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Glycobiology Advance Access originally published online on May 21, 2009
Glycobiology 2009 19(9):971-986; doi:10.1093/glycob/cwp074
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© The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

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

Jennifer T Aguilan2, Subha Sundaram2, Edward Nieves3 and Pamela Stanley1,2

2 Department of Cell Biology
3 Laboratory of Macromolecular Analysis and Proteomics, Albert Einstein College Medicine, New York, NY 10461, USA

1 To whom correspondence should be addressed: Tel: +1-718-430-3346; Fax: +1-718-430-8574; e-mail: stanley{at}aecom.yu.edu

Received on November 3, 2008; revised on May 12, 2009; accepted on May 15, 2009

Inactivating mutations of Large reduce the functional glycosylation of {alpha}-dystroglycan ({alpha}-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, {alpha}-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 {alpha}-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 {alpha}-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 {alpha}-DG. Thus, Large may act on the O-mannose, complex N-glycans and mucin O-GalNAc glycans of {alpha}-DG.

Key words: {alpha}-dystroglycan / CHO mutants / DXD / laminin / Large / mutagenesis


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