JP3156937B2 - Rabbit apoE lipoprotein receptor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel rabbit apoE lipoprotein receptor and its precursor, cDNA encoding the receptor and cD encoding the precursor.
It is about NA. By utilizing the present invention, development of a novel diagnostic kit for arteriosclerosis and myocardial infarction is expected.

[0002]

2. Description of the Related Art Triglycerides and cholesterol are
It is present in the blood in the form of a lipoprotein, and on this lipoprotein molecule, apoE lipoprotein and apoprotein B are present as protein portions. It is known that the entire lipoprotein molecule is taken into the tissue from blood through a receptor that recognizes the apoE lipoprotein and apoprotein B.

[0003] Very low density lipoprotein (Very Low Density Lipoprotein), which is found in blood in hyperlipidemia,
It is called “VLDL”. ), Intermediate-density lipoprotein (Inter-
mediate Density Lipoprotein) and chylomicron remnants are lipoproteins that have apoE lipoprotein in their molecules. These lipoproteins accumulate cholesterol in cells by being taken into cells via receptors. It has been known. Particularly, in the mechanism of cholesterol accumulation in macrophages present in arterial tissues and atherosclerotic layers, the involvement of apoE lipoprotein and its specific apoE lipoprotein receptor is considered to play an important role. Therefore, in order to diagnose the accumulation of cholesterol in macrophages in arterial tissue or atherosclerotic layer, which is a main cause of arteriosclerosis, it is an effective method to investigate the pharmacokinetics of the receptor.

On the other hand, in heart diseases such as myocardial infarction,
It is known that a cell-specific protein is released into a body fluid with the necrosis of a heart cell, and is expected to be used as a diagnostic marker. Therefore, if a novel protein expressed in large amounts in heart cells is found, its application to the diagnosis of heart disease is expected.

[0005] Conventionally, as receptors for plasma lipoproteins, low density lipoprot-
ein: Hereinafter, it is called "LDL". ) Has been found and LD containing apoprotein B-100
L and VLDL containing apoE lipoprotein,
It has been confirmed that these are taken into cells (Scie
nce, 232 , 34-47, (1986): J. Biol. Chem. 263 , 1328.
2-13290, (1988)). However, a receptor specific to lipoprotein itself including apoE lipoprotein has not been found yet.

Generally, in order to measure a protein present in the body or in the blood, an immunological method such as an antigen-antibody reaction,
It is extremely effective to employ a genetic engineering method such as a PCR method. In order to carry out this method, the target protein itself as an antigen and / or the DNA encoding it
Must be obtained.

[0007] Therefore, in order to quantify the apoE lipoprotein receptor present in the body or in the blood by using these methods, the receptor and / or DNA encoding the receptor are obtained from humans or related animals. There is a need.

[0008]

SUMMARY OF THE INVENTION The present inventors obtained mRNA encoding apoE lipoprotein from rabbit tissues,
From this, cDNA was prepared, and apoE lipoprotein was expressed using host cells to complete the present invention.

[0009]

That is, the present invention provides:
(1) Rabbit apoE lipoprotein receptor consisting of the amino acid sequence of amino acid numbers (1) to (846) of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, (2) amino acid shown in SEQ ID NO: 2 in the sequence listing Rabbit apoE lipoprotein receptor precursor consisting of the amino acid sequences of amino acids (-27) to (846) in the sequence, (3) amino acids (1) to (4) of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing DNA encoding the amino acid sequence of (846)
And (4) a DNA sequence encoding the amino acid sequence of amino acid numbers (-27) to (846) among the amino acid sequences shown in SEQ ID NO: 2 in the sequence listing.

The apoE lipoprotein receptor precursor of the present invention consists of 873 amino acid residues and has a calculated molecular weight of 96,279.

The apoE lipoprotein receptor of the present invention has the following structural features. An amino-terminal ligand-binding domain consisting of eight repeating structures containing a large amount of cysteine; a domain homologous to the epidermal growth factor (EGF) precursor that mediates dissociation with the ligand under acidic conditions. It consists of an O-linked sugar domain whose function is unknown, a transmembrane site, and a cytoplasmic domain that assists receptor aggregation.

The cDNA encoding the apoE lipoprotein receptor of the present invention can be produced by the following steps.

(1) Poly (A) ⁺ RN obtained from each tissue of rabbit
A, using the Okayama-Berg method (Mol. Cell. Biol., 2 , 161-1).
70, (1983)) to construct a cDNA library.
Rabbit LDL receptor cDNA (Science, 232 , 1230
-1237, (1986)) and the Okayama-Berg vector include Sal.
Since there is one I site each, the above cDNA library is cut with Sal I and recircularized with T4 DNA ligase to obtain rabbit LDL receptor c.
DNA can be omitted. This LDL receptor
By screening a cDNA library from which cDNA has been excluded using a rabbit LDL receptor cDNA fragment as a probe, a positive clone cross-hybridizing with the probe can be obtained.

(2) The positive clone is transfected into cells lacking the LDL receptor.

(3) The cells are observed under a microscope for the uptake of labeled LDL and VLDL into the cells,
By selecting a clone that takes up VLDL but does not take up LDL, a positive clone containing cDNA encoding the receptor of the present invention can be obtained, and a desired cDNA can be obtained from the clone. Each of these steps will be described in detail below.

The rabbit tissue used for preparing the cDNA library can be any tissue, but is preferably heart, muscle, adipose tissue, brain and spleen, and more preferably heart.

From these tissues, the total RNA fraction,
An mRNA fraction was prepared, a single-stranded cDNA was synthesized using the mRNA as a template and reverse transcriptase, and
Double-stranded DNA can be prepared based on A.

In extracting total RNA from tissue,
Guanidine thiocyanate hot phenol method,
The guanidine thiocyanate guanidine hydrochloride method may be employed, but guanidine thiocyanate pheno-
Le chloroform method (Chomczynski, P. and Sacchi,
N.:Anal. Biochem. 162 , 156, (1987)).

It is known that most of the mRNAs present in the cytoplasm of eukaryotic cells have a poly A sequence at the 3 'end, and this sequence is used for oligo (dT) cellulose column. The mRNA fraction can be purified by adsorbing the mRNA and subsequently eluting it. Further, mRNA can be further fractionated by sucrose density gradient centrifugation or the like.

