Dissection of the Functional Domains of theLeishmania Surface Membrane 3′-Nucleotidase/Nuclease, a 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.

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. 1 This organism possesses a unique bifunctional externally oriented cell surface membrane enzyme 3Ј-nucleotidase/nuclease (Ld3ЈNT/NU), 2 which is involved in the salvage of host-derived purines (1). Purine salvage is critical for these parasites because they are incapable of de novo purine synthesis (2). Based on its biochemical characteristics, this trypanosomatid enzyme was shown to be a member of the class I nuclease family (3,4). Recently, we isolated and characterized the gene encoding the Ld3ЈNT/NU and showed that it had significant sequence homology with two class I nucleases i.e. the S1 and P1 nucleases of Aspergillus and Penicillium (1). 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). Whereas genes for several new members of this enzyme family have recently been identified from various plants and a proteobacterium (6,7) 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) and a putative mechanism of catalysis has been proposed (9), 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 3Ј-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.

EXPERIMENTAL PROCEDURES
Parasite Culture and Transfection-L. donovani promastigotes, 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) derived from this clone were cultured as described previously (11). Log-phase promastigotes (2-4 ϫ 10 7 cells/ml) were harvested by centrifugation at 2100 ϫ g for 10 min at 4°C. Cell pellets were washed in ice-cold phosphate buffer (PBS) (50 mM Na 2 HPO 4 , 150 mM NaCl, 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 Na 2 HPO 4 , 6 mM glucose, pH 7.0) to 10 8 cells/ml. 500 l of cell suspension were added to 2-mm gap electroporation cuvettes (BTX Inc., San Diego, CA) to which 20 l 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 * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  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 g/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 g/ml drug. These drug-resistant cells were used in all subsequent experiments. Nomenclature-The designations used in this report for genes, proteins, and plasmids follows the genetic nomenclature for Trypanosoma and Leishmania as outlined by Clayton et al. (12).
Plasmid Constructs-Based on our previous observations (13) and several preliminary experiments, all constructs described below contained the Ld3ЈNT/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.
[pKS NEO Ld3Јnt/nu s ]-The [pKS NEO] plasmid (14) was used to express a truncated Ld3ЈNT/NU protein in L. donovani promastigotes. To that end, a polymerase chain reaction (PCR) was performed using the Ld3ЈNT/NU gene containing plasmid Cl-2 (1) as template with the following forward primer 1 and reverse primer 2. Primer 1, 5Ј-gg act agt ATG GCT CGA GCT CGT TTC-3Ј, contains a SpeI restriction site (underlined) and 18 nucleotides of the Ld3ЈNT/NU gene sequence (uppercase). Primer 2, 5Ј-gg act agt cta gtg gtg gtg gtg gtg gtg cgg gcc GCT GAT GCC TTT CTG ATC-3Ј, contains 18 nucleotides of the Ld3ЈNT/NU gene sequence (uppercase) in frame with a sequence encoding 6 histidines (italic) and a stop codon followed by a SpeI restriction site (underlined [pKS NEO Ld3Јnt/nu s Asn Ϫ ]-The Ld3ЈNT/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, 5Ј-ccg gta cct ttg gat aaa aga TGG TGG AGC AAG GGC CAC-3Ј, contains a KpnI restriction site (underlined) and 18 nucleotides of Ld3ЈNT/NU gene sequence (uppercase). Primer 4, 5Ј-cca cta gtg gtg gtg gtg gtg gtg ggg ccc GCT GAT GCC TTT CTG ATC-3Ј, contains 18 nucleotides of the Ld3ЈNT/NU gene sequence (uppercase) in frame with a sequence encoding 6 histidines and a stop codon followed by a SpeI restriction site (underlined). The resulting (ϳ1 kb) PCR product was cloned into the [pCRII] plasmid (Invitrogen) to generate plasmid [pCRII3ЈB] (this plasmid was made toward expressing the Ld3ЈNT/NU protein in a yeast expression system). To mutate the single N-linked glycosylation site of the Ld3ЈNT/NU gene, the [pCRII3ЈB] plasmid was further used as template in a PCR using the reverse primer 4 (above) and the forward primer 5. Primer 5, 5Ј-TAC CGC CTG GCC AAG ATG CTG CAG ACG ACG CTG-3Ј, contains a CAG condon (underlined) instead of the AAC encoding the asparagine residue involved in the putative N-linked glycosylation site of the Ld3ЈNT/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 [pCRII3ЈB] above. The ligation reaction product was used directly as template in a PCR with primers 3 and 4.  NEO] were grown in the presence of 250 g/ml G418. Late log-phase cultures were centrifuged at 2100 ϫ g for 10 min at 4°C, and cell-free culture supernatant was removed and used directly or stored at Ϫ20°C. For nickel agarose bead adsorption, the pH of the cell-free culture supernatant was adjusted to pH 8.0 by the addition of 2 M NaOH. Ni 2ϩ -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 M imidazole (Sigma), dialyzed against buffer A, and stored at Ϫ20°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 0.3 M ␣-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.
