DNA encoding human apoB48R: a monocyte-macrophage apolipoprotein B48 receptor gene and protein Number:6,740,735 from the United States Patent and Trademark Office (PTO) owispatent 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 .about.3.8 kb mRNA is expressed primarily by reticuloendothelial cells. Immunohistochemical studies indicate that foam cells of human atherosclerotic lesions express the apoB48R.<H apolipoprotein, macrophage, DNA, encoding, hu, 6740735.html, Patent, pto, 6740735

Title: 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 .about.3.8 kb mRNA is expressed primarily by reticuloendothelial cells. Immunohistochemical studies indicate that foam cells of human atherosclerotic lesions express the apoB48R.

Patent Number: 6,740,735 Issued on 05/25/2004 to Gianturco, et al.

Inventors: Gianturco; Sandra H. (Birmingham, AL), Bradley; William A. (Birmingham, AL)
Appl. No.: 09/583,610

Filed: May 31, 2000

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
130242	Aug., 1998	6194558

Current U.S. Class: 530/350 ; 435/252.3; 435/320.1; 435/6; 435/69.5; 435/7.2; 435/7.21; 436/501; 530/300; 536/23.5

Current International Class: C07K 14/435 (20060101); C07K 14/705 (20060101); A61K 38/00 (20060101)

Field of Search: 530/350,300 536/23.5 436/501 435/6,7.2,7.21,69.5,252.3,320.1 514/2

Primary Examiner: Eyler; Yvonne
Assistant Examiner: Brannock; Michael
Attorney, Agent or Firm: Adler; Benjamin Aaron

Government Interests

FEDERAL FUNDING LEGEND

This invention was produced in part using funds from the Federal government under National Institutes of Health grant HL44480. Accordingly, the Federal government has certain rights in this invention.

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part and claims the benefit of priority under 35 USC .sctn.120 of U.S. patent application Ser. No. 09/130,242, filed Aug. 6, 1998, now U.S. Pat. No. 6,194,558.

Claims

What is claimed is:

1. An isolated monocyte-macrophage cell surface apoB48 receptor protein (apoB48R) having the sequence SEQ ID No. 2.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of molecular biology, cardiovascular medicine and cellular nutrition. More specifically, the present invention relates to DNA encoding the human monocyte-macrophage and placental triglyceride-rich lipoprotein/apolipoprotein B (apoB) receptor gene(s) and protein(s).

2. Description of the Related Art

Hypertriglyceridemia is a common, heterogeneous disorder. When chylomicrons persist in the fasting state, lipid-filled monocyte-macrophage-derived foam cells can accumulate in the spleen, liver, bone marrow, atherosclerotic lesions, and skin (Fredrickson, 1978). Many, but not all, early studies (Carlson, 1972; Brunzell, 1976; Grundy, 1988; Schaefer, 1988; Austin, 1991) indicate elevated plasma triglycerides are a risk factor for coronary heart disease and myocardial infarction, sequelae of atherosclerosis. The possibility that triglyceride-rich lipoproteins (hepatic as well as dietary) are involved in atherosclerosis has been strengthened recently. Both the Procam study and a follow-up of the Helsinki Heart Study implicate elevated triglycerides (and therefore triglyceride-rich lipoproteins) as an important risk factor in atherosclerosis (Assmann, 1992). Havel et al. demonstrated that plasma very low density lipoprotein and intermediate density lipoprotein cholesterol levels correlated with progression of coronary atherosclerosis disease, whereas low density lipoprotein cholesterol level did not (Phillips, 1993). Moreover, very low density lipoprotein-intermediate density lipoprotein particles enter the artery wall and are found in human atherosclerotic plaques (Rapp, 1994). Elevated postprandial chylomicron remnants of S.sub.f <400 are significantly higher in subjects with coronary heart disease but with normal fasting lipid levels than in matched control subjects without this disease (Patsch, 1992; Weintraub, 1996). Thus, there is increasing biochemical as well as epidemiologic evidence that the major carriers of plasma triglycerides, very low density lipoproteins and plasma chylomicrons and their remnants, are atherogenic.

Monocytes and macrophages play a key role in atherogenesis, accounting for many lipid-filled "foam cells" in atherosclerotic lesions (Gerrity, 1981; Faggiotto, 1984). Many studies on foam cell formation have focused on uptake of modified and oxidized low density lipoprotein by the macrophage scavenger receptor and putative oxidized low density lipoprotein receptors (van Berkel, 1994). However, monocytes and macrophages also take up intestinally-derived plasma chylomicrons, which contain apoB48, and hepatically-derived very low density lipoprotein (apoB-100). Zilversmit and colleagues demonstrated extrahepatic uptake of .about.40% of chylomicrons in rabbits (Ross, 1977) that was decreased by inhibition of the reticuloendothelial system (Nagata, 1987). Furthermore, studies in marmosets (a primate) and rabbits demonstrated substantial uptake (20-40% of total) of chylomicrons in vivo by accessible, peripheral macrophages, particularly in bone marrow (both animals) and spleen (marmosets) (Hussain, 1989a, 1989b). This would suggest that triglyceride-rich lipoproteins serve as a non-modified, native source of lipid for monocytes' and macrophages' nutrition in the normal state.

Triglyceride-rich lipoproteins are involved in the pathological conversion of monocytes and macrophages into foam cells in humans, a process seen in bone marrow, spleen, etc. in types 1, 3 and 5 hypertriglyceridemia (Fredrickson, 1978). Triglyceride-rich lipoproteins are also involved in formation of monocyte-macrophage-derived foam cells in eruptive xanthomas in untreated hypertriglyceridemic diabetic subjects. These foam cells contained triglyceride-rich lipoprotein core lipids, triglycerides and cholesteryl esters, following chylomicron uptake (Parker, 1970).

Chylomicrons and hypertriglyceridemic-very low density lipoproteins (including .beta.-very low density lipoproteins) are the only known native human lipoproteins, without modification, which directly cause rapid, receptor-mediated macrophage lipid accumulation in vitro, causing macrophages to resemble foam cells histologically (Gianturco, 1982b, 1986a, 1986b, 1988; Brown et al., 1983; Ostlund-Lindqvist, 1983; Bersot, 1986). The lipid that accumulates in macrophages after receptor-mediated uptake of a lipoprotein in vitro reflects the lipid composition of the lipoprotein (Gianturco, 1982b; Brown et al., 1983). Therefore, as seen in vivo, triglyceride is the predominant lipid which accumulates initially in macrophages exposed to hypertriglyceridemic-very low density lipoproteins or chylomicrons, but cholesterol and cholesteryl esters also accumulate even in short term incubations (Gianturco, 1986a). Triglyceride-rich lipoproteins enter the arterial wall in animals (Nordesgaard, 1994) and in man (Rapp, 1994). Since one triglyceride-rich lipoprotein S.sub.f >100 contains 5 times or more cholesterol and cholesteryl esters than one low density lipoprotein (Shen, 1978), each triglyceride-rich lipoprotein that enters a monocyte, macrophage or the arterial wall is equivalent to 5 or more low density lipoprotein particles in terms of cholesterol delivery.

