BiP and immunoglobulin light chain cooperate to control the folding of heavy chain and ensure the fidelity of immunoglobulin assembly

Abstract

The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP-heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.

Figure 1

Expression of the C_H1-deleted γ heavy chain. (A) COS cells transfected with cDNAs encoding WT γ or γΔC_H1 heavy chains were metabolically labeled for 2 h. Cell lysates (γ) and culture supernatants (γ_m) were prepared and reacted with a goat anti- human γ heavy chain-specific antiserum. Immune complexes were precipitated with protein A-Sepharose beads and analyzed by SDS-PAGE under reducing conditions. (B) COS cells were cotransfected with cDNA for γΔC_H1 heavy chains together with either WT or ATPase mutant (G37) hamster BiP. Cells were metabolically labeled for 45 min in the presence of DTT, washed, and chased in complete media devoid of DTT for 2 h. Cell lysates were prepared and immunoprecipitated with either anti-γ or anti-BiP antisera, and culture supernatants were reacted with anti-γ (γ_m). Immune complexes were reduced and analyzed by SDS-PAGE.

Figure 2

Construction and analysis of truncated γ heavy chain. Cells were cotransfected with cDNAs for the indicated genes and metabolically labeled for 3 h. Lysates were prepared and immunoprecipitated with the indicated antiserum. Culture supernatants (m) were collected and immunoprecipitated with the indicated antiserum (λ_m and HA_m). Isolated proteins were reduced and analyzed by SDS-PAGE.

Figure 3

Folding status of the C_H1 domain in unassembled truncated γ heavy chains. COS cells were transfected with cDNAs encoding the indicated genes. One dish expressing WT BiP and the truncated heavy chain was labeled in the presence of DTT to provide a marker for completely reduced heavy chain (lane 1). After 3 h of labeling, all dishes were washed twice and lysed in the presence of NEM and apyrase. The cell lysates (lanes 2, 3, 6, 7, and 8) and culture supernatant (lanes 4 and 5) were immunoprecipitated with the indicated antibodies or protein A-Sepharose alone (P.A.) and analyzed by SDS-PAGE under nonreducing conditions. The mobilities of the various folding intermediates are indicated.

Figure 4

LC folding requirements for H-L assembly and C_H1 domain folding. COS cells were transfected with cDNAs for the indicated genes. After 3 h of labeling, cell lysates were prepared in the presence of NEM and apyrase and then reacted with the indicated antibodies or protein A-Sepharose alone (P.A.). Immunoprecipitated proteins were analyzed by SDS-PAGE under nonreducing conditions. The mobilities of the various folding intermediates of both the heavy and light chain are shown to the right.

Figure 5

Evaluation of the ability of an ER-targeted C_L domain to bind to heavy chains. COS cells were cotransfected with the truncated heavy chain and either WT λ or the ER-targeted λ constant domain (er-Cλ). Cells were metabolically labeled for 3 h, and cell lysates were prepared and reacted with anti-λ, anti-HA, or protein A-Sepharose alone (PA). Culture supernatants were immunoprecipitated with anti-λ (λ_m). Precipitated proteins were analyzed by SDS-PAGE under reducing conditions.

Figure 6

ATP-mediated in vitro release of BiP from NS-1 LCs and truncated heavy chains and the effect on their folding. COS cells were cotransfected with cDNAs for WT BiP and either NS-1 LC or truncated heavy chain and metabolically labeled for 1 h. Cells were either lysed in the presence of apyrase and NEM or cell lysates were prepared without these agents and incubated with ATP. The resulting lysates were immunoprecipitated with the indicated immune reagents. A dish of COS cells that were triply transfected with WT λ, WT BiP, and the truncated heavy chain was labeled and immunoprecipitated with the anti-HA monoclonal antibody to serve as a control for the migration of partially and completely folded heavy chain (lanes 5 and 10). The samples were analyzed by SDS-PAGE under nonreducing conditions. The migration of BiP and each folding intermediate is indicated to the right.

Figure 7

Schematic of the intermediates in H₂L₂ Ig assembly and role of BiP in the process. Although BiP can associate transiently with other Ig heavy chain domains, the C_H1 domain is the primary site of BiP binding that is responsible for the retention of unassembled heavy chains. Unlike the other heavy chain domains, the C_H1 domain remains unfolded and unoxidized in the absence of LCs. Synthesis and binding of a folding competent LC to the heavy chain promotes BiP release by an undefined mechanism and allows the C_H1 domain to fold. The completely folded and assembled Ig molecule is now transport competent.