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Spatial proteomics reveals differences in the cellular architecture of antibody-producing CHO and plasma-cell derived cells

ABSTRACT: Most of the recombinant biotherapeutics employed today to combat severe illnesses e.g. various types of cancer or autoimmune diseases are produced by Chinese hamster ovary (CHO) cells. To meet the growing demand of these pharmaceuticals, CHO cells are under constant development in order to enhance their stability and productivity. The last decades saw a shift from empirical cell line optimization towards rational cell engineering using a growing number of large omics datasets to alter cell physiology on various levels. Especially proteomics workflows reached new levels in proteome coverage and data quality due to advances in high-resolution mass spectrometry instrumentation. One type of workflow concentrates on spatial proteomics by usage of subcellular fractionation of organelles with subsequent shotgun mass spectrometry proteomics and machine learning algorithms to determine the subcellular localization of large portions of the cellular proteome at a given time. Here, we present the first subcellular spatial proteome of a CHO-K1 cell line producing high titers of recombinant antibody and comparison to the spatial proteome of an antibody-producing plasma cell-derived (PCD) myeloma cell line. Both cell lines show strong correlation of immunoglobulin G chains with chaperones and proteins associated in protein glycosylation within the endoplasmic reticulum compartment. However, we report differences in the localization of proteins associated to vesicle-mediated transport, transcription and translation, which may affect antibody production in both cell lines. Furthermore, pairing subcellular localization data with protein expression data revealed elevated protein masses for organelles in the secretory pathway in MPC-11 cells. Our study highlights the potential of subcellular spatial proteomics combined with protein expression as potent workflow to identify characteristics of highly efficient recombinant protein expressing cell lines.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Cricetulus Griseus (chinese Hamster) (cricetulus Barabensis Griseus) Mus Musculus (mouse)

TISSUE(S): Cell Suspension Culture, Plasma Cell

SUBMITTER: Robin Kretz

LAB HEAD: Dieter Stoll

PROVIDER: PXD029115 | Pride | 2022-08-02

REPOSITORIES: Pride

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Project description:Chinese hamster ovary (CHO) cells have been widely employed for production of recombinant proteins (RP) for research and therapeutic purposes. Indeed, this expression system was used for production of most of approved antibodies (84%) in 2015-2018 period. The success of this cell line for RP production relies on several advantages that comprise but are not limited to a safety viral profile, human compatible glycosylation, development of specific culture mediums and supplements, availability of sub-lines with different capabilities and the improvement in design of DNA vectors and selection strategies that allows to obtain higher producer clones in an easier way. Given the high importance of this cell line, a deeper knowledge of their biology through genomic, transcriptomic, proteomic and metabolomic studies has been gained in last years. These studies have aid in the exploration of molecular and cellular mechanisms that could explain cell behavior and to propose new targets for improved RP production and quality. A special emphasis has been placed on proteomic characterization of subcellular compartments over cellular homogenates, due to some advantages that fractionation techniques offer over the classical approach, which include obtaining datasets that are easier to process and understand. Subcellular proteomics allows monitoring of proteins that shuttle between different compartments, quantification of lower abundance proteins, increase in number of identified proteins, elucidation of function and regulation of proteins, and unraveling of organelle dynamics. The use of subcellular fractionation for proteomic characterization of organelles from CHO cells has been limited to the isolation of one or few organelles in an adherent phenotype, and only few fractionation protocols have been reported for these cells in their suspension format. The high cross-contamination of some isolated fractions due to insufficient fractionation steps, prevents adopting some of these protocols for applications such as proteomics. Thus, since this approach has not been addressed before for the proteomic characterization of CHO cell organelles, subcellular proteomics was harnessed in the present study to provide identification and quantification of proteins from subcellular compartments obtained by differential and isopycnic centrifugation of this cell line. Although several cellular processes and organelles play an important role during expression and secretion of RP, the classical secretion pathway has been recognized as a bottleneck for post-translational modifications, transport, modification and secretion of proteins in mammalian cells. In fact, over-expression of some proteins from endoplasmic reticulum and Golgi Apparatus has increased the specific productivity of HEK293 and CHO cells producing different RP. Thus, in line with the paramount importance of organelles from classical secretion pathway for RP production, a novel discontinuous sucrose gradient for improved separation and enrichment of microsomes has been designed in our laboratory, and incorporated to the proteomic characterization of CHO cell organelles. CRL-12444, a DP-12 derived cell line producing a humanized monoclonal antibody against human IL-8, was used as a model of a recombinant CHO cell line during this study. Our goal is that the processing of raw proteomics data, the classification and mapping of identified proteins will allow to construct proteomic maps for most subcellular compartments, describing the molecular functions, biological processes and pathways from enriched fractions. The identification of proteins in each enriched compartment will improve the knowledge about their protein components, function and interaction, especially from those poorly represented like the proteins in the microsomes, allowing a subsequently expansion of the engineering strategies of CHO cells to increase the titer and quality of RPs.

