Báo cáo Y học: Characterization of the lectin from females of Phlebotomus duboscqi sand flies doc

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Báo cáo Y học: Characterization of the lectin from females of Phlebotomus duboscqi sand flies doc

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Characterization of the lectin from females of Phlebotomus duboscqi sand flies Petr Volf 1 , Sona Skarupova ´ 1 and Petr Man 2,3 1 Department of Parasitology and 2 Department of Biochemistry, Charles University, Prague, Czech Republic; 3 Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic Lectin from females of the important sand fly vector, Phlebotomus duboscqi (Diptera: Psychodidae), was isolated by immunoaffinity chromatography using a minicolumn with immobilized anti-lectin immunoglobulins. Carbohy- drate-binding specificity of active fractions corresponded to that of midgut and salivary gland lysates. Haemagglutina- tion was inhibited by D -glucosamine, D -galactosamine and D -mannosamine. The homogeneity and molecular mass of the purified lectin was examined by SDS/PAGE in both reducing and nonreducing conditions. The active fractions showed one band strongly stained by Coomassie blue or silver nitrate; the molecular mass of the lectin was 42 kDa under nonreducing and 44 kDa under reducing conditions. SDS/PAGE of active fractions from the gel filtration revealed four to six protein bands, but the 42/44-kDa protein present in all active fractions was the only component reacting with specific antibodies in Western blots. Local- ization of the lectin in the gut of females was studied using indirect immunofluorescence on sections. The positive reaction of specific antibodies was localized in the lumen and along the microvillar surfaces of epithelial cells. The lectin was partially sequenced and characterized by MS. Peptide maps were obtained by MALDI-TOF MS, and several sequence tags were identified from tandem mass spectra on an ion trap. These sequences displayed high similarity to salivary protein precursors previously identified in a cDNA library of the sand flies Phlebotomus papatasi and Lutzomyia longipalpis. Two main hypotheses on the role of female lectin in Leishmania development are discussed. Keywords: immunoaffinity chromatography; lectin; Phle- botomus duboscqi;sandfly. Females of the sand fly genera Phlebotomus and Lutzomyia are insect vectors of Leishmania parasites, causative agents of a wide spectrum of human diseases, ranging from self- healing cutaneous lesions (e.g. Leishmania major)to progressive and fatal systemic involvement (e.g. Leishmania donovani). The vector part of the life cycle is crucial for Leishmania circulationinnature;Leishmania develop and multiply in the midgut of female sand flies and are transmitted by bite to mammalian hosts. Identification of molecular interactions at the sand fly–Leishmania interface is fundamental to any study of vector competence; the mechanisms responsible for controlling sand fly susceptibi- lity to Leishmania infections, however, are not fully understood. The interplay between the parasite and the vector appears to include a number of potential barriers to complete parasite development. Midgut digestive enzymes may inhibit the early phase of development [1–3], peritrophic matrix behaves as a physical barrier to parasite migration [2,4,5], and putative receptors specific to the parasite glycoconjugate lipophosphoglycan (LPG) seem to be involved in species-specific binding of parasites to the epithelium of the sand fly midgut [6,7]. Another intrinsic factor of the vector that might be involved in sand fly–Leishmania interaction is the lectin activity present in the sand fly midgut. In insects, lectins act as effector, receptor and regulatory molecules in the processes of self/nonself recognition and innate immunity, cell adhesion and tissue differentiation. They also play a regulatory role in pathogen–vector interactions (for reviews, see [8,9]). In Reduviid bugs or Glossina flies, they are involved in the establishment and maturation of trypanosomatid infections (for reviews, see [10,11]). In sand flies, lectin activity has been demonstrated in lysates of various tissues, including