Printer Friendly

Isolation and characterization of multiple-lectins from serum of the desert locust Schistocerca gregaria (Orthoptera: Acrididae).

Introduction

Lectins can be defined as proteins that recognize specific carbohydrate structures and thereby agglutinate cells by binding to cell-surface sugars, glycoproteins, and other glycoconjugates (Lis & Sharon 1998). They are usually structurally complex molecules with one or more carbohydrate-recognition domains and therefore with possible multiple binding sites (Cillespie et al. 1997, Dodd & Drickamer 2001). Among these lectins, those that require [Ca.sup.2+] for their activity are called C-type lectins (Drickamer 1988). Animal lectins have been found in various invertebrates as well as vertebrates (Barondes 1984), either in soluble or in membrane-bound form. These lectins can play a variety of physiological roles: in particular, they are crucial in the innate immune system where they bind, as referred to above, to the carbohydrates present on the surface of potential pathogens (Rudd et al. 2001).

The most extensively studied functions of hemolymph lectins have been their roles in insect immune defense systems (Ratcliffe & Rowley 1980, Vasta et al. 2007). Furthermore, induced or endogenous hemolymph lectins and/or hemocyte-associated lectins have been shown to be involved in phagocytosis (Kawasaki et al. 1993, Wheeler et al. 1993, Wilson et al. 1999), encapsulation (Komano & Natori 1985), nodule formation (Kyriakides et al. 1993), activation of the prophenoloxidase system (Chenetal. 1995) and hemolymph coagulation (Minnick et al. 1986).

Relatively few insect lectins have been purified and characterized. The majority of insect lectins characterized to date have been detected in the hemolymph and in most cases they are thought to be synthesized in the fat body (Kubo et al. 1984, Amanai et al. 1991) and/or the hemocytes (Amiranti 1976, Stiles et al. 1988, Amanai et al. 1991, Boucias & Pendland 1993). Lectins have been isolated and characterized from some insects, e.g., dipterans (Komano et al. 1980, Stynen et al. 1985, Ingram & Molyneux 1990, McKenzie & Preston 1992, Haq et al. 1996, Chen & Billingsley 1999, Volf et al. 2002), lepidopterans (Pendland & Boucias 1986, Castro et al. 1987, Qu et al. 1987, Amanai et al. 1990, Gul & Ayvali 2002, Ourth et al. 2005, Chai et al. 2008), orthopterans (Stebbins & Hapner 1985, Drif & Brehelin 1994, Ayaad 2004), dictyopterans (Kubo & Natori 1987, Chen et al. 1993), phasmids (Richards et al. 1988), and hemipterans (Comes et al. 1991). Their structure, function, and carbohydrate-binding properties still need further investigation to clarify their role in the innate immune system of insects.

The present work reports on isolation and on the biological, physicochemical and molecular characterization of multiple lectins from serum of the 5th-instar desert locust S. gregaria.

Materials and Methods

Insects, sample preparation and biological and physicochemical tests.--Insect rearing, preparation of hemolymph sera, preparation of erythrocyte suspension, assay of hemagglultinating activity (HA), effect of divalent canons, inhibition assays of sugars and glycoconjugates, and stability tests--were carried out for the whole-hemolymph sera lectins (Ayaad et al. 2009 this issue). Recounted briefly: solutions of the isolated lectins were used instead of the prepared sera. Rabbit erythrocytes, 2% suspension in TBS/[Ca.sup.2+]-[Mg.sup.2+]. (pH 7.0) were used for any of the HA assays. The effect on HA of the bivalent canons (20 mM) [Ca.sup.2+], [Mg.sup.2+] [Zn.sup.2+] and [Mn.sup.2+], and EDTA (5-10 mM) was tested at pH 7.0. The inhibitory effect of carbohydrates on HA was tested in the presence of free and conjugated sugars in TBS/[Ca.sup.2+]-[Mg.sup.2+] at pH 7.0. Heating for 25 min at 60 and at 100[degrees]C, and storage at both 4 and at -20[degrees]C over a period of 1 week to 3 months, were tested for their effect on HA (Ayaad et al. 2009 this issue).

Determination of total protein concentration.--The total protein concentration was determined according to the method of Bradford (1976), using Coomassie Brilliant Blue (COBB). Bovine serum albumin (BSA) fraction V (Sigma-Aldrich), dissolved in 0.15 M NaCl, was used as a protein standard.

Isolation of lectins [[(N[H.sub.4]).sub.2] S[O.sub.4]-salting out of serum proteins].--Insect serum (25 ml) was diluted with an equal volume of 0.02 M TBS pH 7.0 containing 2 mM Ca[Cl.sub.2] and 1 mM Mg[Cl.sub.2] (TBS/[Ca.sup.2+]-[Mg.sup.2+]). Proteins were precipitated by addition of [(N[H.sub.4]).sub.2] S[O.sub.4] (Sigma-Aldrich) at a concentration of 70%, with gentle stirring at 4[degrees]C . The resulting precipitate was collected by centrifugation and dialyzed in Visking[R] dialysis tubing (mol. wt cut-off [MWCO]: 12000-14000 Da) (Serva Electrophoresis CmbH, Germany) against distilled water for 24 h at 4[degrees]C with three changes. The dialyzed solution was then centrifuged at 8000 rpm for 5 min at 4[degrees]C to remove insoluble materials.

