Glycoconjugates play a pivotal role in the survival of Leishmania parasites in destructive surroundings. An important constituent present on many glycoconjugates is sialic acid. By virtue of their peripheral position on oligosaccharide chains of glycoconjugates, sialic acids are well suited as molecular determinants of specific biological processes, including the interaction of pathogenic microorganisms with sialylated cellular receptors. Differences in α2,3- and α2,6-sialoglycan patterns detected in clonal virulent Leishmania dono-vani promastigotes, correlated with the level of α2,3- and α2,6-sialyltransferase activity present in these parasites. The role of macrophage sialic acid-receptors in uptake and survival of L.donovani was studied in the murine macrophage cell line raw 264.7. Macro-phage invasion was dependent on the binding to Siglec-1, while suppression of MAPK signaling was medi-ated through Siglec-5. Sialic acid removal by neura-minidase treatment reduced parasite infectivity. The presence of trypsin resistant sialic acid residues in the neuraminidase treated parasites grown in a serum free medium in presence of sialoglycoconjugates indicated that the parasites could salvage sialic acid from exoge-nous sialoglycans and reutilize it for de novo glycopro-tein sialylation in L.donovani parasites. Thus, our re-sults demonstrate the involvement of sialoglycans in the invasion as well as the survival process of L.donovani parasites. Research Article Subir Karmakar‡, Siddhartha K Bhaumik†‡, Joydeep Paul and Tripti De Division of Infectious Disease and Immunology, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 4 Raja S.C.Mullick Road, Kolkata 700032, India †Current address: Division of Infectious Diseases, Department of Pediatrics, Emory University, Atlanta, GA 30322, USA ‡ Contributed equally to the manuscript Received on September 13, 2011; Accepted on December 19, 2011; Published on February 15, 2012 Correspondence should be addressed to Tripti De; Phone: +91 33 2473 0492, Fax : +91 33 2473 5197, E-mail: triptide@iicb.res.in Leishmania Donovani Cell Surface Sialoglycans Regulate Susceptibility for Siglec Mediated Macrophage Invasion and Parasite Survival Introduction Leishmania are protozoan parasites that cause severe diseases in human and animals. The Leishmania para-sites are enveloped by a dense glycocalyx constituted of the glycosylphosphatidylinositol-anchored glyco-proteins, family of glycoinositol-phospholipids, the lipophosphoglycans, the glycolipids, and the pro-teoglycans (Ferguson 1997). Together they have been implicated in a surprisingly large number of functions for the parasites throughout their life cycle and, there-fore, are key players in their pathogenesis (Guha-Niyogi et al. 2001, Kamhawi 2006). Due to their exposed terminal position on gly-coconjugates, sialic acids (Sia) have been implicated in a variety of biological processes, including cell adhe-sion and immune recognition (Crocker et al. 2001, Schauer & Kamerling 1997). The biosynthesis of sia-loglycans comprises of a chemically and topologically complex set of events involving CMP-Sia, CMP-Sia transporter and the sialyltransferases. The sialoglycan is finally secreted or delivered to the plasma membrane by the secretory machinery (Jacobs et al. 2001). The presence of Sia on the promastigotes of Leishmania donovani and Trypanosoma species has been shown previously (Chang 1981, Chatterjee et al. 2003, Schauer et al. 1983). Unable to synthesize Sia, T.cruzi trypomastigotes acquire Sia from host glycoconjugates by means of a plasma membrane-associated trans-sialidase (Previato et al. 1985) that has the unique abil-ity to efficiently carry out a sialyltyransferase reaction using preformed glycoconjugates (Buschiazzo et al. 2000). This multifunctionality is a common feature of many bacterial sialyltransferases (Cheng et al. 2005, Cheng et al. 2010, Hollister & Jarvis 2001, Hollister et al. 2002, Yu et al. 2005). Though the machinery that incorporates Sia to the parasite surface is present in parasites like Trypanosoma sps (Buschiazzo et al. Abstract Journal of Molecular Biochemistry (2012) 1, 6-20 © The Author(s) 2012. Published by Lorem Ipsum Press. 2000), they are still not reported in Leishmania. Of note, two protein coding putative Sia transporters LINJ_24_0350 and LINJ_24_0360 located at chromo-some Chromosome 24, NC_009408.2 (110565..112238) and chromosome Chromosome 24, NC_009408.2 (118081..119703) respectively have been reported in the L.infantum data base. Here we report for the first time the presence of sialyltransferases in L.donovani suggesting that these sialyltransferases may play an important role in the acquisition of Sia on Leishmania surface. Our re-sults further suggest that, these sialoglycans participate in the invasion process and contribute to macrophage (Mφ) deactivation and to disease pathogenesis. Materials and Methods Infection Virulent L.donovani (MHOM/IN/83/AG83) parasite clones used in this study were isolated from a mixed population of 7th passage AG83 promastigotes as de-scribed (Bhaumik et al. 2008). In vitro infection ex-periments were carried out with macrophages using stationary phase L.donovani clonal promastigotes at a 20:1 parasite/macrophage ratio as described with slight modifications (Karmakar et al. 2011). Bone marrow derived macrophages were replaced by murine Mφ cell line RAW 264.7. Cultures without added parasites were run in parallel. Neuraminidase treatment of Leishmania promas-tigotes Promastigotes (1 x 108) were treated with 25 units of Vibrio cholerae neuraminidase in RPMI-HEPES for 1h at 37°C with slow shaking. Subsequently parasites were washed 3 times in PBS and re-suspended in se-rum free medium. Control cells were washed in the same manner as enzyme-treated cells. The efficacy of neuraminidase treatment was determined by using Maackia amurensis agglutinin (MAA). Use of 1,2-Diamino-4,5-methylene dioxybenzene (DMB)-HPLC analysis of Sia Promastigotes were washed in PBS and suspended in 1 mL PBS. Cells were lysed by sonication (5 pulses of 10s each). Gly-coconjugates were subjected to hydrolysis with 2M propionic acid for 4h at 80oC (Kamerling & Gerwig, 2006). Controls included treatment with neuraminidase in RPMI-10% FCS for 1h at 37oC (2) and N-acetyl neuraminate pyruvate lyase (EC 4.1.3.3) treatment at pH 7.2 for 2h at 37oC. Released Sias were derivatized with DMB and resolved by reverse-phase HPLC (Manzi et al. 1990). Sia standards were isolated from bovine submaxillary mucin (Varki & Diaz 1984). Biotinylation Proteins (1mg/ml in PBS) were directly labeled with biotin using the Pierce EZ-Link Sulfo-NHS-Biotin La-beling Kit. FACS analysis of