A number of plausible mechanisms for the above-described observations exist, many involving apoE. Very low density lipoproteins from hypertriglyceridemic subjects were first shown to be abnormal and potentially atherogenic in studies which showed that very low density lipoproteins from hypertriglyceridemic, but not from normal subjects, deliver cholesterol to cultured fibroblasts via the low density lipoprotein receptor (Gianturco, 1978). The abnormality in hypertriglyceridemic-very low density lipoproteins is primarily in the S.sub.f >60 subfraction which, in contrast to normal very low density lipoproteins fraction S.sub.f >60, contains extra apoE of an accessible conformation that specifically binds to the low density lipoprotein receptor; apoB of S.sub.f >60 particles does not bind to the LDL receptor (Gianturco, 1982a, 1983; Bradley, 1984; Hui, 1984; Krul, 1985; Eisenberg 1988). ApoE also mediates triglyceride-rich lipoprotein binding to other widely-distributed receptors in the low density lipoprotein receptor gene family, such as the low density lipoprotein receptor-related protein/.alpha..sub.2 -macroglobulin receptor (Beisiegel, 1989; Kowal, 1989) and a very low density lipoprotein receptor expressed primarily in heart, muscle, and adipose (Takahashi, 1992). One of these could account for apoE-mediated very low density lipoprotein uptake observed in monocytes and macrophages (Wang-Iverson, 1985).

In contrast, apoB mediates the binding of low density lipoprotein (Goldstein, 1977), intermediate density lipoproteins (S.sub.f 12-20), and the predominant very low density lipoprotein in normal subjects, very low density lipoprotein.sub.3 (S.sup.f 20-60) (Bradley, 1984; Krul, 1985), the only very low density lipoprotein subclass from normal subjects that binds to the low density lipoprotein receptor of fibroblasts (Gianturco, 1980a, 1982a, Eisenberg, 1988) or of U937 monocytes (Sacks and Breslow, 1988). The domain of apoB that binds to the low density lipoprotein receptor is in the C-terminal portion not present in apoB-48 (Yang, 1986; Milne, 1989).

Lipolysis of normal very low density lipoprotein S.sub.f >60 permits binding of the lipolytic remnant to the low density lipoprotein receptor (Catapano, 1979; Schonfeld, 1979). Lipoprotein lipase secreted by macrophages (Khoo, 1981) hydrolyzes very low density lipoproteins and enhances its cellular uptake (Lindquist, 1983). This facilitation may occur through localization of triglyceride-rich lipoproteins to membrane heparin sulfate proteoglycan (Eisenberg, 1992) and/or through binding to low density lipoprotein receptor-related protein (Beisiegel, 1991).

The substantial and rapid uptake of triglyceride-rich chylomicrons in vivo by bone marrow and spleen macrophages in marmosets and rabbits was not accelerated by infusion of apoE (Hussain, 1989a). This is surprising, since apoE is a necessary ligand for the uptake of large triglyceride-rich lipoproteins by members of the low density lipoprotein receptor gene family. Indeed, infused apoE diverted much of the uptake from the peripheral macrophages to the liver, suggesting that the observed peripheral macrophage chylomicron uptake was not mediated by apoE and that these macrophages have an apoE-independent uptake mechanism. The rate and magnitude of triglyceride-rich chylomicron uptake by bone marrow monocytes and macrophages (20-40% of chylomicrons cleared from the plasma at 20 minutes (Hussain, 1989a)) suggests this uptake is receptor mediated. Rapid, receptor-mediated delivery of intestinally-derived, triglyceride-enriched chylomicrons may be necessary to assure delivery of sufficient energy and fat-soluble vitamins and other essential compounds to sustain hematopoiesis. In addition, and in contrast to inactivation of the apoE gene, loss of apoB by homologous recombination caused embryonic lethality in the homozygous state. ApoB is normally expressed early in yolk sak visceral endodermal cells for the synthesis of apoB-containing lipoprotein which are apparently necessary for the transport of lipids and lipid-soluble vitamins to embryonic tissues.

Moreover, homologous recombinant ("knockout") mice that completely lack apoE accumulate very low density lipoprotein and chylomicron remnants in their plasma (Plump, 1992; Zhang, 1992). These mice develop atherosclerosis that is accelerated by high fat diets. The lesions are characterized by monocyte-macrophage-derived foam cells, as in human lesions, demonstrating unequivocally that apoE is not necessary for the conversion of monocytes and macrophages into foam cells in vivo (Nakashima, 1994; Reddick, 1994). Taken together, these in vivo studies suggest strongly the existence of an apoE-independent pathway for the uptake of triglyceride-rich lipoproteins by monocytes and macrophages, which would result in foam cell formation in hypertriglyceridemia.

In vitro evidence for an apoE- and lipoprotein lipase-independent, apoB-mediated triglyceride-rich lipoprotein receptor pathway in murine macrophages has been reported (Gianturco, 1988). Because of the potential importance of an apoE-independent, receptor-mediated pathway for triglyceride-rich lipoproteins in the formation of foam cells in human pathology, particularly in hypertriglyceridemic subjects, the human monocyte-macrophage receptor from the monocytic cell line THP-1 was characterized and purified and receptor-specific antibodies were produced. Briefly, this unique apoE- and lipoprotein lipase-independent pathway and binding site is in murine macrophages, human monocytes and macrophages, and in the human monocytic cell lines THP-1 and U937, but not in human fibroblasts or hepatoma cell lines or in Chinese hamster ovary (CHO) cells (Gianturco, 1988, 1994a). Further, ligand blotting studies in bovine and porcine aortic endothelial cells also were positive. Thus, endothelial cells specifically bound chylomicrons followed by hydrolysis and uptake of their cholesteryl esters (Fielding, 1978) and very low density lipoproteins from hypertriglyceridemic subjects, but not from normal subjects, delivered cholesterol to cultured endothelial cells (Gianturco, 1980).

Since the apoE-independent and lipoprotein lipase-independent receptor also binds .beta.-very low density lipoproteins, but with lower affinity, it was once referred to as a .beta.-very low density lipoprotein receptor (Goldstein, 1980; Gianturco, 1986a). Subsequent studies, however, demonstrated that uptake of triglyceride-rich lipoproteins independent of apoE was not inhibited by anti-low density lipoprotein receptor antibodies that inhibited the low density lipoprotein receptor-mediated uptake of rabbit .beta.-very low density lipoproteins in the same cells, nor did anti-low density lipoprotein receptor antibodies bind to the candidate receptor (Gianturco, 1988). The apoE-independent receptor differs from the low density lipoprotein receptor family or the scavenger receptor family in many properties including (1) unchanged expression during differentiation, (2) slower intracellular ligand degradation, (3) ligand specificity, (4) apparent molecular weight of the candidate receptors, and (5) cellular distribution.

