Mutational and functional analysis of Large in a novel CHO glycosylation mutant

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Inactivating mutations of Large reduce the functional glycosylation of α-dystroglycan (α-DG) and lead to muscular dystrophy in mouse and humans. The N-terminal domain of Large is most similar to UDP-glucose glucosyltransferases (UGGT), and the C-terminal domain is related to the human i blood group transferase β1,3GlcNAcT-1. The amino acids at conserved motifs DQD+1 and DQD+3 in the UGGT domain are necessary for mammalian UGGT activity. When the corresponding residues were mutated to Ala in mouse Large, α-DG was not functionally glycosylated. A similar result was obtained when a DXD motif in the β1,3GlcNAcT-1 domain was mutated to AIA. Therefore, the first putative glycosyltransferase domain of Large has properties of a UGGT and the second of a typical glycosyltransferase. Co-transfection of Large mutants affected in the different glycosyltransferase domains did not lead to complementation. While Large mutants were more localized to the endoplasmic reticulum than wild-type Large or revertants, all mutants were in the Golgi, and only very low levels of Golgi-localized Large were necessary to generate functional α-DG. When Large was overexpressed in ldlD.Lec1 mutant Chinese hamster ovary (CHO) cells which synthesize few, if any, mucin O-GalNAc glycans and no complex N-glycans, functional α-DG was produced, presumably by modifying O-mannose glycans. To investigate mucin O-GalNAc glycans as substrates of Large, a new CHO mutant Lec15.Lec1 that lacked O-mannose and complex N-glycans was isolated and characterized. Following transfection with Large, Lec15.Lec1 cells also generated functionally glycosylated α-DG. Thus, Large may act on the O-mannose, complex N-glycans and mucin O-GalNAc glycans of α-DG.

Original languageEnglish (US)
Pages (from-to)971-986
Number of pages16
JournalGlycobiology
Volume19
Issue number9
DOIs
StatePublished - 2009

Fingerprint

Glycosylation
Functional analysis
Cricetulus
Polysaccharides
Ovary
Uridine Diphosphate Glucose
Glucosyltransferases
Glycosyltransferases
Mucins
Mannose
Transfection
Dystroglycans
Muscular Dystrophies
Blood Group Antigens
Transferases
Endoplasmic Reticulum
Cells
Amino Acids
Mutation
Substrates

Keywords

  • α-dystroglycan
  • CHO mutants
  • DXD
  • Laminin
  • Large
  • Mutagenesis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mutational and functional analysis of Large in a novel CHO glycosylation mutant. / Aguilan, Jennifer T.; Sundaram, Subha; Nieves, Edward; Stanley, Pamela.

In: Glycobiology, Vol. 19, No. 9, 2009, p. 971-986.

Research output: Contribution to journalArticle

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abstract = "Inactivating mutations of Large reduce the functional glycosylation of α-dystroglycan (α-DG) and lead to muscular dystrophy in mouse and humans. The N-terminal domain of Large is most similar to UDP-glucose glucosyltransferases (UGGT), and the C-terminal domain is related to the human i blood group transferase β1,3GlcNAcT-1. The amino acids at conserved motifs DQD+1 and DQD+3 in the UGGT domain are necessary for mammalian UGGT activity. When the corresponding residues were mutated to Ala in mouse Large, α-DG was not functionally glycosylated. A similar result was obtained when a DXD motif in the β1,3GlcNAcT-1 domain was mutated to AIA. Therefore, the first putative glycosyltransferase domain of Large has properties of a UGGT and the second of a typical glycosyltransferase. Co-transfection of Large mutants affected in the different glycosyltransferase domains did not lead to complementation. While Large mutants were more localized to the endoplasmic reticulum than wild-type Large or revertants, all mutants were in the Golgi, and only very low levels of Golgi-localized Large were necessary to generate functional α-DG. When Large was overexpressed in ldlD.Lec1 mutant Chinese hamster ovary (CHO) cells which synthesize few, if any, mucin O-GalNAc glycans and no complex N-glycans, functional α-DG was produced, presumably by modifying O-mannose glycans. To investigate mucin O-GalNAc glycans as substrates of Large, a new CHO mutant Lec15.Lec1 that lacked O-mannose and complex N-glycans was isolated and characterized. Following transfection with Large, Lec15.Lec1 cells also generated functionally glycosylated α-DG. Thus, Large may act on the O-mannose, complex N-glycans and mucin O-GalNAc glycans of α-DG.",
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T1 - Mutational and functional analysis of Large in a novel CHO glycosylation mutant

AU - Aguilan, Jennifer T.

