ISJ 11: 331-336, 2014 ISJ 11: 331-336, 2014 ISSN 1824-307X RESEARCH REPORT Physico-chemical characterization of a natural agglutinin from the hemolymph of a millipede Thyropygus descriptus SrMR Basil-Rose1, MH Ravindranath2, SrPD Mercy1 1Department of Zoology, Holy Cross College, Nagercoil-629 004, India 2Terasaki Foundation Laboratory, Los Angeles, CA, USA Accepted November 12, 2014 Abstract Natural hemagglutinins with specific affinity for the glycocalyx of rabbit erythrocytes is identified in the hemolymph of the millipedes, Thyropygus descriptus, Xenobolus acuticonus, Arthrosphaera disticta and A. craspedota. Of the tested species, maximum hemagglutinability is observed in the hemolymph of the millipede Thyropygus descriptus. Further characterization of the hemolymph agglutinin of Thyropygus descriptus showed optimum agglutinability at pH 6.5 and temperatures 30 - 35 ºC. Starvation up to 20 days had no influence on the hemagglutinability of the hemolymph. Agglutinability was impervious by change in diet, and inclusion of diverse concentrations of cations or chelators in the buffer. The agglutinin, though agglutinates rabbit, rat and human A erythrocytes, when pre-adsorbed with erythrocytes of a particular species, loses its ability to agglutinate erythrocytes of any species suggesting the presence of a single agglutinin in the hemolymph. In general, the agglutinating activity of the agglutinin is inhibited by the glycoproteins porcine stomach mucin, lactoferrin, bovine submaxillary mucin, transferrin, fetuin and the sugars N-acetyl galactosamine, N- acetyl lactosamine, lactose, galactose and N-acetyl neuraminic acid. The sialic acid specificity of the agglutinin is revealed by the reduction in hemagglutination activity when treated with the desialylated rabbit erythrocytes. Key words: millipede; Thyropygus descriptus; agglutinin; hemolymph; erythrocytes; hemagglutination assay; hemagglutination inhibition assay Introduction Lectins, the sugar binding proteins are found in a wide range of organisms including viruses, bacteria, fungi, plants and animals (Sharon, 2008). They react with sugars in glycolipids, glycoproteins, or oligosaccharides and agglutinate erythrocytes via cell surface glycoproteins and glycolipids (Stromberg et al.,1991; Sharon, 2008). Their specificity is usually defined in terms of a monosaccharide(s) or simple oligosaccharides that inhibit lectin-induced agglutination (Sharon and Lis, 1972; Goldstein et al.,1980). An agglutinin may recognize a part of a sugar (Shimizu et al.,1977), a whole sugar (Bretting and Kabat, 1976), their glycosidic linkages (Koch et al., 1982) or a sequence of sugars (Kobiler and Mirelman, 1980; Mauchamp, 1982). Among protostomian invertebrates that are incapable of synthesizing sialic acids, molluscan ___________________________________________________________________________ Corresponding author: Sr. Basil Rose Holy Cross College Nagercoil Tamilnadu-629 004, India Email: basilrosemr@gmail.com and arthropodan agglutinins recognize a unique kind of sugar called sialic acids (Mullainadhan et al., 1984). The type of sialic acids and the glycosidic linkages with adjacent sugar in an oligosaccharide differ among pathogenic bacteria (Ravindranath and Cooper, 1984) and human cancer (Ravindranath et al.,1985b; Higashi et al., 1985; Kawai et al., 1991). Therefore, lectins specifically recognizing various sialic acids and their carbohydrate binding patterns can be used as tools for identifying various sialyl epitopes on pathogens or in biopsy of malignant tumors. A search for such sialic acid specific lectin is made in a millipede, Thyropygus descriptus, a Diplopodan representative of the Super Class Myriapoda, a new group for lectin study. To develop strategies for affinity purification we have studied the physico-chemical properties of the agglutinin. Material and Methods Materials Millipedes Thyropygus descriptus, Xenobolus acuticonus, Arthrosphaera disticta and A. craspedota used in this investigation were collected from the forest region of Kanyakumari (Anayadi and 331 Table 1 Survey of hemagglutinins in the hemolymph of millipedes HA titer Erythrocytes T. descriptus (n = 25) X. acuticonus (n = 25) A. disticta (n = 25) A. craspedota (n = 25) Rabbit 