ISSN 0306-3623/98 $19.00 1 .00 PII S0306-3623(97)00014-1 All rights reserved
Gen. Pharmac. Vol. 30, No. 5, pp. 795–800, 1998 Copyright 1998 Elsevier Science Inc. Printed in the USA.
Adhesins of Immunoglobulin-like Superfamily from Earthworm Eisenia foetida* Maja Popovic´,1 Terezija Hrzˇenjak, † Mira Grdisˇa2 and Snjezˇana Vukovic´1 1
1 Department of Biology, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia and 2Department of Molecular Medicine, Ruder Bosˇkovic´ Institute, Bijenicˇka 54, 10000 Zagreb, Croatia
ABSTRACT. 1. From the biologically active extract (G-90) isolated from the tissue homogenate of Eisenia foetida immunogloubin-like structures were isolated and named G-90/4. 2. G-90/4 in nanogram concentrations stimulated cell proliferation more than did the original G-90. It lyses cells in microgram concentrations. 3. G-90/4 acts as an adhesion molecule between the receptors of adjacent cells. 4. The increase in proliferative activity was accompanied by the elevation of cytoplasmic protein containing tyrosine. 5. Immunohistochemical analyses confirm immunoglobulin-like transmembrane structures in the connective and muscular tissues of E. foetida. gen pharmac 30;5:795–800, 1998. 1998 Elsevier Science Inc. KEY WORDS. Earthworm, adhesins, immunoglobulin-like
INTRODUCTION Adhesion is a property indispensable for the life of each cell. It is manifested through adhesive structures—molecules of originally distinct genetic codes. As transmembrane cell receptors, adhesins maintain cell–cell and cell–extracellular matrix binding. In this respect, adhesins take part in mitosis, formation of multicellularic organisms, embryogenesis, differentiation, morphogenesis, transmission of information, cell movement, aggregation of thrombocytes and dissemination of oncogenically transformed cells (Alberts et al., 1994; Edelman and Crossin, 1991; Hynes, 1992). The mechanisms of receiving and transmitting messages at the cell–cell level are largely uniform in organisms throughout the entire evolutionary system. The reason for this uniformity is the stability of the genetic code for macromolecules mediating forementioned functions. Although mutations—often mere duplications of these codes—generate a number of variants, they have preserved basic properties at the genetic, macromolecular and functional levels. The most typical examples are the members of the immunoglobulin (Ig) superfamily (Hood et al., 1985; Matsunaga, 1985; Oikawa et al., 1987; William, 1984). The first structurally and functionally defined adhesins of the Ig superfamily were the nerve-cell adhesion molecules (N-CAMs) that take part in the transmission of nerve impulses. They are transmembrane receptors where part of the extracellular matrix is built of an Ig-like domain. They are Ca21 independent. We currently know of more than 100 N-CAM homologous structures from different biological sources (Alberts et al., 1994; Cheung et al., 1993; Edelman and Crossin, 1986; Hlavin and Lemmon, 1991). Transmembrane receptors that are Ca21 dependent are cadherins, whose great variety depends on alternative mRNA splicing (Takeichi, 1990; Tarone et *The results of this investigation contain the most important parts of the first author’s Master’s thesis. †To whom correspondence should be addressed. Received 26 July 1996.
