Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2000-06-28
2002-08-20
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S009000
Reexamination Certificate
active
06437215
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention is generally in the area of transgenic animal models of atherosclerosis and methods for screening for inhibitors acting via interaction with the SR-BI scavenger receptor.
The intercellular transport of lipids through the circulatory system requires the packaging of these hydrophobic molecules into water-soluble carriers, called lipoproteins, and the regulated targeting of these lipoproteins to appropriate tissues by receptor-mediated pathways. The most well characterized lipoprotein receptor is the LDL receptor, which binds to apolipoproteins B-100 (apoB-100) and E (apoE), which are constituents of low density lipoprotein (LQL), the principal cholesteryl-ester transporter in human plasma, very low- density lipoprotein (VLDL), a triglyceride-rich carrier synthesized by the liver, intermediate-density lipoprotein (IDL), and catabolized chylomicrons (dietary triglyceride-rich carriers).
All members of the LDL receptor gene family consist of the same basic structural motifs. Ligand-binding (complement-type) cysteine-rich repeats of approximately 40 amino acids are arranged in clusters (ligand-binding domains) that contain between two and eleven repeats. Ligand-binding domains are always followed by EGF-precursor homologous domains. In these domains, two EGF-like repeats are separated from a third EGF-repeat by a spacer region containing ihe YWTD motif. In LRP and gp330, EGF-precursor homologous domains are either followed by another ligand-binding domain or by a spacer region. The EGF-precursor homology domain, which precedes the plasma membrane, is separated from the single membrane-spanning segment either by an O-linked sugar domain (in the LDL receptor and VLDL receptor) or by one (in
C. elegans
and gp330) or six EGF-repeats (in LRP). The cytoplasmic tails contain between one and three “NPXY” internalization signals required for clustering of the receptors in coated pits. In a later compartment of the secretory pathway, LRP is cleaved within the eighth EGF-precursor homology domain. The two subunits LRP-515 and LRP-85 (indicated by the brackets) remain tightly and non-covalently associated. Only partial amino acid sequence of the vitellogenin receptor and of gp330 are available.
LDL receptors and most other mammalian cell-surface receptors that mediate binding and, in some cases, the endocytosis, adhesion, or signaling exhibit two common ligand-binding characteristics: high affinity and narrow specificity. However, two additional lipoprotein receptors have been identified which are characterized by high affinity and broad specificity: the macrophage scavenger receptors class A type I and type II.
Scavenger receptors mediate the endocytosis of chemically modified lipoproteins, such as acetylated LDL (AcLDL) and oxidized LDL (OxLDL), and have been implicated in the pathogenesis of atherosclerosis (Krieger and Herz, 1994
Annu. Rev. Biochem
. 63, 601-637; Brown and Goldstein, 1983
Annu. Rev. Biochem
. 52, 223-261; Steinberg et al., 1989
N. Engl. J. Med.
320, 915-924). Macrophage scavenger receptors exhibit complex binding properties, including inhibition by a wide variety of polyanions, such as maleylated BSA (M-BSA) and certain polynucleotides and polysaccharides, as well as unusual ligand-cross competition (Freeman et al., 1991
Proc. Natl. Acad Sci. U.S.A.
88, 4931-4935, Krieger and Herz, 1994). Several investigators have suggested that there may be at least three different classes of such receptors expressed on mammalian macrophages, including receptors which recognize either AcLDL or OxLDL, or both of these ligands (Sparrow et al., 1989.
J Biol. Chem
264, 2599-2604; Arai et al., 1989
Biochem. Biophys. Res. Commun
. 159, 1375-1382; Nagelkerke et al., 1983
J Biol. Chem
. 258, 12221-12227).
The first macrophage scavenger receptors to be purified and cloned were the mammalian class A type I and II receptors. These are trimeric integral membrane glycoproteins whose extracellular domains have been predicted to include a-helical coiled-coil, collagenous and globular structures (Kodama et al., 1990
Nature
343, 531-535; Rohrer et al., 1990
Nature
343, 570-572; Krieger and Herz, 1994). The collagenous domain, shared by the class A type I and type II receptors, apparently mediates the binding of polyanionic ligands (Acton et al., 1993
J Biol. Chem.
