Procyclic Trypanosoma brucei Expresses Separate Sialidase and trans-Sialidase Enzymes on Its Surface Membrane
2006; Elsevier BV; Volume: 281; Issue: 45 Linguagem: Inglês
10.1074/jbc.m604951200
ISSN1083-351X
AutoresG. Montagna, John E. Donelson, Alberto C.C. Frasch,
Tópico(s)Galectins and Cancer Biology
ResumoThe procyclic stage of Trypanosoma brucei in the insect vector expresses a surface-bound trans-sialidase (TbTS) that transfers sialic acid from glycoconjugates in the environment to glycosylphosphatidylinositol-anchored proteins on its surface membrane. RNA interference against TbTS abolished trans-sialidase activity in procyclic cells but did not diminish sialidase activity, suggesting the presence of a separate sialidase enzyme for hydrolyzing sialic acid. A search of the T. brucei genome sequence revealed seven other putative genes encoding proteins with varying similarity to TbTS. RNA interference directed against one of these proteins, TbSA C, greatly decreased the sialidase activity but had no effect on trans-sialidase activity. The deduced amino acid sequence of TbSA C shares only 40% identity with TbTS but conserves most of the relevant residues required for catalysis. However, the sialidase has a tryptophan substitution for a tyrosine at position 170 that is crucial in binding the terminal galactose that accepts the transferred sialic acid. When this same tryptophan substitution in the sialidase was placed into the recombinant trans-sialidase, the mutant enzyme lost almost all of its trans-sialidase activity and increased its sialidase activity, further confirming that the gene and protein identified correspond to the parasite sialidase. Thus, in contrast to all other trypanosomes analyzed to date that express either a trans-sialidase or a sialidase but not both, T. brucei expresses these two enzymatic activities in two separate proteins. These results suggest that African trypanosomes could regulate the amount of critical sialic acid residues on their surface by modulating differential expression of each of these enzymes. The procyclic stage of Trypanosoma brucei in the insect vector expresses a surface-bound trans-sialidase (TbTS) that transfers sialic acid from glycoconjugates in the environment to glycosylphosphatidylinositol-anchored proteins on its surface membrane. RNA interference against TbTS abolished trans-sialidase activity in procyclic cells but did not diminish sialidase activity, suggesting the presence of a separate sialidase enzyme for hydrolyzing sialic acid. A search of the T. brucei genome sequence revealed seven other putative genes encoding proteins with varying similarity to TbTS. RNA interference directed against one of these proteins, TbSA C, greatly decreased the sialidase activity but had no effect on trans-sialidase activity. The deduced amino acid sequence of TbSA C shares only 40% identity with TbTS but conserves most of the relevant residues required for catalysis. However, the sialidase has a tryptophan substitution for a tyrosine at position 170 that is crucial in binding the terminal galactose that accepts the transferred sialic acid. When this same tryptophan substitution in the sialidase was placed into the recombinant trans-sialidase, the mutant enzyme lost almost all of its trans-sialidase activity and increased its sialidase activity, further confirming that the gene and protein identified correspond to the parasite sialidase. Thus, in contrast to all other trypanosomes analyzed to date that express either a trans-sialidase or a sialidase but not both, T. brucei expresses these two enzymatic activities in two separate proteins. These results suggest that African trypanosomes could regulate the amount of critical sialic acid residues on their surface by modulating