Biosynthesis of immunoglobulin A (IgA) and immunoglobulin M (IgM). Requirement for J chain and a disulphide-exchanging enzyme for polymerization.
Mouse myeloma cells secreting 19S IgM (immunoglobulin M) (MOPC 104E and TEPC 183) or monomer and polymer IgA (immunoglobulin A) (MOPC 315) were incubated with radioactive leucine and the intracellular and secreted immunoglobulins and immunoglobulin subunits were prepared by preparative sucrose-density-gradient centrifugation. Samples were reduced in the presence or absence of isolated J chain, passed over Sephadex G-25 and then incubated at 37 degrees C for 30min with or without a source of disulphide-interchange enzyme. The extent of reassembly of reduced subunits was then evaluated by electrophoresis in polyacrylamide gels. Provided that J chain and the disulphide-interchange enzyme were supplied, both IgM and IgA could be assembled from their respective subunits, obtained by reductive cleavage of polymeric forms. Under similar conditions, assembly of polymeric forms from intracellular or secreted 7S monomer subunits also occurred. Under these conditions polymerization was total, there being no residue of the monomeric form. Reassembly did not occur in the absence of either J chain or the enzyme. All of the J chain released from IgM by reductive cleavage was incorporated back into the reassembled polymer. The J chain is therefore likely to be an essential structural requirement for polymeric immunoglobulins. A variety of controls ruled out non-specific interactions, and further suggested that the amino acid sequence of polypeptide chains determines the specificity of polymerization. The fact that intracellular IgA and IgM monomer subunits known to be deficient in galactose and fucose can be completely polymerized suggests that the addition of carbohydrate does not control polymerization.
D000818:Animals; D002250:Carbon Radioisotopes; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D007070:Immunoglobulin A / Q000096:biosynthesis*; D007144:Immunoglobulin J-Chains / Q000378:metabolism*; D007075:Immunoglobulin M / Q000096:biosynthesis*; D066298:In Vitro Techniques; D007930:Leucine / Q000378:metabolism; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C; D009194:Myeloma Proteins / Q000378:metabolism; D009374:Neoplasms, Experimental / Q000378:metabolism; D014166:Transferases; D014316:Tritium
D002250:Carbon Radioisotopes; D004220:Disulfides; D007070:Immunoglobulin A; D007144:Immunoglobulin J-Chains; D007075:Immunoglobulin M; D046911:Macromolecular Substances; D009194:Myeloma Proteins; D014316:Tritium; D014166:Transferases; D007930:Leucine