Cloning of Trypanosoma brucei and Leishmania major Genes Encoding the GlcNAc-Phosphatidylinositol De-N-acetylase of Glycosylphosphatidylinositol Biosynthesis That Is Essential to the African Sleeping Sickness Parasite

Tunhan Chang,Kenneth G Milne,Maria Lucia Sampaio Güther,Terry K Smith,Michael A.J Ferguson

doi:10.1074/jbc.m208374200

Abstract

The second step of glycosylphosphatidylinositol anchor biosynthesis in all eukaryotes is the conversion of D-GlcNAcalpha1-6-d-myo-inositol-1-HPO(4)-sn-1,2-diacylglycerol (GlcNAc-PI) to d-GlcNalpha1-6-d-myo-inositol-1-HPO(4)-sn-1,2-diacylglycerol by GlcNAc-PI de-N-acetylase. The genes encoding this activity are PIG-L and GPI12 in mammals and yeast, respectively. Fragments of putative GlcNAc-PI de-N-acetylase genes from Trypanosoma brucei and Leishmania major were identified in the respective genome project data bases. The full-length genes TbGPI12 and LmGPI12 were subsequently cloned, sequenced, and shown to complement a PIG-L-deficient Chinese hamster ovary cell line and restore surface expression of GPI-anchored proteins. A tetracycline-inducible bloodstream form T. brucei TbGPI12 conditional null mutant cell line was created and analyzed under nonpermissive conditions. TbGPI12 mRNA levels were reduced to undetectable levels within 8 h of tetracycline removal, and the cells died after 3-4 days. This demonstrates that TbGPI12 is an essential gene for the tsetse-transmitted parasite that causes Nagana in cattle and African sleeping sickness in humans. It also validates GlcNAc-PI de-N-acetylase as a potential drug target against these diseases. Washed parasite membranes were prepared from the conditional null mutant parasites after 48 h without tetracycline. These membranes were shown to be greatly reduced in GlcNAc-PI de-N-acetylase activity, but they retained their ability to make GlcNAc-PI and to process d-GlcNalpha1-6-d-myo-inositol-1-HPO(4)-sn-1,2-diacylglycerol to later glycosylphosphatidylinositol intermediates. These results suggest that the stabilities of other glycosylphosphatidylinositol pathway enzymes are not dependent on GlcNAc-PI de-N-acetylase levels.

Highlights

This paper is dedicated to Professor John S
The situation is less clear in Leishmania sp., where, for example, L. mexicana is infective without lipophosphoglycans, glycoinositolphospholipids, and GPI-anchored glycoproteins, whereas L. major is significantly attenuated in the absence of lipophosphoglycan [12,13,14]
We further demonstrate that membranes from the conditional null mutant are deficient in GlcNAc-PI de-N-acetylase activity under non-permissive conditions and that TbGPI12 is an essential gene in bloodstream form T. brucei

Summary

The sequence of events underlying GPI biosynthesis has

Been studied in T. brucei [15,16,17,18,19,20], Trypanosoma cruzi [21], Toxoplasma gondii [22], Plasmodium falciparum [23], Leishmania sp. (24 –26), Saccharomyces cerevisiae [27, 28], and mammalian cells (29 –31), and references therein. The trypanosomal enzyme can deN-acetylate GlcNAc-PI containing either D- or L-myo-inositol and ␣- or ␤-D-GlcNAc, whereas the human (HeLa) enzyme strictly requires ␣-D-GlcNAc [1,2,3,4,5,6]D-myo-inositol [37, 38] These differences, and the ability of the trypanosomal enzyme to tolerate a C8 O-alkyl substituent on C2 of the D-myo-inositol residue, were recently exploited in the design and synthesis of two parasite-specific GlcNAc-PI de-N-acetylase suicide substrate inhibitors [38]. We further demonstrate that membranes from the conditional null mutant are deficient in GlcNAc-PI de-N-acetylase activity under non-permissive conditions and that TbGPI12 is an essential gene in bloodstream form T. brucei

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

CONCLUSIONS