Carbohydrate digestion in ticks and a digestive α-l -fucosidase

Journal of Insect Physiology 59 (2013) 1069–1075

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Carbohydrate digestion in ticks and a digestive a-L-fucosidase R. Moreti a,b, N.N. Perrella a, A.R. Lopes a,⇑ a b

Laboratory of Biochemistry and Biophysics Instituto Butantan, São Paulo, SP, Brazil Institute of Biomedical Sciences, Universidade de São Paulo, São Paulo, SP, Brazil

a r t i c l e

i n f o

Article history: Received 25 September 2012 Received in revised form 14 August 2013 Accepted 16 August 2013 Available online 29 August 2013 Keywords: Chitinase Glycoside hydrolases a-L-fucosidase Ticks Digestion Fucoidan

a b s t r a c t Digestive carbohydrases are present in many species of hematophagous Arthropoda, including ticks. In this work, Amblyomma cajennense (Ixodidae) midgut digestive carbohydrases were tracked with different substrates, resulting in the identification of a chitinase and an N-acetyl-b-glucosaminidase and the first description of a digestive a-L-fucosidase in ticks. a-L-fucosidases are involved in various physiological processes, and digestive a-L-fucosidases have been shown to be present in other types of organisms. Amblyomma cajennense a-L-fucosidase activity was isolated using acidic and salting-out precipitations and chromatographic steps in hydrophobic and cation-exchange columns. The specificity of the isolated activity as an a-L-fucosidase was confirmed by the hydrolysis of 4-methylumbelliferyl a-L-fucopyranoside and the natural substrate fucoidan and the inhibition by fucose and deoxyfuconojirimycin. The isolated activity of a-L-fucosidase forms oligomers with molecular mass of 140 kDa or 150 kDa as determined by gel filtration and non-reducing SDS–PAGE, respectively. This particular fucosidase has an optimum pH of 5.3, is stable even at high temperatures (stable for at least 2 h at 50 °C), has a Km of 45 lM to the substrate 4-methylumbelliferyl a-L-fucopyranoside and IC 50% of 327 lM to fucose and 42 pM to deoxyfuconojirimycin. The presence of digestive fucosidases in hematophagous Arthropoda may be related to defence mechanisms against host–parasite interactions. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Ticks (suborder Ixodida) are the most important group of vectors of pathogens in the phylum Arthropoda, comparable only to mosquitoes (family Culicidae) (Parola and Raoult, 2001). Ticks and tick-borne diseases cause severe economical losses worldwide by directly affecting animal health and by transmitting a variety of pathogens among animals (Jongejan and Uilenberg, 2004). The currently used chemical acaricides have many limitations, including the emergence of drug-resistant tick populations, environmental contamination, and the potential for chemical residues present in milk and meat. An understanding of the physiology of vital processes such as blood digestion and egg and larvae development can help in the development of new and improved control methods (Willadsen, 2006). Adult ticks of Amblyomma cajennense are commonly known as star ticks or horse ticks. They are three-host ticks (Nunes et al., 2010) of epidemiological importance that can transmit several diseases, including spotted fever, also known as ‘‘fever of the moun-

Abbreviations: MUFUC, 4-methylumbelliferyl a-L-fucopyranoside; E-64, 1-[L-N(trans-epoxysuccinyl)leucyl]amino-4-guanidinobutane; TLC, thin layer chromatography. ⇑ Corresponding author. Tel.: +55 11 2627 9746. E-mail address: [email protected] (A.R. Lopes). 0022-1910/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinsphys.2013.08.008

