UA75319C2 - Polypeptides being coded with gene similar to human lipase gene, compositions and methods - Google Patents

Abstract

Lipoprotein Lipase like polypeptides, nucleic acids encoding said polypeptides, antisense sequences, and antibodies to said polypeptides arc disclosed. Also disclosed are methods for the preparation of said polypeptides in a recombinant system and for the use of said polypeptides to screen for agonists and or antagonists of said polypeptides. Also disclosed are methods and compositions for the treatment of disorders of lipid metabolism.

Description

Description of the invention

This application claims the benefits of two concurrently filed prior applications 60/032254 and 60/032783, 2 filed on December 6, 1996.

This invention relates to polypeptides of the triacylglycerol-lipase family, to nucleic acids encoding said polypeptides, to antisense sequences, derivatives of said nucleic acids, and to antibodies against said polypeptides. This invention also relates to the preparation of the indicated polypeptides using recombinant technologies and to the use of the indicated polypeptides for screening to identify agonists or antagonists of the indicated polypeptides. This invention also relates to methods of therapeutic use of such polypeptides and nucleic acid sequences encoding said polypeptides in pharmaceutical compositions, which include compositions for gene therapy and compositions for the treatment of disorders of lipid and lipoprotein metabolism.

A) Lipids

Lipids are water-insoluble organic biological molecules that are essential participants in a variety of biological functions that include energy storage, transport, and metabolism, and play an important role in membrane structure and fluidity. Lipids contained in the body of humans and other animals are formed from two sources: some lipids come from dietary fats and oils, and other lipids are biologically synthesized by the human or animal body. In mammals, at least 1095 percent of their body weight is lipids, most of which are present in the form of triacylglycerols.

Triacylglycerols, also known as triglycerides or triacylglycerides, consist of three fatty acids esterified with glycerol. Food triacylglycerides accumulate in adipose tissues as energy sources or are hydrolyzed in the digestive tract by triacylglycerol lipases, of which pancreatic lipase is the most important.

Triacylglycerols are transported between tissues in the form of lipoproteins. with

Lipoproteins are micelle-like structures found in plasma that contain varying ratios of different types of lipids and proteins (called apoproteins). There are five main classes of plasma lipoproteins, the main function of which is the transport of lipids. These classes are, in order of increasing density: chylomicrons, very low-density lipoprotein (VLDL), intermediate-density lipoprotein (ILD), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). Although each class of lipoproteins includes many types of lipids, each class mainly transports one type of lipids: "- triacylglycerols, described above, are transported as part of chylomicrons, LDNG and LPG; while phospholipids and cholesterol esters are transported as part of LHG and LNG, respectively. at

Phospholipids are complex esters of a dibasic fatty acid and glycerol phosphate, which also contain a polar group bound to phosphate. Phospholipids are important structural components of cell membranes. Phospholipids are hydrolyzed by enzymes called phospholipases. in

Phosphatidylcholine, a typical phospholipid, is a major component of the membranes of most eukaryotic cells.

Cholesterol is a metabolic precursor of steroid hormones and bile acids and is also a major component of cell membranes. In humans and other animals, cholesterol comes from food and is also "synthesized by the liver and other tissues. Cholesterol is transported between tissues in the form of complex C 50 cholesterol esters, which are part of LNG and other lipoproteins. c Each living cell is surrounded by membranes that serve as a barrier between intracellular and extracellular c» compartments. Membranes also surround the nucleus of a eukaryotic cell, are part of the endoplasmic reticulum, and perform specialized functions, such as those in the myelin sheath that surrounds axons. A typical membrane contains about 4095 lipid and 6095 protein, but there are significant variations. The main lipid components are phospholipids, in particular, phosphatidylcholine and phosphatidylethanolamine, and cholesterol. Physicochemical properties and membranes, such as fluidity, can be altered by modifying or fatty acid profiles that are included in the 4! composition of phospholipids or cholesterol content. Modulation of the composition and structure of membrane lipids also leads to modulation of membrane-associated cellular functions, such as receptor activity, endocytosis, and cholesterol uptake. -k 70 V) Enzymes

Triacylglycerol lipases are a family of enzymes that play several key roles in lipid metabolism in the body. Three members of the triacylglycerol lipase family were described: pancreatic lipase, lipoprotein lipase, and liver lipase (Soidrega, I.9., Ge, M.-A., Sipzbrego, N.M., Kgayev, K.M., "I Ipadgep, ET (1988)

U. Siip. Ipmevi., 81, 561-568; ,, soidrego, 1I.9., I e, M., Raiegpii 9.K., Sipzbregao, N.M., I ipadgep, ET. 5 Vhom/p, M.M. (1982) 29 in, Siip. Ipmevi., 70, 1184-1192; Nide, MU.A., Spap, I, 5 1, M/.-N. (1992) 9. Iiriy. Caves. 33, 167-178). Pancreatic

GF) lipase is mainly responsible for the hydrolysis of food lipids. Variants of pancreatic lipase have been described, but their physiological role has not been determined | 267, 16509-16516). Lipoprotein lipase is the main enzyme responsible for the distribution and utilization of triglycerides in the body. Lipoprotein lipase hydrolyzes triglycerides as part of chylomicrons, 60 of which are LDNG. Liver lipase hydrolyzes triglycerides with the formation of LPG and LPH, which is responsible for remodeling lipoproteins. Liver lipase also acts as a phospholipase and hydrolyzes phospholipids to form

LV.

Phospholipases play an important role in the catabolism and remodeling of the phospholipid component of lipoproteins and phospholipids of membranes. Phospholipases also play a role in the release of arachidonic acid with subsequent formation of prostaglandins, leukotrienes, and other lipids that participate in a number of inflammatory processes.

Lipase polypeptides encoded by lipase genes have a sequence length of approximately 450 amino acids with leader signal sequence peptides to facilitate secretion. Lipase proteins consist of two main domains. (1990) Maishge, 343, 771-774). The amino-terminal domain contains the catalytic center, and the carboxy-terminal domain is apparently responsible for binding to the substrate, cofactor attachment and interaction with cell receptors (Muopao, N., YuOamiv, K.S., Mikazgu, 5.,

Seerag, K.E., 5 Zspoi7, M.S. (1991) Rgos. May Asai. si. OBA 88, 11290-11294; map Tiireigoi, N., Koizvei,

A., Iaotsei, 9U.-M., 5 Satbriyats, S (1994) 9. Vioi. Spent 269, 4626-4633; MUopd, N., Oamiv, K.S., Tpygep, T., 70 boegv, 9O.MU., Mikagu, 9U., MMaye, M., 5 5spoi7, M.S. (1994) 9. Vioi. Spent 269, 10319-10323; SparreiI, BA, Ipopshe,

I, Egu, B.G., Riadei, M.MU., Vozhep, 5.Yi, Imegiiv, R.-N., I aiotsei, 9U.-M. 5 5igiskiapa, O.K. (1994) 3. Vioi. Spet., 269, 18001-18006). The overall level of amino acid homology between members of this family is 22-6595, while there are local areas of high homology corresponding to structural homology associated with enzymatic function.

Natural lipoprotein lipase (IRI) is glycosylated and this glycosylation is necessary for IRI enzymatic activity. Y.S., "5 Spap, HER. (1990) 9. Vioi.

Spet., 265, 5429-5433). Liver lipase and lipoprotein lipase have two M-glycosylation sites, and pancreatic lipase has one M-glycosylation site. In addition, four groups of cysteines form disulfide bridges, which are important in maintaining structural integrity to ensure enzymatic activity (So, ..-U., zt, G.S. 5 Spep, SG. (1995) Viospet. Viorpuz. Kev Sottyp. 206, 266-271; Vgadu, Y., Vg2og2omuzKki, A.M., Oegezhshepaa, 2.5., Yuodvop, E., Yuoavzop, O., ToMPeu, 5., TygKeprigo, 9.R.,

SpNgiviiapzep, I, Nide-depzep V., MogeKom, I, Tit, Y, 5 Mepde, ). (1990) Maishge, 343, 767-770.

Members of the triacylglycerol lipase family share a number of common conserved structural features.

One of these features is the "ХХ5ХО" motif, in which the central serine residue is one of the three residues that make up the "catalytic triad" (M/ipKieg, K., S'Agsu, A., "5 Nipgikeg, MU. ( 1990) Maishge, 343, 771-774;

EaiziipeiMya, R., Ztiy, Y.S., 5 Spap, I. (1992) Viospetivighu, 31, 7219-7223). Other residues of the catalytic triad (8) are conservative aspartate and histidine residues. A short section of 19-23 amino acids (the "lid" section) forms an amphipathic helical structure and covers the catalytic "pocket" of the enzyme |MMipKieg, K.,

SUAgsu, A., b NipliKeg, MU. (1990) Mayte, 343, 771-774). This section differs in different members of this Ge! from the family, and recently it was established that this region gives the enzyme substrate specificity (Oitsadi, K.A., bisnek N.G., 5 Zapiatagipa-Bo|o, 5. (1995) 9. Vioi. Spet., 270, 25396-25401|. The comparison of liver lipase and -- lipoprotein lipase showed that the difference in the activities of triacylglycerol lipase and phospholipase is mediated, partly due to the area of the "lid" (Oydi, K.A., Oispek N.G., Zapiatagip -bo|o, 5. (1995) 9. Vioi. Spet., 270, 25396-254011. o

Triacylglycerol lipases have different degrees of heparin-binding activity. Lipoprotein lipase has the highest affinity for heparin, and, according to the mapping, this region of binding activity extends to positively charged residues in the amino-terminal domain (Ma, U., Nepadegvop, N.E., et al. M.-5., 7papo, N., Rogzuiye, 1.9., SiIagke-I em/iv, I., Naudep, M.K., " VgypgeiYii, 9U.O0., U. Pirii Kev., 35, 2049 -2059).

Localization of lipoprotein lipase on the surface of an endothelial cell | (Spepo, S.R., Oovia, S.M., Vepsadotsp, A., 5 « Kozeprego, K.O. (1981) 9. Vioi. Spet., 256, 12893 -12896), is mediated mainly through the binding of c with surface proteoglycans. V.I. (1981) 3.

And Siip. Ipmevi, 68, 995-1002; Bahepa, O., KiIeip, M.O., 5 Soiidrego, I.9., (1991) U. Vioi. Spet., 226, 17516-17521; and? Eiveprego, 5., Zepauek, E., Oyimesgopa, T., 5 MiodameKku, I. (1992) 9. Siip. Ipmevi,, 90, 2013-2021). It is this binding activity that allows the enzyme to accelerate the absorption of LNG, acting as a bridge between LNG and the cell surface. NamekKez, I.M. (1992) Viospet. - And Viorpuz. Kev. Saute 185, 582-587; Kysheide, 9U.S., 5 Soidrego, 1.9. (1994) U. Iiriy Kev. 35. 1152-1160;

Twisnpiua, 5., Hatabre, M., Matadisni, T., Corauavnpi, U., Coppo, T., «« Tada, K. (1980) Ipi. 93. Sapseg. 26, 171-176). o It is known that both lipoprotein lipase and liver lipase function in combination with coactivator proteins: 2) apolipoprotein SP for lipoprotein lipase and colipase for pancreatic lipase.

Genetic sequences encoding pancreatic lipase, liver lipase, and human lipoprotein lipase are described in the literature (SeprapK, registration numbers M93285, 903540, and M15856, respectively). Informational RNAs

He) liver lipase and human pancreatic lipase are approximately 1.7 and 1.8 thousand base pairs long, respectively.

Two mRNA transcripts with a length of 3.6 and 3.2 thousand base pairs are transcribed from the human lipoprotein lipase gene.

These two transcripts use alternative polyadenylation signals and differ in their translation efficiency |Kapdapainap, B., Op, 9U.M., MykKni, A., Zadpigaden, M., Zitzvoio, K.V., Ratseg, A., Kept, R.A. (1995) 9. Axis. Spet., 270, 7149-7155).

F) C) Physiological processes. Lipid metabolism is carried out through the interaction of lipids, apoproteins, lipoproteins and enzymes.

Liver lipase and lipoprotein lipase are multifunctional proteins that mediate binding, utilization, catabolism and remodeling of lipoproteins and phospholipids. Lipoprotein lipase and liver lipase function by binding to the luminal surface of endothelial cells in peripheral tissues and in the liver, respectively. Both of these enzymes are involved in the reverse transport of cholesterol, which consists in the transfer of cholesterol from peripheral tissues to the liver either for excretion from the body or for a repeated metabolic cycle. It is known that genetic defects of both liver lipase and 65 lipoprotein lipase cause hereditary disorders of lipoprotein metabolism. Defects in lipoprotein metabolism lead to serious metabolic disorders, including hypercholesterolemia, hyperlipidemia, and atherosclerosis.

Atherosclerosis is a complex polygenic disease, which, from a histological point of view, is defined as deposition (lipid or fibrolipid plaques) of lipids and other blood derivatives on the walls of blood vessels, especially large arteries (in the aorta, coronary artery, carotid artery). These plaques, which are more or less calcified according to the stage of the atherosclerotic process, can be associated with lesions and associated with the accumulation of fatty deposits in blood vessels, which consist mainly of cholesterol esters. These plaques are accompanied by thickening of the blood vessel wall, smooth muscle hypertrophy, the appearance of "foamy" cells (lipid-laden cells that are formed as a result of unregulated absorption of cholesterol by "recruited" macrophages) and the accumulation of fibrous tissue. Atheromatous plaques are prominent on the vessel wall, which gives them stenosing properties, which are responsible for vessel occlusion due to atheroma, thrombosis or embolism, which develops in those patients who are most susceptible to these lesions. These lesions can lead to serious cardiovascular pathologies, such as heart attack, sudden death, heart failure and stroke.

The role of triacylglycerol-lipases in vascular pathologies, such as atherosclerosis, is the object of intensive research | see, review (S., 5 Oihimesgopa, p., 5 Oihimesgtopa, T. (1995) Syp. Orip. Giriya b, 291 -305). Basically, the action of triacylglycerol lipases is apparently antiatherogenic, since these enzymes reduce the level of triacylglycerol in the serum and stimulate the formation of LVH. Transgenic animals expressing lipoprotein lipase or human liver lipase showed reduced levels of plasma triglycerides and increased levels of high-density lipoproteins (HDL) , M.,

Ipara, T., Magaki, T., 5 Matada, M. (1993) 9. Vioi. Spent 268, 17924-17929; ish, M.-5., Chk, E.K., and eVoetsi,

N.S., Nepaegzop, N., Sazieyiapi, I.M., Iviv, A. 9., Ma, U., RoggzuiPe, 1.9U., 2papao, N., Kigk, E., VgypgeiY, 9U. O., 5 Naudep,

M.K. (1994) U. Vioi. Spent 269, 11417-11424). It was established that people with genetic defects that lead to a decrease in the level of lipoprotein lipase activity are prone to hypertriglyceridemia, but without the condition of an increased risk of coronary heart disease. It was reported that this is due to the lack of production of atherogenic lipoproteins of intermediate sizes, which can accumulate in the subendothelial and) space (Imegetii, Yu.V. (1973) Sigs. Kev. 33, 633-638).

However, it was suggested that in a localized area of atherosclerotic lesions, the increased level of lipase activity accelerates the atherogenic process of hl/liimegetiya O.V. (1995) Sep. Spent 41, 153-158; Gee! zo 7atrop, A., Toevs, A., Vi)moei, 5., Sapde, S, Mooiapi, 5., Iriep, R.)., Naudep M.K., 5 VgypgeiiI, 9.0. (1993)

Iapsei 341, 1119-1121). This may be due to an increase in the binding and absorption of lipoproteins by vascular tissue, which is mediated by lipases (Eiseprego, 5., Zepauek, E., OiYimesgopa, T., Miodauzku, I. p. (1992) 9. Siip. Ipmevi. 90 , 2013-2021; Tarav, I., (1, I., Vgosia K.MU., Khy, 5.MU., Zmepvop, T.Yi, MMShiatv, K.). (1993) 9. Vioi. Spet. 268, 20419-20432; Mogaezidaaga, V.O., 5 Mieivep, A.O. (1994) Sit. Orip. ) Ay. Tigotr. Apa Maze. Vioi 15, 551-561). In addition, a high local level of lipase activity can lead to cytotoxic levels of fatty acids and lysophosphatidylcholine, which is produced in the precursors of atherosclerotic lesions.

Despite the emergence of a large amount of information about the role played by lipase activity in lipid homeostasis, however, the need to identify additional genes that encode proteins and regulate "lipid metabolism" remains relevant. This invention relates to the detection of a gene similar to the lipase gene (10), to the polypeptide expression products of this gene, as well as to compositions and methods of their use. (| C-polypeptide binds to heparin, has homology to lipoprotein lipase and human liver lipase and includes the catalytic ZOkDa domain of the triacylglycerol lipase family. In another embodiment of the invention, this polypeptide has phospholipase A activity. -I This invention belongs to the isolated polypeptide containing the sequence ZEO IU Mo: 10.

In addition, this invention relates to an isolated polypeptide containing the sequence ZEO IJOO Mo: 8 and having an apparent molecular weight of about 55kDa or b8kDa on a 1095 SDS-PAGE gel. 2) This invention also relates to an isolated polypeptide containing the sequence ZEO IJOO Mo: 6 and having an apparent 5p molecular weight of about 40 kDa on a 1095 SDS-PAGE gel. - In addition, this invention belongs to the antigenic fragment II and c-polypeptide.

Ge) In another aspect, the present invention relates to an isolated nucleic acid encoding a polypeptide having the above sequence.

In another aspect, the present invention relates to a vector comprising the above nucleic acid encoding said polypeptide properly linked to a regulatory region such as a promoter.

In another aspect, this invention relates to a recombinant cell that includes the above (F, vector. ka In another aspect, this invention relates to a method for producing a polypeptide, which involves the cultivation of recombinant cells containing a nucleic acid that encodes the specified polypeptide , in conditions that contribute to the expression of the indicated polypeptide.

In another aspect, this invention relates to an antibody capable of specifically binding to the polypeptides of the present invention and/or neutralizing the biological activity of these polypeptides. Indeed, an additional property of the polypeptide of the present invention is that it specifically binds to the antibody of the present invention, that is, to an antibody that is specific for I | b-polypeptide. 65 In another aspect, this invention relates to a composition comprising a polypeptide, nucleic acid, vector, antisense nucleic acid or antibody of the present invention and a pharmaceutically acceptable carrier.

