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.