Purification, cloning and characterization of a GPI inositol deacylase from Trypanosoma brucei.
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.
Güther
M L
ML
Division of Biological Chemistry and Molecular Microbiology, The Wellcome Trust Biocentre, Dundee DD1 5EH, UK.
Leal
S
S
Morrice
N A
NA
Cross
G A
GA
Ferguson
M A
MA
D020413:3' Untranslated Regions / Q000737:chemistry / Q000235:genetics; D020121:5' Untranslated Regions / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D007531:Isoflurophate / Q000493:pharmacokinetics / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D014353:Trypanosomiasis, African / Q000097:blood; D014357:Trypsin
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D017261:Glycosylphosphatidylinositols; D010446:Peptide Fragments; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D007531:Isoflurophate; C436709:GPIdeAc protein, Trypanosoma brucei; D010744:Phosphoric Monoester Hydrolases; D014357:Trypsin