Immunolocalization of 17 and 21.5 kDa MBP isoforms in compact myelin and radial component

Summary

Our previous biochemical analyses revealed that the levels of the minor MBP isoforms 21.5 and 17 kDa are elevated relative to the 14 and 18.5 kDa MBP isoforms in the fraction of isolated myelin of murine CNS that is enriched in interlamellar junctions (or radial component). To substantiate the localization of 21.5 and 17 kDa MBP in the myelin sheath, we used immunoelectron microscopy on thin-sections of mouse optic nerve. Two different polyclonal antibodies were used to distinguish 21.5 and 17 kDa MBP from 14 and 18.5 kDa MBP: Ab-MBP^21.5, which was raised against a synthetic peptide corresponding to the exon II amino acid sequence 61–83 of mouse 21.5 kDa MBP (LKQSRSPLPSHARSRPGLCHMYK), and Ab-MBP¹⁴, which is immunoreactive to all four isoforms of mouse MBP. Our SDS-PAGE/immunoblotting demonstrated that Ab-MBP^21.5, unlike Ab-MBP¹⁴, recognized only the 21.5 and 17 kDa MBP isoforms from isolated mouse CNS myelin. Immunolabelling of tissue sections indicated that Ab-MBP¹⁴ bound tenfold more to junction-free compact myelin than to radial component, whereas Ab-MBP^21.5 bound about equally to the two regions of the myelin sheath. In addition, within the junction-free compact myelin, both antibodies bound nearly three fold more to the major dense line than to the intraperiod line.