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Originally published In Press as doi:10.1074/mcp.M700497-MCP200 on December 17, 2007.
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Molecular & Cellular Proteomics 7:582-590, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Toward an Understanding of the Molecular Mechanism for Successful Blood Feeding by Coupling Proteomics Analysis with Pharmacological Testing of Horsefly Salivary Glands*,S

Xueqing Xu{ddagger},§,¶, Hailong Yang{ddagger},, Dongying Ma{ddagger},§,¶, Jing Wu{ddagger},§, Yipeng Wang{ddagger},§, Yuzhu Song{ddagger},§, Xu Wang||, Yi Lu||, Junxing Yang{ddagger} and Ren Lai{ddagger},||,**

From the {ddagger} Biotoxin Units of Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China, || Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Life Sciences College of Nanjing Agricultural University, Nanjing, Jiangsu 210095, China, and § Graduate School of the Chinese Academy of Sciences, Beijing 100009, China


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Horseflies are economically important blood-feeding arthropods and also a nuisance for humans and vectors for filariasis. They rely heavily on the pharmacological properties of their saliva to get a blood meal and suppress immune reactions of hosts. Little information is available on antihemostatic substances in horsefly salivary glands; especially no horsefly immune suppressants have been reported. By proteomics or peptidomics and coupling transcriptome analysis with pharmacological testing, several families of proteins or peptides, which act mainly on the hemostatic system or immune system of the host, were identified and characterized from 30,000 pairs salivary glands of the horsefly Tabanus yao (Diptera, Tabanidae). They are: (i) a novel family of inhibitors of platelet aggregation including two members, which possibly inhibit platelet aggregation by a novel mechanism and act on platelet membrane, (ii) a novel family of immunosuppressant peptides including 12 members, which can inhibit interferon-{gamma} production and increase interleukin-10 secretion, (iii) a serine protease inhibitor with 56 amino acid residues containing anticoagulant activity, (iv) a serine protease with anticoagulant activity, (v) a protease with fibrinogenolytic activity, (vi) three families of antimicrobial peptides including six members, (vii) a hyaluronidase, (viii) a vasodilator peptide, which is an isoform of vasotab identified from Hybomitra bimaculata, and interestingly (ix) two metallothioneins, which are the first metallothioneins reported from invertebrate salivary glands. The current work will facilitate the understanding of the molecular mechanisms of the ectoparasite-host relationship and help in identifying novel vaccine targets and novel leading pharmacological compounds.

Hematophagous arthropods have developed effective mechanisms to get a blood meal and overcome their host's immune responses. Such arthropods produce a wide array of antihemostatic and immune suppressant compounds in their salivary glands (1–5). Antihemostatic compounds of blood-sucking arthropods have been distinguished into several groups such as inhibitors of coagulation factors (Factors VII, V, and Xa and thrombin), platelet functions, and fibrinolytic enzymes. Immunosuppressive factors from ticks have been extensively studied (6–8). Tick salivary gland extract (SGE)1 can suppress innate and adaptive immunity. It has been reported that tick SGE inhibits host complement activation and depresses macrophage function by inhibiting lipopolysaccharide (LPS)-induced nitric-oxide synthesis and proinflammatory cytokine production (9–17).

As an important hematophagous arthropod, there have been comparatively few studies on antihemostatic substances in horsefly salivary glands, although anticoagulant activity has been identified. Only a vasoactive peptide (vasotab) was reported from Hybomitra bimaculata (Diptera, Tabanidae) salivary glands (3). No immunosuppressive substances have been described before in horsefly salivary glands. Female horseflies require substantial amounts of blood (up to 0.5 ml) for egg production. They can ingest up to 200 mg of blood within only 1–3 min, suggesting that they must possess very potent antihemostatic mechanisms (3, 18). More than one feeding episode is needed for horseflies, and approximately 10 landings on a host are necessary to complete one blood meal, suggesting that they must possess very potent immunosuppressive mechanisms. To identify and characterize interesting salivary compounds for understanding the molecular mechanisms of the ectoparasite-host relationship and to help in identifying novel vaccine targets, we used proteomics or peptidomics and transcriptome analysis coupled with pharmacological testing of the activities to investigate pharmacological molecules in the salivary glands of the horsefly T. yao Macquart.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Collection of Horsefly—
T. yao Macquart horseflies (about 30,000; average weight, 0.17 g) were collected in Shanxi Province of China in July 2004. Collections were performed between 17:00 and 20:00 during optimal weather (sunny, 30–35 °C, and no wind). All the flies were transported to the laboratory alive and kept at –80 °C.