binding of MAA and SNA to L.donovani promastigotes A fixed number of L.donovani clonal promastigotes (1 X 106) were suspended in PBS containing 5% BSA (PBSB). Biotinylated MAA or SNA (0–50 μg/ml) was added to the cell suspension and incubated at room temperature for 30 min. Cells were washed in PBSB thrice and incubated with FITC-conjugated strepta-vidin for 10 min. Cells were washed in PBSB thrice and fixed in paraformaldehyde (2%). Fluroscence of the cells were analyzed using a FACS Calibur (BD Biosciences) using CELL QUEST software. Unstained cells, or cells stained with FITC-BSA or streptavidin-FITC served as controls. Western blotting analysis Sialoglycan profiles of L.donovani CSA (25 mg/lane) was analyzed before and after sialidase treatment by Western blot using biotin-labeled MAA and Sambucus nigra agglutinin (SNA) (10μg/ml). After washing, the blots were incubated with horseradish peroxidase–avidin complex (Vector, ABC Reagent) and developed using an ECL detection system (Thermo Fisher Scien-tific, Rockford, IL, USA). For MAPK activation studies, cells were lysed in ice-cold lysis buffer (100 mM Tris ,pH 8, 2 mM EDTA, 100 mM NaCl, 1% Triton X-100 containing complete EDTA-free protease inhibitors from Roche Diagnostics, which included 5 mM sodium vanadate, 10 mM sodium fluoride, 10 mM b-glycerophosphate sodium, and 5 mM sodium pyrophosphate). Equal amounts of protein were loaded onto 12.5% SDS-polyacrylamide gels, and then transferred to PVDF membranes. The membranes were blocked with 5% BSA in wash buffer (TBS/0.1% Tween 20) for 1 h at room temperature and probed with primary antibody overnight at 4°C at a dilution recommended by the suppliers. Membranes were incubated with primary antibodies overnight at 4°C. Membranes were washed three times with wash buffer and then incubated with HRP-conjugated secondary antibody and detected by the ECL detection system (Thermo Fisher Scientific, Rockford, IL, USA), according to the manufacturer’s instructions. Knockdown of Siglec-1 and Siglec-5 by siRNA in RAW 264.7 cells RAW264.7 cells cultured in DMEM with 10% fetal bovine serum were transfected with the siRNA for 7 Journal of Molecular Biochemistry, 2012 Siglec-1, Siglec-5 or the scrambled control (40 nM, Santacruz Biotechnology, USA) using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfec-tion, cells were collected and analyzed for Siglec-1 and Siglec-5 proteins by western blotting using the Siglec-1 and Siglec-5 specific polyclonal antibodies (N-20 and M-58 respectively). Fluorescence labeling of parasites FITC-labeling of parasites was carried out as described (Butcher et al. 1992), except that labeling was carried out at 37oC in PBS. Promastigotes (106/ml in PBS) were incubated for 15 min at 37°C with 30 μg/ml FITC. After incubation, parasites were washed twice with PBS to remove excess ligand and analyzed for fluorescence with a Becton-Dickinson FACS Calibur flow cytometer (San Jose, CA). L.donovani-Siglec Binding assays Binding studies were performed with a fixed number of clonal promastigotes (1 X 105) suspended in PBSB. Siglecs (0-10 μg/ml) diluted in 1% BSA/PBS were added to the cell suspension and incubated at 4oC for 2h in a total volume of 0.5ml. Cells were washed in PBSB thrice and incubated with biotinylated Siglec antibody (10 ng/ml in 1% BSA/PBS) for 1h at 4oC. The primary solution was discarded, and the cells were washed with cold PBS three times. Streptavidin cou-pled to horseradish peroxidase (200 ng/ml in PBS/1% BSA) was added to the cells and incubated for 1h at 4°C. Hundred μl freshly prepared staining solution (2% w/v ABTS [2,2’-azinodi(3-ethyl-benzthiazoline-6-sulfonate]) in 100 mM sodium acetate buffer, pH 4.2, and 0.001% v/v H2O2) was added and the reaction was stopped after 15 min by the addition of 0.6% (w/v) SDS and absorbance measured at 405 nm. Promastigote binding studies For promastigote binding studies, RAW 264.7 cells were washed twice with serum-free DMEM and incu-bated with stationary phase FITC-labeled parasites re-suspended in serum-free DMEM at a parasite to macrophage ratio of 5:1 for 30 min at 37°C. Non-adherent parasites were removed by five washings with PBS, followed by a fixation step (0.1 M PIPES–NaOH, pH 7.2; 2% para-formaldehyde, 0.05% glu-taraldehyde, 30 min, 22°C). After five washes with PBS and a 20–40 min incubation with 1 μg/mL 4',6-diamidino-2-phenylindole (DAPI) in PBS, 2% BSA, 50 mM NH4Cl, followed by an additional three washes with PBS, the coverslips were embedded in Mowiol containing 2.5% 1,4-diazobicyclo-[2.2.2]-octane (DABCO, Sigma). Macrophages with bound parasites and the total number of bound parasites were counted by inspection with a fluorescence microscope. SHP-1 immunoprecipitation and phosphatase activ-ity To specifically measure SHP-1 activity, 100 mg of total Mφ protein lysate was subjected to immunopre-cipitation using 20 ml anti-SHP-1 SH2 antibody (C14H6, Cell signaling) as reported (Blanchette et al. 1999). The immunoprecipitates were isolated using protein A-Sepharose and a fraction (20 mL) of the im-munoprecipitate was used to perform SHP-1 phos-phatase activity measurement using 2 mM p-nitrophenyl phosphate as substrate (Sigma Chemical Co., St. Louis, MO, USA). Sialoglycan feeding studies Gangliosides GQ1b, GT1b, GD1a, GD1b, GM1, and GM2, in chloroform:methanol 1:1 were dried by rotary evaporation, re-suspended in serum-free medium, and briefly sonicated to ensure proper micellar suspension. Ganglioside containing medium was filter sterilized (0.22-μm) and diluted with fresh medium to a final 50 μM concentration. Fetuin (50 μM) in serum free me-dium was passed through a 0.45-μm sterilizing filter. Sia was removed from clonal parasites by neuramini-dase treatment as mentioned earlier. Asialo parasites (2 X 106 parasites/200μL medium) were incubated in ganglioside- or fetuin-containing serum free medium for 12h at 22 °C. Parasites were thoroughly washed and divided into two parts. One part was used to infect RAW 264.7 cells as mentioned earlier. The other part (1 X 106 parasites) was used to analyze the Sia content by DMB derivatization and liquid chromatography analysis as described later. Preparation of α2-3- and α2-6-sialylated Fetuin, α1Acid glycoprotein (AGP) and Complete Soluble Antigens (CSA) acceptor substrates CSA was prepared from L.donovani promastigotes in presence of 0.04% Non-idet P-40 essentially as de-scribed previously (De & Roy, 1999). Asialo CSA, asialo Fetuin and asialo α1 Acid glycoprotein (AGP) was prepared from CSA, Fetuin, and AGP respectively by incubation with 200mU Clostridium perfringens α2-3,6-Neuraminidase/mg protein in PBS (pH 6.4) at 37oC for 4h. α2-3-sialylated Fetuin, and