differential expression of each of these enzymes. African trypanosomes are protozoan parasites responsible for sleeping sickness in humans and a similar disease in domestic animals called nagana. Their life cycle alternates between the bloodstream of a mammalian host and the tsetse fly vector (Glossina sp.). The surface of the bloodstream form of the parasite is completely covered with 107 copies of a single variant surface glycoprotein (VSG) 3The abbreviations and trivial names used are: VSG, variant surface glycoprotein; TbTS, T. brucei trans-sialidase; TcTS, T. cruzi trans-sialidase; TrSA, T. rangeli sialidase; dsRNA, double-stranded RNA; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; PLD, phospholipase D; IMAC, iminodiacetic acid metal affinity chromatography; MUNen5Ac, 2′-(4-methylumberifery)-α-d-N-acetylneuraminic acid; Pipes, 1,4-piperazinediethanesulfonic acid; Bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol. 3The abbreviations and trivial names used are: VSG, variant surface glycoprotein; TbTS, T. brucei trans-sialidase; TcTS, T. cruzi trans-sialidase; TrSA, T. rangeli sialidase; dsRNA, double-stranded RNA; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; PLD, phospholipase D; IMAC, iminodiacetic acid metal affinity chromatography; MUNen5Ac, 2′-(4-methylumberifery)-α-d-N-acetylneuraminic acid; Pipes, 1,4-piperazinediethanesulfonic acid; Bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol. bearing a glycosylphosphatidylinositol (GPI) anchor. These bloodstream trypanosomes elude the immune response of the mammalian host by periodically switching from one VSG to another immunologically distinct VSG. The bloodstream form of the parasite differentiates into the procyclic form when ingested by the insect vector. This differentiation involves a remodeling of the surface in which the VSG coat is rapidly shed and replaced with a new set of invariant GPI-anchored glycoproteins known as procyclins (1Borst P. Cell. 2002; 109: 5-8Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Procyclins have an unusual GPI anchor that, unlike the GPI anchor of VSGs, is decorated with branched poly-N-acetyllactosamine repeats capped by sialic acid residues (2Treumann A. Zitzman N. Prescott A.R. Almond A. Sheehan J. Ferguson M.A. J. Mol. Biol. 1997; 269: 529-547Crossref PubMed Scopus (121) Google Scholar). Trypanosomes are unable to synthesize sialic acid, but the procyclic form of the African trypanosome Trypanosoma brucei expresses a specific enzyme, trans-sialidase (TbTS), that transfers sialic acid from sialylated glycoconjugates present in the tsetse fly midgut (such as in the blood meal or on the midgut cells) to acceptor molecules on its surface membrane (such as the side chain of GPI-anchored proteins and free GPIs) (3Vasella E. Butikofer P. Englster M. Jelk J. Roditi I. Mol. Biol. Cell. 2003; 14: 1308-1318Crossref PubMed Google Scholar). Trypanosomes lacking sialic acids due to a defective GPI-anchored trans-sialidase do not survive in the midgut, indicating that sialic acid residues of the GPI are critical for the survival of the parasite in tsetse flies (4Nagamune K. Acosta Serrano A. Uemura H. Kunz-Renggli C. Maeda Y. Ferguson M.A. Kinoshita T. J. Exp. Med. 2004; 199: 1445-1450Crossref PubMed Scopus (74) Google Scholar). Trypanosoma cruzi, the agent of Chagas disease in the Americas, has a different life cycle, and its developmental expression of trans-sialidase shows a different pattern. The noninvasive epimastigote form of T. cruzi in the midgut of its hemipterous blood-sucking insect vector expresses trans-sialidase (TcTS) late in this midgut stage. After transmission to the bloodstream of the mammalian host, T. cruzi parasites continue expressing trans-sialidase, although from a different set of genes, and rapidly acquire sialic acid from sialoconjugates in the host (5Schenkman S. Jiang M.S. Hart G.W. Nussenzweig V. Cell. 1991; 65: 1117-1125Abstract Full Text PDF PubMed Scopus (377) Google Scholar). Surface sialylation in T. cruzi plays a central role in its evasion of the early complement-mediated immune response (6Tomlinson S. Raper J. Parasitol. Today. 