In another aspect, this invention relates to a method of screening for the purpose of detecting agonists or antagonists of the enzymatic activity possessed by the polypeptides of the present invention, wherein said method involves contacting potential agonists or antagonists with said polypeptides and their substrate, and measuring the ability of potential agonists or antagonists to enhance or inhibit this activity.

In another aspect, this invention relates to a method of enzymatic hydrolysis of a phosphatidylcholine ester, which involves contacting the indicated phosphatidylcholine ester with the polypeptide of the present invention.

In another aspect, this invention relates to a method of treating a person or an animal for the purpose of improving the profile of lipid content in the serum of a given person or animal having an undesirable lipid profile, where the specified method involves the introduction of an effective amount of the composition of the present invention.

In another aspect, this invention relates to a method of treating or preventing atherosclerosis in a person or animal, which involves administering to a given person or animal an effective amount of the composition of the present invention.

Other aspects and advantages of the present invention, in addition, are illustrated in the drawings and in the detailed description of preferred variants of its implementation.

Figure 1 shows the sequences (ZEO IJOO Mo: 17-31) of primers used for experimental

RSK amplifications.

Figure 2 shows the nucleotide sequence (ZEO IU Mo: 1) and deduced amino acid sequence (ZE 10 Mo: 2) of the CT-RSK reaction product (carried out by the method of differential mapping) containing a cDNA gene similar to the lipase gene. The sequences corresponding to the two primers used in the amplification are underlined. The stop codon and polyadenylation signal are boxed. The SAATTS motifs and flanking sequence are derived from the rec vector into which this product was cloned.

Figure 3 shows the nucleotide sequence (ZEO IU Mo: 3) and the deduced amino acid sequence (ZEO c

IU Mo: 4) 5KASE-elongation of cDNA for 1/0. The sequences corresponding to the two primers used in the amplification are underlined. The SAATTS motifs and flanking sequence are derived from i) the recI vector into which this product was cloned.

Figure 4 shows the sequence (ZEO IU Mo: 7) of cDNA containing the complete open reading frame of a lipase-like gene, GI OX. The start codon (ATO) and the stop codon (TAS) are marked with a frame. YuOga1-site (T'TAAA) Gee! zo tazt -site (ЗСССОоООС), used in the construction of expressing vectors, are underlined.

Fig. 5 shows the deduced amino acid sequence (ZESO IO Mo: 8) of protein I OXY. The intended (87 signal sequence is underlined. p

Figure 6 shows a comparison of the primary sequences of members of the triacylglycerol-lipase gene family (ZEO

IU Mo:13-15)3. The shaded residues are identical to the protein THEM! (5EO I Mo: 8). Breaks were introduced into the sequences to maximize comparison of the primary sequences using the SI OZTAI program. uh-

Figure 7 shows Northern analysis of II C mRNA in TNR-1 cells. Cells were stimulated either with PMA, or PMA and oxidized I OH (PMA «i oxid. I 0). The numbers on the left indicate the position of the RNA standards (thousand base pairs).

Figure 8 shows the Northern analysis of mRNA from a variety of human tissues probed using cDNA II, lipoprotein lipase (PI) and human beta-actin. The provisions of 4.4 t.p.o. - The RNA standard is indicated to the left of the panels « 20 5 and GRI. in c Figure 9 shows the Northern analysis of the expression of 1/0 and IR in cultured endothelial cells and human TNR-1 cells. These cells were either unstimulated (not treated with PMA) or stimulated with PMA. ;" Fig. 10 shows the sequence of the immunizing peptide (ZEO IUO Mo: 16) and its relationship with the sequence of protein I! OKI This peptide is shown in a shaded box. The terminal cysteine was introduced by attaching the peptide to the carrier protein. -I Figure 11 shows Western analysis of proteins concentrated on heparin-sepharose from the conditioned medium of cultured endothelial cells. The blot was probed using an antiserum against oGB. The numbers on the left indicate the position of standard proteins in kilodaltons. 2) Figure 12 shows Western analysis of proteins bound to heparin-Sepharose from conditioned medium 5or from SOZ-7 cells transiently transfected with an expression vector containing cDNA for POM or || OCI, - or does not contain DNA (Control). Proteins from PMA-stimulated endothelial cells (NSAES and PMA) were

Ge) are included for size comparison. Numbers on the left indicate the apparent molecular weight of the main immunoreactive proteins, determined by comparison with standard proteins.

Fig. 13 shows the sequence of the PCR product of rabbit GO (KI, ZEBEO IU Mo: 12) and the comparison of the primary sequences of the PCR product of rabbit II and the corresponding human cDNA sequence (II 57742A).

Identical nucleotides are shaded.

Ф) Fig. 14 illustrates the phospholipase (A) activity of IRI, IM, and IYOHI. using ka phosphatidylcholine substrate.

Fig. 15 illustrates the phospholipase (A) activity of RI, OM and GI OX. with the use of trioleic boron substrate.

Fig. 16 shows the hybridization of I| (5 and 1 CI probes for genomic DNA originating from different species.

This invention relates to the detection of a gene similar to the lipase gene (1/0) and to the polypeptide products of its expression. These polypeptide products, members of the triacylglycerol lipase family, contain a catalytic domain of approximately 30 kDa, characteristic of the triacylglycerol lipase family, for example, having the sequence ZEO IU 65 Mo: 10. In one of its variants, the present invention relates to a GOM polypeptide having 354 amino acids. In another version, this invention belongs to the GI OX polypeptide, which contains 500 amino acids and has 43905 similarity with human lipoprotein lipase and 3795 similarity with human liver lipase. GI OH polypeptide. has phospholipase (A) activity.

The authors of the present invention isolated incomplete cDNA from the mRNA of TNR-1 cells that were treated with phorbol ester and oxidized LNG. After 5'CASE-elongation of this incomplete cDNA, a slightly smaller alternatively spliced cDNA was isolated. Another larger cDNA was isolated from a human placenta cDNA library.

Northern analysis showed that the GHIZ gene is expressed in endothelial cells. The antiserum produced against the peptide predicted on the basis of the open reading frame of cDNA allowed to find proteins of the expected sizes for IM and I OCI in the conditioned medium from cultured endothelial cells. 70 Treatment of endothelial cells with phorbol esters leads to increased production

Go both at the level of mRNA and at the level of proteins. This protein is the first member of the triacylglycerol lipase family to be found to be expressed by endothelial cells.

A) Definition

Below is a definition of the terms used in the description of the present invention, which may be 7/5 Useful for understanding the essence and application of the present invention. "Polypeptide" is a polymeric compound consisting of covalently linked amino acid residues. Amino acids have the following general structure:

N

' k- 35 5 - с2ОоОоНн сч | about "-

Amino acids have been divided into seven groups according to their K side chains: (1) aliphatic C side chains, (2) side chains containing a hydroxyl group (OH), (3) side chains containing sulfur atoms, (4) side chains containing acidic or amide groups, (5) side chains containing basic groups, (6) side chains containing an aromatic ring, and (7) proline, an amino acid in which the side chain is linked to amino group. "Protein" is a polypeptide that plays a certain structural or functional role in a living cell.

Polypeptides and proteins of the present invention can be glycosylated or non-glycosylated.

The term "homology" refers to the similarity of sequences that reflects their common evolutionary origin. "

Polypeptides or proteins are said to have homology or similarity when a significant number of their amino acids are either (1) identical, or (2) have a chemically similar side chain K. Nucleic acids are said to have homology when a significant number of their nucleotides are identical "Separated polypeptide" or "isolated protein is a polypeptide or protein that is essentially free of those compounds that are normally associated with them in nature (eg, other proteins or polypeptides, nucleic acids, carbohydrates, lipids). The term "isolated" does not mean that it excludes artificial -I or synthetic mixtures with other compounds, or the presence of impurities that do not affect biological activity and which may be present, for example, due to incomplete purification, addition of stabilizers or the manufacture of pharmaceutically acceptable preparations. 2) A molecule is "antigenic" if it is able to specifically interact with an antigen-recognition molecule of the immune system, such as an immunoglobulin (antibody) or a receptor for a T-cell antigen Antigenic - a polypeptide contains at least about 5, and preferably, at least about 10 amino acids The antigenic region of the molecule

Ge) may be the site that is immunodominant for the antibody or for T-cell receptor recognition, or it may be the site used to produce an antibody to a given molecule by conjugating the antigenic site to a carrier molecule for immunization. A molecule that is an antigen does not necessarily have to be immunogenic itself, that is, capable of inducing an immune response in the absence of a carrier. "Polypeptide GOM" and "protein G.M" refer to a polypeptide comprising the sequence ZEO IJOO Mo: 6, where (F, the indicated polypeptide is glycosylated or non-glycosylated. ka "Polypeptide I OXI" and "protein OX" refer to a polypeptide comprising the sequence of ZEO IU Mo: 8, wherein said polypeptide is glycosylated or non-glycosylated, and "| b-polypeptide" mainly belongs to both polypeptide GI OM and polypeptide I G OX.

And the C-polypeptide or protein of the present invention is any analogue, fragment, derivative or mutant derived from the GI C-polypeptide and which retains at least one biological property of the GI polypeptide. In nature, there are various variants of the GI "z-polypeptide." These variants can be allelic variants, which are characterized by differences in the nucleotide sequences of the structural gene encoding this protein, or 65 They can differ by splicing or by post-translational modification. Each specialist can independently obtain variants having one or many amino acid substitutions, deletions, additions or permutations. Such variants can be, ipieg aiya, (a) variants in which one or more amino acid residues are replaced by conservative or non-conservative amino acids, (B) variants in which one or more amino acids are added to II C-polypeptide, (c) variants, in which one or more amino acids

Include a replacement group and (4) variants in which the GI bC polypeptide is linked to another polypeptide, such as serum albumin. Other GI Z-polypeptides of the present invention are variants in which amino acid residues originating from one species are replaced by corresponding amino acid residues from another species either in conservative or non-conservative positions. In another embodiment of the invention, amino acid residues in non-conservative positions are replaced by conservative or non-conservative residues. Methods of obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical and enzymatic methods, are known to any average person skilled in the art.

If such allelic variants, analogs, fragments, derivatives, mutants and modifications, including alternative forms of mRNA splicing and alternative forms of post-translational modification, result in the production of derivatives of II C polypeptide that retain any of the biological properties of this I (with -polypeptide, then all 7/5 They are included in the scope of the present invention. "Nucleic acid" is a polymeric compound consisting of covalently linked subunits called nucleotides. Nucleic acids are polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which can be single-stranded or double-stranded.

DNA can be cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA. The sequence of nucleotides that encodes a protein is called the content sequence. "Antisense nucleic acid" is a sequence of nucleotides that is complementary to the content sequence. The antisense sequence can be used to inhibit or block the expression of the polypeptide encoded by the sense chain.

The term "isolated nucleic acid" refers to a nucleic acid that is essentially free of compounds that are normally associated with it under natural conditions. The term "isolated" does not mean that it excludes artificial or synthetic mixtures with other compounds, or the presence of impurities that do not affect the biological activity and i) which may be present, for example, due to incomplete purification, the addition of stabilizers or the manufacture of pharmaceutically acceptable preparations.

The phrase "nucleic acid that hybridizes under high stringency conditions" means that the hybridized Ge!

Z Nucleic acids are able to withstand washing under conditions of high rigidity. An example of washing under conditions of high rigidity for DNA-DNA hybrids is washing 0.1 x З55С, 0.595 DSN at 682С. Other washings in conditions of high hardness are known to specialists. co

The term "regulatory region" refers to a nucleic acid sequence that regulates the expression of the nucleic acid. The regulatory region may include sequences that by their nature are responsible for the expression of a specific amino acid (homologous region), or it may include sequences of diverse origin (responsible for the expression of different proteins or even synthetic proteins). In particular, such sequences can be sequences of eukaryotic genes or viral genes, or sequences derived from them that stimulate or inhibit gene transcription in a specific or non-specific manner and in an induced or non-inducible manner. Regulatory sites include replication initiation sites, RNA splicing sites, enhancers, transcription termination sequences, signal sequences directing the shsh c polypeptide through the secretory pathway to the target cell, and promoters. and A regulatory region from a "heterologous source" is a regulatory region that under natural conditions "" is not associated with the expressed nucleic acid. Heterologous regulatory regions include regulatory regions of individuals of another species, regulatory regions of another gene, hybrid regulatory sequences and regulatory sequences , which do not occur in nature, but which have been constructed artificially. -i The term "vector" means any sequence designed to carry the nucleic acid of the present invention into a host cell. The term "vector" includes vectors of viral and non-viral origin, which others are used to introduce nucleic acid into a prokaryotic or eukaryotic cell ip miigr, ex mimo

Ge) or ip mimo. Vectors of non-viral origin are plasmids, liposomes, lipids with electric charge (cytofectins), DNA-protein complexes and biological polymers. Viral vectors are retroviruses, - adeno-associated viruses, vectors based on poxviruses, baculoviruses, vaccinia viruses, viruses

Ge) herpes simplex, Epstein-Barr viruses and adenoviruses. In addition to the nucleic acid of the present invention, the vector may also contain one or more regulatory regions and/or selective markers used for selection, measurement and monitoring of nucleic acid transfer results (tissue transfer, duration of expression, etc.). A "recombinant cell" is a cell containing a nucleic acid that is not naturally present in that cell. The term "recombinant cell" includes higher eukaryotic cells such as mammalian cells, lower eukaryotic cells such as yeast cells, prokaryotic cells and archaebacterial cells. "Pharmaceutically acceptable carrier" includes diluents and excipients that are pharmaceutically acceptable for this method of administration and sterile, and may be aqueous or oily suspensions prepared using suitable dispersing or wetting agents and suspending agents. The choice of a specific pharmaceutically acceptable carrier and the ratio of the content of the active compound to the content of this carrier is determined by the solubility and chemical properties of the composition, the specific method of administration and standard pharmaceutical practice. 65 "Lipase" is a protein that can enzymatically split a lipid substrate. "Phospholipase" is a protein that can enzymatically cleave a phospholipid substrate.

"Triacylglycerol lipase" is a protein that can enzymatically cleave the triacylglycerol substrate. "Phosphatidylcholine" is a glycerol-containing phospholipid with the following structure: » i-s-o-sn 2 v- 0-5 0-on . | | Oh! z o 0no-o-v-o-sny-sny--Mo 2 and M NI o where K and K' are hydrocarbon side chains of fatty acids. Phosphatidylcholine is also known as lecithin.

The term "lipid profile" refers to a series of concentrations of cholesterol, triglyceride, lipoprotein cholesterol, and other lipids in a human or animal body. "Undesirable lipid profile" is a condition in which the concentrations of cholesterol, triglyceride or o) lipoprotein cholesterol are outside the standard limits that are determined for this age and sex.

Of course, concentrations of total cholesterol of 220Omg/dl, plasma triglycerides of 20Omg/dl, LDL cholesterol of 13Omg/dl, LDL cholesterol of ZOmg/dl or a ratio of total cholesterol to LDL cholesterol of 24.0 are considered undesirable lipid profile. An adverse lipid profile is associated with a number of pathological conditions, including hyperlipidemia, diabetic hypercholesterolemia, atherosclerosis, and other forms of coronary heart disease. co

B) Polypeptides

This invention belongs to polypeptides that are members of the triglyceride-lipase family and which include i) catalytic ZOkDa domain of the triacylglycerol-lipase family, for example, a domain having the sequence ZEO IU Mo:im 10. One of the variants of the implementation of the present invention is isolated I | b-polypeptide containing the sequence ZEO IU

Mo: 6 and has an apparent molecular mass of about 40 kDa on a 1095 PAAG-DSN gel. Another variant of the implementation of the present invention is the isolated II b-polypeptide containing the sequence ZEO IU Mo:8 and having an apparent molecular weight of about 55 kDa or b8 kDa on a 1095 PAGE-DSN-gel. Polypeptides and proteins of the present invention may be recombinant polypeptides, natural 3s polypeptides or synthetic polypeptides and may originate from humans, rabbits or other animals.

These polypeptides are characterized by reproduced single molecular mass and/or multiple molecular masses, chromatographic reactions and elution profiles, amino acid composition and amino acid sequence, as well as biological activity.

The polypeptides of the present invention can be isolated from natural sources, such as placental extracts, human plasma, or conditioned media from cultured cells, such as macrophages or endothelial cells, using purification procedures well known to those skilled in the art. 1 Alternatively, the polypeptides of the present invention can be obtained using the recombinant DNA method, which involves inserting the nucleic acid encoding this polypeptide into a suitable vector, introducing the resulting vector into a suitable host cell, isolating the polypeptide produced by the resulting host cell and purification of this isolated polypeptide.

Ge) C) Nucleic acids

This invention belongs to isolated nucleic acids encoding I and b-polypeptides.

This invention also belongs to antisense nucleic acids, which can be used to suppress or block the expression of II polypeptides ip miigo, ek mimo or ip mimo.

Recombinant DNA techniques are known to anyone skilled in the art. General methods of cloning and expression (F) of recombinant molecules are described in the guide Mapiaiyz |Moiesshiag Siopipd, Soi4 Zrgipd Nagbrog Iaroghaiyogiyev, ko 19821, and A!yzibei! (Sigtepi Rgoiosoiiv ip Moiesciag Vioodu, MUPeu apa Zopz, 1987), which are incorporated into this description by reference. The nucleic acids of the present invention can be connected to one or more regulatory sites.

The selection of the appropriate regulatory site or regulatory sites is a routine method available to any average specialist. Regulatory regions include promoters and may include enhancers, suppressors, and the like.

