Mouse large can modify complex N- and mucin O-glycans on α-dystroglycan to induce laminin binding

Santosh K. Patnaik, Pamela Stanley

Research output: Contribution to journalArticle

78 Citations (Scopus)

Abstract

The human LARGE gene encodes a protein with two putative glycosyltransferase domains and is required for the generation of functional α-dystroglycan (α-DG). Monoclonal antibodies IIH6 and VIA4-1 recognize the functional glycan epitopes of α-DG that are necessary for binding to laminin and other ligands. Overexpression of full-length mouse Large generated functionally glycosylated α-DG in Pro-5 Chinese hamster ovary (CHO) cells, and the amount was increased by co-expression of protein:O-mannosyl N-acetylglucosaminyltransferase 1. However, functional α-DG represented only a small fraction of the α-DG synthesized by CHO cells or expressed from an α-DG construct. To identify features of the glycan epitopes induced by Large, the production of functionally glycosylated α-DG was investigated in several CHO glycosylation mutants. Mutants with defective transfer of sialic acid (Lec2), galactose (Lec8), or fucose (Lec13) to glycoconjugates, and the Lec15 mutant that cannot synthesize O-mannose glycans, all produced functionally glycosylated α-DG upon overexpression of Large. Laminin binding and the α-DG glycan epitopes were enhanced in Lec2 and Lec8 cells. In Lec15 cells, functional α-DG was increased by co-expression of core 2 N-acetylglucosaminyltransferase 1 with Large. Treatment with N-glycanase markedly reduced functionally glycosylated α-DG in Lec2 and Lec8 cells. The combined data provide evidence that Large does not transfer to Gal, Fuc, or sialic acid on α-DG nor induce the transfer of these sugars to α-DG. In addition, the data suggest that human LARGE may restore functional α-DG to muscle cells from patients with defective synthesis of O-mannose glycans via the modification of N-glycans and/or mucin O-glycans on α-DG.

Original languageEnglish (US)
Pages (from-to)20851-20859
Number of pages9
JournalJournal of Biological Chemistry
Volume280
Issue number21
DOIs
StatePublished - May 27 2005

Fingerprint

Dystroglycans
Laminin
Mucins
Polysaccharides
Cricetulus
Epitopes
Ovary
N-Acetylneuraminic Acid
Mannose
Cells
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Glycosylation
Glycosyltransferases
Glycoconjugates
Fucose
Galactose
Sugars
Muscle Cells
Muscle
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mouse large can modify complex N- and mucin O-glycans on α-dystroglycan to induce laminin binding. / Patnaik, Santosh K.; Stanley, Pamela.

In: Journal of Biological Chemistry, Vol. 280, No. 21, 27.05.2005, p. 20851-20859.

Research output: Contribution to journalArticle

@article{f6b835f78b8c4a648d3fec1276398f37,
title = "Mouse large can modify complex N- and mucin O-glycans on α-dystroglycan to induce laminin binding",
abstract = "The human LARGE gene encodes a protein with two putative glycosyltransferase domains and is required for the generation of functional α-dystroglycan (α-DG). Monoclonal antibodies IIH6 and VIA4-1 recognize the functional glycan epitopes of α-DG that are necessary for binding to laminin and other ligands. Overexpression of full-length mouse Large generated functionally glycosylated α-DG in Pro-5 Chinese hamster ovary (CHO) cells, and the amount was increased by co-expression of protein:O-mannosyl N-acetylglucosaminyltransferase 1. However, functional α-DG represented only a small fraction of the α-DG synthesized by CHO cells or expressed from an α-DG construct. To identify features of the glycan epitopes induced by Large, the production of functionally glycosylated α-DG was investigated in several CHO glycosylation mutants. Mutants with defective transfer of sialic acid (Lec2), galactose (Lec8), or fucose (Lec13) to glycoconjugates, and the Lec15 mutant that cannot synthesize O-mannose glycans, all produced functionally glycosylated α-DG upon overexpression of Large. Laminin binding and the α-DG glycan epitopes were enhanced in Lec2 and Lec8 cells. In Lec15 cells, functional α-DG was increased by co-expression of core 2 N-acetylglucosaminyltransferase 1 with Large. Treatment with N-glycanase markedly reduced functionally glycosylated α-DG in Lec2 and Lec8 cells. The combined data provide evidence that Large does not transfer to Gal, Fuc, or sialic acid on α-DG nor induce the transfer of these sugars to α-DG. In addition, the data suggest that human LARGE may restore functional α-DG to muscle cells from patients with defective synthesis of O-mannose glycans via the modification of N-glycans and/or mucin O-glycans on α-DG.",
author = "Patnaik, {Santosh K.} and Pamela Stanley",
year = "2005",
month = "5",
day = "27",
doi = "10.1074/jbc.M500069200",
language = "English (US)",
volume = "280",
pages = "20851--20859",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "21",

}

TY - JOUR

T1 - Mouse large can modify complex N- and mucin O-glycans on α-dystroglycan to induce laminin binding

AU - Patnaik, Santosh K.

