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Cited by 106Viral Infectious Diseases and Gene Expression in InsectsGlycosylation and Glycoproteins ResearchMonoclonal and Polyclonal Antibodies Research

The DOI matches its Crossref record; 65 of the 65 references it cites were found in a registry or an index.

Production of α2,6-sialylated IgG1 in CHO cells

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Raymond, Céline, Robotham, Anna, Spearman, Maureen, Butler, Michael, Kelly, John, Durocher, Yves·2015·mAbs
DOI 10.1080/19420862.2015.1029215PMC PMC4622614
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Abstract

The presence of α2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated access to well-characterized α2,6-sialylated IgGs would help elucidate the mechanism of this intriguing IgG's effector function. This study presents a method for the efficient Fc glycan α2,6-sialylation of a wild-type and a F243A IgG1 mutant by transient co-expression with the human α2,6-sialyltransferase 1 (ST6) and β1,4-galactosyltransferase 1 (GT) in CHO cells. Overexpression of ST6 alone only had a moderate effect on the glycoprofiles, whereas GT alone greatly enhanced Fc-galactosylation, but not sialylation. Overexpression of both GT and ST6 was necessary to obtain a glycoprofile dominated by α2,6-sialylated glycans in both antibodies. The wild-type was composed of the G2FS(6)1 glycan (38%) with remaining unsialylated glycans, while the mutant glycoprofile was essentially composed of G2FS(6)1 (25%), G2FS(3,6)2 (16%) and G2FS(6,6)2 (37%). The α2,6-linked sialic acids represented over 85% of all sialic acids in both antibodies. We discuss how the limited sialylation level in the wild-type IgG1 expressed alone or with GT results from the glycan interaction with Fc's amino acid residues or from intrinsic galactosyl- and sialyl-transferases substrate specificities.