1998; 14: 354-359Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) and its host cell adhesion/invasion mechanism (7Schenckman S. Eichinger D. Parasitol. Today. 1993; 9: 218-225Abstract Full Text PDF PubMed Scopus (116) Google Scholar).The trans-sialidase from T. cruzi is the one that has been better analyzed, including the determination of its three-dimensional structure (8Schenkman S. Pontes de Carvalho L.C. Nussenzweig V. J. Exp. Med. 1992; 175: 567-575Crossref PubMed Scopus (112) Google Scholar, 9Pereira M.E. Mejia J.S. Ortega-Barria E. Matzilevich D. Priori R.P. J. Exp. Med. 1991; 174: 179-191Crossref PubMed Scopus (113) Google Scholar, 10Parodi A.J. Pollevick G.D. Mautner M. Buschiazzo A. Sanchez D.O. Frasch A.C.C. EMBO J. 1992; 11: 1705-1710Crossref PubMed Scopus (105) Google Scholar, 11Buschiazzo A. Amaya M.F. Cremona M.L. Frasch A.A.C. Alzari P.M. Mol. Cell. 2002; 10: 757-768Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). A related American parasite, Trypanosoma rangeli, expresses a homologous protein (TrSA) with 70% amino acid identity to TcTS, but this enzyme is devoid of trans-glycosidase activity and is strictly a hydrolase (12Buschiazzo A. Campetella O. Frasch A.C.C. Glycobiology. 1997; 7: 1167-1173Crossref PubMed Scopus (43) Google Scholar). In the case of T. brucei, the trans-sialidase has been purified and characterized (13Pontes de-Carvalho L.C. Tomlinson S. Vandekerckhove F. Bienen E.J. Clarkson A.B. Jiang M.S. Hart G.W. Nussenzweig V. J. Exp. Med. 1993; 177: 465-474Crossref PubMed Scopus (85) Google Scholar, 14Englstler M. Reuter G. Schauer R. Mol. Biochem. Parasitol. 1992; 54: 21-30Crossref PubMed Scopus (46) Google Scholar, 15Englstler M. Reuter G. Schauer R. Mol. Biochem. Parasitol. 1993; 61: 1-14Crossref PubMed Scopus (87) Google Scholar), and information on the parasite gene encoding the protein is available (16Montagna G. Cremona M.L. Paris G. Amaya M.F. Alzari P.M. Frasch A.C.C. Eur. J. Biochem. 2002; 269: 2941-2950Crossref PubMed Scopus (53) Google Scholar). It should also be mentioned that the T. brucei and T. cruzi trans-sialidases have been found to be essentially trans-glycosidases. However, in the absence of acceptors of sialic acid in the milieu, they are able to release free sialic acid and thus are, to a much lower extent, hydrolytic enzymes.A comparison of the crystal structures of TcTS and TrSA (17Buschiazzo A. Tavares G.A. Campetella O. Spinelli S. Cremona M.L. Paris G. Amaya M.F. Frasch A.C.C. Alzari P.M. EMBO J. 2000; 19: 16-24Crossref PubMed Scopus (122) Google Scholar) and information derived from several mutagenesis approaches (16Montagna G. Cremona M.L. Paris G. Amaya M.F. Alzari P.M. Frasch A.C.C. Eur. J. Biochem. 2002; 269: 2941-2950Crossref PubMed Scopus (53) Google Scholar, 18Paris G. Cremona M.L. Amaya M.F. Buschiazzo A. Giambiagi S. Frasch A.C.C. Alzari P.M. Glycobiology. 2001; 11: 305-311Crossref PubMed Scopus (47) Google Scholar, 19Paris G. Ratier L. Amaya M.F. Nguyen T. Alzari P.M. Frasch A.C.C. J. Mol. Biol. 2005; 345: 923-934Crossref PubMed Scopus (74) Google Scholar) show that trypanosomal sialidases and trans-sialidases share a similar active site architecture in which several amino acid residues critical for enzyme function are conserved. In the T. cruzi and T. rangeli enzymes, a conserved tryptophan residue (Trp-313) was shown to be implicated in the binding of substrate and to be necessary for the specificity of the enzyme for α-(2,3)-linkage sialic acid (18Paris G. Cremona M.L. Amaya M.F. Buschiazzo A. Giambiagi S. Frasch A.C.C. Alzari P.M. Glycobiology. 