Lipid rafts hinder binding of antibodies to the extracellular segment of the membrane-anchor peptide of mIgA
| Autor: | Alfur F. Hung, Tse Wen Chang, Chien-Sheng Lu, Jiun-Bo Chen, Nien-Yi Chen, Hui-Ming Yu |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
medicine.drug_class
Blotting Western Molecular Sequence Data Immunology B-cell receptor Receptors Antigen B-Cell Enzyme-Linked Immunosorbent Assay Cell Separation Biology Monoclonal antibody Cell Line Membrane Microdomains Antigen medicine Humans Immunoprecipitation Amino Acid Sequence Molecular Biology Peptide sequence Lipid raft B-Lymphocytes Antibodies Monoclonal Flow Cytometry Molecular biology Recombinant Proteins Transmembrane protein Immunoglobulin A Immunoglobulin Isotypes Epitope mapping B cell receptor complex CD79 Antigens Epitope Mapping Protein Binding |
| Zdroj: | Molecular Immunology. 48:1975-1982 |
| ISSN: | 0161-5890 |
| DOI: | 10.1016/j.molimm.2011.06.219 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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