Light chain subunit of a poorly soluble human IgG2λ crystallizes in physiological pH environment both in cellulo and in vitro

https://doi.org/10.1016/j.bbamcr.2021.119078Get rights and content
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Highlights

  • The LC subunit of a poorly soluble model IgG2λ crystallized spontaneously in the ER.

  • LC crystallization was readily reconstituted using purified protein in vitro at physiological pH.

  • Structural determinants underlying the LC crystallization were elucidated.

  • Mutation to the determinant remediated the solubility problem when introduced directly to IgG2λ.

  • The ER can not only detect secretory cargo's high condensation propensity but also guide antibody engineering strategy.

Abstract

Prominent inclusion bodies can develop in the endoplasmic reticulum (ER) when overexpressed antibodies possess intrinsically high condensation propensities. These observations suggest that antibodies deemed to show notable solubility problems may reveal such characteristics preemptively in the form of ER-associated inclusion bodies during antibody overexpression. To define the relationships between solubility problems and inclusion body phenotypes, we investigated the biosynthesis of a model human IgG2λ that shows severe opalescence in an acidic formulation buffer yet retains high solubility at physiological pH. Consistent with the pH-dependent solubility characteristics, the model antibody did not induce notable inclusion body in the physiological pH environment of the ER lumen. However, when individual subunit chains of the antibody were expressed separately, the light chain (LC) spontaneously induced notable crystal-like inclusion bodies in the ER. The LC crystallization event was readily reproducible in vitro by simply concentrating the purified LC protein at physiological pH. Two independent structural determinants for the LC crystallization were identified through rational mutagenesis approach by monitoring the effect of amino acid substitutions on intracellular LC crystallogenesis. The effect of mutations on crystallization was also recapitulated in vitro using purified LC proteins. Importantly, when introduced directly into the model antibody, a mutation that prevents the LC crystallization remediated the antibody's solubility problem without compromising the secretory output or antigen binding. These results illustrate that the ER can serve as a “physiological test tube” that not only reports secretory cargo's high condensation propensity at physiological pH, but also provides an orthogonal method that guides antibody engineering strategy.

Abbreviations

CDR
complementarity-determining region;
DIC
differential interference contrast
ER
endoplasmic reticulum
Fab
antigen binding fragment
Fc
crystallizable fragment
HEK
human embryonic kidney
HC
heavy chain
IF
immunofluorescent
Ig
immunoglobulin
mAb
monoclonal antibody
LC
light chain
LLPS
liquid-liquid phase separation
PBS
phosphate-buffered saline
SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis
SEC
size-exclusion chromatography
TBS
Tris-buffered saline
UPR
unfolded protein response
VH
heavy variable region
VL
light chain variable domain

Keywords

Immunoglobulin
Inclusion body
Protein phase separation
Intracellular protein crystallization
Endoplasmic reticulum
Light chain
Antibody solubility

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