2001; 11: 305-311Crossref PubMed Scopus (47) Google Scholar). Other residues surrounding the active site differ when the structures of sialidase and trans-sialidase are compared. In particular, Tyr-119 (replaced by serine in TrSA) was found to be essential for the transfer reaction and important in the structural environment of the catalytic nucleophile Tyr-342.T. cruzi trans-sialidases and T. rangeli sialidases are both encoded by a multigene family (7Schenckman S. Eichinger D. Parasitol. Today. 1993; 9: 218-225Abstract Full Text PDF PubMed Scopus (116) Google Scholar). T. cruzi has ∼140 trans-sialidase genes, half of which encode proteins lacking activity due to a substitution of Tyr-342 by a histidine residue (20Cremona M.L. Sanchez D.O. Frasch A.C.C. Campetella O. Gene. 1995; 160: 123-128Crossref PubMed Scopus (88) Google Scholar). In contrast, it has been postulated that T. brucei has a much lower number of trans-sialidase genes (16Montagna G. Cremona M.L. Paris G. Amaya M.F. Alzari P.M. Frasch A.C.C. Eur. J. Biochem. 2002; 269: 2941-2950Crossref PubMed Scopus (53) Google Scholar). Recently, two different partial sequences of putative trans-sialidase genes were identified in another African trypanosome species, Trypanosoma congolense (21Tiralongo E. Martensen I. Grotzinger J. Tiralongo J. Schauer R. Biol. Chem. 2003; 384: 1203-1213Crossref PubMed Scopus (22) Google Scholar), supporting the hypothesis that the number of gene copies may be lower in African trypanosomes. In this work, we show that, in contrast to all other trypanosome species examined to date, T. brucei expresses on its surface different proteins bearing trans-sialidase and sialidase activities, an unusual situation that allows the parasite to regulate sialic acid content on its surface through independent enzymes.EXPERIMENTAL PROCEDURESCell Lines and Transfections—Procyclic T. brucei 29-13 cells (T7RNAP NEO TETR HYG) or their derivatives were used for all studies. The T. brucei 29-13 cells were maintained in SDM-79 medium supplemented with 10% fetal calf serum and were transfected with EcoRV-linearized plasmid (5–10 μg) as described previously (22Hill K.L. Hutchings N.R. Russell D.G. Donelson J.E. J. Cell Sci. 1999; 112: 3091-3101Crossref PubMed Google Scholar). Logarithmic phase cells (5 × 106 cells ml–1) were collected by centrifugation, washed with electroporation medium (a 3:1 mixture of Cytomix, 120 mm KCl, 0.15 mm CaCl2, KiHPO4, 25 mm HEPES, 2 mm EDTA, 5 mm MgCl2, pH 7.6) and phosphate solution (277 mm sucrose, 1 mm MgCl2, 7 mm KiPO4, pH 7.4) and suspended in electroporation medium at a concentration of 5 × 107 ml–1. 0.45 ml of cells were mixed with 0.1 ml of linearized DNA in a 0.4-cm electroporation cuvette and subjected to two pulses with a Bio-Rad Gene Pulser electroporator set at 1500 V and 25 microfarads. Stable transformants were selected in 15 μg of G418 ml–1, 50 μg of hygromycin ml–1, and 2.5 μg of phleomycin ml–1. After drug-resistant pooled lines were established, clonal lines were obtained by limiting dilution.RNA Interference (RNAi)—RNAi experiments were performed using the vector p2T7Ti (23LaCount D.J. Bruse S. Hill K.L. Donelson J.E. Mol. Biochem. Parasitol. 2000; 111: 67-76Crossref PubMed Scopus (157) Google Scholar), which allows for stable tetracycline-inducible expression of double-stranded RNA from T7 promoters in procyclic T. brucei 29-13 cells. 