Epitope mapping of the neuronal growth inhibitor Nogo-A for the Nogo receptor and the cognate monoclonal antibody IN-1 by means of the SPOT technique.
Nogo-A is a potent inhibitor of axonal outgrowth in the central nervous system of adult mammals, where it is expressed as a membrane protein on oligodendrocytes and in myelin. Here we describe an attempt to identify linear peptide epitopes in its sequence that are responsible for the interaction either with the Nogo receptor (NgR) or with the neutralizing monoclonal antibody IN-1. Analysis of an array of immobilized overlapping 15 mer peptides covering the entire amino acid sequence of human Nogo-A (1192 residues) revealed a single epitope with prominent binding activity both towards the recombinant NgR and the IN-1 F(ab) fragment. Further truncation and substitution analysis yielded the minimal epitope sequence 'IKxLRRL' (x not equal to P), which occurs within the so-called Nogo66 region (residues 1054-1120) of Nogo-A. The bacterially produced Nogo66 fragment exhibited binding activity both for the recombinant NgR and for the IN-1 F(ab) fragment on the Western blot as well as in ELISA. Unexpectedly, the synthetic epitope peptide and the recombinant Nogo66 showed cross-reactivity with the 8-18C5 F(ab) fragment, which is directed against myelin oligodendrocyte glycoprotein (MOG) as a structurally unrelated target. On the other hand, the recombinant N-terminal domain of Nogo-A (residues 334-966) was shown to specifically interact on the Western blot and in an ELISA with the IN-1 F(ab) fragment but not with the recombinant NgR, which is in agreement with previous results. Hence, our data suggest that there is a distinct binding site for the Nogo receptor in the Nogo66 region of Nogo-A, whereas its interaction with NgR is less specific than anticipated before. Although there probably exists a non-linear epitope for the neutralizing antibody IN-1 in the N-terminal region of Nogo-A, which is likely to be accessible from outside the cell, a previously postulated second binding site for NgR in this region (called Nogo-A-24) remains elusive.
Zander
Hilke
H
Lehrstuhl für Biologische Chemie, Technische Universität München, An der Saatzucht 5, 85350 Freising-Weihenstephan, Germany.
Reineke
Ulrich
U
Schneider-Mergener
Jens
J
Skerra
Arne
A
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D003001:Cloning, Molecular; D020650:Combinatorial Chemistry Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes; D005453:Fluorescence; D058851:GPI-Linked Proteins; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D009185:Myelin Proteins / Q000276:immunology* / Q000378:metabolism; D000070798:Nogo Proteins; D000070816:Nogo Receptor 1; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D010957:Plasmids; D015202:Protein Engineering; D011956:Receptors, Cell Surface / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D058851:GPI-Linked Proteins; D007140:Immunoglobulin Fab Fragments; D009185:Myelin Proteins; D000070798:Nogo Proteins; D000070816:Nogo Receptor 1; D010446:Peptide Fragments; C000603199:RTN4 protein, human; C486580:RTN4R protein, human; D011956:Receptors, Cell Surface