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 in IgA dimer assembly were investigated by coexpression of wild-type and mutant forms of J chain with IgA1 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 C alpha2 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.
Krugmann
S
S
Department of Molecular and Cellular Pathology, University of Dundee, Ninewells Hospital and Medical School, United Kingdom.
Pleass
R J
RJ
Atkin
J D
JD
Woof
J M
JM
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003545:Cysteine / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D019281:Dimerization; D007070:Immunoglobulin A / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D007144:Immunoglobulin J-Chains / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D007137:Immunoglobulin alpha-Chains / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering
D007070:Immunoglobulin A; D007144:Immunoglobulin J-Chains; D007137:Immunoglobulin alpha-Chains; D003545:Cysteine