Promoters that can be used in the present invention are constitutive promoters and β5-regulated (inducible) promoters. These promoters can be prokaryotic or eukaryotic depending on the host. Prokaryotic promoters (including bacteriophage promoters) that can be used for the purposes of the present invention are ad, Ias/, TZ, T7, lambda P,, RI and ig promoters. Eukaryotic promoters (including viral promoters) that can be used for the purposes of the present invention are constitutive promoters (for example, promoters of HRET, vimentin, actin, tubulin); promoters of intermediate filaments (for example, promoters of desmin, neurofilaments, keratin, OEAR); promoters of therapeutic genes (for example, such as IOC,

SETK, MII factor); tissue-specific promoters (for example, the actin promoter in smooth muscle cells or the promoters of Рии and РИК, active in endothelial cells); promoters, which are mainly activated in dividing cells; promoters that are responsive to stimulants (eg, steroid hormone receptor, retinoic acid receptor); transcription modulators regulated by tetracycline; early promoters of 7/0 Cytomegalovirus; long terminal repeats (LTRs) of retroviruses; metallothionein promoters; ZM40 promoters; EIIa promoters and MI promoters. Tetracycline-regulated transcription modulators and SMM promoters are described in MO 96/01313, US Patents 5,168,062 and 5,385,839, the contents of which are incorporated herein by reference.

Viral vectors used in gene therapy are preferably replication-defective, ie, preferably unable to replicate autonomously in target cells. Basically, the genome of the replication-defective viral vectors used in the present invention lacks at least one region necessary for viral replication in infected cells. These sites can either be eliminated (in whole or in part) or rendered non-functional by known means. These methods involve complete removal, replacement (with other sequences, in particular inserted nucleic acid), partial deletion or addition

ONE or more bases to the main (for replication) site. These methods can be carried out by ip mygo (on isolated DNA) or ip zysh, using the technique of gene modification or by treatment with mutagenic agents.

At the same time, it is preferable that a virus defective in replication retains the sequences of its genome, which are necessary for the formation of the capsid of viral particles. with

Retroviruses are viruses that integrate and infect dividing cells. The retrovirus genome includes two I TCs (long terminal repeats), a sequence responsible for capsid formation, and i) three coding regions (dad, ro! and epu). The construction of recombinant retroviral vectors was described:

See, in particular, ER 453242, ER 178220, Vepziewip ei aiYi., Sepei. Epo. 7 (1985) 235; MsSogtisk, VioTesppoiodu Z (1985) 689, etc. In recombinant retroviral vectors, the dad, roI and epu genes are usually deleted, Ge! zo in whole or in part, and replaced by the desired heterologous nucleic acid sequence. These vectors can be constructed from retroviruses of various types, such as MoMy! M ("Moloney murine leukemia virus"), --

MM ("mouse Moloney sarcoma virus"), Nazm ("Harvey sarcoma virus"); MM ("splenic necrosis virus"); KZM c (Rau's sarcoma virus") and Freund's virus.

Mainly, for the construction of recombinant retroviruses containing sequences that encode o

AND! O-polypeptide of the present invention, a plasmid containing I TC, the sequence responsible for the formation of the capsid, and the coding sequence was constructed. This construction was used for transfection of a packaging cell line capable of providing in trans the retroviral functions that are absent in the plasmid. Thus, packaging cell lines are mainly capable of expressing the dad, roi, and epu genes. Such packaging cell lines were described earlier, and, in particular, the RAZI17 cell line (054861719); cellular "

The RviSkIR line (UMO 90/02806), and the bRzhepuAt-12 cell line (MUO 89/07150). In addition, recombinant z c retroviral vectors may contain modifications in GTK to inhibit transcriptional activity, as well as elongation of the sequence responsible for capsid formation and which may include part of the dad gene;" IVepdeg ei aiYi., 9. Migoi, 61 (1987) 1639). Recombinant retroviral vectors are purified using standard techniques well known in the art.

Adeno-associated viruses (AAVs) are relatively small DNA-containing viruses that can integrate in a stable and site-specific manner into the genome of the cells they infect. These viruses are able to infect a wide range of cells without inducing any effect on the growth, morphology or differentiation of cells, and they apparently do not cause any pathologies in humans. The AAV genome was cloned, 2) sequenced and characterized. It contains approximately 4700 bases and contains a region of inverted terminal 5p repeats (ITCs) of approximately 145 bases at each end, which is the site of initiation of viral replication. The rest of the genome is divided into two main sections that have the functions of capsid formation: the left-hand part of the genome, which

Ge) contains the gene ger, which takes part in virus replication and expression of viral genes; and the right-hand part of the genome, which contains the sar gene, which encodes the capsid proteins of the virus.

The use of vectors derived from AAV for the transfer of ip myo and ip mimo genes is described in works in Idiv. UUO91/18088, UUO93/09239; US Patent 4,797,368; ER 488528). These publications describe a variety of AAB-derived constructs in which the her and/or sar genes have been delegated and replaced with the desired gene,

Ф) as well as the use of these constructions for the transfer of the indicated desired gene into the cultured cells or into the body (directly into the body). Replication-defective recombinant AAV of the present invention can be obtained by transfection of a plasmid containing the required nucleic acid sequence flanked by two regions of inverted terminal repeats (ITK) of AAV, and a plasmid carrying the genes responsible for the formation of the AAV capsid (ger and sar genes ), into a cell line infected with a human helper virus (eg, adenovirus). Then, the produced recombinant AAVs are purified by standard methods. Therefore, this invention relates to an AAB-derived recombinant virus, the genome of which includes the coding sequence

And O-polypeptide flanked by AAV-ITK. This invention also relates to a plasmid that includes a sequence that 65 encodes an I O-polypeptide flanked by two TKIs from AAB. Such a plasmid can also be used to transfer the GO sequence, and this plasmid, if necessary, can be introduced into a liposomal vector (pseudovirus).

In a preferred embodiment of the present invention, such a vector is an adenoviral vector.

Adenoviruses are eukaryotic DNA viruses that can be modified to deliver nucleic

Acids of the present invention in cells of various types.

There are different serotypes of adenoviruses. From these serotypes, from the point of view of the present invention, it is better to use human adenoviruses of type 2 or 5 (Ад2 or Авд5) or adenoviruses that originate from animals (see

MO094/26914). Such adenoviruses of animal origin that can be used in the present invention are adenoviruses of dogs, cows, mice (e.g., Mami, Veaga et al., Migoyudu 75 (1990) 81), sheep, pigs, birds, and monkeys (e.g., ZAM ). Preferred animal-derived adenoviruses are canine adenoviruses, preferably adenovirus CAM2, such as the Manhattan strain, or the A26/61 strain, such as ATS MIK-8001.

Preferred replication-defective adenoviral vectors of the present invention contain ITK, the capsid formation sequence and the required nucleic acid. Even better, at least the E1 region of the adenoviral vector is non-functional. At the same time, it is better that the deletion in the EI region covers the region from nucleotide 455 to /5 Nucleotide 3329 in the sequence of adenovirus Aa5. Other plots may also be modified, in particular, plot EZ (МИО95/02697), plot E2 (УУО94/28938), plot E4 (М/О94/28152, МО094/12649 and М/О95/02697) or plot in any from late genes I 1-I 5. Defective retroviral vectors are described in UMO95/02697.

In a preferred embodiment of the invention, the adenoviral vector has a deletion in the E1 and E4 regions. In another preferred embodiment of the invention, the adenoviral vector has a deletion in the ET region into which the E4 region and the GI o coding sequence have been incorporated (see EC94 13355).

Replication-defective recombinant adenoviruses of the present invention can be obtained by any method known to specialists (I emgego ei aiY., bSepe 101 (1991) 195, ER 185573; Stgapat, EMVO .., Z (1984) 2917).

In particular, they can be obtained by homologous recombination between an adenovirus and a plasmid that contains, i.e., the required DNA sequence. This homologous recombination is carried out after cotransfection of cells of the specified adenovirus and plasmid into the appropriate cell line. The cell line used should preferably be such that (i) it is transformed by the specified elements and (ii) it contains sequences complementary to and) parts of the genome of the replication-defective adenovirus, preferably in an integrated form to eliminate the risk of recombination. Examples of cell lines that can be used are the kidney cell line of a human embryo 293 |OSgapat ei ai., 9). Sept. Migoi 36 (1977) 59), which contains the left-hand part of the genome of adenovirus Ge! zo La5 (1290), integrated into its genome; and cell lines capable of complementing E1 and E4 functions, as described in applications U/O094/26914 and UM/095/02697. Recombinant adenoviruses were isolated and purified using standard molecular biology techniques well known to those skilled in the art. with

Inhibition of gene expression using antisense nucleic acids can be achieved at the translational or transcriptional level. The antisense nucleic acids of this invention are preferably fragments of nucleic acids capable of specifically hybridizing with all or part of the nucleic acid encoding GI or the corresponding messenger RNA. These antisense nucleic acids can be synthetic oligonucleotides, optionally modified to increase their stability and selectivity. They can also represent DNA sequences, the expression of which in the cell leads to the production of RNA complementary to all or part of mRNA II 0. Antisense nucleic acids can be obtained by expressing all or part of a sequence selected from the group consisting of 5EO IU Mo: 2, with c ZEO IJU Mo: 3, ZEO IJUO Mo: 7 or ZEO IO Mo: 11, and is in the opposite orientation as described in EP 140308. . An antisense sequence of any length is suitable for the implementation of this invention, provided that it is capable of inhibiting or blocking expression (1/0. It is preferable that the length of the antisense sequence is at least 20 nucleotides. The preparation and use of antisense nucleic acids, DNA encoding antisense RNAs, and the use of oligo- and gene antisense sequences are described in the application -I M/092/15680, the content of which is included in this description by reference.

Or) Antibodies o This invention belongs to antibodies against GI polypeptide. These antibodies can be monoclonal or 2) polyclonal antibodies. The present invention includes chimeric, single-chain, "humanized" antibodies, as well as 5o Eab fragments and expressed Rar fragment library products. - Polyclonal antibodies against antigenic fragment I can be obtained C-polypeptide, as described in

Ge) Example 4A. Antibodies can also be produced against an intact (GI) protein or polypeptide, or against a fragment, derivative or epitope of this protein or polypeptide. Antibodies can be produced after administration of the protein, polypeptide, fragment, derivative or epitope to an animal using techniques and procedures, in Known to specialists.

Monoclonal antibodies can be obtained by the method of Mizpei, V.V., ei ai., Zeiesei Meipodz ip Seiyshag

F) Ittypoiodu |MU.N. Egetap, ed., edited by Egapsizso (1980)). For this purpose, the polypeptide of the present invention is used for the immunization of cells of the spleen of mice VAIV/s. Immunized cells of the spleen are subjected to fusion with myeloma cells. Fusion cells, which have the properties of spleen cells and myeloma cells, are isolated by Cultivation in NAT medium, that is, in an environment in which both parent cells die, but the products of the fusion cells survive and grow.

Monoclonal antibodies of the present invention can be "humanized" to prevent the host from producing an immune response to these antibodies. "Humanized antibody" is an antibody in which the hypervariable regions (which determine the complementarity of the region - SOC) and/or other regions of the framework variable domains 65 of the light and/or heavy chain are derived from an immunoglobulin other than human, and other regions of the molecule are derived from one or several human immunoglobulins. "Humanized" antibodies are also antibodies characterized by having a "humanized" heavy chain associated with a donor or acceptor unmodified light chain or with a chimeric light chain, or vice versa. "Humanization" of antibodies can be carried out by methods known to specialists | see, for example, S.E. Mag 4 EA, Radiap, "Sparieg 4.

Nytapigayop Oi Moposiopa! Apiiirodiev", Te NapdrookK oi Ekhregitepia! Rpagtasoioudu Moi. 113, Zrhipdeg-Megiad,

Mem Hogk, 1994). Transgenic animals can be used to produce "humanized" antibodies.

For the production of single-chain antibodies against the immunogenic polypeptides and proteins of the present invention, methods known to specialists and used for the production of single-chain antibodies can be adapted. 70 Antibodies against GI b can be used in assays to detect or quantify levels (I (5.

In one of the variants of implementation of the present invention, these analyzes will make it possible to carry out clinical diagnosis and evaluation of the levels of HER at different stages of the disease and to monitor the effectiveness of treatment.

E) Screening methods for detecting agonists or antagonists

This invention relates to methods of screening libraries of small molecules or sources of natural products /5 to identify agonists (enhancers or coactivators, including protein coactivators) or antagonists (inhibitors) of GI OCI activity. The putative agonist or antagonist is brought into contact with the protein OX and the GI OX substrate, and the ability of the putative agonist or antagonist to enhance or inhibit the activity of GI OX is evaluated.

The II OX protein used in this method can be produced recombinantly in a number of host cells, including mammalian cells (as shown in Example 7), cells infected with baculovirus, yeast and bacteria. Expression | in stably transfected cells, CHO can be optimized by methotrexate amplification of these cells. G/OCHI protein can also be isolated from natural sources, such as human plasma, placental extracts, or conditioned medium from cultured endothelial cells, TNR-1 cells, or macrophages. high school

Optimization of analytical parameters, including pH, ion concentration, temperature, substrate concentration and emulsification conditions, can be carried out empirically by any expert. and)

Fatty acid substitutes for substrates can vary in chain length, as well as in the degree and position of unsaturation. Substrates can be labeled with a radioactive label in any of several positions. Phospholipid substrates, such as phosphatidylcholine, can be subjected to radioactive labeling, Ge! for example, in the position of fatty acids Zp-1 or zZp-2, or in the glycerol, phosphate or polar head group (choline in the case of phosphatidylcholine). --

As an alternative to radioactive substrates, other classes of labeled substrates, such as fluorescent substrates or thiocontaining substrates, can also be used in screening methods.

Fluorescent substrates are particularly suitable for use in screening assays, since in this case enzymatic catalysis can be assessed continuously by measuring fluorescence intensity without resorting to physical separation (extraction) of products from substrates. An example of a fluorescent phosphatidylcholine substrate is

SeMvO-POI1-acyl-2-|6-(nitro-2,1,3-benzoxadiazol-4-yl)-amino|)caproylphosphatidylcholine).

Thio-containing substrates are 1,2-bis(hexanoyl-thio)-1,2-dideoxy-zp-glycero-3-phosphorylcholine |C.9U. Keupoidvs, "

M.M. UMazprigp, K.A. YuOeeetv, 5 E.A. Yueppiv, 1991, Meijoaz ip Epgutoiodu 197: 3-23; 1. We are 5 E.A. Oeppiv, 1991, nt») with Meypodz ip Epgutoiodu 197: 65-75; I.A. M/ShShepatseg, K. ZpPigai, K.Yi. dasKwap 5 9.0. doppzop, 1984, Viospet.

Viorpuz. Kez. Sottype 118:894-9011. ;" E) Hydrolysis of phosphatidylcholine esters

This invention relates to the method of enzymatic hydrolysis of esters of phosphatidylcholine, for example, for industrial or food processing, or for obtaining detergents for laundry. Polypeptides -I of the present invention can be used for the hydrolysis of esters of phosphatidylcholine in solution or these enzymes can be bound to a solid carrier, which can then be subjected to contact with the substrate. This method can be used to produce lysophospholipids and free fatty acids. 2) Ob) Compositions

This invention belongs to compositions in a biologically compatible solution, which includes polypeptides, nucleic acids, vectors and antibodies of the present invention. A biologically compatible solution is a solution in which

Ge) polypeptide, nucleic acid, vector or antibody of the present invention is maintained in an active form, for example, in a form that is favorable for carrying out biological activity. So, for example, the polypeptide of the present invention must have phospholipase activity, the nucleic acid must be able to replicate and translate the transcript or hybridize with a complementary nucleic acid; the vector must be able to transfect the target cell; and the antibody must bind to the polypeptide of the present invention. Mainly,

F) such a biologically compatible solution is an aqueous buffer, for example, Tris, phosphate or HERE5 buffer containing salt cations. Usually, the concentration of salt ions is similar to physiological levels. In a specific embodiment of the invention, the biologically compatible solution is a pharmaceutically acceptable composition. Biologically compatible solutions may include stabilizing agents and preservatives.

Such compositions can be prepared for local, oral, parenteral, intranasal, subcutaneous and intraocular administration. By parenteral administration is meant intravenous injection, intramuscular injection, intra-arterial injection or infusion. This composition can be administered parenterally in the form of ready-made dosage forms containing standard well-known non-toxic physiologically acceptable carriers, adjuvants and excipients, if necessary.

Preferred sterile preparations for injection may be a solution or suspension in a non-toxic parenterally acceptable solvent or diluent. Examples of pharmaceutically acceptable carriers are saline, buffered saline, isotonic saline (eg, monosubstituted sodium phosphate, disubstituted sodium phosphate, sodium, potassium, calcium, or magnesium chloride, or mixtures of these salts), Ringer's solution, dextrose, water, sterile water , glycerin, ethanol and their combinations.

Of course, 1,3-butanol and sterile fatty oils are used as solvents or suspending media.

For this purpose, any soft fatty oil can be used, including synthetic mono- or diglycerides.

Fatty acids such as oleic acid may also be used in injectable preparations.

The medium for the compositions can also be a hydrogel, which is obtained from any biocompatible 70 or non-toxic (homo- or hetero-) polymer, such as a hydrophilic polymer of polyacrylic acid, which can act as a drug-absorbing sponge. Such polymers were described, for example, in the application

M/093/08845, the content of which is included in this description by reference. Some of them, in particular, such as polymers derived from ethylene and/or propylene oxide, are commercially available. The hydrogel can be deposited directly on the surface of the tissue being treated, for example, during surgery.

In another preferred embodiment, this invention relates to pharmaceutical compositions comprising a replication-defective recombinant virus and a poloxamer. More specifically, this invention relates to a composition containing a replication-defective recombinant virus containing a nucleic acid encoding a GI b-polypeptide and a poloxamer. The preferred poloxamer is Poloxamer 407, which is commercially available (VABZE,

Ragvirrapu, MU), and the best is a non-toxic, biologically compatible polyhydric alcohol. Poloxamer impregnated with a recombinant virus can be deposited directly on the surface of the tissue being treated, for example, during surgery. Poloxamer has basically the same advantages as a hydrogel, but at the same time it has a lower viscosity.

H) Methods of treatment

This invention belongs to methods of treatment that involve the administration of an effective amount of the composition of the present invention to a person or other animal.

