Lectins of general arthropods

Lectins may interact either with ‘self’ glycoconjugates or with glycosylated components of viral, bacterial, protozoan and metazoan pathogens. Soluble or membrane-bound invertebrate lectins take part in the processes of cell adhesion, opsonization, phagocytosis and cytolysis (Vasta & Marchalonis, 1983). They are clearly important molecules involved in humoral and cellular reactions for recognition and defence by opsonization of non-self objects in the inner environment of arthropods. Lectins may also play a part in the transmission of pathogens by vectors, for instance as receptors, homing factors, differentiation factors, etc. A great number of lectins of various invertebrates have been described (Yeaton 1982a,b; Ratcliffe et al. 1985). From the viewpoint of the structure and function, the best known arthropod lectins are those from the horseshoe crabs, Limulus polyphemus and Tachypleus tridentatus, and from the scorpions Centruroides sculpturatus and Parauroctonus (Vasta & Marchalonis, 1984; see Olafsen, 1986, 1996 for reviews). Almost all plasma lectins isolated from the above mentioned group of chelicerates specifically bind N-acetyl neuraminic (sialic) acid and other N-acylamino-carbohydrates. Studies on insect immunity show many molecular aspects common to invertebrate and vertebrate immune responses. For instance, there is a great similarity in the clotting mechanisms of haemolymph and blood. Remarkably, one of the four characterized haemolymph clotting factors of Limulus, factor C (serine protease zymogen), resembles in structure and probable function the selectin family of cell-adhesion molecules. It belongs to C-type (Ca²⁺ ion dependent) chimerolectins (for review see Iwanaga, 1993; Hoffmann, 1995).

Recently, lectins purified from haemolymph of the Japanese horseshoe crab, T. tridentatus, and called tachylectins 5A/5B, have been characterized and cloned (Gokudan et al. 1999). Both tachylectins (Tl-5A/5B) are proteins of high structural and sequentional similarity to mammalian ficolins. However, Tl-5A/5B have no effector collagen domains in their structure and no lectin-associated serine proteases, as found in mammalian ficolins. Tl-5s, together with other defence molecules released from haemocytes, is employed in effective innate immunity against invading pathogens (Kawabata & Tsuda, 2002).

Lectins of disease vectors

Despite the great influence of vector-borne pathogens on humans and domestic animals, relatively little information is available on lectins of invertebrate vectors, with the exception of reduviid bugs, tsetse flies, mosquitoes and sand flies (Pereira, Andrade & Ribeiro, 1981; Wallbanks, Ingram & Molyneux, 1986; Maudlin & Welburn, 1988; Gomes, Furtado & Coelho, 1991; Volf, 1993; Hypša & Grubhoffer, 1995; Ratcliffe et al. 1996; Chen & Billingsley, 1999).

The best studied vector/parasite system are lectins of various tsetse flies (Glossina spp.) (Maudlin & Welburn, 1988; Welburn & Maudlin, 1990; Ingram & Molyneux, 1991). Lectins have been demonstrated in the gut and haemolymph of tsetse flies that can function as signalling factors of maturation for African trypanosome species or, on the contrary, lytic (killing) factors. In this case, an interaction between the gut lectin of the tsetse fly and symbiotic rickettsia-like organisms, or with products of their saccharide metabolism, is a major factor affecting the life cycle of trypanosomes in tsetse flies (Maudlin & Welburn, 1988). A similar interference between symbiotic rickettsia with protozoa was thought to be involved in ticks without considering lectin activities (Smith et al. 1976). Sand flies, the vectors of Leishmania spp., are another group of vector arthropods that have been well studied with regards to midgut lectins and their receptor function in the process of Leishmania transmission (Volf et al. 1994; Palánová & Volf, 1997; Volf, škařupová & Man, 2002).

Lectin–carbohydrate interactions participate in the vector–pathogen/parasite relationship. Tissue-specific lectins of blood sucking arthropods interact with glycosylated molecules of receptors on the surfaces of pathogens or their cells within appropriate developmental stages and vice versa. For instance, bacteria or other intracellular parasites have often employed their surface lectins to enter into phagocytes by the mechanism of lectinophagocytosis (Ofek & Sharon, 1988).