3Ј-Nucleotidase Enzyme Assays-3Ј-Nucleotidase activity was measured in both promastigote cell lysates and in cell-free culture supernatants by test tube assays using 3Ј-adenosine monophosphate (3Ј-AMP, Sigma) as substrate as described previously (1).
Rabbit Antisera-Synthetic peptides were made (Genosys Biotechnologies, The Woodlands, TX) corresponding to amino acid residues 361-375 (CYLPKRDRFGSYEHV) of the C-terminal domain of the Ld3ЈNT/NU. These peptides were conjugated to keyhole limpet hemocyanin and used to immunize a New Zealand White rabbit (No. 1432) as described previously (1). The resulting antiserum (anti-Ld3ЈNT/NU C-terminal specific) was shown in preliminary experiments to specifically react with the parasite Ld3ЈNT/NU by Western blot. A second antiserum (rabbit No. 1336, anti-Ld3ЈNT/NU specific), generated against a single internal Ld3ЈNT/NU peptide (amino acid (aa) residues Glu 201 to Tyr 226 ), was described previously (1) 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.
SDS-PAGE, Western Blotting, and Enzyme Activity Gels-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 g/ml leupeptin (Sigma), pH 8.0. Protein concentrations were determined using the bicinchoninic acid (BCA, Pierce) method (15). 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). Proteins separated by SDS-PAGE were also processed for in situ staining of 3Ј-nucleotidase activity according to Zlotnic et al. (16) or in situ staining of nuclease activity according to Bates (17).
Microscopy-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-Ld3ЈNT/ NU-specific (No. 1336) serum or the anti-Ld3ЈNT/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.
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 flagellated Leishmania promastigotes. Both controls and transfected promastigotes were treated with the anti-Ld3ЈNT/NUspecific antibody (No. 1336) to visualize by indirect immunofluorescence the cell surface localization of the Ld3ЈNT/NU protein. Although both WT and C3PO promastigotes (untransfected) possess externally oriented cell surface membrane 3Јnucleotidase enzyme activity, as previously demonstrated by both subcellular fractionation and fine structure cytochemistry (18,19), 3  detected by the anti-Ld3ЈNT/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 Ld3ЈNT/NU proteins expressed on their cell surfaces. This might also explain the apparent lack of reactivity of the anti-Ld3ЈNT/NU-specific antibody (No. 1336) with untransfected cells. Normal rabbit sera controls showed no reactivity with any cell types used in these experiments.
