Intermolecular disulfide bonding in IgM: effects of replacing cysteine residues in the μ heavy chain

Abstract

The conventional model of polymeric IgM depicts a unique structure in which the μ heavy chains and J chain are joined by well defined disulfide bonds involving cysteine residues at positions 337, 414 and 575 of the μ 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 To test this model, we have used site directed mutagenesis to produce IgM in which these cysteines have been replaced by serine.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 actrivity 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 μ chains are mediated by disulfide bonded Cys337-Cys337, Cys414-Cys414 and Cys575-Cys575 but inciate that the arrangement of these Cys-Cys pairs in series and in parallel varies among and within IgM molecules. Our results suggest that (i) formation of Cys414-Cys414 rigidifies the IgM; (ii) this rigidity is necessary for activation of complement component C1; (iii) assembly of hexameric IgM is associated with formation of all possible Cys414-Cys414; (iv) pentameric IgM lacking J chain can be assembled if no Cys414-Cys414 forms; and (v) pentameric IgM containing J chain is assembled if some, but not all, Cys414-Cys414 form.