MAG does not Require NgR1, PirB or Sialic Acid Binding to Inhibit Neurite Outgrowth

Following injury, axons in the central nervous system (CNS) do not spontaneously regenerate, and this is due to several factors, one of which is the presence of myelin-associated inhibitors. There are three major myelin-associated inhibitors that have been identified, Nogo-66, myelin associated glycoprotein (MAG), and Oligodendrocyte myelin glycoprotein (OMgp). MAG is a member of immunoglobulin (IgG) super-family and contains 5 Ig-like domains in its extracellular domain. Like Nogo-66 and OMgp, MAG binds to a receptor complex consisting of NgR1- p75NTR-Lingo-1 to inhibit neurite outgrowth. MAG is also a sialic acid binding protein and specifically binds to gangliosides GT1b and GD1a. Recently, NgR2 was also shown to be a sialic acid--dependent binding receptor for MAG. Recently, paired immunoglobulin B (PirB) was also identified as a novel receptor for MAG, Nogo-66 and OMgp.;Previously, we showed that the sialic acid binding activity of MAG is not necessary for its inhibitory effects. We mapped the sialic acid binding site on MAG to Arg 118 in the first Ig-domain. When this site is mutated, sialic acid binding is lost but MAG, when expressed by CHO cells, still retains its ability to inhibit neurite outgrowth. Also, we showed that a soluble form of MAG consisting of the MAG extracellular domain fused to the Fc portion of human IgG (MAG-Fc), and a truncated soluble form of MAG consisting only of the first three Ig-like domains (MAG (d1-3)-Fc), both bind to neurons in a sialic acid--dependent manner; however, only MAG-Fc inhibits neurite outgrowth. In addition, MAG mutated at Arg118 (MAG (R118A)-Fc), does not bind to neurons and could not inhibit neurite outgrowth. Recently, we mapped the inhibition site on MAG to Ig-domain 5, which is distinct from the sialic acid binding site.;Others have reported that gangliosides are functional binding partners for MAG and are necessary for inhibition by MAG when expressed in immobilized membranes. They reported that neurons from mice deficient in the B1, 4-N-actylgalactosaminyltransferase (GalNAcT) gene, which lack all complex gangliosides including GT1b and GD1a, are not inhibited by MAG in immobilized membrane. Others have also shown that clustering gangliosides with antibodies in the absence of MAG is sufficient to inhibit neurite outgrowth via a mechanism engaging p75 NTR receptor.;Here we show that clustering MAG (d1-3)-Fc can inhibit neurite outgrowth in neurons from wild type mice but not from GalNAcT deficient mice. We also show that MAG can inhibit neurite outgrowth independent of NgR1, PirB, and sialic acid binding. We show that neurons from GalNAcT deficient mice are inhibited by MAG as effectively as neurons from wild type mice. Also, we show that neurons from NgR1 deficient mice are inhibited by full length MAG and mutated MAG (MAG R118A) that cannot bind sialic acid residues. In addition, in the presence of PirB antibodies, both MAG- and mutated MAG (R118A)-expressing CHO were able to inhibit neurite outgrowth of neurons from NgR1 deficient mice and wild type mice. Taking all these results together, MAG interacts with another as yet unknown receptor(s), in addition to NgR's, PirB and sialic acid to inhibit neurite outgrowth.