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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

2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês

10.1074/jbc.m208374200

ISSN

1083-351X

Autores

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

Tópico(s)

Lysosomal Storage Disorders Research

Resumo

The second step of glycosylphosphatidylinositol anchor biosynthesis in all eukaryotes is the conversion of D-GlcNAcα1–6-d-myo-inositol-1-HPO4-sn-1,2-diacylglycerol (GlcNAc-PI) tod-GlcNα1–6-d-myo-inositol-1-HPO4-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 bruceiand Leishmania major were identified in the respective genome project data bases. The full-length genes TbGPI12and 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 TbGPI12conditional 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 thatTbGPI12 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 processd-GlcNα1–6-d-myo-inositol-1-HPO4-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. The second step of glycosylphosphatidylinositol anchor biosynthesis in all eukaryotes is the conversion of D-GlcNAcα1–6-d-myo-inositol-1-HPO4-sn-1,2-diacylglycerol (GlcNAc-PI) tod-GlcNα1–6-d-myo-inositol-1-HPO4-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 bruceiand Leishmania major were identified in the respective genome project data bases. The full-length genes TbGPI12and 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 TbGPI12conditional 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 thatTbGPI12 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 processd-GlcNα1–6-d-myo-inositol-1-HPO4-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. A significant proportion of eukaryotic cell-surface glycoproteins are attached to the plasma membrane by covalent linkage to a glycosylphosphatidylinositol (GPI) 1The abbreviations used are: GPI, glycosylphosphatidylinositol; GlcN-PI, d-GlcNα1–6-d-myo-inositol-1-HPO4-sn-1,2-diacylglycerol; GlcNAc-PI, d-GlcNAcα1–6-d-myo-inositol-1-HPO4-sn-1,2-diacylglycerol; PI, phosphatidylinositol; ORF, open reading frame; BAC, bacterial artificial chromosome; CHO, Chinese hamster ovary; UTR, untranslated region; HPTLC, high performance thin layer chromatography; DAF, decay-accelerating factor; HYG, hygromycin phosphotransferase; PAC, puromycin acetyltransferase; Ti, tetracycline-inducible1The abbreviations used are: GPI, glycosylphosphatidylinositol; GlcN-PI, d-GlcNα1–6-d-myo-inositol-1-HPO4-sn-1,2-diacylglycerol; GlcNAc-PI, d-GlcNAcα1–6-d-myo-inositol-1-HPO4-sn-1,2-diacylglycerol; PI, phosphatidylinositol; ORF, open reading frame; BAC, bacterial artificial chromosome; CHO, Chinese hamster ovary; UTR, untranslated region; HPTLC, high performance thin layer chromatography; DAF, decay-accelerating factor; HYG, hygromycin phosphotransferase; PAC, puromycin acetyltransferase; Ti, tetracycline-inducible membrane anchor. The structure and biosynthesis of GPI membrane anchors and related molecules have been reviewed recently (1Ferguson M.A.J. J. Cell Sci. 1999; 112: 2799-2808Crossref PubMed Google Scholar, 2Morita Y.S. Acosta-Serrano A. Englund P.T. Ernst P.S. Hart G.W. Oligosaccharides in Chemistry and Biology—A Comprehensive Handbook. Wiley-VCH, Weinheim, Germany2000: 417-433Google Scholar, 3Kinoshita T. Inoue N. Curr. Opin. Chem. Biol. 2000; 4: 632-638Crossref PubMed Scopus (167) Google Scholar, 4McConville M.J. Mullin K.A. Ilgoutz S.C. Teasdale R.D. Microbiol. Mol. Biol. Rev. 2002; 66: 122-154Crossref PubMed Scopus (201) Google Scholar). The basic GPI core structure attached to protein comprises NH2CH2CH2PO4H-6Manα1–2Manα1–6Manα1–4GlcNα1–6-d-myo-inositol-1-HPO4-lipid, where the lipid can be