Effective amounts of the composition may vary depending on the age, type, and severity of the patient's condition, i) what is being treated, as well as body weight, the required duration of treatment, the method of administration, and other parameters. The effective amount is determined by the attending physician and other qualified medical personnel. The polypeptides of the present invention are mainly administered in doses ranging from about 0.01 mg/kg to about 10 Ω/kg, preferably from about 0.1 mg/kg to about 5 Ω/kg, and best of all, from about 1 mg/ kg to about 1Omg/kg of body weight - per day. co

Recombinant viruses of the present invention are usually produced and administered in doses of from about 107 to about 10146 µ.0. In the case of AAV and adenoviruses, it is better to use doses from about 105 to about 10116.u.0. i am

The term "b.uo" (plaque-forming unit) corresponds to the infectious capacity of the vibrio suspension, which is determined by the infection of the corresponding cell culture and measured by the number of formed plaques. Methods of determination of the titer of B.U.O. virus solution are well described in the literature.

This invention belongs to the methods of treatment of atherosclerosis, where the specified atherosclerosis is the result of "excessive, abnormal or inadequate expression of I | (c-polypeptide activity.

In addition, this invention relates to methods of treatment of a person or other animal having an undesirable lipid-c profile, where said undesirable lipid profile is the result of high or inadequate expression and I GO-polypeptide activity. This invention also relates to methods of treating diabetes, hyperlipidemia, intrahepatic cholestasis, or other metabolic disorders, where said diabetes, hyperlipidemia, intrahepatic cholestasis, or other metabolic disorders are the result of high or inadequate expression of GI polypeptide activity. sl 1) Correction of undesirable lipid profiles associated with increased expression of I | b-polypeptide.

Methods of reducing the expression of the GI 3-polypeptide for the correction of the specified conditions, where these conditions or disorders (95) are associated with an undesirable lipid profile, caused by II b-polypeptide activity, include, but are not limited to, the administration of a composition containing an antisense nucleic acid ; administration of a composition containing an intracellular binding protein, such as an antibody; administration of a composition containing iChe) GGOM polypeptide or other fragments of GI, and administration of a composition containing a nucleic acid that encodes

Г GooM-polypeptide or other fragment / I.

In one embodiment of the invention, a composition containing an antisense nucleic acid is used to suppress or block the expression of 10. In one embodiment of the invention, the specified nucleic acid encodes antisense RNA molecules. In this version of the present invention, the nucleic acid is correctly connected to the signal sequences that ensure the expression of the nucleotide sequence, and is inserted into the cell, using, preferably, recombinant vector designs capable of expressing the antisense nucleic acid after the introduction of this vector into the cell. Examples of suitable vectors are plasmids, adenoviruses, adeno-associated viruses, retroviruses and herpes viruses.

Adenovirus is the preferred vector. The best vector is a replication-defective adenovirus that has a deletion in the E1 and/or EZ regions of the virus.

In another preferred embodiment of the present invention, the expression | (c is inhibited or blocked by expression of a nucleic acid sequence encoding an intracellular binding protein capable of selectively interacting with GI 5. References UUO94/29446 and MU/O94/02610, the contents of which are incorporated herein by reference, describe transfection of cells with genes encoding an intracellular binding protein An intracellular binding protein is any protein capable of selectively interacting with or binding to | | (with in the cell in which it is expressed and neutralizing the function of the bound HIs A preferred intracellular binding protein is an antibody or antibody fragment A preferred intracellular binding protein is a single chain antibody.

UMUO94/02610 describes the production of antibodies and the identification of nucleic acid encoding a specific antibody. Using GI C or its fragment, a specific monoclonal antibody can be obtained by methods known to those skilled in the art. For use in the method of the present invention, a vector can then be obtained, which includes a nucleic acid that encodes an intracellular binding protein or its fragment, and is capable of being expressed in the host cell.

Alternatively, GI C activity can be blocked by injecting a neutralizing antibody into the bloodstream.

This neutralizing antibody can be introduced directly as a protein or it can be expressed from a vector (containing a secretory signal).

In another variant of the present invention, GI Chi activity is inhibited by the administration of a composition containing the 7/5 GOM polypeptide or another fragment of II 0. This composition can be administered by a standard method, such as oral, local, intravenous, intraperitoneal, intravenous ulcerative, subcutaneous, intranasal or transdermal administration. This composition can be administered directly or it can be encapsulated (for example, in a lipid system, in amino acid microspheres or in globular dendrimers).

In some cases, this polypeptide can be attached to another polymer, such as serum albumin or polyvinylpyrrolidone.

In another variant of the present invention, GI Chi activity is inhibited by using low molecular weight compounds that suppress its enzymatic properties or prevent its recognition by cellular binding sites.

In another variant of the present invention, GI XII activity is inhibited by using gene therapy, that is, by introducing a compound containing a nucleic acid that encodes and directs the expression of the GOM polypeptide or other fragment 0. In a specific embodiment of the invention, the GOM gene of the present invention also has i) affinity for heparin. II (z-polypeptide, which binds to intracellular heparin in the cavity of blood vessels, allows I | (z) to bind and accelerate the absorption of LNG, acting as a bridging connection between LNG and intracellular heparin. It is assumed that at the site of localization of atherosclerotic In the presence of lesions, the increased level of lipase activity accelerates the atherogenic process |Limegetii, O.V. (1995) Siip.

Spent 41, 153-158; 7atrop, A., Toggevs, A., Wi)moei, 5., Sadpe, S., Mooiapi, 5., Ioriep, R.)., Naudep M.K., 5 -

Vgypgeyi, U.O. (1993) I apsei 341, 1119-1121). This may be due to the increased level of binding and absorption of lipoproteins by vascular tissue, which is mediated by lipases (Eiseprego, 5., Zepauek, E., OiYimesgopa,

T., MiodauzKu, I. (1992) 9. Siip. Ipmevi, 90, 2013-2021; Tarav, I., Ii, I., Vgosia K.MU., Khy, 5M/., Zmepvop, Shcheo,

T.G., M/Shiatv, K.). (1993) 9. Vioi. Spet., 268, 20419-20432; Mogaezidaaga, V.O., K Mieivep, A.O. (1994) Sigt. Orip. uh-

MPria. 5, 252-257; MUShiatve, K.), " Taravz, I. (1995) Ap. Tpgotr. Apa Maze VioI. 15, 551-561). In addition, high local levels of lipase activity can lead to cytotoxic levels of fatty acids and lysophosphatidylcholine produced in precursors of atherosclerotic lesions. This particular activity

Gb can contribute to the development or progression of atherosclerosis, especially in the background of excess lipid levels, "caused by nutrition or genetic factors. Thus, this invention makes it possible to inhibit the accumulation of lipoproteins in c by inhibiting the expression of GIA 3-polypeptide or binding to a lipoprotein (for example, LNG). ;" 2) Correction of undesirable lipid profiles associated with insufficient activity of I| b-polypeptide

Methods of increasing HER expression to correct those conditions in which the activity of the II ("z-polypeptide contributes to the development of diseases or disorders associated with an undesirable lipid profile include, but are not limited to, the administration of a composition containing the ГХХ polypeptide, and the administration of a composition, containing a nucleic acid that encodes a polypeptide / I OX. 1 In one of the variants of the implementation of the present invention, GI OX activity is increased by introducing a composition, 2) containing a GI OX polypeptide. This composition can be administered by a standard route, such as orally,

Local, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or transdermal - administration. This composition can be administered directly or it can be encapsulated (eg, in

He) lipid system, in amino acid microspheres or in globular dendrimers). In some cases, this polypeptide can be attached to another polymer, such as serum albumin or polyvinylpyrrolidone.

In another variant of the present invention, I and Xi activity is increased by using low molecular weight compounds that can enhance the expression of GI OX. at the level of transcription, translation or post-translation.

In another variant of the present invention, the level of HER OCI is increased by introducing a composition containing

F) nucleic acid that encodes and directs the expression of I| Chi polypeptide. Intrahepatic cholestasis can be characterized as an increase in serum cholesterol and phospholipid levels. It was recently reported that a rat model of intrahepatic cholestasis induced by the drug phalloidin demonstrated a significant increase in serum cholesterol and phospholipid levels (Izpigaki, K., Kipraga, 5., Miuagamzha, M., Takeshpi, U., Nigarauazni, M. , Kazai, N., " Agaki,

T. (1997) TohisoYi. IeChegs 90, 29-34). The products of the present invention can be used for the treatment of intrahepatic cholestasis in patients with elevated serum cholesterol and/or phospholipid levels. In addition, this rat model also showed a significant reduction in the rate of 65 cholesterol excretion in bile. I O-polypeptide and nucleic acid products acids of the present invention can be used to treat patients with disorders of the biliary system.

Intrahepatic cholestasis is characterized as a violation of bile secretion from the liver. Recent mapping has shown that loci for progressive hereditary intrahepatic cholestasis (PSHC or Bayler's disease) and benign recurrent intrahepatic cholestasis (RBC)

They are located at 18421-d22 ISagyop, M.E.N., Kpize|u, A.5., 5 Egeiteg, M.V. (1995) Nit. My. Sepoys 4, 1049-1053 5

Noshmjep, K.N., Vapiagioo, 5., ViapKepePpir, K., KaeutaekKegv, R., dUyup, 9., Zapakiy, SA., b Egeiteg, M.V. (1994)

Maitsyge Sepei. 8, 380-386, respectively). Since the YII gene was mapped to this chromosome at site 18421, the Y/0z gene or the products of the present invention can be used to treat patients suffering from intrahepatic cholestasis caused by a mutation or defective expression of the gene(s) responsible for the disease PSVH/ DRVH

In another embodiment of the present invention, the GI 5 gene or polypeptide products of the present invention can be used to treat patients suffering from intrahepatic cholestasis, which is not caused by a defect in the gene responsible for the disease PSVH/DRVH, at site 18421-422. Recent studies have suggested that another locus outside the 18421-422 region may also produce the pPoVX phenotype. M., " Tpotrzap, K.). (1996) W. May. Sepoys 33, 833-836). However, administration of GI C-polypeptide, either directly or through gene therapy, can alleviate the condition caused by this form of the disease.

In gene therapy, one or more nucleic acids encoding a polypeptide, as well as regulatory regions that control its expression, are transferred into target cells of a person or another animal. This transfer is carried out either by the ex mimo method, in which the nucleic acid is transferred into cells in the laboratory, and then the modified cells are injected into a person or another animal, or by the ip mimo method, in which the nucleic acid is injected directly into the cells of a person or other animal. The transfer of nucleic acids can be carried out using either viral or non-viral vectors described above.

Nonviral vectors can be transferred into cells by any method known to those skilled in the art, including co-precipitation with calcium phosphate, lipofection (using synthetic anionic and cationic liposomes), receptor-mediated gene delivery, naked DNA injection, electroporation, and delivery methods i) bioballistics or particle acceleration.

Examples

This invention is illustrated by the following examples. These examples are given only for illustrative purposes Ge! with Purpose and do not limit the scope of claims.

Example 1 -

Identification of differentially expressed k DNA p

A) Production of RNA

Human TNR-1 monocytic cells

Rgomegpapo, M.O., Rciitau, E.K., SoeKe, M.M., OiIvop, V.)., apa Kiepk, O.S. (1985) Apai. Viospet. 150, 76-85) were cultured in KRMI-1640 (SIVSO) medium containing 25 mM NEREZ, 1095 fetal bovine serum, 10 units/ml of sodium-containing penicillin b and 1 TO units/ml of streptomycin sulfate. Cells were seeded in 1 Bcm tissue culture dishes at a concentration of 1.5 x 10 cells/well and treated with 4 ng/ml phorbol 12-myristate-13-acetate (Zidta) for 48 hours to induce cell differentiation. Human low-density lipoproteins (LNG), which are supplied by the Sairiospet company, were subjected to exhaustive dialysis against РВЗ5 at 420. Then the LNG was diluted to 500 μg/ml and dialyzed against 5 μM SyzoO, in РВЗ5 at 372С for 16 hours. To stop oxidation, LNG was subjected to exhaustive dialysis against 150 mM Mass, 0.3 mM EOTA, and then the filter was sterilized. The protein concentration was determined by the BSA method (|ZepyNy,

K.N. (1978) Rgos. May Asad All together. BOTH 75, 3173-3177) (Riegse). The degree of oxidation was determined by the TVAK5Z method. and was 25-3ZOnmol of MOA equivalents/mg of protein. - | Differentiated TNR-1 cells were treated for 24 hours with either BOmkg/ml of oxidized LNG or sl MasI-EOTA-buffer in the medium of KRMI containing 10956 fetal bovine serum with a deficiency of lipoprotein (Zidta). To collect RNA, dishes were washed with 10 ml of RB5Z, and then 14 ml of TKI2OY was added to each dish. (95) IShapo, R. " Ragayee, A.V. (1992) Zsiepse 257, 967-971) (SIVSO). The solution was pipetted several times to obtain 20 ml of the mixture, and then identical samples were combined in centrifuge tubes, 3 ml of chloroform per cup was added and mixed. The tubes were centrifuged for 15 minutes at 12,000 x d. After centrifugation, the upper layer was transferred to a new tube, and then 7.5 ml of isopropanol was added per cup and mixed. The test tubes were centrifuged at 12,000 x d for 20 minutes. The precipitate after centrifugation was washed with ice-cooled 70906 ethanol and dried at room temperature. Pellets were resuspended in 500 μl TE (Tris-EOTA) and treated with 200 units of RNase-free DNase 1 and 200 units of RNase Placental RNase Inhibitor (Rgothead) for 30 minutes at 3720. RNA was purified by sequential phenol extraction, phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:71), and then precipitated with ethanol.

C) cDNA synthesis 60 cDNA synthesis and RSC amplification were carried out in accordance with the instructions attached to the kit for the implementation of the differential display method.

Viosiesppoiodu, Ips.)i. This system is based on the method first described by Giapod and Ragdee | Meaid, SA, Reu, MK,

Neptziadi, S., Magsii, K.A., 5 tii, I.M. (1991) Vio/Hesppoisdu 9657-663)Y. The pairs of primers that gave the cDNA fragment containing the first information about the lipase-like polypeptide gene were forward 65 primer 7 and reverse primer 15. cDNA for amplification was synthesized as described below using RNA derived from THP cells 1, treated with PMA and exposed to either buffer or oxidized LNG: 3 µl of 25 mM forward primer 7 and 7.5 µl of diethylpyrocarbonate (DEP)-treated water were added to 100 ng (33,0 µl) of RNA from any TNR-1 RNA sample. This mixture was heated to 702 for 10 minutes and then cooled on ice. Into this tube was added Zmkl 5 x RSK buffer (250mM Tris-HCl, pH 8.3, 375mM KSI) (SIVSO), Zmkl 25mM

MosSiI", Zmcl 0.1M DTT, 1.2μl 500μM aMtTP, 0.7μl RNasein and 5.bmcl OERS-treated water. The tubes were incubated for 2 minutes at room temperature, after which 1.5 μl (300 units) were added

Ziregzsagiri II RNase H-reverse transcriptase (SIVSO). The tubes were incubated sequentially for 2 minutes at room temperature, 60 minutes at 372C and 5 minutes at 9592C, and then cooled on ice.

RSC amplification was carried out using the Mazieg Mich kit containing 117 μl of 10x RSC buffer (500 mM 70 KSI, 100 mM Tris-HCl pH 8.3, 15 mM MoSi! and 0.0195 (w/v) gelatin), 70 ,2μl of 25MM Moassi", 5.9μl of alpha-ZZR-aATP (1OmCi/ml. OyRopi MEM), 4.7μl of 500mμM aMTP mixture, 11μl of Atria-Tazd DNA polymerase (Bodynyts/μl, Regkeep-EIteg), and 493, 3 μl of OERS-treated water. For each reaction, 12 μl of the Mawsieg Mich kit was added to 2 μl of forward primer y7, 1 μl of cDNA and 2415 reverse primer. The reaction mixtures were heated to 942C for 1 minute, and then subjected to 40 thermocycles with a denaturation stage at 949C for 75 for 15 seconds; with the annealing stage at 402C for 1 minute and with the elongation stage at 722C for

30 seconds. After carrying out 40 cycles, the reaction mixtures were incubated at 72 C for 5 minutes and stored at 1020. RSC reactions were carried out in a thermocell ZepeAtr Buziet 9600 Regkip-EIteg.

Four microliters of the amplification reaction mixture was mixed with an equal volume of loading buffer (0.296 bromophenol blue, 0.295 xylene cyanol, 10 mM EOTA pH 8.0, and 20965 glycerol).

Four microliters of this mixture was subjected to electrophoresis in a non-denaturing acrylamide sequencing gel for 3 hours at 1200 volts (constant voltage). This gel was dried at 802 for 1.5 hours and exposed with Kodak HAK film. An amplified product found only in the reaction mixture containing cDNA from TNR-1 cells treated with oxidized LNG was identified and this product was then excised from the gel. In the microcentrifuge tube containing the excised gel fragment, 10 µl of

of OERS-treated water, and then incubated for 30 minutes at room temperature, and then again on Ho) for 15 minutes at 9590.

For re-amplification of the PCR product, 26.5 microliters of eluted DNA was used in the amplification reaction, which also included 5 μl of 10x RSOK buffer, Zmcl of 25 mM MaoSiI, 5 μl of 500 μM amtR, μl of Fu 2 mM of forward primer 7, 7.5 μl of reverse primer 15 and O,bmcl polymerase Atriad. Parameters

RSkK-cycles and equipment were described above. After amplification, 2 μl of the re-amplified reaction mixture was analyzed on an agarose gel and 4 μl was used for subcloning of RSK products in the pSKII vector using the TA cloning system (Egoptap, M.A., Yuyvy, M.K. 5 Magpip, S.K. (1988) Rgosz. Mai. Asad. Zsi.