268, 3530-3537; Doi et al., 1993
J Biol. Chem.
268, 2126-2133). The class A type I and type II molecules, which are the products of alternative splicing of a single gene, are hereafter designated class A-scavenger receptors (SR-Al and SR-All). The class A receptors, which bind both AcLDL and OxLDL (Freeman et al., 1991), have been proposed to be involved in host defense and cell adhesion, as well as atherogenesis (Freeman et al., 1991; Krieger, 1992
Trends Biochem. Sci.
17, 141-146; Fraser et al., 1993
Nature
364, 343-346; Krieger and Herz, 1994).
Based on models of the predicted quaternary structures of the class A type I and type II macrophage scavenger receptors, both contain six domains, of which the first five are identical: the N-terminal cytoplasmic region, the transmembrane region, spacer, a-helical coil, and collagen-like domains. The C-terminal sixth domain of the type I receptor is composed of an eight-residue spacer followed by a 102-amino acid cysteine-rich domain (SRCR), while the sixth domain of the type II receptor is only a short oligopeptide.
Using a murine macrophage cDNA library and a COS cell expression cloning technique, Endemann, Stanton and colleagues, (Endemann, et al. 1993
J. Biol. Chem.
268, 11811-11816; Stanton, et al.
J. Biol. Chem.
267, 22446-22451), reported the cloning of cDNAs encoding two additional proteins that can bind OxLDL. The binding of OxLDL to these proteins was not inhibited by AcLDL. These proteins are FcgRII-B2 (an Fc receptor) (Stanton et al., 1992) and CD36 (Endemann et al., 1993). The significance of the binding of OxLDL to FcgRII-B2 in transfected COS cells is unclear because FcgRII-B2 in macrophages apparently does not contribute significantly to OxLDL binding (Stanton et al., 1992). However, CD36 may play a quantitatively significant role in OxLDL binding by macrophages (Endemann et al., 1993). In addition to binding oxidized LDL, CD36 binds thrombospondin (Asch et al., 1987
J Clin. Invest.
79, 1054-1061), collagen (Tandon et al., 1989
J Biol. Chem.
264, 7576-7583), long-chain fatty acids (Abumrad et al., 1993
J Biol. Chem.
268, 17665-17668) and
Plasmodium falciparum
infected erythrocytes (Oquendo et al., 1989
Cell
58, 95-101). CD36 is expressed in a variety of tissues, including adipose, and in macrophages, epithelial cells, monocytes, endothelial cells, platelets, and a wide variety of cultured lines (Abumrad et al., 1993; and see Greenwalt et al., 1992
Blood
80, 1105-1115 for review). Although the physiologic functions of CD36 are not known, it may serve as an adhesion molecule due to its collagen-binding properties. It is also been proposed to be a long-chain fatty acid transporter (Abumrad et al., 1993) and a signal transduction molecule (Ockenhouse et al., 1989
J Clin. Invest.
84,468-475; Huang et al., 1991
Proc. Natl. Acad. Sci. USA
88, 7844-7848), and may serve as a receptor on macrophages for senescent neutrophils (Savill et al., 1991
Chest
99, 7 (suppl)).
Modified lipoprotein scavenger receptor activity has also been observed in endothelial cells (Arai et al., 1989; Nagelkerke et al., 1983; Brown and Goldstein, 1983; Goldstein et al., 1979
Proc. Natl. Acad. Sci. U.S.A.
76, 333-337). At least some of the endothelial cell activity apparently is not mediated by the class A scavenger receptors (Bickel et al., 1992
J Clin. Invest.
90, 1450-1457; Arai et al., 1989; Nagelkerke et al., 1983; Via et al., 1992
The Faseb J.
6, A371), which are often expressed by macrophages (Naito et al., 1991
Am. J. Pathol.
139, 1411-1423; Krieger and Herz, 1994). In vivo and in vitro studies suggest that there may be scavenger receptor genes expressed in endothelial cells and macrophages which differ from b
Edelberg Jay M.
Krieger Monty
Trigatti Bernardo
Crouch Deborah
Holland & Knight LLP
Massachusetts Institute of Technology
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