Ion-exchange chromatography of lectins.--Twenty grams of DEAE-cellulose (Amersham Pharmacia Biotech) were washed in 0.5 M NaOH for 10 min, then rinsed in distilled water, followed by 0.8 M HCl, and finally 0.5 M NaOH, to remove contaminants. The washed matrix was thoroughly rinsed free of NaOH with distilled water and mixed with a sufficient quantity of starting buffer (0.01 M TBS containing 0.01 M Ca[Cl.sub.2] and 0.01 M Mg[Cl.sub.2], pH 7.0) to produce a thin suspension. The treated matrix was packed into a column (20 cm x 1.0 cm i.d.) and thoroughly washed with the starting buffer. A sample from the last step of [(N[H.sub.4]).sub.2] S[O.sub.4] fractionation (approximately 20 ml of 2.85 mg protein/ml) was applied to the DEAE-cellulose column. The column was then washed with the starting buffer until the baseline of absorption at 280 nm was achieved, and then elution carried out with a linear gradient of 0-0.25 M NaCl in 200 ml of the starting buffer. The flow rate was adjusted to 10 ml/h. Fractions (tubes) constituting the separated peaks were tested separately for HA against rabbit RBCs; all fractions displaying HA were then pooled for use in affinity chromatography.

Affinity chromatography of lectins.--Lectins of the same peak were isolated from the other proteins contained therein, by use of CNBr-activated Sepharose 4B (Sigma-Aldrich) affinity chromatography as described by Komano et al. (1980) with slight modifications. CNBr-activated Sepharose 4B matrix was packed into a column (10X1 cm i.d.) and extensively washed with about 500 ml of TBS/[Ca.sup.2+]-[Mg.sup.2+], pH 7.0 containing 0.02% Na[N.sub.3] (Sigma-Aldrich). Each separate pool of fractions, having HA from the DEAE-cellulose column, was separately applied (slowly) to the CNBr-Sepharose 4B column at 4 [degrees]C. This latter column was washed successively with TBS, to which 0.5 M NaCl was added, until no further protein was detected in the eluate by monitoring the absorbance at 280 nm. Lectins (the bound material) were then eluted with 0.3 M raffinose solution in TBS/ [Ca.sup.2+]-[Mg.sup.2+]. (Ayaad 2004, Ayaad et al. 2009). The collected fractions of each peak were pooled and each pool separately dialyzed extensively against TBS/ [Ca.sub.2+]-[Mg.sup.2+], then against de-ionized distilled water to remove raffinose; then HA of each pool was assayed against rabbit RBCs. Each pool of fractions of the resulting peaks was lyophilized and stored at -20[degrees]C until use.

IEF of lectins.--The isoelectric points of lectins, contained in pools collected from affinity chromatography columns, were estimated on a pH 3.G to 9.3 gradient polyacrylamide gel, according to the method of O'Farrell (1975), in slab gels (140 x 120 x 0.75 mm). Electrophoresis ran at 350 V for 17-18 h, yielding approximately 0000 V/h at 22[degrees]C. The pI values of the isolated lectins were determined by running a mixture of standard proteins of known isoelectric points on the same gel. We obtained a number of mixtures of proteins with different pI values (IEF MIX-Sigma, USA, pH 3.G-9.3) containing amyloglucosidase (pI 3.6), trypsin inhibitor (pI 4.G), [beta]-lactoglobulin A (pI 5.1), carbonic anhydrase II (bovine, pI 5.9), carbonic anhydrase I (human, pI 6.6), myoglobin (pI 6.8, 7.2), lectin from Lens culinaris (pI 8.2, 8.G, 8.8), and trypsinogen (pI 9.3); identifications of the different pI values were made using an image densitometer G 700 (Bio-Rad, USA).

Native PAGE of lectins.--The identity of the destined lectins contained in the pools collected from affinity chromatography columns (raffinose-eluents) was confirmed by native PACE. Electrophoresis was carried out on 10% polyacrylamide gel under nondenaturating conditions, according to the method of Schagger and von Jagow (1991), using a Tris-glycine running buffer without SDS.

After isolation of protein bands by the nondenaturating PACE, the characteristic bands were detected on the gel by staining and matching procedures, then were sliced away from the remainder of the gel with a sharp razor blade. Each of these specific gel areas was cut into pieces, approximately 1[mm.sup.2] for each, and the pieces of each band soaked in 2 ml of distilled water in a separate tube overnight at room temperature. Each tube was centrifuged at 6000 rpm for 10 min; the eluate (supernatant) was transferred into a new tube. The eluted proteins were concentrated by speed vacuum for 30 min and then stored at -20[degrees]C until used.

SDS/PAGE of lectins (under nonreducing and reducing conditions).--SDS/PACE of isolated lectins was carried out by the discontinuous buffer system of Laemmli (Laemmili 1970). Some samples were denatured with 2% SDS containing 5% [beta]-mercaptoethanol by boiling for 3 min. Treated samples were centrifuged at 10000 g for 5 min before being loaded onto the gels. Electrophoresis was carried out at a constant voltage of 200 V for 90 min. The gels were calibrated with standard molecular weight proteins [New England Biolabs Ltd. (low range: 116, 91, 46.4, 34.3, 28.7, and 21 kDa) and/or Titan Biotech Ltd. (high and low range: 200, 116, 97, 66, 45, and 21 kDa)] and the quantifications of the different molecular weights were made using an image densitometer G 700 (Bio-Rad).

Results and Discussion

The isolated lectins

Isolation of lectins.--The methods and procedures used for isolation of the 5th-instar S. gregaria lectins are presented in Figure 1.

After [(N[H.sub.4]).sub.2] S[O.sub.4] fractionation and DEAE-cellulose chromatography, the HA was found to be present in fractions of peak I (18 ml), peak II (24 ml), and peak III (18 ml), but not in peaks IV and V (Fig. 2). Therefore, fraction pools of each of peaks I, II, and III were indicated to contain lectins. These pools were separately, and slowly applied to the prepared CNBr-activated Sepharose 4B columns for affinity chromatography.