ZA 85, 8998-90021 (Ipiygodep). After ligation overnight at 142C, the ligated products were used for iv) transformation of E. soya. The obtained transformants were collected and Zml of overnight cultures were used for the preparation of plasmid minipreparations. The sizes of the inserts were determined using EsokKiI-hydrolysis of the plasmid, and clones containing inserts approximately the size of the original PCR product were sequenced using terminator reagents and a fluorescent dye (Rgizt, Arriley Viozuvietv), and using

DNA-sequencer of Arielei Viozuzietv 373. The sequence of the RSK-product is shown in Fig.2. The sequence of the 20 amplified primers is underlined. - c C) UKASE reaction

Elongation of the cDNA identified by CT-RSK was carried out using the 5'CASE:z system (Soip, E.M., Eyoi, 9U.E., Smipa, 5., I apieg/ Y, "5 Oamiz, M .M. (1989) Zsiepse 243, 217-219; Bittv, Yu., tsap, M., "

Zyagatap, K., (1991) Rosyz 1399 (5IVSO)Y One microgram of TNR-1 RNA (treated with oxidized LNG), Used first in differential display reactions, used in the 5'KASE reaction: -1 1μl (μg) of RNA was combined with 3 µl (Zpmol) of primer 2a and 11 µl of OERS-treated water, and heated to 7090 for 10 minutes and then kept for 1 minute on ice. Then 2.5 μl of 10 x O buffer for the reaction (200 mM Tris-HCl pH 8.4, 500 mM KCl), 3 μl of 25 mM MacIi, 1 μl of 10 mM aMTP mixture and 2.5 μl of 2) 0.1 mM DTT were added. This mixture was incubated for 2 minutes at 422C, and then μl of the processed transcriptase pz Ziregzsegiri II was added. The reaction mixture was incubated for another 30 minutes at 422C, 15 minutes at 702C and 1 minute on ice.

Then one microliter of RNase H (2 units) was added and the mixture was incubated at 559C for 10 minutes. K DNA and Che) were purified using SiazzMach columns | Zatbgook, U. Rhyvssy, E.B. b Mapiaves T. (1989) MoiIesshag Siopipd:

A Iarogayugu Mapia), zesopa eayyiop, So rgipd Nagrog I arogaiugu Rgezv, Riaiipmiemu, MM), included in the set. This cDNA was eluted from the column in BOmcl of NaOH, lyophilized and resuspended in 21 μl of NaOH.

cDNA chain extension was carried out in the following reaction: 7.5 μl of 20O, 2.5 μl of reaction buffer (200 mM Tris-HCl, pH 8.4, 500 mM KCl), 1.5 μl of 25 mM Masi, 2.5 μl of 2 mM astR, and 1 μl of cDNA incubated at 9492 for 3 minutes and then on ice for 1 minute. Then, μl (10 units) of the final deoxynucleotidyl transferase was added and the mixture was incubated for 10 minutes at 37 C. The enzyme was subjected to heat inactivation by incubation at 70 C for 10 minutes, and then the mixture was placed on ice. 60 RSV amplification of cDNA was carried out at the following stages: 5 μl of cDNA with an extended end was included in the reaction mixture, which also contained 5 μl of 10x PCR buffer (500 mM KCl, 100 mM Tris-HCl pH 8.3, 15 mM MoSiI and 0.0195 (w/v) gelatin), 1 μl of 1 μM aMTP mixture, 2 μl (1 μmol) of anchor primer, 1 μl (20 μmol) of primer Za and 35 μl of an2O. The reaction mixture was heated to 952C for 1 minute, and then 0.9 μl (4.5 units) of Atriad polymerase was added. The reaction was carried out for 40 cycles under the following conditions: 9490 - 5 seconds, 5090 - 20 seconds and 722 - 30 seconds. One microliter of this reaction mixture was used for "nest" re-amplification in order to increase the levels of the specific product for the next isolation. For repeated amplification, the following were used: μl of the mixture for primary amplification, 5 μl of 10 x RSK buffer, μl of 10 mM amMTP mixture, 2 μl (20 pmol) of universal primer for amplification, 2 μl (200 pmol) of primer 4a and 38 μl of ano.

The reaction mixture was heated to 95°C for 1 minute, and then 0.7 μl (3.5 units) of polymerase was added

Atriad. The reaction was carried out for 40 cycles under the following conditions: 942С - 5 seconds, 5090 - 20 seconds and 7290 - 30 seconds.

Amplified products were analyzed by electrophoresis in a 0.895 agarose gel. The predominant product containing about 1.2 t.p.o. was found. Two microliters of the reaction products were cloned into the RecII vector from the TA cloning kit (Ipuygodep) and incubated overnight at 14-20C. These ligation products 70 were used to transform E. soybean. The sizes of the inserts of the obtained transformants were determined by

EsokI-hydrolysis. Clones containing inserts approximately the size of the original PCR product were sequenced using terminator reagents - a fluorescent dye (Rgizt, Arrie Viosuviets) and using the Arrie Viosuviets 373 DNA sequencer. The sequence of the KACE product, including

EsokKiI sites from the TA vector, shown in Fig.3. Amplimer sequences (universal amplified 75 primer and primer complementary to 5'KASE-primer 4a) are underlined.

Example 2

Placement and localization of the GI OX gene. on the chromosome

A) Screening of the cDNA library

Human placental cDNA library (oligo 4T and randomly primed, cat. Mo5014r, lot 552033) was obtained from Siopiesp (Raio Ai, CA). Radioactively labeled probe was created by cutting out the plasmid insert containing the above-described RSC product of the 5'KASE reaction. The probe was radiolabeled using the random priming technique: the DNA fragment (50-100 ng) was incubated with 1 μg of randomized hexamers (sirso) at 9592 for 10 minutes and then on ice for 1 minute. Then, at room temperature, the following were added: Zmkl 10x Klenov's buffer (100mM Tris-HCl pH 7.5, 50mM Masi), 57mM dithiothreitol; Mem Epdiapai "M Violyarv", Zmkl 0.5mM adtR, astrR, attR), 10OmKi o- 2rastr (ZO00OKi/mmol, Mem Epdiapa Misieag) and μl of a fragment of Klenova DNA polymerase I (5 units, Sirso). The reaction mixture was incubated for 2-3 hours at room temperature, and then the reaction was stopped by increasing the volume to 100 μl using TE, pH 8.0, and by adding EUTA to the final concentration of MM. Non-incorporated nucleotides were removed by increasing the volume of the reaction mixture to 10 Ωcl, and passing it through a 5-50 centrifugal column (Voeigipdeg FO

Mapppeit). The obtained probes had a specific activity exceeding 5x108mp/min/μg of DNA. "-

The library was probed by standard methods | (MMaieg, R., Siitoge, K., "Viore!, b. (1984) Se 38, 5-8)

For this, the filters were hybridized for 24 hours at 65 C in 4.8X Z5RE (20X 55RE - 3.6M Mass, 0.02M She

ManPO,X, 0.02M EOTA, pH 7.7), 20mM Tris-HCl pH 7.6, 1x Denhardt's solution (100X - 295 Ficol 400, 2956 polyvinylpyrrolidone, 295 B5BA), 10906 dextran sulfate, 0.195 DSN, 10Omkg /ml of salmon sperm DNA and m 1Xx10bimp/min/ml of radioactively labeled probe. Then the filters were washed three times for 15 minutes at room temperature in 2 x 35C (1 x 55C - 150 mM Mass, 15 mM sodium citrate, pH 7.0), 0.196 sodium dodecyl sulfate (DSN), and then washed three times for 15 minutes at 6593 each with 0.5 x 55С and 0.196 DSN. The phage that hybridized with the probe was isolated and amplified. DNA was purified from the amplified phage using the Gatraazog reagent (Rgoteda) according to the manufacturer's instructions. Inserts were excised from phage DNA in c by hydrolysis with Esoki enzyme. These inserts were subcloned into the EsoKiI site of the plasmid vector (Vipezsgiri II ZK,

Zigaiadepe). By means of the automatic sequencing described above, the sequence of the open reading frame "z" was determined, which is located inside the 2.bp.p.o.-EsoKI-fragment of cDNA. This sequence is shown in Fig.4.

The amino acid sequence of the predicted protein encoded by the open reading frame is shown in Fig. 5 and designated ISOHI It was assumed that the first methionine is encoded by nucleotide pairs 252-254. -I The predicted protein has a length of 500 amino acids. The first 18 amino acids form a sequence characteristic of the secretory signal peptide INiddipe, O., 5 Zpagr, R.M. (1988) Sepe 73, 237-244). The predicted propeptide has a molecular weight of 56,800 Daltons. If we assume that cleavage of the OO signal peptide takes place at position 18, then the molecular weight of the unmodified mature protein is 54,724 Daltons.

The complete similarity between this protein and other known members of the triacylglycerol lipase family is illustrated in Fig.b and Table 1. In the comparison of primary sequences shown in Fig.b6, | 5 represents

He is a polypeptide (ZEO IUO Mo: 6) encoded by cDNA (ZEO IUO Mo: 5), which is described in Example 1, and is further denoted

IHOM According to its amino-terminal 345 residues, this protein is identical to protein GI XII. 9 unique residues follow the stop codon and produce a 39.3kDa polypeptide and a 37.33kDa mature protein. The sequences that are common to COM and I OXI are the nucleic acid sequence 5EO IYUO Mo: 9 and the amino acid sequence ZEO IYUO Mo: 10. (F; It is interesting to note that the position in which GI proteins (ZM and GI OX. differ, is a region known from the structure of another lipase to be located between the amino and carboxy domains of these proteins. Therefore, the protein! Its OM apparently consists of one of the two domains of triacylglycerol lipases. This sequence contains a characteristic bo The motif "vheXxOo" of lipase in positions 167-171, as well as conservative residues of the catalytic triad in beg 169, Avzr 193 and Niv 274. Conservatism of cysteine residues (positions 64, 77, 252, 272, 297, 308, 311, 316, 463 and 483), which participate in disulfide bonds in other lipases, suggests that the protein has structural similarities with other enzymes. There are five putative M-glycosylation sites at amino acid positions 80, 136, 393, 469 and 491. Protein sequences that are listed for comparison, there are 65 human lipoprotein lipase (I RI; Seprapk, accession number ЯМ15856, ZEO IYUO Mo: 13), human liver lipase (NO,

SeprapK, accession number 2ШОЗ540, 5EO IЮО Mo: 14), human pancreatic lipase (RI, SeprapK, accession no.

YAMO93285, 5EO IO Mo: 15), human pancreatic lipase-related protein-1 (RI KR-1; SeprapK, accession no. YAMO93283), human pancreatic lipase-related protein-2 (RI KR-2; SeprapK, accession no.

ЯМО3284), about Tavholyau no ve (viari ria), plex vlovvoyav hav 226 about ais voozav ol 09. z |z|yuv - i go 220.

RO la 228228 - 852 vo

RiyaRi 225 (22 mo 652 c

RiyaR2 226 209220 622 vi. y y y

The percentage of similarity was estimated based on the comparison of base pairs of primary structures using the Siivia algorithm! (Satrv", I|., Keipa. M., Porega, M., Midago, 5., b Oimimesgopa, T. (1990) At. 9. RNuvzioi. 258,

C673-C6811) of the Meaaiidp programs in the set of programs I azegdepe Viosotriyipud Zoimage Ziye (Opaviag).

C) Localization on the chromosome

DNA from clone RI|Ziegprego, M., Kie(Shpeg, U. 5 YuOekKiei, K.Tpe Mem Viioodivi 2; 151-62, 1990), containing genomic DNA 110, was labeled with TK (uridine triphosphate) digoxigenin by "nick"- The labeled probe was combined with fragmented human DNA and hybridized with RNA stimulated by peripheral blood lymphocytes taken from a male donor in a solution containing 5095 formamide, 1095 dextran sulfate, and 2 x 5502. Specific hybridization signals were detected by incubation of hybridized cells with fluorescently labeled anti-digoxigenin antibodies followed by OARI contrast staining.

This preliminary experiment led to the specific labeling of chromosome group E, which, based on (o)pARI staining, is apparently chromosome 18.

Then another experiment was conducted, in which a biotin-labeled probe specific for the centromere of chromosome 18 was hybridized together with the GII b-probe. This experiment resulted in the specific staining of the Phoso centromere of chromosome 18 in red and the long arm of chromosome 18 in green. Measurements of 11 specifically labeled hybridized chromosomes 18 showed that SHE has an RBieg of 0.67 (Frank measurements of 0.38), which corresponds to band 18421. Some hereditary diseases, including intrahepatic cholestasis, cone-rod dystrophy of the retina and Hereditary osteolysis is obviously due to defects in this part of the chromosome.

Example C Io)

Analysis of RNA about | what-

A) RNA I C expression in TRN-1 cells

Analysis of mRNA, from which cDNA is obtained, was carried out by Northern analysis of TNR-1 RNA. RNA from these cells was obtained as described above. This mRNA was isolated from total RNA using the Roi-yT-magnetic sphere system (Roiwangasi Zuviet, Rgoteda). Three micrograms of roiu(A)-containing mRNA was subjected to electrophoresis on a 195 agarose 20% formaldehyde gel. This gel was washed for 30 minutes in 48H2»O. RNA was transferred under vacuum onto a black nylon membrane using an alkaline transfer buffer (3M mass, 8mM Mahon, 2mM sarkosyl). c After transfer, the blot was neutralized by incubation for 5 minutes in 200 mM phosphate buffer, pH 6.68. RNA was cross-linked to the membrane using a UV cross-linking device (5igadape).

The probe was obtained by excision of the plasmid insert containing the 5'VDSE-product of the RSC reaction, described

Above. This probe was radiolabeled using the randomized priming technique described in Example 2.

The filters were pre-hybridized in a solution for rapid hybridization OtsiYikNu" (Zigaiadepe) for 30 minutes at 652С. Radioactively labeled probe (1-2 x 10bimp/min/ml) and ultrasound-treated salmon sperm DNA 2) (final concentration 10 Ωkg/ml) were denatured by heating for 10 minutes to 95 C and quickly cooled for 5 minutes with ice, after which they were transferred to a filter in a solution of OcykNub. Hybridization was carried out from 3 hours - at 6520. The unhybridized probe was removed by washing the filters twice for 15 minutes with 2 x 55C, (Che) 0.195 sodium dodecyl sulfate at room temperature, followed by washing the filters twice for 15 minutes with 0.1 x З55С, 0.196 DSN at 622С. After washing, the filters were quickly dried and then exposed to Kodak ХАК-2 film with intensifying screens at -809С. The results are illustrated in Fig. 7, which shows large MmRNA molecules, which are approximately 4.5 thousand base pairs. Smaller molecules with a length of 4.3 and 1.6 thousand base pairs were also present. The expected size of KDNK GOM was 1.6 bpd. The sequence of GI OX is obviously encoded by large molecules of the detected me NC. eat) C) Expression of RNA I. in various human tissues

A commercially available ready-made filter containing Zmkg of each of the mRNAs from various human tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas) was obtained from Siopeyesi (Sailiod Ya7760-1). This filter was probed and processed as described above. After probing with a radioactively labeled GO fragment and radioautography, the probe was cleaned by washing in boiling 0.1 x Z5C, 0.195 DSN in an incubator at 65923 for 2 x 15 minutes. Then the membranes were probed using a 1.4 kb DNA fragment encoding human lipoprotein lipase. This fragment was obtained b5 by polymerase chain reaction with reverse transcriptase (RT-PCR) of RNA from TRN-1 cells (treated with PMA and oxidized LNG) using the 5' RI - and 3 RI - primers shown in Fig. 1, and the conditions CT-RSK,

described above. After autoradiography, the membranes were re-cleared and re-probed with a radiolabeled human beta-actin cDNA fragment to normalize for RNA content. The results of these analyzes are illustrated in Fig

Fig. 8. The highest levels of HER transcript (with) were detected in RNA of the placenta, and the lowest levels were detected in RNA that

DERIVED from lung, liver and kidney tissues. In accordance with previous studies by other authors

Meghtoemep, A.).M., Chapvzep, N. (1994) Viospet. Viorpuz. Asia 1211, 121-124), the lipoprotein lipase transcript was detected in many tissues, with the highest levels detected in heart and skeletal muscle tissues.

The results of these analyzes showed that the distribution of expression | in tissues is very different from the distribution of I RI.

The nature of its expression also differs from the nature of expression of liver lipase or pancreatic lipase, as reported by other researchers (MUapo, S.-5., and NagivysK, UA. (1993) Viospet. Viorpuz. Asia 1166, 1-19; Betepkomisp, S .R., Spep 5.-MU., Muitv, M., Yitso S.-S, (i, MU.-N., Spap, I. (1989) 9. IIry Kev. 30, 423-431;

Adatzvg, M.O., Kepiamade, A.K., Rividv, S., 5 Mepieg, S. (1993) Mayte Sepei. 4, 256-265).

To determine the pattern of expression in other human tissues, another commercially available membrane was probed using cDNA and OX. This dot-blot (human RNA Mazvieg Vioi, Siopiesp Sai. 27770-1) contained 75.100-500ng of mRNA from 50 different tissues and was normalized to the equivalent expression of "housekeeping" genes | Spap, Y, 5 Mohip, K. ( 1971) Viospet. Viorpuz. Asia 231, 194-1971. 1, bt.p.o. OgaI-5PI-fragment

KDNK OK. labeled ZRaSTR using a randomized system of oligonucleotide priming (Rgite II

And, Bigaiadepe) in accordance with the manufacturer's instructions. After a 30-minute prehybridization at 652, the probe was added to the hybridized solution of SkikKNuv at 1.3x10 bpm/min/ml. Hybridization was carried out for 2 20 hours at 652C. The unhybridized probe was removed by washing the filters twice with 2 x 35S for minutes, then with 0.195 sodium dodecyl sulfate at room temperature, followed by two washes for 15 minutes in 0.1 x 5S, 0.195 DSN at 62 °C. After washing, the filters were subjected to rapid drying, and then exposed with Kodak ХАК-2 film using intensifying screens at -802С, for different periods of time. The resulting images were quantified using densitometry. The results of c are shown in Table 2. The relative expression levels in tissues determined by Nozeran analysis of multiple (3 tissues) and by dot blots obtained from multiple tissues were similar, with the highest levels observed in the placenta and the lowest levels observed in in lung, liver, and kidney Fetal liver, kidney, and lung were also expressed at nearly the same levels as in adult tissues.

Unexpectedly, it was found that of all the tissues represented, thyroid tissues expressed the highest levels, which were 12290 times the level expressed in placental tissues. Although the expression of lipase in the placenta was observed earlier | (1982) 9U. Yum. Reputations 4, 153-159; Meghtoemep, A.).M.,

Sapipa O., 5 Oapvep N. (1994) in Iiriy Kevs. 35, 966-975; Vygop, V.K., MyeiMeg, N. MU. (1980) Viospet. and.

