Biochemical and ultrastructural characterization of a novel cell structure associated with immunoglobulin secretion in B-lymphocytes.

Abstract

In the companion paper, it was established that a secretory form of immunoglobulin, sIg, is present at or near the cell surface. This unexpected occurrence of sIg was postulated to be due to the labelling of sIg which remains temporarily associated with the cell packaged in a vesicle which appears to bud from the plasma membrane at a single pole of the cell. The question that is addressed in this report is whether or not this polar accumulation of sIg represents a common pathway for the exit of this protein which is used by antibody-producing cells. This question is important since, in spite of the fact that the intracellular events associated with immunoglobulin synthesis (processing and movement between subcellular compartments) have been defined, very little data exists on how or where immunoglobulin finally leaves the plasma cell. This question was approached here by first demonstrating that the polar immunoglobulin secretory vesicles (ISV) are associated with several sIg-producing cells including other hybridomas, B-cell lines, and mitogen-activated spleen cells. The second approach was to characterize the ISV on the cell ultrastructurally and to establish that these vesicles are released from the cell carrying with them sIg. Isolated vesicles released from biosynthetically labeled Ig-producing cells were analyzed by SDS-PAGE in order to confirm the presence of sIg and to determine the number of other proteins associated with the ISV, their molecular weights, and the degree of disulfide crosslinking of the molecules comprising this structure. Finally, the kinetics of sIg release was established by a pulse chase protocol for biosynthetically labeled cells, and by monitoring the release of radioactive Ig from surface iodinated cells. As was predicted from our biochemical studies of the ISV, we observed a very slow phase of sIg release as well as a rapid release phase. Our studies have established that at least one of the pathways for the release of Ig from hybridomas, B-cell lines, and normal splenic B-cells is via a polar multivesiculated structure that we have termed ISV, and that the sIg can be released either as a free form of the protein or packaged within a satellite vesicle which may release the sIg later and perhaps at considerable distance from the cell that produced it.