Abstract
Protein synthesis and secretion are essential to cellular life. Although secretory activities may vary in different cell types, what determines the maximum secretory capacity is inherently difficult to study. Increasing protein synthesis until reaching the limit of secretory capacity is one strategy to address this key issue. Under highly optimized growth conditions, recombinant CHO cells engineered to produce a model human IgG clone started housing rod-shaped crystals in the endoplasmic reticulum (ER) lumen. The intra-ER crystal growth was accompanied by cell enlargement and multinucleation and continued until crystals outgrew cell size to breach membrane integrity. The intra-ER crystals were composed of correctly folded, endoglycosidase H-sensitive IgG. Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG, and the crystallization was reproduced in vitro by exposing a high concentration of IgG to a near neutral pH. The striking cellular phenotype implicated the efficiency of IgG protein synthesis and oxidative folding exceeded the capacity of ER export machinery. As a result, export-ready IgG accumulated progressively in the ER lumen until a threshold concentration was reached to nucleate crystals. Using an in vivo system that reports accumulation of correctly folded IgG, we showed that the ER-to-Golgi transport steps became rate-limiting in cells with high secretory activity.
Highlights
Immunoglobulins continue to serve as an important model secretory cargo for investigating biochemical processes of oxidative protein folding and subunit assembly in the ER2 lumen [1]
Even if cells expand their capacity by increasing the number of participating catalytic machines and the frequency of catalytic events, there will eventually be a physical limit that determines the ceiling of secretory capacity, namely how much energy to generate and expend as well as how many proteins to synthesize, fold, assemble, package, traffic, and secrete by individual cells in a given time and space
Formation of Rod-shaped Crystals in Cytoplasm of Recombinant CHO Cells Overexpressing Human IgG—To express transgenes encoding our model human IgG protein, a stable CHO cell line was developed by using a pair of expression vectors with a promoter/enhancer element from human cytomegalovirus immediate-early gene and the expression-augmenting sequence elements that will allow a sustained high level transcription [20]
Summary
Recombinantly produced in variants of CHO cells that were adapted to propagate in suspension culture format. Despite the success in boosting protein expression per se through these expression vector engineering approaches, such enhancements did not translate into higher glycoprotein secretion partly because post-translational events such as protein folding/assembly and intracellular vesicular transport steps along the secretory organelles became the new bottlenecks [13,14,15]. Even if cells expand their capacity by increasing the number of participating catalytic machines (e.g. enzymes and mechanoenzymes) and the frequency of catalytic events, there will eventually be a physical limit that determines the ceiling of secretory capacity, namely how much energy to generate and expend as well as how many proteins to synthesize, fold, assemble, package, traffic, and secrete by individual cells in a given time and space. Intra-ER crystallization required both the unique physicochemical properties of the model IgG and the biosynthetic and protein folding efficiency of the recombinant CHO cells that exceeded the ER export capacity under optimized cell culture conditions. In addition to the intrinsic limitation in intracellular protein trafficking efficiency, physicochemical properties of the secretory cargo itself play critical roles in determining the ceiling of cellular secretory capacity
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
