ℃ for 10 minutes and then cooled on ice. Mu.l of 5 XPCR buffer (250mM Tris-HCl pH8.3, 375mM KCl (GIBCO)), 3. mu.l of 25mM MgCl₂Mu.l 0.1M DTT, 1.2. mu.l 500. mu.M dNTPs, 0.7. mu.l RNase and 5.6. mu.l DEPC treated water. The tube was incubated at room temperature for 2 minutes, then 1.5. mu.l (300 units) of SuperScriptII RNase H reverse transcriptase (GIBCO) was added. The tube was incubated at room temperature for 2 minutes, 37 ℃ for 60 minutes, 95 ℃ for 5 minutes, and then cooled on ice. The PCR solution containing 117. mu.l of 10XPCR buffer (500mM KCl, 100mM Tris-HCl pH8.3, 15mM MgCl)₂And 0.01% (w/v) gelatin), 70.2. mu.l 25mM MgCl₂，5.9μlα-³²PCR amplification was performed on a master mix of P dATP (10 mCi/ml; DuPontNEN), 4.7. mu.l of 500. mu.M dNTP mix, 11. mu.l of Ampli Taq DNA polymerase (5 units/. mu.l, Perkin-Elmer) and 493.3. mu.l of DEPC-treated water. For each reaction, 12. mu.l of the master mix was added to 2. mu.l of the downstream primer #7, 1. mu.l of cDNA and 5. mu.l of the upstream primer # 15. The reaction mixture was heated at 94 ℃ for 1 minute, then subjected to 40 thermal cycles of 94 ℃ denaturation for 15 seconds, 40 ℃ annealing for 1 minute, and 72 ℃ extension for 30 seconds after 40 cycles, the reaction was incubated at 72 ℃ for 5 minutes and stored at 10 ℃. The PCR reaction was performed in a Perkin-Elmer GeneAmp System 9600 thermal cycler.

Mu.l of the amplification reaction was mixed with an equal volume of loading buffer (0.2% bromophenol blue, 0.2% xylene nitrile blue, 10mM EDTA pH8.0 and 20% glycerol). Mu.l of the mixture was electrophoresed on a 6% native acrylamide sequencing gel at 1200 volts (constant pressure) for 3 hours. The gel was dried at 80 ℃ for 1.5 hours and exposed to Kodak XAR film. The amplification product was only found in reactions containing cDNA from THP-1 cells contacted with oxidized LDL and was lifted from the gel. Mu.l of DEPC-treated water was added to the centrifuge tube containing the excised gel fragment, incubated at room temperature for 30 minutes and then at 95 ℃ for 15 minutes.

To re-amplify the PCR product, 26.5. mu.l of the eluted DNA was usedIn a medium containing 5. mu.l of 10xPCR buffer, 3. mu.l of 25mM MgCl₂Mu.l of 500. mu.M dNTPs, 5. mu.l of 2. mu.M reverse primer 7, 7.5. mu.l of forward primer 15 and 0.5. mu.l of Amplitaq polymerase. PCR cycle parameters and instrumentation were as described above. After amplification, 20. mu.l of the re-amplified product was analyzed on an agarose gel, and 4. mu.l were taken for subcloning the PCR product into the vector pCRII (Frohman, M.A., Dush, M.K., and Martin, G.R. (1988) proceedings 85, 8998. sub.9002) in the TA cloning system. After overnight ligation at 14 ℃ the ligation product was used to transform E.coli. The resulting transformants were picked and 3ml of overnight culture was used for plasmid miniprep. The insert size was identified by digesting the plasmid with EcoRI and clones containing the appropriate size of the original PCR product insert were sequenced using a fluorescent dye terminator reagent (Prism, Applied Biosystems) and an Applied System, Biosystems373DNA sequencer. The sequence of the PCR product is shown in FIG. 2. The amplification primer sequences are underlined.

C) 5' RACE reaction

Extension of the identified cDNAs was achieved by RF-PCR using the 5' RACE system (Loh, E.Y., Eliot, J.F., Cwirla, S., Lanier, L.L., and Davis, M.M. (1989) science 243, 217-219; Simms, D., Guan, N., and Sitaraman, K. (1991) Focus 13, 997) (GIBCO). 1mg of THP-1RNA (treated with oxidized LDL) originally used for differential display reactions was used in the 5' RACE method.

Mu.l (1. mu.g) of RNA was mixed with 3. mu.l (3pmol) of primer 2a and 11. mu.l of DEPC water and heated at 70 ℃ for 10 minutes, followed by standing on ice for 1 minute. Mu.l 10 Xreaction buffer (20mM Tris-HCl pH8.4, 500mM KCl), 3. mu.l 25mM MgCl, was added₂Mu.l of 10mM dNTP mix and 2.5. mu.l of 0.1M DTT. The mixture was incubated at 42 ℃ for 2 minutes, then 1. mu.l Superscript II reverse transcriptase was added. The reaction was incubated at 42 ℃ for a further 30 minutes, at 70 ℃ for 15 minutes and on ice for 1 minute. Mu.l RnaseH (2 units) was added and the mixture was incubated at 55 ℃ for 10 min. cDNA was purified using a GlassMax column in a kit (Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) molecular cloning: A laboratory Manual, second edition, Cold spring harbor laboratory Press, Plainview, NY). At 50. mu.l dH₂O elution of cDNA from the column, lyophilization and resuspension in 21. mu. ldH₂And (4) in O. cDNA tailing was performed as follows: mix 7.5. mu. ldH₂O, 2.5. mu.l reaction buffer (200mM Tris-HCl pH8.4, 500mM KCl), 1.5. mu.l 25mM Mgcl₂2.5. mu.l of 2mM dCTP and 10. mu.l of cDNA were incubated at 94 ℃ for 3 minutes, followed by ice-cooling for 1 minute. Mu.l (10 units) of terminal deoxynucleotidyl transferase was added and the mixture was incubated at 37 ℃ for 10 minutes. The enzyme was heat-inactivated by incubation at 70 ℃ for 10 minutes. The PCR amplification of cDNA was performed as follows: mu.l of the tailed cDNA was added to a medium containing 5. mu.l of 10XPCR buffer (500mM KCl, 100mM Tris-KCl pH8.3, 15mM MgCl)₂And 0.01% (w/v) gelatin), 1. mu.l of 10mM dNTP mix, 2. mu.l (10pmol) of the anchor primer, 1. mu.l (20pmol) of primer 3a and 35. mu.l of dH₂In the reaction solution of O. The reaction solution was heated at 95 ℃ for 1 minute, and then 0.9. mu.l (4.5 units) of Ampoitaq polymerase was added thereto. The reaction was cycled 40 times under the following conditions: 94 ℃ for 5 seconds, 50 ℃ for 20 seconds, 72 ℃ for 30 seconds. Mu.l of this reaction was used for nested reamplification to increase the level of specific product for subsequent isolation. The re-amplification reaction includes: mu.l of initial amplification product, 5. mu.l of 10XPCR buffer, 1. mu.l of 10mM dNTP mix, 2. mu.l (20pmol) of universal amplification primer, 2. mu.l (20pmol) of primer 4a and 38. mu.l dH₂And O. The reaction was heated at 95 ℃ for 1 minute, and then 0.7. mu.l (3.5 units) of Amplitaq polymerase was added. The reaction was cycled 40 times under the following conditions: 94 ℃ for 5 seconds, 50 ℃ for 20 seconds, 72 ℃ for 30 seconds. The amplification products were separated by 0.8% agarose gel electrophoresis. A major product of about 1.2kb was detected. Mu.l of the reaction product was cloned into the PCRII vector in the TA cloning cassette and incubated overnight at 14 ℃. Ligation products for transformationEscherichia coli. EcoRI digestion to determine the size of the insert in the resulting transformants. Clones containing appropriately sized PCR product inserts were sequenced using fluorescent dye stop reagents (Prism, Applied Biosystems) and an Applied System Biosystems373DNA sequencer. The sequence of the RACE product containing the EcoRI site from the TA vector is shown in FIG. 3 the sequence of the amplification primers (the universal primer and the sequence complementary to the 5' RACE primer 4 a) are underlined.