Viorpuz. Asia 618, 449-460), but the expression of any lipase in the thyroid gland has not been observed before. yu

These results suggest that Z expression may contribute to the preservation of the placenta, where GI Z can

To serve to release free fatty acids from substrates such as phospholipids as energy sources. And | 5, which is expressed in the thyroid gland, can serve as a precursor for the synthesis of biologically active molecules by this gland. them with With with with mozyuchoe 000004 idbuorieyadro nziecha 00000009 sozhahshka 00006 brain of the fetus 6. with and.

AND; m occipital part (from thick ischka 8 thymus gland 122 bony mossy 0 thymus 69, - (shell on osmotic bladder nzoolin gland naz Zalendie 000007 lion placenta////- 8 s These values represent percentages of expression; at the same time, the levels in the placenta tissues were arbitrarily taken for 100905.

These values represent average values from densitometric measurements obtained from two 22 autoradiographic exposures, n.z. - not found. (FI C) Expression of RNA I C in cultured endothelial cells

Human umbilical vein endothelial cells (HUECs) and human coronary artery endothelial cells (HCECs) were obtained from Siopeisv. NOMES were propagated in a commercially available medium for the growth of endothelial cells (ESM, Siopeiisv) to which was added Zmg/ml of bovine brain extract (Masiad, 60 T., Segipdoiu, 9., IvIeu, 5., KePeu, R.K ., 5 Rogapa, K. (1979) Rgos. Mai. Asad. si, OBA, 76, 5674-5678),

Siopeiisv and NSAECs were propagated in ESM medium, to which was added Zmg/ml of bovine brain extract and 396 fetal bovine serum (at a final concentration of 595). Cells were cultured until confluent, after which the medium was replaced with ESM medium that did not contain bovine brain extract. Cultures were stimulated by adding 100 ng/ml phorbol myristate (Zidta). After a 24-hour incubation, RNA was extracted from the cells using the Trizol method described above.

Twenty micrograms of total RNA was electrophoresed and transferred to a membrane for analysis. Membranes were probed with IR and IR probes as described above. The results are illustrated in Fig.9. Twenty micrograms of total RNA from PMA-stimulated TNR-1 cells were subjected to blot analysis for comparison. RNA that hybridizes

With the HH O-probe, was found in unstimulated and PMA-stimulated NOMES cells. In contrast, levels

Detectable mRNA I 10 was detected only in NSAEC cultures after PMA stimulation. In accordance with previous studies by other authors, any detectable level of lipoprotein lipase mRNA was not found in any of the endothelial RNA |Megpoemep, A.).M., Dapsep, N. (1994) Viospet. Viorpuz. Asia 1211, 121-124). 70 Example 4 Analysis of protein II and II

A) Dressing of antibodies

Antiserum was produced against peptides having a sequence corresponding to the region, which is assumed to be encoded by the open reading frame of cDNA II 0. This particular peptide was chosen because it has a high predicted antigenicity index Chatezop V.A. and MUUoi, N. (1988) Sotryi. Arriis. Ip She 7/5 Viovzsiepsev 4, 181-186). The sequence of the immunizing peptide was not found in any of the proteins or in the translated DNA sequence available in the Seprapk database. The corresponding position of this sequence in the protein (o) is shown in Fig. 10. The carboxy-terminal cysteine of this peptide does not correspond to the residue in the predicted protein Ic, but it was introduced to facilitate binding to the carrier protein. This peptide was synthesized on the Arriei peptide synthesizer Viosuzietv Mode! 433A Two milligrams of the peptide were combined with two milligrams of 20 maleimide-activated snail lymph hemocyanin according to the instructions included with the kit

Itiesii Asiimaiei Ittipodep Sopisdayop Kii (Riegse Spetisa!). After desalting, half of this conjugate was emulsified using an equal volume of complete Freud's adjuvant (Riegse). This emulsified mixture was administered to New Zealand white rabbits. Four weeks after the initial inoculation, a booster inoculation was made, while emulsification was carried out in the same way as described above, except that Freud's incomplete adjuvant (Riegse) was used. Two weeks after the booster inoculation, a blood sample was taken and the titers of specific antibodies were determined using the EPI5ZA assay using the immobilized peptide. and)

The next booster inoculation was carried out one month after the first booster inoculation.

C) Western analysis of medium from endothelial cell cultures

NOME5 and NCEAS cells were cultured and stimulated with PMA as described in Example 3C, except that Ge!

Therefore, the cells were stimulated by PMA for 48 hours. Samples of conditioned medium (ml) were incubated with

BOOmcl of 5096 heparin-sepharose suspension SI-6B in phosphate-buffered physiological solution (РВ5, (97 150 mM sodium chloride, 100 mM sodium phosphate, pH 7.2). For partial purification and concentration of proteins | | (heparin was selected from -Sepharose, since the conservation of residues in the sequence of HYOHI was identified as critical for the heparin-binding activity of RI (Ma, U., Nepaeggop, N.E., ish, M.5., 2papad, Shcheo,

N., Rogguine, 1I.9U., SiIagke-I emgiv, I., Naudep, M.K., 5 VgygeiIi, 9U.O., U. Iiriyd Kevs. 35, 2049-2059; and in Fig. b). uh-

After vortex mixing at 4 2C for 1 hour, the samples were centrifuged for 5 minutes at 150 x 9. Then the medium was aspirated and the sepharose was washed with 1-4 ml of PB5. After centrifugation and suction, the precipitated heparin-sepharose was suspended in 200 μl of 2 x SDS loading buffer (490

SDS, 2095 glycerol, 2905 (3-mercaptoethanol, 0.00295 bromophenol blue and 120 mM Tris, pH 6.8). Samples were heated to 952C for 5 minutes and 40 μl of samples were applied to a 1096th Tris-glycine gel with DNA. After electrophoresis at 1408 for approximately 90 minutes, proteins were transferred to nitrocellulose membranes using a Momech electroblotting apparatus (2108, 1 hour). Membranes were blocked for 30 minutes in "" blocking buffer (595 skim milk powder, 0.195 Tween-20, 150 mM sodium chloride, 25 mM Tris, pH 7.2).

Antipeptide antisera and normal rabbit serum were diluted 1:5000 in blocking buffer and incubated with the membranes overnight at 49C with gentle agitation. Then the membranes were washed 4 times for 15 minutes with TV5T (0.195 Tween-20, 150 mM sodium chloride, 25 mM Tris, pH 7.2). Peroxidase-conjugated goat anti-rabbit sl antiserum (Voephipdeg Mapppeit) was diluted 1:5000 in blocking buffer and incubated with mixing with the membrane for 1 hour. These membranes were washed as described above and then (95) reacted with Kepaiizzapse chemiluminescent reagent (YuRopi MEM) and exposed with Kodak Shu 20 XAK-2 film. The results are shown in Fig.11. Two types of immunoreactive proteins were present in samples from unstimulated NOMES and NSAES cells. The levels of immunoreactive protein in the unstimulated NSAEC samples were much lower than the levels corresponding to the NOMES sample. After PMA stimulation, three immunoreactive proteins were secreted by endothelial cell cultures. PMA treatment significantly increased the level of proteins (I) produced by NSAES cultures. PMA-induction of GI proteins (z) in NOMES cultures did not play a significant role.

Example 5 Recombinant protein production! | at

A) PI O-expressing constructs of cDNA encoding OM and 11 OX proteins were cloned into the mammalian RSOMAZ expressing vector (Ipmygodep). This co vector allows the expression of foreign genes in many mammalian cells due to the use of the cytomegalovirus major late promoter. This GI M 5'KASE product was cloned into the RSOMAZ EsoKI site. kKDNKII OKH. 60 was hydrolyzed with Oga!Y and ZY enzymes to obtain cDNA with a size of 1.55 t.p.o. (see Fig. 4). The resulting vector was hydrolyzed with the restriction enzyme EsokU, and then the vector and the insert were ligated using DNA ligase

T4 and reagents from the kit for rapid ligation of Kariya and Idayop Kiya (Voeyipdeg Mappeit) in accordance with the manufacturer's instructions. These ligated products were used to transform competent E. soybean.

The resulting colonies were screened by restriction analysis and sequenced for the presence and orientation of the 65 insert in the expression vector.

B) Short-term transfection of GI o in cells СО5-7

I/C-expressing vectors were injected into CO5-7 cells using the cationic lipid reagent Lipofectamine (SIVSO). 24 hours before transfection, CO5-7 cells were seeded on bOmm dishes for tissue culture at a density of 2x1 OU cells/dish. These cells were propagated in Eagle's medium modified by Dulbecco's method (OMEM; SIVSO), to which 1095 fetal calf serum, 10 bod/ml penicillin and 10 µg/ml streptomycin were added. One microgram of plasmid DNA was added to 30 µl of serum-free medium Oriette I (Birso). Ten microliters of Lipofectamine reagent was diluted in 100 μl of Oriitet I! and this solution was combined with the DNA solution and left to stir at room temperature for 30 minutes. Then the medium was removed from the dishes and the cells were washed with 2 ml of Oriitet I medium. The DNA solution and 70 Lipofectamine were added to the dishes together with 2.7 ml of Oriitet medium, and these dishes were incubated for 5 hours at 372C. After incubation, the serum-free medium was removed and replaced with OMEM medium to which 2906 EB5 and antibiotics were added. Twelve hours after transfection, a portion of the cultures were treated with either 0.25 mM

Regabiose 5C (Voephipdeg Mappeiit), or a protease inhibitor, or 1Ood/ml of heparin. 30 minutes before collection, heparin-treated samples were treated with another 40 units/ml of heparin. The medium was removed from the cells 60 hours 75 after transfection. Heparin-sepharose SI-48 (200 μl of 5095 suspension in РВ5, pH 7.2) was added to one ml of the medium and stirred at 42С for 1 hour. Sepharose was then precipitated by low-speed centrifugation and washed three times with ml of cold PB5. This sepharose was precipitated and resuspended in 10 µl 2x loading buffer. The samples were heated to 952 for 5 minutes. 40 μl of each sample was applied to a 1095 SDS-PAGE gel. Electrophoresis and Western analysis were performed using antiserum against GI 0 as described above. The results are shown in Fig.12. Proteins from conditioned media from the NSAEP were included for size comparison. (IM migrates at approximately 4 OkDa, which corresponds to the lowest band in the NSAEP.

The medium from CO5 cells transfected with IH OX cDNA contains b8kDa and 40kDa protein types. When these cells were treated with heparin, the amount of b8kDa and 4OkDa proteins isolated from the medium increased sharply, which indicated either the release of the protein bound to the proteoglycan from the cell surface, or the stabilization of these proteins by heparin. When cells were treated with the Reiarios protease inhibitor, the amount of bvkDa protein increased in comparison with the 40kDa type protein. This suggests that the protein with a lower molecular weight, which is produced by these cells, is a product of proteolysis of the larger bvkDa form. The role of the mRNA identified by differential mapping encoding the shorter 40kDa protein species is not yet known. However, it has been reported that an alternatively spliced form of liver lipase has been obtained, (o) which is apparently expressed in a tissue-specific manner and should produce a truncated protein. "-

Example 6

Go in animals of various species

A) Cloning of the rabbit homologue of G and yu

A commercially available lambda cDNA library derived from rabbit lung tissue (Siopies, cat. 1110105) was used to isolate a fragment of the rabbit homologue of gene II 0. Five microliters of the original i-library was added to 45 μl of water and heated to 959C for 10 minutes. . The following materials were added in a final volume of 100 μl: 200 μM amtR, 20 mM Tris-HCII, pH 8.4, 500 mM KCl, 1.5 mM MasI, 100 mM μM of each of the primers OGIR774 and I | sdepga, and 2.5 units of Tad polymerase (SIVSO). The reaction was carried out for 35 thermocycles under the following conditions: 15 seconds - 942C, 20 seconds - 509C, 30 seconds - 7220. 10 microliters of this reaction mixture was analyzed using electrophoresis in an agarose gel. A product of approximately 300 base pairs was found. A portion (4 μl) of this reaction mixture was used to clone the product using the TA cloning system. The insert of the resulting clone was sequenced (ZEO IU Mo: 11). Comparison of the primary deduced rabbit amino acid sequence (ZEO IU Mo: 12) and the corresponding sequence "Human DNA is also shown in Fig. 14. For nucleotides not part of any primer for amplification, 85.89 bp similarity was observed between the rabbit sequence and the human GIS sequence. The predicted protein encoded by this

Sh- rabbit cDNA, has 94,695 identity with the human protein, while the majority of nucleotide substitutions is in the third position or in the positions of non-strict correspondence ("swing") of codons. It should be noted that this region covers the sequence of the "lid" of the predicted proteins 1.Y(z) and is a variable domain in the lipase gene family. Obviously, this ensures a high degree of conservation of these 20 genes for various species.

C) o in other species cf. To demonstrate the presence of HER genes in other species, genomic DNA from different species was hydrolyzed with the restriction enzyme EsokKi!I, separated by electrophoresis in agarose gels and blotted on nitrocellulose membranes.

Membranes were hybridized overnight at 65 C with 2.5x10bimp/min/ml randomly primed

HF) probe Z2R-II Z or Z2rR-I RI. (lipoprotein lipase) in solution for hybridization: 6 x 55C, 10965 dextran sulfate, 5

GF x Denhardt's solution, 195 SDS and 5μg/ml salmon sperm DNA. These membranes were washed with 0.1 x Z55C, 0.596 DSN for 10 minutes at room temperature, and then sequentially for 10 minutes at 40 2C, at 502 and 5590. Autoradiograms of the blots are shown in Fig.16.

Figure 16 illustrates the presence of GI and RI genes in all species evaluated, except that hybridization with the GI probe (with anti-rat DNA) was not observed. The exceptional data obtained for rats may represent an artifact caused by the generation of restriction fragments of abnormal size containing Ii sequences. Such fragments may be beyond the bounds of the agarose gel fractionation or may be the result of inefficient blotting. The different bands found with some probes indicate that IRI and Gb represent separate evolutionarily conserved genes .

Example 7

Enzymatic activity of I and OH.

A) Phospholipase activity

Conditioned media from CO5-7 cells transiently expressing human lipoprotein lipase (RU, COM, or II OX) were assayed for phoslipase activity. Minimal maintenance medium (MEM) containing 1095 IU (MEM) was used as a blank control, and conditioned the medium from CO5-7 cells transfected with the anti-content I and Xi plasmid (A5) was used as a negative control.

A phosphatidylcholine (PHO) emulsion was prepared using 1 μl of phosphatidylcholine (1 mM), 70 4 μl of 14C-phosphatidylcholine, dipalmitoyl (2 μCi), labeled in 1 and 2 positions, and 100 μl of Tris-TSMV (100 mM

Tris, 195 Triton, 5MM Sasi", 200mmMmM mass, 0.195 VBA). The resulting emulsion was evaporated for 10 minutes, and then added to the final volume of IML in Tris-TSMV.

Reactions were performed in duplicate and contained 5 µl RS emulsion and 950 µl medium. The samples were incubated in a water bath with shaking for 2-4 hours at 37 C. The reactions were terminated by adding ml of Mn NOSI, and 75 were then extracted with 4 ml of 2-propanoltexane (1:11). The upper 1.9 ml hexane layer was passed through a column with silica gel and the selected fatty acids, which do not contain "C and which are in the flow-through fraction, were quantified in a scintillation counter. The results of these analyzes are shown in Fig. 14.

B) Triacylglycerol-lipase activity

Conditioned medium from CO5-7 cells transiently expressing human lipoprotein lipase (IP3, OM, or I1OXI) was assayed for triglyceride lipase activity. MEM containing 1095 EB5 was used as a blank control, and conditioned medium from CO5-7 cells , transfected with an anti-content I|XII plasmid (AB), were used as a negative control.

The concentrated substrate was obtained in the form of an anhydrous emulsion of labeled triolein, I9,10-ZH(M)| and unlabeled triolein (final content of complete triolein - 150mg at 6.25X108imp/min), which was stabilized by adding 29 ohms of lecithin to 10095 glycerol. 0.5 bml of ZN-triolein (0.28 mCi) was mixed with 0.17 ml of unlabeled triolein (3) and 9 uOml of lecithin (9 mg). The resulting mixture was evaporated in a stream of nitrogen. The dried lipid mixture was emulsified in 2.5 ml of 10095 glycerol by sonication (30- second pulses at level 2 followed by 2 second cooldown cycles for 5 minutes).

The substrate for analysis was obtained by diluting 1 volume of the concentrated substrate with four F 30 volumes of 0.2 M Tris-HCl buffer (pH 8.0) containing 395 wt/vol of bovine serum albumin, which contained -- no fatty acid. The diluted substrate was intensively stirred for 5 seconds.

The reactions were carried out in duplicate in a total volume of 0.2 ml, containing 0.1 ml of the substrate for analysis and 0 T ml of the indicated conditioned medium. The reaction mixture was incubated for 90 minutes at 372C. Reactions were stopped by adding 3.25 ml of methanol-chloroform-heptane (1.41:1.25:1) (v/v/v), followed by 1.05 ml of 0.1 M m 35 potassium carbonate/borate buffer (pH 10.5 ). After intensive mixing for 15 seconds, the samples were centrifuged for 5 minutes at 71,000 rpm. A 1.0 ml aliquot of the upper aqueous phase was evaluated in a scintillation counter. The results of these analyzes are shown in Fig.15.

Example 8 « du Use of II and o-polypeptide for screening in order to identify agonists or antagonists -

Recombinant II was produced in insect cells infected with baculovirus. Recombinant GI C was purified from serum- or serum-free conditioned media by chromatography on 3-Heparin Sepharose followed by chromatography on cation exchange resin. If it was necessary, a third chromatographic stage, such as gel filtration, was used for its purification. During purification, anti-peptide antibodies were used to monitor GI protein, and phospholipase analysis was used - 395 to determine I | c-activities.

In the fluorescence analysis, the final conditions of the analysis provided for the use of approximately 10 mM Tris-HCl (pH 1 7.4), 100 mM KSI, 2 mM Sasi», MCM of SeMvO-POI1-acyl-2-|6-(nitro-2,1,3- benzoxadiazol-4-yl)|amino)caproyl-phosphatidylcholine, and protein 1 (approx. 1-1400ng). The reaction mixture was subjected to fluorescence excitation at 47 Ohms and monitoring was carried out - enzyme activity, which was measured by fluorescent radiation at 54 Ohms. Then the compounds and/or substances tested for stimulation and/or inhibition (| of C-activity were added as 10-200 mM solutions in dimethylsulfoxide. Compounds that stimulate or inhibit II C-activity were identified as compounds that cause an increase or a decrease in fluorescent radiation at 54 Ohm.

