A Mutation Causing a Reduced Level of Expression of Six β4-Galactosyltransferase Genes Is the Basis of the Lec19 CHO Glycosylation Mutant

JaeHoon Lee, Sung Hae Park, Subha Sundaram, T. Shantha Raju, Nancy L. Shaper, Pamela Stanley

Research output: Contribution to journalArticle

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Abstract

To identify factors required for the synthesis of complex glycans, we have isolated Chinese hamster ovary (CHO) cell mutants resistant to plant lectins. We previously identified Lec19 CHO cells as resistant to the Gal-binding lectins ricin, abrin, and modeccin and hypersensitive to the toxicity of other lectins that bind Gal, including L-PHA and E-PHA. Here we show that Lec19 cell extracts have a decreased ability to transfer Gal to simple sugar, oligosaccharide, and glycopeptide acceptors, particularly to biantennary, GlcNAc-terminated acceptors. RicinII-agarose lectin affinity chromatography, oligo-mapping, and monosaccharide analyses provided evidence that Lec19 N-glycans have fewer Gal residues than CHO N-glycans. MALDI-TOF mass spectra of N-glycans released from Lec19 cell glycoproteins by peptide N-glycanase F revealed species with the predicted masses of neutral N-glycans with few Gal residues. Such truncated species are essentially absent from CHO cell glycoproteins. However, the complement of fully galactosylated or sialylated bi-, tri-, and tetra-antennary N-glycans was largely equivalent in Lec19 and CHO cells. In addition, the coding region sequences of the β4Gal-1, -T-2, -T-3, -T-4, -T-5, and -T-6 genes were identical in CHO and Lec19 cells. However, Northern analyses revealed an ∼2-4-fold reduction in the level of transcripts of all six β4GalT genes in Lec19 cells. Since the recessive Lec19 phenotype is the result of a loss-of-function mutation, the combined data predict the existence of a trans-acting regulator of the steady-state level of transcripts that derive from these six mammalian β4GalT genes.

Original languageEnglish (US)
Pages (from-to)12349-12357
Number of pages9
JournalBiochemistry
Volume42
Issue number42
DOIs
StatePublished - Oct 28 2003

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Glycosylation
Galactosyltransferases
Cricetulus
Polysaccharides
Ovary
Genes
Mutation
Lectins
Glycoproteins
Abrin
Plant Lectins
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Affinity chromatography
Ricin
Glycopeptides
Monosaccharides
Oligosaccharides
Sugars
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Sepharose

ASJC Scopus subject areas

  • Biochemistry

Cite this

A Mutation Causing a Reduced Level of Expression of Six β4-Galactosyltransferase Genes Is the Basis of the Lec19 CHO Glycosylation Mutant. / Lee, JaeHoon; Park, Sung Hae; Sundaram, Subha; Raju, T. Shantha; Shaper, Nancy L.; Stanley, Pamela.

In: Biochemistry, Vol. 42, No. 42, 28.10.2003, p. 12349-12357.

Research output: Contribution to journalArticle

Lee, JaeHoon ; Park, Sung Hae ; Sundaram, Subha ; Raju, T. Shantha ; Shaper, Nancy L. ; Stanley, Pamela. / A Mutation Causing a Reduced Level of Expression of Six β4-Galactosyltransferase Genes Is the Basis of the Lec19 CHO Glycosylation Mutant. In: Biochemistry. 2003 ; Vol. 42, No. 42. pp. 12349-12357.
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abstract = "To identify factors required for the synthesis of complex glycans, we have isolated Chinese hamster ovary (CHO) cell mutants resistant to plant lectins. We previously identified Lec19 CHO cells as resistant to the Gal-binding lectins ricin, abrin, and modeccin and hypersensitive to the toxicity of other lectins that bind Gal, including L-PHA and E-PHA. Here we show that Lec19 cell extracts have a decreased ability to transfer Gal to simple sugar, oligosaccharide, and glycopeptide acceptors, particularly to biantennary, GlcNAc-terminated acceptors. RicinII-agarose lectin affinity chromatography, oligo-mapping, and monosaccharide analyses provided evidence that Lec19 N-glycans have fewer Gal residues than CHO N-glycans. MALDI-TOF mass spectra of N-glycans released from Lec19 cell glycoproteins by peptide N-glycanase F revealed species with the predicted masses of neutral N-glycans with few Gal residues. Such truncated species are essentially absent from CHO cell glycoproteins. However, the complement of fully galactosylated or sialylated bi-, tri-, and tetra-antennary N-glycans was largely equivalent in Lec19 and CHO cells. In addition, the coding region sequences of the β4Gal-1, -T-2, -T-3, -T-4, -T-5, and -T-6 genes were identical in CHO and Lec19 cells. However, Northern analyses revealed an ∼2-4-fold reduction in the level of transcripts of all six β4GalT genes in Lec19 cells. Since the recessive Lec19 phenotype is the result of a loss-of-function mutation, the combined data predict the existence of a trans-acting regulator of the steady-state level of transcripts that derive from these six mammalian β4GalT genes.",
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AU - Shaper, Nancy L.

AU - Stanley, Pamela

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AB - To identify factors required for the synthesis of complex glycans, we have isolated Chinese hamster ovary (CHO) cell mutants resistant to plant lectins. We previously identified Lec19 CHO cells as resistant to the Gal-binding lectins ricin, abrin, and modeccin and hypersensitive to the toxicity of other lectins that bind Gal, including L-PHA and E-PHA. Here we show that Lec19 cell extracts have a decreased ability to transfer Gal to simple sugar, oligosaccharide, and glycopeptide acceptors, particularly to biantennary, GlcNAc-terminated acceptors. RicinII-agarose lectin affinity chromatography, oligo-mapping, and monosaccharide analyses provided evidence that Lec19 N-glycans have fewer Gal residues than CHO N-glycans. MALDI-TOF mass spectra of N-glycans released from Lec19 cell glycoproteins by peptide N-glycanase F revealed species with the predicted masses of neutral N-glycans with few Gal residues. Such truncated species are essentially absent from CHO cell glycoproteins. However, the complement of fully galactosylated or sialylated bi-, tri-, and tetra-antennary N-glycans was largely equivalent in Lec19 and CHO cells. In addition, the coding region sequences of the β4Gal-1, -T-2, -T-3, -T-4, -T-5, and -T-6 genes were identical in CHO and Lec19 cells. However, Northern analyses revealed an ∼2-4-fold reduction in the level of transcripts of all six β4GalT genes in Lec19 cells. Since the recessive Lec19 phenotype is the result of a loss-of-function mutation, the combined data predict the existence of a trans-acting regulator of the steady-state level of transcripts that derive from these six mammalian β4GalT genes.

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