Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-12-10
2001-12-18
Clark, Deborah J. R. (Department: 1633)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007100, C435S007320, C435S007800, C435S029000, C435S253100
Reexamination Certificate
active
06331405
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the discovery that the &agr;-dystroglycan receptor is required for
Mycobaeferium leprae
entry into cells, assay for high throughput screening of drugs for use in treatments against leprosy, and methods for studying the role of the receptor in neurodegenerative and musculodegenerative diseases, and the like.
BACKGROUND OF THE INVENTION
M. leprae
Binding to Target Cells
Pathogenic bacteria are adapted to exploit a variety of host cell functions, and host cell receptors usually serve as the initial target for bacterial interaction with a specific cell type (Finlay and Cossart, Science 276:718, 1997; Falkow, Cell 65:1099, 1991). However, not much is known about the bacterial receptors in the nervous system and how bacteria interfere with these neuronal cell receptor-associated functions.
Mycobacterium leprae
, the causative organism of leprosy, is an intracellular pathogen which invades the Schwann cell of the peripheral nervous system (Falkow, supra). The neural tropism of this disease has been recognized for almost 150 years but has remained unexplained. At present, it is estimated that 2-3 million leprosy patients in the world are physically disabled as a result of damage to peripheral nerves and the attendant sensorimotor loss (Job, Int. J. Lpr. 57:532, 1989; Noordeen et al., Bull. World Health Org. 70:7, 1992).
During infection,
M. leprae
cause significant damage to peripheral nerves leaving patients with disabilities and deformities (Job, supra). Schwann cells are unable to destroy the pathogens that reside intracellularly, and access of therapeutic agents to this site is limited owing to the blood-nerve barrier. Although antibiotic therapy and long-term multi-drug treatment are effective bacteriological cures for leprosy, they do not reverse the nerve function loss in these patients (WHO Weekly Epidemiological Record. Sep. 20, p. 269, 1995). Understanding the mechanisms of
M. leprae
-Schwann cell interaction may yield new therapeutic strategies for the prevention of nerve damage.
In the endoneurium of peripheral nerves, all Schwann cells are characterized by a basal lamina covering. Since
M. leprae
must interact with the basal lamina in order to reach the Schwann cell, tropism to this site and perhaps cellular entry might involve the components of Schwann cell basal lamina. Schwann cell basal lamina is comprised of laminin, type IV collagen, entactin
idogen, and heparin sulfate proteoglycans (Combrooks et al., Proc. Natl. Acad. Sci. USA 80:3850, 1983; Jaakkola et al., J. Neurocytol. 22:215, 1989; Sanes et al., J. Cell Biol. 111:1685, 1990). Although there is evidence that
M. leprae
binds to fibronectin (Schorey et al., Infect. Immun. 63:2652, 1995), this binding may not be relevant for
M. leprae
interaction with Schwann cells in vivo, since both fibronectin mRNA and protein are absent in Schwann cell basal lamina in situ and in primary cultures (Combrooks et al., 1983, supra; Jaakkola et al., 1989, supra). Conversely, considering the continuous presence of laminin around the Schwann cell-axon unit in vivo, laminin appeared to be a candidate as an initial target for
M. leprae.
Laminins (LNs) are glycoproteins comprised of three polypeptide chains, &agr;, &bgr;, and &ggr;. The &agr; chain distinguishes itself by having an extra domain at the C-terminus, i.e., the G domain (Burgeson, et al., Matrix Biol. 14:209, 1994; Timpl and Brown, Matrix Biol. 14:275, 1994). At least 10 genetically distinct LN chains have been identified (&agr;1, &agr;2, &agr;3, &agr;4, &agr;5, &bgr;1, &bgr;2, &bgr;3, &ggr;1, and &ggr;2), which assemble into 11 different LN isoforms (LN-1 to -11), each with restricted tissue distribution (reviewed in Timpl and Brown, supra Engvall and Wewer, J. Cell Biochem. 61:493, 1996). In the Schwann cell basal lamina, the predominant LN variant is LN-2 (merosin), which comprises tissue-specific &agr;2 heavy chain together with the &bgr;1 and &ggr;1 light chains (Leivo and Engvall, Proc. Natl. Acad. Sci. USA 85:1544, 1988; Engvall et al., Cell Regul. 1:731, 1990. LN-2 not only forms a major basement membrane network, but also enables a variety of functions of neural cells (Engvall et al., Exp. Cell Res. 198:115-123 1992; Yurchenco et al., J. Biol. Chem. 268:8356, 1992; Anton et al., Dev. Biol. 164:133, 1994). The major cell receptors for LN are the members of the integrin superfamily (Mercurio, Trends Cell Biol. 5:419, 1995). Several integrin receptors bind to LN, and &agr;
6
&bgr;
1
and &agr;
6
&bgr;
4
integrins, particularly the &bgr;
4
subunit, appear to be involved in the Schwann cell interaction with LN (Einheber et al., J. Cell Biol. 123:1223, 1993; Jaakkola et al., supra; Feltri et al., Development 120:1287, 1994; Niessen et al., Development 120:1287, 1994).
