JP2003520599A - Gene encoding ABC1 paralog and polypeptide derived therefrom - Google Patents

Abstract

  (57) [Summary] The present invention relates to three novel genes encoding PD-ABC proteins and polypeptides therefrom. The present invention relates to the detection of genetic defects in the gene, the subcellular localization of the polypeptide, binding assays linked to chemical databases, gene therapy, and the identification of chemicals that can be used in the treatment of PD-ABC mediated diseases. Methods for using the novel genes and polypeptides are also described.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION

The present invention relates to a PD-ATP binding cassette gene (hereinafter, "PD-ABC gene").
) And polypeptides derived and identified therefrom, and use of the PD-ABC gene for drug screening assays, and expression of mutants or by abnormal levels or activity of the PD-ABC gene The present invention relates to a diagnostic method and a therapeutic method for treating cardiovascular disease or inflammatory disease. The present invention is
The D-ABC gene sequence encodes a polypeptide that is paralogous in the ATP-binding cassette transporter gene family, the human gene is localized to a chromosomal region associated with cardiovascular disease and abnormal HDL metabolism, and
It is based on the finding that the human gene is expressed in cells associated with cardiovascular or inflammatory diseases. In particular, novel human PD-ABC gene sequences encoding human PD-ABC polypeptides and polypeptides derived and identified therefrom are disclosed. Furthermore, chromosomal localization of PD-ABC to human chromosome 19p13.3 and P in spleen, thymus, peripheral blood leukocytes, bone marrow, lymph nodes and further tissues.
The expression of D-ABC is disclosed. The present invention further provides a vector and a host cell containing the PD-ABC gene, a method of using the PD-ABC gene, and PD-ABC.
Antibodies specifically directed against the polypeptides in detection of genetic alterations of genes, subcellular localization of polypeptides, gene therapy applications, or binding assays associated with chemical databases are described. The present invention further provides a proprietary screening strategy for molecules that alter PD-ABC protein activity, a method of diagnosing a syndrome associated with altered PD-ABC protein expression, and PD-ABC gene / protein expression, synthesis or Development of methods of identifying compounds that alter activity and those identified as therapeutic agents in the treatment of PD-ABC mediated diseases including, but not limited to, coronary artery disease (CAD). And the use of such compounds.

[0002]

ATP-binding cassette (ABC) transporters constitute a large family of transmembrane proteins that transport a wide range of substrates across cell membranes (Higgins, C
.F., Annu. Rev. Cell Biol, 1992; 8: 67-113). Members of this transporter family have two hydrophobic domains, each containing 6 transmembrane segments. Furthermore, they have two cytoplasmic ATP binding cassettes or nuclear binding folds at the carboxyl terminus of each hydrophobic domain.
folds) (NBF). ATP binding and hydrolysis in NBF energizes transport activity (Higgins, CF, Annu. Rev. Cell Biol, 1992; 8: 67-113.
). While the various members have significant homology, ATP transporters have different substrate specificities. Multidrug-resistant p-glycoprotein (MDR) transports organic chemicals with irrelevant structure, but a related transporter MRP
Is involved in membrane transport of phospholipids (Gottesman, MM, et al., Annu. Rev. Bio
chem., 1993; 62: 385-428; Smit, J. J, et al., Cell, 1993; 75: 451-462; Ruetz,
S., et al., Cell, 1994; 77: 1071-1081). The physiological importance of ABC transporters is underscored by the finding that some genetic defects in ABC transporters are associated with human disease. Mutations in the cystic fibrosis transmembrane conductance regulator gene are responsible for cystic fibrosis (Riordan, JR, et al., S
cience, 1989; 245: 1066-1073). ABCA (Broccardo, C., et al., Biochim.
Biophys. Acta, 1999; 1461: 395-404) ABC transporter ABC
Gene mutations or truncations in R cause Stargardt disease, a degenerative retinal disease (Allikmets, R., et al., Nat. Genet., 1997; 15: 236-246). ABC1
Is also a member of the ABCA superfamily, but P based on sequence homology.
Isolated from mice by CR (Luciani, MF, et al., Genomics, 1994; 21.
: 150-159). ABC1 was initially found to be required for apoptotic cell uptake and transmembrane anion transport (Luciani, MF, et al., EMBO J, 1996;
15: 226235; Becq, F, et al., J Biol. Chem., 1997; 272: 2695-2699). Human A
Expression of BC1 is regulated during macrophage differentiation and by cholesterol loading (Langmann, T., et al., Biochem. Biophys. Res. Comm., 19
99; 257: 29-33). Recently, ABC1 was identified as a defective gene in Tangier disease (TD), a rare form of lipoprotein deficiency (Bodzioch,
M., et al., Nature Genetics, 1999; 22: 347-351; Broccardo, C, et al., Bioc.
him. Biophys. Acta, 1999; 1461: 395-404; Brooks-Wilson, A., et al., Nature
Genetics, 1999; 22: 336-345; Rust, S., et al., Nature, Genetics, 1999; 22:
352-355). Both genetic and pharmacological evidence suggest that ABC1 transports cholesterol and phospholipids across cell membranes in peripheral tissues (Remaley, AT, et al, Proc. Natl. Acad. Sci. USA, 1999; 96: 12685-126
90; Young, SG, et al., Nature Genetics, 1999; 22: 316-318). A phenotype similar to that in TD patients was observed in ABC1 knockout mice (McNeish, J,
et al., Proc. Natl. Acad. Sci. USA, 2000; 97: 4245-4250; Orso, E., et al.,
Nature Genetics, 2000; 24: 192-196).

The relevance of ABC1 in lipoprotein metabolism explores homologues close to ABC1 that are thought to be involved in cholesterol efflux and reverse cholesterol transport. Here, we describe the isolation and tissue-specific expression of a novel ABC transporter, which is the closest ABC1 homologue to date. Furthermore, the present inventor has also identified other spliced variants with different tissue-specific expression patterns. These findings suggest that PD-ABC may have important physiological roles.

A high risk for CAD is low levels of high density lipoprotein (HDL)
Has been associated with particles. In fact, about one-half of all patients with CAD have low levels of HDL cholesterol. As a result, recent efforts to prevent CAD have focused on ways to increase levels of HDL particles. HDL particles are important in removing excess cholesterol from the body by transporting cholesterol from cells (reverse cholesterol transport) to the liver where cholesterol metabolism and eventual excretion occur. Tangier disease and familial high-density lipoprotein deficiency (FHA) are characterized by extremely low plasma HDL levels and increased cellular cholesterol levels, causing early atherosclerosis (Rogler,
et al., Arterioscler Thromb Vasc Biol, 1995; 15 (5): 68390; Marcil, et al.,
Arterioscler Thromb Vasc Biol, 1999; 19 (l): 15969). Mutations in the ATP-binding cassette transporter 1 (ABC1) gene have recently been identified in patients with TD and FHA (Brooks Wilson, A., et al., Nature Genetics, 1999; 22: 336).
345; Bodzioch, M., et al., Nature Genetics, 1999; 22: 347351). These studies indicate that the ABC1 protein plays a critical role in cholesterol efflux from cells into HDL particles (Marcil, et al., Lancet.
, 1999; 354: 13401346). It has become clear that increasing the activity of ABC1 protein by using small compounds may be one way to increase HDL levels and prevent CAD.

Although the ABC1 gene / protein was recently identified as playing a role in HDL metabolism, it is clear that other genes may also be involved. These other genes have not yet been identified. If such a gene were identified, it would be beneficial. Understanding the biochemical mechanisms behind this pathway may open up new opportunities to treat and diagnose diseases associated with abnormal (high or low) production of HDL particles. In other words, a better understanding of the molecular mechanism of HDL-mediated efflux of intracellular cholesterol is
It will enable improved design of therapeutic agents to treat disorders associated with abnormal levels of cholesterol and HDL production.

Dyslipidemia such as changes in HDL metabolism, or C caused by dyslipidemia
AD has been linked to numerous diseases. Such diseases include diabetes (for review Evans, et al., Curr Opin Lipidol, 1999; 10 (5): 387-391), fatty liver disease (Marchesini, et al., Am J Med, 1999). 107 (5): 450-455), obesity (for a review, Indulski, et al., Cent Eur J Public Health, 1999; 7 (3): 122-.
129), insulin resistance (for review, see Bailey, et al., Biochem Pharmaco
l, 1999; 58 (10): 1511-1520), alcoholism (for a review, see Baraona, e.
t al., Recent Dev Alcohol, 1998; 14: 97-134), retinal degeneration (Gordon, et al.,
Am J Ophthalmol, 1991; 112 (4): 385-391), hypertension (for review, Gianna
ttasio, et. al., Pathol Biol (Paris), 1999; 47 (7): 744-751) and common vascular diseases.

