Genome-scale reconstructions of the mammalian secretory pathway predict metabolic costs and limitations of protein secretion.

In mammalian cells, >25% of synthesized proteins are exported through the secretory pathway. The pathway complexity, however, obfuscates its impact on the secretion of different proteins. Unraveling its impact on diverse proteins is particularly important for biopharmaceutical production. Here we delineate the core secretory pathway functions and integrate them with genome-scale metabolic reconstructions of human, mouse, and Chinese hamster ovary cells. The resulting reconstructions enable the computation of energetic costs and machinery demands of each secreted protein. By integrating additional omics data, we find that highly secretory cells have adapted to reduce expression and secretion of other expensive host cell proteins. Furthermore, we predict metabolic costs and maximum productivities of biotherapeutic proteins and identify protein features that most significantly impact protein secretion. Finally, the model successfully predicts the increase in secretion of a monoclonal antibody after silencing a highly expressed selection marker. This work represents a knowledgebase of the mammalian secretory pathway that serves as a novel tool for systems biotechnology.

High-Throughput Lipidomic and Transcriptomic Analysis To Compare SP2/0, CHO, and HEK-293 Mammalian Cell Lines.

A combined lipidomics and transcriptomics analysis was performed on mouse myeloma SP2/0, Chinese hamster ovary (CHO), and human embryonic kidney (HEK) cells in order to compare widely used mammalian expression systems. Initial thin layer chromatography (TLC) analysis indicated that phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were the major lipid components in all cell lines with lower amounts of sphingomyelin (SM) in SP2/0 compared to CHO and HEK, which was subsequently confirmed and expanded upon following mass spectrometry (MS) analysis. HEK contained 4-10-fold higher amounts of lyso phosphatidylethanolamine (LPE) and 2-4-fold higher amounts of lyso phosphatidylcholine (LPC) compared to SP2/0 and CHO cell lines. C18:1 followed by C16:1 were the main contributors to the difference in both LPE and LPC levels. Alternatively, the SP2/0 cell line exhibited 30-65-fold lower amounts of SM principally in the amount of 16:0. By mapping the transcriptomics data to KEGG pathways, we found expression levels of secretory phospholipase A2 (sPLA2), lysophospholipid acyltransferase (LPEAT), lysophosphatidylcholine acyltransferase (LPCAT), and lysophospholipase (LYPLA) can contribute to the differences in LPE and LPC. Sphingomyelin synthases (SMS) and sphingomyelin phosphodiesterase (SMase) enzymes may play roles in SM differences across the three cell lines. The results of this study provide insights that will aid the understanding of the physiological and secretory differences across recombinant protein production systems.

A Consensus Genome-scale Reconstruction of Chinese Hamster Ovary Cell Metabolism.

Chinese hamster ovary (CHO) cells dominate biotherapeutic protein production and are widely used in mammalian cell line engineering research. To elucidate metabolic bottlenecks in protein production and to guide cell engineering and bioprocess optimization, we reconstructed the metabolic pathways in CHO and associated them with >1,700 genes in the Cricetulus griseus genome. The genome-scale metabolic model based on this reconstruction, iCHO1766, and cell-line-specific models for CHO-K1, CHO-S, and CHO-DG44 cells provide the biochemical basis of growth and recombinant protein production. The models accurately predict growth phenotypes and known auxotrophies in CHO cells. With the models, we quantify the protein synthesis capacity of CHO cells and demonstrate that common bioprocess treatments, such as histone deacetylase inhibitors, inefficiently increase product yield. However, our simulations show that the metabolic resources in CHO are more than three times more efficiently utilized for growth or recombinant protein synthesis following targeted efforts to engineer the CHO secretory pathway. This model will further accelerate CHO cell engineering and help optimize bioprocesses.

Cellular traffic cops: the interplay between lipids and proteins regulates vesicular formation, trafficking, and signaling in mammalian cells.

Protein secretion and vesicular trafficking in mammalian cells rely on several key lipids including sphingolipids, phospholipids, and neutral lipids crucial to protein processing and other intracellular events. Proteins interact with these lipids to alter the shape of lipid bilayer, thereby playing a pivotal role in cellular sorting. Although some efforts have elucidated the role of these components, extensive studies are needed to further decipher the protein-lipid interactions along with the effect of membrane curvature and rafts in sorting of proteins. The regulatory role of proteins in subcellular localization and metabolism of lipids also needs to be described. Recent studies on the role of lipid-protein interactions in modulating membrane shape, signal transduction, and vesicular trafficking are presented in this review.

Elucidation of the CHO Super-Ome (CHO-SO) by Proteoinformatics.