On washing each column successively with TBS to which 0.5 M NaCl was added until no further protein was detected, it was shown that most proteins were not adsorbed to the column, appeared in the through-flow fractions, and had no HA (Fig. 3a, b, c). When the bound material (lectins) was then eluted with 0.3 M raffinose solution in TBS/[Ca.sup.2+]-[Mg.sup.2+], small protein peaks containing HA were realized (Fig. 3 a, b, c). Fractions constituting each peak were considered as different pools [pool 1 ([P.sub.1]), pool 2 ([P.sub.2]), and pool 3 ([P.sub.3])], each containing different lectins. After dialysis to remove raffinose, the three resulting pools exhibited prominent HA; therefore, they seemed to contain different lectins. The specific activity of lectins recovered from serum by use of these methods and procedures showed a 1.5 to 4-fold increase in the specific activity (Table 1), indicating prominent reproducibility.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

HA of the isolated lectin pools.--The determined HA patterns of lectins, of the 5th instar S. gregaria, contained in the three collected pools from affinity chromatography, are presented against a range of vertebrate RBCs, (Table 2).The isolated lectin pools have the highest HA against rabbit RBCs, when compared to the other RBC types, in the order [P.sub.2] > [P.sub.3] > [P.sub.1]. For the other RBC types, sometimes there was a very low level of HA, or even none. These results demonstrate a high specificity of the 5th instar S. gregaria isolated serum lectins to RBCs of rabbit, when compared to those of the other vertebrates (Table 2).

Homogeneity and molecular characteristics of lectins

Homogeneity and molecular characteristics of the isolated lectins contained in the pools [P.sub.1], [P.sub.2] and [P.sub.3] were monitored and confirmed by use of IEF, and both the native PACE and SDS/PACE (under nonreducing and reducing conditions).

Isoelectric focusing (IEF).--The obtained results (Fig. 4) reveal some characteristics of these lectins. [P.sub.2] and [P.sub.3] both focused as single bands at pH 8.11 and pH 6.84, respectively. However, [P.sub.1] showed a major band (~84%) focused at pH 6.39 and a minor band (16%) focused at pH 6.53 and realized in association to the major one. Therefore, each of the [P.sub.2] and [P.sub.3] seems to be formed of a single and separate lectin. The nature of the two-band components of [P.sub.1] were confirmed by native PACE, accompanied with HA determination to each separated band (see below).

The pI values of [P.sub.2] protein appear to lie in the slightly alkaline range, whereas those of both [P.sub.1] and [P.sub.3] appear to lie in the slightly acidic range. In other insects, the pI values range from 4.7 to G.4, as in the orthopterans M. sangunipes (Stebbins & Hapner 1985), and L. migratoria (Drif & Brehelin 1994), the dicryopteran B. discoidalis (Chen et al. 1993), the lepidopterans M. sexta (Minnick et al. 1986), and H. cecropia (Castro et al. 1987), the coleopteran A. dichotoma (Umetsu et al., 1984), and the dipteran G. fuscipes (Ingram & Molyneux 1990).

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Native PAGE.--The identity of the lectins contained in the three pools [P.sub.1], [P.sub.2] and [P.sub.3], collected from affinity chromatography columns (raffinose-eluents) (Fig. 3 a-c) was confirmed by native PACE. The electophoretograph obtained (Fig. 5) shows that [P.sub.2] and [P.sub.3] appear as single bands. This confirms the nature of both [P.sub.2] and [P.sub.3] that appeared on IEF gel. Therefore, each of these two bands was considered as a discrete lectin and they are designated as [Sg.sub.2] and [Sg.sub.3], respectively. Both bands have Rm values of almost 0.32, (Fig. 5) that were revealed in the high molecular weight range.

On the other hand, [P.sub.1] was separated into two bands with different-relative mobility values of 0.38 ([P.sub.1a]) and 0.44 ([P.sub.1b]), respectively. The first band [P.sub.1a] (Rm = 0.38) did not display any HA, whereas the second band [P.sub.1b] (Rm = 0.44) enclosed HA, and was considered as a third lectin in the serum of S. gregaria; it was designated as [Sg.sub.1]. The nonagglutinating band may be a nonlectin glycoprotein that binds to, and is eluted from, the ion exchange and the affinity columns, in association with the isolated lectin [Sg.sub.1]. Co-isolated proteins, during the isolation of multiple lectins, that are eluted from both ion exchange and affinity chromatography, present a case observed before in other insects such as E. tiaratum (Richards et al. 1988) and B. discoidalis (Wilson et al. 1999).

Appearance of native PACE patterns of [Sg.sub.1] ([P.sub.1b]), [Sg.sub.2] and [Sg.sub.3] as a single band with high molecular weight range, indicates that each band may be formed of aggregates (oligomers) of subunits. This assumed oligomeric native form of lectins of the 5th instar S. gregaria serum is reported before in other insects, e.g., the orthopterans M. sangunipes (Stebbins & Hapner 1985), L. migratoria (Drif & Brehelin 1994), and adult S. gregaria (Ayaad 2004), and in the dipteran G. fuscipes (Ingram & Molyneux 1990).

SDS/PAGE.--When the lectins [Sg.sub.1]([P.sub.1b]), [Sg.sub.2] and [Sg.sub.3] were sliced away from the native PACE, eluted, then electrophoresed by SDS/PACE, under both nonreducing (Fig. 6), and reducing (Fig. 7) conditions, using 10% separating gel and 5% stacking gel, additional characteristics were revealed. Under the nonreducing conditions, each of the [Sg.sub.1], [Sg.sub.2], and [Sg.sub.3] was separated by SDS/PACE as a single band with approximate molecular weights of 21.5, 44.5, and 43.9 kDa respectively (Fig. G). This may indicate that subunits of each lectin are of approximately the same molecular weight and are held together by noncovalent interactions. Noncovalent linking of protein subunits was reported in lectins of other insects, e.g., the dipteran S. peregrina (Komano et al. 1980), lepidopterans H. cecropia and S. exigua (Castro et al. 1987, Boucias & Pendland 1993), and the phasmid E. tiaratum (Richards et al. 1988).

