Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-03-05
2003-06-17
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007940, C436S518000, C436S013000, C436S071000, C530S359000, C530S389300
Reexamination Certificate
active
06579682
ABSTRACT:
TECHNICAL FIELD
This invention relates to the disease atherosclerosis, methods of modulating the formation of atherosclerotic lesions, and methods of identifying compounds which modulate atherosclerotic lesion formation. Specifically the invention relates to the reduction of atherosclerosis through the modulation of LDL-proteoglycan binding at Site B (amino acids 3359-3369) of the apo-B100 protein in LDL.
BACKGROUND ART
High levels of LDL are a major risk factor for coronary disease and are the source for most of the cholesterol that accumulates in the arterial wall (Ross, R. 1995
. Annu. Rev. Physiol
. 57:791-804). Subendothelial retention of LDL has been suggested to be a key pathogenic process in atherosclerosis, and several lines of circumstantial evidence suggest that intramural retention of atherogenic lipoproteins involves the extracellular matrix, chiefly proteoglycans (Hurt-Camejo, E. et al. 1997
. Arterioscler Thromb Vasc Biol
. 17:1011-1017; Williams, K. J., and I. Tabas. 1995
. Arterioscier. Thromb. Vasc. Biol
. 15:551-561; and Radhakrishnamurthy, B. et al. 1990
. Eur. Heart J
. 11 Suppl E: 148-157).
The significance of the possible LDL proteoglycan interaction has been highlighted in two recent review articles (Hurt-Camejo, E. et al. 1997
. Arterioscler Thromb Vasc Biol
. 17:1011-1017; and Williams, K. J., and I. Tabas. 1995
. Arterioscier. Thromb. Vasc Biol
. 15:551-561). Williams and Tabas proposed that subendothelial retention of atherogenic lipoproteins is the central pathogenic process in atherosclerosis. Moreover, they hypothesized that retained lipoproteins can directly or indirectly provoke all known features of early lesions, such as lipoprotein oxidation, monocyte migration into the artery wall, macrophage foam cell formation, and cytokine production, and can accelerate further retention by stimulating local synthesis of proteoglycans. Several lines of evidence indicate that the retention of arterial lipoproteins involves the extracellular matrix; proteoglycans in particular have been hypothesized to play an important role (Hurt-Camejo, E. et al. 1997
. Arterioscler Thromb Vasc Biol
. 17:1011-1017; Williams, K. J., and I. Tabas. 1995
. Arterioscier. Thromb. Vasc Biol
. 15:551-561; Camejo, G. et al. 1988
. Arteriosclerosis
. 8:368-377; and Hurt, E., and G. Camejo. 1987
. Atherosclerosis
. 67:115-126). First, purified arterial proteoglycans, especially those from lesion-prone sites (Cardoso, L. E., and P. A. Mourao. 1994
. Arterioscler. Thromb
. 14:115-124; and Ismail, N. et al. 1994
. Atherosclerosis
. 105:79-87), bind atherogenic lipoproteins in vitro, particularly LDL from patients with coronary artery disease (Lindén, T. et al. 1989
. Eur. J Clin. Invest
. 19:38-44). LDL binds with high affinity to dermatan sulfate and chondroitin sulfate proteoglycans produced by proliferating smooth muscle cells (Camejo, G. et al. 1993
. J. Biol Chem
. 268:1413-1437). Second, proteoglycans are a major component of the artery wall extracellular matrix and are available to participate in the interactions of lipoproteins in the earliest stages of atherogenesis. Third, retained apo-B immunologically co-localizes with proteoglycans in early and developed lesions (Walton, K., and N. Williamson. 1968
. J. Atheroscler. Res
. 8:599-624; Hoff, H., and G. Bond. 1983
. Artery
. 12:104-116; Hoff, H. F., and W. D. Wagner. 1986
. Atherosclerosis
. 61:231-236; Nievelstein-Post, P. et al. 1994
. Arterioscler. Thromb
. 14:1151-1161; and Galis, Z. et al. 1993
. Am J. Pathol
. 142:1432-1438). The observation that the arterial wall content of these proteoglycans increases during atherosclerosis and correlates with an increased accumulation of aortic cholesterol also supports the potential importance of the interaction between LDL and proteoglycans (Hoff, H. F., and W. D. Wagner. 1986
. Atherosclerosis
. 61:231-236; Merrilees, M. et al. 1990
. Arteriosclerosis
. 81:245-254).
