Localization of basic proteins in human myelin

The myelin basic protein (MBPs) represent a family of proteins (charge isomers) which account for 35% of the total myelin protein. Localization studies have been inconclusive because MBP is not a single protein. Antibodies obtained by injection of MBP into animals recognized all members of the MBP family. In the studies reported here, we have fractionated the MBPs into specific components or charge isomers. One of these which contains citrulline accounts for about 20% of the total MBP. We report the localization of this single MBP to the intraperiod line of myelin by immunoelectron microscopy. For these studies several specific antibodies were used including antibodies raised against total MBP, specific MBP peptides, and against a tetracitrulline peptide. This latter antibody was specific for component 8 (C-8) of MBP. Since C-8 is the only MBP which contains citrulline it was used to localize this particular form of MBP principally to the intraperiod line by immunogold electron microscopy, while antibody against total MBP (consisting of all charge isomers C-1-->C-8) labelled both the major dense line and the intraperiod line. When the anti-citrulline antibody was used with a 3 nm gold conjugated Fab fragments prepared from the secondary antibody, 66.5% of the gold particles were localized to the intraperiod line, while 11.2% of gold particles were localized to the major dense line. On the other hand, with the monoclonal anti-MBP antibodies reactive with residues 69-74, 59.4% of the gold particles were localized to the major dense line and 23.6% of gold particles at the intraperiod line. Other supporting evidence includes increased labelling of myelin by 125I labelled anti-citrulline IgG when isolated myelin was swollen, a process known to take place at the intraperiod line. Gold particles were demonstrated at the intraperiod line in swollen and recompacted myelin. C-8 was shown to associate preferentially with lipids asymmetrically localized to the intraperiod line.