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Eukaryotic Cell, September 2009, p. 1407-1417, Vol. 8, No. 9
1535-9778/09/$08.00+0     doi:10.1128/EC.00178-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Fate of Glycosylphosphatidylinositol (GPI)-Less Procyclin and Characterization of Sialylated Non-GPI-Anchored Surface Coat Molecules of Procyclic-Form Trypanosoma brucei{triangledown} ,{dagger} ,{ddagger}

Maria Lucia Sampaio Güther,1 Kenneth Beattie,2 Douglas J. Lamont,2 John James,3 Alan R. Prescott,4 and Michael A. J. Ferguson1*

Division of Biological Chemistry and Drug Discovery,1 FingerPrints Proteomics Facility,2 Centre for High-Resolution Imaging,3 Division of Cell Biology and Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom4

Received 21 June 2009/ Accepted 29 June 2009

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-NaB3H4 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-NaB3H4 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.

* Corresponding author. Mailing address: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom. Phone: 44-1382 384219. E-mail: m.a.j.ferguson{at}dundee.ac.uk

{triangledown} Published ahead of print on 24 July 2009.

{dagger} Supplemental material for this article may be found at http://ec.asm.org/.

{ddagger} The authors have paid a fee to allow immediate free access to this article.

Eukaryotic Cell, September 2009, p. 1407-1417, Vol. 8, No. 9
1535-9778/09/$08.00+0     doi:10.1128/EC.00178-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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