diacylglycerol, alkylacylglycerol, or ceramide. This minimal GPI structure may be embellished with additional ethanolamine phosphate groups and/or carbohydrate side-chains in a species-and tissue-specific manner (5Ferguson M.A.J. Brimacombe J.S. Brown J.R. Crossman A. Dix A. Field R.A. Güther M.L.S. Milne K.G. Sharma D.K. Smith T.K. Biochim. Biophys. Acta. 1999; 1455: 327-340Crossref PubMed Scopus (122) Google Scholar). Protozoa tend to express significantly higher densities of cell-surface GPI-anchored proteins than do higher eukaryotes (1Ferguson M.A.J. J. Cell Sci. 1999; 112: 2799-2808Crossref PubMed Google Scholar, 4McConville M.J. Mullin K.A. Ilgoutz S.C. Teasdale R.D. Microbiol. Mol. Biol. Rev. 2002; 66: 122-154Crossref PubMed Scopus (201) Google Scholar, 6Guha-Niyogi A. Sullivan D.R. Turco S.J. Glycobiology. 2001; 11: 45R-59RCrossref PubMed Scopus (121) Google Scholar). For example,Trypanosoma brucei, the causative agent of African sleeping sickness, expresses a dense cell-surface coat consisting of ∼5 × 106 dimers of a GPI-anchored variant surface glycoprotein that protects the parasite from the alternative complement pathway of the host and, through antigenic variation, from specific immune responses (7Cross G.A.M. Bioessays. 1996; 18: 283-291Crossref PubMed Scopus (203) Google Scholar). The related kinetoplastid parasiteLeishmania sp. expresses lower copy numbers of GPI-anchored glycoproteins, such as the promastigote surface protease (Psp or gp63), gp42, and GPI-anchored proteophosphoglycans, but high copy numbers of the GPI-related structures lipophosphoglycan and the glycoinositolphospholipids (4McConville M.J. Mullin K.A. Ilgoutz S.C. Teasdale R.D. Microbiol. Mol. Biol. Rev. 2002; 66: 122-154Crossref PubMed Scopus (201) Google Scholar, 6Guha-Niyogi A. Sullivan D.R. Turco S.J. 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Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10673-10675Crossref PubMed Scopus (61) Google Scholar). 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 (12Garami A. Mehlert A. Ilg T. Mol. Cell. Biol. 2001; 21: 8168-8183Crossref PubMed Scopus (81) Google Scholar, 13Spath G.F. Epstein L. Leader B. Singer S.M. Avila H.A. Turco S.J. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9258-9263Crossref PubMed Scopus (243) Google Scholar, 14Turco S.J. Spath G.F. Beverley S.M. Trends Parasitol. 2001; 17: 223-226Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The sequence of events underlying GPI biosynthesis has been studied inT. brucei (15Masterson W.J. Doering T.L. Hart G.W. Englund P.T. Cell. 1989; 56: 793-800Abstract Full Text PDF PubMed Scopus (211) Google Scholar, 16Masterson W.J. Raper J. Doering T.L. Hart G.W. Englund P.T. Cell. 1990; 62: 73-80Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 17Menon A.K. Schwarz R.T. Mayor S. Cross G.A.M. J. Biol. Chem. 1990; 265: 9033-9042Abstract Full Text PDF PubMed Google Scholar, 18Menon A.K. Mayor S. Schwarz R.T. EMBO J. 1990; 9: 4249-4258Crossref PubMed Scopus (121) Google Scholar, 19Güther M.L.S. Ferguson M.A.J. EMBO J. 1995; 14: 3080-3093Crossref PubMed Scopus (103) Google Scholar, 20Morita Y.S. Acosta-Serrano A. Buxbaum L.U. Englund P.T. J. Biol. Chem. 2000; 275: 14147-14154Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), Trypanosoma cruzi (21Heise N. Raper J. Buxbaum L.U. Peranovich T.M. de Almeida M.L. J. Biol. Chem. 1996; 271: 16877-16887Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar),Toxoplasma gondii (22Striepen B. Dubremetz J.-F. Schwarz R.T. 