[0007]

[Summary of Invention]

To confirm whether there are other genes that may also be important in the regulation of cholesterol levels, a commercially available sequence database was used, 1 (ATP
We searched for genes related to the binding cassette transporter 1, hereinafter "ABC1"). The ABC transporter gene family is the largest known gene family, which ABC transporter genes have different substrates including sugars, amino acids, peptides and antibodies (for review see Croop JM
, Methods Enzymol, 1998; 292: 101-116). By performing a homology search on a proprietary database of clustered EST sequences generated using algorithms and tools from Compugen Systems, Ltd., a novel homologue that is 48% identical and 64% similar to ABC1 at the amino acid level is obtained. The human gene sequence was identified. These genes (hereinafter P
D-ABC Form 1 and Form 2) have been identified so far
It is the closest human paralog of the BC1 gene. PD-A
The BC genes and the polypeptides they encode are expressed in a variety of cells and tissues, and the polynucleotide sequences of full-length genes and any splice variants and proteins encoded by them are both described herein. Has been identified. The polynucleotide sequence of PD-ABC Form 1 is specified in SEQ ID NO: 1, and the amino acid sequence of PD-ABC Form 1 protein encoded by the novel gene is shown in SEQ ID NO: 2. The polynucleotide sequence of PD-ABC Form 2 is specified in SEQ ID NO: 3, and the amino acid sequence of PD-ABC protein encoded by the novel gene is shown in SEQ ID NO: 4.

Furthermore, alignment of these genes to high-throughput genomic sequences in the Genbank database revealed "in silico" localization of these genes to human chromosome 19p13.3. A search of the Online Mendelian Inheritance in Man (OMIM) database found that this same region of human chromosome 19 was genetically associated with atherosclerosis susceptibility (Nishina, et al., PNAS, 1992). 89: 7
08712; Naggert, et al., Clin Genet, 1997: 236-240). Affected individuals from families showing linkage to 19p13.3 have low levels of HDL particles characteristic of mutations in the ABC1 gene. The level of identity of these novel genes to the ABC1 gene and genetic linkage to the loci associated with atherosclerosis susceptibility make these genes targets for drugs that prevent CAD. In other words, PD-ABC
Since the Form 1 and Form 2 proteins have amino acid homology to the ABC1 protein, they are likely to share some structural and functional properties with ABC1.

One aspect of the invention is to provide purified PD-ABC Form 1 and Form 2 proteins. These purified proteins can be obtained from recombinant cells or naturally occurring cells. Purified PD-AB of the present invention
The C protein may be of mammalian origin. PD-derived from primates including humans
ABC proteins are examples of the various proteins specifically provided. The present invention further provides allelic variants and biologically active derivatives of naturally occurring PD-ABC proteins.

Another aspect of the present invention is PD-ABC Form 1 and Form 2 of the present invention.
Providing a polynucleotide encoding a protein and providing a polynucleotide complementary to the polynucleotide coding strand. These polynucleotides of the present invention can be used to provide recombinant expression of PD-ABC proteins. The polynucleotide of the present invention is (2) PD-ABC gene for treating diseases which may be caused by (1) alteration of PD-ABC gene or by alteration of cellular pathway including PD-ABC gene / protein. /
(4) To analyze or change the subcellular localization of PD-ABC polypeptide, for examining the presence or susceptibility of a disease due to protein alteration or deletion, to (4) PD-ABC gene (5) R for changing the level of the PD-ABC gene for cloning or isolating separate classes of similar RNAs.
It can also be used for gene therapy purposes to express distinct classes of NA.

The present invention further provides an oligonucleotide molecule useful as a probe or primer, specific for any nucleotide sequence comprising or related to the PD-ABC gene, in particular the sequences of SEQ ID NOs: 1 and 3. Specifically hybridizing oligonucleotide molecules. These oligonucleotides are useful as primers for use in various methods such as DNA amplification and microsequencing, or as probes for DNA recognition in hybridization analysis. The nucleic acid probe or primer according to the present invention comprises at least 8 contiguous nucleotides of the polynucleotide of SEQ ID NO: 1 or 3, preferably 8 to 200 contiguous nucleotides, more specifically 10, 15, 20 or It comprises 30-100 contiguous nucleotides, more preferably 10-90 nucleotides, and most preferably 20-80 contiguous nucleotides of the polynucleotide of SEQ ID NO: 1 or 2. Preferred probes or primers of the invention include oligonucleotides selected from the group consisting of the oligonucleotides shown in the Examples below.

The present invention further relates to a method for amplifying a region of the PD-ABC gene. In this method, a test sample suspected of containing a PD-ABC sequence of interest or a part thereof is used as a pair of amplification primers as described above, the primers being located on both sides of the PD-ABC nucleotide region to be amplified. And a step of contacting with an amplification reaction reagent containing. The method may further include detecting the amplification product. For example, the amplification product can be detected using a detection probe capable of hybridizing with the internal region of the amplified sequence. Alternatively, the amplification product can be detected using any of the primers used in the amplification reaction itself, optionally in labeled form.

The invention further provides PD-ABC Form 1 or Form 2 DNA in a test sample.
A diagnostic kit for detecting the presence of at least one copy of the above, which comprises a primer according to the present invention, a pair of primers or a probe. In a first aspect, the kit comprises primers as described above, preferably forward and reverse primers, used to amplify the PD-ABC gene or fragments thereof. In a second aspect, the kit comprises a hybridization DNA probe, which is immobilized on the solid support or is finally immobilized and hybridizes with the PD-ABC gene or a fragment thereof. be able to. Methods of immobilizing nucleotide primers or probes on solid supports are well known to those of skill in the art.

The kit of the present invention comprises a suitable amplification reagent such as a DNA polymerase when the kit contains primers, a reagent useful in a hybridization reaction, and a labeled hybridization probe and a PD-ABC gene. Any of the elements can be included, including reagents useful for indicating the presence of the hybridization reaction. Another aspect of the invention is to provide an antibody capable of binding to the PD-ABC protein of the invention. These antibodies may be polyclonal or monoclonal. The present invention further includes in vitro or in vi
for detecting and measuring the expression of PD-ABC protein in vo, or PD-
Methods of using the subject antibodies to detect molecules that modulate either the protein that interacts with the ABC protein or the activity of the PD-ABC protein are provided.

Another aspect of the invention is to provide a detection assay for proteins that interact with the PD-ABC gene / protein using a genetic approach.
A preferred embodiment involves the use of the yeast two hybrid approach for this screen (Bartel and Fields, The Yeast TwoHybrid System, Oxford Univ.
ersity Press, 1997). Another aspect of the invention provides an assay for the detection or screening of therapeutic compounds that interfere with or in any case mimic the interaction between the PD-ABC protein and a ligand that binds the PD-ABC protein. That is.

In a first aspect, such a screening method for candidate substances provides a polypeptide comprising: (a) the amino acid sequence of SEQ ID NO: 2 or 4, or a peptide fragment or variant thereof; (B) obtaining a candidate substance; (c) contacting the polypeptide with the candidate substance; and (d) detecting a complex formed between the polypeptide and the candidate substance.

In one embodiment of the screening method defined above, the complex formed between the polypeptide and the candidate substance is specifically directed to the PD-ABC protein of the invention or to a peptide fragment or variant thereof. Further incubation in the presence of bound polyclonal or monoclonal antibody. Candidate agents or molecules assayed for interaction with a PD-ABC polypeptide include, but are not limited to, naturally occurring or synthetic organic compounds or molecules of biological origin, such as polypeptides. It may have a property.

In another embodiment of the present screening method, when step (c) of this method is carried out, an increasing concentration of a substance that competes with a possible candidate substance for binding to PD-ABC protein is treated as a candidate substance or molecule. At the same time as or before.
By this technique, the detection and optionally quantification of the complex formed between the PD-ABC protein or peptide fragment or variant thereof and the candidate substance or molecule to be screened for can be carried out by the person skilled in the art by means of this PD-ABC. It makes it possible to determine the affinity value of this substance or molecule for the protein or its peptide fragments or variants.

The present invention further relates to kits useful for performing the screening methods previously described herein. Preferably, such a kit comprises a PD-ABC protein having the amino acid sequence of SEQ ID NO: 2 or 4 or a peptide fragment or variant thereof, and optionally a candidate for this PD-ABC protein or peptide fragment or variant thereof. It includes means useful for detecting complexes formed with substances. In a preferred embodiment, this detection means consists of a monoclonal or polyclonal antibody directed against the PD-ABC protein or a peptide fragment or variant thereof.

The assay of the present invention therefore comprises the PD-ABC protein and PD-ABC
Measuring the effect of the compound of interest on binding to a ligand that binds to the protein. Binding can be measured by a variety of methods, including the use of labeled PD-ABC protein or labeled ligand. Another aspect of the invention is to provide an assay for the discovery of proteins that interact directly or indirectly with PD-ABC proteins. These assays of the present invention include methods for detecting such interactions in cells or in biochemical assays. These interactions can be detected by a variety of methods including the use of the cDNA encoding the PD-ABC protein, or the PD-ABC protein itself, or fragments or modifications thereof.

In one preferred embodiment of the present invention, the PD-ABC gene is PD-ABC.
Is a novel target that can be used to develop high throughput screens for the identification of chemicals and interacting proteins that increase the activity of Escherichia coli. Ultimately, compounds that alter the activity of the PD-ABC gene can be tested for efficacy in preventing CAD. The locus can also be used for basic and pharmacological studies related to HDL metabolism. In another preferred embodiment of the present invention, the protein product of PD-ABC gene may serve as a novel therapeutic target for the treatment of CAD and dyslipidemia.

