title: 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.
abstract: Amino acid sequence differences in the variable region of immunoglobulin (Ig) cause wide variations in secretion outputs. To address how a primary sequence difference comes to modulate Ig secretion, we investigated the biosynthetic process of 2 human IgG2κ monoclonal antibodies (mAbs) that differ only by one amino acid in the light chain complementarity-determining region 1 while showing ∼20-fold variance in secretion titer. Although poorly secreted, the lower-secreting mAb of the 2 was by no means defective in terms of its folding stability, antigen binding, and in vitro biologic activity. However, upon overexpression in HEK293 cells, the low-secreting mAb revealed a high propensity to aggregate into enlarged globular structures called Russell bodies (RBs) in the endoplasmic reticulum. While Golgi morphology was affected by the formation of RBs, secretory pathway membrane traffic remained operational in those cells. Importantly, cellular protein synthesis was severely suppressed in RB-positive cells through the phosphorylation of eIF2α. PERK-dependent signaling was implicated in this event, given the upregulation and nuclear accumulation of downstream effectors such as ATF4 and CHOP. These findings illustrated that the underlining process of poor Ig secretion in RB-positive cells was due to downregulation of Ig synthesis instead of a disruption or blockade of secretory pathway trafficking. Therefore, RB formation signifies an end of active Ig production at the protein translation level. Consequently, depending on how soon and how severely an antibody-expressing cell develops the RB phenotype, the productive window of Ig secretion can vary widely among the cells expressing different mAbs.
authors.affiliation: a Department of Therapeutic Discovery , Amgen Inc. , South San Francisco , CA , USA.
authors.forename: Haruki
authors.identifier: 0000-0002-3464-4715
authors.initials: H
authors.lastname: Hasegawa
authors.affiliation: b Department of Therapeutic Discovery , Amgen Inc. , Thousand Oaks , CA , USA.
authors.forename: Ann
authors.initials: A
authors.lastname: Hsu
authors.affiliation: a Department of Therapeutic Discovery , Amgen Inc. , South San Francisco , CA , USA.
authors.forename: Christine E
authors.identifier: 0000-0002-6179-0435
authors.initials: CE
authors.lastname: Tinberg
authors.affiliation: c Department of Cardiometabolic Disorders , Amgen Inc. , South San Francisco , CA , USA.
authors.forename: Karen E
authors.initials: KE
authors.lastname: Siegler
authors.affiliation: b Department of Therapeutic Discovery , Amgen Inc. , Thousand Oaks , CA , USA.
authors.forename: Aaron A
authors.identifier: 0000-0001-7531-6040
authors.initials: AA
authors.lastname: Nazarian
authors.affiliation: b Department of Therapeutic Discovery , Amgen Inc. , Thousand Oaks , CA , USA.
authors.forename: Mei-Mei
authors.identifier: 0000-0002-3052-7853
authors.initials: MM
authors.lastname: Tsai
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D022801:Complementarity Determining Regions / Q000096:biosynthesis* / Q000235:genetics; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D010766:Phosphorylation; D014176:Protein Biosynthesis; D055571:Secretory Pathway
chemical_list: D022801:Complementarity Determining Regions; D015852:Eukaryotic Initiation Factor-2; D007074:Immunoglobulin G
keywords: Russell body; eIF2α; endoplasmic reticulum; immunoglobulin; protein translation; single amino acid substitution