Abstract
In humans, secretory IgA exists as two subclasses, IgA1 and IgA2, which contain distinct □1 and □2 heavy chains, respectively. Both subclasses also have membrane-bound forms (mIgA1 and mIgA2) containing the corresponding m□1 and m□2 heavy chains, which differ from □1 and □2 by an additional “membrane-anchor” peptide segment extending from the CH3 domain of □1 and □2. The membrane-anchor segment has three parts: an extracellular, a transmembrane, and an intracellular segment. The heavy chain m□1 exists in short and long isoforms, referred to as m□1S and m□1L, with the latter containing extra 6 amino acid residues, GSCSVA, at the N-terminus of the extracellular segment (residues 453-458). By studying the genomic and mRNA sequences of m□1 and m□2 from 30 individuals residing in Taiwan, we have found that, in addition to the known m□1 allele, referred to as m□1(456S), m□1 also has a previously unknown allele, referred to as m□1(456C) (GenBank accession no. EU431191). This newly identified allele is present in the donor population at a similar proportion to m□1(456S), and appears to exist only as the long isoform, i.e. m□1L, rather than the short isoform, m□1S. We also confirmed that m□2 exists only as the short isoform. Because the membrane-anchoring peptide segment which is only present on mIgA is unique in amino acid sequences we proposed that the extracellular segment, referred to as the mIg isotype-specific (migis-□) segment of m□ could be a specific antigenic site suitable for isotype-specific targeting of mIgA-expressing B cells by antibodies. We rationalized that the migis-□-specific antibodies could bind to, and in turn, regulate IgA-expressing B cells, by mechanisms such as inducing apoptosis or decreasing the production of IgA. In the study, we developed several anti-migis-□ monoclonal antibodies (mAbs), such as mAb 29C11, specific to a segment towards the N-terminus of the 26 amino acid long migis-□. The mAbs bound strongly to synthetic peptides of migis-□ and to various recombinant proteins containing migis-□ 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-□ region of mIgA in the BCR is associated with lipid rafts, which hinder binding of migis-□-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.