In this thio-assay, the final assay conditions involved the use of approximately 25 mM Tris-HCI (pH 8.5), 100 mM KSI, 1 mM Sasi", 424 mM Triton x-100, 0.5 mM

GF) 1,2-bis(hexanoylthio)-1,2-dideoxy-zp-glycero-3-phosphorylcholine, B5mM 4,4"-dithiobispyridine (with 5OmM of the original

HF solution in ethanol), and 1-10O0ng of recombinant GII. Phospholipase activity was determined by measuring the increase in absorbance at 342 nm. Compounds and/or substances tested for stimulation and/or inhibition in | O-activity was added in the form of 10-200mM solutions in dimethylsulfoxide. Compounds that stimulate or inhibit I and b activity have been identified as compounds that cause an increase or decrease in absorption at

With 42 nm.

Example 9

Transgenic mice expressing I| 5 people

For further investigation of the physiological role-- Transgenic mice expressing human I 65 were produced.

The 1.53-t.p.o.-YuOhai/ZpI restriction fragment encoding GIOHI (see Fig. 4) was cloned into a plasmid vector (rnmMo) below the promoter for the constitutively expressed reductase gene,

3-hydroxy-3-methylglutaryl-conformant A (NMO CoA). Transgenic mice in which different levels were expressed

Human IGOs were obtained by standard methods | see, for example, O.Yi. Tgotrei aiYi., Sepe 1565: 199-205, 1995). These transgenic mice were used to determine the effect of GI OX overexpression. on lipid profile, vascular pathology, rate of development and severity of atherosclerosis, and other physiological parameters.

Example 10

Expression of GI C in atherosclerotic tissues 70 The expression of GI OCHI in atherosclerosis was evaluated by performing reverse transcriptase polymerase chain reaction (RT-PCR) using mRNA isolated from vascular biopsies from four patients with atherosclerosis. Tissue samples were taken from the wall of the aorta (one sample), iliac artery (two samples), and carotid artery (one sample).

Biopsies from atherosclerotic vessels were obtained from the clinic (Sioiseviegpige Koua! Nozria, Epdiapa), after which 7/5 was obtained roiu (AukmRNA and resuspended in diethylpyrocarbonate (OERS)-treated water at a concentration of 0.5μg/μl of mRNA. Reactions with reverse transcriptase were carried out in according to the Birso VKI protocol added to the pre-amplification system (Z,iregzsgiri Rgeatryisabyop Zuviet) for first-strand cDNA synthesis Briefly, cDNA was synthesized as follows: 2 μl of each of the mRNAs was added to 1 μl of ido(at)412.1v8-primer and Uml OSERS-water. Tubes were incubated for 10 minutes at 702C and placed on ice for 1 minute. The following components were added to each tube: 2 μl of 10x ROK buffer, 2 μl of 25 mM MoSi", μl of 10 mM amktR mixture and 2 μl of 0.1 M DTT. After a 5-minute incubation at 422 C, 1 μl (200 units) of Zyreg 5sagiri II reverse transcriptase was added. The reaction mixture was gently mixed and then incubated for 50 minutes at -4220. Reactions were terminated by incubation at for 15 minutes at 70 2С, and then the reaction mixture was placed on ice. The remaining mRNA was destroyed by adding 1 µl of RNase H to each tube, and incubated in He for 20 minutes at 3726. o

PCR amplification was carried out using 2 μl of the cDNA reaction mixture. The following components were added to each of the test tubes: μl of 10x RSK buffer, 5 μl of 2mM amtR mixture, 1 μl of PiIId-dzri primer (2 Opmol/ml, see

Fig. 1), iml of pIId-dzr2a-primer (2Omol/ml, see Fig. 1), 1.5µl of 50MM Masi, 0.5µl of Taz polymerase (5 units/ml) and 1µl of water. After holding the reaction mixtures for 2 minutes at 952С, 30 cycles of RSK FU were performed under the following conditions: 15 seconds at 942С, 20 seconds at 522С and 30 seconds at 7220. The final reactions were carried out at «- for 10 minutes at 722С, and then analysis by electrophoresis in an agarose gel, pPIId-dzr primers are specific for ГІ0 and give the expected product in З00p.o. In parallel RSK, it was also shown that the cDNA synthesis reactions were successful, while using primers that are specific to the Mu gene of "housekeeping" and to OZRON (glyceraldehyde-3-phosphate dehydrogenase) (1 μl of each at a concentration of 20 pmol/ml) . -

O5RON primers (see Fig. 1) gave the expected product in 983 p.o. for all four vascular biopsy specimens. IZ expression was detected in three out of four samples, and in the sample taken from the carotid artery, this expression was not observed. "

All discussed works are incorporated into this description by reference.

It is obvious to anyone skilled in the art that this invention can be easily adapted to achieve the purpose of the invention in c and achieve the desired results and the above-mentioned advantages of the present invention. The peptides, polynucleotides, methods, procedures, and techniques described in this application are presented as preferred embodiments of the invention and are provided for illustrative purposes, and therefore should not be construed as limiting the scope of the invention. Changes and other variants of its use may be made to this invention, however, they should not go beyond the essence and scope of the invention formulated in the attached claims.

Ш- List of sequences «сl (1) General information: (65) (i) Applicant: Chaueh, Misnavi! S. - 50 roap, Kit-App T.

Kgamles, Ddopp A.

Me, Hups, Kemip.).

Atip, Oiiir U. zosh, Misiogia .). o (her Name of the invention: Polypeptides of the HER family of triacylglycerol-lipases, compositions and methods of their use in enzymatic hydrolysis, and therapy using proteins and genes ko (ii) Number of sequences: 31 (m) Postal address: bo (А) Address: Kpope -Roshieps Kogeg Ips.(B) Street: 500 Agsoia Ka. z3s43 (C) City: SoPedemShe (0) State: RA (E) Country: USA 65 (R) Zip Code: 19426 (m) Computer Readable Form:

(A) Media type: floppy disk (B) Computer: PC compatible with IVM (C) Operating system: РО-ЮОБ/М5-БО5 (Ю) Software: Raiepiip Keyeaze 2 1.0, Megaviop Zh 1.30 (mi) Data of this application: (А) Application number: О5 (В) Date of submission: (С) Classification: 70 (my) Information about the attorney/agent: (А) Name, surname: ReyiIpeg RA.O., Ratsi KE. (B) Registration number: 35135 (C) Document/file number: А2582-МО (their) Telecommunications data: (A) Telephone: (610) 454-3839 (B) Telefax: (610) 454-3808 (2) Data sequences of ZEO IO Mo: 1: (Y) Sequence characteristics: (A) Length: 367 base pairs (B) Type: nucleic acid (C) Chainability: double-stranded (OB) Topology: linear (ii) Type of molecule: cDNA (ХХ) Distinctive feature: сч (А) Name/key: СО5 (В) Localization: 22... 180 i) (хи) Description of the sequence of ZEO IYU Mo: 1: in Ryzh ruye buye yiUu siv buh aa sbeye Su vnu Ф - and be i 5: 10 - da las pot at dat os. at BOS TAS VAT spos dja zda ATO Aby BAS 55 o ate Atsi Aks bSuya aby vaye GU CiZh tuye dyan dia: pu puz mes vy vyy yu dao dove Ave VBS LAC AT TAS sta ZaA AOS sov sa bos A esU 00

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Vuz sa Ah Bra au Mas to vna dev. 55 o (2) Sequence data of BEO IYU Mo: 10: o (Y) Sequence characteristics: (A) Length: 345 amino acids 60 (B) Type: amino acid (OB) Topology: linear (y) Type of molecule: protein (khi) Description of the sequence of ZEO IU Mo: 10: b5 -4o-

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With Fr. I 48 "lyu lkCs RN You I AK TA Zh Was yzhr tu SI nish bla Ir Suye ri inv vo same su

Zhchuht Sam det Uai Su liv beye biv res go Sim dno buya bah rye yo in 70. tv o

Mov tne action puyu sny rve Be ate ti Mia SI der VE Mav bag 01u ve - 50 dv tiv ne bi5 dep bu pav, niya duv tesh chai Bag da Pio NIV Tv Ak wa cho ch 25 "am uv Avr Aza Anpouai Uvi Uai Uai Yanr Tkr be rub cen div niv t2b yi28 zo siy na Tuk No lavr yiia CHE dvy avt be dme Mi Uvi biu I yav F yi3b 5 vy IY a u so shtV duye Azhu MEE bi Ar tkV VNU siv Fo shchuz Ayar. yvo Ve. Za She yu z5 145 155 i55 ii m vit ach Uvi Ni Bei ie Stu Tu bej rec si din Ni Mak Aiya: BU p to IT: « to «tut Aia biu Avp VNe Uvi tsuz biu tTbr Ua biu Ak Me Tu Shu bab Z s veins ЖН5 shcha ;z»

Zher Bko Aga Su ge MaYE Ne ti biu Aia dev TE NIV Bu Aku i: - 155 HO etebsya sl shi shko Ashr dyar dyi dir ra tuai dav'tyi Beyu NESHOTVE tu tah already

VIY yb VI 230 Sh y oz - Beh Rre biu shen bek yiit bih Me bip Meb bto Uzi biu Niv piv dya. so ev 859 EE: 2550

F) ime) 60 b5 -d41-

tiesTug Be Java siu Biu Abr Rbe bip Rio Stus suy siu pe and dir.

B. 25o and g Y

Mai this biu bek 116 dia Tut Biu bbh iiv Te obi Mai bai buye Sun

G.I. " 289. 285 in Mer puv Bko Beh bce Ku RBe Bii Suye TK ver beg two Yaku Be buye uv TU Ize Suh This Yaeh Suye A puz Avi Agu puye dey Bezh tiv Su 305 Zb; I VE z put kva Ka tsu was MY Ats АБЙ bue Kto blu bek tuye mak tuk ab; - x 525 839 zav uz Ti dif Viv siu ma ryy Ne Kg o (2) Sequence data BEO IYU Mo: 11: o (Й) Sequence characteristics: 3o (А) Length: 225 base pair -- (B) Type: nucleic acid co (C) Chainability: double-stranded (OB) Topology: linear Io) (i) Type of molecule: "dyk m (ХХ) Distinctive feature: (А) Name/key: СО5 (В) Localization: 1... 225 (хи) Description of the sequence ZEO IYU Mo: 11: « -to oh gos dos sso tat opo dob tuo oso odo sto sto hlotos so 34 - s Zhe stu Bad zha Alya Tukhkobuu TB t Aba BI Chai Uch was "u azh kat as spos osv ot sit sta stu sta rai TS Sto bib vaye sv to 8 nev: Anu vda uvo no rea vva Mav der yav em Uai dar vy YAM av" in Zt5 o rya liv vo de ; ry da ve from Su TU yo she Azpodue NYA Muzh Buv - Y kv yav 896. 3e) from

FIU GIV suzh Bey bag buz Agb By5 Kyavo Nu SU Av BI TIV BIU tuye shchi with ve: 405 in 00» 5 І 426 and (2) Sequence data BEO IYU Mo: 12: (І) Sequence characteristics: (А) Length: 75 amino acids because (B) Type: amino acid

(OB) Topology: linear (s) Type of molecule: protein (hi) Description of the sequence ZEO IYU Mo: 12: y im ish Beh Her Ai Tok SIu tn Tiie ytva shi Me Ua was Sov st niv ia lysh aty uni nih pev Za li direeak eat Uvi Aed bij yvo 70 B: W sh 50. yuuz ygo Sher Vve yia BE BIV was Yanu vo bek y eh Te puv Buv i cha 45

Fu tiassuv Zhvy Bak buye va: ssh; There is a child; ; -: the wife of Stu vteosschu tuye

Bo 5 va yu pet via BU Bush Tit ju Aza. Puz Aro: Kai. Tv (2) Sequence data of ZEO IYU Mo: 13: Ha (Y) Sequence characteristics: (A) Length: 472 amino acids and) (B) Type: amino acid (C) Chainability: (OB) Topology: linear Ge»! (i) Type of molecule: protein "- so

IS) h- « shi s ;» -I 1 (95) - 50 3e)

F) name) 60 b5

Her) Description of the sequence ZEDCHO Mo: 15 mae biy svy that vyv red petsouai ped tn Pen action Ua) bkr ev; white

Shnh em Tv ih Yaeh nd bu bih Mei Bin Diya KT" yo sel sho Yaku 23 y. 89; 70 o

Fat RE tla dar Y15 STI SHA uyu revodIi TZ dr tTme to SIV ver 35 40 -Я8 18 tn Adv siyu AEr Te was niya Bai tae ggo saausmau hii sto bek Uuyh 5o I 56 -Via Zah was niya Ve Ayap in dec deh buh TE va has Mai ie ih vv. t 7 vo btu tar tt Uvi Tr biu Nee tuye bin bej Tut Uvi Ryb puh Bav UVI v ia But oi Tut Jester What fight Btosayar Bak dev Chai Tiv Mai MV Av y 400 105 "o o

Yatroizhv Ber oyto bla biv bij Noya Tut RKO Ma) Zh Aiy putuh Te F zo 115 ho wa: - blew May STU Shiv avb zhi Kt Kzhu BBZh tie dev Ter: Meye Zm siv vza so o az5 y ta.

IC c)

She dya Tuh Bu Petz yav AVa Mai niya pev Bai stu tuye Bach pev stu imm zav BO: EV ze kiv niv div Ki Shiu 116 Ata bud Beh Zhe zhi Zhov buh tsuz Chai dev « y 16 Another 275 with - Zhis Tye Tyh OIu tsem oavr. web give o btu hgo, Av RN SIV buye At SHLE ha vho Ato ceyu Bebg reko KER OR Yata dor te Mai Ar Mah TA niv that 195 Cho: 05 Y -I p «ih fre Tk ge Stu Bei ko Su Am Bav tte BIU NAME ZIB PI RE

Fo 253 5 ' - 829 g! I live in SU, and I'm going to pay for it.

Zhuzh ney piE SHI sii Kiya yiye yge Ua) Ya1e Ala biz Agu B1u tep u 59 y dav: 5 255 ko "dev uv Mer o ben Buv Suh Beh Mi biv At b8E ee Ni She: VYK more. 55 what bo tsk o zyavishyanya ie tr bet yz This ABY BIB Shi dva REO Beh Shuv Aza: uk zhu Su v av vav; b5 byap yku buye kv yiy this sSiu tuye st liwa dev fu Uvi ka Aia yiv: o shi: ZiB 320 1 her - ij as po su called PIRCHYNIA and Zhysh shaye bek tsuv nen here this was te dshu bah bidoMes rgo Tu But.

Sho OO yada la 38: me vne nev tuk bi Chai buh IT Ni be bek btbu TE bi Vat SI t5 Z li . 50 355 "z ab sche. - g ish bek ia Azh Kia Vo WBe- Ne She. rsho Z Mah Bi TE ASHO PIKE:

Ya. i Yaru vo «ng tus bat She pait tiv buk ke sta Mah der IT slu bish ev Beyu o),

For 5 | zvo 98: ob "Mey pos buye gay tu ter opuv bak dyar-bek tuk vne Zak Yar pek Avr. F z Shchi y 405 ada sia

Z. k -- tkr ter bah bah rgo biu Bbe Kia gi Ziv Muye T1e yhya Chai MU KL o. 425 410 yu

HU BT: TVE bid uh bu Ugi ie RVa suye Bak denu 1: Buk Chii Yaeh dya zha in bin pahu lovi yuni de she " du iv mesh bid Ha P1uU puh yla VEB Aa Chai Ve uh. ruye suye niv ani - g» put wah te Yaya le yizya bah su (2) Data of the sequence BEO IYU Mo: 14: o ; : . (Y) Sequence characteristics: (95) (A) Length: 499 amino acids 20 (B) Type: amino acid (C) Chainability: (Che) (OB) Topology: linear (y) Type of molecule: protein (hi) Sequence description ZEO IU Mo: 14:

F) ime) 60 b5 -4B-

Ma de TEH BEE oto: Tsevy shuye RBe eh Hle vy May ki demo suyu DM and Be 129 M.

Y fne yiIie biv Bed bet AK Shvy bij BEZH Shesh Cikh RE ii Web BBa 8: t5 zhh Z "Strved zhi my bu liv bah bi otv Mev Buietne rea niv pi Mak to V: y Ya tdla TE Mk RVE Shey pei be SPU SIV TV sent SIV things that were studied. vo still Because, then tt in you niy ko YAZ Same you bib biy Suk sim re dyad dach Beh 55 bo ov vo.