AGP were prepared from the respective asialo-glycoproteins as described with slight modifications (Azuma et al. 2001). Fetuin and AGP was treated with 100 μU of α1,3/4 Fucosidase (Streptomyces sp. 142, EC 3.2.1.51, Takara Shuzo Co. Berkeley, USA) per mg protein. The purified de-fucosylated asialo glycoproteins were incu-bated with 5 mU α2,3-sialyltransferase (EMD4Biosciences, USA) in 0.1M HEPES buffer (pH Journal of Molecular Biochemistry, 2012 8 7.0) containing 1 mM CMP-NeuAc and 10 mM MgCl2 for 48 h at 37oC. The reaction mixture was spotted on Whatman 3 MM paper. Ascending chromatograms were developed in 1% sodium tetraborate and the en-zymatic product (α2,3 sialylated glycoprotein) that remained at the origin was isolated from the papers. Strips from the origin were cut, washed with PBS, con-taining 0.04% non-idet P-40. Detergent was removed by SM-2 bio-beads and the product (further purified on MAA lectin affinity columns) was used as a substrate for the α2-6 sialyltransferase assay. α2-6-sialylated Fetuin, AGP and CSA were prepared from the respec-tive glycoproteins by incubation with 5 U Streptococ-cus pneumoniae recombinant α2,3-neuraminidase/mg protein at 37oC for 4h. The products were run on SDS-PAGE, blotted onto nitrocellulose and probed with MAA to check the extent of hydrolysis. Localization of α2,3- and α2,6-silyltransferase en-zymes by confocal microscopy α2,3- and α2,6-silyltransferase antibody was directly labeled with FITC using the Pierce EZ-LabelTM-FITC Protein Labeling Kit. The Golgi of live promastigotes was visualized with the Golgi marker BODIPY-TR and ER was visualized with BiP. α2,3- and α2,6-silyltransferase was visualized with anti ST3Gal4 (Sigma Chemical Co., St. Louis, MO, USA) and anti CD-75 (H-70) (Santacruz Biotechnology, USA) anti-bodies respectively. Parasites were stained with the antibodies by using standard procedures. BiP antibody was visualized with PE-conjugated secondary anti-body. Imaging was performed with the Nikon A1R live cell confocal imaging system. Results and Discussions Due to their exposed position and physicochemical properties, Sias play important roles in cellular interac-tions and adhesion processes. Sia is a receptor deter-minant for infection of cells by virions (Winter et al. 2006) and protozoa (Chitnis 2001). It has been sug-gested that terminal Sia residues may represent a shared but tailored invasion pathway among apicom-plexan parasites (Hager & Carruthers 2008). Plasmo-dium falciparum strain W2mef depends on Sia for in-vasion of erythrocytes (Durasingh et al. 2003). Sia as-sociated with the host cell contributes to invasion by T.cruzi (Schenkman et al. 2003) and Toxoplasma gondii (Monteiro et al. 1998). The occurrence of Sia has been reported in a broad range of trypanosomatids (d’Avila-Levy et al. 2004). Though sialoglycans have been reported in Leishmania species (Chatterjee et al. 2003; Vannier-Santos et al. 1991, Dwyer 1977, Gho-shal et al. 2010, Iovannisci et al. 2010), the impact of 9 Journal of Molecular Biochemistry, 2012 sialoglycans in leishmaniasis still remains a poorly understood area. Recently Sia has been implicated in nitric oxide mediated host resistance in leishmaniasis (Ghoshal et al. 2010). This study was undertaken to assess the sialylation pattern of virulent L.donovani AG83 clonal promastigote surface glycans and to elu-cidate their role (a) in the invasion process and (b) in modulating the host cell signaling. Presence of sialoglycans on L.donovani promas-tigotes Glycoconjugates have been shown to play important roles in the infectious cycle of Leishmania (Guha-Niyogi et al. 2001, da Silva et al. 2009). We had ear-lier established that differential expression of galacto-terminal glycoconjugates are implicated in virulent attenuation of L.donovani promastigotes (Bhaumik et al. 2008). The presence of sialic acids on L. donovani promastigotes was demonstrated by performing fluorimetric high-performance liquid chromatography (HPLC) of acid hydrolysates of virulent clonal para-sites. The chromatogram exhibited a well-resolved peak that coincided with N-acetyl neuraminic acid (Neu5Ac) (Figure 1B). There was about 275 ng of Sia in 1X108 cells corresponding to 5.37 X 106 molecules of Sia per cell (Table 1). Neuraminidase treatment completely removed Sia residues in the virulent pro-mastigotes (Figure 1c). A lectin blot assay was per-formed using MAA to confirm that the neuraminidase treatment was effective. Diminution of the correspond-ing peak, following treatment with N-acetyl neurami-nate pyruvate lyase confirmed the presence of Sia on these parasites (inset, Figure 1C). Specificity of sialic acid linkage of L.donovani sialo glycans The cell surface expression of sialoglycans was meas-ured by flow cytometric analysis of the virulent clonal parasites stained with two fluoresceinated plant lectins MAA and SNA with specificity for terminal α2,3- and α2,6- linked Sia respectively. Flow cytometric analysis revealed that the amount of the lectin MAA needed for 50% binding was 9μg/ml. At similar concentrations, SNA bound to < 5% of the promastigotes (Figure 2A). In contrast, Chatterjee et al. observed that MAA showed a relatively lower binding than SNA (Chatterjee et al. 2003). This discrepancy may be ex-plained by the fact that while we are working with virulent clonal parasites, Chatterjee et al were working with a multiclonal population of L.donovani promas-tigotes. In the original paper they have referred to, parasites were sub-cultured every 4-5 days. These cells lose their infectivity to host macrophages after pro-longed cultivation in axenic growth media (De & Roy 1999). Presence of predominantly avirulent variants in the mixed L.donovani population maintained in pro-longed axenic culture would account for the predomi-nance of -2,6 linked Sias that Chatterjee et al. (2003) observed, since we too observed a similar higher level of -2,6 linked Sias as compared to -2,3 linked Sias in a multi-clonal L.donovani AG83 promastigote popula-tion maintained in culture for > 2 weeks (data not shown). Lectin-blot studies were carried out to charac-terize the linkage-specificity of sialoglycans present on the virulent clonal parasite surface. The reactivity of MAA and SNA was examined by western blotting. The western blot studies indicated that the level of 2,3-sialoglycans, was higher than the level of 2,6- sia-loglycans, (Figure 2B). The population structure of Leishmania spp. and other kinetoplastids is mainly clonal and strains isolated from naturally infected hosts are likely composed of multiclonal parasite associa-tions (Garin et al. 2001). Working with a multiclonal population consisting of both virulent and