tains,’’ ‘‘tickfever’’, ‘‘blackfever’’ or ‘‘blue disease’’ caused by the bacteria Rickettsia rickettsii. Many studies have been performed using Amblyomma cajennense salivary glands and fat body (Batista et al., 2008; Maruyama et al., 2010), including infection studies. The digestive system of Amblyomma cajennense has not yet been studied. A series of studies have been performed on tick digestive peptidases, which are expected to be present in hematophagous digestive processes (Clara et al., 2011; Franta et al., 2011; Horn et al., 2009; Sojka et al., 2011). Although it is likely that the major digestive enzymes involved in blood digestion in animals are peptidases, carbohydrases are also present in blood-feeding animals (Souza-Neto et al., 2003, 2007; Terra and Ferreira, 2012). However, studies regarding carbohydrases in ticks are very sparse (Del Pino et al., 1999; Kopacek et al., 1999; Grunclova et al., 2003; Mohamed, 2000, 2005). L-fucose is one of the most common monosaccharides on the non-reducing end of many glycans, and fucosylation is involved in various physiological phenomena (Altmann et al., 2001; Ma et al., 2006; Pedra et al., 2010; Staudacher et al., 1999). Alpha-fucosidases (GH families 29 and 95) catalyse the removal of L-fucose from the non-reducing end when it is bound to a-1,2; a-1,3; a1,4; or a-1,6 of oligosaccharides or glycoconjugates. Digestive afucosidases have been characterised in the molluscans Chamelea gallina (Reglero and Cabezas, 1976), Pomacea canaliculata (Endo et al., 1993) and Pecten maximus (Berteau et al., 2002). The

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physiological role of molluscan digestive fucosidases is very clear. These animals feed on brown algae, which contain many sulphated fucans as components of their cell wall. Bowman (1987) demonstrated the activity of digestive a-fucosidase in the mite Pergamasus longicornis. Our group has shown the presence of digestive a-fucosidases in the hepatopancreas of the spider Nephilengys cruentata and of the scorpion Tityus serrulatus (to be published elsewhere). In this work, we identified the most active digestive carbohydrases in the gut of Amblyomma cajennense. N-acetyl-b-glucosaminidase, chitinase and a-fucosidases are the main enzymes involved in carbohydrate digestion. Digestive Amblyomma cajennense afucosidase activity was isolated and characterised. 2. Materials and methods 2.1. Animals and samples 2.1.1. Amblyomma cajennense Amblyomma cajennense (Arachnida; Ixodidae) were maintained in the laboratory in total darkness at 27 °C and 90% relative humidity in an incubation oven and were allowed to feed on the shaved skin of Oryctolagus cuniculus rabbit backs (Rechav et al., 1997). After 5 days, partially fed females were removed from the rabbits and immobilised on ice, and the tick midguts were appropriately dissected in order to isolate midgut tissue and midgut contents. The extracted samples from midgut tissue and midgut contents were transferred into microtubes and stored at 20 °C until use. The samples were homogenized in ultrafiltered cold water with the aid of a Potter–Elvehjem homogenizer and centrifuged at 16,000g for 20 min at 4 °C. 2.2. Chemicals Buffer salts, detergents, protein inhibitors, fucose, deoxyfuconojirimycin, and substrates were purchased from Sigma–Aldrich (USA). 2.3. Protein determination and glycoside hydrolase assays Protein levels were determined according to Smith et al. (1985) using ovalbumin as a standard. All enzymatic assays were performed at 30 °C. For each measurement, incubations were carried out for four or more different time periods, and the initial rates were calculated. One unit of enzyme (U) is defined as the amount of enzyme that hydrolyses the substrate to generate 1 lmol of product/minute. The glycoside hydrolases tested and their substrate and assay conditions are listed in Table 1. Routine assays of a-L-fucosidase used a solution of 25 lM 4-methylumbelliferyl