head, gut, ovaries, haemolymph [12,13] and salivary glands [14]; the same sugar-binding specificity of activities found in different tissues suggested the presence of the same lectin molecule. The lectin activity is sex-dependent, and high activities were found exclusively in females [15]. In vitro, midgut lysates of female sand flies agglutinated Leishmania promastigotes [12,16], but experiments on inhibition of lectin activity in vivo did not clarify the role of this molecule in the Leishmania life cycle [17]. The main aim of this work was to purify and characterize the lectin from females of Phlebotomus duboscqi,an important vector of L. major in Subsaharian Africa. The small size of sand flies required a simple, preferentially one- step purification technique. Preliminary experiments showed that affinity chromatography is not suitable because of the low affinity of the lectin for simple carbohydrates, and immunoaffinity chromatography was therefore used. Correspondence to P. Volf, Department of Parasitology, Vinicna 7, 128 44 Prague 2, Czech Republic. Fax: + 420 2 24919704, Tel.: + 420 2 21953196, E-mail: volf@cesnet.cz Abbreviation: LPG, lipophosphoglycan. (Received 22 June 2002, revised 16 September 2002, accepted 5 November 2002) Eur. J. Biochem. 269, 6294–6301 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03349.x MATERIALS AND METHODS Sand flies A colony of P. duboscqi (Senegal strain, obtained from R. Killick-Kendrick, Imperial College at Silwood Park, Ascot, Surrey, UK) was reared under standard conditions at 25–26 °C and 14 h : 10 h light/dark photoperiod. Adults were maintained on 50% sucrose and females bloodfed on anaesthetized mice once a week. Dissected midguts, salivary glands or whole bodies of females were homogenized with a Teflon homogeniser in Eppendorf tubes on ice in Tris/NaCl buffer (20 m M Tris/HCl, pH 7.6, 150 m M NaCl). Previous experiments showed that the addition of calcium and other bivalent cations is not required. Samples were centrifuged at 10 000 g for 10 min at 4 °C, and supernatants were collected for subsequent assay. Protein concentration was determined by the Bradford assay (Bio-Rad kit) using BSA (Sigma) as a standard. Haemagglutination assay Sand fly tissue lysates and fractions obtained from chro- matography assays were tested for haemagglutination activity in Tris/NaCl buffer on 96-well U-bottomed micro- titration plates, as described previously [13,18]. Briefly, samples (50 lL) were serially diluted twofold, and an equal volume of 2% (v/v) suspension of washed rabbit erythro- cytes was added. The plates were incubated for 1 h at room temperature, the haemagglutination titre being defined as the reciprocal of the highest dilution showing visual agglutination of erythrocytes. In controls, the lysates or fractions were replaced by Tris/NaCl buffer only. Assay of haemagglutination inhibition Inhibition tests with carbohydrates were performed in microtitration plates as described elsewhere [13,14]. The carbohydrate-binding specificity of the lectin activity was known from previous experiments. Therefore, five noninhibitory monosaccharides, D -glucose, D -galactose, D -mannose, N-acetyl- D -glucosamine and N-acetyl- D -gal- actosamine, and three inhibitory ones, D -glucosamine, D -galactosamine and D -mannosamine, were chosen to compare the binding specificity of gut lysates and active fraction from chromatography techniques. Twofold dilu- tions of carbohydrates were prepared in 50 lLTris/NaCl buffer and mixed with an equal volume of lysate or chromatography fractions adjusted to contain  1.5 haemagglutination units. Then, an equal volume of 2% suspension of rabbit erythrocytes was added to each well. The minimum concentration of inhibitors required to block haemagglutination was determined after 2 h incu- bation at room temperature. Anti-lectin immunoglobulins Antibodies against haemagglutinin of P. duboscqi females were raised in rabbits (female; Great Chinchila;  4 kg) as described by Yeaton [19]. The rabbit was bled from the ear, andwashednativeerythrocyteswereadjustedto2% suspension in sterile Tris/NaCl buffer. Then 15 mL eryth- rocyte suspension was agglutinated by filtered gut lysates of P. duboscqi females for 1 