Example 2 cloning and chromosomal mapping of the LLGXL Gene

A) cDNA library screening

Human placental cDNA library (of oligodeoxynucleotides and random primers, Cat #5014b, Cat #52033) was from Clontech (Palo, Alto, CA). The radiolabeled probe was generated by cleaving the insert of the plasmid containing the 5' RACE reaction PCR product described above. Radiolabelling the probe with random primer technology: the DNA fragment (50-100ng) was incubated with 1ng of random hexamer (Gibco) at 95 ℃ for 10 minutes and then on ice for 1 minute. Adding at room temperature: mu.l 10 XKlenow buffer (100mM Tris-HCl pH7.5, 50mM MgCl)₂57mM dithiothreitol; new England Biolabs), 3. mu.l of 0.5mM dATP, dGT, dTTP, 100. mu. Ci. alpha. -³²P dCTP (3000Ci/mmol, New England Nuclear) and 1. mu.l of Klenow fragment of DNA polymerase I (5 units, Gibco). The reaction was incubated at room temperature for 2-3 hours and stopped by increasing the volume to 100. mu.l with TE (pH8.0) and adding EDTA to a final concentration of 1 mM. The reaction volume was increased to 100. mu.l and passed through a G-50 spin column (Boehringer Mannheim) to remove unincorporated nucleotides. The obtained probe has specific activity greater than 5X 10⁸cpm/ugDNA。

The library was probed using established methods (Walter, P., Gilmore, R., and Blobel, G. (1984) cells 38, 5-8). Briefly, the filters were washed at 4.8X SSPE (20X SSPE ═ 3.6M NaCl, 0.2M NaH₂PO₄0.02M EDTA, ph7.7), 20mM Tris-hclph7.6, 1X Denhardt's solution (100X ═ 2% ficoll 400, 2% polypropylenepoyrrolidone, 2% BSA), 10% dextran sulfate, 0.1% SDS, 100 μ g/ml salmon sperm DNA and 1X 10⁶Hybridization in cpm/ml radioactive probe at 65 ℃ for 24 hours. The membrane was then washed 3 times for 15 minutes at room temperature with 2XSSC (1 XSSC ═ i50mM NaCl, 15mM citric acid pH7.0), 0.1% Sodium Dodecyl Sulfate (SDS), and then 3 times for 15 minutes at 65 ℃ with 0.5 XSSC, 0.1% SDS. Phage that hybridize to the probe are isolated and amplified. DNA was purified from amplified phage using Lambdasorb reagent (Promega) according to the manufacturer's instructions. The insert was cut from the phage DNA by digestion with EcoRI.The insert was subcloned into the EcoRI site of a plasmid vector (Bluescript. II SK, Strotagene). The open reading frame sequence contained within the 2.6kb EcoRI fragment of the cDNA was determined by automated sequencing as described above. The sequence is shown in FIG. 4. The deduced amino acid sequence of the protein encoded by this open reading frame is shown in FIG. 5 and designated LLGXL. It is presumed that the first methionine is encoded by the nucleotide pair at positions 252-254. The predicted protein is 500 amino acids long. The first 18 amino acids form a sequence which is characteristic of the secretion signal peptide (Higgins, D.G. and Sharp, P.M. (1988) Gene 73, 237-244). The molecular weight of the propeptide is predicted to be 56,800 daltons. Assuming cleavage of the signal peptide at position 18, the unmodified mature polypeptide has a molecular weight of 54,724 daltons.

The overall similarity of this protein to other known members of the triacylglycerol lipase family is shown in FIG. 6 and Table 1. In the sequence matching shown in FIG. 6, LLG is a polypeptide (SEQ ID NO: 6) encoded by the cDNA (SEQ ID NO: 5) described in example 1, hereinafter referred to as LLGN. This protein is identical to the amino-terminal 345 residues of the LLGXL protein. The 9 unique residues are followed by a stop codon, yielding a 39.3kD propeptide and a 37.3kD mature protein. The sequence common to LLGN and LLGXL is the nucleic acid sequence SEQ ID NO: 9 and the amino acid sequence SEQ ID NO: 10.

interestingly, it is known from other lipase domains that the LLGN and LLGXL proteins differ in position in the amino-terminal and carboxy-terminal domains of the protein. Therefore, the LLGN protein appears to consist of only one of the two domains of the triacylglycerol lipase. This sequence contains the characteristic "GXSXG" lipase motif at position 167-. Conservation of the disulfide-linked cysteine residues (positions 64, 77, 252, 272, 297, 308, 311, 316, 463 and 483) in other lipases indicates structural similarity of LLGXL to other enzymes. There are 5 putative N-glycosylation sites: at amino acid positions 80, 136, 393, 469, and 491. The protein sequences used for comparison were human lipoprotein lipase (LPL; Genbank accession # M15856, SEQ ID NO: 13), human hepatic lipase (HL; Genbank accession # J03540, SEQ ID NO: 14), human pancreatic lipase (PL; Genbank, accession # M93285, SEQ ID NO: 15), human pancreatic lipase-related protein-1 (PLRP-1; Genbank accession # M93283) and human pancreatic lipase-related protein-2 (PLRP-2; Genbank accession # M93284).

TABLE 1 similarity of triacylglycerol lipase gene families

	LLGXL	LPL	HL	PL	PLRP1	PLRP2
							LLGXL	-	42.7	36.5	24.5	22.5	22.6
LPL	42.7	-	40.0	22.8	22.7	20.9
							HL	36.5	40.0	-	22.8	24.0	22.0
PL	24.5	22.8	22.8	-	65.2	62.2
							PLRP1	22.5	22.7	24.0	65.2	-	61.7
PRLP2	22.6	20.9	22.0	62.2	61.7	-

Percent similarity was calculated from pairwise alignments using the Clustal algorithm in the Megalign program of Lasergene Biocentering Software Suite (Dnastar) (Camps, L., Reina, M., Llobera, M., Vilaro., S., and Olivecrona, T. (1990) journal of physiology 258, C673-C681).