a-L-fucopyranoside (MUFUC) as a substrate. The released 4-methylumbelliferone was measured by the method of Baker and Woo (1992). Fluorescence measurements were carried out in a Gemini XPS spectrofluorimeter (Molecular Devices). Colorimetric measurements were obtained using a SpectraMax 190 spectrophotometer (Molecular Devices). 2.4. Isolation of Amblyomma cajennense a-L-fucosidase activity Amblyomma cajennense midgut with their luminal contents was initially homogenised in ultrapure water (MilliQ) with a Potter–Elvehjem homogeniser. Homogenate samples were centrifuged at 16,100g for 20 min at 4 °C. The soluble portion was then mixed (1:1) with a 3.4 M ammonium sulphate solution containing 100 lM E-64 to avoid proteolysis. This mixture was kept at 4 °C for 22 hs and then centrifuged under the same conditions listed above. Subsequently, the soluble portion was diluted in 0.1 M sodium acetate, pH 3.5, kept for one hour at 4 °C and centrifuged again. The soluble portion after acidic centrifugation was submitted to hydrophobic chromatography on a HiTrap Butyl column (GE Healthcare) in a FPLC system. The column was equilibrated in 50 mM citrate–phosphate buffer at pH 5.0 containing 1.6 M (NH4)2SO4. Proteins were eluted with a 25 mL gradient from 1.6 to 0 M (NH4)2SO4 in the same buffer at a flow rate of 1.0 mL/minute. Fractions of 1.0 mL were collected. Active fractions hydrolyzing MUFUC were pooled, desalted using a HiTrap Desalting column (GE Healthcare) and applied to a Resource S column (Amersham Biosciences) equilibrated with 50 mM citrate-phosphate buffer, pH 5.0. Elution was carried out with a 25 mL 0– 0.6 M NaCl gradient in the same buffer at a flow rate of 1.0 mL/ minute. Fractions of 1.0 mL were collected. Active fractions using MUFUC as substrate were pooled and used to enzyme characterisation. 2.5. Sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) and gel filtration Samples containing a-L-fucosidase (approximately 1 ug of protein) were combined with sample buffer containing 60 mM Tris– HCl buffer at pH 6.8, 2.5% SDS, 10% v/v glycerol, and 0.005% (w/v) bromophenol blue. The samples were loaded onto a 12% (w/v) polyacrylamide gel slab containing 0.1% SDS (Laemmli,1970). Non-reducing gels (without b-mercaptoethanol) were run at a constant voltage of 100 V at 10 °C and silver-stained for proteins (Blum et al., 1987). Non-reducing gels were also submitted to on gel activity methods. Filter paper was moistened in a MUFUC solution as described at enzyme assay item. The filter paper was then superimposed to the gel and incubated for one hour at 30 °C. Fluorescense of free methylumbelliferone was photographed on gel Documentation (MiniBis Pro-DNR Bio-Imaging Systems). Mr values

Table 1 Assay conditions and methods used in the determination of hydrolases from Amblyomma cajennense females*.

*

Enzyme

Substrate

Concentration pH (pH range)

Maltase Amylase Trehalase a-Glucosidase a-L-fucosidase chitinase N-acetyl-b-glucosaminidase a-Galactosidase

Maltose Starch Trehalose 4-MU-a-glucopyranoside 4-MU-a-L-fucopyranoside 4-MU b-D-N,N0 ,N0 0 -triacetylchitotrioside 4-Methylumbelliferyl N-acetyl-b-D-glucosaminide 4-MU-a-galactopyranoside

10 mM 1% 5 mM 0.05 mM 0.05 mM 0.05 mM 0.05 mM 0.05 mM

Group determined

Reference

7.0 Glucose Dahlqvist (1968) 7.0 Reducing groups Noelting and Bernfeld, 1948 7.0 Glucose Dahlqvist (1968) 7.0 4-Methylumbelliferone Baker and Woo (1992) 5.3 (2.5–10) 4-Methylumbelliferone Baker and Woo (1992) 7.0 4-Methylumbelliferone Baker and Woo (1992) 5.6 (2.5–10) 4-Methylumbelliferone Baker and Woo (1992) 7.0 4-Methylumbelliferone Baker and Woo, 1992

Assays were performed at 30 °C at the indicated pH values. The buffers (0.05 M) were used: citrate-phosphate (pH 2.5–5.5); MES (6.0–6.5); Tris–HCl (7.0–9.0), and glycineNaOH (pH 9.0–10). Incubations were carried out for at least four different periods of time and the initial rates calculated. One U of enzyme is defined as the amount that catalyses the cleavage of 1 lmol of substrate (or bond)/min.