attenuated variants of the parasite cells, Chatterjee et al. (2003) reported the presence of both -2,3 and -2,6 linked, as well as 9-O-acetyl linked sialoglycans in the L.donovani promastigotes. We however could not de-tect any Neu5,9Ac2 peak in the acid parasite hydrolys-ates (Figure 1C). It may be mentioned that, Chatterjee et al. (2003) did not detect any Neu5,9Ac2 Sia by mass spectrometry analysis either. Our findings are in agreement to another member of the kinetoplastid fam-ily, the trypanosomes, where -2,3 linked Sias predomi-nate (Engstler et al. 1995). Pre-treatment of the para-sites with Clostridium perfringens α(2-3,6)-neuraminidase abolished lectin binding. Prior incuba-tion of the lectins with fetuin plus sialyllactose also abolished binding (Figure 2A, lane3). Effect of culture conditions on L.donovani sialogly-can expression To rule out the possibility that the sialoglycans were not present as contaminants, cells were grown under different conditions. The binding of SNA or MAA and the total Sia expression on L.donovani promastigotes remained unchanged despite alterations in the media, temperature or pH (Table 1). Similarly, cells grown in different batches of FCS for 72h or different concen-trations of FCS (M 199 medium, 22oC and pH 7.2) did not show any differences in their lectin binding proper-ties and total Sia expression. The mass spectrometry of the per-trimethylsilyl-derivative of the methyl ester demonstrated the presence of Neu5Ac with fragment ions (m/z) at 668, 624, 478, 400, 317, 298, 205, and 173. Only m/z values over 100 are given. Binding of Siglecs to L.donovani promastigotes Cumulative evidence indicates that in trypanosomat-Journal of Molecular Biochemistry, 2012 10 Medium Temp. (0C) pH FCS Source (10%) % Lectin Binding ng acid/108 cellsSialic Number of Sialic acid Molecules/ Cells MAA SNA 1 M199 22 7.2 Invitrogen 99 ± 1 31 ± 4 275 5.37 X 106 2 RPMI 1640 22 7.2 Invitrogen 97 ± 1 29 ± 3 270 5.26 X 106 3 Schneider’s Drosophila medium 22 7.2 Invitrogen 97 ± 3 34 ± 2 290 5.65 X 106 4 M 199 30 7.2 Invitrogen 96.5 ± 3 32 ± 5 265 5.16 X 106 5 M 199 22 6.3 Invitrogen 95 ± 4 31 ± 3 252 4.90 X 106 6 M199 22 7.2 Invitrogen (20%) 96 ± 3 30 ± 2 261 5.08 X 106 7 M 199 22 7.2 SIGMA 94 ± 4 31 ± 4 289 5.63 X 106 8 M 199 22 7.2 PAN Biotech Gmbh 95.5 ± 4 29 ± 4 282 5.49 X 106 9 M 199 22 7.2 HiMedia (India) 97 ± 1 30 ± 4 280 5.45 X 106 Table 1. Binding of MAA and SNA to virulent L. donovani clonal promastigotes. Virulent L.donovani clonal promastigotes were grown under different conditions. The binding of Sia-binding lectins SNA or MAA (50 μg/ml) was measured by flow cytometry. A fixed number of L.donovani clonal promastigotes (1 X 106) were sus-pended in PBS containing 5% BSA (PBSB). Biotinylated MAA or SNA (0–50 μg/ml) was added to the cell suspension and incubated at room temperature for 30 min. Cells were washed in PBSB thrice and incubated with FITC-conjugated streptavidin for 10 min. Cells were washed in PBSB thrice and fixed in paraformaldehyde (2%). Fluroscence of the cells were analyzed using a FACS Calibur (BD Biosciences) using CELL QUEST software. Unstained cells, or cells stained with FITC-BSA or streptavidin-FITC served as controls. Total Sia content was estimated by the Prozyme rapid sialic acid quantitation kit. ids, glycoconjugates are relevant in several phases of host parasite interactions (De Souza 1995). Microbes use their own pathogen associated molecular patterns to gain entry into cells by exploiting the host pathogen recognition receptor. Neisseria gonorrhoeae uses the asialo-lipooligosaccharide receptor on sperms to infect the female reproductive tract (Harvey et al. 2000); mannose/fucose containing glycans on microbes such as Mycobacterium tuberculosis, Helicobacter pylori, and Leishmania is recognized by the DC–SIGN on dendritic cells (Appelmelk et al. 2003). Many human pathogens express Sias that are recognized by several Siglecs. It is possible that Siglec-dependent recogni-tion of these pathogen glycans leads to altered immune responses, either to the advantage or to the detriment of the pathogen (Crocker et al. 2007). Basic structures recognized by Siglecs contain the NeuAc−Gal–HexNAc sequence. Human and mouse Sn/Siglec-1 preferentially binds NeuAcα2-3Galβ1-, while CD22/Siglec-2 binds NeuAcα2-6Galβ1- (Angata 2006). Hence the question that we seeked answer to was- does the parasite utilize Sia for invasion of Mφs? 11 Journal of Molecular Biochemistry, 2012 Figure 1. Chromatograms of DMB derivatives of Neu5Ac obtained from virulent L.donovani. Representative profile of a HPLC chromatogram of fluorescent derivatives of free sialic acids derived from the standard mixtures of Neu5Ac and Neu5Gc obtained from bovine submaxillary mucins (A). L. donovani virulent clonal promastigotes before (B) and after treatment with neuraminidase (C). L. donovani virulent clonal promastigotes after pre-incubation with N-acetyl neuraminate pyruvate lyase (C, inset). The fluores-cence of DMB derivatives was detected at an excitation wavelength of 373 nm and an emission wavelength of 448 nm. Peak 1, Neu5Gc; peak 2, Neu5Ac; peak 3, Neu5,9 Ac2. Data are representative of 3 experiments. Figure 2. (A) Quatitation of Sia specific lectin binding to virulent clonal promastigote surface with MAA and SNA. The percent of cells with specific Sia expression as deter-mined by flow cytometry using MAA and SNA. A fixed number of clonal promastigotes (1 X 106) were incubated with increasing concentrations of biotinylated-MAA or SNA (0–50 μg/ml). Binding of biotinylated-lectins to L.donovani promastigotes was monitored using FITC-labeled streptavidin followed by flow cytometry of gated populations. FITC-BSA, or streptavidin-FITC were used as control antibodies. The data are means ± SD of three inde-pendent assays. (B) Western blot showing the sialoglyco-protein patterns detected in L. donovani virulent clonal pro-mastigotes. Solubilized membrane proteins from neuramini-dase untreated (lane 1) and treated (lane 2) promastigotes were resolved by SDS-PAGE under non-reducing condi-tions (10% gels, 5 μg protein/lane) and electro-transferred to nitrocellulose. Membrane strips were incubated with bio-tin-labeled MAA (a) or SNA (b) and developed as indicated in Materials and Methods. Controls (lane 3) were also car-ried out by incubating the lectins with fetuin plus sialylac-tose prior to membrane incubation. The numbers on the left indicate the relative position of molecular mass markers expressed in kDa. Images are representative of three inde-pendent experiments. We performed direct binding between L.donovani virulent clonal promastigotes and the siglec recptors Sn, Siglec-2, Siglec-3 (CD33), and Siglec-5. Initially it was found that Sn, Siglec-2 and Siglec-5, and not CD33 (Figure 3A) bound to the pro-mastigotes. Unlike human CD33, murine CD33 does not bind to α2,3- or α2,6-Sia on lactosamine unit (Brinkman-Van der Linden et al. 2008). Removal of α2,3-Sia with Streptococcus pneumoniae sialidase abolished Sn:parasite but not Siglec-2:parasite binding, emphasizing the importance of α2,3-Sias and α2,6-Sias in Sn:parasite and Siglec-2:parasite interactions re-spectively. The more promiscuous sialoglycan recog-nition molecule Siglec-5, bound to both, clonal pro-mastigotes and NeuAc(α2,3)--clonal promastigotes. To reiterate Siglec-1:parasite sialoglycan inter-action, promastigote binding to Mφs was also studied either i) in presence of anti Siglec-1 antibody or ii) by knocking down the expression of Mφ Siglec-1 by siRNA. Siglec-1 knock down was confirmed by ana-lyzing the whole cell lysates blotted for Siglec-1. Ex-pression of the house keeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Transient transfection of RAW264.7 cells with Siglec-1-specific siRNA reduced Siglec-1 production in these cells (Figure 3B). Siglec-1 sup-pression either through neutralizing antibody or siRNA knockdown resulted in a reduction in the proportion of Mφs with attached promastigotes and also a reduction in the total number of bound parasites (Figure 3C) in-dicative of an inability of cells lacking Siglec-1 to bind L.donovani promastigotes appropriately. Parasite at-tachment was not reduced in RAW cells expressing a scrambled siRNA (Figure 3C). Replacement of the primary antibody with an equivalent concentration of non-immune rat IgG (used for negative control) did not reduce the binding. Siglec mediated MAPK signaling in Leishmania infected Mφs Glycan binding at the extra-cellular domain of Siglecs and signaling at the cytosolic domain appears to be coupled. The cytoplasmic domains of most Siglecs contain sequence motif(s) involved in signal transduc-tion (Angata & Varki, 2000). Leishmania infection induces defects in the host cell signaling pathways that is required to bring the cells to an activated state. Leishmania is known to silence the MAPKs (mitogen-activated protein kinase) signaling pathways (Ben-Othman et al. 2008). It is suggested that SHP-1 inacti-vates IRAK-1 kinase (interleukin-1 receptor-associated kinase-1) activity upon L.donovani infection through binding to an evolutionary conserved ITIM (immunoreceptor tyrosine-based inhibitory motif) like motif located within IRAK-1’s kinase domain (Abu-Dayyeh et al. 2008). Siglec-5, an inhibitory receptor Journal of Molecular Biochemistry, 2012 12 Figure 3. (A) Binding of virulent clonal L.donovan or NeuAc(α2,3)-virulent clonal L.donovani promastigotes to Siglecs. Clonal promastigotes (1 X 105) were suspended in PBS containing 5% BSA and binding of biotinylated Siglec-1, Siglec-2, Siglec-5, and CD33 (insert) was determined (Materials and Methods). Data shown are mean ± SD of three experiments performed in triplicate. Sn-L.donovani binding was significantly higher than the Sn- NeuAc(α2,3)-L.donovani binding (*,p<0.0001; **,p<0.001); paired two-tailed Student’s t-test. (B) Siglec-1 expression by RAW 264.7 cells. Western blotting; RAW cells (Lane 1 and 4), RAW cells transfected with Siglec-1 siRNA (Lane 2), or scrambled siRNA (3) were lysed, electrophoresed, trans-ferred to PVDF membrane and immunostained with anti-Siglec-1 antibody (Lane 1-3) or control antibody (Lane 4). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in total proteins was used as loading controls. Blots are repre-sentative of three separate experiments. (C) Binding of viru-lent clonal L.donovan promastigote to RAW cells in (a) ab-sence of anti Siglec-1 antibody, (b) in presence of anti Siglec-1 antibody, (c) transiently transfected with Siglec-1 specific siRNA, (d) transiently transfected with scrambled non-specific siRNA, and (e) in presence of non-immune rat IgG (Materials and Methods). The bars represent the aver-age of three experiments. The standard error is indicated. *,p<0.01; **,p=0.001 versus as indicated; paired two-tailed Student’s t-test. containing an ITIM domain, functions in association with SHP-1 and/or SHP-2 to block survival signaling. The established dogma is that phosphorylation of tyro-sine residue in ITIM’s during cellular activation results in docking sites for SH2 (Src Homology-2) domain containing phosphatases. The recruited and activated phosphatases can then dephosphorylate relevant sub-strates in the vicinity and regulate cellular activation. Experiments were conducted to address the hypothesis that Sia molecules on L.donovani surface probably interacts with Mφ Sia-binding receptors to modulate intracellular signaling. Phosphorylation of p38, JNK and ERK1/2 MAPK was analyzed by West-ern blot analysis. It was found that the parasite induced suppression of MAPK’s signaling was reversed by pre-treatment of the cells with either (i) neuraminidase (data not shown) or (ii) by transiently transfecting RAW 264.7 cells with Siglec-5 specific siRNA. RAW 264.7 cells were transiently transfected with siRNA targeted at Siglec-5. To monitor the down-regulation of Siglec-5 expression, protein expressions were ana-lyzed. siRNA incubation reduced the protein expres-sion of Siglec-5 (Figure 4A). Down regulation of Siglec-5 resulted in MAPK activation in the parasite infected RAW 264.7 cells. Kinetics of MAPK phos-phorylation in Leishmania infected RAW264.7 cells from 5 to 120 minutes resulted in the induction of p38 MAPK phosphorylation that peaked at 15 min, fol-lowed by dephosphorylation, and an increase in p44/42 MAPK and JNK (c-Jun N-terminal kinase) phosphory-lation at 5 min, which was followed by a gradual de-crease as infection progressed (Figure 4Ba). In contrast, activation of p38, p44/42 and JNK MAPK signaling pathways on Siglec-5 siRNA trans-fection (Figure 4Bb) was accompanied by a reduced rate of parasite survival within these cells (Figure 4D). MAPK phosphorylation was significantly abrogated in cells treated with control siRNA (Figure 4C). Activa-tion of p38, ERK1/2 and JNK MAPK signaling path-ways on Siglec-5 siRNA transfection was blocked in presence of MAPK inhibitors SB203580, U0126 and SP6000125 (data not shown). Pre-treatment with goat Figure 4. L.donovani induced suppression of MAPKs is reversed by blocking Siglec-5. (A) Siglec-5 expression by RAW 264.7 cells. Western blotting; RAW cells (Lane 1 and 4), RAW cells transfected with Siglec-5 siRNA (Lane 2), or scrambled siRNA (3) were lysed, electrophoresed, transferred to PVDF and immunostained with anti-Siglec-5 antibody (Lane 1-3) or control antibody (Lane 4). GAPDH in total proteins