AU - Stanley, Pamela

PY - 2005/5/27

Y1 - 2005/5/27

N2 - The human LARGE gene encodes a protein with two putative glycosyltransferase domains and is required for the generation of functional α-dystroglycan (α-DG). Monoclonal antibodies IIH6 and VIA4-1 recognize the functional glycan epitopes of α-DG that are necessary for binding to laminin and other ligands. Overexpression of full-length mouse Large generated functionally glycosylated α-DG in Pro-5 Chinese hamster ovary (CHO) cells, and the amount was increased by co-expression of protein:O-mannosyl N-acetylglucosaminyltransferase 1. However, functional α-DG represented only a small fraction of the α-DG synthesized by CHO cells or expressed from an α-DG construct. To identify features of the glycan epitopes induced by Large, the production of functionally glycosylated α-DG was investigated in several CHO glycosylation mutants. Mutants with defective transfer of sialic acid (Lec2), galactose (Lec8), or fucose (Lec13) to glycoconjugates, and the Lec15 mutant that cannot synthesize O-mannose glycans, all produced functionally glycosylated α-DG upon overexpression of Large. Laminin binding and the α-DG glycan epitopes were enhanced in Lec2 and Lec8 cells. In Lec15 cells, functional α-DG was increased by co-expression of core 2 N-acetylglucosaminyltransferase 1 with Large. Treatment with N-glycanase markedly reduced functionally glycosylated α-DG in Lec2 and Lec8 cells. The combined data provide evidence that Large does not transfer to Gal, Fuc, or sialic acid on α-DG nor induce the transfer of these sugars to α-DG. In addition, the data suggest that human LARGE may restore functional α-DG to muscle cells from patients with defective synthesis of O-mannose glycans via the modification of N-glycans and/or mucin O-glycans on α-DG.

AB - The human LARGE gene encodes a protein with two putative glycosyltransferase domains and is required for the generation of functional α-dystroglycan (α-DG). Monoclonal antibodies IIH6 and VIA4-1 recognize the functional glycan epitopes of α-DG that are necessary for binding to laminin and other ligands. Overexpression of full-length mouse Large generated functionally glycosylated α-DG in Pro-5 Chinese hamster ovary (CHO) cells, and the amount was increased by co-expression of protein:O-mannosyl N-acetylglucosaminyltransferase 1. However, functional α-DG represented only a small fraction of the α-DG synthesized by CHO cells or expressed from an α-DG construct. To identify features of the glycan epitopes induced by Large, the production of functionally glycosylated α-DG was investigated in several CHO glycosylation mutants. Mutants with defective transfer of sialic acid (Lec2), galactose (Lec8), or fucose (Lec13) to glycoconjugates, and the Lec15 mutant that cannot synthesize O-mannose glycans, all produced functionally glycosylated α-DG upon overexpression of Large. Laminin binding and the α-DG glycan epitopes were enhanced in Lec2 and Lec8 cells. In Lec15 cells, functional α-DG was increased by co-expression of core 2 N-acetylglucosaminyltransferase 1 with Large. Treatment with N-glycanase markedly reduced functionally glycosylated α-DG in Lec2 and Lec8 cells. The combined data provide evidence that Large does not transfer to Gal, Fuc, or sialic acid on α-DG nor induce the transfer of these sugars to α-DG. In addition, the data suggest that human LARGE may restore functional α-DG to muscle cells from patients with defective synthesis of O-mannose glycans via the modification of N-glycans and/or mucin O-glycans on α-DG.

UR - http://www.scopus.com/inward/record.url?scp=20144366896&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=20144366896&partnerID=8YFLogxK

U2 - 10.1074/jbc.M500069200

DO - 10.1074/jbc.M500069200

M3 - Article

C2 - 15788414

AN - SCOPUS:20144366896

VL - 280

SP - 20851

EP - 20859

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 21

ER -