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were calculated according to Shapiro et al. (1967) using a Rainbow full range marker (GE). Gel filtration chromatography using a Superdex G75 column (GE) was performed in a FPLC system (GE) to determine the molecular mass of a-L-fucosidase and chitinase. The molecular mass standards used were ribo nuclease A (13.7 kDa), soybean trypsin inhibitor (21.5 kDa), ovalbumin (45 kDa), bovine serum albumin (66 kDa), and thyroglobulin (669 kDa). 2.6. Specificity of Amblyomma cajennense a-fucosidase Beyond the assay with MUFUC, the specificity of Amblyomma cajennense a-fucosidase was tested using the natural substrate fucoidan and the competitive inhibitors: fucose and fucunojirimicyn. Fucoidan from Fucus vesiculosus (Sigma) was solubilized in citrate-phosphate 0.05 M pH 5.5 buffer containing 0.02% sodium azide and mixed to Amblyomma cajennense homogenate sample. Hydrolysis was held at 37 °C for 120 h. After that, samples: control (fucoidan at citrate phosphate buffer without enzymes at the same hydrolysis condition), experimental (fucoidan at citrate phosphate buffer with enzyme at the same hydrolysis condition described previously) and standard (fucose 5.0 mM) were boiled for 5 min and lyophilized by vacuum centrifugation. Experimental, control and standard samples were then solubilized in ultrapure water and spotted into a Alugram Sil G TLC plate (Macherey–Nagel) using a mixture of butanol/ethanol/water (50:30:20) as solvent (Genta et al., 2003). After the chromatographic separation the results were revealed with a phenol–sulfuric acid reagent (Stahl, 1969). Inhibition assays were accomplished with the mixture of fractions after separation on a Hitrap Butyl column and inhibitors. A 5.0 mM stock solution of fucose in water was diluted and combined to substrate and sample (final concentration 250 lM). Deoxyfuconojirimycin 5.0 lM was diluted up to a final concentration of 65 nM. MUFUC was used as substrate as described previously. 2.7. Kinetic and thermal inactivation studies The effect of pH variation on a-L-fucosidase activity was determined using at least 16 different pH solutions. The buffers (0.05 M) used were as follows: citrate-phosphate (pH 2.5–5.5), MES (6.0– 6.5), Tris–HCl (7.0–9.0), and glycine-NaOH (9.0–10). The effect of substrate concentration on isolated a-L-fucosidase activity was determined using at least 10 different substrate concentrations. Km and Vmax values (mean and S.E.M.) were determined by linear regression using Enzfitter (Elsevier, Biosoft) software. Half maximal inhibitory concentration (IC50%) was determined using different concentrations of fucose or deoxyfuconojirimycin. Remaining activity was measured and plotted against inhibitor concentration. Thermal inactivation of isolated a-L-fucosidase activity at 65 °C was studied by incubating samples in 100 mM citrate-sodium phosphate buffer at pH 5.0, followed by a determination of the residual activity remaining after different time periods. 3. Results 3.1. Carbohydrate digestion in Amblyomma cajennense Eight different carbohydrase activities were measured in homogenate midgut preparations from ingurgitated Amblyomma cajennense females (Tables 1 and 2). However, no activity was found for a-amylase, maltase, trehalase, a-glucosidase, or a-galactosidase. The activities of N-acetyl-b-glucosaminidase, chitinase, and a-L-fucosidase were quantified (Table 2).

Table 2 Carbohydrase activities in the midgut of female Amblyomma cajennense.a Enzyme

mU. midgut1

mU. mg protein1

N-acetyl-b-glucosaminidase chitinase a-L-fucosidaseb

1200 ± 340 310 ± 90 218 ± 15

47 ± 15 22 ± 15 10 ± 4

a Results are means and range corresponding to determinations in three different biological samples. Substrates used: 4-MU-b-N-acetyl-glucosaminidine; 4-MUchitotrioside and 4-MU- a-L-fucopyranoside. b Activity of a-L-fucosidase was 99% at luminal content.