500- and 1000-bp fragments extending downstream from the initiator methionine codon of the TbTS open reading frame were PCR-amplified using the following primers: Tb oligonucleotide 14 (5′-ATGGAGCTCCAGCAACA-3′) and Tb oligonucleotide 15 (5′-TATTTAGCAACGGTGTCGGTG-3′) for the 500-bp fragment and Tb oligonucleotide 14 and Tb oligonucleotide 16 (5′-GCTCATCAGCAGTTTCCCATTCCAC-3′) for the 1000-bp fragment. The PCR-amplified fragments were cloned into pGEM-T easy (Promega), digested with XbaI, and subcloned into XbaI-digested p2T7Ti/GFP to give p2T7Ti/500 bp and p2T7Ti/1000 bp.The open reading frame of TbSA C was amplified using primers TbSA C NH (5′-ATAGCTAGCATGGCATCCTACATGT-3′) and TbSA C stop (5′-TATAGATCTCTATGCTGACAGTAAC-3′) and cloned into pGEM-T Easy vector (pGEM-TbSA C). A fragment of 615 bp of the TbSA C open reading frame was excised with EcoRI and BamHI from pGEM-TbSA C and ligated into the EcoRI and BamHI sites of pTrcHisC. The resulting plasmid was digested with HindIII and BamHI, and the fragment was subcloned into HindIII/BamHI-digested p2T7Ti, yielding p2T7Ti/TbSA C.The open reading frame of TbSA B was amplified using primers TbSA B 1 (5′-GCTAGCATGAAGCGCCTGCCTGTACG-3′) and TbSA B stop (5′-TATAGATCTTCAGATTATAGTAGAATC-3′) and cloned into pGEM-T Easy vector (pGEM-TbSA B). A fragment of 850 bp of the TbSA B open reading frame was obtained by digestion of pGEM-TbSA B with BglII and PstI and was subcloned into pTrcHisC. The resulting plasmid was digested with BamHI and HindIII, and the fragment containing a partial sequence of TbSA B was ligated into the corresponding sites of p2T7Ti, yielding p2T7Ti/TbSA B.Plasmids p2T7Ti/GFPc, p2T7Ti/TbTSc, p2T7Ti/TbSA Bc, and p2T7Ti/TbSA Cc were generated by deleting the tetracycline operator regions from p2T7Ti/GFP and replacing the GFP gene by the XbaI-digested fragments of TbTS or HinDIII/BamHI-digested fragments of TbSA B and TbSA C, respectively. The tetracycline operator regions were excised with BglII, and the resulting DNA fragments were ligated back together.Northern Blot Analysis—Total RNA was extracted from 2 to 20 × 107 cells (cultures non-induced or tetracycline-induced for 48 h) using the TRIzol (Invitrogen) method according to the manufacturer's instructions. RNA concentration was determined by UV spectrophotometry at 260 nm, and RNA quality was confirmed by gel analysis.For the Northern blots, 20 μg of total RNA was electrophoresed on formaldehyde-agarose gels (1%) and transferred by capillary action onto Zeta Probe nylon membranes (Amersham Biosciences). After cross-linking, membranes were blocked in hybridization buffer (0.5 mm NaH2PO4, 7% SDS, 1 mm EDTA, and 1% bovine serum albumin) for 4 h at 60 °C and hybridized with radio-active probes for 18 h at 62 °C. DNA probes were synthesized by PCR in the presence of [α-32P]dCTP. Membranes were washed once for 20 min in 1× SSC containing 0.1% SDS at 62 °C and twice for 15 min in 0.5× SSC containing 0.1% SDS.The TbTS probe was made with oligonucleotides 3endup3 (5′-ATGGTGTGAGTTGGCATTT-3′, forward) and STOP (5′-GTCAAATCGCCAACACATACAT-3′, reverse). The TbTS C probe was made with oligonucleotides TbSA C 2400 (5′-TGCCGTTTTTGAAATTCACACC-3′) and TbSA C 2600 (5′-TAAACGAAAATCGGGCCACACA-3′). The TbTSA B probe was made with oligonucleotides TbSA B primer NH (5′-GCTAGCATGAAGCGCCTGCCTGTACG-3′) and primer 270 (5′-TTTTCCAACTGCCCTTTTCC-3′). The TbSA B′ probe was made with oligonucleotides B3 UTR (5′-TTCAAAACCCCTATCGTACTGC-3′, forward) and B 3UTR (5′-ACAGCAGCGGATACACCAAC-3′, reverse). The tubulin probe was made with oligonucleotides tubulin (5′-CTCCATCATCCCATCCCCCAA-3′, forward) and tubulin (5′-GAGGACTTGATGTTGTTCGGG-3′, reverse).Cloning of the TbSA C 5′- and 3′-UTRs—RNA was purified using TRIzol reagent (Invitrogen) following the manufacturer's instructions. To obtain the 5′-UTR sequence, first-strand cDNA was prepared with the Superscript II system using an internal primer (5′-CACACTTAAGCATCCCCTCGT-3′) of