Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-07-12
2001-03-06
Eyler, Yvonne (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387900, C530S388100, C530S350000, C530S300000, C424S139100, C424S141100, C435S007100, C435S069100, C536S023100
Reexamination Certificate
active
06197937
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a mammalian receptor for modified low-density lipoprotein (LDL), and in more detail relates to a mammalian vascular endothelial receptor for modified low-density lipoprotein.
BACKGROUND OF THE INVENTION
Vascular endothelial dysfunction has been pointed out as an important index in the early stages of progressive atherosclerosis. The vascular endothelial cell releases many sorts of humoral factors to keep circulatory homeostasis. The vascular endothelial function is inhibited by physical stimuli or various substances including the most important factor, oxidized low-density lipoprotein, which is a kind of modified low-density lipoproteins. For example, the vascular endothelial cell releases nitrogen monoxide as a vasohypotonic factor to adjust vascular tonus. The release of nitrogen monoxide is inhibited by the oxidized low-density lipoprotein.
It has been known that macrophages or vascular endothelial cells internalize the modified low-density lipoprotein through a receptor other than receptors for low-density lipoprotein. The macrophages internalize the modified low-density lipoprotein through scavenger receptors, which have already been structurally analyzed (cf., PCT Patent Japanese Publication Nos. 6(1994)-500765 and 6(1994)-508604, and Japanese Patent Provisional Publication No. 3(1991)-290184). The macrophages are then changed to foam cells, which are specific in arteriosclerotic focus. Since the macrophage scavenger receptors are not found in vascular endothelial cells, it has been anticipated that receptors of another structure are present in the vascular endothelial cells (cf., Hidenori Arai, Toru Kita, Oxidized LDL, Metabolism 28/4, 1991).
For the reasons mentioned above, it is necessary to analyze the structure of a n endothelial receptor for modified low-density lipoprotein, namely t he amino acid sequence of the receptor. However, the structure and the amino acid sequence have not yet been elucidated.
DISCLOSURE OF THE INVENTION
According to study of the present inventors, the structure of the vascular endothelial receptor for modified low-density lipoprotein is now elucidated. The amino acid sequences of the vascular endothelial receptor for modified low-density lipoprotein are set forth in SEQ ID NOS: 2, 4 and 6.
There is provided by the present invention a DNA sequence essentially encoding a mammalian vascular endothelial receptor for modified low-density lipoprotein.
The DNA sequence can be cDNA clones derived from the open reading frame of a gene corresponding to a native mam-malian vascular endothelial receptor for modified low-density lipoprotein. The DNA sequence can also be a sequence which is capable of hybridization to the above-mentioned cDNA clones and encodes a biologically active mammalian vascular endothelial receptor for modified low-density lipoprotein. Further, the sequence can be degenerate as a result of the genetic code to the above-mentioned DNA sequences. The degenerate encodes the same biologically active receptor for modified low-density lipoprotein.
Therefore, the present invention provides a DNA sequence set forth in SEQ ID NOS: 1, 3 or 5 (DNA having the sequence) or an analogue thereof, which corresponds to the region encoding a mammalian vascular endothelial receptor for modified low-density lipoprotein.
The present invention also provides a cDNA clone having a DNA sequence set forth in SEQ ID NOS: 1, 3 or 5 or an analogue thereof, which corresponds to the region encoding a mammalian vascular endothelial receptor for modified low-density lipoprotein.
The present invention further provides a DNA sequence which is capable of hybridization to a cDNA clone of a DNA sequence set forth in SEQ ID NOS: 1, 3 or 5 (DNA having the sequence) in 20% (v/v) formamide at 42° C., and encodes a protein of a mammalian vascular endothelial cell, said protein having a function of binding a modified low-density lipoprotein (namely a receptor thereof).
The present invention furthermore provides a DNA sequence which is degenerate as a result of the genetic code to a DNA sequence set forth in SEQ ID NOS: 1, 3 or 5 (DNA having the sequence), and encodes a protein of a mammalian vascular endothelial cell, said protein having a function of binding a modified low-density lipoprotein (namely a receptor thereof).
Moreover, the present invention relates to an antibody of a receptor for modified low-density lipoprotein corresponding to a DNA sequence set forth in SEQ ID NOS: 1, 3 or 5 or an analogue thereof (or a receptor for modified low-density lipoprotein containing an amino acid sequence set forth in SEQ ID NOS: 2, 4 or 6 or an analogue thereof).
The DNA sequence of the present invention can be integrated into an expression vector. Therefore, the present invention further provides a process for the production of a mammalian vascular endothelial receptor for modified low-density lipoprotein or an analogue thereof, which comprises inserting the recombinant expression vector into a host cell and culturing the cell under expression promoting conditions.
The invention furthermore provides a protein composition containing a biologically active mammalian vascular endothelial receptor for modified low-density lipoprotein or an analogue thereof which is produced as mentioned above.
The obtained protein composition containing a biologically active mammalian vascular endothelial receptor for modified low-density lipoprotein or an analogue thereof is effective in an assay of the mammalian modified low-density lipoprotein. The composition can also be used in preparation of an antibody to the vascular endothelial receptor for modified low-density lipoprotein. The antibody can be used in diagnosis.
It is apparent from the above-described biological activities of the modified low-density lipoprotein and the receptor thereof that an agent containing an antibody to the vascular endothelial receptor for modified low-density lipoprotein is effective in diagnosis of atherosclerosis.
In the present specification, the term “receptor for modified low-density lipoprotein” means proteins which are capable of binding modified low-density lipoprotein molecules and, in their native configuration as mammalian plasma membrane proteins, presumably play a role in transducing the signal provided by a modified low-density lipoprotein to a vascular endothelial cell. In the specification, the term includes analogues of native proteins with an activity of binding a modified low-density lipoprotein or a signal transducing activity.
The term “subtype of a receptor for modified low-density lipoprotein” means molecules of a receptor for modified low-density lipoprotein which show different pharmacological potency rank orders, namely different affinities or selectivities for various isopeptides of modified low-density lipoprotein, such as oxidized low-density lipoprotein or acetylated low-density lipoprotein.
The term “essentially” used in the expression “a DNA sequence essentially encoding a receptor for modified low-density lipoprotein” or the like means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between subject sequence and reference sequence set forth in the SEQ ID NOS:. 1, 3 or 5.
In more detail, the sequence can be modified so long as a protein corresponding to the sequence has a biological activity (described below), namely an activity of binding modified low-density lipoprotein. Therefore, the region encoding a portion of binding modified low-density lipoprotein should be the same as the reference sequence set forth in the SEQ ID NOS: 1, 3 or 5 except for variation due to code degeneracy and substitution of an amino acid to an analogous amino acid. The other region merely requires at least 30% (preferably at least 50%, and more preferably at least 80%) similarity in the sequence.
The above-mentioned substitution to an analogous amino acid means amino ac
Masaki Tomoo
Sawamura Tatsuya
Basi Nirmal S.
Eyler Yvonne
Nippon Chemiphar Co., Ltd.
Reed Smith LLP
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