Detection, purification by immunoaffinity chromatography and properties of β-1,3-glucan-specific lectins from the sera of several insect species

Insect Biochemistry and Molecular Biology 28 (1998) 721–731

Detection, purification by immunoaffinity chromatography and properties of ␤-1,3-glucan-specific lectins from the sera of several insect species Changlin Chen, Andrew F. Rowley, Norman A. Ratcliffe

*

Biomedical and Physiological Research Group, School of Biological Sciences, University of Wales, Swansea, Singleton Park, Swansea SA2 8PP, Wales, UK Received 10 June 1997; accepted 2 July 1998

Abstract A lectin specific for ␤-1,3-glucans has previously been purified from the cockroach, Blaberus discoidalis. Using polyclonal antiserum against the lectin, similar molecules with agglutinating activity were detected in the sera of several cockroach species, namely, B. craniifer, Leucophaea maderae, Gromphadorhina portentosa, and Periplaneta americana, by various methods including immunodiffusion, immunoelectrophoresis and Western blotting. Immunoaffinity chromatography using purified antibody against the B. discoidalis ␤-1,3-glucan-specific lectin immobilized on CNBr-activated Sepharose 4B matrix was successfully employed to purify the equivalent molecules from sera of these cockroaches. The purified lectins have molecular masses of ca. 520 kDa by gel filtration and subunit mass estimates of 80–82 kDa by SDS-PAGE. Moreover, they have a similar overall structure to B. discoidalis lectin when examined by electron microscopy. The purified lectin enhanced the activation of prophenoloxidase by laminarin. Localization of the ␤-1,3-glucan-specific lectin using antibody against the B. discoidalis lectin showed that the molecule was associated with the haemocyte surface as well as in the cytoplasm of these cells of B. craniifer, L. maderae and P. americana.  1998 Elsevier Science Ltd. All rights reserved. Keywords: Insect lectins; Haemocytes; Cockroaches; Immunity

1. Introduction

␤-1,3-glucans, such as laminarin, have been found to induce the activation of different defence processes in both plants and animals. In plants, these carbohydrates induce the synthesis of phytoalexins (Schmidt and Ebel, 1987), whereas in vertebrates, they activate the alternative complement pathway (Reid and Porter, 1981) and the killing of bacteria, parasites and tumour cells by macrophages and monocytes (DiLuzio et al., 1976; Bo¨gwald et al., 1982). Studies with invertebrates have shown

Abbreviations: cac, cacodylate; CRP, C-reactive protein; HLS, haemocyte lysate supernatant; L-DOPA, L-3,4-dihydroxyphenylalanine; proPO, prophenoloxidase; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; TBS/Ca2 + , Tris-buffered saline plus calcium (0.05 M Tris/HCl, 0.077 M NaCl, 0.01 M CaCl2, 0.02% sodium azide, pH 7.4) * Corresponding author. Tel.: + 44-1792-295454; Fax: + 44-1792295447; E-mail: [email protected] 0965-1748/98/$19.00  1998 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 9 8 ) 0 0 0 6 9 - 1

that these molecules activate blood coagulation in the horseshoe crab, Limulus polyphemus (Morita et al., 1981) and the prophenoloxidase (proPO) system in both the crayfish, Pacifastacus leniusculus (Duvic and So¨derha¨ll, 1990) and the insects, Bombyx mori, Blaberus craniifer and Blaberus discoidalis (Ashida et al., 1983; Leonard et al., 1985; So¨derha¨ll et al., 1988; Chen et al., 1995). It has been found that ␤-1,3-glucans stimulate effector cells, such as vertebrate macrophages and monocytes and also invertebrate haemocytes, to display the various immune-related reactions either via their interaction with receptors on the cell surfaces or recognition molecules associated with the membranes (Ochiai and Ashida, 1988; Chen et al., 1995). Recently, receptors for ␤-1,3-glucans in both vertebrate and invertebrate blood cells have been reported. In arthropods, ␤-1,3-glucan binding/recognition proteins have been isolated from the crayfish, P. leniusculus (Duvic and So¨derha¨ll, 1990), and the insects, B. mori and B. craniifer (So¨derha¨ll et al., 1988; Ochiai and Ash-

