http://orcid.org/0000-0002-3464-4715
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ABSTRACT
Full-length immunoglobulins (Igs) are widely considered difficult to crystallize because of their large size, N-linked glycosylation, and flexible hinge region. However, numerous cases of intracellular Ig crystallization are reported in plasma cell dyscrasias. What makes some Ig clones more prone to crystallize during biosynthesis as well as the biochemical and cell biological requirements for this cryptic event are poorly understood. To investigate the underlying process of intracellular Ig crystallization we searched for model IgGs that can induce crystalline inclusions during recombinant overexpression. By testing various subunit combinations through mixing and matching of individual subunit chains derived from a panel of human IgG clones, we identified one secretion competent IgG2λ that induced needle-like crystalline inclusions in transfected HEK293 cells. Ig crystallization rarely occurred at steady-state cell growth conditions but was easily induced when ER-to-Golgi transport was pharmacologically blocked. Homology modeling revealed the presence of a prominent negatively-charged patch on the variable domain surface. The patch was composed of eight aspartic acids, of which five were in the heavy chain variable region and three were in the light chain. Crystallization occurred only when the two subunits were co-transfected and the intracellular crystals co-localized with ER resident proteins. Furthermore, subtype switching from IgG2 to IgG1 and stepwise neutralization of the acidic patch independently abrogated Ig crystallization events. The evidence supported that the formation of needle-like crystalline inclusions in the ER was underscored by multivalent intermolecular interactions between the acidic patch and undefined determinants present on the γ2 subunit constant region.
Abbreviations
| CB | = | crystalline body |
| CDR | = | complementarity-determining region |
| CHO | = | Chinese hamster ovary |
| DIC | = | differential interference contrast |
| ER | = | endoplasmic reticulum |
| Fab | = | antigen binding fragment |
| Fc | = | crystallizable fragment |
| Fv | = | variable fragment |
| HC | = | heavy chain |
| HEK | = | human embryonic kidney |
| Ig | = | immunoglobulin |
| LC | = | light chain |
| mAb | = | monoclonal antibody |
| RB | = | Russell body |
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgments
The authors thank Allison Bianchi and Jane Carter for making mAb-2 expression constructs and Ai Ching Lim for critically reviewing the manuscript.
Author contributions
Study conception and design: HHCritical reagent generation: LL, KG, FJAcquisition of data: HH, MG, RRKAnalysis and interpretation of data: HHDrafting of manuscript: HHCritical revision: HH, RRK, FJFinal approval of manuscript: HH, MG, RRK, LL, KG, FJ