h. Then, agglutinated erythrocytes were washed three times in sterile Tris/NaCl buffer by centrifugation at 750 g for 15 min, the pellet was resus- pended in 2 mL incomplete Freund’s adjuvant (Difco, Detroit, MI, USA) and injected subcutaneously into the same rabbit. Four immunizations at 2-week intervals were followed after 2 months by an intravenous booster (without adjuvant). Immune sera were obtained 1 week after the final booster. IgG fractions of the sera were isolated by rivanol (2-ethoxy-6,9-diaminoacridine lactate hydrate) and ammo- nium sulfate [20]. Purified IgG samples were stored in aliquots at )70 °C and used for Western blotting and immunoaffinity chromatography. Localization of the lectin in midgut tissue Previous studies detected haemagglutination activity in both midgut epithelium and the midgut content of females [15]. In this work, indirect immunofluorescence with anti-haem- agglutinin IgG was used for more precise localization of the lectin in the midgut tissue. P. duboscqi females (4–6-days- old) were fixed in 70% ethanol and embedded in LR White resin according to instructions of the manufacturer (Poly- sciences, Warrington, Lancs, UK). Parasagittal sections, 1–2 lm thick, obtained with an Ultracut E (Reichert Jung, Wien, Austria), were incubated overnight at 4 °C with Tris/ NaCl buffer containing 0.1% (v/v) Tween 20 (Tris/NaCl/ Tween) and 5% (w/v) BSA to prevent nonspecific binding of serum to hydrophobic epitopes of the section. Then, the sections were incubated with immune rabbit serum diluted in Tris/NaCl/Tween, washed, and incubated with fluoresc- ein isothiocyanate-conjugated swine anti-rabbit immuno- globulins (Sevac, Prague, Czech Republic) diluted in Tris/ NaCl/Tween. In control sections, the preimmune serum from the same rabbit was used or the serum incubation step was omitted (control of unspecific binding of the conjugate). Both incubations with sera and conjugate were performed in a moist chamber for 45 min at 37 °C. Sections stained with Evans blue were photographed using a Jenalumar (Karl Zeiss-Jena) fluorescent microscope. Purification of the lectin Medium-pressure liquid chromatography system BioLogic (Bio-Rad) and two different methods, gel filtration and immunoaffinity chromatography, were used for purification of the lectin. Samples were prepared from batches of about 500 P. duboscqi females, 3–8 days old, which had never had a blood meal. Females were homogenized in 500 lLTris/ NaCl buffer as described above, and supernatant containing  1mgÆmL )1 protein was filtered using 0.45 lmMicrocon filters (Amicon) before loading on the chromatography columns. Preliminary experiments with three different gel-filtration columns showed Superose 12 to be the most suitable one; 400 lL filtered supernatant was applied to the column (1 · 40 cm), pre-equilibrated with Tris/NaCl buffer. Elution was carried out with the same buffer at a flow rate of 0.4 mLÆmin )1 . Fractions were examined for haemagglu- tination activity and active fractions were checked for binding specificity using selected carbohydrates (see above). Then the fractions were concentrated (to  0.5 mgÆmL )1 ) Ó FEBS 2002 Characterization of lectin from sand fly females (Eur. J. Biochem. 269) 6295 using centifugation on Microcon YM-10 filters (Amicon), and protein composition was determined by electrophoresis. For immunoaffinity chromatography, purified anti-lectin IgG was immobilized on CNBr-activated Sepharose. Sam- ples with high agglutinating activity from gel filtration or about 500 lL filtered supernatant from homogenized femaleswereloadedontoacolumn(4mL)equilibratedwith Tris/NaCl buffer. After extensive washing with Tris/NaCl buffer (flow rate 0.5 mLÆmin )1 for 70 min), the immunospe- cific bound protein was eluted with a linear pH gradient of citrate buffer (50 m M citrate, 100 m M NaCl, pH 2.6). The eluted fractions were adjusted to pH 7.5 with 1 M Tris, tested for haemagglutinating activity and carbohydrate-binding specificity, concentrated, and analysed electrophoretically. Electrophoresis and Western blots Supernatants of tissue lysates or concentrated fractions obtained by chromatography were boiled for 3 min in sample buffer with or without 2% (v/v) 2-mercaptoethanol