Portal de Periódicos da CAPES

Dissection of the Functional Domains of theLeishmania Surface Membrane 3′-Nucleotidase/Nuclease, a Unique Member of the Class I Nuclease Family

2000; Elsevier BV; Volume: 275; Issue: 21 Linguagem: Inglês

10.1074/jbc.m908725199

1083-351X

Alain Debrabant, Elodie Ghedin, Dennis M. Dwyer,

CRISPR and Genetic Engineering

Class I nucleases are a family of enzymes that specifically hydrolyze single-stranded nucleic acids. Recently, we characterized the gene encoding a new member of this family, the 3′-nucleotidase/nuclease (Ld3′NT/NU) of the parasitic protozoan Leishmania donovani. The Ld3′NT/NU is unique as it is the only class I nuclease that is a cell surface membrane-anchored protein. Currently, we used a homologous episomal expression system to dissect the functional domains of theLd3′NT/NU. Our results showed that its N-terminal signal peptide targeted this protein into the endoplasmic reticulum. UsingLd3′NT/NU-green fluorescent protein chimeras, we showed that the C-terminal domain of the Ld3′NT/NU functioned to anchor this protein into the parasite cell surface membrane. Further, removal of the Ld3′NT/NU C-terminal domain resulted in its release/secretion as a fully active enzyme. Moreover, deletion of its single N-linked glycosylation site showed that such glycosylation was not required for the enzymatic functions of theLd3′NT/NU. Thus, using the fidelity of a homologous expression system, we have defined some of the functional domains of this unique member of the class I nuclease family. Class I nucleases are a family of enzymes that specifically hydrolyze single-stranded nucleic acids. Recently, we characterized the gene encoding a new member of this family, the 3′-nucleotidase/nuclease (Ld3′NT/NU) of the parasitic protozoan Leishmania donovani. The Ld3′NT/NU is unique as it is the only class I nuclease that is a cell surface membrane-anchored protein. Currently, we used a homologous episomal expression system to dissect the functional domains of theLd3′NT/NU. Our results showed that its N-terminal signal peptide targeted this protein into the endoplasmic reticulum. UsingLd3′NT/NU-green fluorescent protein chimeras, we showed that the C-terminal domain of the Ld3′NT/NU functioned to anchor this protein into the parasite cell surface membrane. Further, removal of the Ld3′NT/NU C-terminal domain resulted in its release/secretion as a fully active enzyme. Moreover, deletion of its single N-linked glycosylation site showed that such glycosylation was not required for the enzymatic functions of theLd3′NT/NU. Thus, using the fidelity of a homologous expression system, we have defined some of the functional domains of this unique member of the class I nuclease family. L. donovani 3′-nucleotidase/nuclease phosphate-buffered saline signal peptide polymerase chain reaction kilobase green fluorescent protein transmembrane domain concanavalin A polyacrylamide gel electrophoresis amino acid wild type Leishmania donovani is an important protozoan pathogen of humans that causes severe and often fatal visceral disease (visceral leishmaniasis or Kala azar) in the tropics and neotropics worldwide. 1Tropical Disease Research, progress 1995–96: thirteenth program report of the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases. This organism possesses a unique bifunctional externally oriented cell surface membrane enzyme 3′-nucleotidase/nuclease (Ld3′NT/NU),2which is involved in the salvage of host-derived purines (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar). Purine salvage is critical for these parasites because they are incapable ofde novo purine synthesis (2.Gottlieb M. Parasitol. Today. 