title: Lipid rafts hinder binding of antibodies to the extracellular segment of the membrane-anchor peptide of mIgA.
abstract: Membrane-bound IgA (mIgA) is associated with Igα/Igβ as the B cell receptor (BCR) complex on mIgA-expressing B cells. The α chain of mIgA (mα) contains a C-terminal membrane-anchor peptide, which encompasses extracellular, transmembrane and intracellular segments. The extracellular segment, referred to as the mIg isotype-specific (migis-α) segment or the extracellular membrane proximal domain of mα, has been proposed to be a specific antigenic site suitable for isotype-specific targeting of mIgA-expressing B cells by antibodies. In this study, we developed several anti-migis-α monoclonal antibodies (mAbs), such as mAb 29C11, specific to a segment towards the N-terminus of the 26 amino acid long migis-α. The mAbs bound strongly to synthetic peptides of migis-α and to various recombinant proteins containing migis-α as revealed by ELISA. On B cells, however, flow cytometric analysis suggested that these mAbs did not bind strongly to mIgA. After lipid rafts of B cells were disrupted by cholesterol extraction, the mAbs were able to bind strongly to the treated B cells. Moreover, immunoprecipitation analysis of these mAbs indicated that mIgA could only be pulled down by the mAbs when mIgA-expressing B cells were solubilized by strong detergents, such as sodium dodecyl sulfate (SDS), or when lipid rafts were disrupted. Together, these results suggest that the migis-α region of mIgA in the BCR is associated with lipid rafts, which hinder binding of migis-α-specific antibodies to mIgA on the cell surface. Further studies are in progress to evaluate the suitability of 29C11 or its affinity-improved variants for targeting mIgA-expressing B cells.
authors: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.
authors: Alfur Fu-Hsin
authors: AF
authors: Hung
authors: Jiun-Bo
authors: JB
authors: Chen
authors: Chien-Sheng
authors: CS
authors: Lu
authors: Nien-Yi
authors: NY
authors: Chen
authors: Hui-Ming
authors: HM
authors: Yu
authors: Tse Wen
authors: TW
authors: Chang
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D051925:CD79 Antigens / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D002469:Cell Separation; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D005434:Flow Cytometry; D006801:Humans; D007070:Immunoglobulin A / Q000276:immunology* / Q000378:metabolism; D007132:Immunoglobulin Isotypes / Q000276:immunology / Q000378:metabolism; D047468:Immunoprecipitation; D021962:Membrane Microdomains / Q000276:immunology* / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011947:Receptors, Antigen, B-Cell / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D051925:CD79 Antigens; C497881:CD79A protein, human; C497883:CD79B protein, human; D007070:Immunoglobulin A; D007132:Immunoglobulin Isotypes; D011947:Receptors, Antigen, B-Cell; D011994:Recombinant Proteins