Chinese hamster ovary (CHO) cells are the preferred host cell line for manufacturing a variety of complex biotherapeutic drugs including monoclonal antibodies. We performed a proteomics and bioinformatics analysis on the spent medium from adherent CHO cells. Supernatant from CHO-K1 culture was collected and subjected to in-solution digestion followed by LC/LC-MS/MS analysis, which allowed the identification of 3281 different host cell proteins (HCPs). To functionally categorize them, we applied multiple bioinformatics tools to the proteins identified in our study including SignalP, TargetP, SecretomeP, TMHMM, WoLF PSORT, and Phobius. This analysis provided information on the presence of signal peptides, transmembrane domains, and cellular localization and showed that both secreted and intracellular proteins were constituents of the supernatant. Identified proteins were shown to be localized to the secretory pathway including ones playing roles in cell growth, proliferation, and folding as well as those involved in protein degradation and removal. After combining proteins predicted to be secreted or having a signal peptide, we identified 1015 proteins, which we termed as CHO supernatant-ome (CHO-SO), or superome. As a part of this effort, we created a publically accessible web-based tool called GO-CHO to functionally categorize proteins found in CHO-SO and identify enriched molecular functions, biological processes, and cellular components. We also used a tool to evaluate the immunogenicity potential of high-abundance HCPs. Among enriched functions were catalytic activity and structural constituents of the cytoskeleton. Various transport related biological processes, such as vesicle mediated transport, were found to be highly enriched. Extracellular space and vesicular exosome associated proteins were found to be the most enriched cellular components. The superome also contained proteins secreted from both classical and nonclassical secretory pathways. The work and database described in our study will enable the CHO community to rapidly identify high-abundance HCPs in their cultures and therefore help assess process and purification methods used in the production of biologic drugs.

A multi-pronged investigation into the effect of glucose starvation and culture duration on fed-batch CHO cell culture.

In this study, omics-based analysis tools were used to explore the effect of glucose starvation and culture duration on monoclonal antibody (mAb) production in fed-batch CHO cell culture to gain better insight into how these parameters can be controlled to ensure optimal mAb productivity and quality. Titer and N-glycosylation of mAbs, as well as proteomic signature and metabolic status of the production cells in the culture were assessed. We found that the impact of glucose starvation on the titer and N-glycosylation of mAbs was dependent on the degree of starvation during early stationary phase of the fed-batch culture. Higher degree of glucose starvation reduced intracellular concentrations of UDP-GlcNAc and UDP-GalNAc, but increased the levels of UDP-Glc and UDP-Gal. Increased GlcNAc and Gal occupancy correlated well with increased degree of glucose starvation, which can be attributed to the interplay between the dilution effect associated with change in specific productivity of mAbs and the changed nucleotide sugar metabolism. Herein, we also show and discuss that increased cell culture duration negatively affect the maturation of glycans. In addition, comparative proteomics analysis of cells was conducted to observe differences in protein abundance between early growth and early stationary phases. Generally higher expression of proteins involved in regulating cellular metabolism, extracellular matrix, apoptosis, protein secretion and glycosylation was found in early stationary phase. These analyses offered a systematic view of the intrinsic properties of these cells and allowed us to explore the root causes correlating culture duration with variations in the productivity and glycosylation quality of monoclonal antibodies produced with CHO cells.

Coupling enrichment methods with proteomics for understanding and treating disease.

Owing to recent advances in proteomics analytical methods and bioinformatics capabilities there is a growing trend toward using these capabilities for the development of drugs to treat human disease, including target and drug evaluation, understanding mechanisms of drug action, and biomarker discovery. Currently, the genetic sequences of many major organisms are available, which have helped greatly in characterizing proteomes in model animal systems and humans. Through proteomics, global profiles of different disease states can be characterized (e.g. changes in types and relative levels as well as changes in PTMs such as glycosylation or phosphorylation). Although intracellular proteomics can provide a broad overview of physiology of cells and tissues, it has been difficult to quantify the low abundance proteins which can be important for understanding the diseased states and treatment progression. For this reason, there is increasing interest in coupling comparative proteomics methods with subcellular fractionation and enrichment techniques for membranes, nucleus, phosphoproteome, glycoproteome as well as low abundance serum proteins. In this review, we will provide examples of where the utilization of different proteomics-coupled enrichment techniques has aided target and biomarker discovery, understanding the drug targeting mechanism, and mAb discovery. Taken together, these improvements will help to provide a better understanding of the pathophysiology of various diseases including cancer, autoimmunity, inflammation, cardiovascular disease, and neurological conditions, and in the design and development of better medicines for treating these afflictions.

Physiologic and pathophysiologic consequences of altered sialylation and glycosylation on ion channel function.

