title: Trypanosoma brucei glycoproteins contain novel giant poly-N-acetyllactosamine carbohydrate chains.
abstract: The flagellar pocket of the bloodstream form of the African sleeping sickness parasite Trypanosoma brucei contains material that binds the beta-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 Manalpha1-3(Manalpha1-6)Manbeta1-4-GlcNAcbeta1-4GlcNAc 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 (Galbeta1-4GlcNAc) repeats per glycan, linked mostly by -4GlcNAcbeta1-6Galbeta1-interrepeat linkages, with an average of one -4GlcNAcbeta1-3(-4GlcNAcbeta1-6)Galbeta1- 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 -4GlcNAcbeta1-6Galbeta1- over -4GlcNacbeta1-3Galbeta1- interrepeat linkages. Molecular modeling suggests that -4GlcNAcbeta1-6Galbeta1- 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.
authors.affiliation: Division of Biological Chemistry and Molecular Microbiology, the School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.
authors.forename: Abdelmadjid
authors.initials: A
authors.lastname: Atrih
authors.forename: Julia M
authors.initials: JM
authors.lastname: Richardson
authors.forename: Alan R
authors.initials: AR
authors.lastname: Prescott
authors.forename: Michael A J
authors.initials: MA
authors.lastname: Ferguson
mesh_terms: D000818:Animals; D002236:Carbohydrate Conformation; D002846:Chromatography, Affinity; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D018551:Solanum lycopersicum; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008970:Molecular Weight; D037121:Plant Lectins / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification*; D011485:Protein Binding; D012276:Ricin / Q000737:chemistry / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry; D014346:Trypanosoma brucei brucei / Q000737:chemistry* / Q000502:physiology
chemical_list: D006023:Glycoproteins; D037121:Plant Lectins; D011134:Polysaccharides; C037199:poly-N-acetyllactosamine; D012276:Ricin