Using the obtained mRNA as a template, a reverse transcriptase
To synthesize a single strand, and then, based on this cDNA,
Methods for synthesizing double-stranded DNA include S1 nuclease
Act (Efstratiadis, A.et al. (1976) Cell7, 279-288), Ok
ayama-Berg method (Okayama, H. and Berg, P. (1982) Mol. Cell. Bi
ol.Two, 161-170), Land method (Land, H.et al. (1981) Nucleic
Acids Res.9, 2251-2266), O. Joon Yoo method (Yoo, O.J.et a
l. (1983) Proc.Natl.Acad.Sci.USA79, 1049-1053) etc.
Can be adopted.

Okayama-Berg vector and using it
Details of the method for constructing a DNA library are described in known literature (Mo
l. Cell. Biol., 2 , 161-170, (1983)).

[0022] When the positive clone is co-transfected into cells, any cell can be used as long as it lacks the LDL receptor, and the CHO cell 1d1A-7 strain is particularly preferred. For this co-transfection, pSV2-Neo is used. In addition, a medium containing G418 is used as a selective medium.

As a selection method for obtaining a positive clone, labeled LDL and VLDL can be used to observe the binding activity and uptake of co-transfected cells.

Human LDL, VLDL, and lipoprotein deficient serum can be obtained from healthy subjects who have fasted overnight, and β-VLDL of rabbits can be obtained from plasma of rabbits fed 0.5% cholesterol. Recombinant human Apo E was obtained by the method of Murayama et al. (J. Biotech. 17 , 109-
120).

The fluorescent label of lipoprotein is 1,1'-dioctad
ecil-3,3.3'tetramethyllindocarbocyanine perchlorat
This can be done by adsorbing e on lipoproteins. Further, iodine labeling of the protein can be performed by the Bolton-Hunter method.

According to the above method, the rabbit apo E of the present invention
CDNA encoding the lipoprotein receptor can be obtained.

Determination of the sequence of the DNA thus obtained
For example, the chemical modification method of Maxam-Gilbert (Maxam,
A.M. and Gilbert, W. (1980): “Methods in Enzymology”
65499-559) and dideoxynucleic acid using M13 phage.
Otide chain termination method (Messing, J. and Vieira, J. (1982) Gene1
9, 269-276).

To determine whether the obtained cDNA was a full-length cDNA encoding the entirety of the desired protein, the cDNA was used as a labeling probe and used as the initial material. A total RNA fraction was prepared from the tissue and
Analyze by blotting to find the corresponding mRNA
And the molecular weight of the cDNA.

Further, the DNA of the present invention can be obtained by the method described below.

(A) Prepared by polymerase chain reaction
Screening method using probe Using all or a part of the amino acid sequence of the target protein determined as described above, sense strand and antisense strand oligonucleotides corresponding to a part of the amino acid are synthesized, and these are combined to form a polymerase chain. Reaction (Sai
ki, RK et al . (1988) Science 239 , 487-491).
A DNA fragment encoding the target protein or its equivalent is amplified. The template DNA used here was synthesized by reverse transcription from mRNA of cells producing the protein.
cDNA or genomic DNA can be used.
The DNA fragment thus prepared is labeled with ³² P or ³⁵ S, and colony hybridization or plaque hybridization is performed using this as a probe to select a desired clone.

(B) Selection using an antibody against the target protein
In advance, a cDNA is incorporated into an expression vector, a protein is produced in the transformed strain, a desired protein-producing strain is detected using an antibody against the protein and a secondary antibody against the antibody, and the target strain is selected. I do.

(C) Selective hybridization
The cDNA resulting from the process transformants using a system down-translation, blotted to nitrocellulose filters, etc., after hybridization of mRNA from protein-producing cells of interest to dissociate mRNA bound to the cDNA recovered I do. Recovered mRNA
Into a protein translation system, e.g., injection into Xenopus oocytes, or a cell-free system such as rabbit reticulocyte lysate or wheat germ, detected using an antibody against the target protein, and detected the target strain. select.

The DNA obtained by such various methods
Can be exemplified by a DNA consisting of the nucleotide sequence of nucleotide numbers 1 to 3209 shown in SEQ ID NO: 1 in the sequence listing, but the present invention is not limited thereto, and (1) or (2)
All DNAs encoding the described proteins are included in the present invention.

The fragment containing the gene encoding the rabbit apoE lipoprotein receptor thus cloned is reintegrated into an appropriate vector DNA to transform another prokaryotic or eukaryotic host cell. be able to. Furthermore, by introducing an appropriate promoter and a sequence related to expression into these vectors, the gene can be expressed in each host cell.

As a host for prokaryotic cells, for example, E. coli (E.
scherichia coli) and Bacillus subtilis. To express the gene of interest in these host cells, replicons derived from species compatible with the host,
That is, a host cell may be transformed with a plasmid vector containing an origin of replication and a regulatory sequence. Further, it is desirable that the vector has a sequence capable of imparting phenotypic (phenotypic) selectivity to the transformed cells.

For example, E. coli K12 strain and the like are often used as Escherichia coli, and pBR322 and pUC type plasmids are generally used as vectors, but are not limited thereto, and any of various known strains and vectors may be used. Available.
As a promoter, in E. coli, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan lactose (tac) promoter,
Lipoprotein (lpp) promoter, the lambda-derived bacteriophage (lambda) P _L promoter, polypeptide chain elongation factor Tu (tufB) promoter and the like, any promoter can be used in the production of the protein of the present invention.

As Bacillus subtilis, for example, strain 207-25 is preferable, and as a vector, pTUB228 (Ohmura, K. et al . (1984)).
J. Biochem. 95 , 87-93) and the like, but are not limited thereto. Bacillus subtilis α
-A regulatory sequence of the amylase gene is often used, and if necessary, a DNA sequence encoding a signal peptide sequence of α-amylase is ligated to enable extracellular secretory expression. Eukaryotic host cells include cells of vertebrates, insects, yeasts, and the like.Examples of vertebrate cells include monkey COS cells (Gluzman, Y.
981) Cell 23, 175-182) and the dihydrofolate reductase deficient strain (Urlaub of Chinese hamster ovary cells (CHO),
G. and Chasin, LA (1980) Proc. Natl. Acad. Sci. USA 77 , 4
216-4220) and the like are often used, but are not limited thereto.