The prior art is deficient in the lack of the sequence of the DNA encoding for the monocyte-macrophage apoB receptor gene and protein, in the genomic structure and chromosomal localization and in the understanding of its expression in the placenta, human coronary, carotid, and aortic macrophage-derived foam cells in atherosclerotic lesions and in other immune tissues including peripheral blood leukocytes, bone marrow, spleen, tonsils and appendix. The present invention fulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

Monocyte-macrophage-derived foam cells accumulate in atherosclerotic lesions and throughout the body in some types of hypertriglyceridemia. Uptake of plasma chylomicrons and hypertriglyceridemic triglyceride-rich lipoproteins by an apoE-independent human monocyte and macrophage receptor, distinct from previously-described lipoprotein receptors, may be involved in foam cell formation in vivo. Two cell-surface membrane binding proteins (MBPs) of .about.200 and .about.235 kDa, in human monocytes and macrophages and THP-1 monocytes and macrophages, were characterized as the likely receptors. It was determined that both MBPs share a common .about.200 kDa ligand binding subunit. This ligand-binding subunit was purified and internal tryptic peptide sequences were obtained. Receptor-specific antipeptide antibodies were generated against a 10-residue unique and unambiguous internal sequence (to which no matches were found in GenBank, Swiss Pro, etc) that binds the active receptor forms MBP200, MBP200R and MBP235. Antibodies against the C-terminal .about.47 kDa receptor domain and other domains were produced and shown to bind to all active forms of the receptor. Overlapping partial cDNAs from a .lambda.gt10 THP-1 library and from a .lambda.gt10 human placental library corresponding to the receptor were obtained and sequenced.

The present invention shows that cell-surface MBP200 and MBP235 are unique monocyte, macrophage, placental and endothelial cell receptors for apoB in plasma chylomicrons and some hypertriglyceridemic-triglyceride-rich lipoproteins and their remnants; other apoB-containing lipoproteins also bind to the receptor with varying, generally much lower, affinities. The present invention also shows that said receptors bind to apoB-48 and to the N-terminal portion of apoB-100 at or near the lipoprotein lipase binding site and not in a heparin-binding domain. Normally, the MBPs may be involved in nutrition of circulating monocytes and accessible, peripheral macrophages, e.g. bone marrow; in lipemic states, the pathway can be overwhelmed and contribute to foam cell formation and endothelial cell dysfunction. Therefore, diminished triglyceride-rich lipoprotein uptake by this receptor, due either to receptor defects or to triglyceride-rich lipoprotein defects leading to altered receptor affinity, may be involved with metabolic abnormalities associated with increased risk for cardiovascular disease, such as modest hypertriglyceridemia and small, dense low density lipoproteins (pattern B) and/or persistence of chylomicron-derived, (i.e., apoB-48-containing) lipoproteins in the fasting state. Diminished activity of the receptor in the placenta could result in fetal abnormalities due to reduced delivery of dietary fat-soluble vitamins (A, E, D) and essential fatty acids and other essential nutrients that are carried in chylomicrons.

To clone the cDNA for MBP200R, PCR with degenerate primers were used and a THP-1 .lambda.gt10 cDNA library to produce a 631 bp product (pcr631) (SEQ ID No. 3) which contains three peptide sequences found in amino acid sequence from MBP200R. pcr631 was used to identify several distinct cDNA clones. One clone, THP-1 .lambda.73-3 (SEQ ID No. 9), contains an 1851 bp insert with a 1381 bp open reading frame (ORF) and 470 bp untranslated region including the stop codon, the polyadenylation signal and the poly-A tail. PCR on a 5' stretch human placenta .lambda.gt10 cDNA library using antisense primers derived from the 5' end of THP-1 .lambda.73-3 resulted in a 1466 bp clone with an open reading frame that overlapped the open reading frame of THP-1 .lambda.73-3 (pcr1466) (SEQ ID No. 8) resulting in a 3071 bp sequence with a 2601 bp open reading frame. Glutathione-S-transferase fusion proteins were expressed using the pcr631, THP-1 .lambda.73-3, and pcr1466 pGEX constructs. Polyclonal antibodies were produced to each protein domain. Immunoblots demonstrate that the antibodies specifically recognize the GST-fusion products and all receptor activities (MBP200, MBP235 and MBP200R). Additional 5' sequence obtained by PCR of the human placenta .lambda.gt10 cDNA library with antisense primers from the 5' end of pcr1466 resulted in a 751 bp clone (pcr751) (SEQ ID No. 7) that contained a 10 bp untranslated 5' end, a Kozak consensus start sequence and the initial ATG start codon. The sequences obtained from multiple clones from THP-1 monocyte genomic DNA and the cDNA library result in 3744 bases of cDNA sequence (SEQ ID No. 1) with an open reading frame of 3264 bp encoding a 1088 residue protein for the human monocyte apoB48 receptor. Northern analysis of THP-1s, human placenta, bone marrow, peripheral blood leukocytes, spleen, tonsils, appendix, and lymph node reveal a messenger RNA of approximately 3.8 kb, indicating the complete cDNA sequence has been determined. A full-length cDNA was constructed in a pCDNA vector. Chinese hamster ovary (CHO) cells transfected with the vector containing the receptor cDNA, in contrast to the pCDNA vector alone, expressed full receptor activity as determined by rapid, high affinity binding and uptake of fluorescent DiI-labeled trypsinized VLDL and, in stably transfected CHOs, by rapid cellular triglyceride mass accumulation and by the rapid (.about.1.5 hour) accumulation of cytoplasmic lipid droplets visualized by light microscopy after staining with the neutral lipid Oil Red 0, after incubation with chylomicrons containing apoB48 as the B species, HTG-VLDL and tryp VLDL but not with normal VLDL or LDL.

One object of the present invention is to provide an isolated DNA molecule encoding a monocyte-macrophage cell surface apoB48R binding protein selected from the group consisting of: (a) a DNA molecule comprising a sequence SEQ ID No. 1 and which encodes the monocyte-macrophage cell surface apoB48R binding protein (the apoB receptor) (SEQ ID No. 2) or a portion of the monocyte-macrophage cell surface apoB48R binding protein; 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 the monocyte-macrophage cell surface apoB48R binding protein (SEQ ID No. 2) or a portion of the monocyte-macrophage cell surface apoB48R binding protein. Embodiments of this object of the invention include provisions for a vector containing the isolated DNA molecule encoding a monocyte-macrophage cell surface apoB48R binding protein and regulatory elements necessary for expression of said isolated DNA molecule in a cell, the vector adapted for expression in a recombinant cell, as well as a host cell containing the vector.