These activities were optimal in an acidic pH range of 5.0–5.5 (Figure 1). Tick midgut contents, as described by Horn and coworkers (2009), is an acidic environment favourable to these enzymes. Activity of a-L-fucosidase were also measured in fresh isolated midgut tissue and luminal contents. We have found activity only at the luminal contents. 3.2. Isolation of Amblyomma cajennense a-L-fucosidase activity Samples of midgut and midgut contents homogenate were submitted to a combination of ammonium sulphate and pH precipitation with E-64, which allowed for a 10-fold enrichment of a-Lfucosidase, with a yield of 50%. The samples were then applied to a sequence of chromatographic columns: Hitrap Butyl and Resource S, with a final yield of 47% (Figure 2A, B and C). However, after this fractionation steps we still have a protein contamination identified as rabbit albumin by mass spectrometry, which, probably co-migrate with the monomeric form of a-L-fucosidase. Neverthless, a-L-fucosidase activity is isolated of other enzymatic activities allowing its characterisation. 3.3. Properties of Amblyomma cajennense a-L-fucosidase The isolated a-L-fucosidase activity had a molecular mass of 140 kDa (Figure 3A) or 150 kDa, as determined by gel filtration chromatography or non-reducing SDS-PAGE and activity on gel, respectively (Figure 2D and E). Differences in molecular mass due to oligomerisation have been shown to occur for a-L-fucosidases from the bacteria Thermotoga maritima (Sulzenbacher et al., 2004). Besides that, a-L-fucosidase from Amblyomma cajennense is stable even at high temperatures (stable for at least 2 h at 50 °C) (data not shown) and was able to hydrolyze fucoidan, producing fucose (Figure 3B) featuring an exo-fucosidase activity. The inhibition of partially purified samples with fucose and deoxyfuconojirimycin confirmed the specificity of this enzyme as an a-L-fucosidase (Figures 3C). The isolated digestive a-L-fucosidase from Amblyomma cajennense had a Km of 45 ± 14 lM for MUFUC as a substrate (Figure 3D) and IC50% values of 327 lM by fucose and 42 pM by deoxyfuconojirimycin (Figure 3E and F). 4. Discussion 4.1. Tick carbohydrate digestion The characterisation of carbohydrate digestion in Amblyomma cajennense midgut using distinct substrates led us to the identification of three major enzymes: N-acetyl-b-glucosaminidase, chitinase and a-L-fucosidase. Assays using isolated tissue and midgut contents indicated that the a-L-fucosidase is present in the midgut contents suggesting that carbohydrate digestion may occur at the midgut lumen distinctly of protein digestion in ticks. The carbohydrates identified in Amblyomma cajennense midgut are mainly acidic enzymes. However, pH optimum profile indicates the presence of a mixture of more than one chitinase and N-acetyl-b-glu-

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100

80

80

Relative activity, %

Relative activity, %

A 100

60 40 20 0 2

3

4

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6

7

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60 40 20 0

8

2

4

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10

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80 60 40 20 0 2

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Figure 1. Effect of pH on the activities of (A) a-L-fucosidase (B) chitinase (C) N-acetyl-b-glucosaminidase. Activities were measured using midgut homogenate as sample. Buffers: (j) citrate-phosphate; (s) MES; () Tris; (4) Glycine-NaOH.

cosaminidases. In silico analysis of Ixodes scapularis genome corroborate the data that tick presents a series of distinct carbohydrases since it is possible to identified a series of chitinases, N-acetyl-bglucosaminidases and a-L-fucosidases. Midgut ESTs related to carbohydrate digestion, specifically enzymes of the glycoside hydrolases group, have also been reported for Dermacentor variabilis (Anderson et al., 2008). A genomic search for a-amylase, trehalase, lysozyme, a-glucosidase and b-glucosidase sequences in the genome of Ixodes scapularis resulted in one sequence for a-amylase (45% identity with digestive a-amylase from Tenebrio molitor), one sequence for lysozyme (45% identity with digestive lysozyme from Musca domestica), one sequence for a-glucosidase (47% identity with digestive a-glucosidase from Acyrtosiphum pisum) and no sequences for trehalase or b-glucosidase. In conclusion, although some carbohydrases could not be identified in terms of activity, most likely because they are present at low levels or are not stable in sample conditions, they are probably expressed and used in digestion during some phase of development. Alpha-amylase, for example, has been described in developing embryos (Mohamed, 2000). For the first time, a digestive a-L-fucosidase activity has been described in the tick midgut. This enzyme was thus isolated and characterised in detail. 4.2. Chitinase in tick digestive processes Chitin is a primary component of Arthropoda cuticle and trachea and is also present in fungus cell walls (Kurtti and Keyhani,