B) Chromosome mapping

P from genomic LLG-containing DNA₁Cloned DNA was tagged with nicked translated digoxin UTP (Sternberg, N., Ruether, J. and DeRiel, K. New biologists 2: 151-62, 1990). The labeled probe was mixed with sheared human DNA and hybridized with PHA-stimulated peripheral blood lymphocytes from male donors in a solution containing 50% formamide, 10% dextran sulfate and 2 XSSC. Specific hybridization signals were detected by culturing the hybrid cells in a fluorescein-labeled anti-digoxigenin antibody, followed by counterstaining with DAPI. Initial experiments resulted in specific labeling of chromosomes of group E, which was considered chromosome 18 based on DAPI staining.

A second experiment was performed in which a biotin-labeled 18 chromosome centromere-specific probe was co-hybridized with the LLG probe. This experiment resulted in red labeling of the centromere specific marker of chromosome 18 and green labeling of the long arm of chromosome 18. Measurement of 11 specifically labeled hybrids, chromosome 18, indicated that Flter of LLG was 0.67(Fankle measured as 0.38), corresponding to band 18q 21. Several genetic diseases including intrahepatic cholestasis, vertebral dystrophy (cone rodsytrophy) and familial expansile osteolysis are thought to involve defects in this chromosomal region.

Example 3 LLG RNA analysis

A) Expression of LLG RNA in THP-1 cells

Analysis of cDNA-derived mRNA was performed by Northern analysis of THP-1 RNA. RNA from these cells was prepared as described above. mRNA was purified from total RNA by using the poly-deoxythymidylate magnetic bead system (Polyattract system, Promega). 3mg of polyadenylated mRNA were electrophoresed on a 1% agarose-formaldehyde gel. Gel in dH₂Wash in O for 30 min the RNA was vacuum transferred to a nylon membrane using alkaline transfer buffer (3M NaCl, 8mM NaOH, 2mM sarcosyl). After transfer, the blot was neutralized by incubation in 200mM phosphate buffer pH6.8 for 5 minutes. The RNA was cross-linked to the membrane using a UV cross-linker (Stratagene).

Probes were prepared by excising the insert from the above plasmid containing the 5' RACE reaction PCR product. The probes were radiolabeled using the random primer technique as described in example 2.

The filters were prehybridized in QuikHyb Rapid hybridization solution (Stratagene) for 30 min at 65 ℃. Radiolabeled probe (1-2X 10) prior to addition to the filter in QuikHyb⁶cpm/ml) and sonicated salmon sperm DNA (final concentration 100. mu.g/ml) were denatured by heating at 95 ℃ for 10 minutes and rapidly cooled on ice. Hybridization was carried out at 65 ℃ for 3 hours. The membrane was washed twice with 2XSSC, 0.1% sodium dodecyl sulfate for 15 minutes at room temperature, then twice with 0.1 XSSC, 0.1% SDS for 15 minutes at 62 ℃ to remove non-hybridized probes after washing, the membrane was briefly dried, and then exposed to Kodak XAR-2 film with an intensifying screen at-80 ℃. The results are shown in FIG. 7, which shows about 4.5kb of main mRNA. A small number of fragments of 4.3kb and 1.6kb were also present. The expected size of LLGNcDNA is 1.6 kb. This LLGXL sequence may be encoded by the major mRNA.

B) Expression of LLG RNA in various human tissues

Commercially prepared filters containing 3 μ g of each mRNA from human tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas) were purchased from Clontech (Cat # 7760-1). The filters were probed and treated as described above. After detection and autoradiography with radiolabeled LLG fragments, the probes were removed by washing 2 times with boiled 0.1 XSSC, 0.1% SDS in a 65 ℃ incubator for 15 minutes. The membrane was then probed with a 1.4kb DNA fragment encoding human lipoprotein lipase. This fragment was subjected to RT-PCR using THP-1RNA (treated with PMA and oxLDL) with the 5 'LPL and 3' LPL primers described in FIG. 1 to obtain RT-PCR conditions as described above. After autoradiography, membranes were again selected and re-probed with a radiolabeled fragment of human β actin cDNA to normalize RNA content. The results of these analyses are shown in fig. 8. The highest level of LLG information was detected in placental RNA, with lower levels found in RNA from lung, liver and kidney tissues. Consistent with previous studies by others (verhoven, a.j.m., Jansen, H. (1994) biochem. biophysis. acta 1211, 121-124), lipoprotein lipase information was found in a variety of tissues, with the highest levels found in cardiac and skeletal muscle tissues. The results of the analysis showed that the tissue distribution of LLG expression is very different from LPL. LLG expression patterns differ from hepatic or pancreatic lipase as reported by others (Wang, C.S. and Hartsuck; J.A. (1993) biochem. Biophys acta.1166, 1-19; Semenkovich; C.F., Chen, S. -W.,. Wims, M., Luo. C. -. C., Li, W. -H. and Chan, L. (1989) J. lipid Res.30, 423-.

To determine the expression pattern in other human tissues, another commercial membrane was probed with LLGXL cDNA. This dot Blot (human RNA Master Blot, Clontech catalog #7770-1) contained 100-500ng mRNA of 50 different tissues and had been normalized to comparable housekeeping gene expression (chen, L. and Morin, R. (1971) Biochim. Biophys. acta231, 194-197.) the 1.6kb DraI-SrfI fragment of LLGXL cDNA was used with a random oligonucleotide primer system (Prime ItII, Stratagene) according to the manufacturer's instructions³²PdCTP labeling after 30 min prehybridization at 65 deg.C, probe was run at 1.3X 10⁶cpm/ml QuikHyb hybridization was added. Hybridization was carried out at 65 ℃ for 2 hours. Non-hybridized probes were removed by washing the membrane twice with 2XSSC, 0.1% sodium dodecyl sulfate for 15 minutes at room temperature, and then twice with 0.1 XSSC, 0.1% SDS for 15 minutes at 62 ℃. After washing, the film was briefly dried and then exposed to Kodak XAR-2 film at-80 ℃ using a intensifying screenAt different times. The resulting images were quantified by densitometry. The results are shown in Table 2. The relative expression levels of tissues represented by multiple tissue Northern and multiple tissue dot blots were similar, highest in the placenta and lower in the lungs, liver and kidneys. Fetal liver, kidney and lung were expressed at approximately the same level as adult tissues. Surprisingly, thyroid tissue expression levels were the highest of all the indicated tissues, with expression 122% of the placental tissue. Although lipase expression in placenta has been preferred (Rothwell, J.E., Elphic, M.C. (1982) J.P.C. 159; Verhoeren, A.J.M., Carling D. and Jansen H. (1994) J.P.P.975; Burton, B.K.Mueller, H.W. (1980) Biochim.Biophys Acta 618, 449. 460), thyroid expression of any lipase was not previously known. The results indicate that the expression of LLG may be involved in the maintenance of the placenta, where LLG may play a role in releasing free fatty acids as an energy source from substrates such as phospholipids. Expression of LLG in the thymus may provide a precursor for the gland to synthesize bioactive molecules.