andloadedontoanSDS/10%polyacrylamidegel(thick- ness 0.75 mm). Separations were carried out at a constant 200 V for 50 min using Mini-Protean II apparatus (Bio- Rad). Gels were stained with Coomassie Brilliant blue R-250 or silver nitrate. Proteins separated by SDS/PAGE were transferred to nitrocellulose membrane (0.2 lm; Serva) using a Semiphor unit (Hoefer Scientific Instruments). Blotting was performed for90minat1.5mAÆcm )2 at room temperature. The blot was rinsed in Tris/NaCl/Tween, stained for proteins with 1% (w/v) Ponceau red, and incubated for 2 h in Tris/NaCl/ Tween with 5% (w/v) skimmed milk (Oxoid, UK). The incubation with rabbit immunoglobulins diluted 1 : 200 in Tris/NaCl/Tween (2 h at room temperature) was followed by repeated rinsing in Tris/NaCl/Tween and then by 1 h incubation with swine anti-rabbit immunoglobulins conju- gated with horseradish peroxidase (Sevac; diluted 1 : 1000 in Tris/NaCl/Tween). The peroxidase reaction product was developed in 4-chloro-1-naphthol solution. In-gel digestion and esterification For MS analysis and protein microsequencing, the active fraction from immunoaffinity chromatography was used for electrophoresis on a 12% (w/v) gel. A Coomassie-stained spot was excised from the gel and cut into small pieces. The gel was washed with water. The wash solution was discarded and replaced with 100 m M ethylmorpholine acetate buffer, pH 8.5, in 50% acetonitrile. After complete gel destaining in a sonication bath, the gel pieces were washed with water, shrunk by dehydration in acetonitrile, reswelled in water, and dehydrated again by addition of acetonitrile. The supernatant was removed and the gel was partly dried in a vacuum centrifuge. The gel pieces were then swollen in a digestion buffer containing 50 m M ehylmorph- oline acetate, pH 8.0, 1 m M CaCl 2 , 10% (v/v) acetonitrile and sequencing grade trypsin (trypsin to protein ratio 1 : 75). After overnight digestion (shaking at 37 °C), the resulting peptides were extracted from the gel by increasing the acetonitrile concentration to 50% and by addition of trifluoroacetic acid to a final concentration of 1%. Subse- quently, the tubes were sonicated for 15 min. The liquid phase with the extracted peptides was divided into two tubes, and one was subjected to ethyl esterification in ethanolic HCl prepared by mixing 1 mL ethanol with 160 lL acetyl chloride. The reaction was carried out for 2.5 h and stopped by drying in a SpeedVac concentrator. The second part of the peptide mixture was dried in a SpeedVac concentrator. Both samples were redissolved with 5 lL 50% (v/v) acetonitrile/1% (v/v) trifluoroacetic acid. MALDI-TOF MS A saturated solution of a-cyano-4-hydroxycinnamic acid (Sigma) in aqueous 50% (v/v) acetonitrile/0.2% (v/v) trifluoroacetic acid was used as a MALDI matrix. A 2-lL volume of sample and 2 lL matrix solution were premixed in a tube; 0.5 lL of the mixture was placed on the sample targetandallowedtodryattheambienttemperature. Positive ion MALDI mass spectra were measured on a Bruker BIFLEX reflectron time-of-flight mass spectrometer (Bruker-Franzen, Bremen, Germany) equipped with a SCOUT 26 sample inlet, a gridless delayed extraction ion source, and a nitrogen laser (337 nm) (Laser Science, Cambridge, MA, USA). The ion acceleration voltage was 19 kV, and the reflectron voltage was set at 20 kV. Spectra were calibrated externally using the monoisotopic [M + H] + ion of a-cyano-4-hydroxycinnamic acid and a peptide standard (angiotensin II; Aldrich). lHPLC-nano ESI ion trap MS The tryptic peptides were loaded on to a homemade capillary column (0.18 · 100 mm) packed with reversed- phase resin (MAGIC C-18; 200 A ˚ ;5lm; Michrom Bio- Resources, Auburn, CA, USA) and separated using a gradient from 5% (v/v) acetonitrile/0.5% (v/v) acetic acid to 35% (v/v) acetonitrile/0.5% (v/v) acetic acid for 50 min at a flow rate of 2 lLÆmin )1 . The column was connected directly to an LCQ DECA ion trap mass spectrometer (ThermoQuest, San Jose, CA, USA) equipped with a nanoelectrospray ion source. The spray voltage was held at 1.6 kV and the tube lens potential was )2 V. The heated capillary was kept at 175 °C with a voltage of 13 V. Full-scan spectra were recorded in positive mode