HIGH-RESOLUTION MAPPING OF GENS3 AND B11F7 EPITOPES ON MYELIN-ASSOCIATED GLYCOPROTEIN BY EXPRESSION PCR

The GenS3 and B11F7 monoclonal antibodies (MAbs) have been widely used for biochemical and immunocytochemical experiments on myelin-associat ed glycoprotein (MAG), a cell adhesion molecule mediating the interact ion between myelinating glia and axons. We have mapped the epitopes to within several amino acids on Ig domain 2 (D2) (amino acids 167-77) a nd domain 4 (D4) (amino acids 375-388) for GenS3 and B11F7, respective ly. Domain deletion and substitution mutants of the MAG cDNA were firs t used to map the epitopes to a given domain. In the cases of GenS3, i nsertion mutants were used to resolve the epitope to a small region of D2. For the B11F7 epitope, a novel technique combining PCR and in vit ro transcription and translation was used to generate smalt C-terminal deletions and map the epitope to 13 amino acids. Then, inhibition by peptides corresponding to the GenS3 (ELRPELSWLGHE; amino acids 167-177 )and B11F7 (QLELPAVTPEDDGE; amino acids 375-388) epitopes was used to confirm the position of the epitopes based on the mutant data. Interes tingly, the GenS3 epitope maps to a region predicted to be sequestered within the hydrophobic core of D2. This is consistent with the inabil ity of GenS3 to recognize the epitope in native MAG; GenS3 epitope rec ognition occurs only in denatured MAG, where the epitope is more acces sible. With the definition of the GenS3 and B11F7 epitopes, these anti bodies will be useful for further structure-function studies on MAG.