DNA encoding human apoB48R: a monocyte-macrophage apolipoprotein B48 receptor gene and protein

Abstract

The present invention provides an isolated DNA molecule that codes for a cell-surface binding protein in human monocytes and macrophages. In addition, an amino acid sequence derived from the nucleotide sequence is provided. The newly-identified cell-surface binding protein described herein is instrumental in the apoB-mediated cellular uptake of plasma chylomicrons and remnants and hypertriglyceridemic triglyceride-rich lipoproteins in an ApoE- and lipoprotein lipase- and heparin sulfate proteoglycan-independent pathway. The new human macrophage receptor has been cloned and uniquely, binds TGRLP via apolipoprotein B48, the marker of dietary TGRLP (apoB48R). This process rapidly converts macrophages and apoB48R-transfected Chinese hamster ovary cells in vitro into lipid-filled “foam cells,” hallmarks of atherosclerotic lesions. The apoB48R cDNA (3744 bp) encodes a novel protein with no known homologs. Its ˜3.8 kb mRNA is expressed primarily by reticuloendothelial cells. Immunohistochemical studies indicate that foam cells of human atherosclerotic lesions express the apoB48R.

What is claimed is:  
     
         1 . An isolated DNA molecule encoding a monocyte-macrophage cell surface apoB48 receptor protein (apoB48R), said DNA molecule selected from the group consisting of: 
 (a) a DNA molecule comprising SEQ ID No. 1 encoding said monocyte-macrophage cell surface apoB48 receptor protein as set forth in SEQ ID No. 2 or a fragment thereof; and,    (b) a DNA molecule differing from the DNA molecule of (a) in codon sequence due to the degeneracy of the genetic code, and which encodes said monocyte-macrophage cell surface apoB48 receptor protein as set forth in SEQ ID No. 2 or a fragment thereof.    
     
     
         2 . A vector containing the DNA molecule of  claim 1  and regulatory elements necessary for expression of said DNA in a cell, said vector adapted for expression in a recombinant cell.  
     
     
         3 . A host cell containing the vector of  claim 2 .  
     
     
         4 . An isolated DNA molecule encoding a portion of the apoB48 receptor protein, said DNA molecule selected from the group consisting of SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9.  
     
     
         5 . A vector containing the isolated DNA molecule of  claim 4  and regulatory elements necessary for expression of said DNA in a cell, said vector adapted for expression in a recombinant cell.  
     
     
         6 . A host cell containing the vector of  claim 5 .  
     
     
         7 . An isolated monocyte-macrophage cell surface apoB48 receptor protein (apoB48R) having the sequence SEQ ID No. 2.  
     
     
         8 . An antibody directed to an isolated monocyte-macrophage cell surface apoB48 receptor protein having the sequence encoded by a DNA molecule selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and a fragment thereof.  
     
     
         9 . A method of cell-specific delivery of a therapeutic compound to an individual in need of such treatment, comprising the steps of: 
 providing a recognition compound selected from the group consisting of a peptide, an antibody and an antibody fragment having the ability to bind to the monocyte-macrophage cell surface apoB48 receptor protein of  claim 7  (SEQ ID No. 2), or a portion of said sequence; and    incorporating said recognition compound into a delivery vehicle containing said therapeutic compound.    
     
     
         10 . The method of  claim 9 , wherein said delivery vehicle is a liposome.  
     
     
         11 . The method of  claim 9 , wherein said therapeutic compound is selected from the group consisting of cytocidal drugs, cytotoxic drugs, genes, vitamins, hormones, cytokines, growth factors and growth inhibitors.  
     
     
         12 . The method of  claim 9 , wherein said cell is selected from the group consisting of monocytes, macrophages, endothelial cells, placental cells, peripheral blood leukocytes, bone marrow cells, astrocytes, osteoclasts and other monocyte-derived cells.  
     
     
         13 . The method of  claim 9 , wherein said individual has a disease selected from the group consisting of monocytic leukemia, tuberculosis, leprosy, and AIDS.  
     
     
         14 . The method of  claim 9 , wherein said therapeutic compound delivered to said individual has an effect selected from the group consisting of inhibition of angiogenesis, enhancement of angiogenesis, inhibition of fibrinolysis, enhancement of fibrinolysis, inhibition of tissue factor production and enhancement of tissue factor production.  
     
     
         15 . The method of  claim 9 , wherein said therapeutic compound is delivered to a developing embryo in said individual.  
     
     
         16 . The method of  claim 9 , wherein said therapeutic compound is a label used to locate atherosclerotic plaques.  
     
     
         17 . The method of  claim 9 , wherein said therapeutic compound is a compound used to eliminate atherosclerotic plaques.  
     
     
         18 . A method of inhibiting the binding of lipoproteins to cells, comprising the step of treating said cells with an agent which binds a monocyte-macrophage cell surface apoB48 receptor protein of  claim 7  (SEQ ID No. 2), wherein said binding inhibits the binding of lipoproteins to cells, thereby inhibiting foam cell formation and increased monocyte adhesion to endothelial cells.  
     
     
         19 . The method of  claim 18 , wherein said agent is selected from the group consisting of a peptide, an antibody directed to an isolated apoB48 receptor protein having the sequence encoded by a DNA molecule selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and a fragment thereof.  
     
     
         20 . The method of  claim 19 , wherein said peptide is lipoprotein lipase or a fragment thereof.  
     
     
         21 . The method of  claim 18 , wherein said cells are selected from the group consisting of monocytes, macrophages, endothelial cells, placental cells, peripheral blood leukocytes, bone marrow cells, astrocytes, osteoclasts and other monocyte-derived cells.  
     