over the mass range 350–1300 atomic mass units. MS/MS data were automatically acquiredonthemostintenseprecursorionineachfull- scan spectrum. Acquired MS/MS spectra were interpreted manually. RESULTS Western blots with female tissue For both midgut lysate and salivary gland lysate, the purified IgG fraction of the immune serum specifically recognized a single protein band. The band represented a major salivary protein and a minor midgut protein; its molecular mass was 42 kDa under nonreducing and 44 kDa under reducing conditions (Fig. 1). When the whole immune serum was used, an additional protein band of molecular mass  70 kDa was visualized in the midgut lysate (Fig. 1) but not in salivary glands. Both preimmune serum and the negative control without serum gave no reaction with both antigens. A similar result was observed when midgut lysate of the closely related species Phlebotomus papatasi 6296 P. Volf et al.(Eur. J. Biochem. 269) Ó FEBS 2002 was used: anti-haemagglutinin IgG specifically recognized the 42–44-kDa region (data not shown). Localization of the lectin Anti-haemagglutinin IgG reacted with the content of the midgut lumen and along the surfaces of midgut epithelial cells. A positive reaction was observed in both thoracic and abdominal parts of the midgut (Fig. 2). Antibody binding was specific: no reaction was observed on control sections incubated with preimmune sera or with fluorescein conju- gate only. Purification of the lectin by gel filtration Gel filtration of whole body lysates on a Superose 12 column revealed about six protein peaks. Haemagglutina- tion activity against rabbit erythrocytes was observed between peaks 3 and 4, with a broad maximum in fractions 18–21 (Fig. 3A). The carbohydrate-binding specificity of the active fractions was similar to that of midgut lysates. Inhibition was achieved with D -glucosamine, D -galactosa- mine (both at 20 m M final concentration) and D -mannosa- mine (40 m M ), whereas D -glucose, D -galactose, D -mannose, N-acetyl- D -glucosamine and N-acetyl- D -galactosamine had no inhibitory effect at 160 m M final concentration. The active fractions were concentrated and submitted to SDS/ PAGE under reducing conditions; four to six protein bands were detected in each fraction (Fig. 4). The 44-kDa protein present in all active fractions was the only component that reacted with anti-haemagglutinin immunoglobulins in Western blotting. Antibodies from preimmune rabbit serum gavenoreaction(Fig.4). Isolation of the lectin by immunoaffinity chromatography Fractions with haemagglutinating activity (titres 1 : 8 and 1 : 16) against native rabbit erythrocytes were present in the first peak eluted from the immunoaffinity column by low pH (Fig. 3B). The homogeneity and molecular mass of the purified lectin were examined by SDS/PAGE in both reducing and nonreducing conditions. The active fractions showed one band strongly stained with Coomassie blue or silver nitrate; the molecular mass of the lectin was 42 kDa under nonreducing and 44 kDa under reducing conditions (Fig. 4). The second peak eluted from the column at low pH had no haemagglutinating activity and contained a frag- ment of IgG detached from the column (data not shown). MS and data processing In the first step, we analyzed a tryptic peptide mixture by MALDI-TOF MS. Despite the fact that the spectrum contained a considerable number of fully resolved peaks (Fig. 5A), the approach of peptide mapping gave no positive hit. In the second step, the peptide mixture was analyzed by LC-MS/MS on an ion trap mass spectrometer. In this experiment, we obtained several tandem mass spectra of peptides, which were interpreted manually (Fig. 5B). The sequences were read out from y-ion and b-ion series according to known fragmentation mechanisms proposed and described elsewhere [21]. We also measured the peptide mixture after ethyl esterification and thus were able to assign the number of acidic residues in each peptide. Because the ion trap instrument does not allow detection of low-mass and ammonium ions, we were not able to assign the N-terminal di-residues accurately in all cases. Fig. 2. Parasagittal section of the abdomen of P. duboscqi female under the fluorescent