Network reconstruction of the mouse secretory pathway applied on CHO cell transcriptome data

Abstract

Background: Protein secretion is one of the most important processes in eukaryotes. It is based on a highly complex machinery involving numerous proteins in several cellular compartments. The elucidation of the cell biology of the secretory machinery is of great importance, as it drives protein expression for biopharmaceutical industry, a 140 billion USD global market. However, the complexity of secretory process is difficult to describe using a simple reductionist approach, and therefore a promising avenue is to employ the tools of systems biology.

Results: On the basis of manual curation of the literature on the yeast, human, and mouse secretory pathway, we have compiled a comprehensive catalogue of characterized proteins with functional annotation and their interconnectivity. Thus we have established the most elaborate reconstruction (RECON) of the functional secretion pathway network to date, counting 801 different components in mouse. By employing our mouse RECON to the CHO-K1 genome in a comparative genomic approach, we could reconstruct the protein secretory pathway of CHO cells counting 764 CHO components. This RECON furthermore facilitated the development of three alternative methods to study protein secretion through graphical visualizations of omics data. We have demonstrated the use of these methods to identify potential new and known targets for engineering improved growth and IgG production, as well as the general observation that CHO cells seem to have less strict transcriptional regulation of protein secretion than healthy mouse cells.

Conclusions: The RECON of the secretory pathway represents a strong tool for interpretation of data related to protein secretion as illustrated with transcriptomic data of Chinese Hamster Ovary (CHO) cells, the main platform for mammalian protein production.

Keywords: Chinese hamster ovary cells; Pathway reconstruction; Protein secretion; RNA-Seq; Secretion pathway.

Fig. 1

The reconstruction process of the mouse secretory machinery. The process from the overall secretory pathway with: a defining the subsystems, b classifying functional grouping and protein complexes within the subsystems, to c schematically categorising and adding interactions at the level of sequence, gene, and proteins

Fig. 2

Hierarchical cluster analysis with average-linkage of mouse expression levels. a Dendrogram representing the hierarchical clustering of the gene expression levels of the components from the subsystems of ERAD, PF, proteasome, and translocation. Vertical colour bar: Purple, Proteasome; Blue, ER associated degradation (ERAD); Green, Protein folding (PF) and translocation. b Gene expression levels across all samples within proteasome. c Gene expression levels across all samples within genes with the functional annotation protein folding. d Gene expression levels across all samples within genes related to the ERAD. Blue, ER associated degradation (ERAD); Green, Protein folding (PF) and translocation; Purple, Proteasome. 1: CNS_e11.5-1, 2: CNS_e11.5-2, 3: CNS_e14-1, 4: CNS_e14-2, 5: CNS_e18-1, 6: CNS_e18-2, 7: Placenta_8w-1, 8: Placenta_8w-2, 9: Limb_e14.5-1, 10: Limb_e14.5-2, 11: Wholebrain_e14.5-1, 12: Wholebrain_e14.5-2, 13: Bladder_8w-1, 14: Bladder_8w-2, 15: Cerebellum_8w-1, 16: Cerebellum_8w-2, 17: Liver_e14-1, 18: Liver_e14-2, 19: Liver_e14.5-1, 20: Liver_e14.5-2, 21: Liver_e18-1, 22: Liver_e18-2

Fig. 3

Spearman correlated expression profiles. Expression profiles correlated by Spearman correlation coefficient to the selected protein folding components from Fig. 2c. a Expression profile of correlated gene Rbbp7 (red) across all mouse samples visualised with the expression profiles of Fig. 2c. b Expression profile across all mouse samples of the gene Mecp2 (red) correlated by squared Spearman coefficient visualised with the expression profiles of Fig. 2c

Fig. 4

Hierarchical cluster analysis with average-linkage of CHO cells expression levels. a Dendrogram representing the hierarchical clustering of the gene expression levels of the components from the subsystems of ERAD, protein folding, and translocation and the proteasome protein complex. Vertical colour bar: Purple, Proteasome; Blue, ER associated degradation (ERAD); Green, protein folding, and translocation. b Gene expression levels across all samples for components with the functional annotation proteasome clustering in mouse. c Gene expression levels across all samples for components with the functional annotation protein folding in mouse. Grey shadow indicates the position of the components in the hierarchical clustering of the CHO genes. d Gene expression levels across all samples for components with the functional annotation ERAD that clustered in mouse. Horizontal bar, identifier of samples. Top line: protein expressed; no recombinant proteins (grey), IgG (green), and FVIII high levels (dark purple), FVIII medium levels (purple), FVIII low levels (light purple). Middle line: cultivation phase; exponential growth (light blue), stationary phase (Dark blue). Bottom line: NaBu treatment (red)

Fig. 5

Components of the secretory network gene expression correlated with growth and protein production. The Spearman correlation coefficient is calculated for gene expression level to both growth rate and IgG production rate. Each dot marks a component of the secretory network. The highlighted points in blue are previously described generic targets. A Green circle indicates known targets associated with protein folding and UPR. Red circles indicate known targets associated with activation or inhibition of apoptosis

Fig. 6

Graphical representation of the reconstructed secretory network. The change in differential expression of each components is visualised by the log2 fold change: up-regulation (red) and down regulation (blue). The intensity of the colour indicates the level in fold change. No cut-off to the fold change was added since minor changes in the expression level are important when identifying areas of activity and processes in the secretory network. Nodes are circled by a thicker line if FDR < 0.05. a The complete network graphically visualised in Cytoscape. b Selected protein complexes of OST, COPI, COPII, ESCRT-I, and the functional group of ER glycosylation and proteasome displayed in their position within the secretion pathway. c Proteasome components overlaid with gene expression data with the effect of secretion stress. d Proteasome protein/functional complex overlaid with gene expression data with the difference of exponential growth phase and stationary phase. Nodes: Green, Function; Turquoise, Proteins complex; Yellow, isoprotein; Red, Up-regulated; Blue, Down-regulated