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Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity.

ABSTRACT: Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of altering one specific glycosyltransferase or glycosylation activity on the overall expression of cellular glycans, an analysis of the N-glycans and major O-glycans of a panel of CHO mutants was performed using glycomic analyses anchored by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. We report here the complement of the major N-glycans and O-glycans present in nine distinct CHO glycosylation mutants. Parent CHO cells grown in monolayer versus suspension culture had similar profiles of N- and O-GalNAc glycans, although the profiles of glycosylation mutants Lec1, Lec2, Lec3.2.8.1, Lec4, LEC10, LEC11, LEC12, Lec13, and LEC30 were consistent with available genetic and biochemical data. However, the complexity of the range of N-glycans observed was unexpected. Several of the complex N-glycan profiles contained structures of m/z approximately 13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Gal beta1-4GlcNAc)(n) units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewis(x) and sialyl-Lewis(x) determinants. This analysis reveals the larger-than-expected complexity of N-glycans in CHO cell mutants that may be used in a broad variety of functional glycomics studies and for making recombinant glycoproteins.

SUBMITTER: North SJ

PROVIDER: S-EPMC2820803 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity.

North Simon J SJ Huang Hung-Hsiang HH Sundaram Subha S Jang-Lee Jihye J Etienne A Tony AT Trollope Alana A Chalabi Sara S Dell Anne A Stanley Pamela P Haslam Stuart M SM

The Journal of biological chemistry 20091201 8

Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of a ...[more]

PMID: 19951948

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Project description:The Chinese hamster ovary (CHO) cell is the gold standard for manufacturing of glycosylated recombinant proteins for production of biotherapeutics. The similarity of its glycosylation patterns to the human versions enable the products of this cell line favorable pharmacokinetic properties and lower likelihood of causing immunogenic responses. Because glycan structures are the product of the concerted action of intracellular enzymes, it is difficult to predict a priori how the effects of genetic manipulations alter glycan structures of cells and therapeutic properties. For that reason, quantitative models able to predict glycosylation have emerged as promising tools to deal with the complexity of glycosylation processing. For example, an earlier version of the same model used in this study was used by others to successfully predict changes in enzyme activities that could produce a desired change in glycan structure. In this study we utilize an updated version of this model to provide a comprehensive analysis of N-glycosylation in ten Chinese hamster ovary (CHO) cell lines that include a wild type parent and nine mutants of CHO, through interpretation of previously published mass spectrometry data. The updated N-glycosylation mathematical model contains up to 50,605 glycan structures. Adjusting the enzyme activities in this model to match N-glycan mass spectra produces detailed predictions of the glycosylation process, enzyme activity profiles and complete glycosylation profiles of each of the cell lines. These profiles are consistent with biochemical and genetic data reported previously. The model-based results also predict glycosylation features of the cell lines not previously published, indicating more complex changes in glycosylation enzyme activities than just those resulting directly from gene mutations. The model predicts that the CHO cell lines possess regulatory mechanisms that allow them to adjust glycosylation enzyme activities to mitigate side effects of the primary loss or gain of glycosylation function known to exist in these mutant cell lines. Quantitative models of CHO cell glycosylation have the potential for predicting how glycoengineering manipulations might affect glycoform distributions to improve the therapeutic performance of glycoprotein products.

Project description:Cell surface proteins carrying N-glycans play important roles in inter- and intracellular processes including cell adhesion, development, and cellular recognition. Dysregulation of the glycosylation machinery has been implicated in various diseases, and investigation of global differential cell surface proteome effects due to the loss of N-glycosylation will provide comprehensive insights into their pathogenesis. Cell surface proteins isolated from Parent Pro-5 CHO cells (W5 cells), two CHO mutants with loss of N-glycosylation function derived from Pro-5 CHO (Lec1 and Lec4 cells), were subjected to proteome analysis via high-resolution LCMS. We identified 44 and 43 differentially expressed membrane proteins in Lec1 and Lec4 cells, respectively, as compared to W5 cells. The defective N-glycosylation mutants showed increased abundance of integrin subunits in Lec1 and Lec4 cells at the cell surface. We also found significantly reduced levels of IGF-1R (Insulin like growth factor-1 receptor); a receptor tyrosine kinase; and the GTPase activating protein IQGAP1 (IQ motif-containing GTPase activating protein), a highly conserved cytoplasmic scaffold protein) in Lec1 and Lec4 cells. In silico docking studies showed that the IQ domain of IQGAP1 interacts with the kinase domain of IGF-1R. The integrin signaling and insulin growth factor receptor signaling were also enriched according to GSEA analysis and pathway analysis of differentially expressed proteins. Significant reductions of phosphorylation of ERK1 and ERK2 in Lec1 and Lec4 cells were observed upon IGF-1R ligand (IGF-1 LR3) stimulation. IGF-1 LR3, known as Long arginine3-IGF-1, is a synthetic protein and lengthened analog of insulin-like growth factor 1. The work suggests a novel mechanism for the activation of IGF-1 dependent ERK signaling in CHO cells, wherein IQGAP1 plausibly functions as an IGF-1R-associated scaffold protein. Appropriate glycosylation by the enzymes MGAT1 and MGAT5 is thus essential for processing of cell surface receptor IGF-1R, a potential binding partner in IQGAP1 and ERK signaling, the integral components of the IGF pathway.

Dataset Information

Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity.

Publications

Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity.

Similar Datasets

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