1989; 5: 257-260Abstract Full Text PDF PubMed Scopus (30) Google Scholar). Based on its biochemical characteristics, this trypanosomatid enzyme was shown to be a member of the class I nuclease family (3.Neubert T.A. Gottlieb M. J. Biol. Chem. 1990; 265: 7236-7242Abstract Full Text PDF PubMed Google Scholar, 4.Campbell T.A. Zlotnick G.W. Neubert T.A. Sacci Jr., J.B. Gottlieb M. Mol. Biochem. Parasitol. 1991; 47: 109-117Crossref PubMed Scopus (19) Google Scholar). Recently, we isolated and characterized the gene encoding the Ld3′NT/NU and showed that it had significant sequence homology with two class I nucleasesi.e. the S1 and P1 nucleases of Aspergillus andPenicillium (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar). These two secreted fungal nucleases are the archetype members of this class of enzymes. Further, they function as single strand-specific nucleases that are involved in scavenging phosphate and nucleosides for fungal cell growth (5.Fraser M.J. Low R.L. Linn S.M. Lloyd R.S. Roberts R.J. Nucleases. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 171-207Google Scholar). Whereas genes for several new members of this enzyme family have recently been identified from various plants and a proteobacterium (6.Sullivan J.T. Ronson C.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5145-5149Crossref PubMed Scopus (451) Google Scholar, 7.Aoyagi S. Sugiyama M. Fukuda H. FEBS Lett. 1998; 429: 134-138Crossref PubMed Scopus (118) Google Scholar) based on their deduced amino acid sequence homology with the fungal nucleases, the biochemical properties of these nonfungal nucleases remain to be characterized. Within this class I nuclease family, the leishmanial enzyme is the only one to have been characterized as a cell surface membrane-anchored protein. Although the three-dimensional structure of the P1 nuclease has been determined (8.Volbeda A. Lahm A. Sakiyama F. Suck D. EMBO J. 1991; 10: 1607-1618Crossref PubMed Scopus (256) Google Scholar) and a putative mechanism of catalysis has been proposed (9.Romier C. Dominguez R. Lahm A. Dahl O. Suck D. Proteins. 1998; 32: 414-424Crossref PubMed Scopus (100) Google Scholar), no structure/function studies have been performed with any other member of the class I nuclease family. In this report, we used a homologous leishmanial expression system to dissect and analyze the functional domains of the parasite 3′-nucleotidase/nuclease. These domains included: 1) the N-terminal signal peptide for targeting this enzyme to the endoplasmic reticulum, 2) the C-terminal putative transmembrane domain and its role in anchoring/targeting this enzyme into the parasite cell surface, and 3) the N-linked glycosylation site and its role in enzyme activity. L. donovanipromastigotes, strain 1S, clone 2D (World Health Organization designation: MHOM/SD/62/1S-CL2D), and a lipophosphoglycan-deficient mutant (C3PO, kindly provided by Dr. Salvatore J. Turco, Department of Biochemistry, University of Kentucky Medical Center) (10.McNeely T.B. Tolson D.L. Pearson T.W. Turco S.J. Glycobiology. 1990; 1: 63-69Crossref PubMed Scopus (32) Google Scholar) derived from this clone were cultured as described previously (11.Bates P.A. Hermes I. Dwyer D.M. Mol. Biochem. Parasitol. 