2048 32 512 1024 Rat 128 0 256 128 Human A 64 0 512 128 Human B 0 0 512 128 Human O 0 0 32 16 Ox 0 0 0 0 Horse 0 0 0 0 Pig 0 0 0 0 Mouse 0 0 0 0 Kodayar) and Tirunelveli (Kalacaud and Thalayanai) Districts, Tamil Nadu, India. Hemolymph collection The arthropodial membrane in between the collum and the adjacent segment was punctured after cleaning the area with wet cotton. The exuding hemolymph was collected in 15 ml polypropylene tubes kept on ice and stored in refrigerator. Erythrocyte collection Blood from different mammals was collected by venipuncture of the ear (rabbit), fore arm (Human A, B, O and horse), cardiac puncture (mouse and rat) and from the slaughter house (ox and pig) directly in modified Alsevier's medium (pH 6.1) containing sodium citrate (30 mM), sodium chloride (77 mM), glucose (114 mM), neomycin sulphate (100 μg/ml) and chloramphenicol (330 μg/ml) at a ratio of 2:8. Erythrocytes were suspended and washed three times by centrifugation at 4,000g for 5 min with ten volumes of Tris-Buffered Saline (TBS) pH 6.5 (Tris- HCl 50 mM, NaCl 100 mM, CaCl2 10 mM) and resuspended in the same as 1.5 % suspension. Hemagglutination (HA) Assay The HA activity of the hemolymph agglutinin was assayed by measuring its ability to agglutinate erythrocytes. HA assays were performed at 30 °C by serial dilution of the hemolymph (25 µl) with TBS (25 µl) and mixing with 25 µl of 1.5 % erythrocyte suspension. HA titer was determined by the visual estimation of erythrocyte agglutination on microtiter plates 60 min after adding the cells. The HA titer (the units of agglutinin activity) is the reciprocal of the highest dilution of the sample that gave agglutination. To develop strategies for affinity purification, HA assay was also performed with high agglutinating rabbit erythrocytes at different pH, temperature and using buffer with different concentrations of cations calcium, magnesium and manganese and chelators, EDTA and EGTA. Table 2 Effect of moulting on the biochemical factors and HA titer of the hemolymph of the millipede of Thyropygus descriptus Parameters analysed Premoult (n = 15) Freshmoult (n = 10) Postmoult (n = 10) Intermoult (n = 25) Volume (ml/animal) 2.568±1.229 2.967±1.09 2.78±1. 922 2. 65±1.27 Water content (mg%) 96.887±1.1 98.35±1.218 97.232±1. 29 97.335±1.209 Calcium (mM) 12.983±0.045 13.101±0.321 12.503±0.245 12.801±0.504 Protein (µg/25µl) 0.430±0.031 0.432±0. 5 0.441±0.053 0.461±0.035 HA Titer 1024 4096 2048 2048 332 Fig. 1 Effect of pH, temperature and starvation on hemagglutination assay of the hemolymph agglutinin of the millipede Thyropygus descriptus To study the effect of pH on HA titer, the hemolymph sample was mixed with TBS at specific pH (5 - 9) at 1:1 ratio and incubated for 1 h and serially diluted in microtiter plates having TBS of same pH before adding erythrocyte suspension. To study the effect of temperature on HA titer, the hemolymph sample was incubated for 1 h at specific temperature (10 - 50 °C) and used for HA assay. To study the effect of cations and chelators on HA titer, the hemolymph sample incubated for 1 h in equal volume of TBS containing specific concentration (0.01,0.1,1.0 and 10 mM) of cations (calcium, magnesium and manganese) and chelators (EDTA and EGTA) was used for HA assay. In addition, HA was also carried out in the hemolymph obtained from animals fed with different foods, subjected to starvation and various stages of moult cycle after determining the volume, protein, calcium and water content of the hemolymph. Cross-adsorption test Packed erythrocytes (rabbit/rat/human A) were prepared by repeated washing of erythrocytes in 0.9 % saline by centrifugation at 4,000g for 5 min until we get a clear pellet. Hemolymph was mixed with equal volume of packed rabbit/rat/human A erythrocytes and incubated for 18 h at 4 °C with occasional shaking. After centrifugation, the supernatant was analyzed for HA. Hemagglutination inhibition (HAI) assay Hemolymph (25 µl) diluted to sub agglutination concentration (dilution at which hemolymph was able to provide 2 wells HA) was added to each well containing 25 µl of known concentration of serially diluted inhibitors (glycoproteins, mono and oligosaccharides). After incubation for 1 h, 25 µl of 1.5 % rabbit