THIOL-REDUCING AGENTS AND CALCIUM PERTURBANTS ALTER INTRACELLULAR SORTING OF IMMUNOGLOBULIN-M

The secretory form of IgM (sIgM) undergoes developmentally regulated i ntracellular sorting and transport, as demonstrated by the distinct as sembly patterns and intracellular fate exhibited by its mu heavy chain (mu s) In the 38C B lymphocytes, mu s-containing monomers are retaine d and degraded intracellularly, whereas in the 38C-derived D2 hybridom a, mu s-containing polymers are secreted. Here we show that sorting of sIgM is impaired in the presence of the thiol-reducing agent beta-mer captoethanol or when cellular calcium sequestration is perturbed eithe r with the Ca2+ ionophore A23187 or with thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase. Under these conditions, sIgM evade s retention/degradation in the 38C cells, resulting in its enhanced se cretion. Conversely, in the D2 cells, secretion is strongly attenuated , and sIgM is partly degraded. Both cell types secrete unusually proce ssed 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 mu s. Moreover, secretion is no longer restricted to fully assembled polymeric sIgM, and in both cell types under Ca2+ perturbation preferentially hemimeri c and monomeric assembly intermediates are detected in the medium. Our results suggest that the intracellular fate of sIgM is collectively d etermined by several sorting events that confer sIgM retention/degrada tion, ensure sIgM secretion from D2 cells, and prevent the arrival of assembly intermediates to the cell surface. All of these sorting event s appear to require high Ca2+ concentration and oxidizing thiol redox state, the conditions that prevail in the lumen of the endoplasmic ret iculum. Thus, thiol and calcium are implicated as modulators of intrac ellular traffic.