Salivary Gland Dissection and SGE Preparation—
Horseflies were glued to the bottom of a Petri dish and placed on ice. They were then dissected under a microscope. The salivary gland was excised and transferred into 0.1 M phosphate buffer solution, pH 6.0, and kept in the same solution at –80 °C. 30,000 pairs of horsefly salivary glands were homogenized in 0.1 M phosphate buffer solution, pH 6.0, and centrifuged at 5000 x g for 10 min. The supernatant was termed SGE and lyophilized.

Fractionation of SGE—
The lyophilized SGE sample (2.1 g, total A280 nm of 600) was dissolved in 10 ml of 0.1 M phosphate buffer solution, pH 6.0, and then was applied to a Sephadex G-75 (Superfine, Amersham Biosciences, 2.6 x 100-cm) gel filtration column equilibrated with 0.1 M phosphate buffer, pH 6.0. Elution was performed with the same buffer, collecting fractions of 3.0 ml. The absorbance of the eluate was monitored at 280 nm (see Fig. 1A). Every fraction was subjected to pharmacological testing including inhibition of platelet aggregation, microbe killing, inhibition of serine protease hydrolysis activity on chromogenic substrate, effects on blood coagulation, contraction of isolated rat femoral artery, cytokine secretion, fibrinogenolytic activity, hyaluronidase activity, and {alpha}-amylase activity as indicated under "Pharmacological Testing." The protein peaks containing tested pharmacological activities were pooled and purified further by anionic exchange column, cationic exchange column, or reverse phase (RP) HPLC (Hypersil BDS C4 or C8, 30 x 0.46 cm) column as illustrated in Fig. 1, B–H.


Figure 1
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  		FIG. 1. Fractionation of SGE. A, the lyophilized SGE sample (2.1 g, total A280 nm of 600) was dissolved in 10 ml of 0.1 M phosphate buffer solution, pH 6.0, and then was applied to a Sephadex G-75 (Superfine, Amersham Biosciences, 2.6 x 100-cm) gel filtration column equilibrated with 0.1 M phosphate buffer solution, pH 6.0. Elution was performed with the same buffer, collecting fractions of 3.0 ml. The absorbance of the eluate was monitored at 280 nm. B, peak III from Sephadex G-75 gel filtration was subjected to AKTA fast protein liquid chromatography Mono S (1-ml volume, Amersham Biosciences) cationic exchange equilibrated with 0.02 M phosphate buffer solution, pH 6.0. The elution was performed at a flow rate of 1 ml/min with the indicated NaCl gradient. C, peak IV in A was applied to a DEAE-Sephadex A-50 anionic exchange column (1.6 x 30 cm) equilibrated with 0.05 mM Tris-HCl buffer, pH 7.8. The elution was performed at a flow rate of 10 ml/h with the indicated NaCl gradient. D, peak VI in A was purified further by RP-HPLC (Hypersil BDS C8, 25 x 0.46-cm) column. The elution was performed at a flow rate of 0.7 ml/min with the indicated gradients of acetonitrile in 0.1% (v/v) trifluoroacetic acid in water; fractions PI and S in B and fractions H and PI in C were purified further by RP-HPLC (Hypersil BDS C4, 25 x 0.46-cm) column. The elution was performed at a flow rate of 0.7 ml/min with the indicated gradients of acetonitrile in 0.1% (v/v) trifluoroacetic acid in water (E–H). All the purified proteins were analyzed by reducing SDS-PAGE. AU, absorbance units; mAU, milliabsorbance units.

 
Structural Analysis—
The amino acid sequences of the N terminus and partial interior amino acid fragments recovered from the trypsin hydrolysis were determined by automated Edman degradation on an Applied Biosystems pulsed liquid-phase sequencer, model 491. The samples were placed on a MALDI plate (Applied Biosystems). Mass fingerprints were obtained using a Voyager-DETM MALDI-TOF MS instrument (Voyager DE Pro, Applied Biosystems) in positive ion and linear mode. The specific parameters were as follows. The ion acceleration voltage was 20 kV, the accumulation times of a single scan was 50, and polypeptide mass standard (Applied Biosystems) served as external standard. The accuracy of mass determinations was within 0.1%.