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Chem. 2000; 275: 24458-24465Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and mammalian cells (29Hirose S. Prince G.M. Sevlever D. Ravi L. Rosenberry T.L. Ueda E. Medof M.E. J. Biol. Chem. 1992; 267: 16968-16974Abstract Full Text PDF PubMed Google Scholar, 30Puoti A. Conzelmann A. J. Biol. Chem. 1993; 268: 7215-7224Abstract Full Text PDF PubMed Google Scholar, 31Chen R. Walter E.I. Parker G. Lapurga J.P. Millan J.L. Ikehara Y. Udenfriend S. Medof M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9512-9517Crossref PubMed Scopus (68) Google Scholar), and references therein. In all cases, GPI biosynthesis involves the addition of GlcNAc to phosphatidylinositol (PI) to give GlcNAc-PI, which is then de-N-acetylated byN-acetyl-d-glucosaminylphosphatidylinositol deacetylase (EC 3.1.1.69), referred to here as GlcNAc-PI de-N-acetylase, to form GlcN-PI (32Doering T.L. Masterson W.J. Englund P.T. Hart G.W. J. Biol. Chem. 1989; 264: 11168-11173Abstract Full Text PDF PubMed Google Scholar, 33Milne K.G. Field R.A. Masterson W.J. Cottaz S. Brimacombe J.S. Ferguson M.A.J. J. Biol. Chem. 1994; 269: 16403-16408Abstract Full Text PDF PubMed Google Scholar, 34Nakamura N. Inoue N. Watanabe R. Takahashi M. Takeda J. Stevens V.L. Kinoshita T. J. Biol. Chem. 1997; 272: 15834-15840Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 35Watanabe R. Ohishi K. Maeda Y. Nakamura N. Kinoshita T. Biochem. J. 1999; 339: 185-192Crossref PubMed Scopus (76) Google Scholar). De-N-acetylation is a prerequisite for the mannosylation of GlcN-PI to form later GPI intermediates (34Nakamura N. Inoue N. Watanabe R. Takahashi M. Takeda J. Stevens V.L. Kinoshita T. J. Biol. Chem. 1997; 272: 15834-15840Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 36Sharma D.K. Smith T.K. Crossman A. Brimacombe J.S. Ferguson M.A.J. Biochem. J. 1997; 328: 171-177Crossref PubMed Scopus (42) Google Scholar). The GlcNAc-PI de-N-acetylases from protozoan and mammalian sources are similar with regard to their specificities for the acyl (R) group removed from GlcNR-PI substrates (36Sharma D.K. Smith T.K. Crossman A. Brimacombe J.S. Ferguson M.A.J. Biochem. J. 1997; 328: 171-177Crossref PubMed Scopus (42) Google Scholar), but differ with regard to their specificity for the myo-inositol residue. Thus, the trypanosomal enzyme can de-N-acetylate GlcNAc-PI containing either d- or l-myo-inositol and α- or β-d-GlcNAc, whereas the human (HeLa) enzyme strictly requires α-d-GlcNAc (1Ferguson M.A.J. J. Cell Sci. 1999; 112: 2799-2808Crossref PubMed Google Scholar, 2Morita Y.S. Acosta-Serrano A. Englund P.T. Ernst P.S. Hart G.W. Oligosaccharides in Chemistry and Biology—A Comprehensive Handbook. Wiley-VCH, Weinheim, Germany2000: 417-433Google Scholar, 3Kinoshita T. Inoue N. Curr. Opin. Chem. Biol. 2000; 4: 632-638Crossref PubMed Scopus (167) Google Scholar, 4McConville M.J. Mullin K.A. Ilgoutz S.C. Teasdale R.D. Microbiol. Mol. Biol. Rev. 2002; 66: 122-154Crossref PubMed Scopus (201) Google Scholar, 5Ferguson M.A.J. Brimacombe J.S. Brown J.R. Crossman A. Dix A. Field R.A. Güther M.L.S. Milne K.G. Sharma D.K. Smith T.K. Biochim. Biophys. Acta. 1999; 1455: 327-340Crossref PubMed Scopus (122) Google Scholar, 6Guha-Niyogi A. Sullivan D.R. Turco S.J. Glycobiology. 2001; 11: 45R-59RCrossref PubMed Scopus (121) Google Scholar)d-myo-inositol (37Sharma D.K. Smith T.K. Weller C.T. Crossman A. Brimacombe J.S. Ferguson M.A.J. Glycobiology. 