An additional object of the present invention is to provide a vector comprising an isolated DNA for a monocyte-macrophage cell surface apoB48R GST fusion binding protein having the sequence SEQ ID No. 2 or portions thereof.

A further object of the present invention is to provide a method of cell-specific delivery of therapeutic compounds to human monocytes, macrophages, other reticuloendothelial cells that express the receptor or embryos comprising the steps of: providing a peptide or antibody(s) having the ability to bind to an isolated monocyte-macrophage cell surface apoB48R binding protein having the sequence SEQ ID No.2, or a portion of said sequence or comprising a related protein of the same gene family; and incorporating the peptide into liposomes containing said therapeutic compound. Yet another method of cell-specific delivery may utilize a receptor-specific antibody or an antibody fragment (Fab) that binds to an isolated monocyte-macrophage cell surface apoB48R binding protein having the sequence SEQ ID No.2, or a portion of the sequence, or comprising a related protein of the same gene family; and incorporating the antibody into liposomes.

Yet another object of the present invention is to provide a method of inhibiting foam cell formation and increased monocyte adhesion to endothelial cells, comprising the step of treating a monocyte-macrophage with an agent which binds an isolated monocyte-macrophage cell surface apoB48 receptor protein having the sequence SEQ ID No.2, thereby blocking or inhibiting binding of apoB-containing lipoproteins to the receptor. Gene therapy such as adenoviral delivery of the receptor proteins of the present invention to LDL-receptor deficient subjects is also contemplated.

Another object of the present invention is to provide delivery of the novel sequences disclosed herein, e.g., in an adenoviral vector, to the liver or elsewhere, for the purpose of correcting metabolic defects that cause abnormal accumulation of apoB-containing lipoproteins in the plasma.

Yet another object of the present invention is to provide 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 the plasma of the individual and from a control individual not considered at risk for cardiovascular disease; and (b) comparing the binding affinity (K.sub.d) of the apoB receptor of the monocytes-macrophages for triglyceride-rich lipoproteins between the individual at risk and the control individual, whereby a difference in the binding affinity between the individual at risk and the control individual is indicative of an alteration in either or both the apoB cell-surface receptor protein and triglyceride-rich lipoproteins, and the alteration in the apoB cell-surface receptor protein or triglyceride-rich lipoproteins is indicative of dyslipidemias, abnormal postprandial triglyceride metabolism or Pattern B phenotype in the individual at risk. Alternatively, one may compare the rapid, receptor mediated monocyte-macrophage lipid accumulation induced by standardized tryp VLDL vs. normal TGRLP vs the patients TGRLP by quantifying lipid mass changes and by Oil Red 0 staining.

This objective may also be accomplished by performing a Western blot analysis on proteins of the monocytes-macrophages using an antibody directed towards the protein of SEQ ID No. 2, or fragments thereof, or alternatively performing a Northern blot analysis on RNAs of the monocytes-macrophages 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, whereby a difference in the migration or mobility of the proteins and/or RNAs between the individual at risk and the control individual is indicative of an alteration in the apoB cell-surface receptor protein, and the alteration in the apoB cell-surface receptor protein is indicative of dyslipidemias, abnormal postprandial triglyceride metabolism or Pattern B phenotype in the individual at risk.

As atherosclerotic plaques are enriched with ApoB48-receptor expressing cells, the instant invention may also be directed toward delivery of therapeutic agents to atherosclerotic plaques. These agents may include label to localize said plaques, inhibitors of apoB48 receptor to inhibit further development of the plaques, or agents designed to eliminate the plaques.

Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.

DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention are attained and can be understood in detail, more particular descriptions of the invention may be had by reference to certain embodiments which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

FIG. 1 shows a 2-dimensional SDS-PAGE of MBP200 and MBP235 before and after reduction. Detergent extracts of THP-1 monocytes were electrophoresed in the first dimension in a minigel without reduction. One cm strips containing the MBPs were removed and treated with buffer (panel A) or reductant, 2% 2-mercaptoethanol, (panel B), placed lengthwise on a second 5% slab minigel, electrophoresed and transferred to nitrocellulose for ligand binding analysis. Panel A illustrates that without reduction MBP200 and MBP235 maintain their distinct mobilities (seen on the diagonal), whereas after reduction (R) in the second dimension, both activities now have identical Rfs that are different from their original Rfs. The reduced form of both receptor proteins that retain ligand binding activity was called MBP200R. The single lane on the far left of each 2-D gel contains internal prestained markers, at approximately 200 kDa and 97 kDa.

FIG. 2 shows (2A) ligand binding of MBPs by ligand blot analysis. Concentrations of the hypertriglyceridemic-very low density lipoproteins are given below each lane before (non-reduced) and after (reduced) treatment with 2-mercaptoethanol. Very low density lipoprotein binding was detected by apoB antibodies followed by IgG-specific enzyme-linked antibody and colorimetric substrate. Densitometry and quantification of the images utilized ImageQuant software. FIG. 2B shows the saturation binding of hypertriglyceridemic-very low density lipoprotein to MBP235, MBP200 and MBP200R. The amount of very low density lipoprotein bound to MBP200, MBP235 and MBP200R activities in FIG. 2A was determined by densitometry of each MBP region in the ligand blots using a calibration curve generated from known amounts of the same very low density lipoprotein applied to nitrocellulose and quantified with a purified anti-apoB antibody. The amount of ligand bound to each MBP region, expressed as ng very low density lipoprotein bound, was plotted as a function of the amount of very low density lipoprotein to which the nitrocellulose was exposed. The broken line represents the calculated sum of binding of the very low density lipoprotein to MBP200 and MBP235 at each level of very low density lipoprotein. FIG. 2C shows the data from FIG. 2B after transformation plotted by the method of Scatchard to determine the K.sub.d for each of the MBPs, expressed as .mu.g/ml.

FIG. 3 shows a model of the relationship between MBP235 and MBP200 and how MBP200R is generated from MBP200 and MBP235 by treatment with reducing agents such as 2-mercaptoethanol.

FIG. 4 shows that the ligand binding activity and antipeptide immunoreactivity are coincident before and after reduction of the MBPs, demonstrating that each MBP contains the 10-residue unique sequence obtained by microsequence data from peptides from purified MBP200R. C=preimmune sera at the appropriate concentration as control. Lanes 1-6 contain two levels of the THP-1 detergent extracts: lanes 1, 3 and 5 are a 1:3 and lanes 2, 4 and 6 are 1:5 dilution. Lanes 3, 4 and 8 are at 1:50 and lane 9 is at 1:100 dilution of the antipeptide antibody. Ligand binding activity was visualized by incubation with hypertriglyceridemic-very low density lipoproteins for 2 hours at room temperature and detected with anti-apoB antibodies. Primary antibody binding was then detected with Anti-IgG alkaline phosphatase conjugated antibodies.

