Mechanism and subcellular localization of secretory IgM polymer assembly.
The predominant functions of secreted IgM, complement fixation and transcytosis across epithelial barriers via the poly(Ig) receptor, are properties of IgM polymers; thus, the mechanisms that regulate oligomerization are critical to immune function. We have developed methods to assess the mechanism and subcellular location of IgM polymer formation. Using denaturing agarose/SDS-polyacrylamide gel electrophoresis and sucrose gradient fractionation, we demonstrate that IgM polymers are assembled in a stepwise fashion in which primary intermediates, including heavy chain-light chain complexes, are sequentially incorporated. Assembly intermediates include both covalent and non-covalent components, suggesting that IgM subunit interactions precede covalent assembly. IgM polymers are assembled from primary intermediates containing immature N-linked oligosaccharides. Polymerization is insensitive to brefeldin A and occurs at temperatures that inhibit protein transport along the secretory pathway. Together, these data demonstrate that IgM polymerization occurs early in the secretory pathway, most likely in the endoplasmic reticulum. These results are consistent with a model in which secretory IgM represents an oligomeric protein that is retained in the endoplasmic reticulum and does not mature along the secretory pathway until complete assembly is achieved.
Brewer
J W
JW
Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710.
Randall
T D
TD
Parkhouse
R M
RM
Corley
R B
RB
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A; D002451:Cell Compartmentation; D003517:Cyclopentanes / Q000494:pharmacology; D004220:Disulfides; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006596:Hexosaminidases / Q000494:pharmacology; D006825:Hybridomas; D007071:Immunoglobulin A, Secretory / Q000737:chemistry / Q000378:metabolism*; D007144:Immunoglobulin J-Chains / Q000378:metabolism; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D051379:Mice; D011108:Polymers; D011499:Protein Processing, Post-Translational; D013696:Temperature
D003517:Cyclopentanes; D004220:Disulfides; D007071:Immunoglobulin A, Secretory; D007144:Immunoglobulin J-Chains; D046911:Macromolecular Substances; D011108:Polymers; D020126:Brefeldin A; D006596:Hexosaminidases