Biochemical characterization of the recombinant human Nogo-A ectodomain.
Nogo-A is a physiologically relevant inhibitor of neuronal growth and regeneration in the myelin of the adult human central nervous system and has attracted considerable attention as a molecular target for the treatment of spinal cord injuries. To gain insight into the structural and functional properties of the large extramembrane region that is characteristic for the Nogo-A splice form of this member of the Reticulon family of membrane proteins, we cloned and expressed the region comprising residues 334-966 as a soluble homogeneous protein in the periplasm of Escherichia coli. SDS/PAGE, under nonreducing conditions, and a systematic substitution analysis of all six Cys residues of Nogo-A indicated that this domain forms two structural disulfide bonds among Cys residues 424, 464, 559 and 597, whereas the Cys residues at positions 699 and 912 seem to be dispensable for folding. The occurrence of a hot spot for host cell proteases and a limited proteolysis experiment suggest that the N-terminal region of Nogo-A up to residue 373 is structurally disordered. Although analytical gel permeation chromatography revealed a large apparent molecular size for the recombinant Nogo-A fragment, indicating oligomer formation, data from analytical ultracentrifugation and dynamic light scattering support a stable monomeric quaternary structure. Notably, the CD spectrum is indicative of a high content of random coil, such that Nogo-A exhibits--at least in part--features of a natively unfolded protein. Nevertheless, the protein fragment identified in this study, as well as its biochemical analysis, provide a promising starting point for future investigations to track down globular subdomains and functionally important regions as well as putative receptor-binding sites therein.
Zander
Hilke
H
Lehrstuhl für Biologische Chemie, Technische Universität München, Freising, Weihenstephan, Germany.
Hettich
Eva
E
Greiff
Kathrin
K
Chatwell
Lorenz
L
Skerra
Arne
A
D002942:Circular Dichroism; D003001:Cloning, Molecular; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D009185:Myelin Proteins / Q000737:chemistry*; D000070798:Nogo Proteins; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D004220:Disulfides; D009185:Myelin Proteins; D000070798:Nogo Proteins; C000603199:RTN4 protein, human; D011994:Recombinant Proteins