microscope. Autofluorescence of the cuticular sclerit (sc) surrounding thoracic muscles (mu). Specific reaction of the midgut lumen (lu) and microvillar layer of the midgut epithelium (ep) with purified anti-lectin immunoglobulins. Ft, Fat body. Fig. 1. SDS/PAGE and Western blotting of lysates from salivary glands and midgut of P. duboscqi females. Protein (1–3 lgperlane)was loaded and samples run as described in Materials and methods. Gels werestainedwithsilvernitrate,andreactiononWesternblotswas visualized with 4-chloro-1-naphthol solution. SDS/PAGE: lane 1, protein markers (BenchMark Protein Ladder; Gibco); lane 2, salivary gland lysate under reducing conditions; lane 3, the same salivary gland lysate sample under nonreducing conditions; lane 4, midgut lysate under nonreducing conditions. Western blotting (nonreduced sam- ples): lane 5, reaction of midgut lysate with immune (+) and preim- mune (–) serum; lane 6, reaction of midgut lysate with purified immunoglobulins from immune (+) and preimmune (–) sera. Ó FEBS 2002 Characterization of lectin from sand fly females (Eur. J. Biochem. 269) 6297 The sequences obtained are summarized in Table 1. Searches were carried out against a nonredundant protein database by using MS - BLAST (http://dove.embl-heidelberg. de/Blast2/msblast.html). High similarity was found to a 42-kDa salivary protein from P. papatasi (SwissProt number Q95WD9). DISCUSSION Lectin from P. duboscqi females was purified and charac- terized by liquid chromatography and SDS/PAGE as a 42– 44-kDa protein. Inhibition tests with carbohydrates gave identical results in purified fractions and crude midgut lysates. This confirmed that the purified lectin corresponds to the haemagglutinin present in various sand fly tissues, including the midgut and salivary glands. Similar electro- phoretic migration of the molecule in reducing and nonre- ducing conditions implies a monomer structure. Most insect lectins characterized to date contain polypeptide chains linked by disulfide bridges, and their activity is Ca 2+ dependent [22,23]. In bloodsucking Diptera, namely tsetse flies and mosqui- toes, lectins have been purified from the haemolymph by various chromatographic techniques, including affinity chromatography [23,24]. In midgut tissue, chromatographic isolation has been less successful and therefore erythrocytes have frequently been used as affinity ligands. In the mosquito Anopheles gambiae, Mohamed and Ingram [22] identified a 65-kDa lectin band using adsorption of midgut extracts with human erythrocytes. In tsetse flies, Grubhoffer et al. [25] detected two lectin bands of molecular mass 27 and 29 kDa in Glossina tachinoides midgut using Western blots with anti-haemagglutinin immunoglobulins raised by the technique of Yeaton [19]. In the gut tissue of another tsetse fly, Glossina longipennis,Osiret al.[26]purifieda protein with two subunits of 27 and 33 kDa; the larger was proposed to be an agglutinin with glucosamine-binding lectin activity, while the smaller showed trypsin activity. The lectin from P. duboscqi females was partially sequenced and characterized by MS. Peptide maps were obtained by MALDI-TOF, and several tandem mass spectra were observed using an ion trap. Several sequence tags were identified from the tandem mass spectra. These sequences displayed a high similarity to salivary protein Fig. 4. SDS/PAGE and Western blots of the purified female sand fly lectin. The haemagglutinating fractions from gel-filtration and immu- noaffinity chromatography were concentrated, loaded on the gel, and run under nonreducing conditions as described in Materials and methods. The gel was stained with silver nitrate, and reaction on Western blots was visualized with 4-chloro-1-naphthol solution. Lane 1, protein markers (Bio-Rad); lane 2, active fraction (no. 20) from Superose 12; lane 3, Western blot of fraction 20 with preimmune (–) and immune anti-lectin serum (+); lane 4, active fraction (no.18) from immunoaffinity chromatography. Fig. 3. Purification of P. duboscqi lectin by gel filtration (A) and immunoaffinity chromatography (B). (A) Supernatant from 500 females (500 lL)wasfilteredandloadedontoSuperose12column (1 · 40 cm), pre-equilibrated with Tris/NaCl