722

C. Chen et al. / Insect Biochemistry and Molecular Biology 28 (1998) 721–731

ida, 1988). These proteins, although differing in their biochemical composition, show a similar biological role in enhancing laminarin activation of the proPO system. Previously, a ␤-1,3-glucan-specific lectin was purified to homogeneity from the haemolymph of B.discoidalis and was also identified as an enhancer of laminarin activation of proPO present in blood cells of this species (Chen et al., 1995, Chen et al., 1998). This ␤-1,3-glucanspecific lectin, however, differed from those ␤-1,3-glucan binding proteins reported from B. mori and B. craniifer (Ochiai and Ashida, 1988; So¨derha¨ll et al., 1988) in that it was a bifunctional lectin possessing both agglutinating activity and also enhanced the activation of proPO in the presence of laminarin. More interestingly, it has been found that the B. discoidalis ␤-1,3-glucan-specific lectin has certain similarity at its N-terminal amino acid sequence to the invertebrate C-reactive protein (CRP) from the horseshoe crab, L. polyphemus (Chen et al., 1998), an innate immune component believed functionally homologous to the vertebrate CRP. Thus, these lectins may function similarly in different animal defence mechanisms. In this paper, the presence of ␤-1,3-glucan-specific lectins in other insects, especially phylogeneticallyrelated cockroach species, was investigated using various immunological methods. The lectin-like molecules in these other cockroaches were also purified using an immobilized polyclonal antibody against the B. discoidalis lectin. The significance of these lectins in insect immune reactions is also discussed. 2. Materials and methods 2.1. Insects Cockroaches, B. discoidalis, B. craniifer, G. portentosa, P. americana and L. maderae were maintained in separate stocks at 30 ± 3°C and supplied with dog biscuits and water ad libitum. Adult females and males of all cockroach species were used throughout these experiments. Other insects used to determine the presence of the lectin included the stick insect, Clitumnus extradentatus, the locust, Locusta migratoria, the Chinese silk moth, Antheraea pernyi and the wax moth, Galleria mellonella. Adult C. extradentatus and L. migratoria were reared at 25°C and fed, ad libitum, on bramble leaves and wheat seedlings, respectively. A. pernyi larva were raised from eggs on oak leaves at room temperature (RT) and fourth instar larvae used in experiments, while G. mellonella were fed on honeycomb in glass containers at 25°C and final instar larvae used. 2.2. Bleeding and preparation of insect sera Sera from the cockroaches, B. discoidalis, B. craniifer, G. portentosa, P. americana and L. maderae were col-

lected from the arthrodial membrane of a posterior limb, as described previously (Chen et al., 1993). C. extradentatus and L. migratoria were anaesthetized at − 20°C for 10–20 min, as for cockroaches, but bled by piercing the abdominal arthrodial membrane. A. pernyi and G. mellonella larvae were first chilled at − 20°C for ca. 10 min, then bled by piercing the abdominal body wall and sera prepared, as described above. 2.3. Assay of agglutinating activity Sera were tested for agglutinating activity using baker’s yeast as indicators. These assays were carried out as described previously (Chen et al., 1998). Briefly, 25 ␮l of serum were first serially diluted two-fold in TBS/Ca2 + buffer, pH 7.4 in the wells of V-bottomed microtitre plates, then an equal volume of yeast solution (1% made up in TBS/Ca2 + pH 7.4 containing 2% BSA) was added and incubated at RT for 1 to 2 h. Titres were recorded as the reciprocal of the highest dilution showing agglutination judged microscopically. Controls consisted of TBS/Ca2 + buffer, pH 7.4, omitting serum. 2.4. Assay of lectin enhancement of prophenoxidase activation by laminarin Twenty five microlitres of lectin samples were serially diluted two-fold in TBS/Ca2 + pH 7.4 buffer in flat-bottomed microtitre plates (Nunc Roskilde, Denmark). To each well, 25 ␮l of laminarin (1 mg/ml in TBS/Ca2 + pH 7.4) and 25 ␮l of haemocyte lysate supernatant (HLS, 0.5 mg/ml in cac buffer pH 7.4, prepared as in Chen et al., 1998) were added, mixed, and incubated for 1 h at RT. Finally, 25 ␮l of L-␤-3,4-dihydroxyphenylalanine (L-DOPA, 3 mg/ml in distilled water) were added and incubated for 3 h at RT. The absorbance at 492 nm was measured at 30 min intervals using an ELISA plate reader. Controls were made without either test lectin, laminarin, or HLS, replacing them with TBS/Ca2 + pH 7.4 buffer. 2.5. Protein determination Protein concentrations in TBS/Ca2 + buffer, pH 7.4 or in cac buffer at pH 7.4 were determined using the method of Bradford (Bradford, 1976) with BSA as a standard. 2.6. Purification of the ␤-1,3-glucan-specific lectin and antibody preparation The ␤-1,3-glucan-specific lectin from B. discoidalis was purified by gel filtration on a Bio-gel P300 column and affinity chromatography on a blue Sepharose CL6B column, while polyclonal antiserum to this lectin was

C. Chen et al. / Insect Biochemistry and Molecular Biology 28 (1998) 721–731

raised in a rabbit by intramuscular injection of 480 ␮g of the purified lectin (Chen et al., 1998). 2.7. Electrophoresis of lectins and antiserum This was carried out by the method of Laemmli (1970). Purified lectins or anti-␤-1,3-glucan-specific lectin antibody (1 ␮g each) were run under reducing conditions in 10% polyacrylamide gels in the presence of 1% SDS and 2% ␤-mercaptoethanol or under non-reducing conditions in the presence of 1% SDS. Native gel electrophoresis was performed in accordance with the method of Maurer (1971) in 5% gels using the Bio-Rad mini gel system. The gels were run at a constant voltage of 200 V for ca. 45 min and stained either with silver nitrate or Coomassie Blue R 250. 2.8. Detection of ␤-1,3-glucan-specific lectin in insects by immunoassay The reactivity of the antiserum against the B. discoidalis ␤-1,3-glucan-specific lectin with sera of various cockroach species was also examined by Ouchterlony gel diffusion, immunoelectrophoresis, immunofluorescence of haemocytes and Western blot analysis. 2.8.1. Ouchterlony double gel diffusion Two studies were carried out with gel diffusion tests. The first was designed to study the influence of varying the concentration of the antibody to the B.discoidalis ß1,3-glucan-specific lectin against the different cockroach sera. The second was to study the cross-reactivity of the antigens from other cockroach species with the antibody against the ␤-1,3-glucan-binding lectin from B. discoidalis. These tests followed the method of Carvey et al. (1977). 2.8.2. Immunoelectrophoresis Immunoelectrophoresis was used to detect and identify individual components from insect sera which reacted with antiserum to the B. discoidalis ␤-1,3-glucan-binding lectin. This was carried out with the method previously described (Carvey et al., 1977). Ten microlitres of rabbit antiserum against the purified B.discoidalis ␤-1,3-glucan-specific lectin or antigens (sera) from the cockroaches, B. craniifer, G. portentosa, P. americana, L. maderae, as well as B. discoidalis, were loaded into wells in 1% agarose M coated slides and electrophoresed for 45 min at 45 V. After termination of the electrophoretic separation, a trough was cut in each slide into which 70 ␮l of serum from various insect species or antiserum solutions were placed. The slides were placed on a level surface in a humid chamber at 37°C and the pattern of precipitation lines left to develop for 24 h.

