Multispecific recognition of developmental neuronal antigens and neurite outgrowth inhibition by an anti-myelin proteolipid protein (PLP) peptide monoclonal antibody (mAb)

Abstract

Abstract CNS injury results in the generation of antibodies to self-antigens, including epitopes of PLP, the most abundant CNS myelin protein. We previously found that despite similar recognition of CNS vertebrate myelin, anti-PLP50–69 mAbs F4.4C2 (IgG1κ) and F3.6E9 (IgG2aκ) differ in their recognition of neurons (Greenfield et al, 2006). To identify the neuronal mAb targets and determine the potential pathobiological significance of this finding, we used rat hippocampal precursor cells (RHPC) and PC12 cells, a nerve growth factor (NGF)-responsive neuronal cell line that does not express PLP. Both mAbs bound RHPC and PC12 cells and inhibited their in vitro neurite outgrowth; they differed in recognition of extracted PC12 cell proteins on Western blot and in extent of their recognition of human CNS neurons of different ages and in adult neural stem cell niches (NSCN). Thus, epitope sequence alone did not predict mAb neuronal reactivity. We immunoprecipitated proteins from NGF-treated and -untreated PC12 cell extracts using F4.4C2- and F3.9E9-coated magnetic beads; SDS-PAGE-separated bands of eluates from the beads were cut from gels and analyzed by GeLC-MS/MS. mAb F4.4C2 consistently captured the proteolipid gene family M6 neuronal protein. M6b and PLP50–69 have 45% sequence identity. M6b is required for neurite outgrowth and axon guidance and is expressed in developing human CNS neurons and NSCN. F4.4C2 also immunoprecipitated EphA family proteins and other cell surface growth and differentiation molecules expressed in human NSCN. Thus, certain anti-PLP autoantibodies generated after myelin injury may inhibit neuronal repair in the developing and adult human CNS by multispecific binding to neuronal growth and differentiation molecules.