TABLE 2 expression of LLG mRNA in various human tissues

Whole brain	Not detected	Black texture	Not detected	Uterus	Not detected	Mammary gland	Not detected	Lung (lung)	29
										Tonsil	Not detected	Temporal lobe	Not detected	Prostate gland	5	Kidney (Kidney)	44	Trachea	12
Caudate nucleus	Not detected	Thalamus	Not detected	Stomach (stomach)	Not detected	Liver disease	61	Placenta hominis	100
										Cerebellum	4	Hypothalamic nucleus	Not detected	Testis	9	Small intestine	6	Fetal brain	5
Cortex of brain	Not detected	Spinal cord	Not detected	Ovary (LU) of human	Not detected	Spleen	Not detected	Fetal heart	Not detected
										Frontal lobe	Not detected	Heart and heart	Not detected	Pancreas (pancreas)	Not detected	Thymus	Not detected	Fetal kidney	56
Hippocampus japonicus	Not detected	Aorta	Not detected	Pituitary gland	Not detected	Peripheral lymphocytes	Not detected	Fetal liver	14
										Brain extension	Not detected	Skeletal muscle	Not detected	Adrenal gland	Not detected	Lymph nodes	Not detected	Fetal spleen	Not detected
Pillow leaf	Not detected	Colon	8	Thyroid gland	122	Bone marrow	Not detected	Fetal thymus	Not detected
										Bean shaped core	Not detected	Bladder of urinary bladder	Not detected	Salivary gland	Not detected	Appendix with appendix	7	Fetal lung	8

Values given are percentages of expression, and levels of placental tissue were arbitrarily set at 100%. The values are the average of two autoradiographic densitometric measurements. ND is not detected.

C) Expression of LLGRNA in cultured endothelial cells

Human Umbilical Vein Endothelial Cells (HUVEC) and Human Coronary Artery Endothelial Cells (HCAEC) were from Clonetics. HUVEC were propagated in commercially prepared endothelial cell culture medium (EGM, Clonetics) supplemented with 3mg/ml bovine brain extract (Maciag, T., Cerundolo, J., Iisley, S., Kelley, P.R., and Forand, R. (1979) Proc. Natl. Acad. Sci. USA 76, 5674-5678), while HCAEC were propagated in EGM supplemented with 3mg/ml bovine brain extract and 3% fetal bovine serum (final concentration 5%). After the cells had grown to confluency, the medium was changed to a medium containing no bovine brain extract. Cultures were stimulated by adding 100ng/ml phorbol myristate (sigma). After 24 hours of incubation, the cells were RNA extracted by Trizol as described above. 20mg of total RNA was electrophoretically separated and transferred to a membrane for analysis. Membranes were probed with LLG and LPL as described previously. The results are shown in FIG. 9. 20mg of total RNA from THP-1 cells stimulated with PMA was run on blots for comparison. RNA hybridized to LLG probe was detected in unstimulated and PMA-stimulated HUVEC cells. In contrast, detectable levels of LLG mRNA were only found in HCAEC cultures after PMA stimulation. Consistent with previous studies by others, no detectable levels of lipoprotein lipase mRNA were detected in any endothelial RNA (Verhoeven, a.j.m., Jansen, H. (1994) biochem. biophysis Acta 121, 121-.

Example 4 LLG protein analysis

A) Antibody preparation

This peptide was chosen for its high predicted antigenicity index (Jameson b.a. and Wolf, H. (1988) in silico applications in bioscience 4, 181-186). The sequence of the immunizing peptide is not found in any protein or translated DNA sequence in the gene bank data. Its corresponding position in the LLG protein is shown in FIG. 10. The carboxyl-terminal cysteine of this peptide does not correspond to the residue of the LLG putative protein and was introduced to facilitate coupling to the carrier protein. This peptide was synthesized on an Applied Biosystems Model 433A peptide synthesizer. 2mg of peptide was conjugated to 2mg of maleimide-Activated keyhole limpet hemocyanin according to the method contained in the Imject Activated Immunogen Conjugation kit (PierceChemicals). After desalting, half of the conjugate was emulsified with an equal volume of complete Freund's adjuvant (Pierce). The emulsion was injected into New Zealand white rabbits. 4 weeks after the first inoculation, the inoculation was boosted with an emulsion prepared exactly as described above but using incomplete Freund's adjuvant (Pierce). 2 weeks after the boost inoculation, test blood samples were prepared and the specific antibody titers were determined by ELISA using immobilized peptides. The first boost was followed 1 month after the second boost.

B) Western analysis of media from endothelial cell cultures

HCEAC cells were cultured and stimulated with PMA as described in example 3C, but cells were stimulated with PMA for 48 hours. Conditioned medium (9ml) samples were incubated with 500. mu.l of a 50% heparin-Sepharose CL-6B suspension in phosphate buffered saline (PBS, 150mM sodium chloride, 100mM sodium phosphate, pH 7.2). Since conservation of residues in the LLGXL sequence has been identified as critical for LPL heparin binding activity, heparin-Sepharose was chosen for partial purification and concentration of LLG proteins (Ma, Y., Henderson, H.E., Liu, M.S., Zhang, H.Forsythe, I.J.Clarke-Lewis, I.S., Hayden, M.R., and Brunzell, J.D. J.lipid research journal 35, 2049-. After shaking at 4 ℃ for 1 hour, the sample was centrifuged at 150Xg for 5 minutes. The medium was aspirated and Sepharose washed with 14ml PBS. After centrifugation and aspiration of the supernatant, the precipitated heparin-Sepharose was suspended in 200. mu.l of 2 XSDS loading buffer (4% SDS, 20% glycerol, 2% beta-mercaptoethanol, 0.002% bromophenol blue and 120mM Tris pH 6.8). The samples were heated at 95 ℃ for 5 minutes, 40. mu.l of which were loaded on a 10% Tris-glycine SDS gel. After electrophoresis at 140V for about 90 minutes, the proteins were transferred to nitrocellulose membranes by Novex electroblotter (210V, 1 hour). Membranes were blocked for 30 min in blocking solution (5% skimmed dry milk powder, 0.1% Tween20, 150mM sodium chloride, 25mM Tris pH7.2). The anti-peptide antiserum and normal rabbit serum were diluted 1: 5000 with blocking solution and incubated with the membrane overnight at 4 ℃ with gentle shaking. The membranes were then washed 4 times for 15 minutes each with TBST (0.1% Tween20, 150mM NaCl, 25mM Tris pH7.2). Goat anti-rabbit peroxidase conjugated antibody was diluted 1: 5000 with blocking solution and incubated with membrane for 1 hour with shaking. The membranes were washed as described above and reacted with Renaissance chemiluminescent reagent (DuPont, NEN) and exposed to Kodak XAR-2 film. The results are shown in FIG. 11. Two immunoreactive proteins were present in the unstimulated HUVEC and HCAEC cell samples. The levels of immunoreactive proteins in the unstimulated HCAEC samples were much lower than the corresponding HUVEC samples. Upon stimulation with PMA, endothelial cell cultures secrete three immunoreactive proteins. PMA treatment greatly increased the level of LLG protein produced by HCAEC cultures. PMA induced LLG protein was not as apparent in HUVEC cultures.