723

2.8.3. Detection of the ␤-1,3-glucan-specific lectin in the haemocytes of cockroaches by immunofluorescence Detection of the ␤-1,3-glucan-specific lectin in haemocytes of various cockroach species was as modified from Chen et al. (1993). Monolayers of haemocytes from various cockroach species were set up and fixed with 1% formaldehyde using the method described by Richards et al. (1989). The fixed monolayers were incubated with blocking solution (1% BSA + 1% horse serum in PBS containing 100 mM glycine and 100 mM Tween, pH 7.4 to mask non-specific binding sites) for 1 h at RT and, subsequently, with a 1:500 dilution of rabbit antiserum against the purified B. discoidalis ␤-1,3-glucan-specific lectin in blocking solution for 2 h at RT. The slides were then washed three times with TBS/Ca2 + pH 7.4 (5 min each) prior to being overlaid with a 1:2000 dilution of fluorescein isothiocyanate isomer (FITC)-conjugated goat anti-rabbit IgG antibody for a further 1 h at RT. Cells were examined using a UV microscope after washing away the excess second antibody with TBS/Ca2 + pH 7.4. Control slides were prepared with pre-immunized rabbit serum instead of the first antibody. 2.8.4. Western blot analysis Western blots were used to analyse the cross-reaction between antibody against the purified ␤-1,3-glucan-specific lectin from B. discoidalis and serum proteins from various insect species. Sera (20–30 ␮g protein) from all insect species and 1 ␮g of purified lectin from B. discoidalis were run on SDS-PAGE (Chen et al., 1993). Proteins were electroblotted onto 0.45 ␮m Hybond CSuper nitrocellulose membranes in a transfer buffer containing 25 mM Tris, 192 mM glycine and 20% methanol (Towbin et al., 1979) for 90 min, using a Bio-Rad Transblot system. Any bands equivalent to the B. discoidalis ␤-1,3-glucan-specific lectin were visualized using the specific rabbit, anti-B. discoidalis lectin polyclonal antibody, and peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Sigma) developed with 4-chloro-1naphthol/H2O2 (Hawkes et al., 1982). 2.9. Purification of ␤-1,3-glucan-specific lectin from different cockroach species by immunoaffinity chromatography 2.9.1. Antibody purification and preparation of ␤-1,3glucan-specific lectin affinity column One millilitre of protein A-Sepharose 4B (Sigma) was packed in a 10 ml mini-column. The matrix was washed, sequentially, first with 1 ml 2 M urea, then 10 ml 1 M NaCl and finally with 10 ml 100 mM glycine (pH 2.5), and equilibrated with 100 mM Tris (pH 8.0). Rabbit antiserum against the B. discoidalis ␤-1,3-glucan-specific lectin was adjusted to pH 8.0 by adding 1/10 volume of 1 M Tris (pH 8.0) and the diluted antiserum solution passed through the protein A column at a flow rate of

724

C. Chen et al. / Insect Biochemistry and Molecular Biology 28 (1998) 721–731

0.2–0.5 ml/min. The solution was recycled on the same column three times. The column was then washed with 20 ml of 100 mM Tris (pH 8.0) to return the absorbance to zero and 20 ml of 10 mM Tris (pH 8.0) to decrease the buffering capacity. Finally, the column was eluted stepwise with 500 ␮l per sample of 100 mM glycine (pH 3.0). The eluates (ca. 0.5 ml) were collected in tubes containing 50 ␮l of 1 M Tris (pH 8.0) and mixed gently to bring the pH back to neutral. The IgG-containing fractions were identified by absorbance at 280 nm and pooled. The purity of the purified IgG was checked by SDS-PAGE and gel filtration on an HPLC column (Chen et al., 1993). For the affinity column, approximately 10 mg of purified IgG in 4.5 ml of Tris buffer was extensively dialysed against 200 ml of binding buffer (0.5 M sodium phosphate, pH 7.5) with 3 changes over 24 h at 4°C. The antibody solution was adjusted in sodium phosphate to 2 mg/ml concentration in 0.5 M sodium phosphate by measuring the absorbance at 280 nm. CNBr-activated Sepharose 6B (Sigma, 0.4 g) was prepared, as described by the manufacturer. The gel was filtered free from water, 5 ml of the antibody solution immediately added and the suspension gently mixed at RT overnight to immobilize the antibody onto the gel beads. After incubation, the beads were sequentially washed twice with 0.5 M sodium phosphate (pH 7.5), once with 1 M sodium chloride, once with 0.05 M sodium phosphate (pH 7.5), and then resuspended in 10 ml of the blocking solution (100 mM ethanolamine, pH 7.5). The suspension was incubated at RT for 4 h with gentle mixing. The eluate from the overnight binding was saved and the protein concentration determined to assess the degree of binding. Finally, the beads were washed twice with 20 ml of 0.1 M sodium phosphate (pH 6.8) and packed in a 10 ml minicolumn equilibrated with the same buffer. 2.9.2. Immunoaffinity chromatography Sera from B. discoidalis, B. craniifer, P. americana and G. portentosa (3 ml each) were first adjusted to pH 6.8 with 300 ␮l of 1 M sodium phosphate (pH 6.8) and diluted to 10 ml with 0.1 M sodium phosphate buffer (pH 6.8) before loading onto the pre-equilibrated antibody-Sepharose 6B column. The column was washed with 0.1 M sodium phosphate (pH 6.8), and then eluted, first, with 5 ml of 0.2 M galactose in 0.1 M sodium phosphate buffer (pH 7.5) and, second, with 3 ml of 3.5 M MgCl2 in the same buffer. Lectin activity was monitored by yeast agglutination after extensive dialysis of the eluates against Tris/Ca2 + , pH 7.4, buffer. The column was regenerated with 1 ml 2 M urea, then 10 ml 1 M NaCl, and, finally, 5 ml 100 mM glycine (pH 3.0) before re-equilibration with sodium phosphate buffer (pH 6.8). Electrophoresis and gel filtration were used to check the homogeneity of the purified antibody and for any