TbSA C. Reverse transcription-PCR was carried out with Taq DNA polymerase (Invitrogen) and primers for the T. brucei 39-nucleotide 5′-spliced leader sequence as forward (5′-AACGCTATTATTAGAACAGTTTCTGTACT-3′) and the one used for first-strand synthesis as reverse. To obtain the 3′-UTR sequence, reverse transcription-PCR was performed with Superscript II (Invitrogen) using the oligonucleotide anchor-(dT)18 (5′-GCGACTCCGCGGCCGCG(T)18-3′). PCR was conducted on the first-strand product using the anchor-(dT)18 as the reverse primer. The forward primer was 5′-TACTTTACCTGTTGATGGGTCT-3′. All PCR products were cloned in pGEM-T Easy (Promega) and sequenced using the dideoxychain termination method with Sequenase (USB Corp.).Expression of Sialidase Genes in Bacteria and Protein Purification—In preparation for producing active recombinant proteins, we analyzed TbSA B, TbSA C, and TbTSsh using the iPSORT program (Human Genome Center, Institute of Medical Sciences, University of Tokyo) for predicting the existence of a signal peptide. We also examined the primary sequences of their C-terminal domains to predict the existence of trans-membrane regions. Inserts were designed to encode all of the predicted recombinant proteins for these genes.Plasmids containing the open reading frames of TbSA B and TbSA C (pGEM-TbSA B and pGEM-TbSA C, respectively; see above) were cut with BglII and NheI, and the DNA fragments corresponding to the genes were ligated into expression vector pTrcHis C (Invitrogen). TbSA B constructs starting at three alternative codons and ending at two alternative stop codons were obtained by PCR on the pGEM-TbSA B plasmid using the following primers: TbSA B NH (5′-GCTAGCATGAAGCGCCTGCCTGTACG-3′; NheI restriction site is underlined), TbSABMa(5′-CGCGCTAGCTACTGCTGTGACCTGGTGTCCT-3′), TbSA B 14 (5′-GGTGCTAGCCTTTCTCAGAGGAGCAAA-3′), TbSA B stop TM (5′-TATAGATCTCTAGTCCCCTTCACAATACATC-3′; BglII restriction site is underlined), and TbSA B STOP16 (5′-TATAGATCTTCAGATTATAGTAGAATC-3′).A TbSA C construct starting at the codon for lysine 24 was obtained by PCR on the pGEM-TbSA C plasmid using the primers TbSA C ma (5′-GAGGCTAGCAAAGAAGGTACTAC-3′; NheI restriction site is underlined) and TbSA C STOP (5′-TATAGATCTCTATGCTGACAGTAAC-3′; BglII restriction site is underlined).For the TbTS sh gene starting at the leucine 28 codon, the primers were LTSK (5′-TATGCTAGCTTGACTTCCAAGGCTGCGG-3′; NheI restriction site is underlined) and TbTS sh STOP (5′-AGATCTTTAGTACACCATCACGAGTTGC-3′; BglII restriction site is underlined).After digestion with the corresponding restriction enzymes, the fragments were ligated in the pTrcHisC vector. The His tag encoded in the plasmid vector was used to purify protein. The constructs were introduced in Escherichia coli BL21 (DE3) pLysS cells by the calcium chloride method. Overnight cultures were diluted 1:50 in Terrific Broth and grown at 37 °C to an A600 of 0.8–1.0 with constant agitation at 250 revolutions/min. Bacteria were induced to overexpress recombinant proteins by adding 0.5 mm isopropyl thio-α-d-galactoside (Sigma), and induction was maintained at 18 °C for 12–16 h. Cells were harvested, washed with NaCl/Tris (20 mm Tris/HCl, pH 7.6, and 50 mm NaCl), and frozen (–80 °C) until needed. After thawing, lysis was carried out in 20 mm Tris/HCl, pH 7.6, 30 mm NaCl, 0.5% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 100 μg of DNase I ml–1.Supernatants were centrifuged at 21,000 × g for 30 min and subjected to iminodiacetic metal affinity chromatography (IMAC) (Hitrap Chelating, Amersham Biosciences AB) via Ni2+-charged equilibration in 20 mm Pipes, pH 6.9, and 0.5 m NaCl (buffer IMAC). The column was washed with 30 mm imidazole in buffer IMAC. Elution was achieved using a linear gradient of 30–250 mm imidazole in buffer IMAC.Site-directed Mutagenesis and Mutant Protein Purification—Site-directed point mutagenesis was performed by amplifying the pTrcHisC vector containing the TbTS gene starting at the codon for leucine 28 (16Montagna G. Cremona M.L. Paris G. Amaya M.F. Alzari P.M. Frasch A.C.C. Eur. J. Biochem. 