Zhets Btodei ni Maye Ti: ie Vie biu tr Beh Chai der hu Chai her - bij da tur ihe Ter biv nev vt ki ovDa dev Bu beh biv Ruo Aid . sch 25 he rived in the book Ua SSchu ey au yar tiv Still Tv bbo Kia Mi bno o lak: 125 125

Ji-e Tut tet Ti Kta Mui des le tnh VKF This uki Fu ce pra Ua 130 aB | that F 30 iv BUT rey ro Yabu fer bez siv bin betu Tli biz Shev beh yku Btt IY

The bottom of the horror is not ey Ishe Mu tuye bok Without SIM yiv No Chai bej biu Re shchi poi, yo 115 te "Kin b1u Bek bek yiye bi S1u Tk niya uv tiF stu you go tv tv S1u " - Yanka Zhor Kii DIA b1u wgo dey Ne tv sah Beash-AIA HO; BO Ar: at 15 bu Wei fuo-kvoskzhi Az Gag TKe Uk) Avo div Yi1e Nih She» Ve Tbg - at how 18 225 7 and hv bim niv Meb bu bez Beh Hai F1u Zhe uv Biy Web She b1u niv still and | she "rue dar re Tut kto vp Bu bIiu Bek Be SIV: IN STU SPuz niv janv

Sho -445 255 55

Zeshchsh iv pay here keV snit tiv div sta nia biu Bezhen lin Yshe TV shi She: zhvB ato yuyu stand tk ti mus sSuv bek Niv biz Arsh bek Chai Nie Bosh Beite: yav -: Pi " vh 259 E 395 zob b5

Take g and i5 29 uz and tnE dey pi tuh niya cha! vzhu bij bin jz yo Beh Buv Bej: go uv AJ bas nya bec ai Zhvt Ave Kia bij bek Bgto bne buw Mais Tukh y 340. 545 359 y zva 50 ЗB5

Fin yin Tvho REV NE MeEE Bob rey pei b1u tk shuye b10 Buzh mevooiv tUE te Rub te TNt Bai si uv siu gi action zah la duh Pih Tut i85 ! with sDNSH kov "o

Weight of RMA TA; IZ tet yin Der in Avr ti bi biv pev even Meb Me s 5 I - o Z o ko. dach o F y Chai sid Te tiv 16 Rez Ter Beh nyu sadu o At Nav Bezh tush 518 kb. 45 so

Uch Bai tsue tyaz ti t chni Bi vla soky bin tozh biv od aku Ne k shchi s YOU "55: vo. tyag zna Suz ZBK PI ЖЯav ТНЕ Fat ha Bey spe pay At vEO Tv shiy yv5 . 5 tvo « 47 sings half Be Chai half book Siyu tiv pub back Muz Jiu Bak yuvoyhu she scheche MVV with

Tuye tlie kn, y - (2) Sequence data ZEO IYUO Mo: 15: s (Y) Sequence characteristics: (A) Length: 465 amino acids and (B) Type: amino acid -oUu 70 (C) Chainability: (OB) Topology : linear c (i) Type of molecule: protein (hi) Description of the sequence ZEO IYUO Mo: 15: ime) 60 b5 -A7-

the same song wai buye tuye biz yho Bech siu suh vne shah lion dia bik rEo

And what else?

Zkr Beh biu Tif ba bi ysu bho Men Niv Lived peb he hr bek ro to zhov wav My hevotTne Abu ne Bec bey Tu FBI zai bhysh Av Already two

Same vne hundred Yaia chai Aza Viv dir B Bach shah Tte da tu bok aa 8 ' te i 75: y to

Same was Ts Aep Kk buh Track ju vne te tiv niya su ke tiy bo - VV a | B: Y

Zhuv SU bin biy dey Tu" rech Azh byap fai Suye uz Ana Tsei Rna: y to zav and ZI and lai, b. Ve Mai koi buye 18 sub UVK Sawyer Tk Put Fiu biu' bach yho what. 135

Ua Aa Tut Ve U«! 625 Bve Ben btya bbg aza Vne OIu tup be go F zo shcha5 . 150. shya5 bo - viye Azh chuai niya Ma) tie STI Niya sah pub pi div NE A Mah VU: so - this is ito shlya : yu "a azh vu wk at Tr Ap Ziu You lived bu yge iv bBu bu bez. what's not: That one

Author Rkgo Via bi: Rko was Be biy stI se Wbo biy deychni dvo pey « yu 195 20o zo5 - - dav vko dayu der-kIi puya rRYE Ua DER HiXh dl ViJ Zhbu der pIU da z Mao - de zov vu tu ti uai Ro dvy gustu Ve biu Me: bat Siv Uai Mai bi Viv: - 225 - vva y 238 240. ti zr Ve Ver rko va bu Biu Ua: biv Mav Vt bius sv was yuh o ya va 28o 5 tsu . s dep zhiv tsech det ba tiv ME OAVO BF AB SBU dye Troi vau og gc dir RNE da lin sukh yshi Niv svi dlzhsh bek bukh fun tuzh bukh Shok vu zvo 285 o N

Yaki ev bach iv UVI Akv rko Abr stu Rie Ata Su BVE go Suh Da Wb with BA 85 Cohen Z as two tar vna tn KIV deli muye suyu vne VEB buye W BV bij Bi. 305 Зв и ш оф и ше б5 '

5. and dyav mach under Om ul Ve Tut pe Ako Tvzh ZT KYar dz: Bet Avpovve and another 35th call for zveze ti BU Bin ba Be ah bek geo рн biu y Shuch OKU vyadobey husband

With zt zvo "shti tuye sing ti Feruz biu TBh Bash ruye Bho Anyu bek TI JI Vazh s: B zva y 355 i "vay pi velem eh Achyu Chai yvr zh Biu Ayar Bey biv Mes bi Puye that "7 voB. ko 8. tne Tbzh tku buh yar vai 4 Lives Khyab VZHO Tk Bey re Azh chai. FU about 85 students of the University of Ukraine. with was Bug zZhto biv and Uvi Ahe Siv biv Uvi Mem shen sTvy bno Zno Ver; F

I still came " - bitch!

I - (2) Sequence data of ZEO IYU Mo: 16: (Y) Characteristics of the sequence: (А) Length: 17 amino acids « (В) Type: amino acid (С) Chainability: шщ с (ОБ) Topology: linear и (ии) Type of molecule: peptide "" (y) Type of fragment: internal (hi) Description of the sequence ZEO IYUO Mo: 16: -5 Ba kw sii shi ju pai sla Ave tliai ey Na tuye ve to scha. small town

M Ya. o x5 o A e s schuye 1 Y v -- z i

Ge (2) Data of the sequence of ХЕО ИЮО Мо: 17: (Й) Sequence characteristics: (А) Length: 13 base pairs 5B (В) Type: nucleic acid (С) Chainability: single-stranded (Ф) (ОБ) Topology: linear ka (ii) Type of molecule: other nucleic acid (A) Description: /opis. - "oligonucleotide" in (xi) Description of the sequence of ZEO IU Mo: 17:

ТТТТТТТТТТ ТА 13 (2) Data of the sequence of ZEO IYU Mo: 18: (Y) Sequence characteristics: (A) Length: 10 base pairs 65 (B) Type: nucleic acid (C) Chainability: single-stranded

(OB) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: /opis. - "oligonucleotide"' (xi) Description of the sequence of ZEO IU Mo: 18:

SATSAATSOS 10 (2) Sequence data of BEO IYU Mo: 19: (Y) Sequence characteristics: (A) Length: 23 base pairs 70 (B) Type: nucleic acid (C) Chainability: single-stranded (OB) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: /description. - "oligonucleotide"' (xi) Description of the sequence of ZEO IU Mo: 19:

TASSASATOS ASASTOTAAT STO 23 (2) Sequence data ХЕО ИЮО Mo: 20: (Y) Sequence characteristics: (A) Length: 20 base pairs (B) Type: nucleic acid (C) Chainability: single-stranded (OB) Topology: linear (ii ) Type of molecule: other nucleic acid (A) Description: /opis. - "oligonucleotide"' ch (xi) Description of the sequence of ZEO IU Mo: 20:

SATTOTOSTO SSSASTTSTS 20 i) (2) Sequence data ХЕО ИЮО Mo: 21: (Й) Sequence characteristics: (А) Length: 19 base pairs Ge! zo (B) Type: nucleic acid (C) Chainability: single-stranded -- (OB) Topology: linear zo (ii) Type of molecule: other nucleic acid (A) Description: /opis. - "oligonucleotide" o (xi) Description of the sequence of ZEO IU Mo: 21: cha

САСАСТССАС ССАСТОАДАС 19 (2) Sequence data ХЕО ИЮО Mo: 22: (Y) Sequence characteristics: (А) Length: 48 base pairs " (В) Type: nucleic acid with c (С) Chainability: single-stranded

І (OB) Topology: linear і? (ii) Type of molecule: other nucleic acid (A) Description: /opis. - "oligonucleotide"' (THEM) Distinctive feature: -I (A) Name/key: modified base (B) Localization: 36 o (0) Other information: /mod.base - and 2) (THEM) Distinctive feature : (A) Name/Key: Modified Base - (B) Localization: 37

Ge) (0) Other information: /mod.base - and (THEIR) Distinguishing feature: (А) Name/key: modified base (B) Localization: 41 (0) Other information: /mod.base - and

Ф) (THEM) Distinguishing feature: ka (А) Name/key: modified base (B) Localization: 42 vo (0) Other information: /mod. base - and (THEM) Distinguishing feature: (А) name I/key: modified base (B) Localization: 46 (0) Other information: /mod.base - and 65 (YH) Distinguishing feature: (А) Name/key: modified base -BO0-

(B) Localization: 47 (0) Other information: / mod. base - and (xi) Description of the sequence of ZEO IU Mo: 22:

SPASOASPAS PAOSOSASASOVO sSTOoSASTTAST AesZankoYa MMOossMMe 48 (2) Sequence data ХЕО ІХО Mo: 23: (Y) Sequence characteristics: (A) Length: 28 base pairs (B) Type: nucleic acid 70 (C) Chainability: single-stranded (OB) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: /opis. - "oligonucleotide"' (xi) Description of the sequence of ZEO IU Mo: 23:

САСАСАСАЕЕ ССАСССЗТО АСТАСТАЕС 78 (2) Sequence data ХЕО ИЮО Mo: 24: (Y) Sequence characteristics: (A) Length: 24 base pairs (B) Type: nucleic acid (C) Chainability: single-stranded (OB) Topology: linear (ii ) Type of molecule: other nucleic acid (A) Description: /opis. - "oligonucleotide"' (xi) Description of the sequence of ZEO IU Mo: 24: p

ASASSATTVY ABASSAADOE SB 24 o (2) Sequence data ХЕО ИЮО Mo: 25: (Y) Sequence characteristics: (A) Length: 25 base pairs (B) Type: nucleic acid (o) (C) Chainability: single-stranded (OB) Topology : linear -- (ii) Type of molecule: other nucleic acid co (A) Description: /opis. - "oligonucleotide"' (xi) Description of the sequence of ZEO IU Mo: 25: y

ССАСТТТСАшШ ССТОАСТИСТ TАТТС 25 - (2) Sequence data ХЕО ИЮО Mo: 26: (Y) Sequence characteristics: (А) Length: 21 base pairs « (В) Type: nucleic acid (С) Chainability: single-stranded С с (Юр) Topology : linear "» (ii) Type of molecule: other nucleic acid " (А) Description: /opis. - "oligonucleotide" (chi)

Description of the sequence of ZEO IUO Mo: 26: sastatosSAC TSAASOATET C -i (2) Sequence data of XEO IUO Mo: 27: sl (Y) Sequence characteristics: (A) Length: 22 base pairs (95) (B) Type: nucleic acid shi 20 (C) Chainability: single-stranded (OB) Topology: linear iChe) (i) Type of molecule: other nucleic acid (A) Description: /opis. - "oligonucleotide"' (xi) Description of the sequence ZEO Yu Mo: 27, (2) Sequence data XEO IYUO Mo: 28: o (Y) Sequence characteristics: name) (A) Length: 25 base pairs (B) Type: nucleic acid 60 (C) Chainability: single-stranded (OB) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: /opis. - "oligonucleotide"' (xi) Description of the sequence ZEO IU Mo: 28: 65 shsnzh vit ACE TOSOL STE 025

(2) Sequence data of ХЕО ИЮО Mo: 29: (Й) Sequence characteristics: (А) Length: 25 base pairs (В) Type: nucleic acid (С) Chainability: single-stranded (ОБ) Topology: linear (ии) Type of molecule: other nucleic acid (A) Description: /description. - "oligonucleotide"' 70 (xi) Description of the sequence of ZEO IU Mo: 29:

ААСТСТОААА ОССАТОССТо SOSOP 025 (2) Sequence data ЗЕО ИЮО Mo: 30: (Й) Sequence characteristics: (А) Length: 26 base pairs (В) Type: nucleic acid (С) Chainability: single-stranded (OB) Topology: linear (ии ) Type of molecule: other nucleic acid (A) Description: /opis. - "oligonucleotide"' (xi) Description of the sequence of ZEO IU Mo: 30:

ТтОААСОТСоО АОТОСЛАСИЗА ТИТОКЕЖ 76 (2) Sequence data of BEO IYUO Mo: 31: (Y) Sequence characteristics: (A) Length: 24 base pairs c (B) Type: nucleic acid o (C) Chainability: single-stranded (OB) Topology: linear (ii) Type of molecule: other nucleic acid (A) Description: /opis. - "oligonucleotide"' (o) (xi) Description of the sequence of ZEO IU Mo: 31: -

Formula of the invention

IC) 1. The isolated polypeptide encoded by a gene similar to the lipase gene (II C), where the indicated polypeptide h- (a) binds to heparin; (p) has homology with lipoprotein lipase and human liver lipase; and (c) includes a 39 kDa catalytic domain of the family of triacylglycerol lipases having ZEO IU MO: 10, "where said polypeptide contains an amino acid sequence selected from the group consisting of ZEO IU MO: 20.6, ZBYU MO: 8. - c 2 The isolated polypeptide according to item 1, where the indicated catalytic domain weighing 39 kDa of the family of triacylglycerol lipases has the amino acid sequence zEO IU MO: 10. "» 3. The isolated polypeptide according to item 1, where the specified isolated polypeptide has the amino acid sequence ZEO IU

MO: 8 and an apparent molecular weight of about 55 kDa. 4. The isolated polypeptide according to item 1, where the specified isolated polypeptide has the amino acid sequence ZEO IU -I MO: 8 and an apparent molecular weight of about 68 kDa according to the results of electrophoresis in 1096 SDS-PAGE. 5. The isolated polypeptide according to paragraphs 1-4, where the specified polypeptide has the activity of phospholipase A. and 6. The isolated polypeptide according to item 1, where the specified isolated polypeptide has the amino acid sequence ZEO IU about MO: 6 and an apparent molecular weight of about 40 kDa according to the results of electrophoresis in 1096 DSN -PAAG shu 20 7. The isolated polypeptide according to paragraph 1-6, where said polypeptide is derived from a human. 8. The composition containing the polypeptide according to pp. 1-7 and a biologically compatible solution. (Che) 9. A pharmaceutical composition containing a polypeptide according to pp. 1-7 and a pharmaceutically acceptable carrier. 10. Antigenic fragment of the isolated polypeptide according to paragraphs 1-7, containing the sequence ZEO IO MO: 16. 11. The isolated nucleic acid encoding the polypeptide according to pp. 1-7. 12. Isolated nucleic acid according to item 11, which is cDNA. 13. The isolated nucleic acid according to item 12, where the indicated nucleotide sequence is selected from the group that

F, includes ZEO IU Mo: 5, 7 and 9. ko 14. Isolated nucleic acid according to item 13, which includes nucleotides 252-1754 5EO IU MO: 7. 15. Isolated nucleic acid that hybridizes with nucleic acid having a sequence selected from the group including ZEO IUO MO: 3, 5 and 7 under the conditions under which such hybridized nucleic acid is able to withstand washing under conditions of high stringency: 0.17555, 0.595 SDN at 6896. 16. Isolated nucleic acid according to item 15, which includes 20 nucleotides. 17. The isolated nucleic acid according to claim 15, which is an antisense nucleic acid. 18. The isolated nucleic acid according to claim 17, wherein said antisense nucleic acid sequence 65 is operably linked to the gene expression regulatory region. 19. The isolated nucleic acid according to item 15, which hybridizes under high stringency conditions with the target,

Claims (1)

selected from the group consisting of nucleotides 44-79 of BEO IUO MO: C and nucleotides 1036-1065 ZEO IUO MO: 5. 20. A composition containing a nucleic acid according to claim 17 and a biologically compatible solution. 21. A pharmaceutical composition containing the nucleic acid according to claim 17 and a pharmaceutically acceptable carrier. 22. The vector containing the isolated nucleic acid according to claims 11-14 operatively linked to the regulatory region. 23. The vector of claim 22, wherein said regulatory region is obtained from a heterologous source. 24. The vector according to claim 22 or 23, which is a viral vector. 25. The vector according to claim 24, which is an adenoviral vector. 26. Recombinant host cell containing the vector according to claims 22-25. 70 27. Recombinant cell according to claim 26, where the specified cell is a eukaryotic cell. 28. Recombinant cell according to claim 27, where the specified cell is a CO5-7 cell. 29. Composition containing the vector according to claims 22-25 and a biologically compatible solution. 30. Pharmaceutical composition containing the vector according to claims 22-25 and a pharmaceutically acceptable carrier. 31. The method of obtaining the polypeptide according to claim 1, which involves the cultivation of recombinant cells according to claims 26-28, /5 In conditions that promote the expression of the indicated polypeptide. 32. The polypeptide according to claim 1, obtained by the method according to claim 31. 33. An antibody capable of specifically binding to the polypeptide according to claims 1-7 or 32. 34. An antibody capable of specifically binding to the polypeptide according to claim 5 and neutralizing the phospholipase activity of this polypeptide. 35. The antibody according to claim 33 or 34, which is a monoclonal antibody. 36. The antibody according to claim 33 or 34, which is a polyclonal antibody. 37. A hybridoma cell that produces an antibody according to claim 35. 38. A composition containing an antibody according to claims 33-36 and a biologically compatible solution. 39. A pharmaceutical composition containing an antibody according to claims 33-36 and a pharmaceutically acceptable carrier. high school 40. Screening method for detecting agonists or antagonists of I| b-activity, which involves: (a) contacting potential agonists or antagonists o and substrate and 1 0, and i) (B) measurement of the ability of potential agonists or antagonists to enhance or inhibit C-activity, where the indicated / I 5 is the polypeptide according to claim 1. 41. The method of enzymatic hydrolysis of the complex ester of phosphatidylcholine, which involves contacting Ge! from the specified ester of phosphatidylcholine with a polypeptide according to claims 1-5. 42. The method of improving the lipid profile in the serum of a person or an animal having an undesirable lipid profile, where the specified method involves the introduction of an effective amount of the pharmaceutical composition according to claim 9 or c 21. 43. The method of improving the lipid profile in the serum of a person or an animal having an undesirable lipid profile, where the specified method involves the introduction of an effective amount of the pharmaceutical composition according to claim 39. 44. The isolated polypeptide according to claim 1, wherein said polypeptide originates from a rabbit. 45. The isolated polypeptide according to claim 44, having the amino acid sequence ZEO IU MO: 12. 46. Isolated nucleic acid encoding the polypeptide according to claim 45. 47. Nucleic acid according to claim 46, which has the nucleic acid sequence ZEO IU MO: 11. " 48. A transgenic mouse that expresses the polypeptide according to claims 1-5. with c ;" -I 1 (95) - 50 iChe) F) ime) 60 b5