In another preferred embodiment of the invention, a genetic model for studying CAD and dyslipidemia can be created by altering the PD-ABC gene in animals such as mice. In yet another preferred embodiment of the invention, the polymorphism of PD-ABC gene is CA
Members of the population at risk for D can be identified, and genetic tests can be created to identify such people using the sequences of these genes. In yet another preferred embodiment of the invention, polymorphisms in the sequence of the PD-ABC gene could be used to select the appropriate therapeutic regimen for CAD and dyslipidemia. In yet another preferred embodiment of the invention, the PD-ABC gene sequence is CA
D and dyslipidemia can be used to generate antisense RNA or antibody probes that can subsequently be used for therapeutic treatment. In still another preferred embodiment of the present invention, the sequence of PD-ABC gene is
It can be used to identify interacting genes which themselves can serve as therapeutic targets.

In yet another preferred embodiment of the invention, the nucleotide sequences including and surrounding the PD-ABC genes can be used to identify factors that regulate the levels of these genes. These factors may be therapeutic targets for dyslipidemia and CAD. In yet another preferred embodiment of the invention, the protein product of the PD-ABC gene can be used to identify compounds that are selective for particular members of the ABC transporter family.

Described herein are preferred sequences, polypeptides and methods for making and using the invention. However, it should be understood that the invention is not limited to only the specific sequences, polypeptides and methods described. These sequences, polypeptides and methods may vary, and the nomenclature used herein is to describe particular embodiments. The scope of protection ultimately depends on the claims, and thus, in any case, the foregoing does not limit the invention and should not be construed as limiting. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All US patents and publications mentioned herein are incorporated herein by reference.

[0025]

[0026]

[Chemical 1]

[0027]

[Chemical 2]

[0028]

[Chemical 3]

[0029]

[Chemical 4]

[0030]

[Chemical 5]

FIG. 2: Structure of splice variants and intron / exon organization. Diagram A shows the positions of introns and exons in each variant, as well as alternative splice sites. The transmembrane domain (TM1 = N-terminal, TM2 = C-terminal) and nucleotide binding fold (NBF) are indicated.

[0032]

[Table 1]

Figure 3: Expression of PD-ABC in a number of tissues. A, PD in various human tissues
-Northern blot analysis of ABC expression. Blots with mRNA from the indicated tissues were hybridized with human PD-ABC and GAPDH probes, respectively. Bands for PD-ABC and GAPDH are indicated by arrows.
B, Northern blot analysis of PD-ABC expression in tissues or cells of the immune system. Hybridization was performed as described above, but two forms of PD-ABC are shown.

[0034]

[Table 2]

FIG. 4: Tissue distribution of PD-ABC splice variants. Rapid-Scan Gene Expressi
Using on Panels as a template, reverse transcription-polymerase chain reaction was performed with primer pairs specific for each of the two variants. The PCR product was resolved on an agarose gel; lane 1, fetal liver; lane 2, fetal brain; lane 3, bone marrow; lane 4, PB.
Lane 5, skin; lane 6, prostate; lane 7, uterus; lane 8, ovary; lane 9, pancreas; lane 10, adrenal gland; lane 11, thyroid; lane 12, salivary gland; lane 13, Lane, testis; lane 15, stomach; lane 16, muscle; lane 17, small intestine; lane 18, lung; lane 19, colon; lane 20, liver; lane 21, spleen; lane 22, kidney; lane 23, Heart; lane 2
4, the brain.

[0036]

[Table 3]

[0037]

[Detailed Description of the Invention]

Unless otherwise specified, the technology used in this application is Molecular Cloning: A Lab.
oratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Pr
ess), Gene Expression Technology (Methods in Enzymology, Vol. 185, edite
d by D. Goeddel, 1991. Academic Press, San Diego, CA), "Guide to Protei
n Purification "in Methods in Enzymology (MP Deutshcer, ed., (1990) Ac
ademic Press, Inc.), PCR Protocols: A Guide to Methods and Applications
(Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal
Cells:.. A Manual of Basic Technique, 2 nd Ed (RI Freshney 1987. Liss,
Inc. New York, NY) and Gene Transfer and Expression Protocols, pp.109-
128, ed. EJ Murray, The Humana Press Inc., Clifton, NJ) Sequence Ana
It can be found in any of several well known references such as lysis Primer (Gribskov, et al., 1994, Oxford University Press).

In one aspect, the invention provides a novel isolated and purified polynucleotide encoding a PD-ABC protein, hereafter referred to as the ATP-binding cassette transporter 1 paralog (PD-ABC) gene. Where the polynucleotide sequences are substantially similar to the sequences set forth in SEQ ID NOs: 1 and 3, and the polypeptide sequence is substantially similar to the sequences set forth in SEQ ID NOs: 2 and 4. Are similar to each other. The term "PD-ABC" is used broadly herein. Unless otherwise stated, the term "PD-ABC" refers to
Included are all naturally occurring mammalian forms of PD-ABC and the like. Suitably the term PD-ABC includes all mammals including but not limited to primates and humans.

The provided polynucleotide may encode a PD-ABC protein or a portion thereof. The polynucleotide of the present invention can be produced by various methods including in vitro chemical synthesis by cloning or a combination thereof using well-known solid-phase synthesis methods. The polynucleotide of the present invention may be derived from a cDNA or genomic library. Those skilled in the art are known for the degeneracy of the genetic code, and may encode a PD-ABC protein that has partial or polynucleotide sequence homology to a naturally occurring polynucleotide sequence that encodes a PD-ABC protein. Nucleotides can be easily designed. The polynucleotide of the present invention may be single-stranded or double-stranded. PD-ABC
Polynucleotides complementary to the protein-encoding polynucleotides are also provided.

The polynucleotide encoding the PD-ABC protein can be obtained from a cDNA library produced from a tissue that has the PD-ABC protein or mRNA and is believed to express it at detectable levels. For example, cd
NA libraries can be constructed by obtaining polyadenylated mRNA from cell lines known to express PD-ABC proteins and using that mRNA as a template to synthesize double-stranded cDNA. A cDNA or genomic library is screened with probes designed to identify the gene of interest or the protein encoded by it. For cDNA expression libraries, suitable probes include monoclonal and polyclonal antibodies that recognize and specifically bind PD-ABC protein. For cDNA libraries, suitable probes include
A carefully selected oligonucleotide probe encoding a known or possible portion of the PD-ABC protein from the same or different species (typically about 20
~ 80 bases long) and / or complementary or homologous cDNAs encoding the same or similar genes or fragments thereof, and / or homologous genomic DNAs
Or fragments thereof are included. Screening a cDNA or genomic library with selected probes is described in Chapters 1012 of Sambrook, et al.
al., Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbo
r Laboratory Press, 1989).

The preferred method of practicing the present invention is to use carefully selected oligonucleotide sequences to screen cDNA libraries from various tissues. The oligonucleotide sequence chosen as the probe should be of sufficient length and yet be sufficiently clear to minimize false positives. The actual nucleotide sequence is usually designed based on the region of the PD-ABC gene with the least codon overlap. The oligonucleotides may be degenerate at one or more positions. The use of degenerate oligonucleotides is especially important when screening libraries from species for which the preferential codon usage is unknown.

The oligonucleotide needs to be labeled so that it can be detected by hybridization to the DNA in the screened library. A preferred labeling method is to use ATP (eg T32P) and polynucleotide kinase to radiolabel the 5'end of the oligonucleotide. However, the oligonucleotides may be labeled using other methods, including but not limited to biotinylation or enzyme labeling. The cDNA encoding the PD-ABC protein is described in US Pat. No. 4,683,1.
95, Sambrook, et al., Molecular Cloning: A Laboratory Manual, seco.
Item 1 of nd edition, Cold Spring Harbor Laboratory Press, New York, 1989
4 or Chapters 15 of Current Protocols in Molecular Biology, Ausubel,
et al. eds., Green Publishing Associates and Wiley Interscience 1991, by direct expression cloning or by polymerase chain reaction (PCR).
Can also be identified and isolated by other known techniques of recombinant DNA technology, such as. This method requires the use of oligonucleotide probes that will hybridize to the DNA encoding the PD-ABC protein.

As defined herein, “substantially similar” includes identical sequences as well as any conserved motifs that retain any biologically active portion of the protein product. It includes deletions, substitutions or additions to the DNA, RNA or protein sequences that it has. This includes, but is not limited to, any splice variant of PD-ABC known to exist. Preferably, the DNA sequence according to the invention consists essentially of the DNA sequence SEQ ID NO: 1 or 3. These novel purified and isolated DNA sequences can be used to direct the expression of PD-ABC protein and to analyze mutations of PD-ABC protein function. Mutated sequences according to the present invention can be routinely identified by those of ordinary skill in the art using the content provided herein and techniques well known in the art.

In a preferred embodiment, the invention comprises a nucleotide sequence that hybridizes under high stringency hybridization conditions to the nucleotide sequence set forth in SEQ ID NO: 1 or 3. As used herein, "high stringency hybridization conditions" means about 8-24 to 2x10 ⁸ cpm / μg of the probe of interest in low salt hybridization buffer on a filter support at 65 ° C. After hybridization for 1 hour, 1% SDS, 20 mM
It means washing in phosphate buffer and 1 mM EDTA at 65 ° C. for about 30 minutes to 4 hours. In a preferred embodiment, the low salt hybridization buffer contains 0.5% to 10% SDS and 0.05M to 0.5M sodium phosphate.
In the most preferred embodiment, the low salt hybridization buffer is 7% SDS.
And 0.125 M sodium phosphate.