2002; 269: 2941-2950Crossref PubMed Scopus (53) Google Scholar) with primers YW forward (5′-CAATGTCACGAAGGGGTGGTGCACAACGAAAACAAGG-3′) and YW reverse (5′-CGTTGTTTTCGTTGTGCCACCACCCCTTCGTGACATTG-3′).The PCR product was digested with DpnI, purified, cloned, and introduced in E. coli BL21 (DE3) pLysS cells. Clones were sequenced to confirm the mutation of target sites.The mutant and the wild-type TbTS recombinant proteins were expressed in bacteria and purified by IMAC (see previous section). After IMAC purification, the activity peak was pooled, dialyzed against 20 mm Bistris, pH 7.4, and further purified by fast protein liquid chromatography anion exchange (Mono Q) equilibrated with the same buffer. The protein was eluted by applying a linear gradient of 0–250 mm trisodium citrate. Purified proteins were analyzed by SDS-PAGE under reducing conditions, stained with Coomassie Blue R250, and quantified with Kodak one-dimensional version 3.0 software using purified bovine serum albumin as the standard.GPI-PLD Treatment—For GPI-PLD digestion, 5 × 106 T. brucei procyclic cells were osmotically lysed and the membrane fractions were incubated with GPI-PLD (Boehringer, Mannheim, Germany) at a concentration of ∼5 units ml–1 for 4 h at 37 °Cin100 μl of buffer A (150 mm NaCl, 10 mm Hepes, NaOH, pH 7) in the presence of 0.1% Nonidet P-40 (24Low M.G. Huang K.S. Biochem. J. 1991; 279: 483-493Crossref PubMed Scopus (107) Google Scholar).Enzyme Activity Assays—Neuraminidase activity was determined by measuring the fluorescence of 4-methylumbiliferone released by the hydrolysis of 0.2 mm 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNen5Ac, Sigma). The assay was performed in 50 μl in 20 mm Pipes, pH 6.9. After incubation at 37 °C for 30 min, the reaction was stopped by dilution in 0.2 m sodium carbonate, pH 10, and fluorescence was measured with a DYNAQuant TM 200 fluorometer (Hoefer Pharmacia, Inc). Trans-sialidase activity was measured in 20 mm Pipes, pH 6.9, using 1 mm Neu5Ac-α-(2–3)-lactose as the sialic acid donor and 12 μm [d-glucose-1-14C]lactose (55 mCi mol–1) (Amersham Biosciences) as the acceptor in a 30-μl final volume at 37 °C for 15 min. The reaction was stopped by dilution, and sialyl-[14C]lactose was quantified with a β-scintillation counter as previously described (25Buschiazzo A. Frasch A.C.C. Campetella O. Cell. Mol. Biol. (Noisy-Le-Grand). 1996; 42: 703-710PubMed Google Scholar).Sialic Acid Determination—The total amount of sialic acid in procyclic parasites was determined after hydrolysis in 0.1 m HCl for 1 h at 80 °C using the thiobarbituric acid method and high pressure liquid chromatography analysis (26Powell L.D. Hart G.W. Anal. Biochem. 1986; 157: 179-185Crossref PubMed Scopus (70) Google Scholar).RESULTSIdentification of Genes Coding for Trypanosomal trans-Sialidase-like Proteins—BLAST searches were performed using sequences corresponding to the catalytic domain of TbTS (AF 310232.1, a T. brucei trans-sialidase) in the T. brucei Genome Data Base (Sanger Centre). The search identified seven T. brucei open reading frames with a blast E value between 1 × 10–167 and 0.20. These open reading frames (Fig. 1) were named TbTS for T. brucei trans-sialidase, TbTSsh for a truncated version of TbTS, TbSA B and C for putative T. brucei sialidases B and C, and genes D1, D2, and E (which have much less identity to TbTS, so their products were named TbTS-like proteins). Most of these genes are present as a single