Abstract
Functional recovery from central neurotrauma, such as spinal cord injury, is limited by myelin-associated inhibitory proteins. The most prominent example, Nogo-A, imposes an inhibitory cue for nerve fibre growth via two independent domains: Nogo-A-Δ20 (residues 544–725 of the rat Nogo-A sequence) and Nogo-66 (residues 1026–1091). Inhibitory signalling from these domains causes a collapse of the neuronal growth cone via individual receptor complexes, centred around sphingosine 1-phosphate receptor 2 (S1PR2) for Nogo-A-Δ20 and Nogo receptor 1 (NgR1) for Nogo-66. Whereas the helical conformation of Nogo-66 has been studied extensively, only little structural information is available for the Nogo-A-Δ20 region. We used nuclear magnetic resonance (NMR) spectroscopy to assess potential residual structural propensities of the intrinsically disordered Nogo-A-Δ20. Using triple resonance experiments, we were able to assign 94% of the non-proline backbone residues. While secondary structure analysis and relaxation measurements highlighted the intrinsically disordered character of Nogo-A-Δ20, three stretches comprising residues 561EAIQESL567, 639EAMNVALKALGT650, and 693SNYSEIAK700 form transient α-helical structures. Interestingly, 561EAIQESL567 is situated directly adjacent to one of the most conserved regions of Nogo-A-Δ20 that contains a binding motif for β1-integrin. Likewise, 639EAMNVALKALGT650 partially overlaps with the epitope recognized by 11C7, a Nogo-A-neutralizing antibody that promotes functional recovery from spinal cord injury. Diffusion measurements by pulse-field gradient NMR spectroscopy suggest concentration- and oxidation state-dependent dimerisation of Nogo-A-Δ20. Surprisingly, NMR and isothermal titration calorimetry (ITC) data could not validate previously shown binding of extracellular loops of S1PR2 to Nogo-A-Δ20.
Highlights
Neurons in the central nervous system (CNS) exhibit very limited capacity to regrow upon neurotrauma, preventing them from restoring disrupted networks after a spinal cord or brain injury
In accordance with the circular dichroism (CD) spectrum observed for Nogo-A-Δ20, a 2D-nuclear magnetic resonance (NMR) [15N,1H]HSQC spectrum confirmed the intrinsically disordered character of Nogo-A-Δ20, as deduced from the low chemical shift dispersion in the 1H dimension (Fig 2A)
We investigated Nogo-A-Δ20 using CD and NMR spectroscopy
Summary
Neurons in the central nervous system (CNS) exhibit very limited capacity to regrow upon neurotrauma, preventing them from restoring disrupted networks after a spinal cord or brain injury. This is contrary to the situation in the peripheral nervous system (PNS), where regrowth of nerve fibres can occur to a much higher extent [1, 2]. Neurons express distinct receptors for each of these inhibitory domains, i.e., sphingosine 1-phosphate receptor 2 (S1PR2) together with tetraspanin-3 for Nogo-A-Δ20 and Nogo receptor 1 (NgR1) in association with co-receptors p75, Troy and Lingo-1 for Nogo-66 [12,13,14,15,16,17,18]. S1PR2 and NgR receptor complexes both lead to an activation of RhoA in the neuronal cytoplasm, which in turn causes destabilisation of the actin cytoskeleton and collapse of the neuronal growth cone as well as a general downregulation of the neuronal growth machinery [10]
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