Tick lectins in overview

The lectins of ticks have not received the same level of attention as those of insects. A role for tick haemolymph lectins in transmission of pathogens by ticks has been suggested (Munderloh & Kurtti, 1995). Lectin/haemagglutinin activites in the haemolymph of four tick species, Ixodes ricinus, Ornithodoros tartakovskyi, O. tholozani (papillipes) and Argas polonicus have been described (Vereš & Grubhoffer, 1990; Grubhoffer & Maťha, 1991; Grubhoffer, Vereš & Dusbábek, 1991). The partially characterized tick lectins have affinity for sialic acid and N-acetyl-D-glucosamine but differ in their binding specificity to other sugars. It is postulated that these lectins can interact with glycosylated structures of transmitted pathogens, but this has not yet been proved. I. ricinus haemolymph lectin was immunolocalized in tick tissues and its distribution supports the idea that the lectin is produced and/or stored in haemocytes and contributes to the immune system by recognition of foreign substances (Kuhn, Uhlíř & Grubhoffer, 1996). Like limulin, the sialic acid-specific lectin of the horseshoe crab, Limulus polyphemus, and other sialic acid-specific lectins of chelicerates, the haemolymph lectin of ixodid ticks might also recognize a wide range of Gram-negative bacteria due to its site specificities for N-acetyl-D-glucosamine, D-galactose and 2-keto-3-deoxyoctonate acid (Vasta & Marchalonis, 1983; Grubhoffer et al. 1991; Kuhn et al. 1996). The haemocytes of I. ricinus phagocytose spirochaetes of Borrelia burgdorferi by the coiling method, and coiling phagocytosis is thought to be a lectin-mediated process (Kuhn et al. 1994).

Kamwendo et al. (1993) reported haemagglutination activities in the haemolymph, gut homogenates and salivary glands in the tick Rhipicephalus appendiculatus, the vector of East Coast fever. This is a serious tick-borne disease of cattle caused by the protozoan parasite Theileria parva (see chapter by Bishop et al. in this Supplement). Bovine red blood cells were found as the most sensitive detection system for all tissue homogenates examined with no significant differences between those in extracts from unfed and fed ticks (Kamwendo et al. 1993). However, human erythrocytes of the ABO system and rabbit red blood cells showed significantly higher values of haemagglutination titres in tissue samples coming from fed than unfed ticks, with the highest increase recorded in the gut extract. Later, Kamwendo et al. (1995) focused particularly on a specific functional role of the tick salivary gland haemagglutinin in terms of the tick's capability to transmit T. parva parasites. R. appendiculatus gut lectin activity also influences transmission of the causative agent of theileriosis in Africa with agglutination of piroplasms (E. Sebitosi, personal communication). This has led to the conclusion that the protein–carbohydrate interactions of piroplasms in the inner environment of tick might be a target for the control of theileriosis. Many functional and morphological aspects of tick tissues are involved in the process of parasite transmission (Friedhoff, 1990). However, little is known about the molecular aspects of cell communication and tick interactions with the envelope components of transmitted pathogens. The specific part of the review which follows consists of two sections devoted to tissue specific lectins of the hard tick, Ixodes ricinus, and the soft tick, Ornithodoros moubata. Due to a lack of published data on tick lectins, most results presented are based on the research of the Tick Lectin Group in the Institute of Parasitology České Budějovice, Czech Republic (see Table 1).

