Molecular basis of the interactions of the Nogo-66 receptor and its homolog NgR2 with myelin-associated glycoprotein: Development of NgR^OMNI-Fc, a novel antagonist of CNS myelin inhibition

Myelin-associated glycoprotein (MAG) is a sialic acid-binding Ig-family lectin that functions in neuronal growth inhibition and stabilization of axon-glia interactions. The ectodomain of MAG is comprised of five Ig-like domains and uses neuronal cell-type-specific mechanisms to signal growth inhibition. We show that the first three Ig-like domains of MAG bind with high affinity and in a sialic acid-dependent manner to the Nogo-66 receptor-1 (NgR1) and its homolog NgR2. Domains Ig3-Ig5 of MAG are sufficient to inhibit neurite outgrowth but fail to associate with NgR1 or NgR2. Nogo receptors are sialoglycoproteins comprised of 8.5 canonical leucine-rich repeats (LRR) flanked by LRR N-terminal (NT) and C-terminal (CT)-cap domains. The LRR cluster is connected through a stalk region to a membrane lipid anchor. The CT-cap domain and stalk region of NgR2, but not NgR1, are sufficient for MAG binding, and when expressed in neurons, exhibit constitutive growth inhibitory activity. The LRR cluster of NgR1 supports binding of Nogo-66, OMgp, and MAG. Deletion of disulfide loop Cys³⁰⁹-Cys³³⁶ of NgR1 selectively increases its affinity for Nogo-66 and OMgp. A chimeric Nogo receptor variant (NgR ^OMNI) in which Cys³⁰⁹-Cys³³⁶ is deleted and followed by a 13 aa MAG-binding motif of the NgR2 stalk, shows superior binding of OMgp, Nogo-66, and MAG compared with wild-type NgR1 or NgR2. Soluble NgR ^OMNI (NgR^OMNI-Fc) binds strongly to membrane-bound inhibitors and promotes neurite outgrowth on both MAG and CNS myelin substrates. Thus, NgR^OMNI-Fc may offer therapeutic opportunities following nervous system injury or disease where myelin inhibits neuronal regeneration.