al., 1992). Adhesins with heteromeric transmembrane parts are integrins. They are Ca21 and Mg21 dependent; they bind to target proteins of the extracellular matrix and engage in significant biological processes such as thromboses, inflammations and carcinomas (Hynes, 1992). All these transmembrane receptors have a cytoplasmic part with tyrosine kinase activity, and they enhance the proliferation of cells. Selectins are transmembrane proteins with heterophilic activity, are Ca21 dependent and bind to specific oligosaccharide groups. They are most numerous in blood and endothelial cells (Hynes, 1992; Sharon, 1979). Throughout the phylogenetic tree of the invertebrata, various adhesive structures and their functions have been investigated and described. The importance of adhesion is pronounced in immunoadhesins of the Ig-superfamily structures, which are HIV receptors on CD-4 cells (Chamon et al., 1990). By its structure, the poliovirus receptor also belongs to the Ig superfamily (Morrison and Racaniello, 1992). An adhesin similar to intercellular adhesion molecule 1 (I-CAM 1) was detected in E. coli (Martin et al., 1993). A galactose-specific adhesin was found in Entamoeba histolytica (Petri et al., 1994). Thrichomonas vaginalis binds with adhesins on its surface to target epithelial cells (Arroyo et al., 1992). Proteins with Ig-like domains and a protein-kinase domain are found in the Nematoda (Benian et al., 1993). “Minititins”—Ig-like protein structures binding to myosin—were found in muscles of the Mollusca (Vibert et al., 1993). C-CAM molecules are pronounced in insect cells. They induce the aggregation of cells. They contain four Ig domains (Cheung et al., 1993). The role of adhesins in embryonic development was determined in Drosophila (Zusman et al., 1993). Hemolins are adhesins of the Ig superfamily in insects, binding as a protection to bacterial surfaces (Sun et al., 1990). Proteins with the same adhesion roles, without an Ig-like structure, were also detected in the earthworm Eisenia foetida (Rejnek et al., 1991; Tuckova et al., 1991). Owing to its biological properties and position on the phylogenetic tree, E. foetida is often investigated. Research on its immunological recognition, memory, regeneration, mitogenicity, hemo-
M. Popovic´ et al.
796 lyticity and bacteriostaticity was conducted in its coelomic fluid and tissue extracts (Cooper, 1976; Cotuk and Dales, 1984; Du and Duprat, 1986; Herland and Deligne, 1964; Hirigoyenberry et al., 1990). The tissue extract of E. foetida, named G-90, shows lectin properties and biological activities: mitogenicity, tumorostaticity in vivo and in vitro and bacteriostaticity. It is neither a mutagen nor a carcinogen (Hrzˇenjak et al., 1992). The presence of insulin-like molecules with the property of a growth factor was determined (Hrzˇenjak et al., 1993). Two serine proteases were isolated (Bozˇic´, 1996. G-90 has high anticoagulation and fibrinolytic activity (Popovic´ et al., 1996). The fact that the glycolipoprotein complex G-90 contains macromolecules with significant biological properties inspired the idea of the possible presence of adhesion structures. This possibility is shown in the interaction between the total G-90 and the antibodies to human and animal sera, which, in the method of double gel diffusion, form precipitation arcs as a relation between the receptor and ligand. The present study investigates the macromolecules in G-90 responsible for the previously mentioned effect and their biological potential. MATERIALS AND METHODS
Animals Earthworms, E. foetida, (Annelida, Oligochaeta, Lumbricidae), were used for isolating the biologically active compound.
Isolation of G-90 Glycolipoprotein extract (G-90) was obtained from earthworm tissue as described by Hrzˇenjak et al. (1992).
Determination of protein concentration The protein concentration in a glycolipoprotein mixture of G-90 was determined by the methods of Lowry et al. (1951) and Bradford (1976).
Cell cultures In this study, we used several human cell lines: WI38 (normal fibroblasts), HeLa (cervical carcinoma), MiaPaCa2 (pancreatic carcinoma) and Raji (T-leukemia lymphocytes). The WI38, HeLa and MiaPaCa2 cells were plated at a concentration of 13104 cells/ml in Dulbecco’s Modified Eagle’s Medium DMEM (Gibco, SAD) supplemented with 2% FCS (fetal calf serum; Sigma, St. Louis, MO, USA, SAD) and Raji at a concentration of 13105 cells/ml in RPMI 1690 (Immunological Institute, Zagreb, Croatia) with 2% FCS.
Radial immunodiffusion (Wier, 1978) In these experiments, the commercial agarose plates with incorporated antibodies were used (Immunological Institute, Zagreb, Croatia).