Gut

The sheep tick, Ixodes ricinus, which transmits tick-borne encephalitis (TBE) virus and the Lyme disease-causing spirochaete Borrelia burgdorferi sensu lato is the most important tick vector in Europe. Gut haemagglutinating activity of I. ricinus has been found and partially characterized (Uhlíř, Grubhoffer & Volf, 1996). Native mouse erythrocytes were the most sensitive detection cell system in a wide pH range of 6·5–8·0. Binding specificity analysis showed that simple sugars N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, rhamnose, dulcit and glycoconjugates fetuin, hyaluronic acid, laminarin (β-1,3 glucan) and bacterial LPS strongly inhibit haemagglutination. Binding characteristics of the tick gut agglutinin are consistent with LPS-binding agglutinins in the gut tissue of other blood-sucking arthropods (Grubhoffer et al. 1997). It seems that the midgut agglutinin as a potential LPS-binding protein (LPS-BP) could (in co-operation with digestive enzymes) affect Gram-negative spirochaetes B. burgorferi passing through the gut epithelium. Midgut extracts from unfed ticks lack a haemagglutinating activity, which is consistent with observations of Kamwendo et al. (1993) who worked on R. appendiculatus. Mouse polyclonal antibodies raised against midgut haemagglutinating activity (Yeaton, 1982a) recognized, by western blotting, four proteins with molecular weights of 37, 60, 65, and 73 kDa as putative structural components of the lectin(s) (Uhlíř et al. 1996). Whereas the 37 and 60 kDa proteins are glycoproteins modified by both high mannose and complex N-glycans, the 70 kDa subunit is modified by complex type of glycans only (Uhlíř et al. 1994). Using specific mouse polyclonal antibodies, the midgut haemagglutinin/lectin complex was immunohistochemically localized solely in the midgut cells, and not in the gut content or other tick tissues including haemolymph (Uhlíř et al. 1996).

Two agglutinins/lectins of the gut haemagglutinating complex in I. ricinus were isolated from the gut homogenate after delipidation by acetone extraction (Durnová, 1998). Both agglutinins/lectins were purified by affinity chromatography either on immobilized bovine submaxillary mucine (BSM) or laminarin (β-1,3 glucan). Using SDS–PAGE, a 65 kDa protein was found as the main agglutinin with binding affinity to BSM, and a 37 kDa protein showed strong binding specificity for laminarin (β-1,3 glucan). Although crude gut homogenate had no binding affinity for free sialic acid in the haemagglutination inhibition assay, the delipidated proteinaceous ‘acetone powder’ prepared from the crude gut homogenate showed a strong ability to bind sialic acid. It seems to be likely that lipids, including glycolipids abundant in the gut tissue, hide the lectin binding site for sialic acid. The pure 65 kDa agglutinin/lectin also maintained the affinity for free sialic acid. Mouse antibodies raised against the entire haemagglutinating activity of the crude gut homogenate recognized both proteins. Because, the 37 kDa gut agglutinin/lectin functions as a β-1,3 glucan-binding protein, it might be part of the defence machinery protecting the tick against fungal infections.

Factors taking place in processes of pathogen/parasite transmission by ticks were reviewed by Friedhoff (1990). According to Friedhoff (1990), the midgut agglutinin/LPS-binding protein may belong to factors associated with the development of pathogens/parasites in the digestive tract of the tick. The process of blood digestion in ticks differs from that of haematophagous insects, where protein digestion proceeds rapidly and takes place in the lumen of the gut (Gooding, 1972). Instead, digestion in ticks is intracellular (Balashov, 1972). It proceeds slowly, and the pH optimum of proteases is about 3·0, while the pH of the gut content is 6·5 (Akov, Samish & Galun, 1976). In hard ticks, the blood meal is taken up by the digestive cells by means of fluid-phase endocytosis and via receptor-mediated endocytosis. Large particles such as whole cells are phagocytized. The binding activity of the midgut agglutinin/LPS-BP is pH dependent. At pH 3·0, which is an optimum value for the gut proteases, the gut agglutinin does not work at all whereas at pH 6·5, which corresponds to the gut content, a haemagglutinating activity of this binding protein reaches its optimum (Uhlíř et al. 1996). Therefore, the midgut agglutinin/LPS-BP could be involved in the preparatory phase of blood meal digestion, in addition to its role in defence.

