Antibodies that recognize bisected complex N-glycans on cell surface glycoproteins can be made in mice lacking N-acetylglucosaminyltransferase III

JaeHoon Lee, Sung Hae Park, Pamela Stanley

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

The bisecting GlcNAc is transferred to complex or hybrid N-glycans by the action of N-acetylglucosaminyltransferase III (GlcNAc-TIII) encoded by the Mgat3 gene. CHO cells expressing mouse GlcNAc-TIII were shown by matrix-assisted laser desorption ionization (MALDI) mass spectrometry to produce mainly complex N-glycans with the predicted extra (bisecting) GlcNAc. In order to probe biological functions of the bisecting GlcNAc, antibodies that recognize this residue in the context of complex cell surface glycoconjugates were sought. The LEC10 gain-of-function Chinese hamster ovary (CHO) cell mutant that expresses GlcNAc-TIII and complex N-glycans with the bisecting GlcNAc was used to immunize Mgat3+/+ and Mgat3-/- mice. ELISA of whole sera showed that polyclonal antibodies that bound specifically to LEC10 cells were obtained solely from Mgat3-/- mice. Fluorescence-activated cell cytometry of different CHO glycosylation mutants and western blotting after glycosidase treatments were used to show that anti-LEC10 cell antisera from Mgat3-/- mice recognize cellular glycoproteins with complex N-glycans containing both a bisecting GlcNAc and Gal residues. The polyclonal antibody specificity was similar to that of the lectin E-PHA. IgM-depleted serum containing IgG and IgA antibodies retained full binding activity. Therefore Mgat3-/- mice but not wild type mice can be used effectively to produce polyclonal antibodies that specifically recognize glycoproteins bearing complex N-glycans with a bisecting GlcNAc.

Original languageEnglish (US)
Pages (from-to)211-219
Number of pages9
JournalGlycoconjugate Journal
Volume19
Issue number3
DOIs
StatePublished - Mar 1 2003

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beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase
Membrane Glycoproteins
Polysaccharides
Antibodies
Cricetulus
Ovary
Glycoproteins
Bearings (structural)
Cells
Glycosylation
Glycoconjugates
Glycoside Hydrolases
Antibody Specificity
Lectins
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Immunoglobulin A
Ionization
Mass spectrometry
Immunoglobulin M
Immune Sera

Keywords

  • Bisecting N-acetylgucosamine
  • LEC10 CHO mutant
  • MALDI-TOF mass spectrometry
  • Mgat3 gene
  • N-glycan antibodies

ASJC Scopus subject areas

  • Biochemistry

Cite this

Antibodies that recognize bisected complex N-glycans on cell surface glycoproteins can be made in mice lacking N-acetylglucosaminyltransferase III. / Lee, JaeHoon; Park, Sung Hae; Stanley, Pamela.

In: Glycoconjugate Journal, Vol. 19, No. 3, 01.03.2003, p. 211-219.

Research output: Contribution to journalArticle

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abstract = "The bisecting GlcNAc is transferred to complex or hybrid N-glycans by the action of N-acetylglucosaminyltransferase III (GlcNAc-TIII) encoded by the Mgat3 gene. CHO cells expressing mouse GlcNAc-TIII were shown by matrix-assisted laser desorption ionization (MALDI) mass spectrometry to produce mainly complex N-glycans with the predicted extra (bisecting) GlcNAc. In order to probe biological functions of the bisecting GlcNAc, antibodies that recognize this residue in the context of complex cell surface glycoconjugates were sought. The LEC10 gain-of-function Chinese hamster ovary (CHO) cell mutant that expresses GlcNAc-TIII and complex N-glycans with the bisecting GlcNAc was used to immunize Mgat3+/+ and Mgat3-/- mice. ELISA of whole sera showed that polyclonal antibodies that bound specifically to LEC10 cells were obtained solely from Mgat3-/- mice. Fluorescence-activated cell cytometry of different CHO glycosylation mutants and western blotting after glycosidase treatments were used to show that anti-LEC10 cell antisera from Mgat3-/- mice recognize cellular glycoproteins with complex N-glycans containing both a bisecting GlcNAc and Gal residues. The polyclonal antibody specificity was similar to that of the lectin E-PHA. IgM-depleted serum containing IgG and IgA antibodies retained full binding activity. Therefore Mgat3-/- mice but not wild type mice can be used effectively to produce polyclonal antibodies that specifically recognize glycoproteins bearing complex N-glycans with a bisecting GlcNAc.",
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