Thiol-reducing agents and calcium perturbants alter intracellular sorting of immunoglobulin M.
The secretory form of IgM (sIgM) undergoes developmentally regulated intracellular sorting and transport, as demonstrated by the distinct assembly patterns and intracellular fate exhibited by its mu heavy chain (microseconds). In the 38C B lymphocytes, microseconds-containing monomers are retained and degraded intracellularly, whereas in the 38C-derived D2 hybridoma, microseconds-containing polymers are secreted. Here we show that sorting of sIgM is impaired in the presence of the thiol-reducing agent beta-mercaptoethanol or when cellular calcium sequestration is perturbed either with the Ca2+ ionophore A23187 or with thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase. Under these conditions, sIgM evades retention/degradation in the 38C cells, resulting in its enhanced secretion. Conversely, in the D2 cells, secretion is strongly attenuated, and sIgM is partly degraded. Both cell types secrete unusually processed sIgM, which is completely resistant to endoglycosidase H, unlike the partially sensitive sIgM secreted in the usual manner, indicating terminal glycosylation of all carbohydrate moieties of microseconds. Moreover, secretion is no longer restricted to fully assembled polymeric sIgM, and in both cell types under Ca2+ perturbation preferentially hemimeric and monomeric assembly intermediates are detected in the medium. Our results suggest that the intracellular fate of sIgM is collectively determined by several sorting events that confer sIgM retention/degradation, ensure sIgM secretion from D2 cells, and prevent the arrival of assembly intermediates to the cell surface. All of these sorting events appear to require high Ca2+ concentration and oxidizing thiol redox state, the conditions that prevail in the lumen of the endoplasmic reticulum. Thus, thiol and calcium are implicated as modulators of intracellular traffic.
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel.
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Shachar
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Rabinovich
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Kerem
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Bar-Nun
D000818:Animals; D020126:Brefeldin A; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002451:Cell Compartmentation; D002460:Cell Line; D003517:Cyclopentanes / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007075:Immunoglobulin M / Q000737:chemistry / Q000378:metabolism*; D046911:Macromolecular Substances; D008623:Mercaptoethanol / Q000494:pharmacology; D051379:Mice; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000502:physiology*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D003517:Cyclopentanes; D007075:Immunoglobulin M; D046911:Macromolecular Substances; D011947:Receptors, Antigen, B-Cell; D013438:Sulfhydryl Compounds; D013729:Terpenes; D020126:Brefeldin A; D000001:Calcimycin; D008623:Mercaptoethanol; D019284:Thapsigargin; D002118:Calcium