SDS-PAGE Analysis and Protein Concentration Determination—
SDS-PAGE was performed under reducing conditions. Protein samples were loaded onto a 12% polyacrylamide gel. Protein bands were observed after using a standard Coomassie Blue stain. The protein concentration was determined by a protein assay kit (Bio-Rad) with BSA as a standard.

SMART cDNA Synthesis and cDNA Library Construction—
Total RNA was extracted using TRIzol (Invitrogen) from 30 pairs of horsefly salivary glands of T. yao Macquart. cDNA was synthesized by SMARTTM techniques by using a SMART PCR cDNA synthesis kit (Clontech). The first strand was synthesized by using cDNA 3' SMART CDS Primer II A, 5'-AAGCAGTGGTATCAACGCAGAGTACT30N–1N-3' (N = A, C, G, or T; N–1 = A, G, or C), and SMART II An oligonucleotide, 5'-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3'. The second strand was amplified using Advantage polymerase by 5' PCR primer II A, 5'-AAGCAGTGGTATCAACGCAGAGT-3'. A directional cDNA library was constructed with a plasmid cloning kit (SuperScriptTM Plasmid System, Invitrogen) following the instructions of the manufacturer, producing a library of about 2.3 x 105 independent colonies.

Screening of cDNA—
A PCR-based method for high stringency screening of DNA libraries was used for screening and isolating the clones with some modifications. The specific primers in the sense direction as listed in supplemental Table S1 designed according to the peptide sequences determined by Edman degradation and primer II A as mentioned under ‘SMART cDNA Synthesis and cDNA Library Construction‘ in the antisense direction were used in PCRs. The DNA polymerase was Advantage polymerase from Clontech. The PCR conditions were: 2 min at 94 °C followed by 30 cycles of 10 s at 92 °C, 30 s at 50 °C, and 40 s at 72 °C. DNA sequencing was performed on an Applied Biosystems DNA sequencer, model ABI PRISM 377.

Pharmacological Testing—
Hyaluronidase assays were performed according to the method described by Charlab et al. (2). Serine protease and fibrinogenolytic activity assays were according to the method described by Zhang et al. (19). Anticoagulation and microbe killing were tested according to our previous methods (20). The Gram-positive bacterium Staphylococcus aureus (ATCC2592), Gram-negative bacterium Escherichia coli (ATCC25922), Bacillus dysenteriae, and fungus Candida albicans (ATCC2002) were obtained from Kunming Medical College. Inhibitory activities of platelet aggregation were determined according to the methods described previously (21). Platelet aggregation was monitored by light transmission in an aggregometer (Plisen, Beijing, China) with continuous stirring at 37 °C. The effects on the contraction of isolated rat femoral artery were followed according to the method reported by Takác et al. (3). Cytokines were detected using antibody sandwich ELISAs as reported in Refs. 16 and 17. All the animal experiments were approved by Kunming Institute of Zoology, Chinese Academy of Sciences. The detail description for pharmacological testing can be found in the supplemental materials.

Synthetic Peptides—
All of the peptides used for the bioactivity assays in this study were synthesized by a peptide synthesizer (433A, Applied Biosystems) at AC Scientific Inc. (Xi An, China) and analyzed by HPLC and MALDI-TOF mass spectrometry to confirm that the purity was higher than 95%. All peptides were dissolved in water.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Purification of Pharmacological Molecules from the Horsefly SGE—
As indicated in Fig. 1A, the supernatant of the horsefly salivary gland extract was divided into six peaks after Sephadex G-75 gel filtration. Peak III could inhibit platelet aggregation and hydrolyze fibrinogen and chromogenic substrates for serine proteases. Peak IV could also inhibit platelet aggregation, hydrolyze hyaluronate, and inhibit microbes. Peak VI could inhibit microbe growth and the hydrolysis activity of thrombin on chromogenic substrate S-2238 (H-D-Phe-Pip-Arg-pNA), decrease interferon-{gamma} secretion, and relax the contraction of isolated rat artery. Peaks I, II, and V had strong allergen-like activity, and we did not study them further.2