Example 5 production of recombinant LLG proteins

A) LLG expression constructs

The cDNAs encoding the LLGN and LLGXL proteins were cloned into the mammalian expression vector pCDNA3 (Invitrogen). This vector can express foreign genes in a variety of mammalian cells by using the cytomegalovirus major late promoter. The LLGN 5' RACE product was cloned into the EcoRI site of pCDNA 3. LLGXL cDNA was digested with DraI and SrfI to yield 1.55kbcDNA (see FIG. 4). The vector was digested with the restriction enzyme EcoRV and treated with T in a Rapid ligation kit (Boehringer Mannheim)₄DNA ligase and reagents were ligated to the vector and insert according to the manufacturer's instructions. This ligation product was used to transform competent E.coli. The resulting clones were screened by restriction analysis and sequencing to detect the presence and orientation of the insert in the expression vector.

B) Transient transfection of COS-7 cells with LLG

The LLG expression vector was introduced into COS-7 cells using Lipofectamine cationic lipid reagent (GIBCO). 24 hours before transfection, COS-7 cells were treated with 2X 10⁵The density of cells/plate was seeded on 60mm tissue culture plates. Cells were propagated in Dulbecco's modified Eagle's Medium (DMEM; GIBCO) containing 10% fetal bovine serum, 100U/ml penicillin, 100. mu.g/ml streptomycin. 1mg of plasmid DNA was added to 300. mu.l Optimem I serum-free medium (Gibco). Mu.l Lipofectamine reagent was diluted into 300. mu.l Optimem I medium and mixed with DNA solution, allowed to stand at room temperature for 30 minutes, the medium in the plate was removed and the cells were washed with 2ml Optimem medium. DNA-Lipofectamine solution was added to the plate along with 2.7ml Optimem medium and the plate was incubated at 37 ℃ for 5 hours. After incubation, serum-free medium was removed and replaced with DMEM supplemented with 2% FBS and antibiotics. 12 hours after transfection, some cultures were treated with 0.25mM PefablocSC (Boehringer Mannheim), protease inhibitors or 10U/ml heparin. 30 minutes before harvest, the samples that had been treated with heparin were then treated with 40U/ml heparin.Media was removed from the cells 60 hours after transfection. heparin-Sepharose CL-4B (200. mu.l of a 50% suspension in PBS, pH7.2) was added to 1ml of the medium and mixed at 4 ℃ for 1 hour. Sepharose was precipitated by low speed centrifugation and washed 3 times with 1ml cold PBS. Sepharose was precipitated and suspended in 100. mu.l 2 XLoading buffer. The sample was heated at 95 ℃ for 5 minutes. Mu.l of the sample was loaded on a 10% SDS-PAGE gel. Electrophoresis and Western analysis using anti-LLG antiserum were performed as described above. The results are shown in FIG. 12. Proteins from HCAEC conditioned media were included as size references. LLGN migrated at about the 40kD position, consistent with the HCAEC lowest band. Both 68kD and 40kD bands were contained in the medium from COS-7 cells transfected with LLGXL cDNA. When these cells were treated with heparin, the amounts of 68kD and 40kD proteins recovered from the culture medium increased significantly. When the cells were treated with the protease inhibitor Pefabloc, the amount of 68kD protein increased relative to the 40kD protein. This indicates that the low molecular weight proteins produced by these cells are hydrolysis products of the larger 68kD form. The effect of the identified mRNA encoding the short 40kD protein was shown to be unknown by the differences. However, alternatively spliced forms of hepatic lipase have been reported which are apparently expressed in a tissue-specific manner and which produce truncated proteins.

Example 6 LLG in animal species

A) Cloning of LLG Rabbit homolog

A commercially available lambda cDNA library of rabbit lung tissue (clontech, Cat # TL1010b) was used to isolate fragments of the rabbit homologue of the LLG gene. Mu.l of the stock was added to 45. mu.l of water and heated at 95 ℃ for 10 minutes. The following are added to a final volume of 100. mu.l of 200. mu.M dNTPs, 20mM Tris-HCl pH8.4, 50mM KCl, 1.5mM MgCl₂Primers DLIP774 and LLGgen2a were 100. mu.M each and 2.5U of Taq polymerase (GIBCO). The reaction was thermally cycled 35 times according to the following parameters: 94 ℃ for 15 seconds, 50 ℃ for 20 seconds, 72 ℃ for 30 seconds. Mu.l of the reaction was analyzed by agarose gel electrophoresis. A product of about 300 base pairs was detected. Mu.l of the reaction mixture was used for cloning the product using the TA cloning system. The insert of the resulting clone was sequenced (SEQ ID NO: 11). Deduced rabbit amino acid sequence (S)EQ ID NO: 12) the alignment between the corresponding human cDNA sequences is shown in FIG. 14. The rabbit and human LLG sequences were 85.8% identical in nucleotides other than the two amplification primer portions. The putative rabbit cDNA encoded a protein 94.6% identical to human that, with most nucleotide substitutions in the third or "wobble" position of the codon. Notably, this region spans the putative "cap" sequence of the LLG protein and is the variable region of the lipase gene family. This is also evidence that this gene is highly conserved between species.

B) LLG in other species

To confirm the presence of the LLG gene in other species, genomic DNA from various species was restriction digested with EcoRI, separated by agarose gel electrophoresis and blotted onto nitrocellulose membranes.

Hybridization with 2.5X 10 in a hybridization solution containing 6 XSSC, 10% dextran sulfate, 5 XDardt solution, 1% SDS and 5. mu.g/ml salmon sperm DNA⁶cpm/ml random primer-labeled³²p-LLG or³²The P-LPL (lipoprotein lipase) probe was hybridized to the membrane overnight at 65 ℃. The membrane was washed with 0.1 XSSC, 0.5% SDS at room temperature for 10 minutes, then sequentially at 40 deg.C, 50 deg.C and 55 deg.C for 10 minutes. Autoradiography of the blot is shown in FIG. 16.

FIG. 16 shows that LLG and LPL genes were present in all species tested, and no hybridization was observed with only LLG probe to rat DNA. The exception in rats may be artificially caused by restriction fragments of abnormal size containing LLG sequences, these fragments may be outside the separation range of agarose gel electrophoresis or may be null transfers. The different bands detected by the two probes indicate that LPL and LLG are separate evolutionarily conserved genes.

