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NOGO-A polypeptide fragments, variant NOGO receptor-1 polypeptides, and uses thereof

A technology for connecting polypeptide fragments and polypeptide fragments, which is applied in the field of neurons and compositions used to mediate axon growth, and can solve problems such as undetermined molecular basis

Inactive Publication Date: 2007-09-12
YALE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the receptors for the Amino-Nogo domain and the molecular basis for the interaction of NgR with a variety of ligands have not been identified

Method used

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  • NOGO-A polypeptide fragments, variant NOGO receptor-1 polypeptides, and uses thereof
  • NOGO-A polypeptide fragments, variant NOGO receptor-1 polypeptides, and uses thereof
  • NOGO-A polypeptide fragments, variant NOGO receptor-1 polypeptides, and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0315] Amino Nogo fragment binds NgR

[0316] This example demonstrates that the carboxyl terminus of the Amino-Nogo domain interacts with NgR with high affinity. Several alkaline phosphatase (AP) fusion proteins containing different Nogo-A fragments (derived from the region between the amino terminus and the first hydrophobic fragment) were investigated to identify the Amino-Nogo-A mechanism of action. To generate additional AP fusion proteins, human Amino-Nogo fragments were amplified and ligated with restriction enzymes EcoRI and Xhol as described (Fournier, A.E., et al., Nature 409:341-346 (2001)). Digested pcAP6 vector. The plasmid was then transfected into HEK293T cells, and the conditioned medium was collected 7 days later. None of these fragments bound with high affinity to untransfected COS-7 cells. When examining hypothetical control conditions, we unexpectedly observed that the carboxyl moiety of Amino-Nogo (fragment B) exhibited high affinity bindi...

Embodiment 2

[0323] Inhibition of cell extension and axon outgrowth by Amino Nogo is independent of NgR binding

[0324] It has been recognized that the Amino-Nogo-A protein inhibits non-neuronal cell extension and neurite outgrowth when the protein is substrate bound (Chen, M.S., et al., Nature 403:434-439 (2000); Fournier, A.E. , et al., Nature 409:341-346 (2001)). Work by Oertle et al. showed that the specific aa stretches near the amino terminus and that the middle part of Amino-Nogo-A is responsible for this activity (Oertle, T., et al., J. Neurosci. 23:5393-5406 (2003b)). The latter domain has been named Δ20. To determine whether the NgR-interacting aa of Amino-Nogo-A described in Example 1 regulates cell extension and neurite growth, fragments were expressed as AP fusion proteins and purified from E. coli. To generate the GST fusion protein, the Amino Nogo fragment was cloned in pGEX2T (Amersham Pharmacia). Native and soluble GST fusion proteins were expressed and purified as des...

Embodiment 3

[0330] The carboxyl region of Amino-Nogo-A binds the LRR domain of NgR

[0331] Since Amino-Nogo binding to neuronal NgR does not inhibit neurite outgrowth, we sought to determine whether the specificity of Amino-Nogo for NgR is similar to that of the Nogo-66 domain. As for Nogo-66, MAG and OMgp (Barton, W.A., et al., Embo J.22:3291-3302 (2003); Fournier, A.E., et al., Nature 409:341-346 (2001); Wang, K.C., et al., Nature 417:941-944 (2002b)), deletion of any two LRRs abolishes binding of Amino-Nogo-B4C to NgR (Fig. 3A). Similarly, the cysteine-rich LRR-NT and LRR-CT capping domains are essential for Amino-Nogo-B4C binding. In contrast, deletion of the unique signaling domain of NgR extending from the LRR region to the GPI anchor site (CT domain) did not alter Amino-Nogo-B4C binding. NgR is part of a gene family that includes NgR2 and NgR3. These associated proteins do not bind AP-Nogo-66 or AP-MAG or AP-OMGp when expressed on the surface of COS-7 cells (Barton, W.A., et al...

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Abstract

Nogo, MAG, and OMgp are myelin-derived proteins that bind to a neuronal Nogo-66 Receptor (NgR) to limit axonal regeneration after CNS injury. Nogo-A protein may play the most prominent role in vivo, perhaps because its action is mediated both by NgR and by other receptors. Here, we extend our previous analysis of Nogo-A and NgR functional domains. In addition to a NgR-dependent Nogo-66 inhibitory domain and a NgR-independent Amino-Nogo-A specific domain, we identify a third Nogo-A specific domain that binds to NgR with nanomolar affinity. This third domain of 19 amino acids (aa) does not alter cell spreading or axonal outgrowth. Ala-scanning mutagenesis of surface residues in NgR partially distinguishes ligand binding sites for the two Nogo domains and for MAG, OMgp and Lingo-1. Fusion of the two NgR-binding Nogo-A domains creates a ligand with ten-fold enhanced affinity for NgR and converts a NgR antagonist peptide to an agonist. Thus, inhibition of axonal regeneration by NgR occurs after binding a subnanomolar bipartite Nogo-A ligand at a site partly overlapping with that for MAG and OMgp.

Description

technical field [0001] The present invention relates to neurobiology, neurology and pharmacology. More specifically, the invention relates to neurons and compositions and methods for mediating neurite outgrowth. Background technique [0002] In the adult mammalian brain and spinal cord, axonal connections are static. If the connection is interrupted by injury, there will be little or no axon regeneration. Outside neurons, astrocyte scarring and CNS myelin inhibit axon growth (Homer, P.J. and Gage, F.H., Nature 407:963-970 (2000); McGee, A.W. and Strittmatter, S.M., Trends Neurosci. 26: 193-198 (2003)). If the environment surrounding adult CNS axons is altered, axon growth may occur (Benfey, M. and Aguayo, A.J., Nature 296:150-152 (1982); David, S. and Aguayo, A.J., Science 214:931 -933 (1981); Richardson, P.M., et al., Nature 284:264-265 (1980)). Three proteins, Nogo, MAG and OMgp, capable of inhibiting axon growth in vitro, have been isolated from CNS myelin (McGee, A....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/00
CPCC07K2319/00A61K38/00C07K14/475A61P25/00A61P25/14A61P25/18A61P25/24A61P25/28
Inventor 斯蒂芬·M·斯特里特马特
Owner YALE UNIV