title: In vivo crystallization of human IgG in the endoplasmic reticulum of engineered Chinese hamster ovary (CHO) cells.
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.
authors.affiliation: Department of Protein Science, Amgen Inc., Seattle, Washington 98119, USA. harukih@amgen.com
authors.forename: Haruki
authors.initials: H
authors.lastname: Hasegawa
authors.forename: John
authors.initials: J
authors.lastname: Wendling
authors.forename: Feng
authors.initials: F
authors.lastname: He
authors.forename: Egor
authors.initials: E
authors.lastname: Trilisky
authors.forename: Riki
authors.initials: R
authors.lastname: Stevenson
authors.forename: Heather
authors.initials: H
authors.lastname: Franey
authors.forename: Francis
authors.initials: F
authors.lastname: Kinderman
authors.forename: Gary
authors.initials: G
authors.lastname: Li
authors.forename: Deirdre Murphy
authors.initials: DM
authors.lastname: Piedmonte
authors.forename: Timothy
authors.initials: T
authors.lastname: Osslund
authors.forename: Min
authors.initials: M
authors.lastname: Shen
authors.forename: Randal R
authors.initials: RR
authors.lastname: Ketchem
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D007074:Immunoglobulin G; D011994:Recombinant Proteins