As an expression vector for a vertebrate cell, a vector having a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence and the like which are usually located upstream of the gene to be expressed can be used. It may have a replication origin. Examples of the expression vector include pSV2dh having the SV40 early promoter.
fr (Subramani, S. et al . (1981) Mol. Cell. Biol. 1 , 854-86
4) can be exemplified, but the present invention is not limited to this.

As eukaryotic microorganisms, yeasts are commonly used, and among them, yeasts of the genus Saccharomyces, for example, Saccharomyces cerevisiae are preferred. Examples of expression vectors for eukaryotic microorganisms such as the yeast include, for example, an alcohol dehydrogenase gene promoter (Bennetzen, JLand H
all, BD (1982) J. Biol. Chem. 257 , 3018-3025) and the acid phosphatase gene promoter (Miyanohara, A. et a
l . (1983) Proc. Natl. Acad. Sci. USA 80 , 1-5) and the like can be preferably used.

As an example, when a COS cell is used as a host cell, the expression vector has an SV40 origin of replication, is capable of autonomous growth in COS cells, and has a transcription promoter, a transcription termination signal, and an RNA splice. One having a site can be used. The expression vector is a DEAE-dextran method (Luthman, H. and Magnu
sson, G. (1983) Nucleic Acids Res. 11 , 1295-1308), calcium phosphate-DNA coprecipitation method (Graham, FLand van der
Ed, AJ (1973) Virology 52 , 456-457) and electric pulsed perforation (Neumann, E. et al . (1982) EMBO J. 1 , 841-845). Thus, a desired transformed cell can be obtained. When CHO cells are used as host cells, G4
A vector capable of expressing the neo gene functioning as an 18 resistance marker, for example, pRSVneo (Sambrook, J. et al . (1989):
"Molecular Cloning-A Laboratory Manual" Cold Spr
ingHarbor Laboratory, NY) and pSV2-neo (Southern, PJa
1 , 327-341), etc., and G418-resistant colonies are selected to obtain a receptor capable of binding to apoE lipoprotein. Transformed cells that stably produce can be obtained.

The desired transformant obtained above can be cultured according to a conventional method, and the culture produces the rabbit apoE lipoprotein receptor intracellularly or extracellularly. As the medium used for the culture, various types commonly used depending on the host cell used can be appropriately selected.For example, in the case of the above COS cells, RPMI-1640 medium or Dulbecco's modified Eagle minimum essential medium (DMEM) etc. A medium to which serum components such as fetal bovine serum (FBS) are added as necessary can be used.

As described above, the rabbit apoE lipoprotein receptor produced intracellularly or extracellularly in the transformant is:
The rabbit apoE lipoprotein receptor can be separated and purified from the rabbit apoE lipoprotein receptor by various known separation procedures utilizing physical properties, chemical properties, immunological properties and the like.

Examples of the method include, for example, treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchanger chromatography, affinity chromatography, high-performance liquid Examples thereof include various liquid chromatography such as chromatography (HPLC), dialysis, and combinations thereof.

By the above method, the desired rabbit apoE lipoprotein receptor can be easily produced on an industrial scale with high yield and high purity. Various properties of the rabbit apoE lipoprotein receptor of the present invention thus obtained are described in detail in the following Examples.

[0045]

Various DNAs of the present invention can be prepared, for example, by the phosphite-triester method (Hunkapiller, M. et al . (1984)) based on the above amino acid sequence and nucleotide sequence information.
It can also be produced by chemical synthesis of nucleic acids according to a conventional method such as Nature 310 , 105-111).

The codon for the desired amino acid is known per se, and its selection may be arbitrarily determined. For example, it can be determined according to a conventional method in consideration of the codon usage frequency of the host to be used (Grantham, R. et al . (1981)). ) Nucleic Acids Res. 9 r43-
r74). Furthermore, partial modification of the codons of these nucleotide sequences can be performed by a conventional method using site-specific mutagenesis (site specific mutase) using a primer consisting of a synthetic oligonucleotide encoding the desired modification.
agenesis) (Mark, DF et al . (1984) Proc. Natl. Acad. Sc)
i.USA8 1 , 5662-5666).

The DNA of the present invention has the ability to strongly hybridize across species to mRNA corresponding to the DNA expressed by in vivo tissues, which is associated with arteriosclerosis and myocardial infarction. Therefore, the corresponding mRNA was used in each tissue extracted from the living body using the DNA as a probe.
Examining the degree of expression is effective for early diagnosis of arteriosclerosis and heart disease. As such a tissue, for example, a micro-tissue obtained by manipulating biopsy or the like from human muscle, adipose tissue or spleen, or peripheral blood lymphocytes obtained from human blood can be used.

Further, the protein of the present invention is used as an essential protein when establishing an immunoassay system useful for such early diagnosis.

That is, a monochrome specific to the protein
It is used as an antigen when preparing a null antibody or a polyclonal antibody. Polyclonal antibodies were prepared using animals such as horses, and monoclonal antibodies were prepared using mice,
All can be carried out by using a method such as a rat, which is well known to those skilled in the art. In this case, an antibody against the protein of the present invention can be obtained by immunizing the above-mentioned animals with the above-mentioned various animals in several doses of 10 to 1,000 μg protein.

In such a measurement system, the protein of the present invention is used as a target antigen. In the measurement system, the protein or the polyclonal or monoclonal antibody obtained above is labeled with a fluorescent reagent, an enzyme, a radioisotope, or the like by a method well known to those skilled in the art. Any of a liquid phase method using a competitive reaction or the like, a solid phase method using a sandwich method by a one-step reaction or a two-step reaction, or the like can be employed.