FIG. 5 shows that anti-apoB antibodies block binding of triglyceride-rich lipoproteins to MBP200 and MBP235. Biotinylated very low density lipoproteins were incubated for 0.5 hour with anti-apoB antibodies, lane 2, or with preimmune serum, lane 1, prior to ligand blotting for 1.5 hours. Biotinylated very low density lipoprotein binding was detected with streptavidin-alkaline phosphatase. The blots were imaged, then quantitated with ImageQuant. The relative total binding to the MBPs with the specific apoB antisera and the preimmune sera are as indicated. The insert contains the imaged lanes from the ligand blot.

FIG. 6 shows the analysis of THP-1 monocytes by FACS. Antipeptide antibodies are shown to bind to the surface of THP-1 monocytes using two concentrations of the primary antibody. Goat(Fab').sub.2 rabbit anti-IgG (H+L)-FITC only (used as the negative control) and the alpha chain of the monocyte integrin VLA-4 was used as a known positive control.

FIG. 7 shows the amino acid sequence derived from the 631 base pair PCR product. The carboxyterminal sequence (shaded) represents the peptide sequence used to produce the initial degenerate oligonucleotide primer and is from the same peptide used to develop the anti-peptide antibodies and used for surface labeling of the THP-1 monocytes. The underlined peptides represent sequences found by tryptic peptide mapping and microsequence analysis of MBP200R.

FIG. 8 shows that THP-1 monocytes contain mRNA coding for MBP200R. RT-PCR using primer pairs from the PCR631/MBP200R sequence and mRNA from THP-1 monocytes produced a RT-PCR product of the correct size. The ethidium bromide stained gel (inverse image) of the RT-PCR products is shown. Lane 1: 572 bp product using primers 53-72, sense and 624-604, antisense; Lane 2: Glucose-6-phosphate dehydrogenase (GPDH) control product; Lane 3: MW ladder; Lane 4: second round product generated with internal second antisense primer, 490 bp.

FIG. 9 shows the restriction mapping of the 572 bp RT-PCR product described in FIG. 8 (inverse image). Lane 1: 572 bp product; Lane 2: EcoRI control, no digestion; Lane 3: BamHI; Lane 4: MW ladder 50 bp, 100, 200, 300, 400, 500, 700, 1000, 1500 and 2000 bp; Lane 5: PstI; Lane 6: HindIII; Lane 7: XbaI.

FIG. 10 shows that monospecific rabbit anti-human apoB IgGs specifically inhibit the binding of HTG-VLDL to the monocyte TGRLP receptor (MBP200 or MBP235). THP-1 monocyte aqueous phase extracts were electrophoresed and transferred to nitrocellulose (.about.100 .mu.g/lane). Biotinylated HTG-VLDL S.sub.f 100-400 (0.5 .mu.g/ml) was preincubated with buffer (lane 1) or with 2 levels of anti-apoB (rabbit 1325) 40 .mu.g/mL lane 2; 400 .mu.g/ml lane 3; or two levels of preimmune (rabbit 1325) IgG 90 .mu.g/mL, lane 4 and 400 .mu./ml, lane 5. Lipoproteins and IgGs were preincubated for 30 minutes and then incubated with the nitrocellulose strips for 3 hours at 4.degree. C. After extensive washing, bound lipoprotein was detected with streptavidin linked to alkaline phosphatase followed by colorimetric substrates (the digitized image is shown in (FIG. 10A) and quantified by scanning densitometry (FIG. 10B) using two-dimension area integration and illustrated as VLDL binding in densitometric units (pixels).

FIG. 11 shows that anti-apoB, but not anti-apoE, anti-apoCIII, or nonimmune IgG, inhibits the binding of HTG-VLDL S.sub.f 100-400 to MBP200 or MBP235. THP-1 monocyte aqueous phase extracts were electrophoresed and transferred to nitrocellulose, blocked, and incubated with biotinylated HTG-VLDL and the indicated antibodies at 4.degree. C. Lane 1, buffer; lane 2, 3 mg/ml anti-apoB (1325); lane 3, 2.4 mg/ml nonimmune IgG; lane 4, 2.3 mg/ml anti-apoE; lane 5, 2 mg/ml anti-apoCIII. The IgGs were preincubated with biotinylated HTG-VLDL (2.5 .mu.g/ml) for 30 minutes at 4.degree. C. and then were incubated with the nitrocellulose strips for 3 hours at 4.degree. C. Lipoprotein binding was visualized (FIG. 11A) and quantified (FIG. 11B) by densitometry as described in FIG. 10.

FIG. 12 shows that anti-apoB IgG, but not nonimmune IgGs, inhibit the binding of .sup.125 I-HTG-VLDL to THP-1 macrophages (top panel) but not to human fibroblasts with upregulated LDL receptors. (bottom panel) THP-1 monocyte-macrophages one day after adherence were grown as described. Duplicate dishes of cells and no cells for controls were incubated with .sup.125 I-HTG-VLDL S.sub.f 100-400, 5 .mu.g/ml, alone (none) or in the presence of a 30-fold excess of unlabeled HTG-VLDL (self), in the presence of affinity-purified sheep anti-apoB IgG (anti-apoB), or the equivalent level of sheep nonimmune IgG (Non-Imm) at 4.degree. C. for 16 hours and then incubated with precooled, washed cells for 1.5 hours at 4.degree. C. After extensive washing, the cells were dissolved in 0.1 NaOH for determination of bound .sup.125 I-HTG-VLDL as described. Values represent the average of duplicate dishes corrected for background by subtracting the averages of the no-cell controls and are expressed in terms of percent of control, that is percent of the uninhibited activity (100%). Specific binding activity for .sup.125 I-HTG-VLDL, 5 .mu.g/ml was 24 ng/mg THP-1 cell protein which represented 100% uninhibited activity. (top panel) Human skin fibroblasts were grown to 75% confluency and preincubated with DME containing 5% lipoprotein-deficient serum for 36 h to induce the LDL receptor. The binding and competition protocols were identical to those for the THP-1 cells as described above. Specific binding activity for .sup.125 I-HTG-VLDL, 5 .mu.g/ml was 237 ng/mg fibroblast cell protein which represented 100% uninhibited activity.

FIG. 13 shows the effects of lactoferrin and heparin on binding of HTG-VLDL to MBP200 and MBP235 (these agents fail to inhibit or enhance binding). THP-1 monocyte aqueous phase extracts were electrophoresed and transferred to nitrocellulose. The nitrocellulose strips were incubated for 4 hours at 4.degree. C. with 0.5 .mu.g biotinylated HTG-VLDL/ml in the absence (lane 1) or in the presence of lactoferrin at 50 .mu.g protein/ml (lane 2) or 500 .mu.g protein/ml (lane 3); heparin at 10 U/ml (lane 4) and 100 U/ml (lane 5); or unlabeled HTG-VLDL at 25 .mu.g/ml (lane 6) or 5 .mu.g/ml (lane 7). Biotinylated HTG-VLDL binding was detected with streptavidin linked alkaline phosphatase (digitized image, (FIG. 13A) and quantified by densitometry (FIG. 13B).