Under the reducing conditions, SDS/PACE separation (Fig. 7) showed a single band only from [Sg.sub.1] (21.7 kDa), whereas each of [Sg.sub.2] and [Sg.sub.3] were separated into two bands. Those resulting from [Sg.sub.2] have approximate molecular weight of 32.G and 31.7 kDa, and those of [Sg.sub.3] are of 32.9 and 31.5 kDa. The separation of two different bands with two different molecular weights from each of the [Sg.sub.2] and [Sg.sub.3] (Fig. 8) indicates that each of these latter is formed of two different types of subunits linked with disulphide bonds. This character was reported before for some insect lectins, for example, the orthopterans T. commodus (Hapner & Jermyn 1981), and M. sanguinipes (Stebbins & Hapner 1985), BDL1, and BDL2 of the dicryopteran B. discoidalis (Chen et al. 1993), and allo A-I and -II of the coleopteran A. dichotoma (Umetsu et al. 1984).

The low molecular-weight range of the single-banded [Sg.sub.1] separated by the nonreducing and reducing SDS/PACE (21.5 to 21.7 kDa) is near to those of other insects, for instance, 30-kDa lectin of P. americana (Kubo & Natori, 1987), 20-kDa lectin of S. peregrina (Fujita et al. 1998).

Physicochemical properties of the isolated lectins

Stability.--The obtained data revealed that HA was completely abolished after 25 min exposure to 100[degrees]C, but reduced to 75 only upon exposure to 60[degrees]C for the same time. On the other hand, it was observed that HA was stable when exposed to 25[degrees]C (room temperature) for the same period of time. These observations indicate that lectins of the 5th' instar S. gregaria are heat-labile in nature. Heat instability is characteristic for lectins of some other insects, e.g., the orthopterans T. commodus, (Hapner & Jermyn 1981), and M. sanguinipes (Stebbins & Hapner 1985), the phasmid E. tiaratum (Richards et al. 1988), and the dipteran G. fuscipes (Ingram & Molyneux 1990). However, in contrast to these cases, in the coleopteran L. decemlineata (Minnick et al. 1986), and theorthopteran L. migratoria (Drif & Brehelin 1994) the lectins were reported to be heat resistant when subjected to elevated temperatures of 70-100[degrees]C.

For the storage temperature and period, the HA of lectins of 5th instar S. gregaria were stable when stored at-20[degrees]C, with an extremely slow insignificant decline observed on prolonged storage (3 mo).

[FIGURE 5 OMITTED]

Since the storage medium of the isolated lectins under investigation was without any additive-sugars, it seems that these lectins do not need the presence of sugars for their stability. This situation is analogous to that reported in S. peregrina (Komano et al. 1980). However, in contrast to this case, isolated lectins from other insects require addition of sugar to the storage medium to maintain their activity. For example, in T. commodus (Hapner & Jermyn 1981), the lectin required the addition of galactose. Also the lectins of M. sanguinipes (Stebbins & Hapner 1985), once isolated, were so unstable that disaggregation of the multimer occurred readily in the absence of D-galactose; and in E. tiaratum instability was observed when the lectin was not stored in lactose (Richards et al. 1988).

Divalent cation requirement for HA.--The present data show that the isolated lectins [Sg.sub.1], [Sg.sub.2], and [Sg.sub.3] require [Ca.sup.2+] to express their HA. Some canons such as [Mg.sup.2+] or [Zn.sup.2+] can only partly replace [Ca.sup.2+]; however other divalent canons, such as [Mn.sup.2+], have no effect (Table 3). As expected, addition of EDTA caused complete inhibition to HA. Also removal of [Ca.sup.2+] by dialyzing the lectin solutions against either TBS alone or TBS with 10 mM EDTA, similarly resulted in a complete loss of HA of these lectins. EDTA presumably exerted its effect through chelation of divalent canons that structurally associated with the lectins. The HA of C-type lectins of T. commodus, M. sanguinipes, S. exigua, E. tiaratum and adult S. gregaria were also lost in the presence of EDTA (Pendland & Boucias 1986, Stebbins & Hapner 1985, Ayaad 2004, Richards et al. 1988, Hapner & Jermyn 1981). In most insect lectins a complete loss of HA was observed in the presence of EDTA (Ratcliffe et al. 1985). Therefore, for the lectins isolated from serum of the 5th instar S. gregaria, the observed requirement of [Ca.sup.2+] to enhance and maintain HA of the three lectins [Sg.sub.1], [Sg.sub.2], and [Sg.sub.3], prominently indicates that these lectins are [Ca.sup.2+]--dependent. This property is a general characteristic of a group of lectins denoted as C-type lectins. This type of lectin was originally named to reflect the special importance of [Ca.sup.2+] in the mechanism of carbohydrate binding (Zelensky & Cready 2005). Each of the known C-type lectins has carbohydrate-recognition domain structures containing a conserved [Ca.sup.2+]- binding site; and may also contain a second site (Drickamer 1999).