In such an immunoassay, examining the degree of expression of the protein in each tissue or body fluid extracted from the living body is effective for early diagnosis of arteriosclerosis and myocardial infarction. As such a tissue, for example, a trace tissue obtained by manipulating biopsy or the like from human adipose tissue or a peripheral blood lymphocyte obtained from human blood, and such a body fluid may be serum or the like it can.

[0052]

[0053]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

(1) Isolation of cDNA Encoding ApoE Lipoprotein Receptor In order to search for cDNA encoding ApoE lipoprotein receptor, a cDNA fragment corresponding to the ligand binding domain of rabbit LDL receptor was used as a probe. Was. This region is essential for the binding of the LDL receptor to βVLDL, and is considered to be conserved in other receptors for the apoE protein.

A cDNA library was constructed by the Okayama-Berg method using poly (A) ⁺ RNA extracted from normal rabbit liver, heart and spleen. LDL receptor
cDNA (Science, 232 , 1230-1237, (1986)) and Ok
Since the ayama-Berg vector has one SalI site each, the above cDNA library is digested with SalI and recircularized with T4 DNA ligase to obtain rabbit LDL receptor c from each library.
DNA was omitted.

The spleen cDN excluding the LDL receptor
About 5 × 10 ⁵ colonies obtained from A library,
Hybridization is performed under weak conditions, and LDL
One positive clone cross-hybridizing with the receptor probe was obtained. Hybridization was performed on 5 × 10 ⁵ colonies from the heart cDNA library by the same method under weak conditions, and five positive clones were obtained. However, it was not obtained at all from the liver cDNA library.

These six positive clones were individually:
LDL receptor deficient cell line 1d1A- with pVS2-Neo
CDNA co-transfected to 7. G418 resistant cells were isolated. The lipoprotein binding activity of the cells was examined under a microscope using fluorescently labeled LDL and βVLDL.

One of the positive clones obtained from the heart library (hereinafter referred to as “pApoER1”) was tested for βV.
LDL binding activity was found.

In cells transfected with the LDL receptor, binding activity was found in both LDL and βVLDL, but cells transfected with pApoER1 did not bind to LDL. Control cells transfected with pSV2-Neo were LDL and βVLDL.
Did not bind to any of

The isolated cDNA showed the presence of the apoE lipoprotein receptor specific for lipoproteins having apoE containing βVLDL.

(2) Structure of ApoE Lipoprotein Receptor The sequence of the obtained cDNA showed the following properties of the ApoE lipoprotein receptor. The cDNA had a nucleotide sequence of nucleotide numbers 1 to 3209 shown in SEQ ID NO: 1 in the sequence listing.

ApoE lipoprotein receptor precursor was 873
It is a protein consisting of residues of amino acids, and has a calculated molecular weight of 96,279 (of the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, amino acid numbers (-27) to (846))
Consisting of the amino acid sequence of ). The Apo E lipoprotein receptor has the following structural features. An amino-terminal ligand-binding domain consisting of eight repeating structures containing a large amount of cysteine; a domain homologous to the epidermal growth factor (EGF) precursor that mediates dissociation with the ligand under acidic conditions. It consists of an O-linked sugar domain whose function is unknown, a transmembrane site, and a cytoplasmic domain that assists receptor aggregation.

[0063]

【The invention's effect】

Test Examples (1) Ligand specificity The specificity of apoE lipoprotein receptor expressed in 1d1A cells for ligands was determined by ¹²⁵ I-LDL and ¹²⁵ I-
Analyzed using βVLDL.

FIG. 1 shows the results.

(A) In CHO cells expressing rabbit apoE lipoprotein receptor or human LDL receptor
Figure 2 shows the binding and uptake of ¹²⁵ I-LDL and ¹²⁵ I-β VLDL. Cells transfected with a plasmid encoding the human LDL receptor (pLDLR2) or a plasmid encoding the rabbit apoE lipoprotein receptor (pApoER1) were treated with ¹²⁵ I-LDL (223 cpm /
ng) or ¹²⁵ I-βVLDL (614 cpm / ng). After incubation at 37 ° C for 4 hours, bound and incorporated ¹²⁵ I-LDL and ¹²⁵ I-β
The value of VLDL was measured.

(B) Competition of unlabeled ligand for binding and uptake of ¹²⁵ I-βVLDL in CHO cells expressing rabbit apoE lipoprotein receptor. CHO cells transfected with pApoER1 2.5 μg protein / ml ¹²⁵ I-βVLD in the presence or absence (□) of label / ligand
The cells were incubated in the presence of L (614 cpm / ng). After incubating at 37 ° C. for 4 hours, the value of ¹²⁵ I-βVLDL bound and taken up by the cells was measured.

According to (A), the rabbit apoE lipoprotein receptor binds only to ¹²⁵ I-βVLDL with high affinity, whereas the LDL receptor binds ¹²⁵ I-LDL and ¹²⁵
It is shown to bind with high affinity to I-βVLDL.

(B) shows that ¹²⁵ I-βVLDL against cells transfected with the apoE lipoprotein receptor
Is competitively inhibited by the linear concentration gradient of the unlabeled apoE protein liposome, and
It is shown to recognize proteins.

(2) Tissue specificity Total RNA (20 μg) extracted from each tissue of rabbits was subjected to electrophoresis on an agarose gel, transferred to a nylon membrane, and labeled with ³² P rabbit apoE lipoprotein receptor cDNA. (FIG. 2).

In the Northern hybridization, a band was confirmed around 3.8 kb, and a minor band was also detected around 9.5 kb (FIG. 2).

This minor band is presumed to be the result of separate splicing or polyadenylation.

M encoding the apoE lipoprotein receptor
RNA is most highly expressed in heart and muscle and is also found in adipose tissue, spleen, lung, brain, kidney, adrenal gland, and testis.

On the other hand, it was hardly detected in the liver.

From the above results, the tissue specificity of the apoE lipoprotein receptor of the present invention was confirmed.