Proteoglycans contain long carbohydrate side-chains of glycosaminoglycans, which are covalently attached to a core protein by a glycosidic linkage. The glycosaminoglycans consist of repeating disaccharide units, all bearing negatively charged groups, usually sulfate or carbohydrate groups. In vitro, LDL bind with high affinity to many proteoglycans found in the artery wall, including dermatan sulfate proteoglycans (e.g., biglycan) and chondroitin sulfate proteoglycans (e.g., versican), which are produced by smooth muscle cells in response to PDGF or TGF&bgr; (Schonherr, E. et al. 1991
. J. Biol. Chem
. 266:17640-17647; and Schönherr, E. et al. 1993
. Arterioscler. Thromb
. 13:1026-1036). The interaction between LDL and proteoglycans have been hypothesized to involve clusters of basic amino acids in apo-B100, the protein moiety of LDL, that interact with the negatively charged glycosaminoglycan proteoglycans (Mahley, R. et al. 1979
. Biochem. Biophys. Acta
. 575:81-91; Carnejo, G. et al. 1988
. Arteriosclerosis
. 8:368-377; Weisgraber, K., and S. Rall, Jr. 1987
. J. Biol. Chem
. 262:11097-11103; and Hirose, N. et al. 1987
. Biochemistry
. 26:5505-5512) or by bridging molecules such as apo-E or lipoprotein lipase (Williams, K. J., and I. Tabas. 1995
. Arterioscier. Thromb. Vasc. Biol
. 15:551-561).
Isolation of large fragments of apo-B100 from different regions characterized by concentrations of positive clusters indicated that up to eight specific regions in apo-B100 bind proteoglycans (Camejo, G. et al. 1988
. Arteriosclerosis
. 8:368-377; Weisgraber, K., and S. Rall, Jr. 1987
. J. Biol. Chem
. 262:11097-11103; and Hirose, N. et al. 1987
. Biochemistry
. 26:5505-5512). Weisgraber, K., and S. Rall, Jr. 1987
. J. Biol. Chem
. 262:11097-11103 identified two fragments, residues 3134-3209 and 3356-3489, that bind to heparin with the highest affinity. Recently Camejo and coworkers confirmed this finding and proposed that residues 3147-3157 and 3359-3367 may act cooperatively in the association with proteoglycans (Hurt-Camejo, E. et al. 1997
. Arterioscler Thromb Vasc Biol
. 17:1011-1017; and Olsson, U. et al. 1997
. Arterioscler. Throm. Vasc. Biol
. 17:149-155). However, because these studies were carried out with delipidated apo-B fragments in the presence of urea or with short synthetic apo-B peptides, it is not clear which of the binding sites are functionally expressed on the surface of LDL particles. Some or many of these postulated glycosaminoglycan-binding sites may not be functional when apo-B is associated with LDL. For example, apo-E has two heparin-binding sites, but only one binds to heparin when apo-E is completed with phospholipid (Weisgraber, K. et al. 1986
. J. Biol Chen
261:2068-2076). This heparin-binding site coincides with the LDL receptor-binding site of apo-E.
Although eight potential glycosaminoglycan-binding sites have been identified in apo-B100 (Camejo, G. et al. 1988
. Arteriosclerosis
. 8:368-377; Weisgraber, K., and S. Rall, Jr. 1987
. J. Biol. Chem
. 262:11097-11103; and Hirose, N. et al. 1987
. Biochemistry
. 26:5505-5512), it was not known which of them participate in the physiological binding of LDL to proteoglycans. Previously, we have demonstrated, in conjunction with others, that Site B (residues 3359-3369) is the LDL receptor-binding site, and in the study which generated the present invention we found that it is also the primary binding site to proteoglycans.
Modification of LDL potentially exposes the other proteoglycan-binding sites. Paananen and Kovanen (Paananen, K., and P. T. Kovanene. 1994
. J. Biol. Chem
. 269:2023-2031) noted that proteolysis of apo-B100 strengthened the binding of LDL to proteoglycans, suggesting the exposure of buried heparin binding sites. Likewise, when LDL are fused by sphingomyelinase treatment, the modified lipoproteins bind more avidly to proteoglycans. The finding that multiple heparin molecules bind to LDL (Cardin, A. et al. 1987
. Biochemistry
. 26:5513-5518) may also be explained by a cooperative effect of heparin binding to one site that triggers a conformational change in apo-B100 that enables other sites to participate in the interaction. Thus,
Boren Jan
Innerarity Thomas
Le Long V.
Morrison & Foerster / LLP
The Regents of University of California
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