Peak III from Sephadex G-75 gel filtration was subjected to AKTA fast protein liquid chromatography Mono S (1-ml volume; Amersham Biosciences) cationic exchange as illustrated in Fig. 1B. The eluted fraction at 20.7 min indicated as PI in Fig. 1B could inhibit platelet aggregation. Fraction PI in Fig. 1B was purified further by reverse phase high performance liquid chromatography column as illustrated in Fig. 1E, and the eluted fraction at 32 min indicated as PI1 in Fig. 1E had the ability to inhibit platelet aggregation. The purified inhibitor was named tabinhibitin 1. Fraction FL at 22.9 min in Fig. 1B could hydrolyze fibrinogen, and it was found to be a homogeneous protein (named tabfiblysin) with a molecular mass of 36 kDa determined by non-reducing SDS-PAGE analysis. Fraction S at 30.8 min in Fig. 1B could hydrolyze chromogenic substrates for serine proteases S-2238 and S-4760 (N-succinyl-Ala-Ala-Ala-pNA) and was purified further by RP-HPLC column as illustrated in Fig. 1F; the eluted fraction at 23.2 min indicated as SP in Fig. 1F had the ability to hydrolyze the substrates S-2238 and S-4760. The purified serine protease was named tabserin.

Peak IV in Fig. 1A was applied to a DEAE-Sephadex A-50 anionic exchange column as illustrated in Fig. 1C. Fractions H and PI exerted hyaluronidase and platelet aggregation inhibitor activity, respectively, and were further subjected to RP-HPLC purification as illustrated in Fig. 1, G and H, respectively. The purified hyaluronidase (indicated as HD in Fig. 1G) was named hyaluronidase TY. A platelet inhibitor named tabinhibitin 1 (indicated as PI2 in Fig. 1H) and an antimicrobial protein named attactin TY3 (indicated as AT3 in Fig. 1H) were purified from the PI fraction of Fig. 1C.

Peak VI in Fig. 1A was purified further by RP-HPLC as illustrated in Fig. 1D. More than 50 fractions were eluted. Six pharmacologically active molecules were purified. They are defensin TY1 (indicated as Def in Fig. 1D), tabimmunregulins 1 and 12 (indicated as IR1 and IR2 in Fig. 1D), tabkunin (indicated as SI in Fig. 1D), cecropin TY1 (indicated as CP in Fig. 1D), and vasotab TY (indicated as VL in Fig. 1D). Defensin TY1 and cecropin TY1 are antimicrobial peptides. Tabimmunregulins 1 and 12 could increase interleukin-10 (IL-10) production and decrease interferon-{gamma} (IFN-{gamma}) secretion. Tabkunin is a serine protease inhibitor. Vasotab TY is a vasodilator peptide.

cDNA Cloning, Structure, and Function of Platelet Aggregation Inhibitor from the Horsefly SGE—
Two platelet aggregation inhibitors, tabinhibitins 1 and 2, were purified from T. yao Macquart SGE. Their amino acid sequences of the N-terminal and partial interior amino acid fragments recovered from the trypsin hydrolysis are illustrated in Fig. 2A. Based on the amino acid sequences of the N terminus, degenerate primers were designed to screen the cDNA sequences encoding tabinhibitins 1 and 2. The complete cDNA sequences encoding tabinhibitins 1 and 2 are 868 and 857 bp, respectively (The GenBankTM accession numbers are EU147252 and EU147253, respectively.). They encode two proproteins composed of 255 amino acid (aa) residues including predicted signal peptides (23 aa) and mature tabinhibitins (232 aa) (Fig. 2A) containing the sperm-coating protein (SCP) domain (Scp/TAPS family extracellular domains) found in insect antigen 5 proteins. Mature tabinhibitins 1 and 2 contain 10 half-cystines that possibly form five disulfide bridges. Tabinhibitins 1 and 2 show 20–30% identity with Aedes aegypti venom allergen containing 12 half-cystines (GenBank accession number EAT48176). There is an Arg-Gly-Asp (RGD) sequence at the N terminus of tabinhibitin 2 and an Arg-Gly-Cys-Asp sequence at the N terminus of tabinhibitin 1. Although the primary sequences of tabinhibitins had little homology with other platelet aggregation inhibitors such as variabilin (22), decorsin (23), ornatin (24), and snake disintegrins (25–27), the RGD sequences are conserved in tabinhibitins and are positioned in a loop bracketed by cysteine residues. No other known antigen 5 protein member contains such RGD domains. In most RGD-containing platelet aggregation inhibitors, RGD sequences are positioned in the middle or C terminus of the sequences, whereas the RGD sequences are positioned in the N terminus of tabinhibitin sequences. Most RGD-containing platelet aggregation inhibitors have a high percentage of cysteine residues, such as variabilin (11%), ornatin (12%), decorsin (15%), and disintegrins (16–17%) (Fig. 2B). Tabinhibitins have a much lower content of cystine (4.3%) and are much larger molecules. These are also the first members of the antigen 5 family found in salivary glands of blood-sucking arthropods to have any identified function.


