Secretion of immunoglobulin M assembly intermediates in the presence of reducing agents

Abstract

There are several demonstrations that misfolded or unassembled proteins are not transported along the secretory pathway, but are retained intracellularly, generally in the endoplasmic reticulum. For instance, B lymphocytes synthesize but do not secrete IgM, and only the polymeric form of IgM is secreted by plasma cells. The C-terminal cysteine of the mu heavy chain of secreted IgM (residue 575) is involved in the intracellular retention of unpolymerized IgM subunits. Here we report that the addition of reducing agents to the culture medium, at concentrations which do not affect cell viability, terminal glycosylation, or retention of proteins in the endoplasmic reticulum through the KDEL mechanism, induces secretion of IgM assembly intermediates by both B and plasma cells. Free joining (J) chains, which are not normally secreted by plasma cells unless as part of IgM or IgA, are also secreted in the presence of reducing agents. We propose a role for free thiol groups in preventing the unhindered transport of proteins through the secretory pathway. Under the scheme, assembly intermediates interact through their thiol groups between themselves and/or with unknown proteins of the endoplasmic reticulum. Such interactions may be prevented by altering the intracellular redox potential or by site-directed mutagenesis of the relevant cysteine residue(s).

Eeyarestatin I inhibits Sec61-mediated protein translocation at the endoplasmic reticulum

“…As expected, treatment of the cells with dithiothreitol (DTT) or brefeldin A (BFA) resulted in the complete loss or a substantial reduction of secretory glycoproteins from the media, respectively (Fig. 1B, lanes 1 and 2) (Alberini et al, 1990;Orci et al, 1991).…”

Recombinant human IgA expressed in insect cells.

“…Cells not expressing Ig heavy and light chains failed to secrete J chain (lane 6) except when treated with millimolar amounts of 2-mercaptoethanol (ref. 41 and data not shown); however, J chain did appear in the medium when expressed with IgA (lane 3). Insect cells triply infected with viruses expressing Ig light chain, heavy chain, and J chain were able to assemble dimeric IgA (Fig.…”

From antibodies to adiponectin: role of ERp44 in sizing and timing protein secretion

“…In the early 1990s, studies on IgM led to the identification of yet another QC mechanism, defined thiol-mediated retention (TMR [31,32] 6 'hexamers') are held together by disulphide bonds formed by the C-terminal cysteine (Cys575) in the conserved tailpiece of secretory μ chains (μ s tp). Cys575 acts as a three-way switch, mediating the assembly, retention and degradation of unpolymerized IgM subunits during B cell development [33].…”

“…Whether and how the still misfolded protein is passed to the BiP pathway or shunted to ERAD is not yet clear. Interestingly, the BiP-containing matrix includes UGGT, suggesting a way by which a protein can be passed to the CNX/CRT pathway [25].The choice between the BiP and the calnexin/calreticulin pathway depends on the location of glycans: if close to the N-terminus of the nascent protein, calnexin will have the right of way [30].In the early 1990s, studies on IgM led to the identification of yet another QC mechanism, defined thiol-mediated retention (TMR [31,32] 6 'hexamers') are held together by disulphide bonds formed by the C-terminal cysteine (Cys575) in the conserved tailpiece of secretory μ chains (μ s tp). Cys575 acts as a three-way switch, mediating the assembly, retention and degradation of unpolymerized IgM subunits during B cell development [33].…”