The dispensability of VH-VL pairing and the indispensability of VL domain integrity in the IgG1 secretion process

Abstract

Introduction: The C_H1 domain of IgG antibodies controls assembly and secretion, mediated by the molecular chaperone BiP via the endoplasmic reticulum protein quality control (ERQC) mechanism. However, it is not clear whether the variable domains are necessary for this process. Methods: Here, we generated IgG1 antibodies in which the V domain (V_H and/or V_L) was either removed or replaced, and then assessed expression, assembly, and secretion in HEK293 cells. Results: All Ig variants formed a covalent linkage between the C_γ1 and C_κ, were successfully secreted in an assembled form. Replacement of the cognate V_κ with a non-secretory pseudo V_κ (_ψV_κ) hindered secretion of individual or assembled secretion of neither heavy chains (HCs) nor light chains (LCs). The _ψLC (_ψV_κ-C_κ) exhibited a less folded structure compared to the wild type (wt) LC, as evidenced by enhanced stable binding to the molecular chaperone BiP and susceptibility to proteolytic degradation. Molecular dynamics simulation demonstrated dramatic alterations in overall structure of _ψFab (Fd-_ψLC) from wt Fab. Discussion: These findings suggest that V domains do not initiate HC:LC assembly and secretion; instead, the critical factor governing IgG assembly and secretion is the C_H-C_L pairing. Additionally, the structural integrity of the V_L domain is crucial for IgG secretion. These data offer valuable insight into the design of bioactive molecules based on an IgG backbone.

Keywords: IgG; VH domain; VL domain; assembly; endoplasmic reticulum quality control; secretion.

FIGURE 1

Covalent association between the C_γ1 and C_κ domains is sufficient for secretion of fully assembled Igs, regardless of the presence of the V domains. (A) Schematic representation of the plasmid vectors and Ig chain (or domain) proteins encoded by the corresponding Ig genes. The expected molecular weights of the individual subunits are indicated. The hinge region of human γ heavy chain is shown as a thick black bar upstream of the C_γ2 region. L, leader sequence; P, cytomegalovirus promoter. (B,C) Immunoblot analysis of Ig subunits. The lysates (B) and supernatant (C) of HEK293 transfectants were resolved in reducing and non-reducing SDS-PAGE gels. The resolved Ig proteins were probed with antibodies specific for human IgG-Fc and C_κ region. The bar graph below each gel image displays the relative intensity of bands detected by the specified antibodies under reducing conditions. Lanes 1–7 contain cell lysates obtained after lysis if cells transfected with each plasmids labeled as in (A). Lane C represents the non-transfected control. GAPDH was used as a loading control for protein normalization.

FIGURE 2

Replacement of V_κ with the _ψV_κ domain disrupts secretion of Ig molecules. (A) Nucleotide and amino acid sequences of 2C281 _ψV_κ. CDR residues according to the IMGT (International ImMunoGeneTics) information system shaded yellow. Due to a frame shift mutation in CDR3, the IMGT system does not define CDR3 of 2C281 _ψV_κ. Consequently, regions beyond FR3 are shaded in grey. Mint-colored shading marks the site with presumed base deletion (s). Amino acid sequence affected by base (s) deletion-induced reading-frame shift is underlined. Asterisk indicates stop codon. (B) V_κ sequence alignment. Sequences of 2C281 _ψV_κ and true V_κ regions from 6C407 clone aligned using the Clustal Omega server (https://www.ebi.ac.uk/Tools/msa/clustalo/). Absent residues denoted by dash. Dots indicate the positions of conserved residues between the sequences. (C) Schematic representation of the plasmid vectors and genes encoding the corresponding Ig chain (or domain) proteins. The hinge region of the human γ1 heavy chain is always present upstream of the C_γ2 region, and is shown here as a thick black bar. L, leader sequence; P, cytomegalovirus promoter. (D) Immunoblot analysis of culture supernatants. Lanes 1–4 contain supernatants obtained from cells transfected with each plasmid labeled as in (C). Lane C represents the non-transfected control.