Figure 2
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  		FIG. 2. A, the amino acid sequences of the platelet aggregation inhibitors of T. yao Macquart salivary glands, tabinhibitins 1 and 2, and their comparison with venom allergen from A. aegypti (GenBank accession number EAT48176). The amino acid sequences determined by Edman degradation are underlined, and the predicted signal sequences are italic. B, other aggregation inhibitors containing RGD sequences. The RGD sequences are bold. *, identical amino acid.

 
We investigated the effect of tabinhibitin 2 on platelet aggregation. As illustrated in Fig. 3B, FITC-conjugated tabinhibitin 2 bound to human formalin-fixed platelets in a saturable manner determined by flow cytometric analysis. Platelet aggregation induced by agonists such as ADP, arachidonic acid, TMVA (a snake C-type lectin-like protein from Trimeresurus mucrosquamatus venom that activates platelet via GPIb) (28, 29), stejnulxin (a snake C-type lectin-like protein from Trimeresurus stejnegeri venom that is a potent platelet agonist acting specifically via GPVI) (30), U46619 (a thromboxane A2 analog), and thrombin was inhibited by tabinhibitin 2 as illustrated in Fig. 3, C–H. Platelet aggregation can be induced by a variety of agonists through different pathways, but the final common step of these pathways is the binding of fibrinogen to its receptor, GPIIb/IIIa, on the platelet surface (22). Variabilin, ornatin, decorsin, and most disintegrins have an RGD motif, the well known receptor recognition site present on the receptor GPIIb/IIIa. They can compete with fibrinogen for binding to GPIIb/IIIa and effectively inhibit platelet aggregation induced by agonists such as thrombin, arachidonic acid, ADP, U46619, TMVA, and stejnulxin. Tabinhibitins 1 and 2 have an RGD sequence and possibly act as an antagonist of the GPIIb/IIIa fibrinogen receptor existing in membranes to inhibit platelet aggregation as other RGD-containing platelet aggregation inhibitors do. As a blood-feeding arthropod, it is effective and rational that the horsefly utilizes a GPIIb/IIIa antagonist to inhibit the final common step for platelet aggregation. Nonetheless it is not clear whether tabinhibitin acts on other sites. Further research is required to address this question. Additionally it is also interesting to investigate further the biological significance of the RGD sequence positioned in the N terminus as well as the contribution of the remainder of the molecule to platelet inhibition or additional functions.


Figure 3
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  		FIG. 3. The effect of tabinhibitin 2 on platelet aggregation. Shown are the results of FITC-BSA control (A) or FITC-tabinhibitin 2 (B) binding to platelets by flow cytometric analysis. Platelet aggregation induced by agonists such as ADP (C), arachidonic acid (D), TMVA (E), stejnulxin (F), U46619 (G), and thrombin (H) was inhibited by tabinhibitin 2 at different concentrations (220, 440, or 880 ng/ml).

 
cDNA Cloning, Structure, and Function of Immunosuppressant Peptides from the Horsefly SGE—
Two immunosuppressant peptides, tabimmunregulins 1 and 12, were purified from T. yao Macquart SGE. Their amino acid sequences are GGVSGVSDFEPIEVFGEDYDSDEADEDGKG and GGVSGVGDYKPIVVFGKSFNQFEAAEGAKG, respectively. They are similar to the putative secreted peptide from H. bimaculata (GenBank accession number BAC81345). Twelve related sequences (tabimmunregulins 1–12, GenBank accession numbers EU147264–EU147275) were recovered from the cDNA library of T. yao Macquart salivary glands as illustrated in Fig. 4. Their precursors are composed of 68–73 amino acid residues including a predicted signal peptide followed by an acidic spacer peptide and the mature tabimmunregulin peptides containing 30 or 35 amino acid residues at the C terminus. There is a dibasic cutting site (-37RK38-) for trypsin-like proteases between spacer peptide and mature peptide. Except for tabimmunregulin 11 precursor, all the tabimmunregulin precursors have highly conserved signal peptide sequences.


Figure 4
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  		FIG. 4. The amino acid sequences of tabimmunregulin 1–12. The mature peptides are boxed. The different amino acids are shaded.