was used as loading controls. Blots are representative of three separate experi-ments. (B) RAW 264.7 cells were exposed to virulent clonal L.donovani promastigotes for various times, cell lysates were col-lected and subjected to Western blot analysis to detect phosphorylated and total forms of p38, ERK1/2 (extracellular-signal-regulated kinase) and JNK. LPS-treated RAW 264.7 cells served as positive controls for MAPK induction. (a,c) RAW 264.7 cells or (b) Siglec-5 si-RNA transfected RAW 274.7 cells infected with L.donovani promastigotes. (C) Control siRNA trans-fected RAW 264.7 cells infected with virulent clonal L.donovani promastigotes. Comparable results were obtained in three independent experiments. The data are representative of three independent experiments. Densitometries are shown as bar graphs on the right-hand side of each panel. Leishmania infected RAW 264.7 cells (open bar) or si-RNA transfected RAW cells (black bar). The relative phosphorylation of MAPKs in the LPS induced controls was set at 1, and the data represent mean ± SD of three independent experiments. *, p<0.0001; **, p<0.001; ***, p<0.05 versus LPS stimulated control; paired two-tailed Student’s t-test. 13 Journal of Molecular Biochemistry, 2012 IgG failed to activate MAPK signaling (data not shown). LPS (10 ng/ml) treated cells expressed a strong phosphorylation of p38 MAPK, ERK1/2 and JNK that peaked at 15-30 min. Replacement of the primary antibody with an equivalent concentration of rabbit IgG was used for negative controls. L.donovani sialoglycan induced SHP-1 phosphatase activity To assess the potential association of L.donovani sia-loglycans and inhibitory function of Siglec-5, we measured SHP-1 phosphatase activity. Immediate inhi-bition of SHP-1 activity in RAW 264.7 cells that had been pretreated with anti Siglec-5 confirmed the in-hibitory signaling capacity of Siglec-5 (Figure 5). On the other hand, pretreatment of RAW 264.7 cells with Siglec-1 antibody had no significant effect on SHP-1 activity. To further support our data we also checked SHP-1 activity in RAW 264.7 cells after Siglec-1 or Siglec-5 knockdown with siRNA. We observed de-creased SHP-1 activity after 24 hours in Siglec-5 siRNA transfected but not Siglec-1 siRNA transfected Mφs. Thus, these results suggest that L.donovani para-sites can modulate infection via its sialylated glycopro-tein antigens and Mφ-Siglec receptors. It may be men-tioned that, to date, all Sia-dependent binding of Mφs has been found to be Sn-dependent (Munday et al. 1999), suggesting that, on Mφs, Sn is the major Sia binding receptor responsible for mediating cell-cell interactions and that the primary function of other Siglecs may be in signaling. This corroborated with the finding that Siglec-1 suppression either through neu-tralizing antibody or siRNA knockdown, resulted in a reduction in macrophage L.donovani binding (Figure 4C). Thus it may be concluded from our results that in all probability, following sialoglycan-Sn binding, Siglec-5 dependent inhibition of MAPKs signaling occurred. This study represents a major advance in our understanding of Mφ invasion by L.donovani, by de-lineating sialoglycans as important parasite ligands and the identification of Siglec-1 as its host receptor. Uptake and incorporation of sialic acid by L.donovani promastigotes The two primary routes to obtain Sia in microbes is either de novo biosynthesis or acquisition from the en-vironment (Lauc & Heffer-Lauc 2006). It has been suggested that no de novo biosynthetic machinery ex-ists in L.donovani (Chatterjee et al. 2003). To investi-gate the possibility that gangliosides and sialoglyco-proteins found in serum (Yu & Ledden 1972) may be exploited by the parasites as the supply line for making Sia modified cell surface, parasites were treated with sialidase and re-incubated with different sialoglycans in MEM medium containing no serum (serum free me-dium). V.cholerae sialidase treatment for 1h resulted in a decrease in parasite infectivity. Incubation of the control parasites in RPMI/10% FCS at 37oC did not affect the viability or infectivity of the parasites. As shown in the Figure 6A incubation of the neuramini-dase treated parasites with gangliosides containing the sequence Neu5Acα(2-3)Galβ(1-3)GalNAcβ- (GD1a, and GT1b,) partially restored infectivity and Sia con-tent of the parasites (3-6 X 106 Sia molecules/cell). The ganglioside GQ1b in which the sequence ends with two Sia in a NeuAcα(2-8)NeuAc linkage instead of a single Sia as in GD1a and GT1b, was able to fully restore the infectivity and Sia content of the parasites as compared to wild type AG83 promastigotes. Incu-bation with gangliosides similar in structure to GD1a, GT1b, and GQ1b but lacking the Sia attached to the terminal galactose (i.e., GM1, GM2 and GD1b) had no effect. Incubation with Fetuin, (Sia attached to the ter-minal galactose), also restored the infectivity and Sia content of the parasites. None of the gangliosides or fetuin affected the viability of the cells; the cell viabil-ity was always 92% to 97% of that before culture as examined by a trypan blue exclusion test. The presence of sialoglycans in the re-sialylated parasites were con-firmed by FACS analysis using MAA lectin (Figure Journal of Molecular Biochemistry, 2012 14 Figure 5. Virulent clonal L.donovani induced Mφ SHP-1 activity. Virulent clonal L.donovani infected RAW 264.7 cells were incubated in presence of anti Sn and anti Siglec-5 antibodies for the indicated time points. Siglec-1/Siglec-5 si-RNA transfected RAW 264.7 cells were infected with L.donovani clonal parasites. Cells were lysed, SHP-1 pro-tein immunoprecipitated and used to perform phosphatase assay. Results are the mean ± SD of three experiments per-formed in triplicate. *, p < 0.05; versus as indicated; paired two-tailed Student’s t-test. 6A, hatched bars). Most of the Sia was surface associ-ated since it could be removed by sialidase treatment. Enzymatic treatment did not affect cell morphology as assessed using phase-contrast light microscopy. Since metacyclic promastigotes do not see se-rum in the fly and are only briefly exposed to it follow-ing animal infection, the kinetics of Sia incorporation was studied with fetuin. The time course of uptake of Sia was essentially linear through 120 min of incuba-tion, but then plateaued (Figure 5B). Our earlier work using radiolabeled-parasites had indicated that the L.donovani parasites are cleared from blood within 48 minutes (Bhaumik et al. 2008). It has been suggested that the sialoglycans are adsorbed from the serum (Chatterjee et al. 2003). Non-specifically adsorbed molecules are trypsin sensitive but once they are in-serted into