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Cited by 207Glycosylation and Glycoproteins ResearchViral Infectious Diseases and Gene Expression in InsectsCarbohydrate Chemistry and Synthesis

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Glycomics Profiling of Chinese Hamster Ovary Cell Glycosylation Mutants Reveals N-Glycans of a Novel Size and Complexity

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North, Simon J., Huang, Hung-Hsiang, Sundaram, Subha, Jang-Lee, Jihye, Etienne, A. Tony, Trollope, Alana, Chalabi, Sara, Dell, Anne, Stanley, Pamela, Haslam, Stuart M.·2010·Journal of Biological Chemistry
DOI 10.1074/jbc.m109.068353PMC PMC2820803
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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.

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FIGURE 1. Altered N-glycans and O-GalNAc glycans in the CHO mutants analyzed here. A loss or reduction of a sugar residue at a particular position is indicated with a circled −, and the gain of a sugar residue is shown by a circled +. Symbolic nomenclature is presented as outlined by the Consortium for Functional Glycomics Nomenclature Committee. Full details are available on line.
FIGURE 1. Altered N-glycans and O-GalNAc glycans in the CHO mutants analyzed here. A loss or reduction of a sugar residue at a particular position is indicated with a circled −, and the gain of a sugar residue is shown by a circled +. Symbolic nomenclature is presented as outlined by the Consortium for Functional Glycomics Nomenclature Committee. Full details are available on line.CC BY-NC · PubMed Central
FIGURE 2. MALDI-TOF MS profiles of the permethylated N-linked glycans derived from wild type CHO cells grown in monolayer. For complete annotation of the spectrum, see supplemental Table S1. Data were obtained from the 50% MeCN fraction, and all molecular ions are present in sodiated form ([M + Na]+).
FIGURE 2. MALDI-TOF MS profiles of the permethylated N-linked glycans derived from wild type CHO cells grown in monolayer. For complete annotation of the spectrum, see supplemental Table S1. Data were obtained from the 50% MeCN fraction, and all molecular ions are present in sodiated form ([M + Na]+).CC BY-NC · PubMed Central
FIGURE 3. Comparison between the MALDI-TOF MS profiles of the permethylated O-glycans derived from wild type CHO cells grown in suspension (A) and wild type CHO cells grown in monolayer (B). Data were obtained from the 35% MeCN fraction, and all molecular ions are present in sodiated form ([M + Na]+).
FIGURE 3. Comparison between the MALDI-TOF MS profiles of the permethylated O-glycans derived from wild type CHO cells grown in suspension (A) and wild type CHO cells grown in monolayer (B). Data were obtained from the 35% MeCN fraction, and all molecular ions are present in sodiated form ([M + Na]+).CC BY-NC · PubMed Central
FIGURE 4. Comparison between the MALDI-TOF MS profiles of the permethylated N-glycans derived from wild type CHO cells grown in suspension (A) and wild type CHO cells grown in monolayer (B). Data were obtained from the 50% MeCN fraction and all molecular ions are present in sodiated form ([M + Na]+). The peaks at m/z 2623 and 3054 are highlighted in green to better aid meaningful comparison. The ion at m/z 4040.9 (highlighted yellow) corresponds to the exemplar MS/MS shown in Fig. 5.
FIGURE 4. Comparison between the MALDI-TOF MS profiles of the permethylated N-glycans derived from wild type CHO cells grown in suspension (A) and wild type CHO cells grown in monolayer (B). Data were obtained from the 50% MeCN fraction and all molecular ions are present in sodiated form ([M + Na]+). The peaks at m/z 2623 and 3054 are highlighted in green to better aid meaningful comparison. The ion at m/z 4040.9 (highlighted yellow) corresponds to the exemplar MS/MS shown in Fig. 5.CC BY-NC · PubMed Central
FIGURE 5. MALDI-TOF/TOF MS/MS spectrum of the permethylated N-glycan at m/z 4040. 9, derived from the N-glycan spectrum of wild type CHO cells grown in suspension (Fig. 4, highlighted in yellow). Assignments of the fragment ions generated and alternative antennal arrangements are shown. The ion highlighted in red represents the cleavage of multiple antennae in a fashion that does not facilitate clear annotation.
FIGURE 5. MALDI-TOF/TOF MS/MS spectrum of the permethylated N-glycan at m/z 4040. 9, derived from the N-glycan spectrum of wild type CHO cells grown in suspension (Fig. 4, highlighted in yellow). Assignments of the fragment ions generated and alternative antennal arrangements are shown. The ion highlighted in red represents the cleavage of multiple antennae in a fashion that does not facilitate clear annotation.CC BY-NC · PubMed Central
FIGURE 6. Selected mass range comparison between the MALDI-TOF MS profiles of the permethylated N-glycans derived from wild type sCHO cells (A), Lec2 sCHO (B), and Lec13 sCHO (C). For complete annotation of the spectra, see supplemental Table S1. Data were obtained from the 50% MeCN fraction, and all molecular ions are present in sodiated form ([M + Na]+).
