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Eukaryotic Cell, April 2004, p. 255-263, Vol. 3, No. 2
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.2.255-263.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Defects in the N-Linked Oligosaccharide Biosynthetic Pathway in a Trypanosoma brucei Glycosylation Mutant{dagger}

Alvaro Acosta-Serrano,1,{ddagger} Jessica O'Rear,2,§ George Quellhorst,2,|| Soo Hee Lee,1 Kuo-Yuan Hwa,1,# Sharon S. Krag,2 and Paul T. Englund1*

Department of Biological Chemistry, Johns Hopkins School of Medicine,1 Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 212052

Received 20 December 2003/ Accepted 19 February 2004

Concanavalin A (ConA) kills the procyclic (insect) form of Trypanosoma brucei by binding to its major surface glycoprotein, procyclin. We previously isolated a mutant cell line, ConA 1-1, that is less agglutinated and more resistant to ConA killing than are wild-type (WT) cells. Subsequently we found that the ConA resistance phenotype in this mutant is due to the fact that the procyclin either has no N-glycan or has an N-glycan with an altered structure. Here we demonstrate that the alteration in procyclin N-glycosylation correlates with two defects in the N-linked oligosaccharide biosynthetic pathway. First, ConA 1-1 has a defect in activity of polyprenol reductase, an enzyme involved in synthesis of dolichol. Metabolic incorporation of [3H]mevalonate showed that ConA 1-1 synthesizes equal amounts of dolichol and polyprenol, whereas WT cells make predominantly dolichol. Second, we found that ConA 1-1 synthesizes and accumulates an oligosaccharide lipid (OSL) precursor that is smaller in size than that from WT cells. The glycan of OSL in WT cells is apparently Man9GlcNAc2, whereas that from ConA 1-1 is Man7GlcNAc2. The smaller OSL glycan in the ConA 1-1 explains how some procyclin polypeptides bear a Man4GlcNAc2 modified with a terminal N-acetyllactosamine group, which is poorly recognized by ConA.

* Corresponding author. Mailing address: Department of Biological Chemistry, Johns Hopkins School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Phone: (410) 955-3790. Fax: (410) 955-7810. E-mail: penglund{at}jhmi.edu.

{dagger} We dedicate this work to the loving memory of Viiu Klein.

{ddagger} Present address: Wellcome Centre for Molecular Parasitology, Anderson College, University of Glasgow, Glasgow G11 6NU, Scotland, United Kingdom.

§ Present address: Sunesis Pharmaceuticals, Inc., South San Francisco, CA 94080.

|| Present address: SuperArray Bioscience Corporation, Frederick, MD 21704.

# Present address: Graduate Institute of Medical Technology, Taipei Medical University, Taipei, Taiwan.

Eukaryotic Cell, April 2004, p. 255-263, Vol. 3, No. 2
1535-9778/04/$08.00+0     DOI: 10.1128/EC.3.2.255-263.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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