As is known in the art, a number of equivalent conditions can be used to include low or high stringency conditions. Sequence length and properties (D
NA, RNA, base composition), target properties (DNA, RNA, base composition, presence in solution or immobilization, etc.), and concentrations of salts and other components (eg formamide, dextran sulfate and / or polyethylene glycol Factors such as (present or absent) are contemplated and the hybridization solution may be varied to produce low or high stringency conditions that are different or equivalent to those listed above. As used herein, "stringent conditions" are "stringency" that is within the range of approximately Tm-5 ° C (5 ° C below the melting temperature (Tm) of the probe) to about 20 ° C to 25 ° C below Tm. Is. As will be appreciated by one of skill in the art, the stringency of hybridization may be altered to identify or detect identical or related polynucleotide sequences.

The polynucleotides of the invention have a variety of uses, some of which have been shown or will be discussed in more detail below. The particular application for a given polynucleotide will depend, in part, on the particular polynucleotide embodiment of interest. The polynucleotide of the present invention can be used as a hybridization probe to recover a PD-ABC nucleotide sequence from a gene library. The polynucleotides of the present invention can also be used as primers for the amplification of PD-ABC gene sequences encoding polynucleotides or portions thereof by PCR and other similar amplification methods. The polynucleotide of the present invention can be used as a probe and an amplification primer to treat diseases, especially PD.
-PD-AB that was correlated with disease associated with altered function of ABC protein
It is also possible to detect mutations in the C protein encoding gene. Includes, but is not limited to, the diseases mentioned above.

The present invention further provides various polynucleotide expression vectors containing the subcloned PD-ABC gene or a sequence substantially similar thereto in an extrachromosomal vector. This aspect of the invention allows in vitro expression of the PD-ABC gene, thereby allowing PD-ABC gene regulation and analysis of PD-ABC protein structure and function. The term "extrachromosomal vector" as used herein includes, but is not limited to, plasmids, bacteriophage, cosmids, retroviruses and artificial chromosomes. In a preferred embodiment, the extrachromosomal vector comprises an expression vector that enables PD-ABC protein production when the recombinant DNA molecule is inserted into a host cell. Such vectors are well known in the art and are capable of directing T3 or T7 polymerase promoters, SV40 promoters, CMV promoters, or gene expression, or for the ability to direct gene expression. It includes, but is not limited to, those containing any promoter.

In a preferred embodiment, the expression vector comprises one or more promoter sequences so as to express the PD-ABC protein (or an antisense copy of a sequence suitable for inhibiting the expression of an endogenous gene). In functional combination, it comprises a polynucleotide sequence encoding the PD-ABC protein. These vectors may contain additional polynucleotide sequences for gene expression, regulation, or convenient manipulation of the vector, such additional sequences including terminators, reporters,
Includes enhancers, selectable markers, packaging sites and the like. A detailed description of polynucleotide expression vectors and their use can be found in, among others, Gene Exp.
ression Technology: Methods in Enzymology, Vol 185 Goeddel, ed, Academic
Press Inc., San Diego, CA (1991), Protein Expression in Animal Cells, R
oth, ed., Academic Press, San Diego, CA (1994).

The polynucleotide expression vector of the present invention has various uses. Such applications include genetic engineering of host cells to express PD-ABC proteins. In another aspect, the invention provides a recombinant host cell stably transfected with a recombinant DNA molecule PD-ABC subcloned into an extrachromosomal vector. The host cells of the invention may be from any type including, but not limited to, bacteria, yeast, mammalian cells and Xenopus oocytes. Transfection of host cells with recombinant DNA molecules is well known in the art (Sambrook, et al., Molecular Clon.
^{ing, A Laboratory Manual, 2 nd} ed., Cold Spring Harbor Press, 1989), as used herein, These include calcium phosphate transfection, dextran sulfate transfection, electroporation, lipofection and infection But is not limited to. This aspect of the invention relates to PD-
Allows in vitro and in vivo expression of ABC and their gene products, thus allowing high level expression of PD-ABC proteins. In another aspect of the invention, RNA molecules containing PD-ABC can be injected into Xenopus oocytes, and substrate transport measured using standard electrophysiological techniques. You can

In another aspect of the invention, transgenic animals can be produced by injecting into germ cells the nucleotide sequence of PD-ABC cloned into a suitable expression vector. Other uses of polynucleotide expression vectors, discussed in further detail below, include:
Included are those uses for gene therapy of diseases and conditions where it may be a desirable use to express PD-ABC proteins at levels higher than their naturally occurring expression levels. Alternatively, it may be desirable to use the vector for antisense expression that reduces naturally occurring levels of PD-ABC protein.

The polynucleotide sequences of SEQ ID NOs: 2 and 4 have been mapped to the human chromosome using the nucleotide sequence of the cDNA from the library source that generated the probe. These sequences have been mapped to specific chromosomes or to specific regions of chromosomes using well known techniques. These include in situ hybridization to chromosome distribution,
And PC by amplifying DNA from standard radiation hybrid cell lines
Includes R-based cartography. (Verma, et al., (1988) Human Chromosomes:
A Manual of Basic Techniques, Pergamon Press, NYC).

In another aspect, the invention provides a PD-A set forth in SEQ ID NO: 2 or 4.
Provided is a substantially purified recombinant protein comprising a polypeptide substantially similar to a BC polypeptide. Further, this aspect of the invention is described in several
Allows the use of PD-ABC proteins in in vitro assays. As used herein, the term "substantially similar" refers to SEQ ID NO: 2 or 4 introduced by any in vitro means or by any genetic alteration naturally found in vivo. Includes deletions, substitutions and additions to the sequence. As used herein, the term "substantially purified" means that the protein should be separated from detectable contaminating proteins, while the PD-ABC protein is associated with the interacting protein or It means that it may be co-purified as an oligomer. In the most preferred embodiment, the protein sequence according to the invention comprises the amino acid sequence of SEQ ID NO: 2 or 4. Mutated sequences according to the present invention can be routinely identified by those of ordinary skill in the art using the content provided herein and techniques well known in the art. This aspect of the invention provides novel purified proteins that can be used in in vitro assays and as components of pharmaceutical compositions.

PD-ABC proteins can be used to discover molecules that interfere with its activity. For example, a molecule that interferes with the binding of PD-ABC to a ligand or other molecule. The PD-ABC proteins of the invention have a putative biological activity that modulates cellular efflux of cholesterol. The PD-ABC protein of the present invention is
It can be isolated from various mammalian species. Suitable mammalian species for isolation are primates and humans. The present invention provides allelic variants of the PD-ABC protein,
It is believed that it can be produced from various mammalian tissues. Preferably PD
-ABC protein is obtained from a recombinant host cell genetically engineered to express a significant amount of PD-ABC protein. PD-ABC proteins can be isolated from non-recombinant or recombinant cells by a variety of methods well known to those of skill in the art.

The term “PD-ABC protein” as used herein means not only a protein having the amino acid residue sequence of a naturally occurring PD-ABC protein, but also a naturally occurring PD-ABC protein. Functional derivatives and variants of proteins are also meant. A “functional derivative” of a natural polypeptide is a natural PD-ABC.
A compound that has a qualitative biological activity in common with a protein. Therefore,
A functional derivative of a native PD-ABC protein is a compound that has a qualitative biological activity in common with the native PD-ABC protein, eg, transports a substrate across a biological membrane. "Functional derivatives" include all animal species (including humans)
Natural polypeptide fragments from, and derivatives of natural (human and non-human) polypeptides and fragments thereof, including, but not limited to, the respective native polypeptide and Provided that they have a common biological activity. A "fragment" comprises a region within the sequence of a mature native polypeptide. The term "derivative" is used to define amino acid sequences and glycosylation variants, and covalent modifications of naturally occurring polypeptides,
The term "variant" means amino acid sequences and glycosylation variants within this definition. Preferably, the functional derivative is at least about 70% amino acid sequence similarity to the sequence of the native polypeptide in question, more preferably about 80% amino acid sequence similarity, even more preferably at least 90% amino acid sequence similarity. ,
Most preferred are polypeptides having at least about 99% amino acid sequence similarity. Most preferably, the functional derivative of the PD-ABC protein retains or mimics one or more regions in the native polypeptide sequence that are directly involved in ligand binding. The phrase "functional derivative" specifically refers to natural PD-AB.
Includes peptides and small organic molecules with qualitative biological activity in common with C protein.

“Identity” or “homology” with respect to naturally occurring polypeptides and functional derivatives thereof refers herein to aligning sequences and, if necessary, introducing gaps to reach maximum% homology. It is defined as the percentage of amino acid residues in the candidate sequence that are similar to the residues of the natural polypeptide of interest. N- or C-terminal extension or insertion,
Alternately spliced variants should not be construed as reducing identity or homology. Methods and computer programs for alignment are well known in the art.