FIGURE 6. Selected mass range comparison between the MALDI-TOF MS profiles of the permethylated N-glycans derived from wild type sCHO cells (A), Lec2 sCHO (B), and Lec13 sCHO (C). For complete annotation of the spectra, see supplemental Table S1. Data were obtained from the 50% MeCN fraction, and all molecular ions are present in sodiated form ([M + Na]+).CC BY-NC · PubMed Central
FIGURE 7. Selected mass range comparison between the MALDI-TOF MS profiles of the permethylated N-linked glycans derived from CHO cells grown in suspension (A), the Lec4 mutant cell line (B), and the LEC10 mutant cell line (C). Peaks highlighted in gray or green are presented as such for comparative purposes (see text). For complete annotation of the spectra, see supplemental Table S1. Data were obtained from the 50% MeCN fraction and all molecular ions are present in sodiated form ([M + Na]+).
FIGURE 7. Selected mass range comparison between the MALDI-TOF MS profiles of the permethylated N-linked glycans derived from CHO cells grown in suspension (A), the Lec4 mutant cell line (B), and the LEC10 mutant cell line (C). Peaks highlighted in gray or green are presented as such for comparative purposes (see text). For complete annotation of the spectra, see supplemental Table S1. Data were obtained from the 50% MeCN fraction and all molecular ions are present in sodiated form ([M + Na]+).CC BY-NC · PubMed Central
FIGURE 8. Selected mass range comparison between the MALDI-TOF MS profiles of the permethylated N-linked glycans derived from wild type CHO cells grown in suspension (A), the Lec1 mutant cell line (B), and the Lec3. 2.8.1 mutant cell line (C). For complete annotation of the spectra, see supplemental Table S1. Data were obtained from the 50% MeCN fraction, and all molecular ions are present in sodiated form ([M + Na]+).
FIGURE 8. Selected mass range comparison between the MALDI-TOF MS profiles of the permethylated N-linked glycans derived from wild type CHO cells grown in suspension (A), the Lec1 mutant cell line (B), and the Lec3. 2.8.1 mutant cell line (C). For complete annotation of the spectra, see supplemental Table S1. Data were obtained from the 50% MeCN fraction, and all molecular ions are present in sodiated form ([M + Na]+).CC BY-NC · PubMed Central
FIGURE 9. Selected mass range comparison between the MALDI-TOF MS profiles of the permethylated N-glycans derived from CHO cells grown in suspension (A), the LEC11 mutant cell line (B), the LEC12 mutant cell line (C), and the LEC30 mutant cell line (D). Only structures bearing multiple fucoses in the mutant samples are annotated with structures to simplify the presentation, although all other glycan peaks are labeled with their m/z values alone. For complete annotation of the spectra, see supplemental Table S1. Data were obtained from the 50% MeCN fraction, and all molecular ions are present in sodiated form ([M + Na]+).
FIGURE 9. Selected mass range comparison between the MALDI-TOF MS profiles of the permethylated N-glycans derived from CHO cells grown in suspension (A), the LEC11 mutant cell line (B), the LEC12 mutant cell line (C), and the LEC30 mutant cell line (D). Only structures bearing multiple fucoses in the mutant samples are annotated with structures to simplify the presentation, although all other glycan peaks are labeled with their m/z values alone. For complete annotation of the spectra, see supplemental Table S1. Data were obtained from the 50% MeCN fraction, and all molecular ions are present in sodiated form ([M + Na]+).CC BY-NC · PubMed Central
FIGURE 10. Exemplar MALDI-tof/TOF data of fucosylated N-glycans. A, MS/MS spectra of the permethylated N-glycan at m/z 2779.2, derived from the N-linked spectrum of the LEC11 mutant cell line (Fig. 9B, highlighted), with the two main components of the peak indicated, a biantennary sialyl Lewisx containing structure 1) and a biantennary Lewisx glycan 2). B, MS/MS spectrum of the permethylated N-glycan at m/z 2779.2, derived from the N-linked spectrum of the LEC12 mutant cell line (Fig. 9C, highlighted). C, MS/MS spectrum of the permethylated N-glycan at m/z 3838.9, derived from the N-linked spectrum of the LEC30 mutant cell line (Fig. 9D, highlighted). Assignments of the fragment ions are shown. The ions highlighted in red represent the cleavage of multiple antennae in a fashion that does not facilitate clear annotation.
FIGURE 10. Exemplar MALDI-tof/TOF data of fucosylated N-glycans. A, MS/MS spectra of the permethylated N-glycan at m/z 2779.2, derived from the N-linked spectrum of the LEC11 mutant cell line (Fig. 9B, highlighted), with the two main components of the peak indicated, a biantennary sialyl Lewisx containing structure 1) and a biantennary Lewisx glycan 2). B, MS/MS spectrum of the permethylated N-glycan at m/z 2779.2, derived from the N-linked spectrum of the LEC12 mutant cell line (Fig. 9C, highlighted). C, MS/MS spectrum of the permethylated N-glycan at m/z 3838.9, derived from the N-linked spectrum of the LEC30 mutant cell line (Fig. 9D, highlighted). Assignments of the fragment ions are shown. The ions highlighted in red represent the cleavage of multiple antennae in a fashion that does not facilitate clear annotation.CC BY-NC · PubMed Central

