Intermolecular disulfide bonding in IgM: effects of replacing cysteine residues in the mu heavy chain.
The conventional model of polymeric IgM depicts a unique structure in which the mu heavy chains and J chain are joined by well defined disulfide bonds involving cysteine residues at positions 337, 414 and 575 of the mu chain. To test this model, we have used site directed mutagenesis to produce IgM in which these cysteines have been replaced by serine. In each case the single mutants were able to assemble polymeric IgM, which was analyzed for its size, morphology, J chain content and activity in complement dependent cytolysis. Whereas normal polymeric IgM is composed predominantly of pentameric and hexameric molecules, the mutant IgM-Ser414 is covalently assembled as pentamers and smaller forms; IgM-Ser575 is assembled as covalent hexamers. IgM-Ser337 appears to include the same pentameric and hexameric forms as normal IgM except that, unlike normal polymeric IgM, most pentameric/hexameric IgM-Ser337 is not covalently assembled. J chain is present in polymeric IgM-Ser337 but absent in polymeric IgM-Ser414 and IgM-Ser575. IgM-Ser414 is defective in activating the classical pathway of complement dependent cytolysis. Our observations are consistent with models in which the covalent linkages between mu chains are mediated by disulfide bonded Cys337-Cys337, Cys414-Cys414 and Cys575-Cys575 but indicate that the arrangement of these Cys-Cys pairs in series and in parallel varies among and within IgM molecules.(ABSTRACT TRUNCATED AT 250 WORDS)
Davis
A C
AC
Department of Immunology, University of Toronto, Canada.
Roux
K H
KH
Pursey
J
J
Shulman
M J
MJ
D000818:Animals; D003545:Cysteine; D003602:Cytotoxicity, Immunologic; D004220:Disulfides; D007144:Immunoglobulin J-Chains / Q000032:analysis; D007075:Immunoglobulin M / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure; D007148:Immunoglobulin mu-Chains / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure; D046911:Macromolecular Substances; D051379:Mice; D008854:Microscopy, Electron; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering
D004220:Disulfides; D007144:Immunoglobulin J-Chains; D007075:Immunoglobulin M; D007148:Immunoglobulin mu-Chains; D046911:Macromolecular Substances; D003545:Cysteine