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

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 conditi

Characterization of the lectin from females of Phlebotomus

Petr Volf1, Sona Skarupova´1and Petr Man2,3

1

Department of Parasitology and2Department of Biochemistry, Charles University, Prague, Czech Republic;3Institute 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 byD-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; sand fly

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

Leishmaniacirculation in nature; 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 Glossinaflies, 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 Leishmanialife 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)

M A T E R I A L S A N D M E T H O D S

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 25–26C 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 mM Tris/HCl,

pH 7.6, 150 mM 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 4C, 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 lL Tris/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,

and washed native erythrocytes were adjusted to 2%

suspension in sterile Tris/NaCl buffer Then 15 mL

eryth-rocyte suspension was agglutinated by filtered gut lysates of

P duboscqifemales 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 4C 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 37C 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 lL Tris/ NaCl buffer as described above, and supernatant containing

 1 mgÆmL)1protein was filtered using 0.45 lm Microcon 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)

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

females were loaded on to a column (4 mL) equilibrated with

Tris/NaCl buffer After extensive washing with Tris/NaCl

buffer (flow rate 0.5 mLÆmin)1for 70 min), the

immunospe-cific bound protein was eluted with a linear pH gradient of

citrate buffer (50 mMcitrate, 100 mMNaCl, pH 2.6) The

eluted fractions were adjusted to pH 7.5 with 1MTris, 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

and loaded on to an SDS/10% polyacrylamide gel

(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

for 90 min at 1.5 mAÆcm)2at 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 mM 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 mM

ehylmorph-oline acetate, pH 8.0, 1 mM CaCl2, 10% (v/v) acetonitrile

and sequencing grade trypsin (trypsin to protein ratio

1 : 75) After overnight digestion (shaking at 37C), 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 target and allowed to dry at the ambient temperature 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˚; 5 lm; 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 LCQDECAion 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

175C 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 acquired on the most intense precursor ion in each full-scan spectrum Acquired MS/MS spectra were interpreted manually

R E S U L T S

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

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 mMfinal concentration) andD

-mannosa-mine (40 mM), whereasD-glucose,D-galactose,D-mannose,

N-acetyl-D-glucosamine and N-acetyl-D-galactosamine had

no inhibitory effect at 160 mM 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 gave no reaction (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/PAGEand Western blotting of lysates from salivary

glands and midgut of P duboscqi females Protein (1–3 lg per lane) was

loaded and samples run as described in Materials and methods Gels

were stained with silver nitrate, and reaction on Western blots was

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.

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)

D I S C U S S I O N

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 Ca2+ 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, Osir et al [26] purified a 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/PAGEand 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) was filtered and loaded on to Superose 12 column

(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.

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

longipalpisshowed 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 promastigotes to attach to the 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 sergentiare 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-nano E SI ion trap MS 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.

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–Trypanosomasystem, 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

A C K N O W L E D G E M E N T S

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.

Bezousˇka, Dr I Hrdy´ and R Sˇuta´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).

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