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

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 μ-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.