Sugar specificity and inhibition to HA.-The obtained profile of sugar inhibition to HA of lectins isolated from serum of 5th instar S. gregaria against rabbit RBCs shows certain characteristics. The inhibition of sugars and of glycoconjugates to HA of [Sg.sub.1], [Sg.sub.2] and [Sg.sub.3] showed great resemblance to each other (Table 4). The HA of these lectins was preferentially inhibited ([IC.sub.50] = 6.25 mM) by raffinose, [alpha]-m-nitrophenyl-D-galactose, [alpha]-p-nitrophenyl-D-galactose, and [alpha]-methyl-D-galactose, and the monosaccharide L-(+)-rhamnose. None of these lectins were inhibited by D-(+)-mannose, trehalose and N-acetylglucosamine (200 mM), or laminaran (> 1 %). Low sensitivities ([IC.sub.50] = 50-100 mM) were observed in case of the free sugars D-galactose, D-glucose, sucrose, lactose, and also by N-acetyl-D-galactosamine. These results allow speculation that the carbohydrate-binding site of the carbohydrate recognition domain of these lectins prefers (within the range of the tested sugars) [alpha]-linked-D-galactosides over both the [beta]-form and also the free D-galactose.

[FIGURE 6 OMITTED]

The affinity of the 5th instar isolated S. gregaria serum lectins toward [alpha]-linked galactosides is a feature that has also been reported in other acridids, such as L. migratoria (Drif & Brehelin 1994) and adult S. gregaria (Ayaad 2004). On the other hand, affinity of lectins to [beta]-linked-D-galactosides, such as lactose and lactulose, was recorded in the coleopteran A. dichotoma (Umetsu et al. 1984), its lectins being inhibited by this group of carbohydrates. The latter lectin specificity is also known in vertebrates, where the best ligands are [beta]-galactosides (Barondes 1984). Some lectins purified from other insect species, especially from orthopterans (Stebbins & Hapner 1985, Lackie 1981, Jurenka et al. 1982, Hapner 1983, Drif & Brehelin 1989) show an affinity for a broad spectrum of carbohydrates. Another orthopteran T. commodus (Hapner & Jermyn 1981), and the dipteran P. duboscqi (Volf et al. 2002) possess lectins with amino sugar-binding affinity. In numerous other insect species, mainly lepidopterans, hemolymph lectins show affinity for galactose and lactose (Pendland & Boucias 1986) or to glucosides (Minnick et al. 1986, Qu et al. 1987). Lectins from another group of insects have binding affinity to mannose, e.g., the dictyopteran B. discoidalis (Chen et al. 1993), and the lepidopteran H. virescens (Ourth et al. 2005), and to mannan, e.g., the dipteran A. stephensi (Chen & Billingsley 1999).

[FIGURE 7 OMITTED]

In this context, C-type lectins are known to have a common carbohydrate recognition domain (Weis et al. 1991, Drickamer 1992), or two domains (Yu & Kanost 2000), that may increase their binding affinity to carbohydrates. This domain contains a conserved [Ca.sup.2+]binding site, and may contain a second one (Drickamer 1999). The binding mechanism involves interaction with carbohydrate and the conserved [Ca.sup.2+], which may form hydrogen bonding with acid and amide side groups (Weis & Drickamer 1996).

The ligand specificity of the carbohydrate recognition domain is largely dependent on the position of hydroxyl groups on the free or conjugated sugar, particularly the 3-OH and 4-OH (Ng et al. 1996, Kolatkar & Weis 1996). In addition to these interactions further specificity may be achieved by hydrogen bonds and electrostatic interactions with the surface of the protein (Ng & Weis 1997, Kolatkar et al. 1998). In the present work, agglutination of rabbit RBCs by C-type lectins [Sg.sub.1], [Sg.sub.2], and [Sg.sub.3] of the serum of the 5th' instar S. gregaria was inhibited most efficiently by the [alpha]-linked-D-galactosides followed by L-(+)-rhamnose. However, the free monosaccharide D(+)-galactose and the [beta]-linked-D-galactosides (as in lactose) were of very low efficiency. In the latter cases, the orientation of 3-OH and 4-OH are the same for the same sugar, D-(+)-galactose; and the same was observed for 2-OH and 4-OH in D-(+)-galactose and L-(+)-rhamnose. Therefore, not onlythe orientation of 2-OH, 3-OH and 4-OH, but also the configuration ([alpha] or [beta]) and the nature of the substituents at [C.sub.1] or the functional group at [C.sub.6], seem to affect the binding specificity and affinity of the 5th instar S. gregaria lectins to carbohydrates.

According to these parameters, carbohydrate-binding specificity is crucial in recognition of these cell-surface carbohydrates, including those of the invading pathogens (McGreal et al. 2004). Therefore, animal C-type Lectins are among the important proteins in pathogen recognition and cellular interaction (Rudd et al. 2001, Weis et al. 1998) by binding to the carbohydrate component of the surface molecular patterns of these targets (Janeway 1989).

Abbreviations

HA, hemagglutinating activity

IEF, isoelectric focusing

PACE, polyacrylamide gel electrophoresis

pl, isoelectric point

RM, relative mobility

SDS,Sodium dodecyl sulfate

Accepted March 31, 2009

References

Amanai K., Sakurai S., Ohtaki T. 1990. Purification and characterization of haemagglutinin in the hemolymph of the silkworm, Bombyx mori. Comparative Biochemistry and Physiology 97B: 471-476.

Amanai K., Sakurai S., Ohtaki T. 1991. Site of hemolymph Lectins production and its activation in vitro by 20-hydroxyecdysone. Archives of Insect Biochemistry and Physiology 17: 39-51.

Amiranti G.A. 1976. Production of heteroagglutinins in haemocytes of Leucophaga maderae L. Experientia 32: 526-528.

Ayaad T.H. 2004. Isolation, characterization, and N-terminal amino acid sequence of lectin from plasma of Schistocerca gregaria. Efflatounia 4: 9-22.

