Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1998-06-25
2001-07-03
Ungar, Susan (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S388100
Reexamination Certificate
active
06255457
ABSTRACT:
BACKGROUND OF THE INVENTION
Cholesterol and glycolipids self-associate in lipid bilayers to form organized compositional microdomains (Thompson, T. E., et al.,
Annu. Rev. Biophys. Chem.
14:361 (1985)). Glycosyl-phosphatidylinositol (GPI)-anchored proteins and other lipid-linked proteins may preferentially partition into glycolipid microdomains that are resistant to nonionic detergent solubilization (Schroeder, R., et al.,
Proc. Natl. Acad. Sci. USA.
91:12130 (1994); Brown, D. A. and Rose, J. K.,
Cell
68:533 (1992); Letarte-Murhead, M., et al.,
Biochem. J.
143:51 (1974); Hoessli, D. and Runger-Brandle, E.,
Exp. Cell. Res.
166:239 (1985); Hooper, N. M. and Turner, A. J.,
Biochem. J.
250:865 (1968); Sargiacomo, M., et al.,
J. Cell. Biol.
122:789 (1993); Lisanti, M. P., et al.,
J. Cell. Biol.
123:595 (1993)). GPI-anchored proteins appear to be sorted into glycolipid, detergent-resistant “rafts” in the trans-Golgi network for polarized delivery to the cell surface by caveolin-rich smooth exocytotic carrier vesicles (Brown, D. A. and Rose, J. K.,
Cell
68:533 (1992); Sargiacomo, M., et al.,
J. Cell. Biol.
122:789 (1993); Lisanti, M. P., et al.,
J. Cell. Biol.
123:595 (1993); Brown, D., et al.,
Science
245:1499 (1989); Simons, K. and van Meer, G.,
Biochemistry
27:6197 (1988); Garcia, M., et al.,
J. Cell Sci.
104:1281 (1993); Kurzchalia, T. V., et al.,
J. Cell Biol.
118:1003 (1992); Dupree, P., et al.,
EMBO J.
12:1597 (1993); Hannan, L. A., et al.,
J. Cell. Biol.
120:353 (1993)). On the cell surface, they are thought to reside in smooth membrane invaginations known as caveolae (Rothberg, K. G., et al.,
J. Cell. Biol.
110:637 (1990); Ying, Y., et al.,
Cold Spring Harbor Symp. Quant. Biol.
57:593 (1992); Ryan, U. S., et al.,
J. Appl. Physiol.
53:914 (1982); Stahl, A. and Mueller, B. M.,
J. Cell Biol.
129:335 (1995)), which are apparently also rich in glycolipids, cholesterol, and caveolin (Kurzchalia, T. V., et al.,
J. Cell Biol.
118:1003 (1992); Dupree, P., et al.,
EMBO J.
12:1597 (1993); Parton, R. G.,
J. Histochem. Cytochem.
42:155 (1994); Rothberg, K. G., et al.,
Cell
68:673 (1992); Schnitzer, J. E., et al.,
Proc. Natl. Acad. Sci. USA
92:1759 (1995); Montessano, R., et al.,
Nature
296:651 (1982)). Antibody cross-linking of cell surface glycolipids (Thompson, T. E., et al.,
Annu. Rev. Biophys. Chem.
14:361 (1985)) and GPI-linked proteins (Mayor, S., et al.,
Science
264:1948 (1994)) can increase sequestration into clusters and induce cell activation (Thompson, T. E., et al.,
Annu. Rev. Biophys. Chem.
14:361 (1985); Thompson, L. F., et al.,
J. Immunol.
143:1815 (1969); Korty, P. E., et al.,
J. Immunol.
146:4092 (1991); Davies, L. S.,
J. Immunol.
141:2246 (1988)), apparently through lipid-anchored nonreceptor tyrosine kinases (NRTKs) (Stefanova, I., et al.,
Science
245:1016 (1991); Shenoy-Scaria, A. M., et al.,
J. Immunol.
149:3535 (1992); Thomas, P. M. and Samelson, L. E.,
J. Biol. Chem.
267:12317 (1992); Cinek, T. and Horejsi, V.,
J. Immunol.
149:2262 (1992)). Caveolae have been implicated not only in signaling but also in transport via endocytosis, transcytosis, and potocytosis (Montessano, R., et al.,
Nature
296:651 (1982); Schnitzer, J. E.,
Trends. Cardiovasc. Med.
