TY - JOUR
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
PY - 2009
Y1 - 2009
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.
KW - CHO mutants
KW - DXD
KW - Laminin
KW - Large
KW - Mutagenesis
KW - α-dystroglycan
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U2 - 10.1093/glycob/cwp074
DO - 10.1093/glycob/cwp074
M3 - Article
C2 - 19470663
AN - SCOPUS:68749120840
SN - 0959-6658
VL - 19
SP - 971
EP - 986
JO - Glycobiology
JF - Glycobiology
IS - 9
ER -