␤-1,3-glucan-specific lectins purified from the different cockroach species using immunoaffinity chromatography. These were carried out by both native polyacrylamide gels and SDS-PAGE, and by gel filtration on a Zorbax Bio-series GF-250 column (Chen et al., 1993). 2.10. Electron microscopy of purified lectins Purified samples were prepared for electron microscopy by the one-step method of negative staining with 1% uranyl acetate (Hayat, 1979). The purified lectin samples (80 ␮g/ml) were dialysed extensively against 0.1 M ammonium acetate buffer at pH 6.0, and mixed with an equal volume of 1% uranyl acetate for 90 sec, and then spread on carbon-coated grids. Electron micrographs were obtained with a JEOL-1200ex microscope operated at 80 kV.

3. Results 3.1. Assay of agglutinating activity As seen in Table 1, sera from all the cockroach species, B. discoidalis, B. craniifer, G. portentosa, L. maderae and P. americana, as well as from the stick insect, C. extradentatus, and the locust, L. migratoria, were found to agglutinate baker’s yeast. However, sera from the lepidopterans, G. mellonella and A. pernyi, showed no visible agglutination of yeast cells. 3.2. Immunological detection of lectin activity The interaction of the B. discoidalis ß-1,3-glucanbinding lectin antibody against the different cockroach species sera was examined by Ouchterlony gel diffusion, immunoelectrophoresis, immunofluorescence and Western blotting.

Table 1 Agglutinating activity of various insect sera against baker’s yeast Insects G. portentosa B. discoidalis B. craniifer L. maderae P. americana C. extradentatus L. migratoria G. mellonella A. pernyi

Agglutinating activitya 64 64 16 4 2 16 2 0 0

a Agglutinating activity is expressed as the reciprocal of the titre against yeast cells. Representative data from one experiment repeated three times.

C. Chen et al. / Insect Biochemistry and Molecular Biology 28 (1998) 721–731

725

3.2.1. Ouchterlony double gel diffusion In the first study with various cockroach sera in the central wells and dilutions of antiserum to the B. discoidalis ␤-1,3-glucan-binding lectin in the outer wells, high specificity was observed between the antibody and serum antigens from B. discoidalis, B. craniifer, P. americana, and L. maderae with only a single precipitation line observed (Fig. 1A). In the case of G. portentosa, however, only a faint precipitation line was observed. With reduction in antibody concentrations, the precipitation bands disappeared especially in the case of L. maderae and G. portentosa. In the second study, a 1:8 dilution of antiserum in the central wells and different cockroach sera in the outer wells were used. A complete fused precipitation line was observed between the antibody and sera from both the original “host” insect (B. discoidalis) and other cockroaches, B. craniifer, L. maderae, and G. portentosa (Fig. 1B). In the case of P. americana serum, a small ‘spur’ was present suggesting only partial identity of lectin with B. craniifer (Fig. 1B). There was no reaction between pre-immunized rabbit serum with the sera from the different insect species (Fig. 1A,B). 3.2.2. Immunoelectrophoresis Immunoelectrophoresis detected the components of the sera from the different insect species which reacted with the antibody against the B.discoidalis ß-1,3-glucanbinding lectin. Results of immunoelectrophoresis analysis confirmed the cross-reactivity with high specificity between antibody raised to the ␤-1,3-glucan-binding lectin of B. discoidalis and antigens from other cockroach sera (B. craniifer, P. americana, L. maderae, and P. portentosa) with single precipitation lines observed by antigen electrophoresis then with diffusion of antibody (Fig. 2). The intensity of the reaction in the case of G. portentosa was reduced due to precipitation of serum components in the agarose. There was no reaction between serum from the pre-immunized rabbit with the serum from B. discoidalis (Fig. 2). 3.2.3. Detection of the ␤-1,3-glucan-specific lectin in cockroach haemocytes by immunofluorescence As shown in Fig. 3, molecules which cross-reacted with antibody against the ␤-1,3-glucan-specific lectin from B. discoidalis were detected on the cell surfaces and in the cytoplasm of all haemocytes (both plasmatocyte and granular cell) of three of the cockroach species, namely, B. craniifer, L. maderae, and P. americana. These results are similar to those found with the haemocytes of B. discoidalis (Chen et al., 1998). 3.2.4. Western blot analysis Fig. 4 shows by Western blot analysis, using antibody against the B. discoidalis ␤-1,3-glucan-specific lectin, that the antibody cross-reacted with sera from other