2008). The digestion of chitin depends on two enzymes: chitinase (EC 3.2.1.14), which initiates chitin digestion, and N-acetyl-b-glucosaminidase (EC 3.2.1.30), which catalyses the hydrolysis of chitinase products. Both enzymes were found in Amblyomma cajennense midgut with high activities. Many chitinases have already been characterised for Insecta and Crustacea, and these may generally be involved in the ecdysis process and on digestion. In general, chitinases involved in moulting have high molecular masses (approximately 80 kDa) (Royer et al., 2002), while digestive chitinases have low molecular masses (approximately 40–50 kDa) (Genta et al., 2006; Girard and Jouanin, 1999). Hovewer, mosquitos digestive chitinases also have high molecular masses (Shen and Jacobs-Lorena, 1997). These authors proposed that the differences observed between chitinases involved in moulting and those involved in digestion, may be an adaptation used to protect the peritrophic membrane. The chitinase of Amblyomma cajennense presented a high molecular mass (approximately 140 kDa, data not shown) determined by gel filtration. Thus, it is not possible to make any inference about this chitinase, based on its molecular mass. The presence of a peritrophic membrane has been described in only a few tick species: Ixodes scapularis (Grigor’eva and Amosova, 2004; Rudzinska et al., 1982), Ixodes ricinus (Zhu et al., 1991), Ornithodoros moubata (Grandjean, 1983) and Haemaphysalis longicornis (Matsuo et al., 2003). However, the function of this structure is still not clear. Moreover, the presence of chitinase could be related to the defence against fungus infections in the gut (Kurtti and Keyhani, 2008).

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C

Amblyomma cajennense midgut homogenate

Ammonium sulfate 50% precipitaon

Acidic precipitaon pH 3.5

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150 76 [(NH4)2 SO4] %, protein %

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52 38 24

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Figure 2. Isolation of the alpha-L-fucosidase activity from Amblyomma cajennense. (A) Schematic presentation of the isolation steps. (B) Chromatography of the supernatant from the ammonium sulfate fractionation in a butyl column equilibrated in 50 mM citrate-phosphate buffer. The samples were eluted using a gradient of 1.6–0 M ammonium sulfate (d) in the same buffer, (j,s) represent respectively fractions activity on MUFUC and relative protein profile) (C) Chromatography of the active fractions using MUFUC as substrate on a Resource S column equilibrated with 50 mM citrate-phosphate buffer pH 5.0. The samples were eluted using a gradient of 0–0.6 M NaCl (d) in the same buffer, (j,s) represent respectively fractions activity on MUFUC and relative protein profile). (D) Silver stained non-reducing SDS-PAGE of the samples exhibiting activity on MUFUC after ammonium sulfate fractionation. (E) On gel activity of ammonium sulfate fractionation after non-reducing SDS-PAGE.

4.3. Digestion of fucose In the present work, we isolated the activity of a digestive a-Lfucosidase from Amblyomma cajennense and characterised it. Digestive a-L-fucosidases are well known in molluscs, where they are involved in the degradation of fucoidan, a sulfated carbohydrate constituent of the cell wall of brown algae (Xue et al., 2012). Molluscan digestive fucosidases have molecular masses of approximately 60 kDa and are generally active in acidic pHs. In this group of animals, the importance of a digestive fucosidase is clear. However, the presence of digestive fucosidases has been described in other organisms, such as the mite Pergamasus longicornis (Bowman, 1987), the Diptera Lutzomia longipalpis (Gontijo et al., 1998), Hermetia illucens (Kim et al., 2011) and Culicoides sonorensis (Campbell et al., 2005), the Polychaeta Riftia pachyptila (Boetius and Felbeck, 1995) and the shrimp Penaeus monodon (Chuang et al., 1991). Our group has also observed digestive a-L-fucosidases in the digestive organs of spiders and scorpions (data to be published elsewhere), in addition to the tick digestive a-L-fucosidase described in the present work. Digestive Amblyomma cajennense a-L-fucosidase has a molecular mass of 140 kDa or 150 kDa, as determined by gel filtration or non-reducing SDS-PAGE, respectively, indicating an oligomerisation process. Such oligomerisation has been observed for other GH-29 family enzymes, such as the a-L-fucosidase

