The Neurite Outgrowth Inhibitory Nogo-A-Δ20 Region Is an Intrinsically Disordered Segment Harbouring Three Stretches with Helical Propensity

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.

The Rise of Molecules Able To Regenerate the Central Nervous System

“…MAG, a transmembrane protein and member of the family of sialic acid-binding proteins (Siglec), is produced by oligodendrocytes. ,, NogoA was initially described as having neurite growth inhibitory activity, , and identified as a member of the Reticulon family almost 10 years later. − OMgp is a GPI-anchored glycoprotein expressed by astrocytes, oligodendrocytes, and neurons, and is a component of CNS myelin. − Finally, the fourth class of molecules involved in neurite/axonal outgrowth inhibition are the CSPGs, a class of proteoglycans that is abundant in the CNS. , In the normal, uninjured brain, the main CSPGs found are RPTP-beta, phosphacan, NG2, brevican, aggrecan, and neurocan. Some of them, e.g., RPTP-beta and brevican, are transmembrane proteoglycans, while others, such as phosphacan, are secreted and deposited in the extracellular matrix by oligodendrocytes, astrocytes, and neurons, and are overexpressed after CNS injury. , CSPGs have also been associated with the presence of reactive astrocytes in neurodegenerative diseases, including Alzheimer’s disease. , …”