title: Fate of glycosylphosphatidylinositol (GPI)-less procyclin and characterization of sialylated non-GPI-anchored surface coat molecules of procyclic-form Trypanosoma brucei.
abstract: A Trypanosoma brucei TbGPI12 null mutant that is unable to express cell surface procyclins and free glycosylphosphatidylinositols (GPI) revealed that these are not the only surface coat molecules of the procyclic life cycle stage. Here, we show that non-GPI-anchored procyclins are N-glycosylated, accumulate in the lysosome, and appear as proteolytic fragments in the medium. We also show, using lectin agglutination and galactose oxidase-NaB(3)H(4) labeling, that the cell surface of the TbGPI12 null parasites contains glycoconjugates that terminate in sialic acid linked to galactose. Following desialylation, a high-apparent-molecular-weight glycoconjugate fraction was purified by ricin affinity chromatography and gel filtration and shown to contain mannose, galactose, N-acetylglucosamine, and fucose. The latter has not been previously reported in T. brucei glycoproteins. A proteomic analysis of this fraction revealed a mixture of polytopic transmembrane proteins, including P-type ATPase and vacuolar proton-translocating pyrophosphatase. Immunolocalization studies showed that both could be labeled on the surfaces of wild-type and TbGPI12 null cells. Neither galactose oxidase-NaB(3)H(4) labeling of the non-GPI-anchored surface glycoconjugates nor immunogold labeling of the P-type ATPase was affected by the presence of procyclins in the wild-type cells, suggesting that the procyclins do not, by themselves, form a macromolecular barrier.
authors.affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, United Kingdom.
authors.forename: Maria Lucia Sampaio
authors.initials: ML
authors.lastname: Güther
authors.forename: Kenneth
authors.initials: K
authors.lastname: Beattie
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Lamont
authors.forename: John
authors.initials: J
authors.lastname: James
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; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D008018:Life Cycle Stages; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
chemical_list: D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; C053277:procyclic acidic repetitive protein, Trypanosoma; D019158:N-Acetylneuraminic Acid