AU - Sundaram, Subha

AU - Nieves, Edward

AU - Stanley, Pamela

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N2 - Inactivating mutations of Large reduce the functional glycosylation of α-dystroglycan (α-DG) and lead to muscular dystrophy in mouse and humans. The N-terminal domain of Large is most similar to UDP-glucose glucosyltransferases (UGGT), and the C-terminal domain is related to the human i blood group transferase β1,3GlcNAcT-1. The amino acids at conserved motifs DQD+1 and DQD+3 in the UGGT domain are necessary for mammalian UGGT activity. When the corresponding residues were mutated to Ala in mouse Large, α-DG was not functionally glycosylated. A similar result was obtained when a DXD motif in the β1,3GlcNAcT-1 domain was mutated to AIA. Therefore, the first putative glycosyltransferase domain of Large has properties of a UGGT and the second of a typical glycosyltransferase. Co-transfection of Large mutants affected in the different glycosyltransferase domains did not lead to complementation. While Large mutants were more localized to the endoplasmic reticulum than wild-type Large or revertants, all mutants were in the Golgi, and only very low levels of Golgi-localized Large were necessary to generate functional α-DG. When Large was overexpressed in ldlD.Lec1 mutant Chinese hamster ovary (CHO) cells which synthesize few, if any, mucin O-GalNAc glycans and no complex N-glycans, functional α-DG was produced, presumably by modifying O-mannose glycans. To investigate mucin O-GalNAc glycans as substrates of Large, a new CHO mutant Lec15.Lec1 that lacked O-mannose and complex N-glycans was isolated and characterized. Following transfection with Large, Lec15.Lec1 cells also generated functionally glycosylated α-DG. Thus, Large may act on the O-mannose, complex N-glycans and mucin O-GalNAc glycans of α-DG.

AB - Inactivating mutations of Large reduce the functional glycosylation of α-dystroglycan (α-DG) and lead to muscular dystrophy in mouse and humans. The N-terminal domain of Large is most similar to UDP-glucose glucosyltransferases (UGGT), and the C-terminal domain is related to the human i blood group transferase β1,3GlcNAcT-1. The amino acids at conserved motifs DQD+1 and DQD+3 in the UGGT domain are necessary for mammalian UGGT activity. When the corresponding residues were mutated to Ala in mouse Large, α-DG was not functionally glycosylated. A similar result was obtained when a DXD motif in the β1,3GlcNAcT-1 domain was mutated to AIA. Therefore, the first putative glycosyltransferase domain of Large has properties of a UGGT and the second of a typical glycosyltransferase. Co-transfection of Large mutants affected in the different glycosyltransferase domains did not lead to complementation. While Large mutants were more localized to the endoplasmic reticulum than wild-type Large or revertants, all mutants were in the Golgi, and only very low levels of Golgi-localized Large were necessary to generate functional α-DG. When Large was overexpressed in ldlD.Lec1 mutant Chinese hamster ovary (CHO) cells which synthesize few, if any, mucin O-GalNAc glycans and no complex N-glycans, functional α-DG was produced, presumably by modifying O-mannose glycans. To investigate mucin O-GalNAc glycans as substrates of Large, a new CHO mutant Lec15.Lec1 that lacked O-mannose and complex N-glycans was isolated and characterized. Following transfection with Large, Lec15.Lec1 cells also generated functionally glycosylated α-DG. Thus, Large may act on the O-mannose, complex N-glycans and mucin O-GalNAc glycans of α-DG.

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KW - Laminin

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KW - Mutagenesis

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