The lipid raft hinders antibodies from binding to the external segment of the membrane-anchor peptide of mIgA (52.19)

Abstract

Abstract The mIgA is associated with Igα/Igβ as the B cell receptor (BCR) complex on mIgA-expressing B cells. The α chain of mIgA (mα), comparing to the α chain of secreted IgA, contains an extra C-terminal membrane-anchor peptide, which encompasses an extracellular, a transmembrane, and an intracellular segments. The extracellular segment, referred to as mIg isotype-specific (migis-α) segment or extracellular membrane proximal domain (EMPD), has been proposed as a specific antigenic site for isotype-specific targeting of mIgA-expressing B cells by antibodies. In this study, we developed several anti-migis-α mAbs, such as MAb 29C11, which were specific to a peptide segment toward the N-terminal of migis-α. The mAbs bound strongly to synthetic peptides of migis-α and various recombinant proteins containing migis-α in ELISA. However, the mAbs did not bind to mIgA on B cell lines strongly in fluorescence flow cytometric analysis. After disrupting the lipid raft integrity of the B cells by a typical cholesterol extraction procedure, the mAbs could then bind to the treated B cells strongly. Immunopricipitation analysis with the mAbs indicated only when the B cell lines were solubilized by sufficiently strong detergents, such as sodium dodecyl sulfate (SDS), the mIgA could be pulled down by the mAbs. These results suggest that the migis-α region of mIgA in the BCR is associated with the lipid raft. Future studies will investigate inhibitory effects of mAb 29C11 on mIgA-expressing B cells.