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. 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. J. N. G. W. A. J. L. Biochem. Parasitol. 1998; PubMed Scopus (79) Google Scholar). Based on H. A. Dwyer D. G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) and several experiments, all described the Ld3′NT/NU signal peptide sequence to the into the endoplasmic reticulum. Further, this signal peptide were only in the of The plasmid H. G. Mol. Biochem. Parasitol. PubMed Scopus Google Scholar) was used to a protein in L. donovani that a polymerase chain reaction was performed using gene containing plasmid (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar) as with the and site and of gene sequence of gene sequence in with a sequence encoding 6 and a by site The was into the system, San Diego, CA) to the The was from the plasmid using and into the site of to expression The of the in was by with enzymes. gene containing plasmid above was used as in a with the and a site and of Ld3′NT/NU gene sequence g of the Ld3′NT/NU gene sequence in with a sequence encoding 6 and a by site The was into the plasmid to plasmid plasmid was the Ld3′NT/NU protein in a expression the single N-linked glycosylation site of the Ld3′NT/NU plasmid was used as in a using the 4 and the a of the encoding the involved in the putative N-linked glycosylation site of the Ld3′NT/NU enzyme. This a site The was with to gel and using the The was with the isolated from the plasmid above. The reaction was used as in a with and The was to the The of plasmid above was by isolated from which the The of the was into the site to the The Ld3′NT/NU gene was by from plasmid (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar) using and 2 the for gene with a site is in with a The and The was into the to the The gene was from Inc., using and for with an for with The was into the above. the was into above previously with the enzymes to the Ld3′NT/NU and to the signal peptide and C-terminal The plasmid is and plasmids were then with and into the site of to the and The gene was as above from plasmid using and for with an site and a The was into the Subsequently, the was isolated from that plasmid and into the previously with the enzymes as above. The plasmid was then with and into the site of the to the L. donovani promastigotes with plasmid the plasmid were in the of 250 μg/ml were at 2100 × g for 10 min at 4 and culture was and used or at °C. For the pH of the culture was to pH by the of Inc., CA) were in 20 mm pH and incubated on a at 4 °C with culture were washed with buffer A containing was from these using buffer A containing buffer and at °C. For concanavalin A the pH of the culture was to pH were in a 20 mm pH buffer and incubated at 4 °C with culture as above. were washed in buffer containing and the were using buffer containing was buffer using Inc., purified were by as described was in cell and in culture by using as as described previously (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar). were The to amino acid of the C-terminal domain of theLd3′NT/NU. These were to and used to a as described previously (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar). The C-terminal was shown in to specifically with the by A a single Ld3′NT/NU peptide acid to was described previously (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar) and used in the In a and an protein were used in these For cell promastigotes were harvested by centrifugation as above and washed in ice-cold Cell pellets were in 20 mm μg/ml pH concentrations were determined using the acid Biochem. PubMed Scopus Google Scholar). were by transferred and for with the various above or their as described previously (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar). by were for in of to al. G.W. Gottlieb M. Biochem. Scholar) or in of nuclease to P.A. Lett. PubMed Scopus Google Scholar). L. donovani promastigotes were in suspension in in for 20 min on washed in and were to to with For cells were in were using a Inc., with and a was using Cells were by using a system For cells were for min in in and incubated with the or the in containing in cells were incubated for with a Inc., in containing Cells were washed with and in Cells were by as with 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 a putative an N-linked glycosylation site and a that the Ld3′NT/NU gene was targeted to the cell surface membrane of L. the sequence to the deduced protein shown in A was into the leishmanial expression plasmid H. G. Mol. Biochem. Parasitol. PubMed Scopus Google Scholar). L. donovani wild type and promastigotes with the plasmid were increasing concentrations of G418 to 250 of such cells were by and of showed that the with a single protein in and In this the Ld3′NT/NU The with this in cells the episomal of the Ld3′NT/NU protein 2 results were with and promastigotes in these experiments. showed no in these not expression the of the with × 107 of 1) and plasmid cell surface of the with with the of or C-terminal with protein and of promastigotes with the plasmid 2 or the plasmid cell surface of protein in promastigotes with the plasmid and its in their The L. donovani promastigotes above were by all of these cells showed the of and promastigotes were with the to by the cell surface of the Ld3′NT/NU protein. Although and promastigotes externally oriented cell surface membrane enzyme as previously by and structure M. Dwyer D.M. Mol. Biochem. Parasitol. PubMed Scopus Google D.M. Gottlieb M. Mol. Biochem. Parasitol. 