Project description:Î³-secretase is an intra-membrane-cleaving aspartyl protease implicated in the processing of a wide range of type I membrane proteins including the Notch receptor and the amyloid-Î² precursor protein (APP). It thus regulates a diverse array of cellular and biological processes including the differentiation of neuronal embryonic stem cells, or intestinal stem cells, with the latter controlling the self-renewing of the intestinal epithelium. Indeed, proteolysis of these proteins by Î³-secretase triggers signaling cascades by releasing intracellular domains (ICDs) which, following association with adaptor proteins and nuclear translocation, modulate the transcription of different genes by binding directly to their promoters. The pronounced proliferative and regenerative effects of Notch signaling and its implication in the generation of the AÎ²-peptides, makes Î³-secretase a therapeutic target for several types of cancer and for Alzheimerâs disease. To investigate the broad effects of Î³-secretase activity onto the cellular transcriptome, Chinese hamster ovary (CHO) cells with enhanced Î³-secretase were compared to cells with abolished Î³-secretase activity via a microarray designed for a genetically close species, mouse. Our findings will potentially help to decipher the biology of Î³-secretase, including a better understanding of the roles of this enzyme in gene transcription. We compared the transcriptomes of two CHO cell lines displaying extreme differences in Î³-secretase activity. The S-1 cell line overexpressed the four components of Î³-secretase (NCT, APH1aL, PS1, and PEN2) and was characterized by a marked increase in the level of PS1 heterodimers associated with 8-fold increased Î³-secretase activity compared to untransfected controls. The other cell line consisted of wild type CHO cells incubated with DAPT, a well-known Î³-secretase inhibitor. The two cell lines were used in combination with a mouse microarray to analyze gene transcription under enhanced Î³-secretase. Two samples, S1 and DAPT treated CHO- were used in biological triplicates each.

Project description:Cystic Fibrosis (CF) is a genetic disorder CF is caused by mutations of the gene encoding for the cystic fibrosis transmembrane conductance regulator protein (CFTR), a transmembrane anion channel expressed at the apical membrane of several organs, including the epithelial cells of the airway. CFTR mutations result in dysfunctional ion transport across the apical membrane at the surface of the epithelia, generating thickened and dehydrated secretions. In the lung, this leads to a decrease in the mucociliary clearance, favoring bacterial colonization and progressive obstruction of the duct. Although over 2000 CFTR variants have been identified so far, the most common mutation is a deletion of the phenylalanine in position 508 (F508del), which shows an allelic frequency of around 90% among CF patients. F508del-CFTR is incorrectly folded, causing its retention at the endoplasmic reticulum (ER) and subsequent proteasomal degradation. Among the several drugs available in CF pharmacological treatment, VX-809 (commercial name Lumacaftor) is the most used drug for patients carrying F508del-CFTR mutation. This drug corrects the aberrant folding of F508del-CFTR by favoring the correct intramolecular interactions, thus enabling a higher number of copies of the defective protein to reach the plasma membrane. Localization of Organelle Proteins by Isotope Tagging after Differential ultra-Centrifugation (LOPIT-DC, https://doi.org/10.1038/s41467-018-08191-w) workflow was used to study the spatial localization of proteins in the CFBE41o- cells and how it is impacted by CFTR rescue triggered by VX-809. LOPIT-DC is a powerful and well-established tool for the investigation of the spatial distribution of global proteome within cells, which combines subcellular fractionation based on differential centrifugation, quantitative mass spectrometry, and machine learning. Its application provides a global snapshot of the steady-state subcelluler distribution of proteins. The aim of this project is to identify proteins that change their cellular localization in association with the treatment, to uncover molecules that play a role in the trafficking of the defective CFTR to the plasma membrane.