Amino acid sequence variants of the native PD-ABC protein or PD-ABC protein fragments may be obtained by suitable nucleotide changes in the native or variant PD-ABC protein encoding DNA by methods known in the art. Or by in vitro synthesis of the desired polypeptide. Construction of amino acid sequence variants has two main variables:
The location of the mutation site and the nature of the mutation. Excluding naturally occurring alleles that do not require manipulation of the DNA sequence encoding the PD-ABC protein,
The amino acid sequence variant of the PD-ABC protein preferably contains this DNA
It is constructed by mutating alleles or to reach non-naturally occurring amino acid sequence variants. Alternatively, or additionally, amino acid changes can be made at sites where PD-ABC proteins from different species or highly conserved regions differ depending on the purpose to be achieved.

Such site sites are typically, for example, (1) initially conservatively selected,
Then, depending on the results obtained, substitution with a more radical selection, (2) deleting one or more target residues, or (3) adjacent to the located site, of the same or different class Will be modified sequentially by inserting the residues of One useful technique is the "alanine scanning" Cunningham and Wells, Science, 19
89; 244: 1081-1085. Here, the residue or group of the target residue is identified and replaced by alanine or polyalanine. Domains that are functionally sensitive to alanine substitution are then refined by introducing additional or other substituents at or in place of the alanine substitution site. After identifying one or more desired mutations, the gene encoding the PD-ABC protein variant can be obtained, for example, by chemical synthesis.

More preferably, D encoding the PD-ABC protein amino acid sequence variant
NA is produced by site-directed mutagenesis of DNA encoding the initially produced mutant or non-mutated version of the PD-ABC protein. Site-directed (site-directed) mutagenesis involves the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficiently large number of contiguous nucleotides.
It provides a primer sequence of sufficient size and sequence complexity to allow the production of PD-ABC protein variants, forming stable double helices on either side of the deletion junction being traversed. Typically, primers of about 20-25 nucleotides in length are suitable, with about 5-10 residues altered on either side of the junction of the sequence. In general, the technique of site-directed mutagenesis is described by Edelman, et al., DNA, 1983.
2: 183, etc., and are well known in the art.
As will be appreciated, site-directed mutagenesis techniques typically employ phage vectors that exist in both single- and double-stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. This and other phage vectors are commercially available and their use is well known to those of skill in the art. A versatile and efficient method for the construction of oligodeoxyribonucleotide-directed site-directed mutagenesis in DNA fragments using M13-derived vectors is described in Zoller, MJ and Smith, M., Nucleic A.
Published by cids Res., 1982; 10: 6487-6500. Furthermore, a plasmid vector containing a single-stranded phage origin of replication (Veira, et al., Meth. Enzymol., 1987;
153: 3) can be used to obtain single-stranded DNA. Alternatively, nucleotide substitutions are introduced by synthesizing the appropriate DNA fragment in vitro and amplifying it by PCR methods known in the art.

In general, site-directed mutagenesis refers to DNs encoding relevant proteins.
This can be done by obtaining a double-stranded or single-stranded vector containing the A sequence in the sequence. Oligonucleotide primers having the desired mutated sequence were generally synthesized synthetically, for example, by Crea, et al., Proc. Natl. Acad.
Sci. USA, 1978; 75: 5765. The primer is then annealed with a vector containing the single-stranded protein sequence, and E. It is submitted to a DNA polymerase such as E. coli polymerase I Klenow fragment to complete the synthesis of the mutation-containing strand. Thus, a heteroduplex is formed in which one strand encodes the original non-mutated sequence and the other strand contains the desired mutation. This heteroduplex vector is then used to transform a suitable host cell, such as HB101 cells, and a clone containing the recombinant vector having the mutated sequence arrangement is selected. The mutated region can then be removed and placed in an expression vector suitable for protein production.

PCR techniques can also be used to generate amino acid sequence variants of the PD-ABC protein. When a small amount of template DNA is used as a starting material in PCR, primers having a slightly different sequence from the corresponding region in the template DNA are used, and specific primers that differ from the template sequence only at positions different from the template are used. D
NA fragments can be produced in relatively large amounts. For the introduction of mutations into plasmid DNA, one of these primers was designed to overlap the position of the mutation and to contain the mutation, while the sequences of the other primers were This sequence can be located anywhere along the plasmid DNA, although it must match the extension of the chain sequence. However, the sequence of the second primer is preferably located at the end within the range of 500 to 5000 nucleotides from the first sequence so that the entire amplified region of the DNA bound by the primers can be easily sequenced. is there. PCR amplification with a primer pair such as just described yields a population of DNA fragments that differ at the position of the primer-defined mutation, and possibly at other positions, because template copies are somewhat error prone. .

Further details of the above and similar mutagenesis techniques can be found in common textbooks such as:
Sambrook, et al., Molecular Cloning: H Laboratorv Manual 2nd edition, Co
ld Spring Harbor Press, Cold Spring Harbor (1989), and Current Protocols
in Molecular Biology, Ausubel, et al. eds., John Wiley and Sons (1995)
Etc. Naturally occurring amino acids are divided into groups based on common side chain properties: (1) Hydrophobicity: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophobicity: cys, ser. (3) Acidity: asp, glu; (4) Basicity: asn, gin, his, lys, erg; (5) Residues affecting chain orientation: gly, pro; and (6) aromatic : Trp, tyr, pine.

A conservative substitution involves exchanging a member within one group for another member within the same group, while a non-conservative substitution involves exchanging one member of these classes for another. Would require. The variants obtained by non-conservative substitutions are believed to result in significant changes in the biological properties / functions of the obtained variants, with PD-ABC protein biological activity, ie modulation of cholesterol efflux. It is possible that PD-ABC protein variants will occur. Amino acid positions that are conserved among different species are generally replaced in a relatively conservative manner when the goal is to retain biological function. Amino acid sequence deletions are generally about 1 to 30 residues, more preferably about 1 to 10 residues.
Residues, which are typically contiguous. Deletions can be introduced into regions that are not directly involved in ligand binding.

Amino acid insertions include amino- and / or carboxyl-terminal fusions from 1 residue to polypeptides containing residues of 100 or more, as well as within sequences of one or more amino acid residues. Includes inserts. Intra-sequence insertion (ie PD-A
Insertions within the BC protein amino acid sequence) may generally be about 1-10 residues, more preferably 1-5 residues, more preferably 1-3 residues. Examples of end insertions include
N-terminal methionyl residues, naturally occurring N-terminal signal sequences, direct expression artifacts in recombinant cell cultures of bacteria, and mature PD-A from recombinant host cells.
Heterologous N to the N-terminus of PD-ABC protein that facilitates secretion of BC protein
Included are PD-ABC proteins that contain a fusion of terminal signal sequences. Such a signal sequence will generally be derived from, and thus homologous to, the intended host cell type. Suitable sequences include E. STII or Ip for coli
p, α-factor for yeast, and viral signals such as herpes gD for mammalian cells. Other insertional variants of the native PD-ABC protein molecule include immunogenic polypeptides, eg, bacterial polypeptides such as β-lactamase or E. coli, as described in PCT Published Application WO 89/02922. coldt
Enzyme encoded by rp locus or PD-ABC to yeast protein
N- or C-terminal fusions of proteins and C-terminal fusions with immunoglobulin regions (preferably immunoglobulin constant regions), proteins with long half-lives such as albumin or ferritin are included.

It will be appreciated that it is often difficult to predict the characteristics of mutant PD-ABC proteins, and that screening is required to select the optimal mutant. Biochemical screening assays, such as those described herein below, would be readily available for this purpose. In another aspect, the invention provides antibodies and methods for detecting an antibody that selectively binds a polypeptide comprising an amino acid sequence substantially similar to the amino acid sequence of SEQ ID NO: 2 or 4. As discussed in more detail below, the antibodies of the invention may be polyclonal or monoclonal antibodies, SEQ ID NO:
It is produced by using all or a part of the sequence of 2 or 4, or a modified portion thereof, and can elicit an immune response in a host animal by standard techniques (Harlow and Lane (1988), eds. Antibody: A Laboratory. Manual, Cold Sprin
g Harbor Press). In a preferred embodiment, the entire polypeptide sequence of SEQ ID NO: 2 or 4 is used to elicit production of polyclonal antibodies in a host animal.

A method of detecting PD-ABC antibodies is to contact the cells with an antibody that recognizes the PD-ABC protein and incubate the cells in a manner that allows the detection of the PD-ABC protein-antibody complex. including. Standard conditions for antibody detection of antigens can be used to achieve this aspect of the invention (Harlow and Lane, 1988.
). This aspect of the invention allows detection of PD-ABC proteins both in vitro and in vivo. The present invention provides methods of treating a variety of disorders characterized by undesired abnormal cellular levels of PD-ABC. The disease can be due to in vitro or in vivo gene therapy. Protocols for gene therapy by the use of viral vectors include, among others, Viral Vector Gene Therapy and Neuroscience Applications, Kaplit and
It can be found in Lowry, Academic Press, San Diego (1995). Gene therapy applications typically identify target host cells or tissues in need of treatment, design vector constructs capable of expressing the desired gene product in the identified cells, and generate those constructs. , To provide effective transduction of target cells to those cells. The cells or tissues targeted by gene therapy are
Typically, one is suffering from a disease for which the vector construct is designed to treat.