[0075]

[Sequence list]

 Sequence length: 3209 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Hypothetical sequence: No Antisense: No Sequence characteristics: 21-2639 E CDS 102 -2639 E mat_peptide sequence GCACCATCCA GGCGGGCACC ATG GGC ACG TCC GCG CGC CGG GCG CTC TGG 50 Met Gly Thr Ser Ala Arg Arg Ala Leu Trp -27 -25 -20 -20 CTG CTG CTC GCG CTG TGC TGG GCG CTC CGG GAG AGC GGC GCC ACC GCA 98 Leu Leu Leu Ala Leu Cys Trp Ala Leu Arg Glu Ser Gly Ala Thr Ala -15 -10 -5 ACC GGG AGA AAA ACC AAG TGC GAA GCT TCC CAA TTC CAG TGC ACC AAT 146 Thr Gly Arg Lys Thr Lys Cys Glu Ala Ser Gln Phe Gln Cys Thr Asn 1 5 10 15 GGC CGC TGC ATT ACT CAG CTG TGG AAA TGT GAT GGC GAT GAA GAC TGT 194 Gly Arg Cys Ile Thr Gln Leu Trp Lys Cys Asp Gly Asp Glu Asp Cys 20 25 30 GTG GAT GGC AGT GAT GAA AAG AAC TGT GTA AAG AAG ACA TGT GCT GAG 242 Val Asp Gly Ser Asp Glu Lys Asn Cys Val Lys Lys Thr Cys Ala Glu 35 40 45 TCG GAT TTT GTG TGC AAC AAT GGC CAG TGT ATT CCC AAT CGA TGG CAG 290 Ser Asp Phe Val Cys Asn Asn Gly Gln Cys Ile Pro Asn Arg Trp Gln 50 55 60 TGC GAC GGG GAT CCT GAC TGT GAA GAT GGT TCT GAT GAA AGC CCA GAG 338 Cys Asp Gly Asp Pro Asp Cys Glu Asp Gly Ser Asp Glu Ser Pro Glu 65 70 75 CAG TGC CAT ATG AGA ACA TGC CGC ATA AAT GAA ATC AGC TGT GGC GCC 386 Gln Cys His Met Arg Thr Cys Arg Ile Asn Glu Ile Ser Cys Gly Ala 80 85 90 95 CGT TCT ACT CAG TGC ATC CCA GTG TCC TGG AGA TGT GAT GGT GAA AGT 434 Arg Ser Thr Gln Cys Ile Pro Val Ser Trp Arg Cys Asp Gly Glu Ser 100 105 110 GAT TGT GAC AGT GGA GAA GAT GAA GAA AAC TGT GGC AAT GTA ACG TGC 482 Asp Cys Asp Ser Gly Glu Glu Asp Glu Glu Asn Cys Gly Asn Val Thr Cys 115 120 125 AGC TCT GAC GAG TTC ACT TGC TCC AGC GGC CGC TGC ATC TCC AGG AAC 530 Ser Ser Asp Glu Phe Thr Cys Ser Ser Gly Arg Cys Ile Ser Arg Asn 130 135 140 TTT GTG TGC AAT GGG CAG GAC GAC TGC AGC GAT GGC AGC GAT GAG CTT 578 Phe Val Cys Asn Gly Gln Asp Asp Cys Ser Asp Gly Ser Asp Glu Leu 145 150 155 GAC TGT GCC CCG CCC ACC TGC GGC GCC CAC GAG TTC C AG TGC AGC ACC 626 Asp Cys Ala Pro Pro Thr Cys Gly Ala His Glu Phe Gln Cys Ser Thr 160 165 170 175 TCC TCG TGC ATC CCC ATC AGC TGG GTG TGC GAC GAC GAC GCG GAC TGC 674 Ser Ser Cys Ile Pro Ile Ser Trp Val Cys Asp Asp Asp Ala Asp Cys 180 185 190 TCC GAC CAG TCC GAT GAA TCC CTG GAG CAG TGT GGC CGC CAG CCC GTG 722 Ser Asp Gln Ser Asp Glu Ser Leu Glu Gln Cys Gly Arg Gln Pro Val 195 200 205 ATC CAC ACC AAG TGC CCT GCC AGC GAG ATC CAG TGT GGC TCC GGC GAA 770 Ile His Thr Lys Cys Pro Ala Ser Glu Ile Gln Cys Gly Ser Gly Glu 210 215 220 TGC ATC CAC AAG AAG TGG CGA TGC GAC GGC GAC CCT GAC TGC AAG GAT 818 Cys Ile His Lys Lys Lys Trp Arg Cys Asp Gly Asp Pro Asp Cys Lys Asp 225 230 235 GGT AGC GAT GAG GTC AAC TGT CCT TCT CGA ACC TGC CGA CCT GAC CAA 866 Gly Ser Asp Glu Val Asn Cys Pro Ser Arg Thr Cys Arg Pro Asp Gln 240 245 250 255 TTC GAA TGC GAG GAT GGT AGC TGC ATC CAT GGC AGC AGG CAG TGT AAC 914 Phe Glu Cys Glu Asp Gly Ser Cys Ile His Gly Ser Arg Gln Cys Asn 260 265 270 270 GGT ATC CGC GAC TGT GTC GAT GGT T CG GAT GAA GTC AAC TGC AAG AAC 962 Gly Ile Arg Asp Cys Val Asp Gly Ser Asp Glu Val Asn Cys Lys Asn 275 280 285 GTC AAC CAG TGC CTG GGC CCT GGC AAG TTC AAG TGC AGA AGT GGG GAA 1010 Val Asn Gln Cys Leu Gly Pro Gly Lys Phe Lys Cys Arg Ser Gly Glu 290 295 300 TGC ATA GAT ATC AGC AAA GTG TGT AAC CAG GAG CAG GAC TGC AGG GAC 1058 Cys Ile Asp Ile Ser Lys Val Cys Asn Gln Glu Gln Asp Cys Arg Asp 305 310 315 TGG AGT GAT GAG CCC CTG AAA GAG TGC CAT GTA AAC GAA TGC TTG GTC 1106 Trp Ser Asp Glu Pro Leu Lys Glu Cys His Val Asn Glu Cys Leu Val 320 325 330 335 AAT AAT GGT GGA TGT TCC CAT ATC TGC AAG GAC TCG GTT ATA GGC TAC 1154 Asn Asn Gly Gly Cys Ser His Ile Cys