Haemolymph (plasma and haemocytes)

Most lectins isolated from arthropods are constituents of the haemolymph, the extracellular circulating fluid that fills the body cavity or haemocoel. The haemolymph, especially haemocytes and large protein complexes, is physically isolated from direct contact with body tissues by basal laminae. However, haemocytes, which are cells within the haemolymph, are in direct contact with this fluid (Mullins, 1985). Haemocytes are immunocytes of arthropods (Gupta, 1991); however, they contribute to nutritional functions, too (Gupta, 1985). Haemolymph lectins and other humoral immune factors in the haemolymph are produced and secreted into the haemolymph mainly by haemocytes and fat body cells. Recently, Gudderra et al. (2002) have published a comprehensive review on haemolymph proteins in ticks emphasizing the structural and functional features of vitellogenin and other tick proteins such as macroglobulins, heme-lipoproteins, antimicrobial proteins and lectins.

Haemagglutinating activity from the haemolymph of I. ricinus was characterized as Ca²⁺-dependent binding activity of the lectin with a subunit molecular size of 85 kDa by non-reducing SDS–PAGE, and binding affinity to sialic acid as well as N-acetyl-D-glucosamine and D-galactose (Grubhoffer et al. 1991, and unpublished observations). Sialic acid-binding lectins are common in chelicerates (Mandal & Mandal, 1990; Wagner, 1990; Zeng & Gabius, 1992), and recognize Gram-negative bacteria. Mouse polyclonal and monoclonal antibodies prepared toward a lectin haemagglutinating activity (Yeaton, 1982a) of the tick haemolymph identified a protein of 85 kDa as a structural lectin subunit. A semipurified plasma fraction highly enriched with a haemagglutinating activity was obtained by the gel filtration FPLC on Superose 12 (unpublished observations). The lectin was localized to the haemocytes in I. ricinus (Kuhn et al. 1996). Immunoreactivity with poly- and monoclonal antibodies was detected in the granules of both types of granular haemocytes, on the plasma membrane of haemocytes, and on the basal laminae surrounding the haemocoel (Figs. 1, 2). Furthermore, cells attached to the midgut invaginations of Géné's organ and granular inclusions of nephrocytes were labeled. The haemolymph- and haemocyte-associated immunoreactivity supports the idea that this haemolymph lectin may function as a recognition molecule in the immune system of I. ricinus. Therefore, the haemolymph lectin could be involved in transmission of Borrelia burgdorferi as a defence molecule in tick haemolymph/haemocytes. Johns et al. (2001a,b), studying differences in vector capability of various tick species for Borrelia transmission, have suggested a potential role of immune molecules. Several studies have shown that B. burgdorferi migrate from the midgut across the haemocoel to the salivary glands during tick feeding (Ribeiro et al. 1987; Zung et al. 1989). However, less than 5% of midgut population of spirochaetes survived to reach salivary glands (Coleman et al. 1997). Another function could be in the protection of eggs that are coated with secretions by Géné's organ (Kuhn et al. 1996).

Fig. 1. Indirect immunofluorescence detection of the plasma lectin in the haemocytes Ixodes ricinus tick using mouse monoclonal antibody 3/D3/C5 to the plasma lectin activity of I. ricinus (diluted 1[ratio ]100). GII – type II of granular haemocytes; PL – plasmatocyte; T – trachea; M – midgut. Laser confocal microscopy (courtesy Dr K.-H. Kuhn).

Fig. 2. Electron micrographs of immunogold-silver labeling of haemocytes with mouse polyclonal antibodies to the plasma lectin activity of Ixodes ricinus tick. Granular inclusions of type-II granular haemocytes (courtesy Dr K.-H. Kuhn).