1999; 9: 415-422Crossref PubMed Scopus (36) Google Scholar, 38Smith T.K. Crossman A. Borissow C.N. Paterson M.J. Dix A. Brimacombe J.S. Ferguson M.A.J. EMBO J. 2001; 20: 3322-3332Crossref PubMed Scopus (48) Google Scholar). These differences, and the ability of the trypanosomal enzyme to tolerate a C8 O-alkyl substituent on C2 of thed-myo-inositol residue, were recently exploited in the design and synthesis of two parasite-specific GlcNAc-PI de-N-acetylase suicide substrate inhibitors (38Smith T.K. Crossman A. Borissow C.N. Paterson M.J. Dix A. Brimacombe J.S. Ferguson M.A.J. EMBO J. 2001; 20: 3322-3332Crossref PubMed Scopus (48) Google Scholar). The gene encoding the rat de-N-acetylase (PIG-L) was the first to be cloned (34Nakamura N. Inoue N. Watanabe R. Takahashi M. Takeda J. Stevens V.L. Kinoshita T. J. Biol. Chem. 1997; 272: 15834-15840Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) and a yeast homologue (GPI12) has been shown to complementPIG-L-deficient mammalian cells and vice versa (35Watanabe R. Ohishi K. Maeda Y. Nakamura N. Kinoshita T. Biochem. J. 1999; 339: 185-192Crossref PubMed Scopus (76) Google Scholar). Here, we describe the molecular cloning of the T. brucei andL. major homologues TbGPI12 andLmGPI12, demonstrate functional complementation in aPIG-L-deficient mammalian cell line, and describe the creation of a T. brucei TbGPI12 conditional null mutant. 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. The 401-bp end-sequence of an Institute for Genomic Research genome survey sequence clone (AQ644232), returned from the tBLASTn search with yeast GPI12p (accession number P23797), was used to design a reverse PCR primer (5′-cgcGGATCCtcatgcgacccccaattccttcacttc-3′ (capital letters indicate a BamHI site)) that was used with Pfupolymerase, blood-stream form T. bruceicDNA, and a forward primer based on the 5′ mini-exon (5′-ggcccgctattattagaacagtttctgta-3′) to amplify an ∼0.8-kb fragment containing the entire TbGPI12 ORF. Amplification conditions were 95 °C for 45 s, 60 °C for 1 min, and 72 °C for 3 min for 30 cycles. The PCR product was purified from an agarose gel (QIAEX II kit) and ligated into a pUC18 cloning vector using a SureClone ligation kit (Amersham Biosciences). Twelve representative clones were used for DNA sequencing, revealing a 759-bp ORF. The same PCR product (the TbGPI12 probe) was fluorescein-labeled by random priming (Gene Images kit; Amersham Biosciences) or labeled with32P (Prime-It RmT random primer labeling kit; Stratagene) for use in Southern blotting and for probing a BAC library filter, respectively (see below). A tBlastn search with the sequence LVIAHPDDEAMFFAP, a sequence strictly conserved in rat and human PIG-L and substantially conserved in yeast GPI12, identified an L. major expressed sequence tag sequence (AA728250) with 85% similarity. The corresponding cDNA clone was kindly provided by Prof. J. M. Blackwell (Cambridge University) and fully sequenced. The clone contained the full-length LmGPI12 gene. For expression in mammalian cells, the TbGPI12 gene was PCR-amplified usingPfu in two segments. The 5′-end of the ORF was amplified using forward primer 5′-gagAAGCTTCATATGcatggtgctttggcgtttggg-3′ and reverse primer 5′catggcggaaaagctGgtgaacaatgag-3′ and the 3′-end of the ORF was amplified using forward primer 5′-ctcattgttcacCagcttttccgccatg-3′ and reverse primer 5′-cgGGATCCtcaCAGGTCCTCCTCCGAGATTAGCTTCTGTTCGTTAATTAAtgcgacccccaattcctt-3′. The two PCR products were used together in a furtherPfu PCR reaction to yield a product containing a silent mutation that removed a HindIII site (capital italic letters indicate the mutation), a myc epitope tag fused to the C terminus of TbGPI12 (underlined letters), and 5′-HindIII and 3′-BamHI restriction sites (capital letters). The purified construct was digested withHindIII and BamHI and ligated into the respective cloning sites of the pcDNA3.1/Hygro (+) (Invitrogen) mammalian expression vector. The LmGPI12 gene was also PCR-amplified using Pfuin two segments. The 5′-end of the ORF was amplified using forward