from Thermotoga maritima (Sulzenbacher et al., 2004) and that from Penaeus monodon, which has a native molecular mass of 233 kDa and a molecular mass as determined by SDS-PAGE of 63 kDa (Chuang et al., 1991). The isolated activity of a-L-fucosidase has a good affinity toward MUFUC, as indicated by Km values of 45 ± 14 lM, similar to other previously described fucosidases, such as that from Penaeus monodon (Km = 22 lM) (Chuang et al., 1991), Drosophila ananassae a-L-fucosidase (Km = 125 lM) (Intra et al., 2006) and human hepatic fucosidase (Km = 220 lM) (Alhadeff et al., 1975). IC 50% value for Amblyomma cajennense a-L-fucosidase using deoxyfuconojirimycin as inhibitor is 42 pM which is comparable with Ki values of 0.1 nM from human a-L-fucosidase (Winchester et al., 1990). Besides that, the a-L-fucosidase from Amblyomma cajennense only cleaves, as human and Thermotoga maritima fucosidases, the a-L-fucosyl linkages at the non-reducing termini of the fucoidan. Apparently, there is no activity of fucoidanases in ticks, since fucose was the only hydrolysis product observed in TLC experiments. However, a question still remains: what is the physiological role of secreted digestive a-L-fucosidase in Arthropoda? One possibility is that it removes fucose from carbohydrates, glycoproteins and glycolipids, thus playing a role in the digestion of these compounds. Another possibility is that it is a defensive agent against parasites. Fungus and bacteria contain fucosilated compounds in-

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A

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80 120 160 [fucose], µM

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Figure 3. Properties of the isolated a-L-fucosidase activity from Amblyomma cajennense. (A) Gel filtration of the isolated a-L-fucosidase on a Superdex G75 column. Retention volumes of protein standards: SBTI (13 mL), egg albumin (11.5 mL); bovine serum albumin (10.5 mL) are indicated by arrows. MUFUC was used as substrate. (B) Thin-layer chromatography of the products of fucoidan hydrolysis by Amblyomma cajennense a-L-fucosidase (1: control, 2: product of fucoidan hydrolysis by Amblyomma cajennense a-Lfucosidase). (C) Inhibition profile by 250 lM fucose (d) and 65 nM deoxyfuconojirimycin (}) of active fraction after hydrophobic chromatography on MUFUC (j). (D) Effect of MUFUC concentration on the activity of a-L-fucosidase. Lineweaver-Burk plot of the MUFUC activity. Inset: Michaelis-Menten plot. Inhibition of a-L-fucosidase activity by fucose (E) or by deoxyfuconojirimycin (F). IC 50% values were 327 lM and 42 pM, respectively.

volved in parasite-host interactions (Chessa et al., 2009; Järvinen et al., 2001; Ruiz-Palacios et al., 2003). Pedra and co-workers used Anaplasma phagocytophilum to illustrate the role of fucose in Anaplasma-tick interactions. The authors demonstrated that Anaplasma phagocutophilum modulates the expression of three a1,3fucosyltransferases and uses a-1,3-fucosylation to colonise ticks. The removal of fucose by digestive a-L-fucosidase could decrease these interactions. Transcriptomic and proteomic analysis is now being performed in order to obtain the sequence of this enzyme which will allow its heterologous expression. Complete sequencing and expression will permit specificity studies and, combined to RNAi experiments, the understanding of the physiological role of fucosidases in ticks.

Acknowledgments We are very indebted to Dr. Ana Marisa Chudzinski Tavassi, Dr. Darci Battesti and Dr. Andrea Fogaça, who kindly provided us with the fed Amblyomma cajennense females. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo. Rodrigo Moreti is a graduate fellow of FAPESP, Natalia Nappi Perrella is a graduate fellow of FUNDAP and Adriana R. Lopes is a scientific researcher from Instituto Butantan. References Alhadeff, J.A., Miller, A.L., Wenaas, H., Vedvick, T., O‘Brien, J.S., 1975. Human liver aL-Fucosidase. J. Biol. Chem. 250, 7106–7113.

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