Expression of Ld3ЈNT/NU-GFP Chimeric Proteins by L. donovani Promastigotes-To demonstrate that the C-terminal region of the Ld3ЈNT/NU functions as a membrane anchor domain, Ld3ЈNT/NU-GFP chimeric proteins were expressed in L. donovani promastigotes. To that end, nucleotide constructs encoding the two Ld3ЈNT/NU-GFP chimeric proteins described below were cloned into the [pKS NEO] leishmanial expression plasmid as above. In one of these chimeric proteins (3ЈSP::GFP::TM, Fig. 1B), GFP was substituted for aa residues Glu 52 to Ser 333 of the Ld3ЈNT/NU. Thus, its N terminus contained the first 51 aa residues of the Ld3ЈNT/NU including the SP (aa residues Met 1 to Ala 26 ). The C terminus of this chimeric protein contained the entire C-terminal region (aa residues Ala 334 to Leu 377 ) of the Ld3ЈNT/NU including its putative anchor domain (TM, aa residues Ala 334 to Leu 359 ). The second Ld3ЈNT/NU-GFP chimeric protein (3ЈSP::GFP, Fig. 1C) was identical to the first one except that it lacked the entire Cterminal region (aa residues Ala 334 to Leu 377 ) of the Ld3ЈNT/ NU. Transfected promastigotes were grown under increasing concentrations of G418 up to 250 g/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 ϳ30-kDa E. coli recombinant GFP protein (Fig. 2C, lane 1). That antibody also reacted with a single ϳ30-kDa protein in both WT (not shown) and C3PO (Fig. 2C, lane 2) promastigotes transfected with the [pKS NEO 3ЈSP::GFP] plasmid. The anti-GFP-specific antibody also reacted with an ϳ35-kDa protein and to a lesser extend with an ϳ32-kDa protein in cells transfected with the [pKS NEO 3ЈSP::GFP::TM] plasmid (Fig. 2C, lane 3). The latter presumably represents a proteolytic degradation product of the ϳ35-kDa protein. Further, this ϳ35-kDa protein was also recognized by our anti-Ld3ЈNT/NU C-terminal specific (No. 1432) antibody (Fig. 2C, lane 3Ј) demonstrating that these cells expressed the 3ЈSP::GFP::TM chimeric protein. In contrast, the anti-Ld3ЈNT/NU C-terminal specific (No. 1432) antibody showed no reactivity with either parasites transfected with the [pKS NEO 3ЈSP::GFP] plasmid (Fig. 2C, lane 2Ј) or with the control E. coli recombinant GFP protein (Fig. 2C, lane 1Ј). 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. 2D) promastigotes transfected with the [pKS NEO 3ЈSP::GFP::TM] plasmid had bright cell surface fluorescence. These results demonstrated that the 3ЈSP::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 3ЈSP::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 cul-ture 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 Ld3ЈNT/NU functioned to anchor this enzyme into the cell surface membrane of these parasites.
Expression of a Truncated Ld3ЈNT/NU in L. donovani-To determine whether the C-terminal region (aa residues Ala 334 to Leu 377 ) of the Ld3ЈNT/NU was necessary for its enzymatic activities, L. donovani promastigotes were transfected with an expression plasmid [pKS NEO Ld3Јnt/nu s ] encoding a truncated Ld3ЈNT/NU. In this truncated protein (Ld3Јnt/nu s ), the C-terminal region of the Ld3ЈNT/NU was replaced by six histidine residues (His) 6 (Fig. 1D). Both [pKS NEO Ld3Јnt/nu s ]transfected promastigotes and those transfected with the control [pKS NEO] plasmid were grown in the presence of 250 g/ml G418 and analyzed by SDS-PAGE. Lysates of these cells were probed in Western blots with the rabbit anti-Ld3ЈNT/NUspecific serum (No. 1336) or normal rabbit serum. Results of these assays showed that parasites transfected with the control [pKS NEO] plasmid contained only a single 43-kDa band corresponding to the endogenous Ld3ЈNT/NU (Fig. 3A, lane 1). Promastigotes transfected with the [pKS NEO Ld3Јnt/nu s ] plasmid showed a similar 43-kDa band of endogenous Ld3ЈNT/NU and an additional band of ϳ38 kDa (Fig. 3A, lane  2). The latter demonstrated that such transfectants synthesized the truncated Ld3Јnt/nu s protein encoded by the [pKS NEO Ld3Јnt/nu s ] plasmid. Lysates of neither transfectant showed any reactivity with normal rabbit serum (not shown). 