plasma membrane they become trypsin re-sistant (Schwarzmann et al. 1983). The presence of trypsin resistant α2,3-linked sialoglycans in the asialo-parasites grown overnight in presence of exogenous sialoglycans, in a serum free medium suggests that the parasite sialylation machinery is active in L.donovani promastigotes and that the sialoglycans are not merely adsorbed from the serum. To note, earlier claim that sialoglycans were merely adsorbed (Chatterjee et al. 2003) is in no way supported by the data presented. There is no support-ing evidence to show that the sialoglycans were at-tached to trypsin sensitive membrane components and not inserted into the membrane in the same manner as endogenous sialoglycans. Besides the presence of se-rum derived sialoglycans in the L.donovani mem-branes, these workers also show the presence of addi-tional α2,3-linked sialoglycans that are absent in the serum. No explanation for the presence of these addi-tional sialoglycans is presented. The working model of sialylation suggests that cells can salvage terminal Sia from extracellular sialoglycans and process it to CMP-Sia, which can be utilized by the intracellular silyl-transferase (Aumiller et al. 2003). The ability of viru-lent clonal promastigotes to produce sialylated glycans in presence of exogenous sialoglycans supports the idea that exogenous sialoglycans can provide a source of Sia for de novo glycoprotein sialylation by the para-sites. Neuraminidase treated parasites grown over night in a serum free medium resulted in 52% reduc-tion in infectivity, 55% decrease in percent infected Mφs and complete absence of MAA binding. Figure 6. (A) Exogenous resialylation of virulent clonal L.donovani parasites. Virulent clonal L.donovani parasites, either pre-treated with (+) or without(-) sialidase, were incubated in serum free medium with (+) or without (-) the addition of gan-gliosides (Gg) or Fetuin (Fet/Gp) and analyzed. RAW 264.7 cells were infected with the resialylated virulent clonal L.donovani parasites and the intracellular amastigotes and percent infected Mφs were estimated. The percentage of resialylated Leishmania promastigotes (1 X 106 promastigotes) binding to Biotinylated MAA lectin (50 μg/ml) is shown by the hatched bars. Results are expressed as mean ± SD for 9 stained coverslips for triplicate experiments. Values (mean of triplicates) at the top indicate sialic acid (Sia) content (number of Sia molecules/parasite). nd=not detected; *,p< 0.0001; **,p < 0.005; ***,p < 0.05 com-pared to virulent clonal L.donovani infected control; paired two-tailed Student’s t-test. GM1, Galβ(1-3)GalNAcβ(1-4)[Neu5Acα(2-3)]Galβ(1-4)Glcβ(1-1)Cer; GM2, GalNAcβ(1-4)[Neu5Acα(2-3)]Galβ(1-4)Glcβ(1-1)Cer; GD1a, Neu5Acα(2-3) Galβ(1-3)GalNAcβ(1-4)[Neu5Acα(2-3)]Galβ(1-4)Glcβ(1-1)Cer; GD1b, Galβ(1-3)GalNAcβ(1-4)[Neu5Acα(2-8-)Neu5Acα(2-3)]Galβ(1-4)Glcβ(1-1)Cer; GT1b, Neu5Acα(2-3)Galβ(1-3)GalNAcβ(1-4)[Neu5Acα(2-8-)Neu5Acα(2-3)]Galβ(1-4)Glcβ(1-1)Cer; GQ1b, Neu5Acα(2-8)Neu5Aca(2-3)]Galβ(1-3)GalNAcβ(1-4)[Neu5Aca(2-8)Neu5Aca(2-3)]Galβ(1-4)Glcβ(1-1)Cer; Cer, Ceramide. (B) Time course of Sia incorporation. Asialo virulent clonal L.donovani parasites (2 X 106 parasites/200μl medium) were incubated for the indicated times at 22 °C in fetuin-containing serum free medium. At the indicated time points, the Sia content of the parasites was analyzed by DMB derivatization and liquid chromatography analysis as described in Materials and Methods. The data are representative of three independent experiments; *,p< 0.0001 compared to asialo virulent clonal L.donovani parasites; paired two-tailed Student’s t-test. (B) (A) 15 Journal of Molecular Biochemistry, 2012 L.major (LMJF_15_1055), L.braziliensis (LBRM_24_0440 & __0360); L.mexicana mexicana (LMXM_24_0360), T.brucei (Tb11.01.8280); T.brucei gambiense (Tbg972.11.18660); T.cruzi (Tc00.1047053507089.40, Tc00.1047053504085.60); and a putative UDP-Gal transporter from L.major (LmjF24.0360). In the formation of sialoglycans, Golgi sialyl-transferases need to have access to their glycoconju-gate substrates and nucleotide sugar donor, CMP-Sia; CMP-Sia in turn is transported into the lumen of the Golgi complex through the CMP-Sia transporter (Zhao et al. 2006). Aumiller et al. (2003) have engineered transgenic insect cell lines to express mammalian gly-cosyltransferases to show that, despite the fact that these cells have little intracellular Sia and no CMP-Sia, these cells can salvage Sia for de novo glycoprotein sialylation from extra-cellular sialoglycoproteins. Searching for an answer to the question how these cell lines sialylate their newly synthesized glycoproteins in absence of CMP-Sia, they discovered that these cell lines can produce sialylated glycoproteins when they expressed a silyltransferase and were co-cultured in presence of an exogenous source of Sia. Though, the precise mechanism by which extra-cellular sialoglyco-proteins effectively support sialylation by the silyl-transferase remains to be determined, it has been sug-gested that the cells somehow salvaged Sia from ex-tracellular sialoglycans and used them to produce CMP There was a direct correlation between the de-gree of cell surface sialylation and infectivity of the parasites. The de-sialylated promastigotes in which sialoglycans were absent were found to be less-infective, suggesting that the loss of terminal Sia probably attenuates the promastigotes. This may be due to the exposure of underlying galactose, as it has been established that the presence of galactose termi-nal glycoconjugates is associated with loss of parasite virulence (Bhaumik et al. 2008, Sacks et al. 1985). L.donovani sialyltransferases In T.cruzi, addition of Sia units to glycoconjugates is mediated by a cell surface trans-sialidase and not by an intracellular CMP-Sia dependent transferase (Colli 1993). Till date no sialidase or trans-sialidase has been reported in Leishmania parasites. On the other hand, two protein coding putative CMP-Sia transporters LINJ_24_0350 and LINJ_24_0360 have been reported in the L.infantum data base. Though it shares 40-50% sequence homology with mammalian UDP-Gal and UDP-GlcNAc transporters, Sia transporters have been shown in a number of systems to only transport CMP-Neu5Ac (Berninsone et al. 1997). Gene DB analysis showed that the candidates LINJ_24_0350 and LINJ_24_0360 associated with other transporters from kinetoplastida with specificities for nucleotide sugar transporters as well as CMP-Sia transporters. The clus-ter contained putative CMP-Sia transporters from Journal of Molecular Biochemistry, 2012 16 Acceptor substrate nmol [14C]NeuAc Incorporated/mg protein/hr V-LD asialo Fetuin 5.76 α2-6 sialylated Fetuin 