Ayaad T.H., Dorrah M.A., Mohamed A.A., Bassal T.T.M. 2009. Specificity and developmental changes of hemagglutination activity of serum of the desert locust Schistocerca gregaria (Orthoptera: Acridadae). Journal of Orthoptera Research 18: 51-56.

Barondes S.H. 1984. Soluble lectins: a new class of extracellular proteins. Science 223: 1259-1264.

Boucias D.G., Pendland J.C. 1993. The galactose binding lectin from the beet armyworm, Spodoptera exigua: distribution and site of synthesis. Insect Biochemistry and Molecular Biology 23: 233-242.

Bradford M.M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254.

Castro V.M., Boman H.G., Hammarstrom S. 1987. Isolation and characterization of isolectins with galactose/N-acetyl galactosamine specificity from hemolymph of the giant silkworm Hyalophora cecropia. Insect Biochemistry 17: 513-523.

ChaiL-Q., Tian Y-Y., Yang D-T., Wang J-X., Zhao X-F. 2008. Molecular cloning and characterization of a C-type lectin from the cotton bollworm, Helicoverpa armigera. Developmental and Comparative Immunology 32: 71-83.

Chen C., Billingsley P.F. 1999. Detection and characterization of a mannan-binding lectin from mosquito, Anopheles stephensi (Liston). European Journal of Biochemistry 263: 360-366.

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. Biochemical Journal 310: 23-31.

Chen C., Ratcliffe N.A., Rowley A.F. 1993. Detection, isolation and characterization of multiple lectins from the hemolymph of the cockroach, Blaberus discoidalis. Biochemical Journal 294: 181-190.

Dodd R.B., Drickamer K. 2001. Lectin-like proteins in model organisms: implications forevolution of carbohydrate-binding activity. Glycobiology 11: 71R-79R.

Drickamer K. 1988. Two distinct classes of carbohydrate-recognition domains in animal Lectins. Journal of Biological Chemistry 263: 9557-9560.

Drickamer K. 1992. Engineering galactose-binding activity into a C-type mannose-binding protein. Nature 360: 183-186.

Drickamer K. 1999. C-type lectin-like domains. Current Opinion in Structural Biology 9: 585-590.

Drif L., Brehelin M. 1989. Agglutinin mediated immune recognition in Locusta migratoria (Insecta). Journal of Insect Physiology 35: 729-736.

Drif L., Brehelin M. 1994. Purification and characterization of an agglutinin from the hemolymph of Locusta migratoria (Orthoptera). Insect Biochemistry and Molecular Biology 24: 283-228.

Fujita Y., Kurata S., Homma K., Natori S. 1998. A novel lectin from Sarcophaga its purification, characterization, and cDNA cloning. Journal of Biological Chemistry 273: 9667-9672.

Gillespie J.P., Kanost M., Trenczek T. 1997. Biological mediators of insect immunity Annual Review of Entomology 42: 611-643.

Gomes Y. De M., Furtado A.F., Coelho L.B.B. 1991. Partial purification and some properties of a hemolymph lectin from Panstrongylus megistus (Hemiptera, Reduviidae). Applied Biochemistry and Biotechnology 31: 97-107.

Gul N., Ayvali C. 2002. Purification and determination of the molecular structure of hemolymph lectin of Agrotis segetum (Denis and Schiff.). Turkish Journal of Biology 26: 49-55.

Hapner K.D. 1983. Hemagglutinin activity in the hemolymph of individual Acrididae (grasshoppers) specimens. Journal of Insect Physiology 29: 101-1O6.

Hapner K.D., Jermyn M.A. 1981. Hemagglutinin activity in the hemolymph of Teleogryllus comodus (Walker). Insect Biochemistry 11: 287-295.

Haq S., Kubo T., Kurata S., Kobayashi A., Natori S. 1996. Purification, characterization, and cDNA cloning of a galactose specific C-type lectin from Drosophila melanogaster. Journal of Biological Chemistry 271: 20213-20218.

Ingram G.A., Molyneux D.H. 1990. Lectins (haemagglutinins) in the haemolymph of Glossina fuscipes fuscipes: isolation, partial characterization, selected physico-chemical properties and carbohydrate-binding specificities. Insect Biochemistry 20: 13-27.

Janeway C.A., Jr 1989. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harbor Symposia on Quantitative Biology 54: 1-13.

Jurenka R., Manfredi K., Hapner K.D. 1982. Haemagglutinin activity in Acrididae (grasshopper) haemolymph. Journal of Insect Physiology 28: 177-181.

Kawasaki K., Kubo T., Natori S. 1993. A novel role of Periplaneta lectin as an opsonin to recognize 2-keto-3-deoxy octonate residues of bacterial lipopolysaccharide. Comparative Biochemistry and Physiology 106B: 675-680.

Kolatkar A.R., Weisw. I. 1996. Structural basis of galactose recognition by C-type animal lectins. Journal of Biological Chemistry 271: 6679-6685.

Kolatkar A.R., Leung A.K., Isecke R., Brossmer R., Drickamer K., Weis W.I. 1998. Mechanism of N-Acetylgalactosamine binding to a C-type animal lectin carbohydrate-recognition domain. Journal of Biological Chemistry 273:19502-19508.

Komano H., Natori S. 1985. Participation of Sarcophaga peregrina humoral lectin in the lysis of sheep red blood cells injected into the abdominal cavity of larvae. Developmental and Comparative Immunology 9: 31-40.

Komano H., Mizuno D., Natori S. 1980. Purification of lectin induced in the hemolymph of Sarcophaga peregrina larvae on injury. Journal of Biological Chemistry 255: 2919-2924.