     
         22 . A method of enhancing hepatic uptake and catabolism of apoB48-containing lipoproteins in an individual in need of such treatment, comprising the step of administering to said individual an effective amount of a vector encoding the monocyte-macrophage cell surface apoB48 receptor protein of  claim 7  (SEQ ID No. 2), or a portion of said protein.  
     
     
         23 . The method of  claim 22 , wherein said individual has been diagnosed with a disease selected from the group consisting of Pattern B phenotype, familial combined hyperlipidemia, familial hypercholesterolemia, non-familial hypercholesterolemia, hypertriglyceridemia and low plasma high density lipoprotein levels.  
     
     
         24 . A method of evaluating an individual at risk for cardiovascular disease, comprising the steps of: 
 (a) extracting a sample of monocytes-macrophages and triglyceride-rich lipoproteins from plasma of said individual and from a control individual not considered at risk for cardiovascular disease; and    (b) comparing the binding affinity (K d ) of the apoB48 receptor of  claim 7  between said individual at risk and said control individual, whereby a difference in said binding affinity between said individual at risk and said control individual is indicative of an alteration in either or both the apoB cell-surface receptor protein an d triglyceride-rich lipoproteins, wherein said alteration in said apoB cell-surface receptor protein or triglyceride-rich lipoproteins is indicative of dyslipidemias, abnormal postprandial triglyceride metabolism or Pattern B phenotype in said individual at risk.    
     
     
         25 . A method of evaluating an individual at risk for cardiovascular disease, comprising the steps of: 
 (a) extracting a sample of monocytes-macrophages from plasma of said individual; and    (b) comparing the binding affinity (K d ) of the apoB48 receptor of  claim 7  of said individual at risk and the apoB48 receptor of control THP-1 cells for triglyceride-rich lipoproteins, whereby a difference in said binding affinity between said individual at risk and said control cells is indicative of an alteration in the apoB cell-surface receptor protein in said individual at risk, wherein said alteration in said apoB cell-surface receptor protein is indicative of dyslipidemias, abnormal postprandial triglyceride metabolism or Pattern B phenotype in said individual at risk.    
     
     
         26 . A method of evaluating an individual at risk for cardiovascular disease, comprising the steps of: 
 (a) extracting a sample of monocytes-macrophages and triglyceride-rich lipoproteins from plasma of said individual; and    (b) comparing the binding affinity (K d ) of said triglyceride-rich lipoproteins for the apoB receptor of  claim 7  said individual at risk and the apoB receptor of THP-1 control cells, whereby a difference in said binding affinity between said individual at risk and said control cells is indicative of an alteration in the triglyceride-rich lipoproteins, wherein said alteration in said triglyceride-rich lipoproteins is indicative of dyslipidemias, abnormal postprandial triglyceride metabolism or Pattern B phenotype in said individual at risk.    
     
     
         27 . A method of evaluating an individual at risk for cardiovascular disease, comprising the steps of: 
 (a) extracting a sample of monocytes-macrophages from plasma of said individual; and    (b) performing a Western blot analysis on proteins of said monocytes-macrophages using an antibody directed towards the protein of  claim 7  (SEQ ID No. 2), or fragments thereof, whereby a difference in the migration or mobility of said proteins between said individual at risk and said control is indicative of an alteration in the apoB cell-surface receptor protein, wherein said alteration in said apoB cell-surface receptor protein is indicative of dyslipidemias, abnormal postprandial triglyceride metabolism or Pattern B phenotype in said individual at risk.    
     
     
         28 . The method of  claim 27 , wherein said control is selected from the group consisting of a THP-1 clonal cell line or a sample of monocytes-macrophages from plasma of a control individual not considered at risk for cardiovascular disease.  
     
     
         29 . A method of evaluating an individual at risk for cardiovascular disease, comprising the steps of: 
 (a) extracting a sample of monocytes-macrophages from plasma of said individual; and    (b) isolating and analyzing RNAs of said monocytes-macrophages, whereby a difference in said RNAs between said individual at risk and said control is indicative of an alteration in the apoB cell-surface receptor protein, wherein said alteration in said apoB cell-surface receptor protein is indicative of dyslipidemias, abnormal postprandial triglyceride metabolism or Pattern B phenotype in said individual at risk.    
     
     
         30 . The method of  claim 29 , wherein said analyzing is performed by Northern blot analysis using a DNA probe selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9, or fragments thereof.  
     
     
         31 . The method of  claim 29 , wherein said analyzing is performed by RT-PCR using oligonucleotides complementary to SEQ ID No. 1.  
     
     
         32 . The method of  claim 29 , wherein said control is selected from the group consisting of a THP-1 clonal cell line or a sample of monocytes-macrophages from plasma of a control individual not considered at risk for cardiovascular disease.  
     
     
         33 . A method of evaluating an individual at risk for cardiovascular disease, comprising the steps of: 
 (a) extracting a sample of monocytes-macrophages from plasma of said individual; and    (b) comparing the number of apoB receptors on said monocytes-macrophages between said individual at risk and said control, whereby a difference in said number of receptors between said individual at risk and said control is indicative of an alteration in the apoB cell-surface receptor protein, wherein said alteration in said apoB cell-surface receptor protein is indicative of dyslipidemias, abnormal postprandial triglyceride metabolism or Pattern B phenotype in said individual at risk.    
     
     
         34 . The method of  claim 33 , wherein said control is selected from the group consisting of a THP-1 clonal cell line or a sample of monocytes-macrophages from plasma of a control individual not considered at risk for cardiovascular disease.