1990; 39: 247-255Crossref PubMed Scopus (79) Google Scholar). Log-phase promastigotes (2–4 × 107 cells/ml) were harvested by centrifugation at 2100 × g for 10 min at 4 °C. Cell pellets were washed in ice-cold phosphate buffer (PBS) (50 mm Na2HPO4, 150 mmNaCl, pH 7.4) by centrifugation as above. For transfection experiments, cells were resuspended in electroporation buffer (Hepes (ICN Biomedicals Inc., Aurora, OH), 137 mm NaCl, 5 mm KCl, 0.7 mm Na2HPO4, 6 mm glucose, pH 7.0) to 108 cells/ml. 500 μl of cell suspension were added to 2-mm gap electroporation cuvettes (BTX Inc., San Diego, CA) to which 20 μl of purified plasmid DNA (1 mg/ml in sterile 10 mm Tris, 2 mm EDTA (Quality Biological, Inc., Gaithersburg, MD), pH 8.0) was added. Cells were electroporated using a BTX ECM-600 electroporation system (BTX). Electroporation conditions were: 475 V, 800 microfarads, 13 ohms, single pulse. Electroporated cells were incubated on ice for 10 min and then transferred into 5 ml of culture medium above and incubated at 26 °C for 24 h. Subsequently, the cells were harvested by centrifugation as above and resuspended in fresh culture medium containing 15 μg/ml of Geneticin (G418, Life Technologies, Inc.). These cells were selected for growth in increasing concentrations of G418 over a period of several weeks and then maintained at 250 μg/ml drug. These drug-resistant cells were used in all subsequent experiments. The designations used in this report for genes, proteins, and plasmids follows the genetic nomenclature forTrypanosoma and Leishmania as outlined by Claytonet al. (12.Clayton C. Adams M. Almeida R. Baltz T. Barrett M. Bastien P. Belli S. Beverley S. Biteau N. Blackwell J. Blaineau C. Boshart M. Bringaud F. Cross G. Cruz A. Degrave W. Donelson J. El-Sayed N. Fu G. Ersfeld K. Gibson W. Gull K. Ivens A. Kelly J. Vanhamme L. et al.Mol. Biochem. Parasitol. 1998; 97: 221-224Crossref PubMed Scopus (79) Google Scholar). Based on our previous observations (13.Ghedin E. Charest H. Zhang W.W. Debrabant A. Dwyer D. Matlashewski G. J. Biol. Chem. 1998; 273: 22997-23003Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and several preliminary experiments, all constructs described below contained the Ld3′NT/NU signal peptide sequence to properly target the expressed proteins into the endoplasmic reticulum. Further, similar constructs lacking this signal peptide (SP) were only expressed in the cytoplasm of transfected cells. The [pKS NEO] plasmid (14.Zhang W.W. Charest H. Ghedin E. Matlashewski G. Mol. Biochem. Parasitol. 1996; 78: 79-90Crossref PubMed Scopus (122) Google Scholar) was used to express a truncatedLd3′NT/NU protein in L. donovani promastigotes. To that end, a polymerase chain reaction (PCR) was performed using theLd3′NT/NU gene containing plasmid Cl-2 (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar) as template with the following forward primer 1 and reverse primer 2. Primer 1, 5′-ggact agt ATG GCT CGA GCT CGT TTC-3′, contains aSpeI restriction site (underlined) and 18 nucleotides of theLd3′NT/NU gene sequence (uppercase). Primer 2, 5′-ggact agt cta gtg gtg gtg gtg gtg gtg cgg gcc GCT GAT GCC TTT CTG ATC-3′, contains 18 nucleotides of theLd3′NT/NU gene sequence (uppercase) in frame with a sequence encoding 6 histidines (italic) and a stop codon followed by aSpeI restriction site (underlined). The resulting PCR product (∼1 kb) was initially cloned into the [pCRII]cloning vector (T/A cloning system, Invitrogen Co., San Diego, CA) to generate the [pCRII3′A] plasmid. The insert was excised from the latter plasmid using SpeI and subsequently cloned into the SpeI site of [pKS NEO] to generate the[pKS NEO Ld3′nt/nu s ] expression plasmid. The orientation of the SpeI fragment in [pKS NEO] was verified by digestion with appropriate restriction enzymes. TheLd3′NT/NU gene containing plasmid Cl-2 above was used as template in a PCR with the following forward primer 3 and reverse primer 4. Primer 3, 5′-ccg gta cct ttg gat aaa aga TGG TGG AGC AAG GGC CAC-3′, contains a KpnI restriction site (underlined) and 18 nucleotides of Ld3′NT/NU gene sequence (uppercase). Primer 4, 5′-cca cta gt g gtg gtg gtg gtg gtg ggg ccc GCT GAT GCC TTT CTG ATC-3′, contains 18 nucleotides of the Ld3′NT/NU gene sequence (uppercase) in frame with a sequence encoding 6 histidines and a stop codon followed by aSpeI restriction site (underlined). The resulting (∼1 kb) PCR product was cloned into the [pCRII] plasmid (Invitrogen) to generate plasmid [pCRII3′B] (this plasmid was made toward expressing the Ld3′NT/NU protein in a yeast expression system). To mutate the single N-linked glycosylation site of the Ld3′NT/NU gene, the[pCRII3′B] plasmid was further used as template in a PCR using the reverse primer 4 (above) and the forward primer 5. Primer 5, 5′-TAC CGC CTG GCC AAG ATG CTG CAG ACG ACG CTG-3′, contains a CAG condon (underlined) instead of the AAC encoding the asparagine residue involved in the putative N-linked glycosylation site of the Ld3′NT/NU enzyme. This primer also contains a MscI restriction site (bold). The resulting (174 base pair) PCR fragment was digested with MscI, subjected to agarose gel electrophoresis, and gel-purified using the Sephaglas BandPrep Kit (Amersham Pharmacia Biotech). The latter fragment was subsequently ligated with the 0.9-kb KpnI/MscI fragment isolated from the plasmid [pCRII3′B] above. The ligation reaction product was used directly as template in a PCR with primers 3 and 4. The resulting (∼1 kb) PCR fragment was cloned into[pCRII] to produce the [pCRII3′C] plasmid. The AccI/BamHI fragment of the[pCRII3′A] plasmid above was replaced by theAccI/BamHI fragment isolated from the[pCRII3′C] plasmid, which contained the mutated Asn codon. The SpeI fragment of the resulting [pCRII3′D]plasmid was subsequently cloned into the SpeI site of[pKS NEO] to generate the [pKS NEO Ld3′nt/nu s Asn − ]expression plasmid. The Ld3′NT/NU gene was amplified by PCR from plasmid Cl-2 (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar) using primers 3′-open reading frame 1 and 3′-open reading frame 2 (3′-open reading frame 1, GGG ACT AGT CCA GAC ATG GCT CGA GCT, the 5′-forward primer for theLd3′NT/NU gene with a SpeI site is in bold; 3′-open reading frame 2, GGG ACT AGT CCC CTA GAG GGC GAC GTG CTG, 3′-reverse primer with a SpeI site). The underlined nucleotides represent start and stop codons. The resulting PCR product (1.3 kb) was first cloned into the [pCR2.1]cloning vector (Invitrogen) to produce the [pCR2.1 3′NT/NU] plasmid. The GFP gene was amplified from plasmid[pEGFP-1] (CLONTECH Laboratories, Inc., Palo Alto, CA, USA) using primers GFP1 and GFP2 (GFP1, TGG TGGGCT GAG CCC ATG GTG AGC AAG GGC GAG, 5′-forward primer for GFP with an EspI site; GFP2, CCA AGC GCT CTT GTA CAG CTC GTC CAT G, 3′-reverse primer for GFP with anEco47III site). The 720-base pair PCR product was cloned into the [pCR2.1] cloning vector above. After anEspI/Eco47III digest, the gel-eluted GFP fragment was subcloned into [pCR2.1 3′NT/NU] above previously digested with the same enzymes to exclude the Ld3′NT/NU core region and to retain solely the signal peptide and C-terminal domains. The resulting plasmid is [pCR2.1 3′ SP :: GFP ::TM]. Both [pCR2.1 3′NT/NU] and [pCR2.1 3′ SP :: GFP ::TM] plasmids were then digested with SpeI and subcloned into the SpeI site of the[pKS NEO] vector to produce the [pKS NEO 3′NT/NU] and [pKS NEO 3′ SP :: GFP ::TM] plasmids, respectively. The GFP gene was amplified as above from plasmid [pEGFP-1] using primers GFP1 and GFP3 (GFP3, CCA AGC GCT CTA CTT GTA CAG CTC GTC CAT G, 3′-reverse primer for GFP with an Eco47III site (bold) and a stop codon (underlined)). The resulting PCR product was first cloned into