Kubo T., Natori S. 1987. Purification and some properties of a lectin from the hemolymph of Periplaneta americana (American cockroach). European Journal of Biochemistry 16: 75-82.

Kubo T., Komano H., Okada O., Natori S. 1984. Identification of haemagglutinating protein and bactericidal activity of Sarcophaga peregrinaz haemolymph of adult on injury of body wall. Developmental and Comparative Immunology 8: 283-291.

Kyriakides T.R., McKillip J.I, Spence K.D. 1993. In vivo distribution of immune protein scolexin in bacteria-injected Manduca sexta larvae. Tissue and Cell 25: 423-434.

Lackie A.M. 1981. The specificity of the serum agglutinins of Periplaneta americana and Schistocerca gregaria and its relationship to the insect's immune response. Journal of Insect Physiology 27: 139-143.

Laemmili U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.

Lis H., Sharon N. 1998. Lectins: carbohydrate-specific proteins that mediate cellular recognition. Chemical Reviews 98: 637-674.

McGreal E.P., Martinez-Pomares L., Gordon S. 2004. Divergent roles for C-type lectins expressed by cells of the innate immune system. Molecular Immunology 41:1109-1121.

McKenzie A.N.J., Preston T.M. 1992. Purification and characterization of a galactose-specific agglutinin from the haemolymph of the larval stages of the insect Calliphora vomitoria. Developmental and Comparative Immunology 16: 31-39.

Minnick M.E., Rupp R.A., Scence K.D. 1986. A bacterial-induced lectin which triggers hemocyte coagulation in Manduca sexta. Biochemical and Biophysical Research Communication 137: 729-736.

Ng K.K., Weis W.I. 1997. Structure of a selectin-like mutant of mannose-binding protein complexed with sialylated and sulfated Lewis(x) oligosaccharides. Biochemistry 36: 979-988.

Ng K.K., Drickamer K., Weis W. 1996. Structural analysis of monosaccharide recognition by rat liver mannose-binding protein. Journal of Biological Chemistry 271: 663-674.

O'Farrell P.H. 1975. High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemistry 250: 4007-4021.

Ourth D.D., Narra M.B., Chung K.T. 2005. Isolation of mannose-binding C-type lectin from Heliothis virescens pupae. Biochemical and Biophysical Research Communication 335: 1085-1089.

Pendland J.C., Boucias D.G. 1986. Characteristics of a galactose binding hemagglutinin (lectin) from hemolymph of Spodoptera exigua larvae. Developmental and Comparative Immunology 10: 477-487.

Qu X-M., Zhang C.-F., Komano H., Natori S. 1987. Purification of a lectin from the hemolymph of Chinese oak silkmoth (Antheraea pernyi) pupae. Journal of Biochemistry 101: 545-551.

Ratcliffe N.A., Rowley A.F. 1980. Insect erythrocyte agglutinins. In vitro opsonization experiments with Clitumnus extradentatus and Periplaneta americana haemocytes. Immunology 40: 483-492.

Ratcliffe N.A., Rowley A.F., Fitzgerald S.W., Rhodes C.P. 1985. Invertebrate immunity: basic concepts and recent advances. International Review of Cytology 97: 183-350.

Richards E. H., Ratcliffe N.A., Renwrantz L. 1988. Isolation and characterization of a serum lectin from the giant stick insect Extatosoma tiaratum. Insect Biochemistry 18: 691-700.

Rudd P.M., E1liott T., Cresswell P., Wilson I.A., Dwek R.A. 2001. Glycosylation and the immune system. Science 291: 2370-2376.

Schagger H., von Jagow G. 1991. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Analytical Biochemistry 199: 223-231.

Stebbins M.R., Hapner K.D. 1985. Preparation and properties of haemagglutinin from haemolymph of Acrididae (grasshoppers). Insect Biochemistry 15: 451-462.

Stiles B., Bradley R.S., Stuart G.S., Hapner K.D. 1988. Site of synthesis of the haemolymph agglutinin of Melanoplus differentialis (Acrididae: Orthoptera). Journal of Insect Physiology 34: 1077-1085.

Stynen D., Vansteenwegen K., De Loof A. 1985. Anti-galactose lectin in the haemolymph of Sarcophaga bullata and three other calliphorid flies. Comparative Biochemistry and Physiology 1381: 171-176.

Umetsu K., Kosaka S., Suzuki T. 1984. Purification and characterization of a lectin from the beetle Allomyrina dichotoma. Journal of Biochemistry (Tokyo) 95: 239-246.

Vasta G.R., Ahmed H., Tasumi S., Odom E.W., Saito K. 2007. Biological roles of lectins in innate immunity: molecular and structural basis for diversity in self/non-self recognition, pp. 389-406. In: Lambris, J. D. (Ed.) Current Topics in Innate Immunity. Springer, New York.

Volf P., Skarupova S., Man P. 2002. Characterization of the lectin from females of Phlebotomus duboscqi sand flies. European Journal of Biochemistry 269: 6294-6301.

Weis W.I., Drickamer K., 1996. Structural basis of lectin-carbohydrate recognition. Annual Review of Biochemistry 65: 441-473.

Weis W.I., Kahn R., Fourme R., Drickamer K., Hendrickson W.A. 1991. Structure of the calcium-dependent lectin domain from a rat mannose-binding protein determined by MAD phasing. Science 254: 1608-1615.

Weis W.I., Taylor M.E., Drickamer K. 1998. The C-type lectin superfamily in the immune system. Immunological Reviews 163: 19-34.

Wheeler M.B., Stuart G.S., Hapner K.D. 1993. Agglutinin mediated opsonization of fungal blastospores in Melanoplus differentialis (Insecta). Journal of Insect Physiology 39: 477-483.