3Ј-Nucleotidase activity present in lysates of transfected promastigotes was visualized by in situ staining of SDS-PAGE gels. Results showed that lysates of each transfected cell line contained a 43-kDa band of activity reflecting the endogenous Ld3ЈNT/NU enzyme (Fig. 3B, lanes 1 and 2). In addition, the [pKS NEO Ld3Јnt/nu s ] transfected promastigotes showed an ϳ38-kDa band of 3Ј-nucleotidase activity corresponding to the Ld3Јnt/nu s expressed protein (Fig. 3B, lane 2). Results obtained from similar gels stained for nuclease activity showed that lysates of both transfected cell types contained a 43-kDa band of enzyme activity corresponding to the endogenous Ld3ЈNT/NU (Fig. 3C, lanes 1 and 2). Further, lysates of para- sites transfected with the [pKS NEO Ld3Јnt/nu s ] plasmid also showed a ϳ38-kDa band of nuclease activity corresponding to the Ld3Јnt/nu s expressed protein (Fig. 3C, lane 2). Taken together these results demonstrated for the first time, that the Ld3ЈNT/NU gene encodes a bifunctional protein, which has both 3Ј-nucleotidase and nuclease activities. Further, they demonstrated that the C-terminal domain of this protein was not required for either its 3Ј-nucleotidase or its nuclease activities.
Extracellular Release of the Soluble Ld3Јnt/nu s Expressed Protein-Having established that the C-terminal domain of the Ld3ЈNT/NU was involved in anchoring it into the surface membrane of the parasite, it was hypothesized that the truncated Ld3Јnt/nu s would be a soluble, and possibly, a released/secreted protein. To address this question, cell-free culture supernatants from promastigotes transfected with [pKS NEO Ld3Јnt/ nu s ] or the control [pKS NEO] plasmids were assayed for soluble 3Ј-nucleotidase activity during their growth in vitro. Both transfected cell lines showed very similar rates of growth (Fig.  4A, inset). Results of colorimetric assays showed that only [pKS NEO Ld3Јnt/nu s ]-transfected promastigotes released 3Ј-nucleotidase activity into their culture supernatants (Fig. 4A). Further, soluble 3Ј-nucleotidase activity accumulated in the culture supernatants of these transfectants reaching a maximum at 32 h of cell growth (Fig. 4A). Subsequently, 3Ј-nucleotidase enzyme activity decreased in these cultures when they reached stationary phase and continued to decline over the remaining time course of the experiment. Aliquots of the above cell-free culture supernatants were separated by SDS-PAGE and stained in situ for 3Ј-nucleotidase activity. No activity was detected in culture supernatant samples obtained from the promastigotes transfected with the control [pKS NEO] plasmid (data not shown). However, 3Ј-nucleotidase activity was readily detected in all culture supernatant samples obtained from the [pKS NEO Ld3Јnt/nu s ]-transfected promastigotes (Fig. 4B). These results are in agreement with the colorimetric results shown in Fig. 4A. Similar samples of parasite culture supernatants were assayed by Western blot using the rabbit anti-Ld3ЈNT/NU-specific serum (No. 1336) or normal rabbit serum. Results of these assays showed that the rabbit anti-Ld3ЈNT/ NU-specific serum (No. 1336) reacted with a protein of ϳ38 kDa in all samples from promastigotes transfected with the [pKS NEO Ld3Јnt/nu s ] plasmid (Fig. 4C). The amount of the ϳ38-kDa protein appeared to increase over the time course of this experiment. In addition, an ϳ37-kDa protein was also detected in samples obtained from these cells after 23 h of culture, and it also appeared to accumulate with time. The latter presumably reflects the proteolytic degradation of the mature ϳ38-kDa expressed protein. Such degradation would also account for the decrease in 3Ј-nucleotidase activity observed in culture supernatants after 32 h of cell growth (cf. Fig.  4A). Samples of cell-free culture supernatants from promastigotes transfected with the control [pKS NEO] plasmid showed no reactivity with the rabbit anti-Ld3ЈNT/NU-specific serum (No. 1336, data not shown).