Immunoprecipitation and Western blotting For immunoprecipitation, we used the property of protein A–Sepharose, which binds the Fc fragment of Igs or Ig-like structures. G-90 (15 mg with 600 mg of proteins) was dissolved in 100 ml of PBS buffer, pH 7.6 (0.109 M/L NaCl, 0.20831023 M/L KCl, 0.6331023 Na2HPO4, 0.331023 CaCl2, 0.2431023 MgCl2) and mixed overnight at 148C with 100 ml of 20% protein A–Sepharose in PBS buffer, pH 7.4 (Hjem et al., 1972). After binding, protein A–Sepharose was rinsed five times with cold Ripa buffer, pH 8.6 (0.15 M/L NaCl, 0.01 M/L Tris, 1% Na-deoxycholate, 1% Triton3100, 0.1% SDS), by centrifugation for 10 min at 12,000g. After rinsing, 50 ml of SDS-sample buffer, pH 6.8 (0.0625 M/L Tris, 2% SDS, 10% b-mercaptoethanol, 10% glycerol, 0.04% bromphenol blue) on pellets was added, denatured by heating (5 min/ 1008C) and analyzed by SDS-PAGE. After electrophoresis, Western blotting was performed. Proteins were transferred to nitrocellulose membrane (Immobilon PVDF, Milipore, Bedford, SAD), and Iglike structures were visualized by specific antibodies to human IgG (alkaline phosphatase–labeled goat–anti-human F(ab)9 fragment IgG; Sigma, St. Louis, MO, SAD), IgA (alkaline phosphatase–labeled goat–anti-human F(ab)9 fragment IgA; Sigma, St. Louis, MO, SAD) and IgM (alkaline phosphatase–labeled goat–anti-human IgM; Sigma, St. Louis, MO, SAD), according to the modified method by Stachelin and Gordon (1979).
Effect of G-90 and G-90/4 on elevation level of cytoplasmic proteins with tyrosine The cells (WI38, HeLa, MiaPaCa2; 53105/ml) were plated in Petri dishes in DMEM supplemented with 2% FCS for 24 hr and then for an additional 24 hr in DMEM without FCS. After “starvation,” the G-90 and G-90/4 at concentrations of 10 ng of proteins/ml were added, and samples were taken at 1, 5, 15, 30, 60 and 120 min. Cell suspensions were put on ice, the medium was removed and the cells were rinsed with Dulbecco’s phosphate buffer (0.0081 M/L NaHPO437 H2O, 0.14 M/L NaCl, 0.0005 M/L MgCl236 H2O, 0.00015 M/L KH2PO4, 0.0027 M/L KCl, 0.0009 M/L CaCl237 H2O). The cells were lysed in lysing buffer, pH 8.0 (0.2 M/L Tris, 0.137 M/L NaCl, 0.001 M/L PMSF, 0.001 M/L NaVO4, 1% NP-40, 10% glycerol, 0.15 U/ml Aprotinin) for 15 min. After centrifugation (15 min/13,000g, 148C), the tyrosine kinase activity was determined in cytosol, using specific primary (PY20-mouse monoclonal anti-phosphotyrosine; Transduction Laboratories, Lexington, KY, SAD) and secondary (alkaline phosphatase–labeled goat–antimouse F(ab)9 fragment IgG; Biorad, Richmond, SAD) antibodies.
Immunohistochemical method Localization of Ig-like proteins in E. foetida was performed by the modified method of Pavelic´ et al. (1991), using alkaline phosphatase–labeled goat–anti-human F(ab)9 fragment IgG (Sigma, St. Louis, MO, SAD).
SDS-polyacrylamide electrophoresis (SDS-PAGE) Glycolipoprotein extract (G-90) was separated on gradient (5–15%) polyacrylamide gel (Laemmli, 1970) and stained with Coomassie Brilliant Blue R-250. Low-molecular-weight standards were used (LKB, Pharmacia, Upsala, Sweden).
Statistics The results were analyzed by using two-tailed Mann-Whitney test and Student’s t-test. RESULTS
Dot blotting The experiments were performed according to Towbin and Gordon (1984).
