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. Localizat ion 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 fract ionated the MBPs into specific components or charge isomers. One of th ese 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 spe cific antibodies were used including antibodies raised against total M BP, 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 immunog old electron microscopy, while antibody against total MBP (consisting of all charge isomers C-1-->C-8) labelled both the major dense line an d the intraperiod line. When the anti-citrulline antibody was used wit h a 3 nm gold conjugated Fab fragments prepared from the secondary ant ibody, 66.5% of the gold particles were localized to the intraperiod l ine, while 11.2% of gold particles were localized to the major dense l ine. On the other hand, with the monoclonal anti-MBP antibodies reacti ve 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 li ne. Other supporting evidence includes increased labelling of myelin b y I-125 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 asymmetr ically localized to the intraperiod line. (C) 1993 Wiley-Liss, Inc.