The gene therapy method of the present invention includes the step of introducing a vector for expressing PD-ABC protein (or inhibitory antisense RNA) into patient cells. The patient cells may be internal to the patient, ie in vivo gene therapy, external to the patient and subsequently reintroduced into the patient, ie in vitro gene therapy. . Diseases that can be treated by the present gene therapy methods include, but are not limited to, those associated with dyslipidemia. HD
Dyslipidemia, such as altered L metabolism, or CAD caused by dyslipidemia, is associated with a number of diseases. Such diseases include diabetes, fatty liver disease, obesity, insulin resistance, alcoholism, retinal degeneration, hypertension and common vascular diseases.

In a preferred aspect of the present invention there is provided a method of protecting a mammalian cell from abnormal levels of PD-ABC in the cell, wherein the mammalian cell has a DNA sequence of SEQ ID NO: 1 or 3. An expression vector operably linked to a DNA sequence which promotes the expression of the DNA sequence containing a DNA sequence similar to the above is introduced, and the DNA sequence of SEQ ID NO: 1 or 3 is high in mammalian cells. Methods are provided that include incubating cells under conditions that will be expressed at levels. Suitable expression vectors are as described above. In a preferred embodiment, human PD-ABC
The coding region of the gene is subcloned into an expression vector under the transcriptional control of the cytomegalovirus (CMV) promoter to allow constitutive PD-ABC gene expression.

In another preferred aspect of the invention, a method of treating or preventing abnormal levels of PD-ABC, wherein the antisense sequence is substantially similar to the DNA sequence shown in SEQ ID NO: 1 or 3. An expression vector comprising the DNA of
A method is provided that comprises introducing into a mammalian cell an expression vector operably linked to a DNA sequence that promotes expression of the antisense DNA sequence. The cells are then grown under conditions in which the antisense DNA sequence of SEQ ID NO: 1 or 3 will be expressed at high levels in mammalian cells. In the most preferred embodiment, this DNA sequence consists essentially of SEQ ID NO: 1 or 3. In a more preferred embodiment, the expression vector is PD-ABC cDNA
Comprises an adenovirus vector operably linked to the CMV promoter in an antisense orientation to allow constitutive expression of PD-ABC antisense cDNA in host cells. In a preferred embodiment, the PD-ABC adenovirus expression vector is introduced into cells by injection into a mammal.

Another aspect of the invention is to provide an assay useful for determining whether a compound of interest can bind to PD-ABC protein. This bond is
It may interfere with or mimic the binding of the ligand to ABC1, or this binding may affect the function of PD-ABC in transporting the substrate across the membrane or in modulating cholesterol efflux. There can be This assay involves measuring the binding of a compound of interest to the PD-ABC protein. The PD-ABC protein or the compound of interest to be assayed is labeled with a detectable label, such as a radioactive or fluorescent label, to detect complex formation between the compound of interest and the PD-ABC protein. You may In another embodiment of this assay, these assays involve the interference, ie, competition, of the compound of interest for the binding interaction between the PD-ABC protein and a ligand already known to bind to the ABC1 protein. Including measuring physical binding. For example, the effect of increasing amounts of the compound of interest on complex formation between radiolabeled ligand and PD-ABC protein can be measured by quantifying labeled ligand PD-ABC protein complex formation. In another embodiment of this assay, these assays involve measuring the change in the compound of interest with the activity of the PD-ABC protein, ie, non-competitive inhibition.

Polyclonal antibodies to PD-ABC protein are generally raised in animals by multiple subcutaneous (se) or intraperitoneal (ip) injections of PD-ABC protein and adjuvant. The PD-ABC protein or a fragment containing the target amino acid sequence is added to a protein that is immunogenic to the species to be immunized, eg, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor, bifunctional or Derivatizing agents such as maleimidobenzoylsulfosuccinimide ester (complexing with cysteine residues), N-hydroxysuccinimide (with lysine residues), glutaraldehyde, succinic anhydride,
Combining with SOCl ₂ or R ₁ -N = C = NR, where R and R ₁ are different alkyl groups, can be useful.

Animals are 1 mg or 1 fig complex (for rabbit or mouse respectively)
Are immunized against the immunogenic complex or derivative by mixing 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. After one month, the animals received 1/5 to 1 of the original amount of complex in Freund's complete adjuvant.
/ 10 is boosted at multiple sites by subcutaneous injection. Seven to 14 days later, test animals are bled and the sera are assayed for anti-PD-ABC protein antibody titers. The animals are boosted until the titer plateaus. Preferably, the test animal is boosted with the same complex of PD-ABC proteins, but may be complexed to different proteins and / or by different cross-linking reagents. The complex can also be produced as a protein fusion in recombinant cell culture. In addition, aggregating agents such as alum are used to enhance the immune response.

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, ie
The individual antibodies that make up the population are identical except for the few naturally occurring mutations that may occur. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. For example, the anti-PD-ABC protein monoclonal antibody of the present invention can be prepared by Kohler & Milstein, N.
Nature, 1975; 256: 495, or can be produced using the hybridoma method first described by C., or the recombinant DNA method (Cabilly, et al, US Pat.
No. 16,567).

Antibodies can also be raised using protein-displaying phage. With this approach, a library of random sequence peptides is generated in antibody genes cloned into phage. These phage libraries are screened for antibodies by screening for immobilized proteins (HoogenboomHR, Trends Biotechnol. 1997; 15 (2): 6270). In the case of the hybridoma method, mice, or other suitable host animals such as hamsters, are immunized as described above to produce antibodies that will specifically bind the protein used for immunization. Or withdrawing lymphocytes that can produce. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Coding, Monoclonal Antibodies: P
rinciples and Practice, pp.59-103 [academic Press, 1986]).

The PD-ABC protein-specific antibodies of the present invention have numerous uses. These antibodies can be used to purify PD-ABC proteins from recombinant or non-recombinant cells. The antibody can be used to detect and / or quantify the presence of PD-ABC protein in tissue samples, eg from blood, skin and the like. Quantification of PD-ABC protein can be used to diagnose for diseases and physiological or genetic conditions that have been correlated with specific levels of PD-ABC protein expression levels. In another aspect, the present invention is a diagnostic assay for detecting cells containing a PD-ABC deletion, wherein total genomic DNA is isolated from the cells and the genomic DN thereof is isolated.
A provides a diagnostic assay comprising performing PCR amplification with primers derived from the DNA sequence of SEQ ID NO: 1 or 3.

This aspect of the invention allows the detection of PD-ABC deletions in cells of any type and can be used in genetic testing or as a laboratory tool. These PCR primers have PDs large enough to be detected by gel electrophoresis.
-Any method that allows amplification of the ABC gene fragment can be selected. Detections include, but are not limited to, ethidium bromide staining of agarose or polyacrylamide gels, autoradiographic detection of radiolabeled PD-ABC gene fragments, Southern blot hybridizations and DNA sequence analysis. It can be done in any way. In a preferred embodiment, detection is performed by polyacrylamide gel electrophoresis followed by DNA sequence analysis to verify the identity of the deletion. PCR conditions are routinely determined based on primer length and base content selected by techniques well known in the art (Sambrook, et al., 1989).

Another aspect of the invention is a diagnostic assay for detecting cells containing a PD-ABC deletion, in which total cellular RNA is isolated and the RNA is SEQ ID NO: 1 or 3.
A diagnostic assay comprising performing reverse transcription-PCR amplification using primers derived from the DNA sequence of This aspect of the invention relates to P in cells of all types.
It allows the detection of D-ABC deletions and can be used in genetic testing or as a laboratory tool. Reverse transcription is a standard technique (Ausubel, et al., In Current Protocols in Molec
ular Biology, ed. John Wiley and Sons, Inc., 1994), and PCR is performed as described above. The present invention can be better understood with reference to the accompanying examples, which are merely illustrative and are not to be construed as limiting the scope of the invention as defined by Claim.

Materials and Methods Sequence Analysis A TBLASTN search using ABC1 and ABCR protein sequences as searchers was performed on the clustered EST database generated using the Compugen LEADS ^™ plarform. A single EST cluster consisting of 4 ESTs was identified (accession number AI733552, H21585). The four ESTs show two different isoforms of PD-ABC. The BLASTIN search for the high throughput genomic sequence (HTG) portion of the Genbank ^™ database is PD-
The HTG sequence containing the complete coding region for ABC (Accession No. AC0115)
58).

Two ESTs (IMAGE # 160038 and I in the isolation database of PD-ABC cDNAs )
． M. A. G. E. # 182933) contains a partial open reading frame that shares significant homology with human ABC1 after translation. These ESTs were obtained from ATCC and fully sequenced. These ESTs are each 1.2 kb (IMAGE.
# 160038) and 1.1 kb (IMAGE # 182933) insert size. These two clones match in their overlap region of 1 kb. CDNA clones were isolated from a mixture of three human cDNA libraries, namely adult brain, skeletal muscle and mammary gland, using a region common to both ESTs as a probe (EdgeBiosystems, Gaithersburg, MD).
). DNA sequencing was performed with universal and synthetic primers.