 
We tested the effects of tabimmunregulins 1 and 12 on IL-10 and interferon-{gamma} secretion induced by LPS in mouse splenocytes. As illustrated in Fig. 5, LPS co-culture with tabimmunregulin 1 or 12 in mouse splenocytes could markedly increase the secretion of IL-10 (Fig. 5A), but the secretion of IFN-{gamma} (Fig. 5B) was decreased in a dose-dependent manner. Tabimmunregulin or LPS alone had only a moderate effect on the two kinds of cytokines in comparison with the control untreated mouse splenocytes. All the results suggested that tabimmunregulins in horsefly salivary glands act as an immunosuppressant and likely inhibit the host's inflammatory response by down-regulation of proinflammatory cytokines. IL-10 is a cytokine suppressor of T-cell proliferative and cytokine responses. IL-10 can inhibit the elaboration of proinflammatory cytokines, for example IFN-{gamma} (16, 17, 31). Tabimmunregulins possibly up-regulated the IL-10 production to inhibit IFN-{gamma} secretion in the current experiments.


Figure 5
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  		FIG. 5. The effect of tabimmunregulin 1 or 12 on IL-10 (A) and IFN-{gamma} (B) secretion in mouse splenocytes induced by LPS. TM1, tabimmunregulin 1; TM12, tabimmunregulin 12.

 
It has been reported that horseflies needed approximately 10 landings on a host to obtain a complete meal (8, 18). The multiple landings on a host provide an opportunity for innate and adaptive immune defenses to affect the horseflies. Not surprisingly, horseflies have developed a number of countermeasures to overcome the host immune responses. As observed in our experiments, tabimmunregulin peptides in horsefly salivary glands can suppress the proinflammatory cytokine IFN-{gamma} induced by LPS. This immunosuppression may facilitate the blood feeding and the establishment of horsefly-transmitted pathogens in the host. Some other blood-feeding arthropods such as ticks use a similar measure to overcome the host's immune responses.

cDNA Cloning, Structure, and Function of Antimicrobial Peptides from the Horsefly SGE—
As described in the first part of ‘Results and Discussion,’ three antimicrobial peptides or proteins were purified from the horsefly SGE. They are named defensin TY1, cecropin TY1, and attactin TY3. The cDNAs encoding three attactin analogs (GenBank accession numbers EU147258–EU147260), two insect defensin analogs (GenBank accession numbers EU147256 and EU147257), and a cecropin analog (GenBank accession number EU147251) were screened from the cDNA library of T. yao Macquart salivary glands as illustrated in supplemental Fig. S1. We also noticed that defensin TY1 has a theoretical pI of 5.24, which is different from most antimicrobial peptides. Most antimicrobial peptides are cationic. Defensin TY1, cecropin TY1, and attactin TY3 showed antimicrobial activities against the tested microorganisms as listed in supplemental Table S2. They are the first antimicrobial factors from horsefly salivary glands. In animals (including humans), insects, and plants, antimicrobial peptides contribute significantly to host defense against invasion by microorganisms. Horseflies have many chances to encounter microorganisms because of their special feeding behavior. The antimicrobial peptides in horsefly salivary glands can keep the blood meal sterile and inhibit microorganism growth. In terms of co-evolution, perhaps horseflies have developed multiple antimicrobial factors to protect their hosts from microorganism infection during blood feeding.

cDNA Cloning, Structure, and Function of Serine Protease Inhibitor from the Horsefly SGE—
A serine protease inhibitor named tabkunin was purified from the horsefly SGE. The full-length cDNA sequence (GenBank accession number EU147255) encoding tabkunin precursor was also cloned from the salivary gland cDNA library of T. yao Macquart. Tabkunin precursor is composed 76 amino acid residues including a signal peptide of 20 amino acid residues and mature tabkunin of 56 amino acid residues (supplemental Fig. S2A). The mature tabkunin has a predicted molecular mass of 6 kDa and contains six half-cystines and a conserved Kunitz-like domain (IYGGCGGN) like many other serine protease inhibitors (supplemental Fig. S2B) (32–39). The tabkunin primary sequence is more similar to serine protease inhibitors (AsKC1 and -3) from the sea anemone Anemonia sulcata (33) than other serine protease inhibitors. It also shares similarity with arthropod serine protease inhibitors such as tick boophilin (35), ixolaris-2, and scapularis-S (38).