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  1. Stanley, 1989, Mol. Cell. Biol., 9, 377

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Metadata

Authors
North, S. J., Huang, H.-H., Sundaram, S., Jang-Lee, J., Etienne, A. T., Trollope, A., Chalabi, S., Dell, A., Stanley, P., & Haslam, S. M.
Affiliations
  • Imperial College London (GB)
  • Albert Einstein College of Medicine (US)
Corresponding author
  • Stanley, Pamela
  • Haslam, Stuart M.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2010-02
Volume
285
Issue
8
Pages
5759-5775
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m109.068353
PMC
PMC2820803
OpenAlex
W2033604747
Cited by
207
References
44
Citations by year
2026: 5 · 2025: 7 · 2024: 8 · 2023: 6 · 2022: 10 · 2021: 12 · 2020: 11 · 2019: 15 · 2018: 17 · 2017: 16 · 2016: 8 · 2015: 11 · 2014: 21 · 2013: 17 · 2012: 13
FWCI
5.78
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
  • Wellcome Trust
  • National Institutes of Health
  • Directorate for Biological Sciences
  • U.S. Public Health Service (NCI RO1-36434)
  • Biotechnology and Biological Sciences Research Council (BB/F008309/1, B19088)
  • National Cancer Institute
  • National Institute of General Medical Sciences (GM 62116, U54GM062116)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
  • Physical Sciences › Chemistry › Organic Chemistry › Carbohydrate Chemistry and Synthesis
Keywords
glycosylation mutants, CHO cells, N-glycans, O-glycans, glycomics, lectin resistance, glycosyltransferases, N-acetyllactosamine, fucosylated N-glycans, CD15, sialyl Lewis X
MeSH terms
Animals, Carbohydrate Sequence, Cricetulus, Glycosylation, Cricetinae, Mutation, Polysaccharides, CHO Cells

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