10: PubMed Scopus Google Scholar), M. showed significant with the The signal with this the of protein on the cell surface of these parasites of protein In promastigotes with the and 2 showed cell surface with the the of this protein on the cell surface of these The was using These results that the protein was targeted to the parasite cell surface as by the Further, results of showed that had cell surface The in these the of Ld3′NT/NU on their cell This the of of the with showed no with any cell used in these experiments. that the C-terminal of functions as a membrane anchor were in L. donovani that encoding the two described were into the leishmanial expression plasmid as above. In one of these was for to of the Thus, its the of the Ld3′NT/NU the to The of this protein the C-terminal to of the Ld3′NT/NU its putative anchor domain to The protein was to the one that it the C-terminal to of the promastigotes were increasing concentrations of G418 to 250 and of such cells were by and In such the showed with a protein 2 with a single protein in and 2 2) promastigotes with the The with an protein and to a with an protein in cells with the plasmid 2 The a of the protein. Further, this protein was by C-terminal 2 that these cells protein. In the C-terminal showed no with parasites with the plasmid or with the protein 2 showed no in these not L. donovani and promastigotes with the above plasmids or the expression plasmid were by and that and promastigotes with the plasmid had cell surface These results that protein was targeted to the cell surface membrane of In was to the of these In promastigotes with the plasmid showed only the of the endoplasmic not Further, the into their culture which was by with the not promastigotes with the expression plasmid showed no these results that the C-terminal domain of the Ld3′NT/NU functioned to anchor this enzyme into the cell surface membrane of these the C-terminal of the Ld3′NT/NU was for its enzymatic L. donovani promastigotes were with an expression plasmid encoding a In this protein the C-terminal of was by promastigotes and with the plasmid were in the of 250 μg/ml G418 and by of these cells were in with the or of these showed that parasites with the plasmid only a single to the Ld3′NT/NU with the plasmid showed a of Ld3′NT/NU and an of The that such the protein by the of showed any with in of promastigotes was by in of showed that of cell a of the enzyme In the promastigotes showed an of to the protein from for nuclease showed that of cell a of enzyme to the Ld3′NT/NU and Further, of parasites with the showed a of nuclease to the protein these results for the that the Ld3′NT/NU gene a bifunctional which has and nuclease Further, they that the C-terminal domain of this protein was not required for its or its nuclease that the C-terminal domain of the Ld3′NT/NU was involved in it into the surface membrane of the it was that the be a and a protein. this culture from promastigotes or the plasmids were for their growth in cell showed of growth of showed that only promastigotes into their culture Further, in the culture of these a at of cell growth 4 Subsequently, enzyme in these they and to over the of the of the above culture were by and in activity. was in culture from the promastigotes with the plasmid not was in all culture from the promastigotes 4 These results are in with the results shown in 4 A. of parasite culture were by using the or of these showed that the with a protein of in all from promastigotes with the plasmid 4 The of the protein to over the of this In an protein was in from these cells of and it to with The the of the protein. for the in in culture of cell growth 4 of culture from promastigotes with the showed no with the not shown that a was and only by it was to that it a that culture from promastigotes were with from such was to and by and by in for and nuclease of showed that the with a protein of The protein a of the protein. Further, results from such using in that the protein had 2) and nuclease of with from promastigotes showed no in with the 5 such showed 5 1) nuclease 5 1) in in these results that the a and was the of the Further, signal peptide the protein into the endoplasmic for it to be from these cells into the parasite The Ld3′NT/NU has been shown to be that to (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar, Jr., J.B. T.A. Gottlieb M. Parasitol. 1990; 71: PubMed Scopus Google Scholar). Further, we showed that the Ld3′NT/NU protein a single N-linked glycosylation site at (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar). In the we that the Ld3′NT/NU and nuclease and in that it to to glycosylation was for the enzymatic functions of the an expression plasmid encoding a sequence was In that plasmid the to the putative glycosylation was by a sequence encoding a This single amino acid in the of the single N-linked glycosylation site of L. donovani promastigotes were with the and selected for growth in the of 250 μg/ml culture from these cells and promastigotes were shown to in of these culture were with and with from such was to and by and by in for and nuclease from results showed that the with the protein in the from the cells Further, the in such showed 6 1) and nuclease 6 1) In from the parasites showed no in with the 2) and showed no in in for 2) or nuclease 6 2) activity. The results that protein not to protein was and from as it with the in 6 In such this with a protein and to a with a protein of The protein a of its Further, the and had and nuclease from cells results in and in in these results that the was at and that the of its N-linked was Further, these results that such N-linked glycosylation was not for the enzymatic functions of Class I nucleases are a family of enzymes from and that specifically hydrolyze single-stranded DNA and (3.Neubert T.A. Gottlieb M. J. Biol. Chem. 1990; 265: 7236-7242Abstract Full Text PDF PubMed Google Scholar, 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, T. Linn S.M. Roberts R.J. Nucleases. Cold Spring Harbor Laboratory, Cold Spring Harbor, Scholar). has been that these nucleases a role in cell growth and by nucleosides and phosphate from nucleic (2.Gottlieb M. Parasitol. Today. 