Example 7 enzymatic Activity of LLGXL

A) Phospholipase Activity

Conditioned media from COS-7 cells transiently expressing human lipoprotein lipase (LPL), LLGN, or LLGXL were analyzed for phospholipase activity. MEM (MEM) containing 10% FBS was used AS a blank, while conditioned medium from COS-7 cells transfected with the antisense LLGXL plasmid (AS) was used AS a negative control.

Phosphatidylcholine (PC) emulsions were labelled with 10. mu.l of phosphatidylcholine (10mM), 40. mu.l at positions sn 1 and 2¹⁴C-Phosphatidylcholine, dipalmitoyl (2. mu. Ci) and 100. mu.l Tris-TCNB [100mM Tris, 1% Triton, 5mM CaCl₂200mM NaCl, 0.1% BSA). This emulsion was allowed to evaporate for 10 minutes to give a final volume of 1ml in Tris-TCNB.

The reaction was run in duplicate, containing 50. mu.l of PC emulsion and 950. mu.1 of medium. The samples were incubated for 2-4 hours in a shaking 37 ℃ water bath, quenched by the addition of 1ml 1N HCl and then extracted with 4ml 2-propanol: hexane (1: 1). The upper 1.8ml hexane layer was passed through a silica gel column, free of release contained in the flow-through components¹⁴Fatty acids of C were quantified in a scintillation counter. The results of these experiments are shown in fig. 14.

B) Triacylglycerol lipase Activity

Conditioned media transiently expressing COS-7 from human lipoprotein lipase (LPL), LLGN or LLGXL were analyzed for triacylglycerol lipase activity. MEM containing 10% FBS was used AS a blank, while conditioned medium from COS-7 cells transfected with the antisense LLGXL plasmid (AS) was used AS a negative control.

The concentrated substrate was made anhydrous and labeled [9, 10-³H(N)]Emulsion of triacylglycerol and unlabeled triacylglycerol (final total triacylglycerol 150mg, 6.25X 10)⁸Cpm) was stabilized by adding 9mg lecithin in 100% glycerol. Mixing 0.56ml³H-triolein (0.28mCi) was mixed with 0.17ml of unlabelled triolein and 90. mu.l of lecithin (9 mg). This mixture was volatilized under a nitrogen stream. The dried lipid mixture was emulsified by sonication in 2.5ml 100% glycerol (30 seconds pulse level 2 followed by 2 seconds cooling cycle for 5 minutes).

The experimental substrate was prepared by diluting 1 volume of the concentrated substrate with 4 volumes of 0.2M Tris-HCl buffer (pH8.0) containing 3% (w/v) of bovine serum albumin free of fatty acids. The diluted substrate was shaken vigorously for 5 seconds.

The reaction was carried out in duplicate, containing 0.1ml of the test substrate and 0.1ml of the conditioned medium in a total volume of 0.2 ml. The reaction was incubated at 37 ℃ for 90 minutes. The reaction was stopped by adding 3.25ml of methanol-chloroform-hexane (1.41: 1.25: 1, v/v) followed by 1.05ml of 0.1M carbonate-potassium borate buffer (pH 10.5). After vigorous shaking for 15 seconds, the sample was centrifuged at 1000rpm for 5 minutes. A sample of 1.0ml of the upper aqueous phase was counted in a scintillation counter. The results of these experiments are shown in fig. 15.

Example 8 screening for agonists or antagonists Using LLG Polypeptides

Recombinant LLG is synthesized in baculovirus infected insect cells. The recombinant LLG is purified from serum-containing or serum-free media by chromatography on heparin-Sepharose followed by chromatography on positive ion exchange resin if desired, using a third chromatography step such as molecular sieves in purifying the LLG. During the purification process, the LLG protein was monitored with anti-peptide antibodies and phospholipase experiments were used to follow the LLG activity.

In the fluorescence experiment, the final experimental conditions were about 10mM Tris-HCl (pH7.4), 100mM KCl, 2mM CaCl₂；5μM C₆NBD-PC { 1-acyl-2- (nitro-2, 1, 3, -benzodiazole-4-acyl) amino]Hexanoyl phosphatidylcholine } and LLG protein (about 1-100 ng). The reaction was used for fluorescence excitation at 470nm and the enzyme activity was continuously monitored by measurement of fluorescence emission at 540 nm. The test compound and/or the substrate which stimulates and/or inhibits the LLG activity is added in the form of a 10-200mM solution in dimethyl sulfoxide. Compounds that stimulate or inhibit LLG activity are identified as an increase or decrease in fluorescence emission at 540 nm.

In this experiment, the final experimental conditions were about 25mM Tris-HCl (pH8.5), 100mM KCl, 100mM CaCl₂4.24mM Triton X-100, 0.5mM 1, 2, -bis (hexanoylthio) -1, 2-dideoxy-sn-glycero-3-phosphatidylcholine, 5mM 4, 4' -dithiopyridine (from a 50mM stock solution in ethanol) and 1-100mg recombinant LLG. By measuring the absorbance at 342nmThe increase in phospholipase activity was determined. The test compound and/or substrate which stimulates and/or inhibits LLG activity is added in the form of a 10-200mM solution in dimethyl sulfoxide. Compounds that stimulate or inhibit LLG activity are identified as an increase or decrease in absorbance at 342 nm.

Example 9 transgenic mice expressing human LLG

To further study the physiological effects of LLG, transgenic mice expressing human LLG were prepared.

A1.53 kb DraI/SrfI restriction fragment encoding LLGXL (see FIG. 4) was cloned downstream of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene promoter ubiquitously expressed by the plasmid vector (pHMG). Transgenic mice expressing different levels of human LLGXL were prepared using standard methods (see e.g., G.L. Tromp et al, Gene 1565: 199-. Transgenic mice were used to determine the effect of LLGXL overexpression on lipid profile, vascular pathology, rate and severity of atherosclerosis development and other physiological parameters.

Example 10 expression of LLG in atherosclerotic tissues

LLGXL expression in atherosclerosis was detected by reverse transcription polymerase chain reaction using mRNA isolated from vascular biopsy material from 4 patients with atherosclerosis. Tissue samples were from the aortic wall (1 sample), iliac arteries (2 samples) and carotid arteries (1 sample).

Atherosclerosis biopsy material was obtained from Gloucestershire Royal Hospital, UK, and polyadenylated mRNA was prepared and resuspended in Diethylpyrocarbonate (DEPC) treated water at a concentration of 0.5. mu.g/. mu.l mRNA. The reverse transcriptase reaction was performed according to the Superscript amplification System method used by GibcoBRL for first strand cDNA synthesis. Briefly, the synthesis of cDNA was performed as follows: mu.l of each mRNA was added to 1. mu.l of oligo (dT)_12-18Primers and 9. mu.l DEPC water. The tube was incubated at 70 ℃ for 10 minutes and placed on ice for 1 minute. The following ingredients were added to each tube: 2 μ l10 XPCR buffer, 2 μ l25mM MgCl₂Mu.l of 10mM dNTP mix and 2. mu.l of 0.1M DTT. After 5 minutes at 42 ℃ 1. mu.l (200 units) of SuperScript II reverse transcriptase were added. The reactions were gently mixed and then incubated at 42 ℃ for 50 minutes. The reaction was stopped by incubation at 70 ℃ for 15 minutes and then on ice 1. mu.l of RHAse H was added to each tube and incubated at 37 ℃ for 20 minutes to destroy residual mRNA.

