Unlike RBs that can be directly stained by anti-gamma and anti-kappa, the rod-shaped CBs were not stained by the same set of antibodies due to their crystalline nature that prevents the penetration of staining antisera. inducing RBs. Another reference Necrosulfonamide mAb without notable solution behavior issues did not induce RBs and was secreted abundantly. Intrinsic physicochemical properties of individual IgG clones thus directly affected the biosynthetic steps in the ER, and thereby produced distinctive cellular phenotypes and influenced IgG secretion output. The findings implicated that RB formation represents a phase separation event or a loss of colloidal stability in the secretory pathway organelles. The process of RB induction allows the cell to preemptively reduce the extracellular concentration of potentially pathogenic, highly aggregation-prone IgG clones by selectively storing them in the ER. Keywords:crystalline body, endoplasmic reticulum, gelation, immunoglobulin, phase separation, protein aggregation, protein condensation, protein crystallization, Russell body == Abbreviations == Brefeldin A crystalline body differential interference contrast endoplasmic reticulum; fragment antigen Necrosulfonamide binding heavy chain human embryonic kidney immunoglobulin G light chain; monoclonal antibody Russell body heavy chain variable domain light chain variable domain == Introduction == Although the vast majority of antibodies are known to be highly soluble in aqueous solution, there are groups of immunoglobulins that show remarkable phase behaviors such as crystallization, aggregation, liquid-liquid phase separation, gelation, and fibril formation.1-7While it is difficult to determine what percentage of immunoglobulin clones in a given repertoire exhibits such high protein condensation propensities, every individual is believed to carry those immunoglobulins in circulation.6However, they are normally harmless (thus asymptomatic) because their overall concentrations are usually low,8unless over-produced as paraproteins in the advanced stages of multiple myeloma and other plasma cell dyscrasias where serum IgG concentration can exceed 70 mg/ml.9 Extensive variations in the intrinsic net attractive inter-protein interaction energy (due to diverse surface charge distribution patterns unique to each mAb clone) have been proposed as an underlying cause for the solution behavior differences among distinct monoclonal antibodies (mAbs).3,10Similarly, differential susceptibility to various types of physical stresses appears to originate from the preexisting stability variations inherent to individual mAb clones.11,12Given the sequence diversities in the variable regions of immunoglobulin molecules, some IgG mAb clones are bound to possess prominent aggregation property and stress-susceptibility a priori. Such predisposed propensity then manifests itself whenever Necrosulfonamide the right conditions are presented. But otherwise, such propensities would remain latent. While the different solution behaviors of distinct IgG clones must originate from the interplay between the intrinsic properties embedded in the molecule itself and the various extrinsic environmental parameters, the accurate prediction of such behaviors remains difficult despite its importance in biopharmaceutical manufacturing13and in the pathogenesis of immunoglobulin deposition diseases.6,14 Episodes of immunoglobulin condensation during protein biosynthesis can lead to the formation of Russell body (RB), enlarged globular aggregates of immunoglobulins stored in the endoplasmic reticulum (ER), both in homologous and heterologous cellular systems.15,16Depending on the differences in physicochemical properties embedded within the variable regions of heavy chain (HC) and light chain (LC), some human IgG clones readily aggregate into RB in the ER lumen, whereas other IgG clones are much less prone to induce RB under the same expression conditions.6,15However, the Necrosulfonamide underlying biophysical or biochemical basis for why some IgG clones are far more inclined to aggregate into RBs remains elusive. Likewise, while RB phenotypes have been extensively studied at the cellular level,15-18little is known about the characteristics of the RB-inducing IgG mAb clones after secreted from Necrosulfonamide the cells. Indeed, it is poorly understood whether there are any physiologically relevant differences in aqueous solution behaviors between RB-inducing IgG mAb clones and generic innocuous IgG mAb clones. To explore the link between the intrinsic physicochemical properties of IgG mAb clones and the phenotypes of the cells that overexpress them, we examined the early biosynthetic events of full-length human IgG mAb clones for which high intrinsic aggregation propensities were known a priori. Specifically, we selected (i) 2 IgG2 mAbs with marked low solubility at neutral pH, (ii) 2 IgG2 mAbs with pronounced sensitivity Rabbit polyclonal to WAS.The Wiskott-Aldrich syndrome (WAS) is a disorder that results from a monogenic defect that hasbeen mapped to the short arm of the X chromosome. WAS is characterized by thrombocytopenia,eczema, defects in cell-mediated and humoral immunity and a propensity for lymphoproliferativedisease. The gene that is mutated in the syndrome encodes a proline-rich protein of unknownfunction designated WAS protein (WASP). A clue to WASP function came from the observationthat T cells from affected males had an irregular cellular morphology and a disarrayed cytoskeletonsuggesting the involvement of WASP in cytoskeletal organization. Close examination of the WASPsequence revealed a putative Cdc42/Rac interacting domain, homologous with those found inPAK65 and ACK. Subsequent investigation has shown WASP to be a true downstream effector ofCdc42 to mechanical agitation stress, and (iii) one IgG2 mAb with exceptionally high solution viscosity. As references, we tested (iv) one IgG2 mAb that was.