Fig. 1. Immunodiffusion analysis for the cross-reaction of serum lectins from 4 cockroach species with antibody against ␤-1,3-glucan-specific lectin from B. discoidalis. (A) Centre wells, sera from different cockroaches; outer wells, antibody dilutions clockwise from 1 (top) to 1:32. (a) B. discoidalis; (b) B. craniifer; (c) P. americana; (d) L. maderae; (e) G. portentosa; and (f) B. discoidalis tested with pre-immune rabbit serum as a control. (B) Centre wells, anti-␤-1,3-glucan-specific lectin antiserum dilution (1:8); outer wells, (a and c), whole undiluted sera from different cockroaches; (b and d), Controls with pre-immunized rabbit serum. 1, B. discoidalis; 2, B. craniifer; 3, P. americana; 4, G. portentosa; and 5, L. maderae.

726

C. Chen et al. / Insect Biochemistry and Molecular Biology 28 (1998) 721–731

etically unrelated insects, L. migratoria, C. extradentatus, A. pernyi and G. mellonella showed no cross-reaction with the antibody against the B. discoidalis lectin (lanes c, f–h in Fig. 4). 3.3. Immunoaffinity chromatography of lectin from insect serum 3.3.1. Purification of antibody The antibody against the B. discoidalis ␤-1,3-glucanspecific lectin was purified through a protein ASepharose 4B affinity column. This one-step purification using 1.5 ml of antiserum resulted in ca. 10 mg of pure antibody. The antibody purity was checked by SDSPAGE which showed one strong band (the heavy chain) and one weak band (the light chain) under the reducing conditions, while with HPLC gel filtration, a symmetrical peak with a retention time of ca. 9 min was monitored confirming the purity of the IgG (results not shown).

Fig. 2. Immunoelectrophoretic analysis of the cross-reaction of serum lectins from four cockroach species with antibody against the ␤-1,3glucan-specific lectin from B. discoidalis. Serum samples from different cockroach species were electrophoresed for 45 min at 45 V, and antibody dilutions (1:8) were placed in the troughs. (a) B. discoidalis; (b) B. craniifer; (c) G. portentosa; (d) L. maderae; (e) P. americana; (f) Control, with pre-immunized rabbit serum tested with B. discoidalis serum.

cockroach species. Sera from B. craniifer, P. americana and G.portentosa were found to have high specific reactivity with the antibody binding as a single band with the same Rf value as the immune reactivity to the purified B.discoidalis ␤-1,3-glucan-specific lectin. Less specificity of the antibody with antigens from L. maderae serum was found, although a major band identical in position to that of the purified lectin could be seen. Multiple bands with L.maderae may have resulted from incomplete reduction of the very viscous serum sample from this species. Other antigens (sera) from phylogen-

3.3.2. Immunoaffinity chromatography The purified antibody against B. discoidalis lectin was successfully immobilized on the CNBr-activated Sepharose beads with immobilization of 10 mg protein on 1.2 ml swollen gel beads with coupling efficiency as high as 97%. Immunoaffinity purification of the ␤-1,3glucan-specific lectins using 3 ml of each serum sample from B. discoidalis, B. craniifer, P. americana, and G. portentosa resulted in pure ␤-1,3-glucan-specific lectin protein with different amounts and recovery of activity (Fig. 5, Table 2). SDS-PAGE showed that the proteins purified from the cockroaches, B. discoidalis, B. craniifer and G.portentosa consisted mainly of only one band of Mr 80–82 kDa under non-reducing and reducing conditions, respectively, indicating high homogeneity (Fig. 6). There was, however, a small contamination band with G. portentosa (Fig. 6B,C). The purity of the purified lectins was also examined by native electrophoresis in which single bands were observed for all lectin preparations (Fig. 6), and by HPLC on a Bio-series GF-250 column in which a peak of retention time 7.5 min was detected which co-eluted with the ␤-1,3-glucan-specific lectin of Mr 520 kDa originally purified from B. discoidalis (results not shown). The purified ␤-1,3-glucanspecific lectins retained the biological activity of both agglutination and activation of proPO in all cockroach species (see below in Fig. 8). 3.4. Electron microscopy Purified ␤-1,3-glucan-specific lectins from all the cockroach species were observed as large aggregates in the ammonium acetate buffer at pH 6.0 (Fig. 7). The overall structures of the lectins from B. craniifer, G. portentosa and P. americana were identical to that of the

C. Chen et al. / Insect Biochemistry and Molecular Biology 28 (1998) 721–731

727

Fig. 3. Immunofluorescent localization of the ␤-1,3-glucan-specific lectin within the haemocytes of three cockroach species. Cross-reactivity and distribution of ␤-1,3-glucan-specific lectin in various cockroach species with antibody against B. discoidalis lectin. Cross-reactivity with haemocytes from (A) B. craniifer; (B) L. maderae and (C) P. americana. Left hand side, phase contrast; right hand side, immunofluorescent distribution of the same field of view showing strong reactive product associated with the cell surface and cytoplasm. Cells incubated with pre-immune rabbit serum showed a low level of background fluorescence. Scale bar = 10 ␮m.