Characterization of the Ld3Јnt/nu s Expressed Protein-Having shown that a soluble ϳ38-kDa nucleotidase was synthesized and released only by [pKS NEO Ld3Јnt/nu s ]-transfected promastigotes, it was necessary to demonstrate that it contained a histidine tag. To that end, cell-free culture supernatants from [pKS NEO Ld3Јnt/nu s ]-transfected promastigotes were adsorbed with nickel-agarose beads. Material eluted from such beads was subjected to SDS-PAGE and analyzed by Western blot and by in situ staining for 3Ј-nucleotidase and nuclease activities. Results of Western blots showed that the rabbit anti-Ld3ЈNT/NU-specific serum (No. 1336) reacted with a protein doublet of ϳ38/37 kDa (Fig. 5A, lane 2). The ϳ37-kDa protein presumably reflects a proteolytic product of the mature ϳ38-kDa released protein. Further, results obtained from such samples using in situ stained gels demonstrated that the ϳ38/ 37-kDa protein doublet had both 3Ј-nucleotidase (Fig. 5B, lane 2) and nuclease activities (Fig. 5C, lane 2), respectively. Results of assays with material from control, [pKS NEO]-transfected promastigotes showed no reactivity in Western blots with the rabbit anti-Ld3ЈNT/NU-specific serum (No. 1336, Fig. 5A, lane  1). Similarly, such control material showed neither 3Ј-nucleotidase (Fig. 5B, lane 1) nor nuclease activity (Fig. 5C, lane 1) in in situ stained gels. Taken together, these results demonstrated that the soluble released Ld3Јnt/nu s possessed a histidine tag and therefore was the product of the [pKS NEO Ld3Јnt/nu s ] plasmid. Further, the Ld3Јnt/nu s signal peptide must target the nascent protein into the endoplasmic reticulum for it to be released from these cells presumably via default into the parasite secretory pathway.
Glycosylation of the Ld3Јnt/nu s Expressed Protein-The native Ld3ЈNT/NU has been shown to be an N-linked glycoprotein that binds to ConA beads (1,20). Further, we showed that the Ld3ЈNT/NU gene-deduced protein contained a single N-linked glycosylation site at asparagine 293 ( Fig. 1A) (1). In the current study, we demonstrated that the release/secreted Ld3ЈNT/NU possessed both 3Ј-nucleotidase and nuclease activities and in preliminary experiments found that it bound to ConA beads. Thus to ascertain whether N-linked glycosylation was necessary for the enzymatic functions of the Ld3Јnt/nu s , an expression plasmid encoding a mutated Ld3Јnt/nu s sequence was constructed. In that plasmid [pKS NEO Ld3Јnt/nu s Asn -], the "AAC" codon corresponding to the putative glycosylation site, Asn 293 , was substituted by a "CAG" sequence encoding a Gln residue. This single amino acid substitution should result in the loss of the single N-linked glycosylation site of the Ld3Јnt/ nu s (Fig. 1E). L. donovani promastigotes were transfected with the [pKS NEO Ld3Јnt/nu s Asn Ϫ ] plasmid and selected for growth in the presence of 250 g/ml G418. Cell-free culture supernatants from these transfected cells and from [pKS NEO Ld3Јnt/nu s ]-transfected promastigotes were both shown to contain 3Ј-nucleotidase activity in colorimetric assays. Aliquots of these culture supernatants were also adsorbed with ConA-Sepharose beads and with nickel-agarose beads. Material eluted from such beads was subjected to SDS-PAGE and analyzed by Western blot and by in situ staining for 3Ј-nucleotidase and nuclease activities. As predicted from our preliminary experiments above, Western blot results showed that the rabbit anti-Ld3ЈNT/NU-specific serum (No. 1336) reacted with the ϳ38-kDa Ld3Јnt/nu s protein present in the ConA-eluted material from the [pKS NEO Ld3Јnt/nu s ]-transfected cells (Fig. 6A,  lane 1). Further, the Ld3Јnt/nu s present in such ConA-eluted material showed