3:124 (1993); Oh, P., et al.,
J. Cell Biol.
127:1217 (1994); Schnitzer and Oh, P.,
J. Biol. Chem.
269:6072 (1994); Millci, A. J., et al.,
J. Cell Biol.
105:2603 (1987); Anderson, R. G. W., et al.,
Science
265:410 (1992)). Low density, Triton-insoluble membranes are frequently equated with caveolae (Sargiacomo, M., et al.,
J. Cell. Biol.
122:789 (1993); Lisanti, M. P., et al.,
J. Cell. Biol.
123:595 (1993); Chang, W.-J., et al.,
J. Cell. Biol.
126:127 (1994); Lisanti, M. P., et al.,
J. Cell. Biol.
126:111 (1994)). The physiological functions of, and interrelations between, caveolae, detergent-resistant microdomains, and various lipid-anchored molecules remain undefined.
SUMMARY OF THE INVENTION
The present invention relates to methods of isolating and purifying microdomains or components of the cell surface or plasma membrane; the resulting purified microdomains and components (e.g., proteins, peptides, lipids, glycolipids); antibodies to the purified microdomains and components; and uses therefor. In one embodiment, the present invention relates to methods of purifying microdomains of plasma membranes, including caveolae, microdomains of GPI-anchored proteins (G-domains) and membrane fragments consisting essentially of caveolae and G domains; the resulting purified microdomains and uses therefor. In a second embodiment, the present invention relates to methods of purifying detergent-sensitive (detergent-soluble) microdomains and cytoskeletal components; the resulting purified microdomains and uses for these components.
Plasma membrane components purified by methods of the present invention are useful, directly or indirectly, in the transport of molecules, such as drugs, DNA molecules, or antibodies in various cells (e.g., epithelial, endothelial, fat cells). For example, such agents targeted to caveolae in endothelium will be transported by the caveolae into and/or across the endothelium, and, thus, are useful in breaking through a critical barrier which prevents entry of many molecules, including drugs, into most tissues from the circulating blood. Caveolae and other plasma membrane components identified as described herein can be used to identify mechanisms or routes by which molecules can be delivered into cells, particularly endothelial cells, through the action of caveolae, G domains and other plasma membrane domains and components. For example, molecules residing in caveolae can be targeted by antibodies or natural ligands to caveolar proteins or receptors such as the insulin receptor, thereby bringing agents conjugated to the antibody or ligand into and/or across the endothelium. Alternatively, purified caveolae can be modified to serve as drug delivery vehicles, such as by introducing into them an agent, such as a drug, including a peptide or small organic molecule; a gene encoding a therapeutic or diagnostic peptide/protein; or an antibody. The resulting modified purified caveolae can be introduced into an individual, in whom they act to deliver the agent.
In the present method, plasma membranes are purified from a cell type of interest, such as endothelial cells, using a method such as that described in U.S. Ser. No. 5,281,700 and Schnitzer, J. E. et al.,
Proc. Natl. Acad. Sci., USA,
92:1759-1763 (1995); the teachings of both of these documents are incorporated herein by reference. The purified plasma membranes are then subfractionated into specific components or microdomains. Plasma membrane microdomains include caveolae, microdomains of GPI-anchored proteins (G-domains) and plasma membrane microdomains consisting essentially of caveolae, G domains and caveolae associated with G domains. Other microdomains include detergent-soluble domains and domains comprising cytoskeletal components.
In the method of the present invention, a cell plasma membrane, such as an endothelial cell plasma membrane, is specifically marked by altering a physical characteristic, such as by increasing its density; the change in physical characteristic of the membrane, such as increased density, is used as the basis for separating the plasma membrane from the other cell components (i.e., purifying the plasma membranes). In one embodiment, the present method of purifying caveolae or G domains entails silica coating of plasma membranes and comprises two broad phases: purification of cell plasma membranes and purification of subfractions or microdomains of the plasma membranes.
The purified cell plasma membranes are subfractionated and the desired membrane component or microdomain is isolated from the appropriate subfraction, resulting in isolation of the purified plasma membrane microdomain or components. In this embodiment, the luminal endothelial cell plasma membranes (normally exposed to the circulating blood) are coated with a suspension of
Beth Israel Deaconess Medical Center
Hamilton Brook Smith & Reynolds P.C.
Ungar Susan
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