␤-1,3-glucan-specific lectin originally purified from the serum of B. discoidalis (Chen et al., 1998). 3.5. Lectin enhancement of prophenoxidase activation by laminarin The lectins purified from all the cockroaches by immunoaffinity chromatography were able to enhance

the prophenoloxidase activation by laminarin in B. discoidalis HLS, although with different potential for enhancement of proPO activation with laminarin (B. discoidalis > B. craniifer > G. portentosa > P. americana; Fig. 8).

728

C. Chen et al. / Insect Biochemistry and Molecular Biology 28 (1998) 721–731

Fig. 4. Western blot analysis showing the cross-reaction of sera from a range of insect species with antibody against the ␤-1,3-glucan-specific lectin from B. discoidalis Sera (10 ␮g each) from a range of insect species were denatured by 1% SDS and 2%␤-mercaptoethanol by boiling in the sample buffer (pH 6.8) for 3 min and loaded on a 10% SDS-PAGE. Proteins were then electrically blotted onto a nitro-cellulose membrane and the specific proteins were monitored using antibody against the purified ␤-1.3-glucan-specific lectin from B. discoidalis, as detailed in the Materials and Methods. Lane a, B. discoidalis; lane b, B. craniifer; lane c, L. migratoria; lane d, P. americana; lane e, G. portentosa; lane f, A. pernyi; lane g, C. extradentatus; lane h, G. mellonella; lane i, L. maderae; and lane j, the purified ␤-1,3-glucan-specific lectin from B. discoidalis.

4. Discussion

Fig. 5. Immunoaffinity chromatography of the ␤-1,3-glucan-specific lectin from sera of B. discoidalis. Three millitres of insect serum were first adjusted to pH 6.8 by adding 300 ␮l of 1 M sodium phosphate (pH 6.8) and then loaded on a pre-equilibrated antibody-Sepharose 6B mini-column (1.2 ml). The column was washed extensively with 0.1 M sodium phosphate, pH 6.8 and eluted first with 5 ml 0.2 M galactose in sodium phosphate buffer and then with 3 ml of 3.5 M MgCl2 in the same buffer. Lectin activity was monitored with an agglutinating assay using yeast as indicator after extensive dialysis of the eluates against TBS/Ca2 + , pH 7.4 buffer. The column was regenerated by passing first 1 ml 2 M urea, then 10 ml 1 M NaCl and finally 5 ml 100 mM glycine (pH 5.0) and re-equilibrated with 0.1 M sodium phosphate buffer pH 6.8. Purification of the ␤-1,3-glucan-specific lectins from B. craniifer, G. portentosa and P. americana also gave similar profiles.

Using Western blotting and various immunological detection techniques, ␤-1,3-glucan-specific lectins have been identified in the sera and localized in the haemocytes from four cockroach species, namely, B. discoidalis, B. craniifer, P. americana and L. maderae. The antibody specificity for the lectins from a range of insect species varied according to the taxonomic position of the tested insect, with strong reactions between rabbit anti␤-1,3-glucan antibody against serum from B. discoidalis and B. craniifer and no reaction observed between this antibody and sera from G. mellonella, A. pernyi, C. extradentatus, and L. migratoria. A ␤-1,3-glucan-specific lectin has been shown on the surface of the plasmatocytes of G. mellonella (Matha et al., 1990) and the present results suggest that this has different antigenic properties to the B. discoidalis ␤-1,3-glucan lectins. Immunoaffinity chromatography has proved to be successful in purifying the ␤-1,3-glucan-specific lectins from a range of cockroaches. The procedure described in this paper is simple and rapid (less than 12 hours) with high yields and good recovery of lectin activity. Immunoaffinity chromatography usually requires drastic manipulation of pH and ionic strength to dissociate the antigen–antibody complex. It is important to use a mild method which does not destroy the biological activity of the antigen. In initial experiments, a low pH method with glycine buffers pH 3.0 was used to elute the ␤-1,3-glucan-specific lectin, but the protein was subsequently found to be denatured even after rapidly neutralising to

C. Chen et al. / Insect Biochemistry and Molecular Biology 28 (1998) 721–731

729

Table 2 Summary of the purification of the ␤-1,3-glucan-specific lectin from sera of four cockroach species using immunoaffinity chromatography Step

Vol (ml)

Unfractioned sera of: B. discoidalis 3 B. craniifer 3 P. americana 3 G. portentosa 3 Immunoaffinity chromatography B. discoidalis 1.0 B. craniifer 1.1 P. americana 1.2 G. portentosa 1.6

Protein (mg)

HAa

TA (units)b

55 48 30 72

64 16 16 64

7680 1920 1920 7680

1.9 3.0 0.8 6.9

128 128 32 64

5120 1401 1536 4096

SA (U/mg)c

139.6 40.0 64.0 106.6 2694 1877 1920 593.6

Fold

Recovery (%)