The neural tropism of
M. leprae
involves the bacterial binding to the G domain of the LN-&agr;2 chain, which serves as a bridge between
M. leprae
and the native LN receptors on Schwann cells (Rambukkana et al., Cell 88:811, 1997). The LN-&agr;2G domain serves as both the bacterial and human cell binding site, which is possibly mediated by the &bgr;
4
integrin subunit as a host-cell receptor for LN-&agr;2G-mediated
M. leprae
cell interaction. However, the actual host cell receptor had not been identified. In order to provide more effective treatments for leprosy, and to better understand this disease and other musculo- and neuro-degenerative diseases involving laminins, it is necessary to determine the host cell receptor.
Dystroglicans
Dystroglycan, a component of the dystrophin-glycoprotein complex, is a laminin receptor encoded by a single gene and cleaved into two proteins, peripheral membrane &agr;-dystroglycan and transmembrane &bgr;-dystroglycan, by post-translational processing (Ibraghimov-Beskrovnaya et al., Nature 355:696, 1992; Ervasti and Campbell, Cell 66:1121, 1991; Campbell, Cell 80:675, 1995). While &agr;-dystroglycan interacts with laminin-2 in the basal lamina of Schwann cells, &bgr;-dystroglycan appears to bind to dystrophin-containing cytoskeletal proteins in muscles and peripheral nerves (Ervasti and Campbell, J. Cell. Biol. 122:809, 1993: Suzuki et al., Eur. J. Biochem. 220:283, 1994; Yamada et al., J. Neurochem. 66:1518, 1996; Yang et al., J. Biol. Chem. 270:11711, 1995). Dystroglycan is involved in the formation of neuromuscular junctions in the morphogenesis early development and in the pathogenesis of muscular dystrophies (Gee et al., Cell 77:675, 1994; Durbeej et al., J. Cell. Biol. 130:79, 1995; Williamson et al., Hum. Mol. Genet. 6:831, 1997; Campbell, supra). Loss or a defect of laminin-2-&agr;-dystroglycan interaction causes certain types of muscular dystrophies and peripheral neuropathies (Sunada et al., J. Biol. Chem. 269:13729, 1994).
As noted above, in peripheral nerves, &agr;-dystroglycan appears to link extracellular laminin-2 to the intracellular cytoskeleton through &bgr;-dystroglycan and associated proteins. In addition to playing a structural role, this system also regulates host cell functions (Campbell, supra; Ervasti and Campbell, 1993, supra, Suzuki et al., supra; Yang et al., supra).
There is a need in the art to identify a cell receptor involved in
M. leprae
infectivity and invasion. There is a further need to identify a laminin-receptor that mediates
M. leprae
invasion. The present invention addresses this need by identifying the sought-after receptor. This discovery has unexpected and surprising implications, as elaborated below.
SUMMARY OF THE INVENTION
The present invention advantageously provides a method for identifying a compound that inhibits binding of
M. leprae
to an &agr;-dystroglycan polypeptide. The method comprises detection of inhibition of
M. leprae
to &agr;-dystroglycan in the presence of a test compound. A molecule comprising the laminin-2G-domain is present to mediate the binding.
In a further embodiment the invention provides a method of inhibiting binding of
M. leprae
to a cell. This method comprises administering an inhibitory amount of a compound tha
Campbell Kevin P.
Fischetti Vincent A.
Rambukkana Anura
Clark Deborah J. R.
Darby & Darby
Kerr Janet M
The Rockefeller University
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