Skip Navigation



Glycobiology Advance Access published online on May 21, 2009
Glycobiology, doi:10.1093/glycob/cwp074
This Article
Right arrow Advance Access manuscript (PDF)
Right arrow Supplementary Data
Right arrow All Versions of this Article:
19/9/971    most recent
cwp074v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Aguilan, J. T.
Right arrow Articles by Stanley, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aguilan, J. T.
Right arrow Articles by Stanley, P.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 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. Aguilan1, Subha Sundaram1, Edward Nieves2 and Pamela Stanley1

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

Corresponding Author: Pamela Stanley; Phone: 1-718-430-3346; Fax: 1-718-430-8574; Email: stanley{at}aecom.yu.edu

Received on November 3, 2008; 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: Large / DXD / mutagenesis / {alpha}-dystroglycan / laminin / CHO mutants


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.