While Golgi morphology was affected by the formation of RBs, secretory pathway membrane traffic remained operational in those cells. 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 Tenacissoside G cells expressing different mAbs. KEYWORDS: eIF2, endoplasmic reticulum, immunoglobulin, protein translation, Russell body, single amino acid substitution AbbreviationsCDRcomplementarity-determining regionCHOChinese hamster ovaryDICdifferential interference contrasteIF2eukaryotic initiation factor 2 -subunitERendoplasmic reticulumHEKhuman embryonic kidneyHCheavy chainmAbmonoclonal antibodyLClight chainPBSphosphate-buffered salinePERKprotein kinase R (PKR)-like endoplasmic reticulum kinaseRBRussell body Introduction Single amino acid substitution within the complementarity-determining region (CDR) and framework region can have dramatic effects on the overall physicochemical properties and the antigen-binding characteristics of immunoglobulins. Humoral immunity naturally exploits such structure-function relationships to generate higher affinity immunoglobulins Tenacissoside G by somatic hypermutation on the rearranged variable region genes.1,2 Both during a secondary immune response and during an antibody engineering Casp3 effort, one can envision that some amino acid substitutions may be beneficial in imparting higher affinities toward pathogens or antigens of interest, better physicochemical properties such as higher protein stability, or more efficient biosynthesis resulting in higher secretion outputs. It is also equally likely that amino acid substitutions would produce neutral and deleterious effects on antibody functions or its biosynthetic processes.3-5 Because it is difficult to know Tenacissoside G what types of amino acid substitutions are favored or disfavored in a given immune response, the immune system relies on a Darwinian selection process.6 Namely, by iterating the expansion of reactive B-cell populations, somatic hypermutations, and the selection of beneficial variants, the antibody repertoire is fine-tuned to suit Tenacissoside G the need of imminent situations.6 During the very same selection process, however, B cells that come to express harmful and disadvantageous immunoglobulin variants are directed to wastage pathways.5 Examples of harmful single amino acid substitution that affect the specificity7-12 and the affinity13-15 of antigen binding are well documented. Another class of deleterious amino acid substitutions increases aggregation propensity of immunoglobulin proteins by affecting folding stability.16-18 Other disadvantageous amino acid substitution are known to impair the secretion of immunoglobulins.4,19-21 Although defective subunit chain folding and flawed subunit assembly were proposed as the potential reasons for the deficiencies in those previously reported studies, detailed biochemical basis for how a single amino acid substitution affects the secretory outputs has not been investigated to date. The involved signaling pathways, if any, and the underlying cell physiologic processes are also unknown. Given the importance of recombinant monoclonal antibodies (mAbs) as a modality of human therapeutics, it is critical to understand the cell biologic basis for the oft-observed mAb secretion output variance caused by the primary sequence difference. To investigate the underlying mechanisms for secretion level variance among distinct mAbs, we reasoned that minimizing the sequence difference of the mAbs down to a single amino acid residue would be the most effective approach. During an antibody discovery research program aiming to generate human mAbs that specifically recognize and antagonize human cannabinoid receptor type 1 (CB1), a pair of highly related human IgG2 mAbs were generated. Although the 2 2 mAbs differed only.