buffer. Elution was car- ried out with the same buffer (flow rate 0.4 mLÆmin )1 ). Fractions were examined for haemagglutinating activity as described in Materials and methods. (B) Filtered supernatant from 500 females was loaded on to a minicolumn (4 mL) with anti-lectin immunoglobulins immobilized on CNBr-activated Sepharose. After the column had been washed with Tris/NaCl buffer (buffer A) the immunospecific bound protein was eluted by a linear pH gradient of buffer B (citrate buffer; 50 m M citrate, 100 m M NaCl, pH 2.6). The eluted fractions were adjusted to pH 7.5 with 1 M Tris and tested for haemagglutinating activity as described in Materials and methods. 6298 P. Volf et al.(Eur. J. Biochem. 269) Ó FEBS 2002 precursors found in the cDNA library of the closely related species P. papatasi [27]. The coded proteins, named PpSP42 (Q95WD9) and PpSP44 (Q95WD8) and a similar Yellow protein from salivary glands of another sand fly Lutzomyia longipalpis showed motifs of the major royal jelly proteins of honeybee (Apis mellifera) and Yellow protein of Drosophila [27]. The biological role of these proteins remains unknown; the major royal jelly proteins are believed to play a major role in nutrition because of their high essential amino-acid content [28]. Interestingly, in sand flies these 42–44-kDa salivary proteins represent the main immunogens strongly reacting with antibodies from hosts repeatedly bitten by sand flies [29]. In the gut tissue of females, the lectin is present free in the lumen of thoracic and abdominal parts of the midgut and along the microvillar surface of midgut epithelium. These observations confirmed previous results obtained by haem- agglutination tests. Volf and Killick-Kendrick [15] showed that high haemagglutination activity was present in both parts of the midgut. In unfed females, the activity was almost equally distributed between the epithelium and the midgut content, whereas in fed females the activity titres were elevated in the lumen, and most of the activity was detected in the peritrophic space surrounded by peritrophic matrix. Part of the midgut lectin activity may originate from saliva swallowed during the feeding of the fly. However, the midgut activity peaked not immediately after the blood meal but 48 h later [15], suggesting that most of the lectin present in midgut lumen is secreted by midgut epithelium and passes through peritrophic matrix during blood meal digestion. However, the site of synthesis of sand fly lectin is not necessarily limited to salivary glands and midgut. Biosynthesis of insect lectins takes place mainly in the fat body or haemocytes [30,31]. In sand flies, various levels of the lectin activity were found in different tissues, including the ovaries and haemolymph [13], and hybridization in situ will be required to identify lectin expression sites. Two main hypotheses may be considered for the role of sand fly lectins in Leishmania development: they could be involved in Leishmania attachment to sand fly midgut or they could serve as inhibitors of Leishmania development. The ability of Leishmania promastigotestoattachtothe midgut epithelium of female sand flies is a critical compo- nent of vectorial competence. There is a close evolutionary fit between sand fly vectors and Leishmania parasites in some Old World leishmaniases: P. papatasi and Phleboto- mus sergenti are susceptible only to L. major and Leishma- nia tropica, respectively. The failure of other parasite species to develop in these sand flies coincided with a time of defecation of the blood meal remnants and is correlated with the ability of promastigotes to attach to the sand fly midgut by this time (for a review, see [32]). The attachment is controlled by polymorphic, species-specific structures on the parasite LPG [6,7] and a strong species-specific vector competence of P. papatasi and P. sergenti is explained by the presence of specific LPG-binding receptors on midgut epithelium [32]. Midgut lectin of P. papatasi binds LPG of L. major [13], and part of the activity is associated with the surface of the midgut epithelium (see above). However, it is unlikely that it is involved in the attachment or is identical with the LPG receptor. Lectin