The distribution of lectin immunoreactivity, especially the labeling of membranes and granular inclusions of haemocytes and of the basal laminae surrounding the haemocoel, strengthens the assumption that the haemolymph lectin is produced or stored in haemocytes and contributes to the immune system of I. ricinus. Like limulin, the sialic acid-specific lectin of Limulus polyphemus, and other sialic acid-specific lectins of chelicerates, the haemolymph lectin of I. ricinus might also recognize a wide range of Gram-negative bacteria due to its additional specificities for N-acetyl-D-glucosamine, D-galactose, and unrelated molecules such as 2-keto-3-deoxyoctonate (Vasta & Marchalonis, 1983; Grubhoffer et al. 1991). Moreover, it was shown in in vitro assays that B. burgdorferi is a target for the immune system of I. ricinus and is readily ingested by phagocytic haemocytes (Kuhn et al. 1994). Additionally, Kurtti et al. (1988) have reported that B. burgdorferi spirochaetes readily adhere to tick cells in tissue culture. Therefore, the lectin might mediate the interaction of pathogens like B. burgdorferi or tick-borne encephalitis (TBE) virus with the tissues of the vector. The main binding specificity of the haemolymph lectin is for sialic acid (Grubhoffer et al. 1991), which is present in the wall of B. burgdorferi (Hulínská, Volf & Grubhoffer, 1992). In addition, the haemolymph lectin of the tick can bind complex oligosaccharides lacking sialic acid. Therefore, the protein gpE of TBE virus, the only structural glycoprotein that exhibits biantennary complex oligosaccharides (Grubhoffer et al. 1990) could interact with the haemolymph lectin. The localization of lectin immunoreactivity in diverse tissues and cells of the tick does not imply that all labeled proteins are associated with the same function. Probably, polyclonal antibodies recognize common epitopes in functionally diverse proteins with multiple domains. Calcium-dependent globular lectin domains (C-type lectins) (Drickamer, 1993; Drickamer & Taylor, 1993) that are components of vertebrate haemostasis and complement systems (Thiel & Reid, 1989; Sastry & Ezekowitz, 1993; McEver, 1994) and a related protein, factor C (Muta et al. 1991), and an immune protein of the complement–coagulation system of L. polyphemus (Iwanaga, 1993), are examples of this phenomenon.

A cDNA library was constructed from unfed I. ricinus females and screened using a probe obtained from the lectin gene of B. burgdorferi B31 (unpublished observations). Clones showing the strongest hybridization signals were selected and sequenced. The amino acid sequence of a putative carbohydrate recognition domain of the recombinant clone from an I. ricinus cDNA library was compared with those of other lectins (Fig. 3). The predicted amino acid sequence of one clone, termed r58, revealed strong homology (81%) with four lectins from Periplaneta americana cockroach haemolymph (Kawasaki, Kubo & Natori, 1996).

Fig. 3. Comparison of carbohydrate recognition domain of I. ricinus lectin (r58) with those of different lectins. The lectins compared are: r58, clone from I. ricinus cDNA library (unpublished observations); PL21, 31, 9 & 25, four lectins from Periplaneta americana cockroach hemolymph (Kawasaki et al. 1996); HLEC, human hepatic lectin (Halberg et al. 1987); FCE2, mucose immunoglobulin Fc receptor (Bettler et al. 1989); MABC, rat mannose-binding protein A (Drickamer, Dordal & Reynolds, 1986); and MANR, human macrophage mannose receptor (Taylor et al. 1990). Dark grey represents identitical amino acid residues and light grey represents similar residues.

Salivary glands

Kamwendo et al. (1995) were the first to study the role of the tick salivary gland haemagglutinin of the tick R. appendiculatus in relation to the mechanism of transmission of the protozoan parasite Theileria parva. A significant increase was observed in T. parva acinar infection rates in the salivary glands of R. appendicalatus fed on ears of rabbits infused with melibiose and raffinose (haemagglutination inhibiting sugars). In contrast, mannose and turanose (non-inhibitory sugars) did not affect of T. parva acinar infection rates. Haemagglutinating activity in I. ricinus salivary gland (SG) extracts was highest against mouse red blood cells, and was inhibited by sialic acid, D-fructose, L-rhamnose, D-trehalose and several glycoconjugates (unpublished observations). Mouse polyclonal antibodies raised against the haemagglutinating activity were used in western blotting to identify a 70 kDa protein responsible for SG haemagglutinating activity. It is likely that the lectins have a significant role in the entry of pathogens into the salivary gland and, hence, the salivary gland lectin in I. ricinus will be important in the transmission of TBE and B. burgdorferi.