FIG. 14 shows that lipoprotein lipase inhibits the binding of HTG-VLDL to MBP200 and MBP235. THP-1 monocyte aqueous phase extracts were electrophoresed and transferred to nitrocellulose, blocked, and incubated at 4.degree. C. with biotinylated HTG-VLDL S.sub.f 100-400 (3 .mu.g of protein/ml) in the absence (lane 1) or in the presence of lipoprotein lipase (0.2 .mu.g/ml, lane 2; 2.0 .mu.g/ml, lane 3; 20 .mu.g/ml, lane 4) or in the presence of bovine serum albumin (0.2 .mu.g/ml, lane 5; 2.0 .mu.g/ml, lane 6; 20 .mu.g/ml, lane 7). Binding was detected by incubation with streptavidin-alkaline phosphatase and the image digitized (FIG. 14A) and quantified (FIG. 14B) by densitometry.

FIG. 15 shows immunoblots that demonstrate that plasma chylomicrons of S.sub.f >1100 contain apoB-48 but not apoB-100. Plasma was isolated from a hypertriglyceridemic subject 4 hours after a standardized test fat meal. Total chylomicrons S.sub.f >400 were subfractionated through a salt gradient into 3 subclasses: S.sub.f >3200 (CM I), S.sub.f 1100-3200 (CM II), S.sub.f 400-1100 (CM III). These were electrophoresed at two levels of each (1 and 2 .mu.g total apoprotein/lane) on a 4-20% SDS-PAGE, transferred to nitrocellulose, and probed for apoB (above the line) and apoE (below the line). CM I, lanes 1 and 2; CM II, lanes 3 and 4; and CM III, lanes 5 and 6; and control hypertriglyceridemic VLDL S.sub.f 100-400, containing apoB-100 and apoB-48 and apoE, in lane 7; lane M=prestained protein molecular weight markers.

FIG. 16 shows the binding of chylomicron subspecies to MBP200 and MBP235 (odd lanes) or to the partially purified LDL receptor (even lanes). THP-1 monocyte aqueous phase extracts and the partially purified bovine LDL receptor were electrophoresed in alternating lanes, transferred to nitrocellulose, blocked, and incubated with chylomicron subfractions (10 .mu.g/ml). CM I (S.sub.f >3200) were incubated with lanes 1, 2; CM II (S.sub.f 1100-3200) with lanes 3, 4; and CM III (S.sub.f 400-1100) with lanes 5, 6. Binding was detected with a polyclonal anti-apoB antibody followed by a second antibody linked to alkaline phosphatase. The sharp band in lanes with THP-1 extracts that migrates between MBP200 and the LDL receptor is a nonspecific lipoprotein binding protein apparent in some but not all ligand blots.

FIG. 17 shows that anti-apoB antibodies specifically block the binding of chylomicrons S.sub.f 1100-3200 (CM II) that contain apoB-48 as the only apoB species to MBP200 and MBP235. Thus, this receptor is also referred to as the apoB48 receptor. THP-1 aqueous phase extracts were electrophoresed and transferred to nitrocellulose. Prior to incubation with the nitrocellulose strips for 3 hours at 4.degree. C., biotinylated CM II were preincubated at 4.degree. C. for 30 minutes with buffer (lane 1), with anti-apoB (rabbit 1325) (lane 2), or with an equivalent level of nonimmune IgG (lane 3). Chylomicron binding was visualized after incubation with streptavidin-linked alkaline phosphatase, digitized (FIG. 17A) and quantified (FIG. 17B) by scanning densitometry.

FIG. 18 shows the expression of the 3.8 kb TGRLP/ApoB Receptor mRNA in human placenta and THP-1 monocytes-macrophages.

FIG. 19 shows the expression of TGRLP/ApoB Receptor mRNA in human immune tissues, including spleen, lymph node, thymus, appendix, blood leukocytes and bone marrow.

FIG. 20 shows the relative positions of the overlapping clones used to characterize the full-length human monocyte TGRLP/ApoB Receptor cDNA.

FIGS. 21A-21E show the nucleotide sequence and derived amino acid sequence of the THP-1 monocyte apoB48 receptor cDNA. Nucleotides are numbered at the right on top with every 10 bp indicated by a "V". The amino acid residues are also numbered at the right with 20 residues per line. The ATG start site codon is preceded by 10 bp; a Kozak start sequence is at bp 5-14. The entire cDNA sequence is 3744 bp. The derived protein length is 1088 residues, with the TGA stop codon at bp 3275. The 10-residue unambiguous amino acid sequence (amino acids 910-919) determined from microsequence analysis of the tryptic hydrolysis of purified apoB48 receptor and used to design the degenerate oligonucleotide primers is indicated with a double underline, as are two other tryptic peptides likewise identified. The single underlined 23 amino acid segment (amino acids 751-773) is the putative transmembrane domain. Cysteine residues are bold and underlined. (C); potential coiled-coil interactive domains are bold and italicized (amino acid 155-189; amino acid 473-486; and amino acid 520-533). There are two potential polyadenylation signals beginning at bp 3700 and bp 3708. Both strands of the cDNA were sequenced; additional sequencing runs were carried out in some cases in regions of high CG content.

FIG. 22 shows the localization of the apoB48 receptor gene to chromosome 16p11 by fluorescence in situ hybridization (FISH). Human apoB48R cDNA (1-2614) was labeled with Cy3-dUTP by nick translation and hybridized to normal human metaphase and interphase nuclei from phytohaemagglutinin-stimulated blood lymphocytes. Chromosomes were counterstained with DAPI and the fluorescence images captured with digitized image microscopy. DAPI-stained chromosomes (blue) with Cy3-labeled hybridization signal (red) are paired with the black and white images of the DAPI-stained chromosomes pair for chromosome identification and for hybridization location.

FIG. 23 shows PCR screening of a multiple human tissue cDNA panel. A multiple tissue cDNA panel (MTC.TM., Clontech) containing normalized, first strand cDNA from (in order from left to right) brain, heart, kidney, liver, lung, pancreas, placenta, skeletal muscle, positive control (for combined tissues), no DNA control, and GPDH control were PCR-amplified using apoB48 receptor sequence specific primers overlapping introns 2 and 3 (Table 2) to ensure that only mRNA species are reflected. The expected 455 bp product was found in all but skeletal muscle. The level of apoB48R cDNA appeared greatest in lung and placenta (seen after 25 cycles) and lowest in brain and heart (seen only after 35 cycles). Control PCR analyses of the individual tissues for the GPDH cDNA verified normalization of tissues and reflect relative mRNA abundance in that tissue.