PCR amplification of 5'cDNA ends The longest clone was sequenced and found to lack the 5'end.
A database-searched, high-throughput genomic sequence cluster with identity to the cDNA sequence was obtained. Three pairs of primers were synthesized based on this high throughput genomic sequence. These primer pair regions are:

[0080]

[Table 4]   1. Forward primer       5'-TCTCACCATGGCCTTTCTGGACACAG-3 '       Reverse primer       5'-CACGTAGCGCAGGTCGGTCAGGG-3 '   2. Forward primer       5'-GCTGATGTGAGCCCTGGACAGCCA-3 '       Reverse primer       5'-GTCCACATAGCACGGATAGGGCAT-3 '   3. Forward primer       5'-TCGTTGTACCTGCAAGACCTGGTG-3 '       Reverse primer       5'-CAGAGCCAGGCTCTCGCAGCC-3 '

PCR reactions were performed on human pituitary or thymus Marathon Ready cDNA (Clontech). The reaction starts at 94 ° C for 5 minutes with initial denaturation and then at 94 ° C for 30 seconds at 60 ° C.
28 cycles of 2 minutes at 72 ° C. for 2 minutes at 72 ° C. for a final extension of 10 minutes at 72 ° C. The PCR reaction with 3 pairs of primers had the following sizes: 1.
Three bands were produced, each having 8 kb, 800 bp and 600 bp. These PCR products were ligated into the pCRII-TOPO vector (Invitrogen) and sequenced with universal and synthetic primers.

Northern Blot Analysis PCR with digoxigenin (DIG) labeled probes for Northern blotting, including primers based on the sequence of the partial PD-ABC cDNA fragment.
It was generated using a labeling kit (Boehringer Mannheim). The forward primer is 5'-CAGCTTTCACTCTTGTCCCATTGAG-3 'and the reverse primer is 5'-TTTATGCAGGGTGAGCACCACAT.
It was AG-3 '. The 262 bp PCR product was gel purified and used for Northern blotting. The template for this PCR was PD-ABC partial cDNA fragment or human spleen cDNA (Clontech). 12 Tissue Master Blot (Orig
(ene, Rockville, MD) and a 6-tissue master blot (Clontech) were hybridized with the probe and developed according to the manufacturer's instructions (Boehringer Mannheim). The same blot was taken and hybridized with DIG-labeled GAPDH or β-actin probe for control purposes.

Tissue distribution by RT-PCR Tissue-specific expression of the two PD-ABC variants was performed by reverse transcription-polymerase chain reaction (hereinafter “RT-PCR”). RapidScan Gene Expression Pa
nels (Origene) was used as a PCR template. Primers specific to Form 1 are 5'-CCCCCTCTCCTTTCTTTCACACTAC-3 '(forward primer) and 5'-AGCAGCCCAAAACACTCACCAC-3.
'(Reverse primer); primer specific for Form 2 is 5'-T
GGGAGAGGAGGACGAGGATGTTAG-3 '(forward primer)
And 5'-AGGTGTTCAGTAAAGGATGATGGG-3 '(reverse primer). The PCR reaction was as follows: 95 ° C for 1 minute; 6
2 ° C. 1 minute; 72 ° C. 1 minute was performed for 35 cycles. PCR product is 1% NuSie
Separated on ve gel (FMC).

Results and Discussion Isolation and Primary Structure of PD-ABC ABC1 is a member of the ABCA subfamily (Broccardo, C. et a.
l., Biochim. Biophys. Acta, 1999; 1461: 395-404), TD. Recent pharmacological experiments have shown that ABC1 is involved in cholesterol and phospholipid transport (Lawn, RM, et al., J. Clin. Invest. 104, R25-31, 1999).
. Two additional ABCA subfamily members ABC2 and ABCR have been described (Luciani, MF., Et al., EMBO J, 1996; 15: 226235; Allikmets, R.,
et al., Nat. Genet., 1997; 15: 236246). Although the functions of ABCR and ABC2 are not yet known, ABCR has been reported as the N-retinylidenephosphatidylethanolamine flipase (Weng, L, et al. Cell.
, 1999; 98: 1323). In order to search for other ABC1 homologs, particularly those also involved in cholesterol metabolism, we searched the database for novel sequences with homology to ABC1 and ABCR. Two overlap E
STs have been identified, which contain a partial open reading frame. The 5'end 80% of this open reading frame is similar to ABC1. On the other hand, 20% of the 3'end of the open reading frame has no homology with ABC1. Furthermore, this open reading frame does not contain the corresponding NBF, as expected in ABC1 (Bodzioch
, M., et al., Nature Genetics, 1999; 22: 347-351; Brooks Wilson, A., et al.
, Nature Genetics, 1999; 22: 336-345; Rust, S., et al., Nature Genetics, 1
999; 22: 352-355).

Using a probe based on a sequence homologous to ABC1, the present inventor
A cDNA clone was obtained from the library. I sequenced the longest clone,
It matches the EST sequence in the 5'end. Interestingly, the 3'end of this clone differs from that of EST. In addition, this clone contains a second post-translational N
It contains BF and the entire amino acid sequence has homology to ABC1. The present inventor predicted that there are two forms of PD-ABC derived from alternative splicing. c
The DNA clone is Form 1 containing the second NBF; EST is the second NBF.
Form 2 lacking BF. The inventor has identified another splice site shown in Figure 2 and the intron and exon sizes, as well as the intron-exon junction region, are shown in Tables 1 and 2 below.

[0086]

[Table 5]

[0087]

[Table 6]

[0088]

[Table 7]

[0089]

[Table 8]

Using various techniques (materials and methods), the inventor has obtained the full length PD-ABC coding region. This full-length PD-ABC contains an open reading frame of 2146 amino acids and is a typical ABC transporter. PD-ABC is currently the closest ortholog to ABC1 in public databases.
). This sequence was aligned with ABC1 and ABCR (Figure 1). P
The homology between D-ABC and ABC1 is 66%. The most conserved regions of the PD-ABC sequence correspond to the transmembrane and nucleotide binding domains.

Presence of two splice variants of PD-ABC, in particular Form 2 is the second NB
It is interesting that it does not contain F (Figure 2). With ABC Transporter,
NBF is required for ATP binding and hydrolysis, which provides energy for substrate transport. Lack of secondary NBFs in other ABC transporters usually results in dysfunctional transporters. In certain TD patients, nonsense mutations or deletions at the C-terminus of ABC1 were associated with reduced cholesterol efflux, suggesting that a second NBF is required for transporter activity. There is. Loss of the second NBF in Form 2 PD-ABC could result in a transporter with no activity. This transporter, however, can still serve as a transport regulator by competing with the Form 1 transporter for substrate, since it can still bind to the substrate. Alternatively, the first NBF may be essential for providing transport energy, as in the case of half-size ABC transporters such as ABC8 (Klucken, L, et al., Proc. Natl. Acad. Sci. USA, 2000; 97: 817-822).

Predicted gene structure of PD-ABC A schematic overall structure of PD-ABC is shown in FIG. Using the full-length cDNA sequence,
The inventor has identified two overlapping genomic sequences in a high throughput genomic database that are aligned with the complete PD-ABC coding sequence. PD-ABC
This was shown to be a pseudogene as no stop codon was found in the genomic sequence. The PD-ABC genomic sequences are both human chromosome 19p13.3.
Derived from.

By aligning the PD-ABC cDNA sequences with their genomic sequences, we were able to determine the intron-exon boundaries of that gene (FIG. 2B). The PD-ABC form 1 coding region is contained in 47 exons and covers a 20 kb genomic sequence (FIG. 2). Form 2 of PD-ABC utilizes another polyadenylation signal found in intron # 38. This results in truncation of the PD-ABC transcript. PD-ABC
Intron / Exon boundary. Interestingly, the intron / exon structure of PD-ABC is very similar to that of ABC1 and ABCR (data not shown).

Tissue distribution of PD-ABC Tissue distribution of PD-ABC was examined by Northern blot analysis using a probe common to both Form 1 and Form 2. A band with a size of 8-9 kb was observed (Fig. 3A). Since the transcript was detected only in the spleen, PD-
It was suggested that ABC is specifically expressed in the spleen. Brain, heart, lungs,
No expression was observed in other tissues examined, including liver and muscle. The same blot was further hybridized with the GAPDH probe to show that this spleen-specific expression was not the result of uneven throughput of mRNA samples. This spleen-specific expression tells us that PD-AB in cells or tissues of the immune system.
It was prompted to examine the expression of C. Indeed, the inventor has found that PD-AB in the immune system tissues examined, including lymph nodes, thymus, peripheral blood leukocytes, bone marrow and fetal liver.
We found that C was highly expressed (Fig. 3B). Interestingly, there are two bands in peripheral blood leukocytes and fetal liver. These two transcripts are almost equally expressed in both tissues, but the less message is shown slightly weaker in bone marrow. The two transcripts can be the two variants identified by the inventor (FIG. 3A). To further evaluate PD-ABC expression in a wider range of different tissues, we performed dot blot analysis on human tissues (Table 3).
).

[0095]

[Table 9]

Consistent with that of Figure 3B, PD-ABC is predominantly expressed in the immune system. Furthermore,
It is also highly expressed in the pituitary gland. The expression pattern of PD-ABC in the immune system suggests that PD-ABC may have a physiological role in those tissues or organs. The link of ABC transporters to the immune system has been previously demonstrated. AB
C1 is required for the uptake of apoptotic cells by macrophages. Furthermore, as shown in studies of ABC1 inhibitors, interleukin-1β secretion and AB
There is a close correlation with C1 activity (Harmon, Y., et al., Blood, 1997; 90: 2.
911-2915). These findings suggest that ABC1 is involved in interleukin 1β secretion and may play a role in the inflammatory response. P
Given the great homology between D-ABC and ABC1, the two transporters may have similar biological functions. In contrast to the ubiquitous expression of ABC1, PD-ABC expression is almost immune system specific. This strongly indicates that PD-ABC was involved in certain immunological pathways.