erythrocyte suspension was added. The HAI titer is reported as the reciprocal of the highest dilution of inhibitors giving complete inhibition of agglutination after 60 min. Protease treatment Rabbit erythrocytes washed with TBS by centrifugation at 4,000g were mixed with equal volumes of mg/ml of trypsin and chymotrypsin and 0.25 mg/ml of pronase and incubated for 4 h at 37 °C. The treated cells were pelleted by low speed centrifugation in TBS-BSA and used for HA assay. Sialidase treatment A reaction mixture containing 10 % washed rabbit erythrocytes in TBS-BSA (pH 6.5) and 140 mU sialidase of C. perfringens (Sigma-Type X) was incubated at 37 °C for 4 h. Sialidase treated cells were washed with TBS-BSA three times, pelleted by low speed centrifugation and used for HA assay. 333 Results HA activity of hemolymph The hemolymph of all the millipedes T. descriptus, X. acuticonus, A. disticta and A. craspedota agglutinated erythrocytes with diverse specificity. All the tested species agglutinated rabbit erythrocytes much higher than the other red blood cells tested (Table 1). Among the species assayed, maximum agglutinability was observed in the hemolymph of the millipede T. descriptus. Hence further characterizations were restricted to the hemolymph of the millipede, T. descriptus. Hemolymph had maximum agglutinin activity at pH 6.5 (Fig.1a) and temperature 30 - 35 °C (Fig. 1b). Calcium, magnesium, manganese, EDTA and EGTA at all the concentrations (0.01, 0.1, 1.0, 10.0 mM) did not have any influence on HA. Maximum HA was observed in the freshmoult stages than in the pre and post and intermoult stages (Table 2). Although varieties of food fed to the animals did not alter the HA, prolonged starvation significantly reduced the HA of the hemolymph (Fig. 1c). HA activity after adsorption with different erythrocytes When T. descriptus hemolymph adsorbed to rabbit, rat and human A erythrocytes was used for HA assay with rabbit, rat and human A erythrocytes, it failed to agglutinate the erythrocytes of any other species except rabbit erythrocytes (Table 3). Table 3 Cross adsorption assay of the hemolymph agglutinin of the millipede Thyropygus descriptus Erythrocyte adsorbed (n = 10) HA Titer Rabbit Rat Human A None 2048 128 64 Rabbit 0 0 0 Human A 16 (0) 0 0 Rat 16 (0) 0 0 Values in parenthesis refer to HA titers after second and subsequent adsorptions. Inhibitors of HA N-acetyl lactosamine (LacNAc), N-acetyl galactosamine (GalNAc) and lactose effectively inhibited the hemagglutinating activity. Though sialidase treatment reduced the agglutinability, free sialic acid was a weak inhibitor of the hemolymph agglutinin. Among glycoproteins, porcine stomach mucin (PSM) and lactoferrin inhibited the hemolymph agglutinin strongly (Tables 4). Table 4 Hemagglutination inhibition (HAI) of the hemolymph agglutinin of the millipede Thyropygus descriptus Inhibition by sugars Inhibitors (n = 5) HAI titer Min. con. req. for HAI (mM) Inhibitory potency (%) N-acetyl lactosamine 64 1.062 100 N-acetyl galactosamine 64 1.062 100 Lactose 64 1.062 100 Galactose 16 6.25 25 N-acetyl Neuraminic acid 8 12.5 12.5 Inhibition by glycoproteins Inhibitors HAI titer Min. con. req. for HAI (µg/ml) Inhibitory potency (%) Porcine stomach mucin 2048 4.88 100 Lactoferrin 512 19.53 25 Bovine submaxillary mucin 8 1250 0.39 Transferrin 8 1250 0.39 Fetuin 4 2500 0.195 Thyroglobulin 0 - - 334 Table 5 Effect of enzymatic cleavage of rabbit erythrocytes on hemagglutination assay of the hemolymph agglutinin of the millipede Thyropygus descriptus Enzymes used Site of enzyme action HA Titer None - 2048 Neuraminidase C. perfringens Type X (140 mU) NeuAcα2,3Gal;NeuAc α2,6Gal; NeuAcα2,8Gal; 512 Trypsin (1 mg/ml) Arg-, Lys- 32768 Chymotrypsin (1 mg/ml) Tyr-Trp-Phe-Leu- 32768 Pronase (0.25 mg/ml) All peptide links 65536 Sugars such as N-acetyl glucosamine, glucose, maltose, mannose, xylose, fructose and arabinose and glycoproteins such as bovine and porcine thyroglobulin did not inhibit HA at concentration 50 mM and 2.5 mg/ml respectively. HA activity of hemolymph after enzymatic alteration of erythrocytes HA activity got reduced when tested with neuraminidase (sialidase) treated rabbit erythrocytes and increased when tested with protease treated rabbit