STRUCTURAL REQUIREMENTS FOR ASSEMBLY OF DIMERIC IGA PROBED BY SITE-DIRECTED MUTAGENESIS OF J-CHAIN AND A CYSTEINE RESIDUE OF THE ALPHA-CHAIN CH2 DOMAIN

The structural features of J chain required for interaction with IgA i n IgA dimer assembly were investigated by coexpression of wild-type an d mutant forms of J chain with IgA1 in CHO cells. With wild-type J cha in, a mixture of J chain-containing dimers and monomers was secreted. Substitution of Cys(14) of J chain with Ser resulted in expression of only monomer IgA covalently associated with J chain. Similarly, mutati on of Cys(68) to Ser also resulted in expression predominantly of a mo nomer lgA-J chain species. These results suggest that Cys(14) and Cys( 68) play critical roles in formation of J chain-containing IgA dimers, with each forming a disulfide bridge to an IgA monomer. Substitution of Asn(48) 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 mutat ed Cys(311) on the C alpha 2 domain of the IgA heavy chain to Ser. Whe n coexpressed with wild-type J chain, this mutant was still capable of forming dimers, indicating that this residue was not involved in dime rization. Taken together, our results are consistent with an arrangeme nt in which IgA monomers are linked end-to-end with J chain interposed .