Wilson R., Chen C., Ratcliffe N.A. 1999. Innate immunity in insects: The role of multiple, endogenous serum lectins in the recognition of foreign invaders in the cockroach, Blaberus discoidalis. Journal of Immunology 157: 1590-1596.

Yu X-Q., Kanost M.R. 2000. Immulectin-2, alipopolysaccharide-specific lectin from an insect, Manduca sexta, is induced in response to gram-negative bacteria. Journal of Biological Chemistry 275: 37373-37381.

Zelensky A.N., Gready J.E. 2005. The C-type lectin-like domain superfamily. FEBS Journal 272: 6179-6217.

MOATAZA A. DORRAH, TAHANY H. AYAAD, AMR A. MOHAMED AND TAHA T.M. BASSAL *

Entomology Department, Faculty of Science, Cairo University

* Author for correspondence. Email: tahabasal@gmail.com
Table 1. Purification schedule of the 5th instar
S. gregaria serum lectins.

Purification step               Volume     Protein    Hemagglutinating
                                 (ml)      (mg/ml)     activity (HA)

A- Crude lectins and
  salting out
Whole serum (crude lectins)       25        2.28            512
70% saturated                     150       0.62            256
  [(N[H.sub.4]).sub.2]
  S[O.sub.4]
B- DEAE cellulose
  chromatography

Peak I                            18        1.99             8
Peak II                           24        1.73             16
Peak III                          18        2.04             16

C- Affinity
  chromatography
Pool 1                            7.5       0.85            128
Pool 2                            7.5       0.66            256
Poo13                              6        0.55            256

Purification step                Specific       Yield (%)
                                 activity
                              (HA/mg protein)

A- Crude lectins and
  salting out
Whole serum (crude lectins)        224.5           100
70% saturated                      412.9           50
  [(N[H.sub.4]).sub.2]
  S[O.sub.4]
B- DEAE cellulose
  chromatography

Peak I                             4.02           1.562
Peak II                            9.24           3.125
Peak III                           7.84           3.125

C- Affinity
  chromatography
Pool 1                              150            25
Pool 2                             387.8           50
Pool 3                             465.4           50

Table 2. Hemagglutinating activity (HA) (a) of the 5th instar
S. gregaria serum lectins contained in the collected pools from
affinity chromatography, against a group of vertebrate RBCs.

                                 HA (b)

                [P.sub.1]      [P.sub.2]      [P.sub.3]

Rabbit             128          256-512          256
Sheep               4              32             1G
Human:
  [A.sup.+]         4              4              0
  [B.sup.-]         0              16             0
  [O.sup.-]         4              4              0
Rat                 0              4              0
Cuinea pig          0              0              0
Horse               0              0              0

(a) Measured in a sample pool of 20 insects for each RBC type.
(b) Standard assay condition: using TBS/Ca-Mg. HA expressed as
[log.sub.2] titer.

Table 3. Effects of divalent canons and EDTA (canon chelator)
on hemagglutinating activity (HA) of the lectins [Sg.sub.1],
[Sg.sub.2], and [Sg.sub.3] isolated from serum of 5th instar
S. gregaria.

                                              HA

                            [Sg.sub.1]    [Sg.sub.2]    [Sg.sub.3]
[Ca.sup.2+]-- free medium        0             0             4
20 mM [Ca.sup.2+a]              128         256-512         256
20 mM [Mg.sup.2+]             G4-128          128           128
20 mM [Zn.sup.2+]               128           128           128
20 mM [Mn.sup.2+]                4             4             4
10 mM EDTA                       0             0             0
5 mM EDTA                        4             4             4

(a) Standard assay condition; i. e., reference value of HA.

Table 4. Inhibition by sugars and glycoconjugates to
hemagglutinating activity (HA), of lectins [Sg.sub.1],
[Sg.sub.2], and [Sg.sub.3], isolated from serum of the 5th
instar S. gregaria, against rabbit RBCs.

                                       Minimum concentration (a)
                                      (mM/or %) required for 50
                                   inhibition (I[C.sub.50]) to HA

                                   [Sg.sub.1]  [Sg.sub.1]  [Sg.sub.1]

Monosaccharide
D-(+)-galactose                       100         100         100
D-(+)-glucose                         100          50         100
L-(+)-rhamnose                         25          6           25
Oligosaccharides
Lactose                               100         100         100
Raffinose                              12          6           6
Sucrose                               100         100         100
N-acetylated sugars
N-acetyl-D-galactosamine              100          50          50
Others
[alpha]-methyl-D-galactose             12          12          25
[alpha]-m-nitrophenyl-D-galactose      6           6           6
[alpha]-p-nitrophenyl-D-galactose      6          < 6         < 6

(a) Data presented are from experiments repeated three times.
Values > 200 mM [(D-(+)-mannose, trehalose, and
N-acetyl-D-glucosamine] or >1 % (zymosan and laminarin)
indicate that no inhibition of agglutination was observed. All
inhibitions were of two wells unless otherwise indicated.
COPYRIGHT 2009 The Orthopterists' Society
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2009 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Dorrah, Moataza A.; Ayaad, Tahany H.; Mohamed, Amr A.; Bassal, Taha T.M.
Publication:Journal of Orthoptera Research
Article Type:Report
Geographic Code:7EGYP
Date:Jan 1, 2009
Words:6712
Previous Article:Annotated check-list of Orthoptera of Libya.
Next Article:A new lobeattid insect from the Permo-Carboniferous of Niedermoschel, southwestern Germany (Archaeorthoptera).
Topics:

Terms of use | Privacy policy | Copyright © 2021 Farlex, Inc. | Feedback | For webmasters