Background
The CH1 domain of IgG antibodies controls antibody assembly and secretion through the endoplasmic reticulum protein quality control (ERQC) mechanism, mediated by the molecular chaperone BiP. However, it is unclear whether this process requires the variable domain. On May 2, 2024, researchers from Ajou University in South Korea published a study titled " The dispensability of VH-VL pairing and the indispensability of VL domain integrity in the IgG1 secretion process " in Frontiers in Molecular Biosciences. Their study evaluated the expression, assembly, and secretion of IgG1 antibodies with VH/VL domains removed or replaced in HEK293 cells. They found that the V domain does not initiate HC-LC assembly and secretion; instead, the key factor controlling IgG assembly and secretion is CH-CL pairing. Furthermore, the integrity of the VL domain is also crucial for IgG secretion. These data provide valuable insights into the design of bioactive molecules based on the IgG backbone.
Overview of Antibody Assembly
Antibodies are composed of two heavy chains (HCs) and two light chains (LCs). Their correct folding and assembly are prerequisites for secretion from antibody-producing cells. The endoplasmic reticulum quality control (ERQC) mechanism in these cells ensures that only correctly assembled Ig molecules are secreted, thereby preventing the secretion of misfolded and incorrectly assembled immunoglobulin (Ig) proteins. The ERQC mechanism has been most extensively studied for the IgG isotype. Newly synthesized Ig chains first assemble in the ER as γ-H chain (γ-HC) dimers via interchain disulfide bonds. The cγ-1 domain of these HC dimers remains unfolded and retained in the ER by the molecular chaperone BiP until it covalently binds to the two folded LCs through interaction with the CL domain. The interaction of the folded CL domain with the Cγ1 domain releases BiP, inducing complete folding and oxidation of the Cγ1 domain, allowing the HC and LC to assemble into the IgG (H₂L₂) structure prior to secretion.
(Data source: Feige MJ, et al. Biochim Biophys Acta. 2014)
VH and VL pairing is dispensable for IgG assembly and secretion
The researchers used a single-vector strategy to generate domain-deleted variants co-expressing the HC and LC. Lysates and supernatants from transfected cells were subsequently analyzed under reducing and non-reducing denaturing conditions. Results showed that HC and LC expression levels were similar to those of wild-type IgG HC in the absence of either the VH, VL,or both domains. Analysis of supernatants revealed that Igs lacking either the VH, VL or both domains were secreted in a fully assembled form. This suggests that the VH and VL chains, or their paired form (VH/VL) , are not essential for IgG secretion. Instead, the Cγ1 and Cκ domains are crucial.
Abnormal ψVκ domains disrupt Ig secretion
The native Vκ domain was replaced with a pseudotyped Vκ（ψVκ）derived from the 2C281 hybridoma clone. This pseudotyped Vκ lacks the FR4 segment due to a frameshift mutation in CDR3. Given its location upstream of the Cκ domain, it is hypothesized to significantly influence Cκ structure. LCs carrying the ψVκ were designated Ψlc. Their secretion profiles were then investigated in the supernatants of four HEK293 cell transfections: (HC+LC), (Cγ1-3 chains+LC), (HC+Ψlc), and (Cγ1-3 chains+Ψlc). Co-expression of the HCs and Ψlc revealed that neither HCs nor Ψlc were secreted into the culture supernatant, either alone, as assembly intermediates, or in fully assembled form. This abnormality in the ψVκ, domain negatively alters the downstream Cκ structure, hindering BiP dissociation from the Cγ1 chain and, consequently, impairing proper Cγ1 chain folding, ultimately preventing the secretion of intact IgG antibodies.
Abnormal Vκ domains prevent LC from binding to HC
The researchers evaluated the expression and assembly of four HEK293 cell transfections: HC + LC, (Cγ1-3 chains+LC), (HC+Ψlc), and (Cγ1-3 chains+Ψlc). In cell lysates, the covalent assembly efficiency of HC and Ψlc was lower than that of wild-type IgG, as evidenced by a weakening of the 150 kDa IgG band and the appearance of multiple, fuzzy protein bands larger than IgG. Co-immunoprecipitation and ELISA analyses further confirmed that the binding efficiency of Ψlc to HC (or Cγ1-3 chains) was reduced by 52% and 40% compared to wild-type LC. The abnormal ψVκ domain reduced the binding efficiency between LC and HC, thereby impeding the normal assembly and secretion of intact IgG.
Ψlc interacts with BiP
In a cell line stably expressing BiP fused to an HA tag, genes encoding wtLC, Ψlc and Cκ transfected. Compared to wild-type HEK293 cells (containing only endogenous BiP), HEK293 cells transduced with BiP-HA lentiviral particles showed a 1.5-fold increase in BiP expression (both endogenous BiP and BiP-HA). It was found that Ψlc was essentially unable to be secreted, regardless of the presence of HC. wtLC was secreted as both monomers and dimers, while Cκ was secreted as a dimer. Unlike wtLC (Vκ-Cκ), secretion of the single Cκ domain was nearly undetectable in the absence of HC. Regarding cellular expression, Ψlc exhibited higher expression than wtLC, Co-immunoprecipitation revealed that Ψlc was stably associated with BiP within cells, indicating that the VL domain determines the physical stability of the BiP/LC complex. Protease sensitivity assays were performed to compare the folding states of wtLC and Ψlc in cells. The degradation rate of Ψlc was faster than that of wtLC, indicating that Ψlc was in an unstructured state compared with wtLC.
ψVκ causes Fab structural changes
ψFab proteins were generated using Discovery Studio 2020 software. Measurements of the root mean square deviation (RMSD) and Fab elbow angle compared to the wild-type Fab revealed that the ψFab exhibited a RMSD of 18.3115 Å, indicating significant structural differences. The hinge angle of the wild-type Fab is 146°, while that of the ψFab is 194°. This suggests that the introduction of the ψVκ domain significantly altered the relative position of the V and C regions, leading to a significant rearrangement of the overall Fab structure. The abnormal structure of the ψVκ domain significantly distorts the overall Fab conformation, potentially contributing to the inability of antibodies containing the ψLC to fold and be secreted properly.
Summary
This study found that VH-VL pairing is not essential for IgG1 assembly and secretion. Instead, the key factor controlling IgG assembly and secretion is CH-CL pairing within the IgG molecule, and the structural integrity of the VL region is essential for IgG secretion. These findings provide valuable insights for the design of bioactive IgG-based molecules.