copy in the T. brucei Genome Data Base, except TbSA C, which is present in two tandem copies. The differences in these copies are localized in 38 positions. Various sialidase amino acid motifs, such as the FRIP and Asp box motifs, are conserved in the deduced primary structures of proteins encoded by these TbTS family genes (Fig. 1). The six putative non-TbTS proteins have different levels of identity to TbTS. The catalytic regions of TbSA B and TbSA C are 49 and 46% identical, respectively, to the catalytic domain of TbTS. These two proteins have most of the structurally relevant residues displayed in trypanosomal trans-sialidase, including the essential amino acid residues Pro-283 and Tyr-342 (amino acid positions are those of TcTS; see Fig. 2). Point mutations at these positions of TcTS and TbTS completely abolished both sialidase and trans-sialidase activities (16Montagna G. Cremona M.L. Paris G. Amaya M.F. Alzari P.M. Frasch A.C.C. Eur. J. Biochem. 2002; 269: 2941-2950Crossref PubMed Scopus (53) Google Scholar, 19Paris G. Ratier L. Amaya M.F. Nguyen T. Alzari P.M. Frasch A.C.C. J. Mol. Biol. 2005; 345: 923-934Crossref PubMed Scopus (74) Google Scholar). However, each of these two putative trans-sialidases has a natural mutation in different positions that is relevant for enzymatic activity (Fig. 2). TbSA B has a conserved tyrosine residue in position 224, which is a structural homolog of Tyr-119 of TcTS, whereas TbSA C has a tryptophan residue at this position (Trp-170). This tyrosine residue was found to have a crucial role in the transfer reaction in both T. cruzi and T. brucei trans-sialidases (16Montagna G. Cremona M.L. Paris G. Amaya M.F. Alzari P.M. Frasch A.C.C. Eur. J. Biochem. 2002; 269: 2941-2950Crossref PubMed Scopus (53) Google Scholar, 17Buschiazzo A. Tavares G.A. Campetella O. Spinelli S. Cremona M.L. Paris G. Amaya M.F. Frasch A.C.C. Alzari P.M. EMBO J. 2000; 19: 16-24Crossref PubMed Scopus (122) Google Scholar, 18Paris G. Cremona M.L. Amaya M.F. Buschiazzo A. Giambiagi S. Frasch A.C.C. Alzari P.M. Glycobiology. 2001; 11: 305-311Crossref PubMed Scopus (47) Google Scholar). On the other hand, TbSA C has a conserved tryptophan residue in position 372, which is equivalent to Trp-312 of TcTS, whereas TbSA B has a phenylalanine residue in this position (Phe-421). The exposed aromatic side chain of this tryptophan favors, in the case of microbial sialidases and trans-sialidases, the high specificity for sialyl-α-(2, 3) substrates (11Buschiazzo A. Amaya M.F. Cremona M.L. Frasch A.A.C. Alzari P.M. Mol. Cell. 2002; 10: 757-768Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Critical amino acid residues are not present in the catalytic domains of deduced sequences coded by the D1, D2, and E genes. The TbTS family genes encode the partially conserved subterminal VTVXNVFLYNR motif (shown in Fig. 1) that, in the case of T. cruzi, defines the trypanosome trans-sialidase/sialidase superfamily of surface proteins. Finally, the deduced protein of the truncated TbTSsh gene has 93% identity to the catalytic domain of TbTS, but codon 428 is a stop codon; therefore, the lectin domain is missing. This protein might not be able to properly fold and may lack enzymatic activity (16Montagna G. Cremona M.L. Paris G. Amaya M.F. Alzari P.M. Frasch A.C.C. Eur. J. Biochem. 2002; 269: 2941-2950Crossref PubMed Scopus (53) Google Scholar). We tested TbSA B, TbSA C, and TbTSsh products for enzymatic activities (see "Experimental Procedures"). None of constructs displayed sialidase/trans-sialidase activity when expressed in bacteria (data not shown).FIGURE 2Conserved amino acids among trypanosomal sialidases and trans-sialidases. The amino acid position numbers in TrSA
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