1 1 1 1

100 100 100 100 19.3 46.9 30.0 5.6

67 73 80 53

Agglutinating activity (HA) is expressed as the reciprocal of titre against yeast cells per 25 ␮l sample. TA = total activity. c SA = specific activity, units per milligram of protein. Fold = Purification fold. a

b

pH 6.8 with the introduction of a high buffer capacity of 1 M Tris buffer (pH 6.8). Thus, a method of using 3.0 M MgCl2 as eluate was introduced. This approach was found to be successful not only for lectin elution from the immunoaffinity column but also for maintaining the biological activity of all four affinity purified lectins. The ultrastructure of the purified ␤-1,3-glucan-specific lectins from all cockroaches is similar to B. discoidalis ␤-1,3-glucan-specific lectin (Chen et al., 1998). Under physiological conditions, these proteins are highly aggregated with structural subunits of Y-shaped molecular organisation. The aggregates can also be dissociated by the addition of laminarin (result not shown). It has been suggested that the invertebrate immune response to microbial infection largely relies on socalled pattern recognition to certain macromolecular

components of pathogens, such as LPS and peptidoglycans from bacteria, and ␤-1,3-glucans from fungi (Hoffmann, 1994). The recognition protein located on the cell surface of reactive tissue may interact with microbial ligands and subsequently initiate both cellular and humoral reactions, including phagocytosis, cell degranulation, encapsulation and biochemical proteolytic cascades, such as coagulation and proPO activation, and the production of antibacterial peptides (Hoffmann, 1994). This hypothesis might be true for the cockroaches, since such receptors are clearly demonstrated to be widely present in these insects. We have previously shown that an LPS-binding protein and the ␤-1,3-glucanspecific lectin from B. discoidalis can significantly enhance proPO activation by laminarin (Chen et al., 1995). This is also the case for the ␤-1,3-glucan-specific lectins in the cockroaches, B. craniifer, P. americana

Fig. 6. SDS-PAGE of the ␤-1,3-glucan-specific lectins from sera of four cockroach species purified by immunoaffinity chromatography. (A) native polyacrylamide gel electrophoresis of purified ␤-1,3-glucan-specific lectins from B. craniifer (lane a); P. americana (lane b) and G. portentosa (lane c). Arrow indicates positive-staining bands. (B and C), SDS-PAGE of purified ␤-1,3-glucan-specific lectins from different cockroach species. Protein samples were denatured with 1% SDS under non-reducing conditions (B) or with 1% SDS plus 2% ␤-mercaptoethanol under reducing conditions (C) and 0.1–0.2 mg loaded onto 10% polyacrylamide gels. Gels were stained with Coomassie Blue R250. Lane a, B. discoidalis purified lectin; lane b, B. craniifer lectin; lane c. G. portentosa lectin.

730

C. Chen et al. / Insect Biochemistry and Molecular Biology 28 (1998) 721–731

Fig. 8. Enhancement of laminarin activation of prophenoloxidase by the purified cockroach lectins. Twenty-five microlitres of purified ß1,3-glucan-specific lectins from B.discoidalis (Bd-GSL), B.craniifer (Bc-GSL), P.americana (Pa-GSL) and G.portentosa (Gp-GSL) (all at a final concentration of 100 ␮g/ml) were diluted twofold down a row in a microtitre plate with TBS/Ca2 + pH 7.4 and incubated with 25 ␮l of laminarin (La, 1 mg/ml) and 25 ␮l of B.discoidalis HLS (protein concentration 0.5 mg/ml) for 1 h at RT. Finally, 25 ␮l of L-DOPA (3 mg/ml in distilled water) were added and incubated for a further 5 h. The PO activity was measured at A492 nm for various treatments with HLS plus purified lectins and/or laminarin. Results are given as means ± S.E.M. (n = 4).

Fig. 7. Electron microscopy of the purified cockroach lectins. Lectin samples purified by immunoaffinity chromatography were dialysed extensively against 0.1 M ammonium acetate buffer at pH 6.0 and mixed with an equal volume of 1% uranyl acetate pH 4.2 for 90 sec before spreading on carbon-coated copper grids. (A) B. craniifer lectin; (B) P. americana lectin; (C) G. portentosa lectin.

and G. portentosa, as demonstrated in this paper, and may also be true in the silkworm, B. mori (Ochiai and Ashida, 1988), and the crayfish, P. leniusculus (Duvic and So¨derha¨ll, 1990). The mechanism of the activation of such enzyme cascades by microbial-specific proteins (including lectins) in invertebrates is not fully under-

stood. Interaction between microbial polysaccharides and host binding proteins (lectins), however, is essential for the activation of the enzyme cascade systems (Duvic and So¨derha¨ll, 1992), although the carbohydrate binding and proPO activation properties may be conferred on two separate sites (domains) of the protein (Chen et al., 1995). It is interesting that the proPO activation process is similar to that of the vertebrate complement system activated through the classical pathway and by lectins such as mannose-binding proteins and CRP (Ratcliffe et al., 1985; Jiang et al., 1991; Sim and Malhotra, 1994). In addition to such functional similarity between the ␤1,3-glucan-specific lectin and vertebrate CRPs and immunoglobulins, further evidence on the structural organization has revealed partial amino acid sequence similarities between the insect lectin and CRP, although they are phylogenetically unrelated (Chen et al., 1998). Thus, we suggest that the cockroach ␤-1,3-glucan-specific lectin may serve the role of vertebrate CRP and immunoglobulins, since such molecules are not present in insects. Further studies are needed to determine the molecular organisation of the insect lectin in relation to its functional analogy to vertebrate immune molecules.

C. Chen et al. / Insect Biochemistry and Molecular Biology 28 (1998) 721–731

Acknowledgements This work was supported by a studentship from the Sino-British Friendship Scheme and a grant from the Biotechnology and Biological Sciences Research Council (Grant No. GR/G 40224). We thank Mr I. Tew for technical assistance.