erythrocytes (Table 5). Discussion The hemolymph of all the four millipedes T. descriptus, X. acuticonus, A. disticta and A. craspedota recognized rabbit erythrocytes with great specificity. The ability of the millipede agglutinin to agglutinate rabbit erythrocytes argues for the specific recognition of the sugars constituting the glycocalyx of these erythrocytes, which serve as receptors to ligands as in the eukaryotic cells (Hakomori, 1973). It has been found that different animal species have characteristic receptor determinants on their erythrocyte surface (Yamakawa and Suzuki, 1953) and interspecies variations (Yasue et al., 1978). Reduction in HA following starvation beyond 20 days and stability of HA after feeding different types of food reveals the presence of a natural agglutinin. Identification of maximum hemagglutinability in the freshmoult stage of the millipede suggest defense role of this lectin in protecting the animals from foreign invaders and in the development of the millipede. As the exoskeleton of the freshmoult animals is extremely soft it may remain susceptible to the attack of any pathogen. Hence the presence of high amount of agglutinin in the hemolymph could be a defense mechanism to evade microbial attack. Like agglutinins from many other species (Miller et al., 1972), the T. descriptus agglutinin is also sensitive to pH and temperature. The loss of biological activity of the agglutinin with increased temperature could be related to destabilization of sporadic weak interactions of tertiary structure responsible for native conformation of lectin (Singh and Saxena, 2013). HA assay with different concentrations of cations such as calcium, magnesium and manganese and chelators such as EGTA and EDTA suggests that the lectin is calcium independent. The removal of HA following adsorption of the hemolymph to rabbit erythrocytes suggest the presence of a single hemagglutinin as reported in C. antennarius (Ravindranath et al., 1985a), Scylla serrata (Mercy and Ravindranath, 1992, 1993) and Paratelphusa jaquemontii (Maghil et al., 2003). However, the hemolymph when adsorbed to rat and human A erythrocytes continued to agglutinate rabbit erythrocytes suggesting the presence of remnants of agglutinability in the hemolymph capable of recognizing rabbit erythrocytes even after repeated adsorptions. This is supported by the serological studies which show that activity to one type of erythrocyte can be adsorbed by that type of erythrocytes, leaving residual agglutinating activity to other type of erythrocytes (Noguchi, 1903). The rise in HA activity following treatment of erythrocytes with proteases may be due to the removal of certain proteins which mask the glycocalyx of the rabbit erythrocytes that are specifically recognized by the millipede hemolymph agglutinin. The potent inhibitor of the agglutinin is PSM, a glycoprotein rich in GalNAc residues. Accordingly, GalNAc also inhibits the agglutinating activity at 1.5 mM concentration, but the ability of the hemolymph to agglutinate rabbit erythrocytes and its reduction following desialylation of erythrocytes argues for the affinity of the agglutinin to sialic acids. Among the sialic acid content of PSM, 90 % exist as N- glycolyl- neuraminic acid and 10 % as N-acetyl neuraminic acid and traces as N-O-acetyl neuraminic acid. High HA with rabbit erythrocytes containing NeuGc (Bhavananthan et al., 1964) and inhibition by PSM containing 90 % NeuGc (Schoop and Faillard, 1967) accounts for NeuGc specificity, which can be confirmed only after affinity purification of the lectin. Conclusion In spite of various information, the exact specificity of the agglutinin based on sugar specificity can be clearly stated only upon purification. This study provides the physico- 335 chemical requirements of the hemolymph agglutinin for affinity purification. The presence of sialic acid binding agglutinins in arthropods such as T. descriptus that are incapable of synthesizing sialic acids suggest that these agglutinins may be involved in the innate immunity of these organisms. References Bhavanandan VP, Buddecke E, Carubell R, Gottschalk A. The complete enzymatic degradation of glycopeptides containing O-seryl and O-threonyl linked carbohydrate. Biochem. Biophys. Res. Commun. 