The variable domain of nonassembled Ig light chains determines both their half-life and binding to the chaperone BiP

Abstract

Not much is known about the features that determine the biological stability of a molecule retained in the endoplasmic reticulum (ER). Ig light (L) chains that are not secreted in the absence of Ig heavy (H) chain expression bind to the ER chaperone BiP as partially folded molecules until they are degraded. Although all Ig L chains have the same three-dimensional structure when part of an antibody molecule, the degradation rate of unassembled Ig L chains is not identical. For instance, the two nonsecreted murine Ig L chains, kappaNS1 and lambdaFS62, are degraded with half-lives of approximately 1 and 4 hr, respectively, in the same NS1 myeloma cells. Furthermore, the BiP/lambdaFS62 Ig L chain complex appears to be more stable than the BiP/kappaNS1 complex. Here, we used the ability of single Ig domains to form an internal disulfide bond after folding as a measure of the folding state of kappaNS1 and lambdaFS62 Ig L chains. Both of these nonsecreted L chains lack the internal disulfide bond in the variable (V) domain, whereas the constant (C) domain was folded in that respect. In both cases the unfolded V domain provided the BiP binding site. The stability of BiP binding to these two nonsecreted proteins was quite different, and both the stability of the BiP:Ig L chain complex and the half-life of the Ig L chain could be transferred from one Ig L chain isotype to the other by swapping the V domains. Our data suggest that the physical stability of BiP association with an unfolded region of a given light chain determines the half-life of that light chain, indicating a direct link between chaperone interaction and delivery of partially folded substrates to the mammalian degradation machinery.

Figure 1

Identification of the disulfide bond-containing domain in unassembled κ_NS1 Ig L chains. COS-1 cells were transfected with the various κ_NS1 cDNAs in a transient expression system. Biosynthetically labeled material was immunoprecipitated by anti-κ-antibodies from the lysate prepared in the presence of NEM from cells that expressed the κ_NS1V domain mutant (lane 1), the κ_NS1 wt-Ig L chain (lane 2), the κ_NS1 C domain mutant (lane 3), or no Ig L chains (lane 4). The migration of the completely reduced (no S—S bond, red), partially oxidized (S—S bond only in the C domain, ox 1), and completely oxidized (S—S bond in both domains, ox2) forms of the Ig L chains is indicated. An additional migration form (arrow) was observed with the C domain mutant and most likely represents a molecule containing a disulfide bond in the V domain. Isolation of the polypeptide marked by an asterisk (∗) is most likely due to nonspecific interactions, as it is also seen in the material precipitated from nontransfected cells (lane 4). Various amounts of this protein coisolated with the different Ig L molecules (i.e., compare lanes 2 and 3). Note that lanes 3 and 4 are from a longer exposure of the autoradiograph, because smaller amounts of labeled material was reproducibly recovered with the C domain mutant.

Figure 2

The amount of BiP coimmunoprecipitated with Ig L chains depends on the V domains of the Ig L chains. The amount of immunoprecipitated Ig L chains corresponds to the equivalent of 2 × 10⁵ (lanes 1, 4, 7, and 10), 10⁵ (lanes 2, 5, 8, and 11), or 0.5 × 10⁵ (lanes 3, 6, 9, and 12) X63Ag8.653 cells. Immunoprecipitation as well as immunoblotting of the Ig L chains were performed by using antisera that recognize the C domain but not the V domain of the Ig L chains. BiP was detected with a mouse monoclonal IgG antibody against the C-terminal KDEL sequence. This antibody and the Ig L chains that bear the Cκ domain (VκCκ and VλCκ, respectively, lanes 1–6) were detected with an anti-mouse IgG (H+L) antiserum. The Cλ domain-bearing Ig L chains (VκCλ and VλCλ, respectively, lanes 7–12) were stained by anti-λ antibodies.

Figure 3

Half-life determination of wt and chimeric Ig L chains. Stably transfected X63Ag8.653 cells were starved for 2.5 hr in medium lacking methionine and cysteine and labeled with 100 μCi ³⁵S-Translable for 20 min when the cells expressed Ig L chains bearing the V domain of κ_NS1 (VκCκ and VκCλ, respectively) or 2.5 hr when the cells expressed Ig L chains bearing the V domain of λ_FS62 (VλCλ and VλCκ, respectively). Cells were replated in complete medium, chased for the indicated times, and then lysed. Ig L chains were immunoprecipitated by using antisera that recognize the C domain.