the [pCR2.1] cloning vector. Subsequently, the EspI/Eco47III fragment was isolated from that plasmid and cloned into the [pCR2.1 3′NT/NU] previously digested with the same enzymes as above. The resulting [pCR2.1 3′ SP ::GFP] plasmid was then digested with SpeI and subcloned into the SpeI site of the [pKS NEO] vector to produce the [pKS NEO 3′ SP ::GFP] plasmid. L. donovani promastigotes transfected with plasmid [pKS NEO Ld3′nt/nu s ], [pKS NEO Ld3′nt/nu s Asn − ],or the control plasmid [pKS NEO] were grown in the presence of 250 μg/ml G418. Late log-phase cultures were centrifuged at 2100 × g for 10 min at 4 °C, and cell-free culture supernatant was removed and used directly or stored at −20 °C. For nickel agarose bead adsorption, the pH of the cell-free culture supernatant was adjusted to pH 8.0 by the addition of 2m NaOH. Ni2+-nitrilotriacetic acid-agarose beads (Qiagen Inc., Chatsworth, CA) were equilibrated in 20 mm Hepes, pH 8.0 (buffer A) and incubated overnight on a platform rocker at 4 °C with cell-free culture supernatants. Beads were subsequently washed three times with buffer A containing 0.1% Triton X-100 (protein grade, Calbiochem). Affinity adsorbed material was eluted from these beads using buffer A containing 0.5 mimidazole (Sigma), dialyzed against buffer A, and stored at −20 °C. For concanavalin A (ConA) adsorption, the pH of the cell-free culture supernatant was adjusted to pH 7.5. ConA-Sepharose 4B beads (Amersham Pharmacia Biotech) were equilibrated in a 20 mm Hepes, pH 7.5, buffer (buffer B) and incubated overnight at 4 °C with cell-free culture supernatants as above. ConA-Sepharose beads were washed extensively in buffer B containing 0.1% Triton X-100, and the adsorbed proteins were eluted using buffer B containing 0.3m α-methylmannoside (Sigma). Eluted material was concentrated/dialyzed against buffer B using Centricon-10 concentrators (Amicon Inc., Beverly, MA). Affinity purified proteins were subsequently analyzed by SDS-PAGE as described below. 3′-Nucleotidase activity was measured in both promastigote cell lysates and in cell-free culture supernatants by test tube assays using 3′-adenosine monophosphate (3′-AMP, Sigma) as substrate as described previously (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar). Synthetic peptides were made (Genosys Biotechnologies, The Woodlands, TX) corresponding to amino acid residues 361–375 (CYLPKRDRFGSYEHV) of the C-terminal domain of theLd3′NT/NU. These peptides were conjugated to keyhole limpet hemocyanin and used to immunize a New Zealand White rabbit (No. 1432) as described previously (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar). The resulting antiserum (anti-Ld3′NT/NU C-terminal specific) was shown in preliminary experiments to specifically react with the parasiteLd3′NT/NU by Western blot. A second antiserum (rabbit No. 1336, anti-Ld3′NT/NU specific), generated against a single internal Ld3′NT/NU peptide (amino acid (aa) residues Glu201 to Tyr226), was described previously (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar) and also used in the current study. In addition, a rabbit anti-GFP serum (CLONTECH) and an Escherichia coli recombinant GFP protein (CLONTECH) were also used in these immunoassays. For total cell analysis, promastigotes were harvested by centrifugation as above and washed twice in ice-cold PBS. Cell pellets were lysed in 20 mm Hepes, 0.5% (v/v) Triton X-100, 25 μg/ml leupeptin (Sigma), pH 8.0. Protein concentrations were determined using the bicinchoninic acid (BCA, Pierce) method (15.Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18919) Google Scholar). Proteins were analyzed by SDS-PAGE, transferred onto nitrocellulose, and processed for Western blots analysis with the various rabbit sera above or their preimmune sera (normal rabbit serum) as described previously (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar). Proteins separated by SDS-PAGE were also processed for in situstaining of 3′-nucleotidase activity according to Zlotnic et al. (16.Zlotnick G.W. Mackow M.C. Gottlieb M. Comp. Biochem. Physiol. 1987; 87: 629-635Google Scholar) or in situ staining of nuclease activity according to Bates (17.Bates P.A. FEMS Microbiol. Lett. 1993; 107: 53-58Crossref PubMed Scopus (35) Google Scholar). L. donovani promastigotes were fixed in suspension in 4% (w/v) paraformaldehyde (Sigma) in PBS for 20 min on ice, washed three times in PBS, and were allowed to attach to glass slides precoated with poly-l-lysine (Sigma). For direct fluorescence, cells were mounted in PBS. Images were acquired using a Zeiss Axioplan microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with epifluorescence and a cooled CCD camera (Photometrics, Tucson, AZ). Fluorescence was detected using fluorescein isothiocyanate excitation/barrier filters. Cells were also examined by confocal microscopy using a Zeiss LSM 410 system (Zeiss). For indirect immunofluorescence, cells were blocked for 30 min in 1% (w/v) bovine serum albumin (United States Biochemical Co., Cleveland, OH) in PBS and incubated 1 h with either the anti-Ld3′NT/NU-specific (No. 1336) serum or the anti-Ld3′NT/NU C-terminal-specific serum diluted in PBS containing 1% (w/v) bovine serum albumin. After three washes in PBS, cells were incubated for 1 h with a rhodamine-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted in PBS containing 1% (w/v) bovine serum albumin. Cells were subsequently washed three times with PBS and mounted in antifade (Bio-Rad). Cells were examined by fluorescence microscopy as above, except with rhodamine excitation/barrier filters. Previously, we characterized the gene encoding Ld3′NT/NU (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar). The deduced protein of this gene (Fig. 1 A) contains a putative hydrophobic SP (aa residues Met1-Ala2-Arg3-Ala4-Arg5-Phe6-Leu7-Gln8-Leu9-Leu10-Leu11-Val12-Thr13-Leu14-Thr15-Leu16-Leu17-Ser18-Thr19-Ala20-Ala21-Leu22-Pro23-Val24-Ser25-Ala26), an N-linked glycosylation site (aa residue Asn293), and a TM (aa residues Ala334-Ala335-Val336-Thr337-Ala338-Ile339-Val340-Ala341-Val342-Ala343-Leu344-Phe345-Ile346-Ala347-Gly348-Ile349-Ile350-Ile351-Ala352-Thr353-Leu354-Val355-Val356-Leu357-Ala358-Leu359). To confirm that the cloned Ld3′NT/NU gene product was targeted to the cell surface membrane of L. donovani, the nucleotide sequence corresponding to the deduced protein shown in Fig.1 A was cloned into the [pKS NEO] leishmanial expression plasmid (14.Zhang W.W. Charest H. Ghedin E. Matlashewski G. Mol. Biochem. Parasitol. 1996; 78: 79-90Crossref PubMed Scopus (122) Google Scholar). L. donovani wild type (WT) and C3PO promastigotes transfected with the resulting [pKS NEO 3′NT/NU] plasmid were grown under increasing concentrations of G418 up to 250 μg/ml. Lysates of such cells were analyzed by SDS-PAGE and Western blotting. Results of Western blots showed that the anti-Ld3′NT/NU-specific antibody (No. 1336) reacted with a single 43-kDa protein in both untransfected controls and [pKS NEO 3′NT/NU] transfected promastigotes. In control promastigotes, this 43-kDa band reflects the endogenous Ld3′NT/NU (Fig.2 A, lane 1). The enhanced reactivity seen with this antibody in [pKS NEO 3′NT/NU] transfected cells represents the episomal overexpression of the Ld3′NT/NU protein (Fig. 2 A, lane 2). Identical