AUTHOR=Choi Juho , Jeon Yerin , Roh Youngin , Jang Jeongyun , Lee Eunbin , Villamante Luigie , Kim Minjae , Kwon Myung-Hee 

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

JOURNAL=Frontiers in Molecular Biosciences

VOLUME=11

YEAR=2024

URL=https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2024.1346259

DOI=10.3389/fmolb.2024.1346259

ISSN=2296-889X

ABSTRACT=<p><bold>Introduction:</bold> The C<sub>H1</sub> 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.</p><p><bold>Methods:</bold> Here, we generated IgG1 antibodies in which the V domain (V<sub>H</sub> and/or V<sub>L</sub>) was either removed or replaced, and then assessed expression, assembly, and secretion in HEK293 cells.</p><p><bold>Results:</bold> All Ig variants formed a covalent linkage between the C<sub>γ1</sub> and C<sub>κ</sub>, were successfully secreted in an assembled form. Replacement of the cognate V<sub>κ</sub> with a non-secretory pseudo V<sub>κ</sub> (<sub>ψ</sub>V<sub>κ</sub>) hindered secretion of individual or assembled secretion of neither heavy chains (HCs) nor light chains (LCs). The <sub>ψ</sub>LC (<sub>ψ</sub>V<sub>κ</sub>-C<sub>κ</sub>) 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 <sub>ψ</sub>Fab (Fd-<sub>ψ</sub>LC) from wt Fab.</p><p><bold>Discussion:</bold> 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<sub>H</sub>-C<sub>L</sub> pairing. Additionally, the structural integrity of the V<sub>L</sub> domain is crucial for IgG secretion. These data offer valuable insight into the design of bioactive molecules based on an IgG backbone.</p>