FIGURE 3

The presence of an improper V_κ domain hinders assembly of HC and LC. (A) Schematic representation of the plasmid vectors and genes encoding the corresponding Ig chain (or domain) proteins. The hinge region of human γ1 heavy chain is always present upstream of the C_γ2 region, and is shown here as a thick black bar. L, leader sequence; P, cytomegalovirus promoter. (B,C) Immunoblot and immunoprecipitation (IP) analyses of Ig subunits in cell lysates. Lysates of HEK293 transfectants were immunoprecipitated with KappaXP-Agarose, which captures the C_κ domain. Input samples and IP samples were separated by reducing and/or non-reducing SDS-PAGE, and subsequently analyzed by immunoblotting. (B) The input represents 10% of the total amount of lysate used for IP. GAPDH was used as an internal loading control. (C) Co-IP analysis of HC and LC in lysates from HEK293 transfectants. Numbers below the Western blot images represent protein band intensity, which was analyzed with ImageJ software and normalized to lane 1. The bar graph shows the ratio of the HC band intensity to that of the pulled-down LC. (D) Evaluation of H:L chain association by sandwich ELISA. Lysates of transfectants were placed in wells coated with goat anti-human IgG/Fc, and bound LCs were detected with rabbit anti-human C_κ followed by an AP-conjugated anti-rabbit IgG Ab. The data are expressed as the mean ± standard deviation of triplicate samples, and are representative of a single experiment from a series of three independent experiments.

FIGURE 4

Interaction of LCs with BiP in HEK293 cells stably expressing HA-tagged BiP (HEK293-BiP). (A) Schematic representation of the plasmid vectors and genes encoding the corresponding Ig chain (or domain) proteins. (B) BiP detected using anti-BiP antibody in wild-type HEK293 cells (lane 1) and HEK293 cells transduced with the BiP-HA lentiviral particles (lane 2). The asterisk indicates the band corresponding to the size of BiP (C) Immunoblot analysis of LCs in the culture supernatant of HEK293-BiP. Forty-eight post-transfection culture supernatants from HEK293 transfectants were resolved in reducing and non-reducing SDS-PAGE gels. (D) Co-IP of LC and BiP in lysates from HEK293-BiP cells transfected with LCs. Co-immunoprecipitation was performed using an anti-HA agarose. Proteins in the extract (input = 10% of the lysate) and pull-down fractions (IP) were resolved by reducing SDS-PAGE and probed with Abs specific for human IgG-Fc, the C_κ region, and the HA tag. Lane C₁, non-transfected HEK293 control; Lane C₂, non-transfected HEK293-BiP control. (E) Protease susceptibility assay. wt LCs and _ψLCs were enriched by IP of lysates from the respective HEK293 transfectants using KappaXP-Agarose. The eluates were treated with protease trypsin for the indicated times and resolved by SDS-PAGE, followed by immunoblotting with an anti-C_κ antibody. The intensity of bands from wt LC and _ψLC proteins treated with trypsin was normalized to the untreated lane (0 min) and displayed as a graph alongside the gel image. The graph was generated from the cumulative intensity sum of three protein bands in each lane.

FIGURE 5

Ribbon diagrams illustrating the predicted Fab structures. (A) Superposition of wt Fab and _ψFab structures, modeled using Discovery Studio 2020 (Modeler ver. 9.22). The Fab molecules are represented by blue (wt Fab) and pink (_ψFab) ribbons. (B) The Fab elbow angle was calculated and represented visually by PyMOL software (version 2.5.0). Black dumbbells pass through the center of mass of the V and C domains of each Fab. The orange arc denotes the Fab elbow angles. Green and red dumbbells represent residues used to separate the V and C domains, with a green ball representing the LC and a red ball representing the HC.