results were obtained with both WT (not shown) and C3PO promastigotes in these experiments. Normal rabbit sera showed no reactivity in these Westerns blots (data not shown).Figure 2Episomal expression ofLd3′NT/NU. A, Western blot showing the reactivity of the anti-Ld3′NT/NU specific antibody (No. 1336) with promastigote lysates (2 × 107 cells/lane) of untransfected C3PO (lane 1) and C3PO transfected with[pKS NEO 3′NT/NU] plasmid (lane 2).B, indirect immunofluorescence showing cell surface reactivity of the anti-Ld3′NT/NU-specific antibody (No. 1336) with C3PO transfected with [pKS NEO 3′NT/NU]plasmid. C, Western blot showing the reactivity of either anti-GFP-specific antibody (α-GFP, lanes 1–3) or anti-Ld3′NT/NU C-terminal specific antibody (No. 1432, α-3′NT/C-term, lanes 1′–3′) with E. colirecombinant GFP protein (lanes 1 and 1′), lysates of promastigotes transfected with either the [pKS NEO 3′ SP ::GFP] plasmid (lanes 2 and2′) or the [pKS NEO 3′ SP :: GFP ::TM] plasmid (lanes 3and 3′). D, fluorescence image showing cell surface localization of the3′ SP :: GFP :: TM chimeric protein in C3PO promastigotes transfected with the [pKS NEO 3′ SP :: GFP ::TM] plasmid and its accumulation in their flagellar reservoirs.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The L. donovani promastigotes above were also analyzed by light microscopy. By phase contrast microscopy, all of these transfected cells showed the typical pyriform morphology of flagellatedLeishmania promastigotes. Both controls and transfected promastigotes were treated with the anti-Ld3′NT/NU-specific antibody (No. 1336) to visualize by indirect immunofluorescence the cell surface localization of the Ld3′NT/NU protein. Although both WT and C3PO promastigotes (untransfected) possess externally oriented cell surface membrane 3′-nucleotidase enzyme activity, as previously demonstrated by both subcellular fractionation and fine structure cytochemistry (18.Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1983; 7: 303-317Crossref PubMed Scopus (80) Google Scholar,19.Dwyer D.M. Gottlieb M. Mol. Biochem. Parasitol. 1984; 10: 139-150Crossref PubMed Scopus (53) Google Scholar), 3D. M. Dwyer, unpublished data. neither showed significant fluorescence with the anti- Ld3′NT/NU-specific antibody (No. 1336). The weak fluorescence signal obtained with this antibody presumably reflects the low level of endogenousLd3′NT/NU protein expressed on the cell surface of these parasites (i.e. number of protein copies/cell). In contrast, promastigotes transfected with the [pKS NEO 3′NT/NU]plasmid, WT (not shown), and C3PO (Fig. 2 B) showed bright cell surface immunofluorescence with the anti-Ld3′NT/NU-specific antibody (No. 1336). Such distinct fluorescence presumably reflects the increased copy number of this protein present on the cell surface of these parasites. The latter observation was confirmed using confocal fluorescence microscopy. These results demonstrated that the Ld3′NT/NU-expressed protein was targeted to the parasite cell surface as detected by the anti-Ld3′NT/NU-specific antibody (No. 1336). Further, results of multiple observations showed that C3PO transfectants had significantly more cell surface fluorescence than WT transfectants. The difference in fluorescence intensity between these transfectants could represent the relative amounts of Ld3′NT/NU proteins expressed on their cell surfaces. This might also explain the apparent lack of reactivity of the anti-Ld3′NT/NU-specific antibody (No. 1336) with untransfected cells. Normal rabbit sera controls showed no reactivity with any cell types used in these experiments. To demonstrate that the