For the purposes of this study, a G-90 complex was prepared, with a protein concentration of 40 mg/ml (Hrzˇenjak et al., 1992). By radial immunodiffusion, we obtained precipitation arcs in the gel stem
Adhesins of Immunoglobulin-like Superfamily
FIGURE 1. SDS-PAGE of G-90 and G-90/4 (after separation on protein A–Sepharose). Proteins were separated by gradient gel (5– 15%) in Tris-glycine buffer, pH 8.3, and 0.8% SDS (4 hr, 108C, 40 mA, 240 V). Gel was stained with Coomassie Brillant Blue R-250. (1) Molecular weight standards (LKB, Pharmacia, Sweden); (2) G-90 (50 mg); (3) G-90/4 (60 mg). from the binding of antibodies to serum IgG and IgA and the protein components of the G-90 complex. Dot blotting carried out on G-90 with alkaline phosphatase—marked antibodies to human IgG confirms binding between Ig-like structures in G-90 and antibodies to serum Igs. The analysis of the G-90 protein complex in SDSPAGE showed 17 protein fractions of molecular mass 14–97 kDa. Through affinity chromatography, by the binding of G-90 to protein A–Sepharose, structures were isolated that antigenically resemble serum Igs. In terms of quantity, 10% of the total G-90 proteins were bound through the process. The isolate obtained was named G-90/4. The isolate was electrophoretically analyzed on SDS-PAGE, parallel with the original G-90. G-90/4 gives lines of the following masses: two dominant fractions of 29.5 and 45.6 kDa; two weaker of 15.9 and 75.5 kDa; and three tender of 40.9, 60.8 and 65.9 kDa (Fig. 1). The receptor–ligand relation in the G-90 and G-90/4 interactions with antibodies to serum Igs was tested by Western blotting with marked antibodies to human serum IgG, IgA and IgM. The antibody to serum IgG bound to G-90 by means of the protein fraction of 45.6 kDa and to G-90 by fractions 15.9, 29.5, 40.9 and 45.6 kDa (Fig. 2A). Anti-IgA gives no reaction with G-90, whereas, with G-90/4, it reacts with fractions of 45.6, 60.8 and 65.9 kDa (Fig. 2B). Anti-IgM reacts with G-90 by fractions of 65.9 and 75.5 kDa, and, with G-90/4 it reacts with protein fractions of 45.6, 65.9 and 75.5 kDa (Fig. 2C). In G-90/4, the protein fraction of 45.6 kDa reacts with all three antibodies and, in G-90, only with anti-IgG (Fig. 2). The proliferation activity of total G-90 for the growth of normaland tumor-cell cultures was established earlier (Hrzˇenjak et al., 1992). A part of this activity certainly belongs to earlier isolated structures similar to insulin (Hrzˇenjak et al., 1993). With the use of dot blotting and antibodies specific to insulin (I5-mouse polyclonal antibodies to human insulin; Institute Ruder Bosˇkovic´, Zagreb, Croatia), such structures have not been detected in G-90/4 (as much as 240 mg
FIGURE 2. Detection of Ig-like structures in G-90 and G-90/4. After SDS-PAGE and Western blotting, nitrocellulose membranes were incubated with antibodies specific to human IgG (A), IgA (B) and IgM (C). (1) Molecular weight standards; (2) human IgG (5 mg); (3) G-90 (50 mg); (4) G-90/4 (60 mg); (5) human IgA (5 mg); (6) G-90 (50 mg); (7) G-90/4 (60 mg); (8) human IgM (5 mg); (9) G-90 (50 mg); (10) G-90/4 (60 mg).