Voltage-gated ion channels are transmembrane proteins that regulate electrical excitability in cells and are essential components of the electrically active tissues of nerves, muscle and the heart. Potassium channels are one of the largest subfamilies of voltage sensitive channels and are among the most-studied of the voltage-gated ion channels. Voltage-gated channels can be glycosylated and changes in the glycosylation pattern can affect ion channel function, leading to neurological and neuromuscular disorders and congenital disorders of glycosylation (CDG). Alterations in glycosylation can also be acquired and appear to play a role in development and aging. Recent studies have focused on the impact of glycosylation and sialylation on ion channels, particularly for voltage-gated potassium and sodium channels. The terminal step of sialylation often affects channel activation and inactivation kinetics. The presence of sialic acids on O or N-glycans can alter the gating mechanism and cause conformational changes in the voltage-sensing domains due to sialic acid's negative charges. This manuscript will provide an overview of sialic acids, potassium and sodium channel function, and the impact of sialylation on channel activation and deactivation.

Glycoproteomic and glycomic databases.

Protein glycosylation serves critical roles in the cellular and biological processes of many organisms. Aberrant glycosylation has been associated with many illnesses such as hereditary and chronic diseases like cancer, cardiovascular diseases, neurological disorders, and immunological disorders. Emerging mass spectrometry (MS) technologies that enable the high-throughput identification of glycoproteins and glycans have accelerated the analysis and made possible the creation of dynamic and expanding databases. Although glycosylation-related databases have been established by many laboratories and institutions, they are not yet widely known in the community. Our study reviews 15 different publicly available databases and identifies their key elements so that users can identify the most applicable platform for their analytical needs. These databases include biological information on the experimentally identified glycans and glycopeptides from various cells and organisms such as human, rat, mouse, fly and zebrafish. The features of these databases - 7 for glycoproteomic data, 6 for glycomic data, and 2 for glycan binding proteins are summarized including the enrichment techniques that are used for glycoproteome and glycan identification. Furthermore databases such as Unipep, GlycoFly, GlycoFish recently established by our group are introduced. The unique features of each database, such as the analytical methods used and bioinformatical tools available are summarized. This information will be a valuable resource for the glycobiology community as it presents the analytical methods and glycosylation related databases together in one compendium. It will also represent a step towards the desired long term goal of integrating the different databases of glycosylation in order to characterize and categorize glycoproteins and glycans better for biomedical research.

Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome.

Chinese hamster ovary (CHO) cells, first isolated in 1957, are the preferred production host for many therapeutic proteins. Although genetic heterogeneity among CHO cell lines has been well documented, a systematic, nucleotide-resolution characterization of their genotypic differences has been stymied by the lack of a unifying genomic resource for CHO cells. Here we report a 2.4-Gb draft genome sequence of a female Chinese hamster, Cricetulus griseus, harboring 24,044 genes. We also resequenced and analyzed the genomes of six CHO cell lines from the CHO-K1, DG44 and CHO-S lineages. This analysis identified hamster genes missing in different CHO cell lines, and detected >3.7 million single-nucleotide polymorphisms (SNPs), 551,240 indels and 7,063 copy number variations. Many mutations are located in genes with functions relevant to bioprocessing, such as apoptosis. The details of this genetic diversity highlight the value of the hamster genome as the reference upon which CHO cells can be studied and engineered for protein production.

The emerging CHO systems biology era: harnessing the 'omics revolution for biotechnology.

Chinese hamster ovary (CHO) cells are the primary factories for biopharmaceuticals because of their capacity to correctly fold and post-translationally modify recombinant proteins compatible with humans. New opportunities are arising to enhance these cell factories, especially since the CHO-K1 cell line was recently sequenced. Now, the CHO systems biology era is underway. Critical 'omics data sets, including proteomics, transcriptomics, metabolomics, fluxomics, and glycomics, are emerging, allowing the elucidation of the molecular basis of CHO cell physiology. The incorporation of these data sets into mathematical models that describe CHO phenotypes will provide crucial biotechnology insights. As 'omics technologies and computational systems biology mature, genome-scale approaches will lead to major innovations in cell line development and metabolic engineering, thereby improving protein production and bioprocessing.

Proteomic analysis of Chinese hamster ovary cells.

