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J. Biol. Chem., Vol. 280, Issue 2, 865-871, January 14, 2005

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Trypanosoma brucei Glycoproteins Contain Novel Giant Poly-N-acetyllactosamine Carbohydrate Chains^*

Abdelmadjid Atrih, Julia M. Richardson, Alan R. Prescott, and Michael A. J. Ferguson¶

From the Division of Biological Chemistry and Molecular Microbiology, the School of Life Sciences, University of Dundee, Dundee DD1 5EH, and the School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom

The flagellar pocket of the bloodstream form of the African sleeping sickness parasite Trypanosoma brucei contains material that binds the -D-galactose-specific lectin ricin (Brickman, M. J., and Balber, A. E. (1990) J. Protozool. 37, 219–224). Glycoproteins were solubilized from bloodstream form T. brucei cells in 8 M urea and 3% SDS and purified by ricin affinity chromatography. Essentially all binding of ricin to these glycoproteins was abrogated by treatment with peptide N-glycosidase, showing that the ricin ligands are attached to glycoproteins via N-glycosidic linkages to asparagine residues. Glycans released by peptide N-glycosidase were resolved by Bio-Gel P-4 gel filtration into two fractions: a low molecular mass mannose-rich fraction and a high molecular mass galactose and N-acetylglucosamine-rich fraction. The latter fraction was further separated by high pH anion exchange chromatography and analyzed by gas chromatography mass spectrometry, one- and two-dimensional NMR, electrospray mass spectrometry, and methylation linkage analysis. The high molecular mass ricin-binding N-glycans are based on a conventional Man1–3(Man1–6)Man1–4-GlcNAc1–4GlcNAc core structure and contain poly-N-acetyllactosamine chains. A significant proportion of these structures are extremely large and of unusual structure. They contain an average of 54 N-acetyllactosamine (Gal1–4GlcNAc) repeats per glycan, linked mostly by -4GlcNAc1–6Gal1-interrepeat linkages, with an average of one -4GlcNAc1–3(-4GlcNAc1–6)Gal1- branch point in every six repeats. These structures, which also bind tomato lectin, are twice the size reported for the largest mammalian poly-N-acetyllactosamine N-linked glycans and also differ in their preponderance of -4GlcNAc1–6Gal1- over -4GlcNac1–3Gal1- interrepeat linkages. Molecular modeling suggests that -4GlcNAc1–6Gal1- interrepeat linkages produce relatively compact structures that may give these giant N-linked glycans unique physicochemical properties. Fluorescence microscopy using fluorescein isothiocyanatericin indicates that ricin ligands are located mainly in the flagellar pocket and in the endosomal/lysosomal system of the trypanosome.

Received for publication, September 27, 2004 , and in revised form, October 25, 2004.

^* This work was supported by Wellcome Trust Grants 62387 and 71463. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: University of Dundee School of Life Sciences, Wellcome Trust Biocentre, Dow St., Dundee DD1 5EH, Scotland, United Kingdom. E-mail: M.a.j.ferguson{at}dundee.ac.uk.

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