FIG. 24 shows western and ligand blotting activity detected in apoB48R transfected CHO-K1s. A ligand blot (Gianturco, S. H., et al., 1998) is shown on the left and western blot on the right of detergent extracts of, from outer to inner lanes, THP-1 monocytes as a positive control, CHO-K1's transfected with the empty pcDNA expression vector, and CHO-K1's transfected with the apoB48 receptor minigene that contains the first intron. Tryp-VLDL S.sub.f 100-400 (Gianturco, S. H., et al., 1986) served as the ligand, detected with an anti-apoB antibody and alkaline phosphatase-linked IgG second antibody. The apoB48R protein was visualized using a polyclonal antibody prepared against an apoB48R-GST fusion protein containing amino acids 223 to 710. The THP-1 extracts illustrate the two reported forms of the receptor, the .about.200 kDa ligand binding species and a .about.235 kDa form that contains both the ligand binding .about.200 kD species and a noncovalently bound protein, possibly a chaperone (Ramprasad, M. P. et al., 1995). Neither ligand blotting activity nor receptor immunoreactivity is detected in the vector-transfected negative controls. In contrast, the apoB48R-transfected CHOs exhibit both ligand blotting activity and apoB48R immunoreactivity with identical electrophoretic mobilities. The apparent molecular weight of the transfected apoB48R is slightly less (.about.190 kD) than that of the .about.200 kDa activity in THP-1s. This is likely due to processing differences in CHO cells versus human monocyte-macrophages. Of note, however, the apparent molecular weight of the apoB48R in the CHOs is approximately twice that predicted by the cDNA.

FIGS. 25A and 25B show expression of the apoB48R: FIG. 25A shows TG accumulation in apoB48R-transfected CHO-K1 cells, but not in control cells, incubated with tryp-VLDL S.sub.f 100-400. G418-selected, stable transfected CHOs were grown in 6 well tissue culture dishes. Both apoB48R-transfected and vector with inverted insert-transfected cells were incubated with tryp-VLDL S.sub.f 100-400 (Gianturco, S. H., et al. 1986) at the concentrations indicated for 4 hrs at 37.degree. C. and the cells processed to measure TG mass as previously reported (Gianturco, S. H., et al., 1988). The upper curve (closed squares; apoB48R transfected) indicates a rapid, curvilinear accumulation of TG with increasing levels of tryp-VLDL S.sub.f 100-400 reflective of the receptor-mediated uptake as seen in monocyte-macrophages (Gianturco, S. H., et al., 1994C), while the lower curve (open squares; pcDNA 3.1 vector plus inverted insert transfected) demonstrates a low affinity, linear accumulation, as seen in cells where no apoB48R activity is present, representing low affinity, nonspecific uptake. Values are averages from duplicate dishes that differ by <10%; This is one representative experiment of four experiments with the full length cDNA and four with the cDNA containing the first intron. FIG. 25B shows transfection of the apoB48R and its role in uptake of TGRLP and cellular TG accumulation. ApoB48R was transfected into CHO K1 cells and selected with G418 as described in FIG. 25A. The transfected cells were placed in 6-well plates and tested for their ability to accumulate TG when exposed to different TGRLP as a measure of lipoprotein-specific receptor function, as previously reported (Gianturco, S. H, et al., 1994c). After a 2 hour incubation at 37.degree. C., the transfected cells were washed in cold PBS to remove excess TGRLP, the cellular lipid extracted with hexanes:isopropanol (3:2, v/v) and the TG level measured enzymatically. The bar graph indicates the amount of TG that accumulated per mg of cell protein relative to a buffer control (the buffer level was subtracted from the level for each indicated lipoprotein). This value was also corrected for nonspecific binding of TGRLP to the well by subtracting a no-cell control value at each level of TGRLP used. Like human THP-1 macrophages, the apoB48R-transfected cells accumulate TG when exposed to apoB48-only containing chylomicrons S.sub.f 1100-3200 (CMII) and to the model ligand, tryp-VLDL S.sub.f 100-400 (t-V1) but not to VLDL S.sub.f 100-400 from a subject with normal plasma TG levels (N-V1). The experiment was repeated twice with identical results. Concurrent experiments with receptor-negative, vector-only transfected CHO-K1s detected no significant TG accumulation under the same experimental conditions (data not shown).

FIGS. 26A and 26B show oil-red-O staining of neutral lipid, which was used to assess receptor activity in transfected cells. In FIG. 26A, ApoB48R-transfected CHO-K1 cells show massive accumulation of lipid after a three hour incubation at 37.degree. C. with chylomicrons S.sub.f >400 at 100 .mu.g TG/ml in RPMI medium with no other serum components, as demonstrated by the Oil Red O-positive cytoplasmic lipid droplets in the cells. These chylomicrons contain apoB48 as their only apoB species, consistent with the ligand specificity of this receptor. FIG. 26B demonstrates that vector only-transfected cells showed no appreciable Oil-red-O staining after incubation under identical conditions. Receptor- and vector-transfected cells treated with buffer also showed no lipid staining inclusions (not shown).

FIGS. 27A, 27B, 27C, and 27D show immunohistochemical demonstration of apoB48R in human carotid artery atherosclerotic lesions. Serial sections of carotid arteries obtained by endarterectomy were processed as described. Rabbit polyclonal antibodies produced against an apoB48R-domain specific (amino acid 223-710) GST-fusion protein were used to determine apB48R expression and location. All magnifications are 100.times., except for FIG. 27C, which is 80.times.. FIG. 27A shows a hematoxylin-stained section of an advanced lesion with the lumen oriented toward the top and a lipid core surrounded by foam cells seen to the right and center. FIG. 27B shows staining with a macrophage-specific monoclonal antibody (HAM56), which identifies the macrophage-derived foam cells on the edge of the acellular lipid core. FIG. 27C is a negative control, which used preimmune IgG from the same rabbits used to generate the anti-apoB48R-specific antibodies. In FIG. 27D, anti-apoB48R antibodies indicate apoB48R-expression by lesion macrophages and foam cells.

DETAILED DESCRIPTION OF THE INVENTION

Two major triglyceride-rich lipoprotein membrane binding activities with apparent molecular weights of approximately 200 and approximately 235 kDa (MBP200 and MBP235) were identified by ligand blotting analysis in both normal human blood-borne monocyte-macrophages and the long term human THP-1 and U937 monocytes and macrophages. MBP200 and MBP235 are cell surface proteins that share a common backbone (MBP200) containing the ligand binding domain. MBP235 is comprised of MBP200 plus one (or more) small subunit(s) of .about.35 kDa apparent total mass (as determined by mobilities on SDS-PAGE) that associate(s) noncovalently with MBP200, does not inhibit triglyceride-rich lipoprotein binding, and is immunochemically distinct from the receptor-associated protein (RAP), a 39 kDa protein that modulates ligand binding to low density lipoprotein receptor-related protein and other low density lipoprotein receptor family members (Strickland, 1990).