To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimensional liquid chromatography, and solid phase extraction of glycopeptides (SPEG). From the 120 different mass spectrometry analyses generating 682,097 MS/MS spectra, 93,548 unique peptide sequences were identified with at most 0.02 false discovery rate (FDR). A total of 6164 grouped proteins were identified from both glycoproteome and proteome analysis, representing an 8-fold increase in the number of proteins currently identified in the CHO proteome. Furthermore, this is the first proteomic study done using the CHO genome exclusively, which provides for more accurate identification of proteins. From this analysis, the CHO codon frequency was determined and found to be distinct from humans, which will facilitate expression of human proteins in CHO cells. Analysis of the combined proteomic and mRNA data sets indicated the enrichment of a number of pathways including protein processing and apoptosis but depletion of proteins involved in steroid hormone and glycosphingolipid metabolism. Five-hundred four of the detected proteins included N-acetylation modifications, and 1292 different proteins were observed to be N-glycosylated. This first large-scale proteomic analysis will enhance the knowledge base about CHO capabilities for recombinant expression and provide information useful in cell engineering efforts aimed at modifying CHO cellular functions.

Increased expression of the integral membrane proteins EGFR and FGFR3 in anti-apoptotic Chinese hamster ovary cell lines.

Membrane proteins such as receptor tyrosine kinases (RTKs) have a vital role in many cellular functions, making them potential targets for therapeutic research. In this study, we investigated the coexpression of the anti-apoptosis gene Bcl-x(L) with model membrane proteins as a means of increasing membrane protein expression in mammalian cells. Chinese hamster ovary (CHO) cells expressing heterologous Bcl-x(L) and wild-type CHO cells were transfected with either epidermal growth factor receptor or fibroblast growth factor receptor 3. The CHO-Bcl-x(L) cell lines showed increased expression of both RTK proteins as compared with the wild-type CHO cell lines in transient expression analysis, as detected by Western blot and flow cytometry after 15 days of antibiotic selection in stable expression pools. Increased expression was also seen in clonal isolates from the CHO-Bcl-x(L) cell lines, whereas the clonal cell line expression was minimal in wild-type CHO cell lines. Our results demonstrate that application of the anti-apoptosis gene Bcl-x(L) can increase expression of RTK proteins in CHO cells. This approach may be applied to improve stable expression of other membrane proteins in the future using mammalian cell lines with Bcl-x(L) or perhaps other anti-apoptotic genes.

GlycoFish: a database of zebrafish N-linked glycoproteins identified using SPEG method coupled with LC/MS.

Zebrafish (Danio rerio) is a model organism that is used to study the mechanisms and pathways of human disorders. Many dysfunctions in neurological, development, and neuromuscular systems are due to glycosylation deficiencies, but the glycoproteins involved in zebrafish embryonic development have not been established. In this study, a mass spectrometry-based glycoproteomic characterization of zebrafish embryos was performed to identify the N-linked glycoproteins and N-linked glycosylation sites. To increase the number of glycopeptides, proteins from zebrafish were digested with two different proteases--chymotrypsin and trypsin--into peptides of different length. The N-glycosylated peptides of zebrafish were then captured by the solid-phase extraction of N-linked glycopeptides (SPEG) method and the peptides were identified with an LTQ OrbiTrap Velos mass spectrometer. From 265 unique glycopeptides, including 269 consensus NXT/S glycosites, we identified 169 different N-glycosylated proteins. The identified glycoproteins were highly abundant in proteins belonging to the transporter, cell adhesion, and ion channel/ion binding categories, which are important to embryonic, organ, and central nervous system development. This proteomics data will expand our knowledge about glycoproteins in zebrafish and may be used to elucidate the role that glycosylation plays in cellular processes and disease. The glycoprotein data are available through the GlycoFish database (http://betenbaugh.jhu.edu/GlycoFish) introduced in this paper.

GlycoFly: a database of Drosophila N-linked glycoproteins identified using SPEG--MS techniques.

Protein glycosylation affects cellular functions of the central nervous system (CNS). Its deficiency leads to neurological disorders such as ataxia, paralysis, learning disability, mental retardation, and memory loss. However, the glycoproteins that are responsible for these diseases are not well characterized. In this study, Drosophila melanogaster was used as a model organism to identify the N-glycosylated proteins and N-glycosylation sites of its CNS by means of proteomics. Adult fly heads were digested with chymotrypsin or trypsin and the N-linked glycopeptides were captured using solid phase extraction of N-linked glycopeptides (SPEG) technique followed by mass spectrometry (MS) analysis using LTQ OrbiTrap Velos. Three hundred and thirty new and 147 previously known glycoproteins were identified from 721 uniquely detected peptides that have 740 NXS/T glycosylation sites. The N-glycosylation sites were highly abundant in cell adhesion, ion channel, and ion binding molecules, which are important for nerve maturation, organ development, axon guidance, learning, and memory. Identification of the N-glycosylated sites of these proteins will enhance our knowledge of these proteins and serve as a basis for future studies to address the roles of these proteins in neurological function and disorders. A database for Drosophila N-linked glycopeptides ( http://betenbaugh.jhu.edu/GlycoFly ) has been established in this study as a resource for study of neurological disorders.