Ganglioside inhibition of neurite outgrowth requires Nogo receptor function: identification of interaction sites and development of novel antagonists.
Gangliosides are key players in neuronal inhibition, with antibody-mediated clustering of gangliosides blocking neurite outgrowth in cultures and axonal regeneration post injury. In this study we show that the ganglioside GT1b can form a complex with the Nogo-66 receptor NgR1. The interaction is shown by analytical ultracentrifugation sedimentation and is mediated by the sialic acid moiety on GT1b, with mutations in FRG motifs on NgR1 attenuating the interaction. One FRG motif was developed into a cyclic peptide (N-AcCLQKFRGSSC-NH(2)) antagonist of GT1b, reversing the GT1b antibody inhibition of cerebellar granule cell neurite outgrowth. Interestingly, the peptide also antagonizes neurite outgrowth inhibition mediated by soluble forms of the myelin-associated glycoprotein (MAG). Structure function analysis of the peptide point to the conserved FRG triplet being the minimal functional motif, and mutations within this motif inhibit NgR1 binding to both GT1b and MAG. Finally, using gene ablation, we show that the cerebellar neuron response to GT1b antibodies and soluble MAG is indeed dependent on NgR1 function. The results suggest that gangliosides inhibit neurite outgrowth by interacting with FRG motifs in the NgR1 and that this interaction can also facilitate the binding of MAG to the NgR1. Furthermore, the results point to a rational strategy for developing novel ganglioside antagonists.
Williams
Gareth
G
Wolfson Centre for Age-Related Diseases, King's College London, London SE1 1UL, United Kingdom.
Wood
Andrew
A
Williams
Emma-Jane
EJ
Gao
Ying
Y
Mercado
Mary L
ML
Katz
Alan
A
Joseph-McCarthy
Diane
D
Bates
Brian
B
Ling
Huai-Ping
HP
Aulabaugh
Ann
A
Zaccardi
Joe
J
Xie
Yuhong
Y
Pangalos
Menelas N
MN
Walsh
Frank S
FS
Doherty
Patrick
P
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D019556:COS Cells / Q000378:metabolism; D002522:Chlorocebus aethiops; D016000:Cluster Analysis; D005732:Gangliosides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D009185:Myelin Proteins / Q000378:metabolism*; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D016501:Neurites / Q000378:metabolism; D000070798:Nogo Proteins
D005732:Gangliosides; D009185:Myelin Proteins; D000070798:Nogo Proteins; C000603199:RTN4 protein, human; C000603201:Rtn4 protein, mouse; C016481:trisialoganglioside GT1; D019158:N-Acetylneuraminic Acid