Lys Asp Ser Val Ile Gly Tyr 340 345 350 GAA TGT GAC TGT GCG GCT GGG TTT GAA CTC ATA GAT AGG AAA ACC TGC 1202 Glu Cys Asp Cys Ala Ala Gly Phe Glu Leu Ile Asp Arg Lys Thr Cys 355 360 365 GGA GAT ATT GAC GAA TGC CAA AAT CCG GGA ATC TGC AGT CAA ATT TGT 1250 Gly Asp Ile Asp Glu Cys Gln Asn Pro Gly Ile Cys Ser Gln Ile Cys 370 375 380 380 ATC AAC TTA AAA GGC GGT TAC AAG TGT GAA TGT AGT CGT GGC TAT CAA 1298 Ile Asn Leu Lys Gly Gly Tyr Lys Cys Glu Cys Ser Arg Gly Tyr Gln 385 390 395 ATG GAT CTT GCC ACT GGC GTG TGC AAG GCA GTA GGC AAA GAG CCA AGT 1346 Asp Leu Ala Thr Gly Val Cys Lys Ala Val Gly Lys Glu Pro Ser 400 405 410 415 CTG ATC TTC ACT AAT AGA AGA GAC ATC CGC AAG ATT GGC TTA GAG AGA 1394 Leu Ile Phe Thr Asn Arg Arg Asp Ile Arg Lys Ile Gly Leu Glu Arg 420 425 430 AAA GAA TAC ATC CAA CTG GTT GAG CAG CTG CGA AAC ACC GTG GCT CTT 1442 Lys Glu Tyr Ile Gln Leu Val Glu Gln Leu Arg Asn Thr Val Ala Leu 435 440 445 445 GAT GCT GAT ATT GCA GCT CAG AAA CTG TTC TGG GCT GAC GTC AGC CAG 1490 Asp Ala Asp Ile Ala Ala Gln Lys Leu Phe Trp Ala Asp Val Ser Gln 450 455 460 AAG GCC ATC TTC AGT GCC TCA ATT GAT GAC AAG GTT GGT AGA CAT GTA 1538 Lys Ala Ile Phe Ser Ala Ser Ile Asp Asp Lys Val Gly Arg His Val 465 470 475 AAA ATG ATC GAC AAT GTC TAT AAT CCT GCA GCC ATT GCT GTT GAT TGG 1586 Lys Met Ile Asp Asn Val Tyr Asn Pro Ala Ala Ile Ala Val Asp Trp 480 485 490 495 GTG TAC AAG ACC ATC TAC TGG ACT GAT GCG GCT TCT AGG ACT ATT TCA 1634 Val Tyr Lys Thr Ile Tyr Trp Thr Asp Ala Ala Ser Arg Thr Ile Ser 500 505 510 GTA GCC ACC CTA GAC GGA GCC AAG AGG AAG TTC CTG TTT AAC TCT GAC 1682 Val Ala Thr Leu Asp Gly Ala Lys Arg Lys Phe Leu Phe Asn Ser Asp 515 520 525 TTG CGA GAG CCT GCC TCC ATA GCC GTG GAC CCA CTG TCT GGA TTT GTT 1730 Leu Arg Glu Pro Ala Ser Ile Ala Val Asp Pro Leu Ser Gly Phe Val 530 535 540 TAC TAC TGG TCA GAC TGG GGT GAA CCA GCT AAA ATA GAA AAA GCA GGA ATG 1778 Tyr Trp Ser Asp Trp Gly Glu Pro Ala Lys Ile Glu Lys Ala Gly Met 545 550 555 555 AAT GGA TTT GAT AGA CGC CCA CTG GTG ACA GTG GAC ATC CAA TGG CCC 1826 Asn Gly Phe Asp Arg Arg Pro Leu Val Thr Val Asp Ile Gln Trp Pro 560 565 570 575 575 AAT GGA ATT ACA CTG GAC CTC ATC AAA AGC CGC CTC TAC TGG CTG GAT 1874 Asn Gly Ile Thr Leu Asp Leu Ile Lys Ser Arg Leu Tyr Trp Leu Asp 580 585 590 TCC AAG TTG CAC ATG TTA TCG AGT GTG GAT TTA AAT GGC CAA GAC CGC 1922 Ser Lys Leu His Met Leu Ser Ser Val Asp Leu Asn Gly Gln Asp Arg 595 600 605 AGG ATA GTA CTC AAG TCT CTG GAG TTC CTA GCT CAT CCT CTG GCA CTA 1970 Arg Ile Val Leu Lys Ser Leu Glu Phe Leu Ala His Pro Leu Ala Leu 610 615 620 ACA ATT TTC GAG GAT CGT GTC TAC TGG ATA GAT GGG GAA AAC GAA GCA 2018 Thr Ile Phe Glu Asp Arg Val Tyr Trp Ile Asp Gly Glu Asn Glu Ala 625 630 635 GTC TAT GGT GCC AAT AAG TTC ACT GGA TCA GAA CTG GCC ACT CTA GTC 2066 Val Tyr Gly Ala Asn Lys Phe Thr Gly Ser Glu Leu Ala Thr Leu Val 640 645 650 655 AAC AAC CTT AAT GAT GCC CAA GAC ATC ATT GTG TAT CAT GAA CTT GTG 2114 Asn Asn Leu Asn Asp Ala Gln Asp Ile Ile Val Tyr His Glu Leu Val 660 665 670 CAG CCA TCA GGT AAA AAC TGG TGT GAG GAA GAC ATG GAG AAT GGA GGG 2162 Gln Pro Ser Gly Lys Asn Trp Cys Glu Glu Asp Met Glu Asn Gly Gly 675 680 685 TGT GAA TAC CTA TGC CTG CCA GCT CCA CAG ATT AAT GAG CAC TCT CCA 2210 Cys Glu Tyr Leu Cys Leu Pro Ala Pro Gln Ile Asn Glu His Ser Pro 690 695 700 AAG TAT ACC TGT TCC TGT CCC AAT GGG TAC CAC CTA GAG GAA AAT GGC 2258 Lys Tyr Thr Cys Ser Cys Pro A sn Gly Tyr His Leu Glu Glu Asn Gly 705 710 715 CGA GAA TGC CAA AGT ACT GCA ACT ACT GTG ACT TAC AGT GAG ACA AAA 2306 Arg Glu Cys Gln Ser Thr Ala Thr Thr Val Thr Tyr Ser Glu Thr Lys 720 725 730 735 735 GAT ACC AAC ACT ACA GAA ATT TCA CCA ACT AGT GGA CTA GTT CCT GGA 