Overexpression of cryoglobulin‐like single‐chain antibody induces morular cell phenotype via liquid–liquid phase separation in the secretory pathway organelles

Abstract

Cryoprecipitation of immunoglobulins is often reported in association with B-cell lymphoproliferative disorders and plasma cell dyscrasias. However, the biochemical basis of such cryoglobulin behaviors is not well understood because of a general lack of suitable experimental systems. Here, we report the identification and characterization of a single-chain antibody (scFv-Fc) that recapitulates cryoglobulin-like properties. When model scFv-Fc protein was engineered to multimerize, by appending the secretory tailpiece (stp) of human immunoglobulin l-chain to the C terminus, the resulting oligomeric scFv-Fc-stp protein acquired two unexpected properties: the induction of a morular cell phenotype during protein biosynthesis and the cryoprecipitation of secreted proteins in harvested cell culture media. The turbidity of the culture media and the inclusion bodies that gave morular appearances were attributed to microscopic spherical protein droplet formation, a hallmark characteristic of liquid-liquid phase separation (LLPS) event. Mutagenesis approaches revealed that these two phenomena were independent of covalent protein oligomerization induced by stp. Disruption of the N-linked glycosylation motif in the stp region enhanced morular phenotype propensity but reduced protein secretion. Intermolecular disulfide bonds that stabilize Fc dimers and oligomers were necessary for efficient induction of LLPS, but their simultaneous elimination could not abrogate the LLPS propensity completely. Noncovalent protein-protein interactions between scFv-Fc-stp chains sufficiently established a basis for LLPS induction. Morular cell phenotypes and cryoprecipitation were clearly underpinned by intrinsic physicochemical properties embedded in the overexpressed cargo protein.Overproduction of condensation-prone secretory proteins that culminate in LLPS in the endoplasmic reticulum therefore serves as a path to produce morular Russell body phenotype.

Intermolecular interactions involving an acidic patch on immunoglobulin variable domain and the γ2 constant region mediate crystalline inclusion body formation in the endoplasmic reticulum

“…These spherule formations are the hallmark characteristics of protein condensation via liquid-liquid phase separation in the ER. 24 Of note, morular cell phenotypes are alternatively referred to as grape cells. 25 Because both subunit chains were secretion incompetent to begin with, we did not evaluate the effects of BFA on cell phenotypes.…”

Single amino acid substitution in LC-CDR1 induces Russell body phenotype that attenuates cellular protein synthesis through eIF2α phosphorylation and thereby downregulates IgG secretion despite operational secretory pathway traffic

“…These are crystalline body 29,38 and dense liquid protein droplet. 39 We showed previously that these inclusion body phenotypes were not necessarily associated with low immunoglobulin secretion titers. 29,38,39 If the correlation between an attenuated protein synthesis and a diminished immunoglobulin secretion holds true, the protein translation would remain active in those cells despite the formation of prominent inclusion bodies.…”

“…39 We showed previously that these inclusion body phenotypes were not necessarily associated with low immunoglobulin secretion titers. 29,38,39 If the correlation between an attenuated protein synthesis and a diminished immunoglobulin secretion holds true, the protein translation would remain active in those cells despite the formation of prominent inclusion bodies. As predicted from this view, the level of active protein translation was not affected in the cells that house those inclusion bodies (Fig.…”

“…This study unraveled a sequence of events that apparently linked RB formation and the suppression of immunoglobulin 29 and (B) a scFv-Fc construct that induces morula cell phenotype via intra-ER liquid-liquid phase separation (LLPS). 39 Transfected cells were first labeled for 30 min using Alexa Fluor 488 Click-iT Ò Plus OPP reagent to detect actively ongoing protein translation in situ (see Materials and Methods). The click-labeled cells were then immuno-stained with (A) Alexa Fluor 594-conjugated antihuman IgG (HCL) or (B) Texas Red-conjugated anti-gamma chain polyclonal antibodies.…”

“…For instance, our previous studies showed that recombinant human IgG secretion can be as low as < 1 mg/L or as high as > 300 mg/L depending on the individual mAb clones, despite using the identical HEK293 cell-based batch production method. 24,28,29,38,39 As soon as cells develop RBs, they stop contributing to amass the secreted IgGs in cell culture medium during a cell culture process. Consequently, depending on how many cells are to develop RB phenotype in a given cell population and how early it happens, the volumetric titers can vary widely.…”