PCR amplification was performed with 2. mu.l of cDNA reaction. To each tube was added the following: mu.l 10XPCR buffer, 5. mu.l 2mM dNTPs, 1. mu.l hllg-gspl primer (20pmol/ml, see FIG. 1), 1. mu.l hllg-gsp2a primer (20pmol/ml, see FIG. 1), 1.5. mu.l 50mM MgCl₂0.5. mu.l Taq polymerase (5U/ml) and 34. mu.l water. After the reaction was left at 95 ℃ for 2 minutes, 30 cycles of PCR were carried out under the following conditions: 94 ℃ for 15 seconds, 52 ℃ for 20 seconds, 72 ℃ for 30 seconds. The completed reaction was left at 72 ℃ for 10 minutes before analysis by agarose gel electrophoresis. The hllp-gsp primer is LLG specific and gives the expected product of 300 bp. In parallel PCR, primers specific for the housekeeping gene G3PDH (human glyceraldehyde 3-phosphate dehydrogenase) (1. mu.l each, 20pmol/ml) were used to indicate that the cDNA synthesis reaction was successful.

The G3PDH primer (see FIG. 1) produced the expected product of 983 base pairs in 4 vascular biopsies LLG expression was detected in 3 of 4 samples and no expression was detected in carotid artery samples.

All references discussed herein are incorporated by reference.

Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The peptides, polynucleotides, methods, procedures and techniques described herein are presented as representative of preferred embodiments and are intended to be illustrative and not limiting as to the scope of the invention. Variations thereof and other uses will occur to those skilled in the art and are encompassed within the spirit of the invention or the scope of the invention as defined by the appended claims.

Sequence listing

(1) Basic information

(i) The applicant: jaye, Michel C.

Doan，Kim-Anh T

Krawiec，John A.

Lynch，Kevin J.

South，Victoria J.

(ii) The invention name is as follows: polypeptides encoded by human lipase-like gene and compositions and methods utilizing same

(iii) Sequence number: 31

(iv) Contact address:

(A) name: Rhone-Poulenc Rorer Inc.

(B) Street: 500 Arcola Rd.3C43

(C) City: collegeville

(D) State name: PA

(E) The state is as follows: united states of America

(F) And (4) post numbering: 19426

(v) A computer-readable form:

(A) type of medium: flexible disk

(B) A computer: IBM PC compatible machine

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: patentin Release #1.0, version #1.30

(vi) The current application data:

(A) application No.: US

(B) Day of delivery:

(C) and (4) classification:

(viii) lawyer/attorney information

(A) Name: fehlner ph.d., Paul F.

(B) Registration number: 35,135

(C) Reference/bibliographic number: A2582-WO

(ix) Telecommunication information:

(A) telephone: (610)454-3839

(B) Faxing: (610)454-3808

(2) With respect to SEQ ID NO: 1 information

(i) Sequence characterization

(A) Length: 367 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: wire type

(ii) Molecular type: cDNA

(ix) Is characterized in that:

(A) name/Key: CDS

(B) Position: 22..180

(xi) Description of the sequence: SEQ ID NO: 1:

(2) with respect to SEQ ID NO: 2 information

(i) Sequence characterization

(A) Length: 53 amino acids

(B) Type (2): amino acids

(D) Topological structure: wire type

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 2:

(2) with respect to SEQ ID NO: 3 information

(i) Sequence characterization

(A) Length: 1382 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: wire type

(ii) Molecular type: cDNA

(ix) Is characterized in that:

(A) name/Key: CDS

(B) Position: 312..1370

(xi) Description of the sequence: SEQ ID NO: 3:

(2) with respect to SEQ ID NO: 4 information

(i) Sequence characterization

(A) Length: 353 amino acids

(B) Type (2): amino acids

(D) Topological structure: wire type

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 4:

(2) with respect to SEQ ID NO: 5 information

(i) Sequence characterization

(A) Length: 1065 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: wire type

(ii) Molecular type: cDNA

(ix) Is characterized in that:

(A) name/Key: CDS

(B) Position: 1..1065

(xi) Description of the sequence: SEQ ID NO: 5:

(2) with respect to SEQ ID NO: 6 information

(i) Sequence characterization

(A) Length: 355 amino acids

(B) Type (2): amino acids

(D) Topological structure: wire type

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 6:

(2) with respect to SEQ ID NO: 7 information

(i) Sequence characterization

(A) Length: 2565 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: wire type

(ii) Molecular type: cDNA

(ix) Is characterized in that:

(A) name/Key: CDS

(B) Position: 252..1754

(xi) Description of the sequence: SEQ ID NO: 7:

(2) with respect to SEQ ID NO: 8 information

(i) Sequence characterization

(A) Length: 501 amino acids

(B) Type (2): amino acids

(D) Topological structure: wire type

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 8:

(2) with respect to SEQ ID NO: 9 information

(i) Sequence characterization

(A) Length: 1035 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: wire type

(ii) Molecular type: cDNA

(ix) Is characterized in that:

(A) name/Key: CDS

(B) Position: 1..1035

(xi) Description of the sequence: SEQ ID NO: 9:

(2) with respect to SEQ ID NO: 10 information

(i) Sequence characterization

(A) Length: 345 amino acids

(B) Type (2): amino acids

(D) Topological structure: wire type

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 10:

(2) with respect to SEQ ID NO: 11 information

(i) Sequence characterization

(A) Length: 225 base pairs

(B) Type (2): nucleic acids

(C) Chain type: double chain

(D) Topological structure: wire type

(ii) Molecular type: cDNA

(ix) Is characterized in that:

(A) name/Key: CDS

(B) Position: 1..225

(xi) Description of the sequence: SEQ ID NO: 11:

(2) with respect to SEQ ID NO: 12 information

(i) Sequence characterization

(A) Length: 75 amino acids

(B) Type (2): amino acids

(D) Topological structure: wire type

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 12:

(2) with respect to SEQ ID NO: 13, information:

(i) sequence characterization

(A) Length: 472 base pairs

(B) Type (2): amino acids

(C) Chain type:

(D) topological structure: wire type

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 13:

(2) with respect to SEQ ID NO: 14, information:

(i) sequence characterization

(A) Length: 499 base pairs

(B) Type (2): amino acids

(C) Chain type:

(D) topological structure: wire type

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 14:

(2) with respect to SEQ ID NO: 15 information of

(i) Sequence characterization

(A) Length: 465 base pairs

(B) Type (2): amino acids

(C) Chain type:

