Structural Requirements for Assembly of Dimeric IgA Probed by Site-Directed Mutagenesis of J Chain and a Cysteine Residue of the α-Chain CH2 Domain

The structural features of J chain required for interaction with IgA in IgA dimer assembly were investigated by coexpression of wild-type and mutant forms of J chain with lgA1 in CHO cells. With wild-type J chain, a mixture of J chain-containing dimers and monomers was secreted. Substitution of Cys¹⁴ of J chain with Ser resulted in expression of only monomer IgA covalently associated with J chain. Similarly, mutation of Cys⁶⁸ to Ser also resulted in expression predominantly of a monomer IgA-J chain species. These results suggest that Cys¹⁴ and Cys⁶⁸ play critical roles in formation of J chain-containing IgA dimers, with each forming a disulfide bridge to an IgA monomer. Substitution of Asn⁴⁸ with Ala, to prevent attachment of N-linked carbohydrate to J chain, also resulted in markedly reduced dimer assembly, suggesting a requirement for the sugar moiety in J chain function. We also mutated Cys³¹¹ on the Cα2 domain of the IgA heavy chain to Ser. When coexpressed with wild-type J chain, this mutant was still capable of forming dimers, indicating that this residue was not involved in dimerization. Taken together, our results are consistent with an arrangement in which IgA monomers are linked end-to-end with J chain interposed.