Tabkunin could inhibit the hydrolytic activities of trypsin, thrombin, elastase, and chymotrypsin on chromogenic substrates (supplemental Table S3). Among the tested proteases, it had stronger inhibitory ability against thrombin than against the others. To confirm the anticoagulant activity of tabkunin, the effect of tabkunin on blood clotting was investigated using assays that measure both the intrinsic (recalcification time assay) and the extrinsic pathways (prothrombin time assay). In the recalcification time assay, no clotting was detected when 0.5 µM tabkunin was added even after 24 h. 0.5 µM tabkunin also increased the prothrombin clotting time from 16 (control) to 30 s. All the results confirmed that tabkunin is a potent anticoagulant. The presence of anticoagulant factors in horsefly SGE has been reported previously, but no sequence information is available. A thrombin inhibitor named tabnine (40), isolated from Tabanus bovines, is thought to be ~7-kDa peptide. The tabkunin reported here may be an isoform of tabanine because they have similar thrombin inhibitory activity and molecular mass.

cDNA Cloning, Structure, and Function of Vasoactive Peptide from the Horsefly SGE—
It has been mentioned that a vasoactive peptide, named vasotab TY, was purified from the horsefly SGE (Fig. 1D and supplemental Fig. S3A). Its cDNA (GenBank accession number EU147263) was cloned from the cDNA library of T. yao Macquart salivary glands. The vasotab TY precursor is composed of 76 amino acid residues including a signal peptide of 20 amino acid residues and the mature peptide of 56 amino acid residues. Vasotab TY is highly homologous with the vasotab identified from the horsefly of H. bimaculata Macquart (3). The signal peptides have only one different amino acid residue, and the mature peptides have only four different amino acid residues, suggesting that this family of peptides is highly conserved in different genera of Tabanidae horseflies.

Vasotab has been reported to inhibit vasoconstriction of isolated rat femoral artery induced by phenylephrine. As illustrated in supplemental Fig. S3B, vasotab TY had the same function as vasotab. Platelet aggregation and vasoconstriction are key hemostatic responses, particularly in small wounds. As suggested by Takác et al. (3), horsefly vasotabs likely take part in the antihemostatic responses during blood feeding.

cDNA Cloning, Structure, and Function of Serine Protease from the Horsefly SGE—
A serine protease named tabserin was purified from the SGE of T. yao Macquart (Fig. 1F). Tabserin could hydrolyze chromogenic substrates S-2238 and S-4760. Its molecular mass is ~29 kDa as determined by SDS-PAGE analysis. The cDNA sequence (GenBank accession number EU147250) encoding tabserin precursor was also cloned from the cDNA library of T. yao Macquart salivary glands. The precursor is composed of 248 amino acid residues (supplemental Fig. S4A). It shares identity of 30–38% with other arthropod serine proteases such as the serine proteases (GenBank accession numbers NP_523426, EAT42808.1, and AAD21828.1) from Drosophila melanogaster, A. aegypti, and Ctenocephalides felis, respectively.

Tasbserin could inhibit blood coagulation in a dose-dependent manner as illustrated in supplemental Fig. S4B. The addition of tabserin increased the Ca2+ clotting time of platelet-poor plasma in vitro (supplemental Fig. S4B). At a tabserin concentration of 16 µg/ml, the platelet-poor plasma clotting time was longer than 5 h compared with the control. It has been reported that crude and partially purified horsefly SGEs have both anticoagulant and serine protease activities (8). In the present study we characterized the anticoagulant serine protease tabserin from the horsefly of T. yao. It is not clear how tabserin exerts its anticoagulant function. It is possible that tabserin destroys coagulant factors to inhibit blood coagulation as some serine proteases from wasp venoms do.

Identification and Purification of Fibrinogenolytic Enzyme and Hyaluronidase from the Horsefly SGE—
A protease named tabfiblysin with an approximate molecular mass of 36 kDa and a hyaluronidase named hyaluronidase TY with an approximate molecular mass of 35 kDa were purified from the horsefly SGE as illustrated in Fig. 1, B and G, respectively. They were subjected to N-terminal amino acid sequence analysis. Unfortunately their N termini were blocked. We lacked enough purified samples to analyze their interior amino acid sequences.

By transcriptome analysis, a cDNA clone (GenBank accession number EU147254) encoding a hyaluronidase analog with 269 amino acid residues was screened from the cDNA library of T. yao Macquart salivary glands. The purified hyaluronidase TY could hydrolyze hyaluronate in a time-dependent manner as indicated in supplemental Fig. S5A. Hyaluronidases are found in many venomous snakes and arthropods (41). They can facilitate the spreading of toxic compounds by degradation of the extracellular matrix (see the review by Kreil (42)). In blood-sucking arthropods, the hyaluronidase activity was also detected in the tick Amblyomma hebraeum (43) and the black fly Simulium vittatum (44). The activity is thought to play an important role in blood meal acquisition by increasing the permeability of host tissue for other pharmacological compounds present in saliva.