Project description:Chinese hamster ovary (CHO) cells have been widely employed for production of recombinant proteins (RP) for research and therapeutic purposes. Indeed, this expression system was used for production of most of approved antibodies (84%) in 2015-2018 period. The success of this cell line for RP production relies on several advantages that comprise but are not limited to a safety viral profile, human compatible glycosylation, development of specific culture mediums and supplements, availability of sub-lines with different capabilities and the improvement in design of DNA vectors and selection strategies that allows to obtain higher producer clones in an easier way. Given the high importance of this cell line, a deeper knowledge of their biology through genomic, transcriptomic, proteomic and metabolomic studies has been gained in the last years. These studies have aid in the exploration of molecular and cellular mechanisms that could explain cell behavior and to propose new targets to improve production and quality of RP. Some of these transcriptomic and proteomic profiles have been acquired under low temperature, butyrate addition, hyperosomotic pressure, protein degradation phenotypes, cellular engineering, production stability and productive phenotypes. In case of productive phenotypes, several whole cell proteomic studies have been previously reported, where cell populations secreting fusion proteins, monoclonal antibodies (mAb) and antibody fragments, at different specific productivity (Qp), have been compared. However, because of this information has been obtained from whole cell homogenates, highlighted categories represent abundant proteins and have limited coverage of proteome from some organelles such as the classical secretory pathway. Due to the central role of this pathway in protein and lipid metabolism, organelle biogenesis, cell cycle, apoptosis and proliferation, and to be recognized as a bottleneck for protein secretion in mammalian cells, its characterization through subcellular proteomics represents a promising strategy for identification of key targets. In fact, over-expression of some proteins from endoplasmic reticulum and Golgi Apparatus has increased the Qp of HEK293 and CHO cells producing different RP. Recently, proteomic reports of subcellular compartments from a variety of cells have been extensively used to study the protein composition of organelles, protein dynamics and discovering of novel proteins involved in the secretion pathway. Thus, this approach was applied to the secretory pathway of CHO cells to identify novel proteins linked to Qp in the present study. Differential proteomics from all remaining compartments was also carried out, giving new lights over other cellular mechanisms associated to the higher producer phenotype. The novel differentially expressed proteins (DEP) found will improve the engineering strategies of CHO cells that will have a positive impact on titer and quality of RP. Cell lines CHO DP-12 clone #1933 CRL-12444 and clone #1934 CRL-12445, which secrete a mAb against human interleukin 8 (IL-8), were used as cell models of recombinant CHO cell lines producing the same RP at different Qp. Our goal is that the processing of raw proteomics data and the identification and classification of DEP belonging to the classical secretory pathway will allow to propose a model by which the increase in the productivity of CHO cells has been associated with the deregulation of proteins from this pathway that show differential expression among the clones under study. This strategy of subcellular proteomics allowed us to identify a large number of new targets and cell pathways with respect to previous whole cell proteomic studies, which can improve the understanding of the molecular mechanisms behind RP production and provide the basis to design new sub-lines with stronger capabilities.

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Spatial proteomics reveals differences in the cellular architecture of antibody-producing CHO and plasma-cell derived cells

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