of proteins). The extent of proliferation activity in G-90/4 with respect to the original G-90 was examined on cell lines WI38, HeLa, MiaPaCa2 and Raji. G-90 and G-90/4 acted in concentrations of 1 pg to 10 mg. Reference cultures were grown in the medium containing 2% FCS. Concentrations of 10 ng of G-90/4 have shown the highest proliferation activity. The activity of G-90/4 on all cell lines is higher than the activity of the original G-90. The concentration of 10 mg of G-90 or G-90/4 is the strongest inhibitor of proliferation, depending on cell type. The increase in proliferation of the cells treated with G-90/4 is statistically significant (P,0.05) in relation to proliferation of the cells treated with G-90 (Fig. 3). The events during proliferation were also analyzed microscopically in the cell cultures grown on glass slides. Irrespective of the cell type, G-90 stimulates greater cell growth than does 10% FCS; therefore it was convenient to grow cells in a medium containing 2% FCS (Fig. 4I and IIA, B). The strongest stimulus is a 10-ng concentration of G-90/4, whereas G-90, in the same concentration, provides a much weaker stimulus (Fig. 4I and IID, C). In those cell cultures, the cells form clusters, which is a clear proof of the interaction between cells mediated by added G-90 or G-90/4. The clusters are particularly pronounced in the leukemia T-lymphocyte culture (Raji), which is grown in a suspension. The concentration of 10 mg of both G-90 and G-90/4 inhibits growth in culture (Fig. 4I and IIE, F), and G-90 lyses cell membranes (Fig. 4I and IIG), whereas G-90/4 lyses nuclear membranes and the whole cell (Fig. 4I and IIH). The transmission pathway of a mitogen signal was determined by detecting the activity of proteins with tyrosine. After stimulation of the cells with G-90 (10 ng) or G-90/4 (10 ng), the quantity of cytoplasmic proteins with tyrosine increased. The activity is directly proportional to the duration of stimulation. The reaction is visible with 10% FCS and 10 ng of G-90, whose effect is shown after 120
M. Popovic´ et al.
FIGURE 3. The influence of different concentrations of G-90 and G-90/4 (from 1 pg to 10 mg) on cell proliferation: (A) WI38, (B) HeLa, (C) MiaPaCa2, (D) Raji. The number of cells was determined 48 hr after the addition of G-90 and G-90/4. The results are expressed as the ratio of the number of treated cells to the number of control cells. The significance of the cell proliferation with increasing concentrations of G-90/4 in relation to G-90 was performed by using the t-test (*P,0.05).
min. G-90/4 in 10-ng concentrations shows the same stimulation after 60 min. This finding is valid for cell cultures WI38 and HeLa. The signal is more intense in the cell culture MiaPaCa2: 10% FCS again shows stimulation after 120-min, G-90 after 60-min and G-90/4 after 30-min incubation. Immunohistochemical analyses of cross-sections of E. foetida were obtained by alkaline phosphatase–labeled antibody to human IgG. A positive reaction is visible in the intracellular space of the subepithelial connective tissue and in the adjacent smooth and striated muscular tissue. DISCUSSION It is probable that Ig-like structures exist in the tissue of E. foetida and that their activity consists of biological cell–cell and cell– extracellular matrix interaction (Alberts et al., 1994). Because G-90 is a complex demonstrating high and heterogenous biological potential, it was interesting to isolate and analyze Ig-like structures present in G-90. Affinity chromatography by protein A–Sepharose separated G-90 from all structures having Fc fragments of heavy immunoglobulin chains (Hjem et al., 1972) and named G-90/4. The
analysis of Western blotting indicated a number of proteins of different molecular masses. All antibodies to human serum IgG, IgA and IgM indicated a protein of molecular mass 45.6 kDa. According to its size, this obviously stable structure would be built from several Ig domains. Another protein responsible for binding anti-IgA and antiIgM has a molecular mass of 65.9 kDa. A protein of similar molecular mass was isolated earlier from coelomic fluid by protein A–Sepharose, but it is not homologous to the heavy IgG1 chain (Bilej et al., 1992; Rejnek et al., 1991). Two proteins with a capability of adhesion binding to bacteria also were isolated from coelomic fluid, but their properties do not relate to Igs (Tuckova et al., 1991). The proliferation activity of G-90/4 is higher than that of G-90. Because G-90/4 does not contain insulin-like mitogens, the proliferation activity arises from Ig-like structures. On the bases of available data in the literature and our experiments, we suggest that the contact between cells in cultures is mediated by injection of some G-90/4 structure between the receptors of two cells. Such receptors are most often integrins and might bind ligands that are members of the Ig superfamily. These receptors are sometimes referred to as “counterreceptors” (Alberts et al., 1994; Edelman and Crossin, 1991). Integrins specifically recognize the sequence Arg-Gly-Asp (Hynes, 1992) and