primer 5′-cccAAGCTTgggatgcacagtatcacagtt-3′ and reverse primer 5′-gcaggtggaggatGcctggaggcatgttc-3′ and the 3′-end of the ORF was amplified using forward primer 5′-gaacatgcctccaggCatcctccacctgc-3′and reverse primer 5′-cgcGGATCCgcgctagagctcttcgatctc-3′. The two PCR products were used together in a further Pfu PCR reaction to yield a product containing a silent mutation that removed a BamHI site (capital italic letters indicate the mutation) and 5′-HindIII and 3′-BamHI restriction sites (capital letters). The purified construct was digested withHindIII and BamHI and ligated into the respective cloning sites of pcDNA3.1/Hygro (+). The pcDNA3.1/Hygro (+) plasmids (empty and containingTbGPI12-myc or LmGPI12) were purified (Qiagen Maxi-Prep), precipitated, washed with ethanol, resuspended in sterile water, and used for transient transfections. Trypsin-treatedPIG-L-deficient and CD59- and decay-accelerating factor (DAF)-transgenic CHO-K1 (clone M2S2) cells (34Nakamura N. Inoue N. Watanabe R. Takahashi M. Takeda J. Stevens V.L. Kinoshita T. J. Biol. Chem. 1997; 272: 15834-15840Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) were washed twice and resuspended at 1–2 × 107/ml in ice-cold phosphate-buffered saline. Aliquots of 1 ml were mixed with 50–70 μg of plasmid DNA, incubated for 20 min on ice, and electroporated in a 0.4-cm cuvette at 260 V, 950 μF, with a Bio-Rad gene-pulser. The cells were immediately transferred to 20 ml of Dulbecco's modified Eagle's medium with glutamine, 10% fetal calf serum, 100 units/ml penicillin/streptomycin, and 0.3 mg/ml G418 (to maintain the CD59- and DAF-containing plasmid), and cultured at 37 °C. Two days after transient transfection, the cells were incubated with anti-CD59 (B229; 10 μg/ml) and anti-DAF (813.6; 10 μg/ml) mouse monoclonal antibodies, followed by fluorescein isothiocyanate-conjugated secondary antibody (Dako F0313) and visualized by fluorescence microscopy using an MRC-600 laser scanning confocal imaging system (Nikon Microphot-SA). T. brucei genomic DNA (5 μg/lane) was digested with various restriction enzymes and the products were resolved on a 0.7% agarose gel. After transfer to nitrocellulose and UV-cross-linking, the blot was hybridized with fluorescein-labeled TbGPI12 probe (16 h, 60 °C) and washed twice with 1× SSC, 0.1% SDS for 15 min and twice with 0.5× SSC, 0.1% SDS for 15 min. Blots were developed with horseradish peroxidase-conjugated anti-fluorescein antibody according to the manufacturers instructions (gene images CDP-Star kit; AmershamBiosciences). A T. brucei strain 427 BAC library filter (CHORI RPCI-102), representing 46-fold genome coverage, was probed with a 32P-labeled TbGPI12 probe under the same conditions described for the Southern blot. Fifty positive clones were identified and the corresponding TbGPI12-containing BAC plasmids were purified from 3-ml cultures of four clones, using the "DNA isolation from BAC & PAC clones" protocol recommended by CHORI BACPAC Resources (www.chori.org/bacpac). The presence of theTbGPI12 gene was confirmed by PCR using 5′-gagAAGCTTCATATGcatggtgctttggcgtttggg-3 and 5′-cgcGGATCCtcatgcgacccccaattccttcacttc-3′ forward and reverse primers. One clone was selected and the purified BAC plasmid DNA (1 μg) was used as template for DNA sequencing using primers from within the gene, 5′-catcgcttcatcgtccgggtgtgc-3′ and 5′-attccgccgacctcattgttcaca-3′. Consequently, 466 bp of 5′-UTR and 648 bp of 3′-UTR sequence were obtained. Based on these data, 426 bp of 5′-UTR immediately upstream of the start codon were PCR-amplified using Pfu and genomic DNA template with the forward primer 5′-ataagaatGCGGCCGCcctccccccgcgcctacggatg-3′ and reverse primer 