activity with the same sugar-binding specificity was present in all Phlebotomus and Lutzomyia species studied [13,18], and the same is true for 42–44-kDa Table 1. Sequences obtained from tandem mass spectra using lHPLC- nanoESIiontrapMS.Comparison of data with the similar sequences from salivary protein of P. papatasi. Peptides were separated on a reversed-phase capillary column and analyzed on an ion trap mass spectrometer equipped with a nanoelectrospray ion source (details are given in Materials and methods). Acquired MS/MS spectra were interpreted as depicted in Fig. 5. Numbers assign positions in the polypeptide chain; (I/L) indicates that leucine or isoleucine is present in this position (isobaric amino acids). Other characters in parentheses may be in reverse order. 42-kDa salivary protein precursor of P. papatasi (Q95WD9) Sequences obtained from P. duboscqi females 59-MLFFGIPR-67 M(I/L)FFG(I/L)PR 71-VPITFAQLSTR-81 VP(I/L)TVAQ(I/L)STR 90-NPPLDK-95 DPPLDK 167-NPLGYGGFAVDVVNPK-182 TP(I/L)GYGGFAVD VVNPK 238-FKAGIFGIALGDR-250 (LE)TG(I/L)FG(I/L) A(I/L)GDR 295-TEAIALAYDPETK-307 TEA(I/L)A(I/L)AYDPETK Fig. 5. MS of purified lectin of P. duboscqi females. (A) MALDI-TOF mass spectrum of a tryptic peptide mixture after in-gel digestion. Peaks labelled with an asterisk represent peptides successfully sequenced by lHPLC-nano ESI MS. (B) Sequencing by lHPLC-nano ESI MS, example of the peptide 1184.7. Ó FEBS 2002 Characterization of lectin from sand fly females (Eur. J. Biochem. 269) 6299 protein precursors found by Valenzuela et al. [27]. There- fore, the lectin cannot serve as the species-specific receptor responsible for different vectorial competence of various sand fly species. The second hypothesis is based on similarity to the Glossina–Trypanosoma system, where the lectin activity of the vector was proposed to prevent establishment of parasites in the ectoperitrophic space [33] and trigger cell- suicide pathways in trypanosomes, analogous to apoptosis in metazoa (for a review, see [34]). In addition, Glossina lectins were reported to play a dual role, not only to kill parasites but also to provide a signal for the maturation of established ones [35]. At present, we cannot exclude the possibility that sand fly lectin may affect Leishmania development by similar mechanisms. Purification of the lectin by immunoaffinity chromatography promotes further study of the role of this molecule in sand fly–Leishmania interaction. ACKNOWLEDGEMENTS We thank Professor R. Killick-Kendrick for the P. duboscqi colony and help during sand fly research, and Professor L. Grubhoffer for long- term support of parasite–vector studies. We are also grateful to Dr K. Bezous ˇ ka,DrI.Hrdy´ and R. S ˇ uta ´ k for advice on lectins and chromatography techniques and Vera Volfova ´ for sand fly dissections. This study was supported by the Ministry of Education (projects MSM 113100001 and 113100004) and the Grant Agency of the Czech Republic (project 206/03/0325). REFERENCES 1. 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(1987) Lectin mediated establish- ment of midgut infections of Trypanosoma congolense and Try- panosoma brucei in Glossina morsitans. Trop. Med. Parasitol. 38, 167–170. 34. Welburn, S.C., Barcinski, M.A. & Williams, G.T. (1997) Pro- grammed cell death in Trypanosomatids. Parasitol. Today 13, 22– 26. 35. Welburn, S.C. & Maudlin, I. (1989) Lectin signalling of matura- tion of T. congolense infections in tsetse. Med. Vet. Entomol. 3, 141–145. Ó FEBS 2002 Characterization of lectin from sand fly females (Eur. J. Biochem. 269) 6301 . the site of synthesis of sand y lectin is not necessarily limited to salivary glands and midgut. Biosynthesis of insect lectins takes place mainly in the fat body or haemocytes [30,31]. In sand. inhibition of lectin activity in vivo did not clarify the role of this molecule in the Leishmania life cycle [17]. The main aim of this work was to purify and characterize the lectin from females of Phlebotomus. that sand y lectin may affect Leishmania development by similar mechanisms. Purification of the lectin by immunoaffinity chromatography promotes further study of the role of this molecule in sand

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