Adhesins of Immunoglobulin-like Superfamily
FIGURE 4. Influence of G-90 and G-90/4 on cell proliferation: left - HeLa, right - MiaPaCa2. The cells were stained with hemalauneosin 48 hrs after G-90 or G-90/4 adding. A: 2% FCS (10310); B: 10% FCS (10310); C: 2% FCS110 ng G-90 (10310); D: 2% FCS110 ng G-90/4 (10310); E: 2% FCS110 mg G-90 (10310); F: 2% FCS110 mg G-90/4 (10310); G: 2% FCS110 mg G-90 (10363); H: 2% FCS110 mg G-90/4 (10363). perhaps bind certain structures of G-90/4. The contact integrin– G-90/4–integrin in the cytoplasmic part of the integrins binds to actin filaments, creating “stress fimbre” formations and placing cells in focal contact. This can result in cells approaching from 50 nm to 15 nm (Tarone et al., 1992). This effect was observed in cell cultures grown on glass slides. Cluster formations—adhesion plaques—are visible. They are most pronounced in the culture of T-leukemia lymphocytes (Raji). It is established that ligands for beta-2 integrin in blood cells are proteins of the Ig superfamliy (Staunton et al., 1989, 1990). Assays carried out in vitro indicate that Ig-like structures of G-90/4 proteins react as ligands for some receptors of treated cells. The results also indicate the presence of tyrosine kinase activity in the cytoplasm of treated cells (Urlich and Schlessinger, 1990). Receptor properties and proliferation activity were discovered in twitchin protein isolated from a small nematode, Caenorhabditis elegans (Benian et al., 1993). Besides the Ig-like part, this protein also has fibronectin-like structures and a tyrosine kinase doman. According to the description of the structure, it is built like a receptor for growth factors or N-CAM molecules. When the concentrations of G-90 or G-90/4 in these cultures were raised from nanograms to micrograms, there was lytic activity in all cultures. Data abound in the literature on proteolytic enzymes isolated from the tissue and coelomic fluid of E. foetida, as well as from G-90 (Bozˇic´, 1996). Such an enzyme with fibrinolytic activity, of 34 kDa molecular mass, was isolated from G-90. On the basis of the data in the literature, it can lyse fibrin and other extracellular proteins, such as collagen and laminin (Alberts et al., 1994; Jeon et
al., 1995). The discovery by Bilej et al. (1995) of a protein with cytolytic activity yet without enzymatic properties, present in the tissue of E. foetida, allows the possibility that this lytic factor remained bound in the G-90 complex and subsequently in G-90/4. Immunohistochemical analysis confirms Ig-like structures in the connective and muscular tissues of E. foetida. A stronger reaction in the connective and muscular tissues is visible on the ventral part of the earthworm’s body. A stronger reaction has been observed in the extracellular matrix. We suppose that Ig-like structures with properties of transmembrane receptors are connected to microfilaments of the muscular cell cytoplasm. In that way, they have a role in the locomotion of the earthworm (Royuela et al., 1995; Walker et al., 1993; Weigl, 1994). Such structures in invertebrates have been described and isolated (Benian et al., 1993). Apparently, there are Ig-like adhesin structures in G-90/4 that initiate the transmission of mitogen signals and the lysis of normal and oncogenically transformed cells. These properties, together within the properties of G-90, indicate the advantages leading to the practical application of G-90 or G-90/4. References Alberts B., Brany D., Lewis J., Raff M., Roberts K. and Watson J. D. (1994) Molecular Biology of the Cell, 3rd edn, Gerland, New York, London. Arrayo R., Engbring J. and Ajderete J. F. (1992) Molecular basis of host epithelial cell recognition by Trichomonas vaginalis. Mol. Microbiol. 6, 853–863. Benian G. M., L’hernauls S. W. and Morris M. E. (1993) Additional sequence complexity in the muscle gene, unc-22, and its encoded protein, twitchin, of Caenorhabditis elegans. Genetics 134, 1097–1104. Bilej M., Brys L., Beschin A., Lukas R., Vercauteren E. and Hanusova R.
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