Haemolymph (plasma and haemocytes)

The soft tick Ornithodoros moubata is a vector of African swine fever virus and Borrelia duttoni. Laboratory colonies of this species are valuable experimental models that have been employed to answer questions about the tick physiology and morphogenesis, such as vitellogenin synthesis (Chinzei, Chino & Takahashi, 1983; Chinzei & Yano, 1985; Chinzei, 1988; Gudderra et al. 2002, for review), defensins as immunopeptides (Nakajima et al. 2001), and peritrophic matrix synthesis (Grandjean, 1984). Recently, the immune response of the argasid tick O. moubata induced by infection with the filarial worm Acanthocheilonema viteae has been studied by Hutton, Reid & Towson (2000). A plasma/haemolymph lectin of O. moubata has been thoroughly studied (Grubhoffer & Kovář, 1998; Kovář, Kopáček & Grubhoffer, 2000), though we also have evidence of some lectin activity in the salivary glands and gut of O. moubata (unpublished observations).

A lectin with high haemagglutinating activity, called Dorin M, was identified in the plasma of O. moubata (Grubhoffer & Kovář, 1998; Kovář et al. 2000). The activity of the plasma lectin could be efficiently inhibited by sialic acid, N-acetyl-D-hexosamines and acid (sialo)glycoproteins. Dorin M was purified to homogeneity using two different isolation systems: affinity chromatography on a column of bovine submaxillary mucin conjugated to Sepharose 4B elution with N-acetyl-D-glucosamine (Grubhoffer & Kovář, 1998), or chromatography on Blue-Sepharose followed by anion exchange FPLC on a MonoQ column (Kovář et al. 2000). Dorin M is a glycoprotein with N-type of glycosylation that, in the native state, forms aggregates with molecular mass of about 640 kDa. Non-reducing SDS–PAGE revealed that the lectin consists of two non-covalently bound subunits migrating closely around 37 kDa. After chemical deglycosylation, only one band of about 32 kDa, without haemagglutinating activity, was detected. A tryptic cleavage of purified Dorin M was performed and the amino acid sequence of three resulting internal peptides fragments were determined by Edman degradation. Using degenerate primers and a haemocyte cDNA template, a 290 bp PCR product was cloned and sequenced. The haemocyte origin of the cDNA template demonstrated that haemocytes are a site of Dorin M synthesis (unpublished observations). The sequence of the complete Dorin M cDNA was completed by the method of RACE. The derived amino acid sequence clearly showed that the Dorin M has a fibrinogen-related domain and exhibited the highest significant similarity with tachylectins 5A (38%) and 5B (32%) from the horseshoe crab. Since tachylectins function as non-self recognizing molecules, we believe that Dorin M may play a similar role in innate immunity of the tick and possibly also in pathogen transmission (unpublisahed observations).

More than ten years ago the research on tick haemolymph lectins was initiated with the study of O. tartakovskyi and O. tholozani (papillipes), species closely realated to O. moubata (Vereš & Grubhoffer, 1990; Grubhoffer et al. 1991). Sialic acid-specific lectins are present in Ornithodoros tick plasma/haemolymph with minor differences in binding affinity to other free carbohydrates, such as N-acetyl-D-glucosmine, D-glucosamine and D-galactosamine. Two bands of 30 and 35 kDa in tick haemolymph and a single component of 35 kDa in the haemocyte lysate were distinguished by immunoblotting with polyclonal antibodies. In whole body homogenates, in addition to the 30 kDa protein, a 150 kDa band was identified. Both structural units of haemolymph lectin activity were found to be glycoproteins in the lectin affinoblotting, The 30 kDa protein was modified by the complex type of glycans with terminal D-galactose, whereas the 35 kDa protein was complexing with high mannose type of glycans (Grubhoffer et al. 1991).