References Ashida, M., Ishizaki, Y., Iwahana, H., 1983. Activation of pro-phenoloxidase by bacterial cell walls or ␤-1,3-glucans in plasma of the silkworm Bombyx mori. Biochem. Biophys. Res. Commun. 113, 562–568. Bo¨gwald, J., Johnson, E., Seljelid, R., 1982. The cytotoxic effect of mouse macrophages stimulated in vitro by a ␤-1,3-D-glucan from yeast cell walls. Scand. J. Immunol. 15, 297–304. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principles of protein–dye binding. Anal. Biochem. 72, 248–254. Carvey, V.S., Cremer, N.E. and Sussdorf, D.H., 1977. In: Methods in Immunology, 3rd ed. The Benjamin/Cummings Publishing Company, Menlo Park, California, USA, pp. 313-327. Chen, C., Durrant, H.J., Newton, R.P., Ratcliffe, N.A., 1995. A study of novel lectins and their involvement in the activation of the prophenoloxidase system in Blaberus discoidalis. Biochem. J. 310, 23–31. Chen, C., Newton, R.P. and Ratcliffe, N.A., 1998. Identification, purification and properties of a ␤-1,3-glucan-specific lectin from the serum of the cockroach, Blaberus discoidalis, with similarity to invertebrate C-reactive protein. Comp. Biochem. Physiol. (in press). Chen, C., Ratcliffe, N.A., Rowley, A.F., 1993. Detection, isolation and characterization of multiple lectins from the haemolymph of the cockroach, Blaberus discoidalis. Biochem. J. 294, 181–190. DiLuzio, N.R., McNamee, R.B., Jones, E., Cook, J.A. and Hoffman, E.O., 1979. The employment of glucan and glucan activated macrophages in the enhancement of host resistance to malignancies in experimental animals. In: Fink, M.A. (Ed.), The Macrophage in Neoplasia. Academic Press, New York, pp. 181-198. Duvic, B., So¨derha¨ll, K., 1990. Purification and characterization of a ␤-1,3-glucan binding protein from plasma of the crayfish Pacifastacus leniusculus. J. Biol. Chem. 256, 9327–9332. Duvic, B., So¨derha¨ll, K., 1992. Purification and partial characterization of a ␤-1,3-glucan-binding protein membrane receptor from blood cells of the crayfish Pacifastacus leniusculups. Eur. J. Biochem. 207, 223–228.

731

Hawkes, R., Niday, E., Gordon, J., 1982. A dot-immunobinding assay for monoclonal and other antibodies. Anal. Biochem. 119, 142– 147. Hayat, M.A., 1979. Principles and Techniques of Electron Microscopy: Biological Application, 3rd ed. Macmillan Press Ltd., London. Hoffmann, J.A., 1994. Immune responses of insects. Dev. Comp. Immunol. 18 (S1), 37–38. Jiang, H., Siegel, J.N., Gewurz, H., 1991. Binding and complement activation by C-reactive protein via the collagen-like region of C1q and inhibition of these reactions by monoclonal antibodies to Creactive protein and C1q. J. Immunol. 146, 2324–2330. Laemmli, K.U., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 227, 680–685. Leonard, C., So¨derha¨ll, K., Ratcliffe, N.A., 1985. Studies on prophenoloxidase and protease activity of Blaberus craniifer haemocytes. Insect Biochem. 15, 803–810. Matha, V., Grobhoffer, L., Weyda, F., Hermanova, L., 1990. Detection of ␤-1,3-glucan-specific lectin on the surface of plasmatocyte immunocompetent cells of great wax moth Galleria mellonella L. Cytobios 64, 35–42. Maurer, H.R., 1971. Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis, 2nd ed. Bruyter, Berlin. Morita, T., Tanaka, S., Nakamura, T., Iwanaga, S., 1981. A new (1,3)␤-D-glucan-mediated coagulation pathway found in Limulus amebocytes. FEBS Lett. 190, 118–122. Ochiai, M., Ashida, M., 1988. Purification of a ␤-1,3-glucan recognition protein in the prophenoloxidase activating system from hemolymph of the silkworm Bombyx mori. J. Biol. Chem. 263, 12056–12062. Ratcliffe, N.A., Rowley, A.F., Fitzgerald, S.W., Rhodes, C.P., 1985. Invertebrate immunity: basic concepts and recent advances. Int. Rev. Cytol. 97, 183–350. Reid, K.B.H., Porter, R.R., 1981. The proteolytic activation systems of complement. Annu. Rev. Biochem. 50, 433–464. Richards, E.H., Ratcliffe, N.A., Renwrantz, L., 1989. The binding of lectins to carbohydrate moieties on haemocytes of the insects, Blaberus craniifer (Dictyoptera) and Extatosoma tiaratum (Phasmida). Cell Tissue Res. 257, 445–454. Schmidt, W.E., Ebel, J., 1987. Specific binding of a fungal glucan phytoalexin elicitor to membrane fraction from soybean Glycine max. Proc. Natl. Acad. Sci. USA 84, 4117–4121. Sim, R.B., Malhotra, R., 1994. Interactions of carbohydrates and lectins with complement. Biochem. Soc. Trans. 22, 106–111. So¨derha¨ll, K., Ro¨gner, W., So¨derha¨ll, I., Newton, R.P., Ratcliffe, N.A., 1988. The properties and purification of Blaberus craniifer plasma protein which enhances the activation of haemocyte prophenoloxidase by a ␤-1,3-glucan. Insect Biochem. 18, 323–330. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets, procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354.