5′-gtttaaacttacggaccgtcaagcttgtgtatgagcgactccctcaac-3′. Likewise, 478 bp immediately downstream of the stop codon were PCR-amplified using with the forward primer 5′-gacggtccgtaagtttaaacggatccatcgaagaaatttagcccccgc-3′ and reverse primer 5′-ataagtaaGCGGCCGCcgactccggcatcttgtaaattg-3′. The two PCR products were used together in a further PCR reaction to yield a product containing the 5′-UTR linked to the 3′-UTR by a shortHindIII, PmeI, and BamHI cloning site (underlined letters) and NotI restriction sites at each end (capital letters). Subsequently, the PCR product was cloned into the NotI site of pGEM-5Zf(+) vector (Promega) and the hygromycin phosphotransferase (HYG) and puromycin acetyltransferase (PAC) drug resistance genes were introduced into the targeting vector via theHindIII/BamHI cloning site. The previously described HindIII-silenced, C-terminallymyc-tagged TbGPI12 construct was ligated into theHindIII/BamHI cloning site of the pLew100 tetracycline-inducible expression vector (39Wirtz E. Leal S. Ochatt C. Cross G.A.M. Mol. Biochem. Parasitol. 1999; 99: 89-101Crossref PubMed Scopus (1098) Google Scholar). Plasmids were prepared (Qiagen Maxi-Prep), digested with NotI, precipitated with ethanol, redissolved in sterile water, and used for electroporation of bloodstream form T. brucei strain 427 (variant 221), which are stably transfected to express T7 RNA polymerase and tetracycline repressor protein under continuous G418 selection (39Wirtz E. Leal S. Ochatt C. Cross G.A.M. Mol. Biochem. Parasitol. 1999; 99: 89-101Crossref PubMed Scopus (1098) Google Scholar). Cell culture, transformation and drug selection conditions were as described previously (39Wirtz E. Leal S. Ochatt C. Cross G.A.M. Mol. Biochem. Parasitol. 1999; 99: 89-101Crossref PubMed Scopus (1098) Google Scholar, 40Milne K.G. Güther M.L.S. Ferguson M.A.J. Mol. Biochem. Parasitol. 2001; 112: 301-304Crossref PubMed Scopus (27) Google Scholar, 41Güther M.L.S. Leal S. Morrice N. Cross G.A.M. Ferguson M.A.J. EMBO J. 2001; 20: 4923-4934Crossref PubMed Scopus (28) Google Scholar, 42Roper J.R. Güther M.L.S. Milne K.G. Ferguson M.A.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5884-5889Crossref PubMed Scopus (90) Google Scholar). Tet-system approved fetal calf serum (Clontech) was used in experiments on the effects of tetracycline-removal. Total RNA was prepared (Qiagen RNeasy Protect Midi kit) from 2 × 108 cells. Samples of RNA (5 μg) were run on formaldehyde agarose gel and transferred to Hybond-N nylon membrane (Amersham Biosciences) for hybridization with [α-32P]dCTP labeled TbGPI12 probe (Stratagene Prime-It RmT random primer labeling kit). As a loading control, a β-tubulin probe was used on the same blot. Bloodstream formT. brucei membranes (cell-free system) were prepared (15Masterson W.J. Doering T.L. Hart G.W. Englund P.T. Cell. 1989; 56: 793-800Abstract Full Text PDF PubMed Scopus (211) Google Scholar,19Güther M.L.S. Ferguson M.A.J. EMBO J. 1995; 14: 3080-3093Crossref PubMed Scopus (103) Google Scholar, 43Smith T.K. Cottaz S. Brimacombe J.S. Ferguson M.A.J. J. Biol. Chem. 1996; 271: 6476-6482Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) from wild-type cells and TbGPI12 conditional null mutant cells grown continuously in 1 μg/ml tetracycline and grown tetracycline-free for 48 h. Trypanosome membranes were washed twice and resuspended at 5 × 108 cell-equivalents/ml in 2× incorporation buffer supplemented with 10 mm N-ethylmaleimide or 2 mm dithiothreitol (43Smith T.K. Cottaz S. Brimacombe J.S. Ferguson M.A.J. J. Biol. Chem. 1996; 271: 6476-6482Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar,44Smith T.K. Sharma D.K. Crossman A. Dix