Isolation and Characterisation of the GPI:Protein Transamidase From Leishmania mexicana

Hilley, James D (1999) Isolation and Characterisation of the GPI:Protein Transamidase From Leishmania mexicana. PhD thesis, University of Glasgow.

Abstract

Many eukaryotic cell surface proteins are attached to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. Trypanosomatid parasitic protozoa such as Leishmania and Trypanosoma brucei make extensive use of this method of protein surface attachment. The perceived importance of GPI-anchored proteins makes the GPI biosynthetic pathway a good target for anti-parasite chemotherapy. The terminal step in the pathway is the addition of complete pre-formed GPI anchors to the carboxyl-termini of proteins. This involves replacing a GPI signal sequence with a GPI anchor in a transamidation reaction. Two of the proteins involved in this step have been identified in yeast and mammals. One of these components, GPI8, has significant homology to a family of plant cysteine proteinases, the legumains, and GPI8 is therefore believed to be the catalytic subunit of the transamidase. The work described in this thesis focussed on the cloning of the GPI8 gene from Leishmania mexicana and its characterisation. The predicted protein shares 31% identity with yeast and human homologues. The nucleotide sequence of a fragment of the T. brucei GPI8 gene has also been obtained. Targeted gene replacement of the single copy L. mexicana GPI8 produced GPI8 null mutants. The loss of GPI8 was confirmed by Southern blotting. The phenotype of the GPI8 null mutants was analysed with the following findings: (1) Mutant promastigotes grow well in culture. (2) GP63 is not detected on the promastigote surface and is greatly reduced in promastigote lysates. (3) Episomal re-expression of GPI8 restored GP63 to the cell surface. (4) GPI8 null mutants are able to infect macrophages in vitro to approximately wild type levels, and replicate within macrophages. (5) GPI8 null mutants are capable of forming lesions in mice. These data show that GPI-anchored proteins of L. mexicana are not essential for growth of promastigotes, invasion of macrophages by promastigotes, or infection of mice. It remains to be established if GPI-anchored proteins are required for survival in the sandfly. Sequence comparison of L. mexicana, yeast and human GPI8 proteins identified two potential active site cysteine residues. Mutation in which Cys216 was converted to a glycine led to loss of GPI8 activity, as assessed by GP63 surface expression, indicating that this may be the active site cysteine. Recombinant GPI8 was produced in E. coli and used to inoculate rabbits for the production of anti-serum. These antibodies detected recombinant protein but failed to detect protein in L. mexicana cell lysates. Antibodies raised against a peptide of GPI8 also recognised recombinant GPI8, but not GPI8 in L. mexicana lysates. A protein of 38-40 kDa, however, was detected in cell lysates of T. brucei with the antirecombinant GPI8 antibodies. This antiserum gave a similar immunofluorescence pattern in T. brucei to those of an epitope-tagged ribosomal protein, QM. There is some overlap in fluorescence patterns between tagged QM and the endoplasmic reticulum marker protein BiP. This suggests that GPI8 is located in the ER of T. brucei, the same subcellular location that has been identified in yeast. A fusion protein of GPI8 and GFP was expressed in L. mexicana to attempt to localise GPI8 in this organism. The fusion protein, however, was unable to restore GPI8 function in the null mutant, therefore data on its localisation cannot be inferred.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Jeremy Mottram
• Keywords: Parasitology
• Date of Award: 1999
• Depositing User: Enlighten Team
• Unique ID: glathesis:1999-76120
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 16:37
• Last Modified: 19 Nov 2019 16:37
• URI: https://theses.gla.ac.uk/id/eprint/76120