4.06 α2-3 sialylated Fetuin 1.71 asialo AGP 5.41 α2-6 sialylated AGP 4.42 α2-3 sialylated AGP 1.11 LacCer [Galβ(1-4)Glcβ(1-1)Cer] 0.72 nLcOse4Cer [ Galβ(1-4)GlcNAcβ(1-3) Galβ(1-4)Glcβ(1-1)Cer] 1.05 SA- GM1 [ Galβ(1-3)GalNAcβ(1-4)Galβ(1-4)Glcβ(1-1)Cer] 1.11 GM1 [ Galβ(1-3)GalNAcβ(1-4)[Neu5Acα(2-3)]Galβ(1-4)Glcβ(1-1)Cer] 0.87 asialo -CSA 6.65 Table 2. Acceptor specificity of Sialyltransferases of Leishmania donovani. Sialyltransferase was assayed essentially according to the method of Basu et. al. (Basu et. al, 1982). In brief, complete incuba-tion mixture contained the following components (in μmoles) in a final volume of 0.05mL: acceptor glycosphingolipids, 0.025 or glycoprotein, 0.4 mg/ml; Triton X-100, 0.5%; MgCl2, 0.125; cacodylate-HCl buffer, pH 6.4, 10; EDTA, 0.025; CMP-[14C]NeuAc, 0.015 (2 X 106 cpm/μmole); and enzyme fraction, 100 μg protein. The mixture was incubated at 37oC for 2h and the reaction was stopped with 3 mmol EDTA (protein) or 10 ml chloroform:methanol (C:M, 2:1, v/v; glycolipid). The reaction mixture was spotted on Whatman SG81 paper. Ascending chromatograms were developed in 1% sodium tetraborate for the protein substrates and in C:M-0.2% CaCl2 (55:45:10, v/v/) for glycolipid substrates. The radioactivity of appropriate areas of each chromatograms were quantitated in cocktail “O” (contains per L, 6g of PPO and 0.2g of POPOP) in the liquid scintillation counter (TRI CARB 2100TR; Packard). Figure 7. Localozation of sialyltransferases. Confocal images of L.donovani expressing sialyltransferases. Counter staining is as follows: with DAPI (in blue) for the nuclear DNA and the kinetoplast; with BODIPY TR ceramide (in red) for the Golgi; with BiP (in red) for the ER, with FITC-labeled (green) anti α2,3- and α2,6-silyltransferase antibody for sialyltransferases. Anti BiP was detected using goat anti rat IgG-PE. a,f,k,p, bright field image; b,g,l,q, DAPI; c,m, α2,3-sialyltransferse; h,r, α2,6-sialyltransferase; d,i, BiP; n,s, BODIPY TR; e, overlay of b-d showing non-colocalization of ER and α2,3-sialyltransferase; f, overlay of g-i showing non-colocalization of ER and α2,6-sialyltransferase; o, overlay of l-n showing colocalization of α2,3-sialyltransferse and Golgi and t, overlay of q-s showing colocalization of α2,6-sialyltransferse and Golgi. More than 100 cells were visualized microscopically. Data presented in each panel are representative of at least 90% of the population. SAT3, α2,3-sialyltransferse; SAT6, α2,6-sialyltransferase; N, nucleus; K, kinetoplast. Scale bars, 10mm. 17 Journal of Molecular Biochemistry, 2012 -Sia, which was transported into Golgi apparatus and used for de novo glycoprotein sialylation by the intra-cellular silyltransferase. As already stated, the ability of the promastigotes to produce sialoglycans in the presence of serum or exogenous sialoglycan indicates that the exogenous sialoglycoproteins can provide a source of Sia for the de novo glycoprotein sialylation by L.donovani. In view of these observations, we looked for the presence of sialyltransferases in the virulent clonal promastigotes. Using different acceptor molecules, it was found that both α2,3-sialyltransferase and α2,6-sialyltransferase enzymatic activities could be detected in the virulent clonal parasites (Table 2). The sialyl-transferases in the parasites could transfer [14C]NeuAc more efficiently to α2,6-sialylated fetuin and α2,6-sialylated α1acid glycoprotein (AGP) as compared to α2,3-sialylated fetuin and α2,3-sialylated AGP, indi-cating an elevated α2,3-silyltransferase activity in the virulent parasites. The fact that the enzymes could transfer [14C]NeuAc to the de-sialylated antigen (asialo-CSA) from the virulent parasites indicated the pres-ence of the natural substrates in these parasites. The sialyltransferases could also transfer [14C]NeuAc to glycolipid substrates. Therefore, we conclude that dif-ferential expression of α2,3-silyltransferase and α2,6-silyltransferase resulted in the differential expression of α2,3- and α2,6-linked sialoglycans in the virulent clonal promastigotes. The α2,6-silyltransferase enzyme, regardless of its species or tissue origin, is very specific for Galβ(l-4)GlcNAc and acts with very low activity on Galβ(l-3)GlcNAc and Galβl-3GalNAc, if at all (Weinstein et al. 1982). The natural as well as the recombinant form of the α2,3-silyltransferase enzyme, however, has a high preference for acceptor substrates containing a terminal Galβ(l-3)GlcNAc unit (Weinstein et al. 1982, Wlasichuk et al. 1993). The copious presence of Galβ(1,3) terminal residues in virulent Leishmania parasites (McConville et al. 1990), probably results in the higher expression of α2,3-1inked Sia in the virulent promastigotes. By confocal microscopy, we could show the presence of the α2,3-silyltransferase and α2,6-silyltransferase enzymes mainly in the Golgi of the parasites (Figure 7). All eukaryotic sialyltransferases have in common the presence in their catalytic domain of four conserved peptide regions [sialylmotifs L (large), S (small), motif III, and VS (very small)] that are hallmarks for sialyltransferase identification (Jeanneau et al. 2004, Harduin-Lepers et al. 2005). Since, no antibody against any parasite derived sialyl-transferase is available, and we had previously ob-served that the human β1,4-galactosyltranasferase could recognize L.donovani β1,4-galactosyltranasferase, we used antibodies against the human enzymes to stain the parasite sialyltransferase enzymes. Conclusion Taken together, our data suggests an association be-tween the virulence of L.donovani promastigotes and the expression of sialoglycans, which precludes the activation of signal transduction pathways leading to effective host cell invasion. Data Analysis Data were expressed as mean ± SD and evaluated by the student’s t test. P values of <0.05 were assumed to be statistically significant. Acknowledgements We thank Prof P R Crocker, University of Dundee,UK for the gift of Sn-Fc and Dr. S. Adhya and Dr. A. Banerjee of Indian Institute of Chemical Biology for helping with the confocal microscopy. SK, and JP are the recipients of the Council of Scientific and Indus-trial Research fellowship. Disclosure There is no financial/commercial conflict of interests. References Abu-Dayyeh I, Shio MT, Sato S, Akira S, Cousineau B & Oliver M 2008 Leishmania-induced IRAK-1 inactivation is mediated by SHP-1 interacting with an evolutionary con-served KTIM motif. Plos Negl Trop Dis 2 e305. 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1 doi: 10.1093/glycob/cwp207

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1 doi: 10.1093/glycob/cwp207

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