Expression of two isoforms of PD-ABC The expression of the two variants in tissues was examined by RT-PCR. Although RT-PCR is not completely quantitative, the appearance of PCR products on templates from different tissues can give a general trend for transcript abundance. The present inventor examined the expression of two PD-ABC variants in 24 human tissues and found that their expression patterns were different. Most of the 24 tissues express both Form 1 and Form 2 (Figure 4). However, the prostate and ovary selectively express Form 1. On the other hand, tissues including fetal brain, skin, uterus, pancreas, adrenal gland, salivary gland and colon selectively express Form 2 (FIG. 4). Interestingly, the inventor found a larger band in the bone marrow and peripheral blood leukocytes containing a primer specific for Form 2 (Figure 4). This suggests that there may be another type of PD-ABC that does not contain the second NBF in these tissues. This type of tissue-specific expression may serve a particular physiological purpose, as the lack of a second NBF in Form 2 arguably affects transporter activity.

Conclusions We have identified and isolated a new ABC transporter, the closest ABC1 homolog. In addition, an alternative spliced variant was identified. This transporter is expressed primarily in the immune system and may play a role in the immune response. Furthermore, the expression of the smaller, alternatively spliced transcript of PD-ABC is more restricted than the original form. The tissue-specific expression pattern of PD-ABC and alternative splicing suggests that PD-ABC has a similar function as ABC1, but may be more restricted and regulated. The invention is limited to the precise details of operation or exact compound, composition, method, procedure or aspect shown and described, as obvious modifications and equivalents will be apparent to those skilled in the art. It should be understood that this is not the case, and therefore the present invention should only be limited by the full scope of claims.

[Sequence list]

[Brief description of drawings]

Figure 1: Alignment of the predicted amino acid sequence of human PD-ABC to human ABC1 and ABCR. Amino acids are indicated by the one letter code. Consistent residues along the entire sequence are highlighted in black and homologous residues are shown in shaded areas. Dots indicate gaps introduced in the sequence to maximize alignment.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 3/06 C07K 14/47 4C086 25/08 16/18 4C087 C07K 14/47 C12N 1/15 4H045 16 / 18 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/02 5/10 1/68 A C12P 21/02 Z C12Q 1/02 C12N 15/00 ZNAA 1/68 5/00 AB A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT , SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AU, BA, BB, BG, BR, BZ, CA, CN, CR, CU, CZ, DM, DZ, EE, GD, GE, HR, HU, ID, IL, IN, IS, JP, KP, KR, LC, LK, LR, LT, LV , MA, MG, MK, MN, MX, MZ, NO, NZ, PL, RO, SG, SI, SK, SL, TR, TT, UA, US, UZ, VN, YU, ZA (72) Inventor Toffrey, Sherry Ray 48103, Michigan, USA, Ann Arbor, Aberdeen Drive 4773 (72) Inventor Wang, Mingan 48105, Ann Arbor, Fawn Meadow Court 3130 F Term (Reference) 4B024 AA01 AA11 CA04 CA09 CA11 HA14 HA17 4B063 QA18 QA19 QQ08 QQ43 QQ96 QR08 QR55 QR62 QS25 QS33 QS34 4B064 AG01 BA15 CA19 CC24 DA06 DA13 4B065 AA90 AB01 BA02 CA24 CA44 CA46 4C084 AA02 A16 A14 A02 A16 A14 A02 A06 A16 A02 A06 A02 A06 A02 A06 A02 A06 A02 A06 A02 A06 A02 A02 A02 A02 A06 A02 A06 A02 A06 A02 A02 A02 A02 A02 A02 A06 A02 B23 CA02 NA02 B23 CA01 NA14 BA02 CA02 NA02 B23 CA01 NA14 ZA36 ZB11 ZC33 4C087 AA02 BC83 NA14 ZA06 ZA36 ZB11 ZC33 4H045 AA10 AA11 AA20 AA30 CA40 DA50 DA75 EA23 EA50 FA74 HA06

Claims (23)

[Claims] 1. An isolated and purified DNA sequence substantially similar to the DNA sequence shown in SEQ ID NO: 1 or 3. 2. An isolated and purified DNA sequence which hybridizes under high stringency hybridization conditions to the DNA sequence set forth in SEQ ID NO: 1 or 3. 3. An isolated and purified DNA sequence consisting essentially of the DNA sequence shown in SEQ ID NO: 1 or 3. 4. An isolated and purified DNA sequence having at least 70% identity to a polynucleotide encoding the polypeptide represented by SEQ ID NO: 2 or 4. 5. An isolated and purified DNA sequence which is sufficiently complementary to the DNA sequence shown in SEQ ID NO: 1 or 3. 6. The method according to claim 2, which is subcloned into an extrachromosomal vector.
A recombinant DNA molecule comprising an isolated and purified DNA sequence according to claim 1. 7. A recombinant host cell comprising a host cell transfected with the recombinant DNA molecule of claim 6. 8. A substantially purified recombinant polypeptide, wherein the amino acid sequence of the substantially purified recombinant polypeptide is substantially the same as the amino acid sequence shown in SEQ ID NO: 2 or 4. A substantially purified recombinant polypeptide that is similar. 9. The substantially purified recombinant polypeptide of claim 8, wherein the polypeptide has at least about 70% amino acid sequence similarity to the amino acid sequence set forth in SEQ ID NO: 2 or 4. 10. A substantially purified recombinant polypeptide, wherein the amino acid sequence of the substantially purified recombinant polypeptide consists essentially of the amino acid sequence set forth in SEQ ID NO: 2 or 4. A substantially purified recombinant polypeptide consisting of: 11. An antibody that selectively binds a polypeptide having an amino acid sequence substantially similar to the amino acid sequence of claim 8. 12. A method of detecting PD-ABC protein in a cell, which comprises contacting the cell with the antibody of claim 11 and allowing detection of the PD-ABC protein-antibody complex. A method comprising incubating in a manner such that. 13. A diagnostic assay for detecting cells containing a PD-ABC mutation, wherein total genomic DNA is isolated from said cells and said genomic DNA is isolated according to claim 1, 2 or 3. Subjected to PCR amplification using primers derived from the purified and purified DNA sequence or directly analyzing the genomic DNA by hybridization methods and confirming whether the resulting PCR product contains a mutation A diagnostic assay comprising: 14. A diagnostic assay for detecting cells containing a PD-ABC mutation, wherein total cellular RNA is isolated and said RNA is isolated and purified according to claim 1, 2 or 3. A diagnostic assay comprising subjecting to reverse transcription-PCR amplification using primers derived from different DNA sequences and confirming whether the resulting PCR product contains a mutation. 15. A method for amplifying a region of the DNA sequence according to claim 1, 2 or 3, wherein the test sample is assumed to contain the target sequence according to claim 1, 2 or 3 or a part thereof. A method comprising the step of contacting the above with an amplification reaction reagent. 16. A diagnostic kit for detecting the presence of at least one copy of the DNA sequence according to claim 1, 2 or 3 in a test sample, which comprises a primer,
A kit containing a pair of primers or probes and optionally amplification reagents. 17. The polypeptide according to claim 8, 9 or 10, and
, 9 or 10 for the detection or screening of therapeutic compounds that interfere with or mimic the interaction with a ligand that binds to the polypeptide. 18. The assay comprises: (a) providing the polypeptide of claim 8, 9 or 10; (b) obtaining a candidate substance; (c) the polypeptide being the candidate substance. 18. The assay according to claim 17, comprising the steps of: contacting; and (d) detecting a complex formed between the polypeptide and the candidate substance. 19. A method of protecting a mammalian cell from aberrant calcium flux comprising the isolated and purified DNA sequence of claim 1, 2 or 3 in the mammalian cell. A method comprising introducing an expression vector, the expression vector being operably linked to a DNA sequence that promotes high level expression of the isolated and purified DNA sequence in mammalian cells. 20. A method for treating or preventing epilepsy, which is an expression vector comprising in a mammal the isolated and purified DNA sequence according to claim 1, 2 or 3. The isolated and purified DN in mammalian cells
A method comprising introducing an expression vector operably linked to a DNA sequence that promotes high level expression of the antisense strand of the A sequence. 21. A method of purifying PD-ABC protein from cells, which is operatively linked to a promoter capable of directing gene expression in a host cell. Transfecting a host cell with a vector comprising the isolated and purified DNA sequence; inducing expression of the isolated and purified DNA sequence in the cell; lysing the cell; A PD-ABC protein from the isolate; and purifying the PD-ABC protein from the isolate. 22. A method of treating or preventing a disease selected from the group comprising dyslipidemia-related syndromes, wherein a therapeutically effective amount of the mammal is in need thereof. Or a method comprising the step of administering the polynucleotide according to 3. 23. A method of treating or preventing a disease selected from the group comprising dyslipidemia-related syndromes, wherein the mammal in need thereof has a therapeutically effective amount. Or a method comprising the step of administering the polypeptide of item 10.