Amino acid sequence data obtained from tryptic peptides from purified MBP200R, the reduced ligand-binding subunit of the receptor, had no matches in gene and protein databases, indicating that MBP200 is a unique protein. The sequence data was used to produce antipeptide antibodies that were then found to bind solely to the MBP200, MBP235, and MBP200R receptors, thereby confirming that MBP200 is a unique protein. The receptor-specific antibodies also confirmed the structure, chemistry and relationships of MBPs obtained initially by biochemical and ligand blotting analyses.

The specific, apoE-independent binding of plasma chylomicrons and other apoB-containing lipoproteins to this monocyte-macrophage receptor, coupled with in vivo studies, suggests a role of this pathway in the nutrition of circulating monocytes and accessible macrophages, such as in bone marrow in the postprandial state. Moreover, anti-apoB blocking studies indicate this previously-undiscovered human monocyte-macrophage receptor binds triglyceride-rich lipoproteins via apoB. In addition, apoB-48 appears to be sufficient, and neither apoB-100 nor apoE is necessary for binding. This also suggests a new role for apoB-48; i.e., targeting plasma chylomicrons and their remnants to accessible monocytes and macrophages for uptake while still triglyceride-rich, before reaching the triglyceride-depleted, cholesteryl ester- and apoE-enriched remnant that is targeted by apoE to the liver for uptake by hepatic receptors which bind lipoproteins via apoE. A defect in this monocyte-macrophage receptor or in its ligands could result in aberrant triglyceride-rich lipoprotein metabolism (rerouting of chylomicrons for lipolysis and eventual liver uptake) that would lead to delayed chylomicron clearance and abnormal persistence of chylomicron remnants and/or small, dense low density lipoprotein. This condition has been termed Pattern B phenotype.

The Pattern B phenotype is expressed primarily in adults, is associated with increased risk of cardiovascular disease, and is inherited in an autosomal dominant or codominant manner with varying polygenic effects, including lipoprotein lipase deficiency, insulin resistance, apo CIII, and an as-yet, unidentified gene defect(s) (Krauss, 1994). Since candidate mechanisms for this phenotype include altered triglyceride-rich lipoprotein metabolism and clearance, the monocyte-macrophage receptor of the present invention is a candidate gene for one contributing cause of this pattern. The potential role of apo CIII in modulating the binding of triglyceride-rich lipoproteins to this receptor fits with the observation that CIII-enriched triglyceride-rich lipoproteins are found in Pattern B and that transgenic mice that overexpress CIII or CII are hypertriglyceridemic. The apoCs (especially CIII) could interfere with apoE-mediated uptake mechanisms and also mask the apoB domain(s) that bind to MBP200. Identification of genes related to this phenotype allows identification of subjects before the phenotype is expressed. As subjects with this phenotype are extremely responsive to changes in dietary fat (Krauss, 1994), early identification permits dietary intervention so as to delay atherogenic changes.

The experiments leading to the present invention addressed several interactions of triglyceride-rich lipoproteins with monocytes and macrophages and their relation to lipoprotein metabolism and foam cell formation. Such interactions include a) whether MBP200 and MBP235 are chylomicron/apoB-48 receptors; b) the molecular structure and function of MBP200; c) the structure and function of the small subunit(s) in MBP235 (such as chaperone(s)); d) whether MBP200 and MBP235 are restricted to monocytes, macrophages and endothelial cells; e) the receptor binding domains in apoB; and f) whether apoCIII and apoE modulate receptor binding.

The present invention provides a composition of matter comprising isolated DNA molecules encoding overlapping domains of the monocyte-macrophage and placental cell-surface binding protein and the full-length cDNA construct selected from the group consisting of: (a) a DNA molecule comprising a sequence SEQ ID No. 1 and which encodes the full-length cDNA of said monocyte-macrophage cell surface apoB48R binding protein (SEQ ID No. 2) or a portion of said monocyte-macrophage cell surface apoB48R binding protein domain of the sequence of the monocyte macrophage binding protein; 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 apoB48R binding protein (SEQ ID No. 2) or a portion of said monocyte-macrophage cell surface apoB48R binding protein; an isolated monocyte-macrophage cell surface apoB48R binding protein having the sequence SEQ ID No. 2; a method of cell-specific delivery of therapeutic compounds to human monocytes or macrophages, comprising the steps of: providing a peptide or antibody having the ability to bind to an isolated monocyte-macrophage cell surface apoB48R binding protein having the sequence SEQ ID No.2, or a portion of said sequence; and incorporating said peptide or antibody into liposomes containing said therapeutic compound or directly linking said peptide or antibody to therapeutic compound; and a method of inhibiting foam cell formation and increased monocyte adhesion to endothelial cells, comprising the step of treating a monocyte-macrophage with an agent which binds an isolated monocyte-macrophage cell surface apoB48R binding protein having the sequence SEQ ID No.2.

Further provided are methods of evaluating an individual at risk for cardiovascular disease using the compositions of matter provided herein for examining either the apoB receptor-ligand interaction, or the RNA and/or protein corresponding to apoB in an individual at risk as compared to a control individual, to determine the presence of any abnormalities in the apoB receptor of the individual at risk.

In accordance with the present invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, "Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and II (D. N. Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" [B. D. Hames & S. J. Higgins eds. (1985)]; "Transcription and Translation" [B. D. Hames & S. J. Higgins eds. (1984)]; "Animal Cell Culture" [R. I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984). Therefore, if appearing herein, the following terms shall have the definitions set out below.

A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control. An "origin of replication" refers to those DNA sequences that participate in DNA synthesis. An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "operably linked" and "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

In general, expression vectors containing promoter sequences which facilitate the efficient transcription and translation of the inserted DNA fragment are used in connection with the host. The expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells. The transformed hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth.

A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence. A "cDNA" is defined as copy-DNA or complementary-DNA, and is a product of a reverse transcription reaction from an mRNA transcript. An "exon" is an expressed sequence transcribed from the gene locus, whereas an "intron" is a non-expressed sequence that is from the gene locus.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell. A "cis-element" is a nucleotide sequence, also termed a "consensus sequence" or "motif", that interacts with other proteins which can upregulate or downregulate expression of a specific gene locus. A "signal sequence" can also be included with the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell and directs the polypeptide to the appropriate cellular location. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters often, but not always (especially in monocyte-macrophage specific promoters), contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.

The term "oligonucleotide" is defined as a molecule comprised of two or more deoxyribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide. The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a pu