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Figure 1. Complex biantennary N-linked glycan structures found in the Fc domain of IgGs. All complex glycans are composed of 4 N-acetylglucosamine residues (GlcNAc, blue squares), and 3 mannose residues (green circles). G0, G1, G2 indicate 0, 1 or 2 galactose residues (yellow circles). F indicates the presence of a core-fucose residue (red triangle). S1 and S2 indicate mono- and di-sialylated glycans (sialic acids are represented as purple diamonds). The sialic acid linkage type is indicated when required in parentheses: G1FS(3)1 and G1FS(6)1 designate G1FS1 carrying either a α2,3- or a α2,6-linked sialic acid, respectively. Similarly, G2FS1 may be G2FS(3)1 or G2FS(6)1. G2FS(3,3)2, G2FS(3,6)2 and G2FS(6,6)2 designate G2FS2 carrying 2 α2,3SA, one α2,3SA and one α2,6SA, or 2 α2,6SA, respectively. α1,3 and α1,6 designate the linkage types of the core mannose residues, and by extension refer to the branches initiated by these residues: the α1,3-arm and the α1,6-arm, respectively.
Figure 1. Complex biantennary N-linked glycan structures found in the Fc domain of IgGs. All complex glycans are composed of 4 N-acetylglucosamine residues (GlcNAc, blue squares), and 3 mannose residues (green circles). G0, G1, G2 indicate 0, 1 or 2 galactose residues (yellow circles). F indicates the presence of a core-fucose residue (red triangle). S1 and S2 indicate mono- and di-sialylated glycans (sialic acids are represented as purple diamonds). The sialic acid linkage type is indicated when required in parentheses: G1FS(3)1 and G1FS(6)1 designate G1FS1 carrying either a α2,3- or a α2,6-linked sialic acid, respectively. Similarly, G2FS1 may be G2FS(3)1 or G2FS(6)1. G2FS(3,3)2, G2FS(3,6)2 and G2FS(6,6)2 designate G2FS2 carrying 2 α2,3SA, one α2,3SA and one α2,6SA, or 2 α2,6SA, respectively. α1,3 and α1,6 designate the linkage types of the core mannose residues, and by extension refer to the branches initiated by these residues: the α1,3-arm and the α1,6-arm, respectively.CC BY-NC · PubMed Central
Figure 2. Lectin-blot analyses of TZM from culture supernatants. Various amounts of glycosyltransferases and harvest time were tested for optimal galactosylation and sialylation of the Fc. The lectin-blots show the mAbs’ heavy chain (reducing gels). Ponceau red staining was used to control the protein load. Panel (A) Co-expression of TZM and GT. The DNA mix was composed of 0% to 10% in weight of GT encoding plasmid, the rest being a 6:4 mixture of plasmids coding for TZM light and heavy chains. ECL was used to detect terminal galactosylation. Panel (B) Co-expression of TZM, GT and ST6. The DNA mix was composed of 2% of GT plasmid with 0% to 20% of ST6 coding plasmid, the rest being a 6:4 mixture of plasmids coding for TZM light and heavy chains. SNA was used to detect terminal α2,6-sialylation. Panel (C) HC α2,6-sialylation level from day 2 to day 9 post-transfection.
Figure 2. Lectin-blot analyses of TZM from culture supernatants. Various amounts of glycosyltransferases and harvest time were tested for optimal galactosylation and sialylation of the Fc. The lectin-blots show the mAbs’ heavy chain (reducing gels). Ponceau red staining was used to control the protein load. Panel (A) Co-expression of TZM and GT. The DNA mix was composed of 0% to 10% in weight of GT encoding plasmid, the rest being a 6:4 mixture of plasmids coding for TZM light and heavy chains. ECL was used to detect terminal galactosylation. Panel (B) Co-expression of TZM, GT and ST6. The DNA mix was composed of 2% of GT plasmid with 0% to 20% of ST6 coding plasmid, the rest being a 6:4 mixture of plasmids coding for TZM light and heavy chains. SNA was used to detect terminal α2,6-sialylation. Panel (C) HC α2,6-sialylation level from day 2 to day 9 post-transfection.CC BY-NC · PubMed Central
Figure 3. Capillary IEF (cIEF) electropherograms of TZM constructs. Panel (A) The cIEF profile of TZM is composed of a main charge variant and traces of acidic and basic variants. These traces are not the result of glycosylation since the non-glycosylated mutant TZM N297Q has an identical profile. In contrast, the acidic variants of TZMm are the result of sialylation. Panel (B1) cIEF profiles of purified TZM and TZMm expressed alone or in the presence of GT, ST6, or both. Panel (B2–4) Lectin-blots showing the α2,6-sialylation, α2,3-sialylation and terminal galactosylation levels of TZM heavy chain as revealed by SNA, MALII and ECL, respectively.
Figure 3. Capillary IEF (cIEF) electropherograms of TZM constructs. Panel (A) The cIEF profile of TZM is composed of a main charge variant and traces of acidic and basic variants. These traces are not the result of glycosylation since the non-glycosylated mutant TZM N297Q has an identical profile. In contrast, the acidic variants of TZMm are the result of sialylation. Panel (B1) cIEF profiles of purified TZM and TZMm expressed alone or in the presence of GT, ST6, or both. Panel (B2–4) Lectin-blots showing the α2,6-sialylation, α2,3-sialylation and terminal galactosylation levels of TZM heavy chain as revealed by SNA, MALII and ECL, respectively.CC BY-NC · PubMed Central
Figure 4. Fc/2 molecular weight profiles obtained by LC-ESI-MS analysis. The analysis was performed on 2 independent batches. Secondary peaks were detected at +16 Da for all glycopeptides and were considered to be caused by oxidation.
Figure 4. Fc/2 molecular weight profiles obtained by LC-ESI-MS analysis. The analysis was performed on 2 independent batches. Secondary peaks were detected at +16 Da for all glycopeptides and were considered to be caused by oxidation.CC BY-NC · PubMed Central
Figure 5. Effect of α2,3SA-specific sialidase S treatment on TZM samples. Panel (A) cIEF profiles TZM and TZMm; panels (B–D) HILIC profiles of TZMGTST6, TZMmST6 and TZMmGTST6. HILIC was performed on PNGaseF-released glycans from 2 independent batches (u.g. stands for unidentified glycan). Non-specific digestion was suspected in TZMmST6 (Panel C). Unexpected high absorbance is indicated by (*), and unexpected low absorbance by (**). GlcNAcF refers to the Fucα1–6GlcNAc fragment.
Figure 5. Effect of α2,3SA-specific sialidase S treatment on TZM samples. Panel (A) cIEF profiles TZM and TZMm; panels (B–D) HILIC profiles of TZMGTST6, TZMmST6 and TZMmGTST6. HILIC was performed on PNGaseF-released glycans from 2 independent batches (u.g. stands for unidentified glycan). Non-specific digestion was suspected in TZMmST6 (Panel C). Unexpected high absorbance is indicated by (*), and unexpected low absorbance by (**). GlcNAcF refers to the Fucα1–6GlcNAc fragment.CC BY-NC · PubMed Central
Figure 6. The relative abundances of the sialylated biantennary complex glycans (G1FS1, G2FS1 and G2FS2). The HILIC results were used to calculate the proportion of α2,3- and α2,6-linked sialic acids found in TZMGTST6, TZMmST6 and TZMmGTST6. White:% α2,3SA. Gray:% α2,6SA.
Figure 6. The relative abundances of the sialylated biantennary complex glycans (G1FS1, G2FS1 and G2FS2). The HILIC results were used to calculate the proportion of α2,3- and α2,6-linked sialic acids found in TZMGTST6, TZMmST6 and TZMmGTST6. White:% α2,3SA. Gray:% α2,6SA.CC BY-NC · PubMed Central