16: 353-361, 1964. Bretting H, Kabat, EA. Purification and characterization of agglutinins from the sponge Axinella polypoides and a study of their combining sites. Biochemistry 15: 3228-3236, 1976. Goldstein IJ, Hughes RC, Monsigny M, Osawa T, Sharon N. What should be called a lectin? Nature 285: 66, 1980. Hakomori SI. Glycolipids of tumor cell membrane. Adv. Cancer. Res. 18: 265-315, 1973. Higashi H, Hirabayashi Y, Fukui Y, Naiki M, Matsumoto M, Ueda S, et al. Characterization of N-glycolylneuraminic acid containing gangliosides as tumor associated Hanganutziu- Deicher antigen in human colon cancer. Cancer Res. 45: 3796-3802, 1985. Kawai T, Kato A, Higashi H, Kato S, Naiki M. Quantitative determination of N- glycolylneuraminic acid expression in human cancerous tissues and avian lymphoma cell lines as tumor-associated sialic acid by gas chromatography mass spectrometry, Cancer Res. 51: 1242-1247, 1991. Kobiler D, Mirelman D. Lectin activity in Entamoeba histolytica trophozoites. Infect. Immun. 29: 221- 225, 1980. Koch OM, Lee CK, Uhlenbruck G. Cerianthinlectins: a new group of agglutinins from Cerianthus membranaceus. Immunobiology 163: 53-62, 1982. Maghil D, Mercy PD, Bai RN, Suriya JS. Purification and characterization of a sialic acid specific lectin from the hemolymph of the freshwater crab Paratelphusa jaquemontii. Eur. J. Biochem. 270: 4348-4355, 2003. Mauchamp B. Purification of an N-acetyl-D- glucosamine specific lectin (P.B.A.) from epidermal cell membranes of Pieris brassicae. L. Biochimie 64: 1001-1008, 1982. Mercy Sr. PD, Ravindranath MH. An agglutinin with unique specificity for N-glycolylsialic acid residues of thyroglobulin in the hemolymph of a marine crab Scylla serrata (Forskal). Experientia 48: 498-500, 1992. Mercy Sr. PD, Ravindranath MH. Purification and characterization of N-glycolylneuraminic acid - specific lectin from Scylla serrata. Eur. J. Biochem. 215: 697-704, 1993. Miller H, Ballback S, Tauley GB, Krassner SM. A preliminary physico-chemical characterization of an agglutinin found in the hemolymph of the cray fish Procambarus clarkii. J. Invertebr. Pathol. 19: 83-93, 1972. Mullainadhan P, Ravindranath MH, Wright RK, Cooper EL. Crustacean defense strategies 1, Molecular weight dependent clearance of dyes in the mud crab Scylla serrata (Forskal), (Portunidae:Brachyura). Dev.Comp. Immunol. 8: 41-50, 1984. Noguchi, H. On the multiplicity of the serum hemagglutinin of cold blooded animals. Zentr. Bakt. Abt. 34: 2865, 1903. Ravindranath MH, Cooper EL. Crab lectins: receptor specificity and biomedical applications. Progr. Clin. Biol. Res. 157: 83-96, 1984. Ravindranath MH, Higa HH, Cooper EL, Paulson JC. Purification and characterization of an O-acetyl sialic acid specific lectin from a marine crab Cancer antennarius. J. Biol. Chem. 260: 8850- 8856, 1985a. Ravindranath MH, Paulson JC. Irie RF. Human melanoma antigen O-acetylated ganglioside GD3 is recognized by Cancer antennarius. J. Biol. Chem. 260: 8838-8845, 1985b. Schoop HJ, Faillard H. Contribution to the biosynthesis of the glycolyl group of N - glycolylneuraminic acid. Hoppe Seylers Z. Physiol. Chem. 348: 1518-1524, 1967. Sharon N, Lis H. Cell-agglutinating and sugar- specific proteins. Science 177: 949-959, 1972. Sharon N. Lectins: past, present and future Biochem. Soci. Trans. 36: 1457-1460, 2008. Shimizu S, Ito M. Niwa M. Lectins in the hemolymph of Japanese horseshoe crab, Tachypleus tridentatus. Biochem. Biophys. Acta 500: 71-79, 1977. Singh AP, Saxena KD. Effect of temperature, pH and denaturing agents on biological activity of MCJ lectin. Chem. Sci. Trans. 2: 1508-1512, 2013. Stromberg P, Nyholmt G, Paschert I, Normark S. Saccharide orientation at the cell surface affects glycolipid receptor function, Proc. Natl. Acad. Sci. USA 88: 9340-9344, 1991. Yamakawa T, Suzuki, S. The chemistry of lipids of post hemolytic residue or stroma of erythrocytes. IV. Distribution of lipid hexamine and lipid hemataminic acid in red blood corpuscles of various species of animals. J. Biochem. 40: 7-10, 1953. Yasue S, Honda S, Miyagawa S, Inoue J, Hasegava A, Yamakava T. Difference in the form of sialic acid in red blood cells glycolipids of different breeds of dogs. J. Biochem. 83: 1101-1107, 1978. 336