A. Brimacombe J.S. Ferguson M.A.J. EMBO J. 1997; 16: 6667-6675Crossref PubMed Scopus (78) Google Scholar). The lysates were briefly sonicated and aliquots of 107cell equivalents were added to an equal volume of GDP-[2-3H]Man (0.4 μCi, 22 Ci/mmol; PerkinElmer) or UDP-[6-3H]GlcNAc (1 μCi, 41.6 Ci/mmol; PerkinElmer). When 350 pmol of synthetic GlcN-PI or GlcNAc-PI (45Cottaz S. Brimacombe J.S. Ferguson M.A.J. J. Chem. Soc. Perkin Trans. I. 1993; 1: 2945-2951Crossref Scopus (42) Google Scholar) were used, the GDP-[2-3H]Man solution was supplemented 0.3% (w/v)n-octyl-β-d-glucopyranoside. Samples were incubated for 1 h at 30 °C, and glycolipid samples were recovered for analysis by HPTLC before and after enzyme treatments. Samples were digested with jack bean α-mannosidase and Bacillus thuringiensisphosphatidylinositol-specific phospholipase C (both from Glyko) as described in (46Güther M.L.S. Masterson W.J. Ferguson M.A.J. J. Biol. Chem. 1994; 269: 18694-18701Abstract Full Text PDF PubMed Google Scholar). Samples and glycolipid standards were applied to 10 cm aluminum-backed silica gel-60 HPTLC plates (Merck) and developed with chloroform/methanol/1 m ammonium acetate/13m ammonia/water (180:140:9:9:23, v/v). Radiolabeled components were detected by fluorography at −70 °C after spraying with En3Hance (PerkinElmer) using Kodak XAR-5 film and an intensifying screen. A partial gene sequence was found in The Institute for Genomic Research T. brucei data base with a tBLASTn search, using the Saccharomyces cerevisiaeGPI12 protein sequence as the query. The putativeTbGPI12 gene fragment contained the 3′-end of the gene (377 bp), including a stop codon, followed by 24 bp of putative 3′-UTR. A cDNA clone was obtained by PCR using bloodstream formT. brucei cDNA as the template, a forward primer based on the 5′-spliced leader (a 35-bp sequence trans-spliced onto allT. brucei mRNA), and a reverse primer based on the 3′-end sequence found in the data base. The 0.8-kb product contained a 759-bp ORF (GenBankTM accession number AY157267). A genomic clone was also obtained by PCR using genomic DNA as the template, a forward primer to the 5′-end of the gene based on the cDNA clone, and a reverse primer based on the 3′-UTR sequence found in the data base. Both the cDNA and genomic DNA ORF sequences were identical. A tBlastn search with the sequence LVIAHPDDEAMFFAP, a conserved sequence in rat and human PIG-L that is largely conserved in yeast GPI12 (34Nakamura N. Inoue N. Watanabe R. Takahashi M. Takeda J. Stevens V.L. Kinoshita T. J. Biol. Chem. 1997; 272: 15834-15840Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), identified an L. majorexpressed sequence tag sequence. The corresponding cDNA clone was fully sequenced and found to contain the full-length LmGPI12gene (GenBank™ accession number AY157268). The predicted amino acid sequences of the two parasite putative GlcNAc-PI de-N-acetylases, aligned with related sequences, are shown in Fig. 1. All of the sequences predict proteins with the majority of their sequence in the cytoplasm and anchored to the endoplasmic reticulum via a single N-terminal transmembrane domain, an arrangement that has been demonstrated experimentally for rat PIG-L (34Nakamura N. Inoue N. Watanabe R. Takahashi M. Takeda J. Stevens V.L. Kinoshita T. J. Biol. Chem. 1997; 272: 15834-15840Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The PIG-L/GPI12 sequences have similarity to the pfam02585 family of prokaryote sequences that include Rv1170, a GlcNAcα1–1-d-myo-inositol de-N-acetylase involved in mycothiol synthesis inMycobacterium tuberculosis (47Newton G.L. Av-Gay Y

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