(D) topological structure: wire type

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 15:

(2) with respect to SEQ ID NO: 16 information

(i) Sequence characterization

(A) Length: 17 base pairs

(B) Type (2): amino acids

(C) Chain type:

(D) topological structure: wire type

(ii) Molecular type: peptides

(ii) Fragment type: internal of

(xi) Description of the sequence: SEQ ID NO: 16:

(2) with respect to SEQ ID NO: 17 information of

(i) Sequence characterization

(A) Length: 13 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotides" for de sc "

(xi) Description of the sequence: SEQ ID NO: 17:

(2) with respect to SEQ ID NO: 18 information of

(i) Sequence characterization

(A) Length: 10 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotides" for de sc "

(xi) Description of the sequence: SEQ ID NO: 18:

(2) with respect to SEQ ID NO: 19 information of

(i) Sequence characterization

(A) Length: 23 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotides" for de sc "

(xi) Description of the sequence: SEQ ID NO: 19:

(2) with respect to SEQ ID NO: 20 information of

(i) Sequence characterization

(A) Length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotides" for de sc "

(xi) Description of the sequence: SEQ ID NO: 20:

(2) with respect to SEQ ID NO: information of 21:

(i) sequence characterization

(A) Length: 19 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotides" for de sc "

(xi) Description of the sequence: SEQ ID NO: 21:

(2) with respect to SEQ ID NO: 22 information of

(i) Sequence characterization

(A) Length: 48 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotides" for de sc "

(ix) Is characterized in that:

(A) name/Key: modified _ base

(B) Position: 36

(D) Other information: modified _ base ═ i

(ix) Is characterized in that:

(A) name/Key: modified _ base

(B) Position: 37

(D) Other information: modified _ base ═ i

(ix) Is characterized in that:

(A) name/Key: modified _ base

(B) Position: 41

(D) Other information: modified _ base ═ i

(ix) Is characterized in that:

(A) name/Key: modified _ base

(B) Position: 42

(D) Other information: modified _ base ═ i

(ix) Is characterized in that:

(A) name/Key: modified _ base

(B) Position: 46

(D) Other information: modified _ base ═ i

(ix) Is characterized in that:

(A) name/Key: modified _ base

(B) Position: 47

(D) Other information: modified _ base ═ i

(xi) Description of the sequence: SEQ ID NO: 22:

(2) with respect to SEQ ID NO: 23 information of

(i) Sequence characterization

(A) Length: 28 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotides" for de sc "

(xi) Description of the sequence: SEQ ID NO: 23:

(2) with respect to SEQ ID NO: 24 information

(i) Sequence characterization

(A) Length: 24 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotides" for de sc "

(xi) Description of the sequence: SEQ ID NO: 24:

(2) with respect to SEQ ID NO: 25 of

(i) Sequence characterization

(A) Length: 25 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotides" for de sc "

(xi) Description of the sequence: SEQ ID NO: 25:

(2) with respect to SEQ ID NO: 26 information of

(i) Sequence characterization

(A) Length: 21 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotides" for de sc "

(xi) Description of the sequence: SEQ ID NO: 26:

(2) with respect to SEQ ID NO: 27 information of

(i) Sequence characterization

(A) Length: 22 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotides" for de sc "

(xi) Description of the sequence: SEQ ID NO: 27:

(2) with respect to SEQ ID NO: 28 information of

(i) Sequence characterization

(A) Length: 25 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotides" for de sc "

(xi) Description of the sequence: SEQ ID NO: 28:

(2) with respect to SEQ ID NO: information of 29:

(i) sequence characterization

(A) Length: 25 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotides" for de sc "

(xi) Description of the sequence: SEQ ID NO: 29:

(2) with respect to SEQ ID NO: 30 information of

(i) Sequence characterization

(A) Length: 26 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotides" for de sc "

(xi) Description of the sequence: SEQ ID NO: 30:

(2) with respect to SEQ ID NO: 31 information of

(i) Sequence characterization

(A) Length: 24 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: wire type

(ii) Molecular type: other nucleic acids

(A) The following steps are described: "oligonucleotides" for de sc "

(xi) Description of the sequence: SEQ ID NO: 31:

Claims (27)

1. An isolated polypeptide encoded by a lipase-like gene, wherein the polypeptide is of rabbit origin, and

(a) the heparin binding agent is combined with the heparin,

(b) has homology with human lipoprotein lipase and hepatic lipase, and

(c) comprises a polypeptide having a sequence identical to SEQ ID NO: 10 homologous amino acid sequence of 39kD,

wherein the isolated polypeptide comprises SEQ ID NO: 12.

2. The isolated polypeptide of claim 1, wherein the polypeptide has phospholipase A activity.

3. A composition comprising the polypeptide of claim 1 and a biologically compatible solution.

4. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

5. An isolated nucleic acid encoding the polypeptide of claim 1.

6. The isolated nucleic acid of claim 5 which is a cDNA.

7. The isolated nucleic acid of claim 6, wherein the nucleotide sequence is SEQ ID NO: 11.

8. a pharmaceutical composition comprising the nucleic acid of claim 7 and a pharmaceutically acceptable carrier.

9. A vector comprising the isolated nucleic acid of claim 7 operably linked to a regulatory region.

10. The vector of claim 9, wherein the regulatory region is heterologous.

11. The vector of claim 9, which is a viral vector.

12. The vector of claim 11, which is an adenoviral vector.

13. A recombinant cell comprising the vector of claim 12.

14. The recombinant cell of claim 13 wherein the cell is a eukaryotic cell.

15. The recombinant cell of claim 14 wherein the cell is a COS-7 cell.

16. A composition comprising the carrier of claim 9 and a biologically compatible solution.

17. A pharmaceutical composition comprising the vector of claim 9 and a pharmaceutically acceptable carrier.

18. A method of producing the polypeptide of claim 1, comprising the step of culturing the recombinant cell of claim 13 under conditions that allow expression of the polypeptide.

19. An antibody capable of specifically binding to the polypeptide of claim 1.

20. An antibody capable of specifically binding to the polypeptide of claim 2 and neutralizing the phospholipase activity of the polypeptide.

21. The antibody of claim 19 or 20 which is a monoclonal antibody.

22. The antibody of claim 19 or 20, which is a polyclonal antibody.

23. A hybridoma cell producing the antibody of claim 21.

24. A composition comprising the antibody of claim 19 and a biologically compatible solution.

25. A pharmaceutical composition comprising the antibody of claim 19 and a pharmaceutically acceptable carrier.

26. A method of screening for agonists or antagonists of LLG activity comprising:

(a) contacting the potential stimulant or antagonist with LLG and a substrate for LLG, and

(b) determining the ability of a potential agonist or antagonist to enhance or inhibit the activity of LLG, wherein LLG is the polypeptide of claim 1.

27. A method for the in vitro enzymatic hydrolysis of a phosphatidylcholine ester comprising contacting said phosphatidylcholine ester with a polypeptide according to claim 1.