both 3Ј-nucleotidase (Fig. 6B, lane 1) and nuclease (Fig. 6C, lane 1) activities. In contrast, ConA-eluted material from the [pKS NEO Ld3Јnt/nu s Asn Ϫ ] transfected parasites showed no reactivity in Western blots probed with the rabbit anti-Ld3ЈNT/NU-specific serum (No. 1336, Fig. 6A, lane 2) and showed no reactivity in gels stained in situ for either 3Ј-nucleotidase (Fig. 6B, lane 2) or nuclease (Fig. 6C, lane 2) activity. The latter results indicated that the Ld3Јnt/nu s Asn Ϫ protein did not bind to ConA beads. However, the Ld3Јnt/ nu s Asn Ϫ protein was bound and eluted from nickel-agarose beads as it reacted with the rabbit anti-Ld3ЈNT/NU-specific serum (No. 1336) in Western blots (Fig. 6A, lane 4). In such blots this antibody reacted with a ϳ37-kDa protein and to a lesser extent with a protein of ϳ35 kDa. The ϳ35-kDa protein presumably reflects a proteolytic product of its ϳ37-kDa Ld3Јnt/nu s Asn Ϫ precursor. Further, the ϳ37and ϳ35-kDa proteins had both 3Ј-nucleotidase (Fig. 6B, lane 4) and nuclease activities (Fig. 6C, lane 4), respectively. As expected, nickelagarose-eluted material from [pKS NEO Ld3Јnt/nu s ] cells (i.e. Ld3Јnt/nu s ) gave positive results in Western blots and in in situ stained gels (Fig. 6, A-C, lane 3). Taken together, these results demonstrated that the Ld3Јnt/nu s was glycosylated at Asn 293 and that the apparent molecular mass of its N-linked glycan was ϳ1-2 kDa. Further, these results indicated that such N-linked glycosylation was not essential for the enzymatic functions of the Ld3Јnt/nu s . DISCUSSION Class I nucleases are a family of enzymes from diverse sources (e.g. plants, fungi, and protozoa) that specifically hydrolyze single-stranded DNA and RNA (3,5,21). It has been reported that these nucleases play a major biological role in cell growth and division by generating free nucleosides and phosphate from nucleic acids substrates (2,5). Although various members of this family have been identified, the structure of only one of these has been studied in detail (i.e. the P1 fungal nuclease) (8 -9). To date, however, none of these nucleases have been dissected using molecular/recombinant DNA techniques to address the functionality of their structural constituents. In that regard, we used a homologous episomal expression system from a parasitic protozoan, L. donovani, to delineate the functional domains of one member of this family, the unique L. donovani surface membrane Ld3ЈNT/NU. Further, this homologous transfection system was used to ensure that processing of the expressed Ld3ЈNT/NU protein would be equivalent to that of its native endogenous cell surface homolog. The Ld3ЈNT/NU gene-deduced protein was previously shown to contain sequences reflecting a signal peptide, an N-linked glycosylation site, and a hydrophobic transmembrane domain (1). These sequences were presumed responsible for targeting the nascent protein into the endoplasmic reticulum, facilitating its processing and enzymatic activities, and for anchoring the mature protein into the parasite surface membrane, respectively. To address these predictions experimentally, various Ld3ЈNT/NU gene constructs were expressed in L. donovani promastigotes, and the resulting expressed proteins were analyzed with regard to their localization and enzymatic activities. Further, the Ld3ЈNT/NU signal peptide sequence was included in all constructs to ensure the proper translocation of the ex- pressed proteins into the endoplasmic reticulum. This was done because our previous observations demonstrated that a truncated Ld3ЈNT/NU protein lacking a signal peptide was expressed solely in the cytoplasm of transfected cells and was toxic to these parasites (13).