C-terminal region of theLd3′NT/NU functions as a membrane anchor domain,Ld3′NT/NU-GFP chimeric proteins were expressed in L. donovani promastigotes. To that end, nucleotide constructs encoding the two Ld3′NT/NU-GFP chimeric proteins described below were cloned into the [pKS NEO] leishmanial expression plasmid as above. In one of these chimeric proteins (3′SP::GFP::TM, Fig. 1 B), GFP was substituted for aa residues Glu52 to Ser333 of the Ld3′NT/NU. Thus, its N terminus contained the first 51 aa residues of the Ld3′NT/NU including the SP (aa residues Met1 to Ala26). The C terminus of this chimeric protein contained the entire C-terminal region (aa residues Ala334 to Leu377) of the Ld3′NT/NU including its putative anchor domain (TM, aa residues Ala334 to Leu359). The secondLd3′NT/NU-GFP chimeric protein (3′SP::GFP, Fig.1 C) was identical to the first one except that it lacked the entire C-terminal region (aa residues Ala334 to Leu377) of the Ld3′NT/NU. Transfected promastigotes were grown under increasing concentrations of G418 up to 250 μg/ml, and lysates of such cells were analyzed by SDS-PAGE and Western blotting. In such blots, the anti-GFP-specific antibody showed strong reactivity with a control ∼30-kDa E. colirecombinant GFP protein (Fig. 2 C, lane 1). That antibody also reacted with a single ∼30-kDa protein in both WT (not shown) and C3PO (Fig. 2 C, lane 2) promastigotes transfected with the [pKS NEO 3′ SP ::GFP]plasmid. The anti-GFP-specific antibody also reacted with an ∼35-kDa protein and to a lesser extend with an ∼32-kDa protein in cells transfected with the [pKS NEO 3′ SP :: GFP ::TM] plasmid (Fig. 2 C,lane 3). The latter presumably represents a proteolytic degradation product of the ∼35-kDa protein. Further, this ∼35-kDa protein was also recognized by our anti-Ld3′NT/NU C-terminal specific (No. 1432) antibody (Fig. 2 C, lane 3′) demonstrating that these cells expressed the3′ SP :: GFP :: TM chimeric protein. In contrast, the anti-Ld3′NT/NU C-terminal specific (No. 1432) antibody showed no reactivity with either parasites transfected with the [pKS NEO 3′ SP ::GFP] plasmid (Fig.2 C, lane 2′) or with the control E. coli recombinant GFP protein (Fig. 2 C, lane 1′). Normal rabbit sera controls showed no reactivity in these Western blot assays (data not shown). L. donovani WT and C3PO promastigotes transfected with the above plasmids or the control [pKS NEO] expression plasmid were examined by epifluorescence and confocal fluorescence microscopy. Such observations revealed that WT (not shown) and C3PO (Fig.2 D) promastigotes transfected with the [pKS NEO 3′ SP :: GFP ::TM] plasmid had bright cell surface fluorescence. These results demonstrated that the3′ SP :: GFP :: TM chimeric protein was targeted to the cell surface membrane of transfected parasites. In addition, GFP was also seen to accumulate within the flagellar reservoirs of these transfected cells. In contrast, promastigotes transfected with the [pKS NEO 3′ SP ::GFP] plasmid showed only diffuse intracellular fluorescence reflecting the processing of GFP within the endoplasmic reticulum (data not shown). Further, the latter transfectants released/secreted soluble GFP into their culture supernatant, which was detected by Western blots with the anti-GFP antibody (data not shown). Control promastigotes transfected with the [pKS NEO] expression plasmid alone showed no detectable cellular fluorescence. Cumulatively, these results demonstrated that the C-terminal domain of the Ld3′NT/NU functioned to anchor this enzyme into the cell surface membrane of these parasites. To determine whether the C-terminal region (aa residues Ala334to Leu377) of the L