2354 Asp Thr Asn Thr Thr Glu Ile Ser Pro Thr Ser Gly Leu Val Pro Gly 740 745 750 GAG ATC AAT GTG ACC ACG GCA GTC TCA GAA GTC AGT GTT CCC CCA AAA 2402 Glu Ile Asn Val Thr Thr Ala Val Ser Glu Val Ser Val Pro Pro Lys 755 760 765 GGG ACT TCC GCT GCC TGG GCC ATC CTT CCT CTC TTG CTC TTA GCG ATG 2450 Gly Thr Ser Ala Ala Trp Ala Ile Leu Pro Leu Leu Leu Leu Ala Met 770 775 780 GCA GCA GTG GGT GGC TAC TTG ATG TGG CGG AAC TGG CAA CAC AAG AAC 2498 Ala Ala Val Gly Gly Tyr Leu Met Trp Arg Asn Trp Gln His Lys Asn 785 790 795 ATG AAA AGC ATG AAC TTT GAC AAT CCT GTG TAC TTG AAG ACC ACG GAA 2546 Met Lys Ser Met Asn Phe Asp Asn Pro Val Tyr Leu Lys Thr Thr Glu 800 805 810 815 GAG GAC CTC TCC ATT GAC ATC GGC AGA CAC AGT GCT TCT GTT GGA CAC 2594 Glu A sp Leu Ser Ile Asp Ile Gly Arg His Ser Ala Ser Val Gly His 820 825 830 ACG TAC CCA GCA ATA TCA GTT GTA AGC ACA GAT GAT GAT CTA GCT 2639 Thr Tyr Pro Ala Ile Ser Val Val Ser Thr Asp Asp Asp Leu Ala 835 840 845 TGACTCCTGT TCATGACCTC TGAGGTCTAA CCCAGTAACA CCCCCTCTCC GGAACGGTAA 2699 CCGGGCCAGC AGCTGAAGTC TCTTTCTTCC TGCCATCTGG AAGAACGTCA AGATATCTTT 2759 GCGTGGATCA AGCTTGTGTA CTTGACCGTT TTTATATTAC TTTTGTAAAT ATCCTTGCCC 2819 ACATGCTACT TCAGCTTTGG ATATGGTTAC CAAGTATCTG TAATCTATGA ATTTCTAGAC 2879 AGTATTGCCG CCTCTGGCCA AATATGCACT TTCCCTAGAA AGCCATATTC CAGCAATGAA 2939 ACTTGTGCTA TAGTGTACAC CACTGTACAT ACAGTGTATA GGCCACCTGT AAATACTCCA 2999 GAGAACAATC ACTATTCTTA AGCACTTTGA AAATATTTCT ATGTAAATTA TTGTAAACTT 3059 TTTCAATGGT TGGGACAATG GCAATAGGAT AAAACGGGTT ACTAAGATGA AATTGCCAAA 3119 AAAAATTTGT AAACTAATTT TGTACGTATA AATGAACTCT TTGACCTCAA AGGAGGTTTA 3179 CAAAGACTGA GTGTTCAAAC TACTGTATAT 3209 SEQ ID NO: 2 Sequence length: 87 Class: Protein Hypothetical sequence: No Sequence features: -27-846: Rabbit apoE lipoprotein receptor precursor 1-846: Rabbit apoE lipoprotein receptor-mature sequence Met Gly Thr Ser Ala Arg Arg Ala Leu Trp Leu Leu Leu Ala Leu Cys -27 -25 -20 -15 -15 Trp Ala Leu Arg Glu Ser Gly Ala Thr Ala Thr Gly Arg Lys Thr Lys -10 -5 1 5 Cys Glu Ala Ser Gln Phe Gln Cys Thr Asn Gly Arg Cys Ile Thr Gln 10 15 20 Leu Trp Lys Cys Asp Gly Asp Glu Asp Cys Val Asp Gly Ser Asp Glu 25 30 35 Lys Asn Cys Val Lys Lys Thr Cys Ala Glu Ser Asp Phe Val Cys Asn 40 45 50 Asn Gly Gln Cys Ile Pro Asn Arg Trp Gln Cys Asp Gly Asp Pro Asp 55 60 65 Cys Glu Asp Gly Ser Asp Glu Ser Pro Glu Gln Cys His Met Arg Thr 70 75 80 85 Cys Arg Ile Asn Glu Ile Ser Cys Gly Ala Arg Ser Thr Gln Cys Ile 90 95 100 Pro Val Ser Trp Arg Cys Asp Gly Glu Ser Asp Cys Asp Ser Gly Glu 105 110 115 Asp Glu Glu Asn Cys Gly Asn Val Thr Cys Ser Ser Asp Glu Phe Thr 120 125 130 Cys Ser Ser Gly Arg Cys Ile Ser Arg Asn Phe Val Cys Asn Gly Gln 135 140 145 Asp Asp Cys Ser Asp Gly Ser Asp Glu Leu Asp Cys Ala Pro Pro Thr 150 155 160 165 Cys Gly Ala His Glu Phe Gln Cys Ser Thr Ser Ser Cys Ile Pro Ile 170 175 180 Ser Trp Val Cys Asp Asp Asp Ala Asp Cys Ser Asp Gln Ser Asp Glu 185 190 195 Ser Leu Glu Gln Cys Gly Arg Gln Pro Val Ile His Thr Lys Cys Pro 200 205 210 Ala Ser Glu Ile Gln Cys Gly Ser Gly Glu Cys Ile His Lys Lys Trp 215 220 225 Arg Cys Asp Gly Asp Pro Asp Cys Lys Asp Gly Ser Asp Glu Val Asn 230 235 240 245 Cys Pro Ser Arg Thr Cys Arg Pro Asp Gln Phe Glu Cys Glu Asp Gly 250 255 260 Ser Cys Ile His Gly Ser Arg Gln Cys Asn Gly Ile Arg Asp Cys Val 265 270 275 Asp Gly Ser Asp Glu Val Asn Cys Lys Asn Val Asn Gln Cys Leu Gly 280 285 290 Pro Gly Lys Phe Lys Cys Arg Ser Gly Glu Cys Ile Asp Ile Ser Lys 295 300 305 Val Cys Asn Gln Glu Gln Asp Cys Arg Asp Trp Ser Asp Glu Pro Leu 310 315 320 325 Lys Glu Cys His Val Asn Glu Cys Leu Val Asn Asn Gly Gly Cys Ser 330 335 340 His Ile Cys Lys Asp Ser ValIle Gly Tyr Glu