Purification, cloning and characterization of a GPI inositol deacylase from Trypanosoma brucei

Maria Lucia Sampaio Güther, Simone Leal, Nicholas A. Morrice, George A.M. Cross, Michael A. J. Ferguson

    Research output: Contribution to journalArticle

    25 Citations (Scopus)

    Abstract

    NOTE: THE SPECIAL CHARACTERS IN THIS ABSTRACT CANNOT BE DISPLAYED CORRECTLY ON THIS PAGE. PLEASE REFER TO THE ABSTRACT ON THE PUBLISHER’S WEBSITE FOR AN ACCURATE DISPLAY. Inositol acylation is an obligatory step in glycosylphosphatidylinositol (GPI) biosynthesis whereas mature GPI anchors often lack this modification. The GPI anchors of Trypanosoma brucei variant surface glycoproteins (VSGs) undergo rounds of inositol acylation and deacylation during GPI biosynthesis and the deacylation reactions are inhibited by diisopropylfluorophosphate (DFP). Inositol deacylase was affinity labelled with [3H]DFP and purified. Peptide sequencing was used to clone GPIdeAc, which encodes a protein with significant sequence and hydropathy similarity to mammalian acyloxyacyl hydrolase, an enzyme that removes fatty acids from bacterial lipopolysaccharide. Both contain a signal sequence followed by a saposin domain and a GDSL-lipase domain. GPIdeAc-/- trypanosomes were viable in vitro and in animals. Affinity-purified HA-tagged GPIdeAc was shown to have inositol deacylase activity. However, total inositol deacylase activity was only reduced in GPIdeAc-/- trypanosomes and the VSG GPI anchor was indistinguishable from wild type. These results suggest that there is redundancy in T.brucei inositol deacylase activity and that there is another enzyme whose sequence is not recognizably related to GPIdeAc.
    Original languageEnglish
    Pages (from-to)4923-4934
    Number of pages12
    JournalThe EMBO Journal
    Volume20
    Issue number17
    DOIs
    Publication statusPublished - 2001

      Fingerprint

    Keywords

    • Acyloxyacyl hydrolase
    • Glycosylphosphatidylinositols
    • Inositol deacylase
    • Saposin
    • Trypanosoma brucei

    Cite this