Among the class I nucleases, only the Ld3ЈNT/NU has been shown to be a surface membrane-anchored protein (1,20). To demonstrate that its unique C-terminal domain functioned as a membrane anchor, Ld3ЈNT/NU-GFP chimeric proteins containing this C-terminal domain were expressed in L. donovani promastigotes. Fluorescence microscopy analysis showed that the 3ЈSP::GFP::TM chimera was expressed on the cell surface membrane of transfected parasites. These results demonstrated that the C-terminal portion of the Ld3ЈNT/NU functions as the membrane-anchoring domain for this cell surface protein. With regard to targeting of the 3ЈSP::GFP::TM protein to the parasite cell surface, we hypothesize that it might traffic by default through the parasite secretory pathway or be targeted via Ld3ЈNT/NU signals remaining within this chimeric protein. Further, to determine whether the C-terminal domain of the Ld3ЈNT/NU was involved in the enzymatic functions of this protein, a truncated Ld3ЈNT/NU protein lacking its Cterminal domain was expressed in L. donovani promastigotes. Such transfected cells expressed an ϳ38-kDa protein (Ld3Јnt/ nu s ), which had both 3Ј-nucleotidase and nuclease activities. Further, the latter was released in active form into the culture supernatants of these cells during their growth in vitro. These results demonstrated that the C-terminal domain of the Ld3ЈNT/NU was not required for the enzymatic activities of this protein. Moreover, because the Ld3Јnt/nu s lacked a membrane anchor, we postulate that it was released from transfected cells either via default into the secretory pathway of the parasite or targeted into this pathway by signals encoded within this protein. Similar conclusions pertain to the 3ЈSP::GFP protein expressed by transfected promastigotes. Our cumulative results indicated that the N-terminal Ld3ЈNT/NU signal peptide translocated the various expressed proteins into the endoplasmic reticulum of transfected cells. These results are in agreement with our previous observations concerning the function of the Ld3ЈNT/NU signal peptide (13). A similar hydrophobic signal peptide has been deduced from the gene encoding the secreted S1-nuclease from Aspergillus oryzae (22) and must serve a similar endoplasmic reticulumtargeting function for the processing of this fungal enzyme.
Like most other members of the class I nuclease family, the Ld3ЈNT/NU is a glycoprotein (1,20). This protein contains a single putative N-linked glycosylation site at asparagine residue 293 (Fig. 1A) (1). In the current study, we confirmed that this site was used for N-linked glycosylation and that the size of its glycan was ϳ1-2 kDa. These results are in agreement with those of Campbell et al. (4) who showed that N-glycanase treatment of the native Ld3ЈNT/NU resulted in a decrease of ϳ2 kDa in its apparent molecular mass. These authors also reported that such treatment of the Ld3ЈNT/NU resulted in the loss of its enzymatic activity. They concluded that the glycan was either essential for the enzymatic function of this protein or was necessary for its ability to renature after SDS-PAGE. In contrast, we showed that the Ld3Јnt/nu s Asn Ϫ expressed protein possessed both endogenous 3Ј-nucleotidase and nuclease activities. Thus, our results demonstrated that N-linked glycosylation per se was not absolutely required for the enzymatic functions of the mature Ld3ЈNT/NU. Whereas most other class I nucleases are also glycosylated, whether such carbohydrate side chains play any role in effecting their enzymatic activities remains to be examined. However, N-linked glycosylation could play other important roles in the intracellular processing of the Ld3ЈNT/NU and other class I nucleases, e.g. interactions with endoplasmic reticulum chaperons such as calreticulin or Bip (23)(24)(25). Such interactions are presumably important for the proper folding/trafficking of these proteins and in targeting the Ld3ЈNT/NU to the parasite cell surface membrane.
In this study, using a recombinant molecular approach, we identified some of the functional domains of a unique, surface membrane-anchored, class I nuclease of a parasitic protozoan. A truncated form of this parasite cell surface protein was successfully produced by homologously transfected cells as a released/secreted active enzyme. The availability of such a soluble, extracellular, glycosylated enzyme should prove useful for future immunological and fine structural studies of this unique class I nuclease.