Purified tabfiblysin from T. yao SGE could hydrolyze fibrinogen as illustrated in supplemental Fig. S5B. After co-culture for 1 h with 2 µg of tabfiblysin in 25 µl, 25 µg of fibrinogen was analyzed by reducing SDS-PAGE. The {alpha}-chain of fibrinogen nearly disappeared, suggesting that it was hydrolyzed by tabfiblysin. This result also suggested that tabfiblysin preferentially acted on the {alpha}-chain of fibrinogen. Previously an arthropod fibrinogenolytic factor, lonofibrase, with a molecular mass of 35 kDa was purified from the venom of Lonomia obliqua caterpillars; however, its amino acid sequence is unknown (45). Fibrinolytic and fibrinogenolytic activity was also found associated with tick salivary metalloproteases found in Ixodes scapularis (46). An enzyme with effective fibrinogenolytic activity will possibly consume fibrinogen to inhibit coagulation and facilitate blood meal acquisition. Its effect on fibrin remains to be investigated.

Metallothioneins from the Horsefly SGE—
Two metallothioneins named metallothionein TY1 with an amino acid sequence of MGCKLCENNCKCTSSKCGSVCNCDQSCSCPCKNKSSDQCCK and metallothionein TY2 with an amino acid sequence of MSCGGRHKDCQGTGKKCGPSCQCDDSCKCPCKTASKERCCEGK were identified from the DNA library of T. yao Macquart salivary glands (GenBank accession numbers EU147261 and EU147262). Metallothioneins have been reported to be expressed in mammalian salivary glands (46). Metallothioneins TY1 and TY2 are the first reported metallothioneins identified from invertebrate salivary glands. Metallothioneins are metal-binding proteins that have been regarded as intrinsic factors for protecting cells and tissues from metal toxicity and oxidants (47). Further work is needed to better understand the roles that metallothioneins play in the horsefly salivary glands.

In conclusion, the pharmacological molecular profile of the horsefly salivary glands is complex, even considering that the current study was solely concerned with the pharmacological molecular profile acting on antihemostatic, immunosuppressive, antioxidant, and anti-infection responses. In the current work, platelet aggregation inhibitors (two members), serine protease inhibitor, anticoagulant, fibrinogenolytic enzyme, and hyaluronidase were identified to comprise the molecular array for antihemostatic responses. Most of them were purified and characterized, and their cDNAs were cloned. Three families of antimicrobial peptides (six members), which act as innate antimicrobial defense, were purified and characterized. A family (12 members) of regulatory peptides of cytokines, which act as immunosuppressants, were purified and characterized. Two metallothioneins were also found in the salivary glands of the horsefly. These results increase our knowledge of the salivary gland function in the horsefly, will allow a deeper understanding of the molecular interactions occurring between horseflies and their hosts, and at the same time will lead us to the discovery of novel compounds affecting hemostasis and immunity.


   ACKNOWLEDGMENTS
 
We are grateful to Professor Jose Ribeiro for valuable comments and kind help for the manuscript preparation.


  FOOTNOTES
 
Received, October 12, 2007, and in revised form, December 17, 2007.

Published, MCP Papers in Press, December 17, 2007, DOI 10.1074/mcp.M700497-MCP200

1 The abbreviations used are: SGE, salivary gland extract; LPS, lipopolysaccharide; RP, reverse phase; pNA, p-nitroanilide; IL-10, interleukin-10; IFN-{gamma}, interferon-{gamma}; aa, amino acid(s); GP, glycoprotein; BDS, base-deactivated silica. Back

2 X. Xu, H. Yang, D. Ma, J. Wu, Y. Wang, Y. Song, X. Wang, Y. Lu, J. Yang, and R. Lai, unpublished data. Back

* This work was supported by Grant 2006BAD06B07 from the Ministry of Science and Technology of the People's Republic of China and by Grants KSCX2-YW-R-20 and KSCX2-YW-G-024 from the Chinese Academy of Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTMEBI Data Bank with accession number(s) EU147250–EU147275. Back

These authors made equal contributions to this work. Back

** To whom correspondence should be addressed: Kunming Inst. of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China. Tel.: 86-871-5196202; Fax: 86-871-5191823; E-mail: rlai{at}mail.kiz.ac.cn


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