Cys Asp Cys Ala Ala 345 350 355 Gly Phe Glu Leu Ile Asp Arg Lys Thr Cys Gly Asp Ile Asp Glu Cys 360 365 370 Gln Asn Pro Gly Ile Cys Ser Gln Ile Cys Ile Asn Leu Lys Gly Gly 375 380 385 Tyr Lys Cys Glu Cys Ser Arg Gly Tyr Gln Met Asp Leu Ala Thr Gly 390 395 400 405 Val Cys Lys Ala Val Gly Lys Glu Pro Ser Leu Ile Phe Thr Asn Arg 410 415 420 Arg Asp Ile Arg Lys Ile Gly Leu Glu Arg Lys Glu Tyr Ile Gln Leu 425 430 435 Val Glu Gln Leu Arg Asn Thr Val Ala Leu Asp Ala Asp Ile Ala Ala 440 445 450 Gln Lys Leu Phe Trp Ala Asp Val Ser Gln Lys Ala Ile Phe Ser Ala 455 460 465 465 Ser Ile Asp Asp Lys Val Gly Arg His Val Lys Met Ile Asp Asn Val 470 475 480 485 Tyr Asn Pro Ala Ala Ile Ala Val Asp Trp Val Tyr Lys Thr Ile Tyr 490 495 500 Trp Thr Asp Ala Ala Ser Arg Thr Ile Ser Val Ala Thr Leu Asp Gly 505 510 515 Ala Lys Arg Lys Phe Leu Phe Asn Ser Asp Leu Arg Glu Pro Ala Ser 520 525 530 Ile Ala Val Asp Pro Leu Ser Gly Phe Val Tyr Trp Ser Asp Trp Gly 535 540 540 545 Glu Pro Ala Lys Ile Glu Lys Ala Gly Met Asn Gly Phe Asp Arg Arg 550 555 560 565 Pro Leu Val Thr Val Asp Ile Gln Trp Pro Asn Gly Ile Thr Leu Asp 570 575 580 Leu Ile Lys Ser Arg Leu Tyr Trp Leu Asp Ser Lys Leu His Met Leu 585 590 590 595 Ser Ser Val Asp Leu Asn Gly Gln Asp Arg Arg Ile Val Leu Lys Ser 600 605 610 Leu Glu Phe Leu Ala His Pro Leu Ala Leu Thr Ile Phe Glu Asp Arg 615 620 625 Val Tyr Trp Ile Asp Gly Glu Asn Glu Ala Val Tyr Gly Ala Asn Lys 630 635 640 645 Phe Thr Gly Ser Glu Leu Ala Thr Leu Val Asn Asn Leu Asn Asp Ala 650 655 660 Gln Asp Ile Ile Val Tyr His Glu Leu Val Gln Pro Ser Gly Lys Asn 665 670 675 Trp Cys Glu Glu Asp Met Glu Asn Gly Gly Cys Glu Tyr Leu Cys Leu 680 685 690 Pro Ala Pro Gln Ile Asn Glu His Ser Pro Lys Tyr Thr Cys Ser Cys 695 700 705 Pro Asn Gly Tyr His Leu Glu Glu Asn Gly Arg Glu Cys Gln Ser Thr 710 715 720 725 Ala Thr Thr Val Thr Tyr Ser Glu Thr Lys Asp Thr Asn Thr Thr Glu 730 735 740 Ile Ser Pro Thr Ser Gly Leu Val Pro Gly Glu Ile Asn Val Thr Thr 745 750 755 Ala Val Ser Glu Val Ser Val Pro Pro Lys Gly Thr Ser Ala Ala Trp 760 765 770 Ala Ile Leu Pro Leu Leu Leu Leu Ala Met Ala Ala Val Gly Gly Tyr 775 780 785 Leu Met Trp Arg Asn Trp Gln His Lys Asn Met Lys Ser Met Asn Phe 790 795 800 805 Asp Asn Pro Val Tyr Leu Lys Thr Thr Glu Glu Asp Leu Ser Ile Asp 810 815 820 Ile Gly Arg His Ser Ala Ser Val Gly His Thr Tyr Pro Ala Ile Ser 825 830 835 Val Val Ser Thr Asp Asp Asp Leu Ala 840 845

[Brief description of the drawings]

FIG. 1. Rabbit apoE lipoprotein receptor and human LD
Graph showing ligand specificity for L receptor

FIG. 2: Northern blotting electrophoretogram showing expression of rabbit apoE lipoprotein receptor in tissues

Continuation of front page (56) References Nucleic Acids Res. , 17, p. 1259-1260, 1989 Biol. Chem. , 266, p. 18761-18770, 1991 Proc. Natl. Acad. Sc i. USA, 86 (6), p. 1880-1884, 1989 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 C07K 14/705 GENBANK / EMBL / DDBHJ / GENESEQ PIR / SWISSPROT / GENES EQ BIOSIS / MEDLINE / WPI (STN)

Claims (4)

(57) [Claims] 1. A rabbit apoE lipoprotein receptor comprising the amino acid sequence of amino acid numbers (1) to (846) in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. 2. A rabbit apoE lipoprotein receptor precursor comprising the amino acid sequence of amino acid numbers (-27) to (846) of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. 3. A DNA sequence encoding an amino acid sequence of amino acids (1) to (846) in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. 4. A DNA sequence encoding the amino acid sequence of amino acids (-27) to (846) of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.