Immunolocalization of proteolipid protein peptide 103-116 in myelin

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Abstract
Determination of the topographic orientation of proteolipid protein (PLP) within myelin is part of an overall understanding of the functions of PLP and the roles of its multiple domains in diseases that primarily affect central nervous system (CNS) myelin. As part of an analysis of PLP orientation, two mouse monoclonal antibodies (mAb) and a rabbit antiserum against a synthetic peptide corresponding to PLP residues 103–116 (YKTTICGKGLSATV) were tested for their reactivity on compact CNS myelin. By ELISA, the antibodies react with intact PLP and PLP residues 103–116, but not with other PLP peptides. Ultrathin cryosections of adult rat optic nerve were immunostained and antibody binding was localized using appropriate second antibodies coupled to 1 nm gold particles that were visualized by silver enhancement. Localization of the particles on the major or intermediate dense lines was determined by three in dependent observers. Using the PLP peptide mAb and the polyclonal antibody, we demonstrated that ≥71% of the particles were localized on the major dense line. At least 66% of particles directed against myelin basic protein, which is known to occur on the major dense line, were also found in that location. These semiquantitative morphologic observations suggest that PLP residues 103–116 occur on the cytoplasmic face of the myelin membrane.