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Research works printed but not linked: 4

  1. Scott AM, 2012, Cancer Immun, 12, 14
  2. Ferrara C, 2006, Biotechnol Appl Biochem, 93, 851
  3. 10.1111/j.1537-2995.2011.03517 · DOI
  4. Royle L, 2006, Methods Mol Biol, 347, 125

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Metadata

Authors
Raymond, C., Robotham, A., Spearman, M., Butler, M., Kelly, J., & Durocher, Y.
Affiliations
  • National Research Council Canada (CA)
  • University of Manitoba (CA)
Corresponding author
Durocher, Yves
ORCID
Venue
mAbs
Publisher
Informa UK Limited
Published
2015-04-15
Volume
7
Issue
3
Pages
571-583
ISSN
  • 1942-0862 (ISSN-L)
  • 1942-0870
DOI
10.1080/19420862.2015.1029215
PMC
PMC4622614
OpenAlex
W1592578892
Cited by
106
References
65
Citations by year
2026: 6 · 2025: 5 · 2024: 10 · 2023: 10 · 2022: 6 · 2021: 10 · 2020: 9 · 2019: 11 · 2018: 12 · 2017: 11 · 2016: 9 · 2015: 7
FWCI
5.65
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • mAbs (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • NPARC (submittedVersion)
Funders
Natural Sciences and Engineering Research Council of Canada
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
  • Life Sciences › Immunology and Microbiology › Immunology › Monoclonal and Polyclonal Antibodies Research
Keywords
α2,6-sialylation, IgG1, Fc glycan, sialic acid, beta-1,4-galactosyltransferase, CHO cells, antibody glycosylation
MeSH terms
beta-D-Galactoside alpha 2-6-Sialyltransferase, Animals, Antibodies, Monoclonal, Cricetulus, Cricetinae, Humans, Immunoglobulin G, N-Acetyllactosamine Synthase, Sialic Acids, Sialyltransferases, Antigens, CD, CHO Cells, Amino Acid Substitution, Mutation, Missense

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