1989; 5: 257-260Abstract Full Text PDF PubMed Scopus (30) Google Scholar, 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). Although various members of this family have been the structure of only one of these has been in the P1 fungal (8.Volbeda A. Lahm A. Sakiyama F. Suck D. EMBO J. 1991; 10: 1607-1618Crossref PubMed Scopus (256) Google Scholar, C. Dominguez R. Lahm A. Dahl O. Suck D. Proteins. 1998; 32: 414-424Crossref PubMed Scopus (100) Google Scholar). of these nucleases have been using DNA to the of their In that we used a homologous episomal expression system from a parasitic L. to the functional domains of one member of this family, the unique L. donovani surface Further, this homologous transfection system was used to that of the Ld3′NT/NU protein be to that of its cell surface The Ld3′NT/NU protein was previously shown to a signal glycosylation and a transmembrane domain (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar). These were for targeting the protein into the endoplasmic reticulum, its and enzymatic and for the protein into the parasite surface these various Ld3′NT/NU gene were in L. donovani and the were with to their and enzymatic Further, signal peptide sequence was in all to the of the into the endoplasmic reticulum. This was because that a Ld3′NT/NU protein a signal peptide was in the of cells and was to these parasites H. A. Dwyer D. G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the class I nucleases, only the Ld3′NT/NU has been shown to be a surface membrane-anchored protein (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar, Jr., J.B. T.A. Gottlieb M. Parasitol. 1990; 71: PubMed Scopus Google Scholar). that its unique C-terminal domain functioned as a membrane containing this C-terminal domain were in L. donovani showed that was on the cell surface membrane of These results that the C-terminal of the Ld3′NT/NU functions as the domain for this cell surface protein. to targeting of protein to the parasite cell surface, we that it by the parasite or be targeted Ld3′NT/NU this protein. Further, to the C-terminal domain of the Ld3′NT/NU was involved in the enzymatic functions of this a protein its C-terminal domain was in L. donovani cells an protein which had and nuclease Further, the was in active into the culture of these cells their These results that the C-terminal domain of the Ld3′NT/NU was not required for the enzymatic of this protein. Moreover, because a membrane we that it was from cells into the of the parasite or targeted into this by this protein. to the protein by Our results that the N-terminal Ld3′NT/NU signal peptide the various into the endoplasmic of These results are in with the function of signal peptide H. A. Dwyer D. G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). A signal peptide has been deduced from the gene encoding the secreted from Aspergillus O. C. 1995; PubMed Scopus Google Scholar) and a endoplasmic function for the of this fungal enzyme. other members of the class I nuclease family, is a (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar, Jr., J.B. T.A. Gottlieb M. Parasitol. 1990; 71: PubMed Scopus Google Scholar). This protein a single putative N-linked glycosylation site at (1.Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (68) Google Scholar). In the we that this site was used for N-linked glycosylation and that the of its was These results are in with of al. 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) showed of the Ld3′NT/NU resulted in a of in its These that such of resulted in the of its enzymatic activity. that the was for the enzymatic function of this protein or was for its to In we showed that protein and nuclease Thus, results that N-linked was not required for the enzymatic functions of the Whereas other class I nucleases are such any role in their enzymatic to be N-linked glycosylation other important in the of and other class I nucleases, with endoplasmic such as or M. Dwyer D.M. Mol. Biochem. Parasitol. PubMed Scopus Google Scholar, M. Dwyer D.M. Parasitol. Today. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, L. M.J. J. Cell Sci. PubMed Google Scholar). are important for the of these and in targeting to the parasite cell surface membrane. In this using a we identified some of the functional domains of a surface class I nuclease of a parasitic A of this parasite cell surface protein was by cells as a active enzyme. The of such a enzyme for and studies of this unique